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Synthesis, characterization and cytocompatibility of ZrO2 doped borosilicate bioglasses
Journal of Non-Crystalline Solids 447 (2016) 150–155
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Journal of Non-Crystalline Solids journal homepage: www.elsevier.com/locate/jnoncrysol
Synthesis, characterization and cytocompatibility of ZrO2 doped borosilicate bioglasses Rajkumar Samudrala a, Gajulapalli V.N. Reddy b, Bramanandam Manavathi b, P. Abdul Azeem a,⁎ a b
Department of Physics, National Institute of Technology Warangal, Warangal 506004, India Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India
a r t i c l e
i n f o
Article history: Received 5 January 2016 Received in revised form 26 April 2016 Accepted 1 May 2016 Available online xxxx Keywords: Hardness Bioactivity Hydroxyapatite Cytocompatibility Osteosarcoma cells Biomedical applications
a b s t r a c t Glasses with the composition of 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaO-xZrO2 by varying zirconia content (x = 0, 1, 3, 5) were synthesized via conventional melt-quenching technique. The in-vitro properties of these glasses such as bioactivity, cytocompatibility are described in the present report. In-vitro bioactivity of these glasses was analyzed for various immersion timings by means of X-ray diffraction, Fourier transform infrared (FTIR) and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). Cytocompatibility was assessed by MTT assay with human osteosarcoma cells (MG-63). Micro hardness values of the prepared glasses have been measured with Vickers hardness tester. From the present study, it is observed that micro hardness of the glass samples increased from 5.45 GPa to 6.17 GPa with increasing ZrO2 content and it is also observed that the formation of hydroxyapatite layer on the surfaces of the glasses within 5 days of immersion time. With increasing immersion time, intense hydroxyapatite layer formation on the surface of glasses is observed. Increasing the zirconia content in the glass samples results in increase in hardness and decrease in in-vitro bioactivity. It is also observed that cell viability is not affected by the addition of zirconia. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Bioactive glasses and glass-ceramics have proved to be promising materials in the field of medicine and has been used for hard tissue regeneration in the last few decades because of their distinctive properties of rapid bone bonding, controlled biodegradability and ability to stimulate new bone growth [1,2]. They develop a fast direct bond in any bone defect regions [2]. Because of the unique bonding formation with living tissues, these glasses are used in different clinical applications, such as bone repair and middle ear bone replacement as well as in dental applications [3]. Hench [2] reported for the first time the formation of an apatite layer on bioactive glasses in Na2O–CaO–SiO2–P2O5 system, through in vitro as well as in vivo studies. At present 45S5 glass is the most highly bioactive glass, but it has low mechanical strength [4]. Hench [2] reported that, to generate bioactivity, phosphate ions play an important role in the glass composition. Calcium phosphate is one of the important ingredients present in the bones. Some of the composition forms apatite layer on the surface of the glasses without addition of phosphate content [5–8]. Ryu et al. [9] demonstrated that phosphate free calcium silicate glass also formed an apatite layer when the glass ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (P.A. Azeem).
http://dx.doi.org/10.1016/j.jnoncrysol.2016.05.001 0022-3093/© 2016 Elsevier B.V. All rights reserved.
was exposed to a simulated body fluid. When pure borate glasses are immersed in phosphate solutions or simulated body fluids, they react more rapidly than silicate glasses and also convert completely into hydroxyapatite, leaving some residue of the glass matrix [10, 11]. Higher amount of borate content weakens glass structure [12], by adding silica and CaO the glass structure is strengthened as glass former, modifier respectively. In literature several reports on evaluating the bioactivity were found. Singh et al. [13,14] studied the effect of different dopants on the bioactivity of borosilicate glasses and the results shows that the borosilicate glasses showed bioactivity even in the absence of calcium oxide. Mohini et al. [15] studied the bioactivity of borosilicate glasses with the addition of Al2O3. Devi et al. [16] studied zirconia doped phosphate glasses and glass-ceramics, and evaluated their mechanical properties and the bioactivity. On comparison, glass-ceramics are showing better hardness but bioactivity seems to be reduced. Dibakar Mondal et al. [17] have synthesized zirconia doped silicate glasses and observed an increase in mechanical properties as well as cell proliferation. Borosilicate glasses with high amount of CaO, Na2O and ZrO2 as an minor additive were synthesized. ZrO2 was used as a minor additive in the composition because it is a unique oxide ceramic material with excellent mechanical properties [18] such as higher yield strength and fracture toughness [19,20]. The objective of the present work is
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synthesize borosilicate bioactive glasses doped with ZrO2 at low processing temperature and studies the influence the ZrO2 incorporation into physico-chemical and biological properties. 2. Materials and methods 2.1. Synthesis Glasses of composition 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaOxZrO2 (x = 0, 1, 3, 5) have been prepared by melt quenching technique [21] using analytical grade reagents with 99.9% purity in a platinum crucible in electric furnace for 3 h under the temperature range 10001200 °C. The melts were cast into pre-heated stainless steel moulds, annealed at 350 °C for 2 h. For convenience, these glass systems are labelled Z0, Z1, Z3 and Z5 according to the ZrO2 content in the glass matrix have been shown in Table 1. 2.2. Preparation of SBF Simulated body fluid is prepared in polypropylene bottles by using standard procedure developed by Kokubo [22] et al. (to study the invitro bioactivity), powder to SBF solution ratio was used as 0.1 g of powder in 50 mL in static conditions only [23]. 2.3. Characterization The prepared samples were characterized with an Powder X-ray Diffractometer (Model: PANALYTICAL XPERT POWDER) using CuKα as a radiation source at a scanning angle ranging between 20° and 60°. During measurement the step size and time per step were set to 0.02° and 50 s respectively. The formation of the apatite layer on the surface of glasses after immersion in SBF solution was studied in all specimens using FTIR transmittance spectroscopy (model S 100; PerkinElmer). FTIR spectra were recorded using KBr pellets with a resolution of 4 cm−1. The functional groups in the specimens were obtained in the wavelength range of 4000–400 cm−1. The surface morphologies and elemental composition of the glasses were examined using SEM-EDS (model 5WEGA 3 LMU; TESCAN). The hardness of the glass samples were carried out by using Vickers hardness tester (HMV-2000 SHIMADZU). The applied load was 100 g and loading time was 15 s. Each sample was measured ten times, and the mean value of the test results was taken.
