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Materials Chemistry and Physics 148 (2014) 416e425

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Materials Chemistry and Physics

journal homepage: www.elsevier .com/locate/matchemphys

A preliminary investigation into the structure, solubility andbiocompatibility of solgel SiO2eCaOeGa2O3 glass-ceramics

A.W. Wren a, *, M.C. Jones a, S.T. Misture a, A. Coughlan b, N.L. Keenan a, M.R. Towler c,M.M. Hall a

a Inamori School of Engineering, Alfred University, Alfred, NY 14802, USAb School of Materials Engineering, Purdue University, West Lafayette, IN, USAc Department of Mechanical & Industrial Engineering, Ryerson University, Toronto, Canada

h i g h l i g h t s

A solgel derived glass-ceramic series was synthesized with 5 mol% Ga2O3 doped for the SiO2 and CaO. Characterization included X-ray diffraction, thermal analysis and particle analysis methods. Materials released gallium (Ga), calcium (Ca), silica (Si) and caused an increase in pH over time. Antibacterial effects were observed when tested in E. coli and S. epidermidis. No significant reduction in cell viability was observed with material ionic dissolution species.

a r t i c l e i n f o

Article history:Received 26 February 2014Received in revised form1 August 2014Accepted 3 August 2014Available online 29 August 2014

Keywords:BiomaterialsCeramicsGlassesSol-gel growthCorrosion

* Corresponding author. Tel.: 1 607 871 2183; faxE-mail address: wren@alfred.edu (A.W. Wren).

http://dx.doi.org/10.1016/j.matchemphys.2014.08.0060254-0584/Published by Elsevier B.V.

a b s t r a c t

The proposed study aims to synthesize gallium (Ga3) containing glass-ceramics via the solgel methodand determine their solubility and biocompatibility. Three solgel derived glass-ceramics, two containingGa3 were synthesized by substituting 5 mol% Ga3 for both Ca2 (Si-65) and Si4 (Si-70) and werecompared to a Ga3 free control (control). Glass transition temperatures (Tg) ranged from 641 to 660 Cfor all materials with particle sizes ranging between 1.5 and 2.5 mm. Surface area analysis ranged from 45to 55 m2 g1 and any changes in pH were determined over 0e14 days. Ga3 ion release from Si-65 peakedat 433 mg L1 after 7 days, and Si-70 peaked at 601 mg L1 after 1 day. Both calcium (Ca2) and silica(Si4) were also released from each material. Antibacterial testing against E. coli and S. epidermidisrevealed both bactericidal (maximum inhibition zone of 2.5 0.3 mm) and bacteriostatic effects. Thecontrol material exhibited inhibition zones in both bacteria while bacteriostatic properties were foundpredominantly against E. coli with Si-65 and Si-70. Cytocompatibility testing was conducted in L929mouse fibroblasts and determined no significant reduction in cell viability with respect to the control,with minimal, non-significant reductions for Si-65 and Si-70.

Published by Elsevier B.V.

1. Introduction

Since the invention of Bioglass, bioactive glasses, have beeninvestigated for a range of orthopedic applications as they exhibitexcellent osteoconductive properties [1,2]. Controlled release ofionic dissolution products, in particular, concentrations of solublesilica (Si4) and calcium (Ca2) are known to be essential to thebioactive process [1]. This has led to investigators to synthesize

: 1 607 871 2353.

novel bioactive glasses to improve bioactivity and/or antibacterialproperties [3,4]. Employing solgel processing can lead to positivecharacteristics over melt quenching route, including lower pro-cessing temperatures, uniform phase distribution, newcrystalline/non-crystalline materials [5], high surface area pow-ders [6,7] and greater homogeneity [6] which yields glasseswhich cannot be easily prepared by melt quenching [7,8]. Studieson 70SiO2e30CaO solgel glasses has previously been described byVallet-Regi [9,10] et al. and Saravanapavan et al [11], whichfocused primarily on the synthesis and incubation of these glassesin Simulated Body Fluid (SBF), which presented positive signs ofbioactivity [9e12].

mailto:wren@alfred.eduhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.matchemphys.2014.08.006&domain=pdfwww.sciencedirect.com/science/journal/02540584http://www.elsevier.com/locate/matchemphyshttp://dx.doi.org/10.1016/j.matchemphys.2014.08.006http://dx.doi.org/10.1016/j.matchemphys.2014.08.006http://dx.doi.org/10.1016/j.matchemphys.2014.08.006

Table 1Composition of glass series (Mol. %).

Composition

SiO2 CaO Ga2O3Control 0% Doped 70 30 0Si-65 5% SiO2 Doped 65 30 5Si-70 5% CaO Doped 70 25 5

Fig. 1. Solgel glasses a.) Control, b.) Si-65 and c.) Si-70.

