Osteoconductivity of modified fluorcanasite glass-ceramics for bone tissue augmentation and repair

Osteoconductivity of modified fluorcanasite glass–ceramics for bonetissue augmentation and repair

S. Bandyopadhyay-Ghosh,1,2 P. E. P. Faria,3 A. Johnson,2 D. N. B. Felipucci,4 I. M. Reaney,1

L. A. Salata,5 I. M. Brook,2 P. V. Hatton2

1Department of Engineering Materials, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield S1 3JD,

United Kingdom2Centre for Biomaterials and Tissue Engineering, School of Clinical Dentistry, Claremont Crescent, University of Sheffield,

Claremount Crescent, Sheffield S10 2TA, United Kingdom3Department of Oral and Maxillofacial Surgery, Faculty of Dentistry of Aracatuba, University of the State of Sao Paulo, Sao

Paulo, Brazil4Department of Prosthodontics, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil5Department of Oral and Maxillofacial Surgery, Faculty of Dentistry of Ribeirao Preto, University of Sao Paulo, Sao Paulo,

Brazil

Received 20 February 2009; revised 14 August 2009; accepted 18 August 2009

Published online 24 March 2010 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32750

Abstract: Modified fluorcanasite glasses were fabricated by ei-

ther altering the molar ratios of Na2O and CaO or by adding P2O5

to the parent stoichiometric glass compositions. Glasses were

converted to glass–ceramics by a controlled two-stage heat treat-

ment process. Rods (2 mm � 4 mm) were produced using the

conventional lost-wax casting technique. Osteoconductive 45S5

bioglass was used as a reference material. Biocompatibility and

osteoconductivity were investigated by implantation into healing

defects (2 mm) in the midshaft of rabbit femora. Tissue response

was investigated using conventional histology and scanning elec-

tron microscopy. Histological and histomorphometric evaluation

of specimens after 12 weeks implantation showed significantly

more bone contact with the surface of 45S5 bioglass implants

when compared with other test materials. When the bone contact

for each material was compared between experimental time

points, the Glass–Ceramic 2 (CaO rich) group showed significant

difference (p ¼ 0.027) at 4 weeks, but no direct contact at 12

weeks. Histology and backscattered electron photomicrographs

showed that modified fluorcanasite glass–ceramic implants had

greater osteoconductivity than the parent stoichiometric compo-

sition. Of the newmaterials, fluorcanasite glass–ceramic implants

modified by the addition of P2O5 showed the greatest stimulation

of new mineralized bone tissue formation adjacent to the

implants after 4 and 12 weeks implantation. VC 2010 Wiley Periodi-

cals, Inc. J Biomed Mater Res Part A: 94A: 760–768, 2010

Key Words: fluorcanasite, glass–ceramic, 45S5 bioglass,

osteoconductivity, in vivo biocompatibility, bone tissue

INTRODUCTION

Glasses and ceramics have been widely used as biomaterialsfor bone tissue repair and augmentation. Bioactive glasses(e.g., 45S5 bioglass) have been used in bone augmentationwith some success, although they are used mainly in partic-ulate form as ‘‘fillers’’ for small defects.1,2 The 45S5 bioglasshas the capacity to form a direct interface with both boneand soft tissues,3 it has the highest bioactivity index,4 and itis a mechanically weak material with relatively low fracturetoughness. Calcium phosphate bioceramics, for example, hy-droxyapatite (HA), remain the most popular osteoconductivebone substitute materials that are used as particulate bonefillers or as coatings on bone-anchored prostheses.5

Although both calcium phosphates and the bioactive glassesare widely used as particulate bone graft substitutes, inboth cases their poor mechanical properties make themunsuitable for the fabrication of load-bearing implants. Fur-

ther problems with granular bone substitutes include migra-tion, exfoliation of individual particles, infection, and incon-sistent host tissue response.5 To summarize, all of thecurrently available bone graft substitutes and implant bio-materials suffer from limitations, and therefore, there is aclinical need to develop new materials for use in orthopae-dics, maxillofacial surgery, and dental implantology.

Glass–ceramics represent a promising alternative to thebioactive glasses and calcium phosphate ceramics. Glass–ceramics are polycrystalline solids obtained by the con-trolled crystallization of glasses. Crystallization is accom-plished by subjecting suitable glass compositions to a care-fully controlled heat treatment that results in the nucleationand growth of crystal phases within the glass, resulting insignificantly improved mechanical properties. Specific glass–ceramic compositions have been developed for use both asosteoconductive biomaterials and dental prostheses. The

Correspondence to: S. Bandyopadhyay-Ghosh, Centre for Biocomposites and Biomaterials Processing, University of Toronto, 33 Willcocks

Street, Toronto, Ontario, Canada M5S 3B3; e-mail: [email protected]

Contract grant sponsor: FAPESP, The State of Sao Paulo Research Foundation; contract grant number: 05/58132-3

