Journal of Non-Crystalline Solids 380 (2013) 25–34

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Journal of Non-Crystalline Solids

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Investigating the mechanical durability of bioactive glasses as a functionof structure, solubility and incubation time

Y. Li a, A. Coughlan a, F.R. Laffir b, D. Pradhan a, N.P. Mellott a, A.W. Wren a,⁎a Inamori School of Engineering, Alfred University, Alfred NY, USAb Materials and Surface Science Institute, University of Limerick, Ireland

⁎ Corresponding author at: Inamori School of EngineNew York 14802, USA. Tel.: +1 585 489 4496; fax: +1 6

E-mail address: wren@alfred.edu (A.W. Wren).

0022-3093/$ – see front matter © 2013 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.jnoncrysol.2013.08.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 July 2013Received in revised form 13 August 2013Available online 17 September 2013

Keywords:Bioactive glass;Ion release;Solubility;Crystalline;Hardness

This study focuses on investigating the solubility of a series of bioactive glasses that alternates the sodium (Na+)and strontium (Sr2+) concentration. Additionally, this study investigates the effect of crystallization of theseglasses and the subsequent effect on ion release, pH and mechanical strength as a function of incubation time.Ion release profiles were determined over 1, 7 and 30 days and in each case ion release was greatly reducedwhen the materials were crystallized. Additionally pH changes were reduced with the onset of crystallizationcompared to the amorphous counterparts. The most prominent changes in solubility can be attributed to theNa+ containing glasses, which presented the more soluble glasses and hence higher changes in pH. Regardingthe amorphous materials, hardness was found to significantly reduce with respect to maturation; however thecrystallized analogues presentedmuch higher hardness values that did not reducewith respect to incubation time.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Bioactive glasses/bioceramics arematerials designed to interactwithhard tissues within the human body to promote healing. The mostprominent material to date is Bioglass®, a Na2O–CaO–SiO2–P2O5 basedglass developed by Prof. L. Hench at the University of Florida in 1969[1]. Early studies determined that this specific glass composition, 45S5Bioglass®, forms a permanent bond to bone and can only be removedby breaking the bone [1]. These early studies stimulated interest in de-termining the osteoconductivity (the growth of osseous tissue on ama-terials surface) of bioceramics/bioactive glasses as the osteoconductivepotential of the material is critical in determining the long-termin vivo success of the implant [2]. To date many bioactive glass formula-tions have been conceived including silicates, such as Bioglass®, whichhave numerous specific compositions such as 45S5 (45% SiO2, 24.5%Na2O, 24.5% CaO, 6% P2O5), 52S (52% SiO2, 21% Na2O, 21% CaO, 6%P2O5) and 55S (55% SiO2, 19.5% Na2O, 19.5% CaO, 6% P2O5). A study byVogel et al. determined the osteoconductivity of each of these glassesin in vivo models and found the 45S5 composition to have the highestbone bonding kinetics and the highest degradation rate [3]. Additional-ly, phosphate based glasses (PBGs) have been investigated by Knowleset al. in the system P2O5–CaO–Na2O where PO3−

4 acts as the networkformer substituting for SiO2. Specific PBGs are known to exhibit positivesurface reactivity; however, studies using human bonemarrow derived(hBMSCs) were unfavorable, which was attributed to the high dis-solution rate of these glasses [4,5]. The osteoconductivity of borate-

ering, Alfred University, Alfred,07 871 2353.

ghts reserved.

containing glasses has also been investigated and it has been suggestedthat some borate containing glasses convert faster andmore completelyto hydroxyapatite than 45S5 Bioglass®, in addition to supporting thegrowth and differentiation of human mesenchymal stem cells [6]. Re-garding the silicates, the proposedmechanism bywhich 45S5 Bioglass®bonds to bone can be attributed to dissolution of the particulates uponexposure to physiological medium or body fluids [7]. Soluble silica israpidly released into the local environment in the form of silicic acid,due to ion exchange with H+ and H3O+. Changes in the H+ concentra-tion can influence cell metabolism and function and the subsequentrise in pH (alkalinization) has been cited as being either harmless orpotentially beneficial as it has been suggested to increase collagensynthesis and crosslinking and to promote hydroxyapatite formation[8]. Glasses/glass-ceramics that induce a precipitated hydroxyapatitesurface layer in a physiological medium is regarded as a positive attri-bute and is seen as a precursor to bone bonding in vivo [7].

