R&D Department

Biodentine

Active Biosilicate Technology

Scientific File

Summary
Introduction ................................................................................................................................................................. 4

Active Biosilicate Technology Physico-chemical features

............................................................................................... 5

1.1 - Setting reaction ..................................................................................................................................................... 6 1.2 - Formulation of Biodentine ...................................................................................................................... 7

............................................................................................................... 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

Introduction
Biodentine was developed by Septodonts Research Group as a new class of dental material which could conciliate high mechanical properties with excellent biocompatibility, as well as a bioactive behavior. Several years of active and collaborative research between Septodont and several universities led to a new calcium-silicate based formulation, which is suitable as a dentine replacement material whenever original dentine is damaged. In addition to the chemical composition based on the Ca3SiO5 water chemistry which brings the high biocompatibility of already known endodontic repair cements (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 endodontic procedures, but also for restorative clinical cases of dentine replacement. Sealing ability of this biomaterial was also assessed to be equivalent to glass-ionomers, without requiring any specific conditioning of the dentine surface. Leakage resistance and mechanical strength will improve over the first weeks after placement. Biodentine turns out to be one of the most biocompatible of all the biomaterials in dentistry as demonstrated according to all the ISO standard tests, as well as in the different preclinical and clinical research collaborations. Moreover, reactionary dentine formation was demonstrated in rats, exhibiting high quality and quantity of protective dentine stimulation in indirect pulp capping. In the case of direct pulp capping and pulpotomy in pigs, the compatibility with the pulp enables a direct contact with fibroblasts, with limited inflammatory response compared to controls. Formation of a regular and dense dentine bridge is histologically demonstrated within one month. Besides the usual endodontic indications of this class of calcium-silicate cements (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 clinical study in 400 patients. It was suitable as a permanent dentine substitute and temporary enamel substitute. Restoration of deep or large crown carious lesions provides a very tight seal, without post-operative sensitivity and insures the longevity of restorations in vital teeth. Biodentine has also achieved 100% success in direct pulp capping in adults presenting healthy pulp.

Active Biosilicate Technology

Septodonts initial objective was to develop a material based on the most biocompatible chemistry available for dental materials: calcium silicates, which can set in the presence of water. Although recognized as highly biocompatible and bioactive, all these materials lack reactivity, with very long setting times (more than 2 hours), low mechanical properties and with very difficult handling (depending on the water ratio, from a sandy consistency to a fluid paste). In order to take up the technological challenge of combining this calcium silicate chemistry with the requirements of a formulation compatible with classical restorative and endodontic practice, Septodont developed a new technological platform called Active Biosilicate Technology. This consists in controlling every step of the material formulation 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 natural stone treatment. This implies that all these products inherently contain unpurifiable mixtures of calcium silicates (C3S + C2S), calcium aluminates (C3A), calcium aluminoferrites (C4AF), calcium sulfates (CaSO4 - gypsum), together with low concentrations of metallic impurities coming from the natural minerals used as raw materials. The only way to reach our objectives in terms of purity control, high mechanical strength and short setting times, was to synthesize our own calcium silicate product. The Active Biosilicate Technology is a proprietary technology developed according to our state-of-the-art pharmaceutical background applied to the high temperate ceramic mineral chemistry. Septodont is now able to ensure the purity of the calcium silicate content of the formulation and the absence of any aluminate and calcium sulfate in the final product.

Grinding Firing

Ground powder

Biodentine capsule

1.1 - Setting reaction

The calcium silicate has the ability to interact with water leading to the setting and hardening 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)2 C3S CSH

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

2H2SiO4

Ca O2H CSH CaOH

2+

H2 O

Biodentine Particle
The unreacted tricalcium silicate grains are surrounded by layers of calcium silicate hydrated gel, which are relatively impermeable to water, thereby slowing 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. The hardening process results from of the formation of crystals that are deposited in a supersaturated solution.

Powder before hydration

Deposition of CSH

Biodentine after setting

1.2 - Formulation of Biodentine

In order to reach a formulation with a short setting time (12 minutes) and high mechanical properties 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, which is 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, the shorter the setting. Also, adding calcium chloride to the liquid component accelerates the system. Finally, the decrease of the liquid content in the system decreases the setting time to harden within 9 to 12 minutes.