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(100 μL) and treated with different concentrations of glass particles for 48 h. Then 20 μL of MTT solution (5 mg/mL in PBS) was added into each well and cells were incubated at 37 °C for 4 h. The medium was removed and 100 μL of acidified isopropanol was added into each well. The plate was gently rotated on an orbital shaker for 10 min to completely dissolve the precipitation. The OD (optical density) was measured at 570 nm with a Microplate Reader. The experiments were performed in triplicates at each time point. The percentage of cell viability as calculated as follows: %Cell Viability ¼ ½ODtest =½ODcontrol :
2.5. Statistical analysis One way ANNOVA tests were used to compare the data between the samples. All tests were performed using Prism software (Graph pad 5.0). P value b 0.05 is considered as significant. 3. Results The XRD patterns of all glasses before and after soaking in SBF for 5 and 15 days respectively are shown in Figs.1–3. The XRD analysis of prepared glasses shows no diffraction peaks (Fig. 1) which confirms the amorphous character of the glasses. Fig. 2 and Fig. 3 show the crystalline phases on the glass surfaces after 5 and 15 days of immersion in SBF respectively. The observed diffraction peaks were at 2θ = 25.87°, 31.77° and 46.71°, related to (002), (211) and (222) crystalline planes respectively. On increasing zirconia content, the peak intensity is reduced in all the glass samples. FTIR spectra of all glass samples before soaking in SBF are shown in Fig. 4. From the figure it is observed that in all glass samples wavenumber 470–480 cm−1 corresponds to rocking vibrations of Si-O-Si bridges, [24,25], characteristic wavenumbers 520–530,720–730 cm−1 related to Zr-O presence and B\\O\\B bond bending vibrations of borate network respectively [26]. While, the peak observed at 930–1197 cm−1 is due to stretching vibration of tetrahedral (BO4)− units and band at 1200– 1500 cm−1 B\\O stretching vibrations of trigonal BO3 units respectively [27]. With increasing zirconia content additional shoulder peaks are noticed and 920 cm−1 band also occurred in all zirconia doped glass samples before in vitro studies [28]. With the addition of zirconia in the glass samples, the dual peak at 920 cm−1 and 1034 cm−1 is observed [29].
2.4. Cytotoxicity and MTT assay In vitro cell culture studies were performed using MG-63 human osteosarcoma cell line. MG-63 cells were obtained from the National Centre for Cell Science (NCCS, Pune, India). The cells were grown to confluence in DMEM supplemented with 10% fetal bovine serum (FBS). Then the cells were trypsinized and subcultured for further treatments. Cytotoxicity study was carried out using MTT [3-(4,5dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium] to detect the nontoxic concentration of the zirconia doped borosilicate glasses. MG-63 cells were seeded into 96 well plates at a density of 5 × 103 per well
Table 1 Compositions of undoped and zirconia doped borosilicate glasses. Sample
Composition (mol%) B2O3
SiO2
Na2Co3
CaO
ZrO2
Z0 Z1 Z3 Z5
31 31 31 31
20 20 20 20
24.5 24.5 24.5 24.5
24.5 23.5 21.5 19.5
0 1 3 5
Fig. 1. XRD spectra of undoped and zirconia doped borosilicate glasses before in-vitro studies.
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Fig. 2. XRD spectra of undoped and zirconia doped borosilicate glasses after 5 days of invitro studies.
Fig. 4. FTIR spectra of undoped and zirconia doped borosilicate glasses before in-vitro studies.
From Fig. 5 and Fig. 6 it is observed that the new peak values were positioned at ~561, ~609, ~875, ~1024, ~1639 cm−1 in all glasses after 5 and 15 days of immersion in SBF which confirms the formation of hydroxyapatite layer. The SEM micrograph shows visible evidence of bioactivity of the glasses by the formation of white precipitate on the surface of the glasses. The SEM micrograph of Z3 glass before and after in-vitro studies is shown in Fig. 7. Before in-vitro studies the morphology of the glasses seemed to resemble flake type structure and had no phosphate content, which is confirmed with EDS. Formation of hydroxyapatite layer on the surface of the glass after 5 and 15 days of immersion time is shown in Fig. 7. The measured micro hardness values of these glasses are presented in Fig. 8, the hardness of these glasses increasing with an increase in ZrO2 content. Thus, the high values of micro hardness attained in the present work are significant ones and encouraging compared to those of reported in the literature [16,17].
All glass samples with different amounts of zirconia incorporation were subjected to cytocompatibility study at different dosages (1000 μg/mL to 50 μg/mL) and the results obtained are shown in Fig. 9. As can be seen from this graph, there is no significant change in the cell viability of MG-63 cells, an osteosarcoma cell line, by the dosages used (increase when the dose decreases). Moreover, within the experimental settings, the cell viability is unaffected by the content of zirconia used at different doses (Table 2).
Glass composition 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaO-xZrO2 consists of network formers, network modifiers and intermediates. Network formers, such as SiO2 and B2O3, form a framework of glass structure, and modifiers, such as Na2O, CaO and ZrO2 break the framework and produce non-bridging oxygen [30]. The role of zirconia as a modifier leads to non-bridging oxygen and giving strength to the glass. Boron
Fig. 3. XRD spectra of undoped and zirconia doped borosilicate glasses after 15 days of invitro studies.
Fig. 5. FTIR spectra of undoped and zirconia doped borosilicate glasses after 5 days of invitro studies.
4. Discussion
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Fig. 6. FTIR spectra of undoped and zirconia doped borosilicate glasses after 15 days of invitro studies.
ions in the borosilicate glasses have two different coordination numbers such as three and four, and it has been shown that the fraction of three coordinated borons increased with an increase of B2O3 in the glass composition [31]. Pure borate glass is mostly made up of a random network of boroxyl units with boron in three-fold co-ordination (BO3). CaO enters the glass network structure in the form of a network modifier [32], by producing four-fold coordinated boron (BO4) by cross-linking the planar triangles and tightening and strengthening the network. High amount of B2O3 increases the chemical reactivity [12]. As a result
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Fig. 8. Micro Hardness of undoped and zirconia doped borosilicate glasses before in-vitro studies. The error bars indicate standard deviation.
more Ca2+ ions leached out of the glass, enhancing the hydroxyapatite formation on the surface of glass. (a) Some of the glass components, like B2O3, Na2O dissolve rapidly in the surrounding solution, when the bioactive glass is in contact with the physiological fluids. (b) Loss of silica leads to the formation of silanol groups Si(OH)4 which acts as catalyst to nucleate apatite layer. The hydrolysis reaction can be given as:
Fig. 7. SEM-EDX micrographs of 3 mol% zirconia (Z3) doped borosilicate glass before and after in-vitro studies. (Bar = 5 μm.)
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Fig. 9. Cell viability of undoped and zirconia doped borosilicate glasses with MG-63 osteoblast-like cells using MTT assay. The error bars indicate standard deviation.
