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Bioceramics in Simulated Body Fluid

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Bioceramics in Simulated Body Fluid

  1. 1. 1 23 Journal of Materials Science: Materials in Medicine Official Journal of the European Society for Biomaterials ISSN 0957-4530 J Mater Sci: Mater Med DOI 10.1007/s10856-014-5229-x Investigating the surface reactivity of SiO2– TiO2–CaO–Na2O/SrO bioceramics as a function of structure and incubation time in simulated body fluid Y. Li, A. Coughlan & Anthony. W. Wren
  2. 2. 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.
  3. 3. Investigating the surface reactivity of SiO2–TiO2–CaO–Na2O/SrO bioceramics as a function of structure and incubation time in simulated body fluid Y. Li • A. Coughlan • Anthony. W. Wren Received: 21 January 2014 / Accepted: 21 April 2014 Ó Springer Science+Business Media New York 2014 Abstract This study focuses on evaluating the biocom- patibility of a SiO2–TiO2–CaO–Na2O/SrO glass and glass– ceramic series. Glass and ceramic samples were synthe- sized and characterized using X-ray diffraction. Each material was subject to maturation in simulated body fluid over 1, 7 and 30 days to describe any changes in surface morphology. Calcium phosphate (CaP) deposition was observed predominantly on the Na? containing amorphous and crystalline materials, with plate-like morphology. The precipitated surface layer was also observed to crystallize with respect to maturation, which was most evident in the amorphous Na? containing glasses, Ly-N and Ly-C. The addition of Sr2? greatly reduced the solubility of all sam- ples, with limited CaP precipitation on the amorphous samples and no deposition on the crystalline materials. The morphology of the samples was also different, presenting irregular plate-like structures (Ly-N), needle-like deposits (Ly-C) and globular-like structures (Ly-S). Cell culture analysis presented a significant increase in cell viability with the Na? materials, 134 %, while the Sr2? containing glasses, 60–80 % and ceramics, 60–85 % presented a general reduction in cell viability, however these reduc- tions were not significant. 1 Introduction In recent years bioactive glasses and ceramics have stim- ulated interest as materials that can stimulate the regener- ation of bone tissue [1]. A common and widely studied characteristic of bioactive glasses and glass–ceramics if the formation of a biologically active apatite (A) layer which supports bone bonding which can be evaluated using simulated body fluid (SBF), which is a solution that con- tains an ionic composition similar to that of human blood plasma [2, 3]. Prior to the 1970s, artificial materials that were implanted into the human body, specifically bone defects, resulted in the materials being encapsulated by fibrous tissue which resulted in the materials isolation for the surrounding bone. In the early 1970s, Hench [4, 5] produced glass in the Na2O–CaO–SiO2–P2O5 system that spontaneously bonds to living bone without the formation of surrounding fibrous tissue. Since this development, many different types of ceramics such as sintered hydroxyapatite, sintered b-tricalcium phosphate, A/b-tri- calcium phosphate biphasic ceramics and glass/ceramic A– W (wollastonite) have been shown to bond to living bone [2]. Additionally, many different materials have been pro- duced from bioactive glass and ceramics that can be used for numerous medical applications. These materials include glass–ceramic scaffolds [6–8] for bone repair, glass microspheres for cancer treatment [9–11], composite materials for drug release [12, 13] and also composite materials where bioactive glasses are used to improve bioactivity or mechanical strength [14–17]. Bioactive glass and ceramics are a valuable addition to medical materials as they can incorporate biological compatibility with mechanical strength and bone adhesion, vital components for skeletal repair [18]. However, uncertainties still exists Y. Li Á Anthony. W. Wren (&) Inamori School of Engineering, Alfred University, Alfred, NY 14802, USA e-mail: wren@alfred.edu A. Coughlan School of Materials Engineering, Purdue University, West Lafayette, IN, USA 123 J Mater Sci: Mater Med DOI 10.1007/s10856-014-5229-x Author's personal copy
