Septodont R&D – France November 2009

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 à usage odontologique : action sur quelques souches bactériennes représentatives 2009 E.Valyi, P.Colon, F.Bornand, D.Decoret,

B.Grosgogeat, FranceAbstract - SFBD (Société Francophone des biomatériaux 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 DWorking 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, FranceJournée Scientifique du CNEOC Brest -June 2009

BiodentineTM- RD94, A portland cement, stimulates in vivo reactionary dentin formation

2009 T.Boukpessi, F.Decup, D.Septier, M. Goldberg, C. Chaussain, France

Journée 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 Ca3SiO5-based posterior restorative material

2008 Laurent P, Camps J, De Méo M, Déjou 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,

GoldbergOral 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. DEJOUAbstract :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. KOUBIabstract : 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, Franceabstract : 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,

Franceabstract : European Cells and Materials Vol. 10. Suppl. 4, 2005 (page 22)

PUBLICATIONS AND COMMUNICATIONS ON BIODENTINETM - RD94 - SEPTODONT

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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 In Indirect Pulp-Capping Situation Induces Reactionary Dentin Formation (Joint Meeting of the Continental European, Israeli and Scandinavian Divisions of the IADR (September 10-12, 2009))

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153 RD94 In Indirect Pulp-Capping Situation Induces Reactionary Dentin Formation

Location: Library (Gasteig Convention Center München)

T. BOUKPESSI, F. DECUP, D. SEPTIER, C. CHAUSSAIN, and M. GOLDBERG, University Paris Descartes-Dental School- EA 2496, Montrouge, France

RD94 a new experimental Ca3SiO5-based restorative cement intends to be a glass ionomer cement and composite-resin substitute in restorative dentistry. Objectives:to evaluate in vivo the biocompatibility and bioactivity effects of RD94 as assumed from the formation of reactionary dentin. Methods:Using the rat as an animal model, half-moon cavities were prepared on the mesial aspect of the first maxillary molar without pulp exposure. The cavities were then left unfilled (sham group) or filled either with a glass-ionomer cement (control group) or with RD94 (experimental group). The rats were killed by perfusion through the heart with the fixative solution 8, 15, 30 days, and 3 months after the dental treatment. Block sections including the three maxillary molars were demineralised and processed for light microscopy. Measurements were done on micrographs obtained after histological observations. Results: After 8 days, a slight inflammatory reaction was seen in each group. In the RD94 group, a dentin layer of reactionary dentin starts to be formed, by contrast with the 2 other groups. After 15 days, a tendency of spontaneous repair was observed in the pulps of the sham and control groups. In the RD94 group, the pulp near the cavity retracts, covered by a 40-80 µm thick layer of reactionary dentin. In the RD94 group, after one month, the mesial part of pulp was partially filled with a homogenous dentin-like material (160µm) whereas the rest of pulp appeared normal. After three months, RD94 induced the formation of a homogenous reactionary dentin but the thickness of this layer was unchanged between 1 and 3 months. Conclusions:The present data 1-suggest that RD94 displays novel bioactive properties. 2- This new cement stimulates the formation of reactionary dentin in the rat molar model shortly after a switch on, 3- but there is actually a “switch off”, keeping the remaining pulp alive.

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Ciments alcalins ou acides à usage odontologique : action sur quelques souches bactériennes représentatives 2009 E.Valyi, P.Colon, F.Bornand, D.Decoret, B.Grosgogeat, France Abstract - SFBD (Société Francophone des biomatériaux dentaires). 25-26 juin 2009.

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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

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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

Ca3SiO5-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, Faculté d'Odontologie, Université de la Méditerranée, 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 Black’s 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 Ca3SiO3-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 Ca3SiO5-based restorative cement has been developed, put on the market

under the name of BIODENTINETM (Septodont, Saint Maur des Fosses, France). As the

ProRoot MTA® (Torabinejad et al, 1995a,b; Camilleri et al, 2005) and Portland’s 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 CaCO3 and ZrO2. The liquid

is a solution of CaCl2 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.SiO2)

Calcium carbonate (CaCO3)

Zirconium dioxide (ZrO2)

Liquid

Calcium chloride (CaCl2.2H2O)

Water reducing agent

Water

Properties of the different components

- Tricalcium silicate (3CaO.SiO2): it is the main component of the powder. It regulates the

setting reaction.

- Calcium carbonate (CaCO3): it role is similar to the fillers

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- Zirconium dioxide (ZrO2): it is added to provide the radio-opacity to the cement

- Calcium chloride (CaCl2.2H2O): 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.SiO2) 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 (CaCO3) 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=SiO2, H=H2O). 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.SiO2) + 6H2O 3CaO.2SiO2.3H2O + 3Ca(OH)2

Structure

The surface of the cement observed with the SEM one week after mixing is loaded by calcite

–rich structures (CaCO3) of variable sizes (Figure 2). The calcite is a chemical or

biochemical mineral crystallizing in the rhombohedra system (a=b=c; ,,90). Crystals of

CaCO3 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 CaCO3 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

CaCO3 are filling the spaces between grains of cement. Finally, the calcite (CaCO3) 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.SiO2 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 BIODENTINETM

is time-dependent (Figure 4). This shows that

immediately after mixing, the setting reaction of BIODENTINETM

is not yet achieved. At

least 2 weeks are necessary to reach a final stable stage. The setting reaction of

BIODENTINETM

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 BIODENTINETM

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 BIODENTINETM

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 BIODENTINETM

is superior to conventional GIC but still much lower than the composite resin.

Tooth – BIODENTINETM

– Adhesives Interfaces

Morphological characterization

The SEM microphotographies show BIODENTINETM

- dental structures interfaces (Figures

5-7) and BIODENTINETM

- adhesive systems interfaces (Figures 8, 9). The results show the

occurrence of a cohesive failure within the BIODENTINETM

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 BIODENTINETM

- 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 CaCO3 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 BIODENTINETM

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 BIODENTINETM

-

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 BIODENTINETM

- enamel / dentin was very low

(Table III).

A J0, the seal obtained with the Xeno

III treatment is more important than with G-Bond

.

With time (after 3 months), the sealing ability of G-bond

treatment was improved (Table

IV).

At the interface BIODENTINETM

- adhesive systems, the results display also a very low rate

of penetration. However, the choice of the solvent of the adhesive system seems important to

optimize results. The Xeno

III system adhesive contains 2-HEMA and a solvent-based on

ethanol and water. The G-Bond

system adhesive contains 4-MET and a solvent-based on

acetone and water. The acetone is soluble in water and more sensitive to moisture than

ethanol. Acetone evaporates faster than ethanol, and we have reported previously that water

plays an important role in the setting reaction of the cement. Therefore, the incorporation into

the cement of 2-HEMA associated with ethanol solvent appears favorable to the association

4-MET - acetone solvent. Through contact with water as a function of time, the sealing is

improved in the samples treated with G-Bond.

By combining these results with those obtained with the SEM, we can conclude that this

material has excellent sealing capacities.

Conclusion

The setting reaction of BIODENTINETM

led to the formation of a gel-similar to the CSH gel

described for the Portland cement. The gel is the matrix of a cement that incorporates CaCO3

crystals as "fillers". Following the setting reaction, the cement presents a certain degree of

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porosity. This is gradually reduced by the continuous hydration of cement within a given

period of time. The mechanical properties of the cement improve with the setting period of

time. It is interesting to note that BIODENTINETM

is a material based on calcium salts, which

has the capacity to develop watertight interfaces both with dental structures and with adhesive

systems.

Because of the unaesthetic appearance and the poor resistance to flexural strength,

BIODENTINETM

may be considered mostly as a dentin substitute. The seal of the tooth –

material interface is improving with time, linked with the ability to develop a

micromechanical anchorage. Regarding the biomaterial – dentin bonding interface, the

solvent nature has to be considered, with a net preference for a self-etching adhesive system

using water or ethanol – water solvent.

References

Allen A J, Thomas J J, Jennings H M. Composition and density of nanoscale calcium-silicate-

hydrate in cement. Nature Materials, 2007, 6 : 311-316.

Andrale C, Blanco V, CollazoA, Keddam M, Novoa X.R, Takenouti H. Cement paste

hardening process studied by impedance spectroscopy. Electrochim Acta,1999 ; 44: 4314-

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Cabeza M, Keddam M, Novoa X R, Sanchez I, Takenouti H. Impedance Spectroscopy to

characterize the pore structure during the hardening process of Portland cement paste.

Electrochim Acta, 2006 ; 51 : 1831-1841.

Camilleri J, Montesin F E, Brady K, Sweeney R, Curtis RV, Pitt Ford T R. The constitution

of mineral trioxyde aggregate. Dental Materials, 2005 ; 21, 297-303.

Camilleri J, Montesin F E, Curtis R V, Pitt Ford T R. Characterization of Portland cement for

use as a dental restorative material. Dental Materials 2006, 22 : 569-575.

Davidson C L, Mjör I A. Advances in glass-ionomer cements. Quintessence Publising Co, Inc

1999.

Chessmann C R, Asavapisit S. Effet of calcium chloride on the hydratation and leaching of

lead-retarded cement. Cement and Concrete Research 1999, 29 : 885-892.

Garrault S, Behr T, Nonat A. Formation of the C-S-H during earlt hydraton of tricalcium

silicate grains with different sizes. The Journal of physical chemistry B 2006, 110: 270-275.

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Holland R, Souza V, Nery M J. Calcium salts deposition in rat connective tissue after the

implantation of calcium hydroxide-containing sealers. Journal of Endodontics, 2002; 28,

173-6.

Laurent P, Camps J, De Méo M, Déjou j, About I. Inductionof specific cellresponses to a

Ca3SiO5 – based posterior restorative material. Dental Materials 2008; 24: 1486-94.

Lea S J, Monsef M, Torabinejad M. Sealing ability of a mineral trioxyde aggregate for repair

of lateral root perforations. Journal of Endodontics, 1993 ; 19, 541-5.

O’Brien WJ. Dental Materials and Their Selection, third edition, Quintessence Publishing Co,

Inc 2002, p.380.

Santos A D, Moraes J C, Araujo E B Yukimitu K, Valerio Filho W V. Physio-chemical

properties of MTA and a novel experimental cement. International Endodontic Joural,

2005 ; 38, 443-7.

Taylor H F W. Cement chemistry, 2nd

edition, Thomas Telford Publishing, London 1997,

p.113-126

Torabinejad M, Hong C U, McDonald F, Pitt Ford T R. Physical and chemiacal properties of

a new root-end filling mateial. Journal of Endodontics, 1995a ; 21, 349-53.

Torabinejad M, Rastegar A F, Pitt Ford T R, Kettering J D. Bacterial leakage of mineral

trioxide aggregate as a root-end filling material. Journal of Endodontics, 1995b ; 21, 109-

12.

Figures and tables:

Figure 1: Structure of calcium based cement after crystallisation (Allen et al., 2007).

3CaO.SiO2

Inter - layer water

Adsorbed water

Pores containing water

C-S-H gel

adsorbed water

Inter –layer water

Pore

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Figure 2: Cement surface observed at Day 7 (SEM)

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Figure 3: Cement surface observed at Day7 (SEM)

Rhombohedric calcite crystals (A) and crystal plates (B) suggest the formation of calcium

hydroxide or calcium phosphate.

A

B

B

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Figure 4: Evolution of electrical resistance of BIODENTINE

TM as a function of time

639

999

1157

562528517

384

500425

200

400

600

800

1000

1200

1H 2H 4H 5H 6H 9H 1D 7D 14D

Time

()

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Figure 5: Enamel- BIODENTINETM

interface (SEM)

BIODENTINETM

(A) adheres to enamel (B) after a cohesive fracture

A

B

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Figure 6. Dentine – BIODENTINETM

Interface (SEM)

BIODENTINETM

(A) adheres to dentine (B) after a cohesive fracture

A

B

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Figure 7: A "Mineral Tag"

The crystals (A) have infiltrated the dentine tubule (B): "Mineral Tag"

A

B

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Figure 8: BIODENTINETM

- G bond

Interface (SEM)

Cohesive fracture (B), cement (A), G bond

(C), Composite Resin (D).

