R&D Department

Biodentine™Active Biosilicate Technology™

Scientific File

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Introduction ................................................................................................................................................................. 4

Active Biosilicate Technology™ ............................................................................................... 5 1.1 - Setting reaction ..................................................................................................................................................... 6 1.2 - Formulation of Biodentine™ ...................................................................................................................... 7

Physico-chemical features ............................................................................................................... 8 2.1 - Setting Time ............................................................................................................................................................. 8 2.2 - Density and Porosity ...................................................................................................................................... 10 2.3 - Compressive strength .................................................................................................................................. 11 2.4 - Flexural strength ................................................................................................................................................ 12 2.5 - Vickers micro hardness ............................................................................................................................... 12 2.6 - Radiopacity ............................................................................................................................................................ 13 2.7 - Comparison with Glass Ionomers and ProRoot® MTA ..................................................... 13

Biodentine™ Interfaces ...................................................................................................................... 14 3.1 - Resistance to acid ........................................................................................................................................... 14 3.2 - Resistance to microleakage .................................................................................................................... 16 3.3 - Electron Microscopy ...................................................................................................................................... 18

Outstanding biocompatibility .................................................................................................... 20 4.1 - Cytotoxicity tests (ISO 7405, ISO 10993-5) ............................................................................... 20 4.2 - Sensitization tests (ISO 7405, ISO 10993-1) ............................................................................ 21 4.3 - Genotoxicity tests (ISO 7405, ISO 10993-3, OCDE 471) ............................................... 22 4.4 - Cutaneous irritation tests (ISO 7405, ISO 10993-10) ........................................................ 23 4.5 - Eye irritation tests (OCDE 405) ............................................................................................................. 23 4.6 - Acute toxicity tests (ISO 7405, ISO 10993-11, OCDE 423) ......................................... 23 4.7 - Preclinical safety conclusion .................................................................................................................. 23

Evidence based bioactivity ............................................................................................................ 24 5.1 - In vitro test of direct pulp capping on human extracted teeth .................................. 24 5.2 - In vitro test for angiogenesis .................................................................................................................. 25 5.3 - Stimulation of reactionary dentine in indirect

pulp capping : rat model ............................................................................................................................ 25 5.4 - Calcification as a result of Biodentine™ in a direct

pulp capping and pulpotomy : pig model ................................................................................... 26 5.5 - Overall bioactivity ............................................................................................................................................. 28

Clinical efficacy ................................................................................................................................................. 29 6.1 - Biodentine™ is used as a dentine substitute under a composite .......................... 29 6.2 - Biodentine™ is used as a direct pulp capping material ................................................. 31 6.3 - Biodentine™ is used as an endodontic repair material .................................................... 32

References ................................................................................................................................................................ 33

Summary

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IntroductionBiodentine™ was developed by Septodont’s Research Group as a new class ofdental material which could conciliate high mechanical properties with excellentbiocompatibility, as well as a bioactive behavior. Several years of active andcollaborative research between Septodont and several universities led to a newcalcium-silicate based formulation, which is suitable as a dentine replacementmaterial whenever original dentine is damaged.

In addition to the chemical composition based on the Ca3SiO5 – water chemistrywhich brings the high biocompatibility of already known endodontic repaircements (MTA based), Septodont increased the physico-chemical properties(short setting time, high mechanical strength…) which make Biodentine™clinically easy to handle and compatible, not only with classical endodonticprocedures, but also for restorative clinical cases of dentine replacement. Sealingability of this biomaterial was also assessed to be equivalent to glass-ionomers,without requiring any specific conditioning of the dentine surface. Leakageresistance and mechanical strength will improve over the first weeks afterplacement.

Biodentine™ turns out to be one of the most biocompatible of all the biomaterialsin dentistry as demonstrated according to all the ISO standard tests, as well as inthe different preclinical and clinical research collaborations. Moreover, reactionarydentine formation was demonstrated in rats, exhibiting high quality and quantityof protective dentine stimulation in indirect pulp capping. In the case of directpulp capping and pulpotomy in pigs, the compatibility with the pulp enables adirect contact with fibroblasts, with limited inflammatory response compared tocontrols. Formation of a regular and dense dentine bridge is histologicallydemonstrated within one month.

Besides the usual endodontic indications of this class of calcium-silicatecements (repair of perforations or resorptions, apexification, root-end filling),Biodentine™ has been evaluated for its restorative properties versus composite(Z100™, 3M ESPE) in a three year follow-up, randomized, multicentre clinicalstudy in 400 patients. It was suitable as a permanent dentine substitute andtemporary enamel substitute. Restoration of deep or large crown carious lesionsprovides a very tight seal, without post-operative sensitivity and insures thelongevity of restorations in vital teeth. Biodentine™ has also achieved 100%success in direct pulp capping in adults presenting healthy pulp.

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Septodont’s initial objective was to develop a material based on the most biocompatiblechemistry available for dental materials: calcium silicates, which can set in the presenceof water. Although recognized as highly biocompatible and bioactive, all these materialslack reactivity, with very long setting times (more than 2 hours), low mechanicalproperties and with very difficult handling (depending on the water ratio, from a sandyconsistency to a fluid paste).

In order to take up the technological challenge of combining this calcium silicatechemistry with the requirements of a formulation compatible with classical restorativeand endodontic practice, Septodont developed a new technological platform calledActive Biosilicate Technology™. This consists in controlling every step of the materialformulation beginning with the purity of the raw materials.

Usual dental calcium silicate cements are based on the “Portland Cement” materials,which result from the clinker products manufactured by the building industry from naturalstone treatment. This implies that all these products inherently contain unpurifiablemixtures of calcium silicates (C3S + C2S), calcium aluminates (C3A), calcium alumino-ferrites (C4AF), calcium sulfates (CaSO4 - gypsum), together with low concentrations ofmetallic impurities coming from the natural minerals used as raw materials.

The only way to reach our objectives in terms of purity control, high mechanical strengthand short setting times, was to synthesize our own calcium silicate product.

The Active Biosilicate Technology™ is a proprietary technology developed according toour state-of-the-art pharmaceutical background applied to the high temperate ceramicmineral chemistry.

Septodont is now able to ensure the purity of the calcium silicate content of theformulation and the absence of any aluminate and calcium sulfate in the final product.

Active Biosilicate Technology™

Firing

Grinding Groundpowder

Biodentinecapsule

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1.1 - Setting reaction

The calcium silicate has the ability to interact with water leading to the setting andhardening of the cement. This is a hydration of the tricalcium silicate (3CaO.SiO2 = C3S)which produces a hydrated calcium silicate gel (CSH gel) and calcium hydroxide (Ca (OH)2).

