Professional Documents
Culture Documents
Bio Dentine
Bio Dentine
Biodentine
(RD94)
Publications and
Communications
2005 - 2009
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Nov. 2009
TITLE YEAR AUTHORS REFERENCE
RD94 In Indirect Pulp-Capping Situation Induces Reactionary
Dentin Formation.
2009
T.Boukpessi, F.Decup, D.Septier, C. Chaussain and
M. Goldberg - France
IADR-CED congress in Munich, Germany, 9-
12 September 2009
Ciments alcalins ou acides usage odontologique : action sur
quelques souches bactriennes reprsentatives
2009
E.Valyi, P.Colon, F.Bornand, D.Decoret,
B.Grosgogeat, France
Abstract - SFBD (Socit Francophone des
biomatriaux dentaires). 25-26 juin 2009.
Biocompatibility or cytotoxic effects of dental composites -
Chapter VI Emerging trends in (bio)material research
2009
Goldberg M, Pradelle-Plasse N, Tran XV, Colon P,
Laurent P, Aubut V, About I, Boukpessi T, Septier D
Working group of ORE FDI -edited by
Michael Goldberg
A clinical study of a new Ca3SiO5-based material indicated as
a dentine substitute
2009
G.Weissrock, J.C. Franquin, P. Colon , G.Koubi
University of Paris 7, France
Journe Scientifique du CNEOC Brest -June
2009
Biodentine
TM
- RD94, A portland cement, stimulates in vivo
reactionary dentin formation
2009
T.Boukpessi, F.Decup, D.Septier, M. Goldberg, C.
Chaussain, France
Journe Scientifique du CNEOC Brest -June
2009
A clinical study of a new Ca3SiO5-based material indicated as
a dentine substitute
2009 G.Koubi, J.C. Franquin, P. Colon.
Abstract in Clin. Oral Invest 2009 + poster
Conseuro 2009 (Seville, Spain March 12-14th
2009)
Induction of specific cell responses to a Ca
3
SiO
5
-based
posterior restorative material
2008
Laurent P, Camps J, De Mo M, Djou J, About I.
Marseille, France.
Dent Mater. 2008 Nov;24(11):1486-94.
Microleakage of a new restorative calcium based cement
(Biodentin)
2008 Tran V, Pradelle N, Colon P
Oral presentation PEF IADR Sept 2008
London
RD 94, a Portland cement, stimulates in vivo reactionary
dentine formation
2008
Boukpessi T, Septier D, Decup F, Chaussain-Miller C,
Goldberg
Oral presentation PEF IADR Sept 2008
London
Evaluation of adhesion between composite resins and an
experimental mineral restorative material
2007
C. BOINON, MJ. BOTTERO-CORNILLAC, G. KOUBI
and J. DEJOU
Abstract :European Cells and Materials Vol.
13. Suppl.1
A clinical study of a new Ca3Si05-based material for direct
posterior fillings
2007
S. KOUBI, H.TASSERY, G.ABOUDHARAM, J.L
VICTOR, G. KOUBI
abstract : European Cells and Materials Vol.
13. Suppl.1
Cytotoxicity and genotoxicity of a new material for direct
posterior fillings.
2005
I. ABOUT, A RASKIN, *M. DE MEO, J.DEJOU -
Marseille, France
abstract : European Cells and Materials Vol.
10. Suppl. 4, 2005 (page 23)
Physical, chemical and mechanical behavior of a new material
for direct posterior fillings.
2005
J. DEJOU, J COLOMBANI and I. ABOUT. Marseille,
France
abstract : European Cells and Materials Vol.
10. Suppl. 4, 2005 (page 22)
PUBLICATIONS AND COMMUNICATIONS ON BIODENTINE
TM
- RD94 - SEPTODONT
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4/74
RD94 In Indirect Pulp-Capping Situation Induces Reactionary
Dentin Formation.
2009
T.Boukpessi, F.Decup, D.Septier, C. Chaussain and M.
Goldberg - France
IADR-CED congress in Munich, Germany, 9-12 September
2009
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Paper: RD94 I n I ndirect Pulp- Capping Sit uat ion I nduces React ionar y Dent in Format ion ( Joint Meet ing of t he Cont inent al European, I sraeli and Scandinavian Divisions of t he I ADR ( Sept ember
10- 12, 2009) )
St art | Browse by Day | Aut hor I ndex | Keyword I ndex
153 RD94 I n I ndirect Pulp- Capping Sit uat ion I nduces React ionar y Dent in Format ion
Locat ion: Libr ar y ( Gast eig Convent ion Cent er Mnchen)
T. BOUKPESSI , F. DECUP, D. SEPTI ER, C. CHAUSSAI N, and M. GOLDBERG, Universit y Paris Descart es- Dent al School- EA 2496, Mont rouge, France
RD94 a new experiment al Ca3SiO5- based rest orat ive cement int ends t o be a glass ionomer cement and composit e- resin subst it ut e in rest orat ive dent ist ry. Obj ect ives: t o
evaluat e in vivo t he biocompat ibilit y and bioact ivit y effect s of RD94 as assumed fr om t he format ion of react ionary dent in. Met hods: Using t he r at as an animal model,
half- moon cavit ies were prepared on t he mesial aspect of t he first maxillary molar wit hout pulp exposure. The cavit ies were t hen lef t unf illed ( sham group) or filled
eit her wit h a glass- ionomer cement ( cont r ol group) or wit h RD94 ( experiment al group) . The rat s were killed by perfusion t hrough t he hear t wit h t he fixat ive solut ion 8,
15, 30 days, and 3 mont hs aft er t he dent al t r eat ment . Block sect ions including t he t hree maxillary molars were demineralised and processed for light microscopy.
