Molecular Responses of Osteocytes to Mechanical Loading of Trabecular Bone at Millimeter to Micron Scales

Osteoporosis is the most common metabolic bone disease affecting approximately 200 million people worldwide. One in two women and one in four men above the age of 50 will suffer an osteoporotic fracture in their remaining lifetime. Approximately three hundred thousand people break their hips annually, of which 25% will die within a year. A major feature of osteoporosis is the loss of trabecular bone in vertebrae, proximal femur and distal radius. Trabecular bone is an essential load bearing component at these sites, and decreased mineral density and degraded architecture contribute to fracture risk, which adapt in response to mechanical stimuli. Understanding the molecular biology of this response could help to identify novel therapeutic targets for osteoporosis. However, the mechanics of trabecular bone are difficult to study due to heterogeneous and anisotropic structure. The mechanosensitive osteocytes experience a wide range of strains and respond accordingly. RNA-Sequencing was used to identify the differentially expressed genes (DRG) by osteocytes in response to macroscopic mechanical loading. Trabecular bone explants from porcine vertebrae were cultured in a custom bioreactor and subjected to one or three days of mechanical compression to approximately 4 MPa. There was a 9-fold increase in the number of DEGs with sustained loading, suggesting that loading activates transcriptional responses in osteocytes. The differentially expressed genes were related to osteoarthritis, osteocyte, and chondrocyte signaling pathways. The same experimental model was employed to determine the tissue level strains that cause a change in gene expression in osteocytes. Both deviatoric strain and hydrostatic strain gradient were sensitive and specific predictors of the mechanobiological response for individual genes as well as combinations of genes. SOST expression was highly related to elevated strain gradient, providing evidence that osteocytes respond to fluid flow in the lacuna-canalicular system. Two-point correlation revealed that high strain is more likely to be found near osteocytes with altered gene expression, but there was no relationship between strain or strain gradient and baseline gene expression. Sclerostin, the translated protein encoded by the SOST gene, expression was investigated using the same explant culture model with three days of loading. Sclerostin is well-known to be down-regulated in response to mechanical loading which results in increased bone formation via more permissive osteoblast differentiation. The expression of the protein was found to be decoupled from the expression of the SOST RNA as sclerostin expression downregulated within 5 minutes of loading. Six hours after loading however, sclerostin expression had begun to recover, although it did not reach to the levels of unloaded bone. The research presented in this dissertation has quantified for the first time the precise mechanical stimuli that elicit the initial transcriptomic and proteomic responses in osteocytes in situ. The specific molecular response of osteocytes provides us with new insight into the complex control system that governs bone adaptation. Understanding the response of osteocytes immediately after loading enhances our ability to search for molecules that can be employed as therapies in bone diseases such as osteoporosis, diabetes, anorexia, and cancer metastasis where the body’s natural ability to maintain bone health is compromised.