Review
Caries Res 2016;50:422–431
DOI: 10.1159/000447309
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
Bacteria · Cathepsin · Dental caries · Dentin · Ecological
hypothesis · Matrix metalloproteinases · Proteolysis ·
Root caries
Abstract
Recent advances regarding the caries process indicate that
ecological phenomena induced by bacterial acid production
tilt the de- and remineralization balance of the dental hard
tissues towards demineralization through bacterial acid-in-
duced adaptation and selection within the microbiota –
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;
however, the fact that these tissues contain a considerable
amount of organic material suggests that protein degrada-
tion is involved in caries formation. In this review, we com-
piled relevant histological, biochemical, and microbiological
information about dentin/root caries and refined the hy-
pothesis by adding degradation of the organic matrix (the
proteolytic stage) to the abovementioned stages. Bacterial
acidification not only induces demineralization and expo-
sure of the organic matrix in dentin/root surfaces but also
activation of dentin-embedded and salivary matrix metallo-
proteinases and cathepsins. These phenomena initiate deg-
radation of the demineralized organic matrix in dentin/root
surfaces. While a bacterial involvement has never been con-
© 2016 S. Karger AG, Basel
0008–6568/16/0504–0422$39.50/0
E-Mail karger@karger.com
www.karger.com/cre
Received: March 8, 2016
Accepted after revision: May 31, 2016
Published online: July 27, 2016
firmed in the initial degradation of organic material, the de-
tection of proteolytic/amino acid-degrading bacteria and
bacterial metabolites in dentin and root caries suggests a
bacterial digestion and metabolism of partly degraded ma-
trix. Moreover, bacterial metabolites might induce pulpitis
as an inflammatory/immunomodulatory factor. Root and
dentin surfaces are always at risk of becoming demineralized
in the oral cavity, and exposed organic materials can be de-
graded by host-derived proteases contained in saliva and
dentin itself. New approaches to the prevention and treat-
ment of root/dentin caries are required.
© 2016 S. Karger AG, Basel
Recent advances in our knowledge about the caries
process indicate that ecological phenomena, e.g. bacterial
adaptation to acidic environments (increases in bacterial
acidogenicity and acidurance) and bacterial shifts to a
more acidogenic and aciduric microbiota (increases in
the proportion of acidogenic and aciduric bacteria), are
induced by frequent and prolonged acidification. The re-
sultant environmental acidification can tilt the balance
between demineralization and remineralization toward
demineralization, resulting in the initiation/progression
This paper is based on a presentation held at the 10th European Oral
Microbiology Workshop, Aarhus, Denmark, in May 2014.
Nobuhiro Takahashi
Division of Oral Ecology and Biochemistry
Tohoku University Graduate School of Dentistry
4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan)
E-Mail nobu-t @ dent.tohoku.ac.jp
of caries. The ecological concept of caries was first pro-
posed in the ‘ecological plaque hypothesis’ by Marsh
[1994] and was subsequently extended by Takahashi and
Nyvad [2008, 2011] based on detailed information re-
garding the biochemical responses to environmental
acidification, i.e. the induction of bacterial acidogenicity/
acidurance and the selection of acidogenic/aciduric bac-
teria. This ecological hypothesis forms the basis of our
current understanding of caries, but mainly focusses on
phenomena occurring in enamel caries. Ecological fac-
tors in dentin and root caries have only been scarcely ad-
dressed, possibly because of the complex structural com-
position of these tissues.
The etiology of caries has been debated over centuries,
and three key hypotheses have been proposed: the chem-
ico-parasitic, the chelation, and the proteolysis-chelation
theories. There is no doubt that the etiology of enamel
caries can be mainly explained by the chemico-parasitic
theory, in which the dental biofilm (bacteria) produces
acid from sugar, and the resultant acidic pH promotes
demineralization of the enamel. Dentin and root caries
can also be explained by this theory; however, the fact that
dentin and root surfaces contain a considerable amount
of organic material, such as collagen, has led to the notion
that protein degradation (the proteolysis theory) might
also play a role in caries development in these tissues. In-
deed, a series of publications [Dayan et al., 1983; Tjäder-
hane et al., 1998, 2015; Chaussain-Miller et al., 2006] have
supported this possibility.
