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Septodont R&D France December 2010

Biodentine

Publications and
Communications
2005 - 2010
Dec. 2010
TITLE YEAR AUTHORS REFERENCE
Impedance methodology: A new way to characterize the
setting reaction of dental cements
2010
Cyril Villat, V.X. Tran, Nelly Pradelle-Plasse,d, Pierre
Ponthiaux, Francois Wenger, Brigitte Grosgogeat,
Pierre Colon
Dent Mater 26 (2010) : 1127-1132
Ca

SiO

, CaCO

, ZrO

(Biodentine): a new biomaterial used


as pulp-capping agent in primary pig teeth
2010 A.Shayegan, M. Petein, A. Vanden Abbeele
Poster at IADT 16 th World Congress Dental
Traumatology, June 2010 Verona Italy
Bioactivity Of Biodentine
TM
: a Ca
3
SiO
5
-based Dentin
Substitute
2010 I. About, P. Laurent, and O. Tecles
Oral session, IADR Congress July 2010,
Barcelona, Spain
Antibacterial Activity of New Ca-Based Cement Compared to
Other Cements
2010
E. Valyi, N. Plasse-Pradelle, D. Decore, P. Colon, and
B. Grosgogeat
Oral session, IADR Congress July 2010,
Barcelona, Spain
Interactions Between a Calcium Silicate Cement (Biodentine)
and Its Environment
2010
P. Colon, F. Bronnec, B. Grosgogeat, and N. Plasse-
Pradelle
Oral session, IADR Congress July 2010,
Barcelona, Spain
Physical Properties of a New Ca3SiO5-Based Dentin
Substitute
2010
J.-C. Franquin, O. Marie, M.-J. Bottero, G. Koubi, and
I. About
Poster Sessions, IADR Congress July 2010,
Barcelona, Spain
Dimensional Changes of a Ca3SiO5 Based Cement During
Setting
2010 F. Bronnec, P. Colon, and N. Plasse-Pradelle
Poster Sessions, IADR Congress July 2010,
Barcelona, Spain
Biodentine
TM
Microleakage in Class II Open-sandwich
Restorations
2010 A. Raskin, G. Eschrich, I. About, and J. Dejou
Poster Sessions, IADR Congress July 2010,
Barcelona, Spain
A Clinical Study of a New Ca3SiO5-Based Dentine Substitute 2010 G. Koubi, G. Weisrock, M.O. Faure, P. Colon, and J.-C. Franquin
Poster Sessions, IADR Congress July 2010,
Barcelona, Spain
RD94 In Indirect Pulp-Capping Situation Induces Reactionary
Dentin Formation.
2009
T.Boukpessi, F.Decup, D.Septier, C. Chaussain and
M. Goldberg - France
IADR-CED congress in Munich, Germany, 9-
12 September 2009
Ciments alcalins ou acides usage odontologique : action sur
quelques souches bactriennes reprsentatives
2009
E.Valyi, P.Colon, F.Bornand, D.Decoret,
B.Grosgogeat, France
Abstract - SFBD (Socit Francophone des
biomatriaux dentaires). 25-26 juin 2009.
Biocompatibility or cytotoxic effects of dental composites -
Chapter VI Emerging trends in (bio)material research
2009
Goldberg M, Pradelle-Plasse N, Tran XV, Colon P,
Laurent P, Aubut V, About I, Boukpessi T, Septier D
Working group of ORE FDI -edited by
Michael Goldberg
A clinical study of a new Ca3SiO5-based material indicated as
a dentine substitute
2009
G.Weissrock, J.C. Franquin, P. Colon , G.Koubi
University of Paris 7, France
Journe Scientifique du CNEOC Brest -June
2009
Biodentine
TM
- RD94, A portland cement, stimulates in vivo
reactionary dentin formation
2009
T.Boukpessi, F.Decup, D.Septier, M. Goldberg, C.
Chaussain, France
Journe Scientifique du CNEOC Brest -June
2009
A clinical study of a new Ca3SiO5-based material indicated as
a dentine substitute
2009 G.Koubi, J.C. Franquin, P. Colon.
Abstract in Clin. Oral Invest 2009 + poster
Conseuro 2009 (Seville, Spain March 12-14th
2009)
Induction of specific cell responses to a Ca
3
SiO
5
-based
posterior restorative material
2008
Laurent P, Camps J, De Mo M, Djou J, About I.
Marseille, France.
Dent Mater. 2008 Nov;24(11):1486-94.
Microleakage of a new restorative calcium based cement
(Biodentin
2008 Tran V, Pradelle N, Colon P
Oral presentation PEF IADR Sept 2008
London
RD 94, a Portland cement, stimulates in vivo reactionary
dentine formation
2008
Boukpessi T, Septier D, Decup F, Chaussain-Miller C,
Goldberg
Oral presentation PEF IADR Sept 2008
London
Evaluation of adhesion between composite resins and an
experimental mineral restorative material
2007
C. BOINON, MJ. BOTTERO-CORNILLAC, G. KOUBI
and J. DEJOU
Abstract :European Cells and Materials Vol.
13. Suppl.1
A clinical study of a new Ca3Si05-based material for direct
posterior fillings
2007
S. KOUBI, H.TASSERY, G.ABOUDHARAM, J.L
VICTOR, G. KOUBI
abstract : European Cells and Materials Vol.
13. Suppl.1
Cytotoxicity and genotoxicity of a new material for direct
posterior fillings.
2005
I. ABOUT, A RASKIN, *M. DE MEO, J.DEJOU -
Marseille, France
abstract : European Cells and Materials Vol.
10. Suppl. 4, 2005 (page 23)
Physical, chemical and mechanical behavior of a new material
for direct posterior fillings.
2005
J. DEJOU, J COLOMBANI and I. ABOUT. Marseille,
France
abstract : European Cells and Materials Vol.
10. Suppl. 4, 2005 (page 22)
PUBLICATIONS AND COMMUNICATIONS ON BIODENTINE
TM
- SEPTODONT

Dental Materials 26 (2010) 1127 - 1132

Impedance methodology: a new way to characterize the setting reaction of dental cements

C. Villat, V.X. Tran, N. Pradelle-Plasse, P. Ponthiaux, F. Wenger, B. Grosgogeat, P. Colon.

Objectives. Impedance spectroscopy is a non-destructive, quantitative method, commonly used nowadays
for industrial research on cement and concrete.
The aim of theis study is to investigate the interest of impedance spectroscopy in the characterization of
setting process of dental cements.
Methods. Two types of dental cements are used in this experiment: a new Calcium Silicate cement
Biodentine (Septodont, Saint Maur des Fosss, France) and a glass ionomer cement resin modified or not
(Fuji II

LC improved capsules and Fuji IX

GP Fast set capsules, GC Corp., Tokyo, Japan). The


conductivity of the dental cements was determined by impedance spectroscopy measurements carried out
on dental cement samples immersed in a 0.1M potassium chloride solution (KCl) in a like-permeation cell
connected to a potentiostat and a Frequency Response Analyzer. The temperature of the solution is 37C.
From the moment of mixing of powder and liquid, the experiments lasted 2 weeks.
Results. The results obtained for each material are relevant of the setting process. For GIC, impedance
values are stabilized after 5 days while at least 14 days are necessary for the calcium silicate based cement.
Significance. In accordance with the literature regarding studies of cements and concrete, impedance
spectroscopy can characterize ion mobility, porosity and hardening process of dental hydrogel materials.
Oral Session at IADR Congress July 2010, Barcelona, Spain

Bioactivity Of Biodentine
TM
: a Ca3SiO5-based Dentin Substitute
I. ABOUT, P. LAURENT, and O. TECLES, Laboratoire Interface Matrice Extracellulaire-Biomatriaux (IMEB), Marseille,
France

Objectives: This work was carried out to investigate Biodentine bioactivity by studying its effects on pulp progenitor
cells activation, differentiation and dentin regeneration in entire human tooth cultures. Methods: Human dental pulp
cells from third molars were cultured by the explant outgrowth method and used at 30000/cm. Biodentine was
prepared according to the manufacturer's instructions. Samples of the material were incubated in the culture medium
(DMEM) at determined surface/medium volume ratios. After 24 hours, these conditioned media were incubated with
injured or intact pulp cells. Transforming Growth Factor-b1 (TGF b1) quantification in the supernatants was performed
with ELISA test. Immature third molars were used to investigate the effect of Biodentine on dentin regeneration using
the human entire tooth culture model. A cavity with pulp exposure was performed per tooth under sterile saline
coolant. The cavity was dried and Biodentine was applied as a pulp capping material. The apical part of the treated
teeth was dipped in culture dishes containing DMEM medium. The teeth were cultured for 1, 14 or 28 days then they
were processed for histology and molecular staining. Results: Biodentine significantly increased TGF b1 secretion level
in the conditioned media from injured cells. After tooth pulp capping with Biodentine, dense mineralized foci including
sequestered cells were observed after 14 days just beneath the material in the pulp wound area. Collagen I,
Osteonectin and Dentin Sialoprotein were expressed in the mineralized matrix and in the sequestered cells which also
expressed Nestin. Conclusion: Biodentine induced odontoblast differentiation from pulp progenitor cells. The resulting
mineralized matrix has the molecular characteristics of dentin. The sequestered cells in this matrix express the
molecular markers of odontoblasts. These results strongly suggest that Biodentine is bioactive and stimulates dentin
regeneration.
Oral Session at IADR Congress July 2010, Barcelona, Spain

Antibacterial Activity of New Ca-Based Cement Compared to Other Cements
E. VALYI
1
, N. PLASSE-PRADELLE
2
, D. DECORET
3
, P. COLON
2
, and B. GROSGOGEAT
4
,
1
Universit Claude Bernard
Lyon1, Lyon, France,
2
Department of Operative and Preventive Dentistry University of Paris V, Villeurbanne, France,
3
Universit Claude Bernard Lyon1, UFR d'odontologie, Lyon, France,
4
Universit Claude Bernard Lyon1, Laboratoire des
Multimatriaux et Interfaces (LMI), UFR d'Odontologie UMR CNRS 5615, Lyon, France