Contract grant sponsor: Overseas Research Studentship (ORS) Committee

760 VC 2010 WILEY PERIODICALS, INC.

parent glasses may be cast to shape and, using appropriatecompositions and heat treatments, may achieve bendstrength and fracture toughness in excess of 200 MPa and 3MPa m½, respectively.6 One of the glass–ceramics that hasbeen commercially used in load-bearing orthopaedic appli-cations is apatite-wollastonite (A-W) glass–ceramic.7

Although it has excellent mechanical properties and isosteoconductive, it is relatively difficult to process to formcomplex or custom shapes due to the surface nucleation ofwollastonite. In contrast, high-strength apatite-mullite glass–ceramics are castable and volume nucleate.8 Unfortunately,their high Al2O3 content may have the potential to suppressbioactivity,9,10 and they have not yet been commerciallyexploited. Ideally, a glass–ceramic for biomedical applica-tions should be Al2O3 free, biocompatible, osteoconductive,have appropriate mechanical properties, and could be eco-nomically produced in a variety of custom or complexshapes. One promising glass–ceramic for bone substitutionthat might meet these requirements is fluorcanasite.

Fluorcanasite (Ca5Na4K2Si12O30F4) is a quadruple chainsilicate in which SiO2 rings are crosslinked into groups of fourand are separated by a network composed of Na(O,F)6 andCa(O,F)6 octahedra.11–13 The glass–ceramic has a highly crys-talline microstructure consisting of interpenetrating laths thatresult in a relatively high flexural strength (>300 MPa) andfracture toughness (>5 MPa m½).6,14–16 It also bulk nucleatesand may therefore be suitable for casting into complexshapes. Early published compositions were unfortunately bioi-nert or even associated with inflammation following implanta-tion into bone tissue.17 Since then, modified fluorcanasiteglass–ceramics have been developed18 with the intention ofintroducing bioactivity by changing the molar ratios of CaOand Na2O or by incorporating P2O5. Miller et al.18 predictedtheir osteoconductive potential using a cell-free in vitro apa-tite deposition model. This group reported apatite layer for-mation in simulated body fluid (SBF) within 5 days whencompared with 45S5 bioglass and A-W glass–ceramic. Wehave also reported castabability and good in vitro biocompati-bility of these modified compositions in comparison withother materials.19–22 Consequently, modified fluorcanasiteswere considered to have great potential for use as load-bear-ing, osteoconductive biomaterials in orthopaedics, implantol-ogy, and reconstructive facial surgery. However, there hasbeen no work reported to date on the in vivo biocompatibilityof these promising materials to conclusively demonstrate theirosteoconductive potential. Therefore, the aim of this researchwas to study the in vivo biocompatibility of modified fluorca-nasite glass–ceramics and to investigate the mechanism re-sponsible for osteoconductivity in these systems. The resultspresented here will determine whether fluorcanasite glass–ceramics have the potential for use as biomaterials in therepair and augmentation of bone tissue.

MATERIALS AND METHODS

Sample preparationIn this study, Glass–Ceramic 1 (Stoichiometric), Glass–Ce-ramic 2 (CaO rich), and Glass–Ceramic 3 (�2% P2O5) wereused as implant materials. First, modified fluorcanasite

glasses were produced from the stoichiometric fluorcanasiteglass composition to induce bioactivity.18 Table I shows themolar composition of glasses used in this study. Commercialbioactive 45S5 bioglass was also used as a control material.Glass batches were prepared in house by mixing appropri-ate amount of silica (Loch Aline Sand 99.5%), CaHPO4,Na2CO3, K2CO3 (Standard Laboratory Grade Chemicals,Fisher Scientific, UK), and CaF2 (Aldrich Chemical Company,USA). These glasses were melted in uncovered platinum–rhodium (2%) crucibles at 1450�C for 3 h in an electric fur-nace with a final 2 h agitation with a platinum stirrer (60rpm) to increase homogeneity. For 45S5 bioglass, a meltingtemperature of 1300�C was used and melting time was 4 h.Glasses were then fritted by casting into water.

The in vivo biocompatibility study used a femoral heal-ing defect model in the rabbit (2 mm � 4 mm). Rods forimplantation were cast using the conventional lost-wax cast-ing technique described in previous studies.21,22 Gypsum-bonded investment (Whip-Mix Cristobalite, Whip-Mix Corp.,Louisville, KY) was used as a mold material. Glasses wereconverted to glass–ceramics by a controlled two-stage heattreatment process in the hot zone of a calibrated Lentontube furnace. First, the glasses were heated to 520�C (nucle-ation temperature) at 5�C min�1 and held for 2 h, followedby a ramp at 3�C min�1 to 780�C (crystal growth tempera-ture). Samples were held at this temperature for 2 h andthen cooled at 5�C min�1. Glass and glass–ceramic rodswere then cleaned with water, acetone, and finally with dis-tilled water. Samples were sterilized in an autoclave(Baumer Hi-Vac Cad) at 134�C for 35 min before surgery.

