doi:10.1111/j.1365-2591.2011.01995.

BiodentineTM induces TGF-b1 release from human

pulp cells and early dental pulp mineralization

P. Laurent1,2, J. Camps1 & I. About1,2

1
Laboratoire Interface Matrice Extracellulaire-Biomateriaux (IMEB), Faculté D’Odontologie, Université de la Méditerranée; and
2
Institut des Sciences du Mouvement UMR 6233, Université de la Méditerranée et CNRS, Marseille, France

Abstract medium, while injured cells TGF-b1 level was used as

the baseline value.
Laurent P, Camps J, About I. Biodentine induces TGF-b1
Results Biodentine induced mineralized foci forma-
release from human pulp cells and early dental pulp mineral-
tion early after its application. The mineralization
ization. International Endodontic Journal, 45, 439–448, 2012.
appeared under the form of osteodentine and expressed
Aim To assess the ability of a recently developed markers of odontoblasts. Biodentine significantly
tricalcium silicate-based cement (Biodentine) to increased TGF-b1 secretion from pulp cells (P < 0.03)
induce reparative dentine synthesis and to investi- independently of the contact surface increase. This
gate its capacity to modulate pulp cells TGF-b1 increase was also observed with calcium hydroxide and
secretion. MTA, but not with the resinous XenoIII. The statis-
Methodology Biodentine was directly applied tical analysis showed statistically significant differences
onto the dental pulp in an entire human tooth culture between capping materials and the resinous XenoIII
model. After various culture periods, the interaction of (P < 0.001).
the material with dental pulp tissue was analysed on Conclusions When Biodentine was applied di-
tissue sections. The effect of increasing surface area of rectly onto the pulp, it induced an early form of
this material on TGF-b1 secretion was investigated on reparative dentine synthesis, probably due to a modu-
pulp cell cultures and compared with that of MTA, lation of pulp cell TGF-b1 secretion.
calcium hydroxide and XenoIII adhesive resin. After
Keywords: Biodentine, odontoblast, pulp, reparative
performing artificial injuries on pulp cell cultures, the
dentin, TGF-b1.
materials eluates were added for 24 h and then TGF-b1
secretion was quantified by ELISA. Controls were Received 30 July 2011; accepted 23 November 2011
performed by incubating intact cells with the culture

its biocompatibility and its ability to prevent bacterial

Introduction
microleakage; the outcome also depends on the pulp’s
Maintaining pulp health following carious, traumatic ability to respond to injury (Camps et al. 2000).
or iatrogenic injuries remains a challenge and is of Portland cements such as ProRootMTA (Dentsply
prime importance particularly in immature permanent Tulsa Dental, Johnson City, TN, USA) have been
teeth where pulp vitality allows completion of root developed and used for pulp capping. Recent data
formation. suggest that this biomaterial stimulates reparative
Several biomaterials are used in vital pulp therapies, dentine formation faster than calcium hydroxide
with the prognosis depending on several factors such as cements (Accorinte et al. 2008a) and provokes less
pulpal inflammation (Sawicki et al. 2008). The miner-
alized barrier is thicker with MTA than with Dycal
Correspondence: Imad About, Directeur, Laboratoire Interface and shows fewer tunnel defects (Nair et al. 2008). The
Matrice, Extracellulaire-Biomatériaux (IMEB), Faculté d’Odon-
tologie, Université de la Méditerranée, 27 BD Jean Moulin,
recent direct application onto the pulp of both calcium
13385 Marseille Cedex 5, France (Tel.: 4 86 13 68 59; fax: hydroxide and ProRootMTA in entire human
4 86 13 68 40; e-mail: [email protected]). tooth culture model confirmed their potential in

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 439–448, 2012 439
 Biodentine induces mineralisation and TGF-b1 release Laurent et al.

