doi:10.1111/j.1365-2591.2012.02101.
Tertiary dentinogenesis with calcium hydroxide: A
review of proposed mechanisms
P. Sangwan, A. Sangwan, J. Duhan & A. Rohilla
Department of Conservative Dentistry, Government Dental College, Pt. B.D. Sharma University of Health Sciences, Rohtak,
Haryana, India
Abstract
Sangwan P, Sangwan A, Duhan J, Rohilla A. Tertiary
dentinogenesis with calcium hydroxide: a review of proposed
mechanisms. International Endodontic Journal, 46, 3–19, 2013.
Calcium hydroxide has been used extensively in den-
tistry for a century. Despite its widespread use as a
pulp-capping agent, its mechanisms of action still
remain ambiguous. Understanding its modes of action
will lead to a broader understanding of the mecha-
nisms associated with induced dentinogenesis and
help in optimizing the currently available agents to
target specific regenerative processes to obtain the
best possible clinical outcomes. A literature search
relating to mechanisms of dentinogenesis of calcium
Introduction
Since its introduction into dentistry by Hermann
(1928), calcium hydroxide (Ca (OH)2) has been
widely used as a mineralizing agent as well as an
effective antimicrobial medicament. Despite several
other newer biomaterials being proposed for pulp-cap-
ping procedures, Ca(OH)2 is still considered as a gold
standard for comparison and evaluation of these new
products (Goldberg et al. 2008). Mineral trioxide
aggregate (MTA), a relatively recently introduced
promising bioactive material, has been shown to
release Ca(OH)2 in water as its main chemical compo-
Correspondence: Dr Pankaj Sangwan, Department of Conser-
vative Dentistry, Government Dental College, Pt. B.D. Shar-
ma University of Health Sciences, Rohtak-124001, Haryana,
India (Tel.: +91 9996112202; Fax: +91 1262 213876;
e-mail: drps_1@yahoo.co.in).
© 2012 International Endodontic Journal
hydroxide up to December 2011 was carried out
using pubmed and MEDLINE database searches as
well as manual searching of cross-references from
identified studies. Resulting suggestions regarding
dentinogenic mechanisms of calcium hydroxide range
from direct irritating action of the material to induc-
tion of release of biologically active molecules. The
purpose of this article is to discuss various mecha-
nisms through which calcium hydroxide may induce
tertiary dentinogenesis in the light of observations
made in included studies.
Keywords: calcium hydroxide, dentinogenesis,
mechanism, odontoblast.
Received 8 December 2011; accepted 25 June 2012
nent (Fridland & Rosado 2003). It has even been pro-
posed that both MTA and Ca(OH)2 share their
property of induction of reparative dentinogenesis
mainly because MTA acts as a ‘calcium hydroxide-
releasing material’ (Okiji & Yoshiba 2009). Although
several studies comparing the two materials have
indicated MTA to be superior to Ca(OH)2 (Pitt Ford
et al. 1996, Faraco & Holland 2001, Aeinehchi et al.
2003, Mente et al. 2010), numerous other authors
have reported equal efficacy of both materials (Qudei-
mat et al. 2007, Accorinte et al. 2008, Shayegan
et al. 2009, Dammaschke et al. 2010b,c). The varying
results in different studies may be attributed to the
fact that the biological reactions resulting from appli-
cation of bioactive materials to the pulp are con-
founded by numerous factors such as the formulation
of the applied material, concentration and rate of
release of the active ingredient, distance it has to dif-
fuse in a chemically active form to the target cells,
status of dentine, presence of infection and the health
International Endodontic Journal, 46, 3–19, 2013 3
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Calcium hydroxide: induced dentinogenesis Sangwan et al.

status of the pulp. It has also been suggested that the
type of biomaterial selected is of lesser consequence
and that the quality of the seal of the cavity to pre-
vent microbial ingress is the most important factor
determining the success of the procedure (Cox et al.
1999).
Studies have indicated a declining rate of pulp sur-
vival over time after pulp capping with Ca(OH)2
(Dammaschke et al. 2010a, Willershausen et al.
2011). Numerous factors may be held responsible for
such a pattern in the success rates observed (Fig. 1).
The non-adhesive nature of the cement and its disso-
lution over time (Prosser et al. 1982) may lead to
microleakage and entry of bacteria to the exposure
site (Hørsted-Bindslev & Løvschall 2002). Whilst this
is definitely a disadvantage, it must be borne in mind
that Ca(OH)2 was initially introduced primarily for its
dentinogenic activity – the seal was expected to be
provided by the overlying restoration. The importance
of seal provided by a restoration overlying the cement
is further emphasized in a study comparing the treat-
ment outcome of MTA and Ca(OH)2 (Mente et al.
2010). Bacterial contamination may also occur
through imperfections in dentine bridges, the so-called
‘tunnel defects’, which may provide passage for bacte-
ria from the exposure site to the pulp (Cox et al.
1985). Impaction of particles of the capping agent
(Murray & Garcı́a-Godoy 2006), necrosing action of
Ca(OH)2 up to depths of 1.5 mm (Swift et al. 2003)
compromising vascularity of the pulp and interference
by blood clot (Schröder 1978) are amongst other
factors held responsible for the failure of procedures.
It must also be remembered, however, that the preop-
erative inflammatory status of the pulp is a major fac-
tor influencing the success rate and is extremely
difficult to diagnose accurately. There is a distinct
possibility that some pulps, which are doomed to fail-
ure, are inaccurately diagnosed as savable and fit for
capping procedures and ultimately contribute to the
burden of failure of Ca(OH)2. Despite the limitations of
Ca(OH)2 compared to newer pulp-capping agents,
understanding its mechanism of action is of merit, as
it will lead to a broader understanding of mechanism
of induced dentinogenesis, help in optimizing the cur-
rently available agents to target specific regenerative
processes and obtain the best possible clinical results.
Whilst the mechanisms of antibacterial action of Ca
(OH)2 have been discussed extensively over the past
decade (Estrela et al. 1999, Siqueira & Lopes 1999, A-
thanassiadis et al. 2007), its dentinogenic mechanism
has received little attention. The purpose of present
article is, therefore, to discuss various possible mecha-
nisms through which Ca(OH)2 induces dentinogenesis
during pulp-capping procedures in the light of obser-
vations made in the literature.
Observations in various clinical and experimental
studies have demonstrated that the prime threat to the
process of healing after any injury is of bacterial origin.
Whilst there are several studies that indicate that the
injured pulp has an inherent ability to heal and even

Calcium hydroxide
1 2
6 Necrotic zone
Clot Dentine
4

Tertiary
dentine 3

Bacteria
Ca(OH)2 particles 5

Pulp

Figure 1 A schematic diagram summarizing various factors which may be held responsible for the failure of pulp-capping pro-
cedure with Ca(OH)2. (1) Non-adherence to dentine. (2) Dissolution over time. (3) Tunnel defects. (4) Necrosis of adjacent pulp
tissue, which may lead to compromised vascularity. (5) Dislodgement of particles to within the body of the pulp. (6) Blood clot
present between the material and pulp.

