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Investigating the effect of TiO2on the structure and biocompatibility ofbioactive glass


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Investigating the effect of TiO2on the structure and biocompatibility ofbioactive glass

  1. 1. Investigating the effect of TiO2 on the structure and biocompatibility of bioactive glass Lana M. Placek,1 Timothy J. Keenan,1 Yiming Li,1 Chokchai Yatongchai,1 Dimple Pradhan,1 Daniel Boyd,2 Nathan P. Mellott,1 Anthony W. Wren1 1 Inamori School of Engineering, Alfred University, Alfred, New York 2 Department of Applied Oral Sciences, Dalhousie University, Halifax, Nova Scotia, Canada Received 7 May 2015; revised 4 August 2015; accepted 23 August 2015 Published online 7 September 2015 in Wiley Online Library ( DOI: 10.1002/jbm.b.33521 Abstract: Titanium (Ti41 ) containing materials have been widely used in medical applications due to its associated bio- activity in vivo. This study investigates the replacement of Si41 with Ti41 within the system SiO2-Na2O-CaO-P2O5 to deter- mine its influence on glass structure. This strategy was con- ducted in order to control the glass solubility to further improve the cellular response. Ti41 incorporation was found to have little influence on the glass transition temperature (Tg 5 520 6 88C) and magic angle spinning-nuclear magnetic resonance (MAS-NMR) shifts (280 ppm) up to additions of 18 wt %. However, at 30 wt % the Tg increased to 6008C and MAS- NMR spectra shifted to 288 ppm. There was also an associ- ated reduction in glass solubility as a function of Ti41 incorpo- ration as determined by inductively coupled plasma optical emission spectroscopy where Si41 (1649–44 mg/L) and Na1 (892–36 mg/L) levels greatly reduced while Ca21 (3–5 mg/L) and PO32 4 (2–7 mg/L) levels remained relatively unchanged. MC3T3 osteoblasts were used for cell culture testing and it was determined that the Ti41 glasses increased cell viability and also facilitated greater osteoblast adhesion and prolifera- tion to the glass surface compared to the control glass. VC 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Bio- mater, 104B: 1703–1712, 2016. Key Words: bioactive glass, titanium, ion release, cell culture, solubility How to cite this article: Placek LM, Keenan TJ, Li Y, Yatongchai C, Pradhan D, Boyd D, Mellott NP, Wren AW. 2016. Investigating the effect of TiO2 on the structure and biocompatibility of bioactive glass. J Biomed Mater Res Part B 2016:104B:1703–1712. INTRODUCTION Bioglass is regarded as one of the most widely studied and commercially successful bioceramics applied in orthopedics today. This material was originally developed in 1969 by Prof. Larry Hench at the University of Florida and the sub- sequent class of materials that stemmed from this research, bioactive glasses, has been used to formulate numerous glass-based biomaterials today. Early studies on the original 45S5 Bioglass formulation determined that a permanent sta- ble bond to bone could be established in animal models, which has led to Bioglass being marketed and applied in the medical field as particulates and pastes.1,2 Additional bioactive glass-based materials include glass-ceramic scaf- folds for orthopedic applications,3–5 glass polyalkenoate bone cements,6–8 and glass microspheres for cancer treat- ment.9–11 Since their inception, bioactive glasses have been applied and investigated for hard tissue repair1,2,12 as they stimulate osteogenesis in in vitro models.1,2 Bioactive glasses are characterized by their ability to promote healing within the body due to the dissolution of the glass surface and ion exchange upon exposure to physiological medium or body fluids.13,14 The specific mechanism includes soluble Si41 being released into the surrounding medium in the form of silicic acid due to ion exchange with H1 and H3O1 .12 This subsequently results in the precipitation of an amorphous calcium phosphate layer which crystallizes to a carbonated hydroxyapatite layer (HCA).14,15 Additionally, it has been established that ionic dissolution products from 45S5 Bioglass and other silicate-based glasses can stimulate angiogenesis and expression of several genes of osteoblasts, which suggests their applicability may supersede primarily osseous tissue.15–17 This study aims to determine any changes in the solubil- ity and cytocompatibility of a novel SiO2-Na2O-CaO-P2O5 glass series, where titanium Ti41 is substituted for Si41 in 6 wt % increments. Ti41 has previously been incorporated into numerous medical materials as it is known to promote apatite formation on the materials surfaces when in contact with physiological fluids, that is, Ti-6Al-4V,18–21 Ti-gels,14 coatings,22 and glasses.23–25 Cytocompatibility studies on the effect of Ti41 in cells such as monocytes/macrophages26 and leukocytes have been conducted and resulted in no sig- nificant changes in cell viability.27 Additional studies in monocyte derived dendritic cells (DCs) resulted in increased Correspondence to: A. W. Wren; e-mail: VC 2015 WILEY PERIODICALS, INC. 1703