2(3CaO.SiO2) + 6H2O 3CaO.2SiO2.3H2O + 3Ca(OH)2C3S CSH

This dissolution process occurs at the surface of each grain of calcium silicate. Thehydrated calcium silicate gel and the excess of calcium hydroxide tend to precipitate atthe surface of the particles and in the pores of the powder, due to saturation of themedium. This precipitation process is reinforced in systems with low water content.

The unreacted tricalcium silicate grains are surrounded by layers of calcium silicatehydrated gel, which are relatively impermeable to water, thereby slowing down the effectsof further reactions. The C-S-H gel formation is due to the permanent hydration of thetricalcium silicate, which gradually fills in the spaces between the tricalcium silicategrains. The hardening process results from of the formation of crystals that are depositedin a supersaturated solution.

Biodentine™ Particle

CSH CaOH

H2OH2SiO4

2-

Ca2+ O2H

Powder before hydration Deposition of CSH Biodentine™ after setting

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1.2 - Formulation of Biodentine™

In order to reach a formulation with a short setting time (12 minutes) and high mechanicalproperties in the range of natural dentine, calcium silicates could not be used alone.

Usually calcium silicate cements have setting times in the range of several hours, whichis too long in most of the protocols in clinical practice.

Increasing the setting time was achieved by a combination of different effects. First,particle size greatly influences the setting time, since the higher the specific surface, theshorter the setting. Also, adding calcium chloride to the liquid component acceleratesthe system. Finally, the decrease of the liquid content in the system decreases the settingtime to harden within 9 to 12 minutes.

Reaching high mechanical strength is also quite difficult for these systems. The firstcause of low mechanical properties of Portland cements are the aluminate components,which make the product fragile. Septodont controls the purity of the calcium silicatethrough the Active Biosilicate Technology™ which consists in eliminating aluminates andother impurities.

The second axis of formulation was to adjust the particle size distribution in order toreach an optimal powder density. The additional charge system selected was calciumcarbonate, for both its biocompatibility and calcium content.

The paradox of calcium silicate systems is also that water, which is essential for thehardening of the product, can also affect the strength of the material. On the hand,excess water in the system will create some remaining porosity, significantly degradingthe macroscopic mechanical resistance, but on the other hand decreasing the watercontent leads to reducing the possibility of a homogenous mix. The addition ofhydrosoluble polymer systems described as “water reducing agents” or superplasticizers, helps in maintaining the balance between low water content andconsistency of the mixture.

Radiopacity is obtained by adding zirconium oxide to the final product.

Powder Tri-calcium Silicate (C3S) Main core material

Di-calcium Silicate (C2S) Second core material

Calcium Carbonate and Oxide Filler

Iron Oxide Shade

Zirconium Oxide Radiopacifier

Liquid Calcium chloride Accelerator

Hydrosoluble polymer Water reducing agent

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2.1 - Setting Time

There are several methods to evaluate the setting of dental materials. The first one isbased on the macroscopic evaluation of the resistance of a needle to penetrate thesurface of the material: when the needle does not leave a trace on the surface of thematerial, it corresponds to the setting time. This is the principle of the ISO standard 9917.

An alternative instrumented and more objective method can be used especially to helpin the selection of different formulations: the use of a rheometer (Nonat and Franquin,2006).

Method:

Dynamic rheometry tests were performed to determine the characteristics of eachmaterial during their workability (working and setting times) as well as the rate of buildingearly mechanical resistance. These tests consisted in measuring, by a viscoelastometry,the constraint transmitted by the sample, when a sinusoidal strain is applied. An ARESstrain-controlled rheometer was used (Rheometric Scientific Inc., Piscataway, US). Aftermixing, the sample was inserted between the two striated parallel plates, 6 mm indiameter, with a 2 mm gap. Only the lower plate was maintained at the controlledtemperature of 37.5°C, and a closed chamber maintained the temperature of the entiresample at 100% relative humidity to prevent drying. The experimental conditions wereas follows: oscillation frequency: 1 radian per second, applied strain: 0.0005%. Underthese conditions, the applied strain is less than the critical strain beyond which thestructure of the cement paste is altered (about 0.0015%), and the transmitted stress isproportional to the strain. This system can therefore be used to measure the evolutionof the elastic modulus G’ of the material, without any modification of the structure of thematerial.

This instrumented method was used to determine the setting time of the Biodentine™Formulation (Fig.1) and to compare it to a classical glass ionomer (Fuji IX – GC) andProRoot® MTA (Dentsply). The initial setting time was determined at the moment whenthe elastic modulus begins to increase (10MPa). The final setting time was determinedas the elastic modulus reached 100MPa.

The time between the mixing and the initial setting corresponds to the working time.

Physico-chemical features

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Fig. 1: Dynamic rheometry evaluation of the initial and final setting times.

From these results it can be concluded that the working time of Biodentine™ is up to 6 minutes with a final set at around 10-12 minutes. The classical glass ionomer setsfaster that Biodentine™ in less than 4 minutes. This represents a great improvementcompared to the other calcium silicate dental materials (ProRoot® MTA), which set inmore than 2 hours.

The setting times of Biodentine™ are in the same range as the amalgams.

When tested according to the ISO standard with the Gilmore needle, the working timeis over 1 minute and the setting time is between 9 and 12 minutes.

Biodentine™ has a consistency after mixing which enables manipulation with a spatula,with an amalgam carrier or with carriers which are used for endodontic cements inretrograde fillings (Messing gun, MTA gun).

In all these cases, the instruments should be rinsed with water just after their use in orderto avoid that excess of Biodentine™ will set inside the systems and cause blockage.

Material Setting times (Minutes) Initial Final PMTA 70 (2.58) 175 (2.55) FUJI IX 1 (0.12) 3.4 (0.20) BIODENTINE ™ 6 (0.30) 10.1(1.20)

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2.2 - Density and Porosity

The mechanical resistance of calcium silicate based materials is also dependant on theirlow level of porosity. The lower the porosity, the higher the mechanical strength. Thesuperior mechanical properties of Biodentine™ were determined by the low watercontent in the mixing stage. Two different methods confirmed the low porosity ofBiodentine™.

First a mercury intrusion porosimetry method was used. Mercury, the only known liquidreally suitable for porosimetry type measurements, can be forced into pores. Thepressure required to intrude mercury into a pore is determined by the pore diameter. Thesamples were prepared under the same conditions as those used for CS measurements.Measurements were carried out on fourteen 28-day-old cylinders, dried at 40°C in aprimary vacuum for 12 days to eliminate residual water. The porous volume and thedistribution of pore diameters were determined by mercury intrusion porosimetry(Autopore III, Micromeritics Instruments Corporation, Norcross, USA).