4 International Endodontic Journal, 46, 3–19, 2013 © 2012 International Endodontic Journal

deposit reactionary and reparative dentine, the most
direct evidence in this context is provided by studies
conducted in germ-free environment. A classic study
(Kakehashi et al. 1965) conducted to evaluate the
influence of bacteria on the fate of surgically exposed
dental pulps revealed that in spite of severe surgical
trauma, complicated by impaction of food and debris,
the germ-free rats exhibited healing and a reparative
response with consequent dentinal bridging. In con-
trast, pulps in conventional rats were severely
inflamed and ultimately underwent necrosis. It is
important to note that during this entire experimental
period, no attempts were made to influence the course
of healing by application of therapeutic agents. Other
studies on germ-free animals (Kakehashi et al. 1969,
Tsuji et al. 1987) have similarly indicated that healing
and tertiary dentine formation is a natural process,
which will occur without any medication as long as
bacterial contamination is avoided. Thus, in response
to trauma and caries, and if the pulp is able to main-
tain its vitality post-insult, a protective mechanism is
initiated and tertiary dentine is laid down at specific
locations at the pulp-dentine interface. Although the
end result is deposition of dentine matrix, two biologi-
cally distinct sequences of events occur depending on
whether the existing odontoblasts survive the insult or
perish. To differentiate and recognize the events, two
distinct terms have been employed: reactionary denti-
nogenesis and reparative dentinogenesis. Reactionary
dentinogenesis generally follows an insult of mild nat-
ure in which tertiary dentine matrix is secreted by
focally upregulated pre-existing odontoblasts. On the
other hand, reparative dentinogenesis involves secre-
tion of tertiary dentine matrix by an outright new gen-
eration of secreting cells after the death of primary
odontoblasts in response to a direct pulpal exposure or
greater intensity of insult (Lesot et al. 1993, Smith
et al. 1994).
Although it is difficult to recognize the two entities
clinically as well as histologically, it is important to
differentiate between the two events for exploitation
of our knowledge of molecular and cellular mecha-
nisms of tooth repair and regeneration.
The mechanism of dentinogenesis has been a mat-
ter of considerable interest and numerous studies
have been conducted, which contribute much to our
knowledge of pathways involved in the process. How-
ever, the precise mechanism involved in dentinogene-
sis is still unclear. In fact, there is a huge probability
that several overlapping mechanisms may be involved
in formation of dentine. It is also being suggested that
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Sangwan et al. Calcium hydroxide: induced dentinogenesis
processes of tooth formation and repair are closely
related and strong parallels exist between early devel-
opmental events and repair process. ‘Molecules
involved in cell fate determination, cell adhesion, cell-
cell communication and cytoskeletal reorganization,
which are expressed in dental tissues during odonto-
genesis, are re-expressed in dental tissues after injury’
(Mitsiadis & Rahiotis 2004).
The following observations point towards a close
relationship between the two processes:
1. The same cluster of biomolecules that is involved
in regulation of differentiation and matrix produc-
tion by odontoblasts during developmental stages
has also been shown in several experimental
studies to influence the process of reparative den-
tinogenesis (Mitsiadis & Rahiotis 2004).
2. The bioactive molecules produced during devel-
opmental process play an ‘auto-paracrine’ role in
differentiation and regulation of odontoblasts.
These molecules get sequestered into the den-
tinal matrix during deposition phase and are
re-released during dissolution of dentine by cer-
tain agents. As in the formative stage of tooth,
these molecules released from dentine matrix
have been shown to be involved in regulation of
tertiary dentinogenesis (Smith et al. 2001).
3. Once the primary odontoblasts are lost owing to
injury, long tubular cells morphologically similar
to primary odontoblasts with secretory potential
are seen for resumption of the lost function. The
origin of these cells is sill debated. Although
numerous possibilities may have been suggested,
it is most widely accepted that these originate
from neural crest–derived cells, an origin shared
by primary odontoblasts (Thesleff & Vaahtokari
1992).
With the understanding that the processes during
development and those during repair and regenera-
tion share common pathways, it becomes easier to
comprehend the mechanism of action of Ca(OH)2.
However, it must also be emphasized that several
mechanisms are assumed to play important roles in
dentinogenesis following application of Ca(OH)2 and
the role of any single mechanism cannot be consid-
ered in isolation.
Search strategy
A literature search for relevant articles was performed
up to December 2011 using pubmed and MEDLINE
database searches. The search was based on the
International Endodontic Journal, 46, 3–19, 2013 5

