Primary cilia are sensory organelles that facilitate early skeletal development, as well as maintenance and adaptation of bone later in life. These solitary, immotile organelles are known to be involved in cell differentiation, proliferation, and mechanotransduction, a process by which cells sense and covert external physical stimuli into intracellular biochemical signals. Bone is a metabolically active tissue that continuously recruits osteogenic precursors and relies on osteocytes, the sensory cells of bone, to coordinate skeletal maintenance. Overall bone quality is dependent on the integrity of the initial structure formed, as well as this organ’s ability to adapt to physical loads. Proper differentiation and controlled proliferation of osteogenic progenitors are critical to the initial formation of the skeleton, while osteocyte mechanotransduction is essential for adaptation of developed bone. These phenomena rely on primary cilia, but little is known about the origin of osteogenic precursors and the ciliary mechanisms that promote osteogenesis. In this thesis, we first characterize an osteochondroprogenitor (OCP) population that rapidly and extensively populates skeletal tissues during juvenile skeletal development (Chapter 2). We also demonstrate that the primary cilium is critical for these cells to differentiate and contribute to skeletogenesis. We then show this OCP population is required for adult bone adaptation and is mechanoresponsive (Chapter 3). Again, we demonstrate that primary cilia are necessary for these OCPs to sense physical stimuli and differentiate into active bone-forming cells. Finally, we identify a novel link between ciliary calcium and cAMP dynamics in the osteocyte primary cilium (Chapter 4). Specifically, we show that a calcium channel (TRPV4) and adenylyl cyclases, which produce cAMP, bind calcium to mediate calcium entry and cAMP production, respectively, and these phenomena are critical to fluid flow-induced osteogenesis. Collectively, our results demonstrate that an easily extracted progenitor population is pre-programmed towards an osteogenic fate and extensively contributes to bone generation through primary cilium-mediated mechanisms at multiple stages of life. Furthermore, we identified ciliary proteins that are potentially unique to the osteocyte and can be manipulated to encourage osteogenesis by tuning calcium/ cAMP dynamics. For these reasons, we propose that this OCP population and their primary cilia, as well as osteocyte ciliary proteins that coordinate calcium/ cAMP dynamics, are attractive therapeutic targets to encourage bone regeneration.

Osteoporosis is a devastating condition characterized by decreased bone mass, and affects over 50% of the population over 50 years old. Progression of osteoporosis results in significantly heightened risk of fracture leading to loss of mobility, prolonged rehabilitation, and even mortality due to extended hospitalization. Current therapeutic options exist to combat low bone mass, but these treatments are being met with increasing concern as reports emerge of atypical fractures and necrosis. Thus, new therapeutic strategies are required. Bone is highly dynamic, and it has long been known that physical load is a potent stimulus of bone formation. Despite this, none of the current treatments for bone disease leverage the inherent mechanosensitivity of bone – the ability of bone cells to sense and respond to mechanical forces such as exercise. One potential therapeutic target is the primary cilium. Primary cilia are solitary antenna-like organelles, and over the last 20 years have been identified as a critical cellular mechanosensor. Primary cilia and cell mechanotransduction are critical to the function of numerous cells and tissues. Thus, understanding primary cilia-mediated mechanotransduction has potential applications in treating kidney and liver disease, atherosclerosis, osteoarthritis, and even certain cancers. Previous work from our group has demonstrated that disruption of the cilium impairs bone cell mechanosensitivity, resulting in abrogated whole bone adaptation in response to physical load. In this thesis we examine the potential of targeting the primary cilium to enhance bone cell mechanosensitivity and promote whole bone formation. First, we demonstrate the pharmacologically increasing primary cilia length significantly enhances cell mechanotransduction. Next, we expand our list of candidate compounds to manipulate ciliogenesis through the use of high-throughput drug screening. We developed an automated platform for culturing, staining, imaging, and analyzing nearly 7000 small molecules with known biologic activity, and classify them based on mechanism of action. One of these compounds is then used in a co-culture model to study the effects of manipulating osteocyte primary cilia-mediated mechanosensing on pro-osteogenic paracrine signaling to promote the activity of bone-forming osteoblasts and osteogenic differentiation of mesenchymal stem cells. Finally, we translate our in vitro findings into an in vivo model of load-induced bone formation using the same compound to enhance cell mechanotransduction. We demonstrate that we can sensitize bones to mechanical stimulation to enhance load-induced bone formation in healthy and osteoporotic animals, with minimal adverse effects. Together, this work demonstrates the therapeutic potential and viability of targeting primary cilia-mediated mechanotransduction for treating bone diseases.

Chan Chun Wai. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Bibliography: leaves 169-197. Hypothesises that the physis has an internal mechanism of repair to restore physeal function. Aims to establish a defined degree of deformity by partial growth plate excision, then to examine different methods of reversal of such deformity to observe the process of growth plate repair. A secondary aim was to define the percentage of physis that could be resected yet still enable reversal of deformity.