Measurement s were done on micrographs obt ained aft er hist ological observat ions. Result s: Aft er 8 days, a slight inflammat ory react ion was seen in each group. I n t he
RD94 group, a dent in layer of react ionary dent in st ar t s t o be for med, by cont r ast wit h t he 2 ot her groups. Aft er 15 days, a t endency of spont aneous repair was observed
in t he pulps of t he sham and cont rol groups. I n t he RD94 group, t he pulp near t he cavit y r et r act s, cover ed by a 40- 80 m t hick layer of react ionar y dent in. I n t he RD94
gr oup, af t er one mont h, t he mesial part of pulp was part ially filled wit h a homogenous dent in- like mat erial ( 160m) whereas t he rest of pulp appeared normal. Aft er
t hree mont hs, RD94 induced t he format ion of a homogenous react ionary dent in but t he t hickness of t his layer was unchanged bet ween 1 and 3 mont hs.
Conclusions: The present dat a 1- suggest t hat RD94 displays novel bioact ive propert ies. 2- This new cement st imulat es t he format ion of react ionar y dent in in t he rat
molar model short ly aft er a swit ch on, 3- but t here is act ually a swit ch off , keeping t he remaining pulp alive.
See mor e of: Dent al Mat erials 5
See mor e of: Scient ific Groups
< < Previous Abst ract | Next Abst r act > >
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Ciments alcalins ou acides usage odontologique : action sur
quelques souches bactriennes reprsentatives
2009
E.Valyi, P.Colon, F.Bornand, D.Decoret, B.Grosgogeat,
France
Abstract - SFBD (Socit Francophone des biomatriaux
dentaires). 25-26 juin 2009.
7/74
8/74
Biocompatibility or cytotoxic effects of dental composites -
Chapter VI Emerging trends in (bio) material research
2009
Goldberg M, Pradelle-Plasse N, Tran XV, Colon P, Laurent P,
Aubut V, About I, Boukpessi T, Septier D
Working group of ORE FDI -edited by Michael Goldberg
9/74
10/74
Chapter VI Emerging trends in (bio)material researches:
VI-1-Repair or regeneration, a short review
Michel Goldberg (Univ. Paris Descartes)
VI-2- An example of new material: preclinical multicentric studies on a new
Ca
3
SiO
5
-based dental material.
VI-2-1 Physico-chemical properties.
Nelly Pradelle-Plasse (University Paris 7 Denis Diderot & LGPM,
Ecole Centrale de Paris) France, Xuan-Vinh Tran (University of
Medicine and Pharmacy, Ho Chi Minh city, Vietnam),
& Pierre Colon (University Paris 7- Denis Diderot, & LGPM, Ecole
Centrale de Paris). France
VI-2-2 Biological properties
VI-2-2-1 In vitro studies Patrick Laurent, Virginie Aubut, Imad
About Laboratoire IMEB, Facult d'Odontologie, Universit de la Mditerrane, Marseille,
France
VI-2-2-2 In vivo studies Tchilalo Boukpessi, Dominique Septier &
Michel Goldberg (University Paris Descartes, France)
_________________________________________________________________________
VI- Emerging trends in (bio)material research
VI-1- Repair or regeneration, a short review.
Michel Goldberg (Univ. Paris Descartes)
Where do we come from? What are we? Where are we going? In a famous picture, the painter
Paul Gauguin raised this series of questions, and following the example of Oedipus and the
Sphinx, he gave some answers, valid or not.
Where do we come from?
Fifty years ago silver amalgam was the most common restorative material employed for
posterior teeth, and silicate cements were used for anterior teeth. The development of resin
composites with formulations more adapted to the clinical needs, new generations of
adhesives and the gradual reduction of the gap between the resins and dental tissues has led to
an increased use of resin-containing materials, even for molar restorations.
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Silver amalgam fulfills most of the general criteria that are required for a good restorative
material from a clinical point of view, taking into account both mechanical and biological
properties. It is generally recognized as safe for the patients and so far no adverse effect or
body burden have been identified, except some allergic effects detected in a limited number
of cases. More importantly, degradation in a wet environment provides cariostatic properties
that are due to the corrosion of the metal, a phenomenon inherent to the material.
However, despite its overall qualities, nowadays this material is gradually discarded from the
restorative procedures in dental practice for three main reasons.
-Firstly, the adhesive properties of resin composites allow better preservation of dental
tissues during the preparation of the cavity, reinforcing undercuts. After the opening and the
suppression of un-sustained enamel, followed by the cleaning of the lesion, the preparation of
the cavity is simplified. This procedure allows a smaller size of the cavities, and therefore
favoring a non-reversible evolution toward a minimal dentistry. This opens some gates for
new concepts and principles in prevention, namely by sealing occlusal pits and fissures with
fluoride releasing cements, and in restorative dentistry as well. Although the longevity of
dental restorations is shorter with resin fillings in comparison with metallic restorations, the
tissue economy is obviously better using adhesive materials compared with what was done
when the classical Blacks rules of preparations were applied.
-Secondly, tooth-colored resins are more esthetic or at least less visible than metallic
dental fillings.
-Thirdly, Hg is an agent implicated in soil and water pollution and from an ecological
point of view constitutes a potential danger for the environment. Most of the Hg comes from
the industry, and only a small part comes from the dental practice. Devices separating Hg
residues from the water of the dental unit allow eliminating a large part of the metal, but not
all. Decision to ban Hg from dental therapies was taken in Norway by the Ministry of
Environment and not by the Ministry of Health. Hg may also contribute to select bacteria that
are resistant to some antibiotics.
For these three reasons, the place occupied by silver amalgam filling is gradually reduced and
resin-containing materials are developed as amalgam substitutes. Altogether, resin-containing
restorative materials include composite resins, resin-modified glass ionomer cements and
adhesives. In the previous chapters, different reports have summarized our actual knowledge
both in terms of physico-chemical properties and biological adverse effects.