C

A B

D

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Figure 9: BIODENTINETM

– Xeno

III interface (SEM)

Cohesive fracture (B), cement (A), Xeno

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 / BIODENTINETM

17.65 (4.35) 19.86 (10.72)

Dentin / BIODENTINETM

10.46 (3.23) 14.84 (5)

Table III: Tooth – BIODENTINETM

INTERFACES

% of microleakage

Adhesive systems Dye penetration (%) at D0 Dye penetration (%) at D90

Xeno

III 6.93 (± 3.57) 10.07(± 2.23)

G-Bond

18.64 (± 4.13) 7.68 (± 3.2)

Table IV: BIODENTINETM

/ adhesives INTERFACES

% of microleakage

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VI-2-2 Biological effects

VI-2-2-1 Development of a bioactive Ca3SiO5 based posterior

restorative material (Biodentine TM

).

Patrick Laurent, Virginie Aubut, Imad About Laboratoire IMEB, Faculté d'Odontologie,

Université de la Méditerranée, Marseille, France

Interest of biocompatible materials

Resin composites and amalgams represent the currently used dental restorative materials for

Class I and II cavities (Qvist et al 1990). Due to mercury vapours release from amalgam

restorations (Mitchell et al 2005), direct composite restorations have gradually been used to

replace amalgam for anterior restorations and small-to-moderate sized posterior restorations.

Although resin composites enable micro-mechanical retention by the use of different bonding

techniques, composite resin raise other problems due to polymerization shrinkage with the

subsequent microleakage and unreacted monomers release (Rathbun et al 1991; Geurtsen et

al, 1998).

This explains why recent research focused on use of biocompatible materials such as the

Portland cement. Mineral trioxide aggregate developed as a root-end filling material has a

similar constitution of Portland cement. It is composed primarily of tricalcium and dicalcium

silicate (Camilleri et al 2005) and known as a biocompatible material. This has been shown

by high cell viability with MTA extracts when biocompatibility was investigated with the

methyltetrazoilum (MTT) assay (Keiser et al, 2000; Huang et al, 2003; Camilleri et al, 2005).

Additionally, when used for pulp capping or after partial pulpotomy, MTA stimulated

reparative dentin and complete bridge formation in vivo after 2 months with no signs of

inflammation (Aienehchi et al, 2002; Pittford et al 1996; Faraco and Holland, 2004). In spite

of its biocompatibility, the setting time of MTA is too long (2h 45 min) (Torabinejad et al,

1995) and its mechanical properties are not compatible for use as a dental restorative material.

Tricalcium silicate-based cements as promising materials:

Tricalcium silicate is the main constituent of MTA, and the main raw material in Portland

cements. In addition to the biocompatibility of tricalcium silicate cements, this type of

materials has two major properties:

1) Bioactivity:

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A bioactive material is one that elicits a specific biological response at the interface of the

material, which results in the formation of a bond between the tissue and the material (Hench

and West, 1996). It has long been believed that artificial materials implanted into bone defects

are generally encapsulated by a fibrous tissue, leading to their isolation from the surrounding

environment (reviewed in Kokubo and Takadama, 2006). However, it has been shown that

Bioglasses spontaneously bond to living bone without the formation of surrounding fibrous

tissue (Hench et al, 1972). Since then, several types of materials have been shown to bond to

living tissues. It has been hypothesized that an essential requirement for an artificial material

to bond to living bone for example is the formation of bonelike apatite on its surface when

implanted in the living body (Kokubo, 1991). In vivo, this apatite formation can induce cell

adhesion and differentiation as well as the mineralized tissue directly on the surface of the

material thus reflecting its bioactivity.

A recently developed Ca3SiO5-based bone injectable material has been investigated in

simulated body fluid conditions. The results of this study showed, by X-ray diffraction and

scanning electron microscopy, that Ca3SiO5 stimulated cells growth and induced

Hydroxyapatites (HA) formation on the surface of the material when exposed to the simulated

body fluid (Zhao et al, 2005). HA have been shown to induce bone formation, growth and

maintenance at the bone-material interface in vivo and this can be reproduced and

demonstrated in vitro by soaking HA in vitro in simulated body fluids (Kokubo, 1990;

Greenspan et al, 1994). This is of prime importance during the process of healing as Silica

can induce the mineralisation function of cells by affecting cell proliferation and genes

expression. Indeed, in a study on the effect of three kinds of silica nanospheres with different

nanometer dimensions on a human osteoblast-like cell line (MG-63), the presence of silica

showed higher cell viability and Alcaline Phosphatase activity of treated cells (Feng et al,

2007). This may be due to the fact that the silicon ion can be released from silica. Silicon has

been recognized as an essential element in young bone calcification. The release of soluble

ion of silicon can stimulate osteoblast cells to produce bone (Bielby et al. 2004). In a recent

work, the biocompatibility together with the bioactivity of tricalcium silicate led to its use in

constructing bone scaffolds for the treatment of bone defects. Indeed, bone tissue engineered

silicate-substituted tricalcium phosphate scaffolds were prepared and seeded with human bone

marrow-derived mesenchymal stem cells. The cells seeded onto the scaffolds were then

cultured in a perfusion bioreactor for up to 21 days. During culture, cells from the flow

cultured constructs demonstrated improved proliferation and osteogenic differentiation

demonstrated by a higher expression of several bone markers such as alkaline phosphatase,

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osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. The study

showed that the cells and the synthesized matrix were distributed homogenously throughout

the entire scaffold. This viable and homogenous ex vivo bone construct with osteogenic

properties may provide a replacement for autologous bone grafts in vivo and demonstrate the

bioactivity of such materials for future applications (Bjerre et al, 2008).

2) Self setting and spontaneous development of strength on hydration.

One of the major properties of the Ca3SiO5 is its self setting and development of compressive

strength on setting. However, in spite of the bioactive property of the above described

material, it has a setting time, which is still too long (above 180 min) and its compressive

strength hardly reaches 20.2 MPa after 28 days to meet the need of clinical applications as a

restorative material (Zhao et al, 2005). Calcium chloride is known as an effective accelerator

of hydration and setting in Portland cement pastes. Although its addition up to 15% in the

liquid phase into Ca3SiO5 decreased the final setting time from 180 to 90 min, the

compressive strength remained weak (23.46 MPa) at 7 days (Wang et al 2008). An increase

of compressive strength requires a total reduction of the Ca3SiO5-based material water

content. The use of superplasticisers as very effective dispersing agents to reduce the water

content was used in fast setting Portland cements. This has been shown to lower the setting

time to 7 min but the compressive resistance didn’t exceed 50MPa even after 28 days

(Camilleri et al, 2006).

Development of a biocompatible Ca3SiO5-based material for dental applications

Taking advantage of the Ca3SiO5-based cements self setting and bioactive properties, a new

Ca3SiO5-based cement (Biodentine TM) for direct restorative posterior fillings has been

developed recently. The material is inorganic and non metallic. It is composed of Ca3SiO5,

CaCO3, ZrO2, water and a superplasticising admixture to reduce the water content of the mix

and to retain its workability. This material is presented in the form of a powder and a liquid

and can be prepared by mixing with an amalgamator. Biodentine TM is compatible with

working in clinic. It has a setting time of 10 minutes and was developed to be used in direct

and indirect pulp capping procedures as a single application dentin substitute without any

cavity conditioning treatment. The biological studies performed on this material indicate that

it may be safely and directly applied to the dental pulp.

Indeed, genotoxicity tests were performed on this material in vitro to ensure the safety of its

use in vivo. Ames test performed on 4 S. typhimurium strains (TA97a, TA98, TA100, and

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TA102) failed to detect significant reverse mutations. The micronucleus test was performed

on human lymphocytes in order to detect any structural chromosomal alteration in the host

cells involved in the defense mechanisms. It revealed that no chromosomal damage was found

with the material. The Comet assay was performed on the target cells of the new cement and

did not show significant DNA breaks in human pulp fibroblasts.

The toxicity has been evaluated on L929 cell line as well as on target pulp fibroblast isolated

from human third molars with the MTT test. This test revealed that the new material is non

toxic and comparable to materials such as MTA and Ca(OH)2 which are currently used in

direct pulp capping situations (Laurent et al, 2008).

Bioactivity of Biodentine TM

The effect of Biodentine TM on the specific functions was also investigated in the conditions of

their application in vivo simulating direct pulp capping by incubating its extracts with pulp

fibroblasts. It was also investigated under indirect pulp capping conditions with a dentin slice

interposition with a regular thickness (0.7mm) between the new material and the culture

medium under pulsatile pulp pressure for 24 hours. The resulting conditioned medium was

then put in contact with the target cells. In both direct and indirect application, the new

material didn’t seem to affect the target cells specific functions. A previous work has shown

that pulp fibroblasts were capable of differentiation into odontoblastic cells when cultured

with -glycerophosphate (About et al, 2000). Similarly, the cells incubated with the

conditioned media expressed a high level of odontoblastic cell markers: Collagen I, Dentin

Sialoprotein and Nestin and formed mineralization nodules indicating a mineralization

potential subsequent to odontoblastic differentiation (Laurent et al, 2008). This result is in

agreement with the bioactivity of Ca3SiO5-based materials observed in bone and confirms the

fact that Biodentine TM is bioactive. It induces the synthesis of a dentin-like matrix by human

odontoblast-like cells in the form of mineralization nodules that have the molecular

characteristics of dentin (About et al, 2000; Laurent et al, 2008). Additionally, the FTIR

analysis has previously shown that this mineralized material was a specific deposition, which

had the same mineral and organic composition of dentin (About et al, 2000).

This is of prime importance in clinic. Coronal restorations may be placed on teeth where the

odontoblastic layer is partially destroyed, making the differentiation of secondary

odontoblasts necessary prior to pulp healing. The presence of toxic compounds such as

monomers may interfere with this critical step of pulp healing (About et al, 2005). By

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contrast, the presence of bioactive materials will enhance this step which has a major role in

vital pulp protection and in the prevention of recurrent caries.

This property of the new cement was shown as similar to that of biocompatible materials used

in pulp capping situations such as MTA. The advantage of Biodentine TM over such materials

resides in the fact that in addition to its biocompatibility, its mechanical and physical

properties, strongly suggest its future utilisation as a dentin substitute and not only as a pulp

capping agent.

Conclusions

The results of our study need to be confirmed in vivo and suggest that this new Ca3SiO5

cement could be used as a direct pulp capping agent but also as dentin substitute. This

material would likely induce secretion of reactionary dentin often considered as a preliminary

step for pulp healing after caries removal. The good handling properties of this material

associated with its biological, mechanical and physical properties let us think that Biodentine

TM could be used as pulp capping agent and as a bulk restorative material. The fact that no

preliminary conditioning treatment of the cavities is required with this new cement would

greatly simplify the pulp capping techniques.

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References

About I, Bottero M-J, de Denato P, Camps J, Franquin J-C, Mitsiadis TA. Human dentin

production in vitro. Exp Cell Res, 2000; 258: 33-41.

About I, Camps J, Burger A-S, Mitsiadis TA, Butler W, and Franquin J-C. The effects of

bonding agents on the differentiation in vitro of human pulp cells into odontoblasts. Dent

Mater, 2005; 21 (2): 156-163.

Aienehchi M, Eslami B, Ghanbariha M, Saffar AS. Mineral trioxide aggregate and calcium

hydroxide as pulp capping agent in human teeth: a preliminary report. Int Endod J. 2002 ;

36 :225-231.

Bielby RC, Christodoulou IS, Pryce RS, Radford WJ, Hench LL, Polak JM. Time- and

Concentration-Dependent Effects of Dissolution Products of 58S Sol–Gel Bioactive Glass

on Proliferation and Differentiation of Murine and Human Osteoblasts. Tissue Eng. 2004 ;

10(7-8): 1018-26.

Bjerre L, Bünger CE, Kassem M, Mygind T. Flow perfusion culture of human mesenchymal

stem cells on silicate-substituted tricalcium phosphate scaffolds. Biomaterials. 2008;

29(17):2616-27.

Camilleri J, Montesin FE, Brady K, Sweeney R, Curtis RV, Pitt Ford TR. The constitution of

mineral trioxide aggregate. Dent Mater. 2005; 21,297-303.

Camilleri J, Montesin FE, Curtis RV, Ford TR. Characterization of Portland cement for use as

a dental restorative material. Dent Mater. 2006; 22: 569-75.

Camilleri J, Montesin FE, Di Silvio I, Pitt Ford TR. The chemical constitution and

biocompatibility of accelerated Portland cement for endodontic use. Int Endod J. 2005;

38,834-42.