SiOSi þ H2 O→2SiOH: This silica-rich layer may act as a seed to attract Ca2+ ions and PO3− 4 ions from the solution. Because of the high amount of borate content in the glass very fast dissolution occurred during the in-vitro studies. Because of faster dissolution, an early stage of the reaction, an amorphous calcium phosphate film was first formed on the silica-rich layer. As reaction time increased, the amorphous calcium phosphate by incorporation of OH− and CO2– 3 anions from the solution to converted into a hydroxyapatite crystalline phase [33]. The XRD pattern of the immersed samples (Fig. 2) shows diffractions peaks at 2θ = 25.87°, 31.77° and 46.66° and were matched with the standard JCPDS card number 09-0432 [34–36] regarded as peaks of hydroxyapatite [Ca10(PO4)6(OH)2]. The hydroxyapatite layer formation can be observed on all glass samples but there is a decrease in peak intensity with increase in ZrO2 content. Saturation of hydroxyapatite layer formation in all glasses with the increase of immersion time can be observed from Fig. 3. As shown in Fig. 4, with increasing zirconia content additional shoulder peaks are noticed and 920 cm−1 band (Si\\O bonds with two nonbridging oxygen) also occurred in all zirconia doped glass samples before in vitro studies [28]. With the addition of zirconia in the glass
Table 2 Cell viability of undoped and zirconia doped borosilicate glasses with different dosages (mean ± SD). Dosage (μg/mL)
50 100 200 500 1000
Cell viability of glasses Z0
Z1
Z3
Z5
98.86 ± 1.53 96.42 ± 4.80 94.70 ± 3.48 88.18 ± 1.50 86.24 ± 6.80
89.57 ± 4.20 82.20 ± 0.50 81.32 ± 0.44 76.32 ± 2.16 72.49 ± 8.50
88.60 ± 0.79 82.38 ± 1.70 79.85 ± 2.60 76.59 ± 7.50 72.60 ± 5.10
95.22 ± 6.96 87.38 ± 8.90 82.84 ± 1.30 73.72 ± 0.30 72.42 ± 2.60
samples, the dual peak at 920 cm− 1 and 1034 cm− 1 is observed which is indication of the presence of network modifiers in the structure of glass; i.e. Na and Ca [29]. These bands may not be appeared in Fig. 5 and Fig. 6 because of dissolution of Si-O-NBO and formation of silica rich layer for the immersion times used, further calcium phosphate layer may appeared [23,28]. The appearance of calcium phosphate layer or hydroxyapatite layer on the surface of the glasses after immersion in SBF is confirmed by FTIR data as shown in Fig. 5 and Fig. 6. In all glasses the double peak at ~561, ~609 cm−1 can be observed which is due to P\\O bending vibrations in PO4 tetrahedral and a characteristic band of HA phase [37–39]. The small shoulder around 875cm−1, is related to the stretching vibration of SiO44 − units (Q0) and to A-type carbonate-substituted hydroxyapatite (CHA) phase [27,40]. The band at 1024 cm−1 indicates P\\O bending vibrations in PO4 tetrahedral and characteristic band of the formation of apatite layer [39,41]. The peak observed at 1639 cm−1 is the absorption band of water molecule [42] and decreasing in band intensity with increasing zirconia content is observed in all glasses. After soaking the glasses for 15 days in SBF, widening of transmittance bands is observed (Fig. 6) which in turn indicates a decrease in intensity [42], which is the strong evidence for the saturation of hydroxyapatite layer (results obtain from XRD (Fig. 3)). SEM micrographs (Fig.7) illustrate that the formed HAp was in the form of agglomerated globular crystals similar to the HAp crystals formed on the silica gel [16]. On increasing immersion time for up to 15 days, rich precipitate of hydroxyapatite can be observed on the glass surface, which is strong evidence for the results obtain from XRD analysis and FTIR spectra. Sainz et al. [43] showed that Ca/P ratio increased up to 2.3, leading to formation of carbonate hydroxyapatite. EDS analysis of present study shows that Ca/P ratio is increasing from 1.86 to 2.1 with increase of incubation time from 5 to 15 days, suggesting the formation of carbonate hydroxyapatite [43], which is also confirmed by characteristic wavenumbers of 1420, 1500, 875, 605 cm−1 related to A- and B-type CHA and the peak observed at 2θ = 29° is also evidence for the carbonated hydroxyapatite (Fig. 3). According to biological evaluation of medical devices – Part 5: tests for in vitro cytocompatibility (ISO 10993 - 5: 2009) – if the cell viability of the material is less than 70% then it has cytotoxic potential. From the
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present study it is observed that cell viability is greater than 70% for all concentrations from Z0–Z5 glass samples. The present study confirms that all synthesized samples i.e., from Z0–Z5 exhibit cytocompatibility even for higher (1000 μg/mL) concentrations. 5. Conclusions In-vitro bioactivity, cytocompatibility and micro hardness were studied in zirconia doped borosilicate glasses. The XRD spectra with the maxima at 31.77° confirm the formation of hydroxyapatite layer on the glass surfaces. The FTIR spectra with double shoulder peaks positioned at 561, 609 cm−1 and the band at 1024 cm−1 with high intensity confirms the formation of hydroxyapatite layer. SEM micrographs show the formation of layer on the surface of the glass. With the addition of zirconia content, hardness of the glass samples is increased but the bioactivity is decreased. The cell viability is not affected by the addition of zirconia. The findings in the present report strongly confirm the zirconia doped glasses exhibit cytocompatibility, good bioactivity and better hardness. Hence these glasses may be used as promising bioactive materials for potential applications in the medical field
[16]
[17] [18] [19] [20]
[21]
[22] [23]
[24]
[25]
Acknowledgements
[26]
The authors wish to express their thanks to the Department of Science and Technology, Government of India for the financial support (Order No: SR/S2/CMP/-0090/2010) and sincere gratitude to the Director NIT Warangal, for providing required facilities in the institute to carry out research.
[27]
