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Title: Bioactive Properties of Glasses/glass-ceramics Synthesized from Agricultural and Food Wastes
Authors: Punj, Shivani
Supervisor: Singh, Kulvir
Keywords: Corn husk;Sugarcane leaves;Wheat straw ashes;Egg shell powder;SBF;Bioactivity,biocompatability
Issue Date: 10-Nov-2022
Abstract: Agro-food wastes/ashes such as corn husks, sugarcane leaves, wheat straws, and eggshell powder are used as resource materials to prepare the glasses via the melt-quench technique. These agro-food wastes/ashes/powders contain silica, calcium, magnesium, potassium, and some other trace elements that are usually required to make bioglass/glass-ceramics. Moreover, agro-food wastes are also sustainable, and cost-effective sources to synthesize bioglasses/glass-ceramics. The effect of different agro-food waste ashes like sugarcane leaves ashes, wheat straw ashes, and eggshell powder with fixed corn husk ashes are investigated. The formed glasses/glass-ceramics are investigated for their physical, structural, thermal, and mechanical properties to check their suitability and applicability as biomaterials. Bioactive properties of the glasses/glass-ceramics are investigated in-vitro using simulated body fluid. Further, the biocompatibility of these glasses/glass-ceramics is observed on osteoblastic-like human cell lines (MG-63) using 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay to investigate their applicability. The thesis work is represented in seven chapters. Chapter 1 deals with the basics of glass/glass-ceramics, bioglasses/glass-ceramics, and their basic requirements to qualify as bioactive materials. Different testing methods used to test the bioactivity of glasses/glass-ceramics followed by the mechanism of formation of HAp layer on the glass/glass-ceramics surface after soaking in simulated body fluid (SBF) are also given in this chapter. The biocompatibility testing methods are also given in brief. The 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay is a reliable method. It is directly related to the number of viable (living) cells introduced to test the biocompatibility of glass/glass-ceramics. Further, the potential agro-food wastes and their constituents are also discussed in this chapter. Chapter 2 This chapter contains the literature survey on various agro-food wastes/ powder/ashes and their use as resources to synthesize different bioactive materials. The different agro-waste materials along with their ashes have been studied to extract silica as alternative resource material to commercial silica production to synthesize bioglasses/glass-ceramics. Apart from this, other structural, thermal, mechanical, bioactive, and biocompatible properties of agro-food wastes derived bioglasses/glass-ceramics and calcium phosphate ceramics, silicate-calcium based bioceramics have been summarised. Based on the literature survey, the motivation of the present study followed by objectives are also given in the last of this chapter. Chapter 3 The compositions of raw materials such as corn husk ash (CHA), sugarcane leave ash (SCLA), wheat straw ash (WSA), and eggshell powder (ESP) are analyzed by energy dispersive spectroscopy (EDS). Based on chemical analysis, different compositions with variable agro-food wastes contents are proposed such as [(60) CHA-(40-x) SCLA-(x) ESP] where, x=10, 20, and 30 (wt %) and [(60) CHA-(40-x) WSA-(x) ESP] where, x=10, 20, and 30 (wt %). These compositions are synthesized by recrystallized alumina crucibles via melt quench techniques. The two samples from each series with similar compositions [60 CHA-30SCLA-10 ESP], [60 CHA-10 SCLA-30 ESP] and [60 CHA-30WSA-10 ESP], [60 CHA-10WSA-30 ESP] in wt % are selected to prepare in platinum-rhodium (Pt-Rh) crucible. Agro-food wastes derived glasses are also replicated using conventional chemical oxides based on the best bioactive results for comparison. The chemical compositions of the as-prepared samples have been examined via energy dispersive spectroscopy (EDS). The Archimedes principle has been used to study the density of the as-prepared samples. The structural and thermal properties of the samples are analyzed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The mechanical property such as microhardness of these samples are studied using a Vickers hardness tester. The bioactivity