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Sodium and Strontium -Structure and solubility

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Sodium and Strontium -Structure and solubility

  1. 1. 1 23 Journal of Materials Science: Materials in Medicine Official Journal of the European Society for Biomaterials ISSN 0957-4530 Volume 26 Number 2 J Mater Sci: Mater Med (2015) 26:1-12 DOI 10.1007/s10856-015-5415-5 Investigating the influence of Na+ and Sr2+ on the structure and solubility of SiO2– TiO2–CaO–Na2O/SrO bioactive glass Y. Li, L. M. Placek, A. Coughlan, F. R. Laffir, D. Pradhan, N. P. Mellott & A. 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. BIOMATERIALS SYNTHESIS AND CHARACTERIZATION Investigating the influence of Na+ and Sr2+ on the structure and solubility of SiO2–TiO2–CaO–Na2O/SrO bioactive glass Y. Li • L. M. Placek • A. Coughlan • F. R. Laffir • D. Pradhan • N. P. Mellott • A. W. Wren Received: 3 July 2014 / Accepted: 1 November 2014 Ó Springer Science+Business Media New York 2015 Abstract This study was conducted to determine the influence that network modifiers, sodium (Na? ) and stron- tium (Sr2? ), have on the solubility of a SiO2–TiO2–CaO– Na2O/SrO bioactive glass. Glass characterization deter- mined each composition had a similar structure, i.e. bridging to non-bridging oxygen ratio determined by X-ray photo- electron spectroscopy. Magic angle spinning nuclear mag- netic resonance (MAS-NMR) confirmed structural similarities as each glass presented spectral shifts between -84 and -85 ppm. Differential thermal analysis and hard- ness testing revealed higher glass transition temperatures (Tg 591–760 °C) and hardness values (2.4–6.1 GPa) for the Sr2? containing glasses. Additionally the Sr2? (*250 mg/L) containing glasses displayed much lower ion release rates than the Na? (*1,200 mg/L) containing glass analogues. With the reduction in ion release there was an associated reduction in solution pH. Cytotoxicity and cell adhesion studies were conducted using MC3T3 Osteoblasts. Each glass did not significantly reduce cell numbers and osteo- blasts were found to adhere to each glass surface. 1 Introduction Bioactive glasses have generated considerable interest in the recent past as a medical material. Since the inception of BioglassÒ in the late 1960s by Prof Larry Hench, numerous glass compositions have been investigated for their thera- peutic potential [1]. The original composition (45S5 Bio- glassÒ ), is composed of 45 % SiO2–24.5 % Na2O–24.5 % CaO–6 % P2O5 and it was determined that when implanted as cast glass blocks in a rat femoral implant model, the glass blocks bonded to the surrounding bone [2–4]. Since this discovery, many formulations of bioactive glass have been investigated from a structural aspect to determine their solu- bility, degradability and subsequent therapeutic effect in vivo [4]. Many commercial materials have resulted from this class of materials including bulk implants to replace bones or teeth, coatings to anchor orthopedic or dental devices, or in the form of powders as bone grafts to fill defects in bone [3, 5]. Glass compositions such as BioglassÒ have the highest rates of bioactivity and lead to rapid regeneration of trabecular bone with a composition, architecture and quality that matches the host tissue. The regeneration of bone is due to a combination of processes; termed osteostimulation and osteoconduction [6]. In particular, these reactions involve dissolution of criti- cal concentrations of soluble Si4? and Ca2? ions that gives rise to both intracellular and extracellular responses at the interface of the glass with its physiological environment [4]. These responses result in the rapid formation of osteoid bridges between particles followed by mineralization to produce mature bone structures [3, 4]. The dissolution and subsequent ion release from these materials is known to be the predominant characteristic that initiates the mineralization process as network modifiers (Ca2? , Na? ) from the glass react with H? (H3O) ions from the solution leads to hydrolysis of the silica groups with the Y. Li Á L. M. Placek Á D. Pradhan Á N. P. Mellott Á A. 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 F. R. Laffir Materials and Surface Science Institute, University of Limerick, Limerick, Ireland 123 J Mater Sci: Mater Med (2015) 26:85 DOI 10.1007/s10856-015-5415-5 Author's personal copy