SurgeryAdult female New Zealand white rabbits (n ¼ 15) weighingbetween 3.5 and 4.0 kg were used in this study. Six standar-dized defects were made, three in the left and three in theright femur. Four rods, one of each Glass–Ceramic 1 (Stoichi-ometric), Glass–Ceramic 2 (CaO rich), Glass–Ceramic 3(�2% P2O5), 45S5 bioglass, and one Sham (withoutimplant) were placed randomly in left and right femur ofeach rabbit. The animals were housed with veterinary careand nutritional support in individual cages during the wholeexperimental period in accordance with national regulations.All surgical procedures were performed under aseptic con-ditions by the same surgeon. The animals were anesthetizedusing subcutaneous 1.0 mg kg�1 Acepromazine (RhobifarmaIndustria Farmaceutica, Sao Paulo, Brazil) followed by intra-muscular injection of 3.0 mg kg�1 Xilazine (Calier Laborato-ries, Barcelona, Spain) mixed with 60.0 mg kg�1 Ketamine(Uniao Quımica Farmaceutica Nacional S.A., Embuguaçu, SaoPaulo, Brazil). After giving anesthesia, the animals received

TABLE I. Compositions of Glasses in Mole Percent

Glasses SiO2 CaF2 Na2O K2O CaO P2O5

Glass 1 60.0 10.0 10.0 5.0 15.0 0Glass 2 61.6 10.3 3.8 5.1 19.2 0Glass 3 62.7 8.4 3.9 5.2 17.8 2.145S5 bioglass 46.2 0 24.3 0 26.8 2.6

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 1 SEP 2010 VOL 94A, ISSUE 3 761

0.2 mL kg�1 oxi-tetracycline (Tortuga Comp., Brazil) as pro-phylactic antibiotic therapy.

Linear incision was carried out after a section of the legwas shaved. Holes of �2.2 mm in diameter with a distanceof 1 cm between each other were then drilled through thecortical bone using a surgical burr (Conexao, Sao Paulo, Bra-zil) under controlled rotation and profuse irrigation withsterile saline. Samples were then inserted into the defectsites [Fig. 1(a,b)], and the wound was sutured in layers withVicryl 3–0V

R

; (Ethicon, Johnson & Johnson, Sao Jose dos Cam-pos, Brazil) for the periosteum, and nylon 4–0 (Ethicon,Johnson & Johnson, Sao Jose dos Campos, Brazil) for theskin, with interrupted stitches. After operation, all animalswere intramuscularly given a single dose of FenarenV

R

(sodium diclophenaco 2.0 mg kg�1; Uniao Quım. Farm.Nacional S.A., Sao Paulo, Brazil).

HarvestingPostoperatively, wounds were inspected daily for clinicalsigns of complications or adverse reactions and to monitorhealing. The rabbits were maintained in cages with freeaccess to food and water. Four weeks after surgery, five rab-bits were first anesthetized and then sacrificed by intraperi-toneal administration of an overdose of Thiopentax (5 mL;Cristalia, Brazil). The skin was exposed, and the bone withthe implants was cut using carburundum disc. Periapical ra-diographs (Dabi Atlante, Spectro 70�) were taken immedi-ately of all femora to locate the exact position of theimplants, and subsequently, the bone was cleaned and wasready for fixation. Similarly, five rabbits were further har-vested after 12 weeks.

Preparation for microscopyThe bones and implants were immediately fixed in 4%formaldehyde solution (Casa da Quimica, Brazil) for 10

days. The solutions were changed every 48 h. These speci-mens were then dehydrated through a series of gradedethanol soaks for 15 days and embedded in resin (LR WhiteHard Grade, London, UK) over a period of up to 15 dayswith a resin changing time of 48 h. Each specimen wasplaced in a teflon mold and filled with fresh resin, whichwas polymerized in an oven (Shel Laboratory, Sellex, Brazil)at 60�C for 24 h. The block was cut using a band saw fittedin a precision slicing machine (Microslice 2, Ultra Tec, UK)until the implant was exposed. The surface with the encap-sulated implant was polished using waterproof paper, sili-con carbide paper, and finally polishing cloth with a drop ofethylene glycol (Labsynth Product, Brazil). The polishedblocks were mounted on glass slides using glass bond (PoxiBonder, Loctite, Spain Henkel) and were cut using a dia-mond band saw (Saint Gobain abrasive, D54T00-NP) toleave a section of �1-mm thick. The cut surfaces of the sec-tion were then polished and thinned down to 20–30 lmusing waterproof paper, silicon carbide paper, and finallypolishing cloth (with a drop of ethylene glycol) without dis-ruption of the implant tissue interface. The polished blocksurfaces were then stained with toluidine blue-pyronine.Coverslips were used to protect the sections, and the slideswere examined under a standard light microscope to histo-logical analyses (Leica DMLB, Germany). For histometricevaluation, Leica QWin Plus software version 2.8 (Leica,Germany) was used to measure the percentage of bone/implant contact.

