Biodentine
Biodentine
Biodentine
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R&D Department<br />
<strong>Biodentine</strong> <br />
Active Biosilicate Technology <br />
Scientific File
Summary<br />
Introduction ................................................................................................................................................................. 4<br />
❶ Active Biosilicate Technology ............................................................................................... 5<br />
1.1 - Setting reaction ..................................................................................................................................................... 6<br />
1.2 - Formulation of <strong>Biodentine</strong> ...................................................................................................................... 7<br />
❷ Physico-chemical features ............................................................................................................... 8<br />
2.1 - Setting Time ............................................................................................................................................................. 8<br />
2.2 - Density and Porosity ...................................................................................................................................... 10<br />
2.3 - Compressive strength .................................................................................................................................. 11<br />
2.4 - Flexural strength ................................................................................................................................................ 12<br />
2.5 - Vickers micro hardness ............................................................................................................................... 12<br />
2.6 - Radiopacity ............................................................................................................................................................ 13<br />
2.7 - Comparison with Glass Ionomers and ProRoot ® MTA ..................................................... 13<br />
❸ <strong>Biodentine</strong> Interfaces ...................................................................................................................... 14<br />
3.1 - Resistance to acid ........................................................................................................................................... 14<br />
3.2 - Resistance to microleakage .................................................................................................................... 16<br />
3.3 - Electron Microscopy ...................................................................................................................................... 18<br />
❹ Outstanding biocompatibility .................................................................................................... 20<br />
4.1 - Cytotoxicity tests (ISO 7405, ISO 10993-5) ............................................................................... 20<br />
4.2 - Sensitization tests (ISO 7405, ISO 10993-1) ............................................................................ 21<br />
4.3 - Genotoxicity tests (ISO 7405, ISO 10993-3, OCDE 471) ............................................... 22<br />
4.4 - Cutaneous irritation tests (ISO 7405, ISO 10993-10) ........................................................ 23<br />
4.5 - Eye irritation tests (OCDE 405) ............................................................................................................. 23<br />
4.6 - Acute toxicity tests (ISO 7405, ISO 10993-11, OCDE 423) ......................................... 23<br />
4.7 - Preclinical safety conclusion .................................................................................................................. 23<br />
❺ Evidence based bioactivity ............................................................................................................ 24<br />
5.1 - In vitro test of direct pulp capping on human extracted teeth .................................. 24<br />
5.2 - In vitro test for angiogenesis .................................................................................................................. 25<br />
5.3 - Stimulation of reactionary dentine in indirect<br />
pulp capping : rat model ............................................................................................................................ 25<br />
5.4 - Calcification as a result of <strong>Biodentine</strong> in a direct<br />
pulp capping and pulpotomy : pig model ................................................................................... 26<br />
5.5 - Overall bioactivity ............................................................................................................................................. 28<br />
❻ Clinical efficacy ................................................................................................................................................. 29<br />
6.1 - <strong>Biodentine</strong> is used as a dentine substitute under a composite .......................... 29<br />
6.2 - <strong>Biodentine</strong> is used as a direct pulp capping material ................................................. 31<br />
6.3 - <strong>Biodentine</strong> is used as an endodontic repair material .................................................... 32<br />
References ................................................................................................................................................................ 33<br />
3
4<br />
Introduction<br />
<strong>Biodentine</strong> was developed by Septodont’s Research Group as a new class of<br />
dental material which could conciliate high mechanical properties with excellent<br />
biocompatibility, as well as a bioactive behavior. Several years of active and<br />
collaborative research between Septodont and several universities led to a new<br />
calcium-silicate based formulation, which is suitable as a dentine replacement<br />
material whenever original dentine is damaged.<br />
In addition to the chemical composition based on the Ca 3 SiO 5 – water chemistry<br />
which brings the high biocompatibility of already known endodontic repair<br />
cements (MTA based), Septodont increased the physico-chemical properties<br />
(short setting time, high mechanical strength…) which make <strong>Biodentine</strong><br />
clinically easy to handle and compatible, not only with classical endodontic<br />
procedures, but also for restorative clinical cases of dentine replacement. Sealing<br />
ability of this biomaterial was also assessed to be equivalent to glass-ionomers,<br />
without requiring any specific conditioning of the dentine surface. Leakage<br />
resistance and mechanical strength will improve over the first weeks after<br />
placement.<br />
<strong>Biodentine</strong> turns out to be one of the most biocompatible of all the biomaterials<br />
in dentistry as demonstrated according to all the ISO standard tests, as well as in<br />
the different preclinical and clinical research collaborations. Moreover, reactionary<br />
dentine formation was demonstrated in rats, exhibiting high quality and quantity<br />
of protective dentine stimulation in indirect pulp capping. In the case of direct<br />
pulp capping and pulpotomy in pigs, the compatibility with the pulp enables a<br />
direct contact with fibroblasts, with limited inflammatory response compared to<br />
controls. Formation of a regular and dense dentine bridge is histologically<br />
demonstrated within one month.<br />
Besides the usual endodontic indications of this class of calcium-silicate<br />
cements (repair of perforations or resorptions, apexification, root-end filling),<br />
<strong>Biodentine</strong> has been evaluated for its restorative properties versus composite<br />
(Z100, 3M ESPE) in a three year follow-up, randomized, multicentre clinical<br />
study in 400 patients. It was suitable as a permanent dentine substitute and<br />
temporary enamel substitute. Restoration of deep or large crown carious lesions<br />
provides a very tight seal, without post-operative sensitivity and insures the<br />
longevity of restorations in vital teeth. <strong>Biodentine</strong> has also achieved 100%<br />
success in direct pulp capping in adults presenting healthy pulp.