Powder
Tri-calcium Silicate (C3S) Di-calcium Silicate (C2S) Calcium Carbonate and Oxide Iron Oxide Zirconium Oxide Main core material Second core material Filler Shade Radiopacifier

Liquid
Calcium chloride Hydrosoluble polymer Accelerator Water reducing agent

Reaching high mechanical strength is also quite difficult for these systems. The first cause of low mechanical properties of Portland cements are the aluminate components, which make the product fragile. Septodont controls the purity of the calcium silicate through the Active Biosilicate Technology which consists in eliminating aluminates and other impurities. The second axis of formulation was to adjust the particle size distribution in order to reach an optimal powder density. The additional charge system selected was calcium carbonate, for both its biocompatibility and calcium content. The paradox of calcium silicate systems is also that water, which is essential for the hardening 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 degrading the macroscopic mechanical resistance, but on the other hand decreasing the water content leads to reducing the possibility of a homogenous mix. The addition of hydrosoluble polymer systems described as water reducing agents or super plasticizers, helps in maintaining the balance between low water content and consistency of the mixture. Radiopacity is obtained by adding zirconium oxide to the final product.


Method:

Physico-chemical features

2.1 - Setting Time

There are several methods to evaluate the setting of dental materials. The first one is based on the macroscopic evaluation of the resistance of a needle to penetrate the surface of the material: when the needle does not leave a trace on the surface of the material, 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 help in the selection of different formulations: the use of a rheometer (Nonat and Franquin, 2006).

Dynamic rheometry tests were performed to determine the characteristics of each material during their workability (working and setting times) as well as the rate of building early mechanical resistance. These tests consisted in measuring, by a viscoelastometry, the constraint transmitted by the sample, when a sinusoidal strain is applied. An ARES strain-controlled rheometer was used (Rheometric Scientific Inc., Piscataway, US). After mixing, the sample was inserted between the two striated parallel plates, 6 mm in diameter, with a 2 mm gap. Only the lower plate was maintained at the controlled temperature of 37.5C, and a closed chamber maintained the temperature of the entire sample at 100% relative humidity to prevent drying. The experimental conditions were as follows: oscillation frequency: 1 radian per second, applied strain: 0.0005%. Under these conditions, the applied strain is less than the critical strain beyond which the structure of the cement paste is altered (about 0.0015%), and the transmitted stress is proportional to the strain. This system can therefore be used to measure the evolution of the elastic modulus G of the material, without any modification of the structure of the material. 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) and ProRoot MTA (Dentsply). The initial setting time was determined at the moment when the elastic modulus begins to increase (10MPa). The final setting time was determined as the elastic modulus reached 100MPa. The time between the mixing and the initial setting corresponds to the working time.

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

Material PMTA FUJI IX BIODENTINE

Setting times (Minutes) Initial Final 70 (2.58) 175 (2.55) 1 (0.12) 3.4 (0.20) 6 (0.30) 10.1(1.20)

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 sets faster that Biodentine in less than 4 minutes. This represents a great improvement compared to the other calcium silicate dental materials (ProRoot MTA), which set in more 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 time is 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 in retrograde fillings (Messing gun, MTA gun). In all these cases, the instruments should be rinsed with water just after their use in order to avoid that excess of Biodentine will set inside the systems and cause blockage.

2.2 - Density and Porosity

The mechanical resistance of calcium silicate based materials is also dependant on their low level of porosity. The lower the porosity, the higher the mechanical strength. The superior mechanical properties of Biodentine were determined by the low water content in the mixing stage. Two different methods confirmed the low porosity of Biodentine. First a mercury intrusion porosimetry method was used. Mercury, the only known liquid really suitable for porosimetry type measurements, can be forced into pores. The pressure required to intrude mercury into a pore is determined by the pore diameter. The samples were prepared under the same conditions as those used for CS measurements. Measurements were carried out on fourteen 28-day-old cylinders, dried at 40C in a primary vacuum for 12 days to eliminate residual water. The porous volume and the distribution of pore diameters were determined by mercury intrusion porosimetry (Autopore III, Micromeritics Instruments Corporation, Norcross, USA).
Material Dens. g/cm3 PMTA 1.882(0.002) FUJI IX 2.320(0,002) BIODENTINE 2.260(0.002) Porous characteristics Pore V.cm3/g 0.120(0.002) 0.033(0.002) 0.031(0.002)