Objective: To investigate the antibacterial properties of an in development calcium-based cement; Biodentine
(Ca3SiO2) compared to commercial glass ionomer cements and MTA. Materials and methods: Pellets of GICs (Ionofil
Molar AC Quick , Ketac-Fil Plus Aplicap ), ProRootMTA and Biodentine
TM
were prepared to test the influence of
cements setup process on the growth of five oral bacterial strains: Streptococcus mutans, Enterococcus faecalis,
Actinomyces naeslundii, Lactobacillus casei and Fusobacterium nucleatum. To evaluate the antibacterial activity, both
agar diffusion test and incubation in aqueous solution were used. With agar, the pellet is lodged in seeded plates and
the growth inhibition diameter around the material was measured after 24 to 72h incubation at 37 C. In the aqueous
medium, MTA cement excluded, the pellet is immersed in a bacterial suspension, and samples were taken at 4 and
24h to agar plates to account the colony forming units. Results: None of the studied cements showed a real
antibacterial activity towards S. mutans and E. faecalis. All cements show antibacterial activity towards A. naeslundii.
For L. casei, with agar test, a large inhibition zone around the two Ca-based cements was found but none around
GICs. The in aqueous medium test indicates that the antibacterial activity of Biodentine is very strong, it extends over
time towards this strain and contrary to what might suggest the agar test it also concerns GICs. For F. nucleatum with
agar, a large zone of inhibition around both GICs was found but none around Ca-based cements. In liquid medium,
there is an antibacterial activity that decreases with time for the studied cements in this environment. Conclusion:
Under this study's conditions, there was an antibacterial activity that is more dependent on strain type than material.
Antibacterial properties of the new cement Biodentine are comparable to that of Ca-based cement.
Oral Session at IADR Congress July 2010, Barcelona, Spain

Interactions Between a Calcium Silicate Cement (Biodentine) and Its Environment
P. COLON, F. BRONNEC, B. GROSGOGEAT, and N. PRADELLE-PLASSE, CNRS UMR 5615, Laboratoire Multimateriaux et
Interfaces, Villeurbanne cedex, France

Objectives: To evaluate interactions between a calcium silicate based material, BiodentineTM and its environment in
solutions featuring the oral environment.Methods: Five samples are immersed in two PBS solutions (phosphate
buffered saline) at pH 7.4 and 5.5 for 7 days. The precipitates formed on the surface were observed using binocular
microscope, optical microscope and SEM. They are then analyzed using X-ray diffraction. Results: The optical methods
show that the precipitates formed at the surface of samples immersed in PBS 7.4 present a morphology of hollow
spherules and white cords suggestive of amorphous structure; those formed at the surface of those immersed in PBS
5.5 present clusters of refractile small hexagonal platelets (size 100m) exhibiting a crystalline structure. SEM
observations of the precipitates formed on the first sample (PBS 7.4) show hollow spherules of very large size (>
100m) formed by coalescence of small forms rounded near the micron scale, with entanglement of small plates in the
amorphous structure appearance. Precipitates formed on the second samples (PBS 5.5) consist as large plates, whose
dimensions are about 500m and as smaller clusters formed by small rounded formed similar to the previous. These
compounds are covered by formation of different crystal morphologies: little rhombohedral structures (<10m),
pinacoids and large needles (> 50 microns). X ray diffraction of the precipitates obtained in the pH 7.4 PBS solution is
realized. Three crystalline elements are characterized : calcium hydroxide or portlandite (Ca(OH)2 hexagonal
structure), calcite and poorly crystallized phase "apatite". The structure is close of the hexagonal structure of
hydroxyapatite (Ca10(PO4)3(OH)2)).Conclusions: In contact with phosphate ions, this calcium silicate cement creates
precipitates close of hydroxyapatite.

Poster at IADR Congress July 2010, Barcelona, Spain

Physical Properties of a New Ca3SiO5-Based Dentin Substitute
J.-C. FRANQUIN
1
, O. MARIE
2
, M.-J. BOTTERO
1
, G. KOUBI
1
, and I. ABOUT
1
,
1
Laboratoire IMEB, Marseille, France,
2
R&D
Center Septodont, Saint Maur, France

Objectives: A Ca3SiO5 dental material (BiodentineTM, Septodont) has been developed as dentine substitute in various
indications including posterior restorations, direct pulp capping and root dentinal repair. The main components of this
material are Ca3SiO5, CaCO3, ZrO2 and water. This study compares the material physical characteristics with those of
two other materials, PMTA (Dentsply, USA) and Fuji IX (GC, Japan), which can also be used as base materials.
Methods: The specimens prepared according to the manufacturer's instructions, were stored in distilled water at 37C,
to mimic the clinical conditions. The three materials were tested at 1 hour, and at 1, 7 and 28 days for compressive
strength (CS), and at 1 day for flexural strength (FS). The working and setting times were evaluated through dynamic
rheometry tests. The porosity was studied with the mercury intrusion porosimetry. A one-way analysis of variance,
followed by post-hoc tests, was used to compare the materials in each test. Results: The CS of the new Ca3SiO5-
based material increased by 240% between 1 hour and 28 days to reach a mean value of 316.3 MPa. The new
material CS value is significantly higher than that of the other two materials after 28 days. On the other hand, the
mean flexural strengths measurement is lower than PMTA and Fuji IX. The setting time of BiodentineTM (10 mn) is
compatible with the clinical application: faster than the PMTA (1 hour), but longer than Fuji IX (3.4 min). BiodentineTM
density is higher than PMTA (2.26 vs 1.88 g/cm3) while the porosity (6.8 vs 22.6%) is lower. The density and porosity
values were similar to Fuji IX. Conclusions: This new Ca3SiO5-based material could be used as a bulk dentinal
substitute for definitive dentinal treatment, to replace bases or liners used in class I and II cavities on vital permanent
posterior teeth.
Poster at IADR Congress July 2010, Barcelona, Spain

Dimensional Changes of a Ca3SiO5 Based Cement During Setting
F. BRONNEC, P. COLON, and N. PRADELLE-PLASSE, CNRS UMR 5615, Laboratoire Multimateriaux et Interfaces,
Villeurbanne, France

Objectives: To characterize the dimensional variations of a tricalcium silicate-based cement (BiodentineTM) at the first
stages of the setting reaction using a hydrostatic weighting machine. To evaluate the correlation between enthalpy of
reaction and this dimensional changes using DSC. Methods: The compounds of the cement [powder (0.700mg) and
liquid (0.178mg)] were mixed (30s; 4.200osc/min) in a vibrating device. The samples (n=5) were weighted in the air
(weight=0.553g0.142) and 30s later immersed in Rhodorsil 47V20 silicon oil. They are then weighted in the liquid
(24C0.5) at interval of 5s during 20h. Dimensional variations were expressed in %. The experiment was completed
with a thermodifferential analysis using a DSC. Results: Dimensional variations and heat evolution curves obtained
during cement hydration process showed four distinct phases: a short contraction step corresponding to the
endothermic dissolution of the crystalline compounds during the first 6min; an exothermic expansion phase
corresponding to the hydration reaction between 6 to 30min; a non-linear contraction step (-2.14%1) from 30min to
5h due to the combination of the contraction upon cooling and the endogenous contraction; a trend to recover their
original volume from 5h to the end of the experiment. Conclusion: The results have showed a great reactivity of this
new cement. In fact, the aspects of the curves were similar with those reported in literature for Portland cements but
the setting kinetic of the experimental cement was faster. The samples underwent an initial volume reduction due to
chemical contraction and capillary absorption during the first hours. Then a reversal with a secondary expansion is
observed due to the continuation of the hydration process.

Poster at IADR Congress July 2010, Barcelona, Spain

Biodentine
TM
Microleakage in Class II Open-sandwich Restorations
A. RASKIN, G. ESCHRICH, I. ABOUT, and J. DEJOU, Universit de la Mditerrane, Marseille, France
Objectives: The aim of this study was to investigate the microleakage of Class II sandwich technique with a
biocompatible Ca3SiO5-based dentin substitute (Biodentine
TM
, Septodont) (B). Methods: Mesio-occlusal and disto-
occlusal preparations below cemento-enamel junction were made in 30 extracted molars. The teeth were randomly
assigned one of the following treatments before restoration with Filtek Z250 (3M) composite resin: B; Fuji II LC, GC
(F); B + Optibond Solo Plus (Kerr) (OS) + silane (Si); B + OS; B + Septobond SE (Septodont) (SE); F + OS. The
materials were applied according to the manufacturer's instructions. The teeth were thermocycled for 2500 cycles of
15sec each alternatively at 5 and 55C. Marginal leakage was evaluated using a silver nitrate solution as a penetration
marker. Three sections (six measures) per restoration were realized and the microleakage was assessed
microscopically. The tracer penetration was recorded using a penetration scale from 0 (no penetration) to 3 (high
penetration). The statistical evaluation of the results was performed with Kruskal-Wallis (KW) and Games-Howell (GH)
post hoc test, and the comparison between enamel and dentin microleakage with Wilcoxon test (W) at p level = 0.05.
Results:
Materials Base/Composite
resin
Maximal median scores
Enamel Dentin W
B - 1.0 ab 1.0 NS
F - 1.0 ab 1.5 S (p=0.0431)
B+Si+OS 1.0 1.0 ab 1.0 NS
B+OS 1.0 1.0 a 1.0 NS
B+SE 1.0 2.0 b 1.0 S (p=0.0431)
F+OS 1.0 1.0 a 1.0 NS
KW+GH NS S (0.048) NS
For enamel maximal median scores, the groups with similar letters (vertical reading of the board) are not statistically
different.
Conclusions: The dye penetration obtained with Biodentine is similar to that observed with Fuji II LC at the interface of
the enamel, dentin and dentin bonding agents. Biodentine can be indicated in open-sandwich Class II restorations
without any preliminary treatment.
Poster at IADR Congress July 2010, Barcelona, Spain

A Clinical Study of a New Ca3SiO5-Based Dentine Substitute
G. KOUBI
1
, G. WEISROCK
2
, M.O. FAURE
3
, P. COLON
4
, and J.-C. FRANQUIN
1
,
1
Laboratoire IMEB, Marseille, France,
2
Marseille Dental School, Mediterranean university, Marseille, France,
3
R&D Center Saint Maur France, Saint Maur des
Fosses, France,
4
Department of Operative and Preventive Dentistry University of Paris V, Saint Maur, France

Objectives: A Ca3SiO5-based biocompatible material (BiodentineTM, Septodont, France) has been developed as dentin
substitute for direct and indirect posterior fillings. A three-year follow-up multicentric randomized clinical study was
initiated to evaluate its tolerance and efficacy longevity as restoration vs a hybrid composite (Z100, 3M). Methods:
400 vital first and second permanent premolars and molars with Class I or II lesions were randomized on patients
aged from 18 to 80. Half of the cavities were filled with BiodentineTM and the other half with Z100 according to the
manufacturer's instructions. The restorations were evaluated at the baseline, 15 days, 6, 12 and 24 and 36 months.
Results: 232 cases with a one year follow-up at least were analysed. All restorations evaluated until 6 months
demonstrated acceptable clinical performance. BiodentineTM was easy to handle, has an excellent anatomic form, a
good marginal adaptation and proximal contact at the baseline. During the follow-up, the product was well tolerated in
all cases, but the anatomic form, marginal adaptation and proximal contact started to be deteriorated after 6 months.
As a consequence, a complementary treatment was performed. BiodentineTM was kept as cavity lining as the pulp
vitality test was positive. BiodentineTM has presented a good resistance to cutting, and can be covered by the
composite Z 100. After one year of follow-up, 92.6% (25/27) treated cases presented a good anatomic form and
marginal adaptation. The clinical trial is still on going and the specific analysis of the cases which already attempted 3
years will be analysed. Conclusions: The results indicate that the new Ca3SiO5-based material could be used as dentin
substitute for definitive dentinal treatment in restoration of posterior teeth. Overlaying Z100 on BiodentineTM protects
the product from erosion. BiodentineTM exhibits an efficient and durable protection of the pulp from bacterial invasion
as a dentine substitute.