  2. 2. T-lymphocyte activity.28 Further testing in cell lines more relevant to bone (osteoblast like ROS) resulted in increased expression of ALP, OPN, and osteonectin, thereby indicating their role as promoters of osteoblast differentiation.29,30 Ion concentration from these studies cite that 0.1–10 mg/L Ti41 levels did not significantly alter cell viability, however, decreases were evident when the concentration reached 20 mg/L.31 In relation to incorporating TiO2 into bioactive glasses, the authors have completed studies that investigated the structure and solubility of TiO2 doped silicate glasses. These studies on CaO-SrO-ZnO-SiO2/TiO2 glasses determined Ti41 to act predominantly as a network modifier as its replace- ment for Si41 greatly increased the concentration of non- bridging oxygen (NBO) species within the glass.32 In rela- tion to glass structure it has previously been determined that the concentration of NBO (Si-O-NBO) species within a glass is directly related to its solubility.33 NBO groups, cre- ated by the inclusion of alkali/alkali earth cations, facilitate the ion exchange process and promote the rate of silica dis- solution further increasing bioactivity.33 Cations play a com- plex structural role in oxide glasses, allowing them to influence the physical and chemical properties of the result- ing glass.32,34 Cations are usually classified as network for- mers, which form the basic structure of the glass network, or network modifiers, which de-polymerize the silicate glass network and ensure charge compensation for non- tetravalent network forming elements. Glass properties such as ion release and pH may be strongly influenced depending on the structural role of its cations,34 specifically, with respect to this study, the effect of increasing TiO2 concentra- tion will be investigated as Ti41 can assume a network modifying or network forming role in a glass. Previous stud- ies by the authors on the solubility of Ti41 substituted sili- cates glasses (SiO2-TiO2-Na2O-CaO) was found to greatly reduce the rate of Ca21 release as the concentration of Ti41 increased within the glass which had a positive influence on the viability of L929 fibroblast cells.35 With respect to this study, a TiO2 substituted bioactive glass (SiO2-Na2O-CaO- P2O5) series was produced by the traditional melt-quench method and was investigated with respect the glass struc- ture, solubility, (ion release and pH) and the subsequent effect on MC3T3 osteoblast cells. Controlling the solubility of bioactive materials surfaces, such as glass, can greatly increase the cellular response, in particular growth and adhesion. For this study magic angle spinning-nuclear mag- netic resonance (MAS-NMR) was used to investigate the structure of the glasses while the glass solubility was deter- mined using inductively coupled plasma optical emission spectroscopy (ICP-OES). To conclude, cell viability and adhe- sion studies were conducted on each of the glass composi- tions using appropriate cell lines such as MC3T3 osteoblasts. MATERIALS AND METHODS Glass synthesis Glass powder production. Five glass compositions (BG-1, BG-2, BG-3, BG-4, and BG-5) were formulated for this study with the principal aim being to investigate structure and solubility changes with the substitution of TiO2 for SiO2 within the glass composition (42SiO2224Na2O-21CaO- 13P2O5 wt %) with Ti41 replacing Si41 at 6 wt % (BG-1) increments up to 30 wt % (BG-5). A control glass (Control) was also formulated which did not contain TiO2. Glasses were prepared by weighing out appropriate amounts of ana- lytical grade reagents (SiO2, TiO2, CaCO3, Na2CO3, NH4H2PO4) and ball milling (1 h). The powdered mixes were fired (15008C, 1 h) in platinum crucibles and shock quenched in water. The resulting frits were dried, ground, and sieved to retrieve glass powders ranging from 90 to 710 lm. To ensure consistent surface area/particle size, each glass was ultrasonically cleaned in isopropyl alcohol for 30 min and dried in an oven at 378C overnight. Material characterization X-ray diffraction. Diffraction patterns were collected using a Siemens D5000 X-ray Diffraction (XRD) Unit (Bruker AXS, WI). Glass powder samples were packed into standard stainless steel sample holders. A generator voltage of 40 kV and a tube current of 