  4. 4. relating to the mechanical durability due to their interac- tions within the biological environment, and also the effect of mechanical strain and the dissolution rate of materials, and as such, altering the mechanical properties of a bio- active glass without compromising its bioactivity is of key interest [18]. This concern has previously been highlighted when producing glass–ceramic scaffolds from 45S5 Bio- glassÒ , where it has been suggested that crystallization of 45S5 BioglassÒ can reduce its bioactivity as, post sintering, crystallization turns the material from being a bioactive material into an inert ceramic [19]. However, there have since been studies that suggest the predominant crystal phase of 45S5 BioglassÒ (Na2Ca2Si3O9), can significantly improve the mechanical properties of the material, and that crystallization does not inhibit the bioactivity as precipi- tation of an amorphous calcium phosphate (CaP) occurs in biological fluids [20–22]. This study was conducted to determine any differences in bioactivity and subsequent changes in surface mor- phology, specifically in SBF and cell culture, as a function of material composition (Na? /Sr2? concentration), incu- bation time, and structure (amorphous/crystalline). The starting glass composition, SiO2–TiO2–CaO–Na2O/SrO, alters Na? and Sr2? concentration which will affect the dissolution rate of the glasses. A previous study by the authors on the solubility of these materials determined that (1) crystallization greatly reduces the ion release rate, (2) pH is also reduced with crystallization, (3) the mechanical durability of the materials is greatly enhanced by crystal- lization where no significant changes are observed after 30 days in aqueous media and (4) the Na? containing materials produces higher ion release rates than the Sr2? analogues. The glass composition utilized in this study was selected as Na? is critical in promoting dissolution of the glass as it acts as a network modifier within the glass network [22, 23]. Sr2? also acts as a network modifier within the glass, however, it shares atomic similarities to Ca2? and has been previously investigated and applied to treating postmenopausal women with osteoporosis [24, 25]. Titanium (Ti) has been incorporated as numerous studies cite that Ti containing materials result in the deposition of CaP surface layer when incubated in SBF [26]. That can be attributed to the formation of Ti–OH groups providing favourable conditions for Ca precipitation [26]. 2 Materials and methods 2.1 Glass synthesis Three glass compositions (Ly-N, Ly-C, Ly-S) were formu- lated for this study with the principal aim being to inves- tigate any property changes with the modification of sodium (Na? ) and strontium (Sr2? ) in the glass. A control glass (Ly-C) was also formulated which contained equal quantities of Na? and Sr2? . Glasses were prepared by weighing out appropriate amounts of analytical grade reagents and ball milling (1 h, Table 1). 2.1.1 Glass powder production The powdered mixes were oven dried (100 °C, 1 h) and fired (1,500 °C, 1 h) in platinum crucibles and shock quenched into water. The resulting frits were dried, ground and sieved to retrieve glass powders with a maximum particle size of 45 lm. 2.1.2 Disc sample preparation Disc samples (Ly-N, Ly-C, Ly-S) were prepared by weighing approximately 0.5 g powder into a stainless steel die (sample dimensions 1.5 9 6/ mm) which was pressed under 3 tonnes of pressure. Disc samples were kept amorphous for Ly-N, Ly-C, Ly-S by heat treating the pressed discs below the glass transition temperature for 24 h, and crystalline analogues were heat treated to the sintering temperature for Ly-N (653 °C), Ly-C (713 °C) and Ly-S (825 °C) in order to determine any differences in bioactivity as a result of structure. Disc samples were then used for SBF testing and while 100 ll extracts were used for indirect cell culture analysis. 2.2 Glass characterization 2.2.1 X-ray diffraction (XRD) Diffraction patterns were collected using a Siemens D5000 XRD Unit (Bruker AXS, Inc., WI, USA). Glass powder samples were packed into standard stainless steel sample holders. A generator voltage of 40 kV and a tube current of 30 mA was employed. Diffractograms were collected in the range 10° 2h 80°, at a scan step size 0.02° and a step time of 10 s. Any crystalline phases present were identified using JCPDS (Joint Committee for Powder Dif- fraction Studies) standard diffraction patterns (data repro- duced from previously published manuscript [27], Fig. 1; Table 3). Table 1 Glass composition (mol. fr.) Ly-N Ly-C Ly-S SiO2 0.55 0.55 0.55 TiO2 0.05 0.05 0.05 CaO 0.22 0.22 0.22 Na2O 0.18 0.09 0.00 SrO 0.00 0.09 0.18 J Mater Sci: Mater Med 123 Author's personal copy
