ASSESSMENT OF THE EFFECTS OF VARIOUS PHYTOCHEMICALS AND SYNTHESIZED BIOMATERIAL FOR THE CURE AND MANAGEMENT OF BONE-RELATED DISORDERS

Abstract

The human skeletal system is a multifunctional complex organ system. In general, the skeletal system undergoes the process of renewal through a continuous cycle of resorption and formation by osteoclasts and osteoblasts respectively. The complex balance between the actions of these cells is critical for maintaining bone health through the lifespan of a healthy individual (Bi et al., 2017). While bone loss is inevitably seen during progressing age, several other factors like gender, low calcium and vitamin D in the diet, alcohol/tobacco consumption, long-term use of corticosteroid medications, several malignancies, certain genetic factors and hormonal imbalance can adversely affect the bone mass. In general, bisphosphonates and teriparatide have been used as a gold standard treatment approach for the prevention and treatment of major bone disorders. However, the knowledge regarding their long-term use and efficacy still remains elusive (Lems et al., 2015). Therefore, the current need for skeletal healthcare research is focused on the discovery of novel bone-anabolic agents that can restore the bone mass as well as maintain the physiological balance between bone formation and resorption. For the treatment of fractures and fragility of bone tissues, nowadays researchers are also getting fascinated towards the emerging application of tissue engineering with the use of natural and synthetic compounds for treating several trauma and deformities involved in orthopedic complications. Thus, there is need of strategic synthesis of novel natural or synthetic scaffolds that can be potentially used for bone tissue engineering purpose. The present thesis entitled “Assessment of the effects of various phytochemicals and synthesized biomaterial for the cure and management of bone-related disorders” deals with experiments that provide new insights into our understanding of using various natural compounds and synthesized scaffold in the treatment of various bone disorders like osteoporosis, aberrant bone turnover maintenance and osteosarcoma. The outcomes from all these investigations may pave the pathway for novel therapeutic interventions that can be used for improving major skeletal complications. In the beginning, Chapter 1 introduces briefly about several skeletal disorders that commonly occur in humans. The key mechanisms and cellular signalling pathways driving these pathophysiological conditions are mentioned briefly for better understanding. These mechanisms and their associated triggering elements can be targeted for the discovery of better therapeutic i Abstract regimens for the cure and management of skeletal complications. Finally, the detailed objectives to be attained in this study are mentioned in this chapter. Followed by this, Chapter 2 deals with the detailed review on several bone related pathological conditions and associated molecular mechanisms associated with these aberrations. The brief knowledge about several mechanisms, strategies and limitations from literature review further led us to investigate precisely on novel natural and synthetic compounds in different bone diseases through in vitro and in vivo models. All the materials and experimental methods for achieving our proposed objectives through in vitro and in vivo assays f are elaborated precisely in Chapter 3. Several investigations have suggested that phyto-constituents from several fruits, leaves, roots or plant parts can influence the process of bone formation and bone resorption (Suvarna et al., 2018). To start with the current work, we have screened various plants for their osteogenic effect in in vitro system as mentioned in Chapter 4. We have attempted to investigate their effects in bone metabolism. In this study we have selected two major species of Piperaceae family ;(1) Piper betle and (2) Piper nigrum. For our study, we collected the leaves from both the plants and prepared the methanolic, ethanolic and water (aquous) extracts from these leaves and tested in vitro using C3H/10T1/2 cell line (murine mesenchymal stem cells) to evaluate their cytotoxicity. Further, we screened these extracts for their osteogenic activity and found methanolic extract of piper betel leaves (BLE) as the best identified plant extract for further detailed and mechanistic studies. In the Chapter 5, the selected plant extract i.e methanolic extract of piper betel leaves (BLE), was further studied elaborately for its osteogenic effects and to get a comparative idea, the presence of major phytoconstituent present in betel leaves i.e hydroxychavicol (HCV) were reconfirmed through HPTLC and GC-MS analysis. In order to understand the detailed cellular and molecular mechanism of osteogenesis in response to BLE and HCV, we investigated their effects in in vitro and in vivo models. In this part of study, both BLE and HCV were studied for their cytotoxic effects and whether they could potentially enhance osteogenesis of mesenchymal stem cells. The involvement of major signaling pathway i.e β-catenin-GSK3b signaling cascade was also investigated through immunoblotting, immunofluorescence and siRNA mediated