A.W. Wren et al. / Materials Chemistry and Physics 148 (2014) 416e425 417

The addition of gallium (Ga3) to silicate glasses through solgelprocessing can be used to address problematic areas in orthopedics.Previous studies by Shruti et al. [13] on GaO2 dopedSiO2eCaOeP2O5 solgel glasses presented positive surface reactionsin SBF [13], whereas Bolis et al. reports that GaO2 reduces apatiteformation [14]. However, Ga3 can also be incorporated to eradicatetumor cells and reducing the risk of infection post-surgery. Anti-biotic overuse can promote multi drug-resistant strains of bacteria[15e17], such as MRSA (Methicillin Resistant S. aureus) which hasresulted in the need for antibiotic free materials for treatingpersistent infection such as antibacterial ions (Zn2, Ag) [18e21]and compounds [22,23]. Ga3 is an element that presents multi-therapeutic potential when incorporated into medical materialsand although there is no specific biological role for Ga3 in humans,it has been reported to present beneficial therapeutic effects whenincorporated into materials used to treat disease/infection inhumans. These disorders range from accelerated bone resorption,autoimmune disease and allograft rejection, certain cancers andinfectious disease [24] in addition to treating hypercalcemia ofmalignancy and Pagets disease [24]. Ga3 has also been used tosuppress osteolysis and bone pain associated with multiplemyeloma and bone metastases, in addition to being investigated asa treatment for osteoporosis [24]. Ga3 physiological activity iscited as being due to its atomic similarities (electric charge, ionicdiameter and coordination number [25]) with iron (Fe3), inparticular, regarding protein and chelate binding [24]. Biochemi-cally, the most important difference is that Ga3 is irreducible un-der physiological conditions, whereas Fe3 can be readily reducedto Fe2. This prevents Ga3 from entering Fe2 binding moleculesand prevents it from participating in redox reactions and is knownto be transported through blood plasma by the iron-transportprotein transferrin. Ga3 has a strong affinity for certain tissuessuch as bone andmany tumors and accumulation of Ga3 in tumors(lymphomas) is associated with large amounts of transferrin re-ceptors (TF). However, bone tissue does not generally contain highconcentrations of Fe3 binding proteins and the mechanism ofskeletal Ga3 accumulation remains relatively unknown. Ga3

accumulation in bone tissue has led to treating disorders such asmultiple myeloma [24] and treating infection as Ga3 is known tobe able to disrupt bacterial metabolism [26e28] in species such asM. tuberculosis and M. avium, S. aureus, E. coli, P. aeruginosa, MRSAand C. difficile [26,29e31], which is also related to Ga3 ability toenter microbes through their iron transport mechanisms whichdisrupts iron metabolism and DNA/protein synthesis [24].

Regarding this study, a CaOeGa2O3eSiO2 glass-ceramic serieswas prepared using the solgel processing. The effect of each ma-terials structure and solubility was evaluated against commonbacterium such as E. coli and S. epidermidis, in addition to a standardmammalian cell line (L929 mouse Fibroblasts) to evaluate any po-tential cytotoxicity.

2. Materials & methods

2.1. Glass-ceramic synthesis

2.1.1. MaterialsGlass-ceramic samples were prepared using a solgel method

based upon that described by Saravanapavan and Hench [11]. Thefollowing compounds were used to create CaOeSiO2 andCaOeGa2O3eSiO2 gel glass-ceramics: TEOS (Si(OC2H5)4), nitric acid(HNO3), calcium nitrate tetrahydrate (Ca(NO3)2$4H2O), gallium ni-trate hexahydrate (Ga(NO3)3$6H2O) (reagents, Fisher Scientific),and deionized (DI) water, according to Table 1 compositions. AsTEOS needs an acid or base catalyst, the 2 N nitric acid (HNO3) isadded during sol preparation. The amount of HNO3 is determined

according to the following ratios: a molar ratio of H2O/TEOS is 12:1and a volume ratio of H2O/HNO3 is 6:1 [11].

2.1.2. Gel synthesisThe 2 NHNO3 and the deionizedwater were stirred together in a

Teflon beaker at room temperature for 5 min. Si(OC2H5)4 was addedover a 30-min period and the mixture was stirred for an additional30 min to ensure homogeneity and complete hydrolysis. Ca(N-O3)2$4H2O and, where appropriate, Ga(NO3)3$6H2O, were thenadded and allowed to dissolve. Ga(NO3)3$6H2Owas added over a 5-min period to prevent rapid gelation, which reduces homogeneity.The sol was stirred for 1 h, followed by casting into sealed 38 mmpolypropylene containers which had three 1 mm holes in theirtops to allow release of gases that evolved during drying (Fig. 1).