Porosity % 22.6 (0.2) 7.2 (0.2) 6.8 (0.2)

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

Electrical Resistance Measurements An alternative method to illustrate the hardening process is to examine the mobility of ions which depend of the pore size and number of pores during setting by electrochemical analysis. Impedance spectroscopy technique leads to the increase of the electrical resistance along with the porosity reduction (Fig.2) (Golberg et al., 2009). This shows that even after the initial setting of Biodentine, the material continues to improve in terms of internal structure towards a more dense material, with a decrease in porosity. Biodentine is an evolutive material which improves its mechanical properties with time.
Fig. 2: Electrical resistance () versus time (hours) during setting of Biodentine

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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 each manufacturers instructions. 6 specimens were prepared using cylindrical Teflon moulds, 4 mm in diameter and 6 mm long, removing air bubbles. Specimens were stored in an incubator for 15 minutes in 100% relative humidity (dry) with 37C and then removed from the mould and stored (wet) in distilled water at 37C, for the remaining time (simulation of the clinical application). ProRoot MTA samples were left in the incubator for 24 hours at 37C and 100% relative humidity (dry) to allow complete hardening. Each product was tested at 1 hour, 1 day, 7 days and 28 days. The cylinders were compressed using a Universal Testing Machine (Model 2/M MTS Systems 1400, Eden Prairie, Minneapolis, USA), with a cross-head speed of 0.5 mm by minute and the maximum load was recorded (Fig. 3).
Material PMTA FUJI IX BIODENTINE p value 1h 144.2 (6.3) a 131.5 (7.1)b 0.01 24h 7.5 (5.1) a 188.2 (33.1)b 241.1 (13.3)c 0.001 7d 164.5 (19.3) a 220.6 (16.7)b 253.2 (16.1)c 0.001 28d 139.9 (35.2) a 185.3 (25.9)b 316.4 (8.7)c 0.001

Compressive strength (Mpa)

Time (h) 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 strength reaching more than 100 MPa in the first hour. The mechanical strength continues to improve to reach more than 200 MPa at 24h which is more than most glass ionomers values. A specific feature of Biodentine is its capacity to continue improving with time over several days until reaching 300 MPa after one month. This value becomes quite stable and is in the range of the compressive strength of natural dentine (297 MPa,

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(OBrien 2008)). This maturation process can be related to the decrease of porosity with time, which was illustrated previously. Biodentine is an evolutive biomaterial which improves 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 be observed with Fuji IX but Biodentine is significantly more resistant to compression. With ProRoot MTA, even after 1 day, the material has no mechanical resistance. As classical Portland cement, the mechanical strength develops after several days, reaching 150 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 with dental restorations.

2.4 - 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, the lower the risk of fracture in clinical use. The value of the bending obtained with Biodentine after 2 hours is 34 MPa. Compared with that of other materials: 5-25 MPa (conventional Glass Ionomer Cement), 17-54 MPa (Resin modified GIC), 61-182 MPa (composite resin) (OBrien 2008), it shows clearly that the bending resistance of Biodentine is superior to conventional GIC, but still much lower 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 of a material after indentation or penetration. Measurements at different times have been evaluated 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 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). Calcite is a mineral known to improve the hardness of cements. The formation of 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 the interfaces, compressive strength). The reported micro hardness values for natural dentine are in the range of 60-90 HVN (OBrien 2008). Biodentine has surface hardness in the same range as natural dentine.

