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Review

Caries Res 2016;50:422–431 Received: March 8, 2016


Accepted after revision: May 31, 2016
DOI: 10.1159/000447309
Published online: July 27, 2016

Ecological Hypothesis of Dentin and


Root Caries
Nobuhiro Takahashi a Bente Nyvad b
a
Division of Oral Ecology and Biochemistry, Tohoku University Graduate School of Dentistry, Sendai, Japan;
b
School of Dentistry, Faculty of Health, Aarhus University, Aarhus, Denmark

Key Words firmed in the initial degradation of organic material, the de-
Bacteria · Cathepsin · Dental caries · Dentin · Ecological tection of proteolytic/amino acid-degrading bacteria and
hypothesis · Matrix metalloproteinases · Proteolysis · bacterial metabolites in dentin and root caries suggests a
Root caries bacterial digestion and metabolism of partly degraded ma-
trix. Moreover, bacterial metabolites might induce pulpitis
as an inflammatory/immunomodulatory factor. Root and
Abstract dentin surfaces are always at risk of becoming demineralized
Recent advances regarding the caries process indicate that in the oral cavity, and exposed organic materials can be de-
ecological phenomena induced by bacterial acid production graded by host-derived proteases contained in saliva and
tilt the de- and remineralization balance of the dental hard dentin itself. New approaches to the prevention and treat-
tissues towards demineralization through bacterial acid-in- ment of root/dentin caries are required.
duced adaptation and selection within the microbiota – © 2016 S. Karger AG, Basel
from the dynamic stability stage to the aciduric stage via the
acidogenic stage [Takahashi and Nyvad, 2008]. Dentin and
root caries can also be partly explained by this hypothesis; Recent advances in our knowledge about the caries
however, the fact that these tissues contain a considerable process indicate that ecological phenomena, e.g. bacterial
amount of organic material suggests that protein degrada- adaptation to acidic environments (increases in bacterial
tion is involved in caries formation. In this review, we com- acidogenicity and acidurance) and bacterial shifts to a
piled relevant histological, biochemical, and microbiological more acidogenic and aciduric microbiota (increases in
information about dentin/root caries and refined the hy- the proportion of acidogenic and aciduric bacteria), are
pothesis by adding degradation of the organic matrix (the induced by frequent and prolonged acidification. The re-
proteolytic stage) to the abovementioned stages. Bacterial sultant environmental acidification can tilt the balance
acidification not only induces demineralization and expo- between demineralization and remineralization toward
sure of the organic matrix in dentin/root surfaces but also demineralization, resulting in the initiation/progression
activation of dentin-embedded and salivary matrix metallo-
proteinases and cathepsins. These phenomena initiate deg-
radation of the demineralized organic matrix in dentin/root This paper is based on a presentation held at the 10th European Oral
surfaces. While a bacterial involvement has never been con- Microbiology Workshop, Aarhus, Denmark, in May 2014.

