Sol-Gel Synthesis of Soda Lime Silica-Based Bioceramics Using Waste as Renewable Sources

The purpose of the work is to prepare and assess soda lime silica-based (SiO 2 -CaO-Na 2 O) bioactive ceramics using waste as renewable sources. Thus we produced a SiO 2 -CaO-Na 2 O-based bioactive ceramic by sol-gel process using rice husk and eggshells as sources of silica and calcium oxide, respectively. The precursors such as calcinated eggshell powder, rice husk ash (RHA) and sodium hydroxide (NaOH) were processed by the sol-gel method, resultant in SiO 2 -CaO-Na 2 O-based bioactive ceramics. The gel derived sintered sample showed combeite high (Na 6 Ca 3 Si 6 O 18 ) as a major crystalline phase. Subsequently, the sintered specimens were analyzed from the physical and structural point of view, and in terms of apatite mineralization rate in simulated environments and cytocompatibility in relative to human osteoblast-like cells. The studies showed that the produced crystalline SiO 2 -CaO-Na 2 O-based ceramics showed an average porosity of 45%. In vitro evaluation of the biological properties revealed that the prepared ceramics possesses the mineralization of carbonated hydroxyapatite (CHA) in a simulated environment with good cytocompatibility and controlled degradation rate. Therefore, the results obtained suggest that the prepared SiO 2 -CaO-Na 2 O-based bioactive ceramics using waste as renewable sources might be a low cost ceramics for applications in biomedical field.


Introduction
Bioceramics are defined as the synthetic crystalline inorganic material used for the healing and replacement of injured or diseased parts of the human tissue. When bioceramics are implanted in body tissue defects, the complex chemical and biological process occurs immediately due to the interaction between bioceramics and living cells that determines the progression of the tissue regeneration process [1,2]. Several bioceramics have been explored to date as potential candidates for tissue engineering applications starting from alumina (Al2O3) and zirconia (ZrO2) [3,4] to calcium phosphates and calcium silicates owing to their exceptional osteoconductivity and biocompatibility [5,6]. Wide variety of bioceramics have been designed based on the need and applications, comprised of thin layer coating on metallic implants, microspheres, composites by combined with biopolymer materials, large wellpolished surfaces and porous networks [7]. Furthermore, loading of bioceramic composite scaffolds with drugs for therapeutic purposes such as antibacterial and anti-tumor effects has also been explored [8]. However, due to the lack of quality of food structure with increasing population, bone defects have become common diseases, resulting in increasing clinical demands on tissue regeneration and wound healing. Therefore, improvement of new fabrication procedures of bioceramics is a significant aspect that researchers are exploring and trying to point out.
In general, conventional solid state sintering, melt quenching subsequent crystallization and sol-gel technique have proven to be most commonly used preparation techniques for bioceramics [9,10]. Solid state sintering and melting methods require high temperature processing; during high temperature, evaporation of the volatile component is a drawback of these methods. Whereas, sol-gel technique requires low processing temperatures and it provides high chemical homogeneity [11,12]. This method allows a wide variety of compositions with the production of different shapes like powders, monoliths, coatings or fibers [13]. Moreover, bioceramics obtained from sol-gel technique shows porosity and higher surface areas which are favourable factors for the bioactivity [14,15].
The conventional sol-gel process requires high purity alkoxysilane precursors such as tetramethylorthosilicate (TMOS) and tetraethylorthosilicate (TEOS) as silica precursors and calcium nitrate tetra hydrate (Ca(NO3)2.4H2O) as calcium oxide precursor. Nevertheless, these materials are costly. Rice husk as a silica source and eggshells as a calcium oxide source could be low-cost raw materials for sol-gel processing of bioceramics. Therefore, in the present study, a sol-gel technique was employed to produce soda lime silica-based (SiO2:CaO:Na2O) bioceramics using silica source of rice husk and calcium oxide source of eggshells with the objective of providing a method for producing cost effective soda lime silica-based bioceramics for applications in biomedical field.