❶<br />
Septodont’s initial objective was to develop a material based on the most biocompatible<br />
chemistry available for dental materials: calcium silicates, which can set in the presence<br />
of water. Although recognized as highly biocompatible and bioactive, all these materials<br />
lack reactivity, with very long setting times (more than 2 hours), low mechanical<br />
properties and with very difficult handling (depending on the water ratio, from a sandy<br />
consistency to a fluid paste).<br />
In order to take up the technological challenge of combining this calcium silicate<br />
chemistry with the requirements of a formulation compatible with classical restorative<br />
and endodontic practice, Septodont developed a new technological platform called<br />
Active Biosilicate Technology. This consists in controlling every step of the material<br />
formulation beginning with the purity of the raw materials.<br />
Usual dental calcium silicate cements are based on the “Portland Cement” materials,<br />
which result from the clinker products manufactured by the building industry from natural<br />
stone treatment. This implies that all these products inherently contain unpurifiable<br />
mixtures of calcium silicates (C 3 S + C 2 S), calcium aluminates (C 3 A), calcium aluminoferrites<br />
(C 4 AF), calcium sulfates (CaSO 4 - gypsum), together with low concentrations of<br />
metallic impurities coming from the natural minerals used as raw materials.<br />
The only way to reach our objectives in terms of purity control, high mechanical strength<br />
and short setting times, was to synthesize our own calcium silicate product.<br />
The Active Biosilicate Technology is a proprietary technology developed according to<br />
our state-of-the-art pharmaceutical background applied to the high temperate ceramic<br />
mineral chemistry.<br />
Septodont is now able to ensure the purity of the calcium silicate content of the<br />
formulation and the absence of any aluminate and calcium sulfate in the final product.<br />
Firing<br />
Active Biosilicate Technology <br />
Grinding Ground<br />
powder<br />
<strong>Biodentine</strong><br />
capsule<br />
5
6<br />
1.1 - Setting reaction<br />
The calcium silicate has the ability to interact with water leading to the setting and<br />
hardening of the cement. This is a hydration of the tricalcium silicate (3CaO.SiO2 = C3S)<br />
which produces a hydrated calcium silicate gel (CSH gel) and calcium hydroxide (Ca (OH)2).<br />
2(3CaO.SiO2) + 6H2O 3CaO.2SiO2.3H2O + 3Ca(OH) 2<br />
C3S CSH<br />
This dissolution process occurs at the surface of each grain of calcium silicate. The<br />
hydrated calcium silicate gel and the excess of calcium hydroxide tend to precipitate at<br />
the surface of the particles and in the pores of the powder, due to saturation of the<br />
medium. This precipitation process is reinforced in systems with low water content.<br />
CSH<br />
CaOH<br />
2- H SiO 2 4<br />
Ca2+ O H 2<br />
<strong>Biodentine</strong> Particle<br />
H 2 O<br />
The unreacted tricalcium silicate grains are surrounded by layers of calcium silicate<br />
hydrated gel, which are relatively impermeable to water, thereby slowing down the effects<br />
of further reactions. The C-S-H gel formation is due to the permanent hydration of the<br />
tricalcium silicate, which gradually fills in the spaces between the tricalcium silicate<br />
grains. The hardening process results from of the formation of crystals that are deposited<br />
in a supersaturated solution.<br />
Powder before hydration Deposition of CSH <strong>Biodentine</strong> after setting
1.2 - Formulation of <strong>Biodentine</strong><br />
In order to reach a formulation with a short setting time (12 minutes) and high mechanical<br />
properties in the range of natural dentine, calcium silicates could not be used alone.<br />
Usually calcium silicate cements have setting times in the range of several hours, which<br />
is too long in most of the protocols in clinical practice.<br />
Increasing the setting time was achieved by a combination of different effects. First,<br />
particle size greatly influences the setting time, since the higher the specific surface, the<br />
shorter the setting. Also, adding calcium chloride to the liquid component accelerates<br />
the system. Finally, the decrease of the liquid content in the system decreases the setting<br />
time to harden within 9 to 12 minutes.<br />
Powder<br />
Tri-calcium Silicate (C3S) Main core material<br />
Di-calcium Silicate (C2S) Second core material<br />
Calcium Carbonate and Oxide Filler<br />
Iron Oxide Shade<br />
Zirconium Oxide Radiopacifier<br />
Liquid<br />
Calcium chloride Accelerator<br />
Hydrosoluble polymer Water reducing agent<br />
Reaching high mechanical strength is also quite difficult for these systems. The first<br />
cause of low mechanical properties of Portland cements are the aluminate components,<br />
which make the product fragile. Septodont controls the purity of the calcium silicate<br />
through the Active Biosilicate Technology which consists in eliminating aluminates and<br />
other impurities.<br />
The second axis of formulation was to adjust the particle size distribution in order to<br />
reach an optimal powder density. The additional charge system selected was calcium<br />
carbonate, for both its biocompatibility and calcium content.<br />
The paradox of calcium silicate systems is also that water, which is essential for the<br />
hardening of the product, can also affect the strength of the material. On the hand,<br />
excess water in the system will create some remaining porosity, significantly degrading<br />
the macroscopic mechanical resistance, but on the other hand decreasing the water<br />
content leads to reducing the possibility of a homogenous mix. The addition of<br />
hydrosoluble polymer systems described as “water reducing agents” or super<br />
plasticizers, helps in maintaining the balance between low water content and<br />
consistency of the mixture.<br />
Radiopacity is obtained by adding zirconium oxide to the final product.<br />
7
8<br />
❷ Physico-chemical<br />
2.1 - Setting Time<br />
There are several methods to evaluate the setting of dental materials. The first one is<br />
based on the macroscopic evaluation of the resistance of a needle to penetrate the<br />
surface of the material: when the needle does not leave a trace on the surface of the<br />
material, it corresponds to the setting time. This is the principle of the ISO standard 9917.<br />
An alternative instrumented and more objective method can be used especially to help<br />
in the selection of different formulations: the use of a rheometer (Nonat and Franquin,<br />
2006).<br />
Method:<br />
features<br />
Dynamic rheometry tests were performed to determine the characteristics of each<br />
material during their workability (working and setting times) as well as the rate of building<br />
early mechanical resistance. These tests consisted in measuring, by a viscoelastometry,<br />
the constraint transmitted by the sample, when a sinusoidal strain is applied. An ARES<br />
strain-controlled rheometer was used (Rheometric Scientific Inc., Piscataway, US). After<br />
mixing, the sample was inserted between the two striated parallel plates, 6 mm in<br />
diameter, with a 2 mm gap. Only the lower plate was maintained at the controlled<br />
temperature of 37.5°C, and a closed chamber maintained the temperature of the entire<br />
sample at 100% relative humidity to prevent drying. The experimental conditions were<br />
as follows: oscillation frequency: 1 radian per second, applied strain: 0.0005%. Under<br />
these conditions, the applied strain is less than the critical strain beyond which the<br />
structure of the cement paste is altered (about 0.0015%), and the transmitted stress is<br />
proportional to the strain. This system can therefore be used to measure the evolution<br />
of the elastic modulus G’ of the material, without any modification of the structure of the<br />
material.<br />
This instrumented method was used to determine the setting time of the <strong>Biodentine</strong><br />
Formulation (Fig.1) and to compare it to a classical glass ionomer (Fuji IX – GC) and<br />
ProRoot ® MTA (Dentsply). The initial setting time was determined at the moment when<br />
the elastic modulus begins to increase (10MPa). The final setting time was determined<br />
as the elastic modulus reached 100MPa.<br />
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.<br />
Material Setting times (Minutes)<br />
Initial Final<br />
PMTA 70 (2.58) 175 (2.55)<br />
FUJI IX 1 (0.12) 3.4 (0.20)<br />
BIODENTINE 6 (0.30) 10.1(1.20)<br />
From these results it can be concluded that the working time of <strong>Biodentine</strong> is up to<br />
6 minutes with a final set at around 10-12 minutes. The classical glass ionomer sets<br />
faster that <strong>Biodentine</strong> in less than 4 minutes. This represents a great improvement<br />
compared to the other calcium silicate dental materials (ProRoot ® MTA), which set in<br />
more than 2 hours.<br />
The setting times of <strong>Biodentine</strong> are in the same range as the amalgams.<br />
When tested according to the ISO standard with the Gilmore needle, the working time<br />
is over 1 minute and the setting time is between 9 and 12 minutes.<br />
<strong>Biodentine</strong> has a consistency after mixing which enables manipulation with a spatula,<br />
with an amalgam carrier or with carriers which are used for endodontic cements in<br />
retrograde fillings (Messing gun, MTA gun).<br />
In all these cases, the instruments should be rinsed with water just after their use in order<br />
to avoid that excess of <strong>Biodentine</strong> will set inside the systems and cause blockage.<br />
9
10<br />
2.2 - Density and Porosity<br />
The mechanical resistance of calcium silicate based materials is also dependant on their<br />
low level of porosity. The lower the porosity, the higher the mechanical strength. The<br />
superior mechanical properties of <strong>Biodentine</strong> were determined by the low water<br />
content in the mixing stage. Two different methods confirmed the low porosity of<br />
<strong>Biodentine</strong>.<br />
First a mercury intrusion porosimetry method was used. Mercury, the only known liquid<br />
really suitable for porosimetry type measurements, can be forced into pores. The<br />
pressure required to intrude mercury into a pore is determined by the pore diameter. The<br />
samples were prepared under the same conditions as those used for CS measurements.<br />
Measurements were carried out on fourteen 28-day-old cylinders, dried at 40°C in a<br />
primary vacuum for 12 days to eliminate residual water. The porous volume and the<br />
distribution of pore diameters were determined by mercury intrusion porosimetry<br />
(Autopore III, Micromeritics Instruments Corporation, Norcross, USA).<br />
Material Porous characteristics<br />
Dens. g/cm3 Pore V.cm3/g Porosity %<br />
PMTA 1.882(0.002) 0.120(0.002) 22.6 (0.2)<br />
FUJI IX 2.320(0,002) 0.033(0.002) 7.2 (0.2)<br />
BIODENTINE 2.260(0.002) 0.031(0.002) 6.8 (0.2)<br />
As expected, <strong>Biodentine</strong> exhibits lower porosity than ProRoot ® MTA. The density and<br />
the porosity of <strong>Biodentine</strong> and Fuji IX are equivalent.<br />
Electrical Resistance Measurements<br />
An alternative method to illustrate the hardening process is to examine the mobility of<br />
ions which depend of the pore size and number of pores during setting by<br />
electrochemical analysis. Impedance spectroscopy technique leads to the increase of<br />
the electrical resistance along with the porosity reduction (Fig.2) (Golberg et al., 2009).<br />
Fig. 2: Electrical resistance (Ω) versus time (hours)<br />
during setting of <strong>Biodentine</strong><br />
This shows that even<br />
after the initial setting of<br />
<strong>Biodentine</strong>, the<br />
material continues to<br />
improve in terms of<br />
internal structure towards<br />
a more dense material,<br />
with a decrease in<br />
porosity.<br />
<strong>Biodentine</strong> is an<br />
evolutive material which<br />
improves its mechanical<br />
properties with time.