Due to the success of Bioglass®, numerous glass based materialshave been developed including glass coatings [9], composite materials[10] and Y-TZP bioceramics [11], bioactive glass tubes [12] and glass-ceramic scaffolds (G-CS) [13–16]. It has been suggested that one of thelimiting factors when developing G-CS from Bioglass® in particular, isrelated to sintering of the scaffold. Post sintering of G-CS, crystallizationof 45S5 Bioglass® converts it from a bioactive material into an inertceramic as full crystallization occurs prior to densification. This leadsto compromised strengths within the scaffold struts, and also, crystalli-zation is believed to reduce the bioactivity [14]. However, it hasbeen cited that the crystalline 45S5 Bioglass® glass-ceramics i) formNa2Ca2Si3O9 phases that significantly improve the mechanical propertiesof the material, ii) crystallization does not inhibit bioactivity with bonebonding ability, and iii) when immersed in body fluids the crystalline

26 Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

Na2–Ca2–Si3–O9 decomposes and transits to amorphous calcium phos-phate [17]. Studies by Clupper and Hench determined that the predom-inant crystal phase associated with Bioglass®, Na2Ca2Si3O9 slightlydecreases the formation kinetics of an apatite layer on Bioglass® surfacebut did not totally suppress its formation [18]. Further studies by Filhoand Hench found that there is no compromise in bioactivity for the45S5 glass ceramic system even when 100% crystalline [19]. This studyin particular focused on determining the surface precipitation reactionsin simulated body fluid (SBF) and determined that even by inducingcrystallinity (ranging from 8 to 100%), the materials maintained theirbioactivity in SBF [19].

This study aims to determine the solubility of a series of SiO2–TiO2–

CaO–Na2O/SrO bioactive glasses where ion release, pH and mechanicaldurability of the materials were investigated as a function ofa.) Na+/Sr2+ incorporation, b.) incubation time in an aqueous mediaand c.) atomic arrangement, specifically amorphous and crystallinemorphologies. Conventional methods used to control the solubility ofbioactive glasses include controlling the ratio of network formers to net-work modifiers (Si:M), where M is traditionally Ca2+ or Na+. Depoly-merization of the silicate network (Si–O–NBO) by incorporating alkali/alkali earth cations (network modifiers) results in the breaking ofSi―O bonds resulting in an increase in the solubility [20]. Regardingthis study, these glasses alternate network modifiers such as Na+ andSr2+ and are investigated as Na+ is known to promote dissolution ofbioactive glasses [17] while Sr2+ has been shown to promote osteogen-esis in postmenopausal women treated with Sr-containing compounds[21–23]. Titanium (Ti4+) is added as it is included in manymedical ma-terials as it is known to form Ti–OH groups, which encourages changesin surface chemistry [7,24]. Silica (Si4+) acts as a network former;however, soluble Si4+ can induce hydroxyapatite precipitation by theformation of Si–OH groups which precipitate Ca2+ from the surround-ing aqueous environment [7,23]. It is known that thermal treatment ofbioactive glasses/glass-ceramics can dictate many important featuressuch as microstructure, the degree of crystallinity, mechanical proper-ties and the biological response [25]. With regard to this study, networkmodifier ion and inducing crystallization can be utilized to control thedissolution rate of the glass, in addition to controlling the mechanicalstability of the material.

2. Materials and methods

2.1. Glass synthesis

Three glass compositions (Ly-N, Ly-C, Ly-S) were formulated for thisstudy (Table 1) with the principal aim being to investigate any propertychanges with the substitution of sodium (Na+) and strontium (Sr2+)within the glass. A control glass (Ly-C) was also formulated whichcontained equal quantities of Na+ and Sr2+. Glasses were prepared byweighing out appropriate amounts of analytical grade reagents andball milling (1 h).

2.1.1. Glass powder productionThe powdered mixes were oven dried (100 °C, 1 h) and fired

(1500 °C, 1 h) in platinum crucibles and shock quenched into water.The resulting frits were dried, ground and sieved to retrieve glass pow-ders with a maximum particle size of b45 μm.

Table 1Nominal and XPS-derived (in italics) glass compositions. All compositions provided inmole fraction.

Ly-N Ly-C Ly-S

SiO2 0.55 (0.52) 0.55 (0.51) 0.55 (0.54)TiO2 0.05 (0.03) 0.05 (0.04) 0.05 (0.04)CaO 0.22 (0.22) 0.22 (0.23) 0.22 (0.22)Na2O 0.18 (0.23) 0.09 (0.13) 0.00 (0.00)SrO 0.00 (0.00) 0.09 (0.09) 0.18 (0.19)

2.1.2. Disc sample preparationDisc samples (Ly-N, Ly-C, Ly-S) were prepared by weighing approx-

imately 0.5 g powder into a stainless steel die (sample diameter1.5 × 6 ϕmm) which was pressed under 3 tons of pressure. Disc sam-ples were kept amorphous for Ly-N, Ly-C, and Ly-S by annealing atTg + 10 °C for 24 h. Crystalline discs were produced by heating thesamples to the first crystallization region for Ly-N (591 °C), Ly-C(778 °C) and Ly-S (871 °C) in order to determine any differences inbioactivity as a result of crystallization. Disc samples were then usedfor ion release studies, pH testing and mechanical testing.