2.1.3. Solgel glass synthesisWet gels were dried and aged simultaneously in a program-

mable oven at 60 C for 72 h. Aging and drying ensure completegelation and solidification. Each material was stabilized in Al2O3crucibles in a programmable oven according to the schedule inTable 2 [11]. X-ray diffractionwas conducted on each glass (Control,Si-65 and Si-70) prior to stabilization and each was determined tobe amorphous (data not presented).

2.2. Sample preparation

Each glass-ceramic was ground to a fine powder using a gyro-mill (Glen Creston, UK) for initial characterization. For solubilityand antibacterial testing, each material was tested in two forms,powder form and disc form, to determine if the increased disso-lution of the individual particles in the powder form increases theantibacterial effect. Samples were produced and evaluated asfollows:

Powder form (1 m2 surface area, n 3) Solid pressed disc (4 mm 1.3 mm, 1 m2 surface area, n 3)

To form discs, molds 4 mm were filled with powder andpressed to form discs (1.3 mm thick). For ion release and pH

Table 2Stabilization schedule of gels.

Stage Ramp (C min1) Temperature (C) Dwell (h)

1 1.0 100 0.02 0.5 300 2.03 1.0 600 5.04 5.0 30 0.0

Fig. 2. XRD patterns of i.) Control, ii.) Si-65, and iii.) Si-70.

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analysis, 5 m2 surface area was immersed in 10 ml of sterile de-ionized water for specified time periods, i.e. 1, 7 and 14 days. Forcell culture analysis, 100 ml liquid extracts were removed after 1, 7and 14 days incubation and stored until testing.

2.3. Material characterization

2.3.1. X-ray diffraction (XRD)Diffraction patterns were collected using a Siemens D5000 X-

ray Diffraction Unit (Bruker AXS Inc., WI, USA). Glass powdersamples were packed into standard stainless steel sample holders.A generator voltage of 40 kV and a tube current of 30 mA wasemployed using Cuka x-ray source. Diffractograms were collectedin the range 10 < 2q < 100, at a scan step size 0.02 and a step timeof 10 s. Any crystalline phases present were identified using theICDD (International Centre for Diffraction Data) standard diffrac-tion patterns, and phase quantification was undertaken using in-ternal standard Rietveld analysis. 10 wt.% corundum (Al2O3) wasused added to each specimen as the internal standard, and Rietveldanalysis was performed using Topas software (Bruker-AXS). Theinternal standard Rietveld method provides the quantities of eachcrystalline phase and the quantity of amorphous phase bydifference.

2.3.2. Differential thermal analysis (DTA)A combined differential thermal analyser-thermal gravimetric

analyser (SDT 2960 Simultaneous DSC-TGA, TA Instruments, DW,USA) was used to measure the glass transition temperature (Tg) forboth glasses. A heating rate of 20 C min1 was employed using anair atmosphere with alumina in a matched platinum crucible as areference. Sample measurements were carried out every six sec-onds between 30 C and 1300 C.

2.3.3. Particle size analysis (PSA)Particle size analysis was achieved using a Beckman Coulter

Multisizer 4 Particle size analyzer (BeckmanCoulter, Fullerton, C.A,USA). The glass powder samples (n 3) were evaluated in the rangeof 0.4 mme20.0 mm and the run length took 60 s. The fluid used inthis case was water and was used at a temperature range of10e37 C. The relevant volume statistics were calculated on eachmaterial.

2.3.4. Advanced surface area and porosity (ASAP)In order to determine the surface area of the glass-ceramics, an

Accelerated Surface Area and Porosimetry, ASAP 2010 SystemAnalyser (Micrometrics Instrument Corporation, Norcross, USA)was employed using nitrogen gas. Approximately 60 mg of eachmaterial (n 3) was used to calculate the specific surface areas,1 m2, using multi-point BrunauereEmmetteTeller (BET) method.

2.3.5. Scanning electron microscopy & energy dispersive X-rayanalysis (SEMeEDS)

Backscattered electron (BSE) imaging was carried out with anFEI Co. Quanta 200F Environmental Scanning Electron Microscope.Additional compositional analysis was performed with an EDAXGenesis Energy-Dispersive Spectrometer. All EDS spectra was

collected at 20 kV using a beam current of 26 nA. Quantitative EDSspectra was subsequently converted into relative concentrationdata.

2.4. Evaluation of material solubility

2.4.1. pH analysisChanges in pH of solutions were monitored using a Corning 430

pH meter. Prior to testing, the pH meter was calibrated using pHbuffer solution 4.00 0.02 and 7.00 0.02 (Fisher Scientific,Pittsburgh, PA). Sample solutions were prepared by exposing 5 m2

surface area of each glass-ceramic (Control, Si-65 and Si-70, wheren 3) in 10 ml sterile de-ionized water. Measurements wererecorded over t 1 h, 6 h, 24 h, 7 days and 14 days. Sterile de-ionized water was used as a control and was measured at eachtime period.

