BioRoot™ RCSBioactive Root Canal Sealer

Scientific FileFor distribution in the USA - January 2015

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What is BioRoot™ RCS

BioRoot™ RCS is the latest generation of root canal sealer from Septodont whichbenefits from the unique Active Biosilicate Technology.

BioRoot™ RCS was designed to simplify the obturation techniques of root canal, by itsease of mix, ease of use, its optimized consistency and eliminating the need for a warmgutta percha technique. Using BioRoot™ RCS with both monocone or lateralcondensation will provide for a reproducible seal during obturation.

The Active BioSilicate Technology is free of monomers and provides outstandingbiological and bioactive properties including biocompatibility, hydroxyapatite formation,mineralization of dentinal structure, alkaline pH and sealing properties.

Active BioSilicate technology

With over 10 years of research, the Active BioSilicate Technology from Septodont hasprovided safe and effective clinical solutions for the dental profession. This technologyis based upon the well accepted, highly biocompatible chemistry of calcium silicates.Within clinical dentistry, calcium silicates offer the benefit to set in the presence of water.Septodont controls every step of chemical processing during material developmentstarting from pure raw materials.

The technological challenge was to take a calcium silicate to create a formulation withgood clinical practices in both restorative and endodontic procedures. The result wasthe development of a new technology, Active BioSilicate Technology. Usual dentalcalcium silicate cements are based upon “Portland cement” materials derived from themanufacture of cement clinker by treating and grinding natural stones to make cement.This process implies that all of these products inherently contain unpurified mixtures ofcalcium silicates, calcium aluminates, calcium alumino-ferrites, calcium sulfates, togetherwith low concentrations of metallic impurities coming from the natural materials used asraw materials.

Septodont decided that the only way to reach the objective to have a medical gradelevel of purity control with tailored mechanical property characteristics was to synthesizeour own calcium silicate products in our production facilities.

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

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

Biodentine™ was the first Bioactive Dental Substitute incorporating this technologyleading to outstanding physical properties and biological properties. BioRoot™ RCS isnow released to the dental market, with the background of all the previous experiencefrom Biodentine™ with more than 70 international peer-reviewed publicationsdemonstrating the physical, biological and clinical benefits of the Active BioSilicateTechnology.

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Long lasting tight seal

BioRoot™ RCS is indicated for the permanent root canal filling in combination with gutta-percha points and is suitable for use in single cone technique or cold lateralcondensation (Camilleri, 2015). BioRoot™ RCS was designed to be used by mixingpowder part (1 spoon) with the liquid part (5 drops) by simple spatulation : there is noneed of a mixing machine. The working time is around 15 minutes and the setting timeis less than 4 hours in the root canal (table 1). In addition, BioRoot™ RCS displayed atight seal with the dentin and the gutta-percha (Figure 1) and an appropriate radiopacity(figure 2, Xuereb 2014).

The paste is of smooth consistency with good flow (table 1) and adequate adhesion toinstruments in order to enable an optimal placement in the root canal.

Thanks to the use of Active BioSilicate Technology which is resin free, there is noshrinkage of BioRoot™ RCS during setting for reaching a tight seal of the root canal.

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Figure 1: Interfacebetween BioRoot™ RCSand Gutta Percha pointsor root dentine - SEM(Internal data)

Figure 2: Radiographs of roots obturated by BioRoot™ RCS (Internal data)

Table 1 Working time Setting time Flow Film thickness Radiopacity

Specificationsaccording the

ISO 6876: 2012

Not applicable ifWT > 30 minutes

30 minutes to

72 hours> 17 mm < 50 µm

> 3 mm of aluminium

BioRoot™ RCS 10 min< WT < 4 hours 26 mm 45 µm 5 mm

Gutta BioRoot BioRoot Dentin

30 µm 30 µm

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Push out bond strength (Eldeniz et al., poster presentation at the ESECongress Lisbon 2013)

The presence of smear layer on radicular dentine can prevent the penetration ofintracanal treatments into dentinal tubules and influence the adaptation of filling materialsto canal walls. Therefore, the purpose of this study was to assess the bond strength oftwo new silicate-based sealers (Root Canal Sealer - BioRoot™ RCS from Septodont,France and TechBIOSEALER from Isasan, Italy) in comparison with previous silicate-based sealers (iRootSP from Innovative BioCeramix Inc., Canada and MTA Fillapex fromAngelus, Brasil) after smear layer removal with various irrigants