In this review, we attempt to compile relevant histo-
logical, biochemical, and microbiological information
about dentin and root caries and propose a possible
mechanism for the development of dentin and root caries
by including degradation of the organic matrix with the
cyclic de- and remineralization processes in caries.
Structural Composition of Root and Dentin Surfaces
Dentin and root cementum surfaces are less mineral-
ized than enamel. About 30% of dentin and root cemen-
tum is composed of organic materials, mainly collagen,
while enamel is almost entirely composed of hydroxyapa-
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-
el occurs only after total breakdown of the tissue, root
surfaces become invaded by bacteria already at an early
stage of the caries process [Schüpbach et al., 1989; Nyvad
and Fejerskov, 1990].
Ecological Hypothesis of Dentin/Root
Caries
Ultrastructural studies have suggested that demineral-
ization and breakdown of the organic matrix happens in
two successive stages [Selvig, 1969; Schüpbach et al., 1989;
Nyvad and Fejerskov, 1990]. In the early stages of cemen-
tum and dentin caries, minerals are dissolved by a fine
gradient from the outer surface, while maintaining the
characteristic cross-banding of the collagen fibers [Nyvad
and Fejerskov, 1990; Deyhle et al., 2011]. The demineral-
ized collagen serves as a scaffold for colonizing bacteria.
At more advanced stages the exposed collagen is broken
down by proteolytic enzymes, and collagen fibers lose
their structural characteristics [Kuboki et al., 1977]. How-
ever, a recent study [Tjäderhane et al., 2015] suggests that
collagen cross-banding may be lost at a relatively early
stage of demineralization, in which an exposed telopep-
tide region of the collagen molecule can be degraded by
telopeptidase activity of host-derived collagenolytic en-
zymes. Dentin and root surfaces are more labile than
enamel in acidic conditions [Hoppenbrouwers et al., 1987;
Moreno and Aoba, 1991], possibly because of the smaller
hydroxyapatite crystallites in dentin [Yardeni, 1952].
Higher levels of carbonate and magnesium [Hoppenbrou-
wers et al., 1987] and the tubular structure of dentin
[Shellis, 1996] could also add to the higher solubility.
Root and dentin surfaces are highly reactive in the oral
environment. Even clinically sound root surfaces may
take up minerals from the oral fluids, resulting in a high-
er mineral content in the surface layer compared with the
normal dentin [Selvig, 1969]. Such phenomena are likely
to occur as a result of cyclic demineralization and remin-
eralization processes [Nyvad et al., 1997]. However, root
and dentin surfaces are also vulnerable to physical injury.
Because of the low mineral content, root surfaces are eas-
ily abraded by toothbrushing, and although cementum
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-
mentum is often damaged and/or removed by regular
scaling and root planing [Jepsen et al., 2004]. Therefore,
in clinical circumstances the exposed root often presents
a dentin surface. In favorable conditions a new hypermin-
eralized surface layer may develop within a few months
after over-instrumentation of root surfaces [Selvig, 1969].
