<p>Bone loss diseases, including osteoporosis affect millions of people worldwide. Understanding the underlying mechanisms behind bone homeostasis and adaptation is essential to uncovering new therapeutic targets for the prevention and treatment of bone loss diseases. Primary cilia have been implicated in the development and mechanosensation of various tissue types, including bone. The goal of the studies outlined in this thesis is to determine the mechanosensory role of primary cilia in bone cell function, bone growth, and adaptation. This goal was achieved by exploring two specific scenarios. In the first study, mice models with conditional knockouts of MKS5, a ciliary protein, in osteocytes were utilized to demonstrate that dysfunctional primary cilia in those cells result in impaired loading-induced bone formation. The hypothesis tested is that the existence of functioning primary cilia on osteocytes is crucial for proper bone adaptation following stress. The results of this study support the hypothesis, with the conditional knockout mice showing significantly lower loading-induced bone formation compared to controls. The second study highlighted the importance of the osteoblast primary cilia in bone growth by using mice models with osteoblast-specific deletion of the cilia. The hypothesis tested is that the presence of the primary cilia is crucial for proper bone growth. The results show that conditional knockout mice have lower body weights, decreased femur length, and a significantly lower rate of bone formation, confirming that the primary cilia play a great role in bone growth and development. This study has highlighted the role of primary cilia in bone health and this topic merits further investigation. </p>

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Indiana University-Purdue University Indianapolis (IUPUI) / Tissue cells display mechanosensitivity in their ability to discern and respond to changes in the viscoelastic properties of their surroundings. By anchoring and pulling, cells are capable of translating mechanical stimuli into a biological response through a process known as mechanotransduction, a pathway believed to critically impact cell adhesion, morphology and multiple cellular processes from migration to differentiation. While previous studies on polymeric gels have revealed the influence of substrate elasticity on cellular shape and function, a lack of suitable substrates (i.e. with mobile cell-substrate linkers) has hindered research on the role of substrate viscosity. This work presents the successful design and characterization of lipid-bilayer based cell substrates of tunable viscosity affecting cell-substrate linker mobility through changes in viscous drag. Here, two complementary membrane systems were employed to span a wide range of viscosity. Single polymer-tethered lipid bilayers were used to generate subtle changes in substrate viscosity while multiple, polymer-interconnected lipid bilayer stacks were capable of producing dramatic changes in substrate viscosity. The homogeneity and integrity of these novel multibilayer systems in the presence of adherent cells was confirmed using optical microscopy techniques. Profound changes in cellular growth, phenotype and cytoskeletal organization confirm the ability of cells to sense changes in viscosity. Moreover, increased migration speeds coupled with rapid area fluctuations suggest a transition to a different migration mode in response to the dramatic changes in substrate viscosity.