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What appears now from the literature (What are we? Or where are we?) is obviously the
presence of a large gap between in vitro and in vivo experimental approaches that provide
actual and catastrophic evidences for cell and tissue problems and clinical reports that
minimize the occurrence of public health problems. Along this line of evidences, we know
from laboratory studies that the conversion of resin monomers into inactive polymers is
incomplete, despite the absorption of monomers on the remaining dentin (Ferracane, 1994). It
is also well documented that free monomers are released from resin fillings when they are
exposed to occlusal wear and salivary enzymes, even long after the polymerization (Finer et
al., 2004). In vitro studies provide strong evidence that these monomers are toxic and
allergenic. In addition, they contribute to the development of secondary caries (Hansel et al.,
1998). Many questions arise and they are still a matter of discussion. Actually, it is still
difficult to link the large gap between in vitro data and clinical evaluations. The implicit
recognition of the potential occurrence of problems leads to undertake researches on new
fields: new materials and/or new approaches. Therefore the next question is: where we are
going?
Where are we going?
At the moment, three different tendencies orientate the researches upon investigation in many
laboratories. They pave the way for major improvements in the future focusing either on
repair (new materials, reactionary and reparative dentin), or using biological tools to
regenerate dental tissues.
With respect to repair, the first direction aims to improve resin-containing materials by
-1- Increasing the rate of polymerization of resins with the prospect of reducing or perhaps to
suppress the release of free monomers and consequently their potential noxious effects.
Researches aiming to improve the properties of resin-containing materials are carried out with
nanostructures, bio-mimetic and bio-inspired materials, and intelligent materials releasing
molecules. These later are acting as drugs reinforcing dental tissues and inhibiting bacteria.
-2- Another trend is oriented on the control of the shrinkage of the resin during the
polymerization phase. This would eliminate the formation of a gap, still in the order of 1m, a
width that is largely over the size of bacteria, the diameter of a lactobacillus being around
0.1m.
-3- Enzyme elimination of non-collagenic proteins known to be located in the interfibrillar
spaces or along the collagen fibrils, may contribute to the opening of these spaces, and
consequently to increase the penetration of the flow resin in the subsurface, a process that
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may correlate with the reduction of the gap, contributing to an increased adhesion. Catalytic
enzymes and metalloproteinases, provide potential tools.
Secondly, as another option, researches are carried out on some new formulations of cements
that do not contain any resin additive. Such materials are already present in the market. This is
the case for the Portland cements and other exclusively mineral-based materials that are
aiming to stimulate the formation of reactionary or reparative dentin.
The third direction is oriented on dental tissue regeneration, and is based on tissue
engineering. Embryonic or adult progenitor clones or stem cells have the capacity to
differentiate and to produce extracellular matrix (ECM) molecules that promote the formation
and mineralization of either reactionary or reparative dentin, depending the orientation
selected toward repair or regeneration of dental tissues. Some ECM molecules were also
shown recently to stimulate the commitment, recruitment, proliferation and differentiation of
pulp progenitors in a wounded tissue (Goldberg et al., 2008). Growth factors, transcription
factors and others biological molecules may also contribute to the pulp healing, and to the
formation of biological dentin-like materials either in endogenous (repair) or exogenous
(regeneration) sites. However, these promising experimental approaches need further pre-
clinical studies before to be transferred to the dental practice.
In this context a network of laboratories found some interest in collaborating, the only way to
handle nowadays multicentric researches. These groups decided to study the physico-
chemical and biological properties of a new Ca
3
SiO
3
-based posterior restorative cement. This
innovative material does not contain any resin and consequently avoid the danger of free
monomers release. From two clinical pilot studies that were carried out by two different
groups in Marseille and Paris Diderot, which are still in progress and therefore will not be
reported here, we know that such restorative material may be used successfully either as a
medium-term temporary filling, or as permanent base under resin-containing restorations or
inlays/onlays. This is indicative of the present evolution of materials in dentistry. We will
summarize in the chapter firstly some physico-chemical data (VI-2-1) and secondly the
biological aspects which may be deduced from in vitro and in vivo animal studies (VI-2-2).
References
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Ferracane JL. Elution of leachable components from composites. J Oral Rehabil 21: 441-452,
1994.
Finer Y, Jaffer F, Santerre JP. Mutual influence of cholesterol esterase and
pseudocholinesterase on the biodegradation of dental composites. Biomaterials 25: 1787-
1793, 2004.
Goldberg M, Farges J-C, Lacerda-Pinheiro S, Six N, Jegat N, Decup F, Septier D, Carrouel F,
Durand S, Chaussain-Miller C, DenBesten P, Veis A, Poliard A. Inflammatory and
immunological aspects of dental pulp repair Pharmacological Research
(doi :10.1016/j.phrs.2008.05.013).
Hansel C, Leyhausen G, Mai UE, Geurtsen W. Effects of various resin composite
(co)monomers and extracts on two caries-associated micro-organisms in vitro. J Dent Res
77:60-67, 1998.
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VI-2-1 Physico-chemical properties.
Nelly Pradelle-Plasse (University Paris 7 Denis Diderot & LGPM,
Ecole Centrale de Paris) France, Xuan-Vinh Tran (University of
Medicine and Pharmacy, Ho Chi Minh city, Vietnam), & Pierre Colon
(University Paris 7- Denis Diderot, & LGPM, Ecole Centrale de Paris).
France
Introduction
A new experimental Ca
3
SiO
5
-based restorative cement has been developed, put on the market
under the name of BIODENTINE
TM
(Septodont, Saint Maur des Fosses, France). As the
ProRoot MTA
(Torabinejad et al, 1995a,b; Camilleri et al, 2005) and Portlands cements
(Lea, 1970, Camilleri et al, 2006), it is a calcium-based cement. The main component of the
powder is a tricalcium silicate, with the addition to the powder of CaCO
3
and ZrO
2.