2.3 - Compressive strength<br />
Compressive strength is a classical mechanical evaluation of the dental biomaterials<br />
(ISO 9917:1991). Specimens were mixed at room temperature, according to each<br />
manufacturer’s instructions. 6 specimens were prepared using cylindrical Teflon moulds,<br />
4 mm in diameter and 6 mm long, removing air bubbles. Specimens were stored in an<br />
incubator for 15 minutes in 100% relative humidity (dry) with 37°C and then removed<br />
from the mould and stored (wet) in distilled water at 37°C, for the remaining time<br />
(simulation of the clinical application).<br />
ProRoot ® MTA samples were left in the incubator for 24 hours at 37°C and 100% relative<br />
humidity (dry) to allow complete hardening.<br />
Each product was tested at 1 hour, 1 day, 7 days and 28 days. The cylinders were<br />
compressed using a Universal Testing Machine (Model 2/M MTS Systems 1400, Eden<br />
Prairie, Minneapolis, USA), with a cross-head speed of 0.5 mm by minute and the<br />
maximum load was recorded (Fig. 3).<br />
Material 1h 24h 7d 28d<br />
PMTA 7.5 (5.1) a 164.5 (19.3) a 139.9 (35.2) a<br />
FUJI IX 144.2 (6.3) a 188.2 (33.1) b 220.6 (16.7) b 185.3 (25.9) b<br />
BIODENTINE 131.5 (7.1) b 241.1 (13.3) c 253.2 (16.1) c 316.4 (8.7) c<br />
p value 0.01 ≤ 0.001 ≤ 0.001 ≤ 0.001<br />
Compressive strength (Mpa)<br />
Time (h)<br />
Fig.3: Comparative evolution of compressive strength after setting<br />
of <strong>Biodentine</strong>, Fuji IX and ProRoot ® MTA.<br />
The setting of <strong>Biodentine</strong> is illustrated by a sharp increase in the compressive strength<br />
reaching more than 100 MPa in the first hour. The mechanical strength continues to<br />
improve to reach more than 200 MPa at 24h which is more than most glass ionomers<br />
values. A specific feature of <strong>Biodentine</strong> is its capacity to continue improving with time<br />
over several days until reaching 300 MPa after one month. This value becomes quite<br />
stable and is in the range of the compressive strength of natural dentine (297 MPa,<br />
11
12<br />
(O’Brien 2008)). This maturation process can be related to the decrease of porosity with<br />
time, which was illustrated previously. <strong>Biodentine</strong> is an evolutive biomaterial which<br />
improves its mechanical properties with time.<br />
Comparing the compressive strength of a classical glass ionomer (Fuji IX – GC), at 1 hour,<br />
the compressive strengths are similar. No continuous increase over one month can be<br />
observed with Fuji IX but <strong>Biodentine</strong> is significantly more resistant to compression.<br />
With ProRoot ® MTA, even after 1 day, the material has no mechanical resistance. As<br />
classical Portland cement, the mechanical strength develops after several days, reaching<br />
150 MPa after one week.<br />
This demonstrates the superiority of <strong>Biodentine</strong> for building in short time (9-12 min)<br />
sufficient mechanical resistance to be used as a dentine substitute, compatible with<br />
dental restorations.<br />
2.4 - Flexural strength<br />
The 3 points bending test has a clinical significance and is essential when the material<br />
is used for Class I, II and IV cavities. The higher the resistance to flexural strength, the<br />
lower the risk of fracture in clinical use.<br />
The value of the bending obtained with <strong>Biodentine</strong> after 2 hours is 34 MPa. Compared<br />
with that of other materials: 5-25 MPa (conventional Glass Ionomer Cement), 17-54 MPa<br />
(Resin modified GIC), 61-182 MPa (composite resin) (O’Brien 2008), it shows clearly that<br />
the bending resistance of <strong>Biodentine</strong> is superior to conventional GIC, but still much<br />
lower than the composite resin.<br />
The internal values of the flexural strength were 22MPa, very similar to Glass Ionomers<br />
(15-39MPa).<br />
2.5 - Vickers micro hardness<br />
Hardness can be defined as the resistance to the plastic deformation of the surface of<br />
a material after indentation or penetration. Measurements at different times have been<br />
evaluated<br />
The hardness increases in time when cements are immersed in distilled water (Colon in<br />
(Golberg et al., 2009)). After 2 hours, the hardness of <strong>Biodentine</strong> was 51 HVN and<br />
reached 69 HVN after 1 month. These values are comparable to those obtained with<br />
the resin modified GIC-Fuji II LC (36 HVN), and the composite resin-Post Comp II LC<br />
(97 HVN). Calcite is a mineral known to improve the hardness of cements. The formation<br />
of CSH gel reduces the porosity with time. The crystallization of the latter continues,<br />
therefore improving the hardness as well as other mechanical properties (sealing at the<br />
interfaces, compressive strength…).<br />
The reported micro hardness values for natural dentine are in the range of 60-90 HVN<br />
(O’Brien 2008). <strong>Biodentine</strong> has surface hardness in the same range as natural dentine.