inducing reparative dentine secretion (Tecles et al. It has been hypothesized that bioactive materials
2008). such as calcium hydroxide, MTA and Biodentine
Based on the outstanding biological properties of might locally increase TGF-b1 secretion from the
Portland cements, a new calcium silicate-based cement injured pulp tissue; this might partially explain their
called Biodentine (Septodont, Saint-Maur-des-Fosses, stimulating effect on dentine–pulp complex regenera-
France) has been developed recently. The powder is tion.
mainly composed of tricalcium silicate, calcium car- Thus, the objectives of this study were (i) to
bonate and zirconium oxide. The liquid contains water, determine whether Biodentine induces the reparative
calcium chloride (used as a setting accelerator) and a dentine synthesis in pulp capping situation on entire
modified polycarboxylate (a superplasticising agent). A human teeth culture as reported earlier with calcium
single dose of liquid is dropped in a disposable cap hydroxide and MTA (Tecles et al. 2008) and (ii) to
containing the powder and then mixed with an investigate the capacity of several commonly used
amalgamator for 30 s. The cement can be applied biomaterials including Biodentine to modulate TGF-
directly in the restorative cavity with a spatula and a b1 secretion by pulp cells.
plugger as a bulk dentine substitute without any
conditioning pre-treatment. With improved physical
Materials and methods
properties (Goldberg et al. 2009, Villat et al. 2010) and
reduced setting time to 12 min (Goldberg et al. 2009)
Direct pulp capping with Biodentine using a
as compared to Portland cements, the new biomaterial
human entire tooth culture model
can be used as dentine substitute in several clinical
indications. Investigating its interactions with the pulp Tooth culture
cells demonstrated its biocompatibility and its ability to The detailed procedure of this protocol has been
induce odontoblast differentiation and mineralization described previously (Tecles et al. 2008). Briefly, 15
in cultured pulp cells (Laurent et al. 2008). However, human immature third molars extracted for orthodon-
its capacity to promote reparative dentine remains to be tic reasons were collected in agreement with French
demonstrated in direct pulp capping situations. legislation (informed patients and parents’ consent and
Interactions between pulp capping materials and Institutional Review Board approval of the protocol
injured pulp tissue in the initiation and the development used) and stored for 2 h at 4 C in a Dulbecco’s
of wound healing and regenerative processes remain Modified Eagle medium (DMEM) (Lonza, Vervier, Bel-
unclear. Many hypotheses have been evoked, but recent gium) supplemented with 300 UI mL)1 penicillin,
studies highlighted the role of growth factors in angio- 300 lg mL)1 streptomycin and 0.75 lg mL)1 ampho-
genesis, recruitment of progenitor cells, cell differentia- tericin B (Lonza). A cavity with pulp exposure was
tion and finally mineralization in the pulp area beneath performed on each tooth (Fig. 1a,b). Biodentine (Lot
the biomaterial. Amongst these bioactive molecules, no. 48770) was prepared by squeezing out the liquid of
TGF-b1 is known to be involved as a key factor. Indeed, a single-dose container into the powder-containing
calcium hydroxide has been shown to solubilize bioac- capsule. The capsule was then placed in a mixing
tive molecules such as TGF-b1 from the dentine to signal device and mixed for 30 s at 4000 rpm. The cavity was
reparative dentinogenesis (Graham et al. 2006). Simi- dried with a sterile cotton pellet, and Biodentine was
larly, TGF-b1 release was obtained from powdered applied as a direct pulp capping material without any
human dentine with MTA (Tomson et al. 2007). How- conditioning treatment of enamel/dentine (Fig. 1c). A
ever, these materials are often applied directly onto the sterile metallic wire was sealed on the crown with a
pulp, and it is still unclear whether they affect the little drop of EmbraceTM photopolymerized resin (Pulp-
secretion of this growth factor from pulp cells. dent Company; Watertown, MA, USA). The roots of the
In a previous study, an increase of growth factors treated teeth were suspended into DMEM supplemented
secretion after physical injury to pulp cells was dem- with 10% foetal bovine serum (Lonza), 200 UI mL)1
onstrated (Tran-Hung et al. 2008). The presence of a penicillin, 200 lg mL)1 streptomycin and 0.5 lg mL)1
toxic adhesive resin component (HEMA) decreased the amphotericin B in 12-well cell culture plates (Falcon;
factors secreted by injured pulp cells in culture, and this Becton Dickinson, Franklin Lakes, NJ, USA) (Fig. 1d).
could explain their inhibitory role in the early steps of The culture medium was changed every day. The
pulp wound healing and dentine regeneration (Tran- cultured teeth were incubated for 2 days (n = 5),
Hung et al. 2008). 14 days (n = 5) or 28 days (n = 5).