2.4.2. Ion release profilesEach glass-ceramic (n 3) was immersed in 10 ml sterile de-

ionized water for 1, 7 and 14 days and rotated on an oscillatingplatform at 37 C. The ion release of each glass was measured byInductively Coupled Plasma e Atomic Emission Spectroscopy (ICPe AES) on a PerkineElmer Optima 5300UV (Perkin Elmer, MA, USA)at the Pennsylvania State Materials Characterization Laboratory(MCL). ICP e AES calibration standards for Ca2, Si4 and Ga3 ionswere prepared from a stock solution. Three target calibrationstandards were prepared for each ion and deionized water wasused as a control.

2.5. Antimicrobial and cytocompatibility evaluation

2.5.1. Agar diffusion testThe antibacterial activity of each material was evaluated against

E. coli strain ATCC 8739 and S. epidermidis strain ATCC 14990 usingthe agar diffusion method. Luria agar and broth were used for theculture of E. coli, and S. epidermidis was cultured in BHI agar andbroth. Bacteria were grown aerobically at 37 C in an incubator.Preparation of the agar disc-diffusion plates involved seeding agarwith a sterile swab dipped in a 1/50 dilution of the appropriate 16 hculture of bacteria. Samples of glass powder (n 3) and discs(n 3) of each material were placed on the inoculated plates andthe constructs were then cultured (24 h, 37 C). The agar diffusiontest was performed under standard laboratory sterile conditions ina fumigation hood using sterile swabs for inoculation of bacteria.Each sample was analyzed in triplicate and mean zonesizes standard deviations were calculated. Calipers were used to

Table 3Quantitative XRD data.

Amorphous % Crystalline % Crystal size (nm)

Larnite Calcite

Control 59.3 40.7 (0.2) 0.0 30Si-65 84.3 13.1 (0.5) 2.6 (0.3) 10Si-70 74.9 19.0 (0.3) 6.1 (0.2) 10

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measure zones of inhibition at three different diameters for eachdisc and zone sizes were calculated as follows:

Inhibition Zone mm Halof Discf2

(1)

2.5.2. Preparation of agar specimensAgar strips (3 4 5 mm) were cut from the assay, extending

from the cement disc, through any inhibition zone, into the bac-terial colony. The specimens were then placed on a glass slide andincubated at 37 C in an air assisted oven for 24 h until dry. Analysiswas carried out with an FEI Co. Quanta 200F Environmental SEM(FEI Company, MD, USA) equipped with an EDAX Genesis Energy-Dispersive Spectrometer.

2.5.3. Laserscan profilometryLaser Profilometry was performed on a Solarius Laserscan Pro-

filometer (Solarius, Ca, USA), with a vertical sensor resolution of0.1 mm and a spot size of 2 mm. Analysis of data was performedusing Solar Map Version 5.0.4.5261 software.

2.5.4. Cell culture analysisThe established cell line L-929 (American Type Culture collec-

tion CCL 1 fibroblast, NCTC clone 929) was used in this study asrequired by ISO10993 part 5. Cells were maintained on a regularfeeding regime in a cell culture incubator at 37 C/5% CO2/95% airatmosphere. Cells were seeded into 24 well plates at a density of10,000 cells per well and incubated for 24 h prior to testing withliquid extracts (Section 2.2). The culture media used was M199media (Sigma Aldrich, Ireland) supplemented with 10% fetal bovineserum (Sigma Aldrich, Ireland) and 1% (2 mM) L-glutamine (SigmaAldrich, Ireland). The cytotoxicity of the liquid extracts was evalu-ated using the Methyl Tetrazolium (MTT) assay in 24 well plateswhere 100 ml of liquid extract (10% of the original medium) fromcontrol, Si-65 and Si-70 after 1, 7 and 14 days incubation, wereadded into wells containing L929 cells in culture medium (1 ml).Liquid extracts (n 3) were placed in the plate wells, incubated for

Fig. 3. TGA/DTA curve of a.) Con

24 h at 37 C/5% CO2 prior to testing. The MTTassaywas then addedin an amount equal to 10% of the culture medium volume/well. Thecultures were then re-incubated for a further 3 h (37 C/5% CO2).Next, the cultures were removed from the incubator and theresultant formazan crystals were dissolved by adding an amount ofMTT Solubilization Solution (10% Triton x-100 in Acidic Isopropanol(0.1 N HCI)) equal to the original culture medium volume. Once thecrystals were fully dissolved, the absorbance was measured at awavelength of 570 nm. Extracts (100 ml) of sterile tissue culturewater were used as controls, and control cells (denoted cells inFig. 10) were assumed to have metabolic activities of 100%. Liquidextracts in media without cells were tested and found not tointerfere with the MTT assay.

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was employed tocompare the antibacterial efficacy of the experimental materialsand changes in cell viability as a function of time. Comparison ofrelevant means was performed using the post hoc Bonferroni test.Differences between groups was deemed significant when p 0.05.Statistical analysis was performed using SPSS software for windowsversion 16 (SPSS Inc. Chicago, IL).