Comme tous les marsupiaux, l’opossum Monodelphis domestica nait dans un état très immature – glabre, aveugle, sourd – mais doit grimper sur le ventre de la mère pour trouver une tétine à laquelle il s’accroche afin de poursuivre son développement. Pour ce faire, il rampe à l’aide de ses membres antérieurs, qui sont mobiles contrairement aux membres postérieurs, et se dirige vers une tétine qu’il avale partiellement. Des sens céphaliques sont nécessaires pour qu’il trouve la tétine et s’y accroche. Le toucher est un des sens qui est fonctionnel dès la naissance. Ce sens repose sur des cellules spécialisées qui ont la propriété de percevoir des déformations mécaniques des tissus et d’y répondre en déclenchant l’activation de fibres nerveuses. Cette propriété s’appelle mécanotransduction et est rendue possible par la présence de récepteurs moléculaires à la surface des cellules dont la tâche est de réagir aux stimuli mécaniques afin de provoquer la réponse. Peu de ces récepteurs ont été formellement identifiés et caractérisés. Toutefois, Piezo2 est un canal transmembranaire retrouvé dans une vaste gamme de mécanorécepteurs et qui joue un rôle crucial dans la perception du toucher, en plus d’être impliqué dans la vestibulation (sens de l’équilibre) et l’audition. Le rôle de Piezo2 a surtout été étudié chez des mammifères euthériens matures. Peu d’études ont porté sur son expression développementale, et aucune ne concernait les marsupiaux. L’objectif principal de cette thèse était de décrire l’expression de Piezo2 au niveau de la tête d’opossums en développement. L’expression du gène a été examinée par RT-PCR et hybridation in situ, alors que la présence de la protéine a été démontrée par immunohistochimie. La RT-PCR montre une expression de l’ARNm de Piezo2 à tous les âges à partir de la naissance jusqu’au 21e jour postnatal (P21). L’immunohistochimie n’a pas permis de mettre en évidence Piezo2 dans la peau faciale ni chez les nouveau-nés ni chez l’adulte. Cependant, Piezo2 est présent dans l’oreille interne dès la naissance. Dans le vestibule, le marquage Piezo2 est observé sous la forme de disques à la surface de l’épithélium dans la macule utriculaire dès la naissance et dans la macule sacculaire et les crêtes ampullaires à P7. Ces disques ont une morphologie similaire à ceux formés par l’accumulation d’actine où se développent les cils des cellules ciliées, ce qui permet de penser que Piezo2 se trouve à la surface des cellules ciliées présomptives. Dans la cochlée, la protéine est aussi présente sur toute la surface apicale de l’organe de Corti présomptif. Avec l’âge, le patron de marquage se restreint à la surface des cellules ciliées externes, dont les trois rangées sont visibles à P11. À P14, les disques de marquage Piezo2 sont bien nets dans tous les organes sensoriels de l’oreille interne. Du marquage Piezo2 a aussi été observé dans la membrane tectoriale de la cochlée et les membranes otolithiques des macules vestibulaires, ce qui suggère qu’il joue un rôle dans le développement de ces structures acellulaires. Ces résultats suggèrent que Piezo2 n’est pas impliqué dans la mécanosensation tactile faciale à la naissance et pourrait jouer un rôle mineur dans le toucher chez l’opossum. L’expression de Piezo2 dans l’oreille interne indique qu’une forte maturation des cellules ciliées a lieu au cours de la 1re semaine postnatale dans la macule utriculaire et lors de la 2e semaine pour les autres organes sensoriels vestibulaires. Les cellules ciliées cochléaires auraient une maturation un peu plus tardive, au cours de la 2e semaine postnatale. La forte présence de Piezo2 dans l’épithélium cochléaire dès la naissance, alors que les cellules ciliées sont encore indifférenciées, suggère que cette molécule pourrait jouer un rôle dans la différenciation cellulaire. En résumé, cette étude montre que Piezo2 n’est pas impliqué dans la mécanosensation précoce chez l’opossum, mais qu’il joue un rôle dans le développement de la vestibulation et de l’audition. / Like most marsupials, the opossum Monodelphis domestica is born in a very immature state, ie. blind, glabrous and deaf. To pursue its development and growth, the newborn crawls with its forelimbs on its mother’s belly to find a teat where it attaches. Cephalic senses are needed to find the teat and trigger the attachment. Touch is one of the senses, which depends on mechanoreceptors, sensory cells capable of perceiving the mechanical changes in tissues and to transmit them as neural inputs, a process called mechanotransduction. Of the few molecular receptors underlying mechanotransduction identified so far, Piezo2 is the best candidate. It is a mechanosensitive cation channel found in a wide variety of mechanoreceptors and plays a crucial role in the perception of touch, as well as having been linked to the vestibular and auditory vestibular systems. While having been well characterized in mature eutherian mammals, few studies have looked at its role during ontogenesis and none were done in marsupials. The main objective of this thesis was to describe the developmental expression of Piezo2 in the head of the opossum. Gene expression was examined by RT-PCR and in situ hybridization, while the presence of the protein was demonstrated by immunohistochemistry. RT-PCR has shown that gene expression of Piezo2 is present from birth (postnatal day 0, P0) until P21. Immunohistochemistry did not reveal the presence of Piezo2 in cephalic skin tissues at any stage from birth to adulthood. However, Piezo2 is present in the inner ear from birth onwards. In the vestibular labyrinth, disk-shaped patches of Piezo2 labeling are present in the utricular macula at P0 and can be observed at P7 in the saccular macula and in the crista ampullaris. In all these sensory organs, Piezo2 labeling is similar to that of disk-shaped patches of actin accumulation where the stereocilia of hair cells develop. This suggests that Piezo2 is located at the surface of the hair cells in the inner ear. In the auditory system, the protein is present over the surface of the whole presumptive organ of Corti at P0. With age, Piezo2 labeling was restricted to the apical surface of the outer hair cells by P11. At P14, numerous discs are present in all the sensory organs of the inner ear and the only difference with P21 seems to be an increase in their number. Piezo2 labeling was also observed in the tectorial membrane of the cochlea and the otolithic membranes of the macula, suggesting that it plays a role in the development of these acellular structures. These results indicate that Piezo2 is not involved in skin facial mechanosensation at birth in the opossum and may be less important in the perception of touch in marsupials than in eutherians. The pattern of expression of Piezo2 in the vestibular system suggests that the maturation of the hair cells is important during the first postnatal week in the utricular macula and during the second postnatal week in the other vestibular sensory organs. The hair cells in the organ of Corti are maturating more during the late second postnatal week. Moreover, the strong expression of Piezo2 in the undifferentiated cochlear epithelium during the first postnatal weeks suggests that it may play a role in the differentiation of the cells of the organ of Corti of the opossum Monodelphis domestica. In summary, this study highlights that Piezo2 is not involved in early mechanosensation in opossums but plays a role in the development of both the vestibular and auditory systems.