Preparation of bioceramics
The bioceramic with stoichiometric balance of chemical composition 50% SiO2-25% CaO-25% Na2O (mol%) was produced by sol-gel process using calcinated eggshell powder, rice husk ash (RHA) and sodium hydroxide (NaOH; 99.9% purity, Sigma Aldrich). Eggshells and rice husk were collected from NIT canteen and local rice mill, respectively. Rice husk was cleaned using diluted hydrochloric acid as per the procedure proposed by Abu Bakar et al [16]. The washed rice husk was then calcination at 600 °C for 4 h to achieve high purity amorphous silica (~ 99%) as RHA. Boiled chicken eggshells were washed using distilled water and then dried. Afterwards, the eggshells were made into fine powder. High purity calcium oxide (~ 99%) was obtained by the calcination of fine powder of eggshells at 900 °C for 2 hours. The sol preparation was comprised of mixing of stoichiometric amount of RHA according to the TG-DTA analysis to achieve optimum crystalline ceramics.

Characterization
The typical calcination and sintering temperatures were estimated from the simultaneous thermogravimetric (TG) and differential thermal analysis (DTA) (Model: NETZSCH, Germany) of dried gel powder sample. The phase identification of the synthesized bioceramic samples was carried out by means of powder X-ray diffractometer

Physical properties
The percentage of linear shrinkage (LS) of ceramics was determined by measuring the changes in diameter of the specimens before and after sintering using equation (1). The relative density (ρ) and open porosity (Po) were determined as per the ASTM B962-17 by using Archimedes' principle [17] from equations (2&3).
Where l0 is the diameter of the specimen before sintering, l1 is diameter of the specimen after sintering, ρa is apparent density, ϕ is theoretical density of the material, W1 is weight of sintered specimen in air, W2 is weight of specimen suspended in deionized water and W3 is the weight of specimen after being saturated in deionized water.

In vitro bioactivity
Simulated body fluid (SBF) test was developed as a primary test to evaluate the in vitro bioactivity of the biomaterials. The test was conducted according to the ISO 10993-14.
SBF solution was made in consistent with the process described by Kokubo et al. [18,19]. In order to examine the apatite layer mineralization rate, sintered pellets (3 mm thickness and 10 mm diameter) at 900 °C were ultrasonically rinsed, left to air dry and then placed in torsion beakers containing SBF solution for various time periods at 37 °C. The assessment was carried out under static condition to observe the change in pH level of SBF solution over immersion period. After the prearranged soaking time, the pellets were taken out from the beakers, rinsed with deionised water and then dried. The change in surface microstructure, chemical composition, molecular arrangement and crystalline phase were studied using SEM, EDS, FTIR and XRD, respectively.

In vitro degradation
Degradation behavior of synthesized SiO2-CaO-Na2O-based ceramics was studied

Cytocompatibility test
The cellular behaviour and cytocompatibility potential of prepared bioceramics were   [20]. The next stage of mass reduction which accomplished in the temperature region of 150 -480 °C, exhibited an endothermic peak at ~ 270 °C and a small mass reduction of ~ 5.7%, which was related to chemically adsorbed water dehydration [21]. The final stage of mass reduction which could be detected in the temperature region of 480 -700 °C, exhibited an endothermic peak at ~ 625 °C in DTA curve with more prominent mass reduction of ~ 23.3%. This could be recognized to the elimination of by-products from incomplete condensation of the starting materials, mostly due to the elimination of nitrate ions [22]. Thermogravimetry is a most reliable analyses used to determine the calcination temperature of dried samples derived from sol-gel technique by taking the temperature at which the mass reduction becomes constant [23]. The TGA analysis showed that there was no mass reduction after the temperature reached 700 °C. Therefore, 700 °C temperature was chosen as calcination temperature for the dried powder.

Physical properties
The physical properties such as linear shrinkage, open porosity and relative density of sintered SiO2-CaO-Na2O-based ceramics were evaluated. It was observed that the linear shrinkage and relative density were found to be 2.