Calcium hydroxide: induced dentinogenesis Sangwan et al.
following keywords: ‘calcium hydroxide AND dental
pulp’, ‘calcium hydroxide AND odontoblasts’, ‘calcium
hydroxide AND dental pulp capping’, ‘calcium
hydroxide AND pulpotomy’ and ‘calcium hydroxide
AND vital pulp therapy’. This was followed by a sup-
plementary search carried out on 30 January 2012.
No language restriction was applied. The titles and
abstracts of all the resulting articles were indepen-
dently screened by two reviewers (PS and AS) to
identify the articles pertinent to the subject of the
review. Where the title and abstract were not suffi-
ciently perspicuous to make a clear decision, it was
decided to include the article for subsequent evalua-
tion. The full texts of the identified articles were
obtained and further analysed. Additional manual
search of reference lists of retrieved publications was
carried out to find any potentially relevant articles
not identified during the main search. Altogether,
268 articles were read in full text and evaluated, out
of which 133 were included in this review.
Histological reactions
Depending upon the resiliency of the pulp and the
severity of insult, the pulp either undergoes necrosis
or mounts a successful defensive reaction. Preceded
by classical connective tissue reactions, the pulp
reacts to noxious stimuli in three ways: precipitation
of intratubular calcifications, deposition of peritubular
dentine and formation of tertiary dentine, all aimed at
reducing the permeability of pre-existing dentine and
hence limit the insult to the pulp (Mjor 1985).
Application of Ca(OH)2 in close proximity to pulp
tissue also precipitates a similar set of events. Of
these, formation of tertiary dentine seems to provide
the most significant protection as it may not only
reduce the permeability of dentine but also increase
the distance between the source of irritation and pulp,
thus reducing the amount of damage incurred. This
tertiary dentine has been subdivided into two types
depending on whether the dentine has been secreted
by pre-existing primary odontoblasts or by newly dif-
ferentiated secretory cells, which have migrated to the
region following death of primary odontoblasts.
Whereas reactionary dentinogenesis may be consid-
ered an exaggerated response of odontoblasts already
present in the region manifested as an increase in the
amount of dentinal matrix secreted, reparative denti-
nogenesis involves several intricately interrelated bio-
logical events. As the odontoblasts in the region have
already been lost, new cells capable of secretion of
6 International Endodontic Journal, 46, 3–19, 2013
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biomatrix are required. This involves proliferation of
cells capable of forming the secretory cells, their
migration, adhesion to substrate and finally cytodiffer-
entiation into ‘odontoblast-like cells’. Several other
terms such as neo-odontoblasts and second-genera-
tion odontoblasts have also been used in the litera-
ture. The term odontoblast itself is avoided in such an
event as the biochemical characteristics of this new
generation of cells remain to be specified (Tziafas
1995). The controversy regarding these cells is, how-
ever, not limited to their biochemical specificity. As
mentioned earlier, limited information is available
relating to origin of these cells and the issue remains
contentious. Several theories have been proposed in
this regard. The earliest suggestion included subodon-
toblastic cell-rich layer or zone of Höhl (1896) as a
possible source of these odontoblast-like cells. The
cells in this layer have also been suggested to support
pre-existing odontoblasts in pulp repair (Goldberg &
Smith 2004). Autoradiographic study using thymi-
dine-labelled cells (Fitzgerald 1979) revealed a rise in
fibroblastic activity close to site of exposure indicating
that pulp fibroblasts may be source of these secretory
cells. Another similar study conducted by Fitzgerald
et al. (1990) showed that cells from central pulp tis-
sue proliferated and migrated towards the site of
exposure to form secretory cells. Pericytes and myofi-
broblast transitional cells have also been suggested as
possible sources of odontoblast-like cells (Carlile et al.
2000, Alliot-Licht et al. 2001). A recent study using
genetic lineage tracing (Feng et al. 2011) demon-
strated a dual origin of mesenchymal stem cells in
teeth, pericytes resident as quiescent cells in the pulp
and migration of non-pericyte cells from niche area of
tooth that normally provides a continuous supply of
cells to support tooth growth. The extent of contribu-
tion by pericytes was suggested to be dependent on
extent of vascularity of the tissue. Yet another recent
study (Ishikawa et al. 2010) mapping BrdU label-
retaining dental pulp cells (LRCs) indicated that fol-
lowing an injury, granular LRCs (transit-amplifying
cells) present in the subodontoblastic layer first
migrate and differentiate into odontoblast-like cells
without proliferation to replace the lost original odon-
toblasts. However, if both the transit-amplifying cells
and differentiated odontoblasts degenerate, the slow-
cycling long-term dense LRCs (dental pulp stem cells)
actively proliferate and differentiate into odontoblast-
like cells. Ca(OH)2 has recently been shown to
increase recruitment, migration, proliferation and
mineralization of dental pulp stem cells (Ji et al.
© 2012 International Endodontic Journal