The liquid
is a solution of CaCl
2
with a water reducing agent. As every cement, the setting reaction leads
to a gel structure, which allows possible ionic exchanges. Compared to others Ca based
cements, this material presents two advantages: i) a faster setting time of about 12 minutes
and ii) higher mechanical properties. These physico-chemical properties associated with the
biological behavior (Laurent et al, 2008, and this book: sub-chapters VI-2-2) suggest that it
may be used as a permanent dentine substitute.
Chemistry and structure of the cement
Composition
BIODENTINE
TM
is conditioned in a capsule containing the good ratio of powder and liquid,
as shown in Table 1:
Powder :
Tricalcium silicate (3CaO.SiO
2
)
Calcium carbonate (CaCO
3
)
Zirconium dioxide (ZrO
2
)
Liquid
Calcium chloride (CaCl
2
.2H
2
O)
Water reducing agent
Water
Properties of the different components
- Tricalcium silicate (3CaO.SiO
2
): it is the main component of the powder. It regulates the
setting reaction.
- Calcium carbonate (CaCO
3
): it role is similar to the fillers
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- Zirconium dioxide (ZrO
2
): it is added to provide the radio-opacity to the cement
- Calcium chloride (CaCl
2
.2H
2
O): is an accelerator (Chessmann, 1999)
- Water reducing agent (Superplasticiser): It can reduce the viscosity of cement. It is based on
polycarboxylate but modified to obtain a high short-term resistance. It reduces the amount of
water required by the mix (water / cement), although maintaining the same easiness for
handling.
Setting reaction
The reaction of the powder with the liquid led to the setting and hardening of the
cement. The
hydration of the tricalcium silicate (3CaO.SiO
2
) leads to the formation of a hydrated calcium
silicate gel (CSH gel) and calcium hydroxide (Ca (OH)
2
) (Taylor, 1997). The cement located
in inter-grain areas has a high level of calcite (CaCO
3
) content.
The hydration of the tricalcium silicate is achieved by dissolution of tricalcium silicate and
precipitation of calcium silicate hydrate. In generally it is designated by chemist as C-S-H
(C=CaO, S=SiO
2
, H=H
2
O). The calcium hydroxide takes origin from the liquid phase. C-S-H
gel layers formation is obtained after nucleation and growth on the tricalcium silicate surface.
The unreacted tricalcium silicate grains are surrounded by layers of calcium silicate hydrated
gel, which are relatively impermeable to water, thereby slow down the effects of further
reactions. The C-S-H gel formation is due to the permanent hydration of the tricalcium
silicate, which gradually fills in the spaces between the tricalcium silicate grains (Figure 1).
The complete hydration reaction is summarized by the following formula (Taylor, 1997; Lea,
1970, Allen et al, 2007).
2(3CaO.SiO
2
) + 6H
2
O 3CaO.2SiO
2
.3H
2
O + 3Ca(OH)
2
Structure
The surface of the cement observed with the SEM one week after mixing is loaded by calcite
rich structures (CaCO
3
) of variable sizes (Figure 2). The calcite is a chemical or
biochemical mineral crystallizing in the rhombohedra system (a=b=c; o,|,=90). Crystals of
CaCO
3
diamond-shaped (or rhombohedra form) are observed at the surface. We also observed
crystals shaped as hexagonal plates of Ca(OH)
2
described by Taylor (1997) (Figure 3).
According to this author, calcium hydroxide crystallizes in the form of hexagonal plate or
prism. The surface of CaCO
3
crystals is rough and irregular.
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Therefore, we can consider the CSH gel as the matrix of the cement, and the crystals of
CaCO
3
are filling the spaces between grains of cement. Finally, the calcite (CaCO
3
) has two
distinct functions: 1- as an active agent it is implicated in the process of hydration and 2- as
fillers it improves the mechanical properties of the cement (Garrault et al., 2006).
The hardening process results from of the formation of crystals that are deposited in a
supersaturated solution. We can consider that the setting reaction of the 3CaO.SiO
2
includes
four elements: the unreacted particles of cement, surface products (CSH gel), the content of
the pores (Ca (OH)
2
) and porous capillary space (Figure 1).
The electrochemical properties of cement are due to the solid phase and ion mobility of free
ions inside the pores filled with the electrolyte (Andrale et al, 1999; Cabeza et al 2006).
Impedance spectroscopy is a technique that allows studying the process of hardening of a
cement. This is a non-destructive method that may monitor the hardening process. The
electrical resistance increases when the porosity of the system is reduced. Improvement of the
values measured for BIODENTINE
TM
is time-dependent (Figure 4). This shows that
immediately after mixing, the setting reaction of BIODENTINE
TM
is not yet achieved. At
least 2 weeks are necessary to reach a final stable stage. The setting reaction of
BIODENTINE
TM
leads to the formation of initial porosities that are gradually filled after
several days by new crystal compounds. During this final step, the solid phase is increasing
and finally reach a maximum.
Mechanical properties
Vickers microhardness
The hardness can be defined as the resistance to the plastic deformation of the surface of a
material after indentation or penetration. Measurements at different times have been evaluated
(Table II):
The hardness increases in time when cements are immersed in distilled water. After 2 hours,
the hardness of BIODENTINE
TM
is 51 HVN and reached 69 HVN after 1 month. These
values are comparable to those obtained with the resin modified GIC-Fuji II LC (36 HVN),
and the composite resin-Post Comp II LC (97 HVN) (William et al, 2002). The calcite is a
mineral compound in relation with the hardness of cement. The formation of CSH gel reduces
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the porosity with time. The crystallization of the latter continues, therefore improves the
hardness and probably other mechanical properties.
