Bioactivity of quaternary glass prepared from bentonite clay

Alkoxysilane precursors are the most widely used silica source for sol–gel preparation of silicate-based bioactive glass. However, due to their high cost, alternative sources such as bentonite clay are desirable. In the present work, bentonite clay was reacted with sodium hydroxide (NaOH) to extract sodium metasilicate (Na2SiO3). The obtained Na2SiO3 was converted to gel which was then sintered at 950 ℃ for 3 h to give the bioactive glass in the quaternary composition SiO2–NaO–CaO–P2O5. The resulting glass was incubated in simulated body fluid (SBF) for 0–7 days to evaluate the bioactivity. Furthermore, glass samples were characterized before and after SBF study by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Results obtained showed the presence of Na2Ca2Si3O9 (combeite) crystal as the major crystalline phase and the formation of hydroxyapatite (HA) and hydroxycarbonated apatite (HCA) on the surface of the glass after immersion in SBF. The material showed potentials for application as scaffold in bone tissue repair.


Introduction
The interest in bioactive glass has continued to soar particularly within the orthopaedic biomedical research community because of its ability to promote self-repair of damaged bones when used as temporary scaffold in vivo. The "gold standard" for bone treatment involves autograft and allograft transplantation. However, these methods have severe limitations and thus impose high cost on the health care system. The shortcomings associated with autograft include severe pain, morbidity of the harvested site, structural and anatomical problems, and high rate of resorption during healing [1,2]. Likewise, allograft may elicit disease transmission and rejection, further compounded by shortage of donors [3,4].
The use of bioactive glass for bone restoration represents a paradigm shift from replacement to regeneration through the use of synthetic bone grafts as positive effort to address the aforementioned drawbacks. The unique capability of bioactive glass over other bioceramics includes amongst others the ability to form hydroxyapatite (HA) on the surface when immersed in physiological environment [5,6]. This is beneficial because of the similarity in chemical composition of HA to bone and therefore facilitates the bonding between the material and bone [7]. Other significance of bioactive glass in physiological environment is its ability to elicit osteoblastic activity and improvement of cell adhesion [8]. Indeed, it has been reported that seven families of genes are up-regulated by the ionic dissolution products from  bioactive glass [9].
Generally, sol-gel-derived glass, with its inherent mesoporosity, provides larger surface area and consequently more rapid degradation rate than melt-derived glass of similar composition [10,11]. Additionally, the sol-gel technology provides higher purity, homogeneity, and possibility of increasing the compositional range above 60 mol% while maintaining bioactivity of the glass [12].
Recently, our research group embarked on the search for new silica-based materials as economic synthetic substitutes to the widely used alkoxysilane precursors such as tetraethyl orthosilicate (TEOS) and tetramethyl othosilicate (TMOS). Consequently, we have reported the synthesis of bioactive glass from sand and clay using the sol-gel technique [13,14]. However, in our use of bentonite clay as a novel starting material [14], we did not evaluate the bioactivity of the obtained bioceramic, and to the best of our knowledge, no previous work on the bioactivity of sol-gel-derived bioactive glass from bentonite clay has yet been reported. Therefore, herein we report the bioactivity of quaternary bioactive glass in the system SiO 2 -Na 2 O-CaO-P 2 O 5 obtained from bentonite clay.

2 Methods
To prepare the bioactive glass with composition 46.99 mol% SiO 2 , 24.36 mol% Na 2 O, 25.50 mol% CaO, and 3.15 mol% P 2 O 5 , sodium metasilicate (Na 2 SiO 3 ) was first extracted from bentonite clay as described previously [15]. In a typical sol-gel procedure, appropriate amount of the obtained Na 2 SiO 3 was dissolved in deionized water to give a solution that was then added slowly to 1 M HNO 3 in a beaker furnished with a magnetic stirrer under ambient temperature for 1 h to facilitate complete hydrolysis. Thereafter, H 3 PO 4 , NaNO 3 , and Ca(NO 3 ) 2 ·4H 2 O were added under constant stirring to give a molar ratio 1:10 of each reagent to water in the sol. A reaction time of 45 min was allowed for each reagent before adding the next. After complete addition, the mixture was further stirred for 1 h to afford a gel. The resulting gel was aged at room temperature for 3 days, dried at 60 ℃ for 72 h and 130 ℃ for 42 h, and stabilized at 700 ℃ for 2 h. Finally, the material was sintered at 950 ℃ for 3 h for densification and strengthening of pore struts.