3. Results & discussion

3.1. Gallium glass-ceramic characterization

This study focuses on investigating the structure, solubility andbiocompatibility of Ga-containing bioactive glass-ceramics pro-duced via the solegel route. Ga3 is a known antibacterial cation[29,30], however little has been reported on the processing and ionrelease of Ga-containing bioactive glass-ceramics. Fig. 1 presentsthe X-ray diffraction patterns (Fig. 2) of each material and quanti-tative XRD data (Table 3) of the relevant amorphous/crystallinephases. Larnite (Ca2SiO4) was the predominant phase found in eachmaterial. The control was approximately 59% amorphous with theremaining crystalline content being larnite. Si-65 contained thehighest amorphous (84%) content, with crystalline phasesincluding larnite (13%) and calcite (3%). Si-70 was 75% amorphous,but also contained larnite (19%) and calcite (6%). Previous studies bySaravanapavan [11] and Vallet-Regi [9,10] produced similar70SiO2e30CaO glasses that were fully amorphous, however, thisstudy presented 15e40% crystallization, not unusual in solegelprocessed materials [5]. Ga3 incorporation into the Si-65 and Si-70glasses was found to increase amorphicity when compared to the

trol, b.) Si-65 and c.) Si-70.

Table 4Particle size analysis (PSA) and surface area (SA) of each material.

PSA (m) d10 d50 d90 SA (m2 g1)

Control 1.50 (0.54) 0.75 1.50 2.21 45 (13)Si-65 2.54 (0.74) 1.39 2.71 3.34 47 (8)Si-70 2.15 (1.34) 0.84 1.81 4.25 55 (5)

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control. This may be due to the fact that Ga3 has been reported tohave higher activation energy than Si4 in albite (NaAlSi3O8) melts[32]. The addition of Ga3 may also de-polymerize the Si4

network, particularly in the case of Si-65 preventing crystal for-mation with Ca2 and oxygen. In the case of Si-70, where Ca2 issubstituted, Ga3 addition further increases amorphous content asit does not promote crystallization as readily as Ca2. The glass-ceramics incorporating Ga3 also contained calcite phases whichreduced the crystal size from 30 nm (control) to 10 nm (Si-65, Si-70).DTA determined changes in the glass transition temperature (Tg) asa result of Ga3 addition. Fig. 3 presents the TGA/DTA thermogramprofiles for each material after the stabilization schedule wascomplete. Tg was determined at 660 C (control), 641 C (Si-65) and660 C (Si-70). The lower Tg of Si-65 may be due to the lower Si4

content whereas Si-70 had a similar Tg to the control (660 C). Theserelatively minor changes in Tg may be attributed to the role of Ga3

within the glass. A previous study by Shruti et al. onSiO2eCaOeP2O5 solgel glasses doped with GaO2 determined thestructural role of Ga as a network intermediate [13], which mayexplain the relatively minor changes in Tg, however, further char-acterization techniqueswould be needed to confirm this. Studies bySaravanapavan [11] and Vallet-Regi [9] on 70SiO2e30CaO glasses,

Fig. 4. SEM (5 k, 20 k) and corresponding E

presented Tg at slightly lower temperatures (550 C and 566 Crespectively) than reported here, which may be a result of thepresence of crystallization in the control, Si-65 and Si-70. TGAcurves are also presented in Fig. 3 and presented relatively minorweight changes ranging from 7 to 10% post stabilization, verysimilar to the profile presented by Saravanapavan [11] on stabilized70SiO2e30CaO solgel glasses [11]. Each material underwent parti-cle size analysis to determine mean particle size (Table 4). Thecontrol glass-ceramic had amean particle size of 1.5 mm, while Si-65and Si-70 hasmean particle sizes of 2.5 mmand 2.1 mm, respectively,slightly smaller to that reported by Vallet-Regi on 70SiO2e30CaO at9.3 m [10]. Additional analysis of the glass-ceramics was under-taken by surface area measurements (Table 4) where the controlhad a surface area of 45 m2 g1 while Si-65 and Si-70 exhibitedsurface areas of 47 m2 g1 and 55 m2 g1 respectively. Studies byVallet-Regi on 70SiO2e30CaO determined higher surface areas of126 m2 g1 [9] and 78 m2 g1 [10]. SEM-EDS was employed toinvestigate the morphology and composition of the powders. Fig. 4presents the SEM and corresponding EDS where the surface of thepowders present rod-like epitaxial grown structures, which isparticularly evident in Si-65 and Si-70. The corresponding EDSconfirm the compositions where the control was composed of Ca2

and Si4 and Si-65 and Si-70were composed of Si4, Ca2 and Ga3.Gold (Au) and Palladium (Pd) were also detected as part of thesample preparation. The structures presented in the SEM imagesare indicative of high surface area materials which corresponds tothe literature where glass-ceramics fabricated by solegel are re-ported to have increased bioactivity compared to melt-quenchanalogues, as they are highly mesoporous with high surface areas[33].