Crowns of 48 single-rooted human teeth were removed and root canals prepared withProTaper files to size F3. The specimens were randomly assigned to four groups (n=12)and rinsed with 5.25% NaOCl + 17% EDTA (Group 1), 5.25% NaOCl +10% Citric acid(Group 2), 5.25% NaOCl + saline + QMix2in1(Group 3), 5.25% NaOCl + Saline (Group 4).The samples (n=12) were then divided into 4 experimental groups according to the sealerused for the obturation (n=3): BioRoot™ RCS, TechBIOSEALER, iRootSP , MTA Fillapex.

After 7 days incubation period, six horizontal sections were obtained at thickness of1mm ± 0.1 of each root and were subjected to the push out test method using a universaltest machine

Smear removal with EDTA gave better push-out bond strength values than Qmix2in1and 10% citric acid groups (P<0.05) with comparable results to saline irrigated groups(P>0.05).

BioRoot™ RCS displayed statistically better bond strength values(0,63 ± 0,27 MPa) thanMTA Fillapex (0,28 ± 0,14 MPa) and TechBIOSEALER (0,32 ± 0,32 MPa) whereas inferiorthan iRootSP (0,85 ± 0,44MPa).

Conclusion: BioRoot™ RCS and iRootSP adhere better to dentine than MTA Fillapexand TechBIOSEALER.

Microleakage study: BioRoot™ RCS in a single cone techniquecompared to Pulp Canal Sealer in a warm compaction (Internal data)

Warm gutta percha /ZOE root obturation technique is considered as the « gold standard »for root canal filling. The objective of this study was to compare the sealing of root canalfilling with BioRoot™ RCS used according the single cone gutta-percha cold technique(Group 3 and 4) versus Pulp Canal Sealer (PCS - Kerr) used according the warmcompaction gutta-percha technique (Group 1 and 2).

After obturation of the root canal, the samples were stored in phosphate buffer solutions(pH=7, Lonza) for 7 days (Group 1 and 3) or 30 days (Group 2 and 4) before submissionto a silver nitrate percolation test. The samples were cut on the main root axis and lengthof percolation was measured from the apex.

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After 7 days, the mean percolation length is lower for BioRoot™ RCS group than forPCS group (1.23 mm vs 3.06 mm respectively) . This indicates better early leakageresistance for BioRoot™ RCS with cold technique than for PCS with warm technique.

Long term sealing properties is similar in both groups with close mean percolation lengthvalues (0.80 mm for BioRoot™ RCS versus 1.15 mm for PCS).

Conclusion: This apical sealing study allowed to evaluate the sealing properties of thenew tricalcium silicate based cement (BioRoot™ RCS) used according to the cold singlecone technique compared to the standard root canal filling technique (zinc oxide-eugenolwith warm gutta percha).

Early sealing properties of BioRoot™ RCS with cold technique is better than inZinc/eugenol warm technique.

Long term sealing results with BioRoot™ RCS in single cone technique is at leastequivalent to or better than the standard warm technique with zinc/eugenol sealer.

Vertical-fracture Resistance of Roots Obturated with BioRoot™ RCS:(Eldeniz et al. 2014, abstract presented at the 18th World Congress onDental Traumatology June 14 Istanbul)

The aim of this study was to compare the fracture resistance of roots filled with thematched-taper single-cone or lateral condensation technique and different contemporarycalcium-silicate based root canal sealers.

Eighty-two single-rooted extracted mandibular premolars were decoronated to obtain13-mm root segments and randomly divided into 6 test-groups (n=12) and 2 control-groups (n=5). The buccolingual and mesiodistal diameters were measured. No statisticallysignificant differences were found between the groups in terms of diameters. In positive-control Group, roots were instrumented but not filled and in negative-control group roots

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4,00le

ngth

(mm

)

PCS

7 days 30 days

BioRoot™ RCS PCS BioRoot™ RCS

3,50

3,00

2,50

2,00

1,50

1,00

0,50

0,00G1 G4

30 days

G27 days

G3

Figure 3: Length of dye penetration from the apex

Length of silver nitrate percolation from the apex

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pH

Time24h00 48h00 7 days 30 days

AH PLUS JET12

11

10

9

8

7

6

5

Endosequence BC Sealer

Biodentine™

BioRoot™ RCS

Pulp canal Sealer

Figure 4: pH variationof different root sealersfor 30 days

were neither instrumented nor filled. The canals were instrumented using ProTaper System(Dentsply Maillefer, Ballaigues, Switzerland) up to a master apical size of F3.