30 mA were employed. Diffractograms were collected in the range 108 < 2h < 808, at a scan step size 0.028 and a step time of 10 s. Any crystalline phases present were identified using Joint Committee for Powder Diffraction Studies (JCPDS) standard diffraction patterns. Differential thermal analysis. A combined differential ther- mal analyzer-thermal gravimetric analyzer (DTA-TGA) (Stan- ton Redcroft STA 1640, Rheometric Scientific, Epsom, UK) was used to measure the glass transition temperature (Tg) for all glasses. A heating rate of 108C min21 was employed using an alumina crucible where a matched alumina cruci- ble was used as a reference. Sample measurements were carried out every six seconds between 30 and 13008C. MAS-NMR. 29 Si MAS NMR spectra were recorded using a 14 T (tesla) Bruker Avance III wide-bore FT-NMR spectrometer (Billerica, MA), 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 Si MAS NMR spectra were acquired at 300 K with the transmitter set to 119.26 MHz (2100 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 each glass powder. 29 Si NMR chemical shifts are reported in ppm, with trimethylsilylpro- pionate (TMSP) as the external reference (0 ppm). Data were processed using a 25 Hz Gaussian apodization function followed by baseline correction and were deconvoluted using PeakFit version 4.12. Scanning electron microscopy/energy dispersive X-ray spectroscopy. A Quanta 200F Environmental Scanning Elec- tron Microscope (SEM) was used to image the samples under a vacuum at a pressure of 0.90 torr. The electron beam was used at an accelerating voltage of 20 kV and a 1704 PLACEK ET AL. INVESTIGATING THE EFFECT OF TIO2
  3. 3. spot size of 4.0. Energy dispersive X-ray (EDX) spectroscopy was carried out using an FEI EDAX system equipped with a silicon-drift detector. Surface area analysis. In order to determine the surface area of the glass particles the Advanced Surface Area and Porosimetry, ASAP 2010 System analyzer (Micrometrics Instrument Corporation, Norcross) was employed. Approxi- mately 60 mg of each glass was used to calculate the spe- cific surface areas using the Brunauer-Emmett-Teller (BET) method. Glass solubility Ion release profiles (ICP). Each glass (Control, BG-1, BG-2, BG-3, BG-4, BG-5, n 5 3 where n is the number of samples) was immersed in sterile deionized H2O for 1, 7, and 14 days. Glass particulates (1 m2 surface area) was submerged in 10 mL of deionized H2O and rotated on an oscillating platform at 378C. The ion release profile of each specimen was measured using ICP–atomic emission spectroscopy (ICP–AES) on a Perkin-Elmer Optima 5300UV (Perkin Elmer, MA,). ICP–AES calibration standards for Si, Ti, Na, Ca, and P 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. pH analysis. Changes in solution pH were monitored using a Corning 430 pH meter (Fisher Scientific, Pittsburgh, PA). Sample solutions were prepared by exposing glass particu- late samples (1 m2 surface area) in 10 mL of deionized H2O. Measurements were recorded at 1, 7, and 14 days. Sterile deionized water was used as a control and was measured at each time period. Biocompatibility testing Sample preparation. Glass samples (Control, BG-3, and BG- 5) were analyzed for cytocompatibility using both MTT test- ing (glass particles) and cell adhesion studies (polished glass plates). For cell viability analysis glass particulates were incubated (1 m2 ) in sterile deionized water for 1, 7, and 14 days. Liquid extracts were then removed and used for MTT assay testing. For cell adhesion studies, glass plates (6 mm 3 1.5) were used. Each sample was initially auto- claved and then washed with 10 volumes of a mixture of 50% acetone and 50% ethanol once for 30 min. They were then sonicated in 70% ethanol and rinsed three times with 100% ethanol for 30 min each in an orbital shaker at room temperature. They were then air-dried for 15 min. Glass plates were buffered for 72 h by immersing them in PBS and shaking at 50 rpm, at 378C. The PBS solution was exchanged every 8 h. Alterations in the pH of the buffer was monitored until the pH was 7.6–7.85. Furthering buffering was carried out in Dulbecco’s modified eagle medium for 24 h (n 5 3/sample). The final pH of buffering medium was 7.4–7.5. Buffered glasses were vacuum-dried for 15 min and heated to 1208C for 2 h for further sterilization. MTT assay. The established cell line MC3T3-E1 osteoblasts (ATCC CRL-2593) was employed for biocompatibility analy- sis. Cells were maintained on a regular feeding regime in a cell culture incubator at 378C/5% CO2/95% air atmosphere. Cells were seeded into 96 well plates at a density of 10,000 cells per well and incubated for 24 h prior to testing with glass