References [1] L.L. Hench, J.M. Polak, Third-generation biomedical materials, Science 295 (2002) 1014–1017. [2] L.L. Hench, Bioceramics: from concept to clinic, J. Am. Ceram. Soc. 74 (1991) 1487–1510. [3] M. Vallet-Regi, C.V. Ragel, A.J. Salinas, Glasses with medical applications, Eur. J. Inorg. Chem. (2003) 1029–1042. [4] J.R. Jones, Review of bioactive glass: from hench to hybrids, Acta Biomater. 9 (2013) 4457–4486. [5] K. Ohura, T. Nakamura, T. Yamamuro, Bone-bonding ability of P2O5-free CaO·SiO2 glasses, J. Biomed. Mater. Res. 25 (1991) 357–365. [6] C. Ohtsuki, T. Kokubo, Compositional dependence of bioactivity of glasses in the system CaO-SiO2-Al2O3 : its in vitro evaluation, J. Mater. Sci. Mater. Med. 3 (1992) 119–125. [7] A. Costantini, R. Fresa, A. Buri, F. Branda, Effect of the substitution of Y2O3 for CaO on the bioactivity of 2.5CaO·2SiO2 glass, Biomaterials 18 (1997) 453–458. [8] F. Branda, F. Arcobello-Varlese, A. Costantini, G. Luciani, Effect of the substitution of M2O3 (M = La, Y, In, Ga, Al) for CaO on the bioactivity of 2.5CaO·2SiO2 glass, Biomaterials 23 (2002) 711–716. [9] H.S. Ryu, J.K. Lee, J.H. Seo, H. Kim, K.S. Hong, D.J. Kim, J.H. Lee, D.H. Lee, B.S. Chang, C.K. Lee, S.S. Chung, Novel bioactive and biodegradable glass ceramics with high mechanical strength in the CaO-SiO2-B2O3 system, J. Biomed. Mater. Res. A. 68 (2004) 79–89. [10] S.M. Wiederhorn, Y.-H. Chae, C.G. Simon, J. Cahn, Y. Deng, D. Day, Cell adhesion to borate glasses by colloidal probe microscopy, Acta Biomater. 7 (2011) 2256–2263. [11] W. Huang, D.E. Day, K. Kittiratanapiboon, M.N. Rahaman, Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions, J. Mater. Sci. Mater. Med. 17 (2006) 583–596. [12] M.B. Volf, Chemical Approach to Glass, Glass Science and Technology, Elsevier Science, New York, 1984. [13] K. Singh, I. Bala, V. Kumar, Structural, optical and bioactive properties of calcium borosilicate glasses, Ceram. Int. 35 (2009) 3401–3406. [14] G. Kaur, P. Sharma, V. Kumar, K. Singh, Assessment of in vitro bioactivity of SiO2BaO-ZnO-B2O3-Al2O3 glasses: an optico-analytical approach, Mater. Sci. Eng. C 32 (2012) 1941–1947. [15] G. Jagan Mohini, N. Krishnamacharyulu, G. Sahaya Baskaran, P. Venkateswara Rao, N. Veeraiah, Studies on influence of aluminium ions on the bioactivity of B2O3-
[28] [29]
[30] [31]
[32] [33]
[34]
[35]
[36] [37]
[38] [39] [40]
[41] [42]
[43]
155
SiO2-P2O5-Na2O-CaO glass system by means of spectroscopic studies, Appl. Surf. Sci. 287 (2013) 46–53. A.V.G. Devi, G. Rajkumar, K. Sakthipandi, V. Rajendran, N. Rajendran, M. Rajkumar, Influence of ZrO2 on the physicochemical properties of phosphate-based glasses and glass ceramics, Phosphorus, Sulfur Silicon Relat. Elem. 187 (2012) 1434–1449. D. Mondal, S. So-Ra, B.T. Lee, Fabrication and characterization of ZrO2–CaO–P2O5– Na2O–SiO2 bioactive glass ceramics, J. Mater. Sci. 48 (2012) 1863–1872. R.C. Garvie, R.H. Hannink, R.T. Pascoe, Ceramic steel? Nature 254 (1975) 703–704. C. Piconi, G. Maccauro, Zirconia as a ceramic biomaterial, Biomaterials 20 (1999) 1–25. M. Ferraris, E. Verné, P. Appendino, C. Moisescu, A. Krajewski, A. Ravaglioli, A. Piancastelli, Coatings on zirconia for medical applications, Biomaterials 21 (2000) 765–773. P. Abdul Azeem, M. Kalidasan, R.R. Reddy, K. Ramagopal, Spectroscopic investigations on Tb3+ doped lead fluoroborate glasses, Opt. Commun. 285 (2012) 3787–3791. T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. A. Goel, R.R. Rajagopal, J.M.F. Ferreira, Influence of strontium on structure, sintering and biodegradation behaviour of CaO-MgO-SrO-SiO2-P2O5-CaF2 glasses, Acta Biomater. 7 (2011) 4071–4080. A.M. Abdelghany, H. Kamal, Spectroscopic investigation of synergetic bioactivity behavior of some ternary borate glasses containing fluoride anions, Ceram. Int. 40 (2014) 8003–8011. P. Ducheyne, S. Radin, M. Heughebaert, J.C. Heughebaert, Calcium phosphate ceramic coatings on porous titanium: effect of structure and composition on electrophoretic deposition, vacuum sintering and in vitro dissolution, Biomaterials 11 (1990) 244–254. E.I. Kamitsos, G.D. Chryssikos, Borate glass structure by Raman and infrared spectroscopies, J. Mol. Struct. 247 (1991) 1–16. S. Agathopoulos, D.U. Tulyaganov, J.M.G. Ventura, S. Kannan, A. Saranti, M.A. Karakassides, J.M.F. Ferreira, Structural analysis and devitrification of glasses based on the CaO-MgO-SiO2 system with B2O3, Na2O, CaF2 and P2O5 additives, J. NonCryst. Solids 352 (2006) 322–328. J.M. Oliveira, R.N. Correia, M.H. Fernandes, Effects of Si speciation on the in vitro bioactivity of glasses, Biomaterials 23 (2002) 371–379. F. Pishbin, V. Mouriño, S. Flor, S. Kreppel, V. Salih, M.P. Ryan, A.R. Boccaccini, Electrophoretic deposition of gentamicin-loaded bioactive glass/chitosan composite coatings for orthopaedic implants, ACS Appl. Mater. Interfaces 6 (2014) 8796–8806. M. Tomozawa, Fundamentals of Inorganic Glasses, 1994. R. Martens, W. Müller-Warmuth, Structural groups and their mixing in borosilicate glasses of various compositions - an NMR study, J. Non-Cryst. Solids 265 (2000) 167–175. D. Maniu, T. Iliescu, I. Ardelean, S. Cinta-Pinzaru, N. Tarcea, W. Kiefer, Raman study on B2O3-CaO glasses, J. Mol. Struct. (2003) 485–488. A. Balamurugan, G. Balossier, S. Kannan, J. Michel, A.H.S. Rebelo, J.M.F. Ferreira, Development and in vitro characterization of sol-gel derived CaO-P2O5-SiO2-ZnO bioglass, Acta Biomater. 3 (2007) 255–262. L. Ji, W. Wang, D. Jin, S. Zhou, X. Song, In vitro bioactivity and mechanical properties of bioactive glass nanoparticles/polycaprolactone composites, Mater. Sci. Eng. C. 46 (2015) 1–9. G. Kaur, G. Pickrell, G. Kimsawatde, D. Homa, H.a. Allbee, N. Sriranganathan, Synthesis, cytotoxicity, and hydroxyapatite formation in 27-Tris-SBF for sol-gel based CaOP2O5-SiO2-B2O3-ZnO bioactive glasses, Sci. Rep. 4 (2014) 4392. R. Govindan, E.K. Girija, Drug loaded phosphate glass/hydroxyapatite nanocomposite for orthopedic applications, J. Mater. Chem. B 2 (2014) 5468–5477. S. Srinivasan, R. Jayasree, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Biocompatible alginate/nano bioactive glass ceramic composite scaffolds for periodontal tissue regeneration, Carbohydr. Polym. 87 (2012) 274–283. K. Taek Jung, Y.-H. Chu, S. Haam, Y. Gun Shul, Synthesis of mesoporous silica fiber using spinning method, J. Non-Cryst. Solids 298 (2002) 193–201. I. Rehman, W. Bonfield, Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy, J. Mater. Sci. Mater. Med. 8 (1997) 1–4. Z. Hong, A. Liu, L. Chen, X. Chen, X. Jing, Preparation of bioactive glass ceramic nanoparticles by combination of sol-gel and coprecipitation method, J. Non-Cryst. Solids 355 (2009) 368–372. C.Y. Kim, A.E. Clark, L.L. Hench, Early stages of calcium-phosphate layer formation in bioglasses, J. Non-Cryst. Solids 113 (1989) 195–202. R. Lucacel Ciceo, D.L. Trandafir, T. Radu, O. Ponta, V. Simon, Synthesis, characterisation and in vitro evaluation of sol-gel derived SiO2-P2O5-CaO-B2O3 bioactive system, Ceram. Int. 40 (2014) 9517–9524. M.A. Sainz, P. Pena, S. Serena, A. Caballero, Influence of design on bioactivity of novel CaSiO3-CaMg(SiO3)2 bioceramics: in vitro simulated body fluid test and thermodynamic simulation, Acta Biomater. 6 (2010) 2797–2807.