Microbial Ecosystem of Dentin and Root Caries
The microbiological and structural pattern of coloni-
zation of root surfaces has been extensively reviewed
[Nyvad, 1993]. A few hours after the cleaning of a root
Caries Res 2016;50:422–431 423
DOI: 10.1159/000447309
surface the surface is colonized by pioneer species mainly
belonging to Streptococcus (S. sanguinis, S. oralis, and S.
mitis) and Actinomyces. In situ studies have shown that
the early colonizers do not differ between enamel and
root surfaces [Nyvad and Kilian, 1987], suggesting that it
is the oral environment (saliva and/or gingival crevicular
fluid) rather than differences in composition of the pel-
licle [Rüdiger et al., 2002] that determine the colonization
pattern [Nyvad, 1993]. In this scenario, it is not surprising
that the microbial community on clinically sound root
surfaces include various acidogenic bacteria belonging to
Streptococcus and Actinomyces [van Houte et al., 1994,
1996], many of which are capable of producing acid from
dietary carbohydrates and lowering environmental pH to
<5.5 [van Houte et al., 1994, 1996; Aamdal-Scheie et al.,
1996]. In these mildly acidogenic conditions (pH 5–6),
Actinomyces spp. colonizing the innermost part of the
dental biofilm [Dige et al., 2009] may play an important
role in controlling the mineral loss by pH-modulatory
mechanisms [Takahashi and Yamada, 1999]. In addition
to base production from other bacteria, such metabolic
events may help to balance the demineralization and
remineralization processes on root surfaces in a manner
similar to that described for enamel surfaces in the dy-
namic stability stage [Takahashi and Nyvad, 2008].
However, when the mineral balance tilts toward de-
mineralization (e.g. due to frequent sugar-induced acidi-
fication), the cariogenic processes may progress and re-
sult in the development of a clinically visible root caries
lesion. Bacteria can adapt to the acidic environment and
enhance their acidogenicity and acidurance, as described
in the biochemical process responsible for the develop-
ment of enamel caries (the acidogenic stage) [Takahashi
and Nyvad, 2008]. The acidic environment promotes the
introduction of more acidogenic bacteria, such as low-pH
Streptococcus and Actinomyces strains, which are more
capable of lowering the environmental pH than the Strep-
tococcus and Actinomyces strains found on the surfaces of
clinically sound roots [van Houte et al., 1994, 1996]. Ac-
tive root caries lesions sometimes contain increased num-
bers of aciduric bacteria, such as mutans streptococci,
Lactobacillus, or Bifidobacterium species [Nyvad and
Kilian 1990; van Houte et al., 1994; 1996; Schüpbach et al.,
1996; Brailsford et al., 2001; Mantzourani et al., 2009].
These observations suggest that some active lesions with
an acidic environment can facilitate the establishment of
aciduric bacteria, as described in enamel caries (the aci-
duric stage) [Takahashi and Nyvad, 2008]. However, mu-
tans streptococci do not become dominant; even in ad-
vanced stages of active root caries, Actinomyces and non-
424 Caries Res 2016;50:422–431
DOI: 10.1159/000447309
mutans streptococci species remain dominant [Brailsford
et al., 1999, 2001]. Furthermore, a molecular biology-
based study [Preza et al., 2008] revealed that the micro-
bial communities in root caries are highly complex and
include not only mutans streptococci, Actinomyces, and
Lactobacillus species, but also species of Atopobium, Ol-
senella, Pseudoramibacter, Propionibacterium, and Sele-
nomonas. Yeasts, such as Candida albicans, were also de-
tected in root caries lesions in middle-aged adults and the
elderly [Shen et al., 2002; Zaremba et al., 2006], although
the invasion of these species into carious dentin has been
questioned [Maijala et al., 2007].
Despite similarities between the processes responsible
for enamel and root caries in terms of microbial acid-in-
duced adaptation and selection, local environmental fac-
tors on root surfaces, such as the presence of gingival cre-
vicular fluid, thickness of bacterial deposits, and exposure
to salivary flow, may modify the caries process. Most root
caries lesions are shallow with a tendency to spread later-
ally compared to cavities in enamel, and thinner biofilms
with easy access to salivary clearance of metabolites may
reduce the acidity of the biofilm. Gingival crevicular fluid
may also play a modulatory role in root caries because of its
neutral to weakly alkaline pH [Bickel and Cimasoni, 1985].