2.2. Material characterization

2.2.1. X-ray diffraction (XRD)Diffraction patternswere collected using a SiemensD5000X-rayDif-

fraction Unit (Bruker AXS Inc., WI, USA). Glass powder samples werepacked into standard stainless steel sample holders. A generator voltageof 40 kV and a tube current of 30 mA were employed. Diffractogramswere collected in the range 10° b 2θ b 80°, at a scan step size 0.02°and a step time of 10 s. Any crystalline phases present were identifiedusing JCPDS (Joint Committee for Powder Diffraction Studies) standarddiffraction patterns.

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

Multisizer 4 Particle size analyzer (Beckman Coulter, Fullerton, CA,USA). Glass powder samples were evaluated in the range of 0.4 μm–

100.0 μm with a run length of 60 s through a 100 μm aperture. Thefluid usedwas the supplied electrolyte solution (NaCl) at a temperatureof 25 °C. The relevant volume statistics were calculated on each glass.

2.2.3. X-ray photoelectron spectroscopy (XPS)X-ray photoelectron spectroscopy was performed in a Kratos AXIS

165 spectrometer (Kratos Analytical, Manchester, UK) usingmonochro-matic Al Kα radiation (hυ = 1486.6 eV). Glass rods with dimensions of15 × 3 × 3 mmwere produced from themelt and fractured under vac-uum (~2 × 10−8 torr) to create pristine surfaces with minimum con-tamination. Surface charging was minimized by flooding the surfacewith low energy electrons. The C 1s peak of adventitious carbon at284.8 eV was used as a charge reference to calibrate the binding ener-gies. High resolution spectra were taken at pass energy of 20 eV, withstep size of 0.05 eV and 100 ms dwell time. The error are values calcu-lated from the statistical noise on the data and are between±0.01-0.5%for the compositions calculated in this study.

2.2.4. Scanning electron microscopy (SEM)Sample imaging was carried out with an FEI Co. Quanta 200F Envi-

ronmental Scanning Electron Microscope equipped with an EDAXGenesis Energy-Dispersive Spectrometer. Secondary electron (SE) andbackscattered electron (BSE) images were taken on glass particles andpolished disc surfaces.

2.3. Ion release profiles

Each disc (Ly-N, Ly-C and Ly-S, where n = 3) was immersed in ster-ile de-ionized H2O for 1, 7 and 30 days. Each disc (1.5 × 6 ϕmm) wassubmerged in 10 ml of de-ionized H2O and rotated on an oscillatingplatform at 37 °C. The ion release profile of each specimen was mea-sured using inductively coupled plasma–atomic emission spectroscopy(ICP–AES) on a Perkin-Elmer Optima 5300UV (Perkin Elmer, MA,USA). ICP–AES calibration standards for Ca, Si, Ti and Na/Sr ions wereprepared from a stock solution on a gravimetric basis. Three targetcalibration standards were prepared for each ion and de-ionized waterwas used as a control.

Fig. 1. XPS survey scan of a.) Ly-N, b.) Ly-C and c.) Ly-S.

Fig. 2. SEM images of a.) Ly-N, b.) Ly-C and c.) Ly-S glass particles and particle size analysis data (scale bar = 20 μm).

27Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

28 Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

2.4. pH analysis

Changes in the pH of solutions were monitored using a Corning 430pH meter. Prior to testing, the pH meter was calibrated using pH buffersolution 4.00 ± 0.02 and 7.00 ± 0.02 (Fisher Scientific, Pittsburgh, PA).Sample solutions were prepared by exposing disc samples (n = 3amorphous and n = 3 crystalline) in calculated quantities of sterilede-ionized water. Measurements were recorded at 1, 7 and 30 days.Sterile de-ionized water was used as a control and was measured ateach time period.

2.5. Hardness testing

Hardness testing was completed on the same specimens(1.5 × 6 ϕmm) used for ICP and pH testing where 10 measurementswere taken per disc and 3 discs were used for each composition (totaln = 30/sample). Samples were tested after 1, 7 and 30 days immersionin sterile de-ionized water at 37 °C (Fig. 9). A Shimadzu HMV-2000Hardness testing machine was used with a 500 g load cell with 15 s in-tervals. Each disc (amorphous and crystalline analogues of Ly-N, Ly-Cand Ly-S) were mounted in epoxy resin and polished using 600 gritsilicon carbide polishing paper. Ten Vickers indentations at a load of500 g and a dwelling timeof 15 sweremade to each disc using a univer-sal hardness machine (HMV-200, Shimadzu, MD, USA). Using the at-tached light microscope and computer, the diagonals created by theVickers diamond indenter were measured and the VHN was calculatedusing Eq. (1).