  4. 4. creation of Silanol (Si–OH) [1, 4, 7]. Condensation of an amorphous Si-rich layer (depleted in Ca2? and Na? ), pro- ceeds on the glass surface followed by migration of Ca2? and PO4 3- ions from the glass through the Si-rich layer leading to the formation of an amorphous CaP (ACP) surface layer. Over time the ACP surface layer incorporates ions such as OH- and CO3 2- from the surrounding environment which crystallizes to hydroxyapatite [4, 8]. However, a complication that can contribute to local toxicity in vivo is due to the sol- ubility of these ions and the degradation rate of the glass. By increasing the concentration of ions such as Na? and Ca2? in the glass, local environmental changes can occur, in particular the pH. The biological effects of these changes are difficult to predict and their biological role, toxicity, and removal has not been clearly determined [4, 6]. It is understood that the introduction of network modifiers (Na? , K? , Ca2? ) within the glass can lead to the disruption or the breaking of Si–O–Si bonds within the SiOx tetrahedrons, leading to the development of non-bridging oxygen species (Si–O–NBO- ). It is understood that the dissolution and deg- radation of bioactive glasses are directly related to the con- centration of NBOs within the glass structure, and this is in turn related to the concentration of alkali and alkali earth cations [6, 9]. While studies have been conducted to investi- gate the precise role that network formers contribute to a glass structure [10, 11], this study aims to determine the effect that a monovalent (Na? ) and a divalent (Sr2? ) cation can have on the structure of a bioactive glass, and the subsequent solubility. Na? was selected as the monovalent cation as its role in bio- active glasses has been well described [4, 7], and Sr2? is known to have positive therapeutic effects in vivo, where it has been cited as increasing the proliferation of osteoblasts while reducing osteoclastic activity [12]. This coupling activity has resulted in the development of an anti-osteoporotic drug, strontium ranelate that is used to increase bone mineral density in patients with metabolic bone diseases such as osteoporosis [12, 13]. It is known that both of these cations (Na? , Sr2? ) act predominantly as a network modifier within a glass structure [3, 14], and this study aims to use complementary character- ization techniques such as high resolution XPS, magic-angle spinning nuclear magnetic resonance (MAS-NMR) and Raman spectroscopy to investigate any structural differences within the glass as a result of Na? and Sr2? addition. The subsequent effect on bioactivity, specifically cell viability and cell adhesion will be investigated using MC3T3 Osteoblasts. 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 investigate structural and solubility changes within a bio- active glass as a function of Sodium (Na? , Ly-N) and Strontium (Sr2? , Ly-S) incorporation. 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). Different glass samples were produced for testing throughout this study and are explained as follows. 2.1.1 Glass powder production The powdered mixes were oven dried (100 °C, 1 h) and fired (1,500 °C, 1 h) in a platinum crucible and shock quenched in water. The resulting frits were dried, ground and sieved to retrieve glass powders with a particle size of 45 lm (XRD, DTA, Raman, MAS-NMR, pH, ICP). 2.1.2 Glass rod production The powdered mixes were oven dried (100 °C, 1 h) and fired (1,500 °C, 1 h) in platinum crucibles. Glass castings were produced by pouring the glass melts into graphite molds which were preheated to Tg. The graphite molds were left for 3 h and furnace cooled in order to anneal the glass. The resulting glass casts were then cut with a diamond blade on an Isomet 5000 Linear Precision Saw (1,500 rpm, 0.4 mm/min) and were shaped into rods of 15 9 3 mm using a Phoenix 4000 grinding machine with 60 lm silicon carbide grinding paper, Buehler, IL, USA (High resolution XPS). 2.1.3 Glass plate production The powdered mixes were oven dried (100 °C, 1 h) and fired (1,500 °C, 1 h) in platinum crucibles. Glass plates measuring[18 mm in diameter were produced by pouring molten glass on a graphite plate that was pre-heated to the samples Tg. The glass plates were then annealed for 3 h and furnace cooled (XRF, Hardness). 2.1.4 Glass button production The powdered mixes were oven dried (100 °C, 1 h) and fired (1,500 °C, 1 h) in platinum crucibles. Glass buttons were produced by drilling holes (8 mm) in a flat graphite plate measuring 4 mm in thickness. This mold was placed on another flat graphite plate and heated to the individual samples Tg. Molten glass was poured into each button mold and pressed to form an approximately uniform 8 9 4 mm button. Each button was annealed for 3 h and furnace cooled, and then ground and polished using 60 lm silicon carbide grinding paper (Buehler, IL, USA). Final glass buttons measuring 8 9 2 mm were polished further to a fine surface and ultrasonically cleaned and autoclaved. 6 85 Page 2 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy
  5. 5. buttons were produced per glass composition for cell cul- ture testing (Cytotoxicity, Cell Adhesion). 2.2 Glass characterization 2.2.1 X-ray fluorescence (XRF) X-ray fluorescence was undertaken using the S4 Pioneer (Bruker AXS Inc, MA, USA) to calculate the chemical composition of each glass. Glass plates ([18 mm in diameter) were placed in a holder with an 18 mm mask (thus revealing 18 mm diameter of the glass for testing) and underwent testing using the MultiVac 18 program. The results were quantified using the Spectra Plus Launcher (Bruker) and normalized to 100. 2.2.2 Network connectivity (NC) The network connectivity (NC) of the glasses was calcu- lated with Eq. 1 using the molar compositions of the glass. Network connectivity calculations were performed assuming that Ti performs as a network former and also as a network modifier as Ti is a known network intermediate. NC ¼ No:BOs À No:NBOs Total No: Bridging Species ð1Þ where: NC = network connectivity, BO = bridging oxy- gens, NBO = non-bridging oxygens. 2.2.3 X-ray diffraction (XRD) Diffraction patterns were collected using a Siemens D5000 X-ray diffraction 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 mAwas employed. Diffractograms were collected in the range 10° 2h 70°, at a scan step size 0.02° and a step time of 10 s. 2.2.4 Differential thermal analysis (DTA) A combined differential thermal analyzer-thermal gravimet- ric analyzer (DTA-TGA) (Stanton Redcroft STA 1640, Rheometric Scientific, Epsom, UK) was used to measure the glass transition temperature (Tg) for all glasses. A heating rate of 10 °C/min was employed using a nitrogen atmosphere with an alumina crucible where a matched alumina crucible was used as a reference. Sample measurements were carried out every 6 s between 30 and 1,300 °C. 2.2.5 X-ray photoelectron spectroscopy (XPS) High resolution XPS was performed in a Kratos AXIS 165 spectrometer (Kratos Analytical, Manchester, UK) using monochromatic Al Ka radiation (ht = 1,486.6 eV). Glass rods with dimensions of 15 9 3 9 3 mm were produced from the melt and fractured under vacuum (*2 9 10-8 torr) to create pristine surfaces with minimum contamina- tion. Surface charging was minimized by flooding the surface with low energy electrons. The C 1 s peak of adventitious carbon at 284.8 eV was used as a charge reference to calibrate the binding energies. High resolution spectra were taken at pass energy of 20 eV, with step size of 0.05 eV and 100 ms dwell time. For peak fitting, a mixed Gaussian-Lorenzian function with a Shirely type background subtraction was used. 2.2.6 Raman spectroscopy Raman analysis was performed on a Witec Confocal Raman Microscope CRM200 equipped with Si detectors, green laser with an excitation wavelength of 532 nm and power of 70 mW, and a dispersion grating selected of 600 L/mm. The instrument was calibrated using standard silicon, including a test run on a focus spectrum. This was performed to optimize the intensity of the beam. The characteristic Si line at 520 cm-1 was maximized through optimization of SMA connector. 2.2.7 Magic angle spinning-nuclear magnetic resonance (MAS-NMR) 29 SiMAS NMR spectra were recorded using a 14 T (tesla) Bruker Advance III wide-bore FT-NMR spectrometer (Billerica, MA, USA), equipped with a double broadband tunable triple resonance HXY CP-MAS probe. The glass samples were placed in a zirconia sample rotor with a diameter of 4 mm. The sample spinning speed at the magic angle to the external magnetic field was 10 kHz. 29 SiMAS NMR spectra were acquired at 300 K with the transmitter set to *119.26 MHz (-100 ppm) with a 3.0 us pulse length (pulse angle, p/2), 120-s recycle delays, where the signals from 640 scans were accumulated for Ly-S, Ly-C, and Ly-N, respectively. 29 Si NMR chemical shifts are reported in ppm, with TMSP (trimethylsilylpropionate) as the external reference (0 ppm). Data were processed using a 25 Hz Gaussian apodization function followed by base- line correction. J Mater Sci: Mater Med (2015) 26:85 Page 3 of 12 85 123 Author's personal copy
  6. 6. 2.3 Investigating glass solubility 2.3.1 Particle size analysis (PSA) Particlesizeanalysiswasconductedusinga BeckmanCoulter Multisizer 4 Particle Size Analyzer (Beckman- Coulter, Fullerton, CA, USA). The glass powder samples were eval- uated in the range of 0.4–100.0 lm and the run length took 60 s. The fluid used was water and was used at a temperature range between 10 and 37 °C. The relevant volume statistics were calculated on each glass (where n = 3/sample). 2.3.2 Advanced surface area and porosity (ASAP) In order to determine the surface area of each glass Advanced Surface Area and Porosimetry, Micromeritics ASAP 2020 (Micrometrics Instrument Corporation, Nor- cross, USA) was employed. Approximately 60 mg of each glass sample (Ly-N, Ly-C and Ly-S) was analyzed and the specific surface area was calculated using the Brunauer- Emmett-Teller (BET) method, (where n = 3/sample). 2.3.3 Ion release profiles (ICP) Glass powders (Ly-N, Ly-C and Ly-S, where n = 3/compo- sition) were incubated in 10 mL of sterile de-ionized water with surface areas of 1 m2 for 1, 7, 14 and 21 days. The sterile DI water was exchanged after each time period to determine if depletion in ion release occurs as the glasses incubation time increases. Sample tubes were centrifuged (3,000 rpm, 5 min) prior to removing the fluids, and dried for 12 h in an incubator at 37 °C. Then 10 mL sterile DI water was added to the glass samples and stored on a rotary mixer until the next time period where the process was repeated. All fluids extracted (at 1, 7, 14, 21 days, n = 3) were stored in a fridge until testing and were used for ion release and pH testing at each time period. Concentrations of Sodium (Na? ), Silicon (Si4? ), Titanium (Ti4? ), Calcium (Ca2? ) and Strontium (Sr2? ) were determined using Inductively Cou- pled 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 were prepared from a stock solution on a gravimetric basis. Three target calibration standards were prepared for each ion and de-ionized water was used as a control. 