Such weak acidic environments might facilitate coloniza-
tion and invasion by Actinomyces species [Nyvad and Fe-
jerskov, 1990], which are generally not as acidogenic or ac-
iduric as Streptococcus species [Horiuchi et al., 2009]. Fur-
thermore, proteolytic bacteria, such as Prevotella intermedia,
Prevotella denticola, and Propionibacterium acnes, have
been isolated from root surfaces [Aamdal-Scheie et al.,
1996; Schüpbach et al., 1995, 1996; Hashimoto et al., 2011],
suggesting that nitrogenous substrates, such as proteins,
peptides, and amino acids, which can be supplied from gin-
gival crevicular fluid, are available near the root surface.
In contrast to root surfaces, coronal dentin will only
become colonized by bacteria after total breakdown of the
enamel [Dige et al., 2014]. Dentin caries lesions possess
highly diverse microbiotas [Aas et al., 2008; Obata et al.,
2014; Schulze-Schweifing et al., 2014; Simón-Soro et al.,
2014], which can lead to the development of strictly an-
aerobic conditions [Edwardsson, 1974; Hoshino, 1985].
The microbiotas in dentin caries consist of both acido-
genic/aciduric bacteria, such as Streptococcus mutans,
Lactobacillus, and Bifidobacterium species, and proteolyt-
ic/amino acid-degrading bacteria, such as Prevotella and
Propionibacterium species [Aas et al., 2008]. Reflecting
this microbiological diversity, dentin caries lesions were
reported to contain a variety of organic acids, and their pH
was found to vary markedly [Hojo et al., 1991, 1994].
Takahashi/Nyvad
 Active dentin cavities (which were soft and yellowish-
white-colored with thick carious dentin) demonstrated
an acidic pH of 4.9 ± 0.2, and the dominant acid was lac-
tic acid, while arrested dentin cavities (which were hard
and darkly pigmented with thin carious dentin) had a
weakly acidic pH of 5.6 ± 0.4 and exhibited mixed acid
profiles, which included acetic, propionic, n/i-butyric,
n/i-valeric, and n/i-caproic acids [Hojo et al., 1994]. The
dominance of lactate and the acidic pH observed in the
active cavities might have been due to carbohydrate stag-
nancy and carbohydrate metabolism by lactate-produc-
ing saccharolytic bacteria, including Streptococcus, Acti-
nomyces, and Lactobacillus species. Streptococcus species
are reported to mainly produce lactic acid from carbohy-
drates at acidic pH [Iwami et al., 1992]. Meanwhile, the
mixed acid profiles seen in the arrested cavities might
have been derived from the secondary metabolism of lac-
tate to weaker acids, such as acetate and propionate, by
Veillonella and other species that can neutralize acidic
conditions [Takahashi, 2015]. Furthermore, these acid
profiles can be created by proteolytic/amino acid-degrad-
ing bacteria, such as Propionibacterium and Prevotella
species, from nitrogenous metabolic substrates [Taka-
hashi, 2015], which can be supplied from dentin proteins
by proteolysis, as described in the next section. Such ami-
no acid metabolism can also neutralize acidic conditions
[Takahashi, 2003, 2015]. Hence, the weakly acidic condi-
tions encountered in arrested cavities might indicate a tilt
towards remineralization. However, differences in the
microbiotas between active and arrested cavities have not
yet been elucidated.
Acid-Induced Denaturation of Dentin/Cementum
Matrix Proteins
As described above, dentin and root caries involves
both demineralization and collagen degradation. It is im-
portant to note that protein degradation seems to occur
mainly after demineralization. Proteases cannot reach pro-
teinaceous substrates or function in mineralized tissues,
i.e. their substrates have to be exposed to the aqueous
phase. In addition, proteases have to be activated or de-
tached from inhibitors since most proteases are present in
their inactive forms. It is also worth bearing in mind that
intact collagen molecules are resistant to several proteo-
lytic enzymes (except for collagenases) due to their high-
order conformational structure (triple helix), and thus
their internal structure has to be solubilized and denatured
in the aqueous phase prior to proteolytic degradation.