liquid extracts. The culture media used was Minimum Essential Medium Alpha Media (Fisher Scientific, PA) sup- plemented with 10% fetal bovine serum (Fisher Scientific, PA) and 1% (2 mM) L-glutamine (Fisher Scientific, PA). The cytotoxicity of glass liquid extracts (Control, BG-3 and BG-5) was evaluated using the Methyl Tetrazolium (MTT) assay in 96 well plates. Liquid extracts (10 mL) were added into wells containing osteoblast cells in culture medium (100 mL) in triplicate. Each of the prepared plates was incubated for 24 h at 378C/5% CO2. The MTT assay was then added in an amount equal to 10% of the culture medium volume/ well. The cultures were then reincubated for a further 3 h (378C/5% CO2). Next, the cultures were removed from the incubator and the resultant formazan crystals were dis- solved by adding an amount of MTT Solubilization Solution (10% Triton x-100 in Acidic Isopropanol (0.1N 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 (10 mL) of sterile media were used as a control, and cells were assumed to have metabolic activities of 100%. Osteoblast adhesion procedure. The mouse calvaria- derived MC3T3-E1 preosteoblast cells (ATCC) were cultured in Minimum Essential Medium Alpha Media supplement with 10% fetal bovine serum (Fisher Scientific, PA). Cells were maintained on a regular feeding regime in a cell cul- ture incubator at 378C/5% CO2/95% air atmosphere. After 28–48 h incubation, media was removed 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 and cells were resuspended in culture media. The number of cells was calculated to 10,000 cells per 1 mL media. The condi- tioned glass plates were placed in each well (n 5 3/sample) where 1 mL cell/media solution was seeded onto the sur- face of the glass plates and incubated for 24 h. Sample preparation for SEM. Samples were fixed with 4% (w/v) paraformaldehyde in 13 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). Sam- ples 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 SEM equipped with an EDAX Genesis Energy-Dispersive Spectrometer. Statistical analysis. One-way analysis of variance (ANOVA) was employed to compare the experimental mate- rials in relation to (1) differences cell viability as a function of glass composition, and (2) the effect each glass on cell ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | NOV 2016 VOL 104B, ISSUE 8 1705
  4. 4. viability as a function of maturation in aqueous media. Com- parison of relevant means was performed using the post hoc Bonferroni test. Differences between groups was deemed significant when p 0.05. RESULTS Influence of Ti41 on glass structure Glasses in this study were formulated using the melt- quench method where 6 wt % TiO2 is substituted in incre- mental conditions for SiO2 up to 30 wt % TiO2. Initial char- acterization included XRD and is presented in Figure 1. The Control and BG-1 were found to be fully amorphous while BG-2 and BG-3 presented some minor crystal peaks at 228, 248, 318, 338, 418, 4682h which correspond to Na2Ca4(PO)2- SiO4 (00–033-1229). BG-4 and BG-5 were also found to be fully amorphous with a BG-5 presenting a primary amor- phous hump at approximately 328 2h while a secondary hump was evident at a lower position of approximately 1882h. Additionally, the surface area of the glass particles was relatively consistent and ranged from 0.02 to 0.06m2 /g (Table I). DTA is presented in Figure 2 and was conducted to determine any changes in the glass transition tempera- ture (Tg) as a function of TiO2 concentration. From Figure 2 it is evident Control has a Tg 5 5208C. The addition of 6, 12, and 18 wt % TiO2 resulted in an increase of 108C to 5278C, 5288C, and 5248C, respectively. Higher additions of TiO2 (BG-4, BG-5) resulted in Tg increases to 5938C and 6008C, respectively. Additionally, higher additions of TiO2 resulted in increasing the crystallization transitions where Control first crystallization peaks is present at 6028C while the first crystallization peak attributed to BG-5 is observed at 6528C. The ratio of Si/Ti decreased from 6.0 (6 wt % Ti, BG-1) to 0.6 (30 wt % Ti, BG-5) and this trend is also pre- sented in Figure 2 (Table II). Glass composition, particle size and morphology were evaluated using SEM and energy dispersive X-ray (EDS) analysis. EDS analysis is presented in Figure 3 for Control, BG-1, BG-3, and BG-5 and confirms the elemental composi- tion where Si41 , Ca21 , PO32 4 , and Na1 are present in Con- trol, and the same elements in addition to Ti41 are present in the modified glass series. Figure 4 shows SEM images of each glass where it is evident that the large glass particles are free of smaller particles. The morphology of each parti- cle is