Unstained resin blocks that had been sectioned and pol-ished were cut using a diamond cutter and cleaned with ac-etone, placed on aluminum holders with silver paint, andthen carbon coated using a carbon evaporation unit. Theblocks were then analyzed under a scanning electron micro-scope (SEM; JEOL 6400) at an operating voltage of 20 kVand a working distance of 8 mm in backscattered mode.New bone formation around the implant was investigated inbackscattered mode mainly in three different regions. Thefirst region was the space between the edges of cortex andthe glass–ceramic/glass implant surface. The second regionwas the space between bone marrow and the implant sur-face. The third region was the edge of the glass–ceramic/glass implant in contact with bone marrow to observe anysurface dissolution.

Statistical analysisWhere applicable, the descriptive analysis of the raw datawas performed with statistical software SPSS (Chicago, IL).The pertinent comparisons between relevant variables inthe groups were calculated. The ANOVA test was used forcomparisons between treatment groups and with respect toexperimental time points. The differences were consideredsignificant if p � 0.05.

RESULTS

Observations on surgeryFour animals were sacrificed within 1 week of implantationbecause of surgical complications. No complications werenoted with the remaining animals. Sites of implantation

FIGURE 1. Six standardized defects (2 mm) were repaired using rods

of either Glass–Ceramic 1 (Stoichiometric), Glass–Ceramic 2 (CaO

rich), Glass–Ceramic 3 (�2% P2O5), or 45S5 bioglass. One sham was

left untreated (coagulum only). Materials were randomly distributed

in (a) left and (b) right femur of every rabbit. [Color figure can be

viewed in the online issue, which is available at www.interscience.

wiley.com.]

762 BANDYOPADHYAY-GHOSH ET AL. FLUORCANASITE GLASS–CERAMICS FOR BONE TISSUE AUGMENTATION AND REPAIR

appeared to have healed with no visible signs of infectionor adverse tissue reaction. Radiographs were taken to assistlocating the implants prior to gross reduction of specimens.The sham-operated specimens were not processed for eitherscanning electron or light microscopy. Some rabbits exhib-ited fracture in one leg during the 12-week follow-up pe-riod. This postoperative complication may have affected theoutcome of implant integration. The existence of a fractureon one side leads to the animals overloading the contralat-eral leg, resulting in bony atrophy in the injured leg and hy-pertrophy in the overloaded one. The response to implantedmaterials may have been affected, and these rabbits werereplaced in the study.

Histological evaluationFor histological evaluation, ground sections were viewed atvarious magnifications with light microscopy with a view toassess the interface between implant and bone and to deter-mine the nature of the bone–implant interface at a cellularlevel. The nature of the tissue in this region was deter-mined, and special care was taken to search for the evi-dence of fibrous encapsulation, inflammatory reaction, andbone contact. Implants were observed at 4-week and 12-week time points.

Four weeks. 45S5 bioglass implants were surrounded bynew mineralized bone tissue at 4 weeks after implantation[Fig. 2(a,b)]. This bioactive reference material showed goodintegration and osteoconductivity, but cracking and/or frac-ture of the implant was observed at all time points [Fig.2(c)] when examined at higher magnification. Although the45S5 bioglass surface was homogeneous upon implantation,surface dissolution took place at the edge of the implant–bone interface. Backscattered image [Fig. 2(b)] confirmedthe histological image. SEM image [Fig. 2(b)] and histologi-cal image [Fig. 2(a)] also confirmed that the bone–implantinterface was intimate and not interspersed with the fibroustissue.

Fluorcanasite Glass–Ceramic 1 implants were almostentirely surrounded by soft tissue at 4 weeks after implan-tation. Although the soft tissue around the Glass–Ceramic 1appeared stable, areas of inflammation or granulation tissuewere observed in sites where the glass–ceramic surfaceappeared to be degrading. Figure 3(a,b) shows the poorintegration with a visible space between implant and bone.After 4 weeks implantation period, Glass–Ceramic 1 did notshow any integration [Fig. 3(a,b)]. Ion release studies ofthese materials19 have suggested that Glass–Ceramic 1 inaqueous environments releases large quantities of Na and Siions when compared with other glass–ceramics. The irregu-lar appearance of the Glass–Ceramic 1 implant surfacesuggested that the composition tested was unstable in thebiological environment because of surface dissolution, con-sistent with the ion release data [Fig. 3(b)]. The gradual andsustained surface dissolution of the glass–ceramic implants,as well as the glass–ceramic particles released at theimplant surface, may have been responsible for the recruit-ment of inflammatory cells and the failure of bone to

FIGURE 2. (a) Ground section of femur with 45S5 bioglass implant at

4 weeks after implantation showing a new and old mineralized bone

tissue around the implant. (b) Backscattered SEM photomicrograph of

45S5 bioglass implant in midshaft of rabbit femur at 4 weeks after im-

plantation showing close association between cortical bone and

implant and evidence of surface reactivity of implant material. (c)

Backscattered SEM photomicrograph of 45S5 bioglass implant in mid-

shaft of rabbit femur at 4 weeks after implantation showing cracking

of the implant. Specimen damage occurred either at harvest or

processing. [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


integrate. This material, like the commercial cannasitereported previously,17 interfered with local bone healingand osteogenesis via promotion of inflammation.

