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JournalOfMinimum Intervention In Dentistry

J Minim Interv Dent 2009; 2 (4) 208

Abstract

This in vitro study was designedto determine whether bioactiveglass S53P4 could be used toinduce mineralization in livingconnective tissue, in decalcifieddentin matrix and in dentin withopened dentinal tubules. Mucosalexplants were placed on theirsides over the glass plates,cultured, sectioned and stained.Decalcified dentin matrixsections were prepared, kept incontact with the glass plates andstained. Six extracted thirdmolars were prepared into cup-shaped specimens, filled witheither SBF or saline, immersed ineither a suspension of the glasspowder or saline and kept in ahumidified chamber. The dentinpermeability was examined bychanging the SBF to Nigrosin.The results of the threeexperiments were evaluatedvisually, under light microscopyand SEM. They revealed thatglass S53P4 induced tissuemineralization in mucosalexplants and in dentin both atthe glass-tissue interface and atsites away from it. Our proposedpotential uses of the material areas: a mineralizing agent in cariesprophylactics, a desensitisingagent for teeth with openeddentinal tubules, a coating formetal implants, and a root canalsealer. Further studies of glassS53P4 are needed in order tovalidate its above-mentionedpotential uses. First published inDental Update 1999;6(4): 11-15.

Authors:

1 Faculty of Dentistry, Kuwait University,Kuwait.2 Institute of Dentistry, University of Turku,Finland.3 Bnied Al Gar Dental Center, Ministry ofHealth, Kuwait.

Correspondence address:Prof. Jukka I Salonen DDS, PhDAddress: Faculty of Dentistry, KuwaitUniversity.P.O. Box 24923 Safat 13110, KuwaitFax: +965 532 6049E-mail: jusalon@hsc.kuniv.edu.kw

Introduction

Bioactive glasses (BAGs), as opposedto most technical glasses, arecharacterized by the materials’reactivity in water and in aqueousliquids. The bioactivity of BAGs isderived from their reactions withtissue fluids, resulting in theformation of a hydroxycarbonateapatite (HCA) layer on the glass.When BAGs are brought into contactwith body fluids a rapid leach of Na+

and congruent dissolution of Ca2+,PO4

3- and Si4+ takes place at theglass surface. A polycondensatedsilica-rich (Si-gel) layer is formed onthe glass bulk, which then serves asa template for the formation of acalcium phosphate (Ca/P) layer at itsouter surface1. Eventually, the Ca/Pcrystallizes into HCA, the compo-sition of which corresponds to that ofbone. Because of this phenomenonand their good biocompatibility BAGswere intro-duced in dentistry: assubstitutes for reconstruction ofvoids and defects of facial bones2-4,in rehabilitation of the dentoalveolar

Bioactive glass in dentistry

Salonen JI1, Arjasmaa M2, Tuominen U2,Behbehani MJ1 , Zaatar EI3.

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complex, including BAG implants5

and regeneration of periodontalbone-support6-8. Recently, accumu-lating amount of evidence hasemerged suggesting that certaincompositions of BAGs create anosteoconductive response; aid in thedifferentiation of osteo-progenitorcells to osteoblasts and enhancebone proliferation9. The essentialchemical property of BAGs to releaseSi+, Ca2+ and PO4

3- in the tissue fluid,resulting in the initiation of apatiteformation on the glass surface hasled us to believe that it might also bequite possible to use the materials asvehicles for ectopic mineralization ofthe surrounding tissue. In this casethe BAGs may have therapeuticvalue as mineralizing agents in cariesprophylactics, and also as a desens-itizing agent in clinical situationswhere opened dentinal tubules leadto hypersensitive teeth10. Further-more, in implantology, a coating oftechnically adequate BAG on thefixture surface may serve as ameans to attach mucosal or dermalsoft tissues to the osseointegratedconstruction by a HCA bridge9,11. Inaddition, BAGs may also have anapplication in root canal therapyproviding a biological seal in the formof mineral deposition inducingmaterials in the root canal and at theapex. This in vitro study wasdesigned to find out whetherbioactive glass S53P4 can be used toinduce mineralization in livingconnective tissue, in decalcifieddentin matrix and in natural dentinwith opened dentinal tubules.