Faraco IM, Holland R. Histomorphological response of dogs’ dental pulp capped with white

mineral trioxide aggregate. Brazilian Dental Journal 2004; 15, 104-8.

Feng J, Yan W, Gou Z, Weng W, Yang D. Stimulating effect of silica-containing nanospheres

on proliferation of osteoblast-like cells. J Mater Sci Mater Med. 2007; 18(11):2167-72.

Geurtsen W, Spahl W, Leyhausen G. Residual monomer/additive release and variability in

cytotoxicity of light-curing glass-ionomer cements and compomers. J Dent Res. 1998;

77:2012-9.

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Greenspan DC, Zhong JP, LaTorre GP. Effect of surface area to volume ratio in vitro surface

reactions of bioactive glass particulates. In: Andersson O H, Yli-Urpo A, editors.

Bioceramics, vol. 7. Turku, Finland 1994. p. 55–60.

Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanisms at the interface of

ceramics prosthetic materials. J Biomed Mater Res 1972; 2:117–41.

Hench LL, West JK. Biological applications of bioactive glasses. Life Chem Reports.

1996;13:187–241.

Huang TH, Ding SJ, Hsu TC, Kao CT. Effects of mineral trioxide aggregate (MTA) extracts

on mitogen-activated protein kinase activity in human osteosarcoma cell line (U2OS).

Biomaterials 2003; 24, 3909-13.

Keiser K, Johnson C, Tipton DA. Cytotoxicity of mineral trioxide aggregate using human

periodontal ligament fibroblasts. J endod. 2000; 26:288-291.

Kokubo T Takadama T. How useful is SBF in predicting in vivo bone bioactivity?

Biomaterials. 2006; 27: 2907–2915.

Kokubo T. Bioactive glass ceramics: properties and applications. Biomaterials 1991; 12:155–

63.

Kokubo T. Surface chemistry of bioactive glass-ceramics. J Non-Cryst Solids 1990; 120:138–

51.

Laurent P, Camps J, De Méo M, Déjou J, About I. Induction of specific cell responses to a

Ca(3)SiO(5)-based posterior restorative material. Dent Mater. 2008; 24: 1486-94.

Lutz F, Phillips RW, Roulet J F and Setcos JC. In vivo and in vitro wear of potential posterior

composites, J Dent Res 1984; 63:914–920.

Mitchell RJ, Osborne PB, Haubenreich JE. Dental amalgam restorations: daily mercury dose

and biocompatibility. J Long Term Eff Med Implants. 2005;15(6):709-21.

Pitt Ford TR, Torabinejad M, Abedi H. Using MTA as a pulp capping material. J Amer Dent

Assoc. 1996; 127:1491-1494.

Qvist J, Qvist V, Mjör IA. Placement and longevity of amalgam restorations in Denmark. Acta

Odontol Scand. 1990; 48:297-303.

Rathbun MA, Craig RG, Hanks CT, Filisko FE. Cytotoxicity of a BIS-GMA dental composite

before and after leaching inorganic solvents. J Biomed Mater Res. 1991; 25:443-57.

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Torabinejad M, Rastegar AF, Kettering JD, Pitt Ford DR. Bacterial leakage of minerral

trioxide aggregate as root-end filling material. J Endod. 1995; 21:109-112.

Wang X, Sun H, Chang J. Characterization of Ca(3)SiO(5)/CaCl(2) composite cement for

dental application. Dent Mater. 2008; 24(1): 74-82.

Zhao W, Wang J, Zhai W, Wang Z, Chang J. The self-setting properties and in vitro

bioactivity of tricalcium silicate. Biomaterials. 2005; 26: 6113-21.

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VI-2-2-2 Animal studies

Tchilalo Boukpessi, Dominique Septier, Michel Goldberg

(Université Paris Descartes, France)

Animal models

Experimental approaches on animals are considered to provide data that are closer from the

clinical situation than in vitro studies carried out on cells or tissues (Wennberg et al., 1983).

However, they bear self-limitations firstly because physio-pathological reactions differ

between mammalian teeth. Secondly, and this is a major point, in the clinical situation, the

teeth of patients have been already attacked by carious decays. Consequently, they have

overcome an immune reaction and in many cases they have already produced a reactionary

dentin. Despite these limitations in the interpretation of the results that may be obtained,

animal studies provide some useful data, especially with respect of toxic or noxious effects on

the dental pulp.

Although being both primates, there are many well recognized similarities between monkey

and human, the pulp react differently to dental materials. Apparently the closest to human

would be the pig or the mini-pig. Such animal studies imply large animal houses and the

presence of a veterinarian. This is not affordable by most academic groups of researchers.

Looking for smaller sized animals, guinea pigs and ferrets have also been used for such

purposes, sometime successfully. It is admitted that small animals and namely rats may also

be used for such preclinical investigations (Six et al., 2000). They are more affordable and

resist to repeated anesthesia. In general, mice are too small for such experimental approaches.

The technical difficulties inherent to the small size of the rat were overcome by the

preparation of cavities on the mesial aspect of the first maxillary molar as proposed by

Ohshima (1990). We did some modifications to this protocol. The tongue and cheeks add

some difficulties when mandibular molars are selected, this is why we selected maxillary

molars. The large diastema between the incisor and the first molar provide enough space to

drill a half-moon cavity in the mesial aspect. Moreover, after electrosurgery, the gingiva being

removed in the cervical area of the tooth, the cavity is prepared at the anatomical cervical

junction, indeed the enamel-cementum junction. Fillings at this place better resist to occlusal

pressures, and consequently are not expelled after a few days. In addition, the pulp horns are

spontaneously filled by reactionary dentin, whereas in the cervical pulp the reaction is closer

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to the situation found in human. Each step was critically analyzed, and appropriate answers

were provided by our group (Six et al., 2000; Decup et al., 2000). This experimental approach

was used to evaluate the pulp reaction to BiodentineTM

, a Ca3SiO3-based cement.

Materials and methods

A total of 33 male Sprague Dawley rats, 6-7 weeks old (150g), were used in this experiment

performed as previously described (Decup et al., 2000) under an institutionally approved

protocol for the use of animals in research. Anesthesia in each case was with a single

intraperitoneal injection of Chloral (400 mg/Kg body weight). Electrosurgery of the gingival

tissue was carried out with a Servotom (Satelec, France) to prepare an access to the mesial

aspect of the right and left upper first maxillary molars. Half moon cavities were prepared in

1-2 seconds in the cervical third with high-speed contra-angle working at 120 000 rpm with

tungsten carbide burrs (size 0.6mm, 0.05 ISO). The burs were changed after every four

cavities and two teeth per rat were prepared on the mesial aspect of the first maxillary molars.

The rats were killed by intracardiac perfusion of the fixative solution. The rats were killed by

an intracardiac fixative solution perfusion containing 4% paraformaldehyde buffered with

sodium cacodylate, 0.1M, at pH 7.2-7.4. 8 days, 15 days and 30 days after the preparation of

the cavities, which were filled either with BiodentineTM

(Septodont, France) (30 molars, 10

per each period of time), or with a light curing glass ionomer cement Fuji IX, (GC Eur N.V.

Leuven, Belgium), previously examined for its bioactivity by our group (Six et al., 2000) (24

molars, 8 per each period of time). Twelve cavities were left without restoration, as control (4

per each period of time). The rats were perfused for 10 minutes. Afterward, block sections

including the three maxillary molars and the surrounding periodontal tissues were dissected

out and further immersed in the fixative solution for 4h. They were rinsed in the cacodylate

solution, then demineralized in 4.13% EDTA or in sodium formiate. The tissue was

dehydrated in graded ethanols and embedded in Paraplast (Oxford Labware, St Louis, MO,

USA). Five m thick sections were cut then dewaxed and stained with Masson’s trichrome, or

hematoxylin-eosin.

Results

Examination of the sections shows that after 8 days pulp inflammation was moderate in the

mesial third of the pulp chamber. This was mostly due to the preparation of the cavity since

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the controls (preparation of the cavity alone) displayed the same reaction. The inflammatory

process was resolved at day 15. The mesial horn was gradually filled with reactionary dentin.

The formation of reactionary dentin was identified as a newly formed dentin layer located

between the calciotraumatic line and the predentin. Variations in thickness were seen between

the different aspects of the pulp chamber. It was thicker in the tip of the horns and thinner on

the floor of the pulp chamber, but the strongest reaction was seen in the mesial aspect where

the preparation was made. Reactionary dentin was also seen in the isthmus between the

mesial and central pulp chamber. Compared with the group of teeth filled with Fuji IX the

formation of reactionary dentin was enhanced in teeth filled with BiodentinTM

. The thickness

of reactionary dentin in the mesial third of the pulp chamber was time dependent, and

gradually increased, going from 20-40 m at day 8 to 40-80 m at day 15 and reaching 140 –

280 m at day 30 (Figure 1). These measurements indicated that the formation of reactionary

dentin was enhanced when BiodentineTM

was used as filling, compared with the other group

of teeth, where the formation of this dentin was seen to vary between 10 and 20 m for the

same period of time. These results underline that the Ca3SiO5 based posterior restorative

material display high bioactivity.

Discussion

The formation of reactionary dentin is due to the stimulation of secretory odontoblasts and/or

the celles of the so-called Höehl’s layer, also named sub-odontoblastic layer, which may

differentiate and replace the wounded odontoblasts. “Stains all” did not stain the reactionary

dentin produced in this work after stimulation with BiodentineTM

. This dye reveals the

presence of phosphorylated proteins, as it is the case for the sound primary and secondary

circumpulpal dentin (Takagi & Sasaki, 1986). This suggests either that the proteins of the

SIBLING family are not present in reactionary dentin, leading to impaired mineralization, or

that the proteins are there but underphosphorylated.

Even defective, the formation of this layer increases the remaining dentin thickness (RDT)

between the deeper part of the cavity and the pulp. This is a crucial parameter that reduces the

cytotoxic effects of resin monomers that can be adsorbed on apatitic crystals. Reactionary

dentin may be tubular, or of the osteodentin type with cell inclusions, or atubular. Little is

known on the respective properties of these three types of dentin with respect to their

potential to protect the pulp.

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To conclude this in vivo experimental animal approach, indicates that the bioactive cement

stimulated the formation of reactionary dentin and allowed keeping the pulp alive despite the

preparation of a deep cavity and the placement of a filling material. However, we have now

to investigate up to which point the reactionary dentin is formed. Is there a stop or is it a

continuous reaction? This has yet to be studied on longer term studies, and to be confirmed by

pilot human studies.

References

Decup F., Six N., Palmier B., Buch D., Lasfargues J-J., Salih E., Goldberg M.(2000) Bone

sialoprotein-induced reparative dentinogenesis in the pulp of rat’s molar Clinical Oral

Investigations, 4 : 110-119.

Ohshima H. Ultrastructural changes in odontoblasts and pulp capillaries following cavity

preparation in rat molars Arch Histol Cytol 1990; 53: 423-438.

Six N., Lasfargues J-J., Goldberg M. (2000) In vivo study of the pulp reaction to Fuji IX, a

glass ionomer cement. J Dentisty, 28 : 413-422.

Takagi Y, Sasaki S. Histological distribution of phosphophoryn in normal and pathological

human dentins. J Oral Pathol 1986; 15: 463-467.

Wennberg A, Mjör IA, Hensten-Pettersen A. Biological evaluation of dental restorative

materials- a comparison of different test methods. J Biomat Med Res 1983; 17: 23-36.