As expected, Biodentine™ exhibits lower porosity than ProRoot® MTA. The density andthe porosity of Biodentine™ and Fuji IX are equivalent.

Electrical Resistance Measurements

An alternative method to illustrate the hardening process is to examine the mobility ofions which depend of the pore size and number of pores during setting byelectrochemical analysis. Impedance spectroscopy technique leads to the increase ofthe electrical resistance along with the porosity reduction (Fig.2) (Golberg et al., 2009).

Material Porous characteristics Dens. g/cm3 Pore V.cm3/g Porosity % PMTA 1.882(0.002) 0.120(0.002) 22.6 (0.2)FUJI IX 2.320(0,002) 0.033(0.002) 7.2 (0.2) BIODENTINE ™ 2.260(0.002) 0.031(0.002) 6.8 (0.2)

Fig. 2: Electrical resistance (Ω) versus time (hours)during setting of Biodentine™

This shows that evenafter the initial setting ofBiodentine™, thematerial continues toimprove in terms ofinternal structure towardsa more dense material,with a decrease inporosity.

Biodentine™ is anevolutive material whichimproves its mechanicalproperties with time.

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2.3 - Compressive strength

Compressive strength is a classical mechanical evaluation of the dental biomaterials(ISO 9917:1991). Specimens were mixed at room temperature, according to eachmanufacturer’s instructions. 6 specimens were prepared using cylindrical Teflon moulds,4 mm in diameter and 6 mm long, removing air bubbles. Specimens were stored in anincubator for 15 minutes in 100% relative humidity (dry) with 37°C and then removedfrom the mould and stored (wet) in distilled water at 37°C, for the remaining time(simulation of the clinical application).

ProRoot® MTA samples were left in the incubator for 24 hours at 37°C and 100% relativehumidity (dry) to allow complete hardening.

Each product was tested at 1 hour, 1 day, 7 days and 28 days. The cylinders werecompressed using a Universal Testing Machine (Model 2/M MTS Systems 1400, EdenPrairie, Minneapolis, USA), with a cross-head speed of 0.5 mm by minute and themaximum load was recorded (Fig. 3).

Fig.3: Comparative evolution of compressive strength after setting of Biodentine™, Fuji IX and ProRoot® MTA.

The setting of Biodentine™ is illustrated by a sharp increase in the compressive strengthreaching more than 100 MPa in the first hour. The mechanical strength continues toimprove to reach more than 200 MPa at 24h which is more than most glass ionomersvalues. A specific feature of Biodentine™ is its capacity to continue improving with timeover several days until reaching 300 MPa after one month. This value becomes quitestable and is in the range of the compressive strength of natural dentine (297 MPa,

Material 1h 24h 7d 28d PMTA 7.5 (5.1) a 164.5 (19.3) a 139.9 (35.2) a

FUJI IX 144.2 (6.3) a 188.2 (33.1)b 220.6 (16.7)b 185.3 (25.9)b

BIODENTINE ™ 131.5 (7.1)b 241.1 (13.3)c 253.2 (16.1)c 316.4 (8.7)c

p value 0.01 ≤ 0.001 ≤ 0.001 ≤ 0.001

Time (h)

Compressive streng

th (M

pa)

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(O’Brien 2008)). This maturation process can be related to the decrease of porosity withtime, which was illustrated previously. Biodentine™ is an evolutive biomaterial whichimproves its mechanical properties with time.

Comparing the compressive strength of a classical glass ionomer (Fuji IX – GC), at 1 hour,the compressive strengths are similar. No continuous increase over one month can beobserved with Fuji IX but Biodentine™ is significantly more resistant to compression.

With ProRoot® MTA, even after 1 day, the material has no mechanical resistance. Asclassical Portland cement, the mechanical strength develops after several days, reaching150 MPa after one week.

This demonstrates the superiority of Biodentine™ for building in short time (9-12 min)sufficient mechanical resistance to be used as a dentine substitute, compatible withdental restorations.

2.4 - Flexural strength

The 3 points bending test has a clinical significance and is essential when the materialis used for Class I, II and IV cavities. The higher the resistance to flexural strength, thelower the risk of fracture in clinical use.

The value of the bending obtained with Biodentine™ after 2 hours is 34 MPa. Comparedwith that of other materials: 5-25 MPa (conventional Glass Ionomer Cement), 17-54 MPa(Resin modified GIC), 61-182 MPa (composite resin) (O’Brien 2008), it shows clearly thatthe bending resistance of Biodentine™ is superior to conventional GIC, but still muchlower than the composite resin.

The internal values of the flexural strength were 22MPa, very similar to Glass Ionomers(15-39MPa).

2.5 - Vickers micro hardness

Hardness can be defined as the resistance to the plastic deformation of the surface ofa material after indentation or penetration. Measurements at different times have beenevaluated

The hardness increases in time when cements are immersed in distilled water (Colon in(Golberg et al., 2009)). After 2 hours, the hardness of Biodentine™ was 51 HVN andreached 69 HVN after 1 month. These values are comparable to those obtained withthe resin modified GIC-Fuji II LC (36 HVN), and the composite resin-Post Comp II LC(97 HVN). Calcite is a mineral known to improve the hardness of cements. The formationof CSH gel reduces the porosity with time. The crystallization of the latter continues,therefore improving the hardness as well as other mechanical properties (sealing at theinterfaces, compressive strength…).

The reported micro hardness values for natural dentine are in the range of 60-90 HVN(O’Brien 2008). Biodentine™ has surface hardness in the same range as natural dentine.

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2.6 - Radiopacity

Biodentine™ contains zirconium oxide allowing identificationon radiographs. According to the ISO standard 6876,Biodentine™ displays a radiopacity equivalent to 3.5 mm ofaluminum. This value is over the minimum requirement of theISO standard (3 mm aluminum).

This makes Biodentine™ particularly suitable in theendodontic indications of canal repair.

2.7 - Comparison with glass ionomers and ProRoot® MTA

In order to have a larger knowledge of the physico-chemical behavior of Biodentine™compared to glass ionomers and Portland cement based dental materials (ProRoot®

MTA, Dentsply), we performed several measurements in Septodont’s laboratory:diametral tensile strength (DTS), flexural strength, elastic modulus, compressive strengthat 24h, Vickers microhardness.