440 International Endodontic Journal, 45, 439–448, 2012 ª 2011 International Endodontic Journal
 Laurent et al. Biodentine induces mineralisation and TGF-b1 release

(a) (b)

(c) (d)

Figure 1 Direct Pulp capping with Biodentine on entire tooth culture. A cavity was performed ex vivo with a truncated diamond
bur mounted on a high-speed hand-piece and under sterile saline cooling (a) until the pulp exposure was obtained (b). The cavity
was gently dried and immediately restored with Biodentine (c). A metallic wire was used to suspend the teeth in the culture dish
with their roots dipped into the culture medium (d).

Histology following concentrations: anti-collagen I at

At the end of each culture period, the teeth were fixed 40 lg mL)1, anti-osteonectin at 10 lg mL)1, anti-den-
in 4% formalin solution, demineralised, paraffin embed- tine sialoprotein at 1/200 and anti-nestin antibodies at
ded and routinely processed as described previously 5 lg mL)1. The staining was revealed using the labelled
(Tecles et al. 2005). Five slides per tooth were stained streptavidin–biotin kit (DakoCytomation, Carpinteria,
with haematoxylin and eosin. CA, USA) according to the manufacturer’s instructions.
Controls were performed by omitting primary antibodies
Immunohistochemistry or incubations with unrelated primary antibodies
To assess pulp cell differentiation and newly formed (Cytokeratin 19). All controls gave negative results.
reparative dentine, odontoblast and matrix dentine
molecular markers were investigated. This was per-
TGF-b1 secretion by pulp cells in contact with
formed on rehydrated paraffin-embedded sections with
Biodentine eluates
antibodies against collagen I (Southern Biotechnology
Associates Inc., Birmingham, AL, USA), osteonectin Pulp cell culture
(Takara Shuzo Co. Ltd, Shigo, Japan), dentine sialopro- Human dental pulp cells were cultured as described
tein (Santa Cruz Biotechnology Inc., Santa Cruz, CA, previously (Tran-Hung et al. 2008). Briefly, immature
USA) and nestin (Chemicon International, Temecula, third molars were obtained from 16-year-old adoles-
CA, USA). Primary antibodies were diluted in Dulbecco’s cents in compliance with French legislation (informed
phosphate-buffered saline (Lonza) containing 0.1% patients’ and parents’ consent, and Institutional Re-
bovine serum albumin. The incubation with primary view Board approval of the protocol used). After
antibodies was performed overnight at 4 C at the extraction, the teeth were washed and the apical

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 439–448, 2011 441
 Biodentine induces mineralisation and TGF-b1 release Laurent et al.