Glass–Ceramic 2 not only showed some areas of directbone contact but also evidence of surface dissolution and fi-brous tissue encapsulation as seen with Glass–Ceramic 1.Figure 4(a,b) shows some points of bone tissue contact after4 weeks implantation. Figure 4(a) also shows nonmineral-ized, cellular material between the bone and implant. Anintimate relationship between bone and fluorcanasite Glass–Ceramic 2 without soft-tissue intervention was observed inonly a few regions [Fig. 4(a,b)]. Modified fluorcanasiteGlass–Ceramic 3 (with �2% P2O5) showed significant osteo-conduction after 4 weeks of implantation with intimatebone tissue contact. Glass–Ceramic 3 contained chemicallystable fluorcanasite and fluorapatite crystals in addition tosome frankamenite. Surface dissolution and microcrackingwere not a major issue for this particular glass–ceramic

unlike Glass–Ceramic 1 and 45S5 bioglass. Figure 5(a,b)shows that fluorcanasite–fluorapatite Glass–Ceramic 3implants were surrounded by new mineralized bone tissuein the cortical region after 4 weeks of implantation.

Twelve weeks. The observations of bone tissue response to45S5 bioglass were similar at 4 and 12 weeks, although theamount of bone surrounding the implants had slightlyincreased at the longer implantation time. The majority ofthe surface of the implant was covered by new cortical andtrabecular bone, which had formed on the surface and wasin intimate contact with the implant. The observations of45S5 bioglass implants confirmed its status as a biocompati-ble, osteoconductive implant material and provided evi-dence for the suitability of rabbit femur model to evaluatebiocompatibility and osteoconductivity.

Fluorcanasite Glass–Ceramic 1 implants were almostentirely surrounded by soft tissue at 12 weeks after implan-tation [Fig. 6(a,b)]. Although the soft tissue around the

FIGURE 3. (a) Ground section of femur with Glass–Ceramic 1 implant

at 4 weeks after implantation showing the presence of fibrous tissue

between bone and implant. Note the lack of homogeneity in glass. (b)

Backscattered SEM photomicrograph of Glass–Ceramic 1 implant in

midshaft of rabbit femur at 4 weeks after implantation showing lack

of contact, surface dissolution, and cracking. [Color figure can be


wiley.com.]


at 4 weeks after implantation suggesting modest contact between

new bone tissue and the implant. (b) Backscattered SEM photomicro-

graph of Glass–Ceramic 2 implant in midshaft of rabbit femur at 4

weeks after implantation showing direct contact with new mineralized

bone tissue with implant surface. [Color figure can be viewed in the

online issue, which is available at www.interscience.wiley.com.]


Glass–Ceramic 1 appeared stable, areas of inflammation orgranulation tissue were observed in sites where the glass–ceramic surface appeared to be degrading. Figure 6(b)shows poor integration with a visible space betweenimplant and bone. This fluorcanasite glass–ceramic alsoshowed evidence of gradual surface dissolution. Glass–Ceramic 1 did not show any integration at 12 weeks afterimplantation [Fig. 6(a,b)].

Glass–Ceramic 2 showed that the implant was entirelysurrounded by dense fibrous tissue after 12 weeks of im-plantation (Fig. 7), and lack of contact between bone andimplant was observed. Microcracking in Glass–Ceramic 2dramatically reduced when compared with Glass–Ceramic 1and 45S5 bioglass. Figure 7 shows poor integration with avisible space between implant and cortical bone. Glass–Ce-ramic 2 was not very osteoconductive and appeared to de-grade in the biological environment after long implantationperiod. It is therefore suggested that Glass–Ceramic 2 is

unsuitable for use as an implant. Modified fluorcanasiteGlass–Ceramic 3 (with �2% P2O5) showed an increase inthe amount of new bone formation surrounding theimplants at 12 weeks [Fig. 8(a,b)]. Both backscattered andlight microscopy images showed direct contact of new min-eralized bone and implants without soft-tissue intervention.Figure 8(a) shows that the bone–implant interface was inti-mate and not interspersed with the fibrous tissue. Glass–Ce-ramic 3, therefore, showed improved in vivo biocompatibil-ity with respect to the other fluorcanasite glass–ceramiccompositions reported here and in a previous study.17