© 2016 S. Karger AG, Basel Nobuhiro Takahashi


0008–6568/16/0504–0422$39.50/0 Division of Oral Ecology and Biochemistry
Tohoku University Graduate School of Dentistry
E-Mail karger@karger.com
4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)
www.karger.com/cre
E-Mail nobu-t @ dent.tohoku.ac.jp
of caries. The ecological concept of caries was first pro- Ultrastructural studies have suggested that demineral-
posed in the ‘ecological plaque hypothesis’ by Marsh ization and breakdown of the organic matrix happens in
[1994] and was subsequently extended by Takahashi and two successive stages [Selvig, 1969; Schüpbach et al., 1989;
Nyvad [2008, 2011] based on detailed information re- Nyvad and Fejerskov, 1990]. In the early stages of cemen-
garding the biochemical responses to environmental tum and dentin caries, minerals are dissolved by a fine
acidification, i.e. the induction of bacterial acidogenicity/ gradient from the outer surface, while maintaining the
acidurance and the selection of acidogenic/aciduric bac- characteristic cross-banding of the collagen fibers [Nyvad
teria. This ecological hypothesis forms the basis of our and Fejerskov, 1990; Deyhle et al., 2011]. The demineral-
current understanding of caries, but mainly focusses on ized collagen serves as a scaffold for colonizing bacteria.
phenomena occurring in enamel caries. Ecological fac- At more advanced stages the exposed collagen is broken
tors in dentin and root caries have only been scarcely ad- down by proteolytic enzymes, and collagen fibers lose
dressed, possibly because of the complex structural com- their structural characteristics [Kuboki et al., 1977]. How-
position of these tissues. ever, a recent study [Tjäderhane et al., 2015] suggests that
The etiology of caries has been debated over centuries, collagen cross-banding may be lost at a relatively early
and three key hypotheses have been proposed: the chem- stage of demineralization, in which an exposed telopep-
ico-parasitic, the chelation, and the proteolysis-chelation tide region of the collagen molecule can be degraded by
theories. There is no doubt that the etiology of enamel telopeptidase activity of host-derived collagenolytic en-
caries can be mainly explained by the chemico-parasitic zymes. Dentin and root surfaces are more labile than
theory, in which the dental biofilm (bacteria) produces enamel in acidic conditions [Hoppenbrouwers et al., 1987;
acid from sugar, and the resultant acidic pH promotes Moreno and Aoba, 1991], possibly because of the smaller
demineralization of the enamel. Dentin and root caries hydroxyapatite crystallites in dentin [Yardeni, 1952].
can also be explained by this theory; however, the fact that Higher levels of carbonate and magnesium [Hoppenbrou-
dentin and root surfaces contain a considerable amount wers et al., 1987] and the tubular structure of dentin
of organic material, such as collagen, has led to the notion [Shellis, 1996] could also add to the higher solubility.
that protein degradation (the proteolysis theory) might Root and dentin surfaces are highly reactive in the oral
also play a role in caries development in these tissues. In- environment. Even clinically sound root surfaces may
deed, a series of publications [Dayan et al., 1983; Tjäder- take up minerals from the oral fluids, resulting in a high-
hane et al., 1998, 2015; Chaussain-Miller et al., 2006] have er mineral content in the surface layer compared with the
supported this possibility. normal dentin [Selvig, 1969]. Such phenomena are likely
In this review, we attempt to compile relevant histo- to occur as a result of cyclic demineralization and remin-
logical, biochemical, and microbiological information eralization processes [Nyvad et al., 1997]. However, root
about dentin and root caries and propose a possible and dentin surfaces are also vulnerable to physical injury.
mechanism for the development of dentin and root caries Because of the low mineral content, root surfaces are eas-
by including degradation of the organic matrix with the ily abraded by toothbrushing, and although cementum
cyclic de- and remineralization processes in caries. formation triples throughout life, reaching a thickness of
about 50 μm in the coronal part of the root at the age of
20 [Furseth and Mjör, 1979], exposed cervical root ce-
Structural Composition of Root and Dentin Surfaces mentum is often damaged and/or removed by regular
scaling and root planing [Jepsen et al., 2004]. Therefore,
Dentin and root cementum surfaces are less mineral- in clinical circumstances the exposed root often presents
ized than enamel. About 30% of dentin and root cemen- a dentin surface. In favorable conditions a new hypermin-
tum is composed of organic materials, mainly collagen, eralized surface layer may develop within a few months
while enamel is almost entirely composed of hydroxyapa- after over-instrumentation of root surfaces [Selvig, 1969].
tite with trace amounts of organic materials [Furseth and
Mjör, 1979]. These biochemical differences are reflected
in the pattern of caries. While bacterial invasion of enam- Microbial Ecosystem of Dentin and Root Caries
el occurs only after total breakdown of the tissue, root
surfaces become invaded by bacteria already at an early The microbiological and structural pattern of coloni-
stage of the caries process [Schüpbach et al., 1989; Nyvad zation of root surfaces has been extensively reviewed
and Fejerskov, 1990]. [Nyvad, 1993]. A few hours after the cleaning of a root