Indiana University-Purdue University Indianapolis (IUPUI) / Cellular structural mechanics play a key role in homeostasis by transducing mechanical signals to regulate gene expression and by providing adaptive structural stability for the cell. The alteration of nuclear mechanics in various laminopathies and in natural aging can damage these key functions. Arterial endothelial cells appear to be especially vulnerable due to the importance of shear force mechanotransduction to structure and gene regulation as is made evident by the prominent role of atherosclerosis in Hutchinson-Gilford progeria syndrome (HGPS) and in natural aging. Computational models of cellular mechanics may provide a useful tool for exploring the structural hypothesis of laminopathy at the intracellular level. This thesis explores this topic by introducing the biological background of cellular mechanics and lamin proteins in arterial endothelial cells, investigating disease states related to aberrant lamin proteins, and exploring computational models of the cell structure. It then presents a finite element model designed specifically for investigation of nuclear shear forces in arterial endothelial cells. Model results demonstrate that changes in nuclear material properties consistent with those observed in progerin-expressing cells may result in substantial increases in stress concentrations on the nuclear membrane. This supports the hypothesis that progerin disrupts homeostatic regulation of gene expression in response to hemodynamic shear by altering the mechanical properties of the nucleus.

This study was motivated by a theoretical formulation on mechanobiology of soft and hard skeletal tissue differentiation. To prove this formulation experimentally, I hypothesized that cartilaginous phenotype can be induced in vitro in a seemingly non-cartilaginous cell source from fibrous tissue. In testing this hypothesis, I have focused on cartilage as a target and fibrous tissue as an origin or the source of cell. Four different trials were pursued with one supposition in common, i.e. hydrostatic pressure is one of the main driving forces for chondroinduction in vitro. The first and second trials pertained to the influence of a relatively short and long duration cyclic hydrostatic compression on rat Achilles tendon fibroblasts. The third trial was to examine the effect of two different drugs on cytoskeletal elements of mesenchymal stem cells or mouse embryonic fibroblast lines in pellet cultures combined with the similar duration and/or frequency of cyclic hydrostatic pressure adopted in the aforesaid trials with no pharmacological agents added. Last, attempts were made to implement an advanced technique in molecular biology called 'PCR array' to further quantify expression levels of eighty four pathway-specific genes in mouse TGFbeta/BMP signaling traffic under the same physiological regimen of hydrostatic compression. Results demonstrated that transdifferentation in phenotype from tendon to fibrocartilage may have occurred in vitro in tendon fibroblasts in pellet cultures exposed to hydrostatic pressure. Experiments on the role of the cytoskeleton in mechanotransduction of the applied level of hydrostatic pressure demonstrated that disruption of microfilaments in the presence of cytochalasin-D did not significantly interfere with the anabolic effect of cyclic pressure. However, disruption of microtubule assembly by nocodazole abolished the pressure-induced stimulation in cartilage marker genes. These findings suggest that microtubules, but not microfilaments, are involved in mechanotransduction of hydrostatic pressure by mesenchymal stem cells.

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Chronic obstructive pulmonary disease (COPD) is one of the most common lung diseases and the third leading cause of death in the US, estimated to increase in magnitude in the future. Current treatment approaches are palliative in nature and restricted to controlling symptoms and reducing the risk of complications. Lung transplantation is an option for certain patients, but this option is limited by the shortage of donor organs and the possibility of rejection and the need for life-long immune-suppression. Therefore, current studies focus on cell based therapies for lung repair and regeneration. In addressing the issue of cell sourcing for such approaches, I tested the hypothesis that the efficiency of directed pulmonary differentiation of mouse embryonic stem cells (mESC) can be enhanced by employing certain micro-environmental cues, found in the developing lung. Such micro-environmental cues will provide appropriate physicochemical signals at the right time during the embryonic development and thus modulate fate decisions of progenitor cells during tissue assembly and maturation. In this study, I explored the effects of matrix stiffness on cell fate decisions in mESC, first into definitive endoderm and then into lung alveolar epithelial cells. I engineered bio-activated polyacrylamide (PA) gels with varying elastic moduli, mimicking those of physiologic tissues, and covalently modified the surfaces with fibronectin to provide optimal stem cell adhesion. My studies demonstrated, for the first time, a biphasic stiffness-dependent enhancement of endodermal differentiation of mESCs, with an optimum at ~ 20 kPa. This effect was qualitatively similar in three different mESC lines. By contrast, increasing matrix stiffness favored mESC differentiation towards a mesodermal phenotype. The enhanced endodermal differentiation of mESCs was abolished in the presence of a specific inhibitor of ROCK, suggesting that this process is mediated through cytoskeletal signaling. The subsequent differentiation of mESC-derived endodermal cells towards pulmonary epithelial cells was no longer dependent on the stiffness of the matrix. In this dissertation I demonstrate for the first time the feasibility of utilizing developmental and physiological / physicochemical cues, such as matrix stiffness, to selectively modulate and enhance mESC differentiation towards endodermal and pulmonary lineages. The impact of the results will be relevant for optimizing cell-based lung therapies and for effectively engineering lung and other endoderm-derived organs. / Bioengineering

Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins. In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways. Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.