The Signal Transduction Of Bone Cells In Response To Mechanical Or Electromagnetic Stimulations And Their Mechanisms

Osteoporosis is one of the most common diseases in clinics, characterized by significant bone mass loss and bone microarchitecture deterioration, leading to bone fragility and an increased risk of bone fractures. Lots of risk factors are able to cause the occurrence of osteoporosis, such as aging, deficiency of sex hormone, disuse, and diabetes mellitus etc. As the issue of aging populations is becoming even more serious in our country, the economic and social burdens resulted from osteoporosis are dramatically increased year by year. Therefore, understanding the etiology and exploring more effective therapeutic methods for osteoporosis carries great significance for both the economic and social development.Mechanical loading and electromagnetic fields are the most two common physical factors on the skeletal system, which play critical roles in mediating the bone metabolism and maintaining the bone health of the organism. First, the skeleton acts as the weight bearing organ of human body, and it can adjust and modify its structure in accordance with the stress placed upon it. This concept about bone adaptation was raised by Julius Wolff in1892. Either the insufficient loads or deteriorative response of skeleton to the mechanical stimulus can induce significant bone loss, which is also the major triggering factor for osteoporosis. However, the exact mechanism of bone adaptation is still elusive. Critical questions in bone mechanotransduction regarding how the external mechanical force signal is transformed into that at the cellular level, and how bone cells perceive the stimulation and transduce the physical signals into the intracellular biochemical signals are still unknown. Second, the skeleton is exposed to a complex electromagnetic environment, and the external changing stress can also induce electric and magnetic effects inside the bone microenvironment (piezoelectric effect of bone). In1977, an American scientist Andrew Bassett for the first time applied the exogenous pulsed electromagnetic fields (PEMF) in clinics for the treatment of the nonunion of human tibiae after bone fractures. The exciting and positive therapeutic effects of PEMF were found in his study, and he hypothesized that PEMF might become an easy and noninvasive physical methods for the treatment of osteoporosis in clinics. Although the inhibitive effects of PEMF on osteoporosis have been confirmed by several clinical and experimental studies, the exact mechanisms are still unclear. The major concerns about the target bone cells which PEMF functions and the related exact mechanism of the signal transduction are still unknown to date.In this thesis, we first found that calcium (Ca2+) signaling, a pivotal and ubiquitous second messenger regulating many downstream cellular activities, displayed unique Ca2+oscillations with multiple and robust Ca2+spikes in osteocyte-like MLO-Y4cell networks under fluid flow stimulation, which were more responsive and sensitive than osteoblast networks in response to the fluid shear. These findings provide novel and direct evidence that osteocyte networks act as the major ’mechanical sensor’ in bone. Second, we developed a novel protocol for in situ Ca2+probing and imaging, synchronization technique between controlled mechanical loading and confocal imaging to enable the accurate and systematic investigation of osteocytic Ca2+signaling in mouse long bone under dynamic physiological loading. We for the first time found the load-induced unique repetitive spike-like Ca2+peaks in osteocytes. Our results also indicated that lacuna-canalicular system (LCS) fluid shear act as the major inner driving force to trigger the cellular activities in osteocytes. Our data also provide direct evidence that osteocyte networks possess high sensitivity in detecting and processing mechanical stimuli through LCS fluid shear induced calcium signaling. Third, we applied PEMF stimulations on ovariectomy-induced (high bone turnover osteoporosis model) and diabetes-induced (low bone turnover osteoporosis model) osteoporotic rats, respectively. PEMF exhibited significantly preventive effects on both estrogen-deficient and diabetic bone mass loss, and bone quality deterioration. Furthermore, PEMF mainly regulated the bone formation rather than bone resorption for both two animal models, and the Wnt/β-catenin signaling pathway in osteoblast was the major mechanism for PEMF mediation on bone loss. Taken together, our studies greatly enrich our basic knowledge to the mechanisms of signaling transduction in bone cells in response to the stimulations of physical factors, and also provide critical evidence for the application of mechanical and PEMF stimulations in clinics for inhibiting osteoporosis. The thesis includes three parts:Part I:The spatiotemporal dynamics of Ca2+signaling in osteocytic and osteoblastic networks under fluid shear stimulationBackgrounds. Osteocytes, residing in the LCS microenvironment and forming extensive cell networks in bone matrix, are regarded as the major mechanosensors in bone. However, few studies to date are able to provide direct evidence to confirm this hypothesis, and no study has ever systematically compared the mechanical sensitivity between osteocytes and other major cell types in bone(e.g. osteoblasts).Methods. A novel two-dimensional patterned bone cell network was constructed to mimic the elaborate in vivo osteocytic network topology using microcontact printing and self-assembled monolayers techniques. Osteocytic and osteoblastic cell networks were respectively stimulated under physiological related fluid shear (0.5-4Pa), and real-time Ca2+signals were recorded. A set of novel unsupervised Ca2+signaling analysis algorithm based on independent component analysis (ICA) was developed to extract the Ca2+signaling, and the spatiotemporal characteristics in bone cell networks were also systematically analyzed.Results. Osteocytes in the network displayed unique highly repetitive spikelike Ca2+peaks under fluid shear, whereas most osteoblasts in the network only exhibited one major Ca2+peaks at the onset of fluid shear. Ca2+spikes