Materials and methods

S53P4 Glass production

The composition (by weight) of theglass S53P4 (Abmin TechnologiesLtd, Turku, Finland) is SiO2 (53.0%),CaO (20.0%), Na2O (23.0%) andP2O5 (4.0%). It is produced fromreagent grade Na2CO3, Ca HPO4 +2H2O, CaCO3 and Belgium sand. Theglass is prepared by melting the

ingredients at 1360°C for 3 h. It isthen casted into a mold and allowedto cool down in an annealing ovenfrom 520°C to 220°C at 1°C /minand then air-cooled to roomtemperature. For the experimentsthe material was cut with a diamondsaw into thin plates of 3x4x1 mm orused as dry ground powder with aparticle size <45 µm (average 20µm).

Figure 1. Schematic illustration of a mucosalexplant growing on glass substratum (S). Onbioactive glass S53P4 the epithelium (E)grows predominantly outwards from theexplant, which allows the examination of theglass-connective tissue (CT) interface underSEM and in histological sections.

Tissue culture study

Human masticatory mucosa wasobtained, with informed consent,from 2 young subjects (age 14-16years) undergoing a conventionalsurgical exposure of impactedcanines for orthodontic reasons. Asdescribed in an earlier report12 thetissue was cut, perpendicular to theepithelium, into elongated fragmentsof about l x l x 2 mm. The twelveexplants prepared were then placedon their sides on the S53P4 glassplates (Figure 1) and cultured for 5days at 37° C in 5% CO2 in Eagle’sminimum essential medium (EMEM,Gibco). The culture was supple-mented with Earle’s salt solutioncontaining 2mM L-glutamine, 10%sodium bicarbonate, antibiotics (100IU/ml penicillin G, 100 µg/mlstreptomycin sulphate) and l0% heatinactivated fetal calf serum (FCS,Gibco). For light microscopy, thecultured explants were fixed in 4%neutral formalin, dehydrated andembedded in plastic. The cutting-grinding technique described by

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DONATH and BREUNER13 was used toprepare the specimens for histo-logical examination. Serial sectionswere cut and stained with eithertoluidine blue or von Kossa for thepresence of the hydroxylapatite. Thecontact areas between theconnective tissue (CT) of theexplants and the glass plates wereexamined by making use of scanningelectron microscopy (SEM). Afterculture, the explants were partiallypeeled from the glass, in order toexpose the CT-glass interface andimmediately fixed in bufferedglutaraldehyde. The specimens werethen washed, dehydrated, coatedwith car-bon and examined underSEM (Stereoscan 360, LEO,Cambridge, U.K.).

Dentin matrix study

Dentin matrix was prepared fromfreshly extracted third molars. Theteeth were cut with a low speeddiamond saw into 200 µm slices,which were then decalcified in 0.5MHCl at 25°C for 72 h as described byURIST et al.14. A total of seven slicesof decalcified matrix were kept incontact with the glass plates for 2 to5 days under the same conditions asdescribed for tissue cultures. Thehistological slides were prepared asfor the mucosal specimen13, stainedwith von Kossa for the presence ofthe hydroxylapatite and examinedunder light microscopy.

Dentin permeability study

Dentin permeability was studiedusing both upper third molarsextracted from three subjects.Immediately after extraction, the sixteeth were washed in isotonic saline.The occlusal surface of each toothwas flattened through a slighthorizontal cut, using a low speeddiamond saw, in order to provide a“seat” for the teeth to stand in avertical position during theexperiment. Almost ½ of the rootswere cut away using the diamondsaw and the pulp chamber and the