All root samples were incubated at 37°C and 100% humidity for 2 weeks for completesetting of the sealers. The roots within each group were embedded in acrylic molds andsubjected to a vertical loading force (1mm/min) until fracture. The force required tofracture each sample was recorded and data were statistically (Kruskal-Wallis andMann-Whitney U) analyzed.

The highest fracture resistance was recorded for the negative-control group for which rootswere neither instrumented nor filled (771.5 ± 269.3N), BioRoot™ RCS + LatteralCondensation (624.9 ± 153.7N) and BioRoot™ RCS + Single-cone GP (621.3 ± 145.0N)with no significant difference between them (p>0.05)

The new calcium-silicate based sealer (BioRoot™ RCS) showed potential to strengthenendodontically-treated teeth to a level comparable to that of intact teeth.

Biological and Bioactive Properties

A Root Canal sealer with prolonged alkaline pH (Internal data)

As all calcium silicate cement-based materials, BioRoot™ RCS creates a favorablealkaline pH, which remains high for several days (figure 4). This alkaline pH was alreadyreported for mineral based sealer containing calcium silicate (Gandolfi et al., 2010).

Hydroxyapatite formation at the tooth-sealer interface andmineralization of dentinal structure :

In vitro bioactivity of tricalcium silicate has been reported with the formation of ahydroxyapatite layer in the simulated body fluid (Zhao et al., 2005). Precipitation ofhydroxyapatite like crystals in the presence of phosphate solutions in contact to

pH evolution

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Biodentine was reported in several studies (Dejou et al. 2005, Han et al., 2011, Camilleriet al., 2013). This precipitation of apatite crystals induces a high sealing ability at theinterface between Biodentine and human dentin. Some mineral re-crystallization occursalso at the entrance of the dentin tubules inducing micromechanical anchorage anddefinitive obturation. Similarly, calcium release from BioRoot™ RCS as well asprecipitation of calcium phosphate phase when in contact of physiological fluid wasevidenced (Xuereb et al., 2014).

In addition, the interaction of BioRoot™ RCS in contact with multipotent stem cellsshowed a positive Von Kossa coloration suggesting a formation of mineralization(Goldberg, 2014). This technique is for demonstrating deposits of calcium phosphate intissues section or cells. When investigational samples are treated with a silver nitratesolution and the silver is deposited by replacing the calcium reduced by the strong lightand thereby visualized as metallic silver. This test provides evidence for the formation ofcalcium phosphate, main component of hydroxyapatite.

As a conclusion, BioRoot™ RCS improves mineralization by an increase ofhydroxyapatite formation.

Hypersensitivity (NF EN ISO 10993-10) (internal data)

A maximization study for delayed type hypersensitivity was performed to assess thepotential of the test element to produce sensitization by skin contact in 15 guinea-pigs.

Material and Methods: 10 treated animals were exposed to BioRoot™ RCS, as a polarextract (0.9 % NaCl, 72h, 37°C, 0.2g/ml) by intradermal injection and as prepared (as athick paste containing powdered sealer) by epidermal application. Concurrently, the5 control animals received the vehicle only by intradermal (0.9 % NaCl, 72h, 37°C) andepidermal application (0.9 % NaCl). Following a rest period of 14 days (± 1 day) endingthe induction period, all the animals were exposed to the non-irritant challenge dose ofthe test element (moistened with 0.9 % NaCl). The extent, degree and duration of skinto the challenge exposure in the test animals, were compared with those demonstratedby control animals.

Results showed that none of BioRoot™ RCS treated animals and none reactive treatedanimals presented sensitization reactions.

BioRoot™ RCS was classified as weakly (indeed not significantly) sensitizing by skincontact (class I).

Oral mucosa irritation study (NF EN ISO 10993-10) (Internal data)

The aim of the study was to assess qualitatively and quantitatively the local toleranceand the potential to produce irritation of the tested product, applied as a thick paste tothe oral mucous membrane in the hamster for 2 consecutive days.