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
5/74
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 Mnchen)
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 m 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 (160m) 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 usage odontologique : action sur
quelques souches bactriennes reprsentatives
2009
E.Valyi, P.Colon, F.Bornand, D.Decoret, B.Grosgogeat,
France
Abstract - SFBD (Socit Francophone des biomatriaux
dentaires). 25-26 juin 2009.
7/74
8/74
Biocompatibility or cytotoxic effects of dental composites -
Chapter VI Emerging trends in (bio) material research
2009
Goldberg M, Pradelle-Plasse N, Tran XV, Colon P, Laurent P,
Aubut V, About I, Boukpessi T, Septier D
Working group of ORE FDI -edited by Michael Goldberg
9/74
10/74
Chapter VI Emerging trends in (bio)material researches:
VI-1-Repair or regeneration, a short review
Michel Goldberg (Univ. Paris Descartes)
VI-2- An example of new material: preclinical multicentric studies on a new
Ca
3
SiO
5
-based dental material.

VI-2-1 Physico-chemical properties.
Nelly Pradelle-Plasse (University Paris 7 Denis Diderot & LGPM,
Ecole Centrale de Paris) France, Xuan-Vinh Tran (University of
Medicine and Pharmacy, Ho Chi Minh city, Vietnam),
& Pierre Colon (University Paris 7- Denis Diderot, & LGPM, Ecole
Centrale de Paris). France

VI-2-2 Biological properties
VI-2-2-1 In vitro studies Patrick Laurent, Virginie Aubut, Imad
About Laboratoire IMEB, Facult d'Odontologie, Universit de la Mditerrane, Marseille,
France
VI-2-2-2 In vivo studies Tchilalo Boukpessi, Dominique Septier &
Michel Goldberg (University Paris Descartes, France)

_________________________________________________________________________
VI- Emerging trends in (bio)material research
VI-1- Repair or regeneration, a short review.
Michel Goldberg (Univ. Paris Descartes)
Where do we come from? What are we? Where are we going? In a famous picture, the painter
Paul Gauguin raised this series of questions, and following the example of Oedipus and the
Sphinx, he gave some answers, valid or not.
Where do we come from?
Fifty years ago silver amalgam was the most common restorative material employed for
posterior teeth, and silicate cements were used for anterior teeth. The development of resin
composites with formulations more adapted to the clinical needs, new generations of
adhesives and the gradual reduction of the gap between the resins and dental tissues has led to
an increased use of resin-containing materials, even for molar restorations.
11/74
Silver amalgam fulfills most of the general criteria that are required for a good restorative
material from a clinical point of view, taking into account both mechanical and biological
properties. It is generally recognized as safe for the patients and so far no adverse effect or
body burden have been identified, except some allergic effects detected in a limited number
of cases. More importantly, degradation in a wet environment provides cariostatic properties
that are due to the corrosion of the metal, a phenomenon inherent to the material.
However, despite its overall qualities, nowadays this material is gradually discarded from the
restorative procedures in dental practice for three main reasons.
-Firstly, the adhesive properties of resin composites allow better preservation of dental
tissues during the preparation of the cavity, reinforcing undercuts. After the opening and the
suppression of un-sustained enamel, followed by the cleaning of the lesion, the preparation of
the cavity is simplified. This procedure allows a smaller size of the cavities, and therefore
favoring a non-reversible evolution toward a minimal dentistry. This opens some gates for
new concepts and principles in prevention, namely by sealing occlusal pits and fissures with
fluoride releasing cements, and in restorative dentistry as well. Although the longevity of
dental restorations is shorter with resin fillings in comparison with metallic restorations, the
tissue economy is obviously better using adhesive materials compared with what was done
when the classical Blacks rules of preparations were applied.
-Secondly, tooth-colored resins are more esthetic or at least less visible than metallic
dental fillings.
-Thirdly, Hg is an agent implicated in soil and water pollution and from an ecological
point of view constitutes a potential danger for the environment. Most of the Hg comes from
the industry, and only a small part comes from the dental practice. Devices separating Hg
residues from the water of the dental unit allow eliminating a large part of the metal, but not
all. Decision to ban Hg from dental therapies was taken in Norway by the Ministry of
Environment and not by the Ministry of Health. Hg may also contribute to select bacteria that
are resistant to some antibiotics.

For these three reasons, the place occupied by silver amalgam filling is gradually reduced and
resin-containing materials are developed as amalgam substitutes. Altogether, resin-containing
restorative materials include composite resins, resin-modified glass ionomer cements and
adhesives. In the previous chapters, different reports have summarized our actual knowledge
both in terms of physico-chemical properties and biological adverse effects.

12/74
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 1um, a
width that is largely over the size of bacteria, the diameter of a lactobacillus being around
0.1um.
-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
13/74
may correlate with the reduction of the gap, contributing to an increased adhesion. Catalytic
enzymes and metalloproteinases, provide potential tools.

Secondly, as another option, researches are carried out on some new formulations of cements
that do not contain any resin additive. Such materials are already present in the market. This is
the case for the Portland cements and other exclusively mineral-based materials that are
aiming to stimulate the formation of reactionary or reparative dentin.

The third direction is oriented on dental tissue regeneration, and is based on tissue
engineering. Embryonic or adult progenitor clones or stem cells have the capacity to
differentiate and to produce extracellular matrix (ECM) molecules that promote the formation
and mineralization of either reactionary or reparative dentin, depending the orientation
selected toward repair or regeneration of dental tissues. Some ECM molecules were also
shown recently to stimulate the commitment, recruitment, proliferation and differentiation of
pulp progenitors in a wounded tissue (Goldberg et al., 2008). Growth factors, transcription
factors and others biological molecules may also contribute to the pulp healing, and to the
formation of biological dentin-like materials either in endogenous (repair) or exogenous
(regeneration) sites. However, these promising experimental approaches need further pre-
clinical studies before to be transferred to the dental practice.

In this context a network of laboratories found some interest in collaborating, the only way to
handle nowadays multicentric researches. These groups decided to study the physico-
chemical and biological properties of a new Ca
3
SiO
3
-based posterior restorative cement. This
innovative material does not contain any resin and consequently avoid the danger of free
monomers release. From two clinical pilot studies that were carried out by two different
groups in Marseille and Paris Diderot, which are still in progress and therefore will not be
reported here, we know that such restorative material may be used successfully either as a
medium-term temporary filling, or as permanent base under resin-containing restorations or
inlays/onlays. This is indicative of the present evolution of materials in dentistry. We will
summarize in the chapter firstly some physico-chemical data (VI-2-1) and secondly the
biological aspects which may be deduced from in vitro and in vivo animal studies (VI-2-2).

References
14/74
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.

15/74
VI-2-1 Physico-chemical properties.
Nelly Pradelle-Plasse (University Paris 7 Denis Diderot & LGPM,
Ecole Centrale de Paris) France, Xuan-Vinh Tran (University of
Medicine and Pharmacy, Ho Chi Minh city, Vietnam), & Pierre Colon
(University Paris 7- Denis Diderot, & LGPM, Ecole Centrale de Paris).
France


Introduction
A new experimental Ca
3
SiO
5
-based restorative cement has been developed, put on the market
under the name of BIODENTINE
TM
(Septodont, Saint Maur des Fosses, France). As the
ProRoot MTA

(Torabinejad et al, 1995a,b; Camilleri et al, 2005) and Portlands cements
(Lea, 1970, Camilleri et al, 2006), it is a calcium-based cement. The main component of the
powder is a tricalcium silicate, with the addition to the powder of CaCO
3
and ZrO
2.
The liquid
is a solution of CaCl
2
with a water reducing agent. As every cement, the setting reaction leads
to a gel structure, which allows possible ionic exchanges. Compared to others Ca based
cements, this material presents two advantages: i) a faster setting time of about 12 minutes
and ii) higher mechanical properties. These physico-chemical properties associated with the
biological behavior (Laurent et al, 2008, and this book: sub-chapters VI-2-2) suggest that it
may be used as a permanent dentine substitute.
Chemistry and structure of the cement
Composition
BIODENTINE
TM
is conditioned in a capsule containing the good ratio of powder and liquid,
as shown in Table 1:
Powder :
Tricalcium silicate (3CaO.SiO
2
)
Calcium carbonate (CaCO
3
)
Zirconium dioxide (ZrO
2
)
Liquid
Calcium chloride (CaCl
2
.2H
2
O)
Water reducing agent
Water

Properties of the different components
- Tricalcium silicate (3CaO.SiO
2
): it is the main component of the powder. It regulates the
setting reaction.
- Calcium carbonate (CaCO
3
): it role is similar to the fillers
16/74
- Zirconium dioxide (ZrO
2
): it is added to provide the radio-opacity to the cement
- Calcium chloride (CaCl
2
.2H
2
O): is an accelerator (Chessmann, 1999)
- Water reducing agent (Superplasticiser): It can reduce the viscosity of cement. It is based on
polycarboxylate but modified to obtain a high short-term resistance. It reduces the amount of
water required by the mix (water / cement), although maintaining the same easiness for
handling.

Setting reaction
The reaction of the powder with the liquid led to the setting and hardening of the

cement. The
hydration of the tricalcium silicate (3CaO.SiO
2
) leads to the formation of a hydrated calcium
silicate gel (CSH gel) and calcium hydroxide (Ca (OH)
2
) (Taylor, 1997). The cement located
in inter-grain areas has a high level of calcite (CaCO
3
) content.