Product Lot # DTS, MPa

FlexuralStrength MPa

Modulus GPa

CompressiveStrength at24h(MPa) 0,5 mm/min

MicrohardnessHVN

Natural Dentine

Dental Materials& their selection(O’Brien 2008)

- - 18.5 297 60

Biodentine™193-A-03.11.08167-B-19.01.09

16. 0(1.2) 24.0 (7.3) 22.0 (2.3) 213,7 (26,1) 60.9 (5.0)

3M GlassIonomer

349270 27.7 (1.0) 26.6 (4.5) 14.9 (2.1) 124.7 (10.3) 77.8 (4.6)

VOCO Ionofil®

Molar AC915325 16.4 (1.7) 22.5 (2.5) 10.6 (2.9) 129.9 (17.9) 70.3 (3.9)

GC Fuji IX GPCapsule

902101 16.8 (1.3) 22.8 (1.8) 12.8 (2.8) 130.0 (7.0) 76.8 (3.5)

GC Fuji IX GP(hand mix)

0811141/0811031

16.5 (0.5) 14.5 (2.4) 15.4 (3.5) 122.6 (10.1) 72.2 (3.7)

GC Fuji II LightCure Capsule

812111 38.1 (1.8) 39.1 (5.4) 8.1 (0.3) 162.8 (10.1) 45.6 (3.9)

GC Fuji II LightCure (hand mix)

0902231/0812081

32.1 (4.2) 19.3 (6.2) 6.3 (0.4) 183.4 (14.8) 43.3 (4.5)

ProRoot® MTA08003394/

080849.5 (1.2) Non measurable Non measurable 56.1 (7.2) Non measurable

From this table, it can be concluded that Biodentine™ has a mechanical behavior similarto glass ionomers and is also similar to natural dentine. The mechanical resistance ofBiodentine™ is also much higher than that of ProRoot® MTA.

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The quality and durability of the interface is a key factor for the survival of a restorationmaterial in clinical conditions: the marginal adaptation and the intimate contact with thesurrounding materials (dentine, enamel, composites and other dental materials) aredeterminative features. This was investigated by erosion in acid solutions, electronmicroscopy and microleakage tests.

In the case of Biodentine™, the dissolution/precipitation process, which is inherent tothe setting principle of Calcium silicate cements, will differentiate its interfacial behaviourfrom the already known dental materials (composites, adhesives, glass ionomers).

3.1 - Resistance to acid

Concerning durability of water based cements, in the oral cavity; one of relevantcharacteristics of the dental materials is the resistance to acidic environment. It is knownthat glass ionomers have a tendency to erode under such conditions.

The acid erosion and the effects of aging in artificial saliva on the Biodentine™ structureand composition were investigated (Laurent et al., 2008).

Methods:

The acid erosion test was evaluated daily in lactic acid (0.02M) and sodium lactate (0.1M)aqueous solution (pH 2.74), by measuring the height loss of the Biodentine™ samples(2mm, diameter 30mm) for a week. Aging was evaluated in Meyer-modified Fusayamaartificial saliva containing phosphates (pH 5.3).

The height modification of the material was evaluated for a week. Scanning electronmicroscopy was used to examine and characterise the surface of the sample before andafter Aging. The possible dissolution of the new material in the artificial saliva wasevaluated by measuring the concentration of Si, Ca, Zr, and inorganic carbonate in theartificial saliva after 1, 2, 3 and 4 weeks.

Results :

In the 2.74 pH solution, acid erosion is observed (Fig.4), but this erosion is slower thanwith glass ionomer cement reported by Nomoto (Nomoto and McCabe, 2001).

Biodentine™ Interfaces

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In artificial saliva there was no erosion but deposition of white material on the surface ofthe Biodentine™ sample. Scanning electron microscopic analysis of this material revealedneedle-like crystals with an apatitic appearance. The composition of this deposit by X-diffraction analysis seems to confirm the apatitic composition (ratio Ca/P = 1.6). Thiscorrelates well with the analysis of the elements in the solution, which revealed adecrease of Ca concentration with time, which in turn, corresponds to the precipitationof apatite-like calcium phosphate crystals.

500

50 100 150 200

Biodentine

Ketac Fil

Fuji II

400

300

Depth (µm)

time (h)

200

100

0

Fig.4: Acid erosion profile in pH=2.74, lactic acid/lactate solution

Apatite-like crystal deposition on the surface of Biodentine™ in a phosphate containing artificial saliva solution (pH=5.3)

As a conclusion, the erosion of Biodentine™ in acidic solution is limited and lower thanfor other water based cements (Glass Ionomers). In reconstituted saliva (containingphosphates), no erosion is observed. Instead, a crystal deposition on the surface ofBiodentine™ occurs, with an apatite-like structure.

This deposition process due to a phosphate rich environment is very encouraging interms of improvement of the interface between Biodentine™ and natural dentine. Thedeposition of apatitic structures might increase the marginal sealing of the material.

This type of crystal deposition is already well known for MTA systems.

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3.2 - Resistance to microleakage:

Several studies were performed to evaluate the resistance of Biodentine™ tomicroleakage.

The interface with dentine and enamel was examined using dye penetration methodology(silver nitrate), which is one of the most commonly used assays to assess, in vitro, theinterfacial seal, by measuring the percolation of a dye along the different interfacesstudied (Golberg et al., 2009).

Methods:

Freshly extracted human molars were used to prepare class II cavities both on the mesialand on the distal sides. The prepared teeth were divided into different groups to evaluatethe influence of a pretreatment of the cavity using polyacrylic acid solution (GC Conditioner,GC Corp.) before Biodentine™ placement, the application of a surface varnish (Optiguard®,Kerr) after the Biodentine™ setting to protect from humidity after initial setting and theinfluence of the bonding agent (Xeno® III, Dentsply or G Bond, GC) when placing acomposite (Ceram-X® Mono, Dentsply) over Biodentine™ one day after setting.

Each group was submitted to 2200 thermocycles (5°C – 55°C, 10 seconds for each batchand transfer). The percentage of microleakage was determined on six samples as thelength of dye penetration divided by the length of the interface.

•At the enamel - BIODENTINE™ interface: % Dye penetration = (AA1/AB) * 100%

• At the dentin - BIODENTINE™ interface:% Dye Penetration = (CC1/CD) * 100%

• At the composite - BIODENTINE™ interface:% Dye Penetration = (EE1/EF) * 100%

Results:

No significant difference in the percentage of microleakage was observed at the enamel-Biodentine™ and dentine-Biodentine™ interfaces, with or without polyacrylic acidtreatment. The placement of a protective varnish increases microleakage at the enamelinterface in the early stage, but not at the dentine interface. After 3 months of aging, nosignificant difference could be evidenced in the case of Optiguard® placement or not.