Material &methods : 3 animals were exposed to a crushing of BioRoot™ RCS to obtaina fine powder which was then moistened with about 0.1 ml of 0.9% NaCI. These animals(group 2) received in the right cheek pouch a 0.5g of the test mixture for 15 minutesevery hour for 4 hours (5 applications). The other cheek pouch receiving no treatment

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served as control. Animals receiving in the same conditions cotton-wool pelletimpregnated with 0.5 ml of control element (0.9% NaCI) were used as control animals(group 1). Then, the animals were regularly observed during the 48 hour-experiment andall the local or general effects were noted. At the end of the experiment, a macroscopicexamination of the oral mucosa and a histological examination of the cheek poucheswere performed in ail the animals. The results were expressed by a mean irritation score(M Sc). The oral mucous tolerance and the irritation potential of the test mixture wereassessed by determination of a mean grade based on the erythema scores and of theirritation index obtained from the results of histological examination performed at theend of the experiment on representative sections of the pouches in all the animals.

Results: No macroscopically visible anomaly was recorded on the oral mucous membranein both cheek pouches from the control group animals and from the treated groupanimals. The mean lesion scores obtained for the control group and for the treated groupis equal to 0.0. According to the criteria defined in the standard NF EN ISO 10993-10,the irritation ascribable to the test element was nil.The irritation potential ofBioRoot™ RCS, a mixture of solid and liquid part, applied as a thick paste every hourfor 4 hours was non-irritant for the oral mucous membrane of the hamster.

Implantation in rats (NF EN ISO 10993-6) (internal data)

The objective of the study was to evaluate in the rat the local effects and possiblesystemic effects of BioRoot™ RCS implanted in the subcutaneous tissues for 1 and3 months. The possible systemic effects were determined by weighing and performinghistology of main organs. This was added to the common protocol described inISO 10993-6.

Material and Methods: 12 male SPF Sprague Dawley rats are about 8 weeks old at thetime of implantation. BioRoot™ RCS was placed in two sites in the left flank whereastwo other sites in the right flank were dedicated to the blank control. The test elementwas implanted in subcutaneous tissues of all the animals at the rate of 2 samples ofabout 2 mm x 5 mm (weighing about 15 mg each) per animal. Concurrently, 2 shamimplantation sites were performed on the animals under the same conditions.

During the experimental period, the animals were observed regularly (clinical signs, bodyweights). At the end of each observation period (1 month or 3 months), all the appropriateanimals were sacrificed, main organs were removed and weighed and a detailedhistological examination of the implantation sites (local biological reaction) and of mainorgans (axillary lymph nodes, liver, thymus, adrenals, kidneys, spleen) was performed.

The results of this study shows that BioRoot™ RCS implanted in the subcutaneoustissues of the male Sprague Dawley rat for 1 month or 3 months induced no specificchanges indicative of a systemic toxic effect. Locally, BioRoot™ RCS induced aninflammatory cell reaction with predominance of macrophages and the formation of athin discontinuous band of fibrosis at its contact. According to the microscopicevaluation system, it was classified as moderately then slightly irritant to the tissue afterrespectively 1 month or 3 months implantation periods.

BioRoot™ RCS did not induce any other subcutaneous adverse reaction such asnecrosis, hemorrhage or congestion in contact or at distance from the test element.

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Cytotoxicity and mutagenicity evaluation (Eldeniz et al., Oral presentationat the IADR congress Cape Town, June 14)

The aim of this study was to test possible mutagenicity of various new resin-based andsilicate-based endodontic sealers in comparison with contemporary sealers byevaluating their potential to induce DNA double-strand breaks (DSBs).