Flexural strength
The 3 points bending test has a clinical significance and is essential when the material is used
for Class I, II and IV cavities. The higher the resistance to flexural strength is, the lower is the
risk of cohesive fracture of the shutter and broken edges. The value of the bending obtained
with BIODENTINE
TM
after 2hours is 34 MPa. Compared with that of other materials: 10-20
MPa (conventional GIC), 40-70 MPa (GIC amended the resin), 120-200 MPa (composite
resin) (Davidson et al, 1999), it shows clearly that the bending resistance of BIODENTINE
TM
is superior to conventional GIC but still much lower than the composite resin.
Tooth BIODENTINE
TM
Adhesives Interfaces
Morphological characterization
The SEM microphotographies show BIODENTINE
TM
- dental structures interfaces (Figures
5-7) and BIODENTINE
TM
- adhesive systems interfaces (Figures 8, 9). The results show the
occurrence of a cohesive failure within the BIODENTINE
TM
cement without alteration of the
tooth biomaterial interface, hence providing evidence for the quality of the micromechanical
adhesion. The crack is an artefactual result of the drying process occurring during the SEM
preparation. The interfacial layer BIODENTINE
TM
- dentin may be compared to the hard
tissue layer shown to be formed when using ProRoot MTA, which is considered by several
authors as a dentinal bridge or a precipitation of hydroxyapatite (Holland et al, 1999, Santos
et al, 2005). We also observed that CaCO
3
crystals form after the end of the setting reaction.
This constitute a micromechanical anchorage with the surface of the dentine and the
precipitation inside dentine tubule provides mineral tag that may contribute to the cement
adhesive properties (Figure 7).
It appears that the mechanical adhesion of BIODENTINE
TM
cement to dental surfaces may
result from a physical process of crystal growth within dentine tubules leading to a
micromechanical anchor. The possible ion exchanges between the cement and dental tissues
constitute an alternative hypothesis, or the two processes may well combine, eventually
contributing to the adhesion of the cement, as it appears at the interface BIODENTINE
TM
-
adhesive systems (Figures 8, 9).
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Microleakage
The interfacial watertightness is an important parameter of the functionality and longevity of
a restoration. The phenomenon of percolation linked to defects or to the gap at the interface is
also designated by the term "microleakage". To evaluate this parameter, we have selected the
dye penetration methodology (silver nitrate), which is one of the most commonly used assays
to assess in vitro the interfacial seal by measuring the percolation of a dye along different
interfaces studied.
The result of penetration at the interface BIODENTINE
TM
- enamel / dentin was very low
(Table III).
A J0, the seal obtained with the Xeno
.
With time (after 3 months), the sealing ability of G-bond
Interface (SEM)
Cohesive fracture (B), cement (A), G bond
III (C).
C
A
B
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Time Micro hardness
2H 51.5 ( 1.75)
1D 63.14 ( 1.94)
7D 72.19 ( 6.38)
30D 69.46 ( 1.45)
Table II: Average of hardness (HVN) and standard deviation (into brackets)
Interfaces Dye penetration (%) at D0 Dye penetration (%) at D90
Enamel / BIODENTINE
TM
17.65 (4.35) 19.86 (10.72)
Dentin / BIODENTINE
TM
10.46 (3.23) 14.84 (5)
Table III: Tooth BIODENTINE
TM
INTERFACES
% of microleakage
Adhesive systems Dye penetration (%) at D0 Dye penetration (%) at D90
Xeno
C and pro-
cessed for immunohistochemistry. The effect of the materials
on the cytodifferentiation was evaluated by studying the spe-
cic protein expression of control cells compared to that of
cells cultured with the medium after being in contact with
the test material [20].
2.7.1. Immunohistochemistry
The cells were permeabilizedfor 15 minwith0.5%TritonX-100
in PBS. Primary antibodies were diluted in PBS containing 0.1%
Bovine Serum Albumin (BSA). The incubation with primary
antibodies was performed overnight at 4
C. Anti-collagen I
antibodies were used at 40 g/ml and anti-nestin antibody
at 5 g/ml. Anti-dentin sialoprotein antibody was diluted
1:200 in PBS. Immunostaining was revealed using the labeled
streptavidin-biotin kit (LSAB; Dako Corporation, Carpinteria,
CA, USA) according to the manufacturers instructions. Glyc-
ergel was used as a mounting medium (Dako Corporation).
Controls were performed by omitting primary antibodies or
incubationwithunrelatedprimary antibodies (cytokeratin19).
All controls were negative.
2.8. Genotoxicity assays
2.8.1. Ames test
S. typhimurium TA97a, TA98, TA100, and TA102 strains were
grown overnight from frozen cultures in Oxoid nutrient broth
No. 2 for 1012 h. Mutagenicity assays were performed as
described [21]. The genotype of each S. typhimurium tester
strain was conrmed in each experiment, and negative and
positive controls were routinely included.
After the preparation and setting of the cement, it was
ground to prepare a stock solution prior to testing by adding
60 mg of the cement in 1 ml of Nutrient Broth No. 2 (NB 2)
mediumor DMSOsolvent for 24 hat 37
C
56/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1489
for 48 h and revertant colonies were counted with an auto-
mated colony counter (Spiral System Instruments, Bethesda,
MS, USA). The experiments were carried out in the presence
and in the absence of an S9 fraction isolated from liver of
phenobarbital/-naphtoavone-treated rats. This S9 fraction
(4%) was routinely included in an S9-Mix, and the amount of
protein was adjusted to 1.25 mg protein per plate. A substance
was qualied positive if it induced a dose-related and repro-
ducible increase inthe numbers of revertants or twice as many
spontaneous revertants per plate [22].
2.9. Micronucleus test
This work was performed on lymphocytes obtained by
vein puncture from 6 healthy non-smoking donors, after
informed consent, and collected in glass tubes containing
lithium heparin anticoagulant according to Digue et al. [23].