portions removed. The extirpated dental pulp was • Each experiment was carried out on cultures from
minced, and explants were cultured in 100-mm-diam- three different subjects.
eter culture dishes (Falcon; Becton Dickinson) con-
taining DMEM supplemented with 10% foetal bovine Sandwich enzyme-linked immunosorbent assay (ELISA)
serum, 100 UI mL)1 penicillin, 100 lg mL)1 strepto- ELISA was performed to quantify transforming growth
mycin and 0.25 lg mL)1 amphotericin B. factor-beta1 (TGF-b1) in the cell culture supernatants
Confluent cells from 3rd passage were collected by (Quantikine Cytokine ELISA kit; R&D system, Lille,
trypsination and subcultured in 35-mm-diameter cul- France).
ture dishes (Falcon; Becton Dickinson, Plymouth, UK) Prior to running the assays, samples were treated to
for 24 h. The cell density was 30 000/cm2. activate latent TGF-b1 to the immunoreactive form.
This was achieved by acid activation with 1N HCl and
Conditioned media neutralization with 1.2N NaOH/0.5 mol L)1 HEPES.
All the materials were prepared according manufac- A 96-well polystyrene microplate coated with a
turer’s instructions. White ProRootMTA powder was monoclonal antibody specific for TGF-b1 is provided
mixed for 30 s with sterile water. Biodentine (Lot no with this kit. Standards, controls and activated samples
48770) was prepared as described in the tooth culture were added to the wells and incubated for 2 h at room
section. Calcium Hydroxide XR (Dentsply, Montigny-le- temperature. After washing away any unbound mate-
Bretonneux, France) was a ready-to-use paste. All rial, a horseradish peroxidase–conjugate polyclonal
materials were then put directly in sterile calibrated antibody to TGF-b1 was added to each well and then
silicone moulds. Four drops of each components of the incubated for 2 h at room. After washing, a freshly
self-etching adhesive XenoIII (Dentsply De Trey prepared substrate solution was added to each well and
GmbH, Konstanz, Germany) were mixed before place- then incubated for 20 min in the dark at room
ment in silicone moulds and photo-polymerized in the temperature. The colour development was stopped
absence of oxygen for 1 min. Both materials were then with a diluted hydrochloric acid solution, and the
stored in an incubator at 37 C for 6 h to achieve optical density of each well was determined within
complete setting. Each sample was finally removed 30 min using a microplate reader (Metertech Inc.,
from the moulds and sterilized using UV rays for Taipei, Taiwan) at 450 nm.
15 min. A standard curve was generated for each experiment
Samples of set Biodentine were then incubated in and was used to calculate the TGF-b1 concentration
the culture medium (DMEM supplemented with (pg mL)1).
200 UI mL)1 penicillin, 200 lg mL)1 streptomycin
and 0.5 lg mL)1 amphotericin B) for 24 h. Increasing
Statistical analysis
surface areas were used so that the contact surface
between the material and the culture media volume A Kruskall and Wallis test was performed to compare
was 0.05, 0.5, 5 and 50 mm2 mL)1. Similarly, samples the effect of the presence of Biodentine and increasing
of ProRootMTA, Calcium Hydroxyde XR and XenoIII its surface area on TGF-b1 secretion. Mann and
were also incubated in culture media under similar Whitney U test was performed to compare the effect
conditions at 0.05 mm2 mL)1. of the materials with the negative control and with
each other. The confidence level was set at 95%.
Contact between pulp cells and conditioned medium
Injuries to pulp cells were performed with a sterile
Results
scalpel to disrupt the cell monolayer in 35-mm culture
dishes. Ten straight lines per dishes were performed
Direct pulp capping with Biodentine
immediately after the culture medium removal.
• Injured cells were then incubated for 24 h with Haematoxylin and eosin staining revealed early and
2 mL of each conditioned medium (n = 3). dense mineralized foci formation in the pulp connective
• Injured cells incubated with the unconditioned tissue after culture for 2 days just beneath the capping
medium were used as baseline values (n = 3). material (Fig. 2a,b). These foci were small, dense and
• Intact cells (without lesions) were also incubated amorphous and were observed in 2/5 teeth. After 14
with unconditioned medium and used as the and 28 days, these spherical foci appeared in all teeth,
negative (C-) control (n = 3). increased in size and were more numerous. Small

442 International Endodontic Journal, 45, 439–448, 2012 ª 2011 International Endodontic Journal
 Laurent et al. Biodentine induces mineralisation and TGF-b1 release

(a) (b)

(c) (d)

Figure 2 Pulp mineralization after cap-

ping with Biodentine. Mineralized foci
(arrowheads) appeared just beneath the
material in the pulp wound area after
2 days (a,b). Their number increased
(e) (f)
after 14 days and took the appearance
of osteodentine (c,d). Some particles of
the material (arrows) appeared en-
trapped into the matrix of the foci (b,d).
Collagen I (e) and osteonectin (f) are
expressed in the mineralized matrix and
the sequestered cells. a–d: H&E staining.
D, dentine; P, pulp; M, biomaterial. Scale
bars: a,e = 100 lm; b,d,f = 50 lm;
c = 500 lm.