2010), thus facilitating the whole process of repara-
tive dentinogenesis in such cases.
When Ca(OH)2 is placed against pulp, the high pH
of the material causes irritation and produces a super-
ficial burn at the area of exposure. This zone of coag-
ulation necrosis has been suggested to be vital to
formation of tertiary dentine. Ultrastructural studies
(Yoshiba et al. 1996) have shown that the initial cal-
cification following application of Ca(OH)2 is associ-
ated with the cellular debris and swollen collagen
fibrils at the interface of superficial necrotic layer and
underlying vital pulp tissue. It was suggested that the
coagulation necrosis caused by Ca(OH)2 might be
capable of initiating mineralization process. Further
proof to this effect is provided by studies which have
shown that final differentiation of odontoblast-like
cells and hard tissue apposition is not observed in
teeth capped with inert materials such as Teflon (Cvek
et al. 1987, Heys et al. 1990). This important role
ascribed to tissue necrosis is, however, at odds with
studies reporting absence of any intervening zone of
necrosis in experimental pulp cappings with hard set-
ting Ca(OH)2 where the newly deposited matrix comes
to lie directly against the pulp-capping material (Kita-
sako et al. 2000). It has been suggested that the
width of the necrotic zone depends on the nature of
the material used. Aqueous suspensions are associated
with wider zones of necrosis as compared to setting
type of formulations (Heys et al. 1981). Whatever is
the formulation of Ca(OH)2 used for capping proce-
dure, the irritation caused by the material and result-
ing damage is initially tackled by pulp in the same
way as in any other soft tissue healing process.
Lu et al. (2008) evaluated human pulp tissue
response following direct pulp capping. They observed
appearance of a necrotic layer at the site around
7 days after capping with Ca(OH)2, associated with
slight to moderate inflammatory reaction character-
ized by the infiltration of neutrophils and mononu-
clear cells. The pulp tissue was disorganized with
dilated blood vessels. Dentine matrix appeared around
30 days later once the cellular and vascular inflam-
matory events began to fade and new cells with secre-
tory potential arrived at the region to effect repair. On
the other hand, Fitzgerald et al. (1990) reported
matrix deposition by thymidine-labelled new secretory
cells in primate pulp much earlier at day 8 following
Ca(OH)2 application. They also found an increase in
labelled odontoblast-like cells in the cell-free zone.
These cells appeared to have migrated from the
central pulp. They suggested that at least two DNA
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Sangwan et al. Calcium hydroxide: induced dentinogenesis
replications must occur before the migrating cells dif-
ferentiate into odontoblast-like cells. Similar findings
were reported in another ultrastructural study (Mjor
et al. 1991) of healing of pulp exposures in Rhesus
monkeys where specimens were observed at much
more frequent intervals. It reported that tissue
destruction and haemorrhage, caused owing to
trauma inflicted during the procedure and subsequent
capping, resolved by 5–6 days with concomitant
decrease in inflammation. By the end of second week,
a well-organized layer of cells similar in appearance
to adjacent odontoblasts appeared, bordered with pre-
dentine-like matrix (Fig. 2).
Collagen is laid down approximating the necrotic
zone and mineralized crystals are deposited in the
region. Whilst it has been proposed that calcification
of this initial layer may be dystrophic in nature
(Schröder 1985), other studies (Sela et al. 1981,
Hirschfeld et al. 1982) have reported the presence of
matrix vesicles with filled apatite crystals. The pre-
odontoblasts then attach themselves to this primitive
biomatrix and elaborate fibrodentine. The production
of fibrodentine is almost always observed prior to any
further differentiation into odontoblast-like cells
(Senzaki 1980). It has been suggested that fibroden-
tine may serve the same role as that of basement
membrane: fixation of biomolecules and their proper
stereospatial presentation to suitable cells capable of
differentiating into secretory cells (Ruch 1985). Once
the fibrodentine has been laid down, the secretory
cells orient themselves in a polarized pattern and
begin secretion of tubular matrix. Thickening of bar-
rier and appearance of dentine-like tissue with lim-
ited tubular structure has been reported to be
observed at around 4 weeks after pulp-capping proce-
dure (Yoshiba et al. 1996).
Raised expression of biomolecules
The deposition of dentine as a protective response of
pulp to injury is a well-regulated event guided by a
set of biologically active molecules. Little is known
about these processes because replication of these
events under experimental conditions requires a com-
plex set of molecules to be presented to the cells in a
seemingly specific chronological pattern of appearance
and spatial configuration, which is difficult to achieve.
Multifunctional roles suggested for several of these
molecules further complicate the picture. Although
studies have focused on application of any one such
molecule at a time to look at its influence, the
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Calcium hydroxide: induced dentinogenesis Sangwan et al.

Application of Ca(OH)2

Inflammation, tissue (Immediately after

destruction and hemorrhage application of Ca(OH)2)

Stimulus

Resolution of inflammation (Within first week

following pulp capping)

Division & migration of precursor

cells to substrate surface; adhesion (One to two weeks after
and cytodifferentiation into the procedure)
odontoblast-like cells; Matrix
deposition

Dentine-like tissue with some

(Four weeks to a month
tubular structure observed.
after pulp capping)
Pulp-dentine border regains its
original architecture

Figure 2 Histological events observed following application of Ca(OH)2. High pH of the material produces a superficial zone of
necrosis associated with inflammation. Ca(OH)2 as well as damage caused to the pulp tissue provides stimulus for the precursor
cells to migrate and differentiate into secretory cells. Productive changes at the site begin once the inflammation has resolved.

response also varies depending on various other fac-
tors such as the concentration administered (Thyaga-
rajan et al. 2001) and location of the pulp tissue in
question (Six et al. 2002). Thus, the signalling pro-
cesses to achieve the desired objectives are not well
understood.
Recent works directed at establishing the role of
biomolecules have pointed to transforming growth
factors (Dobie et al. 2002, Huojia et al. 2005), bone
morphogenetic proteins (Nakashima 2005), insulin-
like growth factor (Onishi et al. 1999), fibroblast
growth factor (Kettunen et al. 2000, Kim et al. 2010)
and fibronectin (Mizuno & Banzai 2008) as those
most capable of inducing functional changes in secre-
tory cells and resultant tertiary dentinogenesis.
Fibronectin, a high–molecular weight glycoprotein
found in both plasma as well as tissues, is reportedly
involved in a wide array of cellular events such as
adhesion, chemotaxis and cytodifferentiation (Yoshiba
et al. 1994). It is considered a key molecule that
mediates adhesion of matrix-secreting cells to sub-
strate (Wang et al. 2000), which may be predentine,
dentine, necrotic tissue or cementum. It has been
localized to dental basement membrane where it
mediates cytoskeletal changes and polarization of
odontoblasts (Lesot et al. 1981). Its role in odonto-
blast differentiation was further substantiated when it
was found that cytodifferentiation was inhibited on
application of monoclonal antibody directed against
fibronectin receptor (Lesot et al. 1988). Other than
this direct role, fibronectin also possesses ability to
bind TGFb (Mooradian et al. 1989), which in turn
stimulates cytodifferentiation of odontoblasts and
later, secretion of dentine matrix. Bone morphoge-
netic proteins (BMPs), a group of cytokines, are mem-
ber of TGFb superfamily and play an essential role in
promoting differentiation of odontoblasts (Thesleff
et al. 1995) and induction of dentine formation