The hydration of the tricalcium silicate is achieved by dissolution of tricalcium silicate and
precipitation of calcium silicate hydrate. In generally it is designated by chemist as C-S-H
(C=CaO, S=SiO
2
, H=H
2
O). The calcium hydroxide takes origin from the liquid phase. C-S-H
gel layers formation is obtained after nucleation and growth on the tricalcium silicate surface.
The unreacted tricalcium silicate grains are surrounded by layers of calcium silicate hydrated
gel, which are relatively impermeable to water, thereby slow down the effects of further
reactions. The C-S-H gel formation is due to the permanent hydration of the tricalcium
silicate, which gradually fills in the spaces between the tricalcium silicate grains (Figure 1).
The complete hydration reaction is summarized by the following formula (Taylor, 1997; Lea,
1970, Allen et al, 2007).
2(3CaO.SiO
2
) + 6H
2
O 3CaO.2SiO
2
.3H
2
O + 3Ca(OH)
2

Structure
The surface of the cement observed with the SEM one week after mixing is loaded by calcite
rich structures (CaCO
3
) of variable sizes (Figure 2). The calcite is a chemical or
biochemical mineral crystallizing in the rhombohedra system (a=b=c; o,|,=90). Crystals of
CaCO
3
diamond-shaped (or rhombohedra form) are observed at the surface. We also observed
crystals shaped as hexagonal plates of Ca(OH)
2
described by Taylor (1997) (Figure 3).
According to this author, calcium hydroxide crystallizes in the form of hexagonal plate or
prism. The surface of CaCO
3
crystals is rough and irregular.
17/74
Therefore, we can consider the CSH gel as the matrix of the cement, and the crystals of
CaCO
3
are filling the spaces between grains of cement. Finally, the calcite (CaCO
3
) has two
distinct functions: 1- as an active agent it is implicated in the process of hydration and 2- as
fillers it improves the mechanical properties of the cement (Garrault et al., 2006).

The hardening process results from of the formation of crystals that are deposited in a
supersaturated solution. We can consider that the setting reaction of the 3CaO.SiO
2
includes
four elements: the unreacted particles of cement, surface products (CSH gel), the content of
the pores (Ca (OH)
2
) and porous capillary space (Figure 1).

The electrochemical properties of cement are due to the solid phase and ion mobility of free
ions inside the pores filled with the electrolyte (Andrale et al, 1999; Cabeza et al 2006).
Impedance spectroscopy is a technique that allows studying the process of hardening of a
cement. This is a non-destructive method that may monitor the hardening process. The
electrical resistance increases when the porosity of the system is reduced. Improvement of the
values measured for BIODENTINE
TM
is time-dependent (Figure 4). This shows that
immediately after mixing, the setting reaction of BIODENTINE
TM
is not yet achieved. At
least 2 weeks are necessary to reach a final stable stage. The setting reaction of
BIODENTINE
TM
leads to the formation of initial porosities that are gradually filled after
several days by new crystal compounds. During this final step, the solid phase is increasing
and finally reach a maximum.


Mechanical properties

Vickers microhardness
The hardness can be defined as the resistance to the plastic deformation of the surface of a
material after indentation or penetration. Measurements at different times have been evaluated
(Table II):
The hardness increases in time when cements are immersed in distilled water. After 2 hours,
the hardness of BIODENTINE
TM
is 51 HVN and reached 69 HVN after 1 month. These
values are comparable to those obtained with the resin modified GIC-Fuji II LC (36 HVN),
and the composite resin-Post Comp II LC (97 HVN) (William et al, 2002). The calcite is a
mineral compound in relation with the hardness of cement. The formation of CSH gel reduces
18/74
the porosity with time. The crystallization of the latter continues, therefore improves the
hardness and probably other mechanical properties.

Flexural strength
The 3 points bending test has a clinical significance and is essential when the material is used
for Class I, II and IV cavities. The higher the resistance to flexural strength is, the lower is the
risk of cohesive fracture of the shutter and broken edges. The value of the bending obtained
with BIODENTINE
TM
after 2hours is 34 MPa. Compared with that of other materials: 10-20
MPa (conventional GIC), 40-70 MPa (GIC amended the resin), 120-200 MPa (composite
resin) (Davidson et al, 1999), it shows clearly that the bending resistance of BIODENTINE
TM

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

Tooth BIODENTINE
TM
Adhesives Interfaces

Morphological characterization
The SEM microphotographies show BIODENTINE
TM
- dental structures interfaces (Figures
5-7) and BIODENTINE
TM
- adhesive systems interfaces (Figures 8, 9). The results show the
occurrence of a cohesive failure within the BIODENTINE
TM
cement without alteration of the
tooth biomaterial interface, hence providing evidence for the quality of the micromechanical
adhesion. The crack is an artefactual result of the drying process occurring during the SEM
preparation. The interfacial layer BIODENTINE
TM
- dentin may be compared to the hard
tissue layer shown to be formed when using ProRoot MTA, which is considered by several
authors as a dentinal bridge or a precipitation of hydroxyapatite (Holland et al, 1999, Santos
et al, 2005). We also observed that CaCO
3
crystals form after the end of the setting reaction.
This constitute a micromechanical anchorage with the surface of the dentine and the
precipitation inside dentine tubule provides mineral tag that may contribute to the cement
adhesive properties (Figure 7).
It appears that the mechanical adhesion of BIODENTINE
TM
cement to dental surfaces may
result from a physical process of crystal growth within dentine tubules leading to a
micromechanical anchor. The possible ion exchanges between the cement and dental tissues
constitute an alternative hypothesis, or the two processes may well combine, eventually
contributing to the adhesion of the cement, as it appears at the interface BIODENTINE
TM
-
adhesive systems (Figures 8, 9).

19/74

Microleakage
The interfacial watertightness is an important parameter of the functionality and longevity of
a restoration. The phenomenon of percolation linked to defects or to the gap at the interface is
also designated by the term "microleakage". To evaluate this parameter, we have selected the
dye penetration methodology (silver nitrate), which is one of the most commonly used assays
to assess in vitro the interfacial seal by measuring the percolation of a dye along different
interfaces studied.
The result of penetration at the interface BIODENTINE
TM
- enamel / dentin was very low
(Table III).
A J0, the seal obtained with the Xeno

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 BIODENTINE
TM
- 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 BIODENTINE
TM
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 CaCO
3

crystals as "fillers". Following the setting reaction, the cement presents a certain degree of
20/74
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 BIODENTINE
TM
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,
BIODENTINE
TM
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-
4318

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, Mjr 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.

21/74
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 Mo M, Djou 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.

OBrien 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, 2
nd
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.SiO
2

Inter - layer water
Adsorbed water
Pores containing
water
C-S-H gel
adsorbed water
Inter layer water
Pore
22/74

Figure 2: Cement surface observed at Day 7 (SEM)
23/74

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
24/74

Figure 4: Evolution of electrical resistance of BIODENTINE
TM
as a function of time
639
999
1157
562
528 517
384
500
425
200
400
600
800
1000
1200
1H 2H 4H 5H 6H 9H 1D 7D 14D
Time
(O)


25/74



Figure 5: Enamel- BIODENTINE
TM
interface (SEM)
BIODENTINE
TM
(A) adheres to enamel (B) after a cohesive fracture
A
B
26/74



Figure 6. Dentine BIODENTINE
TM
Interface (SEM)
BIODENTINE
TM
(A) adheres to dentine (B) after a cohesive fracture
A
B
27/74




Figure 7: A "Mineral Tag"
The crystals (A) have infiltrated the dentine tubule (B): "Mineral Tag"

A
B
28/74



Figure 8: BIODENTINE
TM
- G bond

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

(C), Composite Resin (D).


C
A
B
D
29/74



Figure 9: BIODENTINE
TM
Xeno

III interface (SEM)


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

III (C).
C
A
B
30/74

Time Micro hardness
2H 51.5 ( 1.75)


1D 63.14 ( 1.94)
7D 72.19 ( 6.38)
30D 69.46 ( 1.45)



Table II: Average of hardness (HVN) and standard deviation (into brackets)


Interfaces Dye penetration (%) at D0 Dye penetration (%) at D90
Enamel / BIODENTINE
TM
17.65 (4.35) 19.86 (10.72)
Dentin / BIODENTINE
TM
10.46 (3.23) 14.84 (5)
Table III: Tooth BIODENTINE
TM
INTERFACES
% of microleakage


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

III 6.93 ( 3.57) 10.07( 2.23)


G-Bond

18.64 ( 4.13) 7.68 ( 3.2)


Table IV: BIODENTINE
TM
/ adhesives INTERFACES
% of microleakage

31/74


VI-2-2 Biological effects
VI-2-2-1 Development of a bioactive Ca
3
SiO
5
based posterior
restorative material (Biodentine
TM
).
Patrick Laurent, Virginie Aubut, Imad About Laboratoire IMEB, Facult d'Odontologie,
Universit de la Mditerrane, 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:
32/74
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 Ca
3
SiO
5
-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 Ca
3
SiO
5
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,
33/74
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 Ca
3
SiO
5
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 Ca
3
SiO
5
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 Ca
3
SiO
5
-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 didnt exceed 50MPa even after 28 days
(Camilleri et al, 2006).

Development of a biocompatible Ca
3
SiO
5
-based material for dental applications
Taking advantage of the Ca
3
SiO
5
-based cements self setting and bioactive properties, a new
Ca
3
SiO
5
-based cement (Biodentine
TM
) for direct restorative posterior fillings has been
developed recently. The material is inorganic and non metallic. It is composed of Ca
3
SiO
5
,
CaCO
3
,

ZrO
2
, 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 didnt 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 Ca
3
SiO
5
-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 Ca
3
SiO
5
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.
36/74
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 SolGel Bioactive Glass
on Proliferation and Differentiation of Murine and Human Osteoblasts. Tissue Eng. 2004 ;
10(7-8): 1018-26.
Bjerre L, Bnger 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. 5560.
Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanisms at the interface of
ceramics prosthetic materials. J Biomed Mater Res 1972; 2:11741.
Hench LL, West JK. Biological applications of bioactive glasses. Life Chem Reports.
1996;13:187241.
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: 29072915.
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 Mo M, Djou 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; 63:914920.
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, Mjr 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.
39/74

VI-2-2-2 Animal studies
Tchilalo Boukpessi, Dominique Septier, Michel Goldberg
(Universit 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
40/74
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 Biodentine
TM
, a Ca
3
SiO
3
-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 Biodentine
TM
(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 um thick sections were cut then dewaxed and stained with Massons 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
41/74
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 Biodentin
TM
. The thickness
of reactionary dentin in the mesial third of the pulp chamber was time dependent, and
gradually increased, going from 20-40 um at day 8 to 40-80 um at day 15 and reaching 140
280 um at day 30 (Figure 1). These measurements indicated that the formation of reactionary
dentin was enhanced when Biodentine
TM
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 um for the
same period of time. These results underline that the Ca
3
SiO
5
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 Hehls 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 Biodentine
TM
. 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 rats 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, Mjr 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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44/74
A clinical study of a new Ca3SiO5-based material indicated as
a dentine substitute
2009
G.Weissrock, J.C. Franquin, P. Colon , G.Koubi -University
of Paris 7, France
Journe Scientifique du CNEOC Brest -June 2009
45/74

101
A clinical study of a new Ca3SiO5-based material indicated as a dentine substitute
G.Weissrock
1
, J.C. Franquin
1
, P. Colon
2
, G.Koubi
1
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 (Biodentine
TM
, 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 Biodentine
TM
, no special
chemical procedures were applied on the
mineralized walls. All patients treated with
Biodentine
TM
were educated to come back to
the authors 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 Biodentine
TM
filling need
to be replaced, and all restorations evaluated at
d+6 months demonstrated acceptable clinical
performance. After 6 months, Biodentine
TM

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
Biodentine
TM
. 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 Biodentine
TM
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, Biodentine
TM
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 practitioners
equipment.