Hv ¼ 1:854Fd2

ð1Þ

where:

F the applied load (kgf).d diagonal length (mm).

Fig. 3. X-ray diffraction patterns of Ly-S, Ly-C and Ly-N of the a.) amo

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was conducted using SPSSStatistical Software Ver. 17.0 2008 to compare the changes in ion re-lease, pH and hardness of the experimental materials in relation to1) maturation of each composition and structure (amorphous andcrystalline) over 1, 7 and 30 days immersion in sterile de-ionizedwater, 2) differences in structure i.e. amorphous and crystalline ateach individual time period i.e. 1 day, 7 day and 30 day. Comparisonof relevant means was performed using the post hoc Bonferroni test.Differences between groups were deemed significant when p ≤ 0.05.Correlation coefficients were calculated using OriginPro 8SR2 2008and were determined to investigate any relationship between ionrelease rate and maturation i.e. 1, 7 and 30 days.

3. Results

3.1. Composition and structural characterization

Initial characterization was conducted to confirm the batch compo-sitions of the starting glasses, where each glass composition was deter-mined using X-ray photoelectron spectroscopy (XPS) and is presentedin Table 1. Additionally Fig. 1 presents the XPS survey scans of each ma-terial. Fig. 1a presents the survey scan of Ly-N which contains Na, O, Ti,Ca and Si with minor traces of carbon (C). Fig. 1b shows the survey scanof Ly-C which contains Na, O, Ti, Ca, Sr and Si, with minor traces of C.Fig. 1c presents the survey scan of Ly-S which contains O, Ti, Ca, Srand Si also with traces of C. The glass particles were analyzed usingscanning electron microscopy (SEM) and particle size analysis (PSA)and are presented in Fig. 2. PSA determined the mean particle size tobe 4.6 μm for Ly-N, 3.9 μm for Ly-C and 4.6 μm for Ly-S. d10 (2.1–2.3 μm), d50 (3.2–3.9 μm) and d90 (6.4–8.1 μm) values were alsodetermined by PSA for each material.

X-ray diffraction (XRD) patterns for each material are presented inFig. 3 after heat treatment. Table 2 lists the crystal phases for each ofthe crystalline forms of Ly-N, Ly-C and Ly-S. Ly-N and Ly-C contain

rphous and b.) crystalline structures (crystal phases see Table 3).

Fig. 4. SEM images of each disc after 1 day immersed in water, representing Ly-N (a and b), Ly-C (c and d) and Ly-S (e and f) (scale bar = 50 μm).

29Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

sodium calcium silicate phases (combeite) in addition to SiO2. Ly-S wasfound to contain multiple crystal phases and each is listed in Table 2.Additionally, the crystal size for each phase was calculated and for Ly-N the mean crystal size was 601 Å, for Ly-C the crystal size exceeded1000 Å and for Ly-S the mean crystal size was 348 Å. Scanning electronmicroscopy (SEM) backscattered electron imaging of the polished sur-faces of the amorphous and crystalline discs are presented in Fig. 4.Fig. 4a and b show the amorphous and crystalline analogues of Ly-N,and it is evident that the surface of the amorphous Ly-N is similar tothe crystalline analogue. Fig. 4c and d present the Ly-C surfaces. It is ev-ident from Fig. 4c that the irregular large and smaller glass particulatesare not sintered to the same extent as the crystalline analogue or Ly-Namorphous (Fig. 4a). Fig. 4c presents a similar situation where Ly-Samorphous (Fig. 4c) shows a more porous surface than the crystallinecounterpart.

3.2. Effect of material structure on ion release and solubility

Ion release profiles were determined for Ly-N, Ly-C and Ly-S withrespect tomaturation for both the amorphous and crystalline analoguesof each composition, additionally the relevant statics are presented inTable 3. Fig. 5 presents the ion release profile for Ly-N amorphous.With respect to the amorphous structure, Na+ levels increase from122 to 216 mg/L over 1–30 days (p = 0.089). Si4+ also had a high re-lease rate ranging from 61 to 167 mg/L over the same time period(p = 0.010). However, Ca2+ levels did not increase with respect tomaturation (p = 1.000), and ranged from 5 to 7 mg/L even after30 days immersion in water. The crystalline analogues are presentedin Fig. 5b, where Na+ increased from 4 to 19 mg/L over 1–30 days(p = 0.000), Si4+ ranged from 6 to 29 mg/L over the same time period(p = 0.000); however Ca2+ levels ranged from 4 to 10 mg/L and also

Fig. 5. Ion release profiles of Ly-N a.) amorphous and b.) crystalline investigating Ca, Si and Na release with respect to maturation i.e. after 1, 7 and 30 days.

