Zhou Xuedong Editor Principles and Management

Dental Caries

123

Zhou Xuedong

Editor

Principles and

Management

Dental Caries

Zhou Xuedong

Editor

Dental Caries

Principles and Management

ISBN 978-3-662-47449-5 ISBN 978-3-662-47450-1 (eBook)

DOI 10.1007/978-3-662-47450-1

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Editor

Zhou Xuedong

State Key Laboratory of Oral Diseases

West China Hospital of Stomatology

Sichuan University

Chengdu

China

1 Tooth Development: Embryology

of the Craniofacial Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Zheng Liwei , Wang Chenglin , and Ye Ling

2 Bioﬁ lm and Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Xu Xin , Zhou Yuan , Shi Wenyuan , Liu Yaling ,

and Zhou Xuedong

3 Saliva and Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Wang Renke

4 Demineralization and Remineralization . . . . . . . . . . . . . . . . . . 71

Cheng Lei , Li Jiyao , Xu Hockin H. K. ,

and Zhou Xuedong

5 The Diagnosis for Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Yang Liu , Li Boer , Wang Shuang , Zhang Yaru ,

and Peng Li

6 Dental Caries: Disease Burden Versus Its Prevention . . . . . . . 91

Hong Xiao

7 Clinical Management of Dental Caries . . . . . . . . . . . . . . . . . . . 107

Li Jiyao

8 Dental Caries and Systemic Diseases . . . . . . . . . . . . . . . . . . . . . 129

Zou Ling and Hu Tao

9 Models in Caries Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Huang Xuelian , Guo Qiang , Ren Biao , Li Yuqing ,

and Zhou Xuedong

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Contents

vii

Ren Biao State Key Laboratory of Oral Diseases , Sichuan University ,

Chengdu , People’s Republic of China

Li Boer State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Wang Chenglin State Key Laboratory of Oral Diseases, West China

Hospital of Stomatology, Sichuan University, Chengdu, China

Xu Hockin H. K. Biomaterials & Tissue Engineering Division,

Department of Endodontics, Prosthodontics and Operative Dentistry ,

University of Maryland Dental School , Baltimore , MD , USA

Li Jiyao State Key Laboratory of Oral Diseases , Sichuan University ,

Chengdu , People’s Republic of China

Department of Operative Dentistry and Endodontics , West China Hospital

of Stomatology, Sichuan University , Chengdu , People’s Republic of China

Cheng Lei State Key Laboratory of Oral Diseases , Sichuan University ,

Chengdu , People’s Republic of China

Department of Operative Dentistry and Endodontics , West China Hospital

of Stomatology, Sichuan University , Chengdu , People’s Republic of China

Peng Li State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Ye Ling State Key Laboratory of Oral Diseases, West China Hospital

of Stomatology, Sichuan University, Chengdu, China

Zou Ling Department of Conservation Dentistry and Endodontics, West

China Hospital of Stomatology, Sichuan University , Chengdu , People’s

Republic of China

Yang Liu State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Contributors

viii

Zheng Liwei State Key Laboratory of Oral Diseases, West China Hospital

of Stomatology, Sichuan University, Chengdu, China

Guo Qiang State Key Laboratory of Oral Diseases , Sichuan University ,

Chengdu , People’s Republic of China

Wang Renke West China Hospital of Stomatology, Sichuan University ,

Chengdu , People’s Republic of China

Wang Shuang State Key Laboratory of Oral Diseases , West China Hospital

of Stomatology, Sichuan University , Chengdu , People’s Republic of China

H u T a o Department of Preventive Dentistry, West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Shi Wenyuan School of Dentistry , University of California-Los Angeles ,

Los Angeles , CA , USA

Hong Xiao Department of Preventive Dentistry, West China Hospital of

Stomatology, Sichuan University, Chengdu, China

Xu Xin State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Zhou Xuedong State Key Laboratory of Oral Diseases , West China

Hospital of Stomatology, Sichuan University , Chengdu , People’s Republic

of China

Department of Operative Dentistry and Endodontics , West China Hospital

of Stomatology, Sichuan University , Chengdu , People’s Republic of China

Huang Xuelian State Key Laboratory of Oral Diseases , Sichuan

University , Chengdu , People’s Republic of China

Department of Operative Dentistry and Endodontics , West China Hospital

of Stomatology, Sichuan University , Chengdu , China

Liu Yaling State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Department of Oral Biology , College of Dentistry, University of Florida ,

Gainesville , FL , USA

Zhang Yaru State Key Laboratory of Oral Diseases , West China Hospital

of Stomatology, Sichuan University , Chengdu , People’s Republic of China

Zhou Yuan State Key Laboratory of Oral Diseases , West China Hospital of

Stomatology, Sichuan University , Chengdu , People’s Republic of China

Li Yuqing State Key Laboratory of Oral Diseases , Sichuan University ,

Chengdu , People’s Republic of China

Contributors

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_1

Tooth Development: Embryology

of the Craniofacial Tissues

Zheng Liwei , Wang Chenglin , and Ye Ling

1.1 Embryology

of the Craniofacial Tissues

The development of craniofacial tissues is part of

human prenatal development. Generally, human

prenatal development goes through three stages:

the proliferative 2-week period, when cell divi-

sion is prevalent; the embryonic period, which

extends from the second to the eighth weeks; and

the fetal period, from eighth week to birth [ 1 ].

With normal accomplishment of the develop-

ment, human body forms stepwise.

1.1.1 Origin of Human Tissue

The origin of tissue begins with fertilization,

which is the fusion of spermatozoa and ova to

form a zygote. Then the zygote moves to the uter-

ine cavity where it will implant into the wall of

the uterus and, meanwhile, undergoes a series of

rapid divisions that lead to the formation of a

ﬂ uid ﬁ lled hollow ball, termed blastocyst, and

small inner cell mass. When this blastocyst

attaches to the sticky wall of the body of the

uterus, uterine endometrium is digested, allowing

blastocyst embedded in its surface and then

deeper penetration. Implantation takes place.

On either side of the inner cell mass, two small

cavities are formed. A small disk (the embryonic

disk) develops in the center, where they reach

each other. The embryonic disk becomes the

embryo, composed of two layers of cells. One

layer is lined with ectodermal cells, which will

form the outer body covering (epithelium), called

ectodermal layer. The cells on the ventral aspect

are endodermal cells, forming the endodermal

layer. This conﬁ guration is completed in the ﬁ rst 2

weeks, which is termed “proliferative period” [ 1 ].

During the third week, two-layered embryonic

disk is converted to a three-layered disk. Cells

that develop between the ectodermal and endo-

dermal layers become the mesodermal layer.

Next, major tissues and organs, including oral

maxillofacial tissue such as tooth and facial

bones, differentiate from these three layers [

2 ].

Key events are the development of the nervous

system, differentiation of neural crest tissue from

the ectoderm, and folding of the embryo.

1.1.2 The Neural Crest

The nervous system begins with a speciﬁ cation of

the neural plate, which develops as a thickening

within the anterior ectodermal layer. Meanwhile,

the neural plate develops raised folds at its margins.

These folds in turn encompass and fuse so that neu-

ral tube forms and separates from the ectoderm.

Z. Liwei • W. Chenglin • Y. Ling (*)

State Key Laboratory of Oral Diseases , West China

Hospital of Stomatology, Sichuan University ,

Chengdu , 610041 Sichuan , China

e-mail:

[email protected]

Upon closure of the neural tube, a unique

population of cells known as neural crest cells

separate from the lateral aspect of the neural

plate. These cells have the capability of migration

and differentiation. This is especially obvious in

the head and neck region, and neural crest cells

have an important role in the head development.

They contribute to most of the embryonic connec-

tive tissue of facial region, which includes dental

tissues such as the pulp, dentin, and cementum.

Consequently, embryonic connective tissue in the

head is termed as ectomesenchyme, reﬂ ecting its

origin from the ectoderm, whereas connective tis-

sue elsewhere is derived from the mesoderm and

is known as mesenchyme. Although the neural

crest tissues arise from neural ectoderm, they

exhibit properties of mesenchyme [ 2 , 3 ].

1.1.3 Head Formation

The head fold of the three-layered embryo is cru-

cial and produces the primitive stomatodeum or

oral cavity. When the stomatodeum ﬁ rst forms, it

is surrounded by frontal prominence rostrally and

by the cardiac bulge caudally. And it is separated

from the foregut by buccopharyngeal membrane,

a bilaminar structure consisting of ectoderm and

endoderm, which breaks down soon so that the

stomatodeum communicates with the foregut.

Laterally the somatodeum becomes delimited by

the ﬁ rst pair of pharyngeal arches [ 1 , 2 ].

1.2 Enamel Development

Fully mature enamel comprises 80–90 % (v/v)

carbonated calcium hydroxyapatite crystals,

which is in contrast to bone and dentin, both with

10 % and 13 % (v/v) carbonated calcium hydroxy-

apatite crystals, respectively. The mechanisms of

crystal initiation, crystal growth, as well as mor-

phology are related to amelogenesis. In develop-

ing teeth, sequential and reciprocal interactions

occur between the epithelium and the underlying

mesenchyme, which derive from the cranial neu-

ral crest. Enamel formation associates with the

differentiation of the tooth-speciﬁ c cell types, the

epithelial ameloblasts. This chapter will provide a

brief overview in different aspects of tooth enamel

development with a particular emphasis on the

current knowledge of enamel morphogenesis, his-

togenesis, and cytodifferentiation.

1.2.1 Histogenesis

and Morphogenesis

The consecutive phases during tooth morphologic

changes, including lamina, bud, cap, and bell

stages, are characterized by epithelial histogene-

sis. The segmentation of the dental epithelium

occurs in the early tooth initiation, which indi-

cates that a local epithelial thickening corresponds

to the dental lamina. Experiment approaches sug-

gested that Wnt/Shh interactions may regulate the

delimitation between the dental epithelium and

the oral ectoderm. Nevertheless, the molecules

intervene in regulating epithelial cell apoptosis,

and survival or compartmentalization of different

elements is still poorly understood (Fig. 1.1 ).

1.2.1.1 Bud Stage

From the bud stage, the thickened presumptive

dental epithelium, which forms the basal epithe-

lium, can be distinguished from the round inter-

nal cells. Depending on the position, epithelial

cells are evident in changes of the expression of

different molecules, including γ-catenin, desmo-

glein, and P- and E-cadherins.

1.2.1.2 Cap Stage

During the cap stage, the dental epithelium

becomes the enamel organ which consists of four

different cell types: inner and outer dental epithe-

lia, the stellate reticulum, as well as transiently the

primary enamel knot. At this time, the inner

enamel epithelium becomes discernible from the

outer enamel epithelium. The histogenesis of the

inner dental epithelium is coordinated by a change

in the composition of the basement membrane.

The enamel knot is a dynamic transient struc-

ture and appears at the onset of mammalian tooth

shape development, which is in contact with sev-

eral cells, including peripheral cells, internal

round cells, and basement membrane cells.

Z. Liwei et al.

Studies indicated that the primary enamel knot

represents a signaling center in formatting cusps,

which may lead to unequal growth of the enamel

epithelium and induce the formation of second-

ary enamel knots.

It has been suggested that the structure and

organization of primary enamel knot rapidly

change during the time. At the beginning of the

cap stage, it appears as a long cylindrical shape

and the shape will extend along the mesial–distal

axis of the ﬁ rst lower molar. Soon after, some of

internal cells begin apoptosis. While the ﬁ rst

lower molar grows during cap formation, the pri-

mary enamel knot starts to extend in anterior and

posterior directions. It is suggested that in the

primary enamel knot, most cells do not divide and

its proliferation needs the recruitment of cells

within the enamel organ. However, the underlying

mechanism is still not known due to differences in

mouse strains or measuring stages of embryos.

1.2.1.3 Bell Stage

At the bell stage, the enamel organ delimitates the

dental papilla and starts to form cusps. At this

time, the secondary enamel knots form, which

only precede cusp formation by a few hours. The

secondary enamel knots are taken place at the tips

of the forming cusps at the bell stage. The rela-

tionship between primary and secondary enamel

knots has been suggested in several models. The

gene expression patterns elucidated the primary

enamel knot induces the secondary enamel knots

by a reaction–diffusion-related mechanism.

During the bell stage of tooth development,

the shape of the crown is determined. The growth

of crown results from cell division and reorgani-

zation of inner dental epithelium. Furthermore,

the programmed cell death is also accompanied

by the regulation of cell number in the inner den-

tal epithelium, suggesting its role in determining

the ﬁ nal number of functional ameloblast cells.

1.2.2 Cytodifferentiation

From the lamina stage to the bell stage, changes

in reorganization of the epithelium compartment

can be distinguished. They not only regulate his-

togenesis but also determine the ﬁ nal number and

speciﬁ c positioning of functional ameloblasts.

Amelogenesis, or enamel formation, consists

of two main steps. The ﬁ rst step creates partially

mineralized enamel (about 30 %). The second

step involves extreme inﬂ ux of additional mineral

while removing organic material and water to

form more than 96 % mineral contents. The dif-

ferentiation of epithelial cells into functional ame-

loblast cells includes several morphologic changes

that occur in time and involve growth, elongation

of the cytoplasm, polarization, and secretion of

matrix protein. These epithelial cells exhibit a

unique character of progressively changed pheno-

type. Amelogenesis has been described in as

many as six stages but generally is divided into

three functional phases, known as presecretory,

secretory, and maturation stages (Fig.

1.2 ).

ab c d

Fig. 1.1 ( a – d ) Morphogenesis of the tooth development (from bud to bell stage)

1 Tooth Development: Embryology of the Craniofacial Tissues

1.2.2.1 Presecretory Stage

During the presecretory phase, the cells of the

inner enamel epithelium start to differentiate into

ameloblast cells. At the morphogenetic phase,

inner enamel epithelium cells are cuboidal or low

columnar, with large centrally located nuclei and

poorly formed organelles in the proximal portion

of the cells. During differentiation phase, once

stimulated, these cells elongate, their nuclei shift

distally toward the stratum intermedium, and the

Golgi elements increase and migrate distally.

Moreover, the cytoplasm becomes ﬁ lled with

organelles which are needed for the synthesis and

secretion of enamel proteins. At this time, a sec-

ond junctional complex forms at the distal

extremity of the cell, compartmentalizing the

ameloblast cells into a body and a distal exten-

sion called Tomes’ process, against which enamel

develops.

Although the pre-ameloblasts have been

regarded as nonsecreting cells, more and more

researches demonstrate that the enamel protein

secretion starts much earlier, even before the sep-

aration between pre-ameloblasts and pre-

odontoblasts. Ameloblast cells are aligned closely

with each other due to the tight junctional com-

plex or attachment specializations [ 2 ]. These

junctional complexes greatly involved in amelo-

genesis determine what may pass between cells to

enter or leave the enamel at different times.

1.2.2.2 Secretory Stage

The newly formed ameloblasts near the dental

papilla are ﬂ at and the matrix secreted is called

rodless enamel matrix. During the secretory

stage, the ameloblasts exhibit a tall columnar and

polarized morphology and secrete an extracellu-

lar protein-rich matrix. The ﬁ ne structure of

secretory stage ameloblasts indicated their strong

synthetic and secretory activity. The Golgi com-

plex is intense and forms a cylindrical organelle

surrounded by many cisternae of rough endoplas-

mic reticulum. Ameloblast secretion is constitu-

tive, which means the secretion is successively,

and the secretory granules are not stored for pro-

longed periods of time.

When enamel formation begins, Tomes’ pro-

cess comprises only a proximal portion. The

secretory granules are released along the surface

of the process against the newly formed mantle

Fig. 1.2 Ameloblast

differentiation

Z. Liwei et al.

dentin to create an initial layer of the enamel

without enamel rods. The very ﬁ rst hydroxyapatite

crystals formed interdigitate with the dentin crys-

tals. After forming the initial enamel layer, ame-

loblast cells migrate from the dentin surface and

form the distal portion of Tomes’ process as an

outgrowth of the proximal portion. The distal

portion extends into and interdigitates beyond the

initial layer of enamel, while the proximal por-

tion penetrates from the distal junctional com-

plex to the enamel layer surface [ 2 ].

It is believed that the distal portion of Tomes’

process progressively lengthens as the enamel

layer thickens and gradually turns to be thinner as

the rod developing in diameter presses it against

the wall of the interrod cavity. Eventually, the

process is squeezed out of existent, leaving a nar-

row area which is ﬁ lled with organic materials

between the enamel rod and interrod enamel.

When the outer layer of enamel is being formed,

the distal portion of Tomes’ process is altered and

orientation also changed, leading to slightly dif-

ference of enamel rods in the outer third of layer

with a more rectilinear trajectory. Finally, the

ameloblasts become the same overall appearance

as when initial enamel was forming. Without the

distal portion of Tomes’ process, the ﬁ nal enamel

has no rods. Notably, the initial, interrod, and

ﬁ nal enamels are developed by the same secre-

tory surface and, indeed, form a continuum [ 2 ].

1.2.2.3 Maturation Stage

During the maturation stage, the ameloblasts aim

at resorbing much of the water and organic matrix

from the enamel in order to allow enough space

for the growing enamel crystals [ 2 ]. This change

results from the thickness and width growth of

preexisting crystals seeded during amelogenesis

formative stage, not due to additional crystal

accumulation.

It is believed that the stratum intermedium

cells are also related to secretory and absorptive

functions of amelogenesis and desmosomes facil-

itate their close contact with ameloblast cells. The

stratum intermedium cells appear less active as

enamel maturation is near completion [ 4 ].

After immature enamel has fully formed,

ameloblast cells undergo several morphologic

changes in preparing maturing the enamel. At

this time, a short transitional stage appears, dur-

ing which ameloblasts become shorter and their

volume and organelle content decrease. At the

maturation stage, some ameloblast cells undergo

programmed apoptosis; roughly about half of the

ameloblasts is reduced during amelogenesis.

In summary, ameloblasts arise from the inner

enamel epithelial cells and experience multiple

morphologic and functional changes. Following

the deposition of a layer of enamel, ameloblasts

deposit enamel in the form of rods or prisms that

become highly mineralized. The arrangement of

ameloblasts with their Tomes’ process plays a

critical role in the formation of enamel rods. The

process of amelogenesis is a series of successive

phases of proliferation, differentiation, secretion,

and maturation, eventually forming the enamel.

1.2.3 Microstructure of the Enamel

The enamel is a composite structure consisting

of mineral and organic phases. At the nanometer

scale, like most other naturally mineralized tis-

sues, dental enamel has hierarchical structures

and surface features [ 5 , 6 ]. On the microscale,

the enamel consists of highly organized architec-

tural units known as enamel prisms. On the

nanoscale, the enamel consists of highly crystal-

line nanorod- like calcium hydroxyl apatite crys-

tallites that are arranged roughly parallel to each

other [

7 ] (Fig. 1.3 ).

1.2.3.1 Enamel Rod

Using the scanning electron microscope and fol-

lowing a short etching part, enamel rods can be

observed in ground or fractured teeth. The enamel

rod represents the mineralized progress of amelo-

blasts and Tomes’ process. Enamel rods cross one

another and follow an undulating course as they

progress from the DEJ toward the enamel surface.

When the arcades connect to each other, enamel

rods have the features of keyholes or paddles, with

the convex surface of the arcades oriented in an

incisal or cuspal order. The enamel rods run nearly

perpendicular to all parts of tooth surface, stop-

ping at the ﬁ nal layer of aprismatic enamel [ 4 ].

1 Tooth Development: Embryology of the Craniofacial Tissues

1.2.3.2 Enamel Spindle

Enamel spindles are generated during the differ-

entiation stage of amelogenesis. When the initial

enamel is formed, the enamel spindles become

terminal extensions of the primary dentinal

tubule into the enamel matrix. Spindles exhibit

bulbous structures at DEJ region in mature tooth

enamel.

1.2.3.3 Enamel Lamellae and Cracks

It is believed that enamel lamellae are the result

of local failure of the maturation process. Enamel

lamellae include thin sheets of organic materials

that extend throughout the enamel mineralization

and exhibit vertical orientation from incisal or

cuspal regions to cervical area. Cracks share

some similar characters with lamellae in ground

section and usually appear as artifacts during

teeth processing.

1.2.3.4 Enamel Tufts

Enamel tufts originate from the DEJ and extend

1/3 to 1/2 of the thickness of the enamel matrix.

They are formed during Tomes’ process develop-

ment and also during the elaboration of the initial

enamel of the enamel rod. They represent protein-

rich regions that failed to mature in the enamel

matrix.

1.2.3.5 Interpit Continuum

The secretory product is released from the ame-

loblast cells at two preferred sites. The compara-

tively superﬁ cial site forms the majority parts of

all developing enamel surfaces. This site is inter-

ameloblastic and the product determines pits,

naming interpit for this stage. In many circum-

stances, the interpit phase is continuous through-

out vast parts of the tissue [ 8 ]. The second

location at which the enamel matrix is released is

from the secretory pole of Tomes’ process proper,

which aims at ﬁ lling in the pit. At these sites,

crystals may have orientations that merge with

those from the interpit stage, building open-sided

prism boundaries.

1.2.3.6 Functional Aspects of Enamel

Structure

The particular organization of the enamel serves

as enhancing hardness and wear resistance. The

parallel formation of crystals perpendicular to the

surface of the teeth brings about the best way for

dense packing of the crystals as well as obviates

the need to nucleate new crystals during enamel

maturation. It has a microporous structure, which

allows extra mineral ﬂ ow in the crystals for fur-

ther growth while degrading matrix components

to be removed. Although single crystal is too

Fig. 1.3 Enamel structures

Z. Liwei et al.

ﬂ exible, the perpendicular positions enable the

growth of long whisker-like crystals, allowing

the crystals form into larger domains to be stron-

ger and stiffer.

Enamel crystals are the largest crystals found

in the body. The primary structural unit of enamel

is the enamel rod, which is formed by the secre-

tory activity of ameloblasts. The orientation of

crystals and the distribution of organic matrix are

involved in maintaining structural properties of

enamel.

1.2.4 Enamel Matrix Proteins

Enamel formation requires a remarkable orches-

tration of diverse and essential enamel-secreted

proteins, including amelogenin, ameloblastin,

enamelin, amelotin, tuftelin, dentin sialoprotein,

and apin. Studies provide functional data show-

ing that the disruption of synthesizing, secreting,

and processing these genes can cause different

subtypes of amelogenesis imperfecta (AI), indi-

cating the indispensable role for enamel compo-

sition and maturation [ 9 ].

1.2.4.1 Enamelin

A number of studies have suggested that the ﬁ rst

protein to be secreted by ameloblasts at the den-

tin–enamel junction (DEJ) region is enamelin

[ 10 , 11 ]. Enamelin is a novel acidic enamel pro-

tein that has been postulated to play an essential

role in enamel mineralization. By high-resolution

protein-A gold immunocytochemistry, the acidic

feature of enamelin proteins has been reported to

be in line with its capability to bind to enamel

mineral crystallite surfaces [

12 ]. Enamelin is rich

in aspartic acid and could be arranged in β-sheet

conformation that results in nucleation of the

mineral component.

The enamelin proteins initially secreted at the

very early phase of enamel formation are strictly

expressed by ameloblasts and persist throughout

enamel developing and maturing stages [ 10 ]. The

mutations of enamelin such as enamelin-null

phenotype are associated with aberrations of

enamel, causing AIH2. Several studies have

described the mutations of enamelin gene caus-

ing an autosomal-dominant AI phenotype [

13 ]. In

Enam

−/−

mice, the enamel layer is completely

absent. The crust over the dentin is thin, irregular,

and easily abraded [ 10 ]. These analyses indicate

that enamelin is essential for enamel matrix orga-

nization and mineralization.

1.2.4.2 Amelogenin

The amelogenin proteins of developing dental

enamel are tissue-speciﬁ c components, rich in

leucine, histidine, proline, and glutamyl residues.

Among all the ameloblast-speciﬁ c proteins, ame-

logenin is the most abundant extracellular pro-

tein. The initial enamel layer is dominated by

amelogenin protein secretion. In human, the

amelogenin gene has been shown to be located

on both X and Y chromosomes [ 14 ]. Human

amelogenin genes have 7 exons, with the princi-

pal variation of sequence homology occurring

within exon 6, which codes for most amelogenin

core and the C-terminus [ 15 ].

It has been shown that the amelogenin nano-

spheres, the supramolecular assembly of amelo-

genin, such as elastin, appear as a functional

structural protein during enamel formation [ 16 ].

Two human pedigrees with an X-linked AI (AIH1)

phenotype both share the same mutation in the

amino-terminal, tyrosine-rich amelogenin peptide

(TRAP) segment [ 17 , 18 ]. The recombinant pro-

teins of those two AIH1 point mutations have been

compared with wild-type amelogenin, exhibiting

altered nanosphere dimensions and amelogenin

assembly kinetics [

19 , 20 ]. During in vivo enamel

formation, the amelogenin nanosphere also can be

observed adjacent to HAP crystallites [ 21 ].

It has been found that human-inherited enamel

defect AI often associates with alterations in ame-

logenin X chromosome gene [ 22 ]. The mutations

in amelogenin are known to hypoplastic or hypo-

mineralized enamel [ 22 , 23 ]. Amelogenin knock-

out mice also display abnormal teeth with

chalky-white discoloration, broken tips of incisors

and molars, as well as disorganized hypoplastic

enamel, indicating amelogenin proteins play a

major role in the regulation of enamel thickness

and organization of crystal pattern [ 24 ] (Fig. 1.4 ).

1 Tooth Development: Embryology of the Craniofacial Tissues

1.2.4.3 Ameloblastin

Ameloblastin, a cell adhesion molecule, is one of

the unique tooth-speciﬁ c proteins, expressed by

secretory ameloblasts, yet the expression decreases

during enamel maturation [ 25 ]. Shortly after den-

tal epithelium initial differentiation, the cells are

detached from the underlying matrix, resume pro-

liferation, and lose polarity, reversing to undiffer-

entiated one, indicating that ameloblastin

maintains the differentiation state of ameloblasts

at the secretory stage, by binding to ameloblasts

and by inhibiting their proliferation [ 26 ].

At secretory amelogenesis, ameloblastin distri-

bution following the ameloblast cell outline

appears to be a ‘ﬁ shnet’-like partitioning [ 27 ]. The

ameloblastin null mice reveal severe enamel hypo-

plasia, and overexpression of ameloblastin in the

enamel organ inﬂ uences enamel crystallite habit

and enamel rod morphology, resulting in a pheno-

type characteristic of AI. Undoubtedly, these data

all suggest that in the enamel matrix, either gain of

function or loss of function of ameloblastin can

cause enamel alterations. It has also demonstrated

that ameloblastin acts as a nucleator of crystalliza-

tion because it is expressed at mineralization initi-

ation sites within the enamel (Fig.

1.5 ).

1.2.4.4 Amelotin

Murine amelotin has been identiﬁ ed recently,

which is the newest described enamel-speciﬁ c

protein. In developing murine incisors and

molars, expression of amelotin mRNA was

restricted to maturation-stage ameloblasts [ 28 ].

Both murine and human amelotin genes contain

9 exons and 8 introns and are located on chromo-

somes 5 and 4q13.3, respectively, which are close

to the enamelin and ameloblastin genes. The

expression of amelotin mRNA is essentially lim-

ited in postsecretory ameloblasts, experiencing a

dramatic increase from secretory to maturation

phase ameloblasts and subsequently lessening

toward the zone of reduced ameloblast cells. Less

information is available describing whether or

not amelotin is a candidate gene for AI.

1.2.4.5 Tuftelin

Shortly after differentiation, tuftelin, an acidic pro-

tein, is synthesized and secreted. Tuftelin gene

localizes to chromosome 1q21 in human. In secre-

tory stage, the secretory pathway of amelogenin to

the extracellular space is from the Golgi complex

and then to Tomes’ processes [ 29 ]. However,

in vivo tuftelin accumulates in cytoplasmic area

other than the Golgi complex and secretes granules

in both mineralizing and nonmineralizing tissues.

1.2.4.6 Proteolytic Enzymes

There are two major proteinases secreted into the

enamel matrix, including matrix metalloprotein-

ase- 20 (MMP20, enamelysin) and kallikrein-4

(KLK4, enamel matrix serine proteinase-1, or ser-

ine proteinase 17).

Fig. 1.4 Immunostaining of amelogenin

Fig. 1.5 Immunostaining of ameloblastin

Z. Liwei et al.

Matrix Metalloproteinase-20 Human MMP20

gene consists of 10 exons and is part of the MMP

gene clusters. The human MMP20 is located on

chromosome 11q22.3, and an autosomal-

recessive form of AI was recently discovered in a

family that had a mutation in the intron 6 splice

acceptor [ 13 ]. In porcine teeth, both ameloblast

and odontoblast cells express MMP20. During

the early stage of enamel formation, MMP20

activity accounts for virtually all of the known

cleavage sites in amelogenin. The mutation of

MMP20 exhibits hypoplastic enamel with

improperly processed amelogenin and rod pat-

tern [

30 ]. In addition, the homozygous MMP20

mutation family reveals severely pigmented, brit-

tle, and soft enamel, which is characterized by

less radiodense.

Kallikrein-4 Human KLK4 gene is located on

chromosome 19q13.41. KLK4 was ﬁ rst discov-

ered in the teeth, but it also expressed in other

tissues such as the prostate. In the teeth, KLK4 is

secreted by different cell types, including odon-

toblasts and late-secretory and maturation phase

ameloblasts [ 31 ]. KLK4 expression during

enamel maturation correlates with the degrada-

tion of enamel proteins, thus indicating it is nec-

essary for the enamel to achieve the high level of

mineralization. KLK4 mutation was found in a

family with autosomal recessive hypomaturation

AI, showing yellow-brown discolored teeth. The

enamel fractured from the teeth has normal thick-

ness but with a decreased mineral content.

Notably, the affected members are all females, so

it is not sure whether KLK4 has an effect on the

prostate. However, only the teeth were apparently

altered by the homozygous KLK4 mutation [

23 ] .

1.3 Pulpodentin Complex

In a mature tooth, dentin is a unique, avascular

mineralized connective tissue that forms the bulk

of the tooth, and dentin encloses a richly inner-

vated and highly vascularized soft connective tis-

sue, the dental pulp. Dentin and pulp are derived

from the dental papilla, whose cells migrate from

the cranial neural crest. The tissues remain

closely associated during development and

throughout the life of an adult tooth and are hence

most commonly referred to as the “pulpodentin

complex.”

During the process of tooth development, most

attentions are focused on the common themes

about odontoblast differentiation that have

emerged and what is known about the inﬂ uence of

tooth-signaling molecules and transcription fac-

tors on the development and homeostasis of the

pulpodentin complex. In addition, the focus is the

theories about the general principles of dentin

matrix formation, particularly the synthesis and

secretion of extracellular matrix molecules and

their postulated roles in the biomineralization of

dentin, and the theories about the development

and homeostasis of differentiated and undifferen-

tiated or stem cell populations can be translated to

regenerative approaches targeted at restoring the

integrity of the adult pulpodentin complex.

1.3.1 Dentin

Fully mature dentin is composed of approximately

70 % inorganic material and 10 % water by weight.

The principal inorganic component consists of

(PO

)

(OH)

(hydroxyapatite). Organic matrix

accounts for 20 % of dentin. 91 % of organic matrix

is collagen, and most of the collagen is type I, with

a minor component of type V. Noncollagenous

matrix components include phosphoproteins, pro-

teoglycans, gamma-carboxyglutamate- containing

proteins (Gla- proteins), acidic glycoproteins,

growth factors, and lipids. By volume, inorganic

matter makes up 45 % of dentin, while organic

molecules and water 33 % and 22 %, respectively.

A characteristic of human dentin is the presence of

tubules that occupy from 20 to 30 % of the volume

of intact dentin. These tubules house the major cell

processes of odontoblasts. The elasticity of dentin

provides ﬂ exibility for the overlying brittle enamel.

1.3.1.1 Structure of Dentin

Dentinal Tubules The characteristic of dentin

is the presence of tubules, which host the major

cell processes of odontoblasts. Tubules form

1 Tooth Development: Embryology of the Craniofacial Tissues

around the odontoblast processes and thus trans-

verse the entire width of the dentin from the DEJ

or DCJ to the pulp. They are slightly tapered in

the wider portion situated toward the pulp. This

tapering is the result of the progressive forma-

tion of peritubular dentin, which leads to a con-

tinuous decrease in the diameter of the tubules

toward the enamel.

In coronal dentin, the tubules have a gentle S

shape as they extend from the DEJ to the pulp.

The S-shaped curvature is presumably the result

of the crowding of odontoblasts as they migrate

toward the center of the pulp. As they approach

the pulp, the tubules converge because the sur-

face of the pulp chamber has a much smaller area

than the surface of dentin along the DEJ.

The number and diameter of the tubules are

different at various distances from the pulp, and

the mean number and diameter of tubules

decrease following the increased distance

(Fig. 1.6 ) [ 32 ]. Investigators found the number

and diameter of dentinal tubules to be similar in

rats, cats, dogs, monkeys, and humans, indicating

that mammalian orthodentin has evolved amaz-

ingly constantly [ 33 ].

Near the DEJ, the dentinal tubules ramify into

one or more terminal branches; this is due to the

fact that during the initial stage of dentinogene-

sis, the differentiating odontoblasts extended sev-

eral cytoplasmic processes toward the DEJ, but,

as the odontoblasts withdrew, their processes

converged into one major process (Fig. 1.7 ).

Peritubular Dentin Dentin lining the tubules is

termed peritubular dentin , whereas that between

the tubules is known as intertubular dentin

(Fig. 1.8 ). Presumably precursors of the dentin

matrix that is deposited around each odontoblast

process are synthesized by the odontoblast, trans-

ported in secretory vesicles out into the process,

and released by reverse pinocytosis. With the for-

mation of peritubular dentin, there is a correspond-

ing reduction in the diameter of the process.

Peritubular dentin represents a specialized

form of orthodentin not common to all mammals.

The matrix of peritubular dentin differs from that

of intertubular dentin in having relatively fewer

collagen ﬁ brils and a higher proportion of sul-

fated proteoglycans. Because of its lower content

of collagen, peritubular dentin is more quickly

dissolved in acid than is intertubular dentin.

Peritubular dentin is more highly mineralized

and therefore harder than intertubular dentin. The

hardness of peritubular dentin may provide added

structural support for the intertubular dentin, thus

strengthening the tooth. By preferentially remov-

ing peritubular dentin, acid etching agents used

during dental restorative procedures enlarge the

openings of the dentinal tubules, thus making the

dentin more permeable.

Intertubular Dentin Intertubular dentin is

located between the rings of peritubular dentin

and constitutes the bulk of circumpulpal dentin.

Its organic matrix consists mainly of collagen

Fig. 1.6 Diagram illustrat-

ing the difference in size and

number of tubules on the

occlusal surface of coronal

dentin ( A and B ) and at the

cervical region of the root

surface ( C ). This combina-

tion is responsible for the

exponential increase in

dentin permeability with

depth (From Pashley [

32 ],

p. 106, ﬁ gure 2)

Z. Liwei et al.

ﬁ brils having diameters of 500–1000 Å. These

ﬁ brils are oriented approximately at right angles

to the dentinal tubules.

Interglobular Dentin

The term interglobular

dentin refers to organic matrix that remains

unmineralized because the mineralizing globules

Odontoblast process

Rough endoplasmic reticulum

Golgi complex

Mitochondria

Cytoplasm

Cytomembrane

Nucleus

Nucleolus

Nerves

Capillary

Fibre

Fig. 1.7 Diagrammatic

representation of the

differentiated odontoblast

Fig. 1.8 The cross section

of dentinal tubules.

( A ) Peritubular dentin;

( B ) intertubular dentin;

( C ) dentinal tubule

1 Tooth Development: Embryology of the Craniofacial Tissues

fail to coalesce. This occurs most often in the

circumpulpal dentin just below the mantle dentin

where the pattern of mineralization is more likely

to be globular than appositional. In certain dental

anomalies (e.g., vitamin D-resistant rickets and

hypophosphatasia), large areas of interglobular

dentin are a characteristic feature.

1.3.1.2 Types of Dentin

Developmental dentin is that which forms during

tooth development. That formed physiologically

after the root is fully developed is referred to as

the secondary dentin . Developmental dentin is

classiﬁ ed as orthodentin , the tubular form of den-

tin found in the teeth of all dentate mammals.

Mantle dentin is the ﬁ rst formed dentin and is

situated immediately subjacent to the enamel or

cementum. It is typiﬁ ed by its content of the thick

fan-shaped collagen ﬁ bers deposited immedi-

ately subjacent to the basal lamina during the ini-

tial stages of dentinogenesis. Spaces between the

ﬁ bers are occupied by smaller collagen ﬁ brils

lying more or less parallel with DEJ or DCJ. The

width of mantle dentin in human teeth has been

estimated at 80–100 μm [ 34 ].

Circumpulpal dentin is formed after the layer

of mantle dentin has been deposited, and it con-

stitutes the major part of developmental dentin.

The organic matrix is composed mainly of colla-

gen ﬁ brils, approximately 500 Å in diameter that

is oriented at right angles to the long axis of the

dentinal tubules. These ﬁ brils are closely packed

together and form an interwoven network.

Predentin is the unmineralized organic matrix

of dentin situated between the layer of odonto-

blasts and the mineralized dentin. Its macromo-

lecular constituents include type I and type II

trimer collagens, and noncollagen elements con-

sist of several proteoglycans (dermatan sulfate,

heparan sulfate, hyaluronate, keratan sulfate,

chondroitin-4-sulfate, chondroitin-6-sulfate),

glycoproteins, glycosaminoglycans (GAGs),

Gla-proteins, dentin phosphoproteins (DPP), and

a tissue-speciﬁ c molecule which is unique to the

odontoblast cell lineage. DPP is produced by the

odontoblast and transported to the mineralization

front, and it is thought to bind to calcium and

play a role in mineralization.

1.3.1.3 Mineralization of Dentin

Mineralization of dentin matrix commences with

the initial increment of mantle dentin. Calcium

phosphate crystals begin to accumulate in matrix

vesicles within the predentin [ 35 ]. Presumably

these vesicles bud off from the cytoplasmic pro-

cesses of odontoblasts. Although matrix vesicles

are distributed throughout the predentin, they are

most numerous near the basal lamina. The apatite

crystals grow rapidly within the vesicles, and in

time, the vesicles rupture. The crystals thus

released mix with crystals from adjoining vesi-

cles to form advancing crystal fronts that merge

to form small globules. As the globules expand,

they fuse with adjacent globules until the matrix

is completely mineralized.

Apparently matrix vesicles are involved only

in mineralization of initial layer of dentin. As the

process of mineralization progresses, the advanc-

ing front projects along the collagen ﬁ brils of the

predentin matrix. Hydroxyapatite crystals appear

on the surface and within the ﬁ brils and continue

to grow as mineralization progresses, resulting in

an increased mineral content of the dentin.

1.3.1.4 Dentinal Sclerosis

Partial or complete obturation of dentinal tubules

may occur as a result of aging or develop in

response to stimuli such as attrition of the tooth

surface or dental caries [ 36 ]. When tubules

become ﬁ lled with mineral deposits, the dentin

becomes sclerotic. Dentinal sclerosis is easily

recognized in histologic ground sections because

of its translucency, which is due to the homoge-

neity of the dentin since both matrix and tubules

are mineralized. Studies using dyes, solvents, and

radioactive have shown that sclerosis results in

decreased permeability of dentin. By limiting the

diffusion of noxious substances through the den-

tin, dentinal sclerosis helps to shield the pulp

from irritation.

One form of dentinal sclerosis is thought to

represent an acceleration of peritubular dentin

formation. This form appears to be a physiologic

process, and in the apical third of the root, it

develops as a function of age [

36 ]. Dentinal

tubules can also become blocked by the precipi-

tation of hydroxyapatite and whitlockite crystals

Z. Liwei et al.

within the tubules. This type occurs in the trans-

lucent zone of carious dentin and in attrited den-

tin and has been termed “pathological sclerosis.”

1.3.1.5 Dentin Repair

Dentin that is produced in response to the injury

of primary odontoblasts has been known by sev-

eral different names: irregular secondary dentin,

irritation dentin, tertiary dentin, and reparative

dentin. The term most commonly applied to

irregularly formed dentin is reparative dentin,

presumably because it so frequently forms in

response to injury and appears to be a component

of the reparative process. It must be recognized,

however, that this type of dentin has also been

observed in the pulps of normal unerupted teeth

without any obvious injury [ 37 ]. The reasons of

this phenomenon and the difference between the

development and repair of irregular dentin are

still unclear.

It will be recalled that secondary dentin is

deposited circumpulpally at a very slow rate

throughout the life of the vital tooth. In contrast,

the formation of reparative dentin occurs at the

pulpal surface of primary of secondary dentin at

sites corresponding to areas of irritation. For

example, when a carious lesion has invaded den-

tin, the pulp usually responds by depositing a

layer of reparative dentin over the dentinal

tubules of the primary or secondary dentin which

communicate with the carious lesion. Similarly,

when occlusal wear removes the overlying

enamel and exposes the dentin to the oral envi-

ronment, reparative dentin is deposited over the

exposed tubules. In general, the amount of repar-

ative dentin formed in response to caries or attri-

tion of the tooth surface is proportional to the

amount of primary dentin that is destroyed. Thus,

the formation of reparative dentin allows the pulp

to retreat behind a barrier of mineralized tissue.

Compared to primary dentin, reparative dentin

is less tubular and the tubules tend to be more

irregular with larger lumina. In some cases, no

tubules are formed. The cells that form reparative

dentin are not as columnar as the primary odonto-

blasts of the coronal pulp and are often cuboidal.

The quality of reparative dentin (i.e., the extent to

which it resembles primary dentin) is quite vari-

able. If irritation to the pulp is relatively mild, as in

the case of a superﬁ cial carious lesion, the repara-

tive dentin formed may resemble primary dentin in

terms of tubularity and degree of mineralization.

On the other hand, reparative dentin deposited in

response to a deep carious lesion may be relatively

tubular and poorly mineralized with many areas of

interglobular dentin. The degree of irregularity of

reparative dentin is probably determined by fac-

tors such as the amount of inﬂ ammation present,

the extent of cellular injury, and the state of dif-

ferentiation of the replacement odontoblasts.

The poorest quality of reparative dentin is usu-

ally observed in association with marked pulpal

inﬂ ammation. In fact, the dentin may be so poorly

organized that areas of soft tissue are entrapped

within the dentinal matrix. In histologic sections,

these areas of soft tissue entrapment impart a

Swiss cheese appearance to the dentin. As the

entrapped soft tissue degenerates, products of tis-

sue degeneration further contribute to the inﬂ am-

matory stimuli assailing the pulp.

1.3.2 Pulp

The pulp is a soft tissue of mesenchymal origin

residing within the pulp chamber and root canals

of the teeth. The primary role of the pulp is to

produce dentin, by specialized cells, the odonto-

blasts, arranged peripherally in direct contact

with dentin matrix. The close relationship

between odontoblasts and dentin is one of several

reasons why dentin and pulp should be consid-

ered as a functional entity, sometimes referred to

as the pulpodentin complex. Following tooth

development, the pulp retains its ability to form

dentin throughout life. This enables the vital pulp

to partially compensate for the loss of enamel or

dentin caused by mechanical trauma or disease.

How well it serves this function depends on many

factors, but the potential for regeneration and

repair is as much a reality in the pulp as in other

connective tissues of the body.

The dental pulp is in many ways similar to

other connective tissues of the body, but its special

characteristic deserves serious consideration. Even

the mature pulp bears a strong resemblance to

1 Tooth Development: Embryology of the Craniofacial Tissues

embryonic connective tissue. Certain peculiarities

are imposed on the pulp by the rigid mineralized

dentin in which it is enclosed. Thus it is situated

within a low-compliance environment that limits

its ability to increase in volume during episodes of

vasodilatation and increased vascular permeabil-

ity. The pulp houses a number of tissue elements,

including vascular tissues, nerves, connective tis-

sues, ﬁ bers, ground substance, interstitial ﬂ uid,

odontoblasts, ﬁ broblasts, antigen-presenting cells,

and other minor cellular components.

1.3.2.1 Vascular Tissues

The pulp is actually a microcirculatory system

whose largest vascular components are arterioles

and venules. No true arteries or veins enter or

leave the pulp. Unlike most tissues, the pulp lacks

a true collateral system and is dependent upon the

relatively few arterioles entering through the root

foramina and occasional arteriole through a lat-

eral canal. Since with age there is a gradual reduc-

tion in the luminal diameters of these foramina,

the vascular system of pulp decreases progres-

sively. Since the pulp is relative incompressible,

the total volume of blood within the pulp chamber

cannot be greatly increased, although reciprocal

volume changes can occur between arterioles,

venules, lymphatics, and extravascular tissue. In

the pulp, therefore careful regulation of blood

ﬂ ow is of critical importance.

Blood from the dental artery enters the tooth

via the arterioles, and these vessels pass through

the apical foramen or foramina in company with

nerve bundles. Smaller vessels may enter the

pulp via lateral or accessory canals. As the arteri-

oles pass into the coronal pulp, they fan out

toward the dentin, diminish in size, and give rise

to a capillary network in the subodontoblastic

region. This network provides the odontoblasts

with a rich source of metabolites.

Capillary blood ﬂ ow in the coronal portion of

the pulp is nearly twice that in the root portion

[

13 ]. Moreover, blood ﬂ ow in the region of the

pulp horns is greater than in other areas of the

pulp. In young teeth, capillaries commonly

extend into the odontoblast layer, thus assuring

an adequate supply of nutrients for the metaboli-

cally active odontoblasts. The subodontoblastic

capillaries are surrounded by a basement mem-

brane, and occasionally, fenestrations (pores) are

observed in capillary walls. These fenestrations

are thought to provide rapid transport of ﬂ uid and

metabolites from the capillaries to the adjacent

odontoblasts.

1.3.2.2 Nerve Fibers

The pulp is a rather unique sensory organ capable

of transmitting information from its sensory recep-

tor to the central nervous system. Being encased in

a protective layer of dentin, which in turn is cov-

ered with enamel, it might be expected to be quite

unresponsive to stimulation; yet, despite the low

thermal conductivity of dentin, the pulp is undeni-

ably sensitive to thermal stimuli such an ice cream

and hot drinks. The innervation of the pulp

includes both afferent neurons, which conduct

sensory impulses, and autonomic ﬁ bers, which

provide neurogenic modulation of the microcircu-

lation and perhaps regulate dentinogenesis.

Nerve ﬁ bers are usually classiﬁ ed according

to their diameter, conduction velocity, and func-

tion. In the pulp, there are two main types of sen-

sory nerve ﬁ bers, myelinated A ﬁ bers and

unmyelinated C ﬁ bers. The A ﬁ bers include both

A-8 and A-5 ﬁ bers. A-8 ﬁ bers may be slightly

more sensitive to stimulation than the A-5 ﬁ bers,

but functionally these ﬁ bers are grouped together.

Approximately 90 % of the A ﬁ bers are A-8

ﬁ bers, and A-8 ﬁ ber terminals principally local in

region of dentin–pulp junction, and the stimula-

tion threshold of A ﬁ bers is relatively low con-

trast with C ﬁ bers which is probably distribute

throughout the pulp and feel the pain of burning,

aching.

In the human premolar, the number of unmy-

elinated axons entering the tooth at the apex

reached a maximum number shortly after tooth

eruption [

38 ]. At this stage, an average of 1800

unmyelinated axons and more than 400 myelin-

ated axons were observed, although in some teeth,

fewer than 100 myelinated axons were present.

The number of A ﬁ bers gradually increased to

more than 700 ﬁ ve years after eruption. The rela-

tively late appearance of A ﬁ bers in the pulp may

help to explain why the electric pulp test tends to

be unreliable in young teeth.

Z. Liwei et al.

The nerve bundles pass upward through the

radicular pulp together with blood vessels. Once

they reach the coronal pulp, they fan out beneath

the cell-rich zone, branch into smaller bundles,

and ﬁ nally ramify into a plexus of single-nerve

axons known as the plexus of Raschkow (Fig. 1.9 )

[ 39 ]. Full development of this plexus does not

occur until the ﬁ nal stages of root formation.

One study showed that a reduction in pulpal

blood ﬂ ow induced by stimulation of sympathetic

ﬁ bers leading to the pulp results in depressed

excitability of pulpal A ﬁ bers. The excitability of

C ﬁ bers is less affected than that of A ﬁ bers by a

reduction in blood ﬂ ow. Pulpal nerve ﬁ bers con-

tain neuropeptides such as neuropeptide Y, calci-

tonin gene-related peptide (CGRP), vasoactive

intestinal polypeptide (VIP), tyrosine hydroxy-

lase, and substance P. The release of these pep-

tides can be trigged by such things as tissue

injury, complement activation, antigen-antibody

reactions, or antidromic stimulation of the infe-

rior alveolar nerve.

Interestingly, pulpal nerve ﬁ bers are relatively

resistant to pulp necrosis. This is apparently due

to the fact that nerve bundles in general are more

resistant to autolysis than other tissue elements.

Even in degenerating pulps, C ﬁ bers might still

be able to respond to stimulation. Furthermore, it

may be that C ﬁ bers remain excitable even after

blood ﬂ ow has been compromised in the diseased

pulp, for C ﬁ bers are better able to function in the

presence of hypoxia. This may offer an explana-

tion as to why instrumentation of the root canals

of apparently nonvital teeth sometimes elicits a

moderate level of pain.

1.3.2.3 Connective Tissue Fibers

Two types of structural proteins are found in the

pulp: collagen and elastin. Elastin ﬁ bers are

conﬁ ned to the walls of the arterioles and, unlike

collagen, are not a part of the ECM.

A single collagen molecule, referred to as tro-

pocollagen, consists of three polypeptide chains,

designated as either a-1 or a-2 depending on their

amino acid composition and sequence. The dif-

ferent combinations and linkages of chains mak-

ing up the tropocollagen molecule have allowed

collagen ﬁ bers and ﬁ brils to be classiﬁ ed into a

number of types. Type I is found in the skin, ten-

don, bone, dentin, and pulp. Type II occurs in car-

tilage. Type III is found in most unmineralized

connective tissues, and it is a fetal form found in

the dental papilla and the mature pulp. Type IV

and VII collagens are components of basement

membranes. Type V collagen is a constituent of

interstitial tissues. Type I collagen is synthesized

by odontoblasts and osteoblasts, and ﬁ broblasts

synthesize types I, III, V, and VII.

In collagen synthesis, the protein portion of

the molecule is formed by the polyribosomes of

the RER of connective tissue cells. The proline

and lysine residues of the polypeptide chains are

hydroxylated in the cisternae of the RER, and the

chains are assembled into a triple-helix conﬁ gu-

ration in the smooth endoplasmic reticulum. The

product of this assembly is termed procollagen,

and it has a terminal unit of amino acids known

as the telopeptide of the procollagen molecule.

When these molecules reach the Golgi complex,

they are glycosylated and packaged in secretory

vesicles. The vesicles are transported to the

plasma membrane and secreted via exocytosis

into the extracellular matrix, thus releasing the

procollagen. Here the terminal telopeptide is

cleaved by a hydrolytic enzyme, and the tropo-

collagen molecules begin aggregating to form

collagen ﬁ brils. It is believed that aggregation of

tropocollagen is somehow mediated by the

Fig. 1.9 Light microscopy showing the relationship of

parietal layer of nerves (plexus of Raschkow) below to the

cell-rich, cell-free zones, odontoblasts, and dentin at top

of the picture (From Avery [

39 ], p. 208, ﬁ gure 8)

1 Tooth Development: Embryology of the Craniofacial Tissues

GAGs. The conversion of soluble collagen into

insoluble ﬁ bers occurs as a result of cross-linking

of tropocollagen molecules.

In the young pulp, small collagen ﬁ bers stain

black with silver impregnation stains and are thus

referred to as argyrophilic ﬁ ber. They are very

similar, if not identical, to reticular ﬁ bers in other

loose connective tissues in that they are not

arranged in bundles and tend to form delicate net-

works. The presence of collagen ﬁ bers passing

from the dentin matrix between odontoblasts into

the dental pulp has been reported in fully erupted

teeth [ 40 ]; these ﬁ bers are often referred to as von

Korff ﬁ bers . Large collagen ﬁ ber bundles are not

argyrophilic but can be demonstrated with special

histochemical methods such as the Masson tri-

chrome stain or Mallory’s triple connective tissue

stain. These ﬁ bers are much more numerous in the

radicular pulp than in the coronal pulp. The high-

est concentration of these larger ﬁ ber bundles is

usually found in the radicular pulp near the apex.

1.3.2.4 Ground Substance

Connective tissue is a system consisting of cells

and ﬁ bers, both embedded in the pervading

ground substance. Cells that produce connective

tissue ﬁ bers also synthesize the major constitu-

ents of ground substance. The term extracellular

matrix ECM is used to describe ground sub-

stance, regarding it as the material into which

ﬁ bers are deposited. Because of its content of

polyelectric polysaccharides, the ECM is respon-

sible for the water-holding properties of connec-

tive tissues.

Nearly all proteins of the ECM are glycopro-

teins. Proteoglycans are an important subclass of

glycoproteins. These molecules support cells,

provide tissue turgor, and mediate a variety of

cell interactions. They have in common the pres-

ence of GAG chains and a protein core to which

the chains are linked. Except for heparan sulfate

and heparin, the chains are composed of disac-

charides. The primary function of GAG chains is

to act as adhesive molecules that can bond to cell

surfaces and other matrix molecules.

Fibronectin is a major surface glycoprotein

that, together with collagen, forms an integrated

ﬁ brillary network that inﬂ uences adhesion,

motility, growth, and differentiation of cells.

Laminin, an important component of basement

membranes, binds to type IV collagen and cell

surface receptors. Tenascin is another substrate

adhesion glycoprotein.

Degradation of ground substance can occur in

certain inﬂ ammatory lesions in which there is a

high concentration of lysosomal enzymes.

Proteolytic enzymes, hyaluronidases, and chon-

droitin sulfatases of lysosomal and bacterial ori-

gin are examples of acid hydrolytic enzymes that

can attract components of the ground substance.

The pathways of inﬂ ammation and infection are

inﬂ uenced by the state of polymerization of the

ground substance components.

1.3.2.5 Lymphatics

The existence of lymphatics in the pulp has been

a matter of debate, since it is not easy to distin-

guish between venules and lymphatics by ordi-

nary light microscopic techniques, although

some studies utilizing light and electron micros-

copy have described lymphatic capillaries in

human and in cat dental pulps.

1.3.2.6 Accessory Canals

Occasionally during formation of the root sheath,

a break develops in the continuity of the sheath,

producing a small gap. When this occurs, den-

tinogenesis does not take place opposite to the

defect. The result is a small “accessory” canal

between the dental sac and the pulp. An acces-

sory canal can become established anywhere

along the root, thus creating a periodontal–end-

odontic pathway of communication and a possi-

ble portal of entry into the pulp if the periodontal

tissues lose their integrity. In periodontal disease,

the development of a periodontal pocket may

expose an accessory canal and thus allow micro-

organisms or their metabolic products to gain

access to the pulp.

1.3.2.7 Morphologic Zones of Pulp

Odontoblast Layer The outermost stratum of

cells of the healthy pulp is the odontoblast layer.

This layer is located immediately subjacent to the

predentin, with the odontoblast processes passing

on through the predentin into the dentin.

Consequently, the odontoblast layer is actually

Z. Liwei et al.

composed of the cell bodies of the odontoblasts.

Additionally, capillaries, nerve ﬁ bers, and den-

dritic cells may be found among the odontoblasts.

The odontoblast layer in the coronal pulp con-

tains more cells per unit area than in the radicular

pulp. Whereas the odontoblasts of the mature

coronal pulp are usually columnar (Fig.

1.10 ),

those in the midportion of the radicular pulp are

more cuboidal. Near the apical foramen, the

odontoblasts appear as a ﬂ attened cell layer.

Since there are fewer dentinal tubules per unit

area in the root than in the crown of the tooth, the

odontoblast cell bodies are less crowded and are

able to spread out laterally.

Cell-Poor Zone Immediately subjacent to the

odontoblast layer in the coronal pulp, there is

often a narrow zone approximately 40 μm in

width that is relatively free of cells. It is traversed

by blood capillaries, unmyelinated nerve ﬁ bers,

and the slender cytoplasmic processes of ﬁ bro-

blasts. The presence or absence of the cell-poor

zone depends on the functional status of the pulp.

It may not be apparent in young pulps where den-

tin forms rapidly or in older pulps where repara-

tive dentin is being produced.

Cell-Rich Zone Usually conspicuous in the

subodontoblastic area is a stratum containing a

relatively high proportion of ﬁ broblasts compared

with the more central region in the pulp. It is

much more prominent in the coronal pulp than in

the radicular pulp. Besides ﬁ broblasts, the cell-

rich zone may include a variable number of mac-

rophages and lymphocytes.

On this evidence obtained in rat molar teeth, it

has been suggested that the cell-rich zone forms

as a result of peripheral migration of cells popu-

lating the central regions of the pulp, commenc-

ing at about the time of tooth eruption. Although

cell division within the cell-rich zone is a rare

occurrence in normal pulps, death of odonto-

blasts causes a great increase in the rate of mito-

sis. Since irreversibly injured odontoblasts are

replaced by cells that migrate from the cell-rich

zone onto the inner surface of the dentin, this

motility ability is probably the ﬁ rst step in the

formation of a new odontoblast.

Pulp Proper The pulp proper is the central mass

of the pulp. It contains the larger blood vessels

and nerves. The connective tissue cells in this

zone are ﬁ broblasts or pulpal cells.

1.3.3 Cells in the Dental Pulp

1.3.3.1 Odontoblast

Because it is responsible for dentinogenesis both

during tooth development and in the mature

Fig. 1.10 Beside dentin ( 1 )

and predentin ( 2 ), there are

tall columnar odontoblasts ( 3 )

of the coronal pulp. Note the

presence of the cell-rich zone

( 5 ) and cell-poor zone ( 4 )

1 Tooth Development: Embryology of the Craniofacial Tissues

tooth, the odontoblast is the most characteristic

cell of the pulpodentin complex. During dentino-

genesis, the odontoblasts form the dentinal

tubules, and their presence within the tubules

makes dentin a living tissue [ 41 ].

Differentiation of epithelial and mesenchymal

cells into ameloblasts and odontoblasts, respec-

tively, occurs during the bell stage of tooth devel-

opment [ 42 ]. The preameloblasts differentiate at a

faster rate than the corresponding odontoblasts so

that at any given level, mature ameloblasts appear

before the odontoblasts have fully matured. In

spite of this difference in rate of maturation, den-

tin matrix is formed before enamel matrix. As the

ameloblasts undergo differentiation, changes are

happening across the basement membrane in the

adjacent dental papilla. Before differentiation of

odontoblasts, the dental papilla consists of

sparsely distributed polymorphic mesenchymal

cells with wide intercellular spaces. With the

onset of differentiation a single layer of cells, the

presumptive odontoblasts (preodontoblasts) align

themselves along the basement membrane sepa-

rating the inner enamel epithelium from the dental

papilla. These cells stop dividing and elongate

into short columnar cells with basally situated

nuclei (Fig. 1.7 ). Several cytoplasmic projections

from each of these cells extend toward the basal

lamina. At this stage, the preodontoblasts are still

relatively undifferentiated.

Dentinogenesis ﬁ rst occurs in the developing

tooth at sites where the cusp tips or incisal edge

will be formed. It is in this region that odonto-

blasts reach full maturity and become tall colum-

nar cells. The production of the ﬁ rst dentin matrix

involves the formation, organization, and matura-

tion of collagen ﬁ brils and proteoglycans. As

more collagen ﬁ brils accumulate subject to the

basal lamina, the lamina becomes discontinuous

and eventually disappears. This occurs as the col-

lagen ﬁ bers become organized and extend into

the spaces between the ameloblast processes.

Concurrently the odontoblasts extend several

small processes toward the ameloblasts. Some of

these become interposed between the processes

of ameloblasts, resulting in the formation of

enamel spindles (dentinal tubules that extend into

the enamel). Membrane-bound vesicles bud off

from the odontoblast processes and become

interspersed among the collagen ﬁ bers of the

dentin matrix. These vesicles subsequently play

an important role in the initiation of

mineralization. With the onset of dentinogenesis,

the dental papilla becomes the dental pulp.

As predentin matrix is formed, the odonto-

blasts commence to move away toward the central

pulp, depositing matrix at a rate of approximately

4–8 μm per day in their wake. Within this matrix,

a process from each odontoblast becomes accen-

tuated and remains to form the primary odonto-

blast process. It is around these processes that the

dentinal tubules are formed.

Dentinal tubule forms around each of the

major odontoblastic processes which occupy

space within the tubule and somehow mediates

the formation of peritubular dentin. Microtubules

and microﬁ laments are the principal ultrastruc-

tural components of the odontoblast process and

its lateral branches. Microtubules extend from

the cell body out into the process. These straight

structures follow a course that is parallel with the

long axis of the cell and impart the impression of

rigidity. Although their precise role is unknown,

theories as to their functional signiﬁ cance sug-

gest that they may be involved in cytoplasmic

extension, transport of materials, or simply the

provision of a structural framework. The plasma

membrane of the odontoblast process closely

approximates the wall of the dentinal tubule.

Localized constrictions in the process occasion-

ally produce relatively large spaces between the

tubule wall and the process. Such spaces may

contain collagen ﬁ brils and ﬁ ne granular mate-

rial, which presumably represents ground sub-

stance. The peritubular dentin matrix lining the

tubule is circumscribed by an electron-dense lim-

iting membrane.

The odontoblast is considered to be ﬁ xed post-

mitotic cell in that once it has fully differentiated,

it apparently cannot undergo further cell division.

If this is indeed the case, the life-span of the

odontoblast coincides with the life-span of the

viable pulp.

Apparently the odontoblast synthesizes mainly

type I collagen, although small amounts of type V

collagen have been found in the ECM. In addition

Z. Liwei et al.

to proteoglycans and collagen, the odontoblast

secretes phosphophoryn, a phosphoprotein involved

in extracellular mineralization. Phosphophoryn is

unique to dentin and is not found in any other mes-

enchymal cell lines. The odontoblast also secretes

alkaline phosphatase, an enzyme that is closely

linked to mineralization but whose precise role is

yet to be illuminated.

In contrast to the active odontoblast, the rest-

ing or injured odontoblast has a decreased num-

ber of organelles and may become progressively

shorter. These changes can begin with the com-

pletion of root development.

1.3.3.2 Pulp Fibroblast

The most numerous cells of the pulp tissues

appear to be tissue-speciﬁ c cells capable of giv-

ing rise to cells that are committed to differentia-

tion as odontoblasts, given the proper signal.

These cells synthesize type I and III collagen as

well as proteoglycans and GAGs; since they

degrade collagen, they are also responsible for

collagen turnover in the pulp.

Although distributed throughout the pulp,

ﬁ broblasts are particularly abundant in the cell-

rich zone. The early differentiating ﬁ broblasts are

polygonal and appear to be widely separated and

evenly distributed within the ground substance.

Cell-to-cell contacts are established between the

multiple processes that extend out from each of

the cells. Many of these contacts take the form of

gap junctions, which provide for electronic cou-

pling of one cell to another. As they mature, the

cells become stellate in form and the Golgi com-

plex enlarges, the RER proliferates, secretory

vesicles appear, and the ﬁ broblasts take on the

characteristic appearance of protein-secreting

cells. Along the outer surface of the cell body,

collagen ﬁ brils commence to appear. With an

increase in the number of blood vessels, nerves,

and ﬁ bers, there is a relative decrease in the num-

ber of ﬁ broblasts in the pulp.

A colleague once remarked that the ﬁ broblasts

of the pulp are very much like Peter Pan in that

they never grow up. There may be an element of

truth in this statement, for these cells do seem to

remain in a relatively undifferentiated modality as

compared to ﬁ broblasts of most other connective

tissues. This perception has been fortiﬁ ed by the

observation of large numbers of reticulin-like

ﬁ bers in the pulp. However, it has been concluded

that because of distinct histochemical differences,

reticulin ﬁ bers, such as most of gingiva and lym-

phoid organ, are not present in the pulp. In the

young pulp, the nonargyrophilic collagen ﬁ bers

are sparse, but they progressively increase in

number as the pulp ages.

Many experimental models have been devel-

oped to study wound healing in the pulp, particu-

larly dentinal bridge formation following pulp

exposure or pulpotomy. One study demonstrated

that mitotic activity preceding the differentiation

of replacement odontoblasts appears to occur pri-

marily among ﬁ broblasts. Thus, it appears that

pulpal ﬁ broblasts can be regarded as odontopro-

genitor cells.

1.3.3.3 Macrophage

Tissue macrophage or histiocytes are monocytes

that have left the bloodstream, entered the tis-

sues, and differentiated into macrophages. They

are usually found in close proximity to blood ves-

sels. These cells are quite active in endocytosis

and phagocytosis. Because of their mobility and

phagocytic activity, they are able to act as scaven-

gers, removing extravasated red blood cells, dead

cells, and foreign bodies from the tissue ingested

material destroyed by the action of lysosomal

enzymes.

In addition, a proportion of macrophages,

when primed by cytokines, participate in immune

reactions by processing antigen and presenting it

to lymphocytes. The processed antigen is bound

to class II histocompatibility antigens on the

macrophage, where it can interact with speciﬁ c

receptors present on immunocompetent T cells.

Such interaction is obligatory for the induction of

cell-mediated immunity. When activated by the

appropriate inﬂ ammatory stimuli, macrophages

are capable of producing a large variety of solu-

ble factors including interleukin-1, tumor necro-

sis factor, growth factors, and other cytokines.

1.3.3.4 Dendritic Cell

Dendritic cells, like macrophages, are cells of the

immune system. Similar cells are found in the

1 Tooth Development: Embryology of the Craniofacial Tissues

epidermis and mucous membranes, where they are

called Langerhans cells . Dendritic cells are primar-

ily found in lymphoid tissues, but they are also

widely distributed in connective tissues, including

the pulp. These cells are termed antigen- presenting

cells and are characterized by dendritic cytoplasmic

processes and the presence of cell surface class II

antigens. Like macrophages, dendritic cells phago-

cytose and process antigens but are otherwise only

weakly phagocytic. Together with macrophages

and lymphocytes, dendritic cells are believed to par-

ticipate in immunosurveillance in the pulp.