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2.6 - Radiopacity
Biodentine contains zirconium oxide allowing identification on radiographs. According to the ISO standard 6876, Biodentine displays a radiopacity equivalent to 3.5 mm of aluminum. This value is over the minimum requirement of the ISO standard (3 mm aluminum). This makes Biodentine particularly suitable in the endodontic 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 Septodonts laboratory: diametral tensile strength (DTS), flexural strength, elastic modulus, compressive strength at 24h, Vickers microhardness.
Compressive Strength at Microhardness 24h(MPa) HVN 0,5 mm/min
297 60

Product

Lot #

DTS, MPa

Flexural Strength MPa

Modulus GPa

Natural Dentine

Dental Materials & their selection (OBrien 2008) 193-A-03.11.08 167-B-19.01.09 349270

18.5

Biodentine 3M Glass Ionomer VOCO Ionofil Molar AC GC Fuji IX GP Capsule GC Fuji IX GP (hand mix) GC Fuji II Light Cure Capsule GC Fuji II Light Cure (hand mix) ProRoot MTA

16. 0(1.2)

24.0 (7.3)

22.0 (2.3)

213,7 (26,1)

60.9 (5.0)

27.7 (1.0)

26.6 (4.5)

14.9 (2.1)

124.7 (10.3)

77.8 (4.6)

915325

16.4 (1.7)

22.5 (2.5)

10.6 (2.9)

129.9 (17.9)

70.3 (3.9)

902101 0811141/ 0811031 812111 0902231/ 0812081 08003394/ 08084

16.8 (1.3)

22.8 (1.8)

12.8 (2.8)

130.0 (7.0)

76.8 (3.5)

16.5 (0.5)

14.5 (2.4)

15.4 (3.5)

122.6 (10.1)

72.2 (3.7)

38.1 (1.8)

39.1 (5.4)

8.1 (0.3)

162.8 (10.1)

45.6 (3.9)

32.1 (4.2)

19.3 (6.2)

6.3 (0.4)

183.4 (14.8)

43.3 (4.5)

9.5 (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 similar to glass ionomers and is also similar to natural dentine. The mechanical resistance of Biodentine is also much higher than that of ProRoot MTA.

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Biodentine Interfaces

The quality and durability of the interface is a key factor for the survival of a restoration material in clinical conditions: the marginal adaptation and the intimate contact with the surrounding materials (dentine, enamel, composites and other dental materials) are determinative features. This was investigated by erosion in acid solutions, electron microscopy and microleakage tests. In the case of Biodentine, the dissolution/precipitation process, which is inherent to the setting principle of Calcium silicate cements, will differentiate its interfacial behaviour from 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 relevant characteristics of the dental materials is the resistance to acidic environment. It is known that glass ionomers have a tendency to erode under such conditions. The acid erosion and the effects of aging in artificial saliva on the Biodentine structure and 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 Fusayama artificial saliva containing phosphates (pH 5.3).

The height modification of the material was evaluated for a week. Scanning electron microscopy was used to examine and characterise 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.

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

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In artificial saliva there was no erosion but deposition of white material on the surface of the Biodentine sample. Scanning electron microscopic analysis of this material revealed needle-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). This correlates well with the analysis of the elements in the solution, which revealed a decrease of Ca concentration with time, which in turn, corresponds to the precipitation of apatite-like calcium phosphate crystals.

500 400 Depth (m) 300 200 100 0 50 100 time (h)
Fig.4: Acid erosion profile in pH=2.74, lactic acid/lactate solution Biodentine Ketac Fil Fuji II

150

200

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 than for other water based cements (Glass Ionomers). In reconstituted saliva (containing phosphates), no erosion is observed. Instead, a crystal deposition on the surface of Biodentine occurs, with an apatite-like structure. This deposition process due to a phosphate rich environment is very encouraging in terms of improvement of the interface between Biodentine and natural dentine. The deposition 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 to microleakage. 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, the interfacial seal, by measuring the percolation of a dye along the different interfaces studied (Golberg et al., 2009).