  5. 5. 2.2.2 Hot stage microscopy (HSM) A MISURA side view HSM, Expert Systems (Modena, Italy), with image analysis system and electrical furnace, with max temperature of 1,600 °C and max rate of 80 °C/ min. The parameters for this experiment were a heat rate of 20 °C/min from 20 to 500 °C and 5 °C/min from 500 to 1,200 °C. The computerized image analysis system auto- matically records and analyses the sample geometry during heating. 2.2.3 Scanning electron microscopy and energy dispersive X-ray analysis (SEM–EDS) Backscattered electron imaging was carried out with an FEI Co. Quanta 200F Environmental SEM. Additional compositional analysis was performed with an EDAX Genesis Energy-Dispersive Spectrometer. All EDS spectra were collected at 20 kV using a beam current of 26 nA. Quantitative EDS spectra were subsequently converted into relative concentration data. Fig. 1 Scanning electron microscopy of a Ly-N, b Ly-C and c Ly-S including corresponding EDX and quantitative composition in mol% J Mater Sci: Mater Med 123 Author's personal copy
  6. 6. 2.3 Biocompatibility testing 2.3.1 Sample preparation Discs of each glass were autoclaved prior to use and sterile de-ionized water was used as the solvent to prepare extracts. The volume of extract was determined using Eq. 1. Vs is the volume of extract used, Sa is the exposed surface area of the disc. Vs ¼ Sa 10 : ð1Þ Samples (n = 3 amorphous, and n = 3 crystalline) were aseptically immersed in appropriate volumes of sterile de- ionized water and agitated at (37 ± 2 °C) for 1, 7 and 30 days after which 100 ll of fluid was used for cytotox- icity testing. 2.3.2 SBF trial SBF was produced in accordance with the procedure out- lined by Kokubo and Takadama [2]. The composition of SBF is outlined in Table 2. The reagents were dissolved in order, from reagent 1 to 9, in 500 ml of purified water using a magnetic stirrer. The solution was maintained at 36.5 °C. 1 M-HCl was titrated to adjust the pH of the SBF to 7.4. Purified water was then used to adjust the volume of the solution up to 1 l. Glass/ceramic discs (n = 2) were immersed in concentrations of SBF as determined by Eq. 1 and were subsequently stored in for 1, 7 and 30 days in an incubator at 37 °C. A JEOL JSM-840 SEM equipped with a Princeton Gamma Tech energy dispersive X-ray (EDX) system was used to obtain secondary electron images and carry out chemical analysis of the surface of glass and ceramic discs. All EDX spectra were collected at 20 kV, using a beam current of 0.26 nA. Quantitative EDX con- verted the collected spectra into concentration data by using standard reference spectra obtained from pure ele- ments under similar operating parameters. 2.3.3 Cell culture analysis The established cell line L929 (American Type Culture Collection CCL 1 fibroblast, NCTC clone 929) was used in this study as required by ISO10993 part 5. Cells were maintained on a regular feeding regime in a cell culture incubator at 37 °C/5 % CO2/95 % air atmosphere. Cells were seeded into 24 well plates at a density of 10,000 cells per well and incubated for 24 h prior to testing with both extracts and cement discs. The culture media used was M199 media supplemented with 10 % fetal bovine serum (Sigma Aldrich, Ireland) and 1 % (2 mM) L-glutamine (Sigma Aldrich, Ireland). The cytotoxicity of cement extracts was evaluated using the methyl tetrazolium (MTT) assay in 24 well plates. Aliquots (100 ll) of undiluted sample were added into wells containing L929 cells in culture medium (1 ml) in triplicate over 1, 7 and 30 days. Cement discs (n = 3 amorphous, and n = 3 crystalline) were placed in the plate wells and were tested after 24 h. Each of the prepared plates was incubated for 24 h at 37 °C/5 % CO2. The MTT assay was then added in an amount equal to 10 % of the culture medium volume/well. The cultures were then re-incubated for a further 2 h (37 °C/5 % CO2). Next, the cultures were removed from the incubator and the resultant formazan crystals were dissolved by adding an amount of MTT Solubilization solution [10 % Triton X-100 in acidic isopropanol (0.1 N HCI)] equal to the original culture medium volume. Once the crystals were fully dissolved, the absorbance was measured at a wavelength of 570 nm. Aliquots (100 ll) of tissue culture water were used as controls, and cells were assumed to have metabolic activities of 100 %. 