Ecological Hypothesis of Dentin/Root Caries Res 2016;50:422–431 423


Caries DOI: 10.1159/000447309
surface the surface is colonized by pioneer species mainly mutans streptococci species remain dominant [Brailsford
belonging to Streptococcus (S. sanguinis, S. oralis, and S. et al., 1999, 2001]. Furthermore, a molecular biology-
mitis) and Actinomyces. In situ studies have shown that based study [Preza et al., 2008] revealed that the micro-
the early colonizers do not differ between enamel and bial communities in root caries are highly complex and
root surfaces [Nyvad and Kilian, 1987], suggesting that it include not only mutans streptococci, Actinomyces, and
is the oral environment (saliva and/or gingival crevicular Lactobacillus species, but also species of Atopobium, Ol-
fluid) rather than differences in composition of the pel- senella, Pseudoramibacter, Propionibacterium, and Sele-
licle [Rüdiger et al., 2002] that determine the colonization nomonas. Yeasts, such as Candida albicans, were also de-
pattern [Nyvad, 1993]. In this scenario, it is not surprising tected in root caries lesions in middle-aged adults and the
that the microbial community on clinically sound root elderly [Shen et al., 2002; Zaremba et al., 2006], although
surfaces include various acidogenic bacteria belonging to the invasion of these species into carious dentin has been
Streptococcus and Actinomyces [van Houte et al., 1994, questioned [Maijala et al., 2007].
1996], many of which are capable of producing acid from Despite similarities between the processes responsible
dietary carbohydrates and lowering environmental pH to for enamel and root caries in terms of microbial acid-in-
<5.5 [van Houte et al., 1994, 1996; Aamdal-Scheie et al., duced adaptation and selection, local environmental fac-
1996]. In these mildly acidogenic conditions (pH 5–6), tors on root surfaces, such as the presence of gingival cre-
Actinomyces spp. colonizing the innermost part of the vicular fluid, thickness of bacterial deposits, and exposure
dental biofilm [Dige et al., 2009] may play an important to salivary flow, may modify the caries process. Most root
role in controlling the mineral loss by pH-modulatory caries lesions are shallow with a tendency to spread later-
mechanisms [Takahashi and Yamada, 1999]. In addition ally compared to cavities in enamel, and thinner biofilms
to base production from other bacteria, such metabolic with easy access to salivary clearance of metabolites may
events may help to balance the demineralization and reduce the acidity of the biofilm. Gingival crevicular fluid
remineralization processes on root surfaces in a manner may also play a modulatory role in root caries because of its
similar to that described for enamel surfaces in the dy- neutral to weakly alkaline pH [Bickel and Cimasoni, 1985].
namic stability stage [Takahashi and Nyvad, 2008]. Such weak acidic environments might facilitate coloniza-
However, when the mineral balance tilts toward de- tion and invasion by Actinomyces species [Nyvad and Fe-
mineralization (e.g. due to frequent sugar-induced acidi- jerskov, 1990], which are generally not as acidogenic or ac-
fication), the cariogenic processes may progress and re- iduric as Streptococcus species [Horiuchi et al., 2009]. Fur-
sult in the development of a clinically visible root caries thermore, proteolytic bacteria, such as Prevotella intermedia,
lesion. Bacteria can adapt to the acidic environment and Prevotella denticola, and Propionibacterium acnes, have
enhance their acidogenicity and acidurance, as described been isolated from root surfaces [Aamdal-Scheie et al.,
in the biochemical process responsible for the develop- 1996; Schüpbach et al., 1995, 1996; Hashimoto et al., 2011],
ment of enamel caries (the acidogenic stage) [Takahashi suggesting that nitrogenous substrates, such as proteins,
and Nyvad, 2008]. The acidic environment promotes the peptides, and amino acids, which can be supplied from gin-
introduction of more acidogenic bacteria, such as low-pH gival crevicular fluid, are available near the root surface.
Streptococcus and Actinomyces strains, which are more In contrast to root surfaces, coronal dentin will only
capable of lowering the environmental pH than the Strep- become colonized by bacteria after total breakdown of the
tococcus and Actinomyces strains found on the surfaces of enamel [Dige et al., 2014]. Dentin caries lesions possess
clinically sound roots [van Houte et al., 1994, 1996]. Ac- highly diverse microbiotas [Aas et al., 2008; Obata et al.,
tive root caries lesions sometimes contain increased num- 2014; Schulze-Schweifing et al., 2014; Simón-Soro et al.,
bers of aciduric bacteria, such as mutans streptococci, 2014], which can lead to the development of strictly an-
Lactobacillus, or Bifidobacterium species [Nyvad and aerobic conditions [Edwardsson, 1974; Hoshino, 1985].
Kilian 1990; van Houte et al., 1994; 1996; Schüpbach et al., The microbiotas in dentin caries consist of both acido-
1996; Brailsford et al., 2001; Mantzourani et al., 2009]. genic/aciduric bacteria, such as Streptococcus mutans,
These observations suggest that some active lesions with Lactobacillus, and Bifidobacterium species, and proteolyt-
an acidic environment can facilitate the establishment of ic/amino acid-degrading bacteria, such as Prevotella and
aciduric bacteria, as described in enamel caries (the aci- Propionibacterium species [Aas et al., 2008]. Reflecting
duric stage) [Takahashi and Nyvad, 2008]. However, mu- this microbiological diversity, dentin caries lesions were
tans streptococci do not become dominant; even in ad- reported to contain a variety of organic acids, and their pH
vanced stages of active root caries, Actinomyces and non- was found to vary markedly [Hojo et al., 1991, 1994].