Methods: Freshly extracted human molars were used to prepare class II cavities both on the mesial and on the distal sides. The prepared teeth were divided into different groups to evaluate the 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 the influence of the bonding agent (Xeno III, Dentsply or G Bond, GC) when placing a composite (Ceram-X Mono, Dentsply) over Biodentine one day after setting. Each group was submitted to 2200 thermocycles (5C 55C, 10 seconds for each batch and transfer). The percentage of microleakage was determined on six samples as the length 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 enamelBiodentine and dentine-Biodentine interfaces, with or without polyacrylic acid treatment. The placement of a protective varnish increases microleakage at the enamel interface in the early stage, but not at the dentine interface. After 3 months of aging, no significant difference could be evidenced in the case of Optiguard placement or not. At the Biodentine-composite interface (Fig.5), 1 day after placement, the specimens bonded with Xeno III exhibited significantly less microleakage than those bonded with G Bond. After 3 months, the micro leakages of specimens treated with G Bond were lower than at 1 day. At 3 months no significant differences at the composite-Biodentine interface were observed between Xeno III or G Bond and Xeno III + Optiguard.

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Fig. 5: Histogram of mean microleakage % at the Biodentine / adhesive interface

According to this study, the interfaces which are developed between Biodentine and the dental surfaces (enamel and dentine) as well as with adhesive systems (Xeno III or G Bond), are very resistant to micro leakage, with or without pre-treatment by polyacrylic acid solutions. The choice of water based adhesive systems might be preferable when placing a composite over Biodentine. The sealing quality of Biodentine is not influenced by the storage after 3 months. Dejou evaluated the micro leakage resistance of Biodentine in comparison with one of the best sealing systems, resin modified glass ionomers (Fuji II LC, GC Corp.): after 2500 thermo cycles, the dye penetration was evaluated by scoring the depth of penetration of silver nitrate marker (ranging from 0= no penetration to 3= full interface penetration) (Internal report). Mesio-occlusal and disto-occlusal preparations below cementum-enamel junction were made in 42 extracted molars. The teeth were randomly assigned one of the following treatments 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).
5 mm

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

2 mm

6 mm

2 mm

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

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Z 250

6 Optibond Solo+ Biodentine FujillLC

In the sandwich technique groups, at the interface between the base material (Biodentine or Fuji II LC) and the composite, in case of Optibond Solo plus (total etch system), similar micro leakage resistance are obtained (Fig.7).

Only in the case of Septobond SE (self etch bonding), was the percolation at the interface slightly increased, but no significant difference could be evidenced on the maximal median scores.

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

In conclusion, Biodentine has a similar behavior in terms of leakage resistance as Fuji II LC at the interface with enamel, with dentine and with dentine bonding agents. Biodentine is then indicated in opensandwich class II restoration without any preliminary treatment.

3.3 - Electron Microscopy:

Interface between Biodentine (left) and human dentine (right): the two surfaces are in direct and intimate contact. The surface of Biodentine presents some crystal deposition which appeared after the sample cutting due to re-exposition to water environment. Pr Dejou, Dr Raskin

There is a direct contact without a gap between Biodentine and the natural dentine. The crack is observed inside Biodentine caused by dehydration, due to SEM sample preparation under vacuum. This cohesive failure does not affect the dentineBiodentine interface, which indicates the quality of the micro-mechanical adhesion. Pr Colon, Dr Pradelle

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At the entrance of the dentine tubules, some mineral re-crystallisation occurs, creating mineral tags. This induces micromechanical anchorage of Biodentine. This process will continue with time, improving the sealing. Pr Colon, Dr Pradelle

Crystallisation process in the dentine tubule of an extracted wisdom tooth treated with Biodentine, observed after 28 days of storage in distilled water. The dentine tubules are obturated by recrystallisation. Dr Franquin

Comparison of the interface between Biodentine or Fuji II LC and a composite, using Optibond Solo Plus: The interfaces are very similar. Pr Dejou, Dr Raskin

Perfect seal of Biodentine in contact with radicular dentine, as well as between two increments of Biodentine, in an in vitro test of apexification. Dr Bronnec, Pr Colon

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Outstanding biocompatibility

From a regulatory point of view, Biodentine is a calcium silicate based material, used for crown and root dentine repair treatment, involving external contact for a period of more than 30 days. The biocompatibility tests required for the preclinical evaluation of dental products followed the guideline ISO 7405 - 2008. The following sections evaluate the compliance with this standard for the tests carried out on Biodentine. It is considered a device with external contact, for long-term tissue contact (>30 days). In certain indications (radicular, apical obstruction and repair of the pulpal floor), it can be considered an implanted system, according to the ISO classification. 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 cement Dycal induced 22 % of cytotoxicity (Table. 1).