DS of a.) Control, b.) Si-65 and c.) Si-70.

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3.2. Evaluation of material solubility

Ion release profiles were determined for Ga3, Si4 and Ca2 byICP analysis (Fig. 5). As expected, there was no Ga3 release fromthe control glass. Regarding Si-65, Ga3 levels ranged from207 mg L1, 433 mg L1 and 215 mg L1 over 1, 7 and 14 daysrespectively. However Ga3 release from Si-70 was much moreprominent where it peaked after 1 day (601 mg L1), reducingslightly (565-538 mg L1) after 7e14 days. Si4 release was rela-tively low for the control glass, 0.74e0.32 mg L1 (1e14 day). Si-70also exhibited low Si4 release ranging from 1.59 to 0.59 mg L1

(1e14 day). Si-65 achieved the highest Si4 release levels where itpeaked after 1 day (25.5 mg L1) and reduced to 3.5 mg L1 after 14days. With respect to Si4 release, there was observed to be aconsistent reduction in Si4 concentration over time which is likelydue to precipitation in solution. Si4 is an ion that is essential in thebioactive process as it facilitates precipitation of ions, such as Ca2,through the formation of SieOH groups [4]. The formation of thesespecies may explain the gradual reduction in Si release over 1e14days as Ca2 levels are high in the surrounding solution. Theaddition of Ga3 to Si-65 resulted in a higher Si4 release ratewhichmay be due to partial de-polymerization of SieOeSi groups in theglass [4]. Previous ion release studies on GaO2 dopedSiO2eCaOeP2O5 solgel glasses presented Ga3 levels below the

Fig. 5. Ion Release profiles of the a.)

detection limit of the ICP, where Ca2 (280 mg L1) and Si4

(40e60 mg L1) levels were more similar to the levels presentedhere [13]. Studies by Vallet-Regi on 70SiO2e30CaO solgel glassesalso presented Ca2 levels that peaked at 400 mg L1 [9]. Ca2

levels were found to be higher in the control than either of the Ga-doped glass-ceramics which reached 424 mg L1 (7 days).Maximum Ca2 levels from Si-65 and Si-70 were 197e339 mg L1

(after 14 days). Ca2 levels were found to reduce in each case withthe addition of Ga3 which may be partially attributed to the 5 mol% reduction in Ca2 composition in Si-65, but predominantly, theCa2 may be assuming a charge compensation role for the Ga3

content as charge compensation by Ca2 is known to occur inCaAlSiO glasses [34] and is also cited to be occurring in GaO2 dopedSiO2eCaOeP2O5 solgel glasses [13]. Additionally, Ga3 has previ-ously been described as having a similar structural role and can beused to substitute aluminum (Al3) in NaAlSi2O6 glasses, which is anetwork intermediate [32]. Lower Ca2 release experienced by Si-65 and Si-70 may also be as a result of additional calcite crystallinephases present, however detailed analysis of the glass-ceramicswould be required in order to definitively predict the role thatGa3 plays.

In considering a material's antibacterial response, pH can have asignificant effect on the proliferation of microbes. The pH of theglass series was compared against a sterile control sample (de-

Control, b.) Si-65 and c.) Si-70.

Fig. 8. Sample images from E. coli and S. epidermidis agar diffusion antibacterialtesting.Fig. 6. pH analysis of deionized water, Control, Si-65, and Si-70 at t 0 h, 6 h, 24 h,

168 h (7 days) and 336 h (14 days).

A.W. Wren et al. / Materials Chemistry and Physics 148 (2014) 416e425422

ionized water) at each time period (Fig. 6). The pH of the sterile de-ionized water decreased from 7.0 to 6.5 over 14 days. The pH of thecontrol increased from 11.6 to 12.2, Si-65 and Si-70 experiencedslight increases from 11.2 to 11.4 (Si-65) and from 11.5 to 11.7 (Si-70)over the same time period). In each case the pH increased with theaddition of each glass-ceramic suggesting that each material isbasic. Additional comparable studies by Vallet-Regi presented pHvalues of 8.0 [10] and 8.1 [9] after 7 days incubation in simulatedbody fluid. Regarding this study the pH was significantly higher,which may be due to differences in the solvent used, and thestructure of the materials.