Ecological Hypothesis of Dentin/Root
Caries
The conformational structure of collagen molecules
remains stable under physiological conditions since the
denaturation temperature of collagen (the temperature
required to achieve a 5% reduction in the specific viscos-
ity of collagen solution) is over 100 ° C [Cadenaro et al.,
2016]. However, once collagen molecules are exposed,
telopeptidic activity of host-derived collagenase can start
to partly break down the telopeptide region of the mole-
cules, as described in the previous section [Tjäderhane et
al., 2015], and then the host-derived collagenase and pro-
teases can further degrade the molecules and transform
them to more water-soluble and labile components. Solu-
bilized collagen molecules can be denatured efficiently
under acidic conditions and shift their denaturation tem-
perature toward body temperature [Hayashi and Nagai,
1973]. A study by Leikina et al. [2002] demonstrated that
solubilized type I collagen can be denatured to a random
coil structure even at body temperature. Collectively,
these observations suggest that during acid-induced de-
mineralization collagen molecules are exposed to the
aqueous phase. Subsequently, they can be initially de-
graded by host-derived collagenases and acid denatured,
and then the denatured collagen (gelatinized collagen)
can be degraded by various proteases, such as gelatinases
and peptidases, which are widely distributed in both host
and bacteria.
It should be appreciated that dentin organic materials
contain proteases [matrix metalloproteinases (MMPs)
and cysteine cathepsins], which, unlike collagen, are re-
sistant to the acidic environment [Tezvergil-Mutluay et
al., 2013] but can be activated in the acidic environment.
Details of the acid activation are described in the next sec-
tion.
Protein Degradation by Host- and Bacteria-Derived
Proteases
Recent studies have suggested that host-derived MMPs
and cysteine cathepsins are directly involved in dentin
matrix degradation during caries formation. Several types
of MMP, such as MMP-2 (a 72-kDa gelatinase), MMP-8
(collagenase-2), and MMP-9 (a 92-kDa gelatinase) [Tjä-
derhane et al., 1998], as well as cysteine cathepsins B and
K [Nascimento et al., 2011; Vidal et al., 2014], have been
found in dentin caries lesions. The dentin matrix mainly
consists of type I collagen, and MMPs and cathepsins can
degrade such collagen after bacterial acid-induced de-
mineralization, as stated above. These host proteases are
also contained in saliva [Tjäderhane et al., 1998; Nasci-
Caries Res 2016;50:422–431 425
DOI: 10.1159/000447309
mento et al., 2011; Hedenbjörk-Lager et al., 2015] and
usually secreted in their inactive forms; however, they are
automatically activated under acidic conditions (around
pH 4.5). Cysteine cathepsins can function actively under
acidic conditions, while MMPs have an optimal pH
around neutral. In addition, Miyoshi et al. [2010] report-
ed that the proteolytic activity of these proteases was
gradually activated in saliva even at neutral pH and 37 ° C,
   
although the mechanism responsible for this activation
has not been elucidated.
Dentin itself is also known to contain proteases, such
as MMP-2, 3, 8, 9, and 20, and cysteine cathepsins B and
K [Sulkala et al., 2002; Mazzoni et al., 2009; Shimada et
al., 2009; Toledano et al., 2010; Boushell et al., 2011; Bu-
zalaf et al., 2015; Tjäderhane et al., 2015]. These proteases
are produced during dentin formation and then become
embedded in the dentin matrix (in their inactive forms)
together with protease inhibitors, such as tissue inhibitor
of metalloproteinases (TIMP)-1 and -2 [Ishiguro et al.,
1994; Niu et al., 2011]. However, once dentin is deminer-
alized or exposed to the aqueous phase, these proteases
are activated and start to degrade the dentin organic ma-
trix. Cysteine cathepsin B is reported to inactivate TIMP-
1 and -2 [Nagase, 1997].