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Synthesis, characterization and cytocompatibility of ZrO2 doped borosilicate bioglasses Rajkumar Samudrala a, Gajulapalli V.N. Reddy b, Bramanandam Manavathi b, P. Abdul Azeem a,⁎ a b
Department of Physics, National Institute of Technology Warangal, Warangal 506004, India Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India
a r t i c l e
i n f o
Article history: Received 5 January 2016 Received in revised form 26 April 2016 Accepted 1 May 2016 Available online xxxx Keywords: Hardness Bioactivity Hydroxyapatite Cytocompatibility Osteosarcoma cells Biomedical applications
a b s t r a c t Glasses with the composition of 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaO-xZrO2 by varying zirconia content (x = 0, 1, 3, 5) were synthesized via conventional melt-quenching technique. The in-vitro properties of these glasses such as bioactivity, cytocompatibility are described in the present report. In-vitro bioactivity of these glasses was analyzed for various immersion timings by means of X-ray diffraction, Fourier transform infrared (FTIR) and scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). Cytocompatibility was assessed by MTT assay with human osteosarcoma cells (MG-63). Micro hardness values of the prepared glasses have been measured with Vickers hardness tester. From the present study, it is observed that micro hardness of the glass samples increased from 5.45 GPa to 6.17 GPa with increasing ZrO2 content and it is also observed that the formation of hydroxyapatite layer on the surfaces of the glasses within 5 days of immersion time. With increasing immersion time, intense hydroxyapatite layer formation on the surface of glasses is observed. Increasing the zirconia content in the glass samples results in increase in hardness and decrease in in-vitro bioactivity. It is also observed that cell viability is not affected by the addition of zirconia. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Bioactive glasses and glass-ceramics have proved to be promising materials in the field of medicine and has been used for hard tissue regeneration in the last few decades because of their distinctive properties of rapid bone bonding, controlled biodegradability and ability to stimulate new bone growth [1,2]. They develop a fast direct bond in any bone defect regions [2]. Because of the unique bonding formation with living tissues, these glasses are used in different clinical applications, such as bone repair and middle ear bone replacement as well as in dental applications [3]. Hench [2] reported for the first time the formation of an apatite layer on bioactive glasses in Na2O–CaO–SiO2–P2O5 system, through in vitro as well as in vivo studies. At present 45S5 glass is the most highly bioactive glass, but it has low mechanical strength [4]. Hench [2] reported that, to generate bioactivity, phosphate ions play an important role in the glass composition. Calcium phosphate is one of the important ingredients present in the bones. Some of the composition forms apatite layer on the surface of the glasses without addition of phosphate content [5–8]. Ryu et al. [9] demonstrated that phosphate free calcium silicate glass also formed an apatite layer when the glass ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (P.A. Azeem).
http://dx.doi.org/10.1016/j.jnoncrysol.2016.05.001 0022-3093/© 2016 Elsevier B.V. All rights reserved.
was exposed to a simulated body fluid. When pure borate glasses are immersed in phosphate solutions or simulated body fluids, they react more rapidly than silicate glasses and also convert completely into hydroxyapatite, leaving some residue of the glass matrix [10, 11]. Higher amount of borate content weakens glass structure [12], by adding silica and CaO the glass structure is strengthened as glass former, modifier respectively. In literature several reports on evaluating the bioactivity were found. Singh et al. [13,14] studied the effect of different dopants on the bioactivity of borosilicate glasses and the results shows that the borosilicate glasses showed bioactivity even in the absence of calcium oxide. Mohini et al. [15] studied the bioactivity of borosilicate glasses with the addition of Al2O3. Devi et al. [16] studied zirconia doped phosphate glasses and glass-ceramics, and evaluated their mechanical properties and the bioactivity. On comparison, glass-ceramics are showing better hardness but bioactivity seems to be reduced. Dibakar Mondal et al. [17] have synthesized zirconia doped silicate glasses and observed an increase in mechanical properties as well as cell proliferation. Borosilicate glasses with high amount of CaO, Na2O and ZrO2 as an minor additive were synthesized. ZrO2 was used as a minor additive in the composition because it is a unique oxide ceramic material with excellent mechanical properties [18] such as higher yield strength and fracture toughness [19,20]. The objective of the present work is
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synthesize borosilicate bioactive glasses doped with ZrO2 at low processing temperature and studies the influence the ZrO2 incorporation into physico-chemical and biological properties. 2. Materials and methods 2.1. Synthesis Glasses of composition 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaOxZrO2 (x = 0, 1, 3, 5) have been prepared by melt quenching technique [21] using analytical grade reagents with 99.9% purity in a platinum crucible in electric furnace for 3 h under the temperature range 10001200 °C. The melts were cast into pre-heated stainless steel moulds, annealed at 350 °C for 2 h. For convenience, these glass systems are labelled Z0, Z1, Z3 and Z5 according to the ZrO2 content in the glass matrix have been shown in Table 1. 2.2. Preparation of SBF Simulated body fluid is prepared in polypropylene bottles by using standard procedure developed by Kokubo [22] et al. (to study the invitro bioactivity), powder to SBF solution ratio was used as 0.1 g of powder in 50 mL in static conditions only [23]. 2.3. Characterization The prepared samples were characterized with an Powder X-ray Diffractometer (Model: PANALYTICAL XPERT POWDER) using CuKα as a radiation source at a scanning angle ranging between 20° and 60°. During measurement the step size and time per step were set to 0.02° and 50 s respectively. The formation of the apatite layer on the surface of glasses after immersion in SBF solution was studied in all specimens using FTIR transmittance spectroscopy (model S 100; PerkinElmer). FTIR spectra were recorded using KBr pellets with a resolution of 4 cm−1. The functional groups in the specimens were obtained in the wavelength range of 4000–400 cm−1. The surface morphologies and elemental composition of the glasses were examined using SEM-EDS (model 5WEGA 3 LMU; TESCAN). The hardness of the glass samples were carried out by using Vickers hardness tester (HMV-2000 SHIMADZU). The applied load was 100 g and loading time was 15 s. Each sample was measured ten times, and the mean value of the test results was taken.
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(100 μL) and treated with different concentrations of glass particles for 48 h. Then 20 μL of MTT solution (5 mg/mL in PBS) was added into each well and cells were incubated at 37 °C for 4 h. The medium was removed and 100 μL of acidified isopropanol was added into each well. The plate was gently rotated on an orbital shaker for 10 min to completely dissolve the precipitation. The OD (optical density) was measured at 570 nm with a Microplate Reader. The experiments were performed in triplicates at each time point. The percentage of cell viability as calculated as follows: %Cell Viability ¼ ½ODtest =½ODcontrol :
2.5. Statistical analysis One way ANNOVA tests were used to compare the data between the samples. All tests were performed using Prism software (Graph pad 5.0). P value b 0.05 is considered as significant. 3. Results The XRD patterns of all glasses before and after soaking in SBF for 5 and 15 days respectively are shown in Figs.1–3. The XRD analysis of prepared glasses shows no diffraction peaks (Fig. 1) which confirms the amorphous character of the glasses. Fig. 2 and Fig. 3 show the crystalline phases on the glass surfaces after 5 and 15 days of immersion in SBF respectively. The observed diffraction peaks were at 2θ = 25.87°, 31.77° and 46.71°, related to (002), (211) and (222) crystalline planes respectively. On increasing zirconia content, the peak intensity is reduced in all the glass samples. FTIR spectra of all glass samples before soaking in SBF are shown in Fig. 4. From the figure it is observed that in all glass samples wavenumber 470–480 cm−1 corresponds to rocking vibrations of Si-O-Si bridges, [24,25], characteristic wavenumbers 520–530,720–730 cm−1 related to Zr-O presence and B\\O\\B bond bending vibrations of borate network respectively [26]. While, the peak observed at 930–1197 cm−1 is due to stretching vibration of tetrahedral (BO4)− units and band at 1200– 1500 cm−1 B\\O stretching vibrations of trigonal BO3 units respectively [27]. With increasing zirconia content additional shoulder peaks are noticed and 920 cm−1 band also occurred in all zirconia doped glass samples before in vitro studies [28]. With the addition of zirconia in the glass samples, the dual peak at 920 cm−1 and 1034 cm−1 is observed [29].