2.6 - Radiopacity<br />
<strong>Biodentine</strong> contains zirconium oxide allowing identification<br />
on radiographs. According to the ISO standard 6876,<br />
<strong>Biodentine</strong> displays a radiopacity equivalent to 3.5 mm of<br />
aluminum. This value is over the minimum requirement of the<br />
ISO standard (3 mm aluminum).<br />
This makes <strong>Biodentine</strong> particularly suitable in the<br />
endodontic indications of canal repair.<br />
2.7 - Comparison with glass ionomers and ProRoot ® MTA<br />
In order to have a larger knowledge of the physico-chemical behavior of <strong>Biodentine</strong><br />
compared to glass ionomers and Portland cement based dental materials (ProRoot ®<br />
MTA, Dentsply), we performed several measurements in Septodont’s laboratory:<br />
diametral tensile strength (DTS), flexural strength, elastic modulus, compressive strength<br />
at 24h, Vickers microhardness.<br />
Product Lot #<br />
Natural<br />
Dentine<br />
Dental Materials<br />
& their selection<br />
(O’Brien 2008)<br />
<strong>Biodentine</strong> 193-A-03.11.08<br />
167-B-19.01.09<br />
3M Glass<br />
Ionomer<br />
VOCO Ionofil ®<br />
Molar AC<br />
GC Fuji IX GP<br />
Capsule<br />
GC Fuji IX GP<br />
(hand mix)<br />
GC Fuji II Light<br />
Cure Capsule<br />
GC Fuji II Light<br />
Cure (hand mix)<br />
ProRoot ® MTA<br />
DTS,<br />
MPa<br />
Flexural<br />
Strength<br />
MPa<br />
Modulus<br />
GPa<br />
Compressive<br />
Strength at<br />
24h(MPa)<br />
0,5 mm/min<br />
Microhardness<br />
HVN<br />
- - 18.5 297 60<br />
16. 0(1.2) 24.0 (7.3) 22.0 (2.3) 213,7 (26,1) 60.9 (5.0)<br />
349270 27.7 (1.0) 26.6 (4.5) 14.9 (2.1) 124.7 (10.3) 77.8 (4.6)<br />
915325 16.4 (1.7) 22.5 (2.5) 10.6 (2.9) 129.9 (17.9) 70.3 (3.9)<br />
902101 16.8 (1.3) 22.8 (1.8) 12.8 (2.8) 130.0 (7.0) 76.8 (3.5)<br />
0811141/<br />
0811031<br />
16.5 (0.5) 14.5 (2.4) 15.4 (3.5) 122.6 (10.1) 72.2 (3.7)<br />
812111 38.1 (1.8) 39.1 (5.4) 8.1 (0.3) 162.8 (10.1) 45.6 (3.9)<br />
0902231/<br />
0812081<br />
08003394/<br />
08084<br />
32.1 (4.2) 19.3 (6.2) 6.3 (0.4) 183.4 (14.8) 43.3 (4.5)<br />
9.5 (1.2) Non measurable Non measurable 56.1 (7.2) Non measurable<br />
From this table, it can be concluded that <strong>Biodentine</strong> has a mechanical behavior similar<br />
to glass ionomers and is also similar to natural dentine. The mechanical resistance of<br />
<strong>Biodentine</strong> is also much higher than that of ProRoot ® MTA.<br />
13
14<br />
❸<br />
The quality and durability of the interface is a key factor for the survival of a restoration<br />
material in clinical conditions: the marginal adaptation and the intimate contact with the<br />
surrounding materials (dentine, enamel, composites and other dental materials) are<br />
determinative features. This was investigated by erosion in acid solutions, electron<br />
microscopy and microleakage tests.<br />
In the case of <strong>Biodentine</strong>, the dissolution/precipitation process, which is inherent to<br />
the setting principle of Calcium silicate cements, will differentiate its interfacial behaviour<br />
from the already known dental materials (composites, adhesives, glass ionomers).<br />
3.1 - Resistance to acid<br />
Concerning durability of water based cements, in the oral cavity; one of relevant<br />
characteristics of the dental materials is the resistance to acidic environment. It is known<br />
that glass ionomers have a tendency to erode under such conditions.<br />
The acid erosion and the effects of aging in artificial saliva on the <strong>Biodentine</strong> structure<br />
and composition were investigated (Laurent et al., 2008).<br />
Methods:<br />
The acid erosion test was evaluated daily in lactic acid (0.02M) and sodium lactate (0.1M)<br />
aqueous solution (pH 2.74), by measuring the height loss of the <strong>Biodentine</strong> samples<br />
(2mm, diameter 30mm) for a week. Aging was evaluated in Meyer-modified Fusayama<br />
artificial saliva containing phosphates (pH 5.3).<br />
The height modification of the material was evaluated for a week. Scanning electron<br />
microscopy was used to examine and characterise the surface of the sample before and<br />
after Aging. The possible dissolution of the new material in the artificial saliva was<br />
evaluated by measuring the concentration of Si, Ca, Zr, and inorganic carbonate in the<br />
artificial saliva after 1, 2, 3 and 4 weeks.<br />
Results :<br />
<strong>Biodentine</strong> Interfaces<br />
In the 2.74 pH solution, acid erosion is observed (Fig.4), but this erosion is slower than<br />
with glass ionomer cement reported by Nomoto (Nomoto and McCabe, 2001).
In artificial saliva there was no erosion but deposition of white material on the surface of<br />
the <strong>Biodentine</strong> sample. Scanning electron microscopic analysis of this material revealed<br />
needle-like crystals with an apatitic appearance. The composition of this deposit by<br />
X-diffraction analysis seems to confirm the apatitic composition (ratio Ca/P = 1.6). This<br />
correlates well with the analysis of the elements in the solution, which revealed a<br />
decrease of Ca concentration with time, which in turn, corresponds to the precipitation<br />
of apatite-like calcium phosphate crystals.<br />
Depth (µm)<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
50 100 150 200<br />
time (h)<br />
Fig.4: Acid erosion profile in pH=2.74, lactic acid/lactate solution<br />
Apatite-like crystal deposition on the surface of <strong>Biodentine</strong><br />
in a phosphate containing artificial saliva solution (pH=5.3)<br />
<strong>Biodentine</strong><br />
Ketac Fil<br />
As a conclusion, the erosion of <strong>Biodentine</strong> in acidic solution is limited and lower than<br />
for other water based cements (Glass Ionomers). In reconstituted saliva (containing<br />
phosphates), no erosion is observed. Instead, a crystal deposition on the surface of<br />
<strong>Biodentine</strong> occurs, with an apatite-like structure.<br />
This deposition process due to a phosphate rich environment is very encouraging in<br />
terms of improvement of the interface between <strong>Biodentine</strong> and natural dentine. The<br />
deposition of apatitic structures might increase the marginal sealing of the material.<br />
This type of crystal deposition is already well known for MTA systems.<br />
Fuji II<br />
15
16<br />
3.2 - Resistance to microleakage:<br />
Several studies were performed to evaluate the resistance of <strong>Biodentine</strong> to<br />
microleakage.<br />
The interface with dentine and enamel was examined using dye penetration methodology<br />
(silver nitrate), which is one of the most commonly used assays to assess, in vitro, the<br />
interfacial seal, by measuring the percolation of a dye along the different interfaces<br />
studied (Golberg et al., 2009).<br />
Methods:<br />
Freshly extracted human molars were used to prepare class II cavities both on the mesial<br />
and on the distal sides. The prepared teeth were divided into different groups to evaluate<br />
the influence of a pretreatment of the cavity using polyacrylic acid solution (GC Conditioner,<br />
GC Corp.) before <strong>Biodentine</strong> placement, the application of a surface varnish (Optiguard ® ,<br />
Kerr) after the <strong>Biodentine</strong> setting to protect from humidity after initial setting and the<br />
influence of the bonding agent (Xeno ® III, Dentsply or G Bond, GC) when placing a<br />
composite (Ceram-X ® Mono, Dentsply) over <strong>Biodentine</strong> one day after setting.<br />
Each group was submitted to 2200 thermocycles (5°C – 55°C, 10 seconds for each batch<br />
and transfer). The percentage of microleakage was determined on six samples as the<br />
length of dye penetration divided by the length of the interface.<br />
Results:<br />
•At the enamel - BIODENTINE interface:<br />
% Dye penetration = (AA1/AB) * 100%<br />
• At the dentin - BIODENTINE interface:<br />
% Dye Penetration = (CC1/CD) * 100%<br />
• At the composite - BIODENTINE interface:<br />
% Dye Penetration = (EE1/EF) * 100%<br />
No significant difference in the percentage of microleakage was observed at the enamel-<br />
<strong>Biodentine</strong> and dentine-<strong>Biodentine</strong> interfaces, with or without polyacrylic acid<br />
treatment. The placement of a protective varnish increases microleakage at the enamel<br />
interface in the early stage, but not at the dentine interface. After 3 months of aging, no<br />
significant difference could be evidenced in the case of Optiguard ® placement or not.<br />
At the <strong>Biodentine</strong>-composite interface (Fig.5), 1 day after placement, the specimens<br />
bonded with Xeno ® III exhibited significantly less microleakage than those bonded with<br />
G Bond.<br />
After 3 months, the micro leakages of specimens treated with G Bond were lower than<br />
at 1 day. At 3 months no significant differences at the composite-<strong>Biodentine</strong> interface<br />
were observed between Xeno ® III or G Bond and Xeno ® III + Optiguard ® .