1.3.3.5 Lymphocyte

T lymphocytes and B lymphocytes were found in

normal pulps from the human teeth. T

(suppres-

sor) lymphocytes were the predominant T lym-

phocyte subset present in pulps. Lymphocytes

have also been observed in the pulps of impacted

teeth. The presence of macrophages, dendritic

cells, and T lymphocytes indicates that the pulp is

well equipped with cells required for the initia-

tion of immune responses.

1.3.3.6 Mesenchymal Cell

Mesenchymal stem cells (MSCs) are found in

many adult tissues and organs. In tissues that

grow continuously or exhibit high rates of cell

turnover, MSCs provide a renewable source of

progenitor cells to differentiate and replace those

lost. In the majority of tissues, MSCs are found in

small numbers and remain quiescent until mobi-

lized in response to tissue damage. In pulp tissue,

some authors hold the opinion that primordial

mesenchymal cells persist in adult pulp tissues as

“undifferentiated” mesenchymal cells. However,

during wound healing, well-differentiated ﬁ bro-

blasts undergo rapid serial division to give rise to

new ﬁ broblasts. Similarly, replacement odonto-

blasts are derived from mature ﬁ broblasts.

Consequently, there is no need to postulate that in

the pulp new mesenchymal cells arise from cells

other than pulpal ﬁ broblasts. This controversy is

still unclear and needs more researches.

1.3.3.7 Mast Cell

Mast cells are widely distributed in the connec-

tive tissues, where they occur in small groups in

relation to blood vessels. This cell has been the

subject of considerable attention because of its

dramatic role in inﬂ ammatory reactive. But mast

cells also present in the normal pulp tissue,

although they are routinely found in chronically

inﬂ amed pulps.

1.4 Root Development

Root development is one of the important stages

in tooth development process, following the bell

stage. The interaction between dental epithelial

and neural crest-derived mesenchymal cells is

essential for tooth development.

1.4.1 The Initiation of Tooth Root

Development

After the crown formation is nearly complete, the

tooth root begins to develop. Then, the special

bilayered epithelial sheath is formed from the

outer and inner enamel epithelium at the neck

ring of the crown and grows in the apical direc-

tion, termed Hertwig’s epithelial root sheath

(HERS). HERS, dental papilla cells, and dental

follicle cells form the organ primordium of tooth

development together [ 43 ]

HERS is the morphogenesis signal of the ini-

tiation of tooth root development [ 44 ].

Morphologically, the epithelial root sheath is

located between the two regions of neural crest-

derived mesenchyme: the dental papilla and the

dental follicle. When the HERS grows apically,

the dental papilla cells adjacent to the inner epi-

thelial layer of the HERS and the epithelial base-

ment membrane induced to become odontoblasts

and later to form root dentin. After root dentin

formation, the epithelial root sheath enveloping

the root begins to be interrupted or perforated.

The formation of a mesh-like structure in the

HERS allows dental follicle cells to contact the

newly formed root dentin surface through the

epithelial root sheath. These dental follicle cells

differentiate into cementoblasts to form cemen-

tum. In addition, some of the HERS cells undergo

epithelial–mesenchymal transition to become

Z. Liwei et al.

cementoblasts and form cementum [ 13 ]. At the

same time, collagen ﬁ bers secreted by dental fol-

licle cells are embedded into the new cementum

matrix and ﬁ x the root in the jaw bone. Following

tooth root development and elongation, the tooth

erupts into the oral cavity to establish occlusal

contacts with the opposing teeth and performs its

physiological function.

Previous studies have shown that Hertwig’s

epithelial root sheath plays an important role in

root development, but the fate of HERS has

remained unknown. Until now, the fate of HERS

and its function are not clear. At least 6 possible

outcomes of HERS have been proposed: (1) epi-

thelial cell rests of Malassez, (2) apoptosis, (3)

incorporation into the advancing cementum front,

(4) epithelial–mesenchymal transformation, (5)

migration toward the periodontal ligament, and

(6) differentiation into cementoblasts [ 10 ].

1.4.1.1 Epithelial Cell Rests

of Malassez (ERM)

Epithelial cell rests of Malassez (ERM) are

involved in the maintenance and homeostasis of

the periodontal ligament. The ERM have a num-

ber of functions, such as to prevent root resorp-

tion, induce cementum formation, and maintain

the homeostasis of the PDL [ 45 ]. On the other

hand, ERM can be stimulated to proliferate in

response to injury in rats. When stimulated by

inﬂ ammatory cytokines, ERM can proliferate

and differentiate into the lining epithelium of

periapical cysts.

Primary HERS/ERM cells had typical epithe-

lial cell morphology and characteristics, and they

maintained for more than ﬁ ve passages. They

expressed epithelial stem cell-related genes:

ABCG2, ΔNp63, p75, EpCAM, and Bmi-1.

Moreover, the expression of embryonic stem cell

markers such as Oct-4, Nanog, and SSEA-4 was

detected [

46 ]

1.4.1.2 Induction of Differentiation

of Mesenchymal Stem Cells

HERS, morphologically, is a structural boundary

of two dental ectomesenchymal tissues: dental

papilla and dental follicle [ 47 ]. It breaks up into

epithelial rests and cords, allowing other cells to

come in contact with the outer dentin surface.

This sandwich structure plays at least two impor-

tant roles during root formation: biomineraliza-

tion (cementogenesis and dentin formation) and

induction of root organization.

HERS cells may be involved in regulating dif-

ferentiation of periodontal ligament stem cells

(PDLSCs) and forming cementum in vivo [

48 ].

Dental follicle cell sheets induced by HERS cells

are able to produce periodontal tissues through

epithelial–mesenchymal interactions.

HERS cells are detectable on the surface of

the root throughout root formation and do not

disappear. Most of the HERS cells are attached to

the surface of the cementum, and others separate

to become the epithelial rest of Malassez. HERS

cells secrete extracellular matrix components

onto the surface of the dentin before dental folli-

cle cells penetrate the HERS network to contact

dentin. HERS cells also participate in the cemen-

tum development and may differentiate into

cementocytes.

1.4.2 Related Signaling Pathway

of Root Morphogenesis

Recently, several genes were found to play cru-

cial role in the process of root development.

1.4.2.1 TGF-β/BMP Signaling

The TGF-β superfamily of cytokines is composed

of TGF-β, BMPs, activins, and related proteins.

TGF-β signaling plays an important role in devel-

opmental biology, disease, and regeneration [

49 ].

Smad4 is a central mediator of the canonical

TGF-β signaling pathway. Deletion of Smad4

results in the blockage of TGF-β/BMP signaling.

Ablation of Smad4 in the dental mesenchyme

(Osr2-IresCre:Smad4

ﬂ /ﬂ

mice) results in short

root formation and defects in odontoblast differ-

entiation and dentin formation. Moreover, ecto-

pic bone-like structures replaced normal dentin

in the teeth of Osr2-IresCre:Smad4

ﬂ /ﬂ

mutant

mice. Loss of Smad4 results in the upregulation

of canonical Wnt signaling and downregulation

of Dkk1 and Sfrp1, which are Wnt pathway

inhibitors.

1 Tooth Development: Embryology of the Craniofacial Tissues

Comparing different animal models provides

more detail about TGF-β signaling during root

dentin formation. In Osr2-IresCre:Smad4

ﬂ /ﬂ

mice, dental mesenchyme differentiation is

arrested at the late bell stage and secretory stage,

with no detectable expression of Dspp.

Odontoblast differentiation is delayed and Dspp

expression is eventually detectable in mice lack-

ing Tgfbr2 [ 50 ].

Bone morphogenetic protein 4 (BMP4) is

secreted by mesenchymal cells, acting on the dental

epithelium as a regulator of cell differentiation dur-

ing crown formation. Studies assessed the localiza-

tions of BMP4, BMP4 receptor (BMPR-1B), and

BMPR-2 during molar root formation in mouse

[ 51 ]. In K14-Noggin transgenic mice, molar epithe-

lial root sheath cell proliferation is abated, and root

development is retarded [ 52 ].

In the early development of mouse molar root,

mesenchymal progenitor cell markers such as

STRO-1 with BMP receptors are expressed in the

dental follicle, hence the hypothesis that mesen-

chymal STRO-1-positive cells are epithelial root

sheath BMP signaling pathways of target cells.

1.4.2.2 SHH Signaling

Shh, a member of the hedgehog signaling family,

is expressed in the dental epithelium and plays an

essential role during tooth development. During

root development, Shh is strongly expressed in

the HERS, which suggests a function in root for-

mation. Gli1, a transcript activated by Shh, is also

detectable in the root epithelium (HERS) and

mesenchyme [

53 ].

1.4.2.3 Wnt Signaling

The Wnt family of proteins plays an important

role in morphogenesis and cellular differentiation

in many tissues. The canonical Wnt signaling

pathway involves the stabilization and nuclear

accumulation of β-catenin, which activates the

expression of Wnt target genes. It is well known

that Wnt/β-catenin signaling plays multiple roles

in various stages of tooth morphogenesis [ 54 ].

During dentin formation, Wnt10a is expressed in

odontoblast-lineage cells, and Axin2 is also

expressed in developing odontoblasts and dental

pulp cells. In addition, Dkk1, an inhibitor of Wnt

signaling, is strongly expressed in pre-

odontoblasts but is decreased in secretory odon-

toblasts. Moreover, it was recently reported that

Lef-1 overexpression accelerates odontoblast dif-

ferentiation of dental pulp cells and constitutive

β-catenin stabilization in the dental mesenchyme

can lead to excessive dentin formation. β-catenin

has been shown to be strongly expressed in

odontoblast- lineage cells and is required for root

formation. Tissue-speciﬁ c inactivation of

β-catenin in developing odontoblasts produced

molars lacking roots and aberrantly thin incisors.

These reports strongly suggest that modulation of

Wnt/β-catenin signaling may play an important

role in odontoblast differentiation.

1.4.2.4 Notch Signaling

The Notch pathway regulates the renewal and

fate decisions of stem cells in multiple tissues.

Notch1 and Notch2 as well as the Notch target

gene Hes1 are expressed in the putative stem

cells in the continuously growing mouse incisors.

Notch signaling is required for epithelial stem

cell survival and enamel formation in the contin-

uously growing mouse incisor [ 55 ].

At embryonic day 19, the molar and the inci-

sor of rat began differentially developing: the

molar formed double-layered cells of the root

sheath while the incisor formed a cervical loop.

By using the subtractive hybridization method, a

subtraction cDNA library of the rat molar and

incisor tissues was successfully constructed.

Differentially expressed gene clones were evalu-

ated by dot blot and sequencing. Sel1l, Nﬁ c,

Edar, GATA6, and some novel genes were found

differentially expressed, which were strongly

related to the tooth root patterning. The Notch

signaling contributes to the maintenance of cervi-

cal loop stem cell niches. SEL1L, the negative

regulating factor of notch signaling, was strongly

expressed in the molars but weakly expressed in

the incisors.

1.4.3 Tooth Eruption

Tooth eruption is the movement of the tooth from

the original developmental site to the functional

Z. Liwei et al.

position through the alveolar bone and gingiva in

the mouth. It is a continuous process, which can

be divided into 5 stages, respectively for pre-

eruptive movement, intraosseous eruption,

mucosal penetration, preocclusal eruption, and

post-occlusal eruption. Each step involves

intense reciprocal interactions between the tooth

and its surrounding tissues and is temporally and

spatially controlled to coordinate the growth of

the jaw and the position of other teeth. However,

the speciﬁ c cellular, molecular, and genetic

mechanisms governing tooth eruption remain

unclear.

Until now, there are several theories explain-

ing how the tooth erupts [ 56 ]. One of the most

acceptable theories is that asymmetric bone

remodeling around the tooth is responsible for

the teeth moving into the oral cavity. When bone

resorbed on the coronal side, bone formed on the

basal side of the tooth. It is thought that the den-

tal follicle, which is a loose connective tissue sac

surrounding the enamel organ of each tooth,

plays a critical role in regulating osteoclasts and

osteoblasts during this process. In 1980s, it was

ﬁ rstly shown that the removal of the dental folli-

cle from developing premolars of dogs prevented

tooth eruption. Meanwhile, if the tooth germ was

removed and replace with another artiﬁ cial tooth,

which the dental follicle was kept intact in situ,

the artiﬁ cial tooth still erupted on schedule.

Furthermore, removal of either the coronal or

basal halves of the dental follicle also prevented

tooth eruption, with the coronal part controlling

bone resorption and the eruption pathway, as

well as the basal part causing bone formation.

Recently, some important signaling molecules

were identiﬁ ed at both these processes for tooth

eruption, such as RANKL, BMP2, and so on.

These studies provided convincing evidence that

the dental follicle is essential for tooth eruption

and also challenged the previously supposed

requirement of dental pulp, periodontal liga-

ments, and roots in the process of tooth

eruption.

For a long time, root development has been

considered as the main force responsible for the

tooth eruption. However, more and more studies

found that rootless tooth can still erupt into the

oral cavity in many species, including human,

dogs, monkeys, and rodents. In some patients

with special diseases, which may affect root

development, the rootless tooth crown did erupt

into the mouth. Developing premolar teeth with

the removal of roots in dogs can erupt into oral

cavity at normal speed, and the void created by

the absence of roots during eruption was ﬁ lled

with the alveolar bone. All of these indicated that

root formation is not required for tooth eruption.

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1 Tooth Development: Embryology of the Craniofacial Tissues

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_2

Bioﬁ lm and Dental Caries

Xu Xin , Zhou Yuan , Shi Wenyuan , Liu Yaling , and

Zhou Xuedong

2.1 Dental Plaque and Microbial

Bioﬁ lm

2.1.1 Bacterial Bioﬁ lm:

An Advanced Mode of Life

Bacteria grow in two different ways: planktonic

and bioﬁ lm forms. Decades ago, most microbiol-

ogists characterized and studied planktonic bacte-

ria in liquid culture in which bacteria were ﬂ oating

free cells. In this period, plenty of fundamental

work in microbiology was accomplished [ 7 ].

However, it is estimated that only less than 0.1 %

of the microbial consortia in natural environment

grow in planktonic state [ 40 ]. Then how do the

most microbes live and grow? From the colorful

microbial mats in Yellowstone geothermal area to

the sophisticatedly structured microbial commu-

nity on tooth surfaces, we have the answer.

2.1.1.1 The Concept and Discovery

of Bioﬁ lm

Bioﬁ lm is deﬁ ned as aggregates of bacterial cells

attached to a surface and embedded in a poly-

meric matrix that is self-produced and helps the

community to gain tolerance against antimicrobi-

als and host defenses. Usually bioﬁ lm cells are

adhered to a surface, for example, catheter or

tooth surfaces. With the extensive bioﬁ lm stud-

ies, the surface attachment feature is not indis-

pensable for bioﬁ lm recognition now [ 15 ].

The ﬁ rst observation of bacteria on surfaces

was made by Antonie van Leeuwenhoek in 1684

[

15 ]. He observed various types of the numerous

“animalcules” in the tartar taken from his own

teeth directly by microscope. He also found the

preliminary evidence of antimicrobial tolerance

of bioﬁ lm from the fact that the surface-attached

cells on his teeth could survive after rinsing with

vinegar, while cells removed from teeth could

be killed by vinegar [ 146 ]. At that time, the

knowledge and techniques in microbiology

were too limited to understand the little com-

munities on surfaces. Recently with molecular

and bioinformatics techniques, the characteriza-

tion of microbial growth and development in

complex communities on surfaces have been

highly promoted [ 82 ].

X. Xin • Z. Yuan • Z. Xuedong (*)

State Key Laboratory of Oral Diseases ,

West China Hospital of Stomatology,

Sichuan University , No. 14, Section 3,

Renmin South Road , Chengdu 610041 ,

People’s Republic of China

e-mail:

[email protected]

S. Wenyuan

School of Dentistry ,

University of California-Los Angeles ,

Los Angeles , CA , USA

L. Yaling

Advanced Education in General Dentistry ,

Eastman Institute for Oral Health,

University of Rochester , Rochester , NY ,

USA

2.1.1.2 Extracellular Polymeric

Substances of Bioﬁ lm

The structure of bioﬁ lm is highly varied depend-

ing on the growth environment and component

cells. In addition to the bacterial aggregates,

another major component, the structural back-

bone of bioﬁ lm, is the extracellular matrix com-

posed of self-secreted extracellular polymer

substances (EPS) [ 55 ]. EPS is a highly diverse

biopolymers mixture, including mainly polysac-

charides, proteins, lipids, nucleic acids, and other

substances from the environment or host

(Fig. 2.1 ) [ 62 ]. As the immediate microenviron-

ment surrounding cells, EPS serves various func-

tions through the entire life cycle of bioﬁ lm. The

parameters of EPS include molecular concentra-

tion, charge condition, quantity, hydrated level,

components, and so on. These factors, varying

because of the embedded cells, stages of bioﬁ lm,

nutrient sources, and local environment/host, are

essential for EPS functions.

Mechanically, EPS provides architectural sta-

bility for the bioﬁ lm entity, helps bacterial cells

adhering to surfaces, immobilizes cells onto local

substratum, and ﬁ nally forms a three-dimensional

network that supports the bioﬁ lm cells.

Metabolically, at the extracellular retention space,

EPS helps the external substances such as oxygen

and carbon source to diffuse into the bioﬁ lm, as

well as the internal metabolic waste moving out.

From the multicellular and polymicrobial features

of bioﬁ lm, EPS is also an external metabolism

system containing free enzymes, metabolites,

extracellular DNA (eDNA), and other substances

from cell lysis. Therefore, within the matrix, gene

horizontal transfer through eDNA, mutualism or

antagonism interactions, and nutrient accumula-

tion are active [ 62 , 212 ]. By supporting vertical

extension of bioﬁ lm, EPS also reduces the

biomass density to buffer impact from external

substances. Moreover, EPS can protect the

microbial consortia from multiple external chal-

lenges, such as antibiotics, and allow the long-

term existence of bioﬁ lm community. EPS not

only provides a structural framework for bioﬁ lm

but also keeps the homeostasis of microenviron-

ment. This relative stable and interactive space

offers chances for bacteria to survive and even

persist in cruel conditions.

2.1.1.3 Bioﬁ lm Formation

The life cycle of surface-associated bioﬁ lm is

well studied in in vitro models. The process is

promoted by all different members in the com-

munity, regulated by associated genetic networks,

and also inﬂ uenced by the environment. Different

standpoints are taken in recent study models of

bioﬁ lm development, such as developmental

genetic regulation network, environmental adap-

tation, and multispecies interactions.

The most accepted model describes the bioﬁ lm

formation as a highly regulated spatiotemporal

process. The formation of bioﬁ lm is generally

summarized into ﬁ ve dynamic stages as (1) loose

attachment of planktonic cells, (2) irreversible

adherence, (3) microcolony formation, (4) mature

Fig. 2.1 The composition of

extracellular polymeric

substances of bacterial

bioﬁ lms. Bacteria in the

mature bioﬁ lm are embedded

in an EPS matrix, which

consists of polysaccharides,

proteins, extracellular DNA

(eDNA), amyloid ﬁ bers and

bacteriophages, etc.

X. Xin et al.

macrocolony formation, and (5) dispersal. The

developmental model of bioﬁ lm focuses on the

regulated transition through the cascade and

emphasizes genetic pathway involvement in the

group level development. Recent studies on the

proteomics and genetic network of gram- negative

bacteria Pseudomonas aeruginosa ( P. aeruginosa )

bioﬁ lm have indicated that the developmental pro-

cess of bioﬁ lm is sequentially regulated [ 181 ,

196 ]. P. aeruginosa bioﬁ lm is one of the earliest

and most studied in medical microbiology ﬁ eld

[ 114 ]. Stages 3 and 4 of its bioﬁ lm formation

involve type 4 pili-mediated mechanism [ 109 ].

The general dynamic processes can be identiﬁ ed

in most species, whereas distinct mechanisms

reside in different bacteria, even for strains from

the same species. For example, LapA from the

bioﬁ lm-associated-protein (Bap) family is impor-

tant in adhesion and transition from reversible to

irreversible attachment in P. ﬂ uorescens and P.

putida ; conversely, no evidence indicates the

involvement of Bap in the P. aeruginosa bioﬁ lm

formation [ 57 , 159 ].

Environmental impact is crucial to bioﬁ lm

formation. Therefore, environmental adaptation

model is employed to describe the bioﬁ lm forma-

tion. The environmental adaptation model sug-

gests that the phenotypic or species heterogeneity

within the bioﬁ lm is the response to the spatio-

temporal change in the ecological niche. For

instance, oxygen and carbon gradient is one of

the driving forces for bioﬁ lm formation. Here the

developmental stages can be sketched as (1)

planktonic cells attachment; (2) initial seed cells

growth with sufﬁ cient nutrients; (3) formation of

microcolonies where outer cell layers restrain the

nutrient availability of inner layers, thereafter

gradient and heterogeneity showing within each

colonies; (4) formation of macrocolonies, where

some cells express high level of EPS and facili-

tate the vertical growth of community, resulting

in the improvement of nutrient and oxygen distri-

bution; (5) bioﬁ lm formation from mature mac-

rocolonies, where structures like pores and

channels form to further assist inner nutrient dif-

fusion [

159 ]. This model describes the changes in

population and structure from the perspective of

metabolism. The genes that regulate the

environmental adaptation should also be included

in the bioﬁ lm-speciﬁ c gene network as described

above.

In nature, a great portion of bioﬁ lm is com-

posed of diverse species. This polymicrobial fea-

ture makes interspecies interaction an important

factor and variable in bioﬁ lm formation. At the

early stages of bioﬁ lm formation, distribution

and roles of different bacteria and/or fungi

depend on various factors, such as metabolic

requirement, surface structures, mutualism, and

quorum sensing regulatory system. For exam-

ple, metabolic cooperation exists between oral

commensal Streptococcus gordonii and patho-

gen Aggregatibacter actinomycetemcomitans .

Peroxide produced by S. gordonii can be utilized

by A. actinomycetemcomitans for further secre-

tion of complement-resistant protein ApiA that

can limit host complement-mediated killing

[

229 ].

So far, we still have a lot to ask about the

mechanisms of bioﬁ lm formation, especially for

that of in vivo bioﬁ lm. There is a long way to go

to ﬁ nd out whether the discoveries from in vitro

models are consistent with in vivo phenotypes.

2.1.1.4 Survival Advantages of Bioﬁ lm

The bioﬁ lm form of growth with its surface asso-

ciation offers bacteria remarkable advantages to

survive and thrive in harsh environment compared

to the planktonic form. Several hypotheses on

how bioﬁ lm gains beneﬁ cial phenotypes and suc-

ceeds have been proposed. First, the underlying

surface offers a relatively stable space within the

environment and also a substratum for microbes

to grow on, facilitating further communities build-

ing, nutrient accumulation, and interaction of

cells in proximity. Second, microbial communi-

ties in bioﬁ lm mode gain protection and higher

buffer ability against various challenges from the

environment, such as ultraviolet exposure [

56 ],

metal toxicity [ 201 ], ﬂ uctuating pH [ 230 ], desic-

cation [ 231 ], phagocytosis [ 125 ], and antibiotics

and antimicrobial agents [ 69 , 137 , 195 ].

The bioﬁ lm mode of survival has profound

importance for prokaryotes in both the natural

environment and human body, even being recog-

nized as the most successful forms of life on the

2 Bioﬁ lm and Dental Caries

earth. In environmental microbiology, bioﬁ lm

fossil records from as early as 3.2 billion years

ago were identiﬁ ed [ 82 ]. From the standpoint of

evolution, we can speculate that bioﬁ lm as an

advantageous form of growth offered space with

homeostasis for the primitive prokaryotes in the

rigid and ever-changing environment. Thus, now-

adays, bioﬁ lms can be found almost everywhere

even in extreme conditions, such as hot spring,

deep-sea vents, and so on [ 15 ].

This survival advantage exists not only in the

nature but also on and in our body. With the

development of human microbiome project

(HMP), we have recognized that we are living in

close relationship with microbes, which are

termed “the microbial part of ourselves” [ 228 ].

Human microbiome has several major compo-

nents including the oral, skin, vaginal, gut, and

nasal/lung microbiome, affecting the homeosta-

sis of health and disease. Like microbes in nature,

human microﬂ ora usually exists as sessile aggre-

gates of cells with or without a surface. From

thousands of recent in vitro and in vivo bioﬁ lm

studies, we can summarize that bioﬁ lm is closely

associated with chronic and persistent infections.

Because bacterial bioﬁ lm is almost 1000-fold

more tolerant to certain antibiotics compared to

the planktonic state, the persistence of bioﬁ lm

infection is most likely resulted from this impor-

tant feature [ 15 , 58 ].

2.1.2 Dental Plaque as a Typical

Bacterial Bioﬁ lm

In 1886, Black ﬁ rst described the accumulated

bacteria on early carious lesions as plaque [ 18 ].

In oral cavity, tooth as mineralized hard tissue

provides non-shedding surfaces for oral microbes

to colonize. Attached bacteria, embedded in

polymeric matrix, form microcommunities called

dental plaque. In recent years, dental plaque as a

typical bioﬁ lm has been studied extensively. As a

naturally formed bioﬁ lm, dental plaque acts

actively as part of the host defense system against

colonization of exogenous pathogenic microﬂ ora

[ 145 ]. On the other hand, when the homeostasis

of dental plaque is disrupted, bioﬁ lm-associated

diseases like caries and periodontal diseases

develop.

Dental plaque was the ﬁ rst bioﬁ lm being

investigated at both composition and antimicro-

bial sensitivity aspects. With the help of molecu-

lar and bioinformatics techniques, more than

800 phylotypes of bacteria in the oral microbi-

ome, including more than 300 species, have been

identiﬁ ed [

193 ]. The diverse and numerous resi-

dents make dental plaque a highly complex and

varied community. Furthermore, a limited num-

ber of fungi and viruses are also found in oral

cavity. Notably, more than half of oral bacteria

cannot grow in pure culture in vitro, largely lim-

iting our investigation on their physiological

characteristics [ 111 ].

The tooth surfaces above gingival margin are

mainly enamel or dentin, where they directly face

the dynamic oral environment, whereas the sur-

faces below the gingival margin are mainly den-

tin or cementum, which are in close contact with

gingival tissue and relatively less ﬂ uctuant.

According to the location of the attached sur-

faces, dental plaque can be roughly divided into

two categories: supragingival plaque and subgin-

gival plaque. Each type has its own special physi-

ological characteristics and relation to disparate

diseases.

Oral cavity is a unique ecological niche, which

is warm, moist and relatively opens to the outer

environment. Tooth surfaces as well as dental

plaque constantly encounter different challenges

from food intake, speech, oral hygiene proce-

dures, and so on. The oxygen and nutrient levels

are changing constantly at different sites. For

example, the oxygen level varies, from being

anaerobic in subgingival plaque and basal layers

of supragingival plaque to being almost aerobic

in outer layers of supragingival plaque [

142 ]. The

local microbial diversity and metabolism also

change enormously from health to disease. Apart

from some speciﬁ c pathogen-induced diseases,

most oral diseases are caused by or associated

with long-term multispecies microbial activity

within dental plaque [ 111 ]. Here, we will take

supragingival bioﬁ lm, for example, to demon-

strate the association between dental plaque and

caries.

X. Xin et al.

2.1.3 Composition of Dental Plaque

Like other bioﬁ lms, dental plaque contains two

major components: microbes and the biopolymer

matrix. The bacterial species varies signiﬁ cantly at

distinct surfaces, such as pits/ﬁ ssures, smooth sur-

faces, approximal surfaces, and gingival crevice.

The habitats are selective for the preferable bacte-

ria that are able to grow, based on the different

anatomy and biological features in the local envi-

ronment [ 1 , 145 , 169 ]. The studies of microbial

composition of bioﬁ lm have been shifting from

culture-based to molecular approaches. Through

16 s rRNA gene-based sequencing, a plethora of

new taxa have been discovered, although their

physiological characteristics are largely unknown.

In normal microﬂ ora on ﬁ ssures and smooth

surfaces, the predominant species are mostly

gram-positive aerobic or facultative anaerobic

bacteria, mainly streptococci. Other common spe-

cies include Gemella , Granulicatella , Actinomyces ,

Rothia , Veillonella , Neisseria , Prevotella ,

Abiotrophia , Capnocytophaga , Fusobacterium ,

and so on [ 1 ]. In contrast, the subgingival species/

phylotypes are more diverse, from facultative

anaerobic to obligate anaerobic. In a locus with

features of both supra- and subgingival surfaces,

the approximal plaque had an intermediate micro-

bial constitution [ 146 ]. The dental microﬂ ora is

highly varied from person to person, depending on

the complex genetic and environmental variations

in population. Once established, the personal

microbial community stays stable and in a dynamic

balance (or homeostasis). This ﬂ uctuant balance is

achieved by heterogeneous microﬂ ora from both

metabolic mutualism and antagonism within the

community. Staying in a relatively open niche, the

resident community is under constant challenges or

invasions. When the habitats change greatly and

affect local microbial activities, the homeostasis

will shift, and a new microbial composition may be

established to ﬁ t new environmental conditions.

The biochemical constitution of dental plaque

varies for different hosts and bacterial composi-

tion. Dental plaque is highly hydrated and con-

tains about 80 % water. Changing with time

before and after food intake, the glucan takes

10–20 % of plaque dry weight, while fructan

takes 1–2 %. Protein comprises approximately

40 % of fry weight, which is usually from bacte-

rial secretion and saliva. In addition, lipid, nucleic

acid, and minerals like calcium, magnesium,

phosphate, and ﬂ uoride are in varying amounts in

plaque [ 39 , 94 , 167 , 171 ].

The part of plaque without bacteria is the

extracellular polymeric matrix, which is an

essential component of bioﬁ lm [ 18 , 221 ]. The

extracellular matrix, as that of other bioﬁ lm, is a

biopolymeric mixture of various components

from multiple sources. The attached bacteria on

tooth surfaces secrete macromolecules by active

secretion or cell lysis. These molecules along

with salivary proteins, host cells, and intake sub-

stances develop into a complicated three-

dimensional network that surrounds cells and

further forms dental plaque. The structure and

elements of matrix are inﬂ uenced by coresidents

and the local habitat. It can be considered as a

variable that reﬂ ects plaque status.

This matrix aids bacteria to adhere to tooth

surface, promotes coaggregation, reserves nutri-

ents and enzymes, and protects the community

from external harms, such as diffusion of charged

or bioreactive molecules. Besides keeping the

local homeostasis, EPS contains high ratio of

polysaccharides and also acts as a virulence fac-

tor in the pathogenesis of dental caries. The poly-

saccharides in matrix are the resources that

acidogenic bacteria metabolize and generate

acids to initiate further caries progress. For

example, S. mutans and lactobacilli are able to

metabolize sucrose in matrix into glucan or fruc-

tan polymers which are either water soluble or

water insoluble (branching). The insoluble car-

bohydrate polymers produced by S. mutans offer

adhesion sites for S. mutans and thus are impor-

tant in the development and progression of dental

caries [

10 , 18 , 221 ]

2.1.4 Spatiotemporal Development

of Oral Bioﬁ lms

When exposed to the oral environment, for exam-

ple, as soon as a few seconds after the tooth erup-

tion or after brushing, the tooth surfaces are

2 Bioﬁ lm and Dental Caries

coated with a conditioning ﬁ lm consisting of pro-

teins and glycoproteins. This ﬁ lm is called

acquired pellicle, the sources of which are mainly

saliva and gingival crevicular ﬂ uid (GCF) [ 100 ].

Selective salivary components absorb to the tooth

surface, including amylases, mucins, proline-rich

proteins, histatins, statherins, lysozymes, and so

on [ 150 , 190 ]. These molecules attach on tooth

surfaces and offer bioactive sites that facilitate

future colonizers to bind. Hence, acquired pelli-

cle is recognized as the sign of the initiation of

dental plaque formation [ 18 , 68 , 88 ].

Following acquired pellicle formation, a few

bacterial species, mostly Streptococcus spp., start

to absorb and attach loosely to the ﬁ lm by a wide

range of nonspeciﬁ c and relatively weak forces.

These initial colonizers (e.g., S. mitis and S. ora-

lis ) bear speciﬁ c surface-anchored proteins

(adhesins) to form irreversible binding with host

receptors in acquired pellicle. Electron micros-

copy has shown that cocci can be detected on

enamel surfaces at as early as four hours after

cleaning in vivo [ 83 , 155 ]. As the initial attach-

ment and proliferation go on, the bacterial metab-

olism changes the microenvironment in favor of

future residents. As the bioﬁ lm develops, more

early colonizers with surface adhesins or recep-

tors interact and attach to the initial colonized

bacteria, starting the cascade of bioﬁ lm prolifera-

tion and biodiversity ﬂ ourish. In this dynamic

process, coaggregation has been extensively

studied as one of the major mechanisms in early

bioﬁ lm succession. This typical bacterial adhesin

and bacteria/host receptor interaction is often

based on protein-carbohydrate (lectin) reaction

and eventually leads to the construction of a more

stable and diverse network. S. oralis and

Actinomyces oris are one of the most studied pair

of coaggregation partners. The receptor polysac-

charides on the cell surface of S. oralis can inter-

act with type 2 ﬁ mbriae on A. oris surface,

forming potent cell aggregates in vitro [

164 ].

With the involvement of more species, the com-

plexity of bioﬁ lm composition and structure is

ever growing. In some cases, in a single site,

more than 50 different species can be found [ 1 ].

With community multiplication, inter- and

inner- species communications become active.

The quorum sensing regulatory pathway plays a

critical role during the development of dental

plaque community.

In supragingival plaque, oral bacteria are

mostly saccharolytic and able to utilize metabolic

substances from endogenous nutrients. Saliva, as

the major local source of amino acids, proteins,

and glycoproteins, is continuously available,

whereas diet intake has a minor role in nutrient

contribution due to its limited surface contact

time and unavailability of some highly complex

ingredients for bacteria. Salivary proteins and

glycoproteins are essential for initial bioﬁ lm for-

mation and bacterial growth, whereas host food

intake and other activities enormously affect the

maturation and dynamic transition of bioﬁ lm. As

previously mentioned, bacteria produce biopoly-

mers to build the matrix during bioﬁ lm formation.

In health bioﬁ lm, the metabolism with salivary

substrates induces minor change to the local

pH. In contrast, when diet change induces local

sucrose concentration increase, the acid produc-

tion will rise and further cause the homeostasis

shift, leading to the numerical predominance of S.

mutans and ﬁ nally dental caries [

100 , 168 ].

Taken together, dental plaque as a typical

bioﬁ lm possesses the social traits and beneﬁ ts

from this community-based lifestyle. Microbial

cells colonize following their own nutrient and

interactive needs, and a gradient of nutrient, air,

pH, and partners gradually forms in the commu-

nity [

20 , 31 ]. The dental plaque as a microeco-

system can offer a wide range of habitats for

diverse microbes and integrate different resi-

dents into a highly organized and tolerant com-

munity. Within an immediate contact, oral

bacteria have better chances to conduct more

efﬁ cient metabolism through mutualistic inter-

actions [ 170 ]. With the support from extracel-

lular matrix, bacteria in plaque are less sensitive

to inhibitory agents and host defenses [ 93 , 210 ,

213 ]. Driven by environmental cues, dental

plaque would shift to pathogenic bioﬁ lm with

distinct microbial composition and metabolic

activity and thus initiates oral infectious dis-

eases such as dental caries.

X. Xin et al.

2.2 Microbial Etiology of Dental

Caries

2.2.1 Oral Microbiology at

Early Stage

There is a long history of understanding the etiol-

ogy of dental caries. A Sumerian text from 5000

BC describes a “tooth worm” as the cause of car-

ies. The “tooth worm” theory has also been

described in the literature of ancient China, India,

Egypt, and Japan. During the European Age of

Enlightenment, the “tooth worm” theory was

gradually rejected by the European medical com-

munity. Pierre Fauchard, known as the father of

modern dentistry, was one of the ﬁ rst to oppose

the “tooth worm” theory and noted that sugar was

detrimental to the teeth and gingiva.

Around 1680, van Leeuwenhoek (1632–1723)

[ 67 ], a Dutch dry goods merchant, observed and

described ﬁ rst microorganisms in the tartar from

his teeth with his primitive microscopes. In his

notebook, he recorded “I didn’t clean my teeth

for three days and then took the material that has

lodged in small amounts on the gums above my

front teeth…. I found a few living animalcules.”

The microbes sketched in his notebook are now

known as some of the most abundant bacteria

resided within oral cavity, including cocci, spiro-

chetes, and fusiform bacteria. These fascinating

observations at the birth of microbiology had

already signaled the complexity of the oral

microbial community [

91 ].

2.2.2 Dental Caries as an Infectious

Disease

W. D. Miller was arguably one of the most impor-

tant scientists who substantially advanced the

knowledge of dental caries and associated studies

in oral microbiology. As a practicing dentist with

a comprehensive training in natural sciences, he

identiﬁ ed the “germs” responsible for tooth decay.

In his 1890 book titled Microorganisms of the

Human Mouth , he proposed a “chemoparasitic”

theory of dental caries, which suggested that oral

microorganisms can convert carbohydrate into

acid and eventually result in the demineralization

of teeth and development of dental caries in sus-

ceptible hosts. Miller’s chemoparasitic theory,

together with the description of “gelatinous

microbic plaques”, which are now commonly

known as “dental plaque,” by Black and Williams

[

16 , 219 ], has laid the foundation for our modern

knowledge of dental caries etiology.

Due to the limited bacterial isolation and cul-

tivation technique available in the nineteenth cen-

tury, Miller was unable to identify the causative

agent(s) of dental caries. In 1924, Clarke ﬁ rst iso-

lated a bacterial species from human dental car-

ies site and named it S. mutans , which was

capable of fermenting several sugars and produc-

ing a pH of 4.2 in glucose broth [ 38 ].

Unfortunately, Clarke was unable to demonstrate

that this organism actually caused caries.

Since 1950, various experimental animal

models have been employed to better elucidate

the nature and etiology of oral diseases, including

dental caries [ 105 ]. In 1960, two important arti-

cles were published which were considered the

cornerstone of dental caries research. In the ﬁ rst

paper, using hamster as animal model, Keyes

revealed the infectious and transmissible nature

of dental caries [ 108 ]. Then in another elegant

paper, Fitzgerald and Keyes successfully demon-

strated caries induction in an animal harboring a

“conventional” microﬂ ora, by a single type of

streptococcus that had been isolated by Clarke

more than 30 years ago [

61 ].

2.2.3 Dental Plaque as the Cause

of Dental Caries

One of the early achievements in oral microbiol-

ogy is to link dental plaques to dental and peri-

odontal diseases. Dental plaque was one of the

ﬁ rst substances van Leeuwenhoek examined

under his microscope [ 67 ]. The living microor-

ganisms recorded by him revealed for the ﬁ rst

time the complex nature of dental plaque in terms

of its microbial composition. Later on, both Erdi

and Ficinus described the presence of

2 Bioﬁ lm and Dental Caries

microorganisms within the “membrane” on the

teeth [ 197 ]. However, the full implications of

dental plaque were not realized until the publica-

tion of [ 16 ] paper, in which he referred dental

plaque as “gelatinous microbic plaques,” a gela-

tin-like substance that carried microorganisms

[ 16 ]. Black believed that bacteria within dental

plaque generate acids that dissolve the dental

hard tissue. Black’s work, together with Miller’s

chemoparasitic theory, underscored the impor-

tant role of dental plaque in the etiology of dental

caries and has become one of the essential para-

digms of oral biology.

2.2.4 Association of Streptococcus

mutans with Dental Caries

Considering the involvement of S. mutans in the

pathogenesis of dental caries as demonstrated by

early in vitro and in vivo animal models, epide-

miologist tried to predict the development of den-

tal caries by the detected number of S. mutans

present on the teeth. Numerous studies suggested

a positive correlation, and S. mutans was recog-

nized as the chief culprit of dental caries. Hence,

efforts were gradually shifted to the exploration

of the cariogenic abilities of S. mutans .

Researchers in this ﬁ eld proposed that the viru-

lence of S. mutans resides in three core attributes,

including the abilities (i) to metabolize dietary

sucrose to form insoluble polysaccharides that

mediate the persistent colonization of tooth sur-

faces, (ii) to produce large quantities of organic

acids (primarily lactic acid) from a wide range of

carbohydrates (acidogenicity), and (iii) to toler-

ate various environmental stresses, particularly

low pH which are toxic to most of the other bac-

terial species present in the mouth (aciduricity).

The complete genome sequence of S. mutans

published in 2002 further stimulated numerous

studies of this bacterium at genetic level. However,

the correlations between dental caries and S.

mutans were not deﬁ nitive at the level of individu-

als. Today, it is clear that there are individuals and

population groups of high caries susceptibility

with low levels of S. mutans and vice versa. While

there is little doubt about the ability of S. mutans

to cause dental caries in humans or animal models,

its contribution to the pathogenesis and progres-

sion of dental caries is still unclear.

2.2.5 Nonspeciﬁ c or Speciﬁ c Plaque

Hypotheses?

Miller’s “chemicoparasitic theory” proposed a

“nonspeciﬁ c plaque” hypothesis, in which den-

tal caries is the result of the overall interaction

of all the groups of bacteria within plaque.

However, the rat caries model and the positive

correlation of S. mutans with dental caries in

most cases suggested that this proposal might

not be absolutely right. Hence, the “nonspeciﬁ c

plaque” hypothesis has been challenged for

decades. An alternative view is that among the

200–300 indigenous species identiﬁ ed in the

oral cavity, only a ﬁ nite number of them can be

recognized as dental pathogens. Thus, dental

caries can be considered as speciﬁ c, treatable

infections. This proposal had the beneﬁ t of

focusing studies on the control of speciﬁ c

microbial targets. However, although Mutans

streptococci (including S. mutans , S. rattus , S.

sobrinus , and S. cricetus ) are strongly impli-

cated with caries, the association is not unique;

caries can occur in the apparent absence of

these species, while S. mutans can persist with-

out evidence of detectable demineralization.

Indeed, in such circumstances, some acido-

genic, non- S. mutans are implicated with dis-

ease. Due to the imperfection of both theories,

the debate is still ongoing up until today.

2.2.6 Ecological Plaque Hypothesis

Unlike many known medical pathogens that are

“foreign invaders with speciﬁ c virulence factors,”

the oral “pathogens” such as S. mutans are part of

the normal ﬂ ora. From the initial isolation of S.

mutans by J. K. Clarke in 1924 to the latest

metagenomic studies, over 700 bacterial species

have been identiﬁ ed from the human oral cavity.

The oral microﬂ ora has been recognized as one

of the most complex microbial communities in

X. Xin et al.

the human body [ 122 ]. As proposed by Phil

Marsh in his “ecological plaque hypothesis,” it is

not merely the presence of a single organism in a

complex community that determines the proper-

ties of a bioﬁ lm, but it is the interactions between

the bioﬁ lm residents that are crucial. In dental

caries, there is a shift toward community domi-

nance by acidogenic/aciduric gram-positive bac-

teria (e.g., S. mutans and lactobacilli) at the

expense of the acid-sensitive species associated

with sound enamel [ 110 , 143 ].

The introduction of high-throughput metage-

nomic pyrosequencing has profoundly advanced

our understanding of the overall oral microbial

diversity and function. Data obtained from metage-

nomic level favorably support the ecological

plaque hypothesis. A recent metagenomic study

on microbiome in caries cavity showed that the

caries cavities are not dominated by S. mutans , but

are a complex community formed by tens of bacte-

rial species [ 77 ]. The data support the polymicro-

bial etiology nature of caries. Another investigation

on the oral microbiota of children with dental car-

ies revealed that genera Streptococcus , Veillonella ,

Actinomyces , Granulicatella , Leptotrichia , and

Thiomonas in plaques were signiﬁ cantly associ-

ated with dental caries [ 42 ], further supporting the

idea that no one speciﬁ c pathogen but rather patho-

genic populations in plaque correlate with dental

caries. In addition, a recent study on salivary

microbiome of caries-active population further

supports the ecological hypothesis that the shifts in

community structure, instead of the presence or

absence of speciﬁ c groups of microbes, contribute

to the occurrence of dental caries [

232 ].

2.2.6.1 Microbial Ecology in the Oral

Cavity

It has been estimated that the human body is

made up of over 1 × 10

cells, of which 90 % are

the microorganisms that comprise the resident

microﬂ ora of the host. Thus, this resident micro-

ﬂ ora has been currently proposed as a novel

organ in human body. The resident microﬂ ora

dynamically interacts with the human body, con-

tributing directly and indirectly to the normal

development of the physiology, nutrition, and

defense systems of the host [ 143 ].

Despite the continual shift of these microor-

ganisms, the composition of the resident

microﬂ ora is distinct in different habitats/

niches such as the oral cavity, gut, and vagina.

The different key ecological factors present in

these biohabitats have a great impact on the

community structure and metabolic function of

the resident microﬂ ora. Those ecological fac-

tors include appropriate receptors for attach-

ment, essential nutrients and cofactors for

growth, as well as an appropriate pH, redox

potential, and gaseous environment. Take the

oral cavity, for example, the tooth surfaces pro-

vide distinct binding factors for microorgan-

isms. Moreover, the mouth is continuously

bathed with saliva at a temperature of 35–36 °C

and a pH of 6.75–7.25. The nutritional condi-

tion of the oral cavity is often described as

“feast or famine”, further exerting far-reaching

inﬂ uence on the composition of microﬂ ora.

Consistently, our recent study by sampling

microﬂ ora from various oral sites of different

age groups has demonstrated that the oral cav-

ity is a highly heterogeneous ecological system

containing distinct niches with signiﬁ cantly

different microbial communities. More impor-

tantly, the phylogenetic microbial structure

varies with aging, and only a few taxa were

present across the whole populations [

231 ].

The ﬁ rst stages of dental plaque formation

involve the attachment of bacteria to salivary pro-

teins and glycoproteins that are deposited as pel-

licle on the surfaces of teeth/dentures and other

oral tissues. Bacteria that ﬁ rst attach to the sali-

vary pellicle are designated primary colonizers,

including S. oralis , S. mitis , S. sanguinis , S. para-

sanguinis , and S. gordonii . In addition,

Actinomyces , Veillonella , Gemella , Abiotrophia ,

and Granulicatella species are often detected.

These early colonizers proliferate and change

local environmental conditions, making the site

suitable for colonization by more fastidious spe-

cies (e.g., obligate anaerobes). These early colo-

nizers also form the base layers of complex dental

plaque bioﬁ lms. Subsequently, new microbial

cells adhere via similar adhesin- receptor mecha-

nisms (a process termed coaggregation or coad-

hesion). Subsequent colonizers such as

2 Bioﬁ lm and Dental Caries

Fusobacterium and P. gingivalis are especially

effective in attaching to earlier plaque colonizers

and eventually form complex, structured, multi-

species bioﬁ lms. Once established, the microbial

composition of dental plaque remains relatively

stable over time, and this microbial homeostasis

is intricately maintained by the dynamic intermi-

crobial and host- microbial interactions [ 143 ].

2.2.6.2 Genetic and Environmental

Factors and Oral Microbial

Ecology

In order to fully understand the change of oral

microbial community during the occurrence and

development of oral diseases (e.g., dental caries),

it is necessary to completely characterize the

community structure of the oral microbiome and

its inﬂ uencing factors. Previous studies in healthy

individuals have revealed signiﬁ cant inter- and

intraindividual structure and function diversity in

the human oral microbiome. Nurture (environ-

ment) and nature factors (host genotype), such as

diet, hygiene, geography, cultural traditions, age,

gender, and human genotype, work together to

shape the oral microbiome [ 164 ].

Diet is one of the most important environment

factors that impose profound inﬂ uence on the

structure and function of the human oral micro-

biome. Exogenous nutrients provided via the

diet exert strong selection on the composition of

the oral microbiota. Fermentable carbohydrates

are the class of nutrients that most affect the

microbial ecology of the mouth. They are catab-

olized to acids, inhibiting most of the species

while promoting the growth of aciduric organ-

isms. Frequent exposure to low pH can disrupt

microbial homeostasis and lead to the enrich-

ment of acidogenic/aciduric species within the

dental plaque. Other environment factors, such

as oral hygiene, medication, and geography, will

also inﬂ uence the composition of oral microbi-

ota [

143 ].

Host immunity plays an essential role in

shaping oral microbiome. Saliva contains com-

ponents of innate (e.g., lysozyme, lactoferrin,

sialoperoxidase, antimicrobial peptides) and

adaptive immunity (e.g., sIgA), can directly

inhibit some exogenous microorganisms. Host

tissues surrounding the oral cavity may secrete

antimicrobial agents and immune modulators,

which have substantial inﬂ uence on the proper-

ties of dental plaque. In addition to host immu-

nity, the tooth morphology is heavily determined

to be inﬂ uenced by genetic factors also contrib-

utes to the community structure of human oral

microbiome [

143 ].

In fact, most factors involved in the diversity in

oral microbiome cannot be simply classiﬁ ed into

nurtural or natural. Accumulating evidence from

twin-pair model favors the “environment domi-

nates” theory. By comparing the composition varia-

tion of salivary microbiome in a cohort of 27

monozygotic and 18 dizygotic twin pairs, Stahringer

et al. proposed the philosophy of shared environ-

ment serving as the main determinant of microbial

populations [ 193 ]. Nevertheless, a recent report by

the Human Microbiome Project Consortium has

shown that the ethnic or racial background associa-

tion is one of the most robust associations with

microbiome, indicating that the host genotype such

as race and ethnicity may also be one of the major

determinants for the human microbiome diversity

[ 205 ]. Moreover, several recent studies have sug-

gested that ethnicity tunes the oral microbiome at

more speciﬁ c levels [ 147 , 239 ].

2.2.6.3 Interspecies Interactions

and Dental Caries

The viability of oral microbial community is

dependent not only on the host genetic and envi-

ronmental factors but also on interactions

between the microbial residents. The residents in

the complex oral microbial community interact

extensively, forming bioﬁ lm structures, carrying

out physiological functions, and inducing micro-

bial pathogenesis. Therefore, “war and peace” is

usually used to describe the dynamic microbial

interactions within a bioﬁ lm. In general, micro-

bial interactions include (i) competition between

bacteria for nutrients, (ii) synergistic interactions

for the growth or survival, (iii) antagonistic inter-

actions by secondary metabolites production, (iv)

neutralization of a virulence factor produced by

another resident, and (v) interference in the

growth-dependent signaling mechanisms of each

other [

122 ].

X. Xin et al.

S. mutans and other caries-associated organ-

isms such as lactobacilli and Actinomyces species

are capable of expressing certain pathogenic fac-

tors. A dynamic balance of both synergistic and

antagonistic interactions with the coresidents

plays an essential role in determining whether

these pathogenic factors cause damage or not.

Hence, in the case of dental caries, it is now gen-

erally recognized that this disease is not solely

the result of the presence of S. mutans or any

single organism in dental plaque. Rather, it is the

net result of the interaction of multiple acido-

genic/aciduric organisms such as S. mutans with

other commensals within the dental plaque.

Factors Involved in Interspecies Inter-

actions Metabolic interrelationship is one of

the most common interspecies interactions

within dental plaque. Nutrients could be avail-

able from the periodic intake of food, saliva, and

nutrients provided by other organisms as well as

polysaccharides present in dental plaque. S.

mutans metabolizes sucrose to generate acid

more efﬁ ciently compared to other common oral

bacteria [ 83 ]. When sucrose is frequently con-

sumed, S. mutans may take advantage of the sub-

strate competition and acid selection to emerge

as a predominant resident in caries-associated

bioﬁ lms. In addition, mutualism between two

organisms dependent on nutrients is also com-

mon. For example, lactate produced by strepto-

cocci is utilized directly by Veillonella for

growth. As lactate is removed from the immedi-

ate environment by Veillonella , so the ﬂ ux of

glucose to lactate increases, thus in turn enhanc-

ing growth of streptococci [

155 ]. Another exam-

ple is the combined efforts of A. naeslundii and

S. oralis in metabolizing salivary components to

form extensive bioﬁ lms on saliva- coated sur-

faces [ 168 ].

The secondary metabolites of one organism

also have effects on other coresidents within the

same bioﬁ lms. One of the best examples is

between S. mutans and S. sanguinis . The ecologi-

cal antagonism between these two bacteria in the

oral cavity has been noted for decades. Early col-

onization and high levels of S. sanguinis in an

infant’s oral cavity correlate with signiﬁ cantly

delayed colonization by S. mutans . Similarly,

high levels of S. mutans in the oral cavity corre-

late with low levels of S. sanguinis [

132 ]. Animal

study has also demonstrated a so-called competi-

tive exclusion between S. mutans and S. sanguinis

depending on the sequence of inoculation [ 156 ].

The lactic acid produced by S. mutans favors the

growth of itself relative to that of other less acidu-

ric oral streptococci including S. sanguinis . On

the other hand, S. sanguinis can produce antimi-

crobial hydrogen peroxide to antagonize the

growth of S. mutans , which lacks effective sys-

tems to detoxify hydrogen peroxide [ 117 , 119 ]. In

addition to antagonism, metabolic products of one

organism may promote the growth of other organ-

isms. For example, organisms which are able to

metabolize oxygen would favor the growth of

nearby anaerobic organisms [ 46 ].

Bacteriocin is another key factor involved in

the interspecies competition. Bacteriocins are

proteinaceous toxins produced by all major lin-

eages of bacteria. Unlike traditional antibiotics,

bacteriocins often have a narrow killing spectrum

and inhibit the growth of related organisms [ 178 ].

In oral cavity, streptococci and a variety of oral

bacteria including A. actinomycetemcomitans

produce bacteriocins that are lethal to other bac-

teria. S. mutans produces a number of distinct

bacteriocins (also known as mutacins) as an

“arsenal” against its competitors. At least ﬁ ve

different bacteriocins (mutacins I to V) produced

by S. mutans have been identiﬁ ed [ 36 , 174 , 175 ].

In addition, at least nine other putative

mutacin- encoding genes have been annotated in

the genome of S. mutans UA159, suggesting a

large repertoire that can be used against its com-

petitors. Some of these bacteriocins are able to

inhibit the growth of S. sanguinis and thus may

be responsible, in part, for the negative correla-

tion of the presence of S. sanguinis and S. mutans

in the dental plaque [

81 ]. Similarly, the expres-

sion of bacteriocins by some other bioﬁ lm resi-

dents may determine which organisms are

coresidents in these structures.

Interactions mediated by signaling molecules

are also prevalent in the dental bioﬁ lms. The

quorum- sensing regulatory molecule autoin-

ducer-2 (AI-2) is a potential signaling molecule

2 Bioﬁ lm and Dental Caries

between heterogeneous bacteria within bioﬁ lms.

One typical example is that the luxS mutants of P.

gingivalis and S. gordonii cannot form mixed

bioﬁ lms, but a mutation in either strain alone

allows for such a bioﬁ lm formation [ 153 ]. In

addition, AI-2 also mediates mutualistic bioﬁ lm

formation by S. oralis and A. naeslundii strains

[ 177 ]. Hence, AI-2 functions as one of the pri-

mary mediators of interspecies interactions

within oral bioﬁ lms. In addition to AI-2, many of

the oral streptococci, including S. mutans , S. gor-

donii , and S. sanguinis , signal by the competence

stimulating peptide (CSP). Unlike the AI-2, CSP

is highly species speciﬁ c and is not likely to

interfere with the activity of another distinct CSP

molecule. Of note, the CSP still can be indirectly

involved in interspecies interactions. In the case

of S. gordonii vs S. mutans interactions, a prote-

ase (i.e., challisin) expressed by S. gordonii

inhibits CSP-dependent properties of S. mutans

[ 215 ]. In addition, S. mutans may acquire trans-

forming DNA from the coresidents through the

CSP-induced bacteriocin production [ 118 ].

Taken together, microbial homeostasis can only

be achieved when an equilibrium is established

among different species within the same biologi-

cal niche. The aforementioned factors play an

essential role in modulating interspecies interac-

tions within bioﬁ lms, thus shaping the structure of

the oral microbial community. Changes of these

factors can perturb the established equilibrium,

leading to the emergence of newly predominant

bacteria more adaptive to the microenvironment

(niche). In dental caries, this reestablished micro-

bial community structure is characterized by a

numeric predominance of acidogenic/aciduric

bacteria (e.g., S. mutans ) at the expense of the

acid-sensitive commensal species (e.g., S. sangui-

nis ) associated with sound enamel [

110 , 143 ].

The Role of Interspecies Interactions in Dental

Caries The interspecies interactions within den-

tal bioﬁ lms tune microbial composition, struc-

ture, and virulence factors of the oral microbial

community and thus are involved in the onset and

progression of dental caries [ 122 ].

Bacterial interactions can affect the growth of

individual organisms or groups of related

organisms, thus modulating the composition of

the oral microbial community. For example, S.

sanguinis gained competitive edge over mutacin-

generating S. mutans by producing cytotoxic

hydrogen peroxide under certain conditions. On

the other hand, gshAB is essential for the com-

petitiveness and prevalence of S. mutans by

detoxifying hydrogen peroxide produced by S.

sanguinis [

238 ]. The S. sanguinis / S. mutans ratio

resulting from the dynamic competition between

these two bacteria may have ecological signiﬁ -

cance on the occurrence and incidence of dental

caries.

Bacterial interactions could also affect the

overall expression of bacterial virulence of the

bioﬁ lm. Take S. mutans , for example, one of the

core cariogenic virulences of S. mutans is the

ability to produce large quantities of organic

acids by metabolizing carbohydrates. In this

regard, coresidents which are capable of neutral-

izing/depleting the acidic end products of S.

mutans tend to reduce the cariogenicity of the

bioﬁ lm. In dental bioﬁ lms, some commensal bac-

teria such as Veillonella are able to consume the

lactic acid. In addition, some coresidents such as

S. salivarius , S. gordonii , and S. sanguinis can

generate alkali to neutralize the acid, through the

metabolism of either urease by urease or arginine

by arginine deiminase system [ 26 ]. The quorum-

sensing systems also modulate the virulence

properties of oral residents. S. mutans utilizes

CSP-induced bacteriocin production to acquire

transforming DNA from other coresidents [

118 ],

thus achieving a great genomic diversity for bet-

ter environmental adaptation. The CSP-induced

bacteriocin production also modulates the growth

of related noncariogenic streptococci. In addi-

tion, the CSP-mediated quorum-sensing systems

are involved in the antimicrobial resistance of S.

mutans [ 149 ]. Therefore, bioﬁ lm residents which

could affect the levels of the regulatory molecules

present in the bioﬁ lm could indirectly exert pro-

found effects on the bioﬁ lm virulence. Since non-

cariogenic S. gordonii can inactivate the CSP

produced by S. mutans , its presence could thus

modulate the virulence of these bioﬁ lms.

In conclusion, the interaction with the dental

microbial community can not only affect the

X. Xin et al.

growth of certain species but also modulate the

virulence properties of oral residents, thus inﬂ u-

encing the overall pathogenicity of the dental

plaque. The interspecies interactions have eco-

logical signiﬁ cance in the occurrence and pro-

gression of dental caries.

2.3 Dental Caries-Associated

Bacteria

2.3.1 Carbohydrate Metabolism

and Acidogenic Bacteria

Dental caries is a multifactorial and chronic

infectious disease resulting in the localized

destruction of dental hard tissue. Dental caries is

one of the major disease burdens inﬂ icting

humans throughout history. The pandemic of

dental caries has been linked to the two largest

dietary shifts in human evolution in terms of the

consumption of fermentable carbohydrates. With

the advent of Neolithic farming, the increased

consumption of domesticated grains positively

correlates with a marked prevalence of caries.

However, across the Neolithic and medieval

period, the degree of caries was mild and preva-

lence remained relatively stable until 1850. After

1850, a sudden expansion of caries lesion

occurred coinciding with the introduction of

reﬁ ned ﬂ our and sugar due to industrial revolu-

tion. Although the epidemiological history indi-

cates a correlation of sugar with dental caries, the

“culprit” of the dental caries remained enigmatic

until the late nineteenth century, when Miller evi-

denced that the acidic microbial metabolites from

dietary substrates contribute to the development

of dental caries [

157 , 158 ]. These observations

were further conﬁ rmed in 1940s by Stephan, who

reported that microbial metabolism of carbohy-

drate in dental plaque could drive the local pH

values below 3.0 after continuous sugar exposure

[ 194 ]. Among those acidic microbial metabo-

lites, lactic acid has been identiﬁ ed as the major

contributor to the pH decline in dental plaque.

Hence, the acidogenic bacteria are recognized as

the culprit for caries initiation and progression.

The accumulation of organic acids leads to con-

tinuous pH decline to the critical pH, below

which tooth hard tissue demineralization begins

and dental caries gradually occurs [

143 ].

2.3.2 Major Acidogenic Bacteria

2.3.2.1 Streptococcus mutans

S. mutans was ﬁ rst described by J Kilian Clarke

in 1924 [ 38 ]. It is gram-positive facultative coc-

cus commonly arranged in chains. Oral strepto-

cocci are commensal bacterial but can

opportunistically initiate caries. Mutans strepto-

cocci are a group of most important bacteria

highly associated with caries, consisting of S.

mutans , S. sobrinus , S. rattus , S. cricetus , S.

ferus , S. downeii , and S. macaca . Their cario-

genic virulence mainly involves several attri-

butes, including (1) processing adhesins for

initial attachment to the saliva- coated tooth sur-

face; (2) the production of extracellular polysac-

charide, i.e., glucan, fructan, to facilitate retention

on tooth surface and plaque accumulation; and

(3) the production of organic acid to generate

acidic microenvironment and promote enrich-

ment of aciduric microﬂ ora.

2.3.2.2 Lactobacilli

Lactobacillus is a genus of gram-positive faculta-

tive anaerobe or microaerophilic rod-shaped bacte-

ria. Lactobacillus is able to convert lactose and

other sugars to lactic acid, mostly through

homofermentative metabolism. Lactobacillus count

has been used to assess caries activity for years.

Lactobacillus is acid tolerant and can carry out gly-

colysis at pH values as low as 3. However, lactoba-

cilli are poor colonizer of smooth tooth surface.

Therefore, lactobacilli are generally believed to

exacerbate the initial enamel lesion to deep dentine

lesion. After colonizing into the established dental

plaque, the lactobacilli can further acidify the

plaque and suppress the acid susceptible microor-

ganism, further enriching acidogenic and aciduric

bacteria.

2.3.2.3 Actinomyces

Actinomyces is a genus of gram-positive faculta-

tive or strict anaerobic pleomorphic rod-shaped

2 Bioﬁ lm and Dental Caries

bacteria. As a saccharolytic and acidogenic bac-

terium, Actinomyces spp., especially A. naeslun-

dii , has been frequently isolated from both root

caries lesions and sound root surface, suggesting

their association with root caries. However, an in-

depth knowledge about the involvement of indi-

vidual Actinomyces spp. in root caries is still

sketchy.

2.3.3 Acid Tolerance of Acidogenic

Bacteria

The central pathogenesis of dental caries is the

production of organic acid and the resultant

decalciﬁ cation of dental hard tissue. The dental

plaque undergoes rapid, dynamic pH ﬂ uctuations

ranging from pH 7.0 to 3.0 in less than 20 min

upon carbohydrate intake [ 102 ]. The cariogenic

bacteria emerge as numeric predominant species

during this acidiﬁ cation process. Acid tolerance

or acidurity is the most important attribute for

those acidogenic bacteria to prevail in a cario-

genic bioﬁ lm. Acidogenic bacteria such as S.

mutans can function better at pH 6 and can even

carry out glycolysis at pH below 4. Moderate

acidophile, especially Lactobacillus spp., can

also function better and carry out glycolysis at

low pH values of 3–4. To survive and proliferate

in acid conditions, the cariogenic bacteria have

developed a large repertoire of strategies to main-

tain intracellular homeostasis, which involves

both constitutive acid tolerance and acid-induced/

adaptive tolerance [

25 ]. Constitutive acid toler-

ance mainly relies on the F-ATPase proton pumps

in the cell membrane. Taking S. mutans , for

example, although the glycolytic pathway can

catabolize sugars at pH values as low as 4.0, the

S. mutans cell cannot grow at pH values lower

than 5.0. In this case, the F-ATPase will use the

ATP generated by glycolysis to pump out intra-

cellular protons and thus maintain neutral cyto-

solic pH favorable for normal enzymatic function

and cellular viability. In addition, the end metab-

olite, lactate, may also be extruded from cyto-

plasm by a membrane carrier for lactic acid, thus

maintaining a relative neutral cytosolic pH. The

adaptive acid tolerance response indicates the

induction of enhanced survival ability of bacteria

at a pH as low as pH 3.0 after exposure to a sub-

lethal pH of approximately 5.5. The adaptive acid

tolerance involves global cellular response,

including alteration of metabolism, regulation of

quorum sensing system, synthesis of chaperonin

protein for damaged protein, increased activity of

DNA protection/repair system, changes in mem-

brane fatty acid composition, etc. [

148 ]. Overall,

the acid tolerance mechanisms allow the cario-

genic bacteria to outcompete other acid-sensitive

coresidents under acid selection, which would

eventually lead to the enrichment of acidogenic/

aciduric bacteria and continual acidiﬁ cation of

the dental plaque favorable for caries formation.

2.3.4 Base Generation and Caries

Protection

In addition to the aforementioned acid tolerance

mechanisms, alkali generation is widespread

among oral species. Alkali generation is particu-

larly important for the survival of acid-sensitive

commensal bacteria and thus plays an important

role in modulating the microbial ecology within

oral bioﬁ lm. The primary source of alkali pro-

duction is through microbial metabolism of argi-

nine, agmatine, and urea (Fig. 2.2 ). Alkali

generated by these metabolism pathways might

neutralize the acid metabolites produced by car-

iogenic microﬂ ora and thus provides a promising

strategy for the development of ecological ther-

apy against caries (Fig.

2.3 ).