8 International Endodontic Journal, 46, 3–19, 2013 © 2012 International Endodontic Journal

(Nakashima 1994a). The important ones proven to
be involved in the reparative process are BMP-2,
BMP-4 and BMP-7 (Nakashima 1994a,b, Lin et al.
2007). Transforming growth factor b (TGFb), which
is known to play an important role in repair of vari-
ous tissues, too has also been reported to increase the
proliferation, differentiation and mineralization activ-
ity of dental pulp cells (Melin et al. 2000, Nie et al.
2006).
A number of laboratory studies have been con-
ducted that have demonstrated that Ca(OH)2 has the
capacity to induce matrix formation and mineraliza-
tion during dentinogenesis through the release of
these biomolecules. However, owing to their obvious
limitations, caution must be exercised in extrapolating
the results of such studies in general.
In vitro studies
To examine the influence of Ca(OH)2 on fibronectin
synthesis, an in vitro study (Mizuno & Banzai 2008)
was carried out by treating human dental pulp cells
with high concentration of Ca++. The authors con-
cluded that raised concentration of Ca++ in pulp tissue
achieved owing to its release from applied Ca(OH)2-
induced fibronectin synthesis in pulp cells, followed by
accretion of fibronectin in necrotic tissue adjacent to
the healthy pulp. This accumulated fibronectin might
then induce differentiation and secretion in odonto-
blasts. mRNA levels of BMP-2 have also been found to
increase under elevated Ca++ culture conditions in a
study that suggested the Ca++ from Ca(OH)2 specifi-
cally modulated BMP-2 levels in pulp tissue to effect
repair process (Rashid et al. 2003). Alkaline condi-
tions in culture medium also raise expression of
mRNA for BMP-2 in human dental pulp cells (Okabe
et al. 2006). Another recent study (Tada et al. 2010)
conducted on cultured human dental pulp cells sug-
gested that modulation of gene expression for BMP-2
through elevated extracellular Ca++ from Ca(OH)2
may be a crucial mechanism for induction of dentino-
genesis. It was shown that elevated Ca++ levels in the
environment increased the BMP-2 mRNA levels in
human dental pulp cells not only at transcriptional
level but also at post-transcriptional level by increas-
ing mRNA stability. A recent report on the modula-
tion of pulp cells and induction of reparative dentine
formation (Laurent et al. 2012) has reported an
increase in TGFb secretion from pulp cells after inter-
action with Ca(OH)2 in a human tooth culture model.
Some reports have also proposed that Ca(OH)2 may
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Sangwan et al. Calcium hydroxide: induced dentinogenesis
act on pulp cells and cause mineralization by modu-
lating the expression of, amongst certain other bio-
molecules, BSP (Da Silva et al. 2008) and osteopontin
(Rashid et al. 2003) through its released ions.
An increase in activation levels of p-ERK, consid-
ered important in proliferation activities, has also
been demonstrated in human dental pulp cells grown
in culture with Ca(OH)2 (Ji et al. 2010). The authors
indicated that this raised expression level may be
responsible for stem cell proliferation observed during
the experiment.
Whilst the in vitro studies described above are
important in scrutinizing the molecular events tran-
spiring following application of Ca(OH)2, in vivo stud-
ies provide a more rational way of studying these
processes. Owing to limited availability of ‘experimen-
tal material’ and ethical considerations, however, the
number of studies conducted in this direction is few.
In vivo studies
Expression of fibronectin has been demonstrated in
vivo in dental pulps following pulp capping with
Ca(OH)2 (Yoshiba et al. 1996, Piva et al. 2006,
Fernandes et al. 2008, Leites et al. 2011). Another in
vivo study conducted on dog dental pulp tissue to
determine immunolocalization of fibronectin after
interaction of pulp with Ca(OH)2-containing material
showed that fibronectin had a strong association with
microcrystals formed at the surface of the cement
(Tziafas et al. 1995b). Following its adsorption on the
cement, a new biochemically active surface is formed
serving the same role which the basement membrane
plays for cellular mechanics of odontoblasts during
developmental stages. A study (Kaida et al. 2008)
conducted on rat teeth to investigate the wound heal-
ing process following pulpotomy reported an initial
increase in number of IL-1b-expressing cells, followed
by rise in TGFb-expressing macrophages in pulps cov-
ered with Ca(OH)2. After an initial lag, cells express-
ing BMPs gradually increased in number at the
exposure site. Ca(OH)2 has a favourable influence on
alkaline phosphatase, a crucial enzyme in calcification
process. High levels of alkaline phosphatase generally
indicate mineralization activity going on in the tissue.
Rise in its levels has been reported in odontoblastic
and subodontoblastic cell layers approximating the Ca
(OH)2-covered cavity floors of monkey teeth (Hassel-
gren & Tronstad 1977).
Notch signalling genes, which are believed to play
an important role in differentiation of several cell
International Endodontic Journal, 46, 3–19, 2013 9