2.3.4 pH analysis Changes in pH of the ICP solutions were monitored using a Corning 430 pH meter after 1, 7, 14 and 21 days incuba- tion. Prior to testing, the pH meter was calibrated using pH buffer solution 4.00 ± 0.02 (Fisher Scientific, Pittsburgh, PA). Measurements were recorded in triplicate and De- ionized water (pH 7.0) was used as a control and was measured at each time period. 2.4 Hardness testing Hardness testing was completed on glass plates mounted in epoxy resin. A total of 10 measurements were taken on each glass plate and 3 regions on the each glass plate were analyzed (total n = 30/sample). A Shimadzu HMV-2000 Hardness testing machine was used with a 500 g load cell with 15 s intervals. 2.5 Cell culture analysis 2.5.1 Cytotoxicity analysis MC-3T3-E1 Osteoblasts (ATCC CRL-2593) were used for this study and 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 20,000 cells per well and incubated for 24 h prior to testing. The culture media used was Minimum Essential Medium Alpha Media supplement with 10 % fetal bovine serum and 1 % (2 mM) L-glutamine (Camb- rex, MD, USA). Cell culture analysis was conducted using glass buttons as prepared in Sect. 2.1.4. Glass buttons were incubated in 24 well plates for 24 and 48 h in Minimum Essential Medium Alpha Media (n = 3/sample/time per- iod). For cell viability testing, 100 lL of liquid extract was removed (n = 3 per sample well) and these liquid extracts were used for cytotoxicity testing using the Methyl Tetra- zolium (MTT) assay. Extracts (100 lL) of sample (Ly-N, Ly-C and Ly-S at 24 h and 48 h) were added into wells containing MC-3T3-E1 Osteoblasts in culture medium (1 mL) and the 24 well test plates were then incubated for 24 h at 37 °C/5 % CO2. The MTT was 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) after which, 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. Control media and healthy growing cell population (n = 3) were used as a reference. 2.5.2 Osteoblast adhesion procedure The MC3T3-E1 osteoblast cells were cultured as explained in Sect. 2.5.1. After 48 h incubation, media was removed 85 Page 4 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy
  7. 7. and 5 mL trypsin was added to the culture flask. The cells were left to detach for 20 min, after this time, trypsin was removed (centrifuge, 1,500 rpm, 5 min) and cells were re- suspended in culture media. The trypsin was removed and 10 mL media was added. The number of cells was calcu- lated to 20,000 cells per/ml media. The glass buttons were placed in each well where 1 mL cell/media solution was seeded onto the surface of the glass buttons and incubated for 24 h (n = 3 per composition) and 48 h (n = 3 per composition). Glass buttons were extracted after 24 and 48 h and were fixed with 4 % (w/v) paraformaldehyde in 1* PBS buffer for 30 min, and then post-fixed with 1 % osmium tetroxide in distilled water for 1 h. Samples were dehydrated with a series of graded ethanol washes (50/60/ 70/80/90/100 % DI water). Samples were immersed in hexamethyldislizane for 5 min and then transferred to a desiccator for 30 min. The glass plates were then coated in gold and sample imaging was carried out using an FEI Co. Quanta 200F Environmental Scanning Electron Micro- scope equipped with an EDAX Genesis Energy-Dispersive Spectrometer. 2.6 Statistical analysis One-way analysis of variance (ANOVA) was employed to compare the difference in hardness as a function of com- position. Additionally, differences in cell viability was evaluated based on sample composition compared to the healthy growing cell population at both 24 and 48 h. Comparison of relevant means was performed using the post hoc Bonferroni test. Differences between groups was deemed significant when P B 0.05. 3 Results 3.1 Glass characterization A bioactive glass series was produced to investigate the effect that Sodium (Na? ) and Strontium (Sr2? ) have on the glass structure, solubility and subsequent bioactivity. A Na? containing glass (Ly-N), an intermediate glass containing both Na? and Sr2? (Ly-C), and a Sr2? con- taining glass (Ly-S) were synthesized for this study. Initial characterization techniques included X-ray diffraction (XRD) to confirm the amorphous nature of each of the starting glasses (Fig. 1a) while differential thermal analysis (DTA) was used to identify the thermal characteristics of Ly-N, Ly-C and Ly-S. Figure 1b shows the DTA profile for Ly-N, Ly-C and Ly-S. Regarding Ly-N, the glass transition temperature (Tg) was found to be 591 °C while a small endotherm was present at approximately 700 °C with the predominant crystallization peak (Tc1) being at 777 °C. For Ly-C the Tg was 650 °C while Tc1was present at 778 °C. For Ly-S the Tg was considerably higher than both Ly- N and Ly-C at 760 °C while Tc1 was evident at 871 °C. The network connectivity (NC) of each glass was calculated using the original batch calculations and the composition determined by X-ray fluorescence (XRF, Fig. 2a). XRF data (Table 1) determined that the original batch compo- sitions are comparable to the XRF determined composi- tions. NC calculations were conducted as they are a theoretical method of determining the connectivity of the Si–O–Si bonds within a glass and were performed assum- ing that Titanium (Ti4? ) acts as both a network former and also as a network modifier. Assuming Ti4? acts as a net- work modifier, the theoretical calculation predicts a NC of 2.36 for each glass, while XRF data predicts a NC of 2.26 for Ly-N and Ly-C and a NC of 2.42 for Ly-S. This dif- ference is due to the slightly higher Si4? concentration determined by XRF. Assuming Ti4? acts as a network former the NC is calculated to be 2.67, while XRF data predicts a NC of 2.58 for Ly-N and Ly-C, and 2.72 for Ly- S. Hardness testing is presented in Fig. 2b for each glass which shows the Sr2? containing glasses to have signifi- cantly higher hardness values (Ly-C at 6.01GPa, Ly-S at 5.5 GPa) than the Na? glass (Ly-N at 2.2 GPa). Characterization techniques for analyzing glass struc- ture, such as X-ray photoelectron spectroscopy (XPS), Raman spectroscopy and magic angle spinning nuclear magnetic resonance (MAS-NMR) were employed to determine if any significant differences in glass structure were evident as a result of Na? /Sr2? replacement. High resolution X-ray photoelectron spectroscopy (XPS) was conducted on each glass, where the O1s signal is presented in Fig. 3. Figure 3a shows the high resolution O 1s of Ly- N where the spectra was resolved to reveal two peaks at binding energies (B.E.) of 529.7 and 531.3 eV which are representative of the non-bridging oxygen (NBO) and bridging oxygen (BO) concentration respectively. Ly- C presented peaks that were slightly shifted to lower binding energies of 529.9 eV (NBO) and 531.6 eV (BO) while Ly-S experienced a similar shift to 530.1 eV (NBO) and 531.8 eV (BO). Irrespective of composition, the ratio of BO/NBO was consistent as 45:55 suggesting that both Sr2? and Na? assume a similar role (network modifier) in the glass series. High resolution XPS was also conducted on each element and the results are presented in Table 2. Regarding Si 2p there was a slight shift in B.E. from 101.5 eV (Ly-N) to 101.8 eV (Ly-C) and 102.1 eV (Ly-S). High resolution scans of Ca 2p and Ti 2p experienced similar shifts in B.E. from a lower B.E. in Ly-N to a higher B.E. evident in Ly-S. With respect to the Na? containing glasses the Na 1s peak shifted from 1,070.6 eV (Ly-N) to 1,071.2 eV (Ly-C). The Sr2? containing glasses also J Mater Sci: Mater Med (2015) 26:85 Page 5 of 12 85 123 Author's personal copy
  8. 8. experienced a slight shift from 133.2 eV (Ly-C) to 133.5 eV (Ly-S). Raman spectroscopy was conducted on each of the glasses and the resulting spectra are presented in Fig. 4. It is evident from Fig. 4 that the spectra presented similar characteristics for each glass, particularly at lower wave- numbers. Each glass, Ly-N, Ly-C and Ly-S present a similar band at 344 cm-1 and also at approximately 605 cm-1 . A slight shift in wavenumbers was observed within the region of 800–1,000 cm-1 . Ly-N (Fig. 4a) presented a peak at 873 cm-1 within a relatively narrow spectral region between 900 and 1,000 cm-1 when compared to Ly-C and Ly-S. Ly-C (Fig. 4b) presented a broad absorption band at 861 cm-1 which shifted to lower wavenumbers, 852 cm-1 for Ly-S (Fig. 4c) with further broadening of the spectral envelope ranging from 900 to 1,000 cm-1 . An additional peak was observed for each of the glasses which ranged between 1,052 and 1,060 cm-1 . Magic angle spinning- nuclear magnetic resonance (MAS-NMR) was conducted on each of the glasses and the resulting spectra are pre- sented in Fig. 5. Figure 5a presents the spectra of Ly- Fig. 1 X-ray diffraction and thermal profile of Ly-N, Ly-C, Ly-S Fig. 2 Network connectivity of glass series calculated (Calc.) and determined by X-ray fluorescence (XRF) and hardness testing of each glass surface Table 1 Original glass compositions and (composition determined by XRF) all in mol. fraction Ly-N Ly-C Ly-S SiO2 0.55 (0.53) 0.55 (0.53) 0.55 (0.56) TiO2 0.05 (0.05) 0.05 (0.05) 0.05 (0.05) CaO 0.22 (0.23) 0.22 (0.23) 0.22 (0.22) Na2O 0.18 (0.18) 0.09 (0.09) 0.00 (0.00) SrO 0.00 (0.00) 0.09 (0.09) 0.18 (0.17) 85 Page 6 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy
  9. 9. N which exhibited a peak at -84.1 ppm. De-convolution of the peak resulted in a large peak present at -81.8 ppm with a smaller peak present at -90.1 ppm. An additional peak can be identified at -102.1 ppm. Figure 5b shows the spectrum for Ly-C which produced a peak that was slightly shifted in the negative direction and centered at -84.8 ppm. Peak resolution also revealed three peak positions which are also shifted in the negative direction to -83.1, -91.4 and -103.1 ppm respectively. Ly-S (Fig. 5c) experienced a shift further in the negative direction to -85.1 ppm. The three resolved peaks are centered at -83.8, -92.6 and -103.2 ppm respectively. 