Methods: Periodontal ligament cell (PDL) line using lentiviral gene transfer of humantelomerase reverse transcriptase (hTERT) was used and exposed to subtoxicconcentrations of 24h eluates from two epoxy resin–based endodontic sealer (AH PlusJet-Dentsply De Trey, Germany, and Acroseal-Septodont, France), four variousmethacrylate-based endodontic sealers (EndoREZ-Ultradent, USA, RealSeal andRealSeal SE- SybronEndo, CA and MetaSEAL-Parkell, USA) and three silicate-basedsealers (BioRoot™ RCS-Septodont, France, IRootSP-Variodent, Canada and MTAFillapex-Angelus, Brazil). XTT based cell viability assay was used for cytotoxicityscreening of substances. ɣH2AX assay was used for genotoxicity screening. In ɣH2AXimmunofluorescence assay, PDL-hTERT cells were exposed to eluates of the substancesfor 6 h and DNA DSBs were detected microscopically. Induced foci represent doubleDNA strand breaks (DSBs), which can induce ATM-dependent phosphorylation of thehistone H2AX.

Results: The cytotoxicity of the 24- hour eluates could be ranked in the following order:MetaSEAL>RealSEAL>RealSEAL SE>EndoREZ>AH Plus>Acroseal>MTA Fillapex> IRootSP>BioRoot™ RCS. In negative controls (cells which received medium only) 4.97 ± 0.97DSB-foci (mean ± SEM) while in positive controls 10.59 ± 4.18 DSB foci/cell were found.BioRoot™ RCS and RealSeal SE exhibit significant difference in foci formation at1/3EC50 compared to their 1/10EC50 concentration (p<0.05). The yield of the meannumber of induced foci/cell positively correlates with increasing compoundconcentration for BioRoot™ RCS, MTA Fillapex, MetaSEAL, RealSeal and RealSeal SE.

Conclusions: BioRoot™ RCS could be considered as a suitable material for endodonticprocedures as it is not cytotoxic and did not induce DSBs in PDL-hTERT cells.

Activity of a calcium silicate-based endodontic cement (BioRoot™ RCS):interactions with the human apical tissue in vitro (Camps et al, submittedpublication)

The objective of the study was to evaluate the interactions of BioRoot™ RCS with apicaltissues and to compare it with an endodontic standard root canal sealer (Pulp CanalSealer - PCS – Kerr) in human extracted teeth. The activity was determined by evaluationof viability of periodontal cells and by quantification of factors (protein and growth factors).

Material and Methods: Human periodontal fibroblasts were extracted from healthymolars. After sampling of periodontal ligament issue to two apical third of radicular area,explants were cultured in antibiotics supplemented MEM with SVF (10%). Aftersynchronization of cells, 80,000 cells were seeded in direct contact with BioRoot™ RCSand PCS with different conditions (prewashing during 5 days, prewashing during 7 daysor no washing) during 24 and 48hr. Then, cell proliferation and viability test wereperformed. In addition, an indirect contact between cells and sealer was performed withmedium issue of 30 incisors freshly extracted and obturated with BioRoot™ RCS andPCS according manufacturer’s instructions. After 24h, corresponding to time appropriate

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with setting time of PCS, apical area of each tooth was immerged in controlled culturemedium. Tested medium were collected and pooled by material. After centrifugation,supernatant was collected and mixed at maximum tolerated dose with periodontal cellsto test viability and activity of sealers. Cells viability was performed by MTT test.

The results of this study showed that BioRoot™ RCS displays a good biocompatibilityin comparison with PCS since direct contact induced a proliferation and attachment ofperiodontal cells near BioRoot™ RCS.

In vitro evaluation of BioRoot™ RCS, a new canal sealer. (Goldberg etal. 2014 – publication submitted to Biomaterials)

Activity of BioRoot™ RCS was studied using A4 which is a multipotent cell line knownto express markers correlated to mineralized tissue formation. This cell line was used inorder to quantify the osteogenic and dentinogenic proteins and to compare it with PulpCanal Sealer (PCS - Kerr).

Material and methods: A4 cells were cultured in presence of ß-GlyceroPhosphate/Ascorbic Acid/Dexamethasone. The impact of the sealers on the progenitorswas studied after 3, 7 and 14 days of culture, with the culture medium alone (Control),or in the two other groups (BioRoot™ RCS or PCS). The data of mRNA expression andquantity of protein were performed by qPCR and Western Blots compared between thegroups. Confluent cultures form nodules stained with Alizarin Red and Von Kossa, whichare specific staining methods allowing calcium and phosphate detection.

The results of this study suggest that BioRoot™ RCS is biocompatible with A4 cell lineand may induce the release of some extracellular matrix components implicated in bone-like healing or re-formation.