root canal system were opened usinga round bur of 3.2 mm diameter andsaline irrigation. The cervical part ofeach tooth was root planed using aperiodontal curette and treated with38% phosphoric acid (Scotchbondgel, 7523) in order to remove thesmear layer and to expose thedentinal tubules. The equallyprepared cup-shaped specimens(3+3) were then filled up withsimulated body fluid (SBF) andcarefully immersed up to 3/4 of theirheights, in either a suspension ofS53P4 powder in saline (experiment)or in saline alone (control). After aperiod of 5 days in a humidifiedchamber, the tooth surface waswashed in saline in order to dislodgeall loose materials. The permeabilityof the cervical dentin was examinedby changing the SBF in the cup toNigrosin (Fluka) 0,5% + SDS 1% +H2O for 10 min. The penetration ofthe stain through the dentin wasobserved visually and photographed.After fixation and dehydration, theteeth were coated with carbon andexamined under SEM (Stereoscan360, LEO, Cambridge, U.K.).

Results

Tissue culture study

Microscopic examination of thecultured explants revealed normalappearing connective tissue (CT)with cells in between bundles ofcollagen fibers. Epithelial cells fromthe basal layer of the stationaryepithelium had migrated mainlyoutwards from the explant allowing adirect contact between the CT andthe glass. A silica-rich reaction layerwas identified on the glass tissueinterface, which exhibited a stronggranular staining with both toluidineblue and von Kossa. The density ofthe stain was very high, up to 30-40µm into the CT. Individual granuleswere also detected at remote areasup to 120 µm away from the glasssurface (Figure 2A and B). Peeling ofthe explants indicated that the tissue

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was attached to the glass throughfibers, which were identified in theSEM as collagen fibers.

They were partially covered by andembedded in spherical mineralaggregates (Figure 2C).

Figure 2. Light microscopic examination of the explantglass S53P4 interface demonstrates areaction layer (R) on the glass bulk (G). Insert A: abundant staining with toluidine blue (arrowhead) indicating the presence of ectopic material in the connective tissue (CT) adjacent to thereaction layer. 80x, Bar: 50µm. Panel B: A similar distribution of staining with von Kossa (arrowheads) in the soft CT. 160x, Bar: 50µm. Panel C: SEM image of fibers attached to the glass.Mineral formation is seen on individual collagen fibers (arrow) and on the glass surface to theright. 5000x, Bar: 0.5µm.

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Dentin matrix study

In the decalcified dentin matrixsections, a strong staining with vonKossa was observed only in areas,which had been in close contact withthe glass plates (Figure 3A). Nostaining was found at the oppositeside (serving as an internal control)of the 200 µm thick matrix.Furthermore, the staining fadedlinearly outwards, away from theglass-matrix contact area.

The maximal depth of the staining inthe matrix, in the five daysspecimens, was about 150 µm. Ahigher magnification of the glassmatrix interface revealed that thestaining was granular and mostlylocated at the lateral walls of thetubules. Fully occluded tubules weregenerally found only at areas close tothe glass surface. In some areas of 2days specimens, matrix staining andoccluded tubules became evidentonly at a short distance from theglass surface (Figure 3B).

Fig. 3. Panel A: Von Kossa staining (arrowheads) of 200µm thick decalcified dentin matrix (D)after incubation with bioactive glass S53P4 (BAG). 50x. Panel B: A higher magnification of theglass (G)-dentin matrix (D) contact. Von Kossa staining is seen around the tubules in the collagenframework of the matrix as well as inside the dentinal tubules close to the reaction layer (R).Unstained areas were occasionally found along the glass-dentin matrix interface. 250 x, Bar:50µm.

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Dentin permeability study

Consistently, less Nigrosin stain wasobserved leaking out through thecervical dentin of the teeth that weretreated with the aqueous suspensionof the glass S53P4 (Figure 4A).Examination of the root surfaceunder SEM revealed widely openedorifices of dentinal tubules in thecontrol specimens (Figure 4B).

No noticeable difference wasobserved in the width or density ofthe tubules at the cemento-enameljunction. The experimentalspecimens exhibited granular toalmost smooth precipitates on theroot surface, which apparentlyoccluded the dentinal tubules (Figure4C).