30 Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

did not experience a significant change with respect to maturation(p = 0.000). Fig. 6a presents the Ly-C amorphous considering Ca, Si,Na and Sr release. Ca2+ concentration remained relatively constantranging from 13 to 17 mg/L over 1–30 days (p = 1.000). Si4+ levelsranged from 32 to 69 mg/L (p = 0.000) and Na+ levels ranged from15 to 36 mg/L (p = 0.000). Sr2+ levels decreased from 17 to 9 mg/Lover the same time period (p = 0.020). Fig. 6b presents the Ly-C crys-talline where Ca2+ levels increased over 1–30 days ranging from 2 to9 mg/L (p = 0.000). Si4+ levels also increased from 3 to 16 mg/L(p = 0.000). Na+ presented relatively little change from 2 to 5 mg/L(p = 0.000) and Sr2+ increased from 1 to 8 mg/L over the same timeperiod (p = 0.000). When comparing the release of each ion with re-spect to structure (amorphous vs. crystalline) at each time period,only Ca at 1 day (p = 0.515) and 30 days (p = 0.457), and Sr2+ at30 days (p = 0.179) did not show a significant change. Fig. 7a showsthe ion release profiles for the Ly-S amorphous. Ca2+ levels rangedfrom 14 to 17 mg/L (p = 1.000). Si4+ levels increased from 28 to64 mg/L (p = 0.000) while Sr2+ levels also remained relatively con-stant, ranging from 26 to 35 mg/L over 1–30 days (p = 1.000). Fig. 7bpresents Ly-S crystalline where Ca2+ levels increased from 1 to 4 mg/L,Si4+ increased from 2 to 5 mg/L, Sr2+ increased from 1.5 to 4 mg/Lover the time period of 1–30 days, each of which experienced a signifi-cant change (p = 0.000). When comparing the amorphous and crystal-line levels at each time period it was found to be significantly differentfor each ion at each time frame (p = 0.000–0.007). R2 values were cal-culated for each material as a function of maturation and the results

Fig. 6. Ion release profiles of Ly-C a.) amorphous and b.) crystalline investigating C

are presented in Table 4. The pH of each of the liquid extracts was mea-sured after 1, 7 and 30 days. Fig. 8 presents changes in pH of the amor-phous (Fig. 8a) and crystalline (Fig. 8b) analogues. From Fig. 8a it isevident from Ly-N that the pH increased from 11.6 to 12.4 after30 days incubation time. The pH range attributed to Ly-C ranged from11.0 to 11.7 after 30 days and Ly-S ranged from 10.9 to 11.4 over 1–30 days. The crystalline analogues are presented in Fig. 8b where Ly-Nranged from 10.3 to 11.2, Ly-C ranged from 9.4 to 10.3 and Ly-S rangedfrom 8.9 to 9.6 over the period of 1–30 days.

3.3. Effect of material solubility on mechanical durability

The results of the hardness testing are presented in Fig. 9. Fig. 9apresents the Ly-N amorphous; the hardness significantly reduced from2.6 to 1.0 GPa after 30 days (p = 0.000). Ly-C also experienced a similartrendwhere the hardness reduced from3.0 to 0.9 GPa (p = 0.000) overthe same time period and Ly-S, which had the highest initial hardness at3.4 GPa, reduced to 1.7 GPa after 30 days (p = 0.000). Fig. 9b presentsthe hardness testing of the crystalline samples. From Fig. 9b it is evidentthat there is relatively little difference in hardness with respect tomaturation. Ly-N attained a hardness that decreased from 2.0 to1.7 GPa over the period of 1–30 days. Ly-C experienced the highesthardness values which also presented an insignificant decrease from6.0 to 5.9 GPa over the same time period and Ly-S ranged from 4.8 to4.1 GPa after 30 days.

a, Si, Na and Sr release with respect to maturation i.e. after 1, 7 and 30 days.

Fig. 7. Ion release profiles of Ly-S a.) amorphous and b.) crystalline, investigating Ca, Si and Sr release with respect to maturation i.e. after 1, 7 and 30 days.

31Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

4. Discussion

4.1. Composition and structural characterization

It is known that the addition of networkmodifiers such as Ca2+ andNa+ to bioactive glasses encourages dissolution of the glass by promot-ing the formation of non-bridging oxygen species (NBOs) [20]. This pro-motes changes in surface chemistry which can lead to a precipitatedcalcium phosphate surface layer that subsequently crystallizes to crys-talline hydroxyapatite [7,26]. Initial characterization included X-rayphotoelectron spectroscopy (XPS) in order to confirm the glass compo-sitions and to identify any contaminants present (Fig. 1). Table 1presents the starting composition of the glass and the composition asdetermined by XPS. Each individual glass composition (Ly-N, Ly-C andLy-S) was analyzed on a fracture surface in a pristine environmentand confirms the original glass compositions. One observation that canbemade in Table 1 is the excess of Na+detected byXPSwhen comparedto the original compositions in Ly-N and Ly-C. This can be attributed toNa+ being a mobile ion that tends to migrate to fracture/exposed sur-faces on glass. SEM imaging presents a similar particle size of 3.9–4.5 μm for each glass. It is evident from Fig. 2 that a number of particlesare in excess of 20 μm; however, the majority of the particles are muchsmaller and are agglomerated. X-ray diffraction (XRD) patterns confirmthe amorphous structure of Ly-N, Ly-C and Ly-S (Fig. 3a) and the corre-sponding crystalline analogues (Fig. 3a) after heat treatment. Both Ly-N

Fig. 8. pH of extracts from both the a.) amorphous and b.) crystallin

and Ly-C contain Na2Ca2Si3O9, which is the principal crystal phase ofcrystalline Bioglass® [18,19]. Ly-S presented multiple crystal phaseswhich are presented in Table 2. With respect to the mean crystal size,Ly-S presented the smallest crystal size at 348 Å; Ly-N presented amean crystal size of 601 Å, while the crystals present in Ly-C were thelargest and in excess of 1000 Å.

Post heat treatment, polished samples weremounted and imaged todetermine any differences in sintering (Fig. 4). The difference insintering and porosity of the Ly-C and Ly-S amorphous samples mayhave an influence on the solubility. The increase in porosity of thesesamplesmay lead to an increase in exposed surface area and a reductionin density. The lower porosity of Ly-N amorphous when compared tothe Ly-C and Ly-S amorphous samples is likely attributed to the compo-sition of the glass, as the starting particle size for each material wassimilar. Higher Na+ concentration may result in greater diffusion ofthe atomic constituents between particles resulting in a more densematerial. For each of the crystalline samples, pores in the surface areevident in addition to polishing abrasions; however, it is evident thatthe crystalline samples have a greater density/interconnectivity thanthe amorphous analogues.

4.2. Effect of material structure on ion release and solubility

One issuewith the use of Bioglass® andother bioactive glasses is thatthe local biological microenvironment can be influenced significantly by

e samples with respect to maturation i.e. after 1, 7 and 30 days.

Fig. 9. Hardness testing of sintered discs from both a.) amorphous and b.) crystalline samples with respect to maturation i.e. after 1, 7 and 30 days.

32 Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

the materials degradation process. Increases in the concentration ofions such as Na+ and Ca2+ can result in changes in pH [27]. The biolog-ical effect of these changes can be difficult to predict from in vitro exper-iments; however, there are numerous reports in the literature on theeffect of specific ions released from bioactive glasses and the resultingtherapeutic effect. Na+ and Ca2+ in particular are known to be essentialin the precipitation of a crystalline hydroxyapatite surface layer [7,28].Si4+ is also released in the form of silicic acid (SiOH)4 during degrada-tion and is also known to contribute to enhancing bioactivity of glasses[7]. Studies by Tousi et al. on the combinatorial release of Si4+ and Ca2+

from bioactive glasses reports enhanced osteoblast bio-mineralizationthrough enhanced osteocalcin (OCN) expression [29]. Additionally,studies using animal models over a period of 7 months revealed thatSi4+ is harmlessly excreted in a soluble form through the urine [27].With regard to this study, ion release profiles were conducted for boththe amorphous and crystalline analogues of each material and with re-spect to maturation. Statistical comparisons were conducted based oneach individual elements release rate with respect to maturation ofboth the amorphous and crystalline analogues of each composition(1 day vs. 30 day, Table 3a) and also by comparing ion release as a func-tion of structure (amorphous and crystalline structure) at each time pe-riod (Table 3b). From Fig. 5a it is initially evident that the overall releaserates for the amorphous compositions aremuchhigher than the crystal-line counterparts, in particular with respect to Na+ and Si4+. Whencomparing the amorphous and crystalline release rates for each ion ateach time frame, both Na+ and Si4+ experienced a significant changeat each time period (p b 0.05, Table 3b); however, Ca2+ release wasnot significantly different (p N 0.05) at each time point. The high Na+

concentrations likely de-polymerizes the Si–O–Si network resulting ina highly soluble glass, which explains the high Na+ (216 mg/L) release,which is similar to Na+ levels from Bioglass which ranged from 190 to270 mg/L after 30 days [30]. Previous studies by Chen et al. on Na2O

Table 2Crystal phases identified for Ly-N, Ly-C and Ly-S (see Fig. 3).

