Biophysical Regulation of Cellular Function in Bone
Creators: name: Brady, Robert Thomas
Publisher: name: Royal College of Surgeons in Ireland
Year: 2022
Subjects: subject: 110314 Orthopaedics
subject: FOS: Clinical medicine
subject: 100404 Regenerative Medicine (incl. Stem Cells and Tissue Engineering)
subject: FOS: Medical biotechnology

description: Osteoporosis is the most prevalent bone disease and represents a major public health burden. Resulting from a reduction in bone quality and quantity, it can lead to fragility fractures with significant associated morbidity and mortality. Mechanical loading is a potent, albeit incompletely understood, anabolic stimulus that could lead to novel therapies for osteoporosis. With weight bearing, mechanosensing osteocytes will transduce this physical stimulus into trophic factors that will facilitate the strengthening of bone at the site of loading. It is understood that this process will involve the recruitment, proliferation, and differentiation of progenitors thereby enhancing bone formation. The overall goal of this thesis was to investigate the application of biophysical forces that are experienced by primary cells in vivo and apply them to cellular cultures in vitro with the objective of encouraging more physiological responses. Given the critical role of mechanical loading and fluid flow stimulation in bone, the first aim of the thesis was to apply established fluid flow stimulation techniques to mechanosensitive osteocytes and osteoblasts in order to observe the secretion of bioactive soluble factors that could, in turn, modulate the effects of osteoblasts and stem cells. Significantly, paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and also significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors (Chapter 3). One such important soluble factor regulating bone anabolism is sclerostin, produced by osteocytes. Osteocyte physiology has traditionally been challenging to investigate due to its uniquely mineralized extracellular matrix (ECM) leading to the development of cell lines. Importantly, the most widely researched osteocyte cell line: MLO-Y4, does not express sclerostin (Sost) in typical in vitro culture. We theorised that culture in an environment closer to the in vivo osteocyte environment could impact Sost expression. This study investigated the role of composition and dimensionality in directing Sost expression in MLO-Y4 cells using collagen-based ECM analogues. A significant outcome of this study is that MLO-Y4 cells when cultured on a hydroxyapatite (HA) containing two-dimensional (2D) film analogues expressed Sost. Three-dimensional (3D) culture upon HA-containing scaffolds significantly further increased Sost expression. Importantly, Sost expression in MLO-Y4 cells cultured on a 3D collagen-HA scaffold was found to be comparable to physiological levels. Moreover, MLO-Y4 cells cultured in these novel conditions responded accordingly to fluid flow stimulation with a decrease in Sost expression. This study therefore presents a novel culture system for the MLO-Y4 osteocyte cell line, ensuring the expression of an important osteocyte specific gene, Sost, and therefore overcoming a main limitation of this model (Chapter 4). With mechanical loading it is expected that stem cells would be recruited to a site that they would be required, thereby experiencing a change in their microenvironment from a soft marrow to an organic osteoid with stiff mineralising components which may in turn influence their osteogenic capacity. We therefore investigated the effect of ECM analogues, of different compositions and dimensions, in directing stem cell osteogenic lineage commitment in the absence of exogenous osteoinductive factors. A significant finding was that stem cells cultured upon a collagen HA 3D scaffold demonstrated osteogenesis with the absence of exogenous growth factors (Chapter 5). It is hoped that a greater understanding of the processes occurring in mechanical loading-induced bone formation can then be used to help us understand the pathology underpinning bone diseases such as osteoporosis and develop better models for the investigation of bone biology in future.