424 Caries Res 2016;50:422–431 Takahashi/Nyvad


DOI: 10.1159/000447309
Active dentin cavities (which were soft and yellowish- The conformational structure of collagen molecules
white-colored with thick carious dentin) demonstrated remains stable under physiological conditions since the
an acidic pH of 4.9 ± 0.2, and the dominant acid was lac- denaturation temperature of collagen (the temperature
tic acid, while arrested dentin cavities (which were hard required to achieve a 5% reduction in the specific viscos-
and darkly pigmented with thin carious dentin) had a ity of collagen solution) is over 100 ° C [Cadenaro et al.,
   

weakly acidic pH of 5.6 ± 0.4 and exhibited mixed acid 2016]. However, once collagen molecules are exposed,
profiles, which included acetic, propionic, n/i-butyric, telopeptidic activity of host-derived collagenase can start
n/i-valeric, and n/i-caproic acids [Hojo et al., 1994]. The to partly break down the telopeptide region of the mole-
dominance of lactate and the acidic pH observed in the cules, as described in the previous section [Tjäderhane et
active cavities might have been due to carbohydrate stag- al., 2015], and then the host-derived collagenase and pro-
nancy and carbohydrate metabolism by lactate-produc- teases can further degrade the molecules and transform
ing saccharolytic bacteria, including Streptococcus, Acti- them to more water-soluble and labile components. Solu-
nomyces, and Lactobacillus species. Streptococcus species bilized collagen molecules can be denatured efficiently
are reported to mainly produce lactic acid from carbohy- under acidic conditions and shift their denaturation tem-
drates at acidic pH [Iwami et al., 1992]. Meanwhile, the perature toward body temperature [Hayashi and Nagai,
mixed acid profiles seen in the arrested cavities might 1973]. A study by Leikina et al. [2002] demonstrated that
have been derived from the secondary metabolism of lac- solubilized type I collagen can be denatured to a random
tate to weaker acids, such as acetate and propionate, by coil structure even at body temperature. Collectively,
Veillonella and other species that can neutralize acidic these observations suggest that during acid-induced de-
conditions [Takahashi, 2015]. Furthermore, these acid mineralization collagen molecules are exposed to the
profiles can be created by proteolytic/amino acid-degrad- aqueous phase. Subsequently, they can be initially de-
ing bacteria, such as Propionibacterium and Prevotella graded by host-derived collagenases and acid denatured,
species, from nitrogenous metabolic substrates [Taka- and then the denatured collagen (gelatinized collagen)
hashi, 2015], which can be supplied from dentin proteins can be degraded by various proteases, such as gelatinases
by proteolysis, as described in the next section. Such ami- and peptidases, which are widely distributed in both host
no acid metabolism can also neutralize acidic conditions and bacteria.
[Takahashi, 2003, 2015]. Hence, the weakly acidic condi- It should be appreciated that dentin organic materials
tions encountered in arrested cavities might indicate a tilt contain proteases [matrix metalloproteinases (MMPs)
towards remineralization. However, differences in the and cysteine cathepsins], which, unlike collagen, are re-
microbiotas between active and arrested cavities have not sistant to the acidic environment [Tezvergil-Mutluay et
yet been elucidated. al., 2013] but can be activated in the acidic environment.
Details of the acid activation are described in the next sec-
tion.
Acid-Induced Denaturation of Dentin/Cementum
Matrix Proteins
Protein Degradation by Host- and Bacteria-Derived
As described above, dentin and root caries involves Proteases
both demineralization and collagen degradation. It is im-
portant to note that protein degradation seems to occur Recent studies have suggested that host-derived MMPs
mainly after demineralization. Proteases cannot reach pro- and cysteine cathepsins are directly involved in dentin
teinaceous substrates or function in mineralized tissues, matrix degradation during caries formation. Several types
i.e. their substrates have to be exposed to the aqueous of MMP, such as MMP-2 (a 72-kDa gelatinase), MMP-8
phase. In addition, proteases have to be activated or de- (collagenase-2), and MMP-9 (a 92-kDa gelatinase) [Tjä-
tached from inhibitors since most proteases are present in derhane et al., 1998], as well as cysteine cathepsins B and
their inactive forms. It is also worth bearing in mind that K [Nascimento et al., 2011; Vidal et al., 2014], have been
intact collagen molecules are resistant to several proteo- found in dentin caries lesions. The dentin matrix mainly
lytic enzymes (except for collagenases) due to their high- consists of type I collagen, and MMPs and cathepsins can
order conformational structure (triple helix), and thus degrade such collagen after bacterial acid-induced de-
their internal structure has to be solubilized and denatured mineralization, as stated above. These host proteases are
in the aqueous phase prior to proteolytic degradation. also contained in saliva [Tjäderhane et al., 1998; Nasci-