Calcium hydroxide: induced dentinogenesis Sangwan et al.
types, have been observed to be activated in response
to Ca(OH)2 pulp capping in rat molars. Whilst such
activation is also seen in untreated exposed teeth, the
increase in expression is much higher in pulp-capped
teeth (Løvschall et al. 2005).
Indirect evidence also exists, which indicates that
Ca(OH)2 may promote the bioactivity of available
TGFb. It is well known that TGFb is secreted mostly in
latent form, which limits its biological activity in tissue
(Barcellos-Hoff 1996). To interact with cell surface
receptors and exert its influence, the cytokine must be
released from the latent complex. Alkalization of cul-
ture medium has been reported as one of the estab-
lished chemical methods of its activation (Lyons et al.
1988). Theoretically, this alkaline environment can be
provided by Ca(OH)2 thus facilitating conversion of the
molecule to its active form. Although there is no study
directly favouring this hypothesis in dental tissue, one
may refer to a recent in vitro study conducted to inves-
tigate the effect of TGFb in conjunction with Ca(OH)2
on production of TGFb by osteoblasts (Jaunberzins et al.
2000). It was found that, whilst the difference was sta-
tistically insignificant, the levels produced by combina-
tion of two were consistently higher than those with
TGFb alone. Ca(OH)2 had a synergistic effect with the
cytokine on osteoblasts, thereby implying that Ca(OH)2
is capable of promoting its bioactivity.
Thus, although understanding the exact mecha-
nisms of cellular signalling on application of Ca(OH)2
is still unclear, more and more evidence indicates that
Ca(OH)2 can act on dental pulp cells to cause release
of biologically active molecules, which then guide the
entire dentinogenic mechanism.
Release of extracellular matrix molecules
from dentine
Introduction of dentine fragments into pulp tissue has
long been known to induce reparative dentinogenesis
on its surface (Nakagawa et al. 1989). To elaborate
on this issue, the response of dog pulp tissue after
interaction with demineralized dentine, native dentine
or predentine was studied (Tziafas et al. 1992). Whilst
predentine reportedly showed very favourable
response with differentiation of odontoblasts, native
dentine and demineralized dentine were associated
with appearance of new odontoblast-like cells only
after an initial deposition of matrix by spindle-shaped
or polygonal cells.
Dentine is a biologically active, convoluted tissue
secreted by specialized secretory cells – the odonto-
10 International Endodontic Journal, 46, 3–19, 2013
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blasts. Whilst collagen forms the bulk of its matrix,
non-collagenous proteins have also been identified,
which may have an important role in guiding cytodif-
ferentiation, secretion of matrix and its mineralization.
For instance, dentine phosphoproteins and dentine
sialoproteins have been suggested to be involved in
mineralization process (Suzuki et al. 2009). TGFß,
another component of ECM widely studied, has been
reported to have inductive effect on cell differentiation
and matrix secretion (Dobie et al. 2002). There are sev-
eral other reports that include insulin-like growth fac-
tor, fibroblast growth factor, platelet-derived growth
factor, etc. These bioactive molecules are trapped in
dentine during the deposition phase and are protected
from any degradation, which might have occurred
otherwise. On solubilization of the dentine matrix,
these molecules are released into the immediate vicinity
and affect any related ongoing processes.
The sequestered regulating molecules may be
released from the dentine matrix on interaction with
a variety of agents. Acidic demineralization occurring
during the caries process may cause such a release
and aid in protecting the pulp by resultant dentine
deposition (Farges et al. 1993). There is likelihood
that externally applied acids during various dental
procedures may have identical effects (Klont & ten
Cate 1990). Another commonly used chelating agent,
EDTA, has also been exhibited to effect the release of
the bioactive molecules. The application of its extracts
has been reported to expedite the process of dentino-
genesis (Tziafas et al. 1995a, Smith et al. 2001, Chun
et al. 2011). Another recent study has reported that
such EDTA-solubilized matrix components also pro-
mote angiogenic activity and may contribute to pulp
regenerative processes (Zhang et al. 2011).
On similar lines, it has been demonstrated in vitro
that Ca(OH)2 can solubilize bioactive molecules, previ-
ously sequestered in dentine matrix (Graham et al.
2006). The authors suggested that release of these
molecules from dentine by Ca(OH)2 may be one of the
mechanisms through which it exerts its effect on den-
tine regeneration. This suggestion was further corrob-
orated in yet another in vitro study, which showed
dissolution of bioactive dentine matrix components by
mineral trioxide aggregate and Ca(OH)2 (Tomson
et al. 2007). TGFß1, an important regulatory mole-
cule in the process as described earlier, has also been
exhibited to be solubilized on interaction with Ca(OH)2
(Smith & Smith 1998).
Several other authors have also compared the den-
tinogenic response of pulp to Ca(OH)2 and direct
© 2012 International Endodontic Journal

application of various molecules (Goldberg et al.
2001, Kaida et al. 2008). Ca(OH)2 was more or less
consistently reported to have similar, albeit weaker,
effect on dentinogenic processes. The similarity in the
outcomes observed may be a manifestation of biologi-
cal actions of Ca(OH)2 being mediated via release of
biomolecules.
The evidence presented above implies that applica-
tion of Ca(OH)2 as a pulp-capping agent can solubilize
a blend of biologically active molecules from the den-
tine matrix, which in turn alter the gene expression
of the concerned secretory cells to modulate reaction-
ary as well as reparative dentinogenesis.
Role of pH
Traditional view has always attributed mineralizing
action of Ca(OH)2 to release of hydroxyl ions and the
resultant alkaline pH (Glass & Zander 1949, Schröder
& Granath 1971, Stanley & Lundy 1972). A recent
study (Okabe et al. 2006) conducted to investigate
the influence of alkaline pH showed that rise in pH
caused increased alkaline phosphatase, BMP-2 expres-
sion and calcified nodule formation. It confirmed that
a rise in pH within physiological limits enhanced the
mineralization ability of human pulp cells. However,
the pH of Ca(OH)2 has been shown to rise to as high
as 12.5, which is much higher than the optimum
range for survival of odontoblasts. One may argue
that such a high pH may thus be detrimental rather
than beneficial to the cells. It is possible that, after an
initial rise in pH to such high levels, limited buffering
action of dentine (Wang & Hume 1988) owing to
presence of proton donors may bring it down to levels
within the optimum range suitable for upregulation
of odontoblasts.
Further, the formation of dentine matrix by odonto-
blasts is regulated by presence of inhibitory molecules.
These biomolecules may get denatured in an alkaline
environment provided by Ca(OH)2. An important
inhibitor of mineralization found in tissues is pyrophos-
phate ion. Its removal is essential for mineralization to
occur. This is carried out by its hydrolysation by alka-
line phosphatase, which is found in abundance at the
site of mineralization. The same enzyme may also pro-
vide for inorganic phosphate for deposition of mineral
crystals. The optimum pH for in vitro activity of alkaline
phosphatase has been reported to be around 10 (Hei-
thersay 1975). This favourable alkaline environment
in vivo may be provided by Ca(OH)2-containing pulp-
capping agents applied to the pulp.
© 2012 International Endodontic Journal
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Sangwan et al. Calcium hydroxide: induced dentinogenesis
Alkaline pH may also directly neutralize the acids
produced by resorptive cells and prevent progression
of resorption. Similarly, it may counteract the acidic
environment produced by inflammatory reaction,
which is harmful to the healing process per se (Hei-
thersay 1975). A recent study has also shown that
alkaline pH can denature proinflammatory mediators
to help in resolution of detrimental inflammation
(Khan et al. 2008). Similarly, high pH may also cause
denaturation of bacteria-derived lipopolysaccharides
and aid in reducing the severity of inflammation.
Role of calcium ions
Well recognized in regulation of cellular functions of
the body, the biological response of Ca(OH)2 has also
been attributed to Ca++ released from the applied
material. Ca(OH)2 applied to the pulp dissociates into
Ca++ and OH . Continuous dissolution of the material
produces a local microenvironment with high concen-
tration of Ca++. This concentration is highest at the
site of application and goes on decreasing as the dis-
tance increases from the material, thus forming a gra-
dient. It must be mentioned here that such a Ca++
gradient is capable of activating cells such as stem
cells (Adams et al. 2006), osteoblasts (Yamaguchi
et al. 1998) and fibroblasts (Lansdown 2002) and
bring about migration. In addition, the cells responsi-
ble for formation of odontoblast-like cells in reparative
dentinogenesis are located in the central pulp (Fitzger-
ald et al. 1990). The calcium receptors on these cells
can thus similarly act as sensors to this graduated
increase in concentration and actuate chemotaxis
from deeper tissues to the affected site.
Other than chemotaxis, Ca++ has been reported to
be a potent regulator of several other cellular events
like proliferation, differentiation and mineralization
(Torneck et al. 1983, Kulesz-Martin et al. 1984,
Zayzafoon 2006). There is evidence that Ca++ stimu-
lates synthesis of fibronectin in dental pulp cells (Miz-
uno & Banzai 2008). Extracellular Ca++ has also been
demonstrated in a study to upregulate the gene
expression of bone-related proteins like BMP-2 and os-
teopontin in human pulp cells (Rashid et al. 2003).
The authors concluded that extracellular Ca++,
through modulation of such proteins, plays an impor-
tant role in Ca(OH)2-induced mineralization in dental
pulp tissue. Binding of Ca++ to specific receptors
bound to plasma membrane of dental pulp cells has
been proposed to bring about such changes in the cel-
lular functions. However, the precise mechanism of
International Endodontic Journal, 46, 3–19, 2013 11