of these glasses/glass-ceramics are checked through an in-vitro test after soaking the samples in simulated body fluid (SBF) for 7 to 28 days, parameters like weight change of the samples and pH change of the SBF are checked after every 7 days. The soaked glasses in SBF are characterized by XRD, FTIR, scanning, and field emission scanning electron microscope (SEM and FESEM) with EDS and microwave plasma atomic emission spectroscopy (MP-AES) to know the physicochemical reactions take place on the surface of glasses. The biocompatibility of as-prepared glasses/glass-ceramics with concentrations of 1, 2, 5, and 10 mg/ml, are studied on osteoblast-like human cell lines (MG-63) using 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, assay. The cell viability is determined using the Elisa microplate reader. Chapter 4 is related to the obtained results and their interpretations. This chapter is divided into two sections. In the first section, the effect of SCLA on replacing ESP with the fixed composition of CHA is studied, whereas, in the second section the effect of WSA on replacing SCLA in the base glass is discussed in detail. The effect of SCLA and WSA on the physical, structural, thermal, mechanical, bioactive, and biocompatible properties are investigated thoroughly. The XRD patterns show the confirmation of the amorphous nature of the glasses. The microhardness of the SCLA glasses is in the range of 5.64 to 6.26 GPa, which is within the range (4-10 GPa) of reported conventional chemical-derived silicate glasses. The addition of (30 wt %) ESP (CaCO3) at the cost of (10 wt %) SCLA, depolymerize the silica-phosphate glass network and increases the hydrophilic nature of the glasses increasing the surface reactivity and bioactivity of the glasses that are accessed in SBF. Moreover, weak bands 562, 602 cm-1 due to P-O bonding (amorphous) as well as 1423-1465 cm-1 stretching of carbonate bands on the glass surface after soaking in SBF have appeared in the FTIR spectra. The spherical sponge type of amorphous carbonated HAp (c-HAp) adhered on the surface of soaked glasses is confirmed via SEM and EDS. Initially, all glasses show a Ca/P ratio is high 4.91 as hydroxyapatite layer (HAp) is taken place and became less ~ 2 in all the glasses which is higher than standard HAp (1.67). All glasses have shown good (> 90 %) cell viability on human osteoblastic-like cell lines (MG-63) by varying concentrations (1mg/ml to 10 mg/ml) of glass. When SCLA is replaced by WSA, WSA becomes more reactive than SCLA towards recrystallized alumina crucible as determined via EDS analysis. WSA glasses have shown an increase the compactness, rigidity (5.34 to 6.47 GPa), and less depolymerize the silicate-phosphate glass network than SCLA glasses, due to the presence of higher K2O and alumina contents is confirmed by DSC and FTIR. In-vitro studies, the dissolution rate becomes lower due to the incorporation of alumina contents in the glass network that reduce the bioactivity as well as change the morphology of HAp from sponge to flakes type is confirmed by FESEM. The higher alumina (10.8 wt %) containing glass (10 wt % WSA) inhibits the bioactivity as well as become toxic (cell viability ≤ 68 %) at a higher concentration (10 mg/ml). This is due to the increase of a higher amount of alumina which strengthens the glass network and enrichment of the Al-OH group that decreases the release of silicon as well as a lack of silanol groups that inhibit the apatite formation on its surface is confirmed by FTIR and MP-AES. Chapter 5 In this chapter, the effect of Al2O3 on bioactivity has been discussed to synthesize similar compositions in the Pt-Rh crucible. So, this chapter deals with composition [60 CHA-30SCLA-10 ESP], [60 CHA-10 SCLA-30 ESP] in wt % and [60 CHA-30WSA-10 ESP], [60 CHA-10WSA-30 ESP] in wt % are prepared using Pt-Rh crucible. The glasses synthesized in the Pt-Rh crucible have shown a lower amount of alumina (0.78 wt % to 1.02 wt %) in comparison to those synthesized in an alumina crucible (4 wt % to 10.8 wt %) is confirmed by EDS. With the decrease of alumina contents, the glass-forming ability of agro-food wastes derived glasses has reduced on replacing of SCLA with WSA. This is due to the presence of different alkali and alkaline earth metal ions in agro-wastes/ashes that have higher combinational entropy which plays a