2.4. Cytotoxicity and MTT assay In vitro cell culture studies were performed using MG-63 human osteosarcoma cell line. MG-63 cells were obtained from the National Centre for Cell Science (NCCS, Pune, India). The cells were grown to confluence in DMEM supplemented with 10% fetal bovine serum (FBS). Then the cells were trypsinized and subcultured for further treatments. Cytotoxicity study was carried out using MTT [3-(4,5dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium] to detect the nontoxic concentration of the zirconia doped borosilicate glasses. MG-63 cells were seeded into 96 well plates at a density of 5 × 103 per well
Table 1 Compositions of undoped and zirconia doped borosilicate glasses. Sample
Composition (mol%) B2O3
SiO2
Na2Co3
CaO
ZrO2
Z0 Z1 Z3 Z5
31 31 31 31
20 20 20 20
24.5 24.5 24.5 24.5
24.5 23.5 21.5 19.5
0 1 3 5
Fig. 1. XRD spectra of undoped and zirconia doped borosilicate glasses before in-vitro studies.
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Fig. 2. XRD spectra of undoped and zirconia doped borosilicate glasses after 5 days of invitro studies.
Fig. 4. FTIR spectra of undoped and zirconia doped borosilicate glasses before in-vitro studies.
From Fig. 5 and Fig. 6 it is observed that the new peak values were positioned at ~561, ~609, ~875, ~1024, ~1639 cm−1 in all glasses after 5 and 15 days of immersion in SBF which confirms the formation of hydroxyapatite layer. The SEM micrograph shows visible evidence of bioactivity of the glasses by the formation of white precipitate on the surface of the glasses. The SEM micrograph of Z3 glass before and after in-vitro studies is shown in Fig. 7. Before in-vitro studies the morphology of the glasses seemed to resemble flake type structure and had no phosphate content, which is confirmed with EDS. Formation of hydroxyapatite layer on the surface of the glass after 5 and 15 days of immersion time is shown in Fig. 7. The measured micro hardness values of these glasses are presented in Fig. 8, the hardness of these glasses increasing with an increase in ZrO2 content. Thus, the high values of micro hardness attained in the present work are significant ones and encouraging compared to those of reported in the literature [16,17].
All glass samples with different amounts of zirconia incorporation were subjected to cytocompatibility study at different dosages (1000 μg/mL to 50 μg/mL) and the results obtained are shown in Fig. 9. As can be seen from this graph, there is no significant change in the cell viability of MG-63 cells, an osteosarcoma cell line, by the dosages used (increase when the dose decreases). Moreover, within the experimental settings, the cell viability is unaffected by the content of zirconia used at different doses (Table 2).
Glass composition 31B2O3-20SiO2-24.5Na2O-(24.5-x)CaO-xZrO2 consists of network formers, network modifiers and intermediates. Network formers, such as SiO2 and B2O3, form a framework of glass structure, and modifiers, such as Na2O, CaO and ZrO2 break the framework and produce non-bridging oxygen [30]. The role of zirconia as a modifier leads to non-bridging oxygen and giving strength to the glass. Boron
Fig. 3. XRD spectra of undoped and zirconia doped borosilicate glasses after 15 days of invitro studies.
Fig. 5. FTIR spectra of undoped and zirconia doped borosilicate glasses after 5 days of invitro studies.
4. Discussion
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Fig. 6. FTIR spectra of undoped and zirconia doped borosilicate glasses after 15 days of invitro studies.
ions in the borosilicate glasses have two different coordination numbers such as three and four, and it has been shown that the fraction of three coordinated borons increased with an increase of B2O3 in the glass composition [31]. Pure borate glass is mostly made up of a random network of boroxyl units with boron in three-fold co-ordination (BO3). CaO enters the glass network structure in the form of a network modifier [32], by producing four-fold coordinated boron (BO4) by cross-linking the planar triangles and tightening and strengthening the network. High amount of B2O3 increases the chemical reactivity [12]. As a result
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Fig. 8. Micro Hardness of undoped and zirconia doped borosilicate glasses before in-vitro studies. The error bars indicate standard deviation.
more Ca2+ ions leached out of the glass, enhancing the hydroxyapatite formation on the surface of glass. (a) Some of the glass components, like B2O3, Na2O dissolve rapidly in the surrounding solution, when the bioactive glass is in contact with the physiological fluids. (b) Loss of silica leads to the formation of silanol groups Si(OH)4 which acts as catalyst to nucleate apatite layer. The hydrolysis reaction can be given as:
Fig. 7. SEM-EDX micrographs of 3 mol% zirconia (Z3) doped borosilicate glass before and after in-vitro studies. (Bar = 5 μm.)
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Fig. 9. Cell viability of undoped and zirconia doped borosilicate glasses with MG-63 osteoblast-like cells using MTT assay. The error bars indicate standard deviation.