3.3. Antimicrobial and cytocompatibility evaluation

The therapeutic potential of each glass was evaluated withrespect to testing in two bacterium; E. coli and S. epidermidis, and amammalian cell line, L929 mouse fibroblasts. Fig. 7 presents theantibacterial response against E. coli (Fig. 7a)) and S. epidermidis(Fig. 7b)). Testing against E. coli revealed that only the control ma-terials exhibited bactericidal properties (2.0 0.3 mm and1.9 0.2 mm, for disc and powder respectively), however, abacteriostatic effect was observed with the Si-65 and Si-70regarding both disc& powder morphologies. For the testing againstS. epidermidis, the control materials presented bactericidal

Fig. 7. Antibacterial properties of glass ser

inhibition zones of 2.7 0.2 mm (disc) and 2.2 0.3 mm (powder),respectively. Si-70 also exhibited bactericidal properties2.3 0.2 mm (disc) and 1.6 0.2 mm (powder), respectively. Si-65produced inhibition zones of 2.1 0.2 mm (disc) and 2.5 0.3 mm(powder). There were significant differences when comparing thecontrol to the Si-65 and Si-70 disc and powder (p 0.000) as noinhibition was experienced with Si-65 or Si-70 when tested inE. coli, However, both Si-65 and Si-70 exhibited bacteriostatic ef-fects (Fig. 8b) and c)). With respect to testing in S. epidermidis,materials were compared as a function of sample morphology(powder and disc), where the controlwas compared to Si-65 and Si-70 for both powder and disc samples. The powder samples werecompared, and a significant reduction in inhibition zone wasevident when comparing the control to Si-70 (p 0.001) and nosignificant change was observed when comparing the control to Si-65 (p 0.113). When comparing the disc control sample to Si-70(disc) a significant reduction was observed (p 0.013), similarlywhen comparing the control to Si-65 (disc) a significant reduction ininhibition zone was also observed (p 0.000). Similar to previousstudies [30], the control glass also exhibited inhibition zones. It islikely that pH changes experienced by the control exacerbated theantibacterial effect [29,30]. The reduced action of some of the Ga-containing glass-ceramics may be pH attributable; however, it isalso likely that the ions released from these materials do not diffusewell in bacterial medium, which is investigated further in Fig. 9.

ies in a.) E. coli and b.) S. epidermidis.

Fig. 9. Quantitative EDX of agar sections extracted from agar diffusion antibacterialtesting.

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Regarding the antibacterial efficacy, of Ga3, there are numerousreferences to the mechanism of action in the literature[11,25,27,28,30], where it is reported to act as a substitute for Fe3.The metabolism of Fe3 is required for bacterial metabolism,functioning of enzymes, DNA synthesis, electron transport andoxidative stress defenses, and as such, Fe3 substitution with Ga3

can reduce the pathogenesis of bacterial infection [25,30].Energy dispersive x-ray analysis (EDX) was employed (Fig. 9) to

investigate glass-ceramic ion diffusion through the inhibition zone.EDX revealed the presence of concentrations of carbon (C), O andnitrogen (N) from the bacterial medium in addition to Na, Mg,sulphur (S), chlorine (Cl), K and P, which are either also present inthe agar, or are present as a result of decomposition of bacterialcells. High levels of Si4 and Ca2 were found in the inhibition zoneand not present in the control agar, suggesting they were releasedfrom the glass-ceramics. Ga3 ions were found within the inhibi-tion zones of Si-65 and Si-70, although in low concentrations, 0.75%and 0.48% respectively. It would be expected that Si-70 wouldpresent higher levels of Ga3 than both Ca2 and Si4, as evidentwith ion release studies. This suggests that Ga3 ion diffusion

Fig. 10. Sample laser scan of a.) E. coli control disc and b.) E.

through the agar is limited and diffusion may occur much morereadily in an aqueous environment. Studies by Thomas et al. [35]and Gokarn et al. cites factors such as whether linear or branchedpolymer chains are present within the agar [35], macromoleculesize and competition for hydration between ions and water mole-cules for restricting diffusion [36]. Additionally, diffusion of ionscan be influenced by the existence of fixed negative groups on theagarose saccharides. The presence of sulfuric and uronic acid resi-dues is known to contribute to cation exchange and diffusion and assuch the exchange capacity has been directly linked with the sulfurcontent [35]. Additionally, the migration of ions through the agarmay also be influenced by atomic size and charge. Si4 and Ca2