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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 Journée Scientifique du CNEOC Brest -June 2009

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101

A clinical study of a new Ca3SiO5-based material indicated as a dentine substitute G.Weissrock1, J.C. Franquin1, P. Colon2 , G.Koubi1

1 Dpt. of Operative and Endodontics, University of Marseille, F. 2 Dpt. of Operative Dentistry and Endodontics, University of Paris 7, F

INTRODUCTION: A new Ca3SiO5-based material (BiodentineTM, Septodont) has been developed as a dentine substitute for direct and indirect posterior fillings. This new material is in vitro biocompatible [1], and could be used as pulp capping agent and bulk restorative material. A three-year follow-up randomized multicentric clinical study was initiated to evaluate: 1) its longevity and biocompatibility as temporary restoration vs a resin composite (Z100, 3M), and 2) its ability to be combined with an adhesive filling. METHODS: 334 patients (162 female and 172 male), aged from 18 to 80, with a mean age of 47, were selected and distribution of material was randomized by means of a computer-generated randomization list. Vital first and second permanent premolars and molars with Class I or Class II lesions were included. For restorations with BiodentineTM, no special chemical procedures were applied on the mineralized walls. All patients treated with BiodentineTM were educated to come back to the author’s clinic if any incident could diminish the clinical efficacy of the temporary restoration. When necessary, a definitive restoration was applied with Z 100, as previously described. The reasons of the new definitive treatment was noted (wear, fractures, loss of contact point), and the longevity of the temporary restoration. The restorations were evaluated at the baseline, 15 d, and 6, 12, 24, 36 m. Each restoration was evaluated with slightly modified USPHS criteria [2]. Radiographs and intra-oral colour slides were taken at baseline and at each recall period. RESULTS: Longevity and Biocompatibility : Before 6 months, no BiodentineTM filling need to be replaced, and all restorations evaluated at d+6 months demonstrated acceptable clinical performance. After 6 months, BiodentineTM show an occlusal wear, and 64 on 170 restorations were used as base to support a definitive restoration in Z 100. During the course of the study, 24 teeth with deep cavities received a direct pulp capping, with BiodentineTM. At this time, 20 teeth have been

checked, all of them with a positive pulp vitality, after healing periods from 3 to 26 m. Ability to be combined with adhesive filling: None of these 64 retreated patients presented pain, unpleasant physiologic or pathologic sensations. All the retreated teeth present a positive vitality test. No gap or secondary decay was observable between BiodentineTM and the dentinal walls. DISCUSSION & CONCLUSIONS: In the 64 cases, with complementary Z 100 restorations, 27 have been yet controlled and considered satisfactory, after a mean period of 16 months. Three years after the beginning of the study, the results indicate that the new Ca3SiO5-based material could be used as dentin substitute for definitive dentinal treatment, in restoration of posterior teeth. It could be used as a pulp capping agent and as a bulk restorative material at the same time. As marginal leakage and secondary decays remain a concern in adhesive dentistry, BiodentineTM could be preserved if re-intervention is required, and seems compatible with an adhesive filling. This new product seems to include the most important qualities for a dentine substitute: biocompatibility and longevity. It could find rapidly a place in the therapeutic practitioner’s equipment. REFERENCES : 1P.Laurent, et al. (2008) Induction of specific cell responses to a Ca3SiO5-based posterior restorative material. Dent Mater 24:1486-1494. 2 R.Hickel, et al. (2007) Recommendations for conducting controlled clinical studies of dental restorative materials (2007) Clin Oral Investig 11:5-33.

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BiodentineTM- RD94, A portland cement, stimulates in vivo reactionary dentin formation 2009 T.Boukpessi, F.Decup, D.Septier, M. Goldberg, C. Chaussain, France Journée Scientifique du CNEOC Brest -June 2009

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BIODENTINETM- RD94, A PORTLAND CEMENT, STIMULATES IN VIVO REACTIONARY DENTIN FORMATION

T. BOUKPESSI, F. DECUP, D. SEPTIER, M. GOLDBERG, C. CHAUSSAIN EA 2496 Groupe Matrices extracellulaires et biominéralisation, Faculté de Chirurgie Dentaire, Université

Paris Descartes

INTRODUCTION: RD94 is an experimental Portland cement aiming to be a glass ionomer cement and composite- resin substitute in restorative dentistry because of its properties of biocompatibility and bioactivity. The Aim of the study was to evaluate the effects of RD94 on the formation of reactionary dentin. In vivo experiments were carried out on the rat upper as an appropriate model.

METHODS: Half-moon cavities were prepared on the mesial aspect of the molar without pulp exposure. The cavities were then occluded with a conventional glass- ionomer cement. Comparison was made with a sham group (no implantation) and with a group receiving RD 94. After 8, 15, and 30 days, the rats were killed by heart perfusion with the fixative solution. All the blocks that include the three maxillary molars were extracted and processed for light microscopy. Measurements were done on images obtained after histological analysis.

Fig.1: Half-moon cavities were prepared on the mesial aspect of the first molar without pulp exposure

RESULTS: Eight days after tooth preparation, a few inflammatory cells were seen, mostly located in the pulp surface near the cavity. In RD94 group, a 20-40m thick layer of reactionary dentin was formed beneath a calcio-traumatic line, in contrast to the two other groups where the reactionary dentin thickness was about 10m. After 15 days, the inflammatory process was resolved in the pulps of all groups. In the RD 94 group, the outer part of the pulp chamber was filled with a 40-80m thick layer of reactionary dentin beneath the calcio-

traumatic line. After 30 days using RD94 as restorative material, reactionary dentin was about 160m thick, whereas, the rest of the pulp looked normal.

Fig.2: After 30 days using RD94 as restorative material, reactionary dentin (rd) formation was really increased in width, whereas the rest of pulp looked normal.

DISCUSSION & CONCLUSIONS: The present data suggest that RD94 displays novel bioactive properties and is capable to induce the formation of reparative dentinal tissue in the rat molar model. These results suggest that restorative treatment with RD94 provides new prospects for dental therapy.

REFERENCES:

Six N, Lasfargues JJ, Goldberg M. In vitro study of the pulp reaction to Fuji IX, a glass ionomer cement. J.Dent. 2000 Aug;28(6):413-22. Decup F. et al. Bone sialoprotein-induced reparative dentinogenesis in the pulp of rat’s molar. Clin Oral Invest.2000 Jun;4(2):110-9.

ACKNOWLEDGEMENTS: we acknowledge SEPTODONT, France, for their financial support to this investigation.

SHAM

rd

X100 X 100

rd

rd

RD94

X100

X100

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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)

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A Clinical Study of a New CaA Clinical Study of a New Ca 33SiOSiO55--based Material based Material Indicated as a Dentine SubstituteIndicated as a Dentine Substitute

G.F. KOUBI1, J.C. FRANQUIN 1, P. COLON2

1 Dpt. of Operative Dentistry and Endodontics, Univer sity of Marseilles, France. 2 Dpt. of Operative Dentistry and Endodontics, Univer sity of Paris 7, France.

OP065

AimAimA new Ca3SiO5-based material (Biodentine™ RD94, Septodont) has been developed as a dentine substitute before direct and indirect posterior fillings. A recent study

conclude that this new material is in vitro biocompatible. It could be used as a pulp capping agent and as a bulk restorative material at the same time. A three-year follow-

up randomized multicentric clinical study was initiated to evaluate: 1) its longevity and in vivo biocompatibility as temporary restoration vs a resin composite (Z100, 3M),

and 2) its ability to be combined with an adhesive filling.

Materials and MethodsMaterials and MethodsLongevity and Biocompatibility in vivoThree hundred and thirty-four (n = 334) patients (162 female and 172 male), aging from 18 to 80 years, with a

mean age of 47 years, were selected to participate in this study.

Distribution of material (Biodentine RD94 versus Z100) was randomized by means of a computer-generated

randomization list. One dentist, well experienced with both materials, placed all restorations.

Vital first and second permanent premolars and molars with Class I or Class II lesions were included. For

restorations with Z100 (3M, St Paul, MN, USA) inserted according to the manufacturer’s recommendation , no

cavity liners for indirect pulp capping, and no cavity bases were applied in addition to the adhesive (All Bond

2, Bisco, Ill., USA).

Composition of Biodentine RD94 (Septodont, France)

5900 osc./minPurified water,

calcium chlor

LIQUID

200 mL

Mixed 25 s.

Linea Tac

Tricalcium silicate,

calcium carbonate,

calcium oxide

POWDER

1 g

For Biodentine™ RD 94, the new Ca3SiO5-based cement (Septodont, Saint-Maur, France), developed as a bulk dentine substitute before direct and indirect posterior fillings,

no special chemical procedures were applied on the mineralized walls.

Ability to be combine with an adhesive fillingWhen necessary, a definitive restoration was applied with Z100, in the same conditions previously described. The reason of the new definitive treatment was noted (wear,

fractures, loss of contact point), as well as the longevity of the temporary restoration.

Evaluation The restorations were evaluated direct after placement (baseline), 15 days, and 6, 12, 24, 36 months. Each restoration was evaluated with slightly modified USPHS criteria

for the following characteristics: anatomical form, marginal adaptation, colour matching, marginal staining, surface texture, and secondary caries. The cavity form, the

depth and the clinical dimension of the cavities were also noticed. Radiographs were taken for assessments of proximal integrity and presence of recurrent caries. Intra-

oral colour slides were taken at baseline and at each recall period with a Nikon 70, equipped with medical lens Nikon 105 mm.

ResultsResultsThe clinical study is still on-going. These results of the distribution of restorations by patient’s age are summarized in Figure 1, Figure 2, Figure 3.

18-29 y: 21.55%

30-39 y: 20.65%

40-49 y: 22.18%

50-59 y: 16.77%

60-69 y: 15.56%

70-80 Y: 3.29%

Figure 1. Distribution of restorations by patient’ s age Total RD 94 Z 1000

100

200

300

Class I

Class IIClass I

Class II

Figure 2. Distribution of restorations by cavity c lass0

100

200

300

400

Total RD 94 Z 100

PM

M

PM

M

Figure 3. Distribution of restorations by group of teeth

Longevity and BiocompatibilityOn the 334 patients able to be recalled at 6 months, 254 came back for this control, given a recall percentage of 76 % and 48 % for d+1 year.

At the baseline, 24 teeth with deep cavities have to support a pulp capping with Biodentine RD94. At this time, 20 teeth have been checked, all of them with a positive pulp vitality, after

healing periods from 15 days to 26 months

Before 6 months, no Biodentine RD94 need to be replaced, and all restorations evaluated in this study at d+15 and d+6 months demonstrated acceptable clinical performance within the

evaluation period based on the Alfa and Bravo ratings for clinical satisfactory restorations.

After 6 months, some clinical failures were noted with BiodentineTM RD94 because of a too rapid occlusal wear.

48 restorations with BiodentineTM RD94 have to be replaced by a definitive restoration in Z100.

Ability to be combine with Adhesive filling Z100None of these 48 retreated patients has lodged negative complaint for pain, unpleasant physiologic or pathologic sensations. The calcium silicate cement was easily preserved on all

dentinal walls, and the operator could easily prepare well calibrated cavities, without any infiltration or secondary caries. All the retreated teeth present a positive vitality test without any

hypersensitivity, and all of them have kept BiodentineTM RD94 on the dentinal walls, as bulk or liner in a sufficient quantity to permit directly a normal cavity preparation. No gap and no

secondary decay were observable between the temporary product BiodentineTM RD94 and the dentinal walls, and the preparation of the cavity for the definitive restoration was easy.

In these 48 cases, with complementary Z100 restorations, 28 have been yet controlled and considered satisfactory, after a mean period of 14 months. The second series of definitive

restorations in Z100 inserted on temporary filling will be evaluated with the same methods previously described, and then compared to the first series of Z100.

ConclusionConclusionThree years after the beginning of the study, the results seems to indicate that the new Ca3SiO5-based material called Biodentine

TMRD94 could be used as dentin

substitute for definitive dentinal treatment, in restoration of posterior teeth. It could be used as a pulp capping agent and as a bulk restorative material at the same

time. The mean duration of BiodentineTMRD94 restorations was 12 months, with a minimum of 6 months and maxima of 35 months. As marginal leakage and

secondary decays remain a concern in adhesive dentistry, BiodentineTMRD94 can be preserved if re-intervention is required, and seems compatible with an

adhesive filling. This new product seems to include the most important qualities for a dentine substitute : biocompatibility and longevity. It could find rapidly a

place in the therapeutic practitioner’s equipment.

SEVILLE, SPAIN, MARCH12th – 14th 2009

253

137 116

3381

48

RD94 Z100

233

124 10910145 56

RD94 Z100

Biodentine™ as bulk Biodentine™ as liner

BibliographyBibliographyP. Laurent, J. Camps, M. De Méo, J. Dejou, I. About. Induction of specific cell responses to a Ca3SiO5-based posterior restorative material. Dental Materials 2008;24:1486-1494.

C. Boinon, Collage sur un substitut dentinaire à base de silicate de calcium, Thèse de Doctorat en Chirurgie Dentaire, Marseille, Juin 2006.51/74

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Induction of specific cell responses to a Ca3SiO5-based posterior restorative material 2008 Laurent P, Camps J, De Méo M, Déjou J, About I. Marseille, France. Dent Mater. 2008 Nov;24(11):1486-94.