At the Biodentine™-composite interface (Fig.5), 1 day after placement, the specimensbonded with Xeno® III exhibited significantly less microleakage than those bonded withG Bond.

After 3 months, the micro leakages of specimens treated with G Bond were lower thanat 1 day. At 3 months no significant differences at the composite-Biodentine™ interfacewere observed between Xeno® III or G Bond and Xeno® III + Optiguard®.

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According to this study, the interfaces which are developed between Biodentine™ andthe dental surfaces (enamel and dentine) as well as with adhesive systems (Xeno® III orG Bond), are very resistant to micro leakage, with or without pre-treatment by polyacrylicacid solutions. The choice of water based adhesive systems might be preferable whenplacing a composite over Biodentine™. The sealing quality of Biodentine™ is notinfluenced by the storage after 3 months.

Dejou evaluated the micro leakage resistance of Biodentine™ in comparison with oneof the best sealing systems, resin modified glass ionomers (Fuji II LC, GC Corp.): after2500 thermo cycles, the dye penetration was evaluated by scoring the depth ofpenetration of silver nitrate marker (ranging from 0= no penetration to 3= full interfacepenetration) (Internal report).

Mesio-occlusal and disto-occlusal preparations below cementum-enamel junction weremade in 42 extracted molars. The teeth were randomly assigned one of the followingtreatments before restoration with Filtek™ Z250 (3M ESPE) composite resin:Biodentine™; Fuji II LC (GC); Biodentine™ + Optibond® Solo Plus (Kerr); Biodentine™+ Optibond® Solo Plus (Kerr) + silane; Biodentine™ + Septobond SE (Septodont) ; Fuji II LC (GC) + Optibond® Solo Plus (Kerr).

Concerning the first two groups: leakage was evaluatedseparately, in contact with enamel or in contact with dentine(Fig.6). Biodentine™ exhibits better leakage resistance both toenamel and to dentine compared to Fuji II LC.

2 mm

2 mm

6 mm

5 mm

Fig. 5: Histogram of mean microleakage % at theBiodentine™ / adhesive interface

Fig.6: Micro leakage scores of Biodentine™ or Fuji IILC in contact with enamel or dentine

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3.3 - Electron Microscopy:

Interface between Biodentine™ (left) andhuman dentine (right): the two surfaces are indirect and intimate contact. The surface ofBiodentine™ presents some crystaldeposition which appeared after the samplecutting due to re-exposition to waterenvironment.

Pr Dejou, Dr Raskin

There is a direct contact without a gapbetween Biodentine™ and the natural dentine.The crack is observed inside Biodentinecaused by dehydration, due to SEM samplepreparation under vacuum. This cohesivefailure does not affect the dentine-Biodentine™ interface, which indicates thequality of the micro-mechanical adhesion.

Pr Colon, Dr Pradelle

Z 250

OptibondSolo+

BiodentineFujillLC

6

In the sandwich technique groups, at theinterface between the base material(Biodentine™ or Fuji II LC) and the composite,in case of Optibond® Solo plus (total etchsystem), similar micro leakage resistance areobtained (Fig.7).

Only in the case of Septobond SE (self etchbonding), was the percolation at the interfaceslightly increased, but no significantdifference could be evidenced on the maximalmedian scores.

In conclusion, Biodentine™ has a similarbehavior in terms of leakage resistance as FujiII LC at the interface with enamel, with dentineand with dentine bonding agents.Biodentine™ is then indicated in open-sandwich class II restoration without anypreliminary treatment.

Fig.7: Micro leakage scores of Biodentine™ or Fuji IILC in sandwich technique

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Perfect seal of Biodentine™ in contact with radicular dentine, as well as between twoincrements of Biodentine™, in an in vitro test of apexification.

Dr Bronnec, Pr Colon

Comparison of the interfacebetween Biodentine™ or FujiII LC and a composite, usingOptibond® Solo Plus: Theinterfaces are very similar.

Pr Dejou, Dr Raskin

Crystallisation process in the dentinetubule of an extracted wisdom toothtreated with Biodentine™, observed after28 days of storage in distilled water. The dentine tubules are obturated by re-crystallisation.

Dr Franquin

At the entrance of the dentine tubules, somemineral re-crystallisation occurs, creatingmineral tags. This induces micromechanicalanchorage of Biodentine™. This processwill continue with time, improving thesealing.

Pr Colon, Dr Pradelle

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From a regulatory point of view, Biodentine™ is a calcium silicate based material, usedfor crown and root dentine repair treatment, involving external contact for a period ofmore than 30 days. The biocompatibility tests required for the preclinical evaluation ofdental products followed the guideline ISO 7405 - 2008.

The following sections evaluate the compliance with this standard for the tests carriedout on Biodentine™. It is considered a device with external contact, for long-term tissuecontact (>30 days). In certain indications (radicular, apical obstruction and repair of thepulpal floor), it can be considered an implanted system, according to the ISOclassification.

All biocompatibility tests were carried out on the final product Biodentine™.

4.1 - Cytotoxicity tests (ISO 7405, ISO 10993-5)

Different cytotoxicity tests carried out on Biodentine™ are reported.

The first study was performed on human pulpal fibroblasts (human wisdom tooth),comparing Biodentine™, calcium hydroxide and MTA (Dycal®, Dentsply and ProRoot®

MTA Dentsply). The cell viability was determined by MTT incorporation (About, 2003b).Results showed Biodentine™ was non cytotoxic like MTA, whereas the undiluted cementDycal® induced 22 % of cytotoxicity (Table. 1).

Outstanding biocompatibility

Product Cell death (%) Biodentine™ 0±8 MTA 0±9CaOH 22±10

Table 1. Cell death after Dycal®, MTA and BIODENTINE™ contact.

Figure 8. Expression ofcollagen and dentinesialoprotein (DSP) aftercontact with Biodentine™and MTA during 4 weeks.

Moreover, the cell differentiation was evaluatedwith the expression of collagen, dentinesialoprotein (DSP) and osteonectin (OSN).Results showed the expression of thedifferentiation markers, underlining the safetyof Biodentine™ (Fig. 8).

Collagen DSP

Control

Biodentine™(4 weeks)

MTA(4 weeks)

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The second study was performed on L929 fibroblasts comparing Biodentine™,composite resin Filtek™ Z250 and MTA (Franquin, 2001). Samples were extracted 3 h,24 h and 7 days after the setting. The cell viability was determined by MTT incorporation.Results showed Biodentine™ is not cytotoxic (< 10 %) whatever hardening time isconsidered. Filtek™ Z250 resin is slightly cytotoxic (> 20 %) at the 3 observationperiods (Table 2).