X-ray diffraction (XRD)
XRD analysis was performed to study the crystallization of sodium-calcium-silicate phases in sintered ceramic specimens. The X-ray diffractogram for the ceramic specimens after sintering at 900 °C was showed in Fig. 2. It was observed that the diffractogram depicted two evident phases for the sintered samples. The major crystalline phase was detected as combeite high (Na6Ca3Si6O18) (JCPDS 77-2189) [27]. It was also observed that the small amount of 16-sodium Tetracalcium Cyclo-hexasilicate (Na15.6Ca3.84 (Si12O36)) (JCPDS 75-1332) phase was detected as minor phase in the specimens. Further, crystallite size of ceramic was calculated from XRD pattern using Scherrer's equation by taking the value of full width half maxima (FWHM) of high intense representative peaks, which gave an average crystallite size of ~ 43 nm. Fig. 3 demonstrations the X-ray diffractograms of ceramic specimens those incubated in SBF solution for different time intervals. As can be seen from Fig. 3, the crystallinity index for sodium-calcium-silicate phases reduced with an increase in soaking time of SBF solution and the new distinctive hydroxyapatite (Ca10(PO4)6(OH)2) peaks were observed in consistence with JCPDS no: 09-0432 [28], demonstrating that the hydroxyapatite layer mineralization on the sample after soaking in SBF solution. Furthermore, the crystallinity index for hydroxyapatite enhanced with prolonged soaking time in SBF solution.
The band at ~ 3434 cm -1 was evidence of stretching vibration of moisture absorption, while the band at ~ 1642 cm -1 related with absorbed H2O bending vibration [34].

SEM-EDS analysis
The microstructural images of sintered ceramics obtained from SEM can be visualized in Fig. 5. The microstructure of sintered sample clearly showed the intergranular structure with pores. The removal of residual volatile moieties through calcination and sintering originates the pores on the surface. Microstructural observations also evidence that the formation of vermicular structure due to the together sintering of sodium calcium silicate particles. As it is known, sintering of polycrystalline ceramics ensues by diffusional transport of matter along definite paths that define the sintering mechanisms. The sintering occurs when the sodium calcium silicate particles are in contact together. Consequently, the particle sintered in contact points and result in a vermicular structure. This type of microstructural images were described by other studies in calcinated and sintered ceramics [23,35].
Furthermore, the elemental analysis of the synthesized SiO2-CaO-Na2O-based bioceramic was analysed quantitatively using EDS spectra. The EDS spectra confirmed the presence of elements such as Ca, Na, Si and O.
The surface of the ceramics was exposed again to SEM analysis to identify the possible changes in their microstructure upon soaking in SBF solution as an outcome of hydroxyapatite mineralization. The corresponding images are presented in Fig. 6. The high magnification images revealed the presence of fluffy aggregates through radially packed nanosheets on the surface of ceramics, while their quantity became higher and dense as the soaking time increased from 7 to 21 days. Furthermore, at lower magnification, the agglomerated spherical-shaped entities were evidenced. Similar micrographs were described at the surface of SBF immersed 45S5 bioglass derived glass ceramics [26,36]. The results could represent a considerable fast and pronounced capacity of apatite mineralization of prepared SiO2-CaO-Na2O-based bioactive ceramics using waste as renewable sources. These statements were sustained by EDS spectra. In the elemental mapping images, the signals related with P and Ca became dominant and shielded the contributions of the elements found at the surface, emphasizing the quantitative conformation of mineralization of hydroxyapatite layer.

Variation in pH level of SBF solution
The pH changes of SBF solution with the soaking time as a result of ion exchange reactions at the solid-liquid interface are illustrated in Fig. 7. The pH of the SiO2-CaO-Na2Obased ceramic soaked SBF solution was shown to increase from 7.4 to 7.85, followed by initial rapid increase, with small increments with increasing soaking time. After 11 days of soaking, the pH remained relatively stable at around pH of 7.85. According to the mechanism suggested by Hench [37], the apatite mineralization was accompanying to the release of Ca 2+ and Na + ions from the ceramic sample through ion replacement method with H + and H3O + ions from the SBF solution which is responsible for the increase in pH level. The ion replacement leads to the formation of SiO2-rich layer on the sample surface, which provides favourable environments for mineralization of hydroxyapatite layer. Once the SiO2-rich layer formed on the sample surface, the calcium and phosphate ions are migrated onto the surface from the SBF solution to grow into hydroxyapatite. In the present study, the changes in the pH obtained in the range of 7.4 -7.85 when synthesized SiO2-CaO-Na2O-based ceramic was soaked in SBF solution. The results were comparable to the pH range reported for the soda lime silica-based bioglass ceramics in earlier cases [38,39] and it was favourable environment for bone cell culture [40].