Ecological Hypothesis of Dentin/Root Caries Res 2016;50:422–431 425


Caries DOI: 10.1159/000447309
mento et al., 2011; Hedenbjörk-Lager et al., 2015] and Ecological Hypothesis for Dentin and Root Caries
usually secreted in their inactive forms; however, they are
automatically activated under acidic conditions (around Based on the findings described above, the extended
pH 4.5). Cysteine cathepsins can function actively under ecological caries hypothesis [Takahashi and Nyvad, 2008,
acidic conditions, while MMPs have an optimal pH 2011] can be refined to include the etiology of dentin/root
around neutral. In addition, Miyoshi et al. [2010] report- caries (fig. 1), in which a proteolytic stage has been added
ed that the proteolytic activity of these proteases was to the previous hypothesis.
gradually activated in saliva even at neutral pH and 37 ° C,
    In the previous hypothesis, bacteria-induced acidifica-
although the mechanism responsible for this activation tion was considered to be the major ecological driver of
has not been elucidated. caries formation, i.e. acid formation was considered to
Dentin itself is also known to contain proteases, such promote bacterial acid-induced adaptation and selection
as MMP-2, 3, 8, 9, and 20, and cysteine cathepsins B and within the microbiota and to tilt the balance of deminer-
K [Sulkala et al., 2002; Mazzoni et al., 2009; Shimada et alization and remineralization towards demineralization
al., 2009; Toledano et al., 2010; Boushell et al., 2011; Bu- via a process involving dynamic stability and acidogenic
zalaf et al., 2015; Tjäderhane et al., 2015]. These proteases and aciduric stages [Takahashi and Nyvad, 2008, 2011].
are produced during dentin formation and then become In the refined hypothesis, bacterial acidification also in-
embedded in the dentin matrix (in their inactive forms) duces the exposure of organic matrix and the activation
together with protease inhibitors, such as tissue inhibitor of dentin-embedded and salivary MMPs/cathepsins.
of metalloproteinases (TIMP)-1 and -2 [Ishiguro et al., These proteases may initiate a partial degradation of the
1994; Niu et al., 2011]. However, once dentin is deminer- acid-exposed tooth organic materials (mainly collagen),
alized or exposed to the aqueous phase, these proteases which can then be further denatured and broken down by
are activated and start to degrade the dentin organic ma- bacterial acids and proteolysis.
trix. Cysteine cathepsin B is reported to inactivate TIMP- Activated MMPs do not function efficiently under
1 and -2 [Nagase, 1997]. acidic conditions as their optimal pH is usually around
On the other hand, some oral bacteria possess their neutral, while activated cathepsins are active and stable
own proteases, including collagenases, gelatinases, and under acidic conditions around pH 5. However, when the
peptidases. Proteolytic bacteria, such as Prevotella and acidic environment is neutralized by oral clearance, dis-
Propionibacterium species, have been isolated from den- solved tooth mineral, and/or bacterial alkali production/
tin carious lesions [Aas et al., 2008] and root surfaces acid neutralization through amino acid degradation
[Preza et al., 2008; Hashimoto et al., 2011], suggesting [Takahashi, 2015], MMPs start to function efficiently and
that bacteria may also contribute to dentin matrix degra- degrade acid-exposed and acid-denatured proteins. At
dation. In particular, Prevotella intermedia is known to the same time, the de- and remineralization balance may
exhibit protease activity at a wide range of pH (from neu- begin to tilt towards remineralization; however, once or-
tral to acidic) as well as acid-producing activity [Taka- ganic matrices are partly degraded or lost, complete re-
hashi and Schachtele, 1990]. No studies have identified a mineralization may no longer be possible [Kuboki et al.,
bacterial involvement in the initial degradation of dentin 1977]. In addition, bacteria can metabolize partly degrad-
matrix, but the detection of bacterial metabolites in cari- ed proteins via their proteolytic activity, although it is still
ous dentin cavities [Hojo et al., 1991, 1994] suggests that questionable whether bacteria play a role in the initial
bacteria digest partly degraded dentin matrix and metab- stages of the degradation of the organic components of
olize it into organic acids. Nevertheless, bacterial acidifi- teeth. The highly diverse anaerobic microbiotas found in
cation is essential for the acid-induced demineralization dentin and root caries lesions, which include both acido-
of tooth minerals, the acid-induced denaturation of ex- genic/aciduric bacteria, such as mutans streptococci, Lac-
posed and partly degraded proteins, and the acid-induced tobacillus, and Bifidobacterium species, and proteolytic/
activation of host-derived proteases. Furthermore, pro- amino acid-degrading bacteria, such as Prevotella, Propi-
teolytic/amino acid-degrading bacteria can utilize dena- onibacterium, and Fusobacterium species [Aas et al.,
tured and/or partly digested host proteins as metabolic 2008], seem to thrive on their nutritionally rich environ-
substrates [Takahashi and Yamada, 2000; Takahashi et ments, in which both exogenous metabolic substrates (di-
al., 2000; Takahashi and Sato, 2001, 2002; Takahashi, etary carbohydrates, saliva, gingival crevicular fluid, etc.)
2015]. and endogenous metabolic substrates (degraded colla-
gen, etc.) are available.