2.4 Statistical analysis One-way analysis of variance was employed to compare the difference in cell viability as a function of maturation (1, 7 and 30 days) for each material tested. Comparison of relevant means was performed using the post hoc Bonfer- roni test. Differences between groups was deemed signifi- cant when P B 0.05. 3 Results 3.1 Material characterization Characterization techniques utilized for this study includes scanning electron microscopy (SEM/EDX), XRD and HSM. SEM and the corresponding EDX are presented in Fig. 1 for Ly-N, Ly-C and Ly-S. Figure 1a presents Ly- N which confirms the composition contains Si4? , Ca2? , Ti4? and Na? , which has a composition similar to batch Table 2 Ionic composition of SBF [2] Orders Reagents Amount 1 NaCl 7.996 g 2 NaHCO3 0.350 g 3 KCl 0.224 g 4 K2HPO4Á3H2O 0.228 g 5 MgCl2Á6H2O 0.305 g 6 1 M-HCl 40 ml 7 CaCl2 0.278 g 8 Na2SO4 0.071 g 9 NH2C(CH2OH)3 6.057 g J Mater Sci: Mater Med 123 Author's personal copy
  7. 7. calculations. Minor differences include Si4? slightly higher at 60 % and Na? at 11 %. Figure 1b presents Ly-C which has a composition similar to the batch calculations, addi- tionally, minor difference include Si4? at 57 % and Ca2? slightly lower than batch calculations at 19 %. Both Na? and Sr2? levels were determined to be 9 %, similar to batch calculations. Figure 1c presents Ly-S which also has a composition close to the original batch calculation. Very slight compositional differences include Si4? being 56 % and Ca2? being slightly lower than original calculations at 20 %. XRD patterns for each material are presented in Fig. 2 with the complete list of crystal phases and crystal size for Ly-N, Ly-C and Ly-S. Ly-N presented in Table 3. The crystal phases for Ly-C contain sodium calcium sili- cate phases (combeite) in addition to SiO2. Ly-S was found to contain multiple crystal phases and each is listed in Table 3, however as there is no Na? in the starting com- position, no sodium calcium silicate phases (combeite) exist. Additionally, the crystal size for each phase was calculated and for Ly-N the mean crystal size was 601 A˚ , for Ly-C the crystal size exceeded 1,000 A˚ and for Ly-S the mean crystal size was 348 A˚ . HSM data is presented in Fig. 3 and presents the sintering (Ts), softening (Tf) and melting (Tm) temperature of Ly-N, Ly-S and Ly-C. From Fig. 3 it is evident the thermal properties of each material change as the Na? /Sr2? concentration differs. Regarding Ly-N the Ts was found to be 653 °C, however as Sr2? is substituted the Ts increases to 713 °C (Ly-C) and 825 °C (Ly-S). Regarding the Tf it was found to decrease from 866 °C (Ly-N) to 745 °C (Ly-C) and to increase to 1,243 °C (Ly-S) as the Sr2? concentration increased. The Tm for Ly-N and Ly-C were similar at 1,068 and 1,115 °C, respectively, whereas Ly-S was found to be 1,244 °C. 3.2 Evaluation of surface dissolution and reactivity SBF testing was conducted on glass and ceramic disc samples with respect to (1) composition, (2) amorphous and crystalline structure and (3) incubation time, over 1, 7 and 30 days. With respect to Figs. 4, 6 and 8, images are presented at 1, 7 and 30 days for both the amorphous and crystalline analogues, and the corresponding EDX of the 30 day samples. Figure 4 presents the Ly-N SBF results where after 1 day incubation there was no CaP deposition present. It is evident from Fig. 4 that after 7 days Fig. 2 X-ray diffraction of a amorphous materials and b glass– ceramic materials Table 3 Crystal phases identified for Ly-N, Ly-C and Ly-S (see Fig. 1) Phase ID Reference codes Crystal size (A˚ ) Ly-N Combeite - Na2.2Ca1.9Si3O9 04-04–2757 612 Sodium: Na2Ca3Si6O16 04-012–8681 591 Ly-C Combeite: Na4.8Ca3Si6O18 04-007–5453 [1,000 Silicon dioxide: SiO2 04-007–5453 [1,000 Ly-S Strontium silicon: Sr2Si3 01-089–2593 348 Titanium oxide: Ti8O15 04-007–0444 138 Calcium silicon: CaSi2 04-007–0647 229 Strontium silicide: SrSi 01-076–7303 317 Strontium titanium silicate: Sr2TiSi2O8 04-006–7366 261 Silicon oxide: SiO2 00-029–0085 [1,000 Perovskite: CaTiO3 04-015–4851 229 Fig. 3 Hot stage microscopy testing of Ly-N, Ly-C and Ly-S present- ing sintering, softening and melting temperature for each material J Mater Sci: Mater Med 123 Author's personal copy