different role in the formation of glasses is confirmed by DSC. The microhardness of these Pt-Rh synthesized glasses lies in the range of 5.59 to 6.57 GPa. The diffusion of alumina from the crucible is decreased the water adsorption tendency. Since water adsorption tendency enhances the bioactivity of the glasses. In the in-vitro study, more depolymerized the glass network and Si-OH (silanol) groups increased on its surfaces is confirmed by MP-AES and FTIR. The WSA-based glasses are formed globular type amorphous c-HAp with a Ca/P ratio of 1.7 to 4.0 is also confirmed by FTIR and FESEM with EDS. The cell viability of 10 wt % WSA-based glass on MG-63 cell lines has shown > 88 % at a higher concentration of 10 mg /ml, which could be beneficial for bone bonding applications. However, due to the release of more Mg2+ ions in SCLA glasses, bioactivity is decreased and also inhibits to form amorphous c-HAp on the glass surface. SCLA glasses have formed flakes and cauliflower-type amorphous HAp with Ca/P ratio of 1.6 to 3.9 is confirmed by MP-AES, FTIR, and SEM with EDS in these samples. Chapter 6 based on bioactivity, biocompatibility, and other properties, two of the best compositions have been synthesized using conventional chemicals for comparison with agro-food wastes derived glasses. These glasses have been synthesized using the melt-quench technique following similar conditions for comparison and differentiate among conventional and agro-food wastes derived glasses. The glasses are derived from conventional chemicals show more density and rigidity (6.47-6.83 GPa) in the glass structure due to the formation of more covalent bonds Si-O-M or P-O-M ( M=Al or Mg) than glasses derived from agro-food wastes, which is an agreement with a characteristic temperature of the glass determined by DSC and FTIR. In the in-vitro study, the SCLA-based conventional chemical glass has shown to release more Mg2+ ions in the SBF, reduce the overall rate of calcium phosphate precipitates, and slow (28 days) the transformation of amorphous HAp to amorphous c-HAp. It also changed the morphology of HAp from sponge to irregular flakes with a reduced Ca/P ratio of 3.98 to 2.18 than the glass derived from agro-food wastes. The conventional chemical oxide-based glass has also become toxic at a higher concentration (10 mg/ml) of glass, due to leaching out of more Ca2+ ions in SBF than the glass derived from agro-food wastes. However, WSA- based conventional chemical glass has shown similar bioactive and biocompatible properties as observed for agro-food wastes derived glass. Chapter 7 contains the conclusion and future scope of the present work. The results obtained within the present study reveal that the glass synthesized in an alumina crucible using a higher amount of (30 wt %) ESP contents in place of (10 wt %) SCLA has shown good bioactivity. This glass also shows good biocompatibility on human osteoblastic-like (MG-63) cell lines (> 90 % at higher concentration (10 mg/ml)), as compared to the similar composition of glass synthesized in Pt-Rh crucible and using conventional chemical oxides. This is due to the presence of different alkali and alkaline earth metal ions in agro-wastes/ashes controlling the dissolution rate of the glass in comparison to a reported conventional chemical-derived glasses. SCLA-based glass showed a high tendency to form spherical sponge-type amorphous c-HAp within 14 days of soaking in SBF, due to lower release of Mg2+ and higher release of K+ ions also help to incorporate the Ca-P precipitates on the glass surface. However, it inhibits the formation of crystalline HAp on the glass surface. Conventional chemical-based glasses with a Ca/P ratio >1 (for human bone the Ca/P ratio is 1.67) used as implants have been reported in the previous studies. The present glass has shown a Ca/P ratio >1.67, which is expected to be useful for bone bonding applications. On the other hand, WSA (10 wt %) based glasses have also shown good bioactivity and biocompatibility same as SCLA (10 wt %) only when it is synthesized in Pt-Rh crucible. Therefore, it is less cost-effective than the glass synthesized in an alumina crucible. In future, agro-food wastes materials could be used as resource materials in biomedical applications. .
Appears in Collections:Doctoral Theses@SPMS

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