SiOSi þ H2 O→2SiOH: This silica-rich layer may act as a seed to attract Ca2+ ions and PO3− 4 ions from the solution. Because of the high amount of borate content in the glass very fast dissolution occurred during the in-vitro studies. Because of faster dissolution, an early stage of the reaction, an amorphous calcium phosphate film was first formed on the silica-rich layer. As reaction time increased, the amorphous calcium phosphate by incorporation of OH− and CO2– 3 anions from the solution to converted into a hydroxyapatite crystalline phase [33]. The XRD pattern of the immersed samples (Fig. 2) shows diffractions peaks at 2θ = 25.87°, 31.77° and 46.66° and were matched with the standard JCPDS card number 09-0432 [34–36] regarded as peaks of hydroxyapatite [Ca10(PO4)6(OH)2]. The hydroxyapatite layer formation can be observed on all glass samples but there is a decrease in peak intensity with increase in ZrO2 content. Saturation of hydroxyapatite layer formation in all glasses with the increase of immersion time can be observed from Fig. 3. As shown in Fig. 4, with increasing zirconia content additional shoulder peaks are noticed and 920 cm−1 band (Si\\O bonds with two nonbridging oxygen) also occurred in all zirconia doped glass samples before in vitro studies [28]. With the addition of zirconia in the glass
Table 2 Cell viability of undoped and zirconia doped borosilicate glasses with different dosages (mean ± SD). Dosage (μg/mL)
50 100 200 500 1000
Cell viability of glasses Z0
Z1
Z3
Z5
98.86 ± 1.53 96.42 ± 4.80 94.70 ± 3.48 88.18 ± 1.50 86.24 ± 6.80
89.57 ± 4.20 82.20 ± 0.50 81.32 ± 0.44 76.32 ± 2.16 72.49 ± 8.50
88.60 ± 0.79 82.38 ± 1.70 79.85 ± 2.60 76.59 ± 7.50 72.60 ± 5.10
95.22 ± 6.96 87.38 ± 8.90 82.84 ± 1.30 73.72 ± 0.30 72.42 ± 2.60
samples, the dual peak at 920 cm− 1 and 1034 cm− 1 is observed which is indication of the presence of network modifiers in the structure of glass; i.e. Na and Ca [29]. These bands may not be appeared in Fig. 5 and Fig. 6 because of dissolution of Si-O-NBO and formation of silica rich layer for the immersion times used, further calcium phosphate layer may appeared [23,28]. The appearance of calcium phosphate layer or hydroxyapatite layer on the surface of the glasses after immersion in SBF is confirmed by FTIR data as shown in Fig. 5 and Fig. 6. In all glasses the double peak at ~561, ~609 cm−1 can be observed which is due to P\\O bending vibrations in PO4 tetrahedral and a characteristic band of HA phase [37–39]. The small shoulder around 875cm−1, is related to the stretching vibration of SiO44 − units (Q0) and to A-type carbonate-substituted hydroxyapatite (CHA) phase [27,40]. The band at 1024 cm−1 indicates P\\O bending vibrations in PO4 tetrahedral and characteristic band of the formation of apatite layer [39,41]. The peak observed at 1639 cm−1 is the absorption band of water molecule [42] and decreasing in band intensity with increasing zirconia content is observed in all glasses. After soaking the glasses for 15 days in SBF, widening of transmittance bands is observed (Fig. 6) which in turn indicates a decrease in intensity [42], which is the strong evidence for the saturation of hydroxyapatite layer (results obtain from XRD (Fig. 3)). SEM micrographs (Fig.7) illustrate that the formed HAp was in the form of agglomerated globular crystals similar to the HAp crystals formed on the silica gel [16]. On increasing immersion time for up to 15 days, rich precipitate of hydroxyapatite can be observed on the glass surface, which is strong evidence for the results obtain from XRD analysis and FTIR spectra. Sainz et al. [43] showed that Ca/P ratio increased up to 2.3, leading to formation of carbonate hydroxyapatite. EDS analysis of present study shows that Ca/P ratio is increasing from 1.86 to 2.1 with increase of incubation time from 5 to 15 days, suggesting the formation of carbonate hydroxyapatite [43], which is also confirmed by characteristic wavenumbers of 1420, 1500, 875, 605 cm−1 related to A- and B-type CHA and the peak observed at 2θ = 29° is also evidence for the carbonated hydroxyapatite (Fig. 3). According to biological evaluation of medical devices – Part 5: tests for in vitro cytocompatibility (ISO 10993 - 5: 2009) – if the cell viability of the material is less than 70% then it has cytotoxic potential. From the
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present study it is observed that cell viability is greater than 70% for all concentrations from Z0–Z5 glass samples. The present study confirms that all synthesized samples i.e., from Z0–Z5 exhibit cytocompatibility even for higher (1000 μg/mL) concentrations. 5. Conclusions In-vitro bioactivity, cytocompatibility and micro hardness were studied in zirconia doped borosilicate glasses. The XRD spectra with the maxima at 31.77° confirm the formation of hydroxyapatite layer on the glass surfaces. The FTIR spectra with double shoulder peaks positioned at 561, 609 cm−1 and the band at 1024 cm−1 with high intensity confirms the formation of hydroxyapatite layer. SEM micrographs show the formation of layer on the surface of the glass. With the addition of zirconia content, hardness of the glass samples is increased but the bioactivity is decreased. The cell viability is not affected by the addition of zirconia. The findings in the present report strongly confirm the zirconia doped glasses exhibit cytocompatibility, good bioactivity and better hardness. Hence these glasses may be used as promising bioactive materials for potential applications in the medical field
[16]
[17] [18] [19] [20]
[21]
[22] [23]
[24]
[25]
Acknowledgements
[26]
The authors wish to express their thanks to the Department of Science and Technology, Government of India for the financial support (Order No: SR/S2/CMP/-0090/2010) and sincere gratitude to the Director NIT Warangal, for providing required facilities in the institute to carry out research.
[27]