3 Characterization
The density of the bioactive glass glass  was calculated from the mass and dimensions of the sintered glass. The porosity P was then obtained mathematically as follows: Bioglass ® [16]. The microstructure, composition, and pore size distribution of the glass were evaluated in a Phenom Prox (Eindhoven, the Netherlands) scanning electron microscope (SEM) equiped with energy dispersive X-ray analyzer (EDX) before and after immersion in simulated body fluid (SBF). The sample was carbon-coated and observed at an accelerating voltage of 15 kV.
Samples were characterized using X-ray diffraction (XRD) analysis after sintering and after each immersion experiment in SBF with the aim of assessing the major phase present and the formation of hydroxyapatite (HA) crystal on samples' strut surface respectively. The samples were first ground to powder. Then 0.1 g of the powder was measured in a PANalytical Empyrean X-ray diffractometer using Cu Kα radiation source of wavelength λ = 0.154056 nm operated at 40 kV and 40 mA to obtain the diffraction patterns in the 2θ range from 5° to 90°.
Fourier transform infrared spectroscopy (FTIR, Shimadzu 8400S), with wavenumber range of 4000-400 cm 1 employing KBr pellets and operating in a reflectance mode with 4 cm 1 resolution, was employed to monitor the nature of bonds present in the as prepared samples.

4 In vitro bioactivity assessment in simulated body fluid
Evaluation of bone bonding ability was carried out by the standard in vitro procedure [17]. The acellular simulated body fluid (SBF) was prepared using analytical reagent-grade chemicals NaCl, NaHCO 3 , KCl, K 2 HPO 4 ·3H 2 O, MgCl 2 ·6H 2 O, CaCl 2 , trishydroxymethyl aminomethane (Tris-buffer, (CH 2 OH) 3 CNH 2 ), and 1 M HCl with ion concentrations shown in Table 1. Samples were immersed in the SBF solution at a concentration of 0.01 g/mL in clean plastic bottles, which were initially washed using HCl and deionized water. The bottles were placed inside a thermostated incubator at a temperature of 36.5 ℃ and an initial pH of 7.4. The SBF solutions were not refreshed throughout the period of immersion to allow for measurement of pH of the solution at different intervals between 0 and 120 h duration using a pH meter (Hanna, HI96107) previously calibrated with buffer solutions of pH = 4.0, 7.0, and 9.0. The samples were extracted from the SBF solution after 3 and 7 days. The extracted samples were rinsed with deionized water and left to dry at ambient temperature in a desiccator. The formation of apatite layer on the glass surface was monitored by SEM, EDX, XRD, and FTIR.

1 Changes in morphology
The morphology of the glass before and after immersion in SBF is shown in Fig. 1. As observed in Fig. 1(a), before immersion, the glass presents almost uniform porous surface containing mostly cuboid-shaped glass architecture well distributed throughout the material. The porosity of the glass obtained by applying Eq. (1) is 82%. After immersion in SBF for 3 days, the glass morphology changes to fine grain-sized particles, most of which are agglomerated as shown in Fig. 1(b). The EDX spectrum during this period of immersion shows a decrease in the concentrations of Na, Si, and Ca but an increase in P. These changes can be attributed to the formation of HA on the surface of the glass. After immersion for 7 days in SBF (Fig. 1(c)), the micrograph shows dense clusters of HA particles, which also appear coarse. Accordingly, the EDX spectrum records low detection of Si due to increase in population of apatite on the glass surface, while the concentrations of Ca and P increase. Furthermore, a small carbon peak becomes evident indicating the formation of crystalline hydroxycarbonated apatite (HCA).