2.3.4.1 Urease

The physiological concentration of urea present

in saliva and crevicular ﬂ uids ranges from 3 to

10 mmol/L, roughly equivalent to those in serum.

Some oral species, including S. salivarius , A.

naeslundii , and oral haemophili, can convert urea

by urease to ammonia and CO

, thus increasing

plaque pH [ 33 , 129 ]. Urease is a nickel ion-

dependent multisubunit metalloenzyme encoded

by at least seven genes arranged as operon in

most bacteria. ureC , ureA , and ureB encode the

urease apoenzyme, consisting of α, β, and γ sub-

units. These subunits assemble into (αβγ)

oligo-

X. Xin et al.

meric complex with six nickel ions incorporated

into the active site. ureE , ureF , ureG , and ureD

encode chaperone complex essential for the

incorporation of Ni

and CO

into the metallo-

center. Other genes, such as ureM , ureQ , and

ureO , encode a Ni

-speciﬁ c ATP-binding cas-

sette transporter, and ureI encodes urea trans-

porter [ 32 ]. The expression of bacterial urease is

regulated by multiple environmental cues, includ-

ing low pH, the presence of urea, limited nitrogen

source, carbohydrate availability, and rate of

growth [ 32 ].

Urea + H

2NH

+ CO

L-lactate + CO

ATP

ATPase

L-malate

+ CO

+ H

Agmatine

Carbamoyl putrescine

Urease

ADP ATP

Arginine deiminase

Arginine decarboxylase

Agmatine deiminase

malolactic enzyme

Putrescine carbamoyl-

transferase

Ornithine

carbamoyl-

transferase

Arginine

Citrulline

Putrescine

Ornithine

Carbamoyphosphate

Carbamate kinase

Fig. 2.2 Summary of alkali-generating pathways in the oral cavity (Figure is reproduced from Liu et al. [ 131 ] )

Neutral pH

Low pH

Oral environmental

stresses

Oral alkali

generation

Disease

Caries

Acidogenic and aciduric bacteria

Oral commensals

Health

Enamel

Fig. 2.3 The role of alkali generation in caries preven-

tion. Dental bioﬁ lms in a healthy host maintain a micro-

bial and pH homeostasis with a balanced demineralization/

remineralization of dental hard tissue. The environmental

cues, such as frequent sugar exposure, may enrich the

acidogenic/aciduric bacteria via acid selection and further

lead to plaque acidiﬁ cation in favor of demineralization of

dental hard tissue. The continuous plaque acidiﬁ cation

eventually results in the initiation and/or progression of

caries lesion. Alkali generation by bioﬁ lm commensals

can directly neutralize plaque pH, ease the competitive

edge of acidogenic/aciduric bacteria, and restore a healthy

microbial equilibrium (Figure is reproduced under the

permission of Dr. Liu et al. [

131 ] )

2 Bioﬁ lm and Dental Caries

2.3.4.2 Arginine Deiminase

System (ADS)

Arginine is abundant in salivary secretions as

polypeptides with average concentrations in duc-

tal saliva around 50 mmol/L [ 211 ]. Arginine is

primarily metabolized by the microbial ADS to

release ornithine, ammonia, and CO

. ADS has

been identiﬁ ed in many commensal bacteria,

including S. sanguinis , S. gordonii , and S. para-

sanguis . Certain Lactobacillus and Actinomyces

species, other oral streptococci, and some oral

spirochetes have been also identiﬁ ed as argino-

lytic [ 26 , 141 ]. Unlike urea hydrolysis by urease,

arginine catabolism by ADS generates ATP,

which could be further utilized to counter acid

stress through both constitutive and adaptive acid

tolerance pathways. Similar to urease, ADS-

encoding genes are commonly arranged in an

operon [ 47 , 136 ]. arcA gene encodes arginine

deiminase, which hydrolyzes arginine to generate

citrulline and ammonia. arcB gene encodes orni-

thine carbamoyltransferase, which converts citrul-

line to ornithine and carbamoylphosphate. arcC

gene encodes carbamate kinase that transfers a

phosphate group from carbamoylphosphate to

ADP to generate ATP, CO

, and ammonia. Many

organisms also possess an arginine/ornithine anti-

porter ( ArcD ) that is encoded in the same operon,

and arginine aminopeptidases and transcriptional

regulators are often encoded in ADS gene clusters

[ 235 ]. ADS expression is induced by arginine and

low pH. The operon is also sensitive to carbon

catabolite repression (CCR) and can be downreg-

ulated in response to elevated oxygen levels [

48 ].

In addition, ADS activity in S. gordonii has been

reported to be upregulated when it coaggregated

with A. naeslundii [ 99 ], indicating the ecological

involvement of ADS expression in multispecies

bioﬁ lm.

2.3.4.3 Agmatine Deiminase System

(AgDS)

Agmatine present in the oral cavity can be

obtained from foods, such as rice, milk, and beer,

or be produced from arginine by bacterial argi-

nine decarboxylase enzymes. The physiological

concentration of agmatine is 0.75 mmol in dental

plaque and 0.2 mmol in saliva [

76 ]. Agmatine is

primarily metabolized by AgDS to putrescine,

ammonia, CO

, and ATP. AgDS is present in

many oral bacteria, including S. mutans , S. sobri-

nus , S. downeii , S. rattus , S. uberis , S. mitis , and

S. cricetus , as well Lactobacillus salivarius and

L. brevis [ 76 ]. AgDS is also encoded by genes

arranged in operon ( aguBDAC ) [ 74 ]. AguD

encodes agmatine-putrescine antiporter to allow

the entry of free agmatine into the cell. Agmatine

is then hydrolyzed to N-carbamoylputrescine and

ammonia by the agmatine deiminase enzyme

encoded by aguA . The putrescine carbamoyl-

transferase, encoded by aguB , further metabo-

lizes N-carbamoylputrescine to yield putrescine

and carbamoylphosphate. Finally, carbamate

kinase encoded by aguC gene transfers a phos-

phate group from carbamoylphosphate to ADP

to generate ATP, CO

, and NH

. The putrescine

generated can also be used in exchange of agma-

tine via the antiporter [ 74 ]. aguR gene, which is

located upstream of, and in the opposite orien-

tation to, the agu operon in S. mutans , encodes

a transcriptional activator of agu genes [ 75 ]. In

oral streptococci, AgD activity is generally lower

relative to that of arginine deiminase or urease

[ 74 ]. Therefore, AgDS may not be sufﬁ cient to

counter the plaque acidiﬁ cation. On the contrary,

accumulating evidence has indicated that AgDS

may actually favor the acid tolerance of S. mutans

by elevating the cytoplasmic pH and generating

extra ATP. In S. mutans , AgDS activity is growth

phase dependent, and it can also be induced by

the presence of agmatine and other environmen-

tal stresses, including low pH and heat shock

[

75 ]. Multiple two-component systems, includ-

ing CiaRH , ComDE , and VicRK , also involve in

the induction of AgDS genes under low pH or

thermal stress [ 130 ].

2.3.4.4 Alkali Production and Bioﬁ lm

Ecology

Dental bioﬁ lms are complex ecosystems with

hundreds of metabolically and physiologically

diverse species competing for nutrients. The abil-

ity of oral species to metabolize urea, arginine, or

agmatine at low pH favors the growth of these

X. Xin et al.

bacteria by cytoplasm alkalization. The genera-

tion of ATP could further enhance acid tolerance

by providing energy for proton extrusion, growth,

or maintenance [ 75 ]. The contributions of ADS,

urease, and AgDS to oral bioﬁ lm pH homeostasis

and microbial ecology can be quite different. In

some oral commensals, such as S. salivarius and

S. gordonii , urease and ADS can protect the bac-

teria from excessive environmental acidiﬁ cation

[ 33 ]. In the meantime, other less-aciduric coresi-

dents beneﬁ t from the local alkalization by

ammonia generated from arginine or urea break-

down [ 48 ]. On the other hand, the caries patho-

gens S. mutans and S. sobrinus have no urease or

ADS but possess AgDS that is expressed at a

relatively lower level [ 76 ]. Consequently, AgDS

of S. mutans or S. sobrinus probably cannot sig-

niﬁ cantly alkalize the dental plaque, but instead,

it enhances the acid tolerance of these acidogenic

bacteria by neutralizing cytoplasm acid and gen-

erating extra ATP, thus promoting the prevalence

of these bacteria during the development of

caries.

2.3.4.5 Clinical Relevance of Alkali

Production

Alkali production holds the promise to be a

promising strategy for the management of dental

caries. Firstly, it directly increases the pH of den-

tal plaque in favor of dental hard tissue reminer-

alization. Secondly, alkali generation favors the

persistence of healthy commensals while pre-

venting the overgrowth of cariogenic bacteria

(e.g., S. mutans ) dependent on acid selection. The

activity of alkali generation of oral bioﬁ lm has

shown negative correlation with the development

and progression of caries. A genetically modiﬁ ed

strain of S. mutans expressing the urease genes

from S. salivarius showed potent anticaries

effects in a rat caries model [

37 ]. Chronic renal

failure patients with salivary urea levels signiﬁ -

cantly higher than healthy population are more

resistant to caries, despite their high dietary car-

bohydrate intake [ 172 ]. Consistently, caries-

resistant subjects have higher ammonia

concentrations and resting pH in their plaque

compared with those of caries-free individuals

[ 211 ]. Signiﬁ cantly higher levels of salivary ADS

activity and plaque urease activity have been

reported in caries-free population compared with

subjects with dental caries. Salivary arginine lev-

els are also positively correlated with caries resis-

tance [

163 , 188 ]. A longitudinal study on children

has further validated that ammonia generation by

plaque urease is correlated with dental caries

resistance [ 160 ]. The addition of arginine-

bicarbonate to mouth rinse effectively can raise

the plaque pH above the critical pH after a

sucrose challenge [ 216 ]. More importantly, an

increasing number of clinical trials have shown

that arginine-containing oral hygiene products

signiﬁ cantly reduced the incidence of dental car-

ies [ 2 , 3 , 55 ]. Toothpaste containing 1.5 % argi-

nine and 1450 ppm ﬂ uoride in a calcium base has

also been proven more effective in arresting and

reversing early carious lesions compared with

dentifrice containing 1450 ppm ﬂ uoride alone

[ 116 , 192 , 234 ].

2.3.5 Other Caries-Associated

Bacteria

The oral cavity is inhabited by hundreds of bacterial

species, forming complex ecology system [ 45 ]. The

interplay between oral microﬂ ora and the host

orchestrates the status of oral health and disease.

The “speciﬁ c pathogen hypothesis” has led to the

identiﬁ cation of several other species including

Scardovia wiggsiae and Slackia exigua , and

Biﬁ dobacterium dentium as the potential cariogenic

pathogens [

199 ]. In addition, Atopobium , Olsenella ,

Propionibacterium , and Pseudoramibacter genera

are also indicated as bacteria associated with caries

progress [ 173 ]. In contrast, the “nonspeciﬁ c plaque

hypothesis” supports the concept that caries is the

consequence of the overall acid production activity

of the total plaque microﬂ ora rather than a few spe-

ciﬁ c bacteria. The “ecological plaque hypothesis”

further proposes that the bacterial consortium in the

dental plaque can interact in complex synergistic

and antagonistic fashion, and it is the structure and

function shift of the microbial community driven by

environmental cues (e.g., frequent exposure to car-

bohydrate) that eventually lead to the net pathogen-

esis of dental caries [ 144 ].

2 Bioﬁ lm and Dental Caries

2.4 Antimicrobial Approaches

to the Management

of Dental Caries

Dental caries is a bioﬁ lm-mediated disease. The

composition of the bioﬁ lm associated with caries

initiation and progression is diverse. The man-

agement of dental caries is to target the dental

plaque, in particular to restore the microbial dis-

equilibrium within the oral bioﬁ lm. This section

is to provide an overview of various antimicro-

bial strategies applied in dentistry and related

studies.

2.4.1 Chlorhexidine

Chlorhexidine is one of the most tested antiplaque

agents and represents a gold standard against

which the potency of other antiplaque agents is

compared [ 8 , 59 ]. Chlorhexidine molecules are

positively charged (cations). Chlorhexidine binds

strongly to most bacteria and surface structures in

the oral cavity, including tooth surfaces and

mucous membranes which are negatively charged

(anions). When chlorhexidine binds to microbial

cell walls, it destroys the surface structure, leading

to an osmotic imbalance with consequent

precipitation of cytoplasm causing cell death [ 59 ].

Chlorhexidine also possesses very good

substantive properties [ 139 ]. The antimicrobial

effect of chlorhexidine can be retained for up to

12 h or longer depending on the delivery dosage

and form [

4 ].

Chlorhexidine is a strong base, and it acts bac-

teriostatically when administered at low concen-

trations. It disrupts normal membrane functions

or causes leakage of cell constituents [ 95 ]. At

higher concentrations, chlorhexidine is bacteri-

cidal, inducing leakage of low molecular weight

cell constituents and precipitation of cell con-

tents. Chlorhexidine exhibits broad antibacterial

spectrum with gram-positive microorganisms

particularly sensitive than gram-negative

microorganisms.

The efﬁ cacy of chlorhexidine mainly depends

on the concentration and the frequency of appli-

cation. Mouth rinses, sprays, gels and varnishes

are some of the most often used delivery forms of

chlorhexidine [

191 ]. The usually prescribed dos-

age for chlorhexidine mouthrinses has been

10 ml of a 0.2 % solution, with twice-daily

mouthrinses. By using 15 ml of a 0.12 %

chlorhexidine mouthrinse, a comparable efﬁ cacy

can also achieved [ 59 ].

2.4.2 Fluoride

Fluoride has been applied in dentistry for more

than 70 years, and it is now recognized as the

major contributor to the dramatic decline in car-

ies prevalence worldwide [ 28 ]. Fluoride is a dual

functional anticaries agent, acting on both tooth

hard tissue and oral microbes [ 58 , 114 , 202 ].

Fluoride can disrupt enzyme activity and reduce

acid production by oral bacteria [ 84 ], thus sup-

pressing the enrichment of cariogenic bacteria

within dental plaque [ 20 , 21 ]. Amine ﬂ uoride

[ 107 ] and stannous ﬂ uoride [ 151 ] can be bacteri-

cidal at higher concentrations against oral bacte-

ria. Amine ﬂ uoride in a gel formulation can

inhibit the growth of mixed bacterial populations

in subgingival plaque [ 11 , 24 ]. Amine ﬂ uoride

and tin ﬂ uorides can also inhibit the adhesion of

S. sanguinis to glass conditioned with either

saliva or bovine serum albumin in vitro [ 54 ].

Preincubation of hydroxyapatite with amine ﬂ u-

oride can signiﬁ cantly decrease the growth of S.

sobrinus in bioﬁ lm in vitro [ 183 ]. Of note,

in vivo data reported by Weiger et al. [

218 ] have

shown that the amine/stannous ﬂ uoride mouth-

rinse possesses a transient antibacterial effect

but no clear antiadhesive activity against oral

bacteria.

The methods of ﬂ uoride delivery are either

systemic (water, supplements, milk and salt) or

topical (toothpaste, gels, varnishes, paint-on

applications and mouthrinses) [ 59 ]. Although the

role of ﬂ uorides in caries prevention represents

one of the most successful stories in general pub-

lic health, excessive ﬂ uoride intake during the

period of tooth development can cause dental

ﬂ uorosis. In addition, ﬂ uoride could nonselec-

tively suppress the growth of commensal bacteria

and thus disrupt the microbial equilibrium within

X. Xin et al.

the dental plaque. Therefore, cautions should be

taken in the application of ﬂ uorides in order to

maximize the anticaries beneﬁ ts while minimiz-

ing the risk.

2.4.3 Quaternary Ammonium

Compounds

Quaternary ammonium compounds (QACs) are

surface-active agents with a wide antimicrobial

spectrum against both bacteria and fungi [ 64 , 98 ,

123 ]. QACs deliver their antimicrobial activity

by binding to the cell membrane and causing

cytoplasmic leakage, similar to those polyca-

tionic agents.

Cetylpyridinium chloride (CPC) is a com-

monly used QAC in a variety of mouthwashes

over the past decades due to its antimicrobial

property. The typically used concentration (W/V)

of CPC is 0.05 %, although slightly higher con-

centrations (≥0.07) have also been used [ 44 , 80 ,

214 ]. The antiplaque effect of CPC was ﬁ rst

reported by Schroeder and Hirzel [ 79 , 182 ].

Research has demonstrated that CPC mouthrinses

have antiplaque activity when used alone or in

conjunction with toothbrushing [ 79 , 140 , 222 ,

223 ]. Recently, a systematic review has further

validated the plaque- and gingivitis- inhibiting

effect of CPC-containing mouthrinses [ 87 ].

The CPC molecule allows ionic and hydro-

phobic interactions since it belongs to both

hydrophilic and hydrophobic groups. It interacts

with microorganisms via cationic binding, simi-

lar to chlorhexidine. Although CPC shows equal

or better antimicrobial activity against planktonic

culture compared to chlorhexidine, it exhibits an

inferior inhibitory effect against bioﬁ lm [

59 ].

This divergence may be attributed to the monoca-

tionic nature of CPC [ 53 ]. Initial retention of

CPC is higher than that of chlorhexidine, but

CPC is cleared from the oral cavity more rapidly

[ 17 ]. CPC could be incorporated into dental

materials, such as orthodontic adhesives, to con-

trol caries lesion formation around orthodontic

brackets. Although CPC retains its antimicrobial

properties, the clinical effect remains to be

assessed [ 59 ].

Polymers containing QACs have also been

incorporated into dental materials [

34 , 35 , 236 ].

QACs are immobilized in the composite and not

released or lost over time by copolymerizing with

the resin through the formation of a covalent

bond with the polymer network [ 209 ]. Therefore,

the dental material has a durable and long-lasting

antibacterial capability without signiﬁ cantly dis-

turbing the biologic balance in the oral cavity and

material’s mechanic properties [ 96 ]. This is also

due to the nonleaching properties of the material,

since leaching leads to increased water absorp-

tion and solubility and decreased mechanical

properties with time, decreasing the clinical lon-

gevity of these materials [ 227 ]. Previous study

has presented that adhesive system containing

QACs has similar antibioﬁ lm properties and last-

ing at least 6 months of water aging [ 237 ].

Quaternary ammonium polyethylenimine (QAS-

PEI) nanoparticles were incorporated in restor-

ative materials to increase antibacterial action

without further reduction on mechanical proper-

ties [ 12 ]. It has been demonstrated that the incor-

poration of 1 % QAC-PEI nanoparticles in dental

composite resin has a strong antibacterial effect

against S. mutans and can sustain over 1 month

without alteration of the original mechanical

properties [ 13 ]. QAC-PEI nanoparticles were

also reported to have antibacterial effect against

S. mutans and L. casei when incorporated in con-

ventional glass ionomer [ 14 ].

2.4.4 Triclosan

Triclosan is a nonionic antimicrobial agent being

used for more than 30 years as a preservative in

products such as deodorants, soaps, and body

powders. More recently, triclosan has been

applied to dentifrices and mouthrinses as a pro-

phylactic agent with the purpose of reducing den-

tal bioﬁ lm formation and the development of

gingivitis [ 59 ].

Triclosan is active against both gram-positive

and gram-negative microorganisms and fungi.

Oral bacteria such as S. mutans , S. sanguinis , and

S. salivarius are susceptible to low concentra-

tions of triclosan in vitro. At low concentrations,

2 Bioﬁ lm and Dental Caries

triclosan is bacteriostatic [ 59 ]. Triclosan func-

tions by speciﬁ cally inhibiting bacterial lipid

synthesis which further impairs cell membrane

synthesis [ 152 ]. The antimicrobial/antiplaque

properties of triclosan have been demonstrated in

both in vitro and in vivo studies [ 78 , 101 , 176 ,

186 , 198 ]. Triclosan and amine ﬂ uoride also

show combinatory antibacterial and plaque-

reducing effect in vivo [ 6 ].

The major concern of the widespread use of

triclosan-containing products is the possibility of

inducing antimicrobial resistance [ 124 , 233 ].

Although triclosan is bactericidal at high concen-

trations, triclosan-containing products such as

dentifrices leave residues that will dilute to sub-

lethal concentration. Bacteria could develop

reduced susceptibility to triclosan after repeated

exposure to sublethal concentrations of this

agent. A number of studies have veriﬁ ed the

occurrence of triclosan resistance among dermal,

intestinal, and environmental microorganisms

[ 233 ]. More importantly, the widespread use of

triclosan may also lead to the development of

concomitant resistance to other clinically impor-

tant antimicrobials through cross-resistance or

coresistance mechanisms. Although studies

regarding the concomitant resistance to other

antimicrobials are still limited, cautions should

be taken on the widespread use of triclosan [ 59 ].

2.4.5 Xylitol

Xylitol is an effective tooth decay-preventive

pentitol accredited to its non-acidogenic prop-

erty. Xylitol inhibits the growth of several bacte-

rial species, among which S. mutans appear to be

the speciﬁ c target [ 19 , 133 , 208 ]. The S. mutans

uptakes xylitol via a constitutive transport system

speciﬁ c for fructose and enriches xylitol intracel-

lularly as a non- metabolizable metabolite. In this

way, xylitol inhibits the growth of S. mutans and

reduces the amount of plaque and the number of

S. mutans in both the plaque and saliva of xylitol

consumers [ 203 ]. Xylitol is able to disrupt the

bacterial metabolism and consequently halt the

pH drop in the dental plaque. In addition, xylitol

can suppress the critical cariogenic factor of S.

mutans by impairing their polysaccharide forma-

tion. Long-term consumption of xylitol also pro-

motes the selection of xylitol-resistant S. mutans ,

which are believed to be less virulent than xylitol-

sensitive strains [

207 ]. All the aforementioned

mechanisms suggest that xylitol is a promising

cariostatic agent in controlling dental caries.

Xylitol has been incorporated as an active

ingredient in dental hygiene products, such as

xylitol-containing dentifrices currently available

on the market. Anticaries effect of xylitol has

been reported in longitudinal xylitol dentifrice

studies [ 189 ]. Xylitol is an alternative sweetening

agent to substitute sucrose in chewing gums.

Besides the antimicrobial effect of xylitol, chew-

ing itself promotes secretion of saliva and thus

caries prevention. However, further well-

designed randomized clinical studies with proper

controls are needed to validate the caries-

prophylactic effects of xylitol or superiority

claims of xylitol over other polyols [ 59 ].

2.4.6 Phenolic Antiseptics

Phenols, either alone or in combination, have

been known to be bactericidal since Lister used it

as a surgical antiseptic in the 1860s [ 220 ].

Phenols are able to reduce plaque accumulation

when used at high concentrations [ 63 , 135 ].

Listerine, which is an essential oil/phenolic

mouthwash, has been demonstrated in a number

of clinical studies to possess moderate antimicro-

bial effect against dental plaque [

43 , 71 ].

However, Listerine has poor oral retention com-

pared to chlorhexidine and thus lacks profound

plaque inhibitory effect [ 53 ].

2.4.7 Natural Products

Natural products offer a wide variety of structur-

ally different substances with a broad range of

biological activities, which could be applied into

the development of alternative or adjunctive anti-

caries therapies [ 104 ].

X. Xin et al.

The antibacterial effect of Camellia sinensis

(i.e., tea) against caries-related bacteria has been

widely studied, using both in vitro and in vivo

models [ 165 , 166 , 184 , 185 , 228 – 230 ].

Accumulating evidence has indicated that the

bioactive components of green tea, polyphenolic

catechins, in particular (−)-epigallocatechin gal-

late (EGCg) and (−)-epicatechin gallate (ECg),

are able to inhibit the growth of the streptococci

[ 200 ]. Catechins inhibit the growth of S. mutans

and S. sobrinus with MICs ranging between 50

and 1000 μg/ml, which are within the concentra-

tions determined in brewed tea [ 106 , 180 ].

Catechins are also bactericidal at even higher

concentrations. In addition, many of the “ﬂ avor

compounds” (e.g., nerolidol) in green tea,

although they are not antibacterial by themselves,

might act synergistically with the abundant cate-

chins to suppress bacterial growth [ 120 ]. Recent

studies by our group have also demonstrated that

tea catechins, particular EGCg, could not only

inhibit the growth of cariogenic S. mutans at high

concentrations but also suppress cariogenic fac-

tors involved in the acidogenicity, acidurity, and

extracellular polysaccharide synthesis at sub-

MIC levels [ 228 – 230 ]. The bioactivities of tea

catechins at sub-MIC levels further support the

translational application of tea extracts in vivo,

considering the substantivity of these active com-

ponents in the oral cavity after tea consumption.

Essential oils have also been extensively

investigated for their antimicrobial activity

against caries-related bacteria. Essential oils,

odorous and volatile products of plant secondary

metabolism, are typically a complex mixture of

approximately 20–60 compounds that are in

solution at various concentrations [

104 ]. The

main active component of essential oils is terpe-

noids, followed by aromatic and aliphatic con-

stituents [ 9 ]. Thymol and eugenol inhibit the

growth of a wide range of oral microorganisms

including S. mutans [ 184 , 185 ]. Essential oils

appear to act against bacterial viability by dis-

rupting the integrity of the bacterial membrane

and cause the rapid efﬂ ux of intracellular bacte-

rial components [ 185 ].

Propolis is a natural composite balsam collected

by bees from tree buds and mixed with secreted

beeswax [

66 , 89 ]. Propolis not only inhibits the

growth of S. mutans and S. sobrinus but also sup-

presses the production of bacterial polysaccharides

[ 65 ]. The application of propolis extract on rat

molars also reduces the severity of carious lesions

[ 179 ]. Speciﬁ c ﬂ avanones (pinocembrin), dihydro-

ﬂ avonols (pinobanksin- 3-acetate), and terpenoids

(tt- farnesol) are believed to be the bioactive compo-

nents of propolis [ 112 ]. tt-Farnesol is particularly

effective against the proliferation of both planktonic

and bioﬁ lm-associated S. mutans cells [ 113 ]. The

antibacterial activity of tt-farnesol is likely associ-

ated with its lipophilic property and membrano-

tropic effect, which might destroy the integrity of

bacterial cell membrane [ 41 , 97 ] and increase pro-

ton permeability [ 103 ].

Sanguinarine is a benzophenanthridine alkaloid

originated from the alcoholic extraction of pow-

dered rhizomes of the blood-root plant,

Sanguinaria canadensis [ 73 ]. Sanguinarine at a

concentration of 16 μg/ml could completely inhibit

98 % of microbial isolates from human dental

plaque [ 49 ]. A sanguinarine mouthrinse and tooth-

paste regime has been shown to signiﬁ cantly

reduce plaque by 57 % when given for 6 months

during orthodontic treatment [ 86 ]. Another study

of sanguinarine mouthrinse and toothpaste carried

out in 120 subjects showed 13–17 % lower plaque

scores compared with a placebo group after a

6-month treatment period [ 115 ].

There are many other reports available con-

cerning the anticaries properties of various other

plant extracts. Wu-Yuan et al. [

225 ] and Li et al.

[ 126 ] have identiﬁ ed that gallotannins from

Melaphis chinensis and triterpenes (ceanothic

acid and ceanothetric acid) from Ceanothus

americanus possess antimicrobial activity

against S. mutans . Furthermore, a chemically

characterized extract of Galla chinensis (con-

taining gallic acid and methyl gallate) can

inhibit the growth of S. mutans and other caries-

related organisms, including L. rhamnosus and

A. naeslundii , within bioﬁ lms [ 226 ].

Ginkgoneolic acid from Ginkgo biloba has also

been shown to be a potential natural anticario-

genic agent which exhibits antimicrobial activ-

ity against S. mutans and suppresses speciﬁ c

virulence factors associated with its cariogenicity

2 Bioﬁ lm and Dental Caries

[ 90 ]. 7-Epiclusianone, a prenylated benzophe-

none isolated from Rheedia gardneriana , and

some cranberry ﬂ avonoids (e.g., myricetin, pro-

cyanidin A2, and A-type oligomers) have been

shown to inhibit the acid-sensitive intracellular

glycolytic enzymes by increasing the proton

permeability of S. mutans cells [ 72 , 161 ]. A

crude extract of Psidium cattleianum can also

inhibit the expression of proteins involved in

general metabolism, glycolysis, and lactic acid

production of S. mutans [ 23 ].

Despite the aforementioned efforts to identify

the natural substances potentially active against

cariogenic organisms, it is still challenging to

determine the precise mechanisms of action and

efﬁ cacy due to the complexity of chemistry and

isolation procedures. Moreover, few studies have

actually been conducted in vivo (e.g., using

rodent models of dental caries) and even fewer

have been evaluated in clinical trials [ 104 ]. The

implementation of standardized randomly con-

trolled trials is needed to further validate the

application of natural products in the ﬁ eld of car-

ies management.

2.5 Ongoing Direction of Oral

Dental Plaque Study

2.5.1 Metagenomics and Oral

Microbiome

The term “metagenomics” was ﬁ rst invented by

Handelsman [ 85 ], and it is deﬁ ned as “the appli-

cation of modern genomics techniques to the

study of communities of microbial organisms

directly in their natural environments, bypassing

the need for isolation and lab cultivation of indi-

vidual species.” The advances in the reﬁ nements

of DNA ampliﬁ cation, bioinformatics, and

enhanced computational power for analyzing

DNA sequences have enabled the adaptation of

shotgun sequencing, such as chip-based pyrose-

quencing, to metagenomic samples [ 22 , 52 ]. The

approach randomly shears DNA, sequences

many short sequences, and reconstructs them into

a consensus sequence [ 22 ].

By performing metabolic function analyses on

genes identiﬁ ed via metagenomic approach,

researchers are able to retrieve information both

on which organisms are present and, more impor-

tantly, what functions or metabolic processes are

possible in that particular community [

70 ]. Using

comparative genetic studies coupled with expres-

sion experiments such as microarray and pro-

teomics, microbiologist will be able to piece

together a metabolic network that goes beyond

species boundaries and gain valuable insight into

the metabolism within the community. Recently, a

comparative metagenomic study has been initi-

ated, aiming to compare the microbial community

within dental plaque associated with healthy and

diseased sites. It is anticipated that such compari-

son will assist in identifying potential pathogenic

organisms which may not have been detected

using currently available technologies [ 121 ].

Our group has been working on the

comparative study of oral microbiome using

metagenomics- related techniques. By pyrose-

quencing the “hypervariable regions” of bacterial

16S rRNA of biological samples (i.e., saliva,

supragingival plaque, and mucosal plaque)

collected from healthy population at different age

groups, we have demonstrated that the oral cavity

is a highly heterogeneous ecological system con-

taining distinct niches with signiﬁ cantly different

microbial communities. More importantly, the

phylogenetic microbial structure varies with

aging, and only a few taxa were present across

the whole populations [

231 ]. By comparing the

composition of supragingival plaque microbiota

of acute lymphoblastic leukemia (ALL) pediatric

patients with healthy controls, we have

demonstrated that ALL patients have a structural

imbalance of the oral microbiota, characterized

by reduced diversity and abundance alterations,

possibly involved in systemic infections, indicat-

ing the importance of immune status in shaping

the structure of oral microbiota [ 217 ]. By charac-

terizing the phylogenetic and functional gene

differences between periodontal and healthy indi-

viduals, we have shown that the phylogenetic and

functional gene structures of the oral microbiomes

are distinctly different between periodontal and

X. Xin et al.

healthy groups. Speciﬁ cally, a variety of genes

involved in virulence factors, amino acid metabo-

lism, and glycosaminoglycan and pyrimidine

degradation are enriched in periodontitis, while

the genes involved in amino acid synthesis and

pyrimidine synthesis are suppressed in people

with periodontitis compared with healthy indi-

viduals [ 128 ]. Overall, metagenomics-related

work from our group and those from others have

provided new insights into our understanding of

phylogenetic and functional gene structures of

oral microbial communities interacting with the

host. More cohort studies are needed to further

elucidate the contribution of the comparative

microbial difference to the pathogenesis and

prognosis of oral infectious diseases and hence

provide comprehensive knowledge for the devel-

opment of novel approaches to a better control of

dental caries.

2.5.2 Evidence-Based Dental Caries

Diagnosis

Although the infectious nature of dental caries has

been proved for more than 100 years, instead of

treating it as infectious disease, traditional den-

tistry still focuses on treating the symptom (repair

the damaged tooth) via surgical approaches. The

recent advancement in caries pathogenesis allows

us to understand that a comprehensive analysis of

dental caries should be more than detecting tooth

demineralization sites and repairing damaged

teeth with surgical approaches. Instead, it should

include the detection of cariogenic bacteria and

plaque acidogenicity, followed by a comprehen-

sive treatment of dental caries that includes the

elimination of cariogenic bacteria, the reduction of

plaque acidogenicity, and the enhancement of

tooth remineralization [

204 ]. The combination of

accurate detection of oral bacteria and in situ mon-

itoring of plaque pH, such as the polyaniline-based

planer pH sensor developed by scientists at Jet

Propulsion Laboratory and combined NMR con-

focal microscopy from Paciﬁ c Northwest National

Laboratory which can monitor pH gradient within

dental plaque with high sensitivity and in real time

[ 138 ], is opening a new chapter for cariology.

Early detection and quantiﬁ cation of cariogenic

bacteria in plaque or saliva samples can help clini-

cians take preventive measures to stop caries

development, much like early detection of cancer

markers before overt/detectable cancerous lesions

develop. New antibody- or nucleotide-based bac-

terial detection techniques have also been devel-

oped for the detection of cariogenic bacteria in

chair-side or laboratory settings [

187 ]. In conjunc-

tion with nanotechnology development, these tests

can be further developed into different forms of

nanochips for the detection of multiple pathogens

in the clinical settings [ 127 ].

2.5.3 Novel Antimicrobial Therapies

In addition to “traditional” antimicrobial agents

mentioned above, new approaches have been

developed to maintain oral bioﬁ lm homeostasis.

Such strategies are aimed to inhibit plaque bio-

ﬁ lm formation without disturbing the biological

equilibrium within the oral cavity.

2.5.3.1 Probiotics

Probiotics is deﬁ ned by the World Health

Organization as live microorganisms which,

when applied in adequate amounts, will beneﬁ t

the health of the host [ 5 ]. The use of probiotics,

which has been successful established in the

treatment of intestinal diseases, is now also con-

sidered for the treatment of oral diseases [ 162 ,

206 ]. Experimental studies and clinical trials

have recently demonstrated that certain gastroin-

testinal bacteria, including Lactobacillus and

Biﬁ dobacterium spp., may help in controlling the

proliferation of oral microorganisms, including

cariogenic streptococci. L. rhamnosus CG [ 162 ],

L. casei [ 27 ], L. reuteri [ 30 ], and Biﬁ dobacterium

DN-173 010 [ 29 ] have all been shown to have the

potential to alter colonization of cariogenic bac-

teria and thus contributed in the prevention of

dental caries. However, cautions should be taken

when using the lactobacillus species as they can

also produce acids [ 202 ]. Mechanisms of probi-

otic effect within the oral cavity are likely similar

to those proposed in gastrointestinal studies

[ 154 ]. The introduction of microorganisms as a

2 Bioﬁ lm and Dental Caries

therapeutic tool for the management of dental

caries could possibly act through colonization

resistance and/or immune modulation within the

oral environment [ 5 ].

2.5.3.2 Salivary Antimicrobial

Substances

Antimicrobial substances in saliva and their syn-

thetic analogues are also promising in caries

control [ 92 ]. Whole saliva contains two peroxi-

dase that oxidize thiocyanate (SCN

−

) to hypo-

thiocyanite (OSCN

−

) in the presence of hydrogen

peroxide. Hypothiocyanite is antimicrobial and

inhibits some streptococci and lactobacilli

in vitro [ 134 ]. The effect of the salivary peroxi-

dase system is related to the availability of

hydrogen peroxide, which is produced by vari-

ous microorganisms as a metabolic end product

[ 134 ]. Glucose oxidase produces hydrogen per-

oxide from glucose provided by the enzyme

amyloglucosidase. The addition of these

enzymes to oral products is suggested to gener-

ate adequate hydrogen peroxide so as to control

the growth of microorganisms via enhanced per-

oxidase effect [ 59 ].

2.5.3.3 Speciﬁ cally Targeted

Antimicrobial Peptides

(STAMPs)

The beneﬁ ts of probiotics are mainly achieved by

modulating existing microbial ﬂ ora associated

with the host, thus maintaining a balanced and

healthy microbe-host relationship. In addition to

the use of live organisms, microbiologists are

now developing novel techniques and products

that do not involve live bacteria, yet generate tar-

geted effects against pathogenic factors or organ-

isms and achieve similar probiotic effects [

91 ].

One good example is the targeted antimicrobial

therapy via a novel speciﬁ cally targeted antimi-

crobial peptides technology [ 50 , 51 ]. A “STAMP”

is a fusion peptide with two moieties: a killing

moiety made of a nonspeciﬁ c antimicrobial pep-

tide and a targeting moiety containing a species-

speciﬁ c binding peptide. The targeting moiety

provides speciﬁ c recognition of a selected patho-

gen and targeted delivery of an attached antimi-

crobial peptide. A pheromone produced by S.

mutans , namely, competence-stimulating peptide

(CSP), has been used as a STAMP targeting

domain to mediate S. mutans -speciﬁ c delivery of

killing domain. Such STAMPs are potent against

S. mutans grown in liquid as well as in bioﬁ lm

cultures [ 50 ]. Importantly, STAMPs are able to

eliminate S. mutans from multispecies bioﬁ lms

without affecting other noncariogenic commen-

sal residents [ 50 , 51 ]. Hence, these molecules act

as “probiotic” antimicrobials that may selectively

eliminate pathogens while preserving the protec-

tive beneﬁ ts of the normal ﬂ ora. The success in

the development of S. mutans -speciﬁ c STAMPs

may expand further to the design of other

STAMPs speciﬁ cally targeting various pathogens

including periodontal pathogens within oral cav-

ity and provides an ecological approach to the

management of dental caries.

2.5.3.4 Light Active Killing

Light is more effective on bacteria in bioﬁ lm

where chemical agent might encounter diffusion

limitations. Light therapy could be used to reduce

or modify the oral bioﬁ lm and may offer substan-

tial health beneﬁ ts. S. mutans bioﬁ lm cells can be

killed by up to 3 log

folds when treated with

erythrosine and white light (500–650 nm) [ 224 ].

Exposure of S. mutans bioﬁ lm to blue light had

an impeded effect on bacterial viability through-

out the bioﬁ lm and a sustained antibacterial effect

on bioﬁ lm newly formed by previously irradiated

bacteria [ 60 ]. CO

laser (wavelength of 10.6 μm)

under certain conditions is suggested as a poten-

tial novel preventive light therapy against bio-

ﬁ lms. The activity of CO

laser irradiation on the

viability of S. mutans in bioﬁ lm can reach in the

deep layers. However, CO

may potentially dam-

age the tooth surface, which limits its application

in plaque control [ 60 ] .

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Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_3

Saliva and Dental Caries

Wang Renke

3.1 Salivary Flow

and Composition

3.1.1 Formation of Saliva-Salivary

Glands and Secretion

Whole saliva is mainly the mixture of secretion

of salivary glands. It also contains gingival cre-

vicular ﬂ uid, nonadherent microorganisms, food

debris, and human cells including leucocytes and

epithelial cells. In normal physiological condi-

tions, the average daily ﬂ ow of whole saliva is

between 1 and 1.5 L [ 1 ]. About 90 % of the total

volume of saliva is secreted by the three paired

major salivary glands – the parotid glands, sub-

mandibular glands, and sublingual glands. There

are also a number of minor salivary glands situ-

ated on the tongue, palate, and buccal and labial

mucosa which produce only a small percentage

of saliva. However, the function of the minor sali-

vary glands is also important since about 70 % of

the total volume of salivary proteins is secreted

by them.

The parotids are serous glands; upon stimula-

tion, they produce watery saliva with high con-

tent of enzymes like amylase and lipase, while

the secretions of sublingual glands are predomi-

nantly mucous, mucin-rich ﬂ uids, as same as of

those minor salivary glands. And the secretions

of submandibular glands are a mixture of mucous

and serous ﬂ uids. Upon stimulation, the parotid

glands produce watery saliva with high content of

enzymes like amylase and lipase. The different

properties of the secretions of these glands are

determined by their different composition of the

secretory endpieces, also called acini. Salivary

gland ﬂ uid is produced by acini [ 2 ].

3.1.2 Salivary Composition

The normal salivary pH is slightly acidic, about

6–7, but pH in salivary ﬂ ow can range from 5.3

(low ﬂ ow) to 7.8 (peak ﬂ ow). Salivary ﬂ uid con-

sists of approximately 99 % water and 1 % sol-

utes, including electrolytes, glucose, nitrogenous

products like urea and ammonia, and macromol-

ecules. Salivary electrolytes include sodium,

potassium, calcium, magnesium, bicarbonate,

and phosphate. A variety of proteins are also

found in saliva such as enzymes, immunoglobu-

lins, mucins, other antimicrobial factors, mucosal

glycoproteins, traces of albumin, some polypep-

tides, and oligopeptides [ 3 ]. The average concen-

tration and function of saliva composition is

shown in Table 3.1 .

W. Renke

Department of Operative Dentistry and Endodontics ,

West China School of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

3.1.3 Salivary Flow Rate

and Inﬂ uence Factors

There is a broad normal range in individual sali-

vary ﬂ ow rate. The acceptable ﬂ ow rate is no less

than 0.1 mL/min for unstimulated saliva and

0.2 mL/min for stimulated saliva. In normal con-

dition, the unstimulated whole saliva ﬂ ow rate is

0.3–0.5 ml/min, while stimulated whole saliva

ﬂ ow rate is 1.5–2.0 ml/min and both of them have

wide ranges.

The secretion of salivary glands is mainly con-

trolled by parasympathetic impulses from the

salivary nuclei. Several factors inﬂ uence the sali-

vary ﬂ ow rate, including the nature and duration

of stimulus, the emotional state, water balance of

the body, and medication (which will be dis-

cussed later). The salivary ﬂ ow and composition

of saliva display large variations during the day

[ 4 ]. The salivary ﬂ ow rate usually increases dur-

ing the day and reaches the acrophase in the late

afternoon, around 15.00–17.00 h [ 2 ]. It’s almost

Table 3.1 The average concentration and function of saliva composition

Composition Concentration Function

Inorganic Ca

1–2 mM Modulate demineralization and

remineralization

0.2–0.5 mM

6–26 mM

14–32 mM

1–7 mM

−

& HPO

2−

2–23 mM Modulate pH, buffering, modulate

demineralization and

remineralization

−

17–29 mM

HCO

−

2–30 mM Modulate pH, buffering

−

0.5–5 μM Modulate demineralization and

remineralization, antibacterial

action

−

0.1–2 mM Antibacterial action

Organic Urea 2–6 mM Modulate pH, buffering

Uric acid 0.2 mM Indication for kidney function

Amino acids (free) 1–2 mM

Glucose (free) 50 μM

Lactate 0.1 mM

Fatty acids 10 mg/L

Macromolecules Proteins 1.4–2 g/L Lubricant, cleanse, aggregate,

attach to microorganism, dental

plaque metabolism, modulate

demineralization and

remineralization, antibacterial

action

Glycoprotein sugars 0.11–0.3 g/L Cleanse, aggregate, attach to

microorganism, dental plaque

metabolism

Amylase 0.38 g/L Digestion, antibacterial action

Lysozyme 0.11 g/L Antibacterial action

Peroxidase 3 mg/L Antibacterial action

IgA 0.2 g/L Antibacterial action

IgG 14 mg/L Antibacterial action

IgM 2 mg/L Antibacterial action

Lipid 20–30 mg/L

W. Renke

zero during sleep. This rhythm could be related

to change of hormone level during the day and

indicates the importance of taking oral hygiene

procedures before going to sleep. With the

absence of protective effects of saliva, if there is

much remaining food and dental plaque in the

oral cavity, there would be a great chance for den-

tal caries to happen.

Sympathetic impulses are more likely to inﬂ u-

ence salivary composition by increasing exocytosis

from certain cells. Salivary composition may also

be inﬂ uenced by hormones such as androgens,

estrogens, glucocorticoids, and peptide hormones.

3.2 Salivary Inﬂ uences

on Plaque PH and Oral

Microﬂ ora

3.2.1 Salivary Inﬂ uences

on Plaque PH

Acid-producing bacteria, like Streptococcus

mutans and Lactobacillus sp., existed in normal

oral ﬂ ora. These bacteria metabolize fermentable

carbohydrates in food and produce variety of

organic acids, leading to the reduction of pH

value in dental plaque and causing demineraliza-

tion of hard tissue of teeth.

However, because of the powerful salivary

buffering capacity, dental caries is not likely to

happen all the time. There are three major buffer

systems in saliva: the carbonic acid/bicarbonate,

the phosphate, and the protein buffer. The protein

buffer mainly takes place in buffering below pH

5, while the optimal buffering range for the car-

bonic acid/bicarbonate and the phosphate buffer

occurs at pH 6.3 and pH 7.2, respectively [ 5 ]. The

phosphate buffer plays an important role in

unstimulated saliva. The secondary phosphate

ion, HPO

2−

, could bind a hydrogen ion and form

a primary phosphate ion H

−

. During food

intake, its effectiveness is limited due to insufﬁ -

cient concentrations of phosphate in saliva.

The carbonic acid/bicarbonate buffer is the

most important buffering system in saliva during

food intake and mastication. Bicarbonate is

secreted within the ducts and. In resting saliva,

HCO

−

concentration is as low as 1–2 mM, but it

increases with ﬂ ow rate and reaches 60 mM in

stimulated saliva. So during intake, two impor-

tant events happen: (1) bacteria ferment carbohy-

drates and produce organic acrid, causing a drop

in pH, and (2) the increased salivary ﬂ ow rate

leads to an increased HCO

−

concentration. The

carbonic acid/bicarbonate equilibrium (Fig. 3.1 )

shifts to the left to produce more carbonic acid.

With the presence of carbonic anhydrase VI

secreted by the serous acinar cells in parotid and

submandibular glands, carbonic acid is further

driven to be converted into carbon dioxide and

water.

Concerning about caries, the pH of saliva

may not be as important as the pH of dental

plaque. Both fermentable carbohydrate remain-

ders and salivary buffering capacity affected

plaque pH. The resting plaque pH refers to the

ph of plaque 2–2.5 h after the last intake of

dietary carbohydrates and is usually between 6

and 7. Following exposure to fermentable car-

bohydrates, the plaque pH decreases rapidly

during the ﬁ rst 5 min and reaches a minimum

after approximately 5–20 min. Unless there is

additional ingestion of fermentable carbohy-

drates, the plaque pH returns slowly to the start-

ing level over 30–60 min. This plaque pH

change over time is known as the Stephan

curve.

The buffering capacity of saliva affects not

only the rate at which the plaque pH decreases

but also the minimum value of plaque pH and

how long the pH stays at that minimum. The pH

value of 5.6 is called a “critical value” which is

the pH at which the tissues start to dissolve and

cause demineralization. Low buffering capacity

of saliva leads to increased rate of plaque pH fall

after carbohydrate intake and prolonged time

below critical pH, ﬁ nally leading to an increased

risk of dental caries.

Fig. 3.1 The carbonic acid/bicarbonate equilibrium in

saliva

3 Saliva and Dental Caries

Salivary stimulation after food intake also

affects plaque pH. When gum is chewed, the ﬂ ow

of saliva increases from a resting value of 0.4–

0.5 ml/min to approximately 5–6 ml/min. A

series of studies conﬁ rmed an obvious and sus-

tained rise in plaque pH when gum was chewed

after sugar consumption. Both sugar-containing

and sugar-free gum have such effect, but sugar-

free gum displays a more powerful effect [ 6 ].

Chewing other material that could stimulate sali-

vary secretion such as paraﬁ lm also works.

3.2.2 Salivary Inﬂ uences on Oral

Microﬂ ora

Besides affecting plaque pH, saliva has profound

inﬂ uence on oral microﬂ ora. Saliva provides a

nutritious source for oral microﬂ ora. Salivary

proteins, glycoproteins, peptides, and minerals

can stimulate the growth of oral microﬂ ora and

enhance bioﬁ lm formation. On the other hand,

saliva displays antimicrobial activities with the

presence of a series of immunologic (secretory

IgA, IgG, IgM) and non-immunologic factors

(enzymes, mucins, proteins, peptides). Lysozyme,

lactoferrin, and histatins exhibit bacteriostatic,

bacteriocidic activity, while immunoglobulins,

mucins, and salivary agglutinin play a role in the

oral clearance of bacteria by interfering with

receptors on the microbial cell wall.

The immunoglobulins including IgG, IgM,

IgA, and secretory IgA (SIgA) form the speciﬁ c

defense system in saliva against bacteria. SIgA is

the most abundant immunologic component in

saliva, mainly produced by plasma cells located

in minor mucous glands. SIgA is non-detectable

in neonates but become readily detectable 1 week

after birth. It can neutralize pathogenic viruses,

toxins, and enzymes produced by bacteria. SIgA

can prevent bacteria forming colonies or attach-

ing or penetrating host tissues, kill them directly,

or activate complements or provide synergism

with innate defense mechanisms. It’s also able to

aggregate or clump bacteria, promoting oral

clearance. SIgA against streptococcus mutans

can be detected in children at the age of 3 years

old, and the quantity increases with the length of

exposure. Other immunologic components occur

in less quantity in saliva. IgG is the only detect-

able Ig in saliva of neonates and is mainly mater-

nal origin. The concentration of IgG decreases to

non-detectable after a few months after birth and

appears again after tooth eruption. At this time,

IgG mainly comes from gingival crevicular ﬂ uid,

originating from sera.

Lysozyme is a cationic protein with low molec-

ular weight from the basal cells of striated ducts in

parotid glands as well as monocytes and macro-

phages. It is one of the most important nonspeciﬁ c

defense substances in saliva. Lysozyme catalyzes

cleavage of beta-1,4 glycosidic bonds between

muramic acid and N -acetylglucosamine residues

in peptidoglycan; hence it hydrolyzes bacterial

cell wall. Due to the external lipopolysaccharide

layer, Gram-negative bacteria are more resistant to

lysozyme. Furthermore, nonenzymatic bacteri-

cidic effect of lysozyme related to activation of

bacterial autolysins is reported as well. It is also

documented that this enzyme functions to aggre-

gate bacteria and inhibit bacterial adherence.

There are two categories of salivary peroxi-

dases: human salivary lactoperoxidase (HS-LPO),

also termed sialoperoxidase, synthesized in sali-

vary glands, and myeloperoxidase (MPO) formed

in polymorphonuclear leucocytes. Salivary per-

oxidases have enzymatic activity and are able to

catalyze the oxidation of salivary thiocyanate and

hypothiocyanous acid, inhibiting bacterial metab-

olism [ 7 ].

Salivary chitinase secreted by salivary glands

catalyzes the hydrolytic cleavage of chitin. Chitin

is a cellular wall compound of yeast cells; hence,

chitin may play a role in the protection against

yeast.

Mucins are glycoproteins secreted from subman-

dibular and sublingual glands that selectively modu-

late the microorganism adhesion. There are two

genetically distinct mucin groups: highly glycosyl-

ated, high-molecular-weight MG1 (>1000 kDa) and

single-glycosylated peptide chain, low-molecular-

weight MG2 (200–300 kDa). The two groups of

mucins display similar carbohydrate chain makeup

but have different bacterial adherence ability. MG1

binds tightly to tooth surface and takes part in enamel

pellicle formation. MG2 promotes the aggregation

W. Renke

and clearance of oral bacteria, including

Streptococcus mutans . It’s reported that MG2 pre-

dominates in saliva of caries-resistant individuals,

while the level of MG1 is higher in caries- susceptible

individuals [ 8 ].

Salivary agglutinin (SAG) is a high-molecular-

weight (approximately 340 kDa), mucin-like gly-

coprotein aggregate cells in suspension that can

be found in parotid and submandibular saliva.

SAG is highly glycosylated and extremely sticky.

It could bind to hydroxyapatite and is a compo-

nent of the enamel pellicle and may be involved

in the initial adherence of bacteria to the enamel

surface.

SAG binds to streptococci surface receptor

antigen I/II in a calcium-dependent manner.

Furthermore, SAG also binds to SIgA, resulting

in synergistic effect of bacterial aggregation

which promotes clearance of microorganisms

from the oral cavity [ 9 , 10 ].

Lactoferrin is a glycoprotein with a molecular

weight of about 80 kDa. It is a member of a trans-

ferring family and able to link to ferric iron in

saliva and lead to bactericidal or bacteriostatic

effects on bacteria requiring iron for metabolism

including streptococcus mutans by iron- depriving

effect. This process of starving bacteria of vital

nutrients is termed nutritional immunity. It’s

reported that lactoferrin is a multifunctional pro-

tein having bacteriostatic, bactericidic, fungicidal,

antiviral, anti-inﬂ ammatory, and immunomodula-

tory properties [ 11 , 12 ].

Histatins are a family of peptides rich in histi-

dine, arginine, and lysine residues. At least 12

histatin-like peptides are identiﬁ ed in human

saliva. Histatins have antimicrobial activity against

some strains of Streptococcus mutans and yeasts.

They are implicated in pellicle formation, neutral-

ization of lipopolysaccharides of the external

membranes of Gram-negative bacteria, chelation

of metal ions, and inhibition of proteinases includ-

ing metalloproteins and cysteine proteinases.

Saliva provides nutrients to oral microorgan-

isms and supports their growth. On the other

hand, saliva has a protective function in

maintaining oral health and microbial ecological

balance by inhibiting pathogens and regulating

pH in oral environment.

3.3 Xerostomia

and Its Management

3.3.1 Etiology of Xerostomia

Xerostomia, also termed dry mouth or dry mouth

syndrome, is referred to the subjective symptom

of oral dryness, which is frequently, but not

always, associated with salivary gland hypofunc-

tion and a signiﬁ cant reduction of unstimulated

salivary secretion. As mentioned above, the nor-

mal ﬂ ow rate of unstimulated saliva is about

0.3–0.5 ml/min, while that of stimulated saliva is

about 1.5–2.0 ml/min. It would be considered as

hypofunction of salivary glands if unstimulated

ﬂ ow rate is below 0.1 ml/min or stimulated ﬂ ow

rate is below 0.5 ml/min. As there is a wide range

of salivary ﬂ ow rate, a 50 % reduction of indi-

vidual salivary ﬂ ow rate would also be consid-

ered hypofunction [ 13 , 14 ].

Saliva is secreted by three pairs of major sali-

vary glands and the minor salivary glands. Any

factors that could inﬂ uence salivary gland func-

tion, including diseases of salivary glands,

Sjögren’s syndrome, radiotherapy in the head and

neck area, the use of certain drugs, etc., might

lead to the reduced output of salivary secretion,

causing xerostomia. Besides iatrogenic factors,

aging could be another important cause for xero-

stomia. As life expectancy in developed countries

keeps increasing, xerostomia is becoming

increasingly common in the result of an increased

incidence of systemic diseases and a more exten-

sive intake of medication and/or the degeneration

and reduced volume of acini. Table

3.2 shows the

common causes of xerostomia.

A lot of drugs have inﬂ uence on salivary ﬂ ow

rate and composition, including medication for

hypertension, depression, and allergies. Over 500

medications produce xerostomia as a side effect.

A medication which is known to cause xerosto-

mia may be termed xerogenic. Table 3.3 lists

some drugs that may cause xerostomia.

Xerostomia is a common side effect of radia-

tion therapy of tumors in the region of the head

and neck. The parotid gland is the most radiosen-

sitive gland and then the submandibular gland,

then the sublingual gland, and the minor salivary

3 Saliva and Dental Caries

gland. As reported, a single dose above 52 Gy

could lead to severe salivary dysfunction, so con-

ventional radiation treatment of oral carcinoma at

a dose of 60–70 Gy would cause a rapid reduc-

tion of salivary ﬂ ow rate during the ﬁ rst week of

treatment. By 5 weeks of the treatment, the sali-

vary ﬂ ow almost reduces to zero and hardly

recovers completely after the treatment.

Besides the factors mentioned above, there are

a number of additional disorders that may con-

tribute to the presence of xerostomia, including

thyroid dysfunction, Parkinson’s disease, sys-

temic lupus erythematosus, post-traumatic stress

disorders, depression, anxiety, etc. These disor-

ders inﬂ uence the salivary ﬂ ow either through

pathophysiological process or the medical treat-

ment of the disease.

Xerostomia is a common condition in popula-

tion. It’s reported that 25 % of general population

complain of xerostomia or symptoms associated

with it. For elderly, the incidence is as high as

40 %. With such a high incidence, the inﬂ uence

of xerostomia in life quality brings to great atten-

tion. As the quantity of saliva secretion reduced

reversibly or irreversibly, patients could experi-

ence different extent of dysarthria, dysphagia,

mucosal trauma and ulceration, candida infec-

tion, and dental caries. The clinical consequences

and management had been comprehensively dis-

cussed in a review [

15 ]; here we will mainly

focus on dental caries related to xerostomia and

its management [ 15 ].

One of the most important functions of saliva

is to develop dental pellicle on enamel surface,

protecting against demineralization and replen-

ishing tooth surface minerals including calcium

and phosphate. It also provides buffering activity,

antibacterial activity, and effective carbohydrate

clearance. As salivary output decreases, a series

of respondents take place with the outcome of

increased demineralization and decreased remin-

eralization and virtually lead to dental caries

(Fig.

3.2 ).

3.3.2 Management of Xerostomia

Xerostomia patients should be instructed to

observe a variety of caries preventive procedures,

including oral hygiene instruction, plaque con-

trol, low sugar dietary advice, daily use of topical

ﬂ uoride (0.05 %), antimicrobial mouse rinses

(e.g., chlorhexidine), and placement of sealants.

The most important one is to reduce the intake of

Table 3.2 Common causes of xerostomia

Class Cause

Iatrogenic Drugs

Local radiation

Chemotherapy

Chronic graft-versus-host disease

Diseases of

salivary glands

Sjögren’s syndrome

Sarcoidosis

HIV disease

HCV infection

Primary biliary cirrhosis

Cystic ﬁ brosis

Diabetes mellitus

Rare causes Amyloidosis

Hemochromatosis

Wegner’s disease

Salivary gland agenesis (with or

without ectodermal dysplasia)

Triple A syndrome

Others

Cited from Porter et al. [

15 ]

Table 3.3 Medications associated with xerostomia

Class Generic name

Anticonvulsants Atropine, hyoscine

Antipsychotics Phenothiazine

Antidepressants Amitriptyline, ﬂ uoxetine,

lithium, bupropion, dothiepin

Antihistamines Diphenhydramine,

cimetidine

Antihypertensives Terazosin, prazosin,

clonidine, atenolol,

propranolol

Antireﬂ ux drugs Omeprazole

Opioids Meperidine

Antineoplastics Cytotoxic drugs, retinoids,

interleukin-2

Anti-HIV drugs Protease inhibitors,

didanosine

Diuretics Chlorothiazide, furosemide,

antisterone

Antiasthmatic Ephedrine

Anxiolytics Benzodiazepines

W. Renke

sugars. Sugar intake should be conﬁ ned to meals,

and there should be no sugar consumption

between meals. Non-cariogenic sugar substitutes

including xylitol, sorbitol, aspartame, Lycasin,

saccharin, and sucralose, which could not be fer-

mented by acidogenic bacteria into organic acids,

should be used to reduce sugar consumption [ 16 ].

Xerostomia patients should be recommended

to brush teeth twice a day with a soft bristle tooth

brush along with low abrasive, high ﬂ uorinated

toothpaste. Fluoride is also available in other

forms like foam, varnish, rinse, and gel. It is rec-

ommended by ADA that the daily use of ﬂ uoride

rinses and toothpaste accompanied with ﬂ uoride

varnish applied every 3 months is helpful in den-

tal caries inhibition in xerostomia population [ 17 ].

The use of remineralization products is a rela-

tive new strategy against dental caries in xerosto-

mia patients. It provides calcium and phosphate

ions that are lacking because of reduced salivary

output. These products usually contain different

types of calcium and phosphate compound with

or without additional ﬂ uoride in the form of paste

or medication carrier (products without ﬂ uoride).

Applying to the tooth surface, they can provide

and attract calcium and phosphate ions to the

tooth surface, aiding in remineralization.

3.4 Saliva and Caries Risk

Assessment

Dental caries is a multifactorial infectious dis-

ease that is associated with complex interactions

among acid-producing bacteria, fermentable

carbohydrates, and host factors including saliva

status. To estimate the caries risk before disease

has been given more attention in recent years

since dental caries is generally considered as a

kind of progressive disease unless intervened sur-

gically. Several caries risk assessment methods

have been developed as indicators of caries sus-

ceptibility at the individual level. They can be

used to estimate the probability of caries inci-

dence, determine the need for therapeutic inter-

vention, and are a part of treatment planning.

Generally, these tests are derived from the

severity of past and current caries experience,

diet, protective factors including ﬂ uoride, behav-

ioral and physical factors, medical factors, socio-

economic status, measurement of saliva ﬂ ow rate

and buffering capacity, and estimation of caries-

related microorganisms in saliva including

Streptococcus mutans and Lactobacilli

(Tables

3.4 , 3.5 and 3.6 ) [ 19 ].

3.4.1 Caries-Associated Bacteria

There are more than 700 species of microorgan-

isms inhabit the oral cavity. In healthy individuals,

caries-associated bacteria are usually present in

relatively small amount in saliva. But in condi-

tions of biological or environmental change such

as increased frequency of carbohydrate intake or

poor oral hygiene, the etiological balance of oral

microﬂ ora will shift to favor the aciduric bacteria

and acidogenic bacteria, increasing the risk of

dental caries.

Plaque bacterial composition is most related

to dental caries since acid causing demineraliza-

tion is mainly produced by plaque bacteria. Since

Fig. 3.2 The chain reaction

of reduced salivary ﬂ ow rate

3 Saliva and Dental Caries

plaque bacteria can be released into saliva, it is

documented that the level of certain species of

bacteria in saliva can reﬂ ect their level in dental

plaque, making salivary microﬂ ora an effective

biomarker of the health and disease status of oral

cavity [ 20 , 21 ].

Streptococcus mutans and Lactobacilli have

been implicated as important contributory species

in dental caries. It is reported that high level of sali-

vary Streptococcus mutans (>10

CFU/ml) is asso-

ciated with an increased risk of dental caries

[ 22 – 24 ]. Although the amount of lactobacilli is less

sensitive in predicting caries incidence than the

amount of mutans streptococci [ 25 ], the high level

of Lactobacilli (>10

CFU/ml) [ 26 ] could be an

indicator for increased frequency of carbohydrate

consumption [ 27 ] and caries progression [ 28 ].

Most of tests regarding caries-associated bac-

teria require incubation which makes them less

convenient for dental practitioners. Recently, cul-

ture-independent technologies including checker-

board DNA-DNA hybridization, 16S rRNA

sequence analysis, and T-RFLP have been utilized

for dental caries microbial analysis. These molec-

ular methods have not applied clinically in large

scale but deﬁ nitely suggest new possibilities for

fast, convenient, and culture- independent meth-

ods of caries-associated bacteria test.

Table 3.4 ADA caries risk assessment form for age 0–6

Low risk Moderate risk High risk

Contributing conditions

I Fluoride exposure (through

drinking water, supplements,

professional applications,

toothpaste)

Yes No

II Sugary foods of drinks (including

juice, carbonated or non-

carbonated soft drinks, energy

drinks, medical syrups)

Primarily at

mealtimes

Frequent or prolonged

between meal

exposures/day

Bottle or sippy cup

with anytime other

than water at bed

time

III Eligible for government programs

(WIC, Head Start, Medicaid, or

SCHIP)

No Yes

IV Caries experience of mother,

caregiver, and/or other siblings

No carious lesions in

last 24 months

Carious lesions in last

7–23 months

Carious lesions in

last 6 months

V Dental home: established patient

of record in a dental ofﬁ ce

Yes No

General health conditions

I Special health-care needs

(developmental, physical,

medical, or mental disabilities that

prevent or limit performance of

adequate oral health care by

themselves or caregivers)

No Yes

Clinical conditions

I Visual or radiographically evident

restorations/cavitated carious

lesions

No new carious

lesions or

restorations in last

24 months

Carious lesions or

restorations in last

24 months

II Non-cavitated (incipient) carious

lesions

No new lesions in

last 24 months

New lesions in last

24 months

III Teeth missing due to caries No Yes

IV Visible plaque No Yes

V Dental/orthodontic appliances

present ( ﬁ xed or removable)

No Yes

VI Salivary ﬂ ow Visually adequate Visually Inadequate

W. Renke

Table 3.5 ADA caries risk assessment form for age > 6

Low risk Moderate risk High risk

Contributing conditions

I Fluoride exposure (through

drinking water, supplements,

professional applications,

toothpaste)

Yes No

II Sugary foods of drinks (including

juice, carbonated or non-

carbonated soft drinks, energy

drinks, medical syrups)

Primarily at

mealtimes

Frequent or prolonged

between meal

exposures/day

III Caries experience of mother,

caregiver, and/or other siblings

(for patients ages 6–14)

No carious lesions in

last 24 months

Carious lesions in

last 7–23 months

Carious lesions in last 6

months

IV Dental home: established patient

of record in a dental ofﬁ ce

Yes No

General health conditions

I Special health-care needs

(developmental, physical,

medical, or mental disabilities

that prevent or limit performance

of adequate oral health care by

themselves or caregivers)

No Yes ( over age 14) Yes ( ages 6–14)

II Chemo-/radiation therapy No Yes

III Eating disorders No Yes

IV Medications that reduce salivary

ﬂ ow

No Yes

V Drug/alcohol abuse No Yes

Clinical conditions

I Cavitated or non-cavitated

(incipient) carious lesions or

restorations (visually or

radiographically evident)

No new carious

lesions or restorations

in last 36 months

1 or 2 new carious

lesions or restorations

in last 36 months

3 or more carious

lesions or restorations

in last 36 months

II Teeth missing due to caries in

past 36 months

No Yes

III Visible plaque No Yes

IV Unusual tooth morphology that

compromises oral hygiene

No Yes

V Interproximal restorations, 1 or

No Yes

VI Exposed root surfaces present No Yes

VII Restorations with overhangs and/

or open margins; open contacts

with food impaction

No Yes

VII Dental/orthodontic appliances

present (ﬁ xed or removable)

No Yes

IX Severe dry mouth (xerostomia) No Yes

Table 3.6 The strength of association between salivary characteristics and caries risk

Strong association Weak-to-moderate association No association

Flow rate Buffering capacity; calcium/phosphate;

speciﬁ c sIgA immunoglobulin

pH, glucose clearance rate/concentration; other

electrolytes and small organic molecules; total

sIgA; IgG; innate immunity factors

Cited from Leone et al. [

18 ]

3 Saliva and Dental Caries

3.4.2 Chemical and Physical

Aspects of Saliva

Leone et al. had reviewed 96 references and

divided the chemical and physical characteristics

of saliva into three groups according to their

strength of association with dental caries

(Table 3.6 ) [ 18 ].