Bibliography: leaves 169-197. / xiv, 197 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Hypothesises that the physis has an internal mechanism of repair to restore physeal function. Aims to establish a defined degree of deformity by partial growth plate excision, then to examine different methods of reversal of such deformity to observe the process of growth plate repair. A secondary aim was to define the percentage of physis that could be resected yet still enable reversal of deformity. / Thesis (Ph.D.)--University of Adelaide, Dept. of Pathology, 1989

Introduction: Recent reports in exercise related bone research have shown increased bone mineral content (BMC) at the femoral neck for prepubescent children participating in exercise programs consisting of repeated drop landings from a height of 61 cm. Increases in BMC from this type of exercise are believed to be the result of both high rate and magnitude of loading at the proximal femur. However, the dynamic characteristics associated with these landings in children have not been studied. Purpose: To describe the dynamic characteristics of children during landing and to quantify the forces associated with an activity associated with increases in bone mass. Methods: 13 prepubescent children (males=8, females=5, age 9.3 �� 0.7 years) who had previously completed drop landings over a 7 month period as part of an exercise intervention to increase bone mass participated in this research. Each subject performed 100 drop landings onto a force plate from a height of 61 cm. Ground reaction forces and two-dimensional kinematic data were recorded. Hip joint reaction forces were calculated using inverse dynamics based on a four segment rigid body model. Vertical ground reaction force and displacement data were fit to two single degree of freedom models, the Voigt and standard linear solid (SLS). The goodness of fit was quantified using the standard deviation of the error (SDE) between the experimental and the predicted data. Results: Peak vertical ground reaction forces were 8.5 �� 2.2 (mean �� SD) body weights (BW) while hip joint reactions were 6.0 �� 1.8 BW. Loading rates for ground reaction forces during initial impact were in excess of 470 BW/s. Across 100 jump trials, ground reaction forces changed significantly for 5 subjects (4 increase, 1 decrease, p<0.05) but were unchanged as a group. The SLS and Voigt models replicated the displacement traces well (SDE=0.003 m and 0.001 m respectively). However, in fitting force data, the SLS outperformed the Voigt model (SDE=580 N and 493 N respectively), but slightly under-predicted peak forces by 13%. Conclusion: Comparing force characteristics from drop landing to force characteristics known to be osteogenic, we can see how drop landings contribute to the osteogenic stimulus. The models used to represent children during drop landing closely fit displacement data, but did not replicate the time history of the impact force peaks thought to be important to osteogenesis. Quantification of exercises known to increase bone mass provides a basis on which to develop and implement additional exercise interventions for the purpose of increasing bone mass. / Graduation date: 2000

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

When human mesenchymal stem cells (hMSCs) are cultured inside a 3D collagen meshwork, they become a potential tissue engineering bone graft alternative. However, the in vitro osteogenesis rate of hMSCs is slow, leading to a low mineral deposition. To enhance the osteogenic differentiation of hMSCs, low intensity pulsed ultrasound (LIPUS) was employed as an external stumulus. The present study demonstrated the feasibility of employing daily LIPUS exposure for enhancing osteogenesis in vitro. Exposure of seven consecutive days LIPUS, each of 30 minutes duration, did not affect the cell viability, and the organization of hMSCs within the collagen meshwork was not disturbed. The calcium deposition within the collagen meshwork was enhanced after seven days of exposure. The osteoinductivity was also upregulated at the early period of culture. In order to optimizing the enhancement effects of LIPUS, various ultrasound parameters, including intensity, exposure duration and exposure repetition were investigated. Results showed the LIPUS enhancement effects are dose dependent, LIPUS exposure should be longer than 10 minutes/day in order to elicit a significant effect. Calcium deposition was higher when LIPUS exposure was done twice per day instead of one. Although individual variation exists, optimal LIPUS intensity range was between 60-120 mW/cm2 ISATA (Spatial Average Temporal Average Intensity). The interaction mechanism between LIPUS and cells was also investigated. Microbubbles were added to the culture during LIPUS exposure to find out whether cavitation is involved in the interaction. Flow sensor primary cilium was also studied in order to verify that ultrasound is transduced through fluid flow. Results showed cavitation may not be a contributing factor to osteogenesis, and primary may be involved in the transduction of LIPUS stimulation. This study demonstrated that osteogenesis of hMSCs encapsulated in collagen constructs could be enhanced by LIPUS. The LIPUS parameters were also optimized. The LIPUS interaction pathways were also being better understood. This thesis study will be a paradigm for cellular mechanotransduction studies and put an important step forward for therapeutic ultrasound. / published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / Dentistry / Master / Master of Orthodontics