2 Diffraction patterns
The XRD investigation of the glass samples after annealing at 950 ℃ for 3 h reveals the presence of major peaks of combeite (Na 2 Ca 2 Si 3 O 9 ) crystal usually formed from devitrification of quaternary system of SiO 2 -Na 2 O-CaO-P 2 O 5 bioactive glass [18,19] as shown in Fig. 2(a). Both the angular location and intensity of the peaks match the standard PDF #22.1455. As seen, it is obvious that the glass does not fully crystallize judging by the low intensities of the Na 2 Ca 2 Si 3 O 9 peaks at locations other than the one at 2θ  30°. Even before immersion in SBF, a peak identified as apatite is clearly visible at 2θ  32°. This phenomenon has been attributed to the aqueous environment of the sol-gel process which favours the precipitation of HA from the amorphous structure of silica-based bioactive glass [20]. After immersion in SBF, the intensity of the Na 2 Ca 2 Si 3 O 9 peaks decreases gradually as shown in Figs. 2(a)-2(c). The gradual decrease is an indication that the material could have controlled degradation rate in physiological fluids, and hence is able to act as temporary scaffold prior to complete integration of the damaged site, as a major requirement of an ideal scaffold for bone repair [21]. Apatite peaks increase as immersion duration is prolonged. All apatite positions are corroborated by those in the standard PDF JCPDS #9-0432.

3 Assessment of bonds
The FTIR spectrum of the pristine bioactive glass is shown in Fig. 3(a). As observed, there are prominent peaks at 3462, 1684, 1458, 1005, 850, 602, and 457 cm 1 . The broad band between 3300 and 3800 cm 1 centred at 3462 cm 1 is due to OH groups in the material, which is further confirmed by the presence of water absorbed band around 1684 cm 1 . The band at 1458 cm 1 is representative of the absorption of carbonate group (ν 3 ), while the sharp peak at 1005 cm 1 is associated with Si-O-Si vibrational modes [22]. The peak at 850 cm 1 is synonymous with C-O stretching vibration [22], which may be due to adsorption of atmospheric CO 2 during the processing stage of the glass. There is an intense peak at 602 cm 1 which is assigned to P-O bending mode in crystalline calcium phosphate, and another small peak at 457 cm 1 is attributed to Si-O-Si bending vibrations. After immersion for 3 days (Fig. 3(b)), the peak at 1458 cm 1 becomes broader and splits into two modes at 1476 and 1424 cm 1 , and similarly the peak at 602 cm 1 develops into two modes at 604 and 569 cm 1 . These are characteristics of apatite crystalline phase [23], signalling the incorporation of CO 3 2from the SBF solution into HA on the glass surface to form HCA. While after 7-day immersion in SBF ( Fig. 3(c)), the twin peaks at 604 and 569 cm 1 become more intense due to increase in density of HCA on the surface of the glass, thus explaining the appearance of the small carbon peak observed in the EDX spectrum ( Fig. 1(c) inset). Figure 4 shows the pH change of the glass in SBF for the first 120 h, which is in agreement with the reaction sequence of bioactive glass in biological fluids [5]. As observed, there is fast release of alkali and alkaline (2) After 4 h, the pH increases slowly until 48 h because part of the released Ca 2+ from the glass is used to form CaO-P 2 O 5 , thus decreasing the release rate as shown in Fig. 4. As immersion duration reaches 96 h, the pH increase slows further as more Ca 2+ is withdrawn from the SBF solution to develop HCA layer on the surface of the glass. Finally, the pH reaches a saturated value of 11.7 after 96 h without increasing any further due to crystallization of HCA.