It has been discussed earlier in this chapter

that low salivary ﬂ ow rate is a risk factor of dental

caries. Statistical data suggests that unstimulated

salivary ﬂ ow less than 0.3 ml/min and the stimu-

lated salivary ﬂ ow lower than 0.8–1.0 ml/min

indicate increased caries risk strongly. But these

values should not be treated as an absolute stan-

dard for caries risk screening since there is a wide

range of salivary ﬂ ow rate among individuals. An

obvious reduction of salivary output in one indi-

vidual should be paid attention to as well.

A number of studies showed the correlation

between low salivary buffering capacity and den-

tal caries, while high buffering capacity indicates

better neutralizing capacity and more resistance

to demineralization.

The level of speciﬁ c secretory IgA showed a

relationship with caries risk, and the literature is

almost equally divided for and against an anticar-

ies role of speciﬁ c secretory IgA. No evidence has

indicated sufﬁ cient association between caries

risk and salivary innate non-immunoglobulin fac-

tors including lysozyme, lactoferrin, peroxidase/

myeloperoxidase, praline-rich proteins, stather-

ins, and histatins. On the other hand, Mungia et al.

found signiﬁ cant associations between caries and

speciﬁ c individual submandibular/sublingual sali-

vary proteins including lactoferrin, albumin, lyso-

zyme, mucin, and cystatin recently [

29 ]. But if it

is an indicator of caries risk still needs more data

and further investigation.

References

1. Humphrey SP, Williamson RT. A review of saliva:

normal composition, ﬂ ow, and function. J Prosthet

Dent. 2001;85(2):162–9.

2. Edgar WM, Dawes C, O’Mullane DM. Saliva and oral

health. 4th ed. Duns Tew: Stephen Hancocks Limited;

2012.

3. de Almeida Pdel V, Grégio AM, Azevedo LR. Saliva

composition and functions: a comprehensive review.

J Contemp Dent Pract. 2008;9(3):072–80.

4. Dawes C. Circadian rhythms in human salivary ﬂ ow

rate and composition. J Physiol. 1972;220:529–45.

5. Lenander-Lumikari M, Loimaranta V. Saliva and den-

tal caries. Adv Dent Res. 2000;14:40–7.

6. Manning RH, Edgar WM. pH changes in plaque after

eating snacks and meals, and their modiﬁ cation by

chewing sugared or sugar-free gum. Br Dent J. 1993;174:

241–4.

7. Rosin M, Hanschke M, Splieth C, Kramer A. Activities

of lysozyme and salivary peroxidase in unstimulated

whole saliva in relation to plaque and gingivitis scores

in healthy young males. Clin Oral Invest. 1999;3:

133–7.

8. Slomiany BL, Murty VL, Piotrowski J, Slomiany A.

Salivary mucins in oral mucosal defense. Gen

Pharmacol. 1996;27(5):761–71.

9. Lamont RJ, Demuth DR, Davis CA, Malamud D,

Rosan B. Salivary-agglutinin-mediated adherence of

Streptococcus mutans to early plaque bacteria. Infect

Immun. 1991;59(10):3446–50.

10. Oho T, Yu H, Yamashita Y, Koga T. Binding of sali-

vary glycoprotein-secretory immunoglobulin A

complex to the surface protein antigen of Streptococcus

mutans . Infect Immun. 1998;66(1):115–21.

11. Nieuw Amerongen AV, Veerman ECI. Saliva-the

defender of oral cavity. Oral Dis. 2002;8:12–22.

12. Adlerova L, Bartoskova A, Falduna M. Lactoferrin: a

review. Vet Med. 2008;53(9):457–68.

13. Saltana N, Sham ME. Xerostomia: an overview. Int J

Dent Clin. 2011;3(2):58–61.

14. Fox PC, Eversole R. Diseases of the salivary glands.

Essen Oral Med. 2001;260–76.

15. Porter S, Scully C, Hegarty A. An update of the etiology

and management of xerostomia. Oral Surg Oral Med

Oral Pathol Oral Radiol Endod. 2004;97(1):28–46.

16. American Dental Association Council on Scientiﬁ c

Affairs. Professionally applied topical ﬂ uoride:

evidence- based clinical recommendations. J Am Dent

Assoc. 2006;137(8):1151–9.

17. Su N, Marek C, Ching V, et al. Caries prevention for

patients with dry mouth. J Am Dent Assoc. 2011;77:b85.

18. Leone CW, Oppenheim FG. Physical and chemical

aspects of saliva as indicators of risk for dental caries

in humans. J Dent Educ. 2001;65(10):1054–62.

19. American Dental Association caries risk assessment

forms.

www.ada.org/5157.aspx . Accessed 22 Feb 2011

20. Asikainen S, Alaluusua S, Saxen L. Recovery actino-

mycetemcomitans from teeth, tongue and saliva.

J Periodontol. 1991;62(3):203–6.

21. Umeda M, Contreras A, Chen C, Bakker I, Slots J. The

utility of whole saliva to detect the oral presence of

periodontopathic bacteria. J Periodontol. 1998;69(7):

828–33.

22. von Haute J. Microbiological predictors of caries risk.

Adv Dent Res. 1993;7(2):87–96.

23. Thenisch NL, Bachmann LM, Imfeld T, Leisebach

Minder T, Steurer J. Are mutans streptococci detected

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in preschool children a reliable predictive factor for

dental caries risk? A systematic review. Caries Res.

2006;40(5):366–74.

24. Parisotto TM, Steiner-Oliveira C, Sliva CM,

Rodriques LK, Nobre-dos-Santos M. Early childhood

caries and mutans streptococci: a systematic review.

Oral Health Prev Dent. 2010;8(1):59–70.

25. Wilson RF, Ashley FP. Identiﬁ cation of caries risk in

school-children: salivary buffering capacity and bac-

terial counts, sugar intake and caries experience as

predictors of 2-year and 3-year caries increment.

Br Dent J. 1989;167(3):99–102.

26. Larmas M. Saliva and dental caries: diagnostic tests for

normal dental practice. Int Dent Res. 1992;42(4):1990208.

27. Holbrook WP, de Soet JJ, de Graaf J. Prediction of

dental caries in pre-school children. Caries Res.

1993;27(5):424–30.

28. Klock B, Krasse B. Microbiological and salivary con-

ditions in 9-12-year old children. Scand J Dent Res.

1977;85(1):56–63.

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503–8.

3 Saliva and Dental Caries

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_4

Demineralization

and Remineralization

Cheng Lei , Li Jiyao , Xu Hockin H.K. ,

and Zhou Xuedong

Dental caries is caused by acids produced from

bacterial metabolism diffusing into dental hard

tissue and dissolving the mineral. The process of

dental caries is a continuum which results from

many de-/remineralization cycles [ 1 , 2 ]. Dental

hard tissue consists of enamel, dentin, and cemen-

tum. Dental enamel is highly mineralized, and it

comprises 80–90 % by volume of a calcium-deﬁ -

cient carbonate hydroxyl apatite. Other calciﬁ ed

tissues (bone or dentine) contain considerably

lower amounts of inorganic minerals. Mature

human enamel crystallites are 26.3 ± 2.2 nm thick,

68.3 ± 13.4 nm wide, and between 100 and

1,000 nm long [ 3 ]. Dentine and cementum con-

tain a much greater proportion of organic matrix.

Dentine is made up of approximately 50 vol.%

mineral, 30 vol.% collagenous and non-collage-

nous proteins, and 20 vol.% ﬂ uids. The dentinal

matrix is mainly composed of type I collagen

ﬁ brils forming a three-dimensional scaffold

matrix, reinforced by hydroxyl apatite crystal-

lites, measuring approximately 20 nm in size [ 3 ].

Sound enamel and dentine crystals are com-

prised of a hydroxyapatite-like mineral contain-

ing many impurities and inclusions of other ions.

The mineral phase of the dental hard tissues is

not pure hydroxyapatite (HAP = Ca

(PO

)

). Hydrogen phosphate, carbonate, and mag-

nesium ions are incorporated into the HAP lattice

to form a less stable, more soluble apatite. For

example, approximately 1 out of 10 of the phos-

phate ions in enamel is replaced by carbonate

ions and 1 out of 5 in dentine [

4 , 5 ]. So the min-

eral of enamel and dentine is much more soluble

than pure hydroxyapatite or ﬂ uorapatite. But the

partial substitution of ﬂ uoride ions for OH groups

C. Lei (*)

State Key Laboratory of Oral Diseases ,

Sichuan University , Chengdu ,

People’s Republic of China

Department of Operative Dentistry and Endodontics ,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

State Key Laboratory of Oral Diseases ,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

L. Jiyao

State Key Laboratory of Oral Diseases ,

Sichuan University , Chengdu ,

People’s Republic of China

Department of Operative Dentistry and Endodontics ,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

X. Hockin H.K.

Biomaterials and Tissue Engineering Division,

Department of Endodontics,

Prosthodontics and Operative Dentistry ,

University of Maryland Dental School , Baltimore ,

MD 21201 , USA

Z. Xuedong

State Key Laboratory of Oral Diseases ,

West China Hospital of Stomatology, Sichuan

University , Chengdu , People’s Republic of China

in the crystal lattice can stabilize the apatite

structure less susceptible to acid attack.

4.1 Dynamics Process of

De-/Remineralization

Dental caries is a disease that is manifested as a

dynamic process of de-/remineralization in the

mouth (Fig. 4.1 ). Demineralization is a continual

imbalance between pathological and protective

factors that results in the dissolution of apatite

crystals and the net loss of calcium, phosphate,

and other ions from the tooth [ 6 ]. The ﬁ rst stage of

demineralization is occurring at the atomic level

far before it can be seen visually as gross demin-

eralization. During this step, fermentable carbo-

hydrates are metabolized by bacteria in dental

plaque to produce organic acids. The acids diffuse

into the dental hard tissue through the water

among the crystals and could reach a susceptible

site on a crystal surface. Calcium and phosphate

are dissolved into the surrounding aqueous phase

between the crystals [ 2 ]. This is considered as the

ﬁ rst step in the continuum of the dental caries pro-

cess which can eventually lead to cavitation.

The oral ﬂ uids (saliva, bioﬁ lm ﬂ uid) have cal-

cium (Ca) and phosphate (Pi) in supersaturated

concentrations with respect to the mineral com-

position of enamel. At physiological conditions

(a neutral pH of 7), low ion concentrations are

sufﬁ cient to keep dental hard tissues in equilib-

rium. If the pH drops because of acid produced

by the dental plaque, higher ion concentrations

are needed to prevent dissolution of dental hard

tissue. Calcium (Ca) and phosphate (Pi) ions are

continually deposited on the enamel surface or

are redeposited in enamel areas where they were

lost. At a pH of ca. 5.5, undersaturation begins,

that is, the calcium- and phosphate-ion concen-

trations in the plaque ﬂ uid are not sufﬁ cient to

maintain the enamel in stable equilibrium; thus,

the enamel starts to dissolve.

The term “remineralization” is used to

described mineral gain. Remineralization is the

body’s natural repair process for subsurface non-

cavitated carious lesions [ 7 ]. In the process of rem-

ineralization, calcium and phosphate ions are

supplied from a source external to the tooth to pro-

mote ion deposition into crystal voids in deminer-

alized enamel to produce net mineral gain.

De-/remineralization cycles continue in the

mouth as long as there are factors including cario-

genic bacteria, fermentable carbohydrates, and

saliva present. The balance between pathological

factors and protective factors determines whether

demineralization or remineralization is proceed-

ing at any one time [ 2 ].

4.2 Investigations

of De-/Remineralization

4.2.1 Models

The de-/remineralization process can be stud-

ied using different kinds of models, such as

in vitro model, in situ model, animal models, or

Remineralization

Acld reslstant

(PO

)

(F)

flourapatite-like

coating on crystals

Calclum +

Phosphate

+ Fluorlde

Crystal

nucleus

Enamel/dentin

crystal =

Carbonated apatite

Partly dissolved

crystal

ACID

Fig. 4.1 The caries

process including

demineralization and

subsequent

remineralization to form a

low solubility surface on

the crystals [

2 ]

C. Lei et al.

in randomized controlled clinical trials [ 8 – 10 ].

The induction of artiﬁ cial carious lesions in

bovine and human teeth is an important tool to

study strategies for the prevention or treatment

of carious lesions. Various models can be

selected according to different purposes. Each

model has its advantages along with disadvan-

tages. For example, in vitro experiments are the

most commonly applied methods. They can be

performed over a short period of time, require

fewer staff than in situ studies, avoid partici-

pant compliance issues, and are relatively inex-

pensive. But they cannot replicate the oral

environment with all of the biological varia-

tions known to inﬂ uence de-/remineralization

process [ 11 , 12 ].

4.2.1.1 In Vitro Chemical Model

The modern pH-cycling models were ﬁ rst pro-

duced by ten Cate and Duijsters [ 13 ]. In vitro pH-

cycling models are widely used, especially for

the in vitro evaluation of the efﬁ cacy of ﬂ uori-

dated dentifrices for caries control [ 14 ]. They are

also broadly used in proﬁ le studies for rapid and

inexpensive testing of developing and recently

marketed products [ 9 ]. There are several advan-

tages of in vitro pH cycling: (i) the model can

mimic the dynamics of mineral loss and gain

involved in caries formation; (ii) the high level of

scientiﬁ c control and the resulting lower variabil-

ity intrinsic to in vitro models; and (iii) it requires

smaller sample size [ 14 ].

4.2.1.2 In Vitro Bioﬁ lm Model

Dental plaque bioﬁ lms play a pivotal role in the

progression of dental diseases, so in vitro bioﬁ lm

models are developed to produce artiﬁ cial caries

lesions [ 15 ]. Two main aspects should be consid-

ered when the most suitable bioﬁ lm model for a

de-/remineralization investigation was chosen: (1)

to select pure cultures or deﬁ ned communities or

microcosms and (2) to select closed system bio-

ﬁ lm models or open system bioﬁ lm models [ 15 ].

Streptococcus mutans are considered the most

cariogenic microorganisms in dental bioﬁ lm due

to their capacity to use dietary carbohydrates to

synthesize extracellular polysaccharides (EPS)

and because of their acidogenic and aciduric

properties [ 16 ]. And S. mutans bioﬁ lm models as

single-species bioﬁ lm model are widely used in

de-/remineralization investigations [

17 – 19 ].

Multiple species bioﬁ lm models using deﬁ ned

consortia could achieve a high degree of repro-

ducibility between experimental runs which can-

not be relied upon when using complex inocula

[ 15 , 20 , 21 ]. Microcosms are able to maintain

much of the complexity and heterogeneity of the

original sample. Saliva was usually collected to

form microcosm bioﬁ lms to replicate enabling in

situ bacterial community dynamics within the

laboratory environment [ 22 , 23 ].

Bioﬁ lm models can be divided into two groups

according to whether they are “closed” or “open”

with respect to nutritional availability [ 15 ].

Closed system bioﬁ lm models are analogous to

batch culture and usually based on multi-well

plates [ 18 , 24 ]. Silva TC et al. applied an active

attachment bioﬁ lm model as a high-throughput

demineralization bioﬁ lm model for the evalua-

tion of caries-preventive agents (Fig. 4.2a ) [ 18 ].

Zürich bioﬁ lm model is another typical closed

system bioﬁ lm models applied in de-/remineral-

ization experiments [ 21 , 25 ]. Open system bio-

ﬁ lm models are analogous to continuous culture.

McBain AJ. et al. reviewed different kinds of

open system bioﬁ lm models [ 15 ]. The constant

depth ﬁ lm fermentor (CDFF) was widely used

for de-/remineralization investigations [ 23 , 26 ,

27 ]. The CDFF allows the generation of large

numbers of replicate bioﬁ lms which can be main-

tained at a constant depth by static scraper blades

(Fig.

4.2b ).

4.2.1.3 In Situ Model

In situ models are also widely used for de-/remin-

eralization experiments now. In situ models

involve the use of devices creating conditions that

simulate the process of dental caries. Enamel and

dentin samples are the hard tissue substrates used

in in situ models to assess de- and remineraliza-

tion [ 8 ]. The in situ models are designed to simu-

late the natural process of de-/remineralization

and also to provide information in a short period

of time without causing damage to the natural

teeth of volunteers [ 28 ]. These models serve as a

link between the clinical uncontrolled situation

and the highly controlled laboratory experiments.

The in situ caries model designs are highly

4 Demineralization and Remineralization

variable because of the variations of the in situ

study designs. All in situ studies must have appro-

priate controls including a positive and negative

control where possible [ 8 ].

4.2.1.4 Animal Model

For many reasons—particularly time consider-

ations, animal availability, and attendant costs—

rodents have been the most commonly used

species for experimental caries studies [ 21 ].

According to Stookey et al.’s investigations, the

following rat caries models could be used for eval-

uating ﬂ uoride dentifrices: Francis’ hypomineral-

ized area model, Gaffar’s CARA rat model, the

Connecticut model, and the Indiana model [ 29 ].

4.2.2 Detection and Measurement

Methods

Various techniques have been used to investigate the

mineral loss and gain during de-/remineralization

process, including destructive and nondestructive

methods.

Medium inlets

Top plate

Glass housing

Sprung

scraper blade

Turntable

Tie bar

PTFE seal

Bottom plate

Drive shaft

Outlet for waste medium

(expanded, aerial

view of a pen)

Gas

Sampling port

Fig. 4.2 In vitro bioﬁ lm models, ( a ) an active attachment bioﬁ lm model [ 18 ]; ( b ) CDFF [ 15 ]

C. Lei et al.

4.2.2.1 Transversal

Microradiography (TMR)

TMR or contact-microradiography is a highly

sensitive method used to measure the morphol-

ogy of and the change in mineral content of den-

tal hard tissue [ 30 ]. But the method is usually

destructive to dental hard tissue, so it cannot be

used to study any longitudinal mineral changes in

exactly the same lesions [ 31 ]. To prepare the

samples for TMR investigation, thin slices (about

80 μm for enamel samples and 200 μm for den-

tine samples) are cut perpendicularly to the tooth

surface. A microradiographic image is made on

high-resolution ﬁ lm by X-ray exposure of the

sections together with a calibration aluminum

step wedge. The mineral can be automatically

calculated from the gray levels of the images of

section and step wedge. Parameters of interest

are mineral loss (Delta Z in Vol%.μm), lesion

depth (Lesd in μm), ratio or average loss of min-

eral content in the lesion area (Delta Z/Lesd in

Vol%), and the mineral vol% and position of the

subsurface layer and lesion body [ 32 , 33 ].

4.2.2.2 Indentation Techniques

Indentation techniques have been used to measure

the hardness of the dental hard tissue surface.

There are two kinds of indentation techniques for

de-/remineralization investigations: microinden-

tation (surface hardness) [ 32 ] and nanoindenta-

tion (ultra-microindentation) [ 34 ]. During the

process of both microindentation and nanoinden-

tation, a diamond tip of known dimensions is

pressed onto a surface with a given load and dura-

tion. The microindentation technique yields data

in arbitrary units, usually Knoop hardness number

(KHN) or Vickers hardness number (VHN), and

nanoindentation yields hardness and reduced

elastic modulus in the SI unit of Pascals (Nm

−2

)

[ 35 ]. Moron BM et al. revealed that surface hard-

ness analysis should not be interpreted with

respect to dentine mineral loss [ 11 ]. This was

expected as the high organic content, and thus, the

elastic properties of the dentine inﬂ uence the

hardness measurement [ 36 ].

4.2.2.3 Micro-CT

Micro-CT investigation is a nondestructive

method to measure the mineral changes of dental

hard tissue. So it is possible to measure and visu-

alize longitudinal mineral changes during de- and

remineralization in the same lesion [

37 ]. Published

papers proved that micro-CT could offer the

quantitative analysis of the de- and remineraliza-

tion based on CT intensity data [ 31 ]. The key

point of using micro-CT is to ﬁ nd out how to con-

verse the CT intensity into mineral content. Neves

et al. reported a linear correlation between CT

intensity (or gray scale value) and the mineral

density using three apatite phantoms, the linearity

covering a range of 0.25–3.14 g/cm

[ 38 ]. Schwass

et al. found a good linearity using six phantoms

covering a range from 0.07 to 2.95 g/cm

[ 39 ].

4.2.2.4 Confocal Laser Scanning

Microscopy

Confocal laser scanning microscopy (CLSM) is

able to identify tissue-emitting ﬂ uorescent signal

and can be used to detect the mineral loss of den-

tal hard tissue [ 40 – 42 ]. Demineralizing dentin has

a strongly increased autoﬂ uorescence compared

to sound dentine [ 43 ]. Some previous studies

stained thick enamel samples with a ﬂ uorescent

dye (0.1 m M rhodamine B) and analyzed using

CLSM for quantitating demineralization and rem-

ineralization of enamel specimens [ 44 ].

4.2.2.5 Quantitative Light-Induced

Fluorescence

Quantitative light-induced ﬂ uorescence (QLF) is

a quantiﬁ cation system for assessing early demin-

eralization or remineralization of human enamel.

When high-intensity blue light illuminates the

teeth, the resultant autoﬂ uorescence of enamel is

detected by an intraoral camera. The emitted ﬂ uo-

rescence has a direct relationship with the mineral

content of the enamel. The intensity of the tooth

image at a demineralized area is darker than the

sound area. The software of QLF systems can

process the image to provide user quantitative

parameters such as lesion area, lesion depth, and

lesion volume [

45 – 47 ].

4.2.2.6 Optical Coherence Tomography

Optical coherence tomography (OCT) is a three-

dimensional optical imaging technique which

works in a similar way to ultrasound, but uses

high-frequency light (around 820 nm) instead of

high-frequency sound [ 48 ]. It is a noninvasive,

cross-sectional imaging system that can visualize

4 Demineralization and Remineralization

the internal structures nondestructively [ 49 ].

Amaechi et al. developed a quantitative method

to detect the demineralizing lesions of dental

enamel using an OCT system [ 50 ]. This system

was able to collect A-scans (depth versus

reﬂ ectivity curve), B-scans (longitudinal images),

and C-scans (transverse images at constant

depth). The area (R) under the A-scan could be

quantiﬁ ed to indicate the degree of reﬂ ectivity of

the tissue.

4.3 Methods to Inﬂ uence

the De-/Remineralization

Process

4.3.1 Traditional Methods

4.3.1.1 Fluoride

Fluoride was introduced into dentistry over 70

years ago, and it is now recognized as the main

factor responsible for the dramatic decline in car-

ies prevalence that has been observed worldwide

[ 51 ]. Fluoride can be obtained in two forms: sys-

temic and topical. Systemic methods include

water ﬂ uoridation, salt ﬂ uoridation, milk ﬂ uori-

dation, and supplements. Later in the 1940s, the

well-conducted water ﬂ uoridation program was

established in the United States. Though some

dentists and researchers have conﬂ icting opin-

ions about their safety and beneﬁ ts, these sys-

temic methods are still recommended in many

countries and receive support from recognized

international committees and associations. Due

to the widespread application of ﬂ uoride and the

updated knowledge about its mechanisms of

action, topical applications of ﬂ uoride (e.g., ﬂ uo-

ride toothpastes, gels, varnishes, and mouth-

washes) are considered to be more effective

methods for caries prevention than systemic use

of ﬂ uorides [

52 ].

It is believed that ﬂ uoride could inhibit demin-

eralization and enhance remineralization [ 7 , 53 –

55 ]. Numerous studies were designed to investigate

the mechanism of ﬂ uoride in inhibiting demineral-

ization of dental hard tissue. According to previous

studies, ﬂ uoride could incorporate into the enamel

apatite structure, enhance the resistance of the

dental hard tissue to acidic challenges, and thus

inhibit lesion development [

7 , 56 ]. In addition,

calcium-ﬂ uoride- like deposits could form on den-

tal hard tissues and act as a protective barrier on

the surface and serve as a reservoir for ﬂ uoride

[ 53 ]. Other researchers have some different opin-

ions. They demonstrate that the incorporation of

ﬂ uorides into the mineral components of enamel

only slightly reduced its solubility [ 54 , 55 , 57 ].

Small amounts of free ﬂ uoride ions in solution

around the dental hard tissue play a much more

important role in inhibiting demineralization.

These ﬂ uorides have a much greater caries-

protective potential than a large proportion of FAP

incorporated in enamel mineral [ 58 ]. Free ﬂ uoride

ions are in part adsorbed onto the crystalline sur-

face and are in dynamic equilibrium with the ﬂ uo-

ride ions in solution around dental hard tissue. So

it forms an equilibrium or supersaturation relative

to ﬂ uor(hydroxy)apatite, and the adsorption of

ﬂ uoride on the crystals can offer direct protection

from demineralization. Therefore, according to

this theory, ﬂ uoride should be present in the right

place (bioﬁ lm ﬂ uid or saliva) and at the right time

(when bioﬁ lm is exposed to sugar or right after

bioﬁ lm removal), and even small amount of ﬂ uo-

ride (below ppm values) available is effective.

In addition to its ability of inhibiting demineral-

ization, ﬂ uoride is thought to be effective to pro-

mote remineralization. However, ﬂ uoride’s ability

to enhance net remineralization is limited by the

availability of calcium and phosphate ions [ 59 ]. If

adequate salivary or plaque calcium and phosphate

ions are available, ﬂ uoride ions can drive the rem-

ineralization of extant non-cavitated caries lesions.

Fluorhydroxyapatite forms more rapidly even in

slightly acidic condition than do the other calcium

phosphate phases, so ﬂ uoride can accelerate and

promote remineralization of dental hard tissue.

It has been demonstrated that ﬂ uoride used in

enamel remineralization also work in dentine

remineralization, and even their remineralization

mechanisms are similar [

60 , 61 ]. However, dentin

demands a considerably higher ﬂ uoride concen-

tration in its surrounding solution than enamel

does to reach an equivalent degree of demineral-

ization inhibition [ 62 ].

C. Lei et al.

4.3.1.2 Calcium Phosphate

Calcium and phosphate ions play important roles

in enhancing remineralization of dental caries.

Investigators have tried various solutions

containing calcium and phosphate ions in their

experiments, in which solutions contained

between 1 and 3 mM calcium ions with phosphate

ions in the ratio of 1:1 [ 63 , 64 ] or 1.66:1 [ 63 ],often

with the addition of 1 ppm ﬂ uoride ions. Higher

concentrations are difﬁ culty to be used because

of the instability of the solutions. So the low

solubility of calcium phosphates, particularly in

the presence of ﬂ uoride ions, limited the clinical

application of calcium and phosphate

remineralization systems. Therefore, novel

calcium-phosphate-based delivery systems are

developed to combat the demineralization of

dental hard tissue. New commercial products are

available based on three types of novel systems—

crystalline, unstabilized amorphous, or stabilized

amorphous formulations.

4.3.2 Novel Methods

4.3.2.1 CPP–ACP and CPP–ACFP

CPP–ACP has been shown to promote

remineralization of initial enamel lesions and to

prevent demineralization in laboratory, animal,

and human experiments [ 59 , 65 ]. Casein

phosphopeptides (CPP) can stabilize calcium and

phosphate as nanoclusters of ions through the

formation of amorphous nanocomplexes

(diameter of 2.12 nm) in metastable solution

[

66 ]. But it can also prevent the growth of the

nanoclusters to the critical size required for

nucleation and phase transformation [ 65 ]. CPP

contains the active sequence Ser(P)-Ser(P)-

Ser(P)-Glu-Glu. Phosphorylated seryl residues

are regarded as responsible for the interactions

between casein and the calcium and phosphate

ions in the nanocomplexes [ 67 ].

CPPs can bind to the more thermodynami-

cally favored surface of an apatite crystal face in

the caries lesion due to its high binding afﬁ nity

for apatite. And CPPs prefer binding to the (100)

and (010) faces of hydroxyapatite crystals

(Fig. 4.3 ). So crystal growth would be allowed to

continue only at the hydroxyapatite (001) plane

or along the c-axis, which is the pattern of crystal

growth during amelogenesis. Therefore, CPPs

are able to regulate anisotropic crystal growth

and also inhibit crystal demineralization in the

enamel subsurface lesion [

68 ].

The amorphous calcium phosphate (ACP) is an

important compound because it is a precursor that

can convert to apatite, similar to the minerals in

tooth enamel and dentin. ACP is an unstabilized

calcium and phosphate system. When a calcium

salt (e.g., calcium sulfate) and a phosphate salt

(e.g., potassium phosphate) are delivered sepa-

rately, the calcium ions and phosphate ions are

mixed and result in the immediate precipitation of

ACP or, in the presence of ﬂ uoride ions, amor-

phous calcium ﬂ uoride phosphate (ACFP). In the

intraoral environment, these phases (ACP and

ACFP) are potentially very unstable and may rap-

idly transform into a more thermodynamically

stable, crystalline phase (e.g., hydroxyapatite and

ﬂ uorhydroxyapatite). However, before phase

transformation, calcium and phosphate ions should

be transiently bioavailable to promote enamel sub-

surface lesion remineralization.

4.3.2.2 Natural Medicine

Previous studies indicated some nature medicines

were able to inﬂ uence the de-/remineralization

balance of dental hard tissue. The extracts of

Galla chinensis (GCE) could inhibit the deminer-

alization and enhance the remineralization of

enamel and dentin [

69 – 72 ]. In addition, this poly-

phenol compounds had combined effects with tra-

ditional remineralizing agents, like ﬂ uoride [ 69 ],

nano-hydroxyapatite [ 73 ]. However, the mecha-

nism of GCE is still unclear, and more investiga-

tions are still needed.

4.3.2.3 Laser

Several types of lasers, such as erbium-doped

yttrium aluminum garnet (Er:YAG) [ 74 , 75 ],

neodymium-doped yttrium aluminum garnet

(Nd:YAG) [ 74 , 76 ], and carbon dioxide (CO

)

[ 76 – 79 ], with different parameter settings, have

been used for caries inhibition. It is believed that

the use of the high-intensity laser on the dental

structure can lead to a more acid-resistant sur-

4 Demineralization and Remineralization

face, and previous investigations showed that

lasers could inhibit enamel demineralization and

reduce enamel permeability [ 80 ].

4.3.2.4 Nanoparticles

Currently, nanotechnology is experiencing rapid

growth, with many potential applications in car-

ies prevention and treatment. It is deﬁ ned as the

creation of functional materials, devices, or sys-

tems through control of matter on the nanometer

scale (1–100 nm). Nanotechnology has motivated

mimicking of the nanostructural features of natu-

ral human enamel and development of bioin-

spired strategies for remineralization and caries

therapy, respectively [ 81 ]. Previous researches

have tried to apply nanoparticles in dental caries

Fig. 4.3 A molecular model of the Ser(P)-Ser(P)-Ser(P)-

Glu-Glu motif bound onto the face of hydroxyapatite

(HA). The atoms are colorcoded as follows: calcium (1)

atoms are light-blue crosses, calcium (2) atoms are dark-

blue crosses, oxygen atoms are red, phosphorus atoms are

magenta, carbon atoms are green, nitrogen atoms are blue,

and hydrogen atoms are grey. The symbol X indicates a

crystallographic axis projecting into the paper. Four views

are presented: ( a ) a side view along the c-axis, with the

peptide rendered in CPK and the crystal atoms in ‘line’

form; ( b ) as in ( a ), but viewed from above, looking down

on the HA (100) face; ( c ) as in ( b ), with the peptide dis-

played in stick form and the atoms in the HA surface

within 0.25 nm of the peptide rendered in CPK; and ( d ) as

in ( b ), with the peptide displayed in stick form and the

atoms of the peptide within 0.25 nm of the HA surface

rendered in CPK. [

68 ]

C. Lei et al.

prevention and treatment, including spherical,

cubic, and needlelike nanoscaled particles

(approximately 5–100 nm) and near-nanoscaled

devices (up to micrometers) [ 82 ]. When the sizes

of particles are reduced from micrometer to

nanometer, the resultant properties can change

dramatically. For example, hardness, active sur-

face area, chemical reactivity, and biological

activity are all altered [ 83 ]. The application of

nanoparticles in dentistry can be categorized into

two directions: preventive dentistry and restora-

tion dentistry [ 81 ].

In recent years, various types of nano-sized

hydroxyapatite or calcium carbonate are applied

to combat early caries lesions [ 73 , 84 , 85 ]. Some

in vitro studies indicated that 10 % suspension of

nano-hydroxyapatite particles (10–20 nm diame-

ter, 60–80 nm length) promotes remineralization

of the superﬁ cial layer of initial caries lesions

measuring 20–40 μm, but little remineralization

could be obtained by nano- hydroxyapatite in the

body of the lesion [

73 , 84 ]. Carbonate hydroxyl

apatite nanoparticles have also been reported to be

effective in repairing micrometer-sized tooth-sur-

face defects in vitro [ 86 ]. And some nanocrystals

have been incorporated into toothpastes or mouth-

rinsing solutions as commercial products.

Clinical investigations indicate that secondary

caries and restoration fracture are still the main

reasons for dental restoration failure, which lim-

its the longevity of dental restorations, especially

the resin composites [ 87 ]. Approximately half of

all dental restorations fail within 10 years, and

replacing them consumes nearly 60 % of the

average dentist’s practice time [

88 ]. Investigators

try to improve the resin compositions, ﬁ ller par-

ticles, and cure conditions. Calcium phosphate

(CaP) particles and calcium ﬂ uoride particles

have been used as ﬁ llers in resin composites

(Fig. 4.4 ). These resin-based CaP or CaF com-

posites can release calcium (Ca), phosphate

(PO4), or ﬂ uoride ions. These additives enable

the resin composite to release calcium and phos-

phate when the pH is dropped down under in vitro

conditions, providing caries-inhibiting properties

[ 90 ]. These calcium and phosphate or ﬂ uoride

ion-releasing nanoﬁ llers include nanoparticles of

dicalcium phosphate anhydrous (112 nm in size),

amorphous calcium phosphate (ACP) (116 nm in

size), and so on [ 91 – 95 ].

Nanoparticles of ACP can be synthesized via a

spray-drying technique. Brieﬂ y, a spraying

solution was prepared by dissolving calcium

carbonate and dicalcium phosphate anhydrous

(CaHPO

) into an acetic acid solution. Then this

solution was sprayed through a nozzle into a

heated chamber. The water and volatile acid were

evaporated and expelled into an exhaust hood.

The dried particles were collected by an electro-

static precipitator.

CaF

nanoparticles can be synthesized using

the same spray-drying apparatus. A two-liquid

nozzle was employed during the procedures. Two

solutions are mixed during atomization: Ca(OH)

and NH

F. And the two solutions are atomized

leading to the formation of CaF

nanoparticles:

Ca(OH)

+ NH4F → CaF

+ NH

OH. The NH

is removed as NH

and H

O vapors [ 96 ].

acb

Fig. 4.4 TEM micrographs of the spray-dried nanoparticles: ( a ) small ACP nanoparticles, ( b ) ACP cluster, ( c ) CaF

nanoparticles [

89 ]

4 Demineralization and Remineralization

4.3.3 Biomineralization

Biomineralization is the process by which living

organisms produce minerals, often to mineralized

tissues. The formation of teeth is a process of

biomineralization. Though traditional methods

have been proven to be effective to combat dental

caries, dental hard tissue is unable to heal

and repair itself after demineralization of the

surface and subsequent cavitation due to its

non- regenerative nature. Understanding the

mechanism of dental hard tissue inspired the

researchers who studied the remineralization of

dental hard tissue. Numerous studies have tried to

ﬁ nd special templates to achieve biomineraliza-

tion of dental hard tissue in vitro [ 89 ]. There are

numerous evidences indicating that organic tem-

plates or scaffolds would be a prerequisite for

bioinspired formation of enamel-like structures

[ 97 ]. However, there are some problems about

the clinical application of biomimetic approaches

in vitro. Firstly, the suspected biocompatibility of

nonbiological surfactants strongly limits their

clinical application. Secondly, most of biomi-

metic approaches need various hydrothermal

conditions, including nonphysiological tempera-

ture or pressure [ 98 ], which cannot be applied in

clinical practice. So some investigators have

focused on the biomimetic approaches in physio-

logical-like condition. For example, single crys-

talline hydroxyapatite micro-ribbons were used

as substitutes for amelogenin templates to control

HAP crystallization at biophysical conditions at

37 °C [

99 ] .

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4 Demineralization and Remineralization

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_5

The Diagnosis for Caries

Yang Liu , Li Boer , Wang Shuang , Zhang Yaru ,

and Peng Li

According to the glossary of endodontic terms,

caries is deﬁ ned as a localized and progressive

bacterial infection that results in disintegration of

the tooth, usually beginning with the demineral-

ization of enamel and followed by bacterial inva-

sion. It usually takes 6–12 months for caries to

appear. Generally, we can make a correct diagno-

sis with routine inspection. For some difﬁ cult

cases, dental radiographs or other special inspec-

tions are supplementary methods for caries diag-

nosis (Table 5.1 ).

5.1 Conventional Diagnosis

Methods

5.1.1 Inspection

When making an examination for dental caries on

suspected sites, we can ﬁ nd black or chalky area or

a formed cavity. The interproximal marginal ridge

area has ink stain discoloration under the enamel

or a visible cavity. For observation on tooth cervi-

cal areas, the cheek and tongue should be pulled

away to fully expose buccal and lingual surface of

posterior teeth. Depending on the inspection, we

can get a general scope of carious damage.

5.1.2 Probing

The sharp probe is used to inspect the suspicious

areas. With the help of the probe, the depth and

extension of cavity can be examined. If a proxi-

mal cavity is suspected and cannot be located

through an inspection, the probe is useful to

locate affected area when it is hooked by the edge

of the cavity. The probe can also be used on the

tooth surface to locate the area of dentinal hyper-

sensitivity. Pulp exposure can also be located

while examining the deep carious lesion.

5.1.3 Percussion

Caries does not cause periodontal and periapical

inﬂ ammation, so the reaction to percussion is

always negative.

5.2 Special Diagnostic Methods

5.2.1 Radiographic Examination

Radiographic examination can be helpful in locat-

ing proximal caries and undermining caries and

secondary caries [ 1 ]. It can also be used to assess

the proximity of caries to pulp chamber. Periapical

Y. Liu • L. Boer • W. Shuang • Z. Yaru • P. Li (*)

State Key Laboratory of Oral Diseases ,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

and bite-wing radiographs are commonly used for

clinical assessment of caries. Radiolucency on

hard tissue due to demineralization is identiﬁ ed as

carious lesion. A series of researches have

revealed that more than half of proximal caries are

seen on the radiograph. Since the radiograph is a

two-dimensional image, the diagnosis result

should be analyzed and combined with clinical

examination. Proximal caries should be distin-

guished from normal triangular low-density areas

in the cervical region of the tooth. Secondary car-

ies should be differentiated from low-density bas-

ing materials on the ﬂ oor of the cavity.

5.2.2 Cold and Hot Irritation Test

The response of dental pulp toward cold and hot

irritation is examined during cold and hot

irritation test. Examination is done by putting

chloroethane- soaked cotton ball or hot gutta-

percha stick on the surface of the tooth, and the

patient’s response is evaluated. These external

stimuli can elicit acute pain. The pulp is sup-

posed to be healthy if pain disappears right after

removal of stimuli. But if the pain is lingering,

the pulp is likely to be in irreversible inﬂ amma-

tory. When cold water is used as stimulus, it is

Table 5.1 The diagnosis for caries

Conventional diagnosis methods Inspection Black or chalky area or a formed cavity

can be seen

Probing Probe can be used to locate affected area,

the area of dentinal hypersensitivity, and

pulp exposure

Percussion The reaction to percussion is always

negative

Special diagnostic methods Radiographic examination Radiolucency on hard tissue due to

demineralization is identiﬁ ed as carious

lesion

Cold and hot irritation test The response of dental pulp toward cold

and hot irritation can determine the

severity of caries

Dental ﬂ oss examination Dental ﬂ oss can diagnose caries on

proximal contact area

Diagnostic cavity preparation After removing supportless enamel, the

practitioner can obtain well a vision on

hidden carious lesion

The new technology of caries

diagnosis

Fiber-optic transillumination This system uses ﬁ ber transillumination

for potential caries diagnosis

Electrical impedance technology This technology is an alternative way for

occlusal pit and ﬁ ssure caries diagnosis

Ultrasonic technique It is a new method for caries detection by

measuring the wave that reﬂ ects back

from tooth structure

Elastomeric separating modulus

technique

Elastomeric separating modules are used

to separate apart adjacent tooth

temporarily for examination of proximal

surfaces

Staining technique This technique can show the presence of

caries and estimate the depth of carious

lesion

Quantitative laser ﬂ uorescence

technique

Autoﬂ uorescence is light emission

phenomenon of biological structure

The differential diagnosis of the

superﬁ cial caries

Enamel hypocalciﬁ cation The key points of the differential

diagnosis for caries are glossiness and

smoothness, predilection site, symmetry

of the lesion, and progress of the lesion

Enamel hypoplasia

Dental ﬂ uorosis

Y. Liu et al.

important to note that the ﬂ ow of water may

affect the accurate location of carious cavity. At

last, caries should be differentiated from dentin

hypersensitivity.

5.2.3 Dental Floss Examination

Caries on proximal contact area is difﬁ cult to be

examined by inspection and probing. Dental

ﬂ oss can be used as a convenient method. By

putting a dental ﬂ oss across the embrasure of

the suspicious tooth surface and moving the

ﬂ oss horizontally in a seesaw motion, the exam-

iner can experience the roughness of the sur-

face. The ﬂ oss will be torn if caries is present.

Examination with ﬂ oss can be misled by dental

calculus.

5.2.4 Diagnostic Cavity Preparation

After removing supportless enamel, the practi-

tioner can obtain well a vision on hidden carious

lesion. To identify the scope and depth of cavity,

the infectious dentin on the ﬂ oor and wall of the

cavity should be removed completely. Dying the

decalciﬁ ed dentin with 0.5 % basic fuchsin can

help dentists to identify and remove the infec-

tious dentin. After that, the tooth decay area and

pulp condition can be easily deﬁ ned.

5.3 The New Technology

of Caries Diagnosis

Tooth caries is chronic, progressive, and bacterial

diseases. The main characters of tooth caries are

the changes in color, shape, and quality of tooth

hard tissue. The typical pathological changes

have important reference value for caries diagno-

sis. At present, the methods for dental caries

diagnosis are mainly based on clinical inspection

and X-ray examination. However, it is difﬁ cult to

identify early caries which is in the hidden area

of the tooth. With the latest development in sci-

ence and technology, some techniques and meth-

ods are being used for caries diagnosis. These

methods have greatly improved the accuracy and

sensitivity of caries diagnosis.

5.3.1 Fiber-Optic

Transillumination, FOTI

A new diagnostic technique for caries was

called ﬁ ber-optic transillumination. This system

uses ﬁ ber transillumination for potential caries

diagnosis [ 2 , 3 ]. The principle is based on the

fact that the light transillumination index in

decayed tissue is lower than that in normal tis-

sue. Generally, the decayed area shows dark

shadow.

5.3.2 Electrical Impedance

Technology

The electrical impedance technology is an alter-

native way for caries diagnosis by examining

tooth potential difference. The carious cavity is

ﬁ lled with dead and decayed tissue, saliva, and

electrolytes. Therefore, this area becomes more

electric conductive than the normal tissue.

Following this principle, the resistance offered by

the tooth surface is measured under controlled

drying. The conductivity is measured by probe in

occlusal ﬁ ssure, and current passes through the

pulp to the ground through handheld lead form-

ing a circuit.

The electric caries detector device measures

the bulk electric resistance and potential differ-

ence. This method is simple, sensitive, and sta-

ble for occlusal caries detection.

5.3.3 Ultrasonic Technique

The ultrasonic technique is a new method for car-

ies detection by measuring the wave that reﬂ ects

back from the tooth structure. This ultrasonic

wave is received by a sensor when reﬂ ecting back

from the tooth surface. The normal tooth surface

and the decay one are supposed to have different

reﬂ ecting waves. Currently, 18 MHz frequency

wave was used for caries diagnosis.

5 The Diagnosis for Caries

5.3.4 Elastomeric Separating

Modulus Technique

Elastomeric separating modules are used to sepa-

rate apart adjacent tooth temporarily for exami-

nation of proximal surfaces. This method can be

helpful especially when proximal surface caries

was examined.

5.3.5 Staining Technique

The staining technique usually stains the degraded

collagen which is present in the carious cavity but

never stains the intact collagen. For this reason,

the dye is used in carious cavity to stain dead and

decayed dental tissue. With this technique, the

dentist can determine the presence of caries and

estimate the depth of carious lesion. The com-

monly used dye is 1 % basic fuchsin.

5.3.6 Quantitative Laser

Fluorescence Technique

Autoﬂ uorescence is light emission phenomenon

of biological structure. The autoﬂ uorescence of

dental tissue decreases in demineralization of the

tissue. Quantitative laser ﬂ uorescence devices

use high-intensity halogen lamp to stimulate the

tooth to emit the ﬂ uorescence in green spectrum

[ 4 , 5 ]. This reﬂ ected light is detected by spectrum

and recorded in computer and demineralization is

quantiﬁ ed. The related other new technologies

are dye-enhanced laser ﬂ uorescence (DELF),

quantitative light-induced ﬂ uorescence, light

scattering, and confocal laser scanning micro-

scope [

6 , 7 ].

5.4 The Differential Diagnosis

of the Superﬁ cial Caries

The diagnosis of the pit and ﬁ ssure caries can be

done based upon its location. However, the diag-

nosis of the smooth surface caries is more chal-

lenging due to its appearances. Common clinical

presentation of caries is described below.

5.4.1 Enamel Hypocalciﬁ cation

This is a condition where the enamel is formed

without adequate mineralization. The lesion is

characterized by irregular, opaque, and chalky

spots or plaque on tooth surface [ 8 ] (Fig. 5.1a ).

5.4.2 Enamel Hypoplasia

Enamel hypoplasia is a deﬁ cit in enamel forma-

tion. The enamel is thin and deﬁ cient in amount.

It is seen clinically as dot or banded sunken

defects with chalky, yellowish, or brownish dis-

coloration [ 9 ] (Fig. 5.1b ).

5.4.3 Dental Fluorosis

Dental ﬂ uorosis is a developmental disturbance,

due to exposure of tooth bud to high concentra-

tion of ﬂ uoride during its development. In mild

type of ﬂ uorosis, tiny white streaks or specks are

seen in the enamel, but discolored and pitted in

severe type [ 10 ]. The spots and stains left by ﬂ uo-

rosis are permanent and may darken over time

[ 11 ] (Fig. 5.1c ).

5.4.4 The Key Points

of the Differential Diagnosis

for Caries

Glossiness and Smoothness The enamel

lesions that are caused by developmental distur-

bance may have color changes. However, the

glossiness, smoothness, and hardness are not

affected. The caries teeth have chalky or snuff

colored spots without gloss. The enamel surface

can also be rough in appearance.

Predilection Site Pit and ﬁ ssure, proximal sur-

face, and cervical part are caries’ predilection

sites. It could hardly be found on the self- cleaning

areas like cusps or any other smooth surface. The

developmental disturbance is caused by abnor-

mal development or irregular mineralization on

Y. Liu et al.

the tooth surface. Based on the stage of tooth

development, the lesion can be seen on different

areas.

Symmetry of the Lesion The developmental

disturbances affect the tooth during the period of

development. Therefore, developmental lesion

can be found bilaterally and in a similar manner.

Progress of the Lesion

The lesions of develop-

mental origin will become quiescent as soon as

the tooth erupts in the oral cavity. It will neither

develop nor disappear. The caries is progressive

by its nature. The color of carious lesion changes

from chalky white to dark brown with its prog-

ress toward the pulp chamber. The initial enamel

lesion which is chalky white in appearance can

be arrested by the tooth remineralization.

The Differential Diagnosis of the Medium

Caries The teeth with medium size caries are

sensitive to sweet, acid, heat, and cold stimula-

tions. Patients usually complain that they feel pain

with these stimuli. This is different from dentinal

hypersensitivity. The dentin hypersensitivity is

stimulation of nerve endings in dentinal tubule

due to change of temperature or pH in oral cavity.

The sensation can range all the way from irritation

to intense, shooting pain. This sensitivity can also

be found on wear-away tooth, decayed tooth, or

tooth with exposed root surface.

References

1. Madalli VB, Annigeri RG, Basavaraddi SM. The eval-

uation of effect of developer age in the detection of

approximal caries using three speed dental x-ray ﬁ lms:

an in-vitro study [J]. J Clin Diagn Res. 2014;8(3):236.

2. Hintze H, Wenzel A, Danielsen B, et al. Reliability of

visual examination, ﬁ bre-optic transillumination, and

bite-wing radiography, and reproducibility of direct

visual examination following tooth separation for the

identiﬁ cation of cavitated carious lesions in contacting

approximal surfaces [J]. Caries Res. 1998;32(3):204–9.

3. Mitropoulos CM. The use of ﬁ bre-optic transillumi-

nation in the diagnosis of posterior approximal caries

in clinical trials [J]. Caries Res. 1985;19(4):379–84.

4. Tomczyk J, Komarnitki J, Zalewska M, et al.

Fluorescence methods (VistaCam iX proof and

DIAGNODent pen) for the detection of occlusal cari-

ous lesions in teeth recovered from archaeological con-

text [J]. Am J Phys Anthropol. 2014;154(4):525–34.

Fig. 5.1 Different clinical images of tooth diseases.

( a ) Chalky plaques can be seen on the labial surface of the

central incisors, diagnosed as dental hypocalciﬁ cation.

( b ) The yellow circle shows sunken defect with yellow discol-

oration. It is known as enamel hypoplasia. ( c ) White specks

are seen on the labial surface of maxillary anterior teeth

5 The Diagnosis for Caries

5. Hafström-Björkman U, Sundström F, Josselin D, de Jong

E, et al. Comparison of laser ﬂ uorescence and longitudi-

nal microradiography for quantitative assessment of

in vitro enamel caries [J]. Caries Res. 1992;26(4):241–7.

6. Sheehy EC, Brailsford SR, Kidd EAM, et al.

Comparison between visual examination and a laser

ﬂ uorescence system for in vivo diagnosis of occlusal

caries [J]. Caries Res. 2001;35(6):421–6.

7. Lai G, Kaisarly D, Xu X, et al. MicroCT-based compari-

son between ﬂ uorescence-aided caries excavation and

conventional excavation [J]. Am J Dent. 2014;27(1):12.

8. Rushton MA. The surface of the enamel in hereditary

enamel hypocalciﬁ cation [J]. Br Dent J. 1962;112:

24–7.

9. Seow WK. Enamel hypoplasia in the primary denti-

tion: a review [J]. ASDC J Dent Child. 1990;58(6):

441–52.

10. Dean HT. Endemic ﬂ uorosis and its relation to dental

caries [J]. Public Health Rep. 1938;53(33):1443–52.

11. Fejerskov O, Manji F, Baelum V. The nature and

mechanisms of dental ﬂ uorosis in man [J]. J Dent Res.

1990;69:692–700; discussion 721.

Y. Liu et al.

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_6

Dental Caries: Disease Burden

Versus Its Prevention

Hong Xiao

6.1 Global Trends of Caries

Burden

6.1.1 Oral Diseases: One of the Most

Costly Diseases to Treat

Oral diseases, despite their non-life-threatening

nature, have been ranked the fourth most expen-

sive disease to treat in most industrialized coun-

tries. In the high-income industrialized countries,

5–10 % of public health spending is used for oral

health care [ 1 ]. Back in the year 2000, the

European Union spent a total of €54 billion on

oral health care, and in the United States, the

2004 expenditures for oral health care were US$

81.5 billion [ 2 ]. However, such ﬁ gures did not

include the additional costs of dental care per-

formed under the medical umbrella. Among the

variety of oral diseases, dental caries has long

been the main factor attributable as the most

important source of oral disease burden.

Not surprisingly, these costs will require unaf-

fordable ﬁ nancial resources from low- and middle-

income countries. For example, the costs of

treatment to control periodontal diseases in chil-

dren on basis of the Community Periodontal Index

(CPI) of Treatment Needs (CPITN) data greatly

exceeded the total national health-care budget of

Kenya [ 3 , 4 ]. Take Nepal as another example: the

total costs of restoring dental caries cavities of the

child population would exceed the total health-

care budget for children of the entire country [ 5 ].

This fact should be interpreted on the back-

ground that, for low-income countries, a compre-

hensive national essential health-care package at

a cost of US$ 4 will reduce the burden of mortal-

ity and disability among the 0- to 14-year-old

children by 30 %!

However, planning traditional curative interven-

tion strategies entirely on the basis of epidemiologi-

cal data without considering the social, economic,

and political conditions is doomed to fail. In partic-

ular the national economic conditions appear to be

an important determinant of restorative care. If we

take the Care Index (F⁄DMFT*100 %) for 34- to

44-year-olds as an indicator of the country’s restor-

ative care level, it appears that no country with a

gross domestic product (GDP) below US$ 5,000

has a Care Index higher than 30 %, which further

underpins that restorative treatment is unaffordable

in low- income countries [

5 , 6 ].

6.1.2 Uneven Distribution of Oral

Disease Burden Around

the World

In the recent several decades, we have witnessed

a time of dramatic decline of caries status

worldwide. This trend can be attributed to

H. Xiao

Department of Preventive Dentistry ,

West China Hospital of Stomatology,

Sichuan University , Chengdu , China

e-mail:

[email protected]

widespread use of topical ﬂ uoride and increasing

awareness of the importance of maintaining good

oral hygiene. However, in some parts of the

world, dental caries still remains a common dis-

ease affecting children and adults, posing tre-

mendous economic burden.

If the burden of disease is described using

years lived with disability (YLD) per million

people, the highest disease burden of the world

can be found in sub-Saharan Africa closely fol-

lowed by India. Both the countries carry heavy

disease burdens mainly because of communica-

ble diseases. However, in the high-income coun-

tries, noncommunicable diseases are the main

source of disease burden [ 7 ].

When it comes to oral diseases, including car-

ies, periodontal diseases, and edentulism, they

make very little contribution to the total YLD/

million population of each country with the

worldwide average of 1.6 %. While the Middle

Eastern Crescent is found to have the highest oral

disease contribution to the total YLD/million

population, China is found to be among those

countries with lowest oral disease contributions.

With respect to relative contribution of caries

to oral disease burden of different regions of the

world, contribution of caries was found to be over

a half of most countries or regions, ranging from

as low as 46.7 % in established economy market

countries to as high as 89.8 % in Latin America

and Caribbean.

Usually, caries status can be monitored through

national epidemiological surveys by recording the

dmf/DMF indices (decayed, missing, and ﬁ lled

tooth/surface indices). Although the recording of

such indices disregards the impact of perceived

pain and discomfort as a result of dental caries, it

is able to reveal some valuable objective informa-

tion especially from a professional perspective.

The proportion of each component varies greatly

among high-, middle-, and low-income countries.

Data collected between 1990 and 2004 indicate

that, in high- income countries, only about one-

ﬁ fth of the caries lesions in the primary dentition

were treated. Such proportion of treatment dra-

matically decreased to about 5 % in the middle-

income countries. Worst ﬁ ndings were found in

the low- income countries, where the proportion

of treated caries lesions could be totally neglected.

Situations in the permanent dentition told a

better story. The high-income countries had a

Care Index (F/DMF score) of over 50 %, while

the middle-income countries had about 20 %.

However unfortunately, the Care Index was found

to be within 5 % for the low-income countries.

The temporal trends in dental caries experi-

ence of 12-year-old children in developing and

developed countries can be illustrated in Figs. 6.1

and 6.2 . In most developing countries, the levels

of dental caries were low. However, the preva-

lence rates of dental caries seem to increase in

recent years. This might be attributed to increased

consumption of sugar and inadequate use of

ﬂ uoride.

However, the industrialized countries tell a dif-

ferent story. A steady decline of caries experience

is found. This result from a number of public

health measures including effective use of ﬂ uo-

rides, together with changing living conditions,

lifestyles, and improved self-care practices.

Worldwide, the prevalence of dental caries

among adults is high as the disease affects nearly

100 % of the population in the majority of coun-

tries. Figure 6.3 illustrates the levels of dental

caries among 35–44-year-olds, as measured by

the mean DMFT index. Most industrialized

countries and some countries of Latin America

show high DMFT values.

6.1.3 Developing Global Policies

Highlighting the Importance

of Oral Health

The policy of the WHO Global Oral Health

Programme emphasizes that oral health is integral

and essential to general health and that oral health

is a determinant factor for quality of life. The WHO

Global Oral Health Programme has implied that

greater emphasis is put on developing global poli-

cies based on common risk factors and approaches

should be coordinated more effectively with other

programs in public health [ 8 – 10 ]:

• To adopt measures to ensure that oral health is

incorporated as appropriate into policies for

the integrated prevention and treatment of

chronic noncommunicable and communicable

H. Xiao

diseases and into maternal and child health

policies

• To take measures to ensure that evidence-

based approaches are used to incorporate oral

health into national policies as appropriate for

integrated prevention and control of noncom-

municable diseases

• To consider mechanisms to provide coverage

of the population with essential oral health

care, to incorporate oral health in the frame-

work of enhanced primary health care for

chronic noncommunicable diseases, and to

promote the availability of oral health services

that should be directed toward disease preven-

tion and health promotion for poor and disad-

vantaged populations, in collaboration with

integrated programs for the prevention of

chronic noncommunicable diseases

• For those countries without access to optimal

levels of ﬂ uoride, and which have not yet estab-

lished systematic ﬂ uoridation programs, to

consider the development and implementation

of ﬂ uoridation programs, giving priority to

equitable strategies such as the automatic

administration of ﬂ uoride, for example, in

drinking water, salt, or milk, and to provide

affordable ﬂ uoride toothpaste

• To develop and implement the promotion of

oral health and prevention of oral disease for

preschool and school children as part of activ-

ities in health-promoting schools

• To scale up capacity to produce oral health

personnel, including dental hygienists, nurses,

Decayed missing and

filled permanent teeth

Very low: <1.2>

Low: 1.2–2.6

Moderate: 2.7–4.4

High: >4.4

No date available

Fig. 6.1 Dental caries levels (decayed, missing, and ﬁ lled

teeth (DMFT) index) among 12-year-olds worldwide

(Reproduced, with the permission of the publisher, from

Bulletin of the World Health Organization , Petersen et al.

[

7 ]. http://www.who.int/bulletin/volumes/83/9/petersen-

0905abstract/en/

)

DMFT

1981 1983

1982 1984 1986 1988 1990 1992 1994 1996 1998

1985

1987

1989 1991

1993 1995 1997

All countries

Developing countries

Developed countries

Fig. 6.2 Changing levels of dental caries experience

(decayed, missing, and ﬁ lled teeth (DMFT) index) among

12-year-olds in developed and developing countries

(Reproduced, with the permission of the publisher, from

Bulletin of the World Health Organization , Petersen et al.

[

7 ]. http://www.who.int/bulletin/volumes/83/9/petersen-

0905abstract/en/

)

6 Dental Caries: Disease Burden Versus Its Prevention

and auxiliaries, providing for equitable distri-

bution of these auxiliaries to the primary-care

level and ensuring proper service back-up by

dentists through appropriate referral systems

• To incorporate an oral health information sys-

tem into health surveillance plans so that oral

health objectives are in keeping with interna-

tional standards and to evaluate progress in

promoting oral health

• To strengthen oral health research and use evi-

dence-based oral health promotion and disease

prevention in order to consolidate and adapt oral

health programs and to encourage the inter-

country exchange of reliable knowledge and

experience of community oral health programs

• To address human resources and workforce

planning for oral health as part of every

national plan for health

• To increase, as appropriate, the budgetary provi-

sions dedicated to the prevention and control of

oral and craniofacial diseases and conditions

• To strengthen partnerships and shared respon-

sibility among stakeholders in order to

maximize resources in support of national oral

health programs

6.2 Caries Burden in China

6.2.1 The First and Second National

Epidemiological Investigation

of Oral Health in China

China has organized two nationwide epidemiol-

ogy investigations. The ﬁ rst was conducted

between 1982 and 1984, which had examined the

status of caries and periodontal diseases in pri-

mary and middle school students of 29 provinces,

autonomous administration regions, and munici-

palities directly under the central government.

There were altogether 131,340 students exam-

ined. The ﬁ rst national epidemiological investi-

gation of oral health in China is the ﬁ rst

large-scale epidemiological investigation orga-

nized since the establishment of P.R. China.

The second national epidemiological investiga-

tion of oral health in China took place from 1995 to

1998. This investigation had included 6 age groups

covering 5–74-year-olds from 11 provinces, auton-

omous administration regions, and municipalities

directly under the central government. A total of

140,712 people at 396 sample sites were selected.

Decayed missing and

filled permanent teeth

Very low: <5.0

Low: 5.0–8.9

Moderate: 9.0–13.9

High: >13.9

No data available

Fig. 6.3 Dental caries levels (decayed, missing, and ﬁ lled

teeth (DMFT) index) among 35–44-year-olds worldwide

(Reproduced, with the permission of the publisher, from

Bulletin of the World Health Organization , Petersen et al.

[

7 ]. http://www.who.int/bulletin/volumes/83/9/petersen-

0905abstract/en/

)

H. Xiao

Results of these two epidemiology investiga-

tions have provided valuable ﬁ rst-hand data for

the central government to make oral health-

related policies, set up targets for oral health ser-

vice, and planned future human resource

allocation in oral health service.

According to the second national survey

which was conducted in 1995, caries prevalence

rates for 5-year-olds, 12-year-olds, 35–44-year-

olds, and 65–74-year-olds were 76.6 %, 45.8 %,

63.0 %, and 64.8 %, respectively.

6.2.2 The Third National

Epidemiological Investigation

of Oral Health in China

The third national epidemiological investigation

of oral health in China was implemented in 2005.

At the planning period, the sampling methods

have been carefully designed as stratiﬁ ed multi-

level randomized sampling [ 11 ]. A total of 93,826

people were selected covering 5–74-year-olds in

6 age groups. The sample well represents the

overall status of oral health in China. In addition

to dental examinations, a comprehensive ques-

tionnaire investigation was simultaneously con-

ducted to investigate the oral health-related

concepts and behaviors of the selected sample.

Caries status obtained from stratiﬁ ed popula-

tions is detailed as follows.

6.2.2.1 Caries Status of 5-Year-Olds

The 5-year-olds have a caries prevalence rate of

66.0 % in the primary dentition. Children who

live in the cities have a slightly lower caries prev-

alence rate (62.0 %), while in the rural areas,

children have a higher caries prevalence rate

(70.2 %).

Average DMFT of the 5-year-olds is 3.5,

while most of these children have a DMFT of 2,

accounting for 11.1 % of the total subsample.

The constitution of DMFT indices can be viewed

in Fig. 6.4 , which has shown that an astonishing

proportion of carious teeth have never been

treated (96.7 %). Analysis of the frequency

distribution of DMFT in children has aroused our

attention when it is found that 79.3 % of all cari-

ous teeth concentrate in about one-third of the

examined children, who present a signiﬁ cant car-

ies index of 8.33.

Approximately half of the subsample have

their guardians (mostly parents and grandpar-

ents) completed the questionnaire. It has been

found that 13 % children began to brush their

teeth before the age of 3, and the remaining vast

majority has just begun to brush their teeth.

Around one-ﬁ fth of children don’t brush their

teeth.

There were 49 % parents aware of the ﬂ uoride

toothpaste, and 39 % of 5-year-olds use the ﬂ uori-

dated toothpaste when brushing their teeth. It

should be paid attention to that only 22 % children

visit dentists periodically. The most common rea-

sons for dental visits are acute or chronic tooth-

ache and other reasons such as dental emergency.

Multiple regression analysis was performed to

examine the main reasons for caries in children.

It has been found that urban/rural distribution,

sugar consumption before sleeping, toothbrush-

ing starting age, and oral hygiene by parents all

correlate with caries status of children.

6.2.2.2 Caries Status of 12-Year-Olds

Caries prevalence rate of the 12-year-olds is

28.9 %. No difference is observed between urban

and rural areas, and boys have a statistically sig-

niﬁ cant lower caries prevalence rate than girls

(25.4 % vs. 32.6 %).

The most often affected teeth are mandibular

ﬁ rst molars, maxillary ﬁ rst molars, and mandibu-

lar second molars. Unfortunately, 88.8 % of all

carious teeth have not been treated (Fig. 6.5 ). In

China, 1.45 % of 12-year-olds have pit and ﬁ ssure

Decayed 96.7 Missing 0.5

Filled 2.8

Fig. 6.4 Constitution of decayed, missing, and ﬁ lled

teeth in 5-year-olds in China (%)

6 Dental Caries: Disease Burden Versus Its Prevention

sealants, and there is a huge difference between

urban area and rural area (2.75 % vs. 0.19 %).

The investigated 12-year-olds have completed

the questionnaire by themselves. As this age

group is the most important age group, it has

been receiving continuous attention from the

central government. In addition to questions on

oral health-related knowledge and behavior, the

questionnaire has also included questions on den-

tal visits and self-perceived impact of oral health.

It has been found that 82 % of the 12-year- olds

investigated brush their teeth daily; however, only

28 % of them brush their teeth at least twice a day.