Fig. 5: Histogram of mean microleakage % at the<br />
<strong>Biodentine</strong> / adhesive interface<br />
According to this study, the interfaces which are developed between <strong>Biodentine</strong> and<br />
the dental surfaces (enamel and dentine) as well as with adhesive systems (Xeno ® III or<br />
G Bond), are very resistant to micro leakage, with or without pre-treatment by polyacrylic<br />
acid solutions. The choice of water based adhesive systems might be preferable when<br />
placing a composite over <strong>Biodentine</strong>. The sealing quality of <strong>Biodentine</strong> is not<br />
influenced by the storage after 3 months.<br />
Dejou evaluated the micro leakage resistance of <strong>Biodentine</strong> in comparison with one<br />
of the best sealing systems, resin modified glass ionomers (Fuji II LC, GC Corp.): after<br />
2500 thermo cycles, the dye penetration was evaluated by scoring the depth of<br />
penetration of silver nitrate marker (ranging from 0= no penetration to 3= full interface<br />
penetration) (Internal report).<br />
Mesio-occlusal and disto-occlusal preparations below cementum-enamel junction were<br />
made in 42 extracted molars. The teeth were randomly assigned one of the following<br />
treatments before restoration with Filtek Z250 (3M ESPE) composite resin:<br />
<strong>Biodentine</strong>; Fuji II LC (GC); <strong>Biodentine</strong> + Optibond ® Solo Plus (Kerr); <strong>Biodentine</strong><br />
+ Optibond ® Solo Plus (Kerr) + silane; <strong>Biodentine</strong> + Septobond SE (Septodont) ;<br />
Fuji II LC (GC) + Optibond ® Solo Plus (Kerr).<br />
Concerning the first two groups: leakage was evaluated<br />
separately, in contact with enamel or in contact with dentine<br />
(Fig.6). <strong>Biodentine</strong> exhibits better leakage resistance both to<br />
enamel and to dentine compared to Fuji II LC.<br />
Fig.6: Micro leakage scores of <strong>Biodentine</strong> or Fuji IILC in contact with enamel or dentine<br />
2 mm<br />
6 mm<br />
5 mm<br />
2 mm<br />
17
6<br />
18<br />
Z 250<br />
Optibond<br />
Solo+<br />
<strong>Biodentine</strong><br />
FujillLC<br />
Fig.7: Micro leakage scores of <strong>Biodentine</strong><br />
or Fuji IILC in sandwich technique<br />
3.3 - Electron Microscopy:<br />
In the sandwich technique groups, at the<br />
interface between the base material<br />
(<strong>Biodentine</strong> or Fuji II LC) and the composite,<br />
in case of Optibond ® Solo plus (total etch<br />
system), similar micro leakage resistance are<br />
obtained (Fig.7).<br />
Only in the case of Septobond SE (self etch<br />
bonding), was the percolation at the interface<br />
slightly increased, but no significant<br />
difference could be evidenced on the maximal<br />
median scores.<br />
In conclusion, <strong>Biodentine</strong> has a similar<br />
behavior in terms of leakage resistance as Fuji<br />
II LC at the interface with enamel, with dentine<br />
and with dentine bonding agents.<br />
<strong>Biodentine</strong> is then indicated in opensandwich<br />
class II restoration without any<br />
preliminary treatment.<br />
Interface between <strong>Biodentine</strong> (left) and<br />
human dentine (right): the two surfaces are in<br />
direct and intimate contact. The surface of<br />
<strong>Biodentine</strong> presents some crystal<br />
deposition which appeared after the sample<br />
cutting due to re-exposition to water<br />
environment.<br />
Pr Dejou, Dr Raskin<br />
There is a direct contact without a gap<br />
between <strong>Biodentine</strong> and the natural dentine.<br />
The crack is observed inside <strong>Biodentine</strong><br />
caused by dehydration, due to SEM sample<br />
preparation under vacuum. This cohesive<br />
failure does not affect the dentine-<br />
<strong>Biodentine</strong> interface, which indicates the<br />
quality of the micro-mechanical adhesion.<br />
Pr Colon, Dr Pradelle
At the entrance of the dentine tubules, some<br />
mineral re-crystallisation occurs, creating<br />
mineral tags. This induces micromechanical<br />
anchorage of <strong>Biodentine</strong>. This process<br />
will continue with time, improving the<br />
sealing.<br />
Pr Colon, Dr Pradelle<br />
Comparison of the interface<br />
between <strong>Biodentine</strong> or Fuji<br />
II LC and a composite, using<br />
Optibond ® Solo Plus: The<br />
interfaces are very similar.<br />
Pr Dejou, Dr Raskin<br />
Perfect seal of <strong>Biodentine</strong> in contact with radicular dentine, as well as between two<br />
increments of <strong>Biodentine</strong>, in an in vitro test of apexification.<br />
Dr Bronnec, Pr Colon<br />
Crystallisation process in the dentine<br />
tubule of an extracted wisdom tooth<br />
treated with <strong>Biodentine</strong>, observed after<br />
28 days of storage in distilled water.<br />
The dentine tubules are obturated by recrystallisation.<br />
Dr Franquin<br />
19
20<br />
❹ Outstanding<br />
biocompatibility<br />
From a regulatory point of view, <strong>Biodentine</strong> is a calcium silicate based material, used<br />
for crown and root dentine repair treatment, involving external contact for a period of<br />
more than 30 days. The biocompatibility tests required for the preclinical evaluation of<br />
dental products followed the guideline ISO 7405 - 2008.<br />
The following sections evaluate the compliance with this standard for the tests carried<br />
out on <strong>Biodentine</strong>. It is considered a device with external contact, for long-term tissue<br />
contact (>30 days). In certain indications (radicular, apical obstruction and repair of the<br />
pulpal floor), it can be considered an implanted system, according to the ISO<br />
classification.<br />
All biocompatibility tests were carried out on the final product <strong>Biodentine</strong>.<br />
4.1 - Cytotoxicity tests (ISO 7405, ISO 10993-5)<br />
Different cytotoxicity tests carried out on <strong>Biodentine</strong> are reported.<br />
The first study was performed on human pulpal fibroblasts (human wisdom tooth),<br />
comparing <strong>Biodentine</strong>, calcium hydroxide and MTA (Dycal ® , Dentsply and ProRoot ®<br />
MTA Dentsply). The cell viability was determined by MTT incorporation (About, 2003b).<br />
Results showed <strong>Biodentine</strong> was non cytotoxic like MTA, whereas the undiluted cement<br />
Dycal ® induced 22 % of cytotoxicity (Table. 1).<br />
Product Cell death (%)<br />
<strong>Biodentine</strong> 0±8<br />
MTA 0±9<br />
CaOH 22±10<br />
Table 1. Cell death after Dycal ® , MTA<br />
and BIODENTINE contact.<br />
Control<br />
<strong>Biodentine</strong><br />
(4 weeks)<br />
MTA<br />
(4 weeks)<br />
Collagen DSP<br />
Moreover, the cell differentiation was evaluated<br />
with the expression of collagen, dentine<br />
sialoprotein (DSP) and osteonectin (OSN).<br />
Results showed the expression of the<br />
differentiation markers, underlining the safety<br />
of <strong>Biodentine</strong> (Fig. 8).<br />
Figure 8. Expression of<br />
collagen and dentine<br />
sialoprotein (DSP) after<br />
contact with <strong>Biodentine</strong><br />
and MTA during 4 weeks.