The third study was published in Dental Materials on the biological effects ofBiodentine™ (Laurent et al., 2008). They were compared to those induced by thematerials used for pulp capping such as MTA and Dycal®. Several tests were carried out:

• A cytotoxicity test involving indirect contact through a section of dentine: noneof the tested materials was cytotoxic.

• Where there is no dentine interposition, there is a significant difference intoxicity of the different materials: Biodentine™ did not reveal any cytotoxicityalthough more marked cytotoxicity was reported for Dycal® compared to MTA.

• Differentiation of pulp fibroblasts in orthodontoblastic cells was also analysedfor contact with two materials. Pulp fibroblasts secrete a mineralised matrixand cells in contact express differentiation proteins (nestin and dentine sialo-proteins). Once the cells had been in contact with Biodentine™ cement or withMTA, marker expression was important in the pulp cells involving the formationof mineral nodules. Immunological marking was in all cases higher in the cellsforming mineral nodules.

To conclude, these various tests demonstrate that there is no direct cytotoxic effect withBiodentine™ in the form of an extract in contact with L929 fibroblast line cells, dentalspecialised pulp cells and that moreover it does not affect phenotypic pulp expressionof fibroblasts.

4.2 - Sensitization tests (ISO 7405, ISO 10993-1)

Studies were performed on guinea-pigs thanks to a maximisation method (intradermicand topical application with Freund complete adjuvant induction.

The evaluation of oedema and erythema was performed according to a clinical scale(0-4) 24 and 48 h after retrieval of occlusive patches of the challenge phase. Thesensitisation potential is graded (class 0 to 4) according to the percentage of sensitisedanimals (score of more than 2).

Biodentine™ was not sensitizing (Gomond, 2003c).

Product 3 hours 1 day 7 days Filtek™ Z250 23% 25% 26% MTA 0% 14% 8% Biodentine™ 2% 10% 9%

Table 2. Cell death after Filtek™ Z250, MTA and Biodentine™ contact.

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4.3 - Genotoxicity tests (ISO 7405, ISO 10993-3, OCDE 471)

Several genotoxicity tests were performed on the Biodentine™ cement. They werecarried out on extracts of the cement after complete setting.

AMES test performed on Salmonella typhimurium and Escherichia coli. Strains TA98,TA100, TA1537, TA1535, pKM101 in absence or presence of metabolism activator.Results showed that Biodentine™ was not mutagenic (Harmand, 2003).

Another AMES test was performed on 4 strains of Salmonella typhimurium TA97A, TA98,TA100 and TA102. The results showed that cement Biodentine™ does not induce reversemutation in the presence or absence of the metabolic activator S9. Identical results werereported for the four strains of bacteria tested (Laurent et al., 2008).

An in vitromicronucleus test was also carried out using human lymphocytes (Laurent etal., 2008). These were exposed to extracts of Biodentine™ obtained either from a culturemedium or DMSO. Dilutions of 1% to 5% of the extracts were used. After a culture timeof 72 hours, the cells were stained and analysed. 1000 bi-nucleated lymphocytes weretested, to check for a micronucleus. A toxicity index was determined, together with aratio for the number of micronuclei in relation to the negative reference. The resultsshowed that after incubation of the lymphocytes with different dilutions of the extract ofBiodentine™, the number of lymphocytes presenting a micronucleus was similar to thatobtained with the negative reference (3.9% to 4.1%) when concentrations of 1% to 5%in an aqueous or hydrophobic medium were tested. Positive controls produced amicronucleus rate of 16% (Fig. 2)

Finally, the comet test on human pulpfibroblasts was conducted (About, 2003a).The extract of Biodentine™ was prepared inDMSO and a culture medium, at 50 mg/ml for24 hours and at 37°C. The cells were exposeddirectly to increasing dilutions of cementextracts for two hours. Following electro-phoresis, the slides were analysed byfluorescent microscopy (magnification 400)and an automated analyser was used todetermine DNA lesions. The results obtainedshowed that the percentage of tail DNA variedfrom 12.59 for a dilution of 0.1% to 15.58 forthe undiluted medium. It was 13.19 for thenegative control and 46.52 for the positivecontrol (Table 3). In the presence of DMSO,there was no significant difference betweenthe genotoxicity of Biodentine™ and thenegative control (extracted with NaCl andDMSO).

Biodentine™ Micronucleoted lymphocytes (%±SD)1% 4.0±1.12.3% 4.0±1.13.7% 4.0±1.25% 4.2±1.2- control 3.7±1.2+ control 16.0±6.0*

Biodentine™ Tail DNA meandilution (%±SD)0.1% 12.59±0.961% 13.31±0.8810% 14.90±1.06Undiluted 15.58±1.08Negative control 13.19±0.96Positive control 46.52±1.45*

Table 3. Micronucleated lymphocytes after contact with Biodentine™ .

Table 4. Tail DNA mean after contact with Biodentine™.

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4.4 - Cutaneous irritation tests (ISO 7405, ISO 10993-10)

Cutaneous irritation test was performed in the rabbit by direct application. Oedema anderythmea were evaluated 1h, 24h, 48h and 72h after patch removal. Biodentine™ wasshown to be non irritant (Gomond, 2003a).

4.5 - Eye irritation tests (OCDE 405)

The irritation of the liquid part of Biodentine™ was tested on rabbit eye mucosa. Theaim of the study was to assess qualitatively and quantitatively irritation or corrosionand the delay of appearance of the effects after single application of 0.1 ml on eye in3 rabbits. The ocular reactions (redness and chemosis of conjunctivae, iris and cornealesions) were scored 1h, 24h, 48h and 72h after application. The liquid part ofBiodentine™ undiluted was unclassified among the chemicals irritating to eyes (Fagette,2009).

4.6 - Acute toxicity tests (ISO 7405, ISO 10993-11, OCDE 423)

The acute toxicity tests were performed in order to determine on a qualitative andquantitative basis the toxicity signs and their time of appearance after a unique oraladministration of a dose of 2000 mg/kg of the product in rats. Rats were observedimmediately after administration, 1h, 2h, 3h, 4h, and at least once a day during 14 days.The administration by oral route of the 2000 mg/kg dose of Biodentine™ induced noacute toxicity in the rat. The DL50 of Biodentine™ is superior to 2000 mg/kg (Gomond,2003b).