relatively similar and ranges from approximately 500 to 700 lm in diameter. MAS-NMR was conducted to determine any changes in glass structure as a function of Ti41 substitution. Figure 5 presents MAS-NMR spectra of select glasses including Con- trol, BG-1, BG-3, and BG-5. Figure 5(a) presents Control which shows a main peak centered at 286.0 ppm. The main peak was de-convoluted to reveal a peak at 279.3, 286.0, 295.4, and 2107.4 ppm. Figure 5(b) presents BG-1 which presents a main peak at 283.0 ppm and de-convoluted peaks at 280.9, 287.1, and 2106.6 ppm. Figure 5(c) presents the NMR spectra of BG-3 which has a main peak centered at 280.3 ppm, where de-convolution produced peaks centered at 275.9, 280.3, 289.4, and 289.5 ppm. Figure 5(d) present the spectra of BG-5 which shifted in a FIGURE 1. X-ray diffraction of Control and Ti-series. FIGURE 2. Relationship between the glass transition temperature (Tg) and the Si/Ti ratio throughout the Ti-glass series. TABLE I. Surface Area of Prepared and Cleaned Glass Particles Control BG1 BG2 BG3 BG4 BG5 Surface area (m2 /g) 0.054 0.056 0.046 0.038 0.023 0.017 TABLE II. Summary of Ti41 Glass Series Characterization XRD Tg (8C) NMR (ppm) Si:Ti Ratio Q-Structure Control Amorphous 520 286 – Q2 BG1 Amorphous 527 283 6.0 Q2 BG2 Partial Crys. 528 – 2.6 Q2 BG3 Partial Crys. 524 280 1.5 Q2 BG4 Amorphous 593 – 0.9 Q2 BG5 Amorphous 600 288 0.6 Q2/Q3 1706 PLACEK ET AL. INVESTIGATING THE EFFECT OF TIO2
  5. 5. more negative direction, with the main peak centered at 288.0 ppm. De-convolution revealed peaks at 287.1, 297.1, and 2105.0 ppm. Influence of Ti41 on glass solubility and bioactivity Ion release profiles for Silica (Si41 ) and Sodium (Na1 ) are presented in Figure 6. Ion release was measured over 1, 7, and 14 days. Figure 7(a) presents Si41 release which was highest for Control which was found to increase with respect to time, where it increased from 90 to 1649 mg/L over 1–14 days. Si41 levels reduced with respect to compo- sition (i.e., the replacement of Si41 with Ti41 ), where after 14 days incubation Si41 levels were, BG-1 (344 mg/L), BG-2 (243 mg/L), BG-3 (83 mg/L), BG-4 (58 mg/L), and BG-5 (44 mg/L), and each followed a similar trend to Control where Si41 release increased over time. Figure 6(b) presents Na1 release which presented similar trends where Control experienced the highest release rates which peaked at 14 days at 892 mg/L. Na1 levels peaked at 14 days, how- ever, they were reduced as a consequence of modifying the glass composition, BG-1 (224 mg/L), BG-2 (177 mg/L), BG-3 (90 mg/L), BG-4 (33 mg/L), and BG-5 (36 mg/L), and each also followed a similar trend to Control where Na1 release increased over time. Ion release profiles are presented in Figure 7 for Calcium (Ca21 ) and Phosphorus (P) over 1, 7, and 14 days. With respect to Ca21 release, each material reduced with respect to time. Ca21 release attributed to Control was highest at 1 day at 5 mg/L in addition BG-1 at 6 mg/L. However, with increasing Ti41 concentration in the glass, there was an overall reduction in Ca21 release, BG-2 (4 mg/L), BG-3 (2 mg/L), BG-4 (2 mg/L), and BG-5 (2 mg/ L) at 1 day. Release rates at 14 days were 0 mg/L for Con- trol and ranged from 0.2 to 0.5 mg/L, with BG-5 attaining the highest Ca21 release at 0.5 mg/L. PO32 4 release did not present any visible trend with respect to time or Ti41 con- centration. PO32 4 release ranged from 2 to 7 mg/L and was highest for BG-2 at 14 days at 7 mg/L. Ti41 release was not determined for any of the glass compositions at 1, 7, or 14 days incubation. pH changes were monitored and recorded after each time period, that is, 1, 7, 14 days, and are pre- sented in Figure 8. Control produced the highest pH over 1– 14 days (10.9–12.1) and increased with respect to time. As the Ti41 concentration increased in the modified glasses the pH was found to decrease, however, each glass did produce an increase in pH with respect to time. At 14 days pH val- ues were recorded for each material as BG-1 (10.9), BG-2 (11.5), BG-3 (11.1), BG-4 (10.3), and BG-5 (10.4). The exis- tence of low level crystallinity in BG-3 was not found to sig- nificantly influence the solubility profiles in comparison to each of the amorphous counterparts. Biocompatibility testing was conducted using cell viabil- ity (MTT assay) and cell adhesion studies. Cell viability stud- ies were subject to statistical comparison in order to compare differences cell viability as a function of glass com- position, and to determine the influence each glass has on cell viability as a function of maturation in aqueous media (statistics presented in discussion section). Figure 9 shows that Control presented a viability of 75% after 1 day com- pared to the growing cell population (100%). These levels increased to 100% after 7 days and further increased to 115% after 14 days. Regarding the Ti41 containing glasses, BG-1 cell viability increased from 93%, 104% and 115% FIGURE 3. Energy dispersive X-ray analysis of Control and Ti-series. FIGURE 4. Scanning electron microscopy of Control and Ti-series. ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | NOV 2016 VOL 104B, ISSUE 8 1707