Histomorphometric dataThe results of histomorphometric analysis (bone-to-implantcontact) are shown in the Figures 9 and 10. In the period of4 weeks, no statistical difference was found between thegroups. At 12 weeks evaluation, the bone contact on 45S5bioglass surface was statistically significant when comparedwith other materials. When the bone contact for each


at 4 weeks after implantation showing direct contact between bone

and implant surface. (b) Backscattered SEM photomicrograph of

Glass–Ceramic 3 implant in midshaft of rabbit femur at 4 weeks after

implantation showing very good attachment of new mineralized bone

tissue with implant surface. [Color figure can be viewed in the online

issue, which is available at www.interscience.wiley.com.]


at 12 weeks after implantation showing fibrous tissue between the

implant surface and local bone tissue. (b) Backscattered SEM photo-

micrograph of Glass–Ceramic 1 implant in midshaft of rabbit femur at

12 weeks after implantation showing lack of contact, surface dissolu-

tion, and cracking. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


material was compared at experimental time points, theGlass–Ceramic 2 (CaO rich) showed 31.7% at 4 weeksagainst no contact at all at 12 weeks (p ¼ 0.027). Con-versely, the 45S5 bioglass group exhibited 44.4% of bone-to-implant contact after four postoperative weeks andincreased to reach 85.9% after 12 weeks, although no sta-tistical significance could be found (p ¼ 0.052).

DISCUSSION

Glass–Ceramic 1 showed no evidence of osteoconduction ateither time point, with a similar bioinert response to thatreported in a previous study.17 Glass–Ceramic 2 not onlyshowed some points of bone tissue contact but also evi-dence for surface dissolution, microcracking, and a foreignbody response (fibrous tissue encapsulation and inflamma-tion). By 12 weeks, Glass–Ceramic 2 showed no remainingbone-to-implant contact. Both of these fluorcanasite compo-sitions appeared to suffer from surface dissolution in the bi-ological environment without evidence for osteoconduction.Glass–Ceramic 3 and 45S5 bioglass showed osteoconductiv-ity at both time points, with extensive bone tissue contactand the formation of a stable interface. The presence of newlamellar bone around the control 45S5 bioglass implantconfirmed the suitability of the rabbit femur for the in vivoevaluation of osteoconduction, with 85.9% bone tissue con-tact at 12 weeks. Surface changes observed associated withthe 45S5 bioglass were related to the published mechanismfor bioactivity. Glass–Ceramic 3 showed close contactbetween bone and implant (21.3% and 24.8% at 4 and 12weeks, respectively) but, in contrast to the bioglass, with novisible evidence of surface reactivity. This fluorcanasite–fluorapatite glass–ceramic was considered to beosteoconductive.

The essential condition for glass–ceramic to bond withthe bone is usually the formation of a silica-rich layer and

fluorapatite layer in vivo,23 and so it is not essential to con-tain apatite within the material.24–26 The absence of phos-phate from the fluorcanasite composition was therefore notlikely to be responsible for poor biocompatibility of Glass–Ceramic 1 and Glass–Ceramic 2. However, the compositionwas important as a determinant of solubility and hence sta-bility in the biological environment. This premise is corro-borated by the ion release data,19 in which it is evident thatGlass–Ceramic 3 is relatively insoluble when compared withother compositions. Consequently, Glass–Ceramic 3 withphosphate is stable in the biological environment. The com-bination of fluorapatite with low solubility of Glass–Ceramic3 may therefore be the crucial determinants for biocompati-bility in this system.

The mechanism of apatite formation on the surface of45S5 bioglass (CaO�SiO2-based glasses and bioglass–ceramics) has been described. Hydrated silica on the surfa-ces of the glasses provides favorable sites for apatite nuclea-tion. The calcium ions dissolved from the glasses combine

FIGURE 7. Ground section of femur with Glass–Ceramic 2 implant at

12 weeks after implantation showing fibrous tissue between the

implant surface and local bone tissue. Fibrous tissue contains many

mononucleate cells, suggesting inflammation. [Color figure can be


wiley.com.]


at 12 weeks after implantation showing direct bone contact with the

implant surface. (b) Backscattered SEM photomicrograph of Glass–Ce-

ramic 3 implant in midshaft of rabbit femur at 12 weeks after implan-

tation showing very good integration and direct contact with new

mineralized bone. [Color figure can be viewed in the online issue,

which is available at www.interscience.wiley.com.]


with the phosphate ions from the surrounding body fluid tonucleate on the surface and crystallize while incorporatingcarbonate and hydroxyls. Consequently, the apatite nucleiare rapidly formed on their surfaces. Once the apatite nucleiare formed, they spontaneously grow by consuming calciumand phosphate ions from the surrounding body fluid. Thus,45S5 bioglass implants rapidly form a bond with bone tis-sue via this apatite layer.27