3.2 Investigating glass solubility Ion release studies were conducted to determine if any significant changes in ion release occurs as the incubation media is exchanged after 1, 7, 14 and 21 days. To inves- tigate the solubility of these glasses as a function of Na? / Sr2? incorporation, particle characterization was performed prior to ion release studies. Particle size analysis revealed a similar size distribution for each glass (Table 3) which were 3.9 lm (Ly-S), 4.7 lm (Ly-C) and 4.6 lm (Ly- N).Additionally, surface area analysis (Table 3) presented similar values at 0.97 m2 /g (Ly-S), 0.89 m2 /g (Ly-C) and 1.02 m2 /g (Ly-N). Ion release studies were conducted on each glass at 1, 7, 14 and 21 days with exchange of fluids at each time period. Regarding Ly-N (Fig. 6a), Si4? release was initially 852 mg/L (1 day), increased to 1107 mg/L (7 day) and reduced to 664 and 633 mg/L at 14 and 21 days respectively. Na? release from Ly-N presented a consistent reduction in release from 1,006 mg/L (1 day), reduced to 819 mg/L (7 day) and then further to 484 and 451 mg/L at 14 and 21 days respectively.Ca2? release was much lower than Si4? and Na? and was consistent over time where it was 9 mg/L (1 day), 8 mg/L (7 day) and 9 and 10 mg/L at 14 and 21 days respectively. Ion release from Ly-C considered Si4? , Na? , Ca2? and Sr2? and is presented in Fig. 6b. Si4? release was much lower than Ly- N at 241 mg/L (1 day), increased to 298 mg/L at 7 days and reduced to 253 and 279 mg/L at 14 and 21 days respectively. Na? release from Ly-C initially presented Fig. 3 XPS high resolution O 1 s scan of Ly-N, Ly-C, Ly-S Table 2 High resolution XPS data (Binding Energy, eV) Si 2p Ca 2p Ti 2p Na 1s Sr 3d Ly-S 102.1 346.9 458.6 – 133.5 Ly-C 101.8 346.6 458.4 1,071.2 133.2 Ly-N 101.5 346.4 458.2 1,070.6 – Fig. 4 Raman spectroscopy of, a Ly-N, b Ly-C and c Ly-S J Mater Sci: Mater Med (2015) 26:85 Page 7 of 12 85 123 Author's personal copy
  10. 10. similar values to Si4? at 205 mg/L (1 day), but reduced to 134 mg/L (7 day) and then further reduced to 95 and 95 mg/L at 14 and 21 days respectively, a trend similar to the Na? profile of Ly-N. Ca2? release was also much lower than Si4? and Na? and ranged from 5 mg/L (1 day), 18 mg/L (7 day), 20 mg/L (14 day) and 6 mg/L (21 day). Sr2? release was similar to that of Ca2? where it ranged from 3 mg/L (1 day), 20 mg/L (7 day), 27 mg/L (14 day) and 2 mg/L (21 day). The ion release profile for Ly-S con- siders Si4? , Ca2? and Sr2? and is presented in Fig. 6c. Si4? release was found to be consistent over time, similar to Ly- C at 211 mg/L (1 day), 225 mg/L (7 day), 221 mg/L (7 day) and 220 mg/L (14 day). Ca2? release was again much lower than Si4? and was consistent over time where it ranged from 31 mg/L (1 day), 34 mg/L (7 day) and 27 and 25 mg/L at 14 and 21 days respectively. Sr2? release was higher than Ca2? where it ranged from 62 mg/L (1 day), 94 mg/L (7 day), 65 mg/L (14 day) and 53 mg/L (21 day). pH values were recorded at each time period for each glass and are presented in Fig. 7. Considering Ly-N, there were relatively minor changes where the pH changed from 10.6 to 11.3 over 1–14 days and reduced to 10.7 after 21 days. Ly-C experienced a similar trend, however, lower pH values were recorded at 10.3–10.6 over 1–14 days and 9.8 after 21 days. Similarly, pH values attributed to Ly- S experienced lower pH values than Ly-C at 10.1–10.4 over 1–14 days and reduced to 9.8 after 21 days. The effect of Na? and Sr2? incorporation into the glass on living cells was investigated using MC3T3 Osteoblasts to determine if cell adhesion and cell viability was sig- nificantly influenced. Cell viability results are presented in Fig. 8 and revealed that Ly-N presented an insignificant change compared to the control cell population after 24 h (97 %) and 48 h (90 %). Ly-C showed a slight increase at 24 h (107 %) and increased further after 48 h (122 %) and regarding Ly-S, cell viability increased after 24 h (122 %) but decreased after 48 h (91 %). Cell adhesion was also monitored osver 24–48 h and SEM images are presented in Fig. 9. For each glass, Ly-N, Ly-C and Ly-S, there was osteoblast attachment after both 24 and 48 h incubation. 4 Discussion 4.1 Glass characterization This study was conducted to determine the effect of Na? and Sr2? on the structure and dissolution of bioactive glasses (Table 4). XRD revealed each starting material to be amorphous while DTA determined an increase in the Tg from 591 to 760 °C with the substitution of Sr2? for Na? , an observable trend is that the Tg increases; Ly-N Ly- C Ly-S. Further characterization confirmed the glass composition and determined the specific role that Na? and Sr2? play within the glass structure. Both of these ions are known to perform a similar role in a glass where they act as network modifiers which cause de-polymerization of the Si–O–Si bonds resulting in the formation of NBO- species Fig. 5 MAS-NMR spectra of a Ly-N, b Ly-C and c Ly-S Table 3 Particle size and surface area of each glass powder Particle size (lm) (S.D.) Surface area (m2 /g) (S.D.) Ly-S 3.9 (0.14) 0.97 (0.07) Ly-C 4.7 (0.38) 0.89 (0.06) Ly-N 4.6 (0.15) 1.02 (0.10) 85 Page 8 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy
  11. 11. [6]. Confirming Na? and Sr2? precise role in the glass will eliminate differences in solubility based on any glass structure differences. Initially, network connectivity (NC) calculations were used to theoretically predict the approximate NBO- speciation within the glass. For this study NC were used to predict the glass structure assuming TiO2 acts as a network modifier and also as a network former and compared to experimental data collected using x-ray fluorescence. The calculated and predicted NC was determined to be very similar. In order to validate the NC predictions, complementary techniques were used for evaluating glass structure including, high resolution XPS, Raman Spectroscopy and MAS-NMR. With respect to high resolution XPS, deconvolution of the BO signal (531.3–531.8 eV) and the NBO- (529.7–530.1 eV) revealed a slight shift in BE, which was observed at higher BE as Sr2? is increased within the glass, however, the ratio of BO:NBO was similar for each material at 45:55. An established method of representing the atomic structural arrangement or network connectivity of a glass in terms of structural units can be represented by Qn units, where Q represents the Si tetrahedral unit and n the number of bridging oxygens (BO); where n ranges between 0 and 4. Si4? is the central tetrahedral atom which ranges from Q0 (orthosilicates) to Q4 (tectosilicates) and Q1 , Q2 and Q3 structures representing intermediate silicates containing modifying oxides [15]. Determining the Q-structure of the glass yields structural information about the local envi- ronment around the Si atom which can be determined using Raman spectroscopy and MAS-NMR. Considering Raman data, it is also evident that there is a slight shift in the spectral envelope towards higher wavenumbers in the Fig. 6 Ion release of a Ly-N, b Ly-C and c Ly-S over 1, 7, 14 and 21 days Fig. 7 pH of Ly-N, Ly-C and Ly-S over 1, 7, 14 and 21 days Fig. 8 Cell viability of Ly-N, Ly-C and Ly-S after 24 and 48 h in MC 3T3 Osteoblasts J Mater Sci: Mater Med (2015) 26:85 Page 9 of 12 85 123 Author's personal copy
  12. 12. region of 850–875 cm-1 . Mc Millan et al. assigns the wavenumbers representing Q4 to 1060–1,200, Q3 to 1,100–1,050, Q2 to 1,000–950, Q1 to 900 and Q0 to 850 [16]. The Raman data within this region present relatively similar peaks at 850–870 cm-1 , which is indicative of a highly disrupted glass network, 4NBO/Si. However, the broadening of the spectral envelope to higher wavenum- bers, particularly with respect to Ly-S, is indicative increasing BO content. Additional bands located in the region of 600 cm-1 have previously been described by Fig. 9 Cell adhesion of a Ly-N, b Ly-C and c Ly-S over 24 and 48 h using MC-3T3 Osteoblasts Table 4 Summary of glass structure and characterization Net. conn. XPS (BE) Raman (cm-1 ) MAS-NMR (ppm) Q-structure Theo. XRF BO NBO Ly-S 2.36 2.26 531.8 530.1 852 -85.1 Q1/Q2 Ly-C 2.36 2.26 531.6 529.9 861 -84.8 Q1/Q2 Ly-N 2.36 2.42 531.3 529.7 873 -84.1 Q1/Q2 85 Page 10 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy
  13. 13. Aguiar et al. as being related to ring structures, with this specific region being related to three-membered rings [15]. Si29 MAS-NMR data corroborates high resolution XPS and Raman spectroscopy where the peak positions for each material are similar at -84 ppm (Ly-N, Ly-C) and -85 ppm (Ly-S). Previous NMR studies by Galliano et al. and Hayakawa et al. on silicate melts suggest the presence of Q1 , Q2 and Q3 species at -78, -85 and -95 ppm respectively [17, 18]. With respect to the NMR shift evi- dent in this study, the Ly-N produced resolved peaks at lower ppm than Ly-C, with Ly-S presenting ppm shifts in a more negative direction in each case. This suggests that each glass contains a distribution of Q-species, predomi- nantly Q1 /Q2 . This is a positive attribute as dissolution ion exchange from bioactive glass is favored by a high con- centration of NBO- species and low Q-speciation [6]. Glass characterization determined very little difference in the glass structure in relation to the glass network con- nectivity and BO/NBO content, however, DTA and hard- ness testing suggest that the incorporation of Sr2? encourages more resilient bonds within the glass network as evinced by the increase in Tg (Ly-N 591 °C, Ly- S 760 °C) and the significant increase in hardness with Sr2? incorporation, as the hardness associated with Ly- N was significantly lower than Ly-C (P = 0.000) and Ly- S (P = 0.000), however no significant difference exists between Ly-C and Ly-S (P = 0.852). This shift in Tg and the increase in hardness may be due to the fact that monovalent Na? can charge compensate a single NBO- , while a single divalent Sr2? ion can charge compensate 2NBO- . This essentially results in two fold increase in charge compensated Si-NBO- species within the glass with the addition of Sr2? (Scheme 1). 