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d e n t a l m a t e r i a l s 2 4 ( 2 0 0 8 ) 1486–1494

avai lab le at www.sc iencedi rec t .com

journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls /dema

Induction of specific cell responses to a Ca3SiO5-basedposterior restorative material

Patrick Laurenta, Jean Campsa, Michel De Meob, Jacques Dejoua, Imad Abouta,∗

a Laboratoire IMEB - ERT 30, Faculte d’Odontologie, Universite de la Mediterranee, 27 Boulevard Jean Moulin,13355 Marseille Cedex 05, Franceb Laboratoire de Biogenotoxicologie et Mutagenese Environnementale (EA 1784), Faculte de Pharmacie,Universite de la Mediterranee, Marseille, France

a r t i c l e i n f o

Article history:

Received 23 January 2007

Received in revised form

16 December 2007

Accepted 25 February 2008

Keywords:

Ca3SiO5-based dental cement

Biocompatibility

Genotoxicity

a b s t r a c t

Objectives. A Ca3SiO5-based cement has been developed to circumvent the shortcomings

of traditional filling materials. The purpose of this work was to evaluate its genotoxicity,

cytotoxicity and effects on the target cells’ specific functions.

Methods. Ames’ test was applied on four Salmonella typhimurium strains. The micronuclei test

was studied on human lymphocytes. The cytotoxicity (MTT test), the Comet assay and the

effects on the specific functions by immunohistochemistry were performed on human pulp

fibroblasts.

Results. Ames’ test did not show any evidence of mutagenicity. The incidence of lympho-

cytes with micronuclei and the percentage of tail DNA in the Comet assay were similar to

the negative control. The percentage of cell mortality with the new cement as performed

with the MTT test was similar to that of biocompatible materials such as mineral trioxide

aggregate (MTA) and was less than that obtained with Dycal. The new material does not

affect the target cells’ specific functions such as mineralization, as well as expression of

collagen I, dentin sialoprotein and Nestin.

Significance. The new cement is biocompatible and does not affect the specific functions of

target cells. It can be used safely in the clinic as a single bulk restorative material without

any conditioning treatment. It can be used as a potential alternative to traditionally used

posterior restorative materials.

emy

age, and unreacted monomer and toxic ingredient release

© 2008 Acad

1. Introduction

Commonly used direct restorative materials for Class I and IIcavities are resin composites and amalgams [1,2]. In the early1980s, amalgam restorations were reported to release mercuryvapors which may be harmful to the environment, the dentist

as well as the patient [3].

Direct composite restorations have gradually been usedto replace amalgam for anterior restorations and small- to

∗ Corresponding author. Tel.: +33 4 91 80 43 43; fax: +33 4 91 80 43 43.E-mail address: Imad.about@odontologie.univ-mrs.fr (I. About).

0109-5641/$ – see front matter © 2008 Academy of Dental Materials. Pudoi:10.1016/j.dental.2008.02.020

of Dental Materials. Published by Elsevier Ltd. All rights reserved.

moderate-sized posterior restorations. In contrast to amal-gam, resin composites enable micro-mechanical retentionby the use of different bonding techniques. Yet there isstill some concern with composite resin wear resistance inhigh-stress situations, polymerization shrinkage, microleak-

[4–6].Search for a replacement for amalgam and resin compos-

ites has been ongoing for many years. Calcium hydroxide

blished by Elsevier Ltd. All rights reserved.

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Dycal®) is one of the most widely used pulp capping agents. Itsasic pH is the main reason for its apparent toxicity in vitro [7].owever, it has been demonstrated that a dentin bridge forma-

ion can be obtained with this material 3 months after cappinguman teeth with mild to moderate chronic inflammation,ild hyperemia and necrosis [7,8].Recent research focused on the use of biocompatible

aterials such as Portland cement. Mineral trioxide aggre-ate developed in the 1990s as a root-end filling materialas a similar constitution to Portland cement and is com-osed primarily of tricalcium and dicalcium silicate [9]. It

s known as a biocompatible material. In vitro, a high ratef cell viability was reported with MTA extracts with aethyltetrazoilum (MTT) assay [10–12]. Additionally, MTA

sed for pulp capping or partial pulpotomy stimulates repar-tive dentin and complete bridge formation in vivo after

months with no signs of inflammation [8,13,14]. How-ver, the setting time of MTA is 2 h 45 min which is tooong for a material to be used as a dental restorative mate-ial [15]. Moreover, the mechanical properties of both Dycalnd MTA are not compatible for use as dental restorativeaterial.Tricalcium silicate is the main constituent of MTA, and

he main raw material in Portland cement. It is known thata3SiO5 possesses hydraulic property and the spontaneousevelopment of strength on hydration. But its setting time isoo long and its compressive strength hardly reaches 20.2 MPafter 28 days to meet the need of clinical applications asrestorative material [16]. Calcium chloride is one of theost effective accelerators of hydration and setting in Port-

and cement pastes. Although the addition of CaCl2 up to 15%n the liquid phase into Ca3SiO5 decreased the final settingime from 180 to 90 min, the compressive strength remainedeak (23.46 MPa) at 7 days [17]. The use of superplasticis-

rs as very effective dispersing agents to reduce the waterontent was used in fast setting Portland cements. This haseen shown to lower the setting time to 7 min but the com-ressive resistance did not exceed 50 MPa even after 28 days

18].Based on Portland cement properties, a Ca3SiO5-based

aterial for direct restorative posterior fillings has been devel-ped in the authors’ laboratory. The material is inorganic andon-metallic. It is composed of Ca3SiO5, CaCO3, ZrO2, waternd a superplasticising admixture to reduce the water con-ent of the mix and to retain its workability. This material isresented in the form of a powder and a liquid and can berepared by mixing with an amalgamator. The new Ca3SiO5

ement is compatible with working in the clinic. It has a set-ing time of 10 min and was developed to be used in directnd indirect pulp capping procedures as a single applica-ion bulk restorative material without any cavity conditioningreatment. Since it may be directly applied to the dentalulp, its biological properties were compared to biomateri-ls usually used in pulp capping procedures such as MTA andycal.

Since this material belongs to a new class of restorativeaterials, its biocompatibility is questioned and in this paper

ts cytotoxicity and genotoxicity are investigated. The effectt may have on the specific functions of target cells was alsovaluated.

0 0 8 ) 1486–1494 1487

2. Materials and methods

2.1. Reagents

All materials used for culture media preparation werepurchased from Gibco BRL (Life Technologies Inc., GrandIsland, NY, USA) unless otherwise specified. Minimum Essen-tial Medium (MEM) was supplemented with 10% fetalbovine serum; 100 UI/ml penicillin; 100 �g/ml streptomycin(Biowhittaker, Gagny, France) and 0.25 �g/ml amphotericin B(Fungizone®). Chemicals were obtained from Sigma–Aldrich(Sigma Chemicals Corp., St. Louis, MO) unless otherwisestated.

2.2. Teeth

For pulp cell cultures, normal immature third molars freshlyextracted for orthodontic reasons from 16 to 18 year-oldpatients were used after obtaining theirs and their parents’informed consent and was conducted with local ethical com-mittee approval. Additionally, for the preparation of dentinslices, 30 healthy human third molars freshly extracted werestored at 4 ◦C in saline solution and used within 2 h of collec-tion.

S. typhimurium strains TA97a, TA98, TA100, and TA102 werekindly provided by Dr. B.N. Ames (Berkeley, CA, USA).

2.3. Antibodies

Polyclonal antibodies against the type I collagen werepurchased from Southern Biotechnology Associates Inc.(Birmingham, AL, USA). Anti-dentin sialoprotein antibodieswere obtained from WT Butler (UTHSC, Houston, TX, USA).Preparation and characterization of the polyclonal antibodiesagainst dentin sialoprotein (DSP) have been already described[10]. Anti-nestin antibodies were purchased from ChemiconInternational (Temecula, CA, USA).

This work was performed on a new Ca3SiO5-basedcement developed with an industrial partner (LaboratoiresSeptodont, Saint Maur des Fosses, France). MTA (DentsplyTulsa dental, Tulsa, OK, USA) (batch number 0203332604) andDycal (De Trey Dentsply, Milford, DE, USA) (batch number0204000983) were used as a reference material for cytotoxicitytests.

2.4. Toxicity by indirect contact between thebiomaterial and the culture media

2.4.1. Preparation of the dentin slicesFrom the third molars, thirty dentin slices were prepared witha low speed diamond saw (Isomet, Buehler Ltd., Lake Bluff,IL, USA) with water coolant. The dentin sections were fromareas adjacent to the pulp chamber, but they showed no evi-dence of inclusion of a pulpal horn. The dentin slices had athickness of 0.7 ± 0.05 mm. To create a constant dentin sur-

face area, a Plexiglas ring 1 cm thick, 2 cm in diameter with ahole of 0.8 cm in its center was placed on the pulpal side of thedentin slice and was attached with a non-cytotoxic cyanoacry-late glue. This permitted us to reduce and to standardise the

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s 2 4

1488 d e n t a l m a t e r i a l

exposed dentin surface area to 50.24 mm2. The coronal sideof the dentin slice was covered with 1 mm thick MTA (n = 10),new cement (n = 10) and Dycal (n = 10). The reference materi-als were applied according to the manufacturers’ instructions.The new Ca3SiO5 cement was prepared by mixing the recom-mended quantities of liquid and powder and vibrating with anamalgamator. It was applied without any conditioning treat-ment.

2.4.2. Simulation of pulpal pressureThe Plexiglas rings and the dentin slices were placed ina special device used to simulate a pulsatile pulpal pres-sure, as previously described [19]. The Plexiglas device wasused to maintain the dentin slice in such a position thatthe MEM culture medium slightly touched the pulpal sideof the dentin slice while the coronal side was open to theatmosphere. The lower chamber (4 ml), in contact with thepulpal side of dentin contained the culture medium. A pul-satile pulpal pressure (12–18 cm H2O) was applied. The dentinslices were inserted in the Plexiglas device for 24 h. After24 h, the media were collected and called the indirect contactmedia.

2.5. Toxicity by direct contact between the biomaterialand the culture media

Ten samples of each material were prepared according to man-ufacturer recommendations and stored in an incubator priorto sterilization with UV rays. The Ca3SiO5 cement was pre-pared as described above. The samples were stored in 1 mlMEM with 10% foetal calf serum supplemented with penicillin100 IU/ml and streptomycin 100 �g/ml for 24 h. According toISO standards, the ratio between the surface of the sampleand the volume of medium was 0.5 cm2/ml. The resulting pHvalues in the buffered culture medium were: Dycal 9.2; MTA8.1 the Ca3SiO5 cement 8.2. These media were called the directcontact media (n = 10 per material).

2.6. MTT assay

Pulpal fibroblasts were plated at 30,000 cells cm−2 in 96-wellplates (Falcon 3072, Becton Dickinson, Oxford, GB). The 96-well dishes were then placed into a humid incubator withan atmosphere of 5% CO2, 95% air for 24 h prior to use. Afterthis 24 h period, the medium from the 96-well plates wasremoved and replaced by the test medium. At that time, the96-well plates were placed in an incubator again for 24 h. Asuccinyl dehydrogenase assay (MTT) was performed on thedishes after 24 h of incubation (i.e., 48 h after the beginning ofthe experiment). The medium was removed and immediatelyreplaced with 100 �l/well of a 0.5% of 3-(4,5-dimethylthiazol-2-yl)-2,(-diphenyl tetrazolium bromide) in the medium. Afterincubation for 2 h at 37 ◦C, the supernatant was discarded,and the formazan crystals were solubilized with 100 �l/wellof dimethyl sulfoxide (DMSO). The absorbance of each 96-well dish was measured using an automatic microplate

spectrophotometer (E 960, Bioblock, Strasbourg, France)at 550 nm.

For direct and indirect contact media, a two-way analysis ofvariance (medium dilution and material), followed by a Dun-

( 2 0 0 8 ) 1486–1494

can test, was used to compare the cytotoxicity of MTA, the newCa3SiO5 cement and Dycal.

In order to study the long term effects on the pulp fibrob-lasts differentiation, it is known that lower concentrationscan be toxic after long term incubation with cells. Thus, themedium used for the next part of the study was one whichdecreased the MTT activity by less than 5%.