  6. 6. after 1, 7, and 14 days, respectively. BG-3 presented similar levels at 104%, 95% and 110% after 1, 7, and 14 days. BG- 5 presented higher levels of cell viability of 121% after 1 day, 132% after 7 days, and 103% after 14 days. Cell adhe- sion studies are presented in Figures 10 and 11, and shows that osteoblast did not adhere on the surface of Control FIGURE 5. MAS-NMR of a) Control, b) BG1, c) BG3, and d) BG5. FIGURE 6. Ion release of Control and Ti-series, specifically a) Silica and b) Sodium. 1708 PLACEK ET AL. INVESTIGATING THE EFFECT OF TIO2
  7. 7. samples. However, the Ti41 containing glasses that were tested (BG-3 and BG-5) presented osteoblast adhesion on the glass surfaces after 24 h incubation in culture medium. DISCUSSION Influence of Ti41 on glass structure This study was conducted in order to determine the effect that substituting SiO2 with TiO2 has on the structure, solu- bility and biocompatibility of bioactive glass. Initial charac- terization determined that crystal peaks (Figure 1) were identified in BG-2 and BG-3, which is likely an effect of glass processing. DTA determined that the Tg of the glasses con- taining 6, 12 and 18 wt % TiO2 showed relatively little devi- ation compared to the Control. This is likely due to Ti41 structural role in the glass where, up to BG-3 (18 wt %) it may act predominantly as a network modifier. This is fur- ther evidenced by the MAS-NMR spectra (Figure 5) where, with the increase in Ti41 concentration, the NMR spectra shifts from 286.0 ppm (Control) to 283.0 ppm (BG-1, 6 wt % Ti41 ) and finally 280.3 ppm (BG-3, 18 wt % Ti41 ). How- ever, with the addition of 30 wt % Ti41 (BG-5), a spectral shift was observed to occur to 288.0 ppm, suggesting a change in the structural role of Ti41 . An established method of representing the structural arrangement of a glass can be represented by Qn units, where Q represents the Si tetrahe- dral unit and n the number of bridging oxygens (BO); where n ranges between 0 and 4. Si41 is the central tetrahedral atom which ranges from Q0 (orthosilicates) to Q4 (tectosili- cates) and Q1 , Q2 , and Q3 structures representing intermedi- ate silicates containing modifying oxides.36 Previous studies on silicate glasses have identified regions where the chemi- cal shift represents structural changes around the Si41 atom which lie in the region of 260 to 2120 ppm for SiO4 tetra- hedra. Previous NMR studies by Galliano et al.37 and Haya- kawa et al.38 on silicate melts suggest the presence of Q1 , Q2 , Q3 , and Q4 species at 278, 285, 295, and 2105 ppm, respectively. The de-convolution revealed that the Control glass has a greater distribution of Q-species than the BG- Series as spectral resolution ranges from 279.3 to 2107.4 ppm, suggesting each of these Q-species is represented. With respect to BG-1 and BG-3 it is evident that as Ti41 is incorporated into the glass structure, a shift in a positive direction to 283.0 and 280.3 ppm is presented. BG-1 spec- tral shift ranges from 280.9 to 2106.6 ppm, similar to FIGURE 7. Ion release of Control and Ti-series, specifically a) Calcium and b) Phosphate. FIGURE 8. pH changes of Control and Ti-series recorded over 1, 7, and 14 days. FIGURE 9. MTT assay of bioactive glass series over 1, 7, and 14 days using MC3T3 osteoblast cells. ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | NOV 2016 VOL 104B, ISSUE 8 1709
  8. 8. Control, while BG-3 ranges from 275.0 to 289.5 ppm which predominantly represents Q1 -Q3 structure. This shift in ppm is representative of a reduction in BO content and an increase in the concentration of NBO, a characteristic that has been previously identified in CaO-SrO-ZnO-SiO2/TiO2 using X-ray photoelectron spectroscopy.32 The NMR shift presented for BG-5 [Figure 5(d)] differs from each of the other spectra with a center at 288 ppm and de-convoluted peaks ranging from 287.1 to 2105.0 ppm. This suggests a higher concentration of BO with the addition of 30 wt % TiO2. This may be due to the ratio of Si:Ti within the glass. Previous studies on alumino-silicate glasses have deter- mined that Al31 can change state from a network modifier to a network former (as Al31 is considered a network inter- mediate, similar to Ti41 ) when