Calcium hydroxide: induced dentinogenesis Sangwan et al.
signalling process is still poorly understood. Whilst
one report (Mathias et al. 2001) proposed that the
localization of calcium-sensing receptors to predentine
and local accumulation of extracellular Ca++ might
provide a mechanism of modulation of odontoblast
function by Ca++, another (Tada et al. 2010) indi-
cated that dental pulp cells respond to Ca++ in a man-
ner different from calcium-sensing receptors and
specific for Ca++ amongst cations. N-cadherin, a cell
adhesion molecule shown to be an important regula-
tor of odontoblasts in both health and disease (Hey-
mann et al. 2002), is also a calcium-dependent
adhesion molecule, thus emphasizing the importance
of the cation.
Yet another hypothesis suggests that Ca++ may
decrease capillary permeability (Heithersay 1975),
leading to an even further increase in concentration
of local extracellular Ca++ and calcium-dependent
pyrophosphatase, a group of enzymes that metabolize
mineralization-inhibiting pyrophosphates. The entire
process thus facilitates progression of mineralization
in the dental pulp.
Presentation of a biologically active
surface
Whilst the precise mechanism remains unclear, the
presence of a stable mechanical support seems to be
an essential prerequisite for initiation of reparative
dentine formation (Tziafas 2010). Basement mem-
brane during developmental phase or fibrodentine,
superficial necrotic zone, collagenous matrix, cements
or crystals deposited thereon during the reparative
phase may provide for such a support. It appears that
these adsorb bioactive molecules onto the surface thus
facilitating their immobilization and proper spatial
presentation to cells involved in the process of repair.
The substrate also provides a dynamic surface for
competent cells to attach to, orient and deposit repar-
ative matrix in a polar manner.
Whilst Ca(OH)2 applied to the pulp tissue may itself
act as a suitable substrate, it has also been reported
that these materials may react with the surrounding
environment to form inorganic crystal structures.
Eda (1961) reported deposition of such granules on
Ca(OH)2 during histochemical analysis of the dental
pulp in dogs. Similar findings were reported in
another study (Holland et al. 1982) histochemically
analysing the dog’s dental pulp after pulpotomy and
subsequent protection with calcium hydroxide, bar-
ium hydroxide or strontium hydroxide. It further
12 International Endodontic Journal, 46, 3–19, 2013
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demonstrated that the observed larger granules con-
sisted primarily of calcium carbonate precipitations
arising from the pulp-capping material. The authors
suggested that these calcium carbonate granules
could encourage the precipitation of calcium salt
granules by the pulp tissue and make the conditions
more favourable for odontoblast differentiation and
dentine deposition. Yet another study (Tziafas &
Economides 1999) evaluated the surface of Ca(OH)2-
containing materials and showed precipitation of such
crystals in the presence of almost all the storage
media used, distilled water, phosphate-buffered saline
or culture media with or without foetal calf serum.
The crystals were calcium carbonate or calcium phos-
phate in nature and found to be more complex in
presence of foetal calf serum. As carbonated apatite
represents the biological apatite phases seen in bone,
cementum and dentine, these crystals may aid in ini-
tiation of dentinogenesis (Okiji & Yoshiba 2009).
Fibronectin, the adhesion-promoting molecule, has
been found to be strongly linked to the microcrystals
formed on the surface of Ca(OH)2-containing materi-
als (Tziafas et al. 1995b). This molecule seems to
mediate further attachment of other biologically
active molecules and thus aid directly or indirectly in
attachment and differentiation of odontoblast-like
cells. Odontoblast-like differentiation of human pulp
cells has also been demonstrated in vitro in the pres-
ence of calcite microcrystals produced by the reaction
of Ca(OH)2 with the containing culture medium (Seux
et al. 1991).
Thus, it can be stated that Ca(OH)2 provides a sur-
face rich in biologically active molecules, which helps
attract and orient odontoblasts for dentinogenesis to
begin.
Antimicrobial action
It has been long known that healing can occur spon-
taneously in teeth in germ-free animals (Kakehashi
et al. 1965). This implies that healing of pulp-dentine
complex is primarily impeded by bacterial contamina-
tion. Thus, in appropriately indicated cases, elimina-
tion of bacteria should at least partially, if not
completely, aid in the reparative process.
Antimicrobial properties of Ca(OH)2 are well known
and primarily attributed to its highly alkaline pH of
about 12.5 at which very few bacteria can thrive (By-
strom et al. 1985). At such a high pH, it causes dena-
turation of structural as well as enzymatic proteins
with resultant loss of biological activity of enzymes
© 2012 International Endodontic Journal