References [1] L.L. Hench, J.M. Polak, Third-generation biomedical materials, Science 295 (2002) 1014–1017. [2] L.L. Hench, Bioceramics: from concept to clinic, J. Am. Ceram. Soc. 74 (1991) 1487–1510. [3] M. Vallet-Regi, C.V. Ragel, A.J. Salinas, Glasses with medical applications, Eur. J. Inorg. Chem. (2003) 1029–1042. [4] J.R. Jones, Review of bioactive glass: from hench to hybrids, Acta Biomater. 9 (2013) 4457–4486. [5] K. Ohura, T. Nakamura, T. Yamamuro, Bone-bonding ability of P2O5-free CaO·SiO2 glasses, J. Biomed. Mater. Res. 25 (1991) 357–365. [6] C. Ohtsuki, T. Kokubo, Compositional dependence of bioactivity of glasses in the system CaO-SiO2-Al2O3 : its in vitro evaluation, J. Mater. Sci. Mater. Med. 3 (1992) 119–125. [7] A. Costantini, R. Fresa, A. Buri, F. Branda, Effect of the substitution of Y2O3 for CaO on the bioactivity of 2.5CaO·2SiO2 glass, Biomaterials 18 (1997) 453–458. [8] F. Branda, F. Arcobello-Varlese, A. Costantini, G. Luciani, Effect of the substitution of M2O3 (M = La, Y, In, Ga, Al) for CaO on the bioactivity of 2.5CaO·2SiO2 glass, Biomaterials 23 (2002) 711–716. [9] H.S. Ryu, J.K. Lee, J.H. Seo, H. Kim, K.S. Hong, D.J. Kim, J.H. Lee, D.H. Lee, B.S. Chang, C.K. Lee, S.S. Chung, Novel bioactive and biodegradable glass ceramics with high mechanical strength in the CaO-SiO2-B2O3 system, J. Biomed. Mater. Res. A. 68 (2004) 79–89. [10] S.M. Wiederhorn, Y.-H. Chae, C.G. Simon, J. Cahn, Y. Deng, D. Day, Cell adhesion to borate glasses by colloidal probe microscopy, Acta Biomater. 7 (2011) 2256–2263. [11] W. Huang, D.E. Day, K. Kittiratanapiboon, M.N. Rahaman, Kinetics and mechanisms of the conversion of silicate (45S5), borate, and borosilicate glasses to hydroxyapatite in dilute phosphate solutions, J. Mater. Sci. Mater. Med. 17 (2006) 583–596. [12] M.B. Volf, Chemical Approach to Glass, Glass Science and Technology, Elsevier Science, New York, 1984. [13] K. Singh, I. Bala, V. Kumar, Structural, optical and bioactive properties of calcium borosilicate glasses, Ceram. Int. 35 (2009) 3401–3406. [14] G. Kaur, P. Sharma, V. Kumar, K. Singh, Assessment of in vitro bioactivity of SiO2BaO-ZnO-B2O3-Al2O3 glasses: an optico-analytical approach, Mater. Sci. Eng. C 32 (2012) 1941–1947. [15] G. Jagan Mohini, N. Krishnamacharyulu, G. Sahaya Baskaran, P. Venkateswara Rao, N. Veeraiah, Studies on influence of aluminium ions on the bioactivity of B2O3-
[28] [29]
[30] [31]
[32] [33]
[34]
[35]
[36] [37]
[38] [39] [40]
[41] [42]
[43]
155
SiO2-P2O5-Na2O-CaO glass system by means of spectroscopic studies, Appl. Surf. Sci. 287 (2013) 46–53. A.V.G. Devi, G. Rajkumar, K. Sakthipandi, V. Rajendran, N. Rajendran, M. Rajkumar, Influence of ZrO2 on the physicochemical properties of phosphate-based glasses and glass ceramics, Phosphorus, Sulfur Silicon Relat. Elem. 187 (2012) 1434–1449. D. Mondal, S. So-Ra, B.T. Lee, Fabrication and characterization of ZrO2–CaO–P2O5– Na2O–SiO2 bioactive glass ceramics, J. Mater. Sci. 48 (2012) 1863–1872. R.C. Garvie, R.H. Hannink, R.T. Pascoe, Ceramic steel? Nature 254 (1975) 703–704. C. Piconi, G. Maccauro, Zirconia as a ceramic biomaterial, Biomaterials 20 (1999) 1–25. M. Ferraris, E. Verné, P. Appendino, C. Moisescu, A. Krajewski, A. Ravaglioli, A. Piancastelli, Coatings on zirconia for medical applications, Biomaterials 21 (2000) 765–773. P. Abdul Azeem, M. Kalidasan, R.R. Reddy, K. Ramagopal, Spectroscopic investigations on Tb3+ doped lead fluoroborate glasses, Opt. Commun. 285 (2012) 3787–3791. T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 27 (2006) 2907–2915. A. Goel, R.R. Rajagopal, J.M.F. Ferreira, Influence of strontium on structure, sintering and biodegradation behaviour of CaO-MgO-SrO-SiO2-P2O5-CaF2 glasses, Acta Biomater. 7 (2011) 4071–4080. A.M. Abdelghany, H. Kamal, Spectroscopic investigation of synergetic bioactivity behavior of some ternary borate glasses containing fluoride anions, Ceram. Int. 40 (2014) 8003–8011. P. Ducheyne, S. Radin, M. Heughebaert, J.C. Heughebaert, Calcium phosphate ceramic coatings on porous titanium: effect of structure and composition on electrophoretic deposition, vacuum sintering and in vitro dissolution, Biomaterials 11 (1990) 244–254. E.I. Kamitsos, G.D. Chryssikos, Borate glass structure by Raman and infrared spectroscopies, J. Mol. Struct. 247 (1991) 1–16. S. Agathopoulos, D.U. Tulyaganov, J.M.G. Ventura, S. Kannan, A. Saranti, M.A. Karakassides, J.M.F. Ferreira, Structural analysis and devitrification of glasses based on the CaO-MgO-SiO2 system with B2O3, Na2O, CaF2 and P2O5 additives, J. NonCryst. Solids 352 (2006) 322–328. J.M. Oliveira, R.N. Correia, M.H. Fernandes, Effects of Si speciation on the in vitro bioactivity of glasses, Biomaterials 23 (2002) 371–379. F. Pishbin, V. Mouriño, S. Flor, S. Kreppel, V. Salih, M.P. Ryan, A.R. Boccaccini, Electrophoretic deposition of gentamicin-loaded bioactive glass/chitosan composite coatings for orthopaedic implants, ACS Appl. Mater. Interfaces 6 (2014) 8796–8806. M. Tomozawa, Fundamentals of Inorganic Glasses, 1994. R. Martens, W. Müller-Warmuth, Structural groups and their mixing in borosilicate glasses of various compositions - an NMR study, J. Non-Cryst. Solids 265 (2000) 167–175. D. Maniu, T. Iliescu, I. Ardelean, S. Cinta-Pinzaru, N. Tarcea, W. Kiefer, Raman study on B2O3-CaO glasses, J. Mol. Struct. (2003) 485–488. A. Balamurugan, G. Balossier, S. Kannan, J. Michel, A.H.S. Rebelo, J.M.F. Ferreira, Development and in vitro characterization of sol-gel derived CaO-P2O5-SiO2-ZnO bioglass, Acta Biomater. 3 (2007) 255–262. L. Ji, W. Wang, D. Jin, S. Zhou, X. Song, In vitro bioactivity and mechanical properties of bioactive glass nanoparticles/polycaprolactone composites, Mater. Sci. Eng. C. 46 (2015) 1–9. G. Kaur, G. Pickrell, G. Kimsawatde, D. Homa, H.a. Allbee, N. Sriranganathan, Synthesis, cytotoxicity, and hydroxyapatite formation in 27-Tris-SBF for sol-gel based CaOP2O5-SiO2-B2O3-ZnO bioactive glasses, Sci. Rep. 4 (2014) 4392. R. Govindan, E.K. Girija, Drug loaded phosphate glass/hydroxyapatite nanocomposite for orthopedic applications, J. Mater. Chem. B 2 (2014) 5468–5477. S. Srinivasan, R. Jayasree, K.P. Chennazhi, S.V. Nair, R. Jayakumar, Biocompatible alginate/nano bioactive glass ceramic composite scaffolds for periodontal tissue regeneration, Carbohydr. Polym. 87 (2012) 274–283. K. Taek Jung, Y.-H. Chu, S. Haam, Y. Gun Shul, Synthesis of mesoporous silica fiber using spinning method, J. Non-Cryst. Solids 298 (2002) 193–201. I. Rehman, W. Bonfield, Characterization of hydroxyapatite and carbonated apatite by photo acoustic FTIR spectroscopy, J. Mater. Sci. Mater. Med. 8 (1997) 1–4. Z. Hong, A. Liu, L. Chen, X. Chen, X. Jing, Preparation of bioactive glass ceramic nanoparticles by combination of sol-gel and coprecipitation method, J. Non-Cryst. Solids 355 (2009) 368–372. C.Y. Kim, A.E. Clark, L.L. Hench, Early stages of calcium-phosphate layer formation in bioglasses, J. Non-Cryst. Solids 113 (1989) 195–202. R. Lucacel Ciceo, D.L. Trandafir, T. Radu, O. Ponta, V. Simon, Synthesis, characterisation and in vitro evaluation of sol-gel derived SiO2-P2O5-CaO-B2O3 bioactive system, Ceram. Int. 40 (2014) 9517–9524. M.A. Sainz, P. Pena, S. Serena, A. Caballero, Influence of design on bioactivity of novel CaSiO3-CaMg(SiO3)2 bioceramics: in vitro simulated body fluid test and thermodynamic simulation, Acta Biomater. 6 (2010) 2797–2807.