REFERENCES :
1
P.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
Journe Scientifique du CNEOC Brest -June 2009
47/74

99
BIODENTINE
TM
- 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 biominralisation, Facult de Chirurgie Dentaire, Universit
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 rats 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


rd
RD94
X100
X100
48/74
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)
49/74
50/74
A Clinical Study of a New Ca A Clinical Study of a New Ca
3 3
SiO SiO
5 5
- -based Material based Material
Indicated as a Dentine Substitute Indicated as a Dentine Substitute
G.F. KOUBI
1
, J.C. FRANQUIN
1
, P. COLON
2
1
Dpt. of Operative Dentistry and Endodontics, University of Marseilles, France.
2
Dpt. of Operative Dentistry and Endodontics, University of Paris 7, France.
OP065
Aim Aim
A new Ca
3
SiO
5
-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 Methods Materials and Methods
Longevity and Biocompatibility in vivo
Three 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 manufacturers 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./min
Purified water,
calcium chlor
LIQUID
200 mL
Mixed 25 s.
Linea Tac
Tricalcium silicate,
calcium carbonate,
calcium oxide
POWDER
1 g
For BiodentineRD 94, the new Ca
3
SiO
5
-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 filling
When 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.
Results Results
The clinical study is still on-going. These results of the distribution of restorations by patients 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 patients age
Total RD 94 Z 100
0
100
200
300
Class I
Class II
Class I
Class II
Figure 2. Distribution of restorations by cavity class
0
100
200
300
400
Total RD 94 Z 100
PM
M
PM
M
Figure 3. Distribution of restorations by group of teeth
Longevity and Biocompatibility
On 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 Biodentine
TM
RD94 because of a too rapid occlusal wear.
48 restorations with Biodentine
TM
RD94 have to be replaced by a definitive restoration in Z100.
Ability to be combine with Adhesive filling Z100
None 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 Biodentine
TM
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 Biodentine
TM
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.
Conclusion Conclusion
Three years after the beginning of the study, the results seems to indicate that the new Ca
3
SiO
5
-based material called Biodentine
TM
RD94 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 Biodentine
TM
RD94 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, Biodentine
TM
RD94 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 practitioners equipment.
SEVILLE, SPAIN, MARCH
12th 14th 2009
253
137
116
33
81
48
RD94 Z100
233
124 109
101
45 56
RD94 Z100
Biodentineas bulk Biodentineas liner
Bibliography Bibliography
P. Laurent, J. Camps, M. De Mo, 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 base de silicate de calcium, Thse de Doctorat en Chirurgie Dentaire, Marseille, Juin 2006.
51/74

52/74
Induction of specific cell responses to a Ca3SiO5-based
posterior restorative material
2008
Laurent P, Camps J, De Mo M, Djou J, About I. Marseille,
France.
Dent Mater. 2008 Nov;24(11):1486-94.
53/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494
avai l abl e at www. sci encedi r ect . com
j our nal homepage: www. i nt l . el sevi er heal t h. com/ j our nal s/ dema
Induction of specic cell responses to a Ca
3
SiO
5
-based
posterior restorative material
Patrick Laurent
a
, Jean Camps
a
, Michel De M eo
b
, Jacques D ejou
a
, Imad About
a,
a
Laboratoire IMEB - ERT 30, Facult e dOdontologie, Universit e de la M editerran ee, 27 Boulevard Jean Moulin,
13355 Marseille Cedex 05, France
b
Laboratoire de Biog enotoxicologie et Mutagen` ese Environnementale (EA 1784), Facult e de Pharmacie,
Universit e de la M editerran ee, 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:
Ca
3
SiO
5
-based dental cement
Biocompatibility
Genotoxicity
a b s t r a c t
Objectives. A Ca
3
SiO
5
-based cement has been developed to circumvent the shortcomings
of traditional lling materials. The purpose of this work was to evaluate its genotoxicity,
cytotoxicity and effects on the target cells specic functions.
Methods. Ames test was applied on four Salmonella typhimuriumstrains. The micronuclei test
was studied on human lymphocytes. The cytotoxicity (MTT test), the Comet assay and the
effects on the specic functions by immunohistochemistry were performed on human pulp
broblasts.
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 specic functions such as mineralization, as well as expression of
collagen I, dentin sialoprotein and Nestin.
Signicance. The new cement is biocompatible and does not affect the specic 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.
2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Commonly used direct restorative materials for Class I and II
cavities are resin composites and amalgams [1,2]. In the early
1980s, amalgamrestorations were reported to release mercury
vapors which may be harmful to the environment, the dentist
as well as the patient [3].
Direct composite restorations have gradually been used
to 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).
moderate-sized posterior restorations. In contrast to amal-
gam, resin composites enable micro-mechanical retention
by the use of different bonding techniques. Yet there is
still some concern with composite resin wear resistance in
high-stress situations, polymerization shrinkage, microleak-
age, and unreacted monomer and toxic ingredient release
[46].
Search for a replacement for amalgam and resin compos-
ites has been ongoing for many years. Calcium hydroxide
0109-5641/$ see front matter 2008 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.dental.2008.02.020
54/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1487
(Dycal

) is one of the most widelyusedpulpcapping agents. Its


basic pHis the mainreasonfor its apparent toxicity invitro [7].
However, it has beendemonstratedthat adentinbridge forma-
tioncanbe obtained withthis material 3 months after capping
human teeth with mild to moderate chronic inammation,
mild hyperemia and necrosis [7,8].
Recent research focused on the use of biocompatible
materials such as Portland cement. Mineral trioxide aggre-
gate developed in the 1990s as a root-end lling material
has a similar constitution to Portland cement and is com-
posed primarily of tricalcium and dicalcium silicate [9]. It
is known as a biocompatible material. In vitro, a high rate
of cell viability was reported with MTA extracts with a
methyltetrazoilum (MTT) assay [1012]. Additionally, MTA
used for pulp capping or partial pulpotomy stimulates repar-
ative dentin and complete bridge formation in vivo after
2 months with no signs of inammation [8,13,14]. How-
ever, the setting time of MTA is 2 h 45 min which is too
long for a material to be used as a dental restorative mate-
rial [15]. Moreover, the mechanical properties of both Dycal
and MTA are not compatible for use as dental restorative
material.
Tricalcium silicate is the main constituent of MTA, and
the main raw material in Portland cement. It is known that
Ca
3
SiO
5
possesses hydraulic property and the spontaneous
development of strength on hydration. But its setting time is
too long and its compressive strength hardly reaches 20.2 MPa
after 28 days to meet the need of clinical applications as
a restorative material [16]. Calcium chloride is one of the
most effective accelerators of hydration and setting in Port-
land cement pastes. Although the addition of CaCl
2
up to 15%
in the liquid phase into Ca
3
SiO
5
decreased the nal setting
time from 180 to 90 min, the compressive strength remained
weak (23.46 MPa) at 7 days [17]. The use of superplasticis-
ers 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 com-
pressive resistance did not exceed 50 MPa even after 28 days
[18].
Based on Portland cement properties, a Ca
3
SiO
5
-based
material for direct restorative posterior llings has beendevel-
oped in the authors laboratory. The material is inorganic and
non-metallic. It is composed of Ca
3
SiO
5
, CaCO
3
, ZrO
2
, water
and a superplasticising admixture to reduce the water con-
tent 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. The new Ca
3
SiO
5
cement is compatible with working in the clinic. It has a set-
ting time of 10 min and was developed to be used in direct
and indirect pulp capping procedures as a single applica-
tion bulk restorative material without any cavity conditioning
treatment. Since it may be directly applied to the dental
pulp, its biological properties were compared to biomateri-
als usually used in pulp capping procedures such as MTA and
Dycal.
Since this material belongs to a new class of restorative
materials, its biocompatibility is questioned and in this paper
its cytotoxicity and genotoxicity are investigated. The effect
it may have on the specic functions of target cells was also
evaluated.
2. Materials and methods
2.1. Reagents
All materials used for culture media preparation were
purchased from Gibco BRL (Life Technologies Inc., Grand
Island, NY, USA) unless otherwise specied. Minimum Essen-
tial Medium (MEM) was supplemented with 10% fetal
bovine serum; 100 UI/ml penicillin; 100 g/ml streptomycin
(Biowhittaker, Gagny, France) and 0.25 g/ml amphotericin B
(Fungizone

). Chemicals were obtained from SigmaAldrich


(Sigma Chemicals Corp., St. Louis, MO) unless otherwise
stated.
2.2. Teeth
For pulp cell cultures, normal immature third molars freshly
extracted for orthodontic reasons from 16 to 18 year-old
patients were used after obtaining theirs and their parents
informed consent and was conducted with local ethical com-
mittee approval. Additionally, for the preparation of dentin
slices, 30 healthy human third molars freshly extracted were
stored at 4