On the other hand, some oral bacteria possess their
own proteases, including collagenases, gelatinases, and
peptidases. Proteolytic bacteria, such as Prevotella and
Propionibacterium species, have been isolated from den-
tin carious lesions [Aas et al., 2008] and root surfaces
[Preza et al., 2008; Hashimoto et al., 2011], suggesting
that bacteria may also contribute to dentin matrix degra-
dation. In particular, Prevotella intermedia is known to
exhibit protease activity at a wide range of pH (from neu-
tral to acidic) as well as acid-producing activity [Taka-
hashi and Schachtele, 1990]. No studies have identified a
bacterial involvement in the initial degradation of dentin
matrix, but the detection of bacterial metabolites in cari-
ous dentin cavities [Hojo et al., 1991, 1994] suggests that
bacteria digest partly degraded dentin matrix and metab-
olize it into organic acids. Nevertheless, bacterial acidifi-
cation is essential for the acid-induced demineralization
of tooth minerals, the acid-induced denaturation of ex-
posed and partly degraded proteins, and the acid-induced
activation of host-derived proteases. Furthermore, pro-
teolytic/amino acid-degrading bacteria can utilize dena-
tured and/or partly digested host proteins as metabolic
substrates [Takahashi and Yamada, 2000; Takahashi et
al., 2000; Takahashi and Sato, 2001, 2002; Takahashi,
2015].
426 Caries Res 2016;50:422–431
DOI: 10.1159/000447309
Ecological Hypothesis for Dentin and Root Caries
Based on the findings described above, the extended
ecological caries hypothesis [Takahashi and Nyvad, 2008,
2011] can be refined to include the etiology of dentin/root
caries (fig. 1), in which a proteolytic stage has been added
to the previous hypothesis.
In the previous hypothesis, bacteria-induced acidifica-
tion was considered to be the major ecological driver of
caries formation, i.e. acid formation was considered to
promote bacterial acid-induced adaptation and selection
within the microbiota and to tilt the balance of deminer-
alization and remineralization towards demineralization
via a process involving dynamic stability and acidogenic
and aciduric stages [Takahashi and Nyvad, 2008, 2011].
In the refined hypothesis, bacterial acidification also in-
duces the exposure of organic matrix and the activation
of dentin-embedded and salivary MMPs/cathepsins.
These proteases may initiate a partial degradation of the
acid-exposed tooth organic materials (mainly collagen),
which can then be further denatured and broken down by
bacterial acids and proteolysis.
Activated MMPs do not function efficiently under
acidic conditions as their optimal pH is usually around
neutral, while activated cathepsins are active and stable
under acidic conditions around pH 5. However, when the
acidic environment is neutralized by oral clearance, dis-
solved tooth mineral, and/or bacterial alkali production/
acid neutralization through amino acid degradation
[Takahashi, 2015], MMPs start to function efficiently and
degrade acid-exposed and acid-denatured proteins. At
the same time, the de- and remineralization balance may
begin to tilt towards remineralization; however, once or-
ganic matrices are partly degraded or lost, complete re-
mineralization may no longer be possible [Kuboki et al.,
1977]. In addition, bacteria can metabolize partly degrad-
ed proteins via their proteolytic activity, although it is still
questionable whether bacteria play a role in the initial
stages of the degradation of the organic components of
teeth. The highly diverse anaerobic microbiotas found in
dentin and root caries lesions, which include both acido-
genic/aciduric bacteria, such as mutans streptococci, Lac-
tobacillus, and Bifidobacterium species, and proteolytic/
amino acid-degrading bacteria, such as Prevotella, Propi-
onibacterium, and Fusobacterium species [Aas et al.,
2008], seem to thrive on their nutritionally rich environ-
ments, in which both exogenous metabolic substrates (di-
etary carbohydrates, saliva, gingival crevicular fluid, etc.)
and endogenous metabolic substrates (degraded colla-
gen, etc.) are available.