the ratio of Si:Al exceeds 1. In this case, the Si:Ti ratio decreases from 6.0 (BG-1) to 0.9 (BG-4) and 0.6 (BG-5) with the addition of up to 30 wt % Ti41 . As the ratio of Si:Ti decreases, and approaches 1, there are structural changes occurring within the glass as evident by DTA and MAS-NMR data. With respect to BG-4 and BG-5 there is a significant increase in Tg and an associated shift in the negative direction when measured by MAS-NMR for BG-5. This is in contrast to previous studies on TiO2 doped silicate-based glasses studied by the authors where the Tg measurements were found to consistently reduce with increased Ti41 concentration.32,35 The relatively insignificant change in Tg associated with BG-2 and BG-3 may be due to the partial crystallinity (Figure 1) where the crystal struc- tures produced may hinder molecular movement within the glass as these crystal structures are not soluble at this tem- perature (520–5308C). This may result in an expected reduction in Tg (Figure 2) if the NBO concentration is increasing. This may be the reason the Tg does not correlate with MAS-NMR spectra where with an increase in TiO2 con- centration, there is an associated de-polymerization of the Si41 network. It can be observed with the amorphous BG-4 and BG-5 (Figure 1) that there is an increase in Tg which correlates MAS-NMR data for BG-5 particularly. Influence of Ti41 on glass solubility and bioactivity Glass solubility represents an important characteristic of bioactive glasses ability to play a therapeutic role in vivo. A review article by Hoppe et al.15 cites the relevance of inves- tigating the effects of ionic dissolution products from bioac- tive glasses. Si41 release increases bone mineral density while inducing hydroxyapatite (HaP) precipitation and also stimulates collagen I formation and osteoblastic differentia- tion. Ca21 and PO32 4 release are also known to play roles in bone metabolism such as encouraging osteoblast differentia- tion and proliferation and extracellular matrix (ECM) syn- thesis. P has been shown to stimulate expression of matrix proteins critical to bone formation.15 Ion release profiles determined high release rates for Si41 and Na1 which are seen to reduce with the increase in TiO2 concentration within the glass. The reduction in ion release for each TiO2 addition may be attributed to a number of factors. The BG-1 to BG-3 glasses have greater network disruption (from NMR FIGURE 10. Surface of a) Control surface without adhered cells, b) BG-3 with adhered osteoblasts, and c) BG-5 also with adhered osteoblasts. FIGURE 11. Higher resolution SEM image (31000) of osteoblast adhered to surface of BG-5. TABLE III. Growing Cells (G. Cells) Viability Compared to the Cell Viability of Each Glass Composition at 1, 7, and 14 Days Incubation in Aqueous Media (significant when p 0.05) 1 Day 7 Days 14 Days G. Cells Control 0.941 1.000 1.000 BG1 1.000 1.000 1.000 BG3 1.000 1.000 1.000 BG5 1.000 0.203 1.000 1710 PLACEK ET AL. INVESTIGATING THE EFFECT OF TIO2
  9. 9. spectra) thereby reducing the BO species in addition to the reduced Si41 concentration. Ti41 is a highly charged tetrava- lent ion that may be utilizing Na1 as a charge compensator, as Ca21 levels experience relatively minor changes with respect to Ti41 concentration (Figure 7). The slight reduction in Ca21 levels may be attributed to precipitation onto the glass particles surfaces. The reduction in Si41 release is likely due to the reduced Si41 concentration as a function of TiO2 substitution. Previous studies by Talos¸ et al. on solgel derived xerogels investigated Ti41 effect on ion release and deter- mined that the incorporation of TiO2 resulted in improving solgel stability. Stability was achieved as hydration suscepti- ble P-O-P bonds were partially replaced by hydration- resistant P-O-Ti. Since Ti41 ions have a small ionic radius and a large electric charge, they can be easily integrated into the glassy network and form stronger P-O-Ti bonds compared to P-O-P bonds.39 This may explain the lack in change with respect to PO32 4 release. Additional studies on phosphorus- based glasses also experienced similar characteristics as described here when studying the effect on structure and sol- ubility. This study found an increase in Tg (evident in BG-4 and BG-5) as the concentration of Ti41 in the glass increased.21 This may support earlier finding by Talos¸ et al. and in this study, where P-O-Ti are more resistant to hydra- tion breakdown. There was also an associated reduction in the degradation rate and ion release of the glasses which was attributed to an increase in density. This resulted in a reduc- tion in all ions released from the glass.21 Changes