C in saline solution and used within 2 h of collec-


tion.
S. typhimurium strains TA97a, TA98, TA100, and TA102 were
kindly provided by Dr. B.N. Ames (Berkeley, CA, USA).
2.3. Antibodies
Polyclonal antibodies against the type I collagen were
purchased from Southern Biotechnology Associates Inc.
(Birmingham, AL, USA). Anti-dentin sialoprotein antibodies
were obtained from WT Butler (UTHSC, Houston, TX, USA).
Preparation and characterization of the polyclonal antibodies
against dentin sialoprotein (DSP) have been already described
[10]. Anti-nestin antibodies were purchased from Chemicon
International (Temecula, CA, USA).
This work was performed on a new Ca
3
SiO
5
-based
cement developed with an industrial partner (Laboratoires
Septodont, Saint Maur des Fosses, France). MTA (Dentsply
Tulsa dental, Tulsa, OK, USA) (batch number 0203332604) and
Dycal (De Trey Dentsply, Milford, DE, USA) (batch number
0204000983) were used as a reference material for cytotoxicity
tests.
2.4. Toxicity by indirect contact between the
biomaterial and the culture media
2.4.1. Preparation of the dentin slices
Fromthe third molars, thirty dentin slices were prepared with
a low speed diamond saw (Isomet, Buehler Ltd., Lake Bluff,
IL, USA) with water coolant. The dentin sections were from
areas adjacent to the pulp chamber, but they showed no evi-
dence of inclusion of a pulpal horn. The dentin slices had a
thickness 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 a
hole of 0.8 cmin its center was placed on the pulpal side of the
dentinslice andwas attachedwitha non-cytotoxic cyanoacry-
late glue. This permitted us to reduce and to standardise the
55/74
1488 dental materi als 2 4 ( 2 0 0 8 ) 14861494
exposed dentin surface area to 50.24 mm
2
. The coronal side
of 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 Ca
3
SiO
5
cement was prepared by mixing the recom-
mended quantities of liquid and powder and vibrating with an
amalgamator. It was applied without any conditioning treat-
ment.
2.4.2. Simulation of pulpal pressure
The Plexiglas rings and the dentin slices were placed in
a special device used to simulate a pulsatile pulpal pres-
sure, as previously described [19]. The Plexiglas device was
used to maintain the dentin slice in such a position that
the MEM culture medium slightly touched the pulpal side
of the dentin slice while the coronal side was open to the
atmosphere. The lower chamber (4 ml), in contact with the
pulpal side of dentin contained the culture medium. A pul-
satile pulpal pressure (1218 cm H
2
O) was applied. The dentin
slices were inserted in the Plexiglas device for 24 h. After
24 h, the media were collected and called the indirect contact
media.
2.5. Toxicity by direct contact between the biomaterial
and the culture media
Tensamples of eachmaterial were preparedaccording toman-
ufacturer recommendations and stored in an incubator prior
to sterilization with UV rays. The Ca
3
SiO
5
cement was pre-
pared as described above. The samples were stored in 1 ml
MEMwith 10%foetal calf serumsupplemented with penicillin
100 IU/ml and streptomycin 100g/ml for 24 h. According to
ISO standards, the ratio between the surface of the sample
and the volume of medium was 0.5 cm
2
/ml. The resulting pH
values in the buffered culture medium were: Dycal 9.2; MTA
8.1 the Ca
3
SiO
5
cement 8.2. These media were called the direct
contact media (n=10 per material).
2.6. MTT assay
Pulpal broblasts were plated at 30,000 cells cm
2
in 96-well
plates (Falcon 3072, Becton Dickinson, Oxford, GB). The 96-
well dishes were then placed into a humid incubator with
an atmosphere of 5% CO
2
, 95% air for 24 h prior to use. After
this 24 h period, the medium from the 96-well plates was
removed and replaced by the test medium. At that time, the
96-well plates were placed in an incubator again for 24 h. A
succinyl dehydrogenase assay (MTT) was performed on the
dishes after 24 h of incubation (i.e., 48 h after the beginning of
the experiment). The medium was removed and immediately
replaced with 100 l/well of a 0.5% of 3-(4,5-dimethylthiazol-
2-yl)-2,(-diphenyl tetrazolium bromide) in the medium. After
incubation for 2 h at 37

C, the supernatant was discarded,


and the formazan crystals were solubilized with 100 l/well
of 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 andindirect contact media, a two-way analysis of
variance (medium dilution and material), followed by a Dun-
cantest, was used to compare the cytotoxicity of MTA, the new
Ca
3
SiO
5
cement and Dycal.
In order to study the long term effects on the pulp brob-
lasts differentiation, it is known that lower concentrations
can be toxic after long term incubation with cells. Thus, the
medium used for the next part of the study was one which
decreased the MTT activity by less than 5%.
2.7. Inuence of the new Ca
3
SiO
5
cement and MTA on
the differentiation of pulp broblasts
In order to evaluate the effect of the new Ca
3
SiO
5
cement
and MTA on the differentiation of pulp broblasts, the cul-
tured cells were incubated in the conditioned MEM medium
obtained after direct and indirect contact with the materi-
als supplemented with 2 mM -glycerophosphate. The cells
were cultured for 4 weeks in cell culture chambers and the
media were changed every other day. After culture, the cells
were xed with 70% ethanol for one hour at 4

C and pro-
cessed for immunohistochemistry. The effect of the materials
on the cytodifferentiation was evaluated by studying the spe-
cic protein expression of control cells compared to that of
cells cultured with the medium after being in contact with
the test material [20].
2.7.1. Immunohistochemistry
The cells were permeabilizedfor 15 minwith0.5%TritonX-100
in PBS. Primary antibodies were diluted in PBS containing 0.1%
Bovine Serum Albumin (BSA). The incubation with primary
antibodies was performed overnight at 4

C. Anti-collagen I
antibodies were used at 40 g/ml and anti-nestin antibody
at 5 g/ml. Anti-dentin sialoprotein antibody was diluted
1:200 in PBS. Immunostaining was revealed using the labeled
streptavidin-biotin kit (LSAB; Dako Corporation, Carpinteria,
CA, USA) according to the manufacturers instructions. Glyc-
ergel was used as a mounting medium (Dako Corporation).
Controls were performed by omitting primary antibodies or
incubationwithunrelatedprimary antibodies (cytokeratin19).
All controls were negative.
2.8. Genotoxicity assays
2.8.1. Ames test
S. typhimurium TA97a, TA98, TA100, and TA102 strains were
grown overnight from frozen cultures in Oxoid nutrient broth
No. 2 for 1012 h. Mutagenicity assays were performed as
described [21]. The genotype of each S. typhimurium tester
strain was conrmed in each experiment, and negative and
positive controls were routinely included.
After the preparation and setting of the cement, it was
ground to prepare a stock solution prior to testing by adding
60 mg of the cement in 1 ml of Nutrient Broth No. 2 (NB 2)
mediumor DMSOsolvent for 24 hat 37

Cunder mixing. These


stock solutions fromtwo independent experiments were then
tested in triplicate and results from both experiments in NB
2 and DMSO are presented. Increasing volumes of test sam-
ples (4, 6, 8 and 10 l) were incubated with each bacterial
strain for 60 min at 37

C under mixing. The mixture consist-


ing of bacteria and a test compound was plated on plates
in VB medium. The bacteria were then incubated at 37

C
56/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1489
for 48 h and revertant colonies were counted with an auto-
mated colony counter (Spiral System Instruments, Bethesda,
MS, USA). The experiments were carried out in the presence
and in the absence of an S9 fraction isolated from liver of
phenobarbital/-naphtoavone-treated rats. This S9 fraction
(4%) was routinely included in an S9-Mix, and the amount of
protein was adjusted to 1.25 mg protein per plate. A substance
was qualied positive if it induced a dose-related and repro-
ducible increase inthe numbers of revertants or twice as many
spontaneous revertants per plate [22].
2.9. Micronucleus test
This work was performed on lymphocytes obtained by
vein puncture from 6 healthy non-smoking donors, after
informed consent, and collected in glass tubes containing
lithium heparin anticoagulant according to Digue et al. [23].
Briey, cultures were carried out by adding 0.7 ml of whole
blood to 9.3 ml of X-VIVO
TM
Medium (Bio-Whittaker, Bel-
gium) supplemented with 25% fetal calf serum (Gibco, Life
Technologies
TM
, Germany), heparin (50 U/ml), and antibiotics
(penicillin 100 Ul/ml and streptomycin 100g/ml). The cells
were stimulated with phytohemagglutinin (1 mg/ml), a spe-
cic mitogen agent of human T-lymphocytes. The cells were
then cultured for 72 h at 37

C in a humidied atmosphere
containing 5% CO
2
.
The Ca
3
SiO
5
cement extract was prepared as described
above inthe culture mediumor DMSOandaddedtothe culture
at 24 h. The cells were directly exposed to serial dilutions (1%,
2.3%, 3.7%, and 5%) of the cement extracts for 48 h. Negative
control was achieved by adding DMSO at a nal concentration
of 0.1%. MitomycinC, used as a reference genotoxic agent, was
used as positive control 5g/ml. Cytochalasin B was added to
the culture (5g/ml) 44 h after PHA stimulation.
The cultures were stopped at 72 h and the cells harvested
by centrifuging (10 min at 360g). They were then treated
by a mild hypotonic treatment (1 min in KCl 0.075 M) and
immediately xed with methanol:acetic acid (3:1). This x-
ing step was repeated twice after 20-min storage at 4

C. Cells
were smeared on pre-cleaned microscope slides and air-dried.
Staining was performed with 5% Giemsa in Milli-Q water for
15 min.
Stainedslides were codedandscoredby light microscopy at
400 magnication. For each slide, 1000 Giemsa-stained bin-
ucleated lymphocytes with a well-preserved cytoplasm were
scored for the presence of micronuclei. In the micronucleated
binucleated cells, the number of MN per cell was recorded.
Micronuclei were expressed in terms of micronucleated cells
per 1000 binucleated lymphocytes. All the slides were exam-
ined twice by the same scorer. As a measure for toxicity, the
binuclearity index (BI) was determined by scoring the binu-
cleated cells for 1000 lymphocytes (mono- and binucleated
cells) and linked to the percentage of lymphocytes that pro-
duced complete cell division for the different drugs tested,
and then provided an index of cytotoxicity [24]. An extract
of a material was considered positive if at least a three-fold
increase of the numbers of micronuclei over negative controls
was observed at one or more dilutions of the original extract
[25,26].
2.10. Single-cell gel (Comet) assay
The Ca
3
SiO
5
cement extract was prepared and put in MEM
medium (60 mg/ml) for 24 h at 37

C under mixing. The cells


were directly exposedto serial dilutions of the cement extracts
for 2 h. The protocol used for single-cell gel (Comet) assay
followed the guidelines proposed by Tice et al. [27]. Briey a
volume of 10l of cells (10
4
cells) of each treatment was added
to 120 l of 0.5% low-melting-point agarose at 37