426 Caries Res 2016;50:422–431 Takahashi/Nyvad


DOI: 10.1159/000447309
Enamel Dentin/root surface

Dynamic stability stage

(l e

Ne re
si o

t m gre
Dominance of non-mutans streptococci

in ssi
and actinomyces

er on
(l e

al /
N n re
sio

ga arr otei
et g

in es
m res
in si
er on
al /a

t)
Pr
Acidogenic stage
ga rr
in est

n
Increase in ‘low-pH’ non-mutans streptococci

de
and actinomyces

(le ion
gr
)

ad

sio
Proteolytic stage

Ne itia
at

t m tio
in
Acid exposure of collagen

in n/p
(le

Acid activation of MMPs/cathepsins

er ro
sio

al
N niti

Acid denaturation and proteolysis of

lo gre
n
et at

ss ss
collagen
i
m ion
in /p

Aciduric stage
er ro
al g

Defensive reactions in

io
Increase in mutans streptococci
lo re

n)
dentin-pulp complex
ss ss

and non-mutans aciduric bacteria


io
n)

Irreversible pulpitis
Infected root canal

Fig. 1. Ecological caries hypothesis illustrating the mechanism responsible for the development of dentin and
root caries based on the extended ecological caries hypothesis proposed by Takahashi and Nyvad [2008] (for de-
tailed description, see text).

Recently, Simón-Soro et al. [2013] proposed a tissue- of the dentin matrix concurrently with its acid denatur-
dependent hypothesis of dental caries, which states that ation (fig.  1). Subsequently, proteolytic/amino acid-de-
acid-producing bacteria act as a vehicle for penetrating grading bacteria can settle in the modified tissue together
enamel and allow dentin-degrading microorganisms to with acid-producing bacteria. These phenomena happen
expand cavities. However, as described above, the contri- in parallel with a continuous degradation of dentin ma-
bution made by bacteria to the initial degradation of tooth trix by bacterial and host-derived proteases. In vitro de-
organic materials remains questionable, and host-derived mineralized dentin matrix can be self-degraded by en-
proteases seem to be the main contributors to this pro- dogenous matrix-embedded proteases without the pres-
cess. Nevertheless, proteolytic/amino acid-degrading ence of bacteria [Brackett et al., 2015; Seseogullari-Dirihan
bacteria can metabolize proteins, peptides, and amino ac- et al., 2015; Altinci et al., 2016]; however, in in vivo dentin
ids [Takahashi and Yamada, 2000; Takahashi et al., 2000; caries, bacteria are always present, at least in the infected
Takahashi and Sato, 2001, 2002; Takahashi, 2015], which layer of the carious dentin, and inevitably contribute to
are made available via an initial degradation step involv- further degradation of matrix through their proteolytic/
ing host-derived proteases. This modification of the nu- peptidic and metabolic activity. In both enamel and den-
tritional conditions might be an ecological determinant tin caries bacterial acid production initiates the processes
in the establishment of the microbial communities ob- resulting in caries. Furthermore, in dentin caries, host-
served in dentin and root caries, i.e. highly diverse an- derived proteases are deeply involved in the processes.
aerobic microbiotas including both saccharolytic and
proteolytic/amino acid-degrading bacteria. In this con-
text, current thinking regarding the tissue dependency of Beyond Deep Dentin Caries
caries formation might need to be revised.
We hypothesize that bacterial acids demineralize The polymicrobial invasion of dentin and its subse-
enamel/dentin, expose the dentin matrix, and activate quent breakdown are considered to trigger inflammatory
host-derived proteases to promote the initial degradation responses within the dental pulp, resulting in (dental)