irregular cavities were observed in the foci matrix; control, Biodentine significantly increased TGF-b1
some of them contained sequestered cells showing an secretion by pulp cells (P < 0.03). The differences in
osteodentine aspect (Fig. 2c,d). Interestingly, particles TGF-b1 secretion level were not statistically significant
of cement were visible inside the foci, but not in the (ns) amongst the surface areas used (Fig. 4a). It can be
surrounding connective tissue (Fig. 2b,d). concluded that Biodentine increases TGF-b1 secre-
Immunohistochemistry revealed expression of type I tion by pulp cells and that the increase is not dependent
collagen (Fig. 2e) and osteonectin (Fig. 2f). Dentine of the exposed contact surface area between Bioden-
sialoprotein was strongly expressed in the mineralized tine and the injured pulp.
matrix and sequestered cells (Fig. 3a,b). An intense To compare the effect of Biodentine with other
expression of nestin within the sequestered cells in the capping materials, the effect of conditioned media
foci was also observed (Fig. 3c). No labelling was prepared under identical conditions with injured pulp
observed with the negative control (Fig. 3d). cells were investigated (Fig. 4b). When compared to the
negative control, both Biodentine and MTA signifi-
cantly increased TGF-b1 secretion (P < 0.001) and
TGF-b1 secretion by pulp cells
when Biodentine was compared with MTA, the
Intact pulp cells were found to secrete TGF-b1 difference was not significant. Calcium hydroxide
(374 pg mL)1, mean value). This secretion level was slightly increased TGF-b1 secretion, whilst XENOIII
used as the negative control (C-). Artificial injuries did decreased its secretion, but these modifications were
not modify this TGF-b1 secretion level (400 pg mL)1, not statistically significant (ns). When TGF-b1 level
mean value). When compared with the negative with Biodentine, MTA and calcium hydroxide was

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 439–448, 2011 443
 Biodentine induces mineralisation and TGF-b1 release Laurent et al.

(a) (b)

Figure 3 Expression of odontoblast

(c) (d)
markers. Numerous dense foci (arrow-
heads) can be viewed beneath the bio-
material after H&E staining (a). Dentine
sialoprotein (b) and nestin (c) are ex-
pressed in the mineralized foci. No label-
ling is observed when primary antibodies
were replaced with unrelated (Cytokera-
tin 19) ones (d). D, dentine; P, pulp; M,
biomaterial. Scale bars: a = 500 lm;
b,c,d = 100 lm.

compared with that with XenoIII, the difference was obtained after 2 months in human teeth (Aeinehchi
statistically significant (P < 0.001). et al. 2003). Thus, the mineralization obtained in this
work corresponds to an early step of reparative dentine
synthesis reported in vivo. It appears after an equivalent
Discussion
delay and precedes tubular dentine bridge formation.
The results demonstrate that direct pulp capping with Immunohistochemistry revealed that the molecular
Biodentine using an ex vivo human tooth culture markers of dentine were expressed in the mineralized
model gives a similar response to that observed with foci matrix. Moreover, molecular markers of the
MTA and calcium hydroxide. All three materials odontoblasts such as type I collagen, osteonectin,
induced odontoblast-like cell differentiation and miner- dentine sialoprotein and nestin were expressed in the
alization, and this effect might be because of an sequestered cells. Dentine sialoprotein and nestin
increase of TGF-b1 secretion by pulp cells. expression in the sequestered cells is very significant.
The mineralization obtained was observed after Dentine sialoprotein is widely expressed at 14 days.
2 days of culture and was numerous and of various This noncollagenous protein is considered as a specific
sizes at 14 and 28 days. They appeared under the form marker of dentine even if it is expressed at a lower level
of mineralized foci with a morphological appearance of in bone tissue (Qin et al. 2002). Its role in dentine
osteodentine. formation is essential as it initiates and regulates the
This early form of mineralization has already been dentine mineralization (Suzuki et al. 2009). Nestin is
observed after MTA and calcium hydroxide application an intermediate filament protein that has been shown
using the same entire tooth culture model (Tecles et al. to be specific and characteristic of the secretory human
2008). Similar mineralized foci organization were odontoblast (About et al. 2000). Thus, the expression
observed beneath an incomplete dentine bridge in of these proteins in the sequestered cells strongly
human teeth capped with MTA after 1 month (Nair suggests that these cells are odontoblastic and brings
et al. 2008) and during the early phase of dentine a confirmation that the mineralized foci seem to
bridge formation with calcium hydroxide (Schröder correspond to a form of early reparative dentine
1985). Mineralization foci were also observed in vivo production (Tecles et al. 2008). This reparative dentine
after pulp capping with MTA for 2 weeks in dogs where synthesis is directly related to a disruption of the
irregular osteotypic matrix depositions were observed odontoblastic layer, and the subsequent pulp healing
(Tziafas et al. 2002), whilst the reparative tubular requires the recruitment and differentiation of pulp
dentine bridge formation with this material was progenitor cells to protect the underlying pulp tissue