present much higher levels, which is likely a result of the higherconcentration of these ions in the starting materials. With respectto size, the ionic radius of Ga3 (0.62 ) is intermediate betweenSi4 (0.40 ) and Ca2 (0.99 ) suggesting this is unlikely a sig-nificant influence. Ionic charge of Ga3 in contact with chargedmacromolecules, in addition to the lower concentration is likely torestricted Ga3 diffusion. Although some of the Si-65 and Si-70glass-ceramics did not exhibit clear inhibition zones, particularly inE. coli, a bacteriostatic effect was observed (Fig. 10). To investigatethis effect, a laser profilometer was used to scan the inhibition zoneof the control disc sample in E. coli. Fig. 10 displays a sample laserscan image (X 20 mm, Y 20 mm, Z 89 mm) highlighting therandom distribution of the bacterial colonies compared to thesmooth inner inhibition zone. Fig. 10 displays the projected area(X 10 mm, Y 15 mm, Z 72 mm) of the bacteriostatic halopresented by Si-65 disc in E. coli. A gradual increase in bacterialcolony height can be observed as the distance from the disc in-creases. Fig. 11 shows the analysis of the area displayed in Fig. 10.Fig. 11a) shows the scan area, while Fig. 11b) shows the surfacetopography of the scanned area displaying a change in verticalheight difference of 49.9 mm over a distance of 10 mm extendingfrom the disc to the edge of the bacteriostatic halo. Further analysisof the sample area (Fig. 11c)) determined that approximately 23% ofthe total area of the sample (Si-70 disc in E. coli) was 25 mm inheight, 72% was between 25 and 50 mm, and 5% was50 mm, whichwas located at the far edge of the halo. This effect may be attributedto lower levels of antibacterial ions being released from the mate-rials through the bacterial medium, or even as a result of reduced

coli Si-65 disc showing dimensions and inhibition zone.

Fig. 11. Analysis of E. coli Si-65disc (image in Fig. 6b) presenting the a.) projected area, b.) linescan across area and c.) area% scan.

Fig. 12. Liquid extract cell culture testing considering in L929 Mouse Fibroblasts after1, 7 and 14 days incubation.

A.W. Wren et al. / Materials Chemistry and Physics 148 (2014) 416e425424

diffusion, resulting in an insignificant change in the pH. Furtherevaluation of the therapeutic capability of these materials wasevaluated using cell culture analysis. Sample extracts were evalu-ated and compared against a growing control cell population. It isevident from Fig. 12 that when comparing the control cell popu-lation to the control material at each time period, there was nosignificant change in cell viability, 1 day (p 1.000), 7 day(p 0.741) and 14 days (p 0.148). When comparing the controlcell population to Si-65 at each time period, no significant changewas found at 1 day (p 1.000) and 14 days (p 0.304), however, asignificant reductionwas found at 7 days (p 0.001) where the cellviability reduced to 82%. With respect to Si-70, no significantchanges were identified at 1 day (p 0.399), 7 days (p 0.503) or14 days (p 1.000). Cell culture studies proved that the Ga-containing glass ceramics did not significantly reduce cellnumbers even at high release rates like the levels cited here(200e600 mg L1) when compared to the growing cell population.A review by Bernstein cites much lower levels of Ga3 in bloodplasma (for treating hypercalcemia of malignancy, 2 mg L1), andgallium nitrate has been administered clinically at levels rangingbetween 0.1 and 100 mg L1 [24]. However, since the earliest

A.W. Wren et al. / Materials Chemistry and Physics 148 (2014) 416e425 425

clinical studies performed, Ga has been administered to thousandsof patients with no significant toxicity [24]. Additionally, evenhigher levels of Ga3 presented in this study did not prove toxic,suggesting, at least, that these cells may be tolerant of Ga3 withinthese concentration levels.

Ga3 is a known therapeutic agent against certain microbes(bacteria, fungi) and cells (cancer cells) that have not beenconsidered in this preliminary study. This work aimed to synthesizesoluble solgel derived, Ga-containing bioactive glass-ceramics andto investigate their structure, solubility and therapeutic potential.Ga-containing glass-ceramics were produced and proved to behighly soluble. Additionally, the materials did present antibacterialproperties in addition to proving non-toxic to mammalian cells.Future work on these materials will include antibacterial testing inan aqueous environment and to perform additional cell culturetesting on tumor cells to determine the efficacy of high Ga3 releaserates.

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preliminary investigation into the structure, solubility and biocompatibility of solgel SiO2CaOGa2O3 glass-ceramics1 Introduction2 Materials & methods2.1 Glass-ceramic synthesis2.1.1 Materials2.1.2 Gel synthesis2.1.3 Solgel glass synthesis2.2 Sample preparation2.3 Material characterization2.3.1 X-ray diffraction (XRD)2.3.2 Differential thermal analysis (DTA)2.3.3 Particle size analysis (PSA)2.3.4 Advanced surface area and porosity (ASAP)2.3.5 Scanning electron microscopy & energy dispersive X-ray analysis (SEMEDS)2.4 Evaluation of material solubility2.4.1 pH analysis2.4.2 Ion release profiles2.5 Antimicrobial and cytocompatibility evaluation2.5.1 Agar diffusion test2.5.2 Preparation of agar specimens2.5.3 Laserscan profilometry2.5.4 Cell culture analysis2.6 Statistical analysis3 Results & discussion3.1 Gallium glass-ceramic characterization3.2 Evaluation of material solubility3.3 Antimicrobial and cytocompatibility evaluationReferences