Briey, cultures were carried out by adding 0.7 ml of whole
blood to 9.3 ml of X-VIVO
TM
Medium (Bio-Whittaker, Bel-
gium) supplemented with 25% fetal calf serum (Gibco, Life
Technologies
TM
, Germany), heparin (50 U/ml), and antibiotics
(penicillin 100 Ul/ml and streptomycin 100g/ml). The cells
were stimulated with phytohemagglutinin (1 mg/ml), a spe-
cic mitogen agent of human T-lymphocytes. The cells were
then cultured for 72 h at 37
C in a humidied atmosphere
containing 5% CO
2
.
The Ca
3
SiO
5
cement extract was prepared as described
above inthe culture mediumor DMSOandaddedtothe culture
at 24 h. The cells were directly exposed to serial dilutions (1%,
2.3%, 3.7%, and 5%) of the cement extracts for 48 h. Negative
control was achieved by adding DMSO at a nal concentration
of 0.1%. MitomycinC, used as a reference genotoxic agent, was
used as positive control 5g/ml. Cytochalasin B was added to
the culture (5g/ml) 44 h after PHA stimulation.
The cultures were stopped at 72 h and the cells harvested
by centrifuging (10 min at 360g). They were then treated
by a mild hypotonic treatment (1 min in KCl 0.075 M) and
immediately xed with methanol:acetic acid (3:1). This x-
ing step was repeated twice after 20-min storage at 4
C. Cells
were smeared on pre-cleaned microscope slides and air-dried.
Staining was performed with 5% Giemsa in Milli-Q water for
15 min.
Stainedslides were codedandscoredby light microscopy at
400 magnication. For each slide, 1000 Giemsa-stained bin-
ucleated lymphocytes with a well-preserved cytoplasm were
scored for the presence of micronuclei. In the micronucleated
binucleated cells, the number of MN per cell was recorded.
Micronuclei were expressed in terms of micronucleated cells
per 1000 binucleated lymphocytes. All the slides were exam-
ined twice by the same scorer. As a measure for toxicity, the
binuclearity index (BI) was determined by scoring the binu-
cleated cells for 1000 lymphocytes (mono- and binucleated
cells) and linked to the percentage of lymphocytes that pro-
duced complete cell division for the different drugs tested,
and then provided an index of cytotoxicity [24]. An extract
of a material was considered positive if at least a three-fold
increase of the numbers of micronuclei over negative controls
was observed at one or more dilutions of the original extract
[25,26].
2.10. Single-cell gel (Comet) assay
The Ca
3
SiO
5
cement extract was prepared and put in MEM
medium (60 mg/ml) for 24 h at 37
C, layered
onto a pre-coated slide with 1.5%regular agarose, and covered
with a coverslip. After brief agarose solidication in a refrig-
erator, the coverslip was removed and the slides immersed
in lysis solution (2.5 mol/l NaCl, 100 mmol/l EDTA, 10 mmol/l
TrisHCl buffer pH 10, 1% sodium sarcosinate with 1% Triton
X-100, and 10% DMSO) for about 1 h. Prior to electrophore-
sis, the slides were left in alkaline buffer (pH >13) for 20 min
and electrophoresed for another 20 min, at 25 V (0.86 V/cm)
and 300 mA. After electrophoresis, the slides were neutral-
ized in 0.4 mol/l TrisHCI (pH 7.5) xed in absolute ethanol,
and stored at room temperature until analysis blindly in a
uorescence microscope at 400 magnications. In order to
minimize extraneous DNA damage from ambient ultraviolet
radiation, all steps were performed with reduced illumina-
tion. Anautomatic analysis system(Comet Assay II; Perceptive
Instruments, Haverhill, UK) was used to determine DNA dam-
age. Tail moment (product of tail DNA/total DNA by the center
of gravity) was considered to estimate DNA damage from 50
cells per treatment.
3. Results
3.1. Determination of the toxicity with or without
dentin disc interposition
When the toxicity was evaluated indirectly through a dentin
slice, the analysis of variance failed to showa statistical differ-
ence between the new cement, Pro Root MTA, and Dycal (ns)
(Table 1). None of the materials was cytotoxic. However, when
the toxicity was evaluated without dentin slice interposition,
the analysis of variance showed a statistically signicant dif-
ference among the three materials (P <0.001). The Duncan test
Table 1 Cytotoxicity after indirect contact between the
materials and culture medium through a dentin disc
New Ca
3
SiO
5
cement MTA Dycal
Undiluted 0 8% 0 9% 0 8%
50% 0 4% 0 4% 0 4%
10% 0 4% 0 3% 0 4%
The newCa
3
SiO
5
cement, MTA, and Dycal were applied on the coro-
nal side of the dentin slices in Plexiglass devices with pulp pressure
simulation. After 24 h, the culture media in contact with the pul-
pal side of the dentin slices were used to determine cell viability.
The pulp broblasts were incubated with these media (either undi-
luted, or diluted in the culture medium to 50% or to 10%) for 24 h
before applying the MTT test on human pulpal broblasts. Opti-
cal density values of untreated control cultures normalized to 100%
was in the range of 0.90.95. The results are expressed as mean cell
toxicityS.D.
57/74
1490 dental materi als 2 4 ( 2 0 0 8 ) 14861494
Table 2 Cytotoxicity after direct contact between the
material and culture medium
New Ca
3
SiO
5
cement MTA Dycal
Undiluted 0 8% 0 9% 22 10%
50% 0 5% 0 5% 10% 5%
10% 0 4% 0 3% 2 2%
The cytotoxicity of the new cement compared to MTA and Dycal on
human pulp broblasts was evaluated after 24 h contact between
the materials and the culture medium (either undiluted, or diluted
in the culture medium to 50% or to 10%) with the MTT test. Both
were less cytotoxic than Dycal (P <0.001). The results are expressed
as mean cell toxicityS.D.
showed that Dycal displayed a higher cytotoxicity than MTA
and the new Ca
3
SiO
5
cement (Table 2).