Product Biodentine MTA CaOH

Cell death (%) 08 09 2210

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

Moreover, the cell differentiation was evaluated with the expression of collagen, dentine sialoprotein (DSP) and osteonectin (OSN). Results showed the expression of the differentiation markers, underlining the safety of Biodentine (Fig. 8).

Collagen Control

DSP

Biodentine
(4 weeks)
Figure 8. Expression of collagen and dentine sialoprotein (DSP) after contact with Biodentine and MTA during 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 is considered. Filtek Z250 resin is slightly cytotoxic (> 20 %) at the 3 observation periods (Table 2).
Product Filtek Z250 MTA Biodentine 3 hours 23% 0% 2% 1 day 25% 14% 10% 7 days 26% 8% 9%

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

The third study was published in Dental Materials on the biological effects of Biodentine (Laurent et al., 2008). They were compared to those induced by the materials 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: none of the tested materials was cytotoxic. Where there is no dentine interposition, there is a significant difference in toxicity of the different materials: Biodentine did not reveal any cytotoxicity although more marked cytotoxicity was reported for Dycal compared to MTA. Differentiation of pulp fibroblasts in orthodontoblastic cells was also analysed for contact with two materials. Pulp fibroblasts secrete a mineralised matrix and cells in contact express differentiation proteins (nestin and dentine sialoproteins). Once the cells had been in contact with Biodentine cement or with MTA, marker expression was important in the pulp cells involving the formation of mineral nodules. Immunological marking was in all cases higher in the cells forming mineral nodules.

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

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

Studies were performed on guinea-pigs thanks to a maximisation method (intradermic and 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. The sensitisation potential is graded (class 0 to 4) according to the percentage of sensitised animals (score of more than 2). Biodentine was not sensitizing (Gomond, 2003c).

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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 were carried 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 reverse mutation in the presence or absence of the metabolic activator S9. Identical results were reported for the four strains of bacteria tested (Laurent et al., 2008). An in vitro micronucleus test was also carried out using human lymphocytes (Laurent et al., 2008). These were exposed to extracts of Biodentine obtained either from a culture medium or DMSO. Dilutions of 1% to 5% of the extracts were used. After a culture time of 72 hours, the cells were stained and analysed. 1000 bi-nucleated lymphocytes were tested, to check for a micronucleus. A toxicity index was determined, together with a ratio for the number of micronuclei in relation to the negative reference. The results showed that after incubation of the lymphocytes with different dilutions of the extract of Biodentine, the number of lymphocytes presenting a micronucleus was similar to that obtained 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 a micronucleus rate of 16% (Fig. 2)
Biodentine 1% 2.3% 3.7% 5% - control + control Micronucleoted lymphocytes (%SD) 4.01.1 4.01.1 4.01.2 4.21.2 3.71.2 16.06.0*

Table 3. Micronucleated lymphocytes after contact with Biodentine .

Biodentine dilution 0.1% 1% 10% Undiluted Negative control Positive control

Tail DNA mean (%SD) 12.590.96 13.310.88 14.901.06 15.581.08 13.190.96 46.521.45*

Finally, the comet test on human pulp fibroblasts was conducted (About, 2003a). The extract of Biodentine was prepared in DMSO and a culture medium, at 50 mg/ml for 24 hours and at 37C. The cells were exposed directly to increasing dilutions of cement extracts for two hours. Following electrophoresis, the slides were analysed by fluorescent microscopy (magnification 400) and an automated analyser was used to determine DNA lesions. The results obtained showed that the percentage of tail DNA varied from 12.59 for a dilution of 0.1% to 15.58 for the undiluted medium. It was 13.19 for the negative control and 46.52 for the positive control (Table 3). In the presence of DMSO, there was no significant difference between the genotoxicity of Biodentine and the negative control (extracted with NaCl and DMSO).