Takahashi/Nyvad
 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-
dependent hypothesis of dental caries, which states that
acid-producing bacteria act as a vehicle for penetrating
enamel and allow dentin-degrading microorganisms to
expand cavities. However, as described above, the contri-
bution made by bacteria to the initial degradation of tooth
organic materials remains questionable, and host-derived
proteases seem to be the main contributors to this pro-
cess. Nevertheless, proteolytic/amino acid-degrading
bacteria can metabolize proteins, peptides, and amino ac-
ids [Takahashi and Yamada, 2000; Takahashi et al., 2000;
Takahashi and Sato, 2001, 2002; Takahashi, 2015], which
are made available via an initial degradation step involv-
ing host-derived proteases. This modification of the nu-
tritional conditions might be an ecological determinant
in the establishment of the microbial communities ob-
served in dentin and root caries, i.e. highly diverse an-
aerobic microbiotas including both saccharolytic and
proteolytic/amino acid-degrading bacteria. In this con-
text, current thinking regarding the tissue dependency of
caries formation might need to be revised.
We hypothesize that bacterial acids demineralize
enamel/dentin, expose the dentin matrix, and activate
host-derived proteases to promote the initial degradation
of the dentin matrix concurrently with its acid denatur-
ation (fig.  1). Subsequently, proteolytic/amino acid-de-
grading bacteria can settle in the modified tissue together
with acid-producing bacteria. These phenomena happen
in parallel with a continuous degradation of dentin ma-
trix by bacterial and host-derived proteases. In vitro de-
mineralized dentin matrix can be self-degraded by en-
dogenous matrix-embedded proteases without the pres-
ence of bacteria [Brackett et al., 2015; Seseogullari-Dirihan
et al., 2015; Altinci et al., 2016]; however, in in vivo dentin
caries, bacteria are always present, at least in the infected
layer of the carious dentin, and inevitably contribute to
further degradation of matrix through their proteolytic/
peptidic and metabolic activity. In both enamel and den-
tin caries bacterial acid production initiates the processes
resulting in caries. Furthermore, in dentin caries, host-
derived proteases are deeply involved in the processes.
Beyond Deep Dentin Caries
The polymicrobial invasion of dentin and its subse-
quent breakdown are considered to trigger inflammatory
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
short distance to the pulp) and chemical stress (caused by
the presence of bacterial components and metabolites)
induce pulpal inflammation. Bacterial components such
as lipopolysaccharides (from Gram-negative bacteria),
muramyl dipeptide (from both Gram-positive and Gram-
negative bacteria), and lipoteichoic acid (from Gram-
positive bacteria) can be sensed by Toll-like receptors
(TLRs) [Kawai and Akira, 2010] and hence activate the
host defense inflammatory cascade of TLR2 and TLR4,
which are involved in Gram-positive and Gram-negative
bacterial sensing, respectively. These TLRs have been de-
tected in the odontoblast cell membrane [Jiang et al.,
2006; Veerayutthwilai et al., 2007] and are considered to
be involved in sensing microbial invasion and subsequent
odontoblastic defensive responses, such as the produc-
tion of antibacterials (defensins and nitric oxide) and mo-
lecular messaging molecules (chemokines and cytokines)
[for review, see Farges et al., 2015]. On the other hand,
bacterial metabolites, including short-chain fatty acids
(butyrate, etc.), ammonia, sulfur compounds (hydrogen
sulfide and methyl mercaptan), and amines (histamine,
etc.) are known to be cytotoxic and immunomodulatory
[Niederman et al., 1997; Kurita-Ochiai et al., 2008], and
thus probably initiate and promote pulpal inflammatory
responses. These substances are derived from bacterial
peptide/amino acid metabolism [Takahashi, 2015], i.e.