silencing studies. Also, in glucocorticoid induced osteoporotic rat models, we found BLE and HCV could alleviate the disease condition and restore the bone mass as evidenced from ii Abstract microarchitectural analysis, micro-CT scanning, histological examinations and X-ray analysis. Also, the signaling pathways triggered or inhibited by the action of these phytochemicals were studied in details with immunoblotting experiments. Thus, with these evidences, it can be suggested that betel leaf extract and its major phyto-constituent hydroxychavicol, may be used as bone-anabolic agents in the treatment of osteoporosis and other bone-related diseases. In future, novel phyto-formulation may be proposed for effective therapeutic usage. In our subsequent studies, in the Chapter 6, we have tested the anti-osteoclastogenic effects of BLE and HCV in in vitro models. Here we have used the murine macrophage cell line RAW 264.7 and incubated with recombinant RANKL protein (100ng/ml) for generation of in vitro osteoclast model. Further, the effects of BLE and HCV were tested in these osteoclasts. Interestingly, RANKL induced in vitro osteoclastogenesis was inhibited by BLE and HCV. The anti-osteoclastogenic effect involved the down regulation of the key mediators of NF-κB, and MAPK signalling pathway. Also, BLE and HCV blocked RANKL-induced NF-κB p65 translocation from the cytoplasm into the nuclei. Thus, betel leaf extract and its major phyto constituent show anti-osteoclastogenic effect and hence may be used against bone resorption occurring in several pathophysiological conditions. As we had mentioned earlier, the use of scaffolds and the combination with cells or growth factors are encouraged now-a days as novel therapy against treating several orthopedic complications. Thus, in Chapter 7, we synthesized a novel scaffold and characterized it on physical and biological aspects for its use in bone tissue engineering application. In this study, a porous biomimetic scaffold was synthesized using in situ gas foaming method. Briefly, a blend of gelatin and polyvinylpyrrolidone (PVP) was crosslinked with glutaraldehyde and lyophilized. The blending of two polymers were confirmed with FTIR, XRD and TGA analysis. The synthesized scaffold was further characterized for its biocompatibility and osteoinductive potential. The physico-chemical properties like microarchitecture, porosity, water adsorption ability, and mechanical strength were also investigated. Further, the enhanced proliferation and migration of murine mesenchymal stem cells (C3H/10t1/2 cells) through interconnected pores of the scaffold over an extended time period suggested the cytocompatibility of scaffold. Next, the biocompatibility of the scaffold was confirmed by in ovo implantation on the chorioallantoic membrane (CAM) using chicken embryo. In addition, increased matrix mineralization was confirmed by alizarin red staining and with EDX analysis of apatite depositions over the scaffold, when induced with osteogenic media. In summary, these findings demonstrate biocompatibility and osteo-inductive potentials of the gelatin-PVP biomimetic polymer composite scaffold with iii Abstract suitability as bone graft substitute material. Thus, with these supporting evidences, we propose the use of this novel polymer composite scaffold in fulfilling the unmet needs of bone augmentation and skeletal repair through bone tissue engineering applications. Furthermore, another major bone-related disorder was addressed in our study in Chapter 8 i.e. osteosarcoma or bone cancer, commonly occurring as primary bone tumor in children and young adults. In this part, we have studied the effects of a plant alkaloid, berberine in osteosarcoma cells. In our study, we have investigated whether berberine suppressed cell proliferation in osteosarcoma (OS) cells and modulate the epithelial-mesenchymal transition (EMT) in osteosarcoma cells. Our results show that berberine reduced colony formation, wound healing ability and migration of OS cells. Also, berberine significantly reduced the expression of MMP2 suggesting its inhibitory action on the matrix metalloproteinases that are required for cancer cell invasion. The significant reduction in the expression of vimentin, N-cadherin, fibronectin and increased expression of E-cadherin further suggested its role in the inhibition of EMT in osteosarcoma cells. The downregulation of H3K27me3, as well as the decreased expression of the histone methyl transferase enzyme EZH2, further substantiated the fact that the plant alkaloid can be used as an epigenetic modulator in osteosarcoma treatment. From all these above results, it could be concluded that berberine can inhibit proliferation, migration and above all, inhibit EMT of MG-63, osteosarcoma cells with possible modulation of key epigenetic regulators. Altogether, Chapter 9 summarizes all the major findings demonstrating the effects of several phytochemicals in the treatment of bone-related disorders and provides suggestions for future work in these areas. The scientific findings dealt with in this thesis may be of use to the future researchers working in this area. Last but not least, Chapter 10 of this thesis listed the bibliography which was consulted in the course of the present work.