Ninety-three percent of the children report that

they have never used dental ﬂ oss. The percentage

of children who use ﬂ uoridated toothpaste is

46 %. Sixty-nine percent of the children have

daily consumption of sugar-containing food, such

as sweetened milk, dissert, candy, carbonated soft

drink, and sweetened fruit juice. It is satisfactory

that only a small fraction of the investigated chil-

dren smoke (boys 5.7 % and girls 0.5 %) and that

the percentage of daily consumption of cigarette

and alcohol is further reduced to 1.0 % and 0.1 %,

respectively, for boys and girls.

With regard to oral health-related knowledge,

only one-tenth of 12-year-olds understand dental

plaque as the cause for caries and periodontal dis-

eases. However, over 60 % of them have realized

that bleeding during toothbrushing is abnormal.

In the past 12 months, 21 % of 12-year-olds

have visited a dentist. However, 47 % of the total

subsample have never seen a dentist. The most

common reasons for their dental visit are acute or

chronic toothache. It is sad that only 28 % have

formed the habit of frequent dental check-ups.

6.2.2.3 Caries Status

of 35–44-Year-Olds

The 35–44-year-olds is the WHO recommended

age group for adults. It has been found that the

vast majority of this age group (88.1 %), quite

unfortunately, has been affected by caries. Not

much difference was found between the urban

and rural populations (89.1 % vs. 87.1 %).

However, women are found with statistically sig-

niﬁ cant higher caries prevalence rate than men

(91.3 % vs. 84.9 %). The constitution of the

DMFT indices is shown in Fig. 6.6 . However, the

prevalence rate for root caries is near one-third

(32.7 %), signifying bad periodontal status.

All of the investigated subgroup answered the

questionnaire. Among all the 35–44-year-olds,

89 % have reported that they brush their teeth

daily, and 35 % have reported to be brushing their

teeth at least twice a day. Better teeth brushing

habits are found in cities, women than in rural

areas and men.

On a daily basis, 27 % use toothpicks, but over

99 % do not use dental ﬂ oss. Fluoridated tooth-

paste is used in 46 % people. Daily consumption

of sugar is found in 21 % of those investigated.

There are 32 % questionnaire respondents reported

to be smokers. There is a huge gender difference in

daily alcohol consumption, while 23 % men, in

contrast to 2 % women, drink alcohol daily.

Among the 35–44-year-olds, 69 % believe

there is a need for them to receive oral treatment,

Decayed 88.8 Missing 0.6

Filled 10.6

Fig. 6.5 Constitution of decayed, missing, and ﬁ lled

teeth in 12-year-olds in China (%)

Decayed 34.0 Missing 57.6

Filled 8.4

Fig. 6.6 Constitution of decayed, missing, and ﬁ lled

teeth in 35–44-year-olds in China (%)

H. Xiao

and the percentage is found to be statistically

signiﬁ cantly higher in urban populations than

rural (73 % vs. 65 %). However, in the past 12

months, only 16 % of them have been to a den-

tist. Quite surprisingly, even at this age, there are

as many as 46 % 35–44-year-olds who have

never visited a dentist ever. For the remaining

54 %, the majority of them have attributed their

dental attendance to be because of acute or

chronic toothache or other dental situations, and

few of them have formed the habit of regular

dental check-ups.

Multiple regression analysis has revealed that

the caries status of the 35–44-year-olds is closely

related to the use of ﬂ uoride toothpaste, con-

sumption of sugar-containing foods, toothbrush-

ing frequencies, and other behavioral variables.

Those with no use of ﬂ uoridated toothpaste, with

daily consumption of sugar-containing foods,

and who are not brushing their teeth on a daily

basis manifest with high caries risk.

6.2.2.4 Caries Status

of 65–74-Year-Olds

Caries prevalence in the age group of 65–74-year-

olds is found to be very high. The prevalence rate

is as high as 98.4 %, and not much difference is

found between urban and rural populations. Also,

not much difference is discovered between both

genders. The mean DMFT score is 14.7, the

majority of which is missing due to caries

(75.3 %), and only a fraction of the iceberg is

treated (1.9 %) (Fig. 6.7 ).

Root caries is another important issue that

should not be neglected. The age group of

65–74-year-olds are found to be heavily affected

by caries in the root. Rural population have a root

caries prevalence rate of 67.2 %, higher than the

urban population (60.0 %). It is found that 66.1 %

females are affected by root caries, higher than

males (61.1 %). It is further discovered that only

as low as 1.9 % of all the carious lesions in the

root have been treated (Fig. 6.8 ).

About half of the examined subjects have

handed in the questionnaire. It has been found that

three-quarters of the questionnaire respondents

brush their teeth on a daily basis and that slightly

over one-quarter brush their teeth twice a day. This

self-oral hygiene behavior is found to be better in

cities and females. In 27 % of the questionnaire

respondents, ﬂ uoridated toothpaste is used; the

percentage is found to be higher in cities than in

rural areas. There are 26 % of the respondents who

report they use toothpick daily, but almost none of

the respondents have ever used dental ﬂ oss.

Daily sugar-containing food consumption is

found in 27 % questionnaire respondents. An

average of 27 % of the respondent smoke, and

there is a huge difference between the two gen-

ders. And it is found that among those who

smoke, 90 % of them have been smoking for

more than 20 years. Alcohol consumption is

found in 13.6 % respondents; the percentage of

male is almost six times that of the female.

Over half of the elder population believe they

are in need of oral examination and treatment;

however, only 19 % of the respondents report that

Decayed 22.8 Missing 75.3

Filled 1.9

Fig. 6.7 Constitution of decayed, missing, and ﬁ lled

teeth in 65–74-year-olds in China (%)

Decayed 98.1

Filled 1.9

Fig. 6.8 Constitution of decayed and ﬁ lled root caries in

65–74-year-olds in China (%)

6 Dental Caries: Disease Burden Versus Its Prevention

they have visited a dentist within the past 12

months. The most common reason for dental

referral is acute and chronic toothache. However,

it is unfortunate that about one-third of the all the

respondents have never been to a dentist for any

form of oral examination or treatment. For the

expenditure on dental visits, 83 % of the respon-

dents report that they are fully self-ﬁ nanced, not

to be able to ﬁ nd a source to partly cover the bill.

When we analyze the oral health impact on

daily life, it is sad to ﬁ nd that over half the respon-

dents have experienced toothache, and the major-

ity of the elders are not satisﬁ ed with their oral

hygiene conditions.

6.3 Caries Preventive Strategies

Dental caries results from a variety of contribut-

ing factors. There are three most important fac-

tors, namely, cariogenic microﬂ ora (termed as

dental plaque), carbohydrate-rich diet, and sus-

ceptible teeth. Given time, the interrelation and

interaction between these three factors will pro-

duce the chronic oral disease which brings pain

and discomfort to the majority of the population

worldwide since an early age. Therefore, effec-

tive caries-preventive strategies almost always

require removal of any or all of these contributing

factors.

There is a hierarchy of caries-preventive strat-

egies which include the following.

6.3.1 Primary Prevention

Primary prevention is the most important strategy

as it stresses and deals with combating the etio-

logical factors. Self-awareness with regard to car-

ies prevention is often highlighted in this stage.

People are motivated by a variety of means, either

with propaganda via mass media or face-to-face

educational lectures, so that they will be self-

conscious about the importance of keeping good

oral hygiene, restricting the consumption of sug-

ars and frequent dental visits.

Many countries have set up their own launches

or campaigns to deliver the concept of oral health

education. China, for example, has launched its

own national campaign for over a decade. Each

year on the “love teeth day” on September 20th,

there will be dental health educational and pro-

motional activities held across the country. There

is a unique theme every year. Usually manuals

containing educational information are distrib-

uted free of charge. In many cities where there

are dental schools or colleges, free dental check-

ups are available.

Removal of etiological factors is very impor-

tant in this stage. The main methods include den-

tal plaque control, use of ﬂ uoride, and other

preventive methods that are to be introduced in

the following section.

6.3.2 Secondary Prevention

Secondary prevention mainly refers to the early

diagnosis and treatment of lesions found to be

still at their early stages. For a long time, caries

diagnosis mainly relies heavily on the dentists

themselves who can only resort to their naked

eyes and self-practicing experiences. This has

resulted in countless mistaken diagnosis and

omission of countless lesions which had been left

not dealt with. Later, the invention of X-ray has

offered another possibility to visualize lesions

that hide from usual visual examination.

6.3.2.1 Conventional Caries Detection

Methods

Traditional diagnostic methods, such as visual

inspection, appear to have very low sensitivity

and high speciﬁ city in diagnosing caries [ 12 – 14 ].

Continuous attempts to improve the sensitivity

have been made. Ekstrand et al have proposed a

new caries scoring system as presented in

Table 6.1 . Sensitivity and speciﬁ city for detection

of dentinal lesions were found to be ranging

between 0.92 and 0.97 and 0.85 and 0.93, respec-

tively [ 15 ]. A conclusion from one study was that

although good results were obtained regarding

sensitivity and speciﬁ city as well as operator

agreement, it takes more time to learn the method.

Although improvements in visual inspection with

new scoring systems (take, ICDAS, for another

H. Xiao

example) seem promising, further clinical valida-

tion is still needed.

The use of explorers has received some dis-

pute as some believe it cannot increase diagnos-

ing accuracy while tends to damage tooth tissue

and contaminate sound areas. Loesche et al, in a

study on intraoral transmission of pathogenic

microorganisms, showed that sterile ﬁ ssures

might be inoculated by probing after previous

contact with an infected ﬁ ssure [ 16 ]. For detec-

tion of occlusal dentinal lesions, the sensitivity of

the explorer is reported to be only about 0.5–0.6.

A number of reports have demonstrated that

probing with a sharp explorer may cause damage

to newly erupted teeth or even create a cavity at

the site of a superﬁ cial carious lesion. Therefore,

its use has been questioned by several authors.

The use of ﬁ lm radiograph for caries detection

has a long history and is still the most widely

used diagnostic technique. Bitewing radiography

has been found to be useful for dentinal caries

detection on both occlusal and approximal sur-

faces. However, it has no value for occlusal

enamel caries detection and only a limited value

for approximal enamel caries detection.

6.3.2.2 Fiber-Optic Transillumination

(FOTI)

Fiber-optic transillumination (FOTI) is a widely

used caries detection adjunctive to dental profes-

sionals [ 17 – 20 ]. Its use can be traced back to the

1970s. It brings special advantages in diagnosing

caries in the proximal surfaces which are difﬁ cult

to discover with the naked eye under normal

means of illumination. In FOTI, white light from

a cold-light source is passed through a ﬁ ber to an

intraoral ﬁ ber-optic light probe that is placed on

the buccal or lingual side of the tooth. The sur-

face is examined using the transmitted light, seen

from the occlusal view. Decayed tooth structure

manifests itself as the dark shadow under illumi-

nation of ultraviolet. Demineralized areas appear

darker compared with the surrounding sound tis-

sue. The contrast between sound and carious tis-

sue is then used for detection of lesions.

FOTI has been evaluated in a number of stud-

ies for detection of posterior approximal carious

lesions and has shown low-to-good sensitivity

and good speciﬁ city. Cortes et al showed, in an

in vitro study, that a combination of FOTI and

visual inspection was useful for determination of

occlusal lesion depth.

Using the digital image of a tooth (seen from

the occlusal view during transillumination)

through computer image analysis, researchers

have attempted to improve the performance of

FOTI. This quantitative method, digitized ﬁ ber-

optic transillumination (DI-FOTI), has been eval-

uated in a few studies, and the initial results

indicate that both the sensitivity and speciﬁ city

are very high. However, this method needs to be

developed further before it can be applied in clin-

ical situations.

6.3.2.3 New Caries Detection Methods

Till now, many new and high-tech diagnosing

technologies have become available, providing

new possibilities in caries diagnosis at its earliest

stages. Moreover, caries can not only be detected

but also can be quantiﬁ ed. This has very impor-

tant implications as thanks to these high technol-

ogies, longitudinal quantitative monitoring of

carious lesions is now a reality.

New Caries Detection Methods: Optically

Based Light interacts with the dental hard tis-

sues in different ways. It can be either reﬂ ected,

scattered, transmitted, or absorbed. The different

phenomena can occur alone or in combination.

Table 6.1 Detailed criteria for visual inspection of

occlusal surfaces introduced by Ekstrand et al

Classiﬁ cation Visual inspection

0 No or slight change in enamel

translucency after prolonged air

drying (>5 s)

1 Opacity or discoloration hardly

visible on wet surface but distinctly

visible after air drying

2 Opacity or discoloration distinctly

visible without air drying

3 Localized enamel breakdown in

opaque or discolored enamel and/or

grayish discoloration from the

underlying dentin

4 Cavitation in opaque or discolored

enamel exposing the dentin

6 Dental Caries: Disease Burden Versus Its Prevention

100

A possible consequence of absorption is ﬂ uo-

rescence, in which electrons of a lower-energy

status are moved to a higher status. When they

fall back to the original level, energy is emitted in

the form of light, called ﬂ uorescence. In other

words, ﬂ uorescence occurs as a result of the

interaction of electromagnetic radiation with

molecules in the tissue.

The cause of enamel ﬂ uorescence is still

unclear. Most of the ﬂ uorescence is induced by

organic components, proteinic chromophores, but

some can be probably attributable to apatite. It has

been proposed that ﬂ uorescence in dentin is

caused by inorganic complexes, as well as some

organic components. In sound enamel, the path

lengths are long so that there is a high probability

that the photons will hit a chromophore. Thus,

ﬂ uorescence is relatively intense. Demineralization

of dental hard tissue will result in the loss of auto-

ﬂ uorescence (the natural ﬂ uorescence).

The amount of autoﬂ uorescence loss can be

measured by devices to quantify the severity of

tooth decay. Some of these high-tech caries

detection devices are introduced below.

DIAGNOdent DIAGNOdent is developed based

on the ﬁ nding that natural tooth structure will

absorb the 655 nm wavelength light, while

decayed tooth structure produces ﬂ uorescence in

the near-infrared spectrum [ 21 – 23 ]. Intensity of

the near-infrared ﬂ uorescence reﬂ ects the extent

of tooth decay. By comparing the numerical

value of the test site with that of the comparison

site (usually the same location at the contralateral

tooth in the same dentition), one is able to tell

whether the site of examination is diseased or not

and at what severity is the disease. This device is

very sensitive so that it can be used to analyze the

initial stage of caries while causing no damage to

the tooth structure or pain to the patient.

It has been found to be able to help identify

the most caries-susceptible site of pits and ﬁ s-

sures on fully erupted ﬁ rst permanent molars in

both caries-active and caries-free children, thus

providing helpful information to decide cost-

effective, targeted prevention for pediatricians.

The version called DIAGNOdent (Kavo,

Biberach, Germany) is designed to detect occlusal

and smooth surface caries. A new device, named

the DIAGNOdent pen (Kavo), has recently been

developed for the use in approximal and occlusal

surfaces, which has the same mode of function-

ing. Several in vitro and in vivo studies have been

conducted to evaluate the devices. In a study by

Lussi et al, good to excellent sensitivity and

excellent reproducibility were reported.

In recent years, a new approach has been pro-

posed to improve the performance of

DIAGNOdent in detecting early caries lesions by

using ﬂ uorescent dyes. This proposed approach

has been tested by Alencar CJ et al, who had used

a ﬂ uorescent dye (tetrakis N-methylpyridyl por-

phyrin, TMPyP) to enhance the performance of

DIAGNOdent and DIAGNOdent pen. Mineral

loss was determined with gold standard method.

Correlation was observed between the amount of

mineral loss and DIAGNOdent measurements.

They found that DIAGNOdent and DIAGNOdent

pen are capable of identifying demineralization

around brackets bonded with resin-modiﬁ ed

glass ionomer cements. They also found that the

DIAGNOdent pen associated with TMPyP was

more capable of identifying this difference in

mineral loss as well as the gold standard method.

Quantitative Light-Induced Fluorescence (QLF)

Quantitative light-induced ﬂ uorescence (QLF)

also detects caries lesions based on ﬂ uorescence

ﬁ ndings (laser-induced autoﬂ uorescence of natu-

ral tooth structure) [ 24 – 28 ]. The tooth is illumi-

nated by a broad beam of blue- green light which

is transported through a liquid- ﬁ lled light guide.

The source of light illumination can be either an

argon + ion laser, producing diffuse monochro-

matic light at a wavelength of 488 nm, or blue

light from a 50 W xenon microdischarge arc

lamp, which has an optical band- pass ﬁ lter with a

peak intensity of 370 nm. Using a color CCD

video camera and a frame-grabber, ﬂ uorescent-

ﬁ ltered images can be captured and stored in a

computer.

Demineralized areas appear as dark spots

viewed by QLF. Software (Inspektor Research

Systems BV, Amsterdam, the Netherlands) can be

customized to analyze the collected images. After

calculating the loss of ﬂ uorescence which

H. Xiao

101

indicates the severity of a lesion, three measures

are given: lesion area (mm

), ∆F (average change

in ﬂ uorescence, in %), and ∆Q (area*∆F).

Therefore, compared with other caries detection

methods, QLF has a huge advantage in direct

visual demonstration for patients. In addition,

countless studies have conﬁ rmed its accuracy and

reproducibility. Intra-class correlation coefﬁ cients

(ICCs) for each QLF metric were found to be high

with intra-examiner ∆Q 0.91, ∆F 0.80, and area

0.92, according to a study of 91 in vivo samples.

It can be used to reliably monitor the caries status

of an individual over time. However, it seems to

be limited to a lesion depth of about 400 μm.

To now, the QLF method has been found to be

more suitable for smooth surface caries examina-

tions. The possibility of adapting it for occlusal

caries diagnosis as well as secondary caries is

still under development. However, it has already

been employed in a number of clinical studies

which evaluated the anticaries efﬁ cacy of various

kinds of oral hygiene maintaining products such

as toothpaste, mouth rinse, etc.

It has been found that longitudinal measure-

ments of QLF could detect differences in remin-

eralization of early enamel caries on buccal

surfaces of anterior teeth following supervised

daily brushing with sodium ﬂ uoride (NaF;

1,450 ppm F), sodium monoﬂ uorophosphate

(MFP; 1,450 ppm F) dentifrices, or a herbal, non-

ﬂ uoride placebo dentifrice.

New Caries Detection Methods: Electrical

Impedance Based

Electrical caries monitor

(ECM) is developed on the basis of analyzing the

conductivity of tooth structures. Natural enamel

is no good conductor for electricity. However,

under diseased condition, its conductivity is

greatly increased because of expanded intercrys-

tal space and ﬂ uid content inside. The more

demineralized the tissue, the lower the resistance

becomes. Then, by measuring the electric con-

ductivity of the test site and comparing it with

ﬁ ndings of the contralateral or adjacent tooth in

the same dentition, the extent of the carious

lesion can be derived [ 29 ].

Measurements can be performed by closing a

circuit of a very weak alternating current through

the patient. A probe is placed on the site that is to

be measured, an earth-unit is held in the patient’s

hand, and both are connected with the device by

a cord. During examination, the measuring site

should be isolated from saliva. When a tooth sur-

face is of interest, the probe tip can be placed in

an electrolyte which covers the surface.

This equipment is mainly devised to detect

caries lesions at approximal sites of teeth. In a

number of in vitro and in vivo studies, the

reported sensitivity for ECM in diagnosing den-

tinal carious lesions of permanent premolar and

molar teeth ranges from 0.67 to 0.96. And the

speciﬁ city ranges from 0.71 to 0.98, which could

be regarded as acceptable.

However, under some circumstances, false-

positive or false-negative readings may be

recorded. These circumstances include examined

teeth which have only erupted into the oral cavity

for less than 6 months, excessive dehydration of

the adjacent or other reference sites, and occlusal

surface with too complex morphology.

Temperature variations may also inﬂ uence the

outcome of the measurements in various ways.

Other high-tech caries detection technologies

include electrochemical impedance spectroscopy

(EIS), optical coherence tomography (OCT), and

near-infrared (NIR) technology. These technolo-

gies are less frequently used in recent times, and

further studies are still ongoing.

At the same time, latest inventions and break-

throughs in new dental material have tremen-

dously reshaped the conception of dental

treatment for carious lesions. Tooth structures are

no longer subjected to unnecessary damages in

order to compromise for a more stable restora-

tion. In this way, tooth is much better protected

than ever before. All those new possibilities are

unimaginable without the forthcoming of adhe-

sive dental materials.

6.3.3 Tertiary Prevention

Tertiary prevention is a last-resort strategy which

is not intended to be utilized but has to be turned

to when caries at late stages are discovered. At

this stage, when dental caries already come into

6 Dental Caries: Disease Burden Versus Its Prevention

102

being, all that should be done is to prevent the

undesirable complications it will induce if not

dealt with. Among those possible and often clini-

cally observed complications are acute or chronic

pulpitis, alveolar abscess, periapical inﬂ amma-

tion, alveolar osteomyelitis, and a number of other

less-frequently encountered caries derivatives.

When things become worse, damage to the

integrity of dentition occurs as a result. In this

case, prosthetic dentistry is needed to restore a

full dentition and maintain proper masticatory

functions.

6.4 Methods for Caries

Prevention

6.4.1 Dental Plaque Control

Dental plaque control refers to maintaining the

amount of plaque accumulation on tooth surfaces

at a reasonable level which does not allow the ini-

tiation and progression of carious lesions.

Theoretically, caries can be prevented through

perfect oral hygiene. Usually, dental plaque con-

trol is performed by individuals at home. Under

some circumstances, it is performed by a hygien-

ist or dentist as indicated necessary.

Chemical removal of dental plaque can be

achieved by administration of mouth rinses at dif-

ferent frequencies. This often involves different

kinds of key effective chemical components in

the mouth rinse solutions, such as triclosan,

chlorhexidine, essential oil, benzethonium chem-

icals, and many others. However, there are unde-

sirable consequences as microbes can start to

tolerate the effects of these chemicals given

enough time. Therefore, removal of dental plaque

with chemicals can only be used as an adjunctive

to mechanical methods or used as a substitute for

it when mechanical removal is not possible due to

some reasons.

Many mechanical methods can be employed

for this purpose. Among those, toothbrushing is

the most frequently advocated and most widely

exercised [

30 – 32 ]. However, the efﬁ ciency of

toothbrushing is not always satisfactory, when

the level of which is measured by calculating the

plaque removal rates. Therefore, what is to be

stressed not only involves stressing the adequate

frequency of daily toothbrushing and its duration

of time but also includes the correct toothbrush-

ing methods. However very unfortunately, people

seem not to pay enough attention to this fact that

only by performing correct toothbrushing meth-

ods can they effectively remove the maximum

amount of dental plaque. Moreover, brushing the

teeth in a wrong way can also bring about unde-

sirable consequences, such as multiple wedge-

shaped defects, posing potential danger in pulp

exposure or even tooth fracture.

There are a number of toothbrushing methods

to choose, and each method has its own focus.

Horizontal vibrating method (also known as the

Bass method) stresses the removal of plaque in

the gingival sulcus and the interproximal spaces.

However, this method is very much time-

consuming and difﬁ cult to exercise for most peo-

ple, because it demands dynamic equilibrium of

direction and force. Another method to be sug-

gested is reported by Fones. It mainly involves

repeated rotary movements of the toothbrush

between the maxillary and mandibular dentition.

This method is so easy to conquer that it can even

be mastered by children and adolescents to per-

form effective self-oral hygiene.

Despite the brushing technique, the tooth-

brush itself is to be carefully selected. Now there

are different kinds of toothbrushes in the market.

Each kind serves a targeted subpopulation of a

certain age or of particular physical characteris-

tics. Some toothbrushes are specially designed

for different subgroups, such as children or the

disabled who require a toothbrush to have a

smaller head and less-likely-to-slip handle. For

the majority however, a regular-design tooth-

brush with soft round-end bristles is well enough

to maintain everyday oral hygiene.

In addition, electronic toothbrushes are

invented to better serve the disabled and the elder.

Toothbrushing movements of different groups of

bristles have been preprogrammed, and these

movements are often very complex which include

different rhythms of vibration and rotation in all

directions. Although numerous clinical studies

have revealed no statistically signiﬁ cant

H. Xiao

103

differences in the cleaning ability between an

ordinary toothbrush that is powered by hand and

one that is powered by electricity, the target group

are greatly facilitated in maintaining good oral

hygiene.

However, the toothbrush fails to reach some

remote parts of the oral cavity. Therefore, addi-

tional accessories can be utilized to perform the

cleaning. Among the many choices, there are

dental ﬂ oss, toothpicks, interdental brushes, and

electrical dental syringe. Dental ﬂ oss, either

waxed or not, can effectively help remove dental

plaque on interproximal surfaces.

Toothpicks can be used to remove food debris;

however, care is needed so that it won’t hurt the

adjacent soft tissue. For some subgroup of people

who have lost the protection from gingival papil-

lae, e.g., in the case of periodontitis or attachment

loss therefore leaving the individuals at greater

danger of caries attack, interdental brushes can

be used to clean those places.

Electrical dental syringe can produce high-

speed projection of water which contains rich

foam and can remove food debris for hard-to-

reach places, such as the surface under the orth-

odontic wires or partial removable prosthetic

denture.

6.4.2 Restriction on Sugar

Consumption and Use

of Sucrose Substitute

Oral bacteria, especially the species capable of

acid production and acid toleration, metabolize

sugar contained in food debris to get energy to

survive. This process, which often takes place in

deep part of dental plaque where oxygen concen-

tration is much lower than the superﬁ cial part,

produces acid. This metabolism end-product will

accumulate inside dental microﬂ ora and demin-

eralize enamel crystals if the dental plaque is not

wiped away and remain on the tooth surface for a

long time.

Among all kinds of sugar-containing food

(food that contains glucose, fructose, maltose,

lactose, and many others), sucrose is found to be

the most dangerous in causing dental decay. A lot

of studies, both in vivo and in vitro, have proved

that consumption of sucrose-rich diet will result

in the unbalance of oral microbial micro-ecology

where cariogenic species will tremendously out-

number those non-cariogenic species. Therefore,

the initiation of dental decay will continue to

progress once started. Therefore, restricting the

consumption of such cariogenic-potent sugars is

an important caries-preventive strategy. Such

restriction can be applied to either the amount of

consumption or the frequency of consumption or

both.

However, despite of its undesirable carioge-

nicity, sugar is very important for the general

health of human as a vital source of energy and

nutrition. Therefore, it is very difﬁ cult to com-

pletely replace it by other means. Then it comes

the advocacy of the use of sugar, especially

sucrose, substitutes. These sugar substitutes are

sugars in nature but have been found to be hard

for dental microﬂ ora to use or will produce very

limited amount of acid even if some species have

managed to metabolize them. This special group

of sugars includes those with very high sweetness

such as aspartame, benzoic imine, cyclamate, and

stevia sugar and those with low sweetness such as

xylitol, sorbitol, mannitol, maltose, maltulose,

and many others. Recently, these sugar substi-

tutes have also entered the market for the general

public to choose, where xylitol-containing prod-

ucts (mainly in forms of chewing gum or soft

drinks) dominate.

Also in recent years, studies on isomaltitol are

very hot. Isomaltitol is a mixture of α-d -

glucopyranosyl-1,6- d -sorbitol (GPS) and α-d -

glucopyranosyl-1,1- d -sorbitol (GPM) which are

mixed at a mol ratio of 1:1, which has been found

to be able to hydrolyze into glucose, sorbitol, and

mannitol and is hard to be metabolized by cario-

genic bacteria. Hopefully in the future, it might

bring us a new choice of sugar substitute.

6.4.3 Reinforce Tooth Resistance

to Acid

One important aspect as well as an important pre-

requisite for tooth resistance to acid is that ﬁ rst of

6 Dental Caries: Disease Burden Versus Its Prevention

104

all a tooth must have natural anatomy and proper

amount of inorganic and organic components.

Therefore, before any individual is born, the

proper oral health of the mother is of immense

signiﬁ cance. Clinical observations and clinical

studies have revealed the causal relationship

between gingivitis and periodontitis of the mother

and premature delivery as well as low birth

weight, both of which will have substantial

impact on the tooth formation and maturation for

both the primary and permanent dentitions. Also,

malnutrition of the mother will inﬂ uence such

critical tooth formation process of the child

adversely.

After birth, the ﬁ rst few years serves as the

most pivotal stages for a natural dentition to erupt

into the oral cavity gradually and develop various

physical functions of the oral cavity as well as the

masticatory system. However, in such develop-

mental stage, the permanent dentition is still

under formation, while the primary dentition

remains very vulnerable to local contributing fac-

tors to dental decay. Therefore, maintenance of

proper oral hygiene and good general nutrition is

the only way leading to a healthy full permanent

dentition that will manifest itself years later (usu-

ally at the age of 12) and serve the individual life-

long (under ideal conditions).

However, the other side of the story must not

be neglected. Normal anatomy and composition

are no guarantee of the lifelong service of the

dentition. Local contributing factors, including

cariogenic bacteria and sugar-containing food

consumption, exist in the oral cavity continu-

ously. Weapons we have against the war of dental

decay can be a variety of choices other than sim-

ple personal oral hygiene maintenance. Alongside

with regular dental check-ups, ﬂ uoride in its

various forms can provide potent protection to

our teeth.

From the time of its discovery many years

ago, when it is found to be existing in the drink-

ing water which led to mottled enamel, Dr. Dean

had noticed the reverse relationship between its

existence in drinking water and reduced rate of

caries. Further laboratory studies have convinced

such underlying mechanism. Fluoride is proven

to be able to reduce enamel solubility in acid

solutions because it is able to react with tooth

crystals (mainly exist in the form of hydroxyapa-

tite, HA) and form ﬂ uorohydroxyapatite (FHA)

or ﬂ uorapatite (FA), which are very resistant to

acid. At the same time, ﬂ uoride is able to sup-

press bacterial metabolism of carbohydrates;

therefore, the production of lactic acid is greatly

reduced.

Since the discovery of this potent anticaries

chemical, its various forms have been tested and

proven to be suitable for clinical use. Many dif-

ferent kinds of ﬂ uoride are used worldwide,

among which most frequently used includes

sodium ﬂ uoride (NaF), calcium ﬂ uoride (CaF

sodium monoﬂ uorophosphate (SMFP), stannous

ﬂ uoride (SnF

), and silicon ﬂ uoride (SiF

Systemic and topical applications of ﬂ uoride

have been widely accepted and promoted.

Systemic application of ﬂ uoride refers to addi-

tion of ﬂ uorides into various kinds of vehicles so

that the effective component ﬂ uoride ion gets into

the body and continuously secreted into the oral

cavity alongside with saliva. Such vehicles which

have come into being in the western industrial-

ized countries for many decades include water

(ﬂ uoridation of water supply), milk, and salt and

agents such as tablets or drips usually adminis-

tered by professionals. However, developing

countries, which do not have adequate resources

or infrastructure (such as water supply equip-

ment), fail to have this means of ﬂ uoride admin-

istration that will beneﬁ t the general public.

On the other hand, topical ﬂ uoride application

serves as an equally effective means of ﬂ uoride

supply. This category involves a variety of appli-

cation methods that are more feasible and ﬂ exible

than systemic ﬂ uoride administration. These

methods include ﬂ uoridated toothpaste, mouth

rinses, varnish, gel, and foams. The ﬁ rst two

methods are easy for personal use at home and

have been widely accepted as important means of

daily oral hygiene maintenance. The rest are usu-

ally applied by professionals.

Many clinical studies have shown very encour-

aging results with regard to reductions on caries

incidence rates. Two to three decades later, epide-

miological surveys have shown a steady decrease

in caries prevalence rates worldwide, indicating

H. Xiao

105

the tremendous achievements by widespread use

of ﬂ uoride in caries prevention dentistry has ever

managed to achieve.

6.4.4 Pit and Fissure Sealing

Pit and ﬁ ssure are created in the process of tooth

development. They have distinct anatomical fea-

tures that render them very much vulnerable to

caries attack. These depressions of occlusal sur-

face (together with the buccal and lingual sur-

faces of the molars) always have the bottom part

at deep enamel, enamel-dentinal junction (EDJ),

or even at the dentine. What’s worse, these nar-

row places are hard to reach with routine exami-

nation and cleaning methods.

As a result, it is not surprising that there is

almost always presence of accumulation of mul-

tispecies microbial bioﬁ lm, food debris, remains

of enamelogenic epithelium, organic plug, etc.

Therefore, pit and ﬁ ssure are most often discov-

ered as the ﬁ rst site of carious lesions of an indi-

vidual. Data of a clinical study show that about

67 % of caries lesions of children at the age of 3

are pit and ﬁ ssure caries. Another epidemiologi-

cal survey of 25,000 school children revealed that

80 % of all diagnosed carious lesions are located

at pit and ﬁ ssure.

In the 1960s, on the basis of research ﬁ ndings

on enamel etching and adhesion, pit and ﬁ ssure

sealant was invented. The underlying strategic

thinking is that if a posterior tooth erupts fully

into the oral cavity and gets all the vulnerable

caries-susceptible sites concealed by dental

material the pit and ﬁ ssure are no longer open to

the oral cavity and therefore will not be inﬂ u-

enced by the microbes that inhabit the mouth and

their dangerous acidic metabolic products. And

this invention has been proven to be a huge

success.

During the following years, the components

of pit and ﬁ ssure sealants have been reﬁ ned

greatly. Now, the most often used sealants are

resin in nature, often belonging to the Bis-GMA

system which has many advantages under clini-

cal condition, such as they are easy to use and

aggregate, and its volume shrinkage after

aggregation is small. However, in recent years

there is also some dispute about its safety.

A great number of clinical studies have veri-

ﬁ ed that pit and ﬁ ssure sealing can substantially

reduce caries onset. Systematic reviews on ran-

domized clinical trials have found similar encour-

aging results. In this sense, pit and ﬁ ssure sealing

is strongly recommended by FDI, ADA, IADR,

WHO, and many other professional organiza-

tions. And in many countries, it has already been

included as part of governmental budget for car-

ies prevention programs.

6.4.5 Preventive Resin Restoration

Preventive resin restoration is actually a variation

of pit and ﬁ ssure sealing. It involves treatment of

susceptible or early carious lesions in pit and ﬁ s-

sure (preservative cavity preparation and restora-

tion with ﬂ uid resin mainly) and combines it with

subsequent pit and ﬁ ssure sealing. This method

has adopted concepts of minimal invasive den-

tistry (MID) into caries prevention. However, its

application is restricted to early caries lesions only.

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Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_7

Clinical Management of Dental

Caries

Li Jiyao

Dental caries can be classiﬁ ed clinically as

cavitated and noncavitated lesions according to

substantial damage to tooth morphology. The

non-operative approach is usually used for non-

cavitated lesions or as a preventive measure for

susceptible teeth. For caries with substantial loss

of dental tissue, an operative measure such as res-

toration is taken. The development of material

science provides a variety of choices for dental

caries management.

Dental restoration is an operation on an organ

with unique biological properties, which involves

theoretical knowledge such as mechanics, biol-

ogy, materials science, aesthetics, etc. It is possi-

ble that the pulp–dentin complex could be

irritated or damaged in the process of treatment;

hence it is vital and important to protect the

dentin and pulp throughout the process.

7.1 The Development of Caries

Treatment Theory

At the end of nineteenth century, G.V. Black cre-

ated a dental restoration system according to the

susceptible sites and dental anatomical relation-

ship of dental caries, which involved the clinical

demands of retention and resistance, in addition

to the property of amalgam, and laid the founda-

tion for modern restoration dentistry. In the

1950s, Buonocore introduced the acid-etching

technique into restorative dentistry. Through the

acid-etching technique, resin composite was able

to bind with dental hard tissue with a mechanical

lock. The binding mechanism was different from

amalgam. From then on, restorative dentistry

entered the new age of “adhesive dentistry.” After

the twenty-ﬁ rst century, restorative dentistry

entered the era of microdentistry, by proposing

the concept of early diagnosis, prevention, and

micro treatment; the operative dentistry was

directed toward a biological approach, to prevent

the development of caries and to preserve healthy

dental tissue as far as possible [

1 ].

7.1.1 G.V. Black and Modern

Restorative Dentistry

G.V. Black made a huge contribution in forming

the concept and principles of modern dental res-

toration. At the end of the nineteenth century,

with a rigorous scientiﬁ c attitude, he carried out

L. Jiyao

Department of Operative Dentistry and Endodontics ,

West China Hospital of Stomatology ,

No. 14, 3rd Section, Renmin Road South ,

Chengdu 610041 , Sichuan Province , China

e-mail:

[email protected]

108

an in-depth investigation into the damage of car-

ies in susceptible sites and its relationship with

dental anatomy. Combining the demands of

retention and resistance required by restoration,

in addition to the property of amalgam, he cre-

ated a complete restoration system. The system

explained the corresponding principle and devel-

oped a series of basic principles and requirements

of cavity preparation, material mixing, and ﬁ ll-

ing. Restoration dentistry then embarked upon a

scientiﬁ c, standardized pathway and the founda-

tions of modern restoration dentistry were laid.

The content of restoration dentistry proposed

by G.V. Black was to prepare the cavity upon the

already weakened dental tissue or after removing

the damaged dental tissue base on the principle of

retention, resistance, and protection of the pulp–

dentin complex and then restore its original form

and function with a speciﬁ c material through a

certain process.

The knowledge of cavities is a major part of

G.V. Black's contribution to restoration dentistry.

According to the caries location, and in combina-

tion with the tooth structure, in addition to the

characteristics of the design and preparation, he

divided cavities into ﬁ ve categories, which gener-

ally covered the basic types of caries. Two more

types were added afterward to supplement them.

Clinically, this category is still being widely

used.

According to the properties of amalgam, he

also proposed a principle based on the demands

of auxiliary retention and resistance of cavity

preparation, such as the depth, contour of the

cavity, a cavity with a dovetail, a ladder-shape

retention form, the undercut, and the removal of

weakened enamel. He also proposed certain

demands for each category of cavity. The descrip-

tion of these systems laid the foundations for the

development of modern restoration.

7.1.2 Adhesive Bonding Technique

and Dental Restoration

The development of dental restoration and that of

dental material are inseparable. At the end of

the nineteenth century, G.V. Black created a

scientiﬁ c, standardized restorative system, using

mechanical retention. In 1955, for the ﬁ rst time,

Buonocore used acid to process the enamel sur-

face, to increase the bonding between composite

resin and the tooth surface and started to revolu-

tionize dental practice. As the resin material and

bonding material developed, the concept of resto-

ration had already been changed; the preservation

of more dental tissue was widely accepted as a

bonding technique.

In the past 20 years, there has been a great leap

in the development of resin composite material

and the acid-etching technique has matured.

Bonding systems for dentin have been continu-

ously updated, including total etching and self-

etching techniques and the procedure was

simpliﬁ ed as well. The clinical application of

composite resin has become increasingly wide-

spread and the effectiveness of bonding restora-

tion has also been proven because of its long-term

results.

The bonding technique revolutionized tradi-

tional dental restoration. In terms of cavity

design and preparation, it preserved more

healthy dental tissue by discarding the sacriﬁ ce

certain depth of cavity and dove tail, ladder-

shaped retention form. Combined with the the-

ory of bonding resin composite, more careful

preparation of the cavosurface angle and bevel

was required.

7.1.3 The Foundation and Principle

of Minimally Invasive Caries

Treatment

Traditional dental restoration was based on the

principle created by G.V. Black in 1908, remov-

ing a lesion by operation and then restoring the

damaged part. This approach was launched based

on the foundation of amalgam properties, which

could certainly cause vast damage of dental tis-

sue. In the twenty-ﬁ rst century, modern den-

tistry suggested a more reasonable theory, which

was minimally invasive treatment. Minimally

invasive treatment is a branch of preservation

dentistry. In the literature, terms such as mini-

mal intervention dentistry, minimally invasive

L. Jiyao

109

dentistry, and micro dentistry were used. In the

Modern Oxford Dictionary, minimally invasive

dentistry has the same meaning as microden-

tistry. MI is an abbreviation of minimal inter-

vention dentistry, but not minimally invasive

dentistry [ 2 ]. Tyas et al. believed that minimal

intervention dentistry focuses on the knowledge

of how caries develop, including early diagnosis,

prevention, and treatment, and placed emphasis

on the treatment switch from dental operation to

biological method, to prevent the development of

dental caries and preserve as much healthy den-

tal tissue as possible. Peters et al. believed that

minimally invasive dentistry focuses on the pres-

ervation of healthy dental tissue when removing

caries lesions, instead of the “extend to prevent”

principle of G.V. Black.

In terms of the biological study of dental tis-

sue, in addition to the etiology of caries, espe-

cially the process of remineralization, the

revolution of diagnosis measurement, and the

novel view of prevention, the development of

dental material has laid the foundation for mini-

mally invasive dentistry.

Martin et al. proposed four basic principles for

minimal invasive dentistry: lesion control, remin-

eralization of early caries, minimal surgical

trauma, and restoration, but not replacement, of

dental caries lesions.

Modern material science proved that dental

restoration material could not match the healthy

dental tissue in terms of physical, mechanical, and

biological properties. Removing healthy dental

tissue and restoring the cavities with traditional

ﬁ lling material certainly could not meet the func-

tional requirements. With the development of

adhesive material, in addition to the traditional

chemical dentin bonding, there was also microme-

chanical bonding in the hybrid layer. The dentin

bonding system developed from no-etching bond-

ing to total-etching bonding (1980s), total-etching

wet bonding (1990s), self-etching primer adhesive

systems (1990s–2000), and then one-step adhe-

sive systems (2000s). The micromechanical bond-

ing mechanism produced solid bonding with

dental tissue, decreased the sacriﬁ ce of healthy

dental tissue to obtain sufﬁ cient retention, and

decreased the chance of microleakage.

In the past, the process from the demineraliza-

tion of dental hard tissue to the degradation of

dentin collagen and ﬁ nally the formation of cav-

ity was generally considered irreversible. As a

matter of fact, the process of dental tissue gain-

ing and losing calcium and phosphate ions was

carried out alternately; i.e., a demineralization–

remineralization cycle on the surface of the

tooth. When the pH of the interface between den-

tal tissue and plaque was below 5.5, it showed

that the enamel and dentin demineralized. As the

pH recovered, it showed remineralization.

Fluorine played an important role in this cycle. It

could strengthen the absorption of calcium and

phosphate and form ﬂ uorapatite on the tooth sur-

face. Fluorapatite is highly acid-resistant; demin-

eralization starts at pH < 4.5, but when in

ﬂ uorine- containing environments, remineraliza-

tion of the tooth and anticarious ability are

greatly enhanced.

For now, the method of diagnosis for detect-

ing the different statuses of caries, such as early

demineralization, small caries lesions, and small

cavity were varied, including bacterial counts,

saliva buffering capacity, ﬁ ber-optic transillu-

mination technology, electrical impedance tech-

nology, laser ﬂ uorescence technique, nonvisible

light imaging technology, etc. These techniques

were particularly used for diagnosing hidden

caries, early caries, proximal surface caries, and

caries in pits and ﬁ ssures, which greatly

enhanced the sensitivity and accuracy of early

diagnosis.

The new idea about prevention has high-

lighted the importance of the interaction

between doctors and patients when a dental

operation switches to nonsurgical treatment. It

is widely believed and accepted that caries are

initially reversible, mainly caused by bacterial

infection, and constitute a multi-factorial

chronic disease. Treatment from the doctor is

merely a small part of the complete treatment, to

fulﬁ ll caries prevention; long- term cooperation

between doctors and patients must be built to

achieve that.

New techniques of prevention include enamel

angioplasty, pit and ﬁ ssure sealant, and preventive

resin restorations; the purpose is to retain as

7 Clinical Management of Dental Caries

110

much of the healthy tooth structure as possible

and change the conditions for the development of

dental caries, thereby reducing the incidence of

dental caries.

Modern caries treatment pays more attention

to the biological response of the pulp–dentin

complex. At the same time, the relationship

between the restored tooth and periodontal

health, between occlusion and periodontal health,

and the proximal contact between the prosthetic

and the adjacent teeth also needs to be consid-

ered. Minimizing patients’ anxiety and pain

caused by the fear of dental treatment should also

be considered.

Atraumatic restoration, sandblasting caries

removal, chemical–mechanical caries removal,

the laser treatment of dental caries, and other

new technologies, have overcome the excessive

loss of the healthy tooth structure caused by tra-

ditional dental drilling, which can easily cause

complications when near pulp, especially in the

children with an extreme fear of dental treat-

ment, and the risk of cross-infection. New tech-

niques such as amalgam bonding, tooth colored

material, composite resin inlay, use new materi-

als or technologies in cavity restorations, making

it possible to get rid of the traditional preventive

expansion during cavity preparation.[ 3 ]. The

concept of a minimally invasive dental treat-

ment plan is reﬂ ected in the following three

aspects:

1. Early diagnosis and personal treatment.

Through individual caries risk assessment

such as plaque and saliva, and evaluation of

Streptococcus mutans , in addition to early car-

ies monitoring, the establishment of personal-

ized dental ﬁ les, the enhancement of patient

management, an increase in patient communi-

cation, and improvement of patient loyalty.

2. Treatment and effective control. Based on the

diagnosis to build up prevention treatment

programs, to implement minimum interven-

tion, and prevent secondary caries, using

smart materials, to treat rather than simply

replace the damaged tissue, to maximize the

preservation of dental health.

3. Focused prevention and effective interven-

tion. Control of cariogenic bacteria, plaque

control, reduce the amount of sugar intake,

and improve salivary function. Explain to

patients the causes of dental caries, correct-

ing the patient’s poor eating habits and oral

hygiene habits, guide patients to beneﬁ t from

professional dental methods, and to control

the demineralization and promote remineral-

ization. Implement the concept of profes-

sional dental care and early caries

treatment.

Minimally invasive dental operation process:

patient visits; diagnosis through treatment plan-

ning system, assess caries risk, prevent caries

activity through the history of dental caries, gen-

eral oral condition, and to conﬁ rm treatment the

plan by performing observation, prevention, or

treatment.

Lesion control is a prerequisite for successful

remineralization and ﬁ lling. To control lesions,

the presence of pathogens ﬁ rst needs to be con-

trolled. At the early stage of the lesion, through

the effective use of modern diagnostic techniques

and prevention systems to control bacteria before

irreversible loss of dental tissue, thus preventing

the development of dental caries. For patients at a

high risk of caries, the use of mouthwash, a

change of bad oral habits, eating habits, etc. will

be needed to control bacteria or adjust the pH of

the saliva, ﬂ ow, and viscosity to achieve the goal

of caries prevention.

Minimal surgical intervention is the most

effective method of restoration of tooth structure.

When caries progress to dentin, the enamel layer

forms cavities. Plaque accumulates in the cavi-

ties, which affects calcium, phosphorus, ﬂ uoride

ion uptake, and is difﬁ cult to remove.

Remineralization repair is then unlikely to suc-

ceed, and only by the use of dental surgical meth-

ods. The cavity can be roughly divided into two

layers from outside to inside:

1. Infected layer: this layer of the tooth structure

has been completely denatured and bacteria

settled.

2. Demineralized layer: this layer has a certain

level of demineralization, but the collagen scaf-

fold still exists and can be re-mineralized. In

the past it was thought that the demineralized

L. Jiyao

111

layer should be removed, but recent studies

suggest that the demineralized layer is a pre-

carious status instead of caries-active status.

With the promotion of mineralized material to

repair, this layer can be remineralized.

Therefore, the modern view is that the removal

of diseased tooth structure should be limited to

the infected layer.

Based on the understanding regarding the

early diagnosis of dental caries, focused pre-

vention, and minimally invasive treatment, the

twenty-ﬁ rst century has ushered in the era of

the minimally invasive treatment of dental

caries.

7.2 Current Management

of Dental Caries and Its

Development

Under the inﬂ uence of minimally invasive den-

tistry, dental caries treatment has shifted from

dental surgery to biological treatment.

According to the progressive stages of decay,

the development in the treatment of dental car-

ies focuses on the prevention and management

of people susceptible to dental caries, nonsurgi-

cal treatment at an early stage, and the surgical

treatment of the cavity. Accordingly, dental car-

ies treatment technology has also developed

considerably. The clinical application of the

minimally invasive treatment of dental caries

and the systematic evaluation of the effect of

composite resin restoration has provided the

foundation for the application of new technolo-

gies and materials. Current dental caries treat-

ment should be an optimized treatment regime,

using the best technology to achieve the best

treatment outcomes.

7.2.1 Minimally Invasive Treatment

Technique

The minimally invasive treatment of dental caries

emphasizes the preservation of as much of the

tooth structure as possible, keeping surgical

intervention to a minimum. Broadly speaking, all

of the caries treatment techniques should

emphasize a conservative approach. The follow-

ing brieﬂ y summarizes the minimally invasive

treatment techniques; the nonsurgical treatment

technologies are also included. These techniques

are mainly used in the early stages where dental

caries have not yet developed, in people suscep-

tible to dental caries, and in the prevention of car-

ies in sensitive areas.

7.2.2 Minimally Invasive Cavity

Preparation

7.2.2.1 Nonmachinery Preparation

Air Abrasion Air abrasion was ﬁ rst introduced

in 1945 with the ﬁ rst air abrasion machinery

available in 1951. The air abrasion system has

evolved greatly since then. The principle of air

abrasion is to apply highly pressurized, non-

toxic particles, such as aluminum oxide ions, to

accurately remove the enamel, dentin, carious

tissue, and old ﬁ llings. Air abrasion can par-

tially replace the high-velocity gas turbine cav-

ity preparation. It is quieter, more time- and

energy-efﬁ cient, and requires no anesthesia as it

does not produce vibration and heat. It is well

received by patients and maximizes the conser-

vation of the tooth structure. The interior of the

prepared cavity is smooth, making it easier to

ﬁ ll. It reduces the stress between the ﬁ lling and

the tooth structure, reduces the likelihood of

microfracturing, hence prolonging the life of the

ﬁ xture.

To aid early the detection of caries on the

occlusal surface, the blackened part of the cav-

ity should be examined. After visual examina-

tion, the air abrasion system aims strong

abrasive particles toward the affected areas in

the cavity. If the blackened area is a stain, it can

be removed with ease. If it is accompanied by

decay, a beam of strong abrasive particles can

reveal and blast the stain away in addition to the

decay. As dentin is moist, thick, and elastic, the

particles bind on its surface rendering it unable

to exert its effect. Hence, it should be removed

manually or using a mechanical machine before

using air abrasion.

7 Clinical Management of Dental Caries

112

The disadvantage of this method is that

because it is easier to remove dentin than enamel,

it causes the overhang of enamel, which requires

trimming of the enamel with the drill.

Contraindications to air abrasion include patients

with:

1. A severe allergy to dust, asthma, and chronic

obstructive pulmonary disease.

2. Open wound or recent tooth extraction.

3. Active periodontal disease.

4. Recent placement of an orthodontic

appliance

5. Subgingival caries

Air Polishing Air polishing delivers high-

pressure jet of sodium bicarbonate to the surface

of the tooth, producing a cutting effect. Air pol-

ishing differs by using an aqueous friction solu-

tion, which does not cause a signiﬁ cant amount

of sodium bicarbonate aerosol. This technique

was originally designed for stain removal and it is

now also used to remove crown ﬁ llings. Air pol-

ishing is not very selective when cutting the tooth

structure and can damage healthy dentin and

cementum. It is mainly used in the ﬁ nal prepara-

tion of the caries to remove any remaining

decayed dentin.

Lasers Lasers can be used to perform surgeries

on dental soft tissues as they do not cut through

dental hard tissues. Hence, surgeries involving

hard tissues need to employ other types of lasers.

The ideal laser should be able to manage both

dental hard and soft tissues. Clinically used lasers

that can cut through dental hard tissues include

erbium:yttrium-aluminum-garnet (Er:YAG), car-

bon dioxide (CO

), neodymium:yttrium-

aluminum- garnet (Nd:YAG), and Ar:F. They all

have selective abrasive properties whilst conserv-

ing healthy tooth tissue. Er:YAG is the most

selective of all lasers. Laser cavity preparation is

precise, nonvibrating, has no smell, and does not

require anesthetics. As lasers can seal dentinal

tubules, they can also prevent hypersensitivity

postoperatively. On the downside, the machinery

is bulky and expensive, thus limiting its role in

clinical practice.

Chemomechanical Caries Removal

Chemomechanical caries removal (CMCR) uses

chemical agents to soften the dental tissues

before eliminating infected tissue using machin-

ery. In 1985, the ﬁ rst CMCR system, Caridex,

was introduced into dentistry. Caridex involves

the intermittent application of pre-heated

N-chlorinated-DL-2-amino butyric acid

(GL-101E) into the cavities. This solution causes

the partial disintegration of the collagen in the

cavity, accelerating the removal of dental caries.

The uptake of this technology was not great as it

is expensive, time-consuming, and requires a lot

of additional equipment, including memory cell,

a heater, pump, and special hand piece.

Recently, CMCR has been re-introduced to

the dental industry, providing a new alternative to

caries removal and preparation. A new CMCR

system, Carisolv was introduced – Carisolv gel

and Carisolv hand tools. The Carisolv reagent

consists of two component mixtures. One of the

component mixtures is a red gel composed of

leucine, lysine, glutamic, and sodium hypochlo-

rite. After applying the mixed Carisolv reagent to

the cavity, a hand tool can be used to remove the

softened carious tissue. This method can selec-

tively dissolve carious tissue quickly (around

30s), whilst not affecting any healthy dentin.

Compared with other caries removal techniques,

CMCR can effectively remove the smear layer of

the cavity, reinforce the bond between the ﬁ lling

and the tooth, there is no noise, vibration or anes-

thetics, and patient acceptance is high. However,

when compared with the high-velocity turbine,

the operating time is longer and requires alterna-

tive methods to gain access to and repair some

undermining caries.

The CMCR procedure can handle most dental

caries. It can be used alone or combined with

other traditional caries removal techniques. In

those caries that cannot be directly accessed or

have existing ﬁ llings, CMCR can be used in con-

junction with a dental drill to gain access to the

cavity area or remove the old ﬁ lling, before using

Carisolv to remove the caries.

The CMCR method should be ﬁ rst considered

for the following patient group: root/cervical

caries, coronal caries (especially deep coronal

L. Jiyao

113

caries), caries located on the edge of the crown or

bridge abutment, completion of canal prepara-

tion, those in whom anesthetic is contraindicated,

especially needle-phobic patients, those with a

dental phobia, and pediatric patients.

7.2.2.2 Mechanical Rotary Technique

The mechanical rotary technique uses high-

velocity turbine hand tools to prepare dental cavi-

ties. To maximize healthy tooth conservation, burs

in proper size are chosen in practice in relation to

the principles of minimally invasive dentistry.

Tunnel Preparation Tunnel preparation refers

to the occurrence of caries located on the proxi-

mal surface of the teeth, if the lesion is more than

2.5 mm apart from the marginal ridge, cavity

preparation can enter from the occlusal surface,

to maintain the integrity of the marginal ridge but

it is not prepared as the classical proximal–occlu-

sal cavity. If the tooth surface that is adjacent to

the lesion has demineralized but not actually

damaged, and it is necessary to maintain the

integrity of its surface, this is also called internal

preparation. Advantages of this method are:

1. The marginal ridge of the tooth can be kept

intact, thereby enhancing the resistance of the

remaining tooth structure.

2. Injury of the proximal surface of the adjacent

tooth is avoided during cavity preparation.

3. Normal contact relationship with the adjacent

tooth is maintained.

4. Overhanging of repair materials is prevented.

Slot Preparation

Slot preparation, also known

as mini box preparation, is designed mainly for

proximal caries. It can be divided into the follow-

ing cases:

1. Caries is close to the marginal ridge and can-

not preserve its integrity or the marginal ridge

has been destroyed.

2. Caries is located underneath the interproximal

contact. Designing of cavity preparation

focuses on carious tissue; if the marginal ridge

cannot be preserved, the cavity is only pre-

pared into a box shape without a dovetail.

When the caries is underneath the interproxi-

mal contact, carious tissue is approached from

the buccal or lingual surface, and prepared

into a box or disk shape with the auxiliary

help of groove retention. Then glass ionomer

or composite resin is used for restoration. In

most cases, this method can provide normal

interproximal contact between adjacent teeth.

Microscopic Preparation Techniques

To mini-

mize invasive trauma, the technique of using

micro drills for microscopic preparation, under

the microscope or loupe, to obtain precise tooth

preparation is called “microscopic dentistry.”

Micro drills can be round, oval, and conical, and

the operator can select different drills for ﬁ ssure

caries preparation and ﬁ nishing. The shank of the

burs is longer than that of traditional ones; thus,

the operator’s sight is not blocked by the head of

the hand piece. Using this drill together with

tooth-colored bonding materials for repairs can

better reﬂ ect the purpose of minimally invasive

treatment.

7.2.2.3 Minimal Invasive Prevention

Technique

Pit and Fissure Sealing Pit and ﬁ ssure sealing

is an effective method of preventing ﬁ ssure car-

ies. The occlusal surface of the tooth formed a

different shape and varying depths of the ﬁ ssure

during development, and oral bacteria, metabo-

lites, and food residue often accumulate at caries

predilection sites. Pit and ﬁ ssure sealants may

isolate the ﬁ ssure and the oral environment, pre-

venting bacteria and food debris from entering,

to achieve the purpose of caries prevention.

Fluoride sealants can continuously release ﬂ uo-

ride, provide barriers, and promote

remineralization.

Pit and ﬁ ssure sealing is mainly used for suspi-

cious ﬁ ssure and pit caries as well as deep grooves

adjacent to ﬁ lled ﬁ ssure caries on the occlusal

surface. Sealant is mainly made of resin, a dilu-

ent, an initiator, and a number of auxiliary com-

ponents, such as ﬁ llers, ﬂ uoride, dyes, and other

components. The main body of sealant resin

material, bisphenol-A-glycidyl methacrylate

7 Clinical Management of Dental Caries

114

(Bis-GMA), is a commonly used resin with good

performance.

Enameloplasty Noncaries ﬁ ssures were drilled

out to form a shallow plate shape, making it easy

to clean and keep free from caries, i.e., enamelo-

plasty. This method removed minimal tooth

structure, but produced lasting anti-caries effects,

and no worries regarding fallen sealer. A simple

operation, with few technical requirements. The

suitable depth of the ﬁ ssure is no more than half

the enamel thickness. Using a high speed hand

piece with a pear-shaped diamond, ball dia-

monds, or a sand stone point bur the site should

be gently removed. For the grooves on the cusp

surface, a diamond bur can be used to remove

evenly parallel to the surface, until the groove is

no longer brown. Enameloplasty can be used on

ﬁ ssures that are close to or across the molar buc-

cal lingual ridge. The prepared cavity can be

extended to 2 mm away from the buccal–occlusal

junction or lingual–occlusal junction, and the rest

can undergo enameloplasty, but not following the

G.V .Black principle, which extended to the buc-

cal and lingual surface.

Preventive Resin Restorations In 1977,

Simonsen suggested performing “preventive

resin restorations” to treat suspicious ﬁ ssure car-

ies and provides a new approach to the treatment

of ﬁ ssure caries. Preventive resin restorations

only remove the infected enamel or dentin at the

lesions, according to the size of the caries, using

etching technology and the resin material ﬁ lling

up the early ﬁ ssure caries, and the occlusal sur-

face coated with sealant. It is a preventive mea-

sures combined with pits and ﬁ ssure sealing and

ﬁ ssure caries ﬁ lling. Because it does not use the

traditional extension for prevention, only a

amount of carious tissue is removed and restored

with composite resin or glass ionomer, the pit and

ﬁ ssure caries without caries is protected by the

sealant, thus preserving more healthy dental tis-

sue, and is an effective method for preventing the

further development of caries.

The advantage of preventive resin restorations

is using glass ionomer composite resin as ﬁ lling

and binding with enamel mechanically or

chemically, then bonding with sealant by

chemical bonding reduces the possibility of

generating micro-leakage.

Caries Pharmacotherapy Caries pharmaco-

therapy is the use of drug treatment to stop the

development of caries, or get rid of superﬁ cial

caries. Drug therapy is mainly applied to early

enamel caries in teeth on a easy-to-clean smooth

surface (such as buccal, lingual), on which cavi-

ties have not yet formed; superﬁ cial caries of

anterior primary teeth on the proximal surface;

secondary enamel hypoplasia, which causes

extensive shallow caries, and difﬁ culties with

prepare cavities preparation.

Fluoride, such as 75 % sodium ﬂ uoride and

glycerol paste, 8 % stannous ﬂ uoride solution,

2 % sodium ﬂ uoride, sodium monoﬂ uorophos-

phate solution, or a ﬂ uoride gel are commonly

used drugs for the treatment. By local applica-

tion, these ﬂ uorides can penetrate into the enamel

to form insoluble acid ﬂ uorapatite, and promote

remineralization of enamel, and also to prevent

bacterial growth, inhibit bacterial metabolism,

acid and polysaccharide synthesis. Fluoride has

no corrosive stimulation to soft tissue, no tooth

discoloration, and is considered safe and

effective.

The main agents of silver nitrate are 10 % sil-

ver nitrate solution or ammonia silver nitrate

solution. Silver nitrate is a strong corrosive agent,

which can combine with proteins to form precipi-

tates. When in low concentrations, it provides

convergence and an antibacterial effect; in high

concentrations it has a strong corrosive effect and

can kill bacteria. When it is applied to zones of

caries, with the addition of clove or 10 % forma-

lin solution, it can turn into black reduced silver,

and with the addition of 25 % iodine it results in

off-white silver iodide. Both of these two formu-

lations can penetrate into the enamel and dentin

to coagulate proteins, kill bacteria, plugging gaps

in enamel and dentin tubules, thus blocking and

terminating the development of the caries lesion.

Remineralization Treatment

For early enamel

caries that have been demineralized and softened,

the appropriate drug treatment to re-deposit

L. Jiyao

115

calcium and remineralize, thereby removing its

hardness and eliminating caries, is called remin-

eralization treatment.

Early enamel caries on the smooth surfaces

(buccal, labial, lingual, palatal or proximal), such

as white spots, and people susceptible to caries

are suitable for remineralization therapy.

There are many types of mineralized ﬂ uid,

which divided into single component and com-

plex components. The single component is

mainly ﬂ uorine-containing (e.g., NaF: 0.2 g;

O: 1,000 ml), the complex component

mainly containing different ratios of calcium,

phosphate, and ﬂ uoride salts, while calcium or

ﬂ uoride salt is the main ingredient (e.g., CaCl:

9 g; KH

: 6 g; KCl: 1.1 g; KF0.2 g; DH

1,000 ml).

In recent years, a new remineralization agent,

CPP-ACP, has been used clinically. Casein

(casein phosphopeptide, CPPs) used casein as a

raw material, by hydrolysis, separation, and puri-

ﬁ cation to obtain a class of phosphoserine-rich

bioactive peptides. Under neutral or alkaline con-

ditions, CPPs can form soluble chelates with the

amorphous calcium phosphate (ACP), i.e., casein

phosphopeptide-amorphous calcium phosphate

(CPP-ACP). CPP-ACP has a wide range of appli-

cations in biology, including the promotion of

remineralization of the tooth surface and bone

calciﬁ cation, promoting the absorption of miner-

als, and has an effect on cariogenic bacteria.

Currently, CPP-ACP is used in the treatment of

early caries remineralization of dentin hypersen-

sitivity, dental erosion treatment, and as preven-

tion in caries-susceptible patients. The

remineralizing agent with CPP-ACP as the main

ingredient shows broad application prospects in

caries prevention.

Atraumatic Restorative Technique

Atraumatic

restorative technique (ART) refers to using only

hand instruments such as a spoon excavator to

remove caries tissue, then using glass ionomer

cement or other cementitious ﬁ lling materials for

repair.

The ART meets the requirements of modern

minimally invasive restoration, the use of a

bonding of glass ionomer materials, with as

little cavity preparation and tooth damage as

possible, and the best preservation of tooth

structure.

7.3 Current Silver Amalgam

and Techniques for Direct

Restorations [

4 ]

7.3.1 The Controversy Over Silver

Amalgam

Silver amalgam has a history of more than 160

years as a dental restorative material. Because it

has the ideal wear resistance, inoxidizability,

good mechanical strength, and it is inexpensive

and simple to operate, silver amalgam also dis-

plays great longevity and a low rate of secondary

caries in posterior teeth. Although silver amal-

gam is not satisfactory with regard to aesthetics

and conducts heat and electricity, it is still widely

applied in dental practice.

In China, it was recorded that silver paste ﬁ ll-

ing has been in use since the Tang Dynasty. In

1826 in France silver amalgam was used for tooth

repair; in the mid-1930s, it started being applied

in American dental practice. With the develop-

ment of material preparation and the improve-

ment in property, the silver amalgam in restorative

dental material has obtained the approval of

many international health organizations, includ-

ing the WHO.

However, there was doubt regarding silver

amalgam and its application in tooth repair

because of the mercury content. There have been

reports that mercury can cause adverse effects,

such as kidney function damage, reduced immu-

nity, nerve toxicity, etc. However until now, there

still has been no evidence reporting the direct

causal relationship between silver amalgam use

and adverse effects. Moreover, there are also

cases of silver amalgam allergy, the main mani-

festations of which are a lichen planus-like

response affecting the local mucosa and the

occurrence of skin erythema, but this type of

allergy is only limited to the region adjacent to

the silver amalgam, and had no obvious effect on

the body; moreover, the symptoms may be

7 Clinical Management of Dental Caries

116

relieved after the elimination of the silver

amalgam restoration, with no need for special

treatment.

From the perspective of public health, protec-

tion from silver amalgam pollution should be

strengthened, and the presence of mercury vapor

in the clinic should be closely monitored.

Nowadays, the mixing of silver amalgam has

been automated, closed off, and there is minimal

mercury vapor pollution in the oral clinic.

It is noteworthy that with the continuous

development of dental restorative materials and

technology, the new idea of a minimally invasive

technique has been put forward in the twenty-ﬁ rst

century, and the status of silver amalgam in tooth

repair has changed. Because of the shortage of

the physical and chemical properties of compos-

ite resin and glass ionomer cement tooth-colored

material, it still cannot completely replace the sil-

ver amalgam in posterior tooth repair at present,

although some research has been carried out that

has shown that the longevity of composite resin is

greater than for the same sized silver amalgam

restoration [ 5 ].