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 and erythmea were evaluated 1h, 24h, 48h and 72h after patch removal. Biodentine was shown 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. The aim of the study was to assess qualitatively and quantitatively irritation or corrosion and the delay of appearance of the effects after single application of 0.1 ml on eye in 3 rabbits. The ocular reactions (redness and chemosis of conjunctivae, iris and cornea lesions) were scored 1h, 24h, 48h and 72h after application. The liquid part of Biodentine 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 and quantitative basis the toxicity signs and their time of appearance after a unique oral administration of a dose of 2000 mg/kg of the product in rats. Rats were observed immediately 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 no acute 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 of an extract in a saline environment is not a cytotoxic, mutagenic, irritant or sensitising agent. 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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Evidence based bioactivity

Two in vitro tests and two tests in animals were performed in order to demonstrate the bioactivity 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 then filled 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 of Biodentine (Fig 9).

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

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

Figure 10. Observations after 28 days.

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

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5.2 - In vitro test for angiogenesis

A study was conducted on damaged pulp fibroblasts in order to evaluate the Biodentine activity on angiogenesis (About, 2009). This model mimicked the in vivo situations in cases of pulp damage requiring direct pulp capping. Materials such as Biodentine, Calcipulpe, Hydroxide de calcium XR, ProRoot MTA and XenoIII were applied to the cells and growth factors (VEGF, FGF-2, PDGF-AB, TGF-1) concentrations were evaluated by ELISA test. Results showed that none of the products modified the cell structure in this model. Only ProRot MTA and Biodentine were able to stimulate the formation of mineralisation spots. The concentration level of TGF-1 was enhanced by both ProRoot MTA and Biodentine. Moreover, VEGF and FGF-2 were enhanced in presence of Biodentine (150 200% for VEGF and up to 670 % for FGF-2). These results suggest that Biodentine is able to stimulate angiogenesis, in order to heal 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 first maxillary molars were prepared in order to achieve half-moon cavities (class V) on the mesial face. The cavities were filled with Biodentine and with Fuji IX glass ionomer cement and covered with a protective varnish. The teeth were collected and fixed at 8 days, 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 reaction was 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 reactionary dentine was identified. By comparison with the group treated with the glass ionomer cement, the formation of reactionary dentine was greater in the teeth in the presence of Biodentine and its thickness increased over time from 20 to 40 m after 8 days, 40 to 80 m after 15 days and 140 to 280 m after 30 days, although it varied between 10 and 20 m for the reference group. After 3 months, reactionary dentine generated by Biodentine was thick and dense (Fig. 12), enclosing the horn and the mesial pulp whilst for 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 natural barrier against bacterial invasions. The reactionary dentine formation stabilises at 3 months, indicating that the stimulation process is stopped when a sufficient dentine barrier is formed.

5.4 - Calcification as a result of Biodentine in a direct pulp 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 and placement 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 of Biodentine versus formocresol and MTA (5 pigs per group). Histological sections of the teeth were done after a week, a month and 3 months of treatment. The results showed that Biodentine, like White MTA, promoted beneficial calcification after one week, whereas Formocresol induced necrosis and inflammation (Fig. 13).

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Formocresol

WMTA

Biodentine

1 week
Inflammation

10/10 Necrosis and inflamation 6/10 Beginning of calcification 10/10 Calcification 4 weeks
InflammationTissu regeneration transition

4/10 Necrosis and inflamation 7/10 Important calcification 5/10 Infiltration of inflammatory
cells

7/10 Important calcification

12 weeks
Complete healing

7/10 Necrosis and inflamation 1/10 Calcification

10/10 Complete calcification

9/10 Complete calcification

Fig. 13. Summary of pulpotomy results.

To conclude, Biodentine is a suitable material for pulpotomy. The second protocol was an analysis of the pulp reaction after direct capping for different materials (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 hydroxide and MTA (5 pigs in each group) over 3 trial periods of 1 week, 1 month and 3 months (Shayegan 2009).