The second study was performed on L929 fibroblasts comparing <strong>Biodentine</strong>,<br />
composite resin Filtek Z250 and MTA (Franquin, 2001). Samples were extracted 3 h,<br />
24 h and 7 days after the setting. The cell viability was determined by MTT incorporation.<br />
Results showed <strong>Biodentine</strong> is not cytotoxic (< 10 %) whatever hardening time is<br />
considered. Filtek Z250 resin is slightly cytotoxic (> 20 %) at the 3 observation<br />
periods (Table 2).<br />
Product 3 hours 1 day 7 days<br />
Filtek Z250 23% 25% 26%<br />
MTA 0% 14% 8%<br />
<strong>Biodentine</strong> 2% 10% 9%<br />
Table 2. Cell death after Filtek Z250, MTA and <strong>Biodentine</strong> contact.<br />
The third study was published in Dental Materials on the biological effects of<br />
<strong>Biodentine</strong> (Laurent et al., 2008). They were compared to those induced by the<br />
materials used for pulp capping such as MTA and Dycal ® . Several tests were carried out:<br />
• A cytotoxicity test involving indirect contact through a section of dentine: none<br />
of the tested materials was cytotoxic.<br />
• Where there is no dentine interposition, there is a significant difference in<br />
toxicity of the different materials: <strong>Biodentine</strong> did not reveal any cytotoxicity<br />
although more marked cytotoxicity was reported for Dycal ® compared to MTA.<br />
• Differentiation of pulp fibroblasts in orthodontoblastic cells was also analysed<br />
for contact with two materials. Pulp fibroblasts secrete a mineralised matrix<br />
and cells in contact express differentiation proteins (nestin and dentine sialoproteins).<br />
Once the cells had been in contact with <strong>Biodentine</strong> cement or with<br />
MTA, marker expression was important in the pulp cells involving the formation<br />
of mineral nodules. Immunological marking was in all cases higher in the cells<br />
forming mineral nodules.<br />
To conclude, these various tests demonstrate that there is no direct cytotoxic effect with<br />
<strong>Biodentine</strong> in the form of an extract in contact with L929 fibroblast line cells, dental<br />
specialised pulp cells and that moreover it does not affect phenotypic pulp expression<br />
of fibroblasts.<br />
4.2 - Sensitization tests (ISO 7405, ISO 10993-1)<br />
Studies were performed on guinea-pigs thanks to a maximisation method (intradermic<br />
and topical application with Freund complete adjuvant induction.<br />
The evaluation of oedema and erythema was performed according to a clinical scale<br />
(0-4) 24 and 48 h after retrieval of occlusive patches of the challenge phase. The<br />
sensitisation potential is graded (class 0 to 4) according to the percentage of sensitised<br />
animals (score of more than 2).<br />
<strong>Biodentine</strong> was not sensitizing (Gomond, 2003c).<br />
21
22<br />
4.3 - Genotoxicity tests (ISO 7405, ISO 10993-3, OCDE 471)<br />
Several genotoxicity tests were performed on the <strong>Biodentine</strong> cement. They were<br />
carried out on extracts of the cement after complete setting.<br />
AMES test performed on Salmonella typhimurium and Escherichia coli. Strains TA98,<br />
TA100, TA1537, TA1535, pKM101 in absence or presence of metabolism activator.<br />
Results showed that <strong>Biodentine</strong> was not mutagenic (Harmand, 2003).<br />
Another AMES test was performed on 4 strains of Salmonella typhimurium TA97A, TA98,<br />
TA100 and TA102. The results showed that cement <strong>Biodentine</strong> does not induce reverse<br />
mutation in the presence or absence of the metabolic activator S9. Identical results were<br />
reported for the four strains of bacteria tested (Laurent et al., 2008).<br />
An in vitro micronucleus test was also carried out using human lymphocytes (Laurent et<br />
al., 2008). These were exposed to extracts of <strong>Biodentine</strong> obtained either from a culture<br />
medium or DMSO. Dilutions of 1% to 5% of the extracts were used. After a culture time<br />
of 72 hours, the cells were stained and analysed. 1000 bi-nucleated lymphocytes were<br />
tested, to check for a micronucleus. A toxicity index was determined, together with a<br />
ratio for the number of micronuclei in relation to the negative reference. The results<br />
showed that after incubation of the lymphocytes with different dilutions of the extract of<br />
<strong>Biodentine</strong>, the number of lymphocytes presenting a micronucleus was similar to that<br />
obtained with the negative reference (3.9% to 4.1%) when concentrations of 1% to 5%<br />
in an aqueous or hydrophobic medium were tested. Positive controls produced a<br />
micronucleus rate of 16% (Fig. 2)<br />
<strong>Biodentine</strong> Micronucleoted<br />
lymphocytes (%±SD)<br />
1% 4.0±1.1<br />
2.3% 4.0±1.1<br />
3.7% 4.0±1.2<br />
5% 4.2±1.2<br />
- control 3.7±1.2<br />
+ control 16.0±6.0*<br />
Table 3. Micronucleated lymphocytes<br />
after contact with <strong>Biodentine</strong> .<br />
<strong>Biodentine</strong> Tail DNA mean<br />
dilution (%±SD)<br />
0.1% 12.59±0.96<br />
1% 13.31±0.88<br />
10% 14.90±1.06<br />
Undiluted 15.58±1.08<br />
Negative control 13.19±0.96<br />
Positive control 46.52±1.45*<br />
Table 4. Tail DNA mean after contact<br />
with <strong>Biodentine</strong>.<br />
Finally, the comet test on human pulp<br />
fibroblasts was conducted (About, 2003a).<br />
The extract of <strong>Biodentine</strong> was prepared in<br />
DMSO and a culture medium, at 50 mg/ml for<br />
24 hours and at 37°C. The cells were exposed<br />
directly to increasing dilutions of cement<br />
extracts for two hours. Following electrophoresis,<br />
the slides were analysed by<br />
fluorescent microscopy (magnification 400)<br />
and an automated analyser was used to<br />
determine DNA lesions. The results obtained<br />
showed that the percentage of tail DNA varied<br />
from 12.59 for a dilution of 0.1% to 15.58 for<br />
the undiluted medium. It was 13.19 for the<br />
negative control and 46.52 for the positive<br />
control (Table 3). In the presence of DMSO,<br />
there was no significant difference between<br />
the genotoxicity of <strong>Biodentine</strong> and the<br />
negative control (extracted with NaCl and<br />
DMSO).