7.3.2 Indications

and Contraindications

Silver amalgam can be used in almost all poste-

rior teeth restoration, unless the preparation is not

appropriate for the retention form, or if there is a

mercury allergy. However, with composite resin-

bonded ﬁ xed technology widely used in dental

restoration, the developed countries in Europe

have been gradually reducing the use of amal-

gam. It is believed in the following situations that

amalgam is preferred: a large complicated cavity

in a molar, the need to restore the tooth cusps,

and direct restorations of teeth. If retention is

expected to increase, the bonded amalgam should

preferably be used and/or a box-shaped, grooved,

circular retention groove retention, and then con-

sider dentin pins should be considered. If there is

large area of crown damage, the cementing mate-

rial post and core restoration is superior to the

silver amalgam. Afterward, the entire crown is

used to cover it. Moreover, the use of cementing

material is not suggested for repairing cracked

spots in silver amalgam restoration. The silver

amalgam may also be used as post and core mate-

rial. Then, the amalgam-fractured parts repair,

the repair parts are not comfortable using a bond-

ing material.

However, it is not recommended to use silver

amalgam in the following situations: early tooth

decay; premolar and molar occlusal surface or

adjacent surface cavity of small to medium size;

premolar large occlusal surface or interproximal

caries; all tooth neck cavity repair; root canal ﬁ ll-

ing; pregnant women.

7.3.3 Silver Amalgam Restoration

Technique

7.3.3.1 Cavity Shape Preparation

The principle of cavity shapes of silver amalgam

restorations consists of removing the decayed tis-

sue, outlining the cavity border, removing all

unsubstantial enamel and sharp ridges, empha-

sizing the protection of the dental pulp–dentin

complex and the functional reconstruction after

ﬁ lling, without damaging the periodontal

tissues.

The cavity shape for silver amalgam restora-

tion requires a high level of resistance and reten-

tion. It is the basic requirement that tooth tissues

and silver amalgam restorations should not frac-

ture or deform and restorations should not be

taken off, shift, under normal chewing forces

with proper chewing movements.

The amalgam restorations must have a certain

thickness to ensure basic resistance. The depth of

the occlusal cavity of posterior teeth should be at

least 1.5–2 mm. The distance of healthy tooth tis-

sue between two amalgam restorations on the

same tooth should be at least 1 mm.

7.3.3.2 Silver Amalgam Filling

After the cavity preparation the rubber dam and

suction are used to isolate the operation area from

moisture and saliva.

To conﬁ ne the silver amalgam to the cavity

and complete the compression backﬁ ll, a box-

shaped cavity with a restrictive wall and bottom

L. Jiyao

117

is needed. For a class I cavity on only one tooth

surface, the structure itself has satisﬁ ed this

request. However, for a cavity involving two or

more surfaces, it is essential to use a matrix bond

surrounding to turn it into a box shape. Before

the ﬁ lling, the matrix bond is used to develop the

artiﬁ cial wall. On one hand, it is conducive to the

compressive backﬁ ll of silver amalgam; simulta-

neously, it may also effectively prevent the silver

amalgam from overﬂ owing and periodontal

overhang, which stimulates the periodontal

tissue.

For a class II cavity of proximal tooth dam-

age, the use of a forming piece is extremely con-

venient if it has proximal tooth support, but for

severe tooth body damage, the proximal tooth

cannot play a supportive role; thus, other meth-

ods are needed to assist. The wedge is a com-

mon piece of auxiliary equipment used in

the forming of the proximal surface. When the

matrix band cannot ﬁ t the tooth neck well, the

wedge may be inserted between the necks of

the teeth. Because the matrix band with the teeth

closed, in the case of the silver amalgam pressed

out to form an overhang from the gingiva. The

shape and size of the wedge should be appropri-

ate. If it is too small it is not easy to set tight,

while if it is too big it will affect the adjacent

shape of the restoration.

After cleaning, disinfecting, and drying, the

cavity can be ﬁ lled. Before silver amalgam ﬁ ll-

ing, coating and painting can reduce the occur-

rence of secondary caries; the commonly used

varnish is made from resin materials. Using the

silver amalgam autostirrer completes the attri-

tion evenly. When backﬁ lling, the principle of

little by little ﬁ lling, of layer upon layer com-

pression, and of the dotted angle to the line angle

should be followed. The backﬁ ll process makes

silver amalgam go into the cavity and ﬁ t with the

tooth body closely, and a silver amalgam plug-

ger is used to carry out the process. When com-

pression causing the unnecessary mercury to

squeeze out and causing the air which mixes

with it to discharge, it is more advantageous to

the completion of the operation, thus strengthen-

ing the intensity of the restoration and the

mechanical properties. In class II cavity ﬁ lling

of the side of the cavity shape, the surgical prin-

ciple is lateral compression, and layer upon

layer compaction. When the cavity shape is

large, more attention should be paid to the prin-

ciple of layer upon layer compression. It is gen-

erally believed that a thickness of 1 mm is able

to provide full compression. When applying

pressure, in addition to considering the vertical

direction, attention should also be paid to both

the horizontal and the lateral direction to ensure

that the silver amalgam restoration has a three-

dimensional adaptation.

When ﬁ lling is completed, polishing should

be carried out promptly, using an ovoid hoe light

modulator to act in mesiodistally and buccolin-

gually to remove excess silver amalgam and fur-

ther compact the edge of the silver amalgam

restoration. Then, the restoration should be

carved according to the anatomical form and

adjacent relations. Usually, an instrument with a

sharp edge can be used. A special carving instru-

ment is available, but a digging machine and hoe

can also be used. Silver amalgam carving should

be done in accordance with the anatomical shape,

forming the ideal tooth shape and bulge, but not a

deep pit and ﬁ ssure. It should not appear sparse

or feather-edged on the border of the restoration

with the tooth tissue. The sculpting of the silver

amalgam should be performed before removal of

the forming sheet. This is advisable before the

silver amalgam is fully solidiﬁ ed, generally using

a short push and pull, but attention should be paid

to the direction, which should be from the tooth

body to the restoration. In trimming the edge of

the crest of the adjacent and occlusal cavity,

attention should be paid to the height of the edge

of the crest of the adjacent tooth, and then to

forming an outreach gap. Next, the occlusion

should be checked and adjusted to prevent a high

bite, and the early contact point of middle occlu-

sion, lateral occlusion, and stretching occlusion

should also be removed. General occlusal adjust-

ment can be divided into two steps, namely

immediately after the ﬁ lling is completed and the

second day after the ﬁ lling. The subsequent pol-

ishing, called polishing after carving, is to ensure

that the surface of the silver amalgam restoration

is smooth, to improve the corrosion resistance of

7 Clinical Management of Dental Caries

118

the silver amalgam restoration, and to prolong

the life of the restoration.

7.4 Resin Composites and Direct

Bonding Restoration

Technique

Resin composites are types of polymers that are

used in dentistry as a restorative material devel-

oped based on acrylate. They are mainly com-

posed of Bis-GMA monomers or some Bis-GMA

analog, a ﬁ ller material, and a photo initiator.

The properties of resin composites have been

greatly improved since Bowen invented them in

1962. In particular, the development of bonding

technology has led to resin composites becom-

ing more widely used and becoming the ideal

tooth- colored restorative materials at currently

available.

In 1955, Buonocore ﬁ rst treated the tooth sur-

face with acid to promote the bonding between

resin and the tooth surface, which improved the

stability of restorations. This technique was then

used in clinical practice and universally

acknowledged at the annual meeting of the

International Society of etching techniques in

1974, which brought about great changes in

dental practice.

7.4.1 Resin Composites

There are many kinds of resin composites, which

can be divided into anterior tooth type, posterior

tooth type, and universal, according to the posi-

tions in which they are used in the clinic. Anterior

resin composites emphasize color and polishing,

while posterior tooth type enhances the mechani-

cal strength and abrasion resistance. According to

the inorganic ﬁ ller particle size, resin composites

can be divided into macroﬁ ll resin composite,

microﬁ ll resin composite, microhybrid resin

composite, hybrid resin composite, and nano-

composite. For macroﬁ ll resin composite, the

ﬁ ller particle size ranges from 10 to 100 μm and

the ﬁ ller volume fraction is 70–80 %. As early

products, such materials possess good mechanical

properties, but poorer surface polishing charac-

teristics, so that they are less frequently used at

present. For the microﬁ ll resin composite, the

ﬁ ller particle size ranges from 0.01 to 0.1 μm and

the ﬁ ller volume fraction is 35–50 %. This kind

of material, which was produced in the late 1970s

has great color and polishability, but poor

mechanical properties; thus, they are suitable for

class III, IV, and V restorations, tiny tooth reshap-

ing, and partial discolored tooth restoration.

Microhybrid resin composite refers to the com-

pound ﬁ ller of large particles and small particles.

The ﬁ ller particle size ranges from 0.04 to 4 μm

and the ﬁ ller volume fraction is 56–66 %.

Because small particles ﬁ ll the voids between the

large particles, the microhybrid resin composite

has better abrasion resistance and transparency;

however, they are not as good as ultra-microﬁ ll

resin composites. They are suitable for class III,

IV and V restorations, resin veneers, dental mor-

phology shaping, discolored teeth restorations,

and also posterior teeth restorations. For hybrid

resin composite (namely universal resin compos-

ite), the ﬁ ller particle size is 1–3 μm, the ﬁ ller

volume fraction is 70–77 %, and they are suitable

for class I, II, and V restorations, and dentin res-

toration for classes III and IV. However, their sur-

face polishing performance is inferior to that of

microﬁ ll and nanocomposite. The nanocompos-

ite was ﬁ rst reported in 2002, and the ﬁ ller parti-

cle size ranges from 25 to 75 nm with a precise

arrangement. This composite has excellent

mechanical properties, improved surface polish-

ability, and decreased polymerization shrinkage.

Thus, they have been considered to be the best

universal composites, which are suitable for

repairing all kinds of cavities, resin veneers, den-

tal morphology shaping, and discolored teeth

restorations.

7.4.2 Etching Adhesive Systems

and Bonding Mechanisms

Modern etching systems can be divided into the

total-etch systems and the self-etch systems

based on processing for smear layer by etching

systems [ 6 ]. The total-etch system is also called

L. Jiyao

119

“etch and rinse adhesive” because there is a sep-

arate etching step that completely removes the

smear layer by an acidic gel and demineralizes

hydroxyapatite under the smear layer of the

dentin. The self-etch system only makes the

smear layer permeable without completely

removing it because of the lack of the separate

etching step.

The development of dentine adhesives has

changed a lot to simplify the procedure, for

example, the combination of etching and pre-

treatment. However, the various simpliﬁ ed

methods are all from the two systems. The clas-

siﬁ cation of dentin adhesive systems is shown in

Fig. 7.1 .

7.4.3 Total-Etch Systems

The total-etch system completely removes the

smear layer using an acidic gel (usually phos-

phate acid) and demineralizes hydroxyapatite

under the smear layer of the dentin. The adhe-

sive of the one bottle etching system is synthe-

sized by dissolving the resin monomer to form

an organic solvent. After etching and rinsing,

the adhesive was applied to the treated tooth

surface; then, the resin monomers penetrate into

the demineralized dentin through the gap, which

is ﬁ lled with water between the dentin collagen

(this gap was originally occupied by the

hydroxyapatite) to form a “hybrid layer”. The

hybrid layer is between the adhesive and dentin

and is composed of collagen, resin monomer,

residual hydroxyapatite, and water. The hybrid

layer helps to reduce postoperative sensitivity

and form a better marginal seal; at the same

time, buffering the shrinkage stress produced by

the polymerization of the resin composites as an

“elastic buffer”.

7.4.4 Self-Etch Systems

A self-etch system combines the etching and pre-

treatment as one step when processing enamel

and dentin without the separate etching step. The

mixture combined with etch and primer pene-

trates into and dissolves a part of the smear layer

to form a “hybrid zone” with hydroxyapatite so

that the “hybrid zone” is composed of the hybrid

layer and the residual smear layer.

For both the etch and the primer in the two-

step system and the self-etch adhesive in the one-

step system, the composition is substantially the

same, i.e., a mixture of water and the acidic

monomer. The acid monomer is typically phos-

phate ester or carboxylic acid ester, and its pH is

above that of the phosphoric acid gel. Water is a

key component of self-etch systems because it is

involved in the ionization of acidic components.

According to its acidic strength, self-etching

adhesive can be divided into three categories:

mild (pH > 1.5), medium (1.0 < pH < 1.5), and

strong (pH < 1.0).

7.4.5 Enamel Bonding

Etching is a key step in enamel bonding. There

are large quantities of hydroxyapatite in enamel,

in which the surface layer turns into water- soluble

monocalcium phosphate during phosphoric acid

treatment. Meanwhile, the dental plaque, material

Multiple-bottles system:

etch+primer+adhesive

Total-

etch

systems

Self-

etch

systems

Two-step system:

etch/primer+adhesive

One-bottle system: etch

+adhesive

One-bottle system:

etch/primer/adhesive

Etching

systems

Fig. 7.1 Classiﬁ cation of

dentin adhesive systems

7 Clinical Management of Dental Caries

120

alba, and food residues attached to the tooth sur-

face are removed, thereby exposing a clean fresh

surface layer. Owing to the orientation of

hydroxyl and amino on the tooth surface after

phosphoric acid treatment, a polarized surface is

formed. The increased surface energy of enamel

is beneﬁ cial for wetting and penetrating of the

adhesive. It is generally accepted that 30–50 % of

phosphoric acid enables even demineralization of

the enamel surface; thus, total-etch systems are

considered a better adhesive system for enamel

bonding.

After the etching process, the wettability of

the rough enamel surface is improved so that the

adhesive more easily penetrating the micro-

structure of the tooth surface, thus strengthening

the interaction of adhesive and fresh enamel.

When the adhesive is cured, the bonding inter-

face generates a considerable mechanical inter-

locking force. It can be observed by SEM that a

large quantity of cured adhesive ﬁ lled in the

demineralized interprismatic area of the enamel,

and countless micro-protuberances (usually

called resin tags) are formed on the enamel side

of the bonding interface. These mechanical

anchored structures formed by resin tags and

enamel provide the prime bonding force for the

materials and enamel.

Etching technology is the general method for

performing enamel bonding. In this method,

mechanical interlocking is obtained by etching

treatment, and the formation of resin tags is the

main adhesive mechanism. The etched enamel

prisms and interprismatic areas are demineral-

ized, and the low-viscosity adhesive penetrates

into the micropores of the enamel via the capil-

laries. Then, the polymerization of the adhesive

occurs, which forms the resin tags that can gener-

ate the micromechanical interlocking system.

For the two-step self-etch system, the demin-

eralized enamel layer is thinner than that treated

by phosphoric acid gel because of the higher pH

of adhesive. There are two methods of improving

the bonding strength of self-etch systems:

1. Removing the rodless enamel to obtain a

rough enamel surface.

2. Using phosphate acid to pre-etch the enamel

before self-etching. The bonding strength is

clinically acceptable for dentin and etched

enamel treated using the self-etch method, but

it is insufﬁ cient for untreated enamel and scle-

rotic cementum.

For the one-step self-etch system, the bonding

strength is very low and there is no enamel pre-

etching. However, the bonding strength is accept-

able when the enamel is beveled and prepared.

In spite of the self-etch system gaining in pop-

ularity, phosphoric acid etching of the enamel is

still the gold standard for testing new bonding

material.

7.4.6 Dentin Bonding

The surface property and interior structure of the

dentin is sophisticated. The dentin contains a lot

of organic components, water, dentinal tubule

connected with pulp, and liquid effused from the

tubules. In addition, there is a smear layer caused

by instrumental cutting of the dentin. Therefore,

it is difﬁ cult to perform the treatment of dentin.

The smear layer is caused by the formation of

metamorphic organics and inorganics during the

bonding surface preparation, and its thickness is

0.5–15 μm. The degree of treatment of the smear

layer directly inﬂ uences the bonding effect of

dentin. In the total-etch system, the smear layer

can be thoroughly removed, but probably leads to

excessive opening of the dentinal tubule, which

will increase the postoperative sensitivity.

In the case of the self-etch system, the weak

acid can partially remove the smear layer and

lead to the appropriate opening of the dentinal

tubules, thus bringing about the beneﬁ t for dentin

bonding. Consequently, whether the one-step or

the two-step self-etch system is used, the treat-

ment of dentin includes three aspects: removal of

the smear layer; improving the surface activity of

dentin; promoting the penetration and bonding

strength of the adhesive. It is widely believed that

the prime mechanism of dentin bonding is the

formation of a hybrid layer or hybrid zone.

The treatment of dentin is described as

follows. At ﬁ rst, the dentin surface is treated

by the etch so that the smear layer can be par-

tially removed. After demineralization of the

L. Jiyao

121

intertubular dentin, the microporous stent of

collagenous ﬁ ber is exposed to form a porous

belt and the opening of dentinal tubules; then,

the primer is used. It can become wet and pen-

etrate into the micropores of collagen ﬁ bers and

dentinal tubules to facilitate the subsequent pen-

etration of the adhesive; at last, the adhesive is

coated. After the primer and the adhesive are

cured in situ, they form the hybrid layer (or

hybrid zone) with dentin collagen ﬁ bers, which

will obtain a solid bond with the dentin because

this zone contains many resin micro-protrusions

and large resin protrusions of the dentinal

tubules. Meanwhile, the residual unsaturated

ethylene of the adhesive copolymerizes with the

resin monomer; thus, the resin composites can

be bonded to dentin. Along with the develop-

ment of the dentin bonding system, the pro-

cesses mentioned above are simpliﬁ ed into a

one-step or two-step procedure.

The durability of the self-etch bonding system

is a signiﬁ cant issue. At the early stage, the bond-

ing strength is acceptable. As time goes on, the

bonding strength is continuously decreased,

especially for the one-step self-etch system,

owing to the hydrophilicity of the acidic mono-

mer in addition to the high water content to main-

tain ionization of the acidic monomer, and this

may ultimately even affect the bonding to enamel.

Meanwhile, the inadequate penetration of the

resin into the tooth structure may also accelerate

the degradation of the bonding interface.

Shrinkage stress caused by the polymerization of

resin composites, which act on the bonding inter-

face, results in reduced dentin bond strength if

the dentin bond strength cannot resist it. This will

bring about the formation of gaps or edges result-

ing in secondary caries and dentin sensitivity.

7.5 Resin Composite Bonding

Restoration Technique

7.5.1 Indications

and Contraindications

Currently, resin composite has been widely used

indirect restorative dentistry. Almost all the den-

tal defects can be repaired via a resin composite,

which can also be used for an abnormal shape or

the color of teeth in cosmetic restoration, in addi-

tion to the restoration of endodontic treatment

teeth.

The following situations should be taken into

account: in the anterior restoration of a class IV

cavity, except for a crossbite and clenching, the

teeth deﬁ cit, which does not exceed one half, can

be considered a direct composite restoration. For

posterior teeth restoration, a severe attrition and

cusp defect need to be excluded. We do also not

use resin composite if the cavity cannot be com-

pletely isolated from saliva, gingival crevicular

ﬂ uid, and blood.

7.5.2 Requirements for Restoration

Design

Acid etching followed by bonding provides

retention, while increasing the resistance of the

remaining tooth structure. The tooth types,

the position of the teeth in the dental arch, the

size and type of defect, whether the treatment

is for the placement of the original prosthesis,

the occlusal function, and the relationship

between the edges of the tooth preparation

need to be considered in bonding restoration.

What is more, the quantity and quality of the

remaining hard tissue also need to be consid-

ered, the mechanical force of remaining tooth

structure is exposed to the defect, and the

reserve area extends to the range of the sound

dental tissue.

7.5.3 Cavity Preparation

The principles of cavity preparation for resin

composite restoration are based on the principles

of amalgam cavity preparation, combined with

the characteristics of bonding restoration. The

principles emphasize preserving as much of the

tooth structure as possible in the premise of

removal of infected tissue and caries staining.

Cavity shape is determined by the area of the

lesion, and retention of the restoration relies on

the etching. The extension for prevention is not

needed.

7 Clinical Management of Dental Caries

122

Class I The cavity shape only involves the cari-

ous parts and developmental defects. For enamel

caries, the depth of the cavity should be limited to

the enamel, without proceeding to the dentin, or

increasing the supporting retention; to remove a

large shallow dish caries, the cavity should be

extended at the buccal and tongue groove, and

then prepare the bevel at the edge of the cavity,

adding the auxiliary retention ditch at the bottom

and side walls. However, at the occlusal contact

points in the occlusal cusp the edge bevel is not

needed.

Class II

The abrasion of composite resin mate-

rial is not as good as that for the silver amalgam.

Therefore, the occlusal factors should be con-

sidered, especially the functional occlusal tip

occlusion. On the occlusal surface, the cavity

preparation should embody the preservation

principle, and the cavity edge and line angle

should be more obtuse than with silver amal-

gam, to facilitate closing together. For the occlu-

sal cavity, the beveled edge could increase the

cavity width, which means the wear of the pros-

thesis is greater than for the conventional cavity;

however, at these parts of the restoration, espe-

cially the edges, ﬂ akes often form and breakage

is easy by force. Therefore, the preparation of

the bevel at the non-occlusal contact at the

occlusal surface remains controversial, and in

contact with occlusal cusp, the bevel should be

avoided.

For the proximal cavity, buccal and lingual

walls should be introverted, and the enamel

bevel edge can be prepared, without extending

to the self-cleaning area. An additional retention

groove could be prepared at the axiofacial and

axiolingual line angles, in the same way as the

silver amalgam. There are pulp walls at the

occlusal surface and an axial wall in the proxi-

mal surface, and a large part is involved in den-

tin; thus, less enamel is available in the proximal

cavity, which is not conducive to bonding resto-

rations. Therefore, to preserve as much tooth

tissue as possible, especially the enamel thick-

ness, for the gingival wall parts, carious tissue

should be removed without extension to the root

side.

Class III

Cavity preparation should be started

from the lingual surface, trying to save the lip

surface integrity; if the labial enamel has been

stained, or the edge of the facial surface has been

damaged, the preparation can be entered directly

from the facial surface. Small to medium-sized

cavities, should be designed as conservatively as

possible, without making a special cavity shape,

or as an aid to the retention form. For enamel car-

ies, the retention depends mainly on the retention

wall and resin bonding. Therefore, the bevel edge

should be prepared, without going deep into the

dentin. For a large area of caries, the retention

groove should be made in the axiogingival line

angle, and the undercut should be prepared in the

axiofaciopulpal point angle, while the bevel is at

the enamel wall. The gingival wall, stretching to

the root surface, where there is no enamel, should

not be beveled. Withstanding a greater bite force

at the tongue surface, the tongue edge does not

make a beveled edge.

Class IV

The beveled edges and the size of

the dental defects in class IV cavities should

be considered for resin composite restoration.

For the tooth in which defect is limited to one

side and the incisal part is intact, the carious

structure and weak enamel should be removed,

and all the enamel edges beveled. In cases

with defects exceed mesiodistally half the

width of the incisal part, or the distance

between the incisal and gingival surface

exceeds 2/3 of the crown length, with intact

pulp, only a short bevel of at least 1.0 mm

around the cavity needs to be prepared.

Increasing the width of the bevel could

enhance enamel bond strength, and the spread-

ing of the material along the plane can also

attain a better aesthetic effect.

Class V

For small to medium-sized class V cav-

ities, preparation should be as conservative as

possible, with no special shape, and the bevel

edge only needs to be prepared for the enamel

wall slope. Generally, the retention groove is not

needed, and when there is no enamel on the gin-

gival wall, the slide ditch should be prepared at

the axiogingival line angle. Retention grooves

L. Jiyao

123

not only increase retention, but can also reduce

polymerization shrinkage and micro-leakage in

the bite force of the resin.

Class V cavities, in which the area is large and

involves the root surface, need to be prepared

routinely, similar to the box-like cavity in silver

amalgam restorations. The retention groove

should be prepared at the angle of the gingival

and occlusal axis, while the bevel edge should be

shaped on the occlusal, mesial and the distal

edges of the wall.

7.5.4 The Importance

of Postprocessing Decoration

After the restoration, contouring, blending,

grinding, and polishing are needed. For a long

time, this part was not given serious clinical

attention. Very simply, by removing the ﬂ ash side

at the adjacent surface, trimming the shape care-

fully, and polishing the smooth surface, the

occlusal surface and the adjacent surface from

coarse to ﬁ ne, can form a smooth surface, a suit-

able edge, and a good occlusal relationship,

which can achieve minimal plaque adhesion,

easy bacteria removal, and better aesthetics. The

success of grinding and polishing of the resin

composite is determined by the repair materials

and devices. The surface quality of the resin is

not only impacted by the quality of the polishing

apparatus and polishing paste, but also by the rel-

evant component and ﬁ lling characteristics of the

resin. A new composition with small particles

and micro-mixed resin has changed the ﬁ ller for-

mulation. The size, shape, orientation, and the

aggregation of the resin have been enhanced, and

the physical and mechanical properties improved,

while the character of the polishing is better. The

hardness of the inorganic ﬁ ller and the matrix is

different, which is caused by the difference in the

wear rate of these two components, and this

results in a surface roughness. The surface gloss

of restoration materials should be similar to that

of the dental interface, because it affects the

color-matching of the restoration. At present,

high-quality restoration not only requires decora-

tion for the anatomical shape, contour, marginal

integrity, occlusal function, and improvement of

the smoothness of the surface, but also pays

attention to the concept of aesthetics. For the best

bonding aesthetic, the surface should be smooth

to prevent plaque accumulation and dyeing; the

restoration should have a perfect contour and

form, to improve the organizational ﬂ exibility.

The appropriate dressing also makes the anatom-

ical shape of the prosthesis adapt to occlusal

requirements, and match the color of the sur-

rounding teeth. The integrity and the adaptability

of the edge is also improved. Therefore, the resto-

ration is durable and aesthetically pleasing, and

the life of the restoration is extended.

7.5.5 Problems of Direct Resin

Composite Restoration

7.5.5.1 Polymerization Shrinkage

Polymerization shrinkage stress is one of the

main problems that affects the longevity of direct

composite resin restoration. The dental compos-

ite resin is mostly formed of dimethacrylate mol-

ecules, whose polymerization reaction produces

a polymer network and volumetric contraction.

Owing to the restrictions of the cavity, the volu-

metric contraction resin showed that the volume

changes the effect on the tooth structure and

bonding interface stress, causing tooth deforma-

tion and resulting in bond failure, postoperative

sensitivity, microleakage, secondary caries, and

other adverse consequences. The volumetric con-

traction in the bonded resin composite restora-

tions may cause shrinkage stress at the resin

composite/tooth structure interface and/or within

the tooth or the resin composite. The resulting

shrinkage stress may result in adhesive failure,

tooth deformation, postoperative hypersensitiv-

ity, secondary caries or microleakage. Shrinkage

stress is not only dependent on the characteristics

of the composite resin, but also on the tooth

structure, cavity shape, characteristics, and resto-

ration techniques. Therefore, the correct under-

standing of resin composite polymerization

signiﬁ cantly offers numerous clinical advantages

in reducing the shrinkage stress generated in

polymerizing dental composites.

7 Clinical Management of Dental Caries

124

Material related factors Resin composite con-

sists of organic matrix and inorganic ﬁ llers. The

polymerization shrinkage is mainly caused by the

volumetric contraction of the organic matrix,

with an average of 2.6 % ~ 7.1 vol% [ 7 ]. In this

process, the space occupied by inorganic ﬁ llers is

not involved in the shrinkage. Therefore, the

main strategy in reducing polymerization shrink-

age in methacrylate-based composites focused on

increasing the ﬁ ller load. Compared with resin

composites with a low ﬁ ller load, the resin com-

posites with a high ﬁ ller load have some good

physical properties, lower shrinkage, and greater

hardness. With a constant amount of shrinkage,

the resulting shrinkage stress grows with the

increase in the elastic modulus of the composite

resin.

Conventional composite resins have ﬁ ller par-

ticles larger than 400 nm, but nanocomposites

contain ﬁ ller particles between 20 and 100 nm.

Compared with hybrid and microﬁ lled resins,

nanocomposite resin particles have a high ﬁ ller

load and extensive ﬁ ller distribution, resulting in

low polymerization shrinkage. Compared with

conventional composite resin, ﬂ owable compos-

ite resin contains less inorganic ﬁ ller, which

reduces the mechanical performance, increases

the polymerization shrinkage, and produces more

shrinkage stress at the interface. However, owing

to the reduction of inorganic ﬁ ller during the cur-

ing process, concave deformation at the surface

of a ﬂ owable resin composite can compensate for

the volume contraction within the resin; thus, the

ﬂ owable resin composite can be placed between

the tooth surface and the nonﬂ owable composite

as a stress buffer layer. Currently, there are differ-

ences of opinion about the ﬂ owable resin com-

posite used as a base material to reduce

polymerization shrinkage.

C-factor

The conﬁ guration factor is the ratio of

the bonded to unbonded surface of the restora-

tion. As the C-factor decreases, the polymeriza-

tion shrinkage is limited to only one direction,

and at the early stage of polymerization, the resin

ﬂ ows freely to prevent polymerization shrinkage

stress. As the C-factor increases, the resin is lim-

ited in direction and becomes less ﬂ owable at the

early stage of polymerization; therefore, the

shrinkage stress increases, the bond strength with

dentin declines, resulting in greater marginal

microleakage.

Incremental techniques are always recog-

nized as a major factor in the reduction of

C-factor and shrinkage stress. Incremental layer-

ing is considered to be the conventional tech-

nique for reducing polymerization shrinkage,

compared with the bulk technique. For incre-

mental layering, the shrinkage stress of each

increment can be compensated for by the next

increment. Because of the reduction of C-factor,

the contact surface between the resin and the

cavity walls is minimized and the resin is rela-

tively ﬂ owable during polymerization, which

can decrease the shrinkage further. Incremental

layering techniques include horizontal occluso-

gingival layering, wedge-shaped oblique layer-

ing, and the successive cusp build-up technique.

In recent years, the use of incremental layering

techniques for the purpose of reducing polymer-

ization shrinkage has been questioned by some

authors, who argued that different restoration

techniques had no signiﬁ cant difference on the

shrinkage stress of composite resin. Nevertheless,

incremental layering techniques are still widely

used in clinics.

The polymerization rate is the rate at which

the monomer of composite resin is converted to

polymer. The higher the rate, the more polymer-

ized monomers there are, the higher the polym-

erization stress. The use of a high-intensity

curing light can increase the polymerization

rate and mechanical properties of composite

resin, but the polymerization shrinkage stress is

high. Slowing down the curing process can

release stress during polymerization. Therefore,

a new "soft" curing technique has been reported.

A relative low-intensity curing light was used

during the ﬁ rst few seconds of the light curing

(10 s), and then a high-intensity light is applied

for the ﬁ nal curing. For this technique, the light

intensity is low at the beginning, which makes

the surface polymer sufﬁ ciently cross- linked,

but prolongs the polymerization of the underly-

ing resin to inhibit potential stress. In addition

to the curing methods, the curing units also

L. Jiyao

125

have different effects on polymerization shrink-

age stress. Compared with the traditional halo-

gen unit, a light emitting diode unit using

low-intensity light can effectively reduce the

shrinkage stress.

7.5.5.2 Technique Sensitivity

Technique sensitivity, which is totally depen-

dent on the practitioner, includes the failures

caused by improper selection of indications,

inadequate understanding of material and mech-

anism, neglect of the product manual, and incor-

rect operation by not following the

manufacturer’s instructions. Technique sensitiv-

ity is an important factor that affects the result of

direct composite resin restorations. Any care-

lessness in procedure and difference in technical

proﬁ ciency may lead to the failure of

restoration.

First of all, selection of the appropriate resin

composite and adhesive according to the operat-

ing instructions is essential. It requires consid-

eration of the tooth position, location, and

volume of the cavity, ﬁ ller types of resin to meet

the requirements of the mechanical characteris-

tics of the posterior teeth, or the aesthetic prop-

erties of the anterior teeth, or both. Total-etch

adhesive is recommended for bonding with the

enamel surface, and for bonding with the den-

tine surface, use of a self-etching adhesive is

recommended. Strictly following the product

manual and operating instructions is the founda-

tion of successful direct composite resin

restorations.

The selection of the appropriate indications

is also very important. Direct resin restoration

will deﬁ nitely fail if inappropriate cases are

selected, such as cusp defect, severe attrition, or

a subgingival cavity restored without gingival

retraction. Cavity preparation is another impor-

tant factor. Although cavity preparation of direct

resin restoration does not need strict retention

and resistance form, there are certain principles

that need to be followed. If the position and

angle of the cavity walls, and the bevel of the

cavity edge, are not properly prepared, adhesive

failure will result. In addition, the adhesive

surface must be thoroughly cleaned. Debris,

plaque, pigment or carious tissue have an inﬂ u-

ence on the etching and binding effect on the

healthy tooth tissue.

7.5.5.3 Postoperative Sensitivity

Postoperative sensitivity is described as a moder-

ate pain, of short duration, that is initiated by

mastication, hot and cold stimulus, immediately

after resin restoration or later. Typically, there are

two types of postoperative sensitivity. First, when

the cavity is deep and there is not enough base

material, the cavity need re-ﬁ lling with sufﬁ cient

base material and resin. Second, when the cavity

is shallow but reaches the enamel–dentin junc-

tion (EDJ), the sensitivity may be due to opened

dentinal tubules caused by the etching process, or

insufﬁ cient marginal seal if the adhesive is too

thick, or shrinkage stress-induced microleakage,

or exposed dentin surface due to excessive occlu-

sional adjustment. The incidence of such sensi-

tivity is high and it is difﬁ cult to solve.

7.6 The Prospect

of the Treatment of Dental

Caries

7.6.1 Individualized Ideas

of Treatment

Scholars describe dental treatment in the twenty-

ﬁ rst century as micro-invasive dentistry, cosmetic

dentistry, and adhesive dentistry. These concepts

reveal that modern dental treatment, especially

treatment of dental caries, requires as little trauma

as possible from the treatment, the best func-

tional reconstruction, and the best aesthetic per-

formance. Above are the foundations for the

individualized treatment of dental caries.

7.6.2 The Importance

of Individualized Treatment

of Dental Caries

Dental caries is a multi-factorial disease based on

the ecological plaque hypothesis. Factors includ-

ing individual systemic health, social economic

7 Clinical Management of Dental Caries

126

status, sucrose intake, the buffer capacity of

saliva, the implementation of oral health care, the

past history of caries, and the colonization of

caries-related microbes are closely related to

dental caries. Individualized treatment plans for

dental caries against speciﬁ c risk factors are pro-

posed, after integrated evaluation of the factors

described above. For example, the risk of caries

among children and young people lies in deep

pits and ﬁ ssures, while among middle-aged and

elderly people gingival recession and root expo-

sure contribute to dental caries. Some individuals

have caries because of a preference for sweets

and a tendency to ignore oral health, while others

suffer from caries because of the local accumula-

tion of plaque due to poor restoration. Speciﬁ c

measures and targeted intervention should be

taken against different risk factors for dental car-

ies to obtain the best effect.

7.6.3 The Risk Evaluation Is

the Premise of Individualized

Treatment of Dental Caries

The evaluation of the risk of dental caries is an

assessment of the degree of risk of developing

new caries or continuing the progress of existing

caries under the circumstances of fairly constant

etiological factors, including diet, time, a suscep-

tible tooth surface, and plaque levels of an indi-

vidual at the time. The functions of the risk

evaluation of dental caries are listed as follows:

1. To control dental caries effectively. Recent epi-

demiological studies show that caries activity is

not evenly distributed among the general popu-

lation and a small proportion of people with

high caries activity suffer from most of the den-

tal caries among the general population. More

than 60 % of dental caries occur in only 20 %

of the overall population. Taking precautions in

the general population can lower the overall

prevalence of dental caries, but the efﬁ cacy of

precautions cannot be noticeably increased.

Filtering people at a high risk of dental caries

and combining multiple prophylaxes before the

occurrence of dental caries can greatly enhance

the efﬁ cacy of the prevention of dental caries.

2. To make suitable precautionary treatment plan

for individuals. As dental caries is a multi-

factorial disease, the risk evaluation can help

us to deﬁ ne risk factors for an individual so

that speciﬁ c treatment and precautionary

plans can be made.

3. To cure caries at an early stage. The han-

dling of incipient caries has been paid

increasing attention in the individualized

treatment of dental caries. Remineralization

is one of the effective measures. The risk

evaluation of dental caries is the major tool

for predicting the success rate during han-

dling incipient caries using the remineraliza-

tion method. The success rate is relatively

low if the individual is at a high risk of car-

ies; otherwise, the success rate is relatively

high.

7.6.4 The Development

of Technology and Material

Provides a Guarantee

for the Individualized

Treatment of Dental Caries

The development of technologies, including

early diagnosis, risk evaluation, micro-invasive

treatment, colony prevention, and the progress

of dental adhesive restorative materials, pro-

vide the premise and guarantee for the individ-

ualized treatment of dental caries. The risk

evaluation of dental caries predicts the risk of

caries of an individual by combining analysis

of the results of multiple risk factors to achieve

the goal of early detection, early prevention,

and early treatment of individual caries, focus-

ing on populations at a high risk of caries.

Therefore, the appearance and development of

a highly sensitive and speciﬁ c screening tech-

nology to ﬁ lter population at risk of caries, a

quantitative technology of the early diagnosis

of caries, a micro-invasive treatment technol-

ogy of caries, and a preventive technology with

low side effects, together with deﬁ nite efﬁ cacy,

provide a guarantee for the realization of the

real sense of individualized treatment of

caries.

L. Jiyao

127

Application of tissue engineering in the ﬁ eld

of tooth restoration includes terminating or

avoiding the progress of caries by replacing the

composition of oral microbes with lower-toxicity

or inactivated strains constructed by gene

engineering technology, inducing the regenera-

tion of dentin through the slow release of a sus-

tained release carrier into dental tissue by adding

various growth factors to the ﬁ lling materials,

inducing the production of a new tooth structure

Degree of tooth

damage Minor Severe

Parts of active

tissue lost

Most of active tissue

lost

Active tissue lost

completely

Common cases Incipient nonstatic

caries

Arrested caries Arrested or

chronic deep

caries involving

pulp

Pulp exposed

because of caries or

trauma

Total cavitation

or total

dislocation

accidentally

Common ﬁ lling

restoration at

present

Pit and ﬁ ssure

sealant or ﬁ lling

after removal of

lesion

Filling after

removal of

lesion

Removing

softened dentin

fractionally or

pulp capping

Pulp capping, root

canal treatment or

tooth extraction

Implant or

restoration after

debridement

Possible tissue

engineering

technology

Replacement

therapy by

constructing

lower-toxicity or

inactivated strains

using genetic

engineering

technology to

change the

composition of the

oral microbe

Inducing the

pulp–dentin

complex to

defend outer

stimuli and

produce

reparative

dentin using

growth factor

Promoting

regeneration of

pulp tissue and

repair of dentin

structure using

growth factor

Reconstructing

missing pulp and

hard tissue of the

tooth by implanting

stem cells

Implanted teeth

induced in vitro

to restore missing

teeth

7.7 Biological Treatment

Methods

Nowadays, there are two directions of research into

the regeneration of tooth tissue: ﬁ rst, achieving the

goal of regeneration of tooth tissue by mimicking

biomineralization through the design of organic

matrix using bionic theories; second, realizing the

regeneration of tooth tissue by cultivating stem cells

in scaffold material using tissue engineering meth-

ods. These ideas and studies provide an exciting

future for restorative dentistry [ 8 ].

7.7.1 Restorative Therapy Based

on Tissue Engineering

of Tooth Regeneration

The development of tissue engineering for tooth

regeneration has made the self-repair of dental

damage, including cavitated and noncavitated,

or even missing teeth, possible. By directly

implanting teeth cultivated in vitro instead of

traditional implants or dentures to restore miss-

ing parts of teeth or by conditioning the

microenvironment of tooth damage in situ can

help us to realize the goal of the self-repair of

tooth damage.

There is currently no direct evidence sup-

porting the introduction of the speciﬁ c technol-

ogy of tissue engineering into the ﬁ eld of

tooth-ﬁ lling restoration, or related reports on the

ﬁ nal effect. However, lots of related reviews

have pointed out that tissue engineering may

promote tooth ﬁ lling treatment under speciﬁ c

circumstances.

Classifying tooth-ﬁ lling restoration technolo-

gies by the degree of tooth damage may predict

the indication for tissue engineering-related tech-

nology in the restoration of tooth damage. The

chart below summarizes accessible traditional

ﬁ lling restoration and the possible tissue engi-

neering technology against tooth damage of dif-

ferent degrees.

7 Clinical Management of Dental Caries

128

by implanting multifunctional stem cells for teeth

severely cavitated or severely damaged by

trauma, and implanting tooth tissue cultivated

in vitro to restore the integrity of dentition for

teeth that are missing or that cannot be restored

through the method of ﬁ lling.

Tissue engineering has provided an exciting

future for tooth restoration, but related biosecu-

rity problems, technological problems, and rela-

tively high costs have limited its possibility of

wide deployment. It seems that we can only rely

on the optimization of traditional treatment meth-

ods to improve prognosis.

7.7.2 Restorative Therapy Based

on Bionics

Nowadays, composite resin, metal, and ceramics

are the main materials in clinical application.

However, insolvable problems such as margin

leakage, aging of the materials, and the lack of

fracture toughness remain. Also, removal of a

large amount of tooth tissue during surgery

causes the patient a lot of pain. Therefore, regen-

erating new tooth tissue where the tooth tissue is

damaged in situ has become the dream of dental

scholars. At present, designing organic matrix to

conduct biomineralization using bionic theories

may enable us to regenerate teeth. Therefore,

research into bionic materials to simulate tooth

tissue using a bionic methods has been a hotspot

of current studies. For example, in vitro studies to

simulate the biomineralization of enamel and

dentin have enabled simple nucleation and sedi-

mentation of hydroxyapatite and are going deeper

toward the highly densely arrayed and regular

structures such as enamel. When it comes to

tooth tissue mineralization in situ, we are trying

to implement the mineralization procedure, in

which crystal grows layer by layer in a well-

organized way and closely controlled by organic

matrix, while the exploration of forming enamel

on the surface of dentin continues. As studies on

the forming mechanism of enamel and dentin go

deeper and frontier science such as the self-

assembly of the supermolecule develops, we

believe that we can obtain tooth tissue that is

highly bionic in form and function and that we

will ﬁ nally implement the restoration of tooth

damage in situ.

References

1. Summit JB, Robbins JW, Schwartz RS, et al.

Fundamentals of operative dentistry. 2nd ed. Chicago:

Quintessence Publishing Co Inc; 2001.

2. Tyas MJ, Anusavice KJ, Frencken JE, et al. Minimal

intervention dentistry – a review. FDI commission

Project 1–97. Int Dent J. 2000;50(1):1–12.

3. Featherstone JD. Remineralization, the natural caries

repair process – the need for new approaches. Adv

Dent Res. 2009;21(1):4–7.

4. Fedorowicz Z, Nasser M, Wilson N. Adhesively

bonded versus non-bonded amalgam restorations for

dental caries. Cochrane Database Syst Rev.

2009;(4):CD007517. Published by John Wiley& Sons,

Ltd

5. Dunne SM, Gainsford ID. Current materials and tech-

niques for direct restorations in posterior teeth.

I. Silver amalgam. Int Dent J. 1997;47:123–36.

6. Perdigao J. New developments in dental adhesion.

Dent Clin N Am. 2007;51:333–57.

7. Chen Y, Zhou L. Control of polymerization shrinkage

stress in resin composite restoration. Int J Stomatol.

2008;35(4):393–5.

8. Murray PE, Windsor LJ, Smyth TW, et al. Analysis of

pulpal reactions to restorative procedures, materials,

pulp capping, and future therapies. Crit Rev Oral Biol

Med. 2002;13:509–20.

L. Jiyao

129

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_8

Dental Caries and Systemic

Diseases

Zou Ling and Hu Tao

As stated by World Health Organization, oral

health is fundamental to overall health and well-

being and a determinant of quality of life [ 1 ].

According to “Oral Health in America: A Report of

the Surgeon General,” the mouth and face are mir-

rors of health and disease. A physical examination

of the mouth and face can reveal signs of general

health status. Imaging of the oral and craniofacial

structures (x-ray, MRI, SPECT) may provide early

signs of skeletal changes such as those occurring

with osteoporosis and musculoskeletal disorders

and salivary, congenital, neoplastic, and develop-

mental disorders. For example, the research group

of Dr. David Wong from UCLA has initiated a

series of concerted efforts to spearhead the scien-

tiﬁ c and translational frontiers of salivary diagnos-

tics. The potential use of saliva, a totally noninvasive

bioﬂ uid without the limitations and difﬁ culties of

obtaining blood and urine, for oral and systemic

disease detection, disease progression, and thera-

peutic monitoring is a highly desirable goal

[ 2 , 3 ]. In other words, oral health refers to the

health of our mouth and, ultimately, supports and

reﬂ ects the health of the entire body [ 4 ]. In a sense,

oral disease is not just a minor ailment of the soft

and hard tissues of the mouth, and it may be a dis-

ease of the body that happens to begin in the mouth.

If left unchecked, oral disease can contribute to

other more harmful diseases that can seriously

affect the quality of life [ 5 ].

As Hani T. Fadel from University of

Gothenburg wrote in his doctoral thesis, “the link

between oral and general health has been sug-

gested since early times, almost as early as history

itself. The concept of local or systemic diseases

secondary to a localized chronic infection (e.g., in

the oral cavity) is usually called focal infection.

Its origin can probably be traced back to the time

of Hippocrates” [ 6 , 7 ]. Recently, a report titled

“Links between oral health and general health –

the case for action” from Dental Health Services

Victoria summarized that oral health and general

health are related in four major ways:

1. Poor oral health is signiﬁ cantly associated

with major chronic diseases.

2. Poor oral health causes disability.

3. Oral health issues and major diseases share

common risk factors.

4. General health problems may cause or worsen

oral health conditions.

Dental caries and periodontal disease are the two

biggest threats to oral health and are by far the most

Z. Ling (*)

Department of Conservation Dentistry and

Endodontics, West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

H . T a o

Department of Preventive Dentistry,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

130

common oral infection diseases in the United States

and Australia [ 8 ]. It has been well proven that the

oral cavity contains some of the most varied and

vast ﬂ ora in the entire human body, not only includ-

ing those linked to dental caries and periodontal

disease but also including systemic diseases that

affect general health. In addition to bacterial organ-

isms, oral microorganisms can include fungal, pro-

tozoal, and viral species. It is well accepted that our

body is negatively affected by infection of any kind,

no matter where it is located. Moreover, the more

serious the infection and the longer it is present, the

greater its potential for affecting systemic health.

Infection can also seriously stress the immune sys-

tem and diminish its ability to deal with other infec-

tions and diseases. Its effect on the immune system

is directly related to the extent, type, and duration of

the infection [ 5 ]. Over 100 years ago the theory of

focal sepsis, although lacking empirical scientiﬁ c

evidence, hypothesized that chronic infections in

the mouth caused systemic diseases [ 9 ]. The con-

cept has been neglected for several decades and still

is a subject of controversy [ 10 ]. Since many teeth

were extracted without evidence of infection,

thereby providing no relief of symptoms, the theory

was discredited and largely ignored for many years

[ 11 ]. Interestingly, increasing evidence over the

past 30 years suggests that, due to dental bactere-

mia, the oral cavity can indeed serve as a reservoir

for systemic dissemination of pathogenic bacteria

and their toxins, leading to infections and inﬂ am-

mation in distant body sites, especially in immuno-

compromised hosts such as patients suffering from

malignancies, diabetes, or rheumatoid arthritis or

having corticosteroid or other immunosuppressive

treatment [

12 ].

Most studies stated above concerning the rela-

tionship between oral infection and systemic dis-

eases are related to periodontal disease [ 13 ]. And

according to Thomas McGuire, the most impor-

tant of oral diseases in regard to their impact on

general health are:

(a) Periodontal disease

(b) Infected root canals

(d) Other diseases of the oral cavity, such as oral

cancer [ 5 ]

“Dental caries” cannot be directly found in this

list. Thomas McGuire explained the reasons as the

following: certainly, dental caries can have an effect

on a person’s overall health. For example, it can

interfere with the mastication process and thereby

affect digestion. It can cause tooth loss, again affect-

ing digestion. The main difference is that, unlike

periodontal disease, dental caries is not an infection

that has access to the systemic body. Clearly, it can

contribute to systemic health problems, but its

effects on overall health are signiﬁ cantly less than

the effects of periodontal disease [

5 ].

As mentioned before, dental caries is one of

the most common causes of pulpitis and periapi-

cal diseases by penetrating through the enamel

and dentin to reach the pulp. Untreated decay can

become so advanced that the tooth must be

removed (extraction). Most studies supported that

dental caries was the main cause for tooth loss,

but a few studies revealed that a greater propor-

tion of tooth extractions were due to periodontal

disease, especially in patients over 40 years old.

Overall, 70 % of tooth loss is due to tooth decay,

20 % due to periodontal diseases, and 10 % due to

other causes [ 14 , 15 ]. It was reported that caries

accounted for a higher proportion of extractions

than periodontitis at all ages over 20 years in

1968 and only up to 45 years of age in 1988 [ 16 ].

According to another study, although there is an

increase in orthodontic extractions or a decline in

extractions for caries in under-21-year-olds, when

extractions from the population as a whole are

considered, caries and its sequelae remain the

principal reason for loss of all tooth types apart

from lower incisors which were extracted mainly

for periodontal reasons [

17 ]. In 2012, a quantita-

tive study evaluated the prevalence and factors

related to tooth loss due to dental caries among

workers in industrial estates in central Thailand.

There were 457 adult (283 males; 174 females)

between 19 and 53 years participants. The results

showed that 62.2 % participants had tooth loss

due to caries [ 18 ]. The latest study also proved

that dental caries and its complications were the

leading reasons for extraction. Their study

included a total of 2,620 teeth extracted from

1,382 patients. The highest rate (36.9 %) of

extraction occurred for those of 41–60 years of

Z. Ling and H. Tao

131

age. Tooth loss due to caries was 51 %; periodon-

tal disease was 14.4 %; and supernumerary and

tooth impaction were 13.9 %. Although 86 % of

teeth extracted for periodontal disease were in

patients over 40 years of age, caries was still the

main reason for extraction even in elderly patients,

but to a less degree than in younger ones [ 19 ].

Our goal of this chapter is to discuss the rela-

tionship between dental caries and general health;

we will summarize the limited recent advances in

this topic. Since the effect of dental caries on the

overall quality of health and well-being has not

been well studied, in order to enrich the content

of this chapter, studies associating systemic dis-

eases with periapical diseases, tooth loss, root

canal treatment, and other conditions caused by

dental caries directly are also included [ 20 ].

This chapter explores what the dental caries

can reveal about general health, describes the role

the mouth plays as a portal of entry for infection,

and concludes with studies that are associating

oral infections with serious systemic diseases and

conditions. Following this introduction and over-

view, the remainder of the chapter is organized as

follows: ﬁ rst deﬁ ning dental caries and bactere-

mia, head and neck cancer, and children growth;

then brieﬂ y describing dental caries and athero-

sclerosis, cardiovascular disease, and heart attack;

next discussing dental caries and immune system

disease and kidney diseases; and last describing

dental caries and gastrointestinal diseases, diabe-

tes mellitus, and respiratory infections.

8.1 Dental Caries

and Bacteremia

Bacteremia is an invasion of the bloodstream by

bacteria. The blood is normally a sterile environ-

ment [ 21 ]. So the detection of bacteria in the

blood (most commonly accomplished by blood

cultures) is always abnormal. This may occur

through a wound or infection or through a surgical

procedure or injection when other foreign bodies

are entering the arteries or veins. Bacteremia may

cause no symptoms and resolve without treat-

ment, or it may produce several consequences like

fever and other symptoms of infection. In some

cases, the immune response to the bacteria can

cause sepsis and septic shock, a potentially life-

threatening condition which has a relatively high

mortality rate. Bacteria can also use the blood to

spread to other parts of the body (which is called

hematogenous spread), causing infections away

from the original site of infection.

The oral cavity is intensely colonized by bac-

teria. Recent advances in bacterial identiﬁ cation

methods, particularly culture-independent

approaches such as 16S rRNA gene sequencing,

have shown that the oral cavity is inhabited by

more than six billion bacteria representing in

excess of 700 species belonging to at least nine

different phyla [

22 ]. Bacteremia occurs with vari-

ous frequencies following dental procedures and

has been well documented. As early as 1990,

Heimdahl et al. detected the patients with bacte-

remia after dental extraction, third-molar surgery,

dental scaling, endodontic treatment, and bilat-

eral tonsillectomy by means of lysis ﬁ ltration of

blood samples with subsequent aerobic and

anaerobic incubation. Their results showed that

bacteremia was observed in 100 % of patients

after dental extraction, 55 % of patients after

third-molar surgery, 70 % of patients after dental

scaling, 20 % of patients after endodontic treat-

ment, and 55 % of patients after bilateral tonsil-

lectomy. And anaerobic microorganisms were

isolated more frequently than aerobic microor-

ganisms [ 23 ]. Transient bacteremia is produced

not only as a result of dental manipulation. Even

daily life activities such as eating, chewing gum,

brushing the teeth, or using toothpicks also

induce bacteremia detectable by means of blood

cultures in a variable percentage of subjects [

24 ].

Three mechanisms or pathways linking oral

infections to secondary systemic effects have

been proposed for several years [ 6 ]. Li et al. sum-

marized the mechanisms as the following: meta-

static spread of infection from the oral cavity as a

result of transient bacteremia, metastatic injury

from the effects of circulating oral microbial tox-

ins, and metastatic inﬂ ammation caused by

immunological injury induced by oral microor-

ganisms [ 11 ].

Till now, there is no direct evidence to prove

the connection between the dental caries and

8 Dental Caries and Systemic Diseases

132

bacteremia, but we can ﬁ nd some clues from

published papers. Debelian et al. used phenotypic

and genetic methods to trace microorganisms

released into the bloodstream during and after

endodontic treatment back to the root canal.

Microbiological samples were taken from the root

canals of 26 patients with asymptomatic apical

periodontitis of single-rooted teeth. The blood of

the patients was drawn during and 10 min after

endodontic therapy. The results found that

microorganisms from the root canal and blood

presented identical phenotype and genetic charac-

teristics within the patients examined,which dem-

onstrated that endodontic treatment can be the

cause of anaerobic bacteremia and fungemia.

Interestingly, some cariogenic bacteria were also

isolated from the blood, such as Streptococcus

sanguinis [ 10 ]. Streptococcus mutans and

Streptococcus sanguinis are most consistently

been associated with the initiation of dental caries.

The results not only illustrated that dental caries is

the most common cause of pulpitis and periapical

diseases but also showed a clue that cariogenic

bacteria may be related to bacteremia.

These bacteria are normally harmless as long

as they are kept in check by the body’s natural

barriers and the immune system. In the oral cav-

ity there are several barriers to bacterial penetra-

tion from dental plaque into the tissue: a physical

barrier composed of the surface epithelium;

defensins, which are host-derived peptide antibi-

otics, in the oral mucosal epithelium; an electri-

cal barrier that reﬂ ects the Eh difference between

the host cell and the microbial layer; an immuno-

logical barrier of antibody-forming cells; and the

reticuloendothelial system (phagocyte barrier)

[

25 ]. However, once the equilibrium is disturbed

by an overt breach in the physical system (e.g.,

trauma) or immunological barriers (e.g., through

neutropenia, AIDS), organisms can propagate

and cause both acute and chronic infections with

increased frequency and severity [ 25 , 26 ]. In

addition, medical treatment (e.g., immunosup-

pressant therapy) may bring a person in contact

with new types of bacteria that are more invasive

than those already residing in that person’s body,

further increasing the likelihood of bacterial

infection.

8.2 Dental Caries and Head

and Neck Cancer

8.2.1 Dental Caries and Head

and Neck Cancer Treatment

Head and neck cancer accounts for more than

550,000 cases annually worldwide. The inci-

dence rate in males exceeds 20 per 100,000 in

regions of France, Hong Kong, the Indian sub-

continent, central and eastern Europe, Spain,

Italy, and Brazil and among African Americans

in the Unites States. Mouth and tongue cancers

are more common in the Indian subcontinent

[ 27 ]. Surgical resection, radiotherapy, and che-

motherapy, either used singly or in combination,

are the three most common modalities used in

head and neck cancer treatment. Despite their

effects in eradicating the tumor, they also

negatively impact the normal head and neck

structures surrounding the tumor. Surgical resec-

tion removes abnormal tissue, while radio- and

chemotherapy frequently cause direct damage to

the oral soft and hard tissue, and indirect damage

may also arise from systemic toxicity.

Firstly, we will discuss the radiotherapy

because radiation caries is a common disease in

clinic. We all know that saliva in the oral cavity

protects hard tissues against acid attacks and

demineralization. Salivary glands are very sus-

ceptible to radiation, and any disturbances in

their function are detrimental to the hard tissues

in the oral cavity. Radiation caries is mainly an

indirect effect of irradiation-induced changes in

salivary gland tissue that result in hyposalivation

[

28 ]. Hyposalivation leads to accelerated dental

caries through changes in salivary composition,

a shift in oral ﬂ ora toward cariogenic bacteria,

and dietary changes [ 29 ]. It is reported that the

initial caries usually occur around the third week

of treatment [ 30 ]. Mohammadi et al. [ 31 ]

reviewed 27 cases with head and neck cancers

undergoing radiotherapy. Of these cases, class V

dental caries of posterior teeth were evaluated in

three intervals: before treatment, 3 weeks after

the initiation of the treatment, and at the end of

the treatment. The baseline is that there were no

class V decays prior to radiotherapy. Their

Z. Ling and H. Tao

133

results found that mean percentages of class V

caries 3 weeks after radiotherapy and at the end

of radiotherapy were 28.42 % ± 14.41 and

67.05 % ± 19.02, respectively. These ﬁ ndings are

in accordance with the results of other studies

[ 28 , 32 ]. Since the severity of xerostomia is

related to the radiation dose, dose rate, and

amount of salivary tissue irradiated, the authors

also pointed out that further studies should eval-

uate the effects of new techniques such as inten-

sity-modulated radiotherapy on occurrence of

dental caries, in which a higher dose is beamed

at the tumor site without increased received dose

of the surrounding tissues [ 31 ].

Secondly, we talk about which one of the

three modalities, either used singly or in combi-

nation, is the most common cause of dental car-

ies after therapy. In order to determine the

prevalence of dental caries in cancer survivors,

Catherine et al. conducted a systematic literature

search with assistance from a research librarian

in the databases MEDLINE/PubMed and

Embase for articles [ 33 ]. Finally, 64 published

papers between 1990 and 2008 were reviewed.

Dental caries was assessed by the present (Y/N),

DMFT/dmft, and DMFS/dmfs indexes if avail-

able. Their results showed that the weighted

overall prevalence of dental caries was 28.1 %

and was determined from 19 studies. The

weighted prevalence of dental caries in patients

who received only chemotherapy was 37.3 %.

The weighted prevalences of dental caries in

patients who were post- radiotherapy and those

who were post- chemotherapy and post-radio-

therapy were 24 and 21.4 %, respectively. The

authors attributed the discrepancy to the distinct

differences in the dental management of patients

prior to radiotherapy versus those being prepared

for chemotherapy. Another explanation for the

unanticipated caries prevalence may be because

12 of the 19 studies included were carried out on

children undergoing hematologic malignancies

who were treated largely by curative chemother-

apy. They could have higher caries activity

because of the need to frequently consume

highly cariogenic dietary supplements for weight

maintenance or are taking sucrose-rich medica-

tions. In addition, their oral hygiene may be

ignored. In contrast to the caries prevalence, the

DMFT index is expectedly highest in patients

who were post-radiation therapy compared to

patients who were post- chemotherapy and

healthy controls [ 33 ].

8.2.2 Dental Caries and Head

and Neck Squamous Cell

Carcinoma

Recently, an interesting paper published online

in JAMA Otolaryngology – Head and Neck

Surgery showed that the bacteria that caused

tooth decay are linked to an immune response,

which may be protective against cancer [ 34 ].

The researchers from the University at Buffalo,

NY, set out to determine if there is a signiﬁ cant

link between dental cavities and head and neck

squamous cell carcinoma (HNSCC). The study

involved 399 patients newly diagnosed with

HNSCC and 221 participants without the cancer

who were all selected from the Department of

Dentistry and Maxillofacial Prosthetics at

Roswell Park Cancer Industry between 1999 and

2007. The dental history of all patients, particu-

larly their history of dental cavities, was ana-

lyzed by measuring the number of decayed,

missing, and ﬁ lled teeth. Of the 399 patients

with HNSCC, 146 (36.6 %) had oral cavity squa-

mous cell carcinoma (SCC). Oropharyngeal

SCC occurred in 151 (37.8 %) patients, while

102 (25.6 %) had laryngeal SCC. The results of

the study overall showed that those who had high

cavity numbers were less likely to have HNSCC,

compared with participants who had low cavity

numbers. The authors explained that “Caries is a

dental plaque-related disease. Lactic acid bacte-

ria cause demineralization (caries) only when

they are in dental plaque in immediate contact

with the tooth surface. The presence of these

otherwise beneﬁ cial bacteria in saliva or on

mucosal surfaces may protect the host against

chronic inﬂ ammatory diseases and HNSCC. We

could think of dental caries as a form of ‘collat-

eral damage’ and develop strategies to reduce its

risk while preserving the beneﬁ cial effects of the

lactic acid bacteria” [

34 ].

8 Dental Caries and Systemic Diseases

134

8.2.3 Tooth Loss and Head

and Neck Cancer Risk

As previously mentioned, caries is one of the

most common reasons of tooth loss; we will

include the relationship between tooth loss and

tumors in this chapter. Actually, multiple epide-

miologic studies regarding the potential associa-

tion of tooth loss with head and neck cancer risk

have been published nowadays [ 35 – 38 ]. But con-

sidering the modest sample size and different

study designs, the evidence still remains contro-

versial. Therefore, a quantitative and systematic

summary of the evidence using rigorous methods

is necessary. We know that meta-analysis is the

use of statistical methods to combine results of

individual studies. This allows us to make the

best use of all the information we have gathered

in our systematic review, and by statistically

combining the results of similar studies, we can

improve the precision of our estimates of treat-

ment effect and assess whether treatment effects

are similar in similar situations. Recently, some

Chinese researchers from Guangxi Medical

University conducted a meta-analysis involving

5,204 patients and 5,518 controls to assess the

inconsistent results from published studies on the

association of tooth loss with head and neck can-

cer risk [ 39 ]. Their overall estimates provided

evidence that tooth loss was signiﬁ cantly associ-

ated with increased risk of head and neck cancer.

In addition, the moderate [ 6 – 15 ] tooth loss and

the severe (>15) tooth loss experienced a signiﬁ -

cantly increased risk of head and neck cancer by

18 and 54 %, respectively. Furthermore, the mod-

erate [

6 – 15 ] tooth loss was associated with a

45 % increase in the risk of larynx cancer. The

authors also summarized that several plausible

mechanisms may explain why a signiﬁ cant

increased association of tooth loss with head and

neck cancer was observed in their analysis.

Whether tooth loss is an independent risk fac-

tor of head and neck cancer is an interesting

question. But the answers have not reached con-

sistent conclusions yet. Guha et al. observed that

missing 6–15 teeth increased the odds ratio of

esophageal squamous cell carcinoma by more

than twofold in both Latin America and central

Europe. However, when missing teeth were more

than 15 in number, no increase risk was observed

[ 40 ]. On the other hand, Wang et al. found that

moderate and severe tooth loss did not change

such an association, suggesting that tooth loss is

probably an independent risk factor of head and

neck cancer [ 39 ].

8.2.4 Cariogenic Bacteria and Oral

Cancer

Alcohol is one of the main risk factors for oral

cancer. Alcohol itself is not carcinogenic, but it is

oxidized to carcinogenic acetaldehyde in saliva

by the ADH enzyme of some oral microbes of the

normal oral microﬂ ora. Oral streptococci, espe-

cially S. mutans , are the primary pathogens caus-

ing dental caries, and Neisseria strains are related

to the early stage of caries. About a decade

before, some Neisseria strains are found to be

able to produce signiﬁ cant amounts of acetalde-

hyde, probably via their high alcohol dehydroge-

nase (ADH) activity [ 41 ]. Neisseria strains are

considered to be part of the normal oral ﬂ ora, but

they are found only in low numbers in the oral

cavity. Later, in 2007, oral streptococci were

proved to contribute signiﬁ cantly to the normal

individual variation of salivary acetaldehyde lev-

els after alcohol drinking and thereby also to the

risk of oral cancer [ 42 ]. We believe that the effect

of cariogenic bacteria on oral cancer provides

some evidence between caries and cancer from

another side.

8.3 Dental Caries and Children

Growth

There’s no doubt that dental caries constitutes the

single most common chronic disease of child-

hood: as many as 60 % of school children have

experienced dental caries, and the data can reach

as high as 90 % in some countries according to

the report of World Health Organization (WHO)

[ 43 ]. Among 5- to 17-year-olds, dental decay is

ﬁ ve times as common as asthma and seven times

as common as hay fever [ 44 ]. Current evidences

Z. Ling and H. Tao

135

show that dental caries is a multifactorial disease

and complexly modulated by genetic, behavioral,

social, and environmental factors [ 45 ]. A recent

descriptive cross-sectional study assessed dental

caries experience among 12-year-old school chil-

dren from low socioeconomic status background

attending public primary schools in Zimbabwe.

The results showed that there was a high preva-

lence of dental caries in both urban (59.5 %) and

rural (40.8 %) children [ 46 ]. While most people

in rural areas in Zimbabwe cannot afford and per-

ceive these sugary products as non-beneﬁ cial,

affording them is often considered as a symbol of

higher socioeconomic status. Another retrospec-

tive cohort study gave support to the idea that

children who lived in urban areas showed 75 %

greater probability of presenting caries when

compared to those children residing in rural areas

[ 47 ]. This disparity between urban and rural chil-

dren has been partially attributed to increased

access and consumption of high sugar-containing

foods and beverages in urban areas [ 48 ]. Based

on the recent studies, socioeconomic status has

been shown to be a major risk for caries inci-

dence. Children living in poverty represent a

large population of high-risk individuals who

have undiagnosed and untreated diseases coupled

with limited access to care. Nearly twice the pro-

portion of US children with family incomes less

than the federal poverty level (FPL) show decay

of the primary or permanent dentition (55 %),

compared to those whose family incomes are

greater than 200 % of the FPL (31 %). Low-

educated and low-income families that pay less

attention to the dental hygiene of their children

may be one of the reasons [

49 ].