Ecological Hypothesis of Dentin/Root Caries Res 2016;50:422–431 427


Caries DOI: 10.1159/000447309
pulpitis (fig.  1). Both physical stress (attributed to the stages of pulp infection [Nadkarni et al., 2010], although
short distance to the pulp) and chemical stress (caused by their specific pathogenic mechanisms are unclear. As
the presence of bacterial components and metabolites) pulp infections progress, the microbiota within the le-
induce pulpal inflammation. Bacterial components such sion is reported to shift to an anaerobe-dominant poly-
as lipopolysaccharides (from Gram-negative bacteria), microbial composition (as represented by Coriobacteria-
muramyl dipeptide (from both Gram-positive and Gram- ceae and Lachnospiraceae species) [Nadkarni et al., 2010],
negative bacteria), and lipoteichoic acid (from Gram- which resembles the microbiotas seen in infected and ne-
positive bacteria) can be sensed by Toll-like receptors crotic root canals, indicating that the environment is
(TLRs) [Kawai and Akira, 2010] and hence activate the suitable for promoting the growth of these bacteria rath-
host defense inflammatory cascade of TLR2 and TLR4, er than pathogenic activity.
which are involved in Gram-positive and Gram-negative The caries process, from the formation of dentin/root
bacterial sensing, respectively. These TLRs have been de- caries to infection of the root canal via pulpitis, involves
tected in the odontoblast cell membrane [Jiang et al., various physiological phenomena associated with ecolog-
2006; Veerayutthwilai et al., 2007] and are considered to ical polymicrobial transition. These processes seem to be
be involved in sensing microbial invasion and subsequent continuous; however, it is still unclear which mechanisms
odontoblastic defensive responses, such as the produc- are responsible for the microbial transition and the con-
tion of antibacterials (defensins and nitric oxide) and mo- comitant transition from a reversible to an irreversible
lecular messaging molecules (chemokines and cytokines) pulpitis. Importantly, as the dentin is broken down dur-
[for review, see Farges et al., 2015]. On the other hand, ing caries, bioactive molecules released from the dentin
bacterial metabolites, including short-chain fatty acids matrix itself have the capacity to stimulate regeneration
(butyrate, etc.), ammonia, sulfur compounds (hydrogen of the dentin-pulp complex [for review, see Smith et al.,
sulfide and methyl mercaptan), and amines (histamine, 2012] (fig. 1). If these modulatory responses are within
etc.) are known to be cytotoxic and immunomodulatory the capacity of the pulp to recover (or heal) pulpitis would
[Niederman et al., 1997; Kurita-Ochiai et al., 2008], and be abrogated.
thus probably initiate and promote pulpal inflammatory
responses. These substances are derived from bacterial
peptide/amino acid metabolism [Takahashi, 2015], i.e. Control of Dentin and Root Caries
the bacterial metabolic processes of the partly degraded
dentin organic matrix. The observation that proteolytic/ According to our refined hypothesis for dentin and
amino acid-degrading anaerobes, such as Prevotella, Fu- root caries the caries processes are reversible (fig. 1). Case
sobacterium, and Porphyromonas species, were frequent- studies have demonstrated that root and dentin caries le-
ly detected in advanced dentin caries lesions with pulpitis sions can be controlled/arrested for years by daily me-
[Martin et al., 2002; Chhour et al., 2005; Rôças et al., 2015] chanical cleaning and use of fluoride toothpaste [Nyvad
may support this notion. Furthermore, dentinal fluid and Fejerskov, 1986; Kidd et al., 2015], even in cases of
contains gelatinolytic enzymes [Mazzoni et al., 2012; Pes- manifest cavity formation into dentin [Nyvad and Fejer-
soa, 2013] which increase in pulpal inflammation skov, 1997]. Success of the treatment requires that the le-
[Zehnder et al., 2011] and MMP-20 [Sulkala et al., 2002]. sion can be accessed for cleaning. Evidence supporting
It is possible that these host-derived enzymes, together the effect of such nonoperative treatments came from ex-
with microbial proteases, promote the degradation of perimental studies in which natural root caries lesions
dentin matrix in deep caries lesions. were arrested in situ over 3 months by daily cleaning with
In deep progressive dentin caries, polymicrobial in- a fluoride toothpaste combined with two professional
fections occur in the dental pulp. These infections in- topical fluoride applications by 2% NaF [Nyvad et al.,
volve a subset of the genera encountered in carious den- 1997]. The dual fluoride treatment resulted in a mineral
tin including Actinobacteria (Actinomyces, Bifidobacte- gain in the surface layer and in a zone next to the lesion
rium, Parascardovia, and Propionibacterium) and front. A recent review suggested that higher fluoride con-
Firmicutes (Lactobacillus and Streptococcus) [Nadkarni centrations may be needed to control caries in root sur-
et al., 2010; Chalmers et al., 2015], taxa that have also faces compared to enamel surfaces [Wierichs and Meyer-
been recovered from the advancing front of carious den- Lueckel, 2015]. Further studies should explore the use of
tin [Edwardsson, 1974]. Among them, Lactobacillus spe- fluoride in the arrest of root and dentin caries. Such stud-
cies were reported to play a prominent role in the initial ies might also include evaluation of the caries-controlling