4.4 - Cutaneous irritation tests (ISO 7405, ISO 10993-10)<br />
Cutaneous irritation test was performed in the rabbit by direct application. Oedema and<br />
erythmea were evaluated 1h, 24h, 48h and 72h after patch removal. <strong>Biodentine</strong> was<br />
shown to be non irritant (Gomond, 2003a).<br />
4.5 - Eye irritation tests (OCDE 405)<br />
The irritation of the liquid part of <strong>Biodentine</strong> was tested on rabbit eye mucosa. The<br />
aim of the study was to assess qualitatively and quantitatively irritation or corrosion<br />
and the delay of appearance of the effects after single application of 0.1 ml on eye in<br />
3 rabbits. The ocular reactions (redness and chemosis of conjunctivae, iris and cornea<br />
lesions) were scored 1h, 24h, 48h and 72h after application. The liquid part of<br />
<strong>Biodentine</strong> undiluted was unclassified among the chemicals irritating to eyes (Fagette,<br />
2009).<br />
4.6 - Acute toxicity tests (ISO 7405, ISO 10993-11,<br />
OCDE 423)<br />
The acute toxicity tests were performed in order to determine on a qualitative and<br />
quantitative basis the toxicity signs and their time of appearance after a unique oral<br />
administration of a dose of 2000 mg/kg of the product in rats. Rats were observed<br />
immediately after administration, 1h, 2h, 3h, 4h, and at least once a day during 14 days.<br />
The administration by oral route of the 2000 mg/kg dose of <strong>Biodentine</strong> induced no<br />
acute toxicity in the rat. The DL50 of <strong>Biodentine</strong> is superior to 2000 mg/kg (Gomond,<br />
2003b).<br />
4.7 - Preclinical safety conclusion<br />
The tests carried out on <strong>Biodentine</strong> have shown that the material tested in the form of<br />
an extract in a saline environment is not a cytotoxic, mutagenic, irritant or sensitising<br />
agent. It is devoid of oral toxicity at a dose of 2000 mg/kg. In conclusion, <strong>Biodentine</strong><br />
is safe. Compared to well known dental materials such as Dycal ® (calcium hydroxide),<br />
<strong>Biodentine</strong> exhibits less cytotoxicity. Moreover, when compared to ProRoot ® MTA,<br />
<strong>Biodentine</strong> demonstrates at least equivalent biocompatibility.<br />
23
24<br />
❺ Evidence<br />
based bioactivity<br />
Two in vitro tests and two tests in animals were performed in order to demonstrate the<br />
bioactivity of <strong>Biodentine</strong> in clinical situations.<br />
5.1 - In vitro test of direct pulp capping on human<br />
extracted teeth<br />
Human teeth were extracted in order to make exposed pulp cavities which were then<br />
filled with <strong>Biodentine</strong> (About, 2007). The teeth (n = 15) were cultured for 24 hours<br />
(n = 5), 14 days (n = 5) and 28 days (n = 5) in order to determine the bioactivity of<br />
<strong>Biodentine</strong> (Fig 9).<br />
A<br />
B C<br />
Figure 9. Exposed pulp cavities (A) obturated with <strong>Biodentine</strong> (B) and cultured (C).<br />
At the end of the culture and after demineralisation, histological sections were done. The<br />
results showed good preservation of the pulp up to 28 days. Near the capped area, a<br />
change in the pulp tissue was reported, with the neo-formation of reparatory dentine<br />
comparable to that observed with MTA (Fig 10). This corresponds to the first signs of<br />
the formation of a dentine bridge.<br />
Figure 10. Observations after 28 days.<br />
To conclude, <strong>Biodentine</strong> is able to stimulate initiation and development of mineralization.
5.2 - In vitro test for angiogenesis<br />
A study was conducted on damaged pulp fibroblasts in order to evaluate the<br />
<strong>Biodentine</strong> activity on angiogenesis (About, 2009). This model mimicked the in vivo<br />
situations in cases of pulp damage requiring direct pulp capping. Materials such as<br />
<strong>Biodentine</strong>, Calcipulpe ® , Hydroxide de calcium XR, ProRoot ® MTA and Xeno ® III were<br />
applied to the cells and growth factors (VEGF, FGF-2, PDGF-AB, TGF-β1) concentrations<br />
were evaluated by ELISA test.<br />
Results showed that none of the products modified the cell structure in this model. Only<br />
ProRot ® MTA and <strong>Biodentine</strong> were able to stimulate the formation of mineralisation<br />
spots. The concentration level of TGF-β1 was enhanced by both ProRoot ® MTA and<br />
<strong>Biodentine</strong>. Moreover, VEGF and FGF-2 were enhanced in presence of <strong>Biodentine</strong><br />
(150 à 200% for VEGF and up to 670 % for FGF-2).<br />
These results suggest that <strong>Biodentine</strong> is able to stimulate angiogenesis, in order to<br />
heal pulp fibroblasts.<br />
5.3 - Stimulation of reactionary dentine in indirect pulp<br />
capping : rat model<br />
A study was conducted on the maxillary molars of adult rats (Golberg, 2009). The first<br />
maxillary molars were prepared in order to achieve half-moon cavities (class V) on the<br />
mesial face. The cavities were filled with <strong>Biodentine</strong> and with Fuji IX glass ionomer<br />
cement and covered with a protective varnish. The teeth were collected and fixed at 8<br />
days, 15 days, 30 days and 3 months after filling. The results showed that after 8 days,<br />
pulp inflammation was moderate in the mesial third of the pulp chamber. This reaction<br />
was also observed on the reference teeth (Fig. 11).<br />
Figure. 11. <strong>Biodentine</strong> stimulates reactionary dentine (rd).<br />
25
26<br />
The inflammatory process had disappeared after 15 days. The newly formed reactionary<br />
dentine was identified. By comparison with the group treated with the glass ionomer<br />
cement, the formation of reactionary dentine was greater in the teeth in the presence of<br />
<strong>Biodentine</strong> and its thickness increased over time from 20 to 40 µm after 8 days, 40 to<br />
80 µm after 15 days and 140 to 280 µm after 30 days, although it varied between 10<br />
and 20 µm for the reference group. After 3 months, reactionary dentine generated by<br />
<strong>Biodentine</strong> was thick and dense (Fig. 12), enclosing the horn and the mesial pulp whilst<br />
for Fuji IX, this was less dense, only partially covering the mesial cervical area of the pulp<br />
(Golberg, 2009).<br />
Fig. 12. Formation of a thick reactionary dentine in presence of to <strong>Biodentine</strong> in comparison to Fuji IX<br />
To conclude, <strong>Biodentine</strong> was able to stimulate a reactionary dentine which is a natural<br />
barrier against bacterial invasions. The reactionary dentine formation stabilises at<br />
3 months, indicating that the stimulation process is stopped when a sufficient dentine<br />
barrier is formed.<br />
5.4 - Calcification as a result of <strong>Biodentine</strong> in a direct<br />
pulp capping and pulpotomy : pig model<br />
Two protocols were set up in pigs (Shayegan A, 2009).<br />
The first protocol was the analysis of the pulp reaction following pulpotomy and<br />
placement of different materials (15 deciduous teeth, 15 pigs):<br />
• Formocresol<br />
• White MTA<br />
• <strong>Biodentine</strong><br />
The follow-up was performed during 1, 4 and 12 weeks.<br />
Pulp chamber was excised in 15 pigs in a comparative study of the efficacy of<br />
<strong>Biodentine</strong> versus formocresol and MTA (5 pigs per group). Histological sections of<br />
the teeth were done after a week, a month and 3 months of treatment. The results<br />
showed that <strong>Biodentine</strong>, like White MTA, promoted beneficial calcification after one<br />
week, whereas Formocresol induced necrosis and inflammation (Fig. 13).
1 week<br />
Inflammation<br />
4 weeks<br />
Inflammation-<br />
Tissu regeneration<br />
transition<br />
12 weeks<br />
Complete healing<br />
To conclude, <strong>Biodentine</strong> is a suitable material for pulpotomy.<br />
The second protocol was an analysis of the pulp reaction after direct capping for different<br />
materials (15 deciduous teeth, 15 pigs):<br />
• Ca (OH)2<br />
• White MTA<br />
• <strong>Biodentine</strong><br />
Formocresol WMTA <strong>Biodentine</strong><br />
Fig. 13. Summary of pulpotomy results.<br />
The follow-up was performed during 1, 4 and 12 weeks.<br />
Pulp exposure was performed via a class V vestibular cavity in 15 pigs who were<br />
4 months old, in order to compare the efficacy of <strong>Biodentine</strong> against calcium hydroxide<br />
and MTA (5 pigs in each group) over 3 trial periods of 1 week, 1 month and 3 months<br />
(Shayegan 2009).<br />
1 week<br />
Inflammation<br />
4 weeks<br />
Inflammation-<br />
Tissu regeneration<br />
transition<br />
12 weeks<br />
Complete healing<br />
10/10 Necrosis and inflamation 6/10 Beginning of calcification 10/10 Calcification<br />
4/10 Necrosis and inflamation 7/10 Important calcification 7/10 Important calcification<br />
5/10 Infiltration of inflammatory<br />
cells<br />
7/10 Necrosis and inflamation 10/10 Complete calcification 9/10 Complete calcification<br />
1/10 Calcification<br />
Calcium hydroxyde WMTA <strong>Biodentine</strong><br />
2/10 Calcification 7/10 Calcification 9/10 Calcification<br />
7/10 Important calcification 10/10 Important calcification 10/10 Important calcification<br />
5/10 Partial calcification<br />
10/10 Important calcification 10/10 Important calcification 9/10 Important calcification<br />
Fig.14. Summary of direct pulp capping results.<br />
27
28<br />
To conclude, <strong>Biodentine</strong> enhances the formation of a dentine barrier after direct pulp<br />
capping confirming it has good potential in this indication. In the first month, the quality<br />
of the dentine bridge formed with <strong>Biodentine</strong> is of better quality than with the reference<br />
dental technique (calcium hydroxide). The performance of <strong>Biodentine</strong> is at least<br />
equivalent to White MTA.<br />
5.5 - Overall bioactivity<br />
Pulp capping and pulpotomy studies showed that <strong>Biodentine</strong> was very well tolerated.<br />
Moreover, <strong>Biodentine</strong> was able to promote mineralisation, generating a reactionary<br />
dentine as well as a dense dentine bridge. These phenomena illustrate the great potential<br />
for <strong>Biodentine</strong> to be in contact to the pulp, by demonstrating its bioactivity in several<br />
indications.<br />
As a conclusion, <strong>Biodentine</strong> is bioactive.