Apart from structurally weakening teeth, den-

tal caries can lead to infection, pain, abscesses,

chewing problems, poor nutritional status, and

gastrointestinal disorders. Moreover, serious car-

ies can damage a child’s sense of self-esteem,

which in turn may affect his or her school perfor-

mance, ability to learn, and potential to thrive

[ 50 ]. Speciﬁ cally, in young children, there is a

relationship between dental caries and childhood

obesity [ 51 , 52 ]. Dental caries can also contribute

to poor nutritional status and affect the growth of

adult teeth [ 53 ]. In addition, children with

extensive dental caries may need to undergo

treatment under general anesthesia in hospital.

This is a signiﬁ cant side effect of childhood car-

ies that is widely acknowledged by the experts. It

is essential to remember that dental caries is one

of only very few common childhood diseases

which cause large numbers of the child popula-

tion to undergo general anesthesia.

The relationship between dental caries and

child’s body weight was ﬁ rstly noticed by Miller

30 years ago [

54 ]. Caries of the primary teeth or

“early childhood caries” (ECC) is one of the most

prevalent health problems in infants and toddlers

[ 55 ]. A recent study found a positive correlation

between severe early childhood caries (S-ECC)

and body mass index (BMI) of 3- and 6-year-old

children, which means the mean BMI of S-ECC

children is signiﬁ cantly more than the caries-free

children [ 56 ]. We know that if caries involve the

pulp, the eating of some foods will cause pain;

therefore, toothache and infection alter eating

and sleeping habits, dietary intake, and metabolic

processes [ 57 ]. For example, some of the patients

may thereby avoid certain nutritious foods and

select high-calorie, high-fat food, which is recog-

nized as risk factors for obesity. On the other

hand, some patients cannot pulverize the foods

well and may have an adverse effect on the inter-

nal absorption of nutrients. But if such bad oral

condition has been changed, the children’s

growth will be better. In 2009, Malek et al. con-

ducted a longitudinal clinical trial study to exam-

ine whether the removal of carious teeth affected

children’s growth relative to that of a standard

population. Five- and six-year-old children who

attended for extraction of carious teeth under

general anesthesia took apart in this study. The

children’s dental caries levels, weight, and height

were measured prior to extraction using standard

criteria and a single trained examiner, and they

were then remeasured 6 months later. The partici-

pants had a mean dmft of 7.18 (SD 3.27) at base-

line, and at follow-up children showed a

statistically signiﬁ cant gain in BMI SDS and a

small gain in height SDS [

58 ]. In their another

longitudinal birth cohort, Kay et al. found that

children who had caries at 61 months had slower

increases in weight and height than those without

8 Dental Caries and Systemic Diseases

136

decay at the same age [ 59 ]. These observations

were consistent with a recent study which exam-

ined the association between untreated dental

caries in primary and permanent teeth with age-

adjusted height and weight among 6–12-year-old

children in Bangladesh [ 60 ].

However, the relationship between dental car-

ies and child’s growth is inconclusive so far. A

research from the department of cardiology,

endodontology, and pedodontology in Academic

Centre for Dentistry Amsterdam (ACTA) has

been published in Clin Oral Investig in 2011. The

study has two objectives: ﬁ rst, to assess the rela-

tion between dental caries and body proportions

cross-sectionally in a Suriname caries child pop-

ulation and, second, to investigate whether dental

treatment had a signiﬁ cant inﬂ uence on body

growth of these children in a randomized con-

trolled trial using different treatment strategies.

Three hundred eighty 6-year-old children with

untreated dental decay participated in the study.

Participants were evaluated after 6 months and 1,

2, and 3 years. However, negative correlations

were observed between anthropometric measures

and the number of untreated carious surfaces and

caries experience of the children. Next, no sig-

niﬁ cant differences in growth pattern between the

treatment groups were observed. Thus, the

authors suggested that caries activity is a negative

predictor for body growth in children, and dental

intervention does not show signiﬁ cant improve-

ment within 3 years [ 61 ]. Later, Merrilyn et al.

undertook an updated systematic review of the

relationship between body mass index and dental

caries in children and adolescents. The authors

searched MEDLINE, ISI, Cochrane, Scopus,

Global Health, and CINAHL databases and con-

ducted lateral searches from reference lists for

papers published from 2004 to 2011, inclusive.

Finally, a total 48 studies were included. Three

main patterns of relationships were found

between dental caries and BMI: 23 of the 48

studies found no association between BMI and

dental caries, 17 found a positive relationship

between BMI and dental caries, and 9 found an

inverse relationship. The reasons that authors

analyzed may be method of dental examination,

sample differences, dental caries prevalence, and

BMI distribution. And they also recommend that

future research investigate the nature of the asso-

ciation between body mass index and dental car-

ies in samples that include a full range of body

mass index scores and explore how factors such

as socioeconomic status mediate the association

between body mass index and dental caries [ 62 ].

8.4 Dental Caries

and Atherosclerosis,

Cardiovascular Disease,

and Heart Attack

Atherosclerosis (also known as arteriosclerotic

vascular disease or ASVD) is a speciﬁ c form of

arteriosclerosis in which an artery wall thickens as

a result of the accumulation of fatty materials such

as cholesterol and triglyceride. Cardiovascular

disease ( CVD ) is the broad term used to categorize

any abnormal condition characterized by dysfunc-

tion of the heart and blood vessel system, princi-

pally referring to cardiac disease, vascular diseases

of the brain and kidney, and peripheral arterial dis-

ease. Evidence suggests a number of traditional

risk factors for atherosclerosis and CVD: age, gen-

der, high blood pressure, high serum cholesterol

levels, tobacco smoking, excessive alcohol con-

sumption, sugar consumption [ 63 ], family history,

obesity, lack of physical activity, psychosocial fac-

tors, diabetes mellitus, and air pollution [ 64 ].

However, these factors cannot explain all the

deaths from CVD. For example, about 40 % of

coronary heart disease (CHD) deaths occur in peo-

ple with cholesterol levels that are lower than the

population average [

65 ]. Therefore, medical

researchers’ attention has focused in recent years

on identifying additional risk factors that are non-

traditional but may play major roles in explaining

some of the variability in atherosclerosis and CVD

risk.

During the last three decades, there has been

an increasing interest in the impact of oral health

on atherosclerosis and subsequent cardiovascular

disease (CVD). Just as Meurman et al. wrote in

their paper which was published in Crit Rev Oral

Biol Med : “chronic infections caused by a variety

of micro-organisms are thought to be involved in

Z. Ling and H. Tao

137

the etiopathogenesis of CVD by releasing cyto-

kines and other pro-inﬂ ammatory mediators that

may initiate a cascade of biochemical reactions

and cause endothelial damage and facilitate cho-

lesterol plaque attachment. Yet, due to the multi-

factorial nature of dental infection and CVD,

conﬁ rming a causal association is difﬁ cult, and

the published results are conﬂ icting. The main

deﬁ cit in the majority of these studies has been

the inadequate control of numerous confounding

factors, leading to an overestimation and the

imprecise measurement of the predictor or over

adjustment of the confounding variables, result-

ing in underestimation of the risks” [ 66 ].

8.4.1 Dental Caries

and Atherosclerosis

and Cardiovascular Disease

Many studies have looked at poor dental care as a

risk factor for cardiovascular disease (CVD). The

results have been inconsistent, but most studies

support a modest association between them [ 67 ].

Mattila et al. may be one of the ﬁ rst researchers

to indicate a relationship between orofacial infec-

tions and cardiovascular disease. In 1989, they

published an article in British Medical Journal

(BMJ) and reported that there was an unexpected

correlation between dental disease and systemic

disease. After adjusting for age, exercise, diet,

smoking, weight, blood cholesterol level, alcohol

use, and health care, people who had caries and

periodontal disease had a signiﬁ cantly higher

incidence of acute myocardial infarction [

68 ].

Another prospective cohort study, published in

1993, found that patients with periodontal dis-

ease had a 25 % increase in CVD, and men

younger than 50 years had a signiﬁ cantly higher

risk. However, no association between extent of

active dental decay and risk of coronary heart dis-

ease was observed. Since tooth loss in people

under 60 is usually caused by dental caries, the

authors said they cannot rule out the possibility

that the increased risk of coronary heart disease

among young men with no teeth may have been

related to previous dental decay [ 69 ]. These

important discoveries resulted from the study is

not the only reason that makes it noteworthy.

Actually, the 9,760 subjects included in this work

make it the largest sample size of its kind at that

time. Since then, investigating the relationship

between dental disease and CVD has become a

priority.

Later, in 2001, a prospective cohort study in

Stockholm, Sweden, followed 1,393 individuals

for 27 years and concluded that oral health was a

risk factor for death due to CVD, especially in

combination with smoking, another risk factor.

In this investigation, a signiﬁ cant correlation

between caries and death due to CVD when

adjusted for age and gender was demonstrated,

indicating that this possible etiological pathway

should be further investigated in the future. And

the number of tooth surfaces with caries and pres-

ence of plaque were signiﬁ cantly increased for

smokers compared to nonsmokers [

69 ].

Maharaj and Vayej studied 44 black patients

with severe rheumatic heart disease before they

had cardiac surgery in 2012. Abnormalities were

detected in the panoramic radiographs of 84.1 %

of patients. The most frequent lesion was caries,

present in 56.8 % of patients, followed by miss-

ing teeth in 54.5 %, and impacted teeth in 25 % of

patients. Retained roots were present in 22.7 %

and periapical pathology was detected in 18.1 %

of patients [ 70 ].

It is clear that minimal carious lesions, caries

with and without involvement of the pulpal cavity,

and chronic apical periodontitis (CAP) represent

different stages of the same inﬂ ammatory pro-

cess. A recent study shows for the ﬁ rst time that

dental caries, pulpal caries, and chronic apical

periodontitis are associated positively, while res-

torations are associated inversely, with aortic ath-

erosclerotic burden [

71 ]. The authors’ result

showing that not only CAP but also caries with

pulpal decay or no visible pulpal decay was asso-

ciated with a greater atherosclerotic burden was

somewhat surprising. We know that early stage of

caries is an inﬂ ammatory process localized in the

oral cavity that does not affect the pulpal cavity or

the bone, indicating that a lesser extent of associa-

tion of the early stage of caries with the athero-

sclerotic burden was expected than with the other

two serious stages. One obvious explanation for

8 Dental Caries and Systemic Diseases

138

this ﬁ nding may be the covariance of these fac-

tors, as pulpal caries and CAP occur primarily in

patients with extensive tooth decay. The initial

carious lesion and caries not yet affecting the

pulpal cavity exist for a longer period compared

with the pulpal decay, which can precede pulpal

decay by a number of years. An explanation other

than the disease lasting many years is that even

forms of caries not yet involving the pulp are not

merely local inﬂ ammatory lesions but rather dis-

ease affecting the entire body. The authors sug-

gested that prospective studies are required to

conﬁ rm these observations and answer the ques-

tion of possible causality [ 71 ].

8.4.2 Root Caries and Cardiac

Dysrhythmia

and Gerodontology

Cardiac dysrhythmias, especially atrial ﬁ brilla-

tion, are known to cause ischemic heart disease.

Many studies suggested that inﬂ ammation plays

a prominent role in the onset of atrial ﬁ brillation

[ 72 ]. With respect to the result of logistic regres-

sion analysis, cariogenic bacteria have a speciﬁ c

impact on the pathogenesis of cardiac diseases,

especially dysrhythmias [ 73 ]. In 2005, research-

ers from University of Copenhagen designed a

cross-sectional study to examine whether caries

is associated with cardiac arrhythmias in

community- dwelling people aged 80 and older.

The primary ﬁ nding of the multivariate logistic

regression analysis was that persons with three or

more active root caries lesions had more than

twice the odds of cardiac arrhythmias than per-

sons without active root caries. The ﬁ ndings indi-

cated that there may be a link between active root

caries and cardiac arrhythmias in the oldest old

[

74 ]. In order to explain the link, we should turn

to the immune response because several studies

have reported that an increase in dental caries is

associated with a heightened immune response.

In addition, dental caries affects the production

of IgG and induces acute-phase proteins. The

inﬂ ammatory-mediated cytokines and acute-

phase proteins are practical markers of increased

risk of cardiovascular disease, such as C-reactive

protein (CRP), interleukin-6 (IL-6), and tumor

necrosis factor-α (TNF-α) [

75 ]. Bacterial species

lying within the root surfaces supporting struc-

tures induce systemic inﬂ ammation and immune

response, thereby increasing levels of serum CRP

and serum IgG. In 2011, Kaneko et al. conducted

a longitudinal study to elucidate the relationship

between root caries and the onset of dysrhyth-

mias on electrocardiographs in community-

dwelling persons aged 75 and older. Serum CRP

level was used as a variable to link root caries

with dysrhythmias directly. They found that a

high mean CRP serum level group had a signiﬁ -

cantly higher number of sites with root caries

than a low CRP group. Moreover, number of sites

with root caries events was signiﬁ cantly associ-

ated with cardiac dysrhythmia among nonsmok-

ers. These results conﬁ rmed that root caries is

related to the incidence of dysrhythmias in non-

smokers [ 73 ].

8.4.3 Streptococcus mutans

and Atherosclerosis

We know that Streptococcus mutans ( S. mutans )

is a major cariogenic pathogen that is a normal

inhabitant of the oral cavity in most individuals.

S. mutans has also been isolated from the blood

of patients with infective endocarditis (IE), which

strongly suggests a close relationship of the

pathogen with IE [ 76 , 77 ]. Ullman et al. pointed

out that their experience agrees with the literature

and indicates that S. mutans is primarily a patho-

gen in elderly patients with heart disease and may

be associated with IHSS [

78 ]. In 2006, Nakano

from Osaka University Graduate School of

Dentistry and coworkers published the ﬁ rst study

to analyze the presence of streptococcal species

in diseased heart valve and atheromatous plaque

specimens, as well as in dental plaque samples

from the same subjects by a PCR method.

Unexpectedly, S. mutans was detected at high

frequencies and quantities in both heart valve tis-

sues and atheromatous plaque samples in their

study. Their conclusion indicated that S. mutans

is a possible causative agent of cardiovascular

disease [ 79 ]. In addition, when using DNA

Z. Ling and H. Tao

139

ﬁ ngerprinting to compare S. mutans isolated

from dental plaque and an infected heart valve

from a patient who underwent heart surgery,

Nomura and colleagues demonstrated that the

oral isolates differed from those found in the

heart valve [ 80 ]. Three years later, Nakano et al.

published another paper titled “Detection of oral

bacteria in cardiovascular specimens” in Oral

Microbiology and Immunology . This time, they

found that S. mutans was the most frequently

detected species in the cardiovascular specimens,

followed by Aggregatibacter actinomycetem-

comitans . Furthermore, the positive rate of

S. mutans in cardiovascular specimens from

patients whose dental plaque specimens were

also positive for S. mutans was 78 %, which was

signiﬁ cantly higher than any other tested species

when the same analysis was performed [ 81 ].

Collectively, these ﬁ ndings lend credence to the

idea that there are subpopulations of S. mutans

carried in humans that, while not necessarily

associated with caries, may have an enhanced

capacity to interact, and possibly invade, the cells

of the cardiovascular system [ 82 ]. S. mutans is

classiﬁ ed into four serotypes (c/e/f/k) based on

the chemical composition of its cell surface sero-

type-speciﬁ c rhamnose–glucose polymers

(RGPs), which form a backbone of rhamnose

polymers with side chains of glucose polymers.

Serotype c is reported to be the most prevalent in

oral isolates at approximately 70–80 %, followed

by e, f, and k. Serotypes e and f have been found

to invade endothelial cells [

82 ]. Serotype k, with

a defect of the glucose side chain in RGPs, was

found to show low cariogenicity but high viru-

lence in blood as compared to the other sero-

types, due to alterations of several cell surface

structures [ 83 ]. When it comes to the possible

reasons of the link between S. mutans and cardio-

vascular system diseases, it may be summarized

as the following:

1. One crucial step for the development of ath-

eromatous plaque lesions is formation of foam

cells, which are macrophages that accumulate

from excess cholesterol, and S. mutans strain

GS-5 has been shown to enhance their forma-

tion [ 84 ].

2. In addition, S. mutans were shown to induce

platelet aggregation, which presumably lead to

thrombus formation. It was also found that S.

mutans cells bind to extracellular matrix mol-

ecules and ﬁ brinogen with contribution from

the major surface protein antigen Pac [

85 , 86 ] .

3. The infection with S. mutans expressing

collagen- binding protein (CBP) is a potential

risk factor for hemorrhagic stroke [ 87 ]. Lately,

two types of cell surface collagen-binding

proteins, Cnm and Cbm, have been studied to

see if they play a role in S. mutans attachment

and invasion of human umbilical vein endo-

thelial cells (HUVEC). The results found that

most of the Cbm-positive strains showed

higher levels of binding to type I collagen as

well as higher rates of adhesion and invasion

with HUVEC as compared to the Cnm-

positive strains. Furthermore, the gene encod-

ing Cbm was detected signiﬁ cantly more

frequently in heart valve specimens from IE

patients than from non-IE patients [ 88 ].

8.4.4 Tooth Loss

and Cardiovascular Disease

and Stroke

While some studies have shown that decay is not

a direct risk factor, it can and does cause tooth

loss, which has been demonstrated to be a greater

risk for cardiovascular disease [ 89 , 90 ]. Recent

evidence showed a direct link between oral health

and CVD and that the number of teeth can be

used to assess increased risk of CVD in adults.

They drew the conclusion from a fairly large

( n = 7,647), prospective study with a long follow-

up period (1976–2002) that presents for the ﬁ rst

time a dose-dependent relationship between

number of teeth and both all-cause and CVD

mortality. The authors found that a person with

fewer than 10 of their own teeth remaining is

seven times more likely to die of coronary dis-

ease than someone with more than 25 of their

own teeth [

91 ].

As pointed out by Watt et al., although there is

increasing epidemiological evidence linking poor

oral health with the development of chronic diseases

8 Dental Caries and Systemic Diseases

140

and mortality, these associations are still doubtful

due to imprecise measurement of important risk

factors of systemic disease. Indeed, most previous

studies exploring the link between tooth loss and

systemic disease have been conducted in selected

samples and have failed to control adequately for

socioeconomic, behavioral, and general health sta-

tus [ 92 ]. Thus, their recent prospective cohort study

of a national sample of Scottish adults published in

PLOS ONE caught our attention. The sample con-

sisted of 12,871 participants and they were followed

for 8.0 (SD: 3.3) years. During 103,173 person-

years, there were 1,480 cases of all-cause mortality,

498 of CVD, and 515 of cancer. After adjusting for

demographic, socioeconomic, behavioral, and

health status, edentate subjects had signiﬁ cantly

higher risk of all-cause and CVD mortality com-

pared to subjects with natural teeth only. Their ﬁ nd-

ings conﬁ rm previous studies which have shown a

small but signiﬁ cant association between tooth loss

and all-cause and CVD deaths after controlling for

a range of potential confounding factors [ 92 ].

However, there were different opinions from

other studies, such as the study using the data from

the Glasgow Alumni Cohort to investigate whether

oral health in young adulthood is independently

associated with cause‐speciﬁ c mortality after

accounting for childhood socioeconomic back-

ground and other risk factors in young adulthood.

Over 12,000 subjects (30 years or younger at base-

line) were traced during up to 57 years of follow‐

up, and 1,432 deaths occurred among subjects

with complete data, including 509 deaths from

CVD and 549 from cancer. When the number of

missing teeth was treated as a categorical variable,

there was evidence that students with nine or more

missing teeth at baseline had an increased risk of

CVD compared with those with fewer than ﬁ ve

missing teeth. When the number of missing teeth

was transformed using fractional polynomials,

there seemed to be a nonlinear relation between

missing teeth and CVD mortality [

93 ].

Stroke remains the third leading cause of death

(after heart disease and cancer) in most developed

countries. Cerebrovascular ischemic strokes are

the commonest kind of stroke and occur as a result

of an obstruction, usually a clot, within a blood

vessel supplying blood to the brain. Heitmann and

Gamborg examined if the number of remaining

teeth was associated with the development of car-

diovascular morbidity and mortality over 5–12

years. The prospective observational study

among1474 men and 1458 women born 1922,

1932, 1942 or 1952 from The Danish MONICA

follow up study (monitoring trends in and determi-

nants of cardiovascular disease) in 1987–88 and

1993–94. Their results showed that tooth loss was

strongly associated with incidence of stroke and,

to a lesser extent, incidence of cardiovascular dis-

ease and coronary heart disease, during averagely

7.5 years of follow-up [

94 ]. Choe et al. conducted

a prospective cohort study of stroke in Korea on

hypertension, diabetes, smoking, and tooth loss to

characterize their independent effects and interac-

tions. They conﬁ rm that tooth loss is independently

associated with increased risk of stroke, and hyper-

tension does interact antagonistically, particularly

for hemorrhagic stroke [ 95 ]. A recent study found

that stroke patients in their 50s and 60s had signiﬁ -

cantly fewer remaining teeth than did patients hos-

pitalized for other conditions in the corresponding

age groups. Moreover, the number of remaining

teeth was signiﬁ cantly lower among stroke patients

in their 50s than data reported for that age group in

the Survey of Dental Diseases, suggesting the pos-

sibility that stroke patients may have lost teeth at a

younger age. The authors also pointed out that the

association between stroke and tooth loss can be

explained by common risk factors associated with

lifestyle such as hypertension, diabetes, smoking,

and alcohol intake. It is quite difﬁ cult to rule out all

common risk factors as confounding variables;

therefore, the exact mechanisms of the relationship

between stroke and tooth loss are difﬁ cult to iden-

tify [

96 ]. Interestingly, periapical lesions, normally

resulting from an infected root canal (caused by

caries), are also a factor in stroke risk. This is

another example of how dental caries can play a

role, however indirectly, in heart disease [ 5 ].

8.4.5 Pulpal Periapical Diseases

and Coronary Heart Diseases

Endodontic inﬂ ammation occurs after bacteria or

their byproducts enter a tooth’s pulp chamber.

Z. Ling and H. Tao

141

Apical periodontitis is an acute or chronic

inﬂ ammatory lesion around the apex of a tooth root

which is caused by bacterial invasion of the pulp of

the tooth. Despite numerous differences between

chronic inﬂ ammatory disease of periodontal and

endodontic origins, Caplan et al. summarized their

similarities, primarily that: (1) both conditions share

a common microbiota that often is associated with

Gram-negative anaerobic bacteria, and (2) elevated

systemic cytokine levels have been observed in con-

junction with both disease processes [ 97 ]. Since

several epidemiologic investigations have uncov-

ered relationships between chronic periodontal dis-

ease and coronary heart disease, links between

endodontic inﬂ ammation and cardiovascular out-

comes are biologically plausible. Mechanisms link-

ing endodontic disease to CHD might be similar to

those hypothesized for associations between peri-

odontal disease and CHD, in which a localized

inﬂ ammatory response to bacterial infection leads

to release of cytokines into the systemic circulation

and to subsequent deleterious vascular effects [ 98 ].

Frisk and colleagues are one of the ﬁ rst groups who

speciﬁ cally studied endodontic variables as risk fac-

tors for the development of CHD. However, they

did not reveal a signiﬁ cant association between end-

odontically treated teeth and CHD nor between

teeth with periapical disease and CHD from their

population-based study of Swedish women [ 99 ].

Joshipura et al. found a possible modest association

between pulpal inﬂ ammation and CHD, although

dental caries was not associated with CHD. They

observed a greater CHD incidence among men with

a positive self-reported history of ET in the Health

Professionals Follow-Up Study [

100 ].

8.4.6 Summary

As stated above, several epidemiologic investiga-

tions have uncovered relationships between den-

tal caries (including its subsequent pulpal

periapical diseases and tooth loss) and coronary

heart disease. However, other studies have not

found signiﬁ cant relationships, sparking ques-

tions about the proposed association. In the

future, more direct evidence should be found to

support this connection.

8.5 Dental Caries and Immune

System Disease

It’s no doubt that infection stresses the immune

system. So dental infections, especially long-

term disease (such as periapical abscesses),

should have a deleterious effect on the immune

system. Dental caries is an infectious disease that

occurs because of imbalance in the homeostasis

between the host and microbiota [ 101 ]. Salivary

innate and adaptive immune defenses may inﬂ u-

ence in the bacterial colonization, and some dis-

orders can affect these systems such as general

immune deﬁ ciencies associated with malnutri-

tion, inherited or medication disorders, or other

factors that affect salivary ﬂ ow and saliva compo-

sition [ 102 ].

8.5.1 Salivary Immunoglobulin A

Oral microorganisms and aerodigestive antigens

are continuously inﬂ uenced by the two major anti-

body classes in saliva: SIgA and IgG. The former

is the dominant immunoglobulin in the healthy

mouth which is produced by gland- associated

immunocytes. It can agglutinate oral bacteria,

modulate enzyme activity, and inhibit the adher-

ence of bacteria to the buccal epithelium and to

enamel. It does well at interfering with the initial

colonization of caries-associated microﬂ ora on the

tooth surface. Thus, a protective role for salivary

sIgA was postulated [

103 – 105 ]. However, about

the relationship between Salivary IgA and dental

caries, different studies got contrary results and

conclusions. In 1978, Challacombe S. J. did not

ﬁ nd that salivary antibodies in man play a role in

protection against caries [ 106 ]. Several years later,

Gregory et al. found that caries-free subjects or

individuals with low caries susceptibility exhibited

signiﬁ cantly higher levels of naturally occurring

salivary immunoglobulin A (IgA) and serum IgG,

IgA, and IgM antibodies to a Streptococcus mutans

ribosomal preparation than subjects with high car-

ies susceptibility [ 107 ]. Recently, some Indian

researchers from MAR Dental College carried out

a protocol-driven cross-sectional pilot study to

check the purported association between salivary

8 Dental Caries and Systemic Diseases

142

sIgA and dental caries with special reference to

RA. Rheumatoid arthritis (RA) is an autoimmune

disease that results in a chronic, systemic inﬂ am-

matory disorder that may affect many tissues and

organs, but principally attacks ﬂ exible (synovial)

joints. It can be a disabling and painful condition,

which can lead to substantial loss of functioning

and mobility if not adequately treated. Forty-eight

patients with RA and 102 non-RA, healthy case

controls were taken part in the study. The decay,

missing teeth, ﬁ lled teeth (DMFT) index was used

to classify caries. Whole unstimulated saliva was

collected to assay sIgA using a commercial ELISA

kit. The results showed that there were no statisti-

cally signiﬁ cant differences between RA and non-

RA subjects with respect to salivary sIgA and the

extent of caries [ 104 ]. It’s hard to explain all the

contrary results, since all the studies had their limi-

tations, such as the study designs, if the sample

size was insufﬁ cient or not, or technical variables

also inﬂ uence biomarkers and antibodies in oral

secretions. Further studies should be carried out to

ﬁ nd the truth.

8.5.2 HIV and Dental Caries

According to the World Health Organization,

globally, 34.0 million [31.4–35.9 million] people

were living with HIV at the end of 2011. In 2004,

Phelan J. A. et al. published a paper in Journal of

Dental Research titled “Dental caries in HIV-

seropositive women.” The authors conducted the

study to determine if there was an association

between HIV infection and dental caries among

women enrolled in the Women’s Interagency HIV

Study. Subjects included 538 HIV+ and 141

HIV− women at baseline and 242 HIV+ and 66

HIV− women at year 5. Caries indices included

DMFS and DFS (coronal caries) and DFSrc (root

caries). Cross-sectional analysis of coronal caries

data revealed a 1.2-fold-higher caries prevalence

among HIV+ women compared with HIV−

women. Longitudinally, DMFS increased with

increasing age and lower average stimulated sali-

vary volume. Root caries results were not signiﬁ -

cant except for an overall increased DFSrc

associated with smoking [

108 ]. At the same year,

Mulligan R. et al. also reported that seropositive

women who ﬁ t the Center for Disease Control

(CDC) AIDS criteria were also more likely to

have more DMF teeth ( P = 0.004), DMF surfaces

( P = 0.003), and decayed and/or ﬁ lled (DF) root

surfaces ( P = 0.0002) compared to seropositive

women without AIDS [

109 ].

It is estimated worldwide that there are 2.3

million human immunodeﬁ ciency virus (HIV)-

positive children from 0 to 14 years infected by

mothers [ 110 ]. However, prior to 1992, informa-

tion about dental caries in HIV-infected children

was very limited. Between 1992 and 1996, there

were three published cross-sectional studies of

dental caries in the primary teeth of HIV-infected

children [ 111 ]. These studies showed that there

was a higher prevalence of dental caries (includ-

ing early childhood caries (ECC)) in the primary

dentition of HIV-infected children as compared

to healthy children [ 112 – 114 ]. However, it has

not always been clear from past studies exactly

what factors bring about these differences that

exist in dental caries between HIV-infected chil-

dren and a noninfected control group. In order to

exclude the common environmental factor,

Tofsky et al. compared the baseline prevalence

and 2-year incidence of dental caries found in

both the primary and secondary dentition among

a group of HIV-infected children as compared to

their HIV-negative household peers. They found

that HIV-infected children have a high prevalence

of dental caries in the primary teeth and a low

prevalence in permanent teeth, while the inci-

dence of permanent tooth dental caries is less

than that of a group of noninfected household

peers [

115 ]. And reasons are still unknown.

It has been hypothesized that immunodeﬁ -

ciency and a progressive decrease in CD4+

T-lymphocytes resulting from HIV infection

might alter salivary ﬂ ow rate and impair the

secretory immune system, thus contributing to

increased bacterial colonization in the oral cavity

implying that cariogenic bacteria may also

increase in the oral cavity [ 116 ]. Those changes

could be contributing factors for the development

of HIV-associated oral diseases, including

increased prevalence of dental caries. A recent

study by Liu and coworkers examined the effect

Z. Ling and H. Tao

143

of HIV infection on the level and genotypic char-

acteristics of S. mutans colonization. They found

that HIV+ individuals experienced signiﬁ cantly

higher levels of S. mutans . Interestingly, the level

of S. mutans signiﬁ cantly correlated with CD8+

count, but not with viral load or CD4+ counts,

which is clearly suggesting that other HIV-

associated factors mechanistically mediate S.

mutans colonization in saliva. And they also

revealed decreased salivary ﬂ ow rate in the HIV+

group [ 117 ]. Since more dental caries was evi-

denced in HIV+ individuals from this study and

others, additional studies are required to eluci-

date and understand the correlation between the

colonization of other cariogenic microbes,

including S. mutans , and the status of immuno-

suppression at the advanced stages of HIV

infection.

8.6 Dental Caries and Kidney

Diseases

Chronic kidney disease (CKD), also known as

chronic renal disease, is a progressive loss in

renal function over a period of months or years.

The symptoms of worsening kidney function are

nonspeciﬁ c and might include feeling generally

unwell and experiencing a reduced appetite.

Often, chronic kidney disease is diagnosed as a

result of screening of people known to be at risk

of kidney problems, such as those with high

blood pressure or diabetes and those with a blood

relative with chronic kidney disease. Chronic

kidney disease may also be identiﬁ ed when it

leads to one of its recognized complications, such

as cardiovascular disease, anemia, or pericarditis.

In addition to the systemic manifestations and

complications that arise from the disease and its

treatment, changes in the mouth are common in

patients with chronic kidney disease (CKD).

Poor oral health in CKD patients may thus repre-

sent an important, but often overlooked, problem

[

118 , 119 ].

Several studies have demonstrated higher

rates of oral pathology in dialysis patients with

one or more oral symptoms such as xerostomia,

taste disturbances, uremic odor, tongue coating,

mucosal inﬂ ammation, oral ulceration, or enamel

hypoplasia. We know that xerostomia (or dryness

of the mouth) may predispose to caries and gingi-

val inﬂ ammation as well as contribute to difﬁ cul-

ties with speech, denture retention, mastication,

dysphagia, sore mouth, loss of taste, and infec-

tions. Studies in the general population suggest

that edentulous subjects are prone to have an

inappropriate dietary intake (such as ingesting

too little protein and too much calorie-rich, high-

fat food) as compared with dentated persons.

Whereas the number of teeth is of importance for

masticatory function, having premolar and molar

teeth is especially important for nutritional sta-

tus. The increased periodontitis and dental caries

rates of CKD patients lead to tooth loss, which

may result in chewing difﬁ culties because of

inadequate occlusive surfaces or the limitations

of prostheses. On the other hand, approximately

30 % of patients with advanced CKD are reported

to have a “bad” or a “metallic” taste in their

mouths, which has been associated with meta-

bolic changes, diverse drugs, a reduced number

of taste buds, and changes in both salivary ﬂ ow

rate and composition. Increased dental calculus

has been observed, perhaps as a consequence of a

high salivary urea and phosphate levels.

When it comes to the relationship between

dental caries and CKD, different researches got

different results, and sometimes the conclusions

were contradictory. Some studies showed that

uremic patients have higher rates of decayed,

missing, and ﬁ lled teeth, loss of attachment, and

periapical and mucosal lesions than the general

population. The consequences of poor oral health

may be more severe in CKD patients because of

advanced age, common existing additional dis-

eases such as diabetes, concurrent medications,

and a state of reduced immune function that may

increase the risk for consequences of periodonti-

tis and other oral and dental conditions [ 120 ].

Some researchers reported that the prevalence of

dental caries was low in children with renal dis-

ease [ 121 , 122 ]. As early as 1985, Peterson and

his colleagues got data to support the hypothesis

that the relative paucity of caries in patients with

chronic renal failure results from alteration of

plaque by metabolic end products of urea

8 Dental Caries and Systemic Diseases

144

metabolism. The data further suggested that

transplanted patients whose renal function is nor-

mal may be at increased risk of caries, especially

if enamel hypoplasia is present and oral hygiene

is poor [ 123 ]. Others have not found any evi-

dence that the prevalence of dental caries in per-

manent teeth is signiﬁ cantly different in CKD

children when compared with healthy children

[ 124 , 125 ].

A recent systematic review published in

Pediatric Nephrology tried to determine whether

there is any evidence in the literature referring to

a lower prevalence of dental caries in children

and adolescents with chronic kidney disease

(CKD) compared to healthy individuals. After

the evaluation of title, keywords, and abstracts of

the articles selected, six articles met the inclusion

criteria. Three of these six articles included stud-

ies which showed susceptibility to bias and pos-

sible confounding factors. A subsequent

assessment of the six studies revealed that the

mean caries indices in both primary (dmf) and

permanent (DMF) teeth were lower in the chil-

dren and adolescents with CKD compared with

healthy individuals. In these patients, the low

prevalence of dental caries may be associated

with salivary characteristics, especially the neu-

tralization of end products of bacterial plaque

due to the increased pH resulting from urea

hydrolysis in the saliva. So the authors concluded

that data in the literature weakly support a lower

prevalence of caries in children and adolescents

with CKD than in their healthy counterparts

[

126 ]. There is still a lack of well-designed stud-

ies that provide better scientiﬁ c evidence in sup-

port of this conclusion.

8.7 Dental Caries

and Gastrointestinal

Diseases

To date, the most signiﬁ cant relationship between

dental caries and gastrointestinal diseases is from

chewing pain and tooth loss. As we mentioned

before, carious teeth become pulpitic; the eating

of some foods will cause pain; therefore tooth-

ache and infection alter eating and sleeping

habits, dietary intake, and metabolic processes.

Moreover, the edentulous patient without den-

tures is the most vulnerable to gastrointestinal

and other related problems. In the edentulous

person with a deﬁ cient masticatory performance,

reduced consumption of ﬁ ber-rich foods that are

hard to chew could provoke gastrointestinal dis-

turbances. One study provided a sound basis for

why the denture wearer does not achieve the nec-

essary breakdown of food substances. The

research indicated that the chewing efﬁ ciency of

those wearing dentures was about one-sixth that

of a person with natural teeth. In addition, evi-

dence suggests that nutritional deﬁ ciencies,

regardless of their cause, are associated with

impaired immune responses [

5 ].

There are also some interesting researches

that connected dental caries and gastrointestinal

diseases, such as gastroesophageal reﬂ ux disease

and the effect of S. mutans on the ulcerative

colitis.

8.7.1 Dental Caries

and Gastroesophageal Reﬂ ux

Disease

Gastroesophageal reﬂ ux disease (GERD) is a

chronic symptom of mucosal damage caused by

stomach acid coming up from the stomach into

the esophagus [ 127 ]. GERD is usually caused by

changes in the barrier between the stomach and

the esophagus, including abnormal relaxation of

the lower esophageal sphincter, which normally

holds the top of the stomach closed, impaired

expulsion of gastric reﬂ ux from the esophagus, or

a hiatal hernia. These changes may be permanent

or temporary. The oral lesions resulting from

GERD are not usually noticed until they cause

signiﬁ cant damage. Despite the pruritus and

burning on the oral mucosa, tooth sensitivity,

aphthae, sour taste, and decrease in the vertical

dimension of occlusion to irreversible damage,

dental caries is still our focus in this chapter.

However, the results have been contradictory.

Some studies on the oral health of patients with

chronic reﬂ ux reported an inverse relation

between caries and gastroesophageal reﬂ ux and

Z. Ling and H. Tao

145

showed a small number of caries in individuals

with GERD as compared to control groups [ 128 ].

They attributed this to the low prevalence of bac-

teria (lactobacilli and streptococci) observed in

the saliva of patients with chronic reﬂ ux [ 129 ].

On the other hand, Linnett et al. conducted for 52

children (31 boys and 21 girls) with a deﬁ nitive

history of GERD. They found that caries experi-

ence was higher in GERD patients compared to

controls. Although there were more subjects with

Streptococcus mutans in the GERD group com-

pared to the control group (42 % vs 25 %), the

difference was not statistically signiﬁ cant [ 130 ].

Silva et al. [ 23 ] did not ﬁ nd relationship between

GERD and changes in the oral cavity by saliva

tests, oral clinical examination, or histopatho-

logic examination of the palatal mucosa [ 131 ].

The differences among the different studies may

be explained by the different research design, the

patients from different areas, and sample size.

Future studies are needed to explore the truth.

8.7.2 S. mutans and Ulcerative

Colitis

Ulcerative colitis (Colitis ulcerosa, UC) is a

chronic, or long-lasting, disease that causes

inﬂ ammation and sores in the inner lining of the

large intestine. The main symptom of active dis-

ease is usually constant diarrhea mixed with

blood, of gradual onset. The cause of UC is

unknown though theories exist. People with UC

have abnormalities of the immune system, but

whether these problems are a cause or a result of

the disease is still unclear. Current theories sug-

gest that indigenous gut microbiota play a key

role in the pathogenesis of inﬂ ammatory bowel

disease. Moreover, regulation of mucosal immune

response to unidentiﬁ ed components of normal

intestinal microbiota in a genetically susceptible

host is at the core of these diseases [

132 , 133 ].

In 2011, Nakano et al. found the speciﬁ c

strains of S. mutans that express collagen-binding

protein (CBP) caused hemorrhagic damages in

the murine brain and other tissues because of the

ability to bind the collagen and resistance to

phagocytosis [ 87 ]. In normal situations, only a

limited number of strains are possible risk factors

for aggravation of UC caused by S. mutans -

induced bacteremia. In contrast, the detection

frequency of speciﬁ c strains of S. mutans , such as

CBP-positive and phagocytosis-resistant strains

from UC patients, was extremely higher than in

non-UC control subjects. Thus the authors specu-

lated that such speciﬁ c strains of S. mutans may

be involved in the pathogenesis of UC. So the

next year, in 2012, they published another paper

to show that infection of speciﬁ c strains of

S. mutans is one of the risk factors in aggravating

inﬂ ammation of UC. They stated that it’s the ﬁ rst

paper describing the involvement of oral bacteria

in UC pathology [ 134 ].

8.8 Dental Caries and Diabetes

Mellitus

It’s no doubt that diabetes mellitus is a rapidly

growing health concern in both developed and

developing nations. According to the World

Health Organization (WHO), in 2011, approxi-

mately 364 million people globally suffer from

diabetes mellitus (DM), with projections that

DM-related deaths will double from 2005 to

2030 [ 135 ]. The Center for Disease Control

(CDC) estimates that in the United States alone,

25.8 million Americans, or 8.3 % of the popula-

tion, suffer from DM, with 7 million currently

undiagnosed [ 136 ]. Diabetes mellitus is classi-

ﬁ ed into four broad categories: type 1, type 2,

gestational diabetes, and “other speciﬁ c types.”

Type 1 diabetes mellitus is characterized by loss

of the insulin-producing beta cells of the islets of

Langerhans in the pancreas, leading to insulin

deﬁ ciency. Type 2 diabetes mellitus is character-

ized by insulin resistance, which may be com-

bined with relatively reduced insulin secretion.

Type 2 diabetes is the most common type. In

China, type 2 diabetes mellitus affects almost

92.4 million (9.7 %) Chinese adults, and 148.2

million adults are in the prediabetes. Based on

case numbers in 2007 and projected case num-

bers in 2030, Wang W. et al. estimated that the

direct medical costs of T2DM and its complica-

tions were estimated to be 26.0 billion USD in

8 Dental Caries and Systemic Diseases

146

2007 and were projected to be 47.2 billion USD

in 2030. The results indicated that T2DM con-

sumes a large portion of healthcare expenditures

and will continue to place a heavy burden on

health budgets in the future [ 137 ]. In 2008, Patiño

et al. carried out a cross-sectional study involving

175 subjects to determine the frequency of caries,

periodontal disease, and tooth loss in patients

affected by diabetes mellitus types 1 and 2. Their

results showed a difference between the two

types among the study variables [ 138 ]. Since we

care about the overall association between diabe-

tes mellitus and dental caries prevalence, we did

not make a distinction between type 1 and type 2

diabetes mellitus in this chapter.

It is widely understood that diabetes patients

are at an increased risk for oral complications

such as candidiasis, erosion, xerostomia, and

periodontal disease [ 139 , 140 ]. Studies have

reported that patients with diabetes are suscepti-

ble to oral sensory, periodontal, and salivary dis-

orders, which could increase their risk of

developing new and recurrent dental caries [ 141 ].

However, although the relationship between dia-

betes and dental caries has been investigated

since the last century, no clear association has

been clariﬁ ed till now [ 142 ].

8.8.1 Epidemiological Studies

of Diabetes and Dental Caries

In 2004, Taylor et al. reviewed the post-1960

English-language literature on the relationship

between diabetes and oral health, speciﬁ cally

focusing on periodontal disease, dental caries,

and tooth loss. The literature does not describe a

consistent relationship between type 2 diabetes

and dental caries. It reports increased, decreased,

and similar caries experiences between those

with and without diabetes. This review suggests

that currently there is insufﬁ cient evidence to

determine whether a relationship between diabe-

tes and risk for coronal or root caries exists [ 143 ].

Here are two studies that reported a greater

history of dental caries in people with diabetes.

In 2002, a study evaluated the caries incidence in

64 children and adolescents (8–15 years of age)

with type 1 diabetes mellitus over a 3-year period

from the onset of the disease in relation to meta-

bolic control and to caries-associated risk factors.

Results showed that patients with less good meta-

bolic control exhibited higher glucose levels in

resting saliva and a signiﬁ cantly higher caries

incidence compared to those with good meta-

bolic control. The most inﬂ uential determinants

for high caries development during the 3-year

follow-up period were metabolic control, poor

oral hygiene, previous caries experience, and

high levels of salivary lactobacilli [

144 ]. Later,

Miralles et al. conducted the other work which

comprised 90 type 1 diabetics between 18 and 50

years of age, and a group of non-diabetic controls

matched for age and sex. Their results showed

that under similar conditions of oral hygiene and

salivary ﬂ ow, the diabetic group showed a higher

incidence of caries than the control group.

Likewise, on speciﬁ cally analyzing the diabetic

group, the metabolic control of the disease, the

duration of diabetes, and the existence of compli-

cations of the disease exerted an inﬂ uence upon

the development of dental caries [ 145 ].

Other researchers, however, have detected no

signiﬁ cant difference or even a decrease in caries

susceptibility between diabetic and nondiabetic

patients. A decade ago, Moore et al. published a

paper to describe the prevalence of coronal and

root caries in an adult type 1 diabetic population

and evaluate demographic, dietary, behavioral,

physiologic, salivary, and medical variables asso-

ciated with decayed and ﬁ lled surfaces in the

crown (DFS) or root (RDFS). The authors found

that adult type 1 diabetic subjects did not have

signiﬁ cantly higher DFS rates as compared with

their control subjects or published age-adjusted

NHANES III ﬁ ndings. However, the prevalence

of RDFS was higher in the diabetic subjects as

compared to recruited control subjects [

146 ]. In

2006, another study in Lithuania comprised 68

10–15-year-old diabetics and 68 age- and gender-

matched nondiabetic controls. Diabetics were

categorized into well-to-moderately controlled

and poorly controlled groups. They found that

diabetics had fewer caries and plaque, lower sali-

vary ﬂ ow rates and buffer effect, and more fre-

quent growth of yeasts than their nondiabetic

Z. Ling and H. Tao

147

controls. Well-to-moderately controlled diabetics

had fewer decayed surfaces and lower counts of

mutans streptococci and yeasts than poorly con-

trolled diabetics, but the level of metabolic con-

trol of diabetes had no inﬂ uence on salivary ﬂ ow

rates and buffer effect. High caries levels in dia-

betics were signiﬁ cantly associated with age,

plaque score, and decreased unstimulated sali-

vary ﬂ ow rate but were not associated with the

level of metabolic control of diabetes. High car-

ies experience in this study population could be

related to plaque accumulation and/or to changes

in saliva induced by diabetes mellitus [ 147 ]. Two

years later, the same authors published another

paper to analyze possible associations between

caries increments and selected caries determi-

nants in children with type 1 diabetes mellitus

and their age- and sex-matched nondiabetic con-

trols, over 2 years. A total of 63 (10–15 years old)

diabetic and nondiabetic pairs were examined for

dental caries, oral hygiene, and salivary factors.

Salivary ﬂ ow rates, buffer effect, concentrations

of mutans streptococci, lactobacilli, yeasts, total

IgA and IgG, protein, albumin, amylase, and glu-

cose were analyzed. Means of 2-year decayed/

missing/ﬁ lled surface (DMFS) increments were

similar in diabetics and their controls. No differ-

ences were observed in the counts of lactobacilli,

mutans streptococci, or yeast growth during fol-

low- up, whereas salivary IgA, protein, and glu-

cose concentrations were higher in diabetics than

in controls throughout the 2-year period. Their

results also suggested that, in addition to dental

plaque as a common caries risk factor, diabetes-

induced changes in salivary glucose and albumin

concentrations are indicative of caries develop-

ment among diabetics [

148 ]. In 2009, in her the-

sis for Master degree, Abay conducted a study

that aimed to answer the research question, “Is

there an association between diabetes mellitus

and prevalence of severe dental caries in adults?”

Data of 701 subjects with diabetes and 3,636 sub-

jects without diabetes from the National Health

and Nutrition Examination Survey (NHANES)

conducted from 2003 to 2004 were used. Findings

of her study suggested that prevalence of severe

caries, prevalence of severe untreated caries, and

prevalence of at least one root surface with caries

or ﬁ lling did not differ between adults with and

without diabetes [

149 ].

In experimental diabetic rodent animals, there

are also contradictory reports; some studies

reported that diabetes enhanced the incidence of

dental caries whereas another did not. As early as

1957, Nichols and Shaw reported that in terms of

carious molars and carious legions, normal rats

did not differ from caries-susceptible rats that

received intravenous injections of alloxan mono-

hydrate [ 150 ]. However, Hartles and Lawton

reported that the mean number of carious teeth

per rat and the mean caries score were signiﬁ -

cantly higher for the injected animals than for the

controls. And the authors proposed that the most

probable way in which the injection of alloxan

can inﬂ uence the incidence of dental caries is by

causing an alteration in the salivary secretions,

either directly or indirectly [ 151 ]. A recent study

published by some Japanese authors also con-

ﬁ rmed that diabetic conditions enhance dental

caries in WBN/KobSlc rats [ 152 ].

As stated above, results of these studies have

shown conﬂ icting conclusions. In spite of the dif-

ference between diabetes mellitus types 1 and 2,

the reasons may be methodological difﬁ culties,

such as small sample size, the absence of stan-

dard criteria for caries evaluation, and noncon-

ventional cutoffs to classify good and poor

diabetes control [ 149 ]. Thus, further studies of

the potential association between diabetes melli-

tus and dental caries are suggested.

8.8.2 Root Caries and Diabetes

According to its location, dental caries can be

divided into coronal caries and root caries. The

latter poses a complex challenge for dental practi-

tioners, which is different to those challenges pre-

sented by the former [ 153 ]. Beck et al. found that

coronal and root caries do tend to appear together

in the same individuals. They also found that peo-

ple who experience both types of caries had more

gingival recession at baseline [ 154 ]. As world’s

population ages and retention of teeth increases,

there will be increasing numbers of older patients

at risk of root-surface caries. Root caries has a

8 Dental Caries and Systemic Diseases

148

higher prevalence among older adults than any

other age group. Many of these individuals may

be more likely to have chronic systemic disease.

So we discuss the relationship between root caries

and diabetes separately. Currently, insufﬁ cient

evidence exists to support or refute an association

between diabetes and root caries [ 155 ].

As we mentioned before, Moore et al.

described the prevalence of coronal and root car-

ies in an adult type 1 diabetic population in 2001.

Although no signiﬁ cantly higher DFS (decayed

and ﬁ lled surfaces in the crown) rates were

noticed, the prevalence of RDFS (decayed and

ﬁ lled surfaces in the root) was higher in the dia-

betic subjects as compared to recruited control

subjects [ 146 ]. In 2007, another stratiﬁ ed cross-

sectional study was conducted in Thailand to

determine the effect of type 2 diabetes mellitus

on coronal and root-surface caries. Subjects of

105 type 2 diabetic patients and 103 nondiabetic

at the same age and gender were included. Their

results found that type 2 diabetic patients com-

pared with nondiabetic subjects had a higher

prevalence of root-surface caries and a higher

number of decayed/ﬁ lled root surfaces. The

authors also found that the factors associated

with root-surface caries included type 2 DM, a

low saliva buffer capacity, more missing teeth,

and existing coronal caries [ 156 ].

To analyze the possible factors connected the

root caries and diabetes, we have to talk about

periodontal diseases, ﬁ rstly. Diabetes has been

found to be bidirectionally linked with periodon-

tal disease and subsequent loss of attachment. As

a result, gingival recession will cause the expos-

ing of tooth’s root and contributing to the risk of

root caries [

157 , 158 ]. Secondly, considering that

saliva is responsible for establishing protective

environment against dental caries, we will talk

about the effect of salivary factors like salivary

ﬂ ow rate and adequate level of calcium, phos-

phate, and ﬂ uoride in diabetes mellitus. Recently,

Jawed et al. evaluated the possible protective role

of salivary factors in diabetes mellitus type 2

patients with dental caries. In their study, a total

of 398 diabetes mellitus type 2 patients with den-

tal caries and 395 age- and sex-matched nondia-

betic subjects with dental caries were included as

controls. The blood glucose, HbA1c, and DMFT

indices were found to be signiﬁ cantly high, while

the salivary ﬂ ow rate, calcium, phosphate, and

ﬂ uoride were found to be signiﬁ cantly low in dia-

betic patients as compared to controls [

159 ].

Salivary ﬂ ow is known to be reduced in long-

standing diabetes. This is thought to be due to

neuropathy affecting the salivary glands as a

result of chronic hyperglycemia [ 159 , 160 ].

Therefore, in diabetes, periodontal disease and

associated attachment loss and gingival recession

may mediate increased root caries, compounded

by reductions in salivary ﬂ ow and elevated gingi-

val crevicular glucose levels in people with

poorly controlled diabetes [ 155 ].

Since oral microbiota plays an important role in

the root caries process, the change of microbiology

in the oral cavity of diabetes patients may also have

relationship with root caries. Root caries was

thought to be associated with Streptococcus mutans ,

Lactobacillus (spp.), and Actinomyces (spp.) based

on the culture method [ 161 , 162 ]. So far, the use of

culture-independent methods has played a key role

in the discovery of previously unrecognized species

in the oral cavity as well as in redeﬁ ning the patho-

genesis of the major oral infections. And the authors

found that the microbial ﬂ ora associated with root

caries was far more complex than previously

assumed. Except for these three bacterial species

we just mentioned, additional species, such as

Atopobium spp., Olsenella spp., Pseudoramibacter

alactolyticus , and Propionibacterium sp. strain

FMA5, were also commonly found [

163 ]. The

study showed that signiﬁ cantly more diabetic sub-

jects had higher levels of Treponema denticola ,

Prevotella nigrescens , Streptococcus sanguinis ,

Streptococcus oralis , and Streptococcus interme-

dius in their supragingival plaque than nondiabetic

subjects. Root-surface caries was associated with an

increased count of mutans streptococci, lactobacilli,

and yeasts in saliva and of Streptococcus mutans in

supragingival plaque samples [ 164 ].

8.8.3 Tooth Loss and Diabetes

The results of Lin et al.’s study suggested that

diabetes and poor glycemic control may not be

Z. Ling and H. Tao

149

associated with an increased prevalence of past

coronal and root-surface caries experience in

older adults, but there is a tendency for more

active caries lesions and missing teeth [ 142 ]. In

2011, a Korean study has identiﬁ ed a relationship

between total tooth loss from any cause and dia-

betes [ 165 ]. In addition, a recent study found that

having 19 or fewer teeth was associated with high

HbA1c among men aged 40–64 years but not

among those aged 65–79 years. Since diabetes is

a major risk factor for periodontal disease, the

signiﬁ cant associations of tooth loss with HbA1c

among the middle-aged men may reﬂ ect associa-

tions of periodontal disease with HbA1c,

although they had no information on the causes

of tooth loss [ 166 ].

8.9 Dental Caries

and Respiratory Infections

The anatomical continuity between the lungs and

the oral cavity makes the latter a potential reser-

voir of respiratory pathogens. It is well known

that the respiratory system includes the nasal and

oral cavity: the sinuses and larynx as the upper

airway and the trachea, bronchi, bronchioles, and

alveoli as the lower airway. Thus it’s not surpris-

ing that many of the diseases that occur in the

oral cavity could be also found in the upper air-

way regions. However, it’s not easy for an infec-

tive agent to reach the lower respiratory tract. It

must defeat sophisticated immunological and

mechanical defense mechanisms. The latter is so

efﬁ cient that, in healthy patients, the distal air-

way and lung parenchyma are sterile, despite the

heavy bacterial load found in the upper airway.

An infection occurs when the host’s defenses are

compromised, the pathogen is particularly viru-

lent, or the inoculum is overwhelming. The

microorganisms may enter the lung by inhala-

tion, but the most common route of infection is

aspiration of what pneumologists have long

referred to as oropharyngeal secretions.

Therefore, it is plausible that oral microorgan-

isms might infect the respiratory tract [

166 ].

Dental plaque and poor oral health have been

associated with nosocomial pneumonia and chronic

obstructive pulmonary disease (COPD) [

167 ].

Since community-acquired pneumonia and lung

abscesses may be due to anaerobic bacteria, there

are lots of anaerobes implicated in the destruction

of periodontal tissues that have also been isolated

from infected lungs, for example, Actinobacillus

actinomycetemcomitans , Fusobacterium nuclea-

tum , and Pseudomonas aeruginosa . Pascual-

Ramos et al. found a strong association between

third-grade caries and pneumonia in 30 consecu-

tive women with SLE, hospitalized because of

pneumonia, compared with two groups of patients

with SLE, hospitalized and ambulatory, matched to

cases by age, sex, and hospitalization date.

Compared with ambulatory controls, the oral

health of patients with pneumonia was worse as

reﬂ ected by a higher frequency of periapical lesions

and cervical and third-grade caries and a higher

number of caries per patient [ 168 ]. There is also

evidence that the occurrence of respiratory tract

infections during the ﬁ rst year of life is associated

with a signiﬁ cantly increased risk for developing

early childhood caries during subsequent years

[ 169 ]. A research conducted by Eldem et al. from

Turkey showed the possible association between

poor oral hygiene and upper respiratory tract infec-

tion (URTI) rates. Children without any systemic

disease were enrolled in the study and divided into

two groups: 100 children with dental caries as

patient group and another 100 children without car-

ies as control group. URTI rates and antibiotic

usage in both groups since birth were identiﬁ ed

according to the medical records. Dental caries was

scored according to decayed, missing, and ﬁ lled

teeth index. And their results showed that the URTI

rates were signiﬁ cantly higher among children

with poor oral hygiene and dental caries [

170 ].

Pascual-Ramos et al. made a summary of the dif-

ferent mechanisms that have been proposed to

explain the potential role of oral bacteria in the

pathogenesis of respiratory infections. Among

them are the aspiration of oral pathogens into the

lungs, the modiﬁ cation of mucosal surfaces by

periodontal disease-associated enzymes that pro-

mote adhesion and colonization by respiratory

pathogens, the destruction of salivary pellicles by

periodontal disease-associated enzymes that mod-

ify clearance of pathogenic bacteria from the

8 Dental Caries and Systemic Diseases

150

mucosal surface, and alteration of the respiratory

epithelium by cytokines originating from peri-

odontal tissues to promote infection by respiratory

pathogens [ 168 , 171 ].

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8 Dental Caries and Systemic Diseases

157

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1_9

Models in Caries Research

Huang Xuelian , Guo Qiang , Ren Biao , Li Yuqing ,

and Zhou Xuedong

1. In vitro models in caries research

2. In situ models in caries research

3. Animal models in caries research

Clinical trials are always large, time-

consuming, and costly; furthermore, due to ethi-

cal problems, some studies are not available in

human subjects [ 1 ]. Therefore, models have

played a substantial role in caries research, which

help to establish the multifactorial etiology of

dental caries, deﬁ ne the impact of numerous fac-

tors contributing to the initiation and progression

of dental caries, and identify agents or measures

with the ability to prevent or reduce the incidence

of dental caries [ 2 ]. Models in caries research can

be divided into three categories: in vitro model,

in situ model, and animal model [ 3 ].

9.1 In Vitro Models in Caries

Research

In vitro models or laboratory models are the most

common models applied in dental research with

several advantages [ 4 ]:

1. Less costly and comparatively rapid

2. Can carry out single-variable experiments

under highly controlled conditions, which are

more sensitive and precise

3. Best approach to screen large numbers of agents

and to determine their modes of action [ 5 ]

However, in vitro models have signiﬁ cant limita-

tions, mostly related to their inability to simulate the

complex biological processes involved in caries.

As dental caries results from an ecological

imbalance in the physiological equilibrium

between tooth minerals and oral microbial bio-

ﬁ lms, two basic methods are developed to study

dental caries: de- and remineralization in the teeth

(e.g., in vitro chemical models) and microbial

systems on the teeth (e.g., in vitro microbial mod-

els) [

6 ]. Recently, microbial-based de- and remin-

eralization models are also developed, which are

closer to the tooth decay process in oral cavity.

9.1.1 In Vitro Chemical Models

Chemical induction of caries by organic acids is

one of the principal approaches to study the

H. Xuelian

State Key Laboratory of Oral Diseases ,

Sichuan University , Chengdu ,

People’s Republic of China

Department of Operative Dentistry and Endodontics ,

West China Hospital of Stomatology,

Sichuan University , Chengdu ,

People’s Republic of China

e-mail:

[email protected]

G. Qiang • R. Biao • L. Yuqing

State Key Laboratory of Oral Diseases ,

Sichuan University , Chengdu ,

People’s Republic of China

Z. Xuedong (*)

State Key Laboratory of Oral Diseases ,

West China Hospital of Stomatology, Sichuan

University , Chengdu , People’s Republic of China

158

mechanisms in de- and remineralization of

enamel and dentin. In vitro chemical models

allow strict control of the experimental environ-

ment and are relatively simple and cost-effective;

however, their applicability is limited to factors

which directly inﬂ uence the de- and remineral-

ization process. Between de- and remineraliza-

tion models, substrates are one of the major

differences. Different from remineralization

methods which typically use lesions as substrate,

demineralization models utilize a wider variety

of substrates, including those pretreated and rem-

ineralized with agents, on natural enamel and

dentin [ 5 ]. (For details, please also see “deminer-

alization and remineralization” part.)

9.1.1.1 Demineralization Models

Tooth decay is the result of progressive mineral

loss from dental tissues. In vitro demineralization

models enable researchers to examine those fun-

damental processes, and their applications

include [ 5 ] mechanistic studies of solution/sub-

strate factors affecting demineralization, studies

of factors contributing to the intrinsic resistance

of mineralized tissue to acid demineralization,

and efﬁ cacy evaluation of caries-preventive

agents (e.g., ﬂ uoride, natural products [ 7 , 8 ]) or

the application of laser irradiation [ 9 ] which may

inhibit enamel and dentin dissolution in acid

attack.

9.1.1.2 Remineralization Models

In vitro remineralization models can be applied

to investigate the mechanism involved in caries

lesion repair and evaluating the efﬁ cacy of treat-

ments or agents which are favorable to enhance

remineralization. Remineralization protocols can

be grouped into three general categories:

In pH-Lattice Ion “Drift” Protocol

Substrates are exposed to constant-volume super-

saturated remineralization solutions. They have

the advantage of direct chemical measurement of

remineralization within a given exposure period

[ 10 ]; however, in remineralization solutions, cal-

cium and phosphate ion concentration will

decrease, and the pH will also decrease, if the

systems are not well buffered. Those changes

cannot only decrease the amount of mineral

formation but also change both the type and loca-

tion of mineral deposition [

5 , 11 ].

Constant Composition Protocols

Lattice ion concentrations and pH remain con-

stant throughout remineralization. In details, two

techniques were involved: ﬂ ow-through tech-

niques and titration-controlled techniques. In the

former, supersaturation is kept constantly by

means of a high volume of remineralization

medium, and it permits multi-group studies to be

carried out. The measurement of remineraliza-

tion is conﬁ ned to substrate changes [ 5 ]. In the

latter, controlled addition of calcium phosphate

lattice ions and buffer titrants monitored potenti-

ometrically by pH and/or calcium ion-selective

electrodes was involved. The measurement of

remineralization is used titrant [ 5 ].

“pH Cycling” Protocols

The “pH cycling” protocols consist of numerous

cycles of demineralization and remineralization,

which are correlated to alternant acidiﬁ cation and

alkalinization phases in oral cavity. The genesis

of modern pH cycling protocols was produced by

ten Cate and Duijsters [ 10 ], which have become

favorable choice to study remineralization

in vitro, because they provide better simulation

of the caries process for both mechanistic studies

and for evaluations of some caries-preventive

agents. Recently, a particular in vitro remineral-

ization model, called Featherstone pH cycling

model, was recommended as an appropriate

alternative to animal testing, particularly for ionic

ﬂ uoride based dentifrices, for the reason that it

demonstrated excellent correlation with the cur-

rently accepted animal caries models [

12 ].

9.1.2 In Vitro Microbial Model

In vitro microbial models provide means for

studying complex microbial ecosystems on the

teeth and their roles on the development of dental

caries [ 6 ]. Microbial models can be used to (1)

investigate prevention of carious lesions through

antimicrobial agents or measures, (2) compare the

cariogenic potential of different microorganisms,

and (3) assess the cariogenicity of carious diets.

H. Xuelian et al.

159

Microbial models can be divided into two main

classiﬁ cations: closed (batch) system and open

system (continuous culture). The closed system,

in which there is no ﬂ ow into or out of the reactor

during the cultivation and microorganisms are

provided with ﬁ nite nutrients and growth rates are

rapid, is comparatively rare in nature [ 13 , 14 ]. It is

the simplest and most frequently used in vitro

microbial models. During the growth process, the

environment in the enclosed system will change

(e.g., nutrients become depleted, signaling mole-

cules and metabolites accumulate), unless the

ﬂ uid is regularly replaced. This system is far from

physiological; however, better repeatability, less

contamination, less cost, and high throughput are

its marked advantages. This system also allows

researchers to easily vary multiple parameters

including the composition of growth media, incu-

bation temperatures, humidity, presence or

absence of shear stress, and O

and CO

concen-

trations. Those features make it valuable system

for initial screening assays [ 14 , 15 ].

In dynamic continuous system, fresh medium

ﬂ ows into the bioreactor continuously, and part of

the medium in the bioreactor is withdrawn from

the fermenter at the same ﬂ ow rate of the inlet

ﬂ ow. Then some metabolites can be eliminated

from the bioreactor, and it is more like oral cavity

than batch cultivation [ 16 ]. This system enables

better control of growth rates and other variables

[ 13 ]; however, it is more likely contaminated and

not easy to repeat the results.

9.1.2.1 Inoculum

An important factor in the design of in vitro

microbial models is the choice of inoculum [ 3 ].

Pure culture, deﬁ ned consortium, and microcosm

are all used currently. Pure culture is broadly

used in vitro, in which physiological studies are

normally done. It is much easier to manipulate

the variation of the single test organism. However,

it is far from mimicking the oral cavity.

Microcosm is used to closely mimic the physi-

cochemical, microbiological, and nutrient condi-

tions present in oral cavity [ 13 ]. In in vitro

microbial models for dental caries, dental plaque

or saliva can be used. They have the advantage of

maintaining the complexity and heterogeneity and

enabling in situ bacterial community dynamics to

be replicated in laboratory environment. However,

there are still some disadvantages. Firstly, it is hard

to ﬁ nd out a medium to support all organisms in

the plaque or saliva to grow as they do in oral cav-

ity. Secondly, they are often poorly characterized.

As the system is so complex, it is hard and costly

to analyze its metabolism, composition shift, and

interaction within the ecosystems. Thirdly, it

would have been difﬁ cult to standardize the plaque

inoculum in replicate experiments and to manipu-

late its composition for experimental purposes.