  8. 8. incubation, the amorphous Ly-N surface was completely covered in CaP. The crystalline counterpart did experience CaP deposition, however it was minimal compared to the amorphous Ly-N. However, after 30 days the surfaces of the amorphous and crystalline analogues of Ly-N were fully covered in CaP. The corresponding EDX detected the presence of phosphate at *9 and *4 wt% for the amor- phous and crystalline Ly-N, respectively. Figure 5 presents the 30 days SEM images of the amorphous and crystalline Ly-N at 1, 5 and 50k magnification. With respect to Ly-C (Fig. 6) the surface of the amorphous Ly-C is similar to Ly-N amorphous; however the crystalline counterpart lacks the porous surface presented by Ly-N crystalline. CaP deposition can be seen on the 7 days Ly-C amorphous and the surface is fully covered after 30 days. Corresponding EDX from 30 day samples presents high P levels in the Ly- C amorphous, however a relatively low P signal was present for Ly-C crystalline. In order to investigate any changes in the surface of the materials with respect to incubation time, XRD was con- ducted on the samples that presented complete coverage by CaP both after 30 days incubation in SBF. Figure 7 pre- sents diffraction patterns of Ly-N (amorphous and crystal- line) and Ly-C (amorphous) before and after 30 days immersion in SBF. From Fig. 7a is evident that after 30 days crystal peaks are forming from the initially glassy structure, which can be directly attributed to the deposition of CaP as the starting material is amorphous. Crystal peaks were identified as CaP (PDF 04-014-2292, Ca3(PO4)2) which suggests that the crystal phase is an immature form of hydroxyapatite. Figure 7b presents the crystalline ana- logue of Ly-N which is initially predominantly crystalline. However, after 30 days in SBF, the region corresponding to 5–30° 2h exhibit a relatively minor amorphous region in addition to the loss of a number of peaks in the 5–30° 2h, and between 50 and 75° 2h. This is indicative of a poorly Fig. 4 SEM images of amorphous and crystalline Ly-N after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces J Mater Sci: Mater Med 123 Author's personal copy
  9. 9. crystalline CaP surface layer which is predominantly amorphous with minor crystal formation. Attempts to identify any newly formed crystal regions were inconclu- sive. Figure 7c presents Ly-C amorphous which also pre- sents a number of minor crystal peaks which are present at 55 and 60° 2h. However, this was less pronounced than Ly-N amorphous which made phase identification difficult. Regarding Ly-S, SEM imaging and the corresponding 30 days EDX are presented in Fig. 8. With respect to the amorphous samples, CaP deposition can be observed at each time period (1, 7 and 30 days), however to a much lesser degree than the Na? containing glasses. It is evident from the EDX that after 30 days CaP is localized, and is not as prevalent in density as Ly-N amorphous and Fig. 5 Low and high magnification SEM images of Ly-N amorphous and crystalline samples after 30 days in SBF J Mater Sci: Mater Med 123 Author's personal copy
  10. 10. crystalline and Ly-C amorphous. With respect to the crystalline analogue of Ly-S, there was relatively minor detection of P at less than 0.5 wt% for each time period. When comparing the CaP structures formed with respect to each material (Fig. 9), it is evident that different mor- phologies exist for both Na? and Sr2? containing materials. Figure 9a presents Ly-N at 7 days displaying full surface coverage with highly irregular, plate-like crystal formation. Figure 9a presents the intermediate glass (Ly-C) at 7 days which predominantly displays needle-like crystals that seem to be growing epitaxially, in addition, it is evident that precipitation is beginning on the particles on the left of the same image. This may be the beginning the formation of plate-like crystal similar to the Ly-N as after 30 days the final morphology is similar to that of Ly-N at 30 days. With respect to Ly-S, CaP deposition presented spherical glob- ular like projections that were far less abundant that the plate-like projections presented on Ly-N and Ly-C. 3.3 Evaluation of cytocompatibility Regarding cell culture analysis, Fig. 10a presents the cytocompatibility of the amorphous samples while Fig. 10b presents the crystalline analogues. It is evident from Fig. 10a that Ly-N presented a steady increase in cell viability. The increase was not deemed significant at 1 day (94 %, P = 1.000) or 7 day (111 %, P = 0.900), however at 30 days a significant increase was observed (134 %, P = 0.0001). Ly-C amorphous remained relatively con- stant over each time period with no significant change (P = 0.037–0.197) when compared to the growing cell population. The Sr2? containing series, Ly-S, produced significantly lower cell viability at 1 day (70 %, P = 0.007) and 7 day (59 %, P = 0.0001) however, no significant change in viability (68 %) was observed at 30 days. With respect to the crystalline analogues there was no significant increase in cell viability for any of the Fig. 6 SEM images of amorphous and crystalline Ly-C after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces J Mater Sci: Mater Med 123 Author's personal copy
