ASSESSMENT OF VARIOUS FACTORS REGULATING OSTEOBLAST DIFFERENTIATION OF STEM CELLS

Abstract

Osteoblasts arise from common progenitors with chondrocytes, muscles and adipocytes, and various hormones and local factors regulate their differentiation. The aim of the present thesis entitled “Assessment Of Various Factors Regulating Osteoblast Differentiation Of Stem Cells” was to enhance the current knowledge about the mechanisms involved in the interactions between several factors suggested to be involved in the regulation of osteoblast differentiation. At the beginning Chapter 1 introduces briefly the various types of bone cells which control the bone remodeling process and how dysfunction of these cells leads to various bone disorders. It also discussed the present scenario of the chemotherapeutic drugs available to treat these disorders. Further, it also dealt with the key factors present in bones that need to be targeted to overcome the bone loss. Finally detailed goal to be attained in this study is specified here. Followed by this, Chapter 2 presents (i) the detailed review on various aspects of bone remodeling process; (ii) complete mechanism of osteoblast and osteoclast differentiation process; (iii) a vivid description of several exogenous and endogenous factors known to regulate osteoblast and osteoclast differentiation process which led us to select few promising synthetic, natural and molecular targets to explore in present thesis and finally (iv) the hypothetic idea behind this thesis which may lead ultimately to cure the bone loss or other bone related disorders. Each of the hypotheses is explored in subsequent chapters of the thesis. The main objective behind the present work was to enhance the rate of bone formation so that the bone loss during several pathological disorders can be recovered. There are several in vitro assays which are already established to detect the rate of bone formation and bone resorption, such as, alkaline phosphatase assay, mineralization test, analysis of marker genes specific to osteoblast and osteoclasts etc. All these parameters are elaborated in Chapter 3 of the thesis. Further several cell lines used for the study have been discussed in detail. This chapter also mentioned all the molecular biology techniques used in the present study to explore the mechanism of osteoblast differentiation process. Research in stem cell biology is an important and necessary requirement for the better understanding of cell differentiation and formation of tissues, while also contributing to the field of regenerative medicine. Embryonic and mesenchymal stem cells are powerful tools in ______________________________________________________________________Abstract ii regenerative medicine. Chapter 4 of this thesis described the establishment and optimization of culture protocol of mouse bone marrow mesenchymal stem cells, as well as embryonic stem cells. To establish the various hypotheses, the first part of thesis focused on the investigation of effects of several selected synthetic and natural compounds on differentiation of undifferentiated cells towards osteoblast lineage. In recent time several herbal antidiabetic drugs are proven to be a potent osteogenic agent. In addition, vanadium compounds are also being used as antidiabetic drug and in orthopedic implants. However, the exact role of this incorporated vanadium in improving the quality of bone structure and morphology is not known. Hence in Chapter 5 data on the screening of some potent herbal osteogenic agents were presented and also the impact of vanadium ion was studied and compared to other trace metal ions with respect to the proliferation and osteoblast differentiation of C3H10t1/2 cells. After preliminary study, out of all tested compounds, rutin (among herbal compounds) and vanadium ion (among all trace ions) were found to be most potent osteogenic agents. The detailed effects of both the agents were tested in next chapter of the thesis. Rutin is common flavonoid in fruits and vegetables which is a glycoside of a parent flavonoid known as quercetin. The effect of several flavonoids varies with their aglycone and glycone nature. After the initial screening and selection of rutin in chapter 5, in Chapter 6, role of both rutin and its aglycone quercetin on osteoblast differentiation of bone marrow stem cells were tested. Both quercetin and rutin were found to up regulate the osteoblast differentiation albeit to a lesser extent in case of former than that of latter. The data suggests that certain classes of flavonoids like rutin and quercetin can be used in the cure and management of osteodegenerative disorders due to their osteoblast specific differentiation activities. Vanadium compounds are reported to have insulin mimicking action, which renders them to show excellent osteogenic activity. However, the toxicity and less bioavailability associated with vanadium limits its role as a therapeutic osteogenic agent. Based on these issues several organic vanadium complexes with better bioavailability and reduction of vanadium-associated toxicity are now been designed and evaluated as potential osteogenic agents. In chapter 7 the effect of various vanadium compounds on osteoblast differentiation of C3H10t1/2 cells, were investigated and analyzed the underlying mechanism of vanadium ______________________________________________________________________Abstract iii compounds for its action. This study showed that vanadium compounds are potent as osteogenic agent. Also, the experiments demonstrated profound differences between osteogenic activities of vanadium (IV) and vanadium (V) compounds. Murine embryonic stem cells (mESCs) have the potential to differentiate into almost any type of cell, and hence, represent a useful biological resource for tissue engineering. The differentiation of mESCs into osteoblasts in vitro is usually dampened by simultaneous differentiation of adipocytes. Insulin exerts a profound effect on bone development through increased differentiation of osteoblasts and concurrent formation of adipocytes. Comparatively, Sirt1, which plays a crucial role in osteoblast differentiation, has been reported to down regulate adipocyte formation during osteoblast differentiation. Hence, it was hypothesized that simultaneous activation of Sirt1 and insulin signalling would increase the number differentiated osteoblast cells among the pool of heterogeneous population of various lineages in differentiated mESCs, by decreasing the number of adipocytes formed. The experiemtns as reported in Chapter 8 were conducted to understand the role of these two signalling factors in osteoblast differentiation. Results showed that by targeting the distinct pathways of insulin and Sirt1, the efficiency of mESC differentiation toward the osteoblast lineage can be improved. Another factor which affects bone remodeling is the state of inflammation. Inflammatory processes in, or in close vicinity of the skeleton often lead to loss of bone tissues. Different cytokines have been shown to be involved as stimulators of inflammation induced osteoclastic bone resorption. Among these cytokines, bradykinin (BK) is well reported as a mediator of pain and inflammation and is involved in bone metabolism. The aim of the next part of the study was to delineate the probable mechanisms involved in the interactions between BK and various cell types suggested to be involved in controlling the bone remodeling processes. In Chapter 9 the role of BK in osteoblast lineage commitment of bone marrow derived mesenchymal stem cells and its mode of action in regulation of osteoblast differentiation is evaluated. Among various agents one of the major target of bones are estrogen and evidence shows that several sex steroids promote cartilage morphogenesis and bone maturation. Estrogen is one such sex steroid which has been reported to regulate embryonic stem cell proliferation and differentiation. Naturally, understanding the role of estrogen in the regulation of osteoblast differentiation of embryonic stem cells may provide a clue about their effects on bone ______________________________________________________________________Abstract iv formation. Further, another factor, the homeobox gene, nanog, is a key intrinsic determinant of self renewal in ESCs, and its repression leads ES cells to selectively differentiate into primitive endoderm. So we hypothesized that there should be some crosstalk between estardiol and nanog gene. To check this hypothesis, a study design as described in Chapter 10 was conducted to show initially the effect of estradiol on osteoblast differentiation of mouse ESCs. In the next stage, the crosstalk between estradiol and nanog were investigated using a combination of genetic and biochemical approaches. The data showed that estrogen up regulated osteoblast differentiation and nanog is a critical target for estrogen which mediated its action involving both genomic and non-genomic pathways. Finally, Chapter 11 summarizes the complete work in brief and the future prospects that is still to be ventured. The scientific findings dealt within this thesis may be of use to the future research work in understanding the mechanisms of osteoblast differentiation. Finally the list of bibliographies which was references in course of the present work is enlisted