The additions of P2O5 to Glass–Ceramic 3 gave rise tothe crystallization of fluorapatite as well as fluorcanasite.Hence, the formation of the surface apatite layer occurreddirectly and without the involvement of a Si-rich layer.18

Glass–Ceramic 3 (fluorcanasite and fluorapatite) exhibited invivo bioactivity via a different mechanism to 45S5 bioglass.The mechanism for osteoconduction of Glass–Ceramic 3appeared more closely when related to a calcium phosphatebioceramic rather than 45S5 bioglass. Fluorapatite glass–ceramics already have apatite present at the surface to formthis direct bond to bone. The mechanism reported here istherefore apatite-like and different to that of bone bondingin classical bioactive glasses.

Miller et al.18 predicted osteoconductivity of both Glass–Ceramic 2 and Glass–Ceramic 3 modified fluorcanasiteglass–ceramics using an in vitro apatite deposition model inSBF. Her findings, however, were not entirely reflected bydata presented here. Glass–Ceramic 3 with P2O5 showedgood osteoconduction, confirming the data from the SBFstudy. Glass–Ceramic 2 (CaO rich) ultimately showed poorbiocompatibility and no osteoconduction in contrast to thepublished SBF data. It remains unclear why this is the case,but this is the first report of SBF failing to predict osteocon-ductivity in a biomaterial. It is most likely related to thecomplexity of a glass–ceramic when compared with a mono-lithic biomaterial. Conceptually, it is possible that a bioactiveglass–ceramic with a bioactive residual glass might never befabricated, as early dissolution and lath/crystal releasemight always initiate an inflammatory response that ineffect becomes a ‘‘chain reaction’’ leading to chronic inflam-mation before a surface reactive layer can form.

CONCLUSIONS

These in vivo studies have shown that novel fluorcanasiteglass–ceramic implants were more osteoconductive than thecommercial fluorcanasite compositions reported previously.Modified fluorcanasite glass–ceramic implants with P2O5

that crystallized to fluorapatite were the most osteoconduc-tive biomaterial, with evidence of extensive bone tissue con-tact at 4 and 12 weeks. Histologically, the mechanism forosteoconduction was most like that observed with calciumphosphate biomaterials, as micrographs did not show evi-dence for a surface reaction layer as seen on 45S5 bioglass.In this system, it is therefore the introduction of apatiterather than the generation of a residual bioglass phase thatis responsible for the substantially improved biologicalresponse. This is the first demonstration of the effect ofthese compositional modifications to fluorcanasite in vivoand provides convincing evidence that relatively simplechanges can have profound consequences in terms of bio-logical response. This research suggests that novel biocom-patible, osteoconductive fluorcanasite–fluorapatite glass–ceramics have significant potential for the fabrication of cus-tom prostheses for bone tissue repair and augmentation.

ACKNOWLEDGMENTS

The authors thank Mr. Ian Watts and Mr. Dean Haycock fromthe University of Sheffield, UK, for their help in glass melting.They also thank Mr. Sebastiao Carlos Bianco from the Univer-sity of Sao Paulo, Brazil, for his help in surgery and samplepreparation.

REFERENCES1. Hall EE, Meffert RM, Hermann JS, Mellonig JT, Cochran DL. Com-

parison of bioactive glass to demineralized freeze-dried bone allo-

graft in the treatment of intrabony defects around implants in the

canine mandible. J Periodontol 1999;70:526–535.

2. Wheeler DL, Stokes KE, Hoellrich RG, Chamberland DL, McLough-

lin SW. Effect of bioactive glass particle size on osseous regenera-

tion of cancellous defects. J Biomed Mater Res 1998;41:527–533.

3. Hench LL, Splinter RJ, Allen WC, Greenlee TK Jr. Bonding mecha-

nisms at the interface of ceramic prosthetic materials. J Biomed

Mater Res 1972;2:117–141.

FIGURE 9. Bar chart showing the percentage of bone-to-implant con-

tact (BIC) after 4 postoperative weeks. No significant statistical differ-

ence among groups could be found. Values are presented as mean 6SD. [Color figure can be viewed in the online issue, which is available

at www.interscience.wiley.com.]

FIGURE 10. Bar chart showing the percentage of bone-to-implant con-

tact (BIC) after 12 postoperative weeks. Significant statistical differ-

ence was found between 45S5 Bioglass and the other groups (*).

Values are presented as mean 6 SD. [Color figure can be viewed in

the online issue, which is available at www.interscience.wiley.com.]

ORIGINAL ARTICLE


4. Hench LL, Willson J. An Introduction to Bioceramics. Singapore:

World Scientific; 1993. 386 p.

5. Hench LL, Etheridge EC. Biomaterials: An interfacial approach.

New York: Academic Press; 1982. p 1–7.