  11. 11. Fig. 7 XRD of samples with surface changes in SBF before and after 30 days incubation Fig. 8 SEM images of amorphous and crystalline Ly-S after 1, 7 and 30 days in SBF and 30 day EDX of the amorphous and crystalline surfaces J Mater Sci: Mater Med 123 Author's personal copy
  12. 12. materials. There was a significant decrease in viability for Ly-C at 7 days (59 %, P = 0.000), and 30 days (71 %, P = 0.001). Ly-S also presented an overall reduction in cell viability; however this change did not reach significance. 4 Discussion 4.1 Material characterization SEM coupled with EDX was used to image the glass particles and estimate the glass composition. The mean particle size was previously determined for each glass, which were similar at 4.6 lm for Ly-N, 3.9 lm for Ly-C and 4.6 lm for Ly-S [27]. Particle size distribution can significantly influence biocompatibility through chan- ges in exposed surface area and the subsequent particle dissolution rate. It can also be observed that the Na? containing glasses (Ly-N, Ly-C) were found to have smaller particles agglomerated to larger particles, in particular with Ly-N. This agglomeration of the Na? containing glasses may be due to electrostatic charge build up during pro- cessing. With respect to glass composition, the batch calculation closely resembled the data acquired by EDX. XRD was conducted on each material to confirm the amorphous/crystalline state and to determine the phases present in the crystalline materials. Sodium calcium silicate phases were found to exist in Ly-N and Ly-C, whereas numerous phases were detected in Ly-S (Table 3). Previous studies suggest that crystallization converts glass from being a bioactive to an inert material and, in the case of glass–ceramic scaffolds, the mechanical integrity is also compromised. However additional studies by Chen et al. [28] suggest that crystalline 45S5 BioglassÒ forms Na2- Ca2Si3O9 phases that can significantly improve the mechanical properties of the material, that crystallization does not inhibit bioactivity with respect to bone bonding ability and when immersed in body fluids the crystalline Na2Ca2Si3O9 decomposes and transits to amorphous CaP. Studies by Clupper and Hench [20] determined that the predominant crystal phase associated with BioglassÒ , Na2Ca2Si3O9 slightly decreases the formation kinetics of an A layer on BioglassÒ surface but did not totally suppress its formation. Further studies by Filho et al. [21] found that there is no compromise in bioactivity for the 45S5 glass– ceramic system even when 100 % crystalline. Their study Fig. 9 SEM images of CaP deposition on a Ly-N, b Ly-C and c Ly-S Fig. 10 Cell viability of amorphous and crystalline samples J Mater Sci: Mater Med 123 Author's personal copy
  13. 13. in particular focused on determining the surface precipi- tation reactions in SBF and determined that by inducing crystallinity (ranging from 8 to 100 %), the materials maintained their bioactivity in SBF [21]. HSM determined that differences in the thermal properties of the materials existed where the sintering temperature increases with an increase in Sr2? concentration, i.e. Ly-N Ly-C Ly-S. This may be due to the divalent nature of Sr2? , charge neutralizing intermittent non bridging oxygen sites in the glass, where two Na? atoms are required to fulfill the same vacancy. This would likely result in higher temper- atures to decompose the Ly-S glass particles to the extent required for sintering, as compared to Ly-N. 4.2 Evaluation of surface characteristics and reactivity With respect to the amorphous surface for each material, differences in surface morphology exist where the particles are fused together resulting in a ‘cobblestone’ like surface. The crystalline analogues for each material formed a