4.2 Investigating glass solubility Particle size analysis and surface area analysis proved that there were no significant differences in particle size/surface area that would contribute to difference in ion release data. Additionally, glass characterization determined that the relative concentration of BO to NBO was similar for each glass, hence the dissolution of the glass should be based on the characteristics of the ions (Na? , Sr2? ) present and not related to significant differences in the glass structure or particle effects. The Na? release profiles demonstrated here are higher for Ly-N compared to BioglassÒ , (190–270 mg/ L after 30 days) however, Ly-C and Ly-S are comparable [19]. Regarding Ly-N, the highest ion release rates were recorded for Na? which was found to reduce with each fluid exchange up to 21 days. A similar trend was present with Ly-C which suggests that Na? depletion from the glass particles is occurring. Si4? release from bioactive glass is essential for the formation and calcification of bone tissue and is known to increase bone mineral density. Aqueous Si4? is also known to induce Hap precipitation and Si(OH)4 stimulates collagen I formation and osteo- blastic differentiation [20]. Si4? release from BioglassÒ determined levels much lower than reported here at 5 mg/L after 1 day, 20 mg/L at 7 days and 45 mg/L after 30 days [19]. Si4? release was greatly reduced with the addition of Sr2? to the glass, Ly-N (800–1,100 mg/L), whereas Ly-C, Ly-S, (200–300 mg/L). This is likely due to Sr2? providing a more stable bond between NBO- groups within the glass which essentially forms cross-bridges that stabilize the Si4? tetrahedron. This is also supported by the differences in Tg and hardness between Ly-N and Ly-C/Ly-S. Ca2? release from bioactive glass is known to promote dissolu- tion of the glass surface. This characteristic is essential for encouraging precipitation of a calcium phosphate surface layer in vivo [21]. Ca2? is also cited to encourage osteo- blast proliferation, differentiation and extracellular (ECM) mineralization [6, 20]. Regarding this study, Ca2? release proved to be relatively consistent within each glass, and did not decrease even with fluid exchange at each time period. This suggests that Ca2? may be reaching a solubility limit which ranges from 9 to 10 mg/L (Ly-N), 5–20 mg/L (Ly- C) and 25-34 mg/L (Ly-S). Ca2? release from BioglassÒ are within the approximate levels cited here at 7.5 mg/L (1 day), 10 mg/L (7 days) to 16 mg/L (30 days) [19]. Sr2? release ranged from 2 to 27 mg/L in Ly-C, however, it increased to 53–94 mg/L in Ly-S which is likely due to the increase in Sr2? concentration in the glass. An associated influence of the glass solubility in addition to ion release is changes in solution pH. The solution pH was found to decrease as the Na? concentration in the glasses is reduced and/or eliminated. The lowest pH values were recorded with Ly-C and Ly-S after 21 days. In the case of Ly-C this Scheme 1 a Sodium and b strontium charge compensating NBO- species in a silicate glass network J Mater Sci: Mater Med (2015) 26:85 Page 11 of 12 85 123 Author's personal copy
  14. 14. effect is likely due to the reduction in Na? release, and with regard to Ly-S the likely reason is that the Sr2? levels are lowest at 21 days at 53 mg/L. The biocompatibility of each glass was evaluated using cytotoxicity analysis and cell adhesion studies. Cytotoxicity analysis at 24 h deter- mined that there was no significant difference in cell via- bility when comparing the growing cell population to Ly- N (P = 1.000) or Ly-C (P = 1.000), however, Ly-S pre- sented a significant increase in cell viability (P = 0.012). Regarding the 48 h samples, there was no significant dif- ference between the growing cell population and Ly- N (P = 1.000), Ly-C (P = 1.000) and Ly-S (P = 1.000). To further support the lack of cytotoxicity, cell adhesion studies determined that each composition studied sup- ported the adherence of osteoblast cells to the materials surface. The cell culture data determined that the materials under evaluation did not prove toxic to osteoblast cells after 24 or 48 h despite the difference in glass solubility. 5 Conclusion Substituting Na? and Sr2? within this glass system resulted in insignificant changes in glass structure as determined by XPS, Raman Spectroscopy and MAS-NMR, however, the addition of Sr2? greatly increased bond strength within the glass resulting in a higher Tg and hardness values. The additions of Sr2? also greatly reduced the solubility of the glass and reduced the solution pH, however, there were no significant difference in cell viability and adhesion asso- ciated with the difference in glass solubility. Future work will aim to look at how the difference in glass solubility influences precipitation of a calcium phosphate layer in simulated body fluid on glass plates, and to quantitatively determining preference for cell adhesion on solid glass samples. References 1. Jones JR. 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Hoppe A, Guldal NS, Boccaccini AR. A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics. Biomaterials. 2011;32:2757–74. 21. Kokubo T, Takadama H. How useful is SBF in predicting in vivo bone bioactivity. Biomat. 2006;27:2907–15. 85 Page 12 of 12 J Mater Sci: Mater Med (2015) 26:85 123 Author's personal copy

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