6. Beall GH. Chain silicate glass–ceramics. J Non-Cryst Solids 1991;

129:163–173.

7. Kokubo T. A-W glass–ceramic: Processing and properties. In:

Hench LL, Wilson J, editors. An Introduction to Bioceramics. Sin-

gapore: World Scientific; 1993. p 75–89.

8. Hill RA, Wood D. Apatite-mullite glass–ceramics. J Mater Sci:

Mater Med 1995;6:311–318.

9. Ohtsuki C, Kokubo T, Yamamuro T. Mechanism of apatite forma-

tion on CaO–SiO2–P2O5 glasses in a simulated body fluid. J Non-

Cryst Solids 1992;143:84–92.

10. Ohura K, Nakamura T, Yamamuro T, Ebisawa Y, Kokubo T,

Kotoura Y, Oka M. Bioactivity of CaO�SiO2 glasses added with var-

ious ions. J Mater Sci: Mater Med 1992;3:95–100.

11. Chiragov MI, Mamedov KHS, Belov NV. Crystal structure of cana-

site Ca5Na4K2(Si12O30)(OH, F)4. Dokl Acad Nauk USSR 1969;185:

96–99.

12. Beall GH. Alkali metal, calcium fluorosillicate glass–ceramic

articles. US Patent 4386162, 1983.

13. Beall GH. Method of making alkali metal, calcium fluorosilicate

glass–ceramic articles. US Patent 4397670, 1983.

14. Miller CA, Reaney IM, Hatton PV, James PF. Crystallisation of

canasite/frankamenite glass–ceramics. Chem Mater 2004;16:

5736–5743.

15. Kanchanarat N, Miller CA, Hatton PV, James PF, Reaney IM. Early

stages of crystallisation in canasite-based glass–ceramics. J Am

Ceram Soc 2005;88:3198–3204.

16. Kanchanarat N, Bandyopadhyay-Ghosh S, Reaney IM, Brook IM,

Hatton PV. Microstructure and mechanical properties of fluorca-

nasite glass–ceramics for biomedical applications. J Mater Sci

2008;43:759–765.

17. da Rocha Barros VM, Salata LA, Sverzut CE, Xavier SP, van Noort

R, Johnson A, Hatton PV. In vivo bone tissue response to a cana-

site glass–ceramic. Biomaterials 2002;23:2895–2900.

18. Miller CA, Kokubo T, Reaney IM, Hatton PV, James PF. Formation

of apatite layers on modified canasite glass–ceramics in simulated

body fluid. J Biomed Mater Res A 2002;59:473–480.

19. Bandyopadhyay-Ghosh S, Reaney IM, Brook IM, Hurrell-Gilling-

ham K, Johnson A, Hatton PV. In vitro biocompatibility of fluorca-

nasite glass–ceramics for bone tissue repair. J Biomed Mater Res

A 2007;80:175–183.

20. Bandyopadhyay-Ghosh S, Reaney IM, Johnson A, Brook IM, Hurrell-

Gillingham K, Hatton PV. Castability and biocompatibility of novel

fluorcanasite glass–ceramics. Key Eng Mater 2006;309:293–296.

21. Bandyopadhyay-Ghosh S, Reaney IM, Johnson A, Hurrell-Gilling-

ham K, Brook IM, Hatton PV. The effect of investment materials

on the surface of cast fluorcanasite glasses and glass–ceramics.

J Mater Sci: Mater Med 2008;19:839–846.

22. Bandyopadhyay-Ghosh S, Reaney IM, Hurrell-Gillingham K, Brook

IM, Hatton PV. Castability and biocompatibility of novel fluorcana-

site glass–ceramics. Key Eng Mater 2006;309–311:293–296.

23. Hench LL. The compositional dependence of bioactive glass,

glass–ceramics. Presented at the Seventh CIMTEC World Ceramic

Congress, Montecatini Terme, Italy, 1990.

24. Kokubo T, Hvang ZT, Hayashi T, Sakka S, Kitsugi T, Yamamuro T.

Ca, P-rich layer formed on high strength bioactive glass–ceramic

A-W. J Biomed Mater Res 1990;24:331–343.

25. Kokubo T, Kushitani H, Sakka S, Kitsugi T, Yamamuro T. Solu-

tions able to reproduce in vivo surface-structure changes in bioac-

tive glass-ceramic A-W. J Biomed Mater Res 1990;24:721–734.

26. Kokubo T. Surface chemistry of bioactive glass–ceramics. J Non-

Cryst Solids 1990;120:138–151.

27. Bosetti M, Cannas M. The effect of bioactive glasses on bone

marrow stromal cells differentiation. Biomaterials 2005;26:

3873–3879.


Osteoconductivity of modified fluorcanasite glass-ceramics for bone tissue augmentation and repair

Documents

Transcript of Osteoconductivity of modified fluorcanasite glass-ceramics for bone tissue augmentation and repair