Figure 4. Panel A: Penetration of Nigrosin stain through the cervical dentin (open arrow head) ofroot planed third molars. The molar to the right is treated with the BAG S53P4 in vitro; the one tothe left is control. Panel B: SEM image of the control shows open dentinal tubules (T). Panel C: SEMimage of the S53P4 treated tooth demonstrates a precipitate (P), which covers the tubules. 1000x,Bar: 5µm

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Discussion

Mucosal explants grown in contactwith the S53P4 glass showed calciumphosphate (Ca/P) precipitation notonly at the glass-tissue interface butalso within the living tissue furtheraway from the glass surface. It wasevident that ions released from theglass had precipitated on the Mucosalexplants grown in contact with theS53P4 glass showed calciumphosphate (Ca/P) precipitation notonly at the glass-tissue interface butalso within the living tissue furtheraway from the glass surface. It wasevident that ions released from theglass had precipitated on the silica-rich glass surface as previouslyshown15,16, and that a part of theseions had diffused away from theglass inducing mineralization atectopic locations. These precipitateswere found as far as 120 µm fromthe glass surface. Since softconnective tissues (CT) do notnormally have the capacity to acceptmineral precipitation17, a direct rolefor the glass has to be considered forthe mineral formation both at theglass surface and within the CT.Attempts to mechanically remove theexplants from the S53P4 substratumshowed that the tissue had becomeattached to the glass. Examination ofthis attachment under SEM demons-trated that individual collagen fiberswere embedded in Ca/P aggregates,which were formed on the silica-richlayer on the glass surface. Similarspherical aggregates were also seenon and around individual collagenfibers. The shape and distribution ofthese precipitates under SEMexhibited a clear correlation to thetypical granular staining for thehydroxylapatite (von Kossa) seen inthe histological sections. Themechanism of the attachment of CTby its collagen fibers to the glasssurface appeared to be comparableto that of the attachment of theSharpey’s fibers to the rootcementum18. However, it has to beshown whether implants coated withbioactive glass can provide a similar

attachment in vivo. Such anattachment could make, at least atemporary19 structural seal securingthe implant-gingival junction againstbacterial invasion11. Results from theexperiments with the dentin matrixshowed that Ca/P formation wasdependent on the close contactbetween the sections and the glasssurface. The histological distributionof the stained granules demonstratedthat the nucleation started at thelateral surfaces of the tubules as wellas within the collagen framework ofthe matrix. The general appearanceof the stained granules agreed withthose seen in the mucosal explants.This agreement suggests role of thecollagen fibers in the initiation of themineralization process. It appearsthat the collagen fibers provide asurface for mineral deposition fromthe supersaturated Ca, P anddissolved silica-containing solutioncaused by the presence of thebioactive glass. The biologicalmineralization of hard tissues is,however, always associated with thepresence of polyanionic, calciumbinding non-collagenous proteins(NCPS) such as phosphophoryns andspecific proteoglycans17. Since themineral formation, described here,was similarly evident both in themucosal collagen matrix and in thedentin matrix, the role of specific“natural” nucleators in the describedmineralization process appears to beinsignificant. In contrast, themechanism of the mineral formation,reported here, agrees with theprinciples of the biomimetic processdescribed by ABE et al.20 and HATAet al.21. The biomimetic mineral-ization is based on the dissolution ofcalcium and silica from the bioactiveglass. Calcium increases the ionicactivity product of the apatite in thesurrounding body fluid. When thedissolved silica condenses andbecomes attached to a suitablesurface, it provides favorable sitesfor apatite nucleation. Once theapatite nuclei are formed, they growspontaneously (homogeneous nuc-leation) by consuming calcium and