2.7. Influence of the new Ca3SiO5 cement and MTA onthe differentiation of pulp fibroblasts

In order to evaluate the effect of the new Ca3SiO5 cementand MTA on the differentiation of pulp fibroblasts, the cul-tured cells were incubated in the conditioned MEM mediumobtained after direct and indirect contact with the materi-als supplemented with 2 mM �-glycerophosphate. The cellswere cultured for 4 weeks in cell culture chambers and themedia were changed every other day. After culture, the cellswere fixed with 70% ethanol for one hour at 4 ◦C and pro-cessed for immunohistochemistry. The effect of the materialson the cytodifferentiation was evaluated by studying the spe-cific protein expression of control cells compared to that ofcells cultured with the medium after being in contact withthe test material [20].

2.7.1. ImmunohistochemistryThe cells were permeabilized for 15 min with 0.5% Triton X-100in PBS. Primary antibodies were diluted in PBS containing 0.1%Bovine Serum Albumin (BSA). The incubation with primaryantibodies was performed overnight at 4 ◦C. Anti-collagen Iantibodies were used at 40 �g/ml and anti-nestin antibodyat 5 �g/ml. Anti-dentin sialoprotein antibody was diluted1:200 in PBS. Immunostaining was revealed using the labeledstreptavidin-biotin kit (LSAB; Dako Corporation, Carpinteria,CA, USA) according to the manufacturer’s instructions. Glyc-ergel was used as a mounting medium (Dako Corporation).Controls were performed by omitting primary antibodies orincubation with unrelated primary antibodies (cytokeratin 19).All controls were negative.

2.8. Genotoxicity assays

2.8.1. Ames testS. typhimurium TA97a, TA98, TA100, and TA102 strains weregrown overnight from frozen cultures in Oxoid nutrient brothNo. 2 for 10–12 h. Mutagenicity assays were performed asdescribed [21]. The genotype of each S. typhimurium testerstrain was confirmed in each experiment, and negative andpositive controls were routinely included.

After the preparation and setting of the cement, it wasground to prepare a stock solution prior to testing by adding60 mg of the cement in 1 ml of Nutrient Broth No. 2 (NB 2)medium or DMSO solvent for 24 h at 37 ◦C under mixing. Thesestock solutions from two independent experiments were thentested in triplicate and results from both experiments in NB2 and DMSO are presented. Increasing volumes of test sam-

ples (4, 6, 8 and 10 �l) were incubated with each bacterialstrain for 60 min at 37 ◦C under mixing. The mixture consist-ing of bacteria and a test compound was plated on platesin VB medium. The bacteria were then incubated at 37 ◦C

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4 ( 2

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(Table 1). None of the materials was cytotoxic. However, whenthe toxicity was evaluated without dentin slice interposition,the analysis of variance showed a statistically significant dif-ference among the three materials (P < 0.001). The Duncan test

Table 1 – Cytotoxicity after indirect contact between thematerials and culture medium through a dentin disc

New Ca3SiO5 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 new Ca3SiO5 cement, MTA, and Dycal were applied on the coro-nal side of the dentin slices in Plexiglass devices with pulp pressuresimulation. 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 fibroblasts were incubated with these media (either undi-luted, or diluted in the culture medium to 50% or to 10%) for 24 h

d e n t a l m a t e r i a l s 2

or 48 h and revertant colonies were counted with an auto-ated colony counter (Spiral System Instruments, Bethesda,S, USA). The experiments were carried out in the presence

nd in the absence of an S9 fraction isolated from liver ofhenobarbital/�-naphtoflavone-treated rats. This S9 fraction

4%) was routinely included in an S9-Mix, and the amount ofrotein was adjusted to 1.25 mg protein per plate. A substanceas qualified positive if it induced a dose-related and repro-ucible increase in the numbers of revertants or twice as manypontaneous revertants per plate [22].

.9. Micronucleus test

his work was performed on lymphocytes obtained byein puncture from 6 healthy non-smoking donors, afternformed consent, and collected in glass tubes containingithium heparin anticoagulant according to Digue et al. [23].riefly, cultures were carried out by adding 0.7 ml of wholelood to 9.3 ml of X-VIVOTM Medium (Bio-Whittaker, Bel-ium) supplemented with 25% fetal calf serum (Gibco, LifeechnologiesTM, Germany), heparin (50 U/ml), and antibiotics

penicillin 100 Ul/ml and streptomycin 100 �g/ml). The cellsere stimulated with phytohemagglutinin (1 mg/ml), a spe-

ific mitogen agent of human T-lymphocytes. The cells werehen cultured for 72 h at 37 ◦C in a humidified atmosphereontaining 5% CO2.

The Ca3SiO5 cement extract was prepared as describedbove in the culture medium or DMSO and added to the culturet 24 h. The cells were directly exposed to serial dilutions (1%,.3%, 3.7%, and 5%) of the cement extracts for 48 h. Negativeontrol was achieved by adding DMSO at a final concentrationf 0.1%. Mitomycin C, used as a reference genotoxic agent, wassed as positive control 5 �g/ml. Cytochalasin B was added tohe culture (5 �g/ml) 44 h after PHA stimulation.

The cultures were stopped at 72 h and the cells harvestedy centrifuging (10 min at 360 g). They were then treatedy a mild hypotonic treatment (1 min in KCl 0.075 M) andmmediately fixed with methanol:acetic acid (3:1). This fix-ng step was repeated twice after 20-min storage at 4 ◦C. Cellsere smeared on pre-cleaned microscope slides and air-dried.taining was performed with 5% Giemsa in Milli-Q water for5 min.

Stained slides were coded and scored by light microscopy at00× magnification. For each slide, 1000 Giemsa-stained bin-cleated lymphocytes with a well-preserved cytoplasm werecored for the presence of micronuclei. In the micronucleatedinucleated cells, the number of MN per cell was recorded.icronuclei were expressed in terms of micronucleated cells

er 1000 binucleated lymphocytes. All the slides were exam-ned twice by the same scorer. As a measure for toxicity, theinuclearity index (BI) was determined by scoring the binu-leated cells for 1000 lymphocytes (mono- and binucleatedells) and linked to the percentage of lymphocytes that pro-uced complete cell division for the different drugs tested,nd then provided an index of cytotoxicity [24]. An extract

f a material was considered positive if at least a three-fold

ncrease of the numbers of micronuclei over negative controlsas observed at one or more dilutions of the original extract

25,26].

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2.10. Single-cell gel (Comet) assay

The Ca3SiO5 cement extract was prepared and put in MEMmedium (60 mg/ml) for 24 h at 37 ◦C under mixing. The cellswere directly exposed to serial dilutions of the cement extractsfor 2 h. The protocol used for single-cell gel (Comet) assayfollowed the guidelines proposed by Tice et al. [27]. Briefly avolume of 10 �l of cells (104 cells) of each treatment was addedto 120 �l of 0.5% low-melting-point agarose at 37 ◦C, layeredonto a pre-coated slide with 1.5% regular agarose, and coveredwith a coverslip. After brief agarose solidification in a refrig-erator, the coverslip was removed and the slides immersedin lysis solution (2.5 mol/l NaCl, 100 mmol/l EDTA, 10 mmol/lTris–HCl buffer pH 10, 1% sodium sarcosinate with 1% TritonX-100, and 10% DMSO) for about 1 h. Prior to electrophore-sis, the slides were left in alkaline buffer (pH >13) for 20 minand 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 Tris–HCI (pH 7.5) fixed in absolute ethanol,and stored at room temperature until analysis blindly in afluorescence microscope at 400× magnifications. In order tominimize extraneous DNA damage from ambient ultravioletradiation, all steps were performed with reduced illumina-tion. An automatic analysis system (Comet Assay II; PerceptiveInstruments, Haverhill, UK) was used to determine DNA dam-age. Tail moment (product of tail DNA/total DNA by the centerof gravity) was considered to estimate DNA damage from 50cells per treatment.

3. Results

3.1. Determination of the toxicity with or withoutdentin disc interposition

When the toxicity was evaluated indirectly through a dentinslice, the analysis of variance failed to show a statistical differ-ence between the new cement, Pro Root MTA, and Dycal (ns)

before applying the MTT test on human pulpal fibroblasts. Opti-cal density values of untreated control cultures normalized to 100%was in the range of 0.9–0.95. The results are expressed as mean celltoxicity ± S.D.

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Table 2 – Cytotoxicity after direct contact between thematerial and culture medium

New Ca3SiO5 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 onhuman pulp fibroblasts was evaluated after 24 h contact betweenthe materials and the culture medium (either undiluted, or diluted

in the culture medium to 50% or to 10%) with the MTT test. Bothwere less cytotoxic than Dycal (P < 0.001). The results are expressedas mean cell toxicity ± S.D.

showed that Dycal displayed a higher cytotoxicity than MTAand the new Ca3SiO5 cement (Table 2).

According to this study, a dilution of 10% was chosen forstudying the materials’ effects on fibroblasts specific functionsbecause it has biological effects without being toxic.

3.2. Influence of the two materials on pulp fibroblastsdifferentiation into odontoblastic cells

Control cells expressed collagen I, dentin sialoprotein andNestin. Pulp fibroblasts secreted a mineralizd matrix and thecells, particularly those contacting the mineralizd matrix,expressed Nestin (Figs. 1 and 2).

Fig. 1 – Effect of the new Ca3SiO5 cement on pulp fibroblastspecific gene expression. Immunohistochemistry was usedto evaluate the effect of the new Ca3SiO5 cement and MTAon pulp cells specific genes expression. Control culturesexpress collagen type I (a) and dentin sialoprotein (b).When the media containing the new Ca3SiO5 cement (cand d) and MTA (e and f) extracts were added to thecultures for 4 weeks, collagen I (c and e) and dentinsialoprotein (d and f) were also expressed at a high level inthe pulp cells. Original magnifications = ×10.

Fig. 2 – Effect of the new Ca3SiO5 cement on pulp cellsmineralization. Immunohistochemistry was used toevaluate the effect of the new Ca3SiO5 cement and MTA onpulp cells differentiation and mineralization. Controlcultures express Nestin and secrete a mineralized matrix inthe form of nodules (a). When the media containing thenew cement (b) or MTA (c) extracts were added to thecultures for 4 weeks, a mineralized matrix deposition wasalso observed. Nestin was also expressed at a high level in

pulp cells and its expression was stronger in the mineralnodules forming cells. Original magnifications = ×10.

After adding the media containing extracts of the newCa3SiO5 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 in cellsforming the mineral nodules than the cells away from thesenodules.

3.3. Genotoxicity

Ames’ test did not show any evidence of mutagenicity of

the Nutrient Broth No 2 medium after being in contact withthe new cement, whatever the dilution of the test medium(Table 3). The mutations observed with the new cement werecomparable to the spontaneous reverse mutations obtained in

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Table 3 – Mutation frequencies of Ames tester strains using the liquid preincubation assay

Metabolic activation(S9 mixa)

Product Volume (�l) Number of revertants/plate (mean ± S.D.)