C, layered
onto a pre-coated slide with 1.5%regular agarose, and covered
with a coverslip. After brief agarose solidication in a refrig-
erator, the coverslip was removed and the slides immersed
in lysis solution (2.5 mol/l NaCl, 100 mmol/l EDTA, 10 mmol/l
TrisHCl buffer pH 10, 1% sodium sarcosinate with 1% Triton
X-100, and 10% DMSO) for about 1 h. Prior to electrophore-
sis, the slides were left in alkaline buffer (pH >13) for 20 min
and electrophoresed for another 20 min, at 25 V (0.86 V/cm)
and 300 mA. After electrophoresis, the slides were neutral-
ized in 0.4 mol/l TrisHCI (pH 7.5) xed in absolute ethanol,
and stored at room temperature until analysis blindly in a
uorescence microscope at 400 magnications. In order to
minimize extraneous DNA damage from ambient ultraviolet
radiation, all steps were performed with reduced illumina-
tion. Anautomatic analysis system(Comet Assay II; Perceptive
Instruments, Haverhill, UK) was used to determine DNA dam-
age. Tail moment (product of tail DNA/total DNA by the center
of gravity) was considered to estimate DNA damage from 50
cells per treatment.
3. Results
3.1. Determination of the toxicity with or without
dentin disc interposition
When the toxicity was evaluated indirectly through a dentin
slice, the analysis of variance failed to showa statistical differ-
ence between the new cement, Pro Root MTA, and Dycal (ns)
(Table 1). None of the materials was cytotoxic. However, when
the toxicity was evaluated without dentin slice interposition,
the analysis of variance showed a statistically signicant dif-
ference among the three materials (P <0.001). The Duncan test
Table 1 Cytotoxicity after indirect contact between the
materials and culture medium through a dentin disc
New Ca
3
SiO
5
cement MTA Dycal
Undiluted 0 8% 0 9% 0 8%
50% 0 4% 0 4% 0 4%
10% 0 4% 0 3% 0 4%
The newCa
3
SiO
5
cement, MTA, and Dycal were applied on the coro-
nal side of the dentin slices in Plexiglass devices with pulp pressure
simulation. After 24 h, the culture media in contact with the pul-
pal side of the dentin slices were used to determine cell viability.
The pulp broblasts were incubated with these media (either undi-
luted, or diluted in the culture medium to 50% or to 10%) for 24 h
before applying the MTT test on human pulpal broblasts. Opti-
cal density values of untreated control cultures normalized to 100%
was in the range of 0.90.95. The results are expressed as mean cell
toxicityS.D.
57/74
1490 dental materi als 2 4 ( 2 0 0 8 ) 14861494
Table 2 Cytotoxicity after direct contact between the
material and culture medium
New Ca
3
SiO
5
cement MTA Dycal
Undiluted 0 8% 0 9% 22 10%
50% 0 5% 0 5% 10% 5%
10% 0 4% 0 3% 2 2%
The cytotoxicity of the new cement compared to MTA and Dycal on
human pulp broblasts was evaluated after 24 h contact between
the materials and the culture medium (either undiluted, or diluted
in the culture medium to 50% or to 10%) with the MTT test. Both
were less cytotoxic than Dycal (P <0.001). The results are expressed
as mean cell toxicityS.D.
showed that Dycal displayed a higher cytotoxicity than MTA
and the new Ca
3
SiO
5
cement (Table 2).
According to this study, a dilution of 10% was chosen for
studying the materials effects onbroblasts specic functions
because it has biological effects without being toxic.
3.2. Inuence of the two materials on pulp broblasts
differentiation into odontoblastic cells
Control cells expressed collagen I, dentin sialoprotein and
Nestin. Pulp broblasts secreted a mineralizd matrix and the
cells, particularly those contacting the mineralizd matrix,
expressed Nestin (Figs. 1 and 2).
Fig. 1 Effect of the new Ca
3
SiO
5
cement on pulp broblast
specic gene expression. Immunohistochemistry was used
to evaluate the effect of the new Ca
3
SiO
5
cement and MTA
on pulp cells specic genes expression. Control cultures
express collagen type I (a) and dentin sialoprotein (b).
When the media containing the new Ca
3
SiO
5
cement (c
and d) and MTA (e and f) extracts were added to the
cultures for 4 weeks, collagen I (c and e) and dentin
sialoprotein (d and f) were also expressed at a high level in
the pulp cells. Original magnications =10.
Fig. 2 Effect of the new Ca
3
SiO
5
cement on pulp cells
mineralization. Immunohistochemistry was used to
evaluate the effect of the new Ca
3
SiO
5
cement and MTA on
pulp cells differentiation and mineralization. Control
cultures express Nestin and secrete a mineralized matrix in
the form of nodules (a). When the media containing the
new cement (b) or MTA (c) extracts were added to the
cultures for 4 weeks, a mineralized matrix deposition was
also observed. Nestin was also expressed at a high level in
pulp cells and its expression was stronger in the mineral
nodules forming cells. Original magnications =10.
After adding the media containing extracts of the new
Ca
3
SiO
5
cement or MTA to the cultured pulp cells, collagen I,
dentin sialoprotein were strongly expressed by the pulp cells
(Fig. 1). Mineral nodule formation was also observed (Fig. 2).
Nestin was expressed by the cells but not in the mineral nod-
ules. The immunostaining intensity was always higher incells
forming the mineral nodules than the cells away from these
nodules.
3.3. Genotoxicity
Ames test did not show any evidence of mutagenicity of
the Nutrient Broth No 2 medium after being in contact with
the new cement, whatever the dilution of the test medium
(Table 3). The mutations observed with the new cement were
comparable to the spontaneous reverse mutations obtained in
58/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1491
Table 3 Mutation frequencies of Ames tester strains using the liquid preincubation assay
Metabolic activation
(S9 mix
a
)
Product Volume (l) Number of revertants/plate (meanS.D.)
TA 97a TA 98 TA 100 TA 102
+ NB No. 2 10 171 9 243 1364 382 17
+ DMSO 10 166 7 251 12513 355 16
NB No. 2 5 183 13 265 13511 402 18
DMSO 5 191 11 274 1389 423 26
+
New Ca
3
SiO
5
cement (NB No. 2 extract) 4 162 14 301 13514 360 10
6 177 5 261 1202 397 15
8 177 4 295 1325 351 7
10 192 4 274 15013 345 2

New Ca
3
SiO
5
cement (NB No. 2 extract) 2 215 11 251 16110 500 24
3 223 9 253 17221 424 36
4 225 15 251 16035 439 3
5 205 23 231 18212 517 44
+
New Ca
3
SiO
5
cement (DMSO extract) 4 170 19 292 1193 334 49
6 189 3 252 12613 376 3
8 175 2 288 1451 336 24
10 164 23 437 1365 314 11

New Ca
3
SiO
5
cement
(DMSO extract)
2 193 2 352 1493 421 5
3 186 5 375 1178 445 42
4 224 17 273 1406 463 26
5 173 8 301 1443 435 36
+ B[a]P 0.5 g 112137 42326 100087 679 28
ICR 191 0.02 g 55321 NT NT NT
2,4,7 TNFone 0.02 g NT 1653 NT NT
NaN3 0.5 g NT NT 58512 NT
MitC 0.2 g NT NT NT 365854
After preparation and setting of the cement, it was grinded prior to testing. 60 mg of the cement were placed in 1 ml of Nutrient Broth No 2
or DMSO solvent for 24 h at 37

C under mixing. The stock solutions from two independent experiments were tested in triplicate, and results
from both experiments in NB 2 and DMSO are presented. Increasing volumes of test samples (4, 6, 8 and 10l) were incubated with the each
of the bacterial strains for 60 min at 37

C under mixing. The mixture consisting of bacteria and a test compound was plated on plates in VB
medium at 37 C for 48 h and revertant colonies were counted. The experiments were carried out in the presence and in the absence of an S9
fraction. The test was qualied positive if it induced a dose-related and a reproducible increase of the numbers of revertants or twice higher
than the spontaneous revertants per plate. All data are expressed as means S.D. Positive controls were Benzo[a]pyrene (0.5g) with S9 MIX for
all strains. Positive controls were 2-methoxy-6-chloro-9-(3-(2-chloro-ethyl)aminopropylamino)acridine (ICR 191, 0.1g) for TA97a; 2,4,7-trinitro-
9-uorenone (2,4,7-TNFone, 0.02g) for TA98; sodium azide (NaN
3
, 1 g) for TA100 and mitomycin C (MitC, 0.05 g) for TA102 without S9 MIX.
NT: non-tested.
a
The S9 MIX included 4% S9, 4.2 mM NADP and 5.2 mM G6P.
the controls performed with the NB 2 and DMSO solvent. The
results show that the new Ca
3
SiO
5
cement does not induce
reverse mutations either withor without the S9 metabolic acti-
vation system. Similar results were obtained with all bacterial
strains tested.
Table 4 Micronucleated human lymphocytes count in
Ca
3
SiO
5
cement-treated cultures
Ca
3
SiO
5
cement dilution Micronucleated
lymphocytes (%) S.D.
1% 4.0 1.1
2.3% 4.0 1.1
3.7% 4.0 1.2
5% 4.2 1.2
Negative control
a
3.7 1.2
Positive control
b
16.0 6.0
***
Comparison with the control:
***
P <0.001.
a
Culture medium X-VIVO 10.
b
Mitomycin C 5g/ml.
The micronuclei test revealed that after incubating the
lymphocytes with different dilutions of the new cement, the
rate of lymphocytes with micronuclei was similar to that
obtained with the negative control. It ranged from 3.9% to
4.1% with increasing concentrations (15%) in aqueous or
hydrophobic medium. The positive control showed a rate of
16% (Table 4).
The Comet assay performedwithserial dilutions of the new
Ca
3
SiO
5
cement on human pulp broblasts revealed that the
percentage of DNA in the tail ranged from 12.59 for the 0.1%
dilution to 15.58 with undiluted medium. This percentage was
13.19 with the negative control and 46.52 with the positive
control (Table 5).
4. Discussion
The biocompatibility of the newcement is shown in this study
by the absence of cytotoxicity and genotoxicity and the fact
that the new material does not affect the cytodifferentiation
of human pulp broblasts in odontoblastic cells.
59/74
1492 dental materi als 2 4 ( 2 0 0 8 ) 14861494
Table 5 Comet assay on human pulp broblasts
Ca
3
SiO
5
cement dilution Tail DNA (%) meanS.D.
0.1% 12.59 0.96
1% 13.31 0.88
10% 14.90 1.06
Undiluted 15.58 1.08
Negative control
a
13.19 0.96
Positive control
b
46.52 1.45
***
Comparison with the control: ***P <0.001. NS: non-signicant.
a
0.1% DMSO.
b
H
2
O
2
(13.2 mM).
Although Portland cements are known as non-toxic, in this
work, 3 tests were performed to evaluate the genotoxicity of
the new Ca
3
SiO
5
cement after solubilisation in hydrophilic or
hydrophobic conditions. These tests were performed because
the cement developed here contains a modied polycar-
boxylate in the superplastisizer. It has been reported that
polycarboxylate (Aqualox

) elicited mutagenic effects on S.