❻6.1<br />
- <strong>Biodentine</strong> is used as a dentine substitute under<br />
a composite<br />
A clinical investigation, 04/001, aimed to assess the acceptability of <strong>Biodentine</strong> as a<br />
new restoration of the posterior teeth: a first-in-man study.<br />
Among the products already used in dentistry, one product shares similar properties with<br />
<strong>Biodentine</strong>. This product, MTA, Mineral Trioxide Aggregate, sold by Dentsply under<br />
the brand name ProRoot ® MTA, is a derivative of Portland cement, with the same<br />
chemical properties. It was developed as a product for radicular repair only, due to a<br />
low compressive strength incompatible with restorative indications. The biological<br />
properties of this product allow its use in the capping of dental pulp tissue, in the filling<br />
of the radicular apical part by a retrograde approach or in the closure of perforations, to<br />
promote the restoration of the original tissue in contact with the pulp tissue and radicular<br />
tissue.<br />
<strong>Biodentine</strong> can be defined as a special micronised concrete derived from the main<br />
component of Portland cement, tricalcium silicate. With physical properties far superior<br />
to those of MTA, especially in terms of setting time and compressive strength, it exhibits<br />
the same characteristics of biocompatibility and sealing ability, after setting in an alkaline<br />
pH, with controlled (size and spatial organisation) formation of calcium salts. This product<br />
exclusively composed of mineral components, was initially designed to replace dentine<br />
in restorations.<br />
In this study, <strong>Biodentine</strong> is compared to Filtek Z100, which is used for dental<br />
restorations and requires an adhesive for the bonding the composite on the tooth.<br />
<strong>Biodentine</strong> is applied directly to contact with the tooth, without adhesive or conditioner.<br />
This clinical investigation is a multicentre, randomised, prospective study, which required<br />
the inclusion of 400 patients and a 3-year observation period.<br />
The interim report is based on 232 cases with a minimum one year follow-up: 116 were<br />
treated with <strong>Biodentine</strong> and 116 with Filtek Z100. Among the 116 restorations done<br />
with <strong>Biodentine</strong>, 20 involved a direct pulp capping.<br />
The study planed a follow-up at baseline, 15 days, 6, 12, 24 and 36 months. The analysis<br />
of the cases showed:<br />
At D0, <strong>Biodentine</strong> showed:<br />
• Easy handling.<br />
• Excellent anatomic form.<br />
• Very good marginal adaption.<br />
• Very good interproximal contact.<br />
Clinical efficacy<br />
29
30<br />
During the follow-up, the restoration with <strong>Biodentine</strong> in comparison to Filtek Z100:<br />
• Was well tolerated in all cases.<br />
• The anatomic form, the marginal adaptation and the interproximal contact<br />
started to degrade after 6 months.<br />
• Due to the degradation, a complementary treatment was performed. In 93.8%,<br />
cases needed a retreatment (92/116); <strong>Biodentine</strong> was kept as dentine<br />
substitute as the pulp vitality test was positive. <strong>Biodentine</strong> presented a good<br />
resistance to burring and the composite Filtek Z100 was applied on the top.<br />
The tolerance was evaluated for up to 3 years.<br />
• Was safe for the patient, as the same number of adverse events was observed<br />
in <strong>Biodentine</strong> group (4/116) as in Filtek Z100 group (3/116).<br />
As a conclusion, <strong>Biodentine</strong> was applied in 116 patients with at least one year follow-up.<br />
Thanks to its excellent biocompatibility, <strong>Biodentine</strong> is very well tolerated and can be<br />
used as cavity lining with a permanent composite restoration (Fig.15).<br />
D0 : Patient restoration D0 : Amalgam removal D0 : <strong>Biodentine</strong> application<br />
6 months later<br />
16 months:<br />
<strong>Biodentine</strong> reshaping<br />
Fig. 15.Restoration with <strong>Biodentine</strong><br />
(Courtesy of Prof. KOUBI, Marseille).<br />
30 months later:<br />
<strong>Biodentine</strong> under Filtek Z100
6.2 - <strong>Biodentine</strong> is used as a direct pulp capping<br />
material<br />
In the same clinical trial, 04/001, <strong>Biodentine</strong> was also used as direct pulp capping<br />
material. <strong>Biodentine</strong> showed:<br />
• An excellent tolerance.<br />
• The ability to save pulp vitality even in difficult cases: the vitality test was<br />
positive at each recall.<br />
Moreover, <strong>Biodentine</strong> can be used in direct pulp capping indications with a good<br />
success rate (Fig. 16). It is important to underline that <strong>Biodentine</strong> was used in contact<br />
with pulp tissue in a patient older than 21 and maintained the pulp alive.<br />
D0 : Radiography D0 : Exposed pulp<br />
D0 : <strong>Biodentine</strong> application<br />
D0 Three years later: <strong>Biodentine</strong> covered<br />
by Filtek Z100.<br />
Fig. 16. Direct pulp capping with <strong>Biodentine</strong> (Courtesy of Prof. KOUBI, Marseille).<br />
31
32<br />
6.3 - <strong>Biodentine</strong> is used as an endodontic repair<br />
material<br />
The endodontic indications of <strong>Biodentine</strong> are similar to the usual calcium silicate based<br />
materials, like the Portland cements (i.e. ProRoot ® MTA). This type of product is already<br />
well documented.<br />
Several physical, chemical and biological properties are comparable as summarised in the<br />
preclinical section. However, <strong>Biodentine</strong> has some features which are superior to MTA.<br />
• <strong>Biodentine</strong> consistency is better suited to the clinical use than MTA’s.<br />
• <strong>Biodentine</strong> presentation ensures a better handling and safety than MTA.<br />
• <strong>Biodentine</strong> does not require a two step obturation as in the case of MTA.<br />
As the setting is faster, there is a lower risk of bacterial contamination than<br />
with MTA.<br />
Adding to its ability to be used as dentine substitute, <strong>Biodentine</strong> could safely be used<br />
for each indication where dentine is damaged. Therefore, it is an advantage for the<br />
clinician and the patient (Machtou, 2009b).<br />
Moreover, a clinical trial, 09/001, aimed at assessing the tolerance and efficacy of<br />
<strong>Biodentine</strong> in 6 endodontic procedures, after 3 months and after 2 years follow-up is<br />
in progress:<br />
• Direct pulp capping following carious pulp exposure<br />
• Direct pulp capping following dental trauma/injury to healthy pulp (partial<br />
pulpotomy)<br />
• Repair of perforated root canals and/or pulp chamber floor<br />
• Retrograde endodontic surgery<br />
• Pulpotomy in primary molars<br />
• Apexification<br />
Ten patients per indication are required in this multi-centre and open-label clinical trial<br />
(Machtou, 2009a).
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