According to this study, a dilution of 10% was chosen for
studying the materials effects onbroblasts specic functions
because it has biological effects without being toxic.
3.2. Inuence of the two materials on pulp broblasts
differentiation into odontoblastic cells
Control cells expressed collagen I, dentin sialoprotein and
Nestin. Pulp broblasts secreted a mineralizd matrix and the
cells, particularly those contacting the mineralizd matrix,
expressed Nestin (Figs. 1 and 2).
Fig. 1 Effect of the new Ca
3
SiO
5
cement on pulp broblast
specic gene expression. Immunohistochemistry was used
to evaluate the effect of the new Ca
3
SiO
5
cement and MTA
on pulp cells specic genes expression. Control cultures
express collagen type I (a) and dentin sialoprotein (b).
When the media containing the new Ca
3
SiO
5
cement (c
and d) and MTA (e and f) extracts were added to the
cultures for 4 weeks, collagen I (c and e) and dentin
sialoprotein (d and f) were also expressed at a high level in
the pulp cells. Original magnications =10.
Fig. 2 Effect of the new Ca
3
SiO
5
cement on pulp cells
mineralization. Immunohistochemistry was used to
evaluate the effect of the new Ca
3
SiO
5
cement and MTA on
pulp cells differentiation and mineralization. Control
cultures express Nestin and secrete a mineralized matrix in
the form of nodules (a). When the media containing the
new cement (b) or MTA (c) extracts were added to the
cultures for 4 weeks, a mineralized matrix deposition was
also observed. Nestin was also expressed at a high level in
pulp cells and its expression was stronger in the mineral
nodules forming cells. Original magnications =10.
After adding the media containing extracts of the new
Ca
3
SiO
5
cement or MTA to the cultured pulp cells, collagen I,
dentin sialoprotein were strongly expressed by the pulp cells
(Fig. 1). Mineral nodule formation was also observed (Fig. 2).
Nestin was expressed by the cells but not in the mineral nod-
ules. The immunostaining intensity was always higher incells
forming the mineral nodules than the cells away from these
nodules.
3.3. Genotoxicity
Ames test did not show any evidence of mutagenicity of
the Nutrient Broth No 2 medium after being in contact with
the new cement, whatever the dilution of the test medium
(Table 3). The mutations observed with the new cement were
comparable to the spontaneous reverse mutations obtained in
58/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1491
Table 3 Mutation frequencies of Ames tester strains using the liquid preincubation assay
Metabolic activation
(S9 mix
a
)
Product Volume (l) Number of revertants/plate (meanS.D.)
TA 97a TA 98 TA 100 TA 102
+ NB No. 2 10 171 9 24 3 1364 382 17
+ DMSO 10 166 7 25 1 12513 355 16
NB No. 2 5 183 13 265 13511 402 18
DMSO 5 191 11 274 1389 423 26
+
New Ca
3
SiO
5
cement (NB No. 2 extract) 4 162 14 301 13514 360 10
6 177 5 26 1 1202 397 15
8 177 4 29 5 1325 351 7
10 192 4 27 4 15013 345 2
New Ca
3
SiO
5
cement (NB No. 2 extract) 2 215 11 251 16110 500 24
3 223 9 25 3 17221 424 36
4 225 15 251 16035 439 3
5 205 23 231 18212 517 44
+
New Ca
3
SiO
5
cement (DMSO extract) 4 170 19 292 1193 334 49
6 189 3 25 2 12613 376 3
8 175 2 28 8 1451 336 24
10 164 23 437 1365 314 11
New Ca
3
SiO
5
cement
(DMSO extract)
2 193 2 35 2 1493 421 5
3 186 5 37 5 1178 445 42
4 224 17 273 1406 463 26
5 173 8 30 1 1443 435 36
+ B[a]P 0.5 g 112137 42326 100087 679 28
ICR 191 0.02 g 55321 NT NT NT
2,4,7 TNFone 0.02 g NT 1653 NT NT
NaN3 0.5 g NT NT 58512 NT
MitC 0.2 g NT NT NT 365854
After preparation and setting of the cement, it was grinded prior to testing. 60 mg of the cement were placed in 1 ml of Nutrient Broth No 2
or DMSO solvent for 24 h at 37
C under mixing. The stock solutions from two independent experiments were tested in triplicate, and results
from both experiments in NB 2 and DMSO are presented. Increasing volumes of test samples (4, 6, 8 and 10l) were incubated with the each
of the bacterial strains for 60 min at 37
C under mixing. The mixture consisting of bacteria and a test compound was plated on plates in VB
medium at 37 C for 48 h and revertant colonies were counted. The experiments were carried out in the presence and in the absence of an S9
fraction. The test was qualied positive if it induced a dose-related and a reproducible increase of the numbers of revertants or twice higher
than the spontaneous revertants per plate. All data are expressed as means S.D. Positive controls were Benzo[a]pyrene (0.5g) with S9 MIX for
all strains. Positive controls were 2-methoxy-6-chloro-9-(3-(2-chloro-ethyl)aminopropylamino)acridine (ICR 191, 0.1g) for TA97a; 2,4,7-trinitro-
9-uorenone (2,4,7-TNFone, 0.02g) for TA98; sodium azide (NaN
3
, 1 g) for TA100 and mitomycin C (MitC, 0.05 g) for TA102 without S9 MIX.
NT: non-tested.
a
The S9 MIX included 4% S9, 4.2 mM NADP and 5.2 mM G6P.
the controls performed with the NB 2 and DMSO solvent. The
results show that the new Ca
3
SiO
5
cement does not induce
reverse mutations either withor without the S9 metabolic acti-
vation system. Similar results were obtained with all bacterial
strains tested.
Table 4 Micronucleated human lymphocytes count in
Ca
3
SiO
5
cement-treated cultures
Ca
3
SiO
5
cement dilution Micronucleated
lymphocytes (%) S.D.