444 International Endodontic Journal, 45, 439–448, 2012 ª 2011 International Endodontic Journal
 Laurent et al. Biodentine induces mineralisation and TGF-b1 release

Figure 4 Effect of Biodentine and other biomaterials on TGF-b1 release from pulp cells. (a) Injuries to pulp cells were performed
with sterile scalpels. The injured cells were cultured with conditioned media obtained after incubation with increasing
Biodentine surfaces. After 24 h, ELISA was performed to quantify the secreted TGF-b1. A statistically significant difference in
TGF-b1 level was obtained when pulp cells were incubated with all Biodentine eluates (P < 0.03). No statistical difference was
observed upon increasing the Biodentine contact surface. (b) Comparison of Biodentine effect on TGF-b1 secretion with that of
other biomaterials (0.05 mm2 mL)1). Biodentine and ProRootMTA significantly increased TGF-b1 secretion level as compared
to the negative control (P < 0.001). There was a statistically significant difference between Biodentine, MTA and calcium
hydroxide when compared to XenoIII (P < 0.001). All the results were normalized and expressed as percentage of TGF-b1
concentration in injured cells, but without any contact with the conditioned media.

(Fitzgerald 1979). Previous work has shown that This is because of the fact that the ex vivo model used
perivascular progenitor pulp cells can be activated here has some drawbacks such as a culture period
and migrate to the injury site after pulpal injury in limited to 1 month, absence of noxious components
human teeth (Tecles et al. 2005). This work provides clearance, absence of circulation and a limited inflam-
further evidence that, in entire human tooth cultures, matory reaction. However, it allows investigating the
these cells can differentiate into odontoblast-like cells early steps of dentine regeneration in a whole-tooth
and secrete a form of reparative dentine after capping environment. It also allows prediction of dental pulp
with Biodentine. This form of mineralization was not cells behaviour after application of restorative materi-
the typical tubular one usually observed after longer als, thus reducing the use of animal experiments before
delays. studies on human beings.

ª 2011 International Endodontic Journal International Endodontic Journal, 45, 439–448, 2011 445
 Biodentine induces mineralisation and TGF-b1 release Laurent et al.