On-going Clinical Study : Open, Randomized Trial Evaluating the Efficacy and theClinical Tolerance of PA0903 (BioRoot™ RCS) as Root Canal Sealer (NCT01728532,www.clinicaltrials.gov)

Methodology:

This study is performed only in adults who required a non-surgical root canal obturation.The studied indication is the root canal sealing.

This randomized open-label study comprises two groups corresponding to tested rootcanal sealer (a reference product and investigational product).

The investigational product which is a dental cement based on BioSilicate Technology(BioRoot™ RCS) is applied with lateral compaction or single cone technique. On thecontrary, the reference product which is a zinc oxide-eugenol cement used in dentalpractice (Pulp Canal Sealer, Kerr) is be applied with a single wave technique.

Study duration: 2 years after the end of inclusion period

Objectives: The trial objective is to illustrate the clinical and radiographic outcome ofBioRoot™ RCS in the described indication.

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The primary objective is to evaluate the clinical and radiographic outcomes of root canaltreatment at two years when using BioRoot™ RCS as an endodontic sealer with gutta-percha. The success rate is defined as no pain (score of 0 at the VAS scale) and no LEO(fulfilment of Strindberg's criteria) Treatment is considered successful when the contours,width, and structure of the periodontal margins are normal. All cases in which thosecriteria are not fulfilled are judged as unsuccessful. A follow-up of two years is describedas the predictive time of success.

The secondary objectives are to evaluate the following criteria and in an indicative way,to compare them between patients receiving the BioRoot™ RCS and those receivingthe reference product:

• The success rate at each time point and also the components of this compositevariable (i.e. pain and fulfillment of Strindberg's criteria ).

• The handling, consistency and physical characteristics of BioRoot™ RCS.

• The longevity and safety of the product associated with BioRoot™ RCS after twoyears.

Trial population and number of patients:

about 60 patients will be included in the study with a ratio 2 (BioRoot™ RCS): 1(reference). Adults will be included without gender distinction.

Duration of the treatment:

The product is applied on D0 (inclusion) of the study. The follow-up period includes2 years with 4 visits: Visit 1(Week 2), Visit 2 (Month 6), Visit 3 (Year 1) and Visit 4 (Year 2).

This clinical trial is on-going. Inclusions are done and follow –up is on-going.

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Bibliographic references

About I. et al. 2014 Bioactivity of a calcium silicate-based endodontic cement(BioRoot™ RCS): interactions with the human apical tissue in vitro– publicationsubmitted

Camilleri J et al., Investigation of the hydration and bioactivity of radiopacifiedtricalcium silicate cement, Biodentine™ and MTA Angelus. Dent. Mater. 2014 ; 29 (5)580-93

Camilleri J., Sealers and warm gutta-percha obturation techniques, JOE 2015 ; 41 (1)72-78

Dejou J, 2005 Physical, chemical, mechanical behaviour of a new material for directposterior fillings. Communication Centre Francais des Biomatériaux Dentaires (CFBD)

Eldeniz A, et al. DNA Double-strand Breaks Caused by New and ContemporaryEndodontic Sealers 2014; Oral presentation at the IADR congress Cape Town, June14 (https://iadr.confex.com/iadr/14iags/webprogram/Paper187460.html)

Eldeniz et al., Vertical-fracture Resistance of Roots Obturated with Novel Calcium-silicate-based Sealers. 2014, presentation at the 18th World Congress on DentalTraumatology June 14 Istanbul

Gandolfi MG et al. Kinetics of apatite formation on a calcium-silicate cement for root-end filling during ageing in physiological-like phosphate solutions. Clin. Oral.,Investig. 2010; 14 (6) 659-668

Goldberg et al., In vitro evaluation of the bioactivity of BioRoot™ RCS, a new canalsealer. 2014 Publication submitted to Biomaterials.

Han L, Okiji T Bioactivity evaluation of three calcium silicate based endodonticmaterials. Int. Endod. J. 2013; 46 (9): 808-14.

Xuereb et al., 2014 In Situ Assessment of the Setting of Tricalcium Silicate–basedSealers Using a Dentin Pressure Model, J Endod 2014. Article in press.

Zhao W et al. The self-setting properties and in-vitro bioactivity of tricalcium silicate,Biomaterials 2005; 26 (31): 6113-6121

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