TA 97a TA 98 TA 100 TA 102

+ NB No. 2 10 171 ± 9 24 ± 3 136 ± 4 382 ± 17+ DMSO 10 166 ± 7 25 ± 1 125 ± 13 355 ± 16− NB No. 2 5 183 ± 13 26 ± 5 135 ± 11 402 ± 18− DMSO 5 191 ± 11 27 ± 4 138 ± 9 423 ± 26+ New Ca3SiO5 cement (NB No. 2 extract) 4 162 ± 14 30 ± 1 135 ± 14 360 ± 10

6 177 ± 5 26 ± 1 120 ± 2 397 ± 158 177 ± 4 29 ± 5 132 ± 5 351 ± 7

10 192 ± 4 27 ± 4 150 ± 13 345 ± 2− New Ca3SiO5 cement (NB No. 2 extract) 2 215 ± 11 25 ± 1 161 ± 10 500 ± 24

3 223 ± 9 25 ± 3 172 ± 21 424 ± 364 225 ± 15 25 ± 1 160 ± 35 439 ± 35 205 ± 23 23 ± 1 182 ± 12 517 ± 44

+ New Ca3SiO5 cement (DMSO extract) 4 170 ± 19 29 ± 2 119 ± 3 334 ± 496 189 ± 3 25 ± 2 126 ± 13 376 ± 38 175 ± 2 28 ± 8 145 ± 1 336 ± 24

10 164 ± 23 43 ± 7 136 ± 5 314 ± 11− New Ca3SiO5 cement

(DMSO extract)2 193 ± 2 35 ± 2 149 ± 3 421 ± 53 186 ± 5 37 ± 5 117 ± 8 445 ± 424 224 ± 17 27 ± 3 140 ± 6 463 ± 265 173 ± 8 30 ± 1 144 ± 3 435 ± 36

+ B[a]P 0.5 �g 1121 ± 37 423 ± 26 1000 ± 87 679 ± 28− ICR 191 0.02 �g 553 ± 21 NT NT NT− 2,4,7 TNFone 0.02 �g NT 165 ± 3 NT NT− NaN3 0.5 �g NT NT 585 ± 12 NT− MitC 0.2 �g NT NT NT 3658 ± 54

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 2or DMSO solvent for 24 h at 37 ◦C under mixing. The stock solutions from two independent experiments were tested in triplicate, and resultsfrom both experiments in NB 2 and DMSO are presented. Increasing volumes of test samples (4, 6, 8 and 10 �l) were incubated with the eachof 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 VBmedium 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 S9fraction. The test was qualified positive if it induced a dose-related and a reproducible increase of the numbers of revertants or twice higherthan the spontaneous revertants per plate. All data are expressed as means ± S.D. Positive controls were Benzo[a]pyrene (0.5 �g) with S9 MIX forall strains. Positive controls were 2-methoxy-6-chloro-9-(3-(2-chloro-ethyl)aminopropylamino)acridine (ICR 191, 0.1 �g) for TA97a; 2,4,7-trinitro-9-fluorenone (2,4,7-TNFone, 0.02 �g) for TA98; sodium azide (NaN3, 1 �g) for TA100 and mitomycin C (MitC, 0.05 �g) for TA102 without S9 MIX.

trrvs

NT: non-tested.a The S9 MIX included 4% S9, 4.2 mM NADP and 5.2 mM G6P.

he controls performed with the NB 2 and DMSO solvent. The

esults show that the new Ca3SiO5 cement does not induceeverse mutations either with or without the S9 metabolic acti-ation system. Similar results were obtained with all bacterialtrains tested.

Table 4 – Micronucleated human lymphocytes count inCa3SiO5 cement-treated cultures

Ca3SiO5 cement dilution Micronucleatedlymphocytes (%) ± S.D.

1% 4.0 ± 1.12.3% 4.0 ± 1.13.7% 4.0 ± 1.25% 4.2 ± 1.2Negative controla 3.7 ± 1.2Positive controlb 16.0 ± 6.0***

Comparison with the control: ***P < 0.001.a Culture medium X-VIVO 10.b Mitomycin C 5 �g/ml.

The micronuclei test revealed that after incubating thelymphocytes with different dilutions of the new cement, therate of lymphocytes with micronuclei was similar to thatobtained with the negative control. It ranged from 3.9% to4.1% with increasing concentrations (1–5%) in aqueous orhydrophobic medium. The positive control showed a rate of16% (Table 4).

The Comet assay performed with serial dilutions of the newCa3SiO5 cement on human pulp fibroblasts revealed that thepercentage of DNA in the tail ranged from 12.59 for the 0.1%dilution to 15.58 with undiluted medium. This percentage was13.19 with the negative control and 46.52 with the positivecontrol (Table 5).

4. Discussion

The biocompatibility of the new cement is shown in this studyby the absence of cytotoxicity and genotoxicity and the factthat the new material does not affect the cytodifferentiationof human pulp fibroblasts in odontoblastic cells.

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Table 5 – Comet assay on human pulp fibroblasts

Ca3SiO5 cement dilution Tail DNA (%) mean ± S.D.

0.1% 12.59 ± 0.961% 13.31 ± 0.8810% 14.90 ± 1.06Undiluted 15.58 ± 1.08Negative controla 13.19 ± 0.96Positive controlb 46.52 ± 1.45***

Comparison with the control: ***P < 0.001. NS: non-significant.

The expression of these specific proteins by human pulpal

a 0.1% DMSO.b H2O2 (13.2 mM).

Although Portland cements are known as non-toxic, in thiswork, 3 tests were performed to evaluate the genotoxicity ofthe new Ca3SiO5 cement after solubilisation in hydrophilic orhydrophobic conditions. These tests were performed becausethe cement developed here contains a modified polycar-boxylate in the superplastisizer. It has been reported thatpolycarboxylate (Aqualox®) elicited mutagenic effects on S.typhimurium TA 98 and TA 1535. In the presence of S9 fraction,Aqualox® elicited weak mutagenic effects on S. typhimurium TA1535 and dose-dependent mutagenic effects on S. typhimuriumTA 98 [28]. Here, Ames’ test performed with and without the S9fraction on 4 different bacterial strains including TA 98 failedto detect significant reverse mutations.

While Ames’ test was performed on prokaryotic cells, themicronucleus test and the Comet assay were performed oneukaryotic cells. The micronucleus test was important to per-form in order to detect any structural chromosomal alterationin the host cells involved in the defense mechanisms. Itrevealed that no chromosomal damage was found with thematerial. The Comet assay was developed as reliable biochem-ical technique for evaluating DNA damage and breaks in singlemammalian cells [27]. This test was performed on the tar-get cells of the new cement and did not show significantDNA breaks in human pulp fibroblasts. These results may beexplained either by the fact that the modification of polycar-boxylate suppressed its mutagenic effects or by the fact that itsconcentration is too low in the cement to have any mutageniceffect.

The new material was developed as a restorative materialboth for direct and indirect pulp capping. That is why toxic-ity was investigated under two conditions: indirectly throughdentin discs and directly by applying the medium containingthe new cement extract on the target cells. The new cementwas not toxic to the cells under either condition even whentested undiluted.

The toxicity of the new cement was compared to materi-als used in pulp capping situations. This study confirms theabsence of MTA toxicity. This material was introduced in the90s and is well accepted by endodontists as an excellent mate-rial for retrofilling, perforation repair and apexification. Thissuccess is due, in part, to the sealing properties of the mate-rial [15] but mainly to its biocompatibility [29,30]. It has beenshown that using the same MTT assay that MTA was non-toxic

to periodontal ligament fibroblasts [10] and human gingivalfibroblasts [31]. The current results corroborate those of twoother indirect contact studies using agarose superimposition

( 2 0 0 8 ) 1486–1494

[32] or millipore filter [33]. This total absence of toxicity possi-bly explains the adhesion of human osteoblasts to the materialsurface [34].

Dycal was slightly cytotoxic in direct contact. This con-firms previous work [7] and may be due to the solubility ofsalt resulting from the reaction between salicylic acid andzinc oxide releasing zinc ions and non-reacting hydroxideions. It is possible that this is clinically irrelevant because20% cell death without pulpal clearance does not representharmful behavior of the material. In vivo, Dycal does not elicitan inflammatory reaction after intramuscular implantationin rats [35] and induces slight inflammation after direct pulpcapping [36]. The toxicity decrease after dentin disc interpo-sition is in agreement with previous work on the importanceof dentin thickness and hydraulic conductance on restorativematerial toxicity [37].

All studies comparing the effects of MTA versus Dycal con-cluded a higher efficiency of MTA. Direct pulp capping withMTA gave better results that Dycal at 4 months on humanwisdom teeth [8] and at 2 months in dog teeth [38].

Absence of toxicity with the new cement was comparableto that of MTA and this was the case either with or withoutdentin slice interposition. Additionally, both the new cementand MTA do not seem to affect the odontoblastic specific pro-tein expression or mineralization.

In previous work, the authors have shown that pulpcells cultured with �-glycerophoshate secrete an extracel-lular matrix deposit which progressively forms nodules ofmineralized material. FTIR analysis showed that it was a spe-cific deposition which had the same mineral composition ofdentin [39]. The cultured cells, particularly those involved inmineral nodule formation, express a high level of alkalinephosphatase activity indicating high mineralization potentialof these cells. In addition, the cells involved in the miner-alization express the type I collagen, osteonectin, DSP andNestin. In this work, the cells treated with the new cementor MTA expressed collagen I, dentin sialoprotein and Nestinand synthesized a mineralized matrix. Colagen I is the majordentin matrix organic protein [40]. DSP which is expressedduring human tooth development is a 53-kDa glycoproteinaccounts for 5–8% of the dentin extracellular matrix. It is local-ized mainly in dental tissues and its expression was reportedto be localized and confined to differentiating odontoblasts,with a transient expression in the presecretory ameloblasts[41]. However, odontoblasts express DSP to a much greaterextent than other cell types [42]. Additionally, Nestin which is ahuman odontoblast specific intermediate filament protein [43]was expressed in these cells after adding �-glycerophoshatewith a stronger expression in the cells contacting the mineralnodules.

This is of prime importance in the clinic. Coronal restora-tions may be placed on teeth where the odontoblastic layeris partially destroyed, making the differentiation of secondaryodontoblasts necessary prior to pulp healing. The presence oftoxic compounds such as monomers may interfere with thiscritical step of pulp healing [44].

fibroblasts in the presence of MTA has never been reported,but the potential of MTA to induce cell cytodifferentiation hasalready been shown in animal studies. The root end closure

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d e n t a l m a t e r i a l s 2

ith MTA [45] and growth of new cementoblasts in direct con-act with MTA used as a retrofilling material have been shownn dogs [46] and monkeys [47] and reparative dentin can beeen after direct pulp capping with MTA [13,38,48]. The advan-age of the new material over both Dycal® and MTA resides inhe fact that, in addition to its biocompatibility, its mechanicalnd physical properties strongly suggest its future utilisations a bulk restorative material and not only as a pulp cappinggent.

. Conclusions

he results of the current study need to be confirmed in vivond suggest that this new Ca3SiO5 cement could be used as airect pulp capping agent but also as a lining agent. This mate-ial would possibly induce the secretion of reactionary dentinften considered as a preliminary step for pulp healing afteraries removal. The good handling properties of this materialssociated with its biological, mechanical and physical prop-rties let us think that this material could be used as a pulpapping agent and as a bulk restorative material at the sameime. In addition, no preliminary conditioning of the cavitiess required with this new cement. This would greatly simplifyulp capping techniques.

cknowledgements

his work was supported by institutional funding from therench “Ministere de l’education nationale, de l’enseignementuperieur et de la recherche”. The authors wish to thank Dr.ean-Charles Gardon for providing the third molars used inhis work.

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Microleakage of a new restorative calcium based cement (Biodentin®) 2008 Tran V, Pradelle N, Colon P Oral presentation PEF IADR Sept 2008 London

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0283 Microleakage of a new restorative calcium based cement (Biodentin ®)

V. TRAN, University of medecine and Pharmacy in Ho Chi Minh, Vietnam, Ho Chi Minh City, Vietnam, P. COLON, faculté de Chirurgie Dentaire Paris 7, France, and N. PRADELLE-PLASSE, Faculté de Chirurgie Dentaire Paris 7, France

Objectives: The aim of this study was to evaluate the ability of a new restorative calcium based material Biodentin® (Septodont, France) to be used as class II restoration recording the microleakage by a dye penetration. methodology.

Methods: 18 freshly extracted human molars were used for this study. Standardized class II cavities in the mesial (enamel margin) and distal (dentin margin) surfaces were restored with Biodentin®. The teeth were randomly divided into 3 groups : 1 : direct application of Biodentin® ; 2 : treatment of the wall of cavity with polyacrylic acid before restoration ; 3 : application of Optiguard on Biodentin® surface. The specimens were stored at 37°C one day, thermocycled, stained, sectioned twice longitudinally in the mesio-distal direction. The silver nitrate penetration was measured.

Results:

Groups enamel margin (%) dentin margin (%)

1 : 17,65(± 4,38)(a) - 10,46 (± 3,23)

2 : 8,53 (± 7,44) (b) - 1,83 (± 3,94)

3 : 46,5 (±10,55)(a)(b) - 8,31 (± 4)

Same letters indicate significant differences (p < 0.05)

There was no statistically significant difference in microleakage between with and without treatment of enamel/dentine surface with polyacrylic acid. The microleakage at the enamel - Biodentin® interface with treatment of Optiguard® is more important than without (p<0.05). The cement particles are hydrophilic. After 1 day, the cement reaction was not achieved yet. It was observed that the Optiguard® layer limits the water contact with cement preventing the hydration phenomenon. The surface of the dentine is wetter than that of enamel explaining why no influence of Optiguard® on the microleakage at the Biodentin® - dentine interface was observed.

Conclusion: These data show the good sealing ability of this new material. However the setting time as to be considered regarding the short term results.