Surprisingly, particles of Biodentine were en- tine eluates (P < 0.03) whatever the surface contact
trapped in the newly formed foci, but not in the between the material and the culture medium. The
surrounding pulp tissue. Similar particles were observed clinical relevance of this result is that whatever the size
after pulp capping with calcium hydroxide and self- of the pulp exposure, its direct capping with Bioden-
etching adhesive resin, but these particles were tine induces a significant increase of TGF-b1 secretion.
entrapped into macrophages in the vicinity of the To better understand the modulatory effect of pulp
capping materials (Kitasako et al. 2006). It is important capping materials on this growth factor, the effect of
to note that the particles of Biodentine seemed to be Biodentine on TGF-b1 secretion was compared with
completely integrated in the newly formed mineralized that of other direct pulp capping materials and to an
structures, suggesting that their physicochemical prop- adhesive resin. Interestingly, all direct pulp capping
erties might promote the mineralization process as materials induced an increase in TGF-b1 secretion as
shown with MTA-based cements (Gandolfi et al. 2010). compared to the baseline values. This increase was
To further investigate the interactions between statistically significant only with Biodentine and
Biodentine and injured pulp tissue, it was hypothe- ProRootMTA (P < 0.01). When the results obtained
sized that TGF-b1 concentration could increase locally, with Biodentine, ProRootMTA or calcium hydrox-
and thus contribute to odontoblast-like cells differenti- ide were compared with those obtained with XenoIII,
ation and mineralization. The role of this growth factor there was a significant difference between the three
has been widely studied in pathological simulation mineral-based materials and XenoIII (P < 0.001).
implicating reactionary dentine (Smith et al. 2001), but This might explain, at least in part, the successful
its functions and mechanism of action in direct pulp therapeutic use of pulp capping materials such as
capping situation, when native odontoblast layer is Biodentine, ProRootMTA or calcium hydroxide, and
destroyed, remain unclear. the fact that adhesive resins such as XenoIII are not
The hypothesis of this study was based on the fact that recommended for this purpose.
it has been well demonstrated that TGF-b1 acts as a The decrease in TGF-b1 secretion obtained with
modulator of many reparative processes in various XenoIII is in agreement with previous long-term
tissues. In the dental pulp, TGF-b1 promotes progenitor human clinical pulp capping studies with adhesive
cell migration (Howard et al. 2010) and odontoblast resins where no reparative dentine formation has been
differentiation (Begue-Kirn et al. 1992). Additionally, observed (Gwinnett & Tay 1998, Hebling et al. 1999,
previous work has shown that dentine matrix contains Pereira et al. 2000, de Souza Costa et al. 2001,
sequestered TGF-b1 (Finkelman et al. 1990) and other Accorinte et al. 2008b, Fernandes et al. 2008). Several
bioactive molecules including angiogenic growth factors reasons were used to explain this failure: difficulties to
(Roberts-Clark & Smith 2000). During dentine demin- obtain blood clot formation, no antibacterial activity,
eralization through carious lesions or acidic etchant incomplete polymerization and cell toxicity with adhe-
application, these growth factors are solubilized and can sive resin. The present study revealed another putative
be released to the pulp tissue where they could mediate reason: the inhibition of TGF-b1 secretion by pulp cells
dentine regeneration processes. Additionally, pulp cap- subjected to these resinous materials.
ping materials such as MTA (Tomson et al. 2007) and The effect of TGF-b1 on pulp cells has been studied in
calcium hydroxide (Graham et al. 2006) have been vitro. The results obtained using an organ culture where
shown to solubilize TGF-b1 from dentine. However, after dental pulp cells treated with TGF-b1 differentiated into
surgical pulp amputation and application of capping odontoblast-like cells expressing dentine relative pro-
materials directly onto the pulp, TGF-b1 release from teins such as dentine sialophophoprotein and dentine
dentine dissolution cannot explain the observed odon- matrix protein-1 and formed a pulp–dentine complex
toblastic differentiation and mineralization in the whilst when the dental pulp cells were incubated with
injured pulp tissue just beneath the applied material. TGF-b1, they formed mineralization nodules (Nie et al.
With this regard, the study revealed that cultured 2006). These observations might explain the indirect
human pulp cells secrete TGF-b1. After artificial injury, effects of ProRootMTA and Biodentine on the
these cells continued to secrete TGF-b1 almost at the increase in TGF-b1 secretion and consequently odonto-
same level, indicating that the mechanical stress did not blast differentiation and mineralization.
seem to affect this growth factor secretion by the Whilst the signalling processes involved in this
surrounding cells. However, TGF-b1 secretion signifi- odontoblast-like cell differentiation under these materials
cantly increased after incubating the cells with Bioden- are still not completely elucidated, a recently developed

446 International Endodontic Journal, 45, 439–448, 2012 ª 2011 International Endodontic Journal
 Laurent et al. Biodentine induces mineralisation and TGF-b1 release

calcium silicate-based bone cement with similar com- l’enseignement supérieur et de la recherche’ and by a
position to that of Biodentine has been investigated in partial support from Septodont. The authors thank Dr
simulated body fluid conditions. Scanning electron Jean-Charles Gardon for providing the third molars
microscopy and X-ray diffraction showed that calcium used in this work.
silicate-based cement stimulated cell growth and [Correction added after online publication, 23 January
induced hydroxyapatite formation on the surface of 2012: Grant information added to Acknowledgements
the material (Zhao et al. 2005). It has been suggested (Septodont).]
that alterations in calcium levels in the cellular
environment might invoke odontoblast-like cells differ-
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