428 Caries Res 2016;50:422–431 Takahashi/Nyvad


DOI: 10.1159/000447309
effect of fluoride-releasing dental materials, although the The prevalence of root caries is expected to increase in
clinical relevance of fluoride release from dental materials the coming years because populations get older and retain
is still debatable [Dionysopoulous, 2014; Cury et al., an increased number of teeth with gingival recession.
2016]. When root surfaces are exposed to the oral cavity, they
Our refined hypothesis highlights the vulnerability of will always run a risk of becoming demineralized, and ex-
exposed root surfaces. However, once the root/dentin posed organic materials can be degraded by host-derived
surface has been demineralized protease inhibitors can be proteases contained in saliva and dentin itself. This pro-
effective at preventing the degradation of dentin organic cess of root surface loss seems to be very slow but inevi-
materials. MMP inhibitors, such as nonantimicrobial table. New approaches to the prevention and treatment of
chemically modified tetracyclines and 2-(imidazole- dentin and root caries based on the etiology of the condi-
1-yl)-1-hydroxyethane-1,1-bisphosphonic acid (zoledro- tion are required, especially for the elderly/medically
nate), inhibited caries progression in rats [Tjäderhane et compromised patient.
al., 1999; Sulkala et al., 2001]. Other reagents, including
chlorhexidine [Pashley et al., 2004; Garcia et al., 2009],
fluoride [Kato et al., 2014], silver diamine fluoride [Mei Acknowledgment
et al., 2012], doxycycline [Osorio et al., 2011], ethylene
This study was supported in part by a KAKENHI grant (No.
diamine tetraacetic acid [Thompson et al., 2012], benzal-
60183852) from the Japan Society for the Promotion of Science.
konium chloride [Tezvergil-Mutluay et al., 2011b], qua-
ternary ammonium compounds [Tezvergil-Mutluay et
al., 2011a], various metal ions such as zinc [Toledano et Author Contributions
al., 2012] and iron [Kato et al., 2010], and natural sub-
stances [Chaussain-Miller et al., 2006] have been reported Both authors participated in the preparation of the manuscript.
to inhibit host proteases such as MMPs and cathepsins in
vitro. Recently, hesperidin was reported to reduce the
degradation of human dentin matrix (acid-demineral- Disclosure Statement
ized dentin) in situ [van Strijp et al., 2015]. These obser-
vations suggest that novel approaches for the protection The authors declare no conflicts of interest.
of the organic matrix based on protease inhibitors might
refine and improve therapies for root and dentin caries in
the future.

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Caries DOI: 10.1159/000447309