Borosilicate bioactive glasses (BBGs) have been attracting wide attention in bone tissue engineering due to their relatively rapid degradation that may be used to achieve a more controlled release of specific component ions. Controlled release of ions may enhance their biological activity, such as the increased rate of hydroxyapatite formation. However, high degradation rates can cause cytotoxic effects when BGGs are implanted [1, 2]. This work aimed to synthesise and characterise the thermal and chemical properties of three novel borosilicate-based glasses and to investigate if crystallisation of the BBGs modifies the cytotoxic effects.
Materials and Methods
The BBGs of general formula of 0.05Na2O × xMgO × yCaO × (0.35-x-y)SrO × 0.20B2O3 × 0.40SiO2 (molar ratio, where x, y = 0.35 or 0.00, and x ≠ y) were synthesised by melt quenching, ground and sieved to < 63 μm. The three borosilicate based glass compositions where confirmed by X-ray fluorescence (XRF) spectroscopy and the phase evolution was assessed by differential thermal analysis (DTA) with a heating rate of 10 ºC min-1. Fast-quenched glass samples were heat-treated with a heating rate of 10 ºC min-1, holding for 120 min at each midpoint of the glass transition and at the peak crystallisation temperatures before cooling to room temperature. X-ray diffraction analysis (XRD) and Fourier transform infrared (FTIR) spectroscopy were used to evaluate the glasses before and after heat-treatments (glass-ceramics). The in vitro cytotoxicity evaluation was performed following the international guidelines of ISO 10993-5:2009 using mouse fibroblast cell line (L929). Either, glass or glass-ceramic samples (concentration range from 10 to 200 mg/ml) were incubated for 24 h in culture medium and the conditioned medium was used to assess the possible toxic effect of those samples for a period time of 3 days. The cellular metabolic activity and proliferation rate were evaluated using PrestoBlue® (PB) and PicoGreen (PG) assays, respectively.
Results and Discussion
The DTA patterns identified the glass transition and more than one exothermic peaks for each sample. The XRD patterns after the first heat treatment (Tg + Tc1) showed to be predominantly amorphous with residual crystalline phases. For the second and third heat treatments (Tg + Tc2 or Tc3) we observed predominantly crystalline phases rich in borate and silicate. After heat treatment, as expected, the phases formed depended on the composition. For example, Mg-containing glass-ceramic revealed the presence of magnesium silicate (Mg2(SiO3)2, JCPDS Card No. 86-433) and magnesium borate (Mg2B2O5, JCPDS Card No. 73-2232). Ca-containing glass-ceramics were rich in wollastonite-2M (CaSiO3, JCPDS Card No. 76-186) and calcium borate (Ca(BO2)2, JCPDS Card No. 32-155). Finally, Sr-containing glass-ceramic revealed strontium silicate SrSiO3, JCPDS Card No. 87-474) and strontium borate (Sr2B2O5, JCPDS Card No. 19-1268) phases. The FTIR confirmed that borosilicate glass structure presented new and different molecular bonds after Tc2 and Tc3 heat treatments.
The in vitro cytotoxicity evaluation demonstrated that L929 cells have good proliferation rates and metabolic activity for concentrations equal or lower than 50 mg/mL, for all glass and glass-ceramic extracts. Furthermore, all borosilicate samples exhibited less toxic effects in respect with the Bioglass 45S5 (positive control). Concerning the Mg-containing glasses there was an improvement for Tc2 treated glass-ceramic in comparison with untreated and Tc1 treated glass ones on the toxic effects onto cells. However, for Ca- and Sr-containing glasses the improvement of the toxic effect was observed for Tc1 treated glass-ceramic in comparison with untreated and Tc2 and Tc3 treated glass-ceramics.
Three BBGs with different glass modifiers were successfully synthesised by melt quench. DTA analysis allowed the identification of two or more exothermic peaks indicative of crystallisation. After heat treatment at these temperatures, XRD and FTIR suggested that only the samples that were treated at the higher crystallisation temperatures produced crystalline phase(s). This study demonstrated that controlled crystallisation of borosilicate glasses might produce glass-ceramics with less cytotoxic effects.