Phase ID

Ly-N Combeite — Na2.2Ca1.9 Si3O9

Sodium— Na2Ca3Si6O16

Ly-C Combeite — Na4.8Ca3Si6O18

Silicon dioxide — SiO2

Ly-S Strontium silicon — Sr2Si3Titanium oxide— Ti8O15

Calcium silicon — CaSi2Strontium silicide— SrSiStrontium titanium silicate — Sr2TiSi2O8

Silicon oxide — SiO2

Perovskite — CaTiO3

containing bioactive glasses/glass-ceramics confirmed the enhancedbioactivity of Na2O containing bioactive glass-ceramics over 70Si–30Ca glass-ceramics. The addition of Na2O encourages biodegradableNa–Ca–Si–O phases that enhances bioactivity [17]. Na2O free glassespredominantly produce inert crystalline phases such as CaSiO3. Also,the amorphous analogue of 70Si–30Ca was found to present minor hy-droxyapatite precipitation when immersed in SBF [17]. Si4+ release(167 mg/L) levels for Ly-N were higher than Bioglass® (3.5–43 mg/Lover 1–30 day [30]) and the relatively low Ca2+ release experiencedin each starting glass may be attributed to the inclusion of Ti in theglass. The Ti4+ ion has a small ionic radius and a large electric chargeand they can be incorporated into the Si network (Si–O–Ti). This is like-ly the reason that no Ti4+ is released from these glasses. Previous stud-ies by Talos et al. on similar compositions suggest that the inclusion ofTi4+ has no effect on Si, but does restrict P and Ca release [31]. With re-gard to this study Ca2+ levels increase in Ly-C and Ly-N to a maximumof 17 mg/L after 30 days (similar to Bioglass® at 7.5–17 mg/L over 1–30 day [30]), which may be due to the incorporation of Sr2+. Sr2+

and Ca2+ are known to have similar charge and ionic radius whichenables direct substitution [32], and as such, Sr2+ may be partially ful-filling the role of Ca2+ as a charge compensator. However, further char-acterization studies will be required to prove this. The crystallineanalogues for each material present much lower release levels whichcan be attributed to the formation of relatively insoluble crystallinestructures post heat treatment. The highest release rates are presentwith the incorporation of Na+ (Ly-N, Ly-C), where Ca2+ and Sr2+ levelswere b10 mg/L for each material after 30 days. The correlation coeffi-cient for each material was analyzed to determine if the ion releaserate for each element presented a linear increase with respect to time(1, 7 and 30 days). The crystalline version of each material producedconsistently higher R2 values than the amorphous analogues (Table 4)which may be attributed to the long range atomic order of crystalline

Reference code Crystal size (Å)

(Ref: 04-04-2757) 612(Ref: 04-012-8681) 591(Ref: 04-007-5453) N1000(Ref: 04-007-5453) N1000(Ref: 01-089-2593) 348(Ref: 04-007-0444) 138(Ref: 04-007-0647) 229(Ref: 01-076-7303) 317(Ref: 04-006-7366) 261(Ref: 00-029-0085) N1000(Ref: 04-015-4851) 229

Table 4R2 values calculated for each element with respect to maturation.

Calcium Silica Sodium Strontium

Amor. Crys. Amor. Crys. Amor. Crys. Amor. Crys.

Ly-N −0.31 −0.34 0.21 0.89 0.81 0.93 – –

Ly-C 0.96 0.89 0.43 0.86 0.66 0.84 0.77 0.85Ly-S −0.40 0.93 0.40 0.91 – – −0.96 0.98

Table 3a.) Means comparison of each ion release profiles, including amorphous and crystallinestructures (in parentheses), with respect to maturation, and b.) comparison of each ionat 1 day, 7 day and 30 day with respect to structure.

a. Maturation b. Amorphous vs. crystalline

Ion 1 day vs. 30 day 1D 7D 30D

Ly-N Ca 1.000 (0.240) 0.476 1.000 1.000Na 0.089 (*0.000) *0.003 *0.000 *0.001Si *0.010 (*0.000) *0.000 *0.000 *0.001

Ly-C Ca 1.000 (*0.000) *0.001 0.515 0.457Na *0.001 (*0.000) *0.000 *0.000 *0.000Si *0.000 (*0.000) *0.000 *0.000 *0.000Sr *0.020 (*0.000) *0.000 *0.001 0.179

Ly-S Ca 1.000 (*0.000) *0.000 *0.000 *0.000Si *0.000 (*0.000) *0.000 *0.000 *0.000Sr 1.000 (*0.000) *0.000 *0.000 *0.007

p ≤ 0.05* indicates significance.