4.7 - Preclinical safety conclusion

The tests carried out on Biodentine™ have shown that the material tested in the form ofan extract in a saline environment is not a cytotoxic, mutagenic, irritant or sensitisingagent. It is devoid of oral toxicity at a dose of 2000 mg/kg. In conclusion, Biodentine™is safe. Compared to well known dental materials such as Dycal® (calcium hydroxide),Biodentine™ exhibits less cytotoxicity. Moreover, when compared to ProRoot® MTA,Biodentine™ demonstrates at least equivalent biocompatibility.

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Two in vitro tests and two tests in animals were performed in order to demonstrate thebioactivity of Biodentine™ in clinical situations.

5.1 - In vitro test of direct pulp capping on human extracted teeth

Human teeth were extracted in order to make exposed pulp cavities which were thenfilled with Biodentine™ (About, 2007). The teeth (n = 15) were cultured for 24 hours (n = 5), 14 days (n = 5) and 28 days (n = 5) in order to determine the bioactivity ofBiodentine™ (Fig 9).

Evidence based bioactivity

Figure 9. Exposed pulp cavities (A) obturated with Biodentine™ (B) and cultured (C).

Figure 10. Observations after 28 days.

At the end of the culture and after demineralisation, histological sections were done. Theresults showed good preservation of the pulp up to 28 days. Near the capped area, achange in the pulp tissue was reported, with the neo-formation of reparatory dentinecomparable to that observed with MTA (Fig 10). This corresponds to the first signs ofthe formation of a dentine bridge.

To conclude, Biodentine™ is able to stimulate initiation and development of mineralization.

AB C

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5.2 - In vitro test for angiogenesis

A study was conducted on damaged pulp fibroblasts in order to evaluate theBiodentine™ activity on angiogenesis (About, 2009). This model mimicked the in vivosituations in cases of pulp damage requiring direct pulp capping. Materials such asBiodentine™, Calcipulpe®, Hydroxide de calcium XR, ProRoot® MTA and Xeno®III wereapplied to the cells and growth factors (VEGF, FGF-2, PDGF-AB, TGF-β1) concentrationswere evaluated by ELISA test.

Results showed that none of the products modified the cell structure in this model. OnlyProRot® MTA and Biodentine™ were able to stimulate the formation of mineralisationspots. The concentration level of TGF-β1 was enhanced by both ProRoot® MTA andBiodentine™. Moreover, VEGF and FGF-2 were enhanced in presence of Biodentine™(150 à 200% for VEGF and up to 670 % for FGF-2).

These results suggest that Biodentine™ is able to stimulate angiogenesis, in order toheal pulp fibroblasts.

5.3 - Stimulation of reactionary dentine in indirect pulp capping : rat model

A study was conducted on the maxillary molars of adult rats (Golberg, 2009). The firstmaxillary molars were prepared in order to achieve half-moon cavities (class V) on themesial face. The cavities were filled with Biodentine™ and with Fuji IX glass ionomercement and covered with a protective varnish. The teeth were collected and fixed at 8days, 15 days, 30 days and 3 months after filling. The results showed that after 8 days,pulp inflammation was moderate in the mesial third of the pulp chamber. This reactionwas also observed on the reference teeth (Fig. 11).

Figure. 11. Biodentine™ stimulates reactionary dentine (rd).

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The inflammatory process had disappeared after 15 days. The newly formed reactionarydentine was identified. By comparison with the group treated with the glass ionomercement, the formation of reactionary dentine was greater in the teeth in the presence ofBiodentine™ and its thickness increased over time from 20 to 40 µm after 8 days, 40 to80 µm after 15 days and 140 to 280 µm after 30 days, although it varied between 10and 20 µm for the reference group. After 3 months, reactionary dentine generated byBiodentine™ was thick and dense (Fig. 12), enclosing the horn and the mesial pulp whilstfor Fuji IX, this was less dense, only partially covering the mesial cervical area of the pulp(Golberg, 2009).

Fig. 12. Formation of a thick reactionary dentine in presence of to Biodentine™ in comparison to Fuji IX

To conclude, Biodentine™ was able to stimulate a reactionary dentine which is a naturalbarrier against bacterial invasions. The reactionary dentine formation stabilises at 3 months, indicating that the stimulation process is stopped when a sufficient dentinebarrier is formed.

5.4 - Calcification as a result of Biodentine™ in a directpulp capping and pulpotomy : pig model

Two protocols were set up in pigs (Shayegan A, 2009).

The first protocol was the analysis of the pulp reaction following pulpotomy andplacement of different materials (15 deciduous teeth, 15 pigs):

• Formocresol

• White MTA

• Biodentine™

The follow-up was performed during 1, 4 and 12 weeks.

Pulp chamber was excised in 15 pigs in a comparative study of the efficacy ofBiodentine™ versus formocresol and MTA (5 pigs per group). Histological sections ofthe teeth were done after a week, a month and 3 months of treatment. The resultsshowed that Biodentine™, like White MTA, promoted beneficial calcification after oneweek, whereas Formocresol induced necrosis and inflammation (Fig. 13).

27

To conclude, Biodentine™ is a suitable material for pulpotomy.

The second protocol was an analysis of the pulp reaction after direct capping for differentmaterials (15 deciduous teeth, 15 pigs):

• Ca (OH)2

• White MTA

• Biodentine™

The follow-up was performed during 1, 4 and 12 weeks.

Pulp exposure was performed via a class V vestibular cavity in 15 pigs who were 4 months old, in order to compare the efficacy of Biodentine™ against calcium hydroxideand MTA (5 pigs in each group) over 3 trial periods of 1 week, 1 month and 3 months(Shayegan 2009).

Fig. 13. Summary of pulpotomy results.

Fig.14. Summary of direct pulp capping results.

Formocresol WMTA Biodentine™

1 weekInflammation

4 weeksInflammation-Tissu regenerationtransition

10/10 Necrosis and inflamation 6/10 Beginning of calcification 10/10 Calcification

4/10 Necrosis and inflamation 7/10 Important calcification 7/10 Important calcification

5/10 Infiltration of inflammatory

cells

7/10 Necrosis and inflamation 10/10 Complete calcification 9/10 Complete calcification

1/10 Calcification12 weeksComplete healing

Calcium hydroxyde WMTA Biodentine™

1 weekInflammation

4 weeksInflammation-Tissu regenerationtransition

2/10 Calcification 7/10 Calcification 9/10 Calcification

7/10 Important calcification 10/10 Important calcification 10/10 Important calcification

5/10 Partial calcification

10/10 Important calcification 10/10 Important calcification 9/10 Important calcification12 weeksComplete healing

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To conclude, Biodentine™ enhances the formation of a dentine barrier after direct pulpcapping confirming it has good potential in this indication. In the first month, the qualityof the dentine bridge formed with Biodentine™ is of better quality than with the referencedental technique (calcium hydroxide). The performance of Biodentine™ is at leastequivalent to White MTA.