Calcium hydroxyde

WMTA

Biodentine

1 week
Inflammation

2/10 Calcification 4 weeks

InflammationTissu regeneration transition

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

9/10 Calcification 10/10 Important calcification 9/10 Important calcification

7/10 Important calcification 5/10 Partial calcification 10/10 Important calcification

12 weeks
Complete healing

Fig.14. Summary of direct pulp capping results.

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To conclude, Biodentine enhances the formation of a dentine barrier after direct pulp capping confirming it has good potential in this indication. In the first month, the quality of the dentine bridge formed with Biodentine is of better quality than with the reference dental technique (calcium hydroxide). The performance of Biodentine is at least equivalent 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 reactionary dentine as well as a dense dentine bridge. These phenomena illustrate the great potential for Biodentine to be in contact to the pulp, by demonstrating its bioactivity in several indications. As a conclusion, Biodentine is bioactive.

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Clinical efficacy

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 a new restoration of the posterior teeth: a first-in-man study. Among the products already used in dentistry, one product shares similar properties with Biodentine. This product, MTA, Mineral Trioxide Aggregate, sold by Dentsply under the brand name ProRoot MTA, is a derivative of Portland cement, with the same chemical properties. It was developed as a product for radicular repair only, due to a low compressive strength incompatible with restorative indications. The biological properties of this product allow its use in the capping of dental pulp tissue, in the filling of the radicular apical part by a retrograde approach or in the closure of perforations, to promote the restoration of the original tissue in contact with the pulp tissue and radicular tissue. Biodentine can be defined as a special micronised concrete derived from the main component of Portland cement, tricalcium silicate. With physical properties far superior to those of MTA, especially in terms of setting time and compressive strength, it exhibits the same characteristics of biocompatibility and sealing ability, after setting in an alkaline pH, with controlled (size and spatial organisation) formation of calcium salts. This product exclusively composed of mineral components, was initially designed to replace dentine in restorations. In this study, Biodentine is compared to Filtek Z100, which is used for dental restorations 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 required the 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 were treated with Biodentine and 116 with Filtek Z100. Among the 116 restorations done with Biodentine, 20 involved a direct pulp capping. The study planed a follow-up at baseline, 15 days, 6, 12, 24 and 36 months. The analysis of the cases showed: At D0, Biodentine showed: Easy handling. Excellent anatomic form. Very good marginal adaption. Very good interproximal contact.

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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 contact started 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 dentine substitute as the pulp vitality test was positive. Biodentine presented a good resistance 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 observed in 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 be used as cavity lining with a permanent composite restoration (Fig.15).

D0 : Patient restoration

D0 : Amalgam removal

D0 : Biodentine application

6 months later

16 months: Biodentine reshaping 30 months later: Biodentine under Filtek Z100

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

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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 capping material. Biodentine showed: An excellent tolerance. The ability to save pulp vitality even in difficult cases: the vitality test was positive at each recall. Moreover, Biodentine can be used in direct pulp capping indications with a good success rate (Fig. 16). It is important to underline that Biodentine was used in contact with pulp tissue in a patient older than 21 and maintained the pulp alive.

D0 : Radiography

D0 : Exposed pulp

D0 : Biodentine application

D0

Three years later: Biodentine covered by Filtek Z100.

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

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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 based materials, like the Portland cements (i.e. ProRoot MTA). This type of product is already well documented. Several physical, chemical and biological properties are comparable as summarised in the preclinical section. However, Biodentine has some features which are superior to MTA. Biodentine consistency is better suited to the clinical use than MTAs. 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 than with MTA. Adding to its ability to be used as dentine substitute, Biodentine could safely be used for each indication where dentine is damaged. Therefore, it is an advantage for the clinician and the patient (Machtou, 2009b). Moreover, a clinical trial, 09/001, aimed at assessing the tolerance and efficacy of Biodentine in 6 endodontic procedures, after 3 months and after 2 years follow-up is in progress: Direct pulp capping following carious pulp exposure Direct pulp capping following dental trauma/injury to healthy pulp (partial pulpotomy) 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).

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