typhimurium TA 98 and TA 1535. In the presence of S9 fraction,
Aqualox

elicitedweakmutagenic effects onS. typhimuriumTA


1535 and dose-dependent mutagenic effects on S. typhimurium
TA98 [28]. Here, Ames test performed withand without the S9
fraction on 4 different bacterial strains including TA 98 failed
to detect signicant reverse mutations.
While Ames test was performed on prokaryotic cells, the
micronucleus test and the Comet assay were performed on
eukaryotic cells. The micronucleus test was important to per-
form 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 developed as reliable biochem-
ical technique for evaluating DNAdamage andbreaks insingle
mammalian cells [27]. This test was performed on the tar-
get cells of the new cement and did not show signicant
DNA breaks in human pulp broblasts. These results may be
explained either by the fact that the modication of polycar-
boxylate suppressedits mutagenic effects or by the fact that its
concentration is too low in the cement to have any mutagenic
effect.
The new material was developed as a restorative material
both for direct and indirect pulp capping. That is why toxic-
ity was investigated under two conditions: indirectly through
dentin discs and directly by applying the medium containing
the new cement extract on the target cells. The new cement
was not toxic to the cells under either condition even when
tested undiluted.
The toxicity of the new cement was compared to materi-
als used in pulp capping situations. This study conrms the
absence of MTA toxicity. This material was introduced in the
90s and is well accepted by endodontists as an excellent mate-
rial for retrolling, perforation repair and apexication. This
success is due, in part, to the sealing properties of the mate-
rial [15] but mainly to its biocompatibility [29,30]. It has been
shownthat using the same MTTassay that MTAwas non-toxic
to periodontal ligament broblasts [10] and human gingival
broblasts [31]. The current results corroborate those of two
other indirect contact studies using agarose superimposition
[32] or millipore lter [33]. This total absence of toxicity possi-
blyexplains the adhesionof humanosteoblasts tothe material
surface [34].
Dycal was slightly cytotoxic in direct contact. This con-
rms previous work [7] and may be due to the solubility of
salt resulting from the reaction between salicylic acid and
zinc oxide releasing zinc ions and non-reacting hydroxide
ions. It is possible that this is clinically irrelevant because
20% cell death without pulpal clearance does not represent
harmful behavior of the material. In vivo, Dycal does not elicit
an inammatory reaction after intramuscular implantation
in rats [35] and induces slight inammation after direct pulp
capping [36]. The toxicity decrease after dentin disc interpo-
sition is in agreement with previous work on the importance
of dentin thickness and hydraulic conductance on restorative
material toxicity [37].
All studies comparing the effects of MTA versus Dycal con-
cluded a higher efciency of MTA. Direct pulp capping with
MTA gave better results that Dycal at 4 months on human
wisdom teeth [8] and at 2 months in dog teeth [38].
Absence of toxicity with the new cement was comparable
to that of MTA and this was the case either with or without
dentin slice interposition. Additionally, both the new cement
and MTA do not seem to affect the odontoblastic specic pro-
tein expression or mineralization.
In previous work, the authors have shown that pulp
cells cultured with -glycerophoshate secrete an extracel-
lular matrix deposit which progressively forms nodules of
mineralized material. FTIR analysis showed that it was a spe-
cic deposition which had the same mineral composition of
dentin [39]. The cultured cells, particularly those involved in
mineral nodule formation, express a high level of alkaline
phosphatase activity indicating high mineralization potential
of these cells. In addition, the cells involved in the miner-
alization express the type I collagen, osteonectin, DSP and
Nestin. In this work, the cells treated with the new cement
or MTA expressed collagen I, dentin sialoprotein and Nestin
and synthesized a mineralized matrix. Colagen I is the major
dentin matrix organic protein [40]. DSP which is expressed
during human tooth development is a 53-kDa glycoprotein
accounts for 58%of the dentinextracellular matrix. It is local-
ized mainly in dental tissues and its expression was reported
to be localized and conned to differentiating odontoblasts,
with a transient expression in the presecretory ameloblasts
[41]. However, odontoblasts express DSP to a much greater
extent thanother cell types [42]. Additionally, Nestinwhichis a
humanodontoblast specic intermediate lament protein[43]
was expressed in these cells after adding -glycerophoshate
with a stronger expression in the cells contacting the mineral
nodules.
This is of prime importance in the clinic. Coronal restora-
tions 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 [44].
The expression of these specic proteins by human pulpal
broblasts in the presence of MTA has never been reported,
but the potential of MTA to induce cell cytodifferentiation has
already been shown in animal studies. The root end closure
60/74
dental materi als 2 4 ( 2 0 0 8 ) 14861494 1493
with MTA [45] and growth of newcementoblasts in direct con-
tact with MTA used as a retrolling material have been shown
in dogs [46] and monkeys [47] and reparative dentin can be
seen after direct pulp capping with MTA[13,38,48]. The advan-
tage of the new material over both Dycal

and MTA resides in


the fact that, in addition to its biocompatibility, its mechanical
and physical properties strongly suggest its future utilisation
as a bulk restorative material and not only as a pulp capping
agent.
5. Conclusions
The results of the current study need to be conrmed in vivo
and suggest that this new Ca
3
SiO
5
cement could be used as a
direct pulp capping agent but also as a lining agent. This mate-
rial would possibly induce the 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 prop-
erties let us think that this material could be used as a pulp
capping agent and as a bulk restorative material at the same
time. In addition, no preliminary conditioning of the cavities
is required with this new cement. This would greatly simplify
pulp capping techniques.
Acknowledgements
This work was supported by institutional funding from the
French Minist ` ere de l education nationale, de lenseignement
sup erieur et de la recherche. The authors wish to thank Dr.
Jean-Charles Gardon for providing the third molars used in
this work.
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62/74
Microleakage of a new restorative calcium based cement
(Biodentin)
2008
Tran V, Pradelle N, Colon P
Oral presentation PEF IADR Sept 2008 London
63/74

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, facult de Chirurgie Dentaire Paris 7, France, and N. PRADELLE-
PLASSE, Facult 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 37C 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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64/74
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
65/74
0299 RD94, a Portland Cement, Stimulates in Vivo
Reactionary Dentin Formation
T. 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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66/74
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
67/74
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.Boinon
1
, MJ.Bottero-Cornillac
12
, G. Koubi
12
& J. Dejou
1

1
Laboratoire IMEB-ERT 30,
2
Dpartement dodontologie conservatrice-endodontie, UFR
dOdontologie, Universit de la Mditerrane, Marseille, FRANCE

INTRODUCTION: The purpose of this study
was to evaluate the ability of new Ca
3
SiO
5
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 55C. 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
m
e
d
i
a
n

s
c
o
r
e
A
A
C C C

Fig1. Interfacial microleakage according to
surface treatment.
0
5
10
15
20
25
ESA SA A EA C
M
P
a
AB AB A B B

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), silaneadhesive
resin (SA), etching-adhesive resin (EA) and
etching-silane-adhesive resin (ESA).

DISCUSSION & CONCLUSIONS: Etching the
surface of the experimental cement with a H
3
PO
4

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
29
SI and
13
C solid-state NMR. Journal of
Colloid and Interface Science 11903, 1-11

68/74
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
69/74
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
Dpartement dOdontologie 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-
Grnberg (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.




70/74
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)
71/74
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. About
1
, A Raskin
1
, M. De Meo
2
, and J. Djou
1
.
1
Laboratoire IMEB-ERT 30, Facult dOdontologie,
2
Facult de Pharmacie,
Universit de la Mditerrane, 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)
H
2
O
2 (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



72/74
Physical, chemical and mechanical behavior of a new material
for direct posterior fillings.
2005
J. DEJOU, J COLOMBANI and I. ABOUT. Marseille, France
abstract : European Cells and Materials Vol. 10. Suppl. 4,
2005 (page 22)
73/74
European Cells and Materials Vol. 10. Suppl. 4, 2005 (page 22) ISSN 1473-2262

Physical, Chemical and Mechanical Behavior of a New Material for Direct
Posterior Fillings.
J. Djou, A Raskin, J Colombani & I. About

Laboratoire IMEB-ERT 30, UFR dOdontologie, Universit de la Mditerrane, Marseille,
FRANCE

INTRODUCTION: A new ceramic material
for direct restorative posterior fillings, a
Ca
3
SiO
5
-based Portland cement, has been
developed to circumvent the shortcomings of
the traditional filling materials. The purpose of
this work was to evaluate the marginal sealing
efficiency, the acid erosion and the effects of
aging in artificial saliva on its structure,
composition and compressive strength.
METHODS: The marginal sealing was
evaluated by the silver nitrate penetration
method without any surface treatment, with or
without aging in Fusayama artificial saliva.
The acidic erosion was evaluated daily in lactic
acid (0.02M) and sodium lactate (0.1M)
aqueous solution (pH 2.74) by measuring the
height loss, for a week.
Aging was evaluated in Meyer-modified
Fusayama artificial saliva1 (pH 5.3).
The height modification of the material was
evaluated for a week. Scanning electron
microscopy was used to examine and
characterize the surface of the sample before
and after aging. The possible dissolution of the
new material in the artificial saliva was
evaluated by measuring the concentration of
Si, Ca, Zr, and inorganic carbonate in the
artificial saliva after 1, 2, 3 and 4 weeks. The
compressive strength was measured 24 hours
after setting and after aging for seven and 28
days.
RESULTS: No difference in marginal sealing
was revealed between the new biomaterial and
the Z250-Optibond solo plus adhesive
restorative system. The same results were
obtained after aging for one week in artificial
saliva. The acid erosion increased with time.
This increase was less rapid than that obtained
with glass ionomer cement reported by
Nomoto R2,3. In artificial saliva there was no
erosion but deposition of white material on the
surface of the material. Scanning electron
microscopic analysis of this material revealed
needle-like crystals with an apatitic appearance
(figure.1).

Fig. 1: Needle-like crystals on the surface of
the material after aging in artificial saliva
The composition of this deposit determined by
X-diffraction analysis seems to confirm the
apatitic composition (ratio Ca/P = 1.6). This
correlates well with the analysis of the
elements in the solution, which reveals a
decrease of Ca concentration with time. There
was a slight but not significant release of Si.
The compressive strength was 136 (20.10) at
24 hours, increased to 169.74 (16.92) after 7
days and then was stable until day 28.

DISCUSSION & CONCLUSIONS: The
marginal sealing without any surface treatment
or adhesive system was equivalent to that of
the reference material used. In spite of the
acidic pH of the artificial saliva, the new
material showed no erosion and an increase in
the compressive strength. The deposition of
apatitic structures might increase the marginal
sealing of the material.

REFERENCES:
1
Reclaru L, Meyer JM.
(1994). Study of corrosion between a titanium
implant and dental alloys. J Dent; 22:159-68.
2
Nomoto R, McCabe JF. (2001). A simple acid
erosion test for dental water-based cements.
Dent Mater ;17(1):53-9.
3
Nomoto R, Uchida K, Momoi Y, McCabe JF.
(2003). Erosion of water-based cements
evaluated by volumetric and gravimetric
methods. Dent Mater.;19(3):240-4.
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