In order to overcome these problems, inocu-

lum with a deﬁ ned consortium has been con-

structed by pooling pure cultures of plaque

bacteria in various combinations. The communi-

ties that develop from them are stable with time

and establish reproducibly in replicate experi-

ments. It also allows detailed control and study of

the properties of the individual bacterial species

present. Deﬁ ned inoculum comprised of two or

more organisms in combination, with organisms

chosen for their relevance in health and disease

and for their ease of identiﬁ cation; subsequently,

individual species can be added or deleted for

experimental purposes. One of the good exam-

ples is often referred to as the “Marsh Consortium”

which is composed of nine bacteria [

17 ]. In

another batch culture study, a mixture containing

Actinomyces naeslundii , Veillonella dispar ,

Fusobacterium nucleatum , Streptococcus sobri-

nus , and Streptococcus oralis was cultivated to

form supragingival plaque, which was used to

evaluate antimicrobial agents. A yeast species,

Candida albicans , was added later, for the reason

that a valid prescreening test should rule out the

possibility of fungal overgrowth due to selective

interference with bacterial ecology [

18 , 19 ]. In a

root caries model system developed in artiﬁ cial

mouth, four putative root-caries pathogens,

Streptococcus mutans , Streptococcus sobrinus ,

Actinomyces naeslundii , and Lactobacillus rham-

nosus , form multispecies consortia bioﬁ lms and

furthermore form the caries lesion [ 20 ].

9.1.2.2 Closed (Batch) System

Microbial Models

In closed system microbial models, some small

reactor vessels and test tubes were frequently

used for planktonic culture, and microtiter plate

9 Models in Caries Research

160

(MTP)-based system is among the most fre-

quently used bioﬁ lm model systems. In MTP-

based system, bioﬁ lms are either grown on the

bottom and the walls of the microtiter plate (most

commonly a 96-well microtiter plate) or on the

surface of a substrata (glass, hydroxyapatite,

enamel, or dentin disks) placed in the wells of the

microtiter plate (most commonly a 6-, 12-, or

24-well microtiter plate). The former is generally

based on bacterial sedimentation, in which the

metabolites accumulate, and the latter can form

through attachment, which is more close to the

reality than the former.

Some modiﬁ cations were developed for the

96-well microtiter plate, during which transfer-

able solid phase (TSP) bioﬁ lm model was used to

screen antimicrobial activity [ 21 ]. TSP is com-

mercially available and contains a 96-well

microtiter plate and a lid with 96 pegs placed on

the plate. Bioﬁ lm can form on the surface of pegs

through attachment. Another MTP-based com-

mercially available method is the Bioﬁ lm Ring

Test, which provides a kit including microplates

(12 polystyrene strips of 8 wells), toner (magnetic

bead solution), contrast liquid (a nontoxic and

inert opaque oil used for reading step), dedicated

block test (magnet support), and plate reader

(scanner) [ 22 ]. With this technology, the immobi-

lization of inert paramagnetic beads included in

the culture medium during the formation of the

bioﬁ lm is measured. A magnet is used to collect

the non-immobilized beads into a single spot

which is then quantiﬁ ed through specialized

image algorithms [

14 ]. This model was once used

to conﬁ rm that AI-2-based quorum sensing affects

bioﬁ lm formation in Streptococcus mutans [ 23 ].

Zürich bioﬁ lm model, a multispecies model,

based on 24-well plates, is a classical batch cul-

ture approach, in which the host of environmental

variables can be rigorously controlled. At present,

six microorganism representatives are used to

generate bioﬁ lms for supragingival plaque, which

are Streptococcus oralis , Streptococcus sobrinus ,

Actinomyces naeslundii , Veillonella dispar ,

Fusobacterium nucleatum , and Candida albicans

[ 18 , 19 ]. Cells are cultivated anaerobically in a

saliva-based medium on substrata coated with

a salivary pellicle. As shear forces are absent in a

batch culture system, those disks are dipped in

saline three times daily, and at each time point, the

bioﬁ lms are dipped three times in saline, thereby

being subjected to passages through an air-liquid

interface [

18 ]. The validation of the in vitro caries

model was assessed, which conﬁ rms the repeat-

ability of bioﬁ lm formation after 40.5 h and 64.5 h

in repeated independent trials, and demonstrated

the produced losses in viability from brief expo-

sures of bioﬁ lms to chlorhexidine or triclosan

were similar to those observed in vivo, inferring

that this bioﬁ lm model was very useful for pre-

clinical testing of prospective antiplaque agents at

clinically relevant concentrations [

24 ]. Actually,

the main application of this model was to evaluate

antimicrobial compounds. The model could also

be used to achieve demineralization and reminer-

alization of bovine enamel under bioﬁ lms [ 18 ].

More recently, a novel high-throughput active

attachment model (Fig. 9.1 ) was also used to eval-

uate antimicrobial compounds and contributing

factor on dental caries [ 7 , 26 ]. The model consisted

Fig. 9.1 Pictures of the bioﬁ lm model used in this study.

( a ) Custom-made stainless steel lid on which 24 clamps

are ﬁ xed. Substrata glass cover slips or HA disks are

shown. ( b ) Position of the substrata (HA disks) in the

24-well plate at the time of bioﬁ lm growth [

25 ]

H. Xuelian et al.

161

of a custom-designed lid containing substrata that

ﬁ t on top of standard 24-well plates. Single spe-

cies bioﬁ lms and polymicrobial bioﬁ lm derived

from saliva can be formed in the systems [ 25 ].

This model is also applicable for evaluating novel

caries-preventive agents on both bioﬁ lm and

demineralization inhibition at the same time when

bovine dentin disks were used as substrata [

27 ].

9.1.2.3 Open (Continuous Culture)

System Microbial Models

Flow Cell Bioﬁ lm Model and Modiﬁ ed

Robbins Device

Flow cell bioﬁ lm model and modiﬁ ed Robbins

device [ 28 ] (MRD, Fig. 9.2 ) shared similar prin-

ciples for the operation, whereby culture ﬂ uid is

passed through a tube or cell and bioﬁ lms may be

monitored microscopically (in ﬂ ow cells) or

formed on coupons (in some ﬂ at plate ﬂ ow cells)

or pegs (Robbins devices) [ 29 ]. Flow cells are

commercially available devices with glass

chambers that are particularly well suited for

real-time nondestructive microscopic analyses of

bioﬁ lms [

14 ]. This model is not inexpensive, and

the design can be versatile in the selection of

material for the substratum [ 30 ]. They still have

some disadvantages: Firstly, sterilization can be

difﬁ cult because many of the common materials

used for ﬂ ow cell construction do not respond

well to autoclaving, especially to repeated auto-

claving. Secondly, peristaltic pumps can produce

some pulsation in liquid delivery. Finally, in

cases of high biomass within the ﬂ ow cell [

30 ], it

is conceivable that a gradient in nutrients could

be established over the length of the ﬂ ow cell. As

commonly used bioﬁ lm models, ﬂ ow cells can be

used for single-species bioﬁ lm or multispecies

bioﬁ lm. The co-adhesion of Streptococcus gor-

donii with Streptococcus oralis was studied in a

two-species in vitro oral bioﬁ lm ﬂ ow cell system,

in which green ﬂ uorescent protein was used as a

species-speciﬁ c marker [ 31 ]. In MRD, the tube

can be plastic or metal, into which pegs can be

Media in

Waste

Peristaltic

pumps

Modified Robbins

Device (MRD)

mounted on

a hot plate

(37 °C)

Waste

Side

arm

Bubble

trap

14 µm filter

Fig. 9.2 Modiﬁ ed Robbins

device [

28 ]

9 Models in Caries Research

162

inserted so that when in place, the end of the peg

forms part of the wall of the tube [ 13 ]. In a typical

experiment, the MRD is ﬁ lled with a suspension

of microorganisms and is ﬂ ipped over to improve

the adhesion of the planktonic cells to the disks

[ 14 ]. After the adhesion phase, the pump is

started to allow a continuous ﬂ ow of the growth

medium and bioﬁ lm development on the disks.

The advantage of this system is that the bioﬁ lms

can be sampled by removing plugs at any time,

once they are replaced to maintain a closed sys-

tem. This model was established as an appropri-

ate bioﬁ lm model for susceptibility testing of oral

microorganisms [

32 ].

Drip-Fed Bioﬁ lm Model

Among drip-fed bioﬁ lm models, drip ﬂ ow bioﬁ lm

reactors are a simple sort, in which bioﬁ lms are

grown on angled slides continuously irrigated

with small volumes of (inoculated) media, thereby

providing a low-shear environment with disper-

sive mixing (Fig. 9.3 ) [ 14 , 33 , 34 ]. This system

has been developed as an interproximal labora-

tory model to compare the potential effectiveness

of powered brushing to remove bioﬁ lm plaque

from interproximal spaces beyond the reach of

bristles [

35 ]. Different substrata can be used such

as hydroxyapatite or teeth tissues. Care is required

in experimental design and in sampling because

medium ﬂ ow over the surface of the slide may not

be uniform and hence there may be signiﬁ cant

aerial heterogeneity over the surface of the sub-

stratum [ 13 ]. In constant depth ﬁ lm fermenter

(CDFF), bioﬁ lms are fed by the drip-wise addi-

tion of growth medium onto the turntable, and

excess and/or spent medium ﬂ ows downward

through a waste outlet [

13 ]. The bioﬁ lm devel-

oped on a surface is limited to a predetermined

depth by mechanically removing excess bioﬁ lm, a

situation mimicking the movement of the tongue

over the teeth [ 14 ]. In this system, 200 or 300 μm

is most commonly used for maintenance of den-

tal plaques [ 13 ]. This system can develop single-

species bioﬁ lm, deﬁ ned consortium bioﬁ lm, or

microcosm bioﬁ lm to evaluate antibacterial agent

and explore the etiology of dental caries [ 36 , 37 ].

Perfused Bioﬁ lm Fermenters

Perfused bioﬁ lm fermenters (PBF) are con-

structed such that nutrients are supplied by con-

tinuous perfusion of growth medium, which is

pumped through the substratum (a permeable

Influent

Needle

O-ring

Coupon

Adjustable

leg

Effluent

Bacterial

air vent

Mininert

top

Septum

10°

Fig. 9.3 Schematic diagram of

a drip ﬂ ow reactor [

33 , 34 ]

H. Xuelian et al.

163

membrane) and hence through the bioﬁ lm [ 13 ].

The media ﬂ ow can be accurately controlled, the

growth rate of the bioﬁ lm can also be well modu-

lated, and dynamic steady states can be achieved.

The multiple Sorbarod device (MSD) is one kind

of PBF which is proved to be an ideal system to

grow oral bioﬁ lms by McBain and co-workers. It

uses a simple two-piece stainless steel housing,

yields relatively large amounts of biomass, and

enables continuous monitoring of population

dynamics through the analysis of perfusates

(spent culture ﬂ uid) [

38 ]. It has been validated for

the maintenance of complex salivary microbial

ecosystems and for the in vitro reproduction of

interindividual variation within oral microbiotas.

Disadvantages of MSD are related to the devel-

opment of heterogeneous bioﬁ lms [ 13 ].

Artiﬁ cial Mouth

Artiﬁ cial mouth models mimic the in vivo oral

niches and habitats to act as a laboratory “micro-

cosm” [ 39 ]. It consists of a vessel in which a sur-

face (or surfaces) is inoculated and supplied with

a continuous or intermittent nutrient supply; dur-

ing such experimental procedures, real-time

growth and development of dental plaque/bioﬁ lm

can be investigated (Fig. 9.4 ) [ 3 , 39 , 40 ]. This

system has progressed from simple and basic

apparatus to the currently available, highly

sophisticated, computer-controlled, multi-station

artiﬁ cial mouth systems. The advanced multiple

artiﬁ cial mouth (MAM) system which was devel-

oped by Sissons and his co-workers could be

employed for the long-term growth of multiple

plaque samples within a standardized, simulated

Simulated oral

fluid line

Sucrose line

Micro-reference

electrode

Electrode and

inoculation port

Sample holder

Thermometer

Clamp

Fluid delivery

head port

Gas inlet

Dental

plaque

electrode

Screwcap with septum

Experimental treatments

line

Fluid delivery

head

Endplate

Waste

Fig. 9.4 Artiﬁ cial mouth [ 39 , 40 ].

( a ) Cross-section of bioﬁ lm growth

station, ( b ) longitudinal section of

culture chamber

9 Models in Caries Research

164

oral environment generated by computer-

controlled facilities [ 40 ]. The environment and

the bioﬁ lm pH range can be controlled and manip-

ulated, and oral ﬂ uids and periodic pulses of

sucrose to model meals were simulated. A chemi-

cally deﬁ ned saliva-like oral ﬂ uid analogue is

named deﬁ ned medium with mucin (DMM),

which can work well as a “saliva substitute” [ 41 ].

Plaque samples grown over several weeks in this

system exhibited metabolic behavior and pH pro-

ﬁ les typical of natural plaque. It was possible to

analyze aliquots during plaque development with-

out contaminating the mature samples [ 40 ]. These

current applications contain evaluating microbial

interactions in simulated dental plaque and simi-

lar bioﬁ lms and monitor their physical, chemical,

biological, and molecular features to a very high

degree of accuracy, evaluating potential antimi-

crobial agent [ 40 , 42 ]. Besides, several artiﬁ cial

mouth models have been established to study fac-

tors inﬂ uencing the development of primary cari-

ous lesions and evaluate caries-preventive agents,

which will be represented in “microbial-based min-

eralization model” part in detail.

Chemostat

Chemostat can provide a homogeneous liquid

environment for microbial growth, under highly

deﬁ ned and controllable conditions [ 3 ]. Bacteria

can be grown at ﬁ xed growth rates, enabling sin-

gle parameters to be varied independently so that

true cause-and-effect relationships can be estab-

lished; the culture can also be sampled repeat-

edly, facilitating statistical analysis. However, if

excessive bacteria grew in chemostat, operational

problems may arise from blocked tubing [

3 ].

Prevention of wall growth (change of cells from

planktonic to bioﬁ lm forms adhering to the che-

mostat vessel) is also very important, which will

cause deviations from “steady-state” growth

[ 43 ]. Pure culture and mixed culture can both

grow in chemostat systems. Marsh developed a

consortium, composed of nine bacteria to investi-

gate carbohydrate pulses and pH on population

shifts within oral microbial communities [ 44 ]

and later focus on pH-driven disruption [ 17 ];

both of the two studies are very important to his

“ecological plaque hypothesis.” The control of

pH at neutral values during sugar pulsing was

possible only with this type of laboratory model;

this experiment could not have been performed in

animal or human model studies because of the

inevitable pH change when carbohydrates are

metabolized [

3 ]. The applications of chemostat

for the usual microbiology laboratory are costly

and have space considerations. A lot of medium

are needed, especially when multiple parallel cul-

tures are needed. Nowadays, some modiﬁ cations

are developed to alleviate the hurdles [ 43 ]. For

classic chemostat, one of the shortcomings is that

the organisms are not in a bioﬁ lm; however,

recently, there are some modiﬁ cations for chemo-

stat to involve relevant surfaces to form bioﬁ lms.

Sterile hydroxyapatite rods or disk can be inserted

into chemostat head and immersed in steady-

state planktonic cultures for certain periods [ 45 ].

Chemostat has also been linked to ﬂ ow cells to

form bioﬁ lm. S. Herles and co-worker developed

a chemostat ﬂ ow cell system (Fig. 9.5 ), in which

the chemostat was provided with a continuous

source of ﬁ ve species of oral bacteria grown in an

artiﬁ cial “saliva-like” medium. This mixture was

pumped through six ﬂ ow cells, each containing

two types of surfaces in which plaque formed,

and was subsequently used to compare the anti-

plaque agents [ 46 ]. Furthermore, chemostat can

also be used to inoculate a novel bioﬁ lm generat-

ing model system, the thin ﬁ lm fermenter, in

which bioﬁ lms of a predetermined depth can be

generated, of which a key feature is that bioﬁ lms

of a predetermined depth can be generated [

3 ].

9.1.3 Microbial-Based De-

and Remineralization Model

The use of microbial-based de- and remineraliza-

tion model permits more clinical relevant in vitro

investigations of dental caries etiology and the

properties of caries-preventive agent. In this sys-

tem, primary caries, secondary caries, pit and ﬁ s-

sure caries, root caries, and so on can be

developed. Batch culture techniques can be used,

and continuous culture techniques were also

applied in this system, during which “artiﬁ cial

mouth” was generally involved.

H. Xuelian et al.

165

In an in vitro secondary caries model, the

occlusal surface of the tooth was sealed with a

conventional resin-based ﬁ ssure sealant; during

the process, certain part of the tooth was slightly

moistened (contaminated) with saliva to produce

marginal gaps, the teeth were infected with

Streptococcus mutans in artiﬁ cial mouth, and the

secondary caries can be developed in contami-

nated regions [

47 ]. This model can also be used

to study the relationship of gap size and second-

ary caries, in which gaps were created by insert-

ing shim stocks with different thickness at the

tooth/resin interfaces and Streptococcus mutans

was also used as inoculum [ 48 ].

Katz and his colleagues developed a pit and

ﬁ ssure caries in artiﬁ cial mouth. They selected

extracted human premolars and molars and inoc-

ulated pit and ﬁ ssures with a concentrated

Streptococcus mutans inoculum, which was

overlaid with a nutrient layer of 15 % agar, 15 %

glycerin, and 5 % sucrose. A ﬁ lter paper and a

thin layer of collodion were placed over the arti-

ﬁ cially created plaque. These specimens were

incubated at 37 °C and continuously washed with

artiﬁ cial saliva (pH neutral) at a regular rate. The

acrylic blocks with the tooth specimens were

removed daily, and 0.24 % sodium ﬂ uoride denti-

frice was applied for 3 min with a toothbrush.

The latter procedure was repeated 5 days a week,

and the whole experiment was conducted for 8

weeks. Subsequently, stereomicroscopic evalua-

tions revealed that the artiﬁ cial lesions were very

similar to those of natural pit and ﬁ ssure caries.

This in vitro caries model contributed somewhat

to the study of pit and ﬁ ssure caries including the

study of remineralization due to agents incorpo-

rated in toothpaste [ 40 ].

When root tissue (disks) was used and puta-

tive root-caries pathogens were used, root caries

can be modeled in the bacterial system. M. Shu

Chemostet

Mixed

culture

oral

microorganisms

Modified BM media

conteining mucin

Supplementery

BM media

Flow cells

From flow cells

Flow cells (open view)

Flow cells (assembled)

Germanium

prism

Waste

HAP

disks

Overflow

Mixing chamber

Vent

Pump

Fig. 9.5 Chemostat ﬂ ow cell

system [

46 ]. A Chemostat

containing a mixed culture of

5 oral bacteria, B Vessel

containing a supply of

supplementary BM medium

(without glucose), C The

mixing chamber containing

the ﬂ ow from A (1 ml/min)

plus 5 ml/min from B, D The

pump supplying ﬂ ow (1 ml/

min) from the chemostat to 6

low cells (1ml/min), E Flow

cells containing the HAP

disks and germanium ATR

prisms

9 Models in Caries Research

166

and colleagues developed such root caries model

using four putative root-caries pathogens,

Streptococcus mutans , Streptococcus sobrinus ,

Actinomyces naeslundii , and Lactobacillus rham-

nosus [ 20 ]. Also, saliva can also be used as inoc-

ulum to develop the root caries [ 49 ].

9.2 In Situ Model in Caries

Research

In situ caries models involve the use of appli-

ances or devices which create deﬁ ned conditions

in human mouth to simulate the natural process

of dental caries. These models attempt to provide

clinically relevant information in a relatively

short period without causing irreversible tissue

changes in the natural dentition [ 50 ]. The advan-

tages of in situ caries model systems compared

with clinical trials include [ 1 , 51 ]:

1. They have fewer ethical and logistical problems.

2. They are less costly.

3. Experimental design can be more ﬂ exible,

allowing hypotheses to be tested.

4. The results are acquired in much shorter time.

5. Better control with the study subjects and bet-

ter compliance.

Compared with in vitro caries models, in situ

caries model is complicated by dietary eating

habits, the presence of physiologically secreted

saliva, plaque of varying composition and thick-

ness, and a pellicle-coated tooth surface [

50 ],

which make it more close to oral cavity than

in vitro caries models.

Compared with animal caries model, in situ

caries models are conducted in human beings,

while animal caries models are conducted in ani-

mals. Due to so many differences between ani-

mals and human beings, some of the results from

animal caries models may not extrapolate to

human beings.

The disadvantages of in situ caries model are

still obvious:

1. The number of subjects in in situ caries model

is generally limited. Whether the small study

population can represent the general popula-

tion is raised as a question.

2. The validation of the studies is generally

heavily dependent on compliance of the test

subjects.

By using in situ caries models, we have the

potential to study both fundamental aspects of the

caries process, such as to form artiﬁ cial carious

lesions [

52 ], to study de- and remineralization,

and to obtain dental bioﬁ lm directly in the human

mouth [ 53 ], as well as more applied research

problems such as the effect of food on dental car-

ies and the role of caries-preventive agent in car-

ies prevention [ 54 , 55 ] in human subjects without

actually causing caries in the natural dentition

[ 50 ]. In situ study designs are highly variable,

with models using different hard tissue substrates,

a variety of intraoral sites, different exposure peri-

ods, the inclusion or exclusion of participants’

diet, the use of different depth recesses and gauze

to encourage plaque retention, the use of mesh to

protect surface from mechanical disturbance and

allow plaque accumulation, and the use of various

mineral quantiﬁ cation methods [ 52 , 56 ].

Both enamel and dentin can be used as hard

tissue substrates. For enamel, both the natural

enamel surface, which is more suitable for for-

mation of dental plaque bioﬁ lm [ 57 ], and enamel

slab (pieces of extracted teeth), which is suitable

for mineralization study [ 54 ] to make similar

baseline, can be used.

When the in situ caries model was used to

assess de- and remineralization, enamel speci-

mens were more frequently used than dentin;

however, dentin is useful to simulate root caries.

In remineralization study, it is better to produce

standardized demineralized lesions in laboratory

ﬁ rstly. These lesions can be sectioned to provide

one half lesion for the intraoral appliance and the

other half used as the baseline lesion control [

56 ].

However, the outcome of in situ caries models

may differ substantially depending on their

design, and therefore, the choice of model may

signiﬁ cantly inﬂ uence the conclusions drawn

from such studies [ 58 ]. For example, the micro-

bial composition of plaque will vary among dif-

ferent teeth in the same mouth, on the same tooth

H. Xuelian et al.

167

in different people, and even on different surfaces

on the same tooth. So the selection of patients for

in situ caries model should be carefully done. It

makes sense to recruit subjects with similar sali-

vary ﬂ ow and buffer capacity, with desired micro-

biological pattern, and even with a preferred

immunological proﬁ le [ 3 ]. Also, plaque that

forms with the aid of gauze does not fully resem-

ble “natural” plaque, in either structure or micro-

bial composition [ 59 ].

9.2.1 Classiﬁ cation of In Situ

Models

In 1990, Wefel grouped in situ model systems

into three general types: removable appliances,

single-section models, and banding models [ 60 ].

Nowadays, some new models were developed,

and some models were less used; in situ caries

models can be roughly divided into two catego-

ries: removable appliances and ﬁ xed appliances,

in terms of the mobility characteristic.

9.2.1.1 Removable Appliances

Removable appliance is the widely used in situ

caries model which can be constituted with acrylic

appliances or denture and hard tissue substrates.

The model can be exposed extraorally to the chal-

lenge, or to a therapeutic regime, to decrease vari-

ability in individual mouths. This kind of treatment

method can permit the testing of agents or proce-

dures which might be harmful to the natural denti-

tion or ethically unacceptable [

61 ]. If dietary

challenges were not considered or oral hygiene is

not the inﬂ uence factor, the model can be taken

out of the mouth when subjects are eating or car-

rying out oral hygiene, to give a high degree of

control and to diminish compliance. In an in situ

artiﬁ cial dentin carious lesion study, acrylic pala-

tal appliances containing two bovine dentin speci-

mens, protected with a plastic mesh to allow

bioﬁ lm development, were involved. The volun-

teers dripped a 20 % sucrose solution on each

specimen four times a day for 14 days; ﬁ nally, the

in situ model produced a deep lesion with a high

R value but with a thin surface layer [ 52 ].

A removable appliance with three 200-μm-wide

grooves cut into bovine dentin disks was used to

accumulate plaque, which was later treated with

chlorhexidine. Then the in situ plaque with and

without chlorhexidine treatment can be well

investigated to extrapolate the similar conditions

in oral cavity [ 53 ].

9.2.1.2 Fixed Appliances

Fixed appliances in in situ caries model are the

appliances that can only be removed in the end of

the study. They have various forms: banding model

such as orthodontic band model which can develop

orthodontic non-cavitated (white spot) lesion and

can produce a plaque accumulation niche for

demineralization. The crown single- section model

relies mainly on placement of the sections in

plaque-retentive areas below contact points [ 60 ].

The enamel can even be bonded to the teeth

directly to collect natural plaques on the natural

enamel surface [ 57 ]. Orthodontic non- cavitated

(white spot) lesion models have been used regu-

larly for testing the efﬁ cacy of novel remineraliza-

tion agents; however, this approach cannot be

extrapolated to model other types of non-cavitated

lesions, for after removing the band, the lesion will

not continue to develop [ 62 ]. A special in situ car-

ies model is that non- cavitated lesions formed

under plaque retention sites on the teeth that are

destined for extraction, and the effects of reminer-

alization agents can be tested [ 56 ].

9.3 Animal Model in Caries

Research

In 1995, the US Food and Drug Administration

(FDA) issued the Anticaries Drug Products for

Over-the-Counter (OTC) Human Use ﬁ nal rule,

establishing the requirement that “all OTC anti-

caries dentifrice drug product formulations be

tested in the animal caries reduction test” [ 12 ].

Animal caries models have a long history of

successful use in caries research. They are invalu-

able tools to simulate the natural progression of

caries under true biological conditions. Unlike in

situ and in vitro systems, which measure isolated

components of caries process, animal caries

models truly measure caries [ 4 ]. Animal caries

9 Models in Caries Research

168

model has played a critical role in caries research

due to its unique advantages. Compared with in

situ caries model and in vitro caries model, it is

the closest caries model. The whole saliva is

present, and it can provide components of the

host defenses and simulate more accurately the

clearance of test compounds [ 3 ]. However, there

are also limitations, including differences on the

composition of the oral ﬂ ora and dental plaque,

eating habits, saliva, food retention, dentition,

and the morphology of the teeth [ 2 , 50 ]. It may be

difﬁ cult to inoculate and establish some human

bacterial strains in animals, while the pattern of

caries in rodents is different from that observed in

humans.

Various experimental animal species have

been used in animal caries model, including non-

human primates, rats, hamsters, and mice, during

which the rodent models are the commonest. The

development of genetically modiﬁ ed animals

makes utility of the model more broadly. With

respect to the general considerations in animal

caries models, William H. Bowen has a very

excellent review [ 29 ]. In short, several important

considerations should be paid attention to: selec-

tion of animals, litter effect, age of animals, sex,

caging of animals, and diet. When those consid-

erations are deliberately under control, which

makes the baseline the same, the animal caries

model may show the effect brought by the experi-

mental factors. Animal caries models are very

valuable to study the etiology of dental caries and

evaluate the anticaries products.

9.3.1 Study on Etiology of Dental

Caries

Animal caries models can be used to evaluate the

cariogenicity of diet. The ﬁ rst recorded use of

rats in caries research was published in 1922 by

McCollum et al., who was primarily interested in

the role of diet in the etiology of dental caries.

Most of the early studies were focused on the

inﬂ uence of diet [ 29 ]. Nowadays, some special

food was still evaluated by animal model. For

example, cariogenicity of milk and formula was

compared in Wistar rat. Sucralose, a sugar

substitute, was found to have low cariogenicity in

Sprague-Dawley rat. In SPFOM rat, high-glucose

diet was found to have higher cariogenic capacity

than pure starch [

63 ].

As the inherent difference in the dentition

between animals and humans, the host factors

investigated in animal caries models mainly

focus on the saliva factor. Data from the animal

models, especially desalivated animal models,

have enhanced our understanding of the impor-

tance of saliva in maintaining oral health. The

incidence of caries increased signiﬁ cantly in rats

which had their salivary glands removed surgi-

cally [ 29 ]. Conventionally, rats can be desalivated

partially or completely by surgery to test the role

of saliva in the development of caries or to make

caries formation faster. An alternative strategy is

to genetically modify the test animal system to

exclude the host factor of interest. In mice with

targeted deletion of the gene encoding aquapo-

rin- 5 ( Aqp

−/−

), a water channel involved in the

production of saliva, there was a signiﬁ cant

increase in caries, indicating that caries

susceptibility increases with a reduced salivary

ﬂ ow that is associated with decreased water con-

tent of saliva [ 64 ]. The interaction of bacteria and

host can also be evaluated in genetically manipu-

lated rats. In NOD/SCID. e2f1

−/−

mice that show

hyposalivation, lower salivary antibody, and an

extended life span compared to the parent strain,

the roles of several salivary components in

Streptococcus mutans colonization in mice were

evaluated, suggesting that there are multiple

effects exerted by sIgA in Streptococcus mutans

colonization, with synergistic effects evident

under the condition of sIgA and limited nutrients

on colonization in NOD/SCID. e2f1

−/−

[ 65 ].

Comparing the lesion formation in germfree

and conventional rodents can make sure the role

of microorganism in dental caries, and animal

caries models are a good tool to study the micro-

bial etiology of dental caries. In addition, animals

can be inoculated with mutants of cariogenic

bacteria that lack putative virulence traits or have

putative anticaries traits, so that this type of

model can be valuable in determining factors

involved in the pathogenesis of caries [ 3 ]. A

strain of Streptococcus mutans , which lacks

H. Xuelian et al.

169

urease, was genetically engineered to express the

urease genes of Streptococcus salivarius . Rats

were infected with the parent strain or the alkali-

producing S. mutans and fed a cariogenic diet

supplemented with urea. The rats infected with

the recombinant strain had a dramatically lower

incidence and severity of all types of caries com-

pared with controls, showing that alkali genera-

tion inhibits caries [ 66 ]. Furthermore, the

development of transgenic animals together with

genetic manipulation of microorganisms will

facilitate the utility of the model greatly and may

lead to development of novel approaches to the

prevention of disease [ 29 ].

9.3.2 Evaluate Anticaries Agent

The beneﬁ ts of animal caries models also lie in

their role in evaluating ﬂ uoride and antimicrobial

compounds and vaccination on plaque formation

and caries development. It has been pointed out

that no caries-protective agent currently in human

use has failed in a rodent test. However, it has

been argued that the experimental conditions in

animal caries model may be too severe to mimic

the condition in human being, for example, a

56 % sucrose diet with ad libitum feeding, and

infection with S. sobrinus , which is particularly

adapted to colonizing and causing caries on

smooth surfaces. The outcome is that some prom-

ising agents may be incorrectly discarded [ 3 ].

Animal caries models have been demonstrated

to be suitable to evaluate the caries-preventive

effect of ﬂ uoride, for those reasons: These mod-

els develop incipient and more advanced coronal

caries which resemble clinical caries structurally

and etiologically, and the response of animal car-

ies models to ﬂ uoride demonstrates dose

responses [

2 ].

There are several models that have been used

to evaluate the efﬁ cacy of ﬂ uoride-containing

dentifrices, such as Francis’ hypomineralized

area (HMA) model, Gaffar’s CARA rat caries

model, Connecticut rat caries model, and Indiana

rat caries model. G.K. Stookey and co-workers

have reviewed those models in detail [ 2 ]. Shortly

speaking, HMA model is designed to assess the

impact on incipient enamel lesions, while the

other three models utilize more overt caries

lesions. HMA model relies upon an indigenous

ﬂ ora; the other three models involve the infection

of the animals with cariogenic microorganisms.

It is also possible to develop animal caries

models to evaluate non-ﬂ uoride antimicrobial

agents. As antimicrobial agents utilize different

mechanism with ﬂ uoride, the former mainly

work on microorganism, and the latter mainly

work on mineralization. It is wise to modify the

animal caries model.

9.4 The Role of Saliva in Caries

Models

The periods of alkalinization in dental bioﬁ lm,

which promote remineralization and restore the

integrity of the enamel, are primarily attributable

to diffusion of acids from the bioﬁ lms, buffering

by salivary bicarbonate, salivary peptides, bacte-

rial cells, and bacterial metabolism of urea and

arginine [ 66 ]. In this process, the role of saliva is

very important. This role is also demonstrated by

a lot of clinical evidence.

In animal caries model and in situ caries mod-

els, in which the whole saliva is present, salivary

factor is inevitably involved, while desalivated

animals (salivary glands are partially or com-

pletely removed or drug-induced hyposalivation)

can mimic the extreme cariogenic challenge com-

monly observed in patients suffering from sali-

vary hypofunction [

64 ]. Also, current techniques

to alter gene expression in animals allow direct

analysis of the saliva in caries development [ 64 ].

In in situ caries models, it is well realized that

salivary composition and ﬂ ow differ at different

sites, which may lead to ﬂ uctuating results. So it

is better to put the model in the same dentition

site, even if there are still some differences among

different individuals. To overcome this problem,

it is better to recruit subjects with similar salivary

ﬂ ow and buffer capacity. Those are all important

considerations in designing in situ caries study.

Furthermore, the role of ﬂ uoride in saliva is

also well realized. Fluoride may reach saliva

directly from the ingesta or from topical

9 Models in Caries Research

170

treatments, or indirectly from the bloodstream

via the salivary glands or gingival crevicular

ﬂ uid, or from temporary intraoral reservoirs of

ﬂ uoride, including surface deposits on the teeth

of calcium ﬂ uoride-like material and dental

plaque [ 67 ]. After local application of ﬂ uoride

and initial rapid clearance phase, the saliva can

have a low concentration of ﬂ uoride over long

periods of time, which is as important as a brief

exposure to relatively high ﬂ uoride concentra-

tions for shorter periods of time [ 68 ]. So it is nec-

essary to consider this factor related to saliva,

when evaluating the caries-preventive role of

ﬂ uoride in caries model. Then another question is

also raised, whether other caries-preventive

agents have their “reservoirs” in oral cavity.

However, in in vitro caries models, the role of

saliva was not always emphasized. In some cases,

the effect of saliva was objected, for the reason

that caries lesions form only in “stagnant” sites

where the beneﬁ t of saliva is not working and

thus that models where salivary inﬂ uences are

excluded will best represent the conditions of

caries lesion formation [ 67 ].

On the other hand, there are some models

focusing on the saliva’s role in the oral cavity and

further on its role in dental caries, such as saliva-

plaque interface, salivary clearance of bacterial

substrates, ﬂ uoride, and acid [ 69 , 70 ]. The stud-

ies of the Stephan curve showed that if salivary

stimulation is mimicked, the rate of pH rise in a

model plaque is highly dependent upon the bicar-

bonate concentration and the velocity of the ﬁ lm

of saliva in both thick and thin plaques, indicat-

ing that salivary beneﬁ ts can be exerted even with

thick plaques [

67 ].

Even in caries models considering the effect

of salivary factor, saliva tends to be considered

more as a nonspeciﬁ c diluent or sink rather than

as a ﬂ uid with a complex chemistry that may

interact with the plaque and the teeth in signiﬁ -

cant ways [ 67 ]. Saliva possesses an array of

activities that appear to have been seldom consid-

ered to in many caries models. However, those

bioactivities should not be ignored as the follow-

ing facts.

Firstly, saliva has some properties regarding de-

and remineralization of hard tissue of teeth, such

as the saturation of calcium and phosphate ions,

calcium-ion binding by salivary macromolecules,

and some precursors for the adsorbed protein ﬁ lms

or pellicles, found on teeth surfaces, which have a

signiﬁ cant effect on the interactions of dental min-

eral with overlying ﬂ uids, particularly with respect

to diffusion rates of acids into and calcium and

phosphate ions out of the enamel [

71 ].

Secondly, it is well demonstrated that alkali

generation from salivary substrates, especially

arginine (or polypeptides and proteins) and urea,

could play major roles in plaque pH homeostasis

and in the inhibition of dental caries [ 66 ].

Arginine and urea can be secreted by salivary

glands, and polypeptides and proteins containing

arginine residue belong to saliva proteins.

Interestingly, the presence of saliva itself will

favor the base formation and then the pH rise

[ 72 ]; the reason may be attributed to its buffer

capacity [ 26 ].

Thirdly, salivary proteins adsorbed onto sur-

faces of apatitic minerals profoundly affect bac-

terial adhesion onto those surfaces; those effects

may signiﬁ cantly inﬂ uence the initial bacterial

colonization of teeth and, therefore, the microbial

nature of dental plaque.

Fourthly, there are some antibacterial systems

in saliva which contain sialoperoxidase, lyso-

zyme, lactoferrin, histatins, peroxidases, and

other basic polypeptides, which have less speciﬁ c

antibacterial effect and also other bioactivities.

Collectively, in oral cavity, saliva plays a cru-

cial role in the initiation and progression of den-

tal caries, chemical and physiological process of

de- and remineralization of the teeth, dental bio-

ﬁ lm formation and metabolism, clearance, buffer

and neutralization of acid, and so on. As a result,

in dental caries model, saliva should not be

ignored.

In summary, much of our present understand-

ing of the etiology and initiation and progression

of dental caries as well as the identiﬁ cation of

caries-preventive agents or measures is attributed

to the ﬁ ndings of studies on models. However, no

single, ideal model is optimal for studying all

aspects of caries, and different models have

speciﬁ c roles in studying speciﬁ c aspects. They

have their advantage and disadvantage from both

H. Xuelian et al.

171

experimental design and experiment cost. Models

must be modiﬁ ed or reﬁ ned to achieve the desired

goals.

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175

Z. Xuedong (ed.), Dental Caries: Principles and Management, DOI 10.1007/978-3-662-47450-1

Academic Centre for Dentistry Amsterdam (ACTA) , 136

Accessory canals , 16

ACFP. See Amorphous calcium ﬂ uoride phosphate (ACFP)

Acid-etching technique , 107, 108

Acidogenic bacteria

acid tolerance of , 40

Actinomyces , 39–40

carbohydrate metabolism and , 39

Lactobacilli , 39

Streptococcus mutans , 39

A C P . See Amorphous calcium phosphate (ACP)

Actinomyces

A. actinomycetemcomitans , 37

acidogenic bacteria , 39–40

A. naeslundii , 37, 40, 47

A. oris , 32

arginine deiminase system , 42

Acute lymphoblastic leukemia (ALL) , 48

ADA caries risk assessment , 65–67

Adhesive dentistry , 107

ADS. See Arginine deiminase system (ADS)

Aggregatibacter actinomycetemcomitans , 29, 139

Agmatine deiminase system (AgDS) , 42

Air abrasion , 111–112

Air polishing , 112

Alkali production

ADS , 42

AgDS , 42

and bioﬁ lm ecology , 42–43

in caries prevention , 41

clinical relevance of , 43

urease , 40–41

ALL. See Acute lymphoblastic leukemia (ALL)

Ameloblastin , 8

Amelogenin , 7–8

Amelotin , 8

Amorphous calcium ﬂ uoride phosphate (ACFP) , 77

Amorphous calcium phosphate (ACP) , 77, 79, 115

Animal model

de-/remineralization , 74

in research , 167

anticaries agent evaluation , 169

etiology study , 168–169

saliva role , 169

Anticaries agent, animal model , 169

Antimicrobial approaches

chlorhexidine , 44

ﬂ uoride , 44–45

natural products , 46–48

phenolic antiseptics , 46

quaternary ammonium compounds , 45

triclosan , 45–46

xylitol , 46

Antimicrobial substances, in saliva , 50

Apical periodontitis , 141

Arginine deiminase system (ADS) , 42

A R T . See Atraumatic restorative technique (ART)

Arteriosclerotic vascular disease (ASVD). See

Atherosclerosis

Artiﬁ cial mouth

open system microbial models , 163–164

Streptococcus mutans in , 165

Atherosclerosis , 136–138

Streptococcus mutans and , 138–139

Atopobium , 43

Atraumatic restorative technique (ART) , 115

Bacteremia , 131–132

Bacterial bioﬁ lm

concept and discovery , 27

extracellular polymeric substances , 28

formation , 28–29

survival advantages of , 29–30

Bap family. See Bioﬁ lm-associated-protein

(Bap) family

Bell stage , 3

Bicarbonate , 61

Biﬁ dobacterium spp. , 49

B. dentium , 43

Bioﬁ lm-associated-protein (Bap) family , 29

Bioﬁ lm ecology, alkali production and , 42–43

Bioﬁ lm model

drip-fed , 162

ﬂ ow cell , 161–162

systems , 160–161

TSP , 160

Zürich bioﬁ lm model , 160

Index

176

Biological treatment methods , 127–128

Biomineralization , 80

Bionics, and restorative dentistry , 128

Bis-GMA system , 105

Bitewing radiography, for caries detection , 99

Black, G.V., and restorative dentistry , 107–108

B lymphocytes , 20

Body mass index (BMI) , 135–136

British Medical Journal (BMJ) , 137

Bud stage , 2

Calcium ﬂ uoride particles , 79

Calcium phosphate (CaP) , 77

particles , 79

stage , 2–3

Camellia sinensis , 47

Candida albicans , 159

CaP. See Calcium phosphate (CaP)

Carbohydrate metabolism , 39

Carbonate hydroxyl apatite nanoparticles , 79

Cardiac dysrhythmias , 138

Cardiovascular disease (CVD) , 136–140

Care Index, oral diseases , 91

Caries-associated bacteria , 65–66

Caries pharmacotherapy , 114

Caries-preventive strategies

dental plaque control , 102–103

pit and ﬁ ssure sealing , 105

preventive resin restoration , 105

primary prevention , 98

reinforce tooth resistance to acid , 103–105

secondary prevention

conventional caries detection methods , 98–99

DIAGNOdent , 100

electrical caries monitor , 101

FOTI , 99

QLF , 100–101

sugar restriction and substitute for , 103

tertiary prevention , 101–102

Cariogenic bacteria

logistic regression analysis , 138

oral cancer , 134

Casein phosphopeptide (CPP) , 115

CPP-ACFP , 77

CPP-ACP , 77

Cavity preparation

minimally invasive treatment

air abrasion , 111–112

air polishing , 112

atraumatic restorative technique , 115

caries pharmacotherapy , 114

chemomechanical caries removal , 112–113

enameloplasty , 114

lasers , 112

mechanical rotary technique , 113

microscopic preparation techniques , 113

nonmachinery preparation , 111–113

pit and ﬁ ssure sealing , 113–114

preventive resin restorations , 114

remineralization treatment , 114–115

slot preparation , 113

tunnel preparation , 113

resin composite , 121–123

CBP. See Collagen-binding protein (CBP)

CDC. See Center for Disease Control (CDC)

CDFF. See Constant depth ﬁ lm fermenter (CDFF)

Ceanothus americanus , 47

Cell-poor zone of pulp , 17

Cell-rich zone of pulp , 17

Center for Disease Control (CDC) , 142, 145

Cetylpyridinium chloride (CPC) , 45

CHD. See Coronary heart disease (CHD)

Chemical models, in research , 157–158

Chemomechanical caries removal (CMCR) , 112

Chemoparasitic theory , 33, 34

Chemostat

ﬂ ow cell system , 165

open system microbial models , 164

Childhood, dental caries , 134–136

Chlorhexidine , 44

Chronic apical periodontitis (CAP) , 137–138

Chronic kidney disease (CKD) , 143–144

Chronic obstructive pulmonary disease

(COPD) , 149

Chronic renal disease , 143–144

Circumpulpal dentin , 12

CKD. See Chronic kidney disease (CKD)

Clin Oral Investig in 2011 , 136

Closed system microbial models, in research , 159–161

CMCR. See Chemomechanical caries removal (CMCR)

Cold and hot irritation test , 86–87

Colitis ulcerosa. See Ulcerative colitis (UC)

Collagen-binding protein (CBP) , 139, 145

Community Periodontal Index (CPI) of

Treatment Needs (CPITN) , 91

Competence-stimulating peptide (CSP) , 38, 50

Confocal laser scanning microscopy (CLSM) , 75

Connective tissue ﬁ bers , 15–16

Constant depth ﬁ lm fermenter (CDFF) , 73, 162

Contact-microradiography. See Transsal

microradiography (TMR)

Coronal caries, cross-sectional analysis of , 142

Coronary heart disease (CHD) , 136, 140–141

CPC. See Cetylpyridinium chloride (CPC)

CPP. See Casein phosphopeptide (CPP)

Craniofacial tissues embryology

head formation , 2

neural crest , 1–2

origin of tissue , 1

C-reactive protein (CRP) , 138

CSP. See Competence-stimulating peptide (CSP)

CVD. See Cardiovascular disease (CVD)

Cytodifferentiation, enamel development , 3

maturation stage , 5

presecretory stage , 4

secretory stage , 4–5

Index

177

Decayed, missing, and ﬁ lled teeth (DMFT) index , 92–97,

133, 142

5-year-olds , 95

12-year-olds , 93, 95–96

35–44-year-olds , 94, 96–97

65–74-year-olds , 97–98

salivary immunoglobulin A , 142

Demineralization models, caries research , 158

Dendritic cell, in pulp , 19–20

Dental caries

5-year-olds , 95

12-year-olds , 95–96

35–44-year-olds , 96–97

65–74-year-olds , 97–98

assessment , 133

burden in China , 94–98

detection methods , 98–101

DMFT index , 92–97, 133, 142

Nepal example , 91

prevalence , 92, 133

Dental drilling , 110

Dental enamel , 71

Dental ﬂ oss examination , 87

Dental ﬂ uorosis , 88

Dental microﬂ ora , 31

Dental plaque

and bacterial bioﬁ lm , 30

as cause of dental caries , 33–34

composition , 31

control , 102–103

ecological plaque hypothesis , 34–35

nonspeciﬁ c/speciﬁ c plaque , 34

Dentin

bonding , 120–121

mineralization , 12

repair , 13

sclerosis , 12–13

structure , 9–12

types , 12

Dentinal tubules , 9–10

Dentin-enamel junction (DEJ) , 7

Dentin phosphoproteins (DPP) , 12

De-/remineralization

biomineralization , 80

calcium phosphate , 77

CPP-ACFP , 77

CPP-ACP , 77

detection and measurement methods , 74

CLSM , 75

indentation techniques , 75

micro-CT , 75

OCT , 75–76

QLF , 75

TMR , 75

dynamics process of , 72

ﬂ uoride , 76

investigations models , 72–73

animal model , 74

in situ model , 73–74

in vitro bioﬁ lm model , 73

in vitro chemical model , 73

laser , 77–78

nanoparticles , 78–79

natural medicine , 77

Detection methods, dental caries , 98

DIAGNOdent , 100

electrical caries monitor , 101

ﬁ ber-optic transillumination , 99

quantitative light-induced ﬂ uorescence , 100–101

Diabetes mellitus (DM) , 145–146

epidemiological studies of , 146–147

root caries , 147–148

tooth loss and , 148–149

DIAGNOdent , 100

Diagnosis for caries

cold and hot irritation test , 86–87

dental ﬂ oss examination , 87

dental ﬂ uorosis , 88

diagnostic cavity preparation , 87

elastomeric separating modulus technique , 88

electrical impedance technology , 87

enamel hypocalciﬁ cation , 88, 89

enamel hypoplasia , 88, 89

ﬁ ber-optic transillumination , 87

glossiness and smoothness , 88

inspection , 85

lesion

progress of , 89

symmetry of , 89

medium caries , 89

percussion , 85

predilection site , 88–89

probing , 85

quantitative laser ﬂ uorescence technique , 88

radiographic examination , 85–86

staining technique , 88

ultrasonic technique , 87

Diagnostic cavity preparation , 87

Diet , 36, 103, 166, 168, 169

Digitized ﬁ ber-optic transillumination (DI-FOTI) , 99

Direct bonding restoration technique , 118

DMFT index. See Decayed, missing, and ﬁ lled teeth

(DMFT) index

DPP. See Dentin phosphoproteins (DPP)

Drip-fed bioﬁ lm model , 162

Early childhood caries (ECC) , 135

ECM. See Electrical caries monitor (ECM)

Ecological plaque hypothesis , 34–35

EDJ. See Enamel–dentinal junction (EDJ)

eDNA. See Extracellular DNA (eDNA)

Elastomeric separating modulus technique , 88

Electrical caries monitor (ECM) , 16, 101

Electrical impedance technology , 87

Electron microscopy , 32

Index

178

Embryology of craniofacial tissues

head formation , 2

neural crest , 1–2

origin of tissue , 1

Enamel bonding , 119–120

Enamel–dentinal junction (EDJ) , 105, 125

Enamel development

cytodifferentiation , 3

maturation stage , 5

presecretory stage , 4

secretory stage , 4–5

enamel matrix proteins

ameloblastin , 8

amelogenin , 7–8

amelotin , 8

enamelin , 7

proteolytic enzymes , 8–9

tuftelin , 8

histogenesis and morphogenesis

bell stage , 3

bud stage , 2

cap stage , 2–3

microstructure

enamel lamellae and cracks , 6

enamel rod , 5

enamel spindle , 6

enamel tufts , 6

functional aspects , 6–7

interpit continuum , 6

Enamel hypocalciﬁ cation , 88, 89

Enamel hypoplasia , 88, 89

Enamelin , 7, 8

Enamel lamellae and cracks , 6

Enamel matrix proteins

ameloblastin , 8

amelogenin , 7–8

amelotin , 8

enamelin , 7

proteolytic enzymes , 8–9

tuftelin , 8

Enameloplasty , 114

Enamel rod , 5

Enamel spindle , 6

Enamel tufts , 6

Endodontic inﬂ ammation , 140

Environmental factors, oral microbial ecology , 36

Environment dominates theory , 36

Epithelial cell rests of Malassez (ERM) , 21

Erbium:yttrium-aluminum-garnet (Er:YAG) , 77, 112

Essential oils , 47

Etching adhesive systems, bonding mechanisms , 118–119

Evidence-based dental caries diagnosis , 49

Extracellular DNA (eDNA) , 28

Extracellular polymer substances (EPS) , 28

Extracellular polysaccharides (EPS) , 73

Featherstone pH cycling model , 158

Fertilization, craniofacial tissues , 1

Fiber-optic transillumination (FOTI) , 87, 99

Fibronectin , 16

Film radiograph, for caries detection , 99

Fixed appliances, in situ caries models , 167

Flow cell bioﬁ lm model , 161–162

Fluorapatite , 109

Fluorescence, in dental caries , 100

Fluoride , 44–45, 76, 114

saliva role in research , 169–170

Focal infection , 129

Focal sepsis theory , 130

FOTI. See Fiber-optic transillumination (FOTI)

Fusobacterium , 36

Galla chinensis (GCE) , 77

Gastroesophageal reﬂ ux disease (GERD) , 144–145

GCF. See Gingival crevicular ﬂ uid (GCF)

Gelatinous microbic plaques. See Dental plaque

Genetic factors, oral microbial ecology , 36

GERD. See Gastroesophageal reﬂ ux disease (GERD)

Gerodontology , 138

Gingival crevicular ﬂ uid (GCF) , 32

Ginkgo biloba , 47

Glossiness, and smoothness , 88

Head and neck cancer

tooth loss with , 134

treatment , 132–133

Head and neck squamous cell carcinoma (HNSCC) , 133

Head formation, craniofacial tissues , 2

Hertwig’s epithelial root sheath (HERS) , 20–21

Histatins , 63

Histogenesis, enamel development

bell stage , 3

bud stage , 2

cap stage , 2–3

HIV , 142–143

HNSCC. See Head and neck squamous cell carcinoma

(HNSCC)

Human microbiome project (HMP) , 30

Human salivary lactoperoxidase (HS-LPO) , 62

Human umbilical vein endothelial cells

(HUVEC) , 139

Hypomineralized area (HMA) model , 169

Hyposalivation , 132

IE. See Infective endocarditis (IE)

Immune system disease

effect on , 130

HIV , 142–143

salivary immunoglobulin A , 141–142

Indentation techniques , 75

Individualized treatment

importance , 125–126

risk evaluation , 126

technology and material , 126

Index

179

Infectious disease, dental caries as , 33

Infective endocarditis (IE) , 138

Inoculum, microbial models , 159

In situ model

de-/remineralization , 73–74

in research

classiﬁ cation , 167

disadvantages , 166

outcome , 166

saliva role , 169

Inspection of caries , 85

Interglobular dentin , 11–12

Interspecies interactions

and dental caries , 36–39

factors involved in , 37–38

metabolic interrelationship , 37

Intertubular dentin , 10–11

Intra-class correlation coefﬁ cients (ICCs),

QLF , 101

In vitro bioﬁ lm model, de-/remineralization , 73, 74

In vitro chemical model, de-/remineralization , 73

In vitro models, in research

chemical models , 73, 157–158

microbial-based de-and remineralization model ,

164–166

microbial models , 158–164

saliva role , 170

JAMA Otolaryngology–Head and Neck

Surgery , 133

Kallikrein-4 , 9

Lactic acid bacteria , 133

Lactobacillus , 31, 61, 66

acidogenic bacteria , 39

arginine deiminase system , 42

L. casei , 49

L. reuteri , 49

L. rhamnosus , 47

probiotics , 49

Lactoferrin , 63

Laminin , 16

Langerhans cells , 20

Lasers

cavity preparation , 112

de-/remineralization , 77–78

Lesion

control , 110

progress of , 89

symmetry of , 89

Light active killing , 50

Lymphatics , 16

Lymphocyte, in pulp , 20

Lysozyme , 62

Macrophage, in pulp , 19

MAM system. See Multiple artiﬁ cial mouth (MAM)

system

Mantle dentin , 12

Marsh Consortium , 159

Mast cell, in pulp , 20

Matrix metalloproteinase-20 (MMP20) , 9

Maturation stage, enamel development , 5

Mechanical rotary technique , 113

Melaphis chinensis , 47

Mesenchymal stem cells (MSCs)

induction of differentiation , 21

in pulp , 20

Mesenchyme , 2

Metagenomics, oral microbiome , 48–49

Microbial-based de-and remineralization model ,

164–166

Microbial ecology, in oral cavity , 35–36

Microbial models, in research , 158

classiﬁ cations , 159

closed system , 159–161

inoculum , 159

open system

artiﬁ cial mouth , 163–164

chemostat , 164

drip-fed bioﬁ lm model , 162

ﬂ ow cell bioﬁ lm model , 161–162

modiﬁ ed robbins device , 161–162

perfused bioﬁ lm fermenters , 162–163

Micro-CT investigation , 75

Microindentation technique , 75

Microorganisms of the Human Mouth (Miller) , 33

Microscopic preparation techniques , 113

Microtiter plate (MTP)-based system , 159–160

Mineralization of dentin , 12

Mini box preparation. See Slot preparation

Minimally invasive treatment , 108–111

cavity preparation

air abrasion , 111–112

air polishing , 112

atraumatic restorative technique , 115

caries pharmacotherapy , 114

chemomechanical caries removal , 112–113

enameloplasty , 114

lasers , 112

mechanical rotary technique , 113

microscopic preparation techniques , 113

nonmachinery preparation , 111–113

pit and ﬁ ssure sealing , 113–114

preventive resin restorations , 114

remineralization treatment , 114–115

slot preparation , 113

tunnel preparation , 113

demineralized layer , 110–111

early diagnosis and personal treatment , 110

infected layer , 110

prevention and effective intervention , 110

treatment and effective control , 110

MMP20. See Matrix metalloproteinase-20 (MMP20)

Modiﬁ ed robbins device (MRD) , 161–162

Index

180

Morphogenesis, enamel development

bell stage , 3

bud stage , 2

cap stage , 2–3

Morphogenetic protein 4 (BMP4) , 22

MSCs. See Mesenchymal stem cells (MSCs)

MSD. See Multiple Sorbarod device (MSD)

MTP-based system. See Microtiter plate (MTP)-based

system

Mucins , 62

Multiple artiﬁ cial mouth (MAM) system , 163

Multiple regression analysis , 95, 97

Multiple Sorbarod device (MSD) , 163

Mutacins , 37

Myeloperoxidase (MPO) , 62

Nanoindentation technique , 75

Nanoparticles , 78–79

National Health and Nutrition Examination Survey

(NHANES) , 147

Natural medicine, de-/remineralization , 77

Neisseria strains , 134

Nerve ﬁ bers, pulp , 14–15

Neural crest , 1–2

Notch signaling, for root development , 22

OCT. See Optical coherence tomography (OCT)

Odontoblast , 16–19

Olsenella , 43

Open system microbial models, in research

artiﬁ cial mouth model , 163–164

chemostat , 164

drip-fed bioﬁ lm model , 162

ﬂ ow cell bioﬁ lm model , 161–162

modiﬁ ed robbins device , 161–162

perfused bioﬁ lm fermenters , 162–163

Optical coherence tomography (OCT) , 75–76

Oral bioﬁ lms, spatiotemporal development of , 31–32

Oral cancer, cariogenic bacteria and , 134

Oral cavity , 30, 130

Care Index , 91, 92

CPITN , 91

high-income industrialized countries , 91

microbial ecology in , 35–36

uneven distribution , 91–92

YLD/million population , 92

Oral ﬂ uids , 72

Oral haemophili urease , 40

Oral health , 129

epidemiological investigation in China , 94–98

global policies , 92–94

Oral Health in America: A Report of the Surgeon

General , 129

Oral microbial ecology , 36

Oral microbiology, at early stage , 33

Oral microbiome , 48–49

Oral microbiota , 148

Oral microﬂ ora, saliva on , 62–63

Orthodentin , 12

PBF. See Perfused bioﬁ lm fermenters (PBF)

PCR method , 138

Pediatric Nephrology , 144

Percussion for caries , 85

Perfused bioﬁ lm fermenters (PBF) , 162–163

Periapical diseases , 130

Periodontal disease , 129–130, 149

Peritubular dentin , 10, 11

“pH cycling” protocols , 158

Phenolic antiseptics , 46

Pit and ﬁ ssure sealant , 105, 113–114

Plaque pH, salivary inﬂ uences on , 61–62

Polymerization shrinkage , 123–125

conﬁ guration factor , 124–125

material related factors , 124

Porphyromonas gingivalis , 36

Postoperative sensitivity , 125

Predentin , 12

Predilection site , 88–89

Presecretory stage, enamel development , 4

Preventive resin restorations , 105, 114

Probing for caries , 85

Probiotics , 49–50

Proliferative period , 1

Propionibacterium , 43

Propolis , 47

Proteolytic enzymes , 8–9

Pseudomonas

P. aeruginosa , 29

P. ﬂ uorescens , 29

P. putida , 29

Pseudoramibacter , 43

Pulp , 13–14

accessory canals , 16

cells in

dendritic cell , 19–20

lymphocyte , 20

macrophage , 19

mast cell , 20

mesenchymal cell , 20

odontoblast , 17–19

pulp ﬁ broblast , 19

connective tissue ﬁ bers , 15

ﬁ broblast , 19

ground substance , 15–16

lymphatics , 16

morphologic zones of pulp , 16–17

nerve ﬁ bers , 14–15

proper , 17

vascular tissues , 14

Pulpal periapical diseases , 140–141

Pulpitis , 130

Pulpodentin complex

cells in dental pulp

dendritic cell , 19–20

lymphocyte , 20

Index

181

macrophage , 19

mast cell , 20

mesenchymal cell , 20

odontoblast , 17–19

pulp ﬁ broblast , 19

dentin

mineralization , 12

repair , 13

sclerosis , 12–13

structure , 9–12

types , 12

pulp , 13–14

accessory canals , 16

connective tissue ﬁ bers , 15

ground substance , 15–16

lymphatics , 16

morphologic zones of pulp , 16–17

nerve ﬁ bers , 14–15

vascular tissues , 14

Quantitative laser ﬂ uorescence technique , 88

Quantitative light-induced ﬂ uorescence (QLF) , 75,

100–101

Quaternary ammonium compounds

(QACs) , 45

RA. See Rheumatoid arthritis (RA)

Radiation caries , 132

Radiographic examination for caries , 85–86

Remineralization

models in research , 158

treatment , 114–115

Removable appliances, in situ caries models , 167

Resin composite bonding restoration technique

cavity preparation , 121–123

dentin bonding , 120–121

enamel bonding , 119–120

etching adhesive systems , 118–119

indications and contraindications , 121

polymerization shrinkage , 123–125

postoperative sensitivity , 125

postprocessing decoration , 123

requirements for , 121

self-etch systems , 119

technique sensitivity , 125

total-etch system , 118–119

Resin composites , 118

Respiratory infections , 149–150

Restorative dentistry , 107

based on bionics , 128

biological treatment methods , 127–128

Black, G.V. and , 107–108

bonding technique and , 108

direct bonding restoration technique , 118

individualized treatment , 125–126

minimally invasive treatment ( see Minimally invasive

treatment)

resin composite

cavity preparation , 121–123

dentin bonding , 120–121

enamel bonding , 119–120

etching adhesive systems , 118–119

indications and contraindications , 121

polymerization shrinkage , 123–125

postoperative sensitivity , 125

postprocessing decoration , 123

requirements for , 121

self-etch systems , 119

technique sensitivity , 125

total-etch system , 118–119

resin composites , 118

silver amalgam

cavity shape preparation , 116

controversy , 115–116

ﬁ lling , 116–118

indications and contraindications , 116

Rheedia gardneriana , 48

Rheumatoid arthritis (RA) , 142

Root caries

cardiac dysrhythmias , 138

diabetes , 147–148

Root development

ERM , 21

initiation , 20–21

mesenchymal stem cells , 21

signaling pathway

notch signaling , 22

SHH signaling , 22

TGF-β/BMP Signaling , 21–22

Wnt signaling , 22

tooth eruption , 22–23

16S rRNA gene sequencing , 131

SAG. See Salivary agglutinin (SAG)

Saliva , 73

carbonic acid/bicarbonate equilibrium in , 61

caries-associated bacteria , 65–66

characteristics and caries risk , 67

chemical and physical aspects , 68

composition , 59–60

critical value , 61

ﬂ ow rate , 60–61, 65

formation and secretion , 59

low buffering capacity , 61

on oral microﬂ ora , 62–63

on plaque pH , 61–62

potential use of , 129

role in research

animal model , 169

ﬂ uoride , 169–170

oral cavity , 170–171

in situ model , 169

in vitro models , 170

xerostomia

etiology of , 63–64

management of , 64–65

Index

182

Salivary agglutinin (SAG) , 63

Salivary antimicrobial substances , 50

Salivary glands , 132

Salivary immunoglobulin A (SIgA) , 141–142

Sanguinaria canadensis , 47

Sanguinarine , 47

Scardovia wiggsiae , 43

S-ECC. See Severe early childhood caries (S-ECC)

Secondary dentin , 12

Secretory IgA (SIgA) , 62, 68

Secretory stage, enamel development , 4–5

Self-awareness, caries prevention , 98

Self-etch systems , 119

Severe early childhood caries

(S-ECC) , 135

SHH signaling, for root development , 22

Signaling pathway, for root development

notch signaling , 22

SHH signaling , 22

TGF-β/BMP Signaling , 21–22

Wnt signaling , 22

Silver amalgam , 115–116

cavity shape preparation , 116

controversy , 115–116

ﬁ lling , 116–118

indications and contraindications , 116

Slackia exigua , 43

Slot preparation , 113

Smoothness, glossiness and , 88

Speciﬁ cally targeted antimicrobial peptides

(STAMPs) , 50

Staining technique , 88

Stephan curve , 61

Streptococcus

S. gordonii , 29, 38, 42, 161

S. mutans , 31, 45, 61, 63, 66, 132

acidogenic bacteria , 39

acid tolerance of , 40, 42

AgDS activity , 42

in artiﬁ cial mouth , 165

atherosclerosis , 138–139

in bioﬁ lm , 50, 73, 160

catechins inhibit growth of , 47

colonization in mice , 168

colonization in saliva , 143

ecological plaque hypothesis , 34–35

genetically modiﬁ ed strain , 43

in GERD group , 145

inoculum , 165

interspecies interactions , 37–39

involvement , 34

risk assessment , 110

salivary immunoglobulin A , 141

salivary level , 66

SIgA against , 62

STAMPs targeting , 50

strain of , 168–169

ulcerative colitis , 145

xylitol , 46

S. oralis , 32, 37, 161

S. parasanguis , 42

S. salivarius , 38, 40, 169

S. sanguinis , 38, 132

arginine deiminase system , 42

interspecies interactions , 37

Stroke , 139–140

Survey of Dental Diseases , 140

T2DM. See Type 2 diabetes mellitus

(T2DM)

Technique sensitivity , 125

TGF-β/BMP signaling, for root

development , 21–22

Tissue engineering , 127–128

T lymphocytes , 20

TMR. See Transsal microradiography (TMR)

Tomes’ process , 4, 5

Tooth eruption , 22–23

Tooth loss , 130–131

diabetes , 148–149

head and neck cancer risk , 134

Tooth regeneration , 127–128

Tooth worm , 33

Total-etch system , 118–119

Transferable solid phase (TSP) bioﬁ lm model , 160

Transversal microradiography (TMR) , 75

TRAP segment. See Tyrosine-rich amelogenin peptide

(TRAP) segment

Triclosan , 45–46

Tuftelin , 8

Tunnel preparation , 113

Type 2 diabetes mellitus (T2DM) , 145–146

Tyrosine-rich amelogenin peptide

(TRAP) segment , 7

Ulcerative colitis (UC) , 145

Ultrasonic technique , 87

Upper respiratory tract infection

(URTI) , 149

Vascular tissues, pulp , 14

Veillonella, interspecies interactions , 37, 38

Visual inspection

classiﬁ cation , 99

improvements in , 98

von Korff ﬁ bers , 16

Index

183

WHO Global Oral Health Programme , 92

Wnt signaling, for root development , 22

Xerostomia

causes of , 64

etiology of , 63–64

management of , 64–65

medications , 64

severity of , 133

sugar intake , 65

Xylitol , 46

Zürich bioﬁ lm model , 160

Index