Degradation studies
The degradation behaviour of prepared SiO2-CaO-Na2O-based bioactive ceramics was evaluated in SBF solution as well as in Tris-HCl buffer solution. Fig. 8 shows Tris-HCl is a plain buffer solution that contains no ions therefore it shows minimal reprecipitation activity and maximum solubility of the material [41]. Dissolution rate is an important feature that a bioactive substance must fulfil. The material dissolution produces ions that act as enhancements to the medium stimulating osteogenesis, though the dissolution rate of a bioactive material and the kinetics of mineralization of apatite need to be in the same order for safe implantation [36]. In general, alkali-based bioactive materials possess a slightly fast dissolution rate which leads to the abrupt changes in the pH values of local physiological microenvironment [42]. The dissolution rate of these alkali-based bioceramics can be improved on ion-based modification by doping the less soluble phases like Zr, Zn, Mg, etc.
On the other hand, the degradation study results of the this study exhibited that the synthesized SiO2-CaO-Na2O-based ceramic using waste as renewable sources demonstrated considerable controlled dissolution rate in SBF solution.

Cytocompatibility
The in vitro biocompatibility of prepared SiO2-CaO-Na2O-based bioactive ceramics was analyzed using MG 63 osteoblast-like cells. Specifically, cytocompatibility was assessed by determining the percentage of cell viability 24 hours after incubation of SiO2-CaO-Na2Obased bioactive ceramics with osteoblast-like MG 63 cells by MTT assay. The corresponding cell viability profile is presented in Fig. 9. As can be understood from the cell viability profile, the relative percentage of cell viability slightly decreased after treating with different concentrations (31.25 μg/mL -2000 μg/mL) of bioceramic particle, but not less than 75%.
ISO 10993-5 says that the material can be considered cytotoxic when it losses 30% of its cell viability [43]. In the current study, the relative cell viability is greater than 75% even at higher concentration of bioceramics. Therefore, cytocompability evaluation results could demonstrate that SiO2-CaO-Na2O-based bioactive ceramics synthesized using waste as renewable sources have pronounced cytocompability potential with human osteoblast-like cells.

Conclusion
Soda lime silica-based (SiO2-CaO-Na2O) bioceramic was produced via sol-gel process, utilizing rice husk ash and eggshells as renewable sources for silica and calcium oxide, respectively. Combeite high (Na6Ca3Si6O18) was determined as a major crystalline phase in the sol-gel derived sintered sample at 900 °C with an average porosity of 45%. The hydroxyapatite mineralization ability of resultant ceramic was confirmed using simulated body fluid (SBF). The results of in vitro bioactivity test showed that the ceramics exhibited excellent bioactivity with mineralization of carbonated hydroxyapatite on its surface within 3 days of SBF immersion. Degradation study results showed that the soda lime silica-based ceramics thus produced has substantial controlled degradation rate. Cytocompability evaluation results demonstrated the pronounced cytocompability potential at different dosages (31.25 μg/mL -2000 μg/mL) with human osteoblast-like MG-63 cells. Therefore, SiO2-CaO-Na2O-based ceramics prepared using waste as renewable sources might be prospective low cost ceramics for use in biomedical applications. The work also established believably that, the synthesis method is potential beneficial for the production of low cost soda lime silica-based bioceramics.

Ethical approval:
This article does not contain any studies with human participants or animals performed by any of the authors. FTIR spectra of sintered ceramic before and after soaking in SBF solution The surface microstructure and elemental analysis of sintered ceramic Figure 6 The surface microstructure and elemental analysis of sintered ceramic after soaking in SBF solution Change in pH of SBF solution during bioactivity study Figure 8 Degradation behaviour of prepared SiO2-CaO-Na2O-based bioactive ceramics in SBF and in Tris-HCl buffer solution Figure 9 Cell viability of osteoblast-like MG 63 cell when exposed to the bioceramic particles

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