and eventual cell death. It also damages cytoplasmic
membrane and genetic material thereby inhibiting
cellular activities. As Ca(OH)2 has limited solubility
and diffusibility, its antimicrobial activity is limited to
superficial layers of pulp. Thus, one may infer that
use of Ca(OH)2 may be successful only in cases of
minimal bacterial invasion. This is evident clinically
when pulp capping succeeds only when bacterial con-
tamination is limited to superficial soft tissue and
inflammatory reaction is mild and of relatively short
duration. If the bacteria have penetrated to great
depths in pulp leading to severe inflammation, the
procedure generally fails (Goldberg et al. 2008).
All the above observations indicate that Ca(OH)2
may aid in repair process by eliminating bacterial
contamination to provide a germ-free environment
with minimal inflammation.
Anti-inflammatory action
Although protective in nature at the beginning,
inflammation in pulpal tissue can also have certain
deleterious effects. Enclosed in a rigid, non-yielding
structure, any significant increase in the pressure
may lead to compression of the vessels and undermin-
ing of the much needed blood flow. Also, cells cause
resorption by secretion of hydrogen ions and genera-
tion of local microdomains of low pH. This undesir-
able pH may be counteracted by the alkaline
environment created by Ca(OH)2, thus preventing
destruction of the tissues.
Endogenous inflammatory mediators like interleu-
kin-1a and tumour necrosis factor-a play a decisive
role in regulation of inflammation and the consequen-
tial tissue destruction (Cochran 2008, Gabay et al.
2010). A recent in vitro study (Khan et al. 2008) con-
cluded that Ca(OH)2 causes denaturation of such pro-
inflammatory cytokines. The resultant significant
reduction in their levels may be a potential mecha-
nism by which Ca(OH)2 exerts its immunomodulatory
effect.
Matrix deposition and its consequent mineralization
have been observed to be suppressed when odonto-
blasts are challenged with bacterial by-products such
as lipopolysaccharides and lipoteichoic acid (Durand
et al. 2006, Nomiyama et al. 2007). These by-products
can cause further damage to the pulpal tissue by aug-
menting the inflammatory reaction (Saluk-Juszczak &
Wachowicz 2005). Biological properties of such com-
ponents are altered by Ca(OH)2, thus removing the
inhibitory effect from the cells with reduction in inflam-
© 2012 International Endodontic Journal
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Sangwan et al. Calcium hydroxide: induced dentinogenesis
mation and facilitation of dentinogenesis (Safavi & Nic-
hols 1993, Barthel et al. 1997, Baik et al. 2011).
An in vitro study conducted to understand the role
of Ca(OH)2 in the inflammatory, healing and biomin-
eralization processes found that it induced proinflam-
matory cytokine up-regulation for up to 3 days.
However, at the same time, overexpression of IL-10,
which is known to have strong anti-inflammatory
properties, was also observed which signalled the sub-
sequent decrease in proinflammatory cytokine produc-
tion after 3 days. It was suggested by the authors
that overexpression of IL-10 in tissues in contact with
the material lent support to the view that Ca(OH)2
promotes an anti-inflammatory effect (Reyes-Carmona
et al. 2011). Further evidence of its anti-inflammatory
action is provided by another in vitro study conducted
to evaluate the direct action of Ca(OH)2 on inflamma-
tory cells (Segura et al. 1997). It was reported that
Ca(OH)2 significantly inhibited the substrate adher-
ence capacity of inflammatory macrophages. The
authors suggested that this effect could explain, at
least in part, the mineralizing property of the mate-
rial: action of osteoclasts and dentinoclasts, the
macrophage-derived cells, would decrease and the
balance would tilt in favour of osteogenic/odontogenic
mechanisms.
Conclusion
Although substantial volume of research is being car-
ried out to understand the mechanism of tertiary den-
tinogenesis especially in relation to application of
biomaterials like Ca(OH)2, the precise details still
remain unknown. To conclude, it may be stated that
whilst some of non-specific actions of calcium hydrox-
ide may aid in repair by providing an optimal envi-
ronment for the pulp to best utilize its inherent
healing capacity, ample evidence is available to indi-
cate that the material causes tertiary dentinogenesis
through activation of certain specific pathways in the
pulp (Fig. 3). There is an interesting possibility that
these pathways are not exclusive, but are rather
interrelated. Several of the studies discussed have
been conducted either in vitro or in vivo using non-
human models and hence should be interpreted with
caution for clinical situations involving human sub-
jects. Continued research is required to achieve a
comprehensive picture of the response of pulp tissue
to such biomaterials to best understand the optimum
concentration, formulation, delivery mechanism of the
material to achieve optimum repair and regeneration.
International Endodontic Journal, 46, 3–19, 2013 13
 13652591, 2013, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2591.2012.02101.x by Nat Prov Indonesia, Wiley Online Library on [20/12/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Calcium hydroxide: induced dentinogenesis Sangwan et al.

Migration of progenitor Antibacterial activity Non specific

cells along calcium
concentration gradient Neutralization of acidic effects which
↑ Mineralization inflammatory pH provide
optimal
Denaturation of
healing
↑ Concentration of Ca ++ proinflammatory
environment
and Ca ++ dependent cytokines
pyrophosphatase

↓ Capillary OH’
permeability Ca++ Ca(OH)2 (↑pH)

Provision of stable mechanical support by forming a Coagulation Dissolution of

dynamic zone of microcrystals for adsorption of necrosis dentine ECM
biomolecules & attachment of secretory cells

↑ Fibronectin ↑ BMP Release of ↑ BMP Activation ↑ ALP

synthesis Synthesis Deposition of fibronectin sequestered Synthesis of TGF activity
NCPs

↑ Concentration of biomolecules in local environment

Recruitment, migration, proliferation, cytodifferentiation of secretory cells

TERTIARY DENTINOGENESIS

Figure 3 Hypothetical model of mechanisms by which Ca(OH)2 induces tertiary dentine formation. Release of bioactive mole-
cules, either through direct stimulation of cells or by solubilization of dentine extracellular matrix, is vital for biological effects
of Ca(OH)2. Calcium as well as hydroxyl ions released from the material regulate the events leading to tertiary dentinogenesis.
Ca(OH)2-containing cement, together with microcrystals deposited on its surface, provides a biologically active substrate for
adsorption of biomolecules and adhesion of odontoblasts. In addition, non-specific, antimicrobial and anti-inflammatory effects
of Ca(OH)2 on dental pulp may succour the process of mineralization.

Baik JE, Jang KS, Kang SS et al. (2011) Calcium hydroxide

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