the bacterial metabolic processes of the partly degraded
dentin organic matrix. The observation that proteolytic/
amino acid-degrading anaerobes, such as Prevotella, Fu-
sobacterium, and Porphyromonas species, were frequent-
ly detected in advanced dentin caries lesions with pulpitis
[Martin et al., 2002; Chhour et al., 2005; Rôças et al., 2015]
may support this notion. Furthermore, dentinal fluid
contains gelatinolytic enzymes [Mazzoni et al., 2012; Pes-
soa, 2013] which increase in pulpal inflammation
[Zehnder et al., 2011] and MMP-20 [Sulkala et al., 2002].
It is possible that these host-derived enzymes, together
with microbial proteases, promote the degradation of
dentin matrix in deep caries lesions.
In deep progressive dentin caries, polymicrobial in-
fections occur in the dental pulp. These infections in-
volve a subset of the genera encountered in carious den-
tin including Actinobacteria (Actinomyces, Bifidobacte-
rium, Parascardovia, and Propionibacterium) and
Firmicutes (Lactobacillus and Streptococcus) [Nadkarni
et al., 2010; Chalmers et al., 2015], taxa that have also
been recovered from the advancing front of carious den-
tin [Edwardsson, 1974]. Among them, Lactobacillus spe-
cies were reported to play a prominent role in the initial
428 Caries Res 2016;50:422–431
DOI: 10.1159/000447309
stages of pulp infection [Nadkarni et al., 2010], although
their specific pathogenic mechanisms are unclear. As
pulp infections progress, the microbiota within the le-
sion is reported to shift to an anaerobe-dominant poly-
microbial composition (as represented by Coriobacteria-
ceae and Lachnospiraceae species) [Nadkarni et al., 2010],
which resembles the microbiotas seen in infected and ne-
crotic root canals, indicating that the environment is
suitable for promoting the growth of these bacteria rath-
er than pathogenic activity.
The caries process, from the formation of dentin/root
caries to infection of the root canal via pulpitis, involves
various physiological phenomena associated with ecolog-
ical polymicrobial transition. These processes seem to be
continuous; however, it is still unclear which mechanisms
are responsible for the microbial transition and the con-
comitant transition from a reversible to an irreversible
pulpitis. Importantly, as the dentin is broken down dur-
ing caries, bioactive molecules released from the dentin
matrix itself have the capacity to stimulate regeneration
of the dentin-pulp complex [for review, see Smith et al.,
2012] (fig. 1). If these modulatory responses are within
the capacity of the pulp to recover (or heal) pulpitis would
be abrogated.
Control of Dentin and Root Caries
According to our refined hypothesis for dentin and
root caries the caries processes are reversible (fig. 1). Case
studies have demonstrated that root and dentin caries le-
sions can be controlled/arrested for years by daily me-
chanical cleaning and use of fluoride toothpaste [Nyvad
and Fejerskov, 1986; Kidd et al., 2015], even in cases of
manifest cavity formation into dentin [Nyvad and Fejer-
skov, 1997]. Success of the treatment requires that the le-
sion can be accessed for cleaning. Evidence supporting
the effect of such nonoperative treatments came from ex-
perimental studies in which natural root caries lesions
were arrested in situ over 3 months by daily cleaning with
a fluoride toothpaste combined with two professional
topical fluoride applications by 2% NaF [Nyvad et al.,
1997]. The dual fluoride treatment resulted in a mineral
gain in the surface layer and in a zone next to the lesion
front. A recent review suggested that higher fluoride con-
centrations may be needed to control caries in root sur-
faces compared to enamel surfaces [Wierichs and Meyer-
Lueckel, 2015]. Further studies should explore the use of
fluoride in the arrest of root and dentin caries. Such stud-
ies might also include evaluation of the caries-controlling
Takahashi/Nyvad
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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