por- ous/dense or irregular surface morphology relatively spe- cific to the materials composition. CaP deposition on the Ly-N supports earlier findings by Clupper and Hench, where the formation of an A layer was decreased but not totally suppressed. The higher resolution images (Fig. 5) confirm the complete coverage of both Ly-N amorphous and crystalline surfaces with CaP. Higher resolution SEM images (50k) present a floral-plate like CaP present for both materials, which is similar in structure to A deposition on A–W glass–ceramic [2]. This suggests that the CaP deposited is similar irrespective of amorphous/crystalline structure, with the only difference being the time required for this surface layer to deposit. Ly-C amorphous presented CaP deposition the covered the surface after 30 days. The lack of surface precipitation on Ly-C crystalline may be due to a number of factors. The lower Na? concentration in the starting glass results in reduced solubility, which reduces the rate of ion release from the material. Previous ion release studies on these materials by the authors resulted in the Na? containing materials being more solu- ble than the Sr2? (i.e. Ly-N [ Ly-C [ Ly-S) [27]. In addition, the amorphous materials were found to be far more soluble than the crystalline analogues [27]. XRD conducted on samples with full CaP surface coverage determined that the evolution of crystal structures was identified predominantly in samples containing Na? . Although the amorphous samples Ly-N and Ly-C induced complete surface coverage after 30 days, the crystalline analogue of Ly-N produced the same surface morphology which took longer to produce as there was no significant coverage after 7 days, suggesting that a solubility limit exists. The precipitation of the CaP crystals support this where the Na? containing materials produced plate-like crystals (Fig. 9) after 30 days, whereas Ly-S produced globular-like crystals which were only sporadically dis- tributed on the surface after 30 days. Also, it seems the reduced solubility in addition to the inclusion of Sr2? , results in an overall reduction in CaP deposition. 4.3 Evaluation of cytocompatibility A general trend that can be observed with the cell viability data is that as Sr2? is incorporated into the glass, there is a general reduction in cell viability. Ly-N produced the highest cell viability after 30 days at 134 %, which is likely attributed to the solubility [27] and the release of Na? . Na? is well known to be critical to cellular metabo- lism including providing the required ion concentration gradients between the interstitial fluids and the cytoplasm. As such, cells have the ability to tightly control the influx/ efflux of ions like Na? , K? and Ca2? . The solubility of the Ly-C and Ly-S is lower [27] than Ly-N which may be a contributing factor to the reduction/insignificant change in cell viability. In particular with Ly-S, it is also possible that Sr2? hinders the metabolic process in this particular cell line, as positive reports have been established regarding Sr2? use in osteoblasts [24]. With respect to the crystalline analogues, which were much less soluble than the amor- phous counterparts [27], there were relatively insignificant changes in cell viability. This may be due to a combination of the reduced ion release rate, in addition to these cells not requiring Sr2? for metabolism and ion homeostasis. 5 Conclusion The work determined that the Na? containing glasses and ceramics consistently produced CaP surface layers more readily than the Sr2? containing materials. However, dif- ferent morphologies of CaP was observed with differences in glass composition. Additionally, the CaP surface layer was observed to crystallize after 30 days with the Na? containing materials. Testing of the liquid extracts in cell culture was observed to significantly increase cell viability in the Na? containing glasses, while no significant toxicity was experienced with the Sr2? and crystalline analogues of each material. This study concludes that the inclusion of Na? significantly enhances the surface reactivity of bioceramics, and that the addition of ions with different electronic states can significantly influence the ion release rate, atomic arrangement and morphology of the precipi- tated surface A layer, and the associated cellular response. J Mater Sci: Mater Med 123 Author's personal copy
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