1 Composition
Bentonite clay, a naturally occurring sedimentary clay with the chemical formula (OH) 4 Si 8 Al 4 O 20 • nH 2 O, has a 3-layer structure displaying one aluminium oxide sheet sandwiched by two silicon oxide sheets. The arrangement is such that Mg 2+ ions may substitute for Al 3+ ions leaving a net negative charge on the sheets [14]. The clay is reacted with NaOH to extract the Si 4+ ions from the sheets to form Na 2 SiO 3 as filtrate and Al(OH) 3 as residue as shown in a previous reaction scheme [14]. The obtained Na 2 SiO 3 then serves as precursor for SiO 2 and Na 2 O for preparing the glass. The EDX result of the sintered glass as shown in Fig. 5 confirms the presence of all the elements in their appropriate ratios as prepared. Additionally, a residual peak of Al observed in the spectrum may be due to incomplete removal of Al(OH) 3 from the Na 2 SiO 3 filtrate during the extraction stage. Hence a more efficient filtration method, such as vacuum filtration, may be necessary. Apart from bentonite clay, kaolinite clay (Al 2 Si 2 O 5 (OH) 4 ) could serve as another cheap alternative source of silica.

2 Microstructure
The glass exhibits sub-micron pore diameter of mean value 2.54 μm ( Fig. 6(a)), while the average pore area is 0.29 μm 2 ( Fig. 6(b)). This is expected as clay particles themselves have dimensions on nanoscale [24,25], thus arrangement of these particles leads to evolution of large surface area associated with sub-micron pore structure as observed in Fig. 6. The microstructure of the glass as well as its porosity of 82% are crucial textural parameters for enhanced cell adhesion, vascularisation, infiltration, and osteointegration on the glass material [26][27][28][29][30][31].

3 Na 2 Ca 2 Si 3 O 9 crystalline phase
The Na 2 Ca 2 Si 3 O 9 crystal detected in the XRD spectrum (Fig. 2) is the major crystalline phase, and displays cuboidal architecture as seen in the SEM micrograph shown in Fig. 1(a). During sintering, Na 2 O, CaO, and SiO 2 present in the composition react to form Na 2 Ca 2 Si 3 O 9 . Mechanical strength is a significant factor in the choice of an ideal scaffold to serve as temporary support for new bone growth [31]. While there have been concerns about the biodegradability of crystalline Na 2 Ca 2 Si 3 O 9 , some authors have reported different scaffold designs and synthetic route optimization to improve degradation behaviour of Na 2 Ca 2 Si 3 O 9 in physiological fluids [31][32][33]

4 pH change in SBF
The steep rise in pH during the first 4 h of soaking in SBF indicates high reactivity of the glass. The large surface area conferred on the glass by well distributed particles and sub-micron-sized pores presents optimal sites for ion-exchange reaction to occur. This phenomenon explains why apatite nucleation occurs at a shorter time, even without immersion in SBF, as observed in the spectra for XRD and FTIR in Fig. 2(a) and Fig. 3(a) respectively. At the end of 120 h (5 days), the pH reaches a value of 11.7 from the initial 7.4 during which apatite colony increases on the surface of the glass as evident by the low detection of Si recorded by EDX shown in Fig. 1(a). The basis of bonding property of bioactive glass has been anchored on the chemical reactivity in physiological body fluids (in vitro and in vivo), which leads to the formation of HCA layer to which bone can bond.

Conclusions
The highly bioactive glass in quaternary composition has been prepared starting with bentonite clay as silica source instead of conventional alkoxysilane precursors. The large surface area and micron-sized pore structure obtained through the sol-gel processing method were key in accelerating the reactivity of the glass in SBF to form apatite. The crystalline Na 2 Ca 2 Si 3 O 9 formed from sintering the glass is important in reinforcing the strength of the material during application as scaffold in bone repair. Interestingly, the crystalline phase did not hinder the biodegradability of the glass in SBF.
With continuing efforts geared towards cost effective design of scaffolds for optimal bioactivity, bentonite clay may be a potential cheap raw material for the preparation of bioactive glass.