in pH were experienced with each material, in particular as a function of incubation time (1, 7 and 14 days). Control experienced the highest pH at 12.2 after 14 days. BG-1, BG-2. and BG-3 presented pH ranging from 11.1- 11.6 after the same time period, while BG-4 and BG-5 presents pH of 10.3 and 10.4, respectively (Figure 8). This trend closely follows changes in structure and ion release where Control, which has the lowest Tg, greater distribution of Q-species and highest ion release rate, experiences the highest pH. The changes in pH are likely related to Si41 and Na1 release as Ca21 and PO32 4 release levels are low in comparison and do not experience significant changes with respect to time. Cell viability studies (Figure 9) were con- ducted on each glass and compared to a healthy growing cell population with respect to maturation (1, 7, and 14 days). Statistical comparisons (Tables III and IV) were con- ducted between glasses at each time period and as a func- tion of maturation time. After 1 day incubation, cell viability was reduced by 25% by the Control glass compared to the growing cells, however, this reduction was not deemed sig- nificant (p 5 0.941). There was also no significant differ- ence for BG-1, BG-3, and BG-5 (p 5 1.000), even though BG- 5 increased cell viability by 21%. This initial reduction in cell viability by the Control glass may be attributed to the higher solubility, as evident with ion release (particularly Si41 and Na1 release) and pH studies. Regarding the 7 day samples, Control, BG-1 and BG-3 presented similar cell via- bility levels which deviated from the growing cell popula- tion by 65%. At 7 days, BG-5 presented cell viability 32% greater than the growing cell population, however, this dif- ference was also not significant compared to the growing cells (p 5 0.203). At 14 days, all samples compared to the growing cell population did not prove to be significant (p 5 1.000). Comparing 1 and 14 day samples for each glass, there was no significant difference attributed to Control (p 5 0.215), BG-1 (p 5 0.941), BG-3 or BG-5 (p 5 1.000). Cell culture studies determined that no cell toxicity was experi- enced by any of the glass compositions evaluated. Cell adhe- sion studies (Figures 10 and 11) supported viability studies where the higher solubility of the Control glass after 24 h resulted in reduced viability, in addition to no osteoblasts adhering and proliferating on the glass surface. BG-3 and BG-5 presented an increasing number of osteoblasts as the solubility of the glass decreased. It has previously been shown that ionic dissolution products from bioactive glasses influence the behavior of cells in vivo by stimulating the genes of the affected cells toward self-repair. It has been shown that these ionic dissolution products from glasses can stimulate osteogenesis and angiogenesis, processes that make these materials highly desirable for bone tissue repair.15 An additional characteristic which makes these materials unique, is that there still exists a degree of uncer- tainty as to which ionic dissolution products cause their associated therapeutic responses, also, in what concentra- tion, and what combination of released elements contribute to these effects. CONCLUSIONS Incorporating Ti41 in a 42SiO2224Na2O-21CaO-13P2O5 wt % bioactive glass resulted in modifying the structure of the glasses, particularly at higher concentrations of 25 and 30 wt % TiO2. MAS-NMR data in particular, indicates a shift in the role Ti41 plays within the glass at higher concentrations. In addition, the solubility of the Ti41 containing glasses was greatly reduced compared to the Control glass which also resulted in reducing the solution pH as the Ti41 concentra- tion increased. Additionally, cell viability was improved at earlier time periods with the increase in Ti41 , which also providing a more amenable surface for adhesion of MC-3T3 osteoblasts. Future work on these compositions will include analyzing surface changes in simulated body fluid in order to determine if the reduction in Si41 levels influences the formation of an apatite layer on the glasses surface. ACKNOWLEDGMENTS The authors thank the following people for their assistance with this project: C.M. Smith, C.M. Clark, M.W. Strohmayer, N.L. Keenan, H.A. Liggett, H.F. Haddad, L.E. Jaeger, and L.R. Boutelle. TABLE IV. Comparison of 1 and 14 Day Cell Viability Sam- ples for Each Individual Glass Composition (Significant when p 0.05) 1 vs. 14 Days Control 0.215 BG1 0.941 BG3 1.000 BG5 1.000 ORIGINAL RESEARCH REPORT JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | NOV 2016 VOL 104B, ISSUE 8 1711
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