in osteocytes were more repetitive and robust than that in osteoblasts in response to fluid flow. The Ca2+oscillatory nature in osteocytes and osteoblasts was highly positively correlated with the fluid flow levels. The present study also revealed a dramatic spatiotemporal difference in Ca2+signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. Osteocytes exhibited dramatically higher intracellular Ca2+oscillatory behaviors and intercellular Ca2+coordination than osteoblasts. The spatial intercellular synchronous activities of Ca2+signaling in osteocyte cell networks were also negatively correlated with the intercellular distance. Also, the ICA-based technology yield higher signal fidelity and save much more human efforts than the manual region of interest (ROI) method, with the potential to be widely employed in the Ca2+signaling studies.Conclusion. Osteocytes possess much higher mechanical sensitivity than osteoblasts in detecting and processing the external mechanical loading signals. Osteocytes’ unique living microenvironment and high sensitive nature to fluid shear endow the capacity of them to act as the major mechanosensing system in bone. This study provides evidence that osteocytes are qualified as a critical coordinator in bone modeling and remodeling process.Part Ⅱ:Ex vivo Ca2+oscillations in live osteocytes in intact mouse long bones under dynamic mechanical loading and the related mechanismsBackgrounds. Osteocytes are encapsulated in a fluid-filled mineralized bone matrix. Questions pertaining to how external mechanical signals are transformed into the cell levels and how osteocytes decode these signals and transduce them into the intracellular biochemical signals are still unknown. Systematic and comprehensive investigations for the mechanisms of oseocyte mechanotransduction and bone adaptation carry great scientific and clinical significance.Methods. We designed an ex vivo murine tibia mechanical loading system. Mouse tibiae were sterilely dissected and cultured. A synchronization technique between controlled mechanical loading and confocal imaging was developed to avoid the focus shift during mechanical loading. The combination with both strain gauge and μCT based finite element analysis enables accurate measurement of load-induced resultant strain on the bone surface. The shear stress on osteocytes in the lacuna-canalicular system (LCS) was measured and estimated via a combination of fluorescence recovery after photobleaching (FRAP) imaging and LCS modeling techenique. Furthermore, various pathway inhibitors essential for Ca2+signaling were employed to evaluate the exact mechanisms of in situ osteoycte Ca2+oscillations.Results:Autonomous Ca2+responses with1-2robust spikes were found in13%osteoblasts, but only in1.3%osteocytes. Mechanical loading didn’t induce significant changes in osteoblasts. However, osteocytes reacted to the mechanical loading with unique Ca2+oscillations with multiple and robust Ca2+spikes. Cyclic mechanical loading could induce fluid shear through the bone LCS, which was also highly positively correlated with the tissue strains. The load-induced fluid shear on the cell dendrites was significantly higher than that on the cell body. All Ca2+dynamic parameters were highly positively correlated with the tissue strains, as well as with the LCS shear stress. Pathway studies showed that the intracellular Ca2+store ER and ATP-related signaling pathway played major roles in osteocytic Ca2+signaling.Conclusion:Interstitial fluid flow act as the inner driving force by converting the external load signals to the intracellular signaling responses in osteocytes. Osteocyte networks process mechanical stimuli through fluid flow induced uniqe Ca2+signaling with multiple Ca2+spikes. Extracellular ATP and its related P2R/PLC/IP3signaling pathway play major roles in regulating the Ca2+signaling in osteocytes.Part III:In vivo investigation for the inhibitive effects of pulsed electromagnetic fields on bone loss in osteoporotic rats and the related mechanismsBackgrounds. Bone cells are exposed to a complex electric and magnetic environment, and their biological functions are also regulated by the electromganetic fields. Substantial and growing evidences have proven that exogenous PEMF stimulation was able to promote osteogenesis both in vivo and in vitro. Several clinical trials also demonstrated that PEMF could significantly inhibit bone loss in patients with OP. However, systematic and scientific investigations for the effects of PEMF on OP are still lacking, and the therapeutic target of PEMF on bone cells and its related mechanism of the signaling pathway transduction is still unknown. These issues all limit the widely and scientific applications of PEMF in clinics for the treatment of OP.Methods. We designed a PEMF exposure system, which was composed of a signal generator together with a Helmholz coil assembly with three-coil array. Ovariectomy-induced and type I diabetes-induced OP models were established, respectively. The parameters including the body mass, serum biochemical indices reflecting bone turnover, bone mechanical strength, microstructure of trabecular and cortical bone, and mRNA expression of Wntl, Lrp5, β-catenin, RANK and RANKL were analyzed to eveluate the inhibitive effects of PEMF on estrogen-deficient and diabetic OP.Results:PEMF was capable of significanly inhibiting bone mass loss and trabecular microstructure deterioration induced by either ovariectomy or type I diabetes in rats, as well as improving the mechanical strength of bone. The serum biochemical analysis results showed that PEMF was able to regulate the bone formation activities in both of bone loss animal models, whereas showed minor mediation roles in the bone resorption. Furthermore, PEMF dramatically increased the gene expression of Wntl, LRP5and β-catenin in the rat tibia, and showed no obvious effects on RANKL and RANK levels in both OP animal models.Conclusion:PEMF, as an easy, safe and non-invasive physical treatment method, has demonstrated its great potential to reverse both the estrogen-deficient and insulin-deficient bone loss. The major regulatory target of PEMF in bone is probably the Wnt/Lrp5/β-catenin signaling pathway in OB.