Seq #27 - Marginal Integrity - Oral 3:30 PM-5:30 PM, Wednesday, September 10, 2008 Queen Elizabeth II Conference Centre Theatre H

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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

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0299 RD94, a Portland Cement, Stimulates in Vivo Reactionary Dentin FormationT. BOUKPESSI, F. DECUP, D. SEPTIER, M. GOLDBERG, and C. CHAUSSAIN-MILLER, University Paris Descartes, Dental School, Montrouge, France

RD94 is a novel experimental Portland cement aiming to be a glass ionomer cement and composite- resin substitute in restorative dentistry. Objectives: To explore the effects of RD94 on the formation of reactionary dentine, in vivo experiments were carried out on the rat upper molars.

Methods: Half-moon cavities were prepared on the mesial aspect of the first molar without pulp exposure. Comparison was made with a sham group (preparation of cavities alone), with a group of molars where the cavities were occluded with a conventional glass-ionomer cement and with a group where cavities were filled with RD 94. After 8, 15 and 30 days, the rats were killed by heart perfusion with the fixative solution. Measurements were done on images obtained after histological analysis.

Results: Eight days after tooth preparation, a few inflammatory cells were seen, mostly located in the pulp surface near the cavity. In the RD94 group, a 20-40 mm thick layer of reactionary dentin was formed beneath a calico-traumatic line, in contrast to the two other groups where the reactionary dentin thickness was about 10mm. After 15 days, the inflammatory process was resolved in the pulps of all the groups. In the RD94 group, the outer part of the pulp chamber was filled with a 40-80 mm thick layer of reactionary dentin beneath the calciotraumatic line. After 30 days using RD94 as restorative material, reactionary dentin was about 160mm thick, whereas the rest of pulp looked normal.

Conclusion: The present data show that the novel RD94 cement displays good pulp biocompatibility, and has bioactive properties by stimulating the formation of reactionary dentine in the rat molar model. These results suggest that restorative treatment with RD94 provides new prospects for dental therapy.

We acknowledge SEPTODONT, France, for their financial support to this investigation.

Seq #29 - Oral Tissues - Regeneration and Repair 3:30 PM-5:30 PM, Wednesday, September 10, 2008 Queen Elizabeth II Conference Centre Theatre J

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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

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European Cells and Materials Vol. 13. Suppl. 1, 2007 (page 17) ISSN 1473-2262

Evaluation of adhesion between composite resins and an experimental mineral restorative material.

C.Boinon1, MJ.Bottero-Cornillac12, G. Koubi12 & J. Dejou1 1Laboratoire IMEB-ERT 30, 2 Département d’odontologie conservatrice-endodontie, UFR

d’Odontologie, Université de la Méditerranée, Marseille, FRANCE INTRODUCTION: The purpose of this study was to evaluate the ability of new Ca3SiO5 based cement, used as a base in sandwich technique restorations, to bond to restorative composite resins. Adhesion was studied by evaluating marginal microleakage and shear bond strength of samples of composite resins bonded to the experimental cement with several different surface treatments. METHODS: A three-step adhesive system (AllBond 2®, Bisco) and a silane coupling agent (porcelain primer, Bisco) were used to bond the composite resin (Enamel plus HFO GE3, Micerium) according to 9 different procedures (n=5). The marginal seal was evaluated by the silver nitrate penetration method after 3500 thermocycling cycles at 5 and 55°C. Shear bond strengths were evaluated on samples treated according to only five procedures (n= 10) two hours after bonding. Kruskall Wallis non parametric tests and Games-Howell post hoc tests were used to evaluate statistical differences between the experimental groups. RESULTS: Figures 1 and 2 summarize the results. Groups with the same letter did not differ significantly.

0

1

2

3

4

ESA EA C SA A

med

ian

scor

e

A

A

C C C

Fig1. Interfacial microleakage according to surface treatment.

0

5

10

15

20

25

ESA SA A EA C

MPa

AB AB ABB

Fig. Mean shear bond strength according to surface treatment The results presented here are those obtained with the five following procedures: control (no treatment) (C), adhesive resin (A), silane–adhesive resin (SA), etching-adhesive resin (EA) and etching-silane-adhesive resin (ESA). DISCUSSION & CONCLUSIONS: Etching the surface of the experimental cement with a H3PO4 gel for 15s, then applying a silane coupling agent, before the adhesive resin, led to both the highest shear bond strength [18.57(3.04)MPa] and the lowest microleakage (median score = 0). This procedure seems to be the best when a composite resin has to be bonded to the experimental cement. REFERENCES: 1. Antonucci JM, Dickens SH, Fowler BO, Hockin HK, McDonough WG. 2005 Chemistry of silanes: Interfaces in Dental Polymers and Composite, Journal of Research of the National Institute of Standards and Technology. 110, 541-558 2. Valentin JL, Lopez-Manchado MA, Posadas P, Rodriguez A, Marcos-Fernandez A, Ibarra L. 2006 Characterization of the recativity of a silica derived from acid activation of sepiolite with silane by 29SI and 13C solid-state NMR. Journal of Colloid and Interface Science 11903, 1-11

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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

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European Cells and Materials Vol. 13. Suppl. 1, 2007 (page 18) ISSN 1473-2262 A clinical study of a new Ca3SiO5-based material for direct posterior fillings S. Koubi, H. Tassery, G. Aboudharam, J.L. Victor, G. Koubi Département d’Odontologie Conservatrice, Centre Dentaire Gaston Berger, 17-19 rue Mireille Lauze 13010 Marseille F

INTRODUCTION: A new cement-based material for direct restorative posterior fillings (RD 94, Septodont, France) has been developed to circumvent the shortcomings of the traditional filling materials. This material is inorganic and non-metallic, and the main components are Ca3SiO5, CaCO3, ZrO2, and water. After evaluation of the genotoxicity, the cytotoxicity, the effects on the specific functions of target cells, and the marginal sealing of this new material, a multicentric clinical study was initiated to evaluate, in a three-year follow-up, the performance of this experimental calcium silicate cement (RD 94), versus a traditional resin composite (Z 100, 3M, US), in Class I and Class II restorations.

METHODS: From June 2005, every patient from 18 to 80 years old who, at the examination performed at the authors' University Clinic, needed a posterior restoration, was invited to join this randomized trial, under cover of Huriet's law. Each patient provided informed consent to participate in the study, which was approved by the ethics committee of the University of Marseilles (CCPPRB 1). All the patients invited participated in the study.

Two operators, both familiar with the new material, placed all restorations. All treated teeth were in occlusion, and the cavities were prepared with slightly convergent cavity walls, without bevels, and under rubber dam isolation. In Class II cavities, a thin metallic matrix band and wood wedges were used. All cavities were sprayed with water, and for the RD 94, no conditioning of the cavity or base material was recommended by the manufacturer. The restorations were finished after two weeks with polishing stones and strips. An examination book was created for each restoration, and a slight modification of the USPHS (United States Public Health System) criteria was used to evaluate the quality of the restorations by two calibrated observers [1-3]. Periapical radiographs and color slides were taken of all restorations, at dates D 0, D +15 days and D +6 months.

RESULTS: After ten months, 140 restorations had been performed in the Marseilles Dental School,

70 with RD 94 and 70 with Z 100. Forty-two of the restorations were Class I and ninety-eight Class II cavities. Ninety-four molars were treated, and forty-six premolars. Eighty teeth were treated in the upper maxillary and sixty in the lower.

In April 2006, thirty restorations had been evaluated at six months, including 11 Z 100 and 19 RD 94 restorations, and no non-acceptable clinical result was observed. Post-operative sensitivity was reported for two Z 100 restorations. A very good marginal adaptation and surface finish was observed on RD 94 restorations, although the color match of this new product was not yet perfect. None of the nineteen patients treated with RD 94 has lodged a complaint for pain, unpleasant physiologic or pathologic sensations, or objective or subjective reactions related to the material.

DISCUSSION AND CONCLUSIONS: Randomized clinical trials are considered the optimal way to validate the outcome of dental materials. However, randomized control groups require broad patient support, and are therefore time consuming and extremely demanding to conduct, thus contributing to the expensiveness of such studies. Nevertheless, the biological properties of this new material, combined with its interesting physicochemical characteristics, and with these hopeful preliminary results, justify the follow-up of this clinical study. After six months, the results indicated no significant differences, for direct restorations in medium sized cavities in posterior teeth, between a classical resin composite and the new Ca3SiO5-based material under test.

REFERENCES: 1 N.J. Opdam, B.A. Loomans, F.J. Roeters, E.M. Bronkhorst (2004) J Dent 32: 379-383. 2 J.W. Van Dijken and K.Sunnegard-Grönberg (2005) Swed Dent J. 29:45-51. 3 R.C. Spreafico, I. Krejci and D. Dietschi (2005) J Dent 33:499-507.

ACKNOWLEDGEMENTS: The authors thank Mrs. D. Leblanc and Mr. O. Marie, from Septodont Laboratories, and Pr. J.C. Franquin, for their help.

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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)

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European Cells and Materials Vol. 10. Suppl. 4, 2005 (page 23) ISSN 1473-2262 Cytotoxicity and Genotoxicity of a New Material for Direct Posterior Fillings.

I. About1, A Raskin1, M. De Meo2, and J. Déjou1. 1 Laboratoire IMEB-ERT 30, Faculté d’Odontologie, 2Faculté de Pharmacie,

Université de la Méditerranée, Marseille, FRANCE

INTRODUCTION: A new ceramic material for direct restorative posterior fillings, a Ca3SiO5-based Portland cement, has been developed to circumvent the shortcomings of the traditional filling materials. The purpose of this study was to evaluate its genotoxicity, cytotoxicity, and its effects on the specific functions of target cells.

METHODS: 1) Genotoxicity: An Ames test was performed on four different species of salmonella typhimurium. A micronuclei test was performed on fresh human lymphocytes and a comet test was performed on human pulpal fibroblasts. 2) The cytotoxicity was tested according to ISO 10993 standards immediately after the preparation and after 1 and 7 days with the MTT assay on human pulpal fibroblasts. 3) The effects on the specific functions of human pulp fibroblasts were investigated by studying the expression of collagen type I, Osteonectin, Dentin Sialoprotein and Nestin.

RESULTS: 1) Genotoxicity The Ames test did not show any evidence of mutagenicity whatever the dilution of the test medium. The proportion of lymphocytes with micronuclei was similar to that obtained with the negative control. It ranged from 3.9% to 4.1% with increasing concentrations (1 to 5%) in aqueous or hydrophobic medium. The percentage of DNA in the queue with the comet test ranged from 12.59 for the 0.1% dilution to 15.58 with pure medium (figure 1 and table 1).

Fig.1: Comet test on human pulp fibroblasts with the new material

% ADN Queue Sample

dilution Mean (sd) Median %ADN_Chi2 (sd) Control (pure)

13.19 (0.96) 10.31 3.32 (0.22)

H2O2 (pure) 46.52 (1.45) 45.34 10.69 (0.69)***

0.1% 12.59 (0.96) 10.92 2.79 (0.16)

1% 13.31 (0.88) 11.62 3.66 (0.24)

10% 14.90 (1.06) 13.75 3.61 (0.10)

100% 15.58 (1.08) 13.70 3.78 (0.24)

Table 1: Comet test on human pulp fibroblasts with the new material at different dilutions (***: p<0.0001) 2) Cytotoxicity The cytotoxicity of the material ranged from 10% at 1 day to 7% at 7 days, which was similar to MTA®, but less than Z 250®. 3) The specific functions of human pulp fibroblasts were not altered by the new material (figures 2& 3)

Fig. 2: Collagen I expression with the new material land MTA®

Fig. 3: Dentin sialoprotein expression with the new material and MTA® DISCUSSION & CONCLUSIONS: The material was non cytotoxic and non genotoxic for pulp fibroblasts at any concentration. The specific functions of these cells were not modified.

Exp material MTA®

Exp material MTA®

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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)

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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. Déjou, A Raskin, J Colombani & I. About

Laboratoire IMEB-ERT 30, UFR d’Odontologie, Université de la Méditerranée, Marseille, FRANCE

INTRODUCTION: A new ceramic material for direct restorative posterior fillings, a Ca3SiO5-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: 1Reclaru L, Meyer JM. (1994). Study of corrosion between a titanium implant and dental alloys. J Dent; 22:159-68. 2Nomoto R, McCabe JF. (2001). A simple acid erosion test for dental water-based cements. Dent Mater ;17(1):53-9. 3Nomoto 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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