33Y. Li et al. / Journal of Non-Crystalline Solids 380 (2013) 25–34

materials, or the lower release rates determined for the crystalline ana-logues. Ca2+ release for each material proved to have lower R2 valueswith the exception of Ly-C where both the amorphous and crystallineversion experienced high R2 values, 0.96 and 0.89 respectively.With re-spect to Si4+, the crystalline version of eachmaterial produced R2 valuesabove 0.85 whereas the amorphous analogues for eachmaterial rangedbetween 0.21 and 0.43. Similarly, both the Na+ crystalline (0.84–0.93)and the Sr2+ crystalline (0.85–0.98) sample sets produced higherR2 values than the amorphous counterparts (Na+ 0.66–0.81 and Sr2+

−0.96 to 0.77). Regarding Ly-N the increase in pH closely follows Na+

release from this glass, which ranges from 122 to 216 mg/L, a similarobservation to previous studies by Chen et al. [17]. The pH range attrib-uted to Ly-C is slightly lower with a narrower distribution ranging from11.0 to 11.7 after 30 days, which is also likely due to the lower Na+

release which ranges from 15 to 36 mg/L. The lower release rates andrelatively little change in pH associated with Ly-S can be attributed tothe insignificant changes in alkali earth ion release Ca2+ 16–17 mg/Land Sr2+ 35–31 mg/L over 1–30 days. With respect to maturation(1 day vs. 30 day), there was a significant increase in pH for each ele-ment and for each structure (p = 0.000–0.022). Similarly, when com-paring the amorphous and crystalline pH values at each time period,there was also found to be a significant change for each sample tested(p = 0.000). Additionally, ion release and pH changes in crystallized45S5 Bioglass® followed a similar trend to the experimental glasses/glass-ceramics investigated here, where the crystalline versions experi-enced lower ion release rates and pH changes when compared tothe amorphous analogues [17]. Previous studies cite pH values forBioglass® ranging from 9 to 11, similar to the values cited here [17].Additionally, studies on the osteoconductivity of crystalline materialssuch as synthetic hydroxyapatite (HA) and apatite–wollastonite glass-ceramics (AW-GC) have determined both materials to bond to chemi-cally to bone through a bone-like apatite layer. In order to comparethe osteoconductive potential of each material, the affinity index, orthe percentage of bone at the material-tissue interface is considered areasonable parameter for comparison [33]. Regarding this study, theosteoconductive potential of the crystalline materials (glass-ceramics)will be investigated using simulated body fluid (SBF), which will beconducted in a follow up study.

4.3. Effect of material solubility on mechanical durability

Since bioactive glasses were developed one persistent challenge re-lated to their interactionwith bodyfluids is due touncertainty related totheir chemo-mechanical (changes inmechanical properties due to reac-tions with a biological environment) behavior and mechano-chemical(effect of mechanical strain on dissolution rate) behavior. As a result,there still exists a lack of understanding of the relationship betweenthe mechanical properties of bioactive glasses and their bioactivity[25]. For this work, hardness testing (Fig 9) was conducted in order to

determine any changes inmechanical durability as a function of incuba-tion time in an aqueous environment, and also as a function of structure(amorphous vs. crystalline). It is evident from Fig 9 that each of theglassy materials experiences a significant reduction in hardness after30 days (p = 0.000). This reduction in hardness can be attributed tothe dissolution of the glass particles, which is evident by the ion releaseand pH profiles of the amorphous materials. In contrast to this finding,the crystalline counterparts did not experience a significant reductionin hardness after 30 days. The highest hardness values were attributedto Ly-C (6.0 GPa) and Ly-S (4.8 GPa). These higher values both containSiO2 crystal phases with a crystal size N1000 Å, which may be relatedto the higher strengths. Previouswork suggests that Na2Ca2Si3O9 phasesincrease the mechanical stability of the ceramic 45S5 Bioglass®. Fromthis study it is evident that the mechanical strength increases 3-foldwith the addition of Sr2+. The increase in hardness was particularly ev-ident in Ly-C when both Na+ and Sr2+ are present suggesting that thisintermediate glass/glass-ceramic may present a high strength, durableglass-ceramic that can aid in the long-term bone repair process.

5. Conclusion

This study was conducted to determine the solubility and mechani-cal stability of a series of bioactive glasseswith respect toNa+/Sr2+ con-tent, incubation time and crystallization. Ion releasewas higher for eachof the amorphousmaterials with a corresponding elevation in pH; how-ever, the solubility negatively affects the mechanical durability as thereis a significant reduction in hardness over time. The crystalline counter-parts experienced a lower ion release rate and pH but proved to be farmore stable in an aqueous environment with respect to time. This sug-gests that crystallization can be used to control the solubility and pro-long the mechanical durability of the bioactive glasses/glass-ceramics.Future work on these particular materials will include simulated bodyfluid testing and cell culture analysis to determine the biological effectof these structures, in addition to controlling the amorphous/crystallinecontent by quantitative X-ray diffraction and determining the solubilityas a function of time.

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