5.5 - Overall bioactivity

Pulp capping and pulpotomy studies showed that Biodentine™ was very well tolerated.Moreover, Biodentine™ was able to promote mineralisation, generating a reactionarydentine as well as a dense dentine bridge. These phenomena illustrate the great potentialfor Biodentine™ to be in contact to the pulp, by demonstrating its bioactivity in severalindications.

As a conclusion, Biodentine™ is bioactive.

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6.1 - Biodentine™ is used as a dentine substitute under a composite

A clinical investigation, 04/001, aimed to assess the acceptability of Biodentine™ as anew restoration of the posterior teeth: a first-in-man study.

Among the products already used in dentistry, one product shares similar properties withBiodentine™. This product, MTA, Mineral Trioxide Aggregate, sold by Dentsply underthe brand name ProRoot® MTA, is a derivative of Portland cement, with the samechemical properties. It was developed as a product for radicular repair only, due to alow compressive strength incompatible with restorative indications. The biologicalproperties of this product allow its use in the capping of dental pulp tissue, in the fillingof the radicular apical part by a retrograde approach or in the closure of perforations, topromote the restoration of the original tissue in contact with the pulp tissue and radiculartissue.

Biodentine™ can be defined as a special micronised concrete derived from the maincomponent of Portland cement, tricalcium silicate. With physical properties far superiorto those of MTA, especially in terms of setting time and compressive strength, it exhibitsthe same characteristics of biocompatibility and sealing ability, after setting in an alkalinepH, with controlled (size and spatial organisation) formation of calcium salts. This productexclusively composed of mineral components, was initially designed to replace dentinein restorations.

In this study, Biodentine™ is compared to Filtek™ Z100, which is used for dentalrestorations and requires an adhesive for the bonding the composite on the tooth.Biodentine™ is applied directly to contact with the tooth, without adhesive or conditioner.

This clinical investigation is a multicentre, randomised, prospective study, which requiredthe inclusion of 400 patients and a 3-year observation period.

The interim report is based on 232 cases with a minimum one year follow-up: 116 weretreated with Biodentine™ and 116 with Filtek™ Z100. Among the 116 restorations donewith Biodentine™, 20 involved a direct pulp capping.

The study planed a follow-up at baseline, 15 days, 6, 12, 24 and 36 months. The analysisof the cases showed:

At D0, Biodentine™ showed:

• Easy handling.

• Excellent anatomic form.

• Very good marginal adaption.

• Very good interproximal contact.

Clinical efficacy

30

During the follow-up, the restoration with Biodentine™ in comparison to Filtek™ Z100:

• Was well tolerated in all cases.

• The anatomic form, the marginal adaptation and the interproximal contactstarted to degrade after 6 months.

• Due to the degradation, a complementary treatment was performed. In 93.8%,cases needed a retreatment (92/116); Biodentine™ was kept as dentinesubstitute as the pulp vitality test was positive. Biodentine™ presented a goodresistance to burring and the composite Filtek™ Z100 was applied on the top.The tolerance was evaluated for up to 3 years.

• Was safe for the patient, as the same number of adverse events was observedin Biodentine™ group (4/116) as in Filtek™ Z100 group (3/116).

As a conclusion, Biodentine™ was applied in 116 patients with at least one year follow-up.Thanks to its excellent biocompatibility, Biodentine™ is very well tolerated and can beused as cavity lining with a permanent composite restoration (Fig.15).

Fig. 15.Restoration with Biodentine™(Courtesy of Prof. KOUBI, Marseille).

30 months later: Biodentine™ under Filtek™ Z100

6 months later

D0 : Patient restoration D0 : Amalgam removal D0 : Biodentine™ application

16 months: Biodentine™ reshaping

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6.2 - Biodentine™ is used as a direct pulp capping material

In the same clinical trial, 04/001, Biodentine™ was also used as direct pulp cappingmaterial. Biodentine™ showed:

• An excellent tolerance.

• The ability to save pulp vitality even in difficult cases: the vitality test waspositive at each recall.

Moreover, Biodentine™ can be used in direct pulp capping indications with a goodsuccess rate (Fig. 16). It is important to underline that Biodentine™ was used in contactwith pulp tissue in a patient older than 21 and maintained the pulp alive.

Fig. 16. Direct pulp capping with Biodentine™ (Courtesy of Prof. KOUBI, Marseille).

D0 : Radiography D0 : Exposed pulp

D0 Three years later: Biodentine™ coveredby Filtek™ Z100.

D0 : Biodentine™ application

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6.3 - Biodentine™ is used as an endodontic repair material

The endodontic indications of Biodentine™ are similar to the usual calcium silicate basedmaterials, like the Portland cements (i.e. ProRoot® MTA). This type of product is alreadywell documented.

Several physical, chemical and biological properties are comparable as summarised in thepreclinical section. However, Biodentine™ has some features which are superior to MTA.

• Biodentine™ consistency is better suited to the clinical use than MTA’s.

• Biodentine™ presentation ensures a better handling and safety than MTA.

• Biodentine™ does not require a two step obturation as in the case of MTA. As the setting is faster, there is a lower risk of bacterial contamination thanwith MTA.

Adding to its ability to be used as dentine substitute, Biodentine™ could safely be usedfor each indication where dentine is damaged. Therefore, it is an advantage for theclinician and the patient (Machtou, 2009b).

Moreover, a clinical trial, 09/001, aimed at assessing the tolerance and efficacy ofBiodentine™ in 6 endodontic procedures, after 3 months and after 2 years follow-up isin progress:

• Direct pulp capping following carious pulp exposure

• Direct pulp capping following dental trauma/injury to healthy pulp (partialpulpotomy)

• Repair of perforated root canals and/or pulp chamber floor

• Retrograde endodontic surgery

• Pulpotomy in primary molars

• Apexification

Ten patients per indication are required in this multi-centre and open-label clinical trial(Machtou, 2009a).

References

33

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2. About I 2003b Etude in vitro sur culture cellulaire de la biocompatibilité du produit RD94:étude des fonctions spécifiques des fibroblastes pulpaires humains. Report RG EN RA EXT-RD94/054.

3. About I 2007 Coiffage pulpaire direct de RD94 à l’aide du modèle de culture de dententière. Report RD EN RA EXT-RD94/096.

4. About I 2009 Effets des matériaux bioactifs Biodentine TM et Calcipulpe® sur les étapesprécoces de la régénération dentinaire. Report RD RA DEV 94-013.

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