1% 4.0 1.1
2.3% 4.0 1.1
3.7% 4.0 1.2
5% 4.2 1.2
Negative control
a
3.7 1.2
Positive control
b
16.0 6.0
***
Comparison with the control:
***
P <0.001.
a
Culture medium X-VIVO 10.
b
Mitomycin C 5g/ml.
The micronuclei test revealed that after incubating the
lymphocytes with different dilutions of the new cement, the
rate of lymphocytes with micronuclei was similar to that
obtained with the negative control. It ranged from 3.9% to
4.1% with increasing concentrations (15%) in aqueous or
hydrophobic medium. The positive control showed a rate of
16% (Table 4).
The Comet assay performedwithserial dilutions of the new
Ca
3
SiO
5
cement on human pulp broblasts revealed that the
percentage of DNA in the tail ranged from 12.59 for the 0.1%
dilution to 15.58 with undiluted medium. This percentage was
13.19 with the negative control and 46.52 with the positive
control (Table 5).
4. Discussion
The biocompatibility of the newcement is shown in this study
by the absence of cytotoxicity and genotoxicity and the fact
that the new material does not affect the cytodifferentiation
of human pulp broblasts in odontoblastic cells.
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1492 dental materi als 2 4 ( 2 0 0 8 ) 14861494
Table 5 Comet assay on human pulp broblasts
Ca
3
SiO
5
cement dilution Tail DNA (%) meanS.D.
0.1% 12.59 0.96
1% 13.31 0.88
10% 14.90 1.06
Undiluted 15.58 1.08
Negative control
a
13.19 0.96
Positive control
b
46.52 1.45
***
Comparison with the control: ***P <0.001. NS: non-signicant.
a
0.1% DMSO.
b
H
2
O
2
(13.2 mM).
Although Portland cements are known as non-toxic, in this
work, 3 tests were performed to evaluate the genotoxicity of
the new Ca
3
SiO
5
cement after solubilisation in hydrophilic or
hydrophobic conditions. These tests were performed because
the cement developed here contains a modied polycar-
boxylate in the superplastisizer. It has been reported that
polycarboxylate (Aqualox
Exp material MTA
72/74
Physical, chemical and mechanical behavior of a new material
for direct posterior fillings.
2005
J. DEJOU, J COLOMBANI and I. ABOUT. Marseille, France
abstract : European Cells and Materials Vol. 10. Suppl. 4,
2005 (page 22)
73/74
European Cells and Materials Vol. 10. Suppl. 4, 2005 (page 22) ISSN 1473-2262
Physical, Chemical and Mechanical Behavior of a New Material for Direct
Posterior Fillings.
J. Djou, A Raskin, J Colombani & I. About
Laboratoire IMEB-ERT 30, UFR dOdontologie, Universit de la Mditerrane, Marseille,
FRANCE
INTRODUCTION: A new ceramic material
for direct restorative posterior fillings, a
Ca
3
SiO
5
-based Portland cement, has been
developed to circumvent the shortcomings of
the traditional filling materials. The purpose of
this work was to evaluate the marginal sealing
efficiency, the acid erosion and the effects of
aging in artificial saliva on its structure,
composition and compressive strength.
METHODS: The marginal sealing was
evaluated by the silver nitrate penetration
method without any surface treatment, with or
without aging in Fusayama artificial saliva.
The acidic erosion was evaluated daily in lactic
acid (0.02M) and sodium lactate (0.1M)
aqueous solution (pH 2.74) by measuring the
height loss, for a week.
Aging was evaluated in Meyer-modified
Fusayama artificial saliva1 (pH 5.3).
The height modification of the material was
evaluated for a week. Scanning electron
microscopy was used to examine and
characterize the surface of the sample before
and after aging. The possible dissolution of the
new material in the artificial saliva was
evaluated by measuring the concentration of
Si, Ca, Zr, and inorganic carbonate in the
artificial saliva after 1, 2, 3 and 4 weeks. The
compressive strength was measured 24 hours
after setting and after aging for seven and 28
days.
RESULTS: No difference in marginal sealing
was revealed between the new biomaterial and
the Z250-Optibond solo plus adhesive
restorative system. The same results were
obtained after aging for one week in artificial
saliva. The acid erosion increased with time.
This increase was less rapid than that obtained
with glass ionomer cement reported by
Nomoto R2,3. In artificial saliva there was no
erosion but deposition of white material on the
surface of the material. Scanning electron
microscopic analysis of this material revealed
needle-like crystals with an apatitic appearance
(figure.1).
Fig. 1: Needle-like crystals on the surface of
the material after aging in artificial saliva
The composition of this deposit determined by
X-diffraction analysis seems to confirm the
apatitic composition (ratio Ca/P = 1.6). This
correlates well with the analysis of the
elements in the solution, which reveals a
decrease of Ca concentration with time. There
was a slight but not significant release of Si.
The compressive strength was 136 (20.10) at
24 hours, increased to 169.74 (16.92) after 7
days and then was stable until day 28.
DISCUSSION & CONCLUSIONS: The
marginal sealing without any surface treatment
or adhesive system was equivalent to that of
the reference material used. In spite of the
acidic pH of the artificial saliva, the new
material showed no erosion and an increase in
the compressive strength. The deposition of
apatitic structures might increase the marginal
sealing of the material.
REFERENCES:
1
Reclaru L, Meyer JM.
(1994). Study of corrosion between a titanium
implant and dental alloys. J Dent; 22:159-68.
2
Nomoto R, McCabe JF. (2001). A simple acid
erosion test for dental water-based cements.
Dent Mater ;17(1):53-9.
3
Nomoto R, Uchida K, Momoi Y, McCabe JF.
(2003). Erosion of water-based cements
evaluated by volumetric and gravimetric
methods. Dent Mater.;19(3):240-4.
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