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phosphate ions from the surroundingfluid21. Poly-condensated silica is alsosuggested to act as a heterogeneousnucleator, which stabilizes formingCa/P nuclei in bacterial plaque22.Under suitable conditions, deter-mined by pH and ionic strength,dissolved monosilicic acid poly-merises and forms concentrated fociof silica (at about pH 7.2) withdifferent particle sizes, porosity anddegree of particle aggregation22. Theconspicuously granular appearanceof the Ca/P precipitates, seen in thehistological sections both in bothmucosal CT and in dentin matrix,agrees with the idea of a role forpolycondensated silica in theirformation mecha-nism. The fact thatthere were areas of the dentinmatrix, close to the glass, which didnot exhibit the typical granularstaining with von Kossa, may be dueto regional variations of the pH levelalong the substratum. The Na+ ionsleached from the bioactive glassesare known to raise the pH level(pH>9.5) at the vicinity of the glass(23). Therefore, it is quite possiblethat the pH level in certain areasalong the glass surface was too highto allow the condensation of thedissolved monosilicic acid into silicaon the collagen fibers. As shown bythe experiments making use ofextracted teeth, the treatment withbioactive glass S53P4 significantlydelayed the penetration of Nigrosinstain through dentin. According tothe SEM examination, the reductionof permeability appeared to be dueto precipitates, which occluded thedentinal tubules. These primaryprecipitates appeared to be rathersuperficial. However, if given moretime, they may have spontaneouslyproceeded deeper into the tissue, bythe mechanism of homogeneousnucleation for hydroxylapatite asproposed by HATA et al.21. Theobserved regional differences in theleakage of the Nigrosin stain,through the cervical dentin, may bedue to variations in its thickness. Itmay also be due to other factors,such as remnants of pulpal soft

tissue or dentin smear, which mayhave partially occluded the tubules atthe inside of the mechanicallyprepared experimental cups.According to the results of this study,the bioactive glass S53P4 was ableto induce tissue mineralization inmucosal explants as well as indentin. Previous reports have shownthat the material does not haveharmful effects on human tissuesand that it can, in fact, enhancehuman bone formation24,25. It hasalso been shown that the materialhas a significant anti-bacterialeffect26. Based on these results, thematerial has potential to bedeveloped to dental health careproducts. This can be in the form ofa mineralizing agent in cariesprophylactics or a desensitizingagent in the treatment of hyper-sensitive teeth caused by openeddentinal tubules. In root canaltherapy, a potential application ofthe glass S53P4 as a root canalsealing material can also beconsidered. If a biological HCA sealin the canal and at the root apex canbe achieved, the glass is proved tomeet the requirements of suchmaterials. Further studies of the BAGS53P4 are needed in order tovalidate its above-mentionedpotential uses.

S53P4

6SBF

SBF

S53P4-

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S53P4

Dental Update1999;6(4): 11-15.

Resumen

Este estudio in vitro tuvo elpropósito de determinar laposibilidad de utilizar el vidriobioactivo S53P4 para inducirmineralización en tejido vivoconectivo en la matriz de dentinadescalcificada y en dentina contúbulos dentinarios abiertos. Secolocaron sobre las placas devidrio explantes mucosales decostado, cultivados, seccionadosy teñidos. Se prepararonsecciones de matriz de dentinadescalcificada, las que semantuvieron en contacto con lasplacas de vidrio y se tiñeron. Seprepararon seis terceros molaresextraídos, en especimenescupuliformes; se llenaron confluido corporal simulado (SBF) ocon solución salina, sesumergieron ya sea en unasuspensión del polvo vítreo osalina, y se guardaron en unacámara húmeda. Se examinó lapermeabilidad de la dentinacambiando el SBF a Nigrosin.Los resultados de los tresexperimentos fueron evaluadosvisualmente, bajo microscopio deluz y SEM. Estos revelaron que elvidrio S53P4 indujo la mineral-ización de tejido en explantesmucosales y dentina, tanto en lainterfase vidrio-tejido como enlos puntos alejados de estainterfase. Los usos potencialesdel material que proponemos soncomo: agente mineralizador enprofilaxia de caries; agentedesensibilizador para dientes contúbulos dentinarios abiertos;reves-timiento para implantes de

metal; y como sellante de canalradicular. Se requiere deestudios adicionales del vidrioS53P4 para validar sus usospotenciales anteriormente men-cionados. Publicado primero enDental Update 1999; 6(4): 11-15.

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