Mechanosensitivity of osteocytes in their native matrix

Osteocytes sense and transduce mechanical signals into biochemical signals, thereby regulating the adaptation of bone to mechanical loading. Their deep location in hard mineralized bone matrix hinders their isolation from bone, and thereby limits studies on osteocytes in vitro. In chapter 2, we reviewed two-dimensional (2D) and three-dimensional (3D) in vitro models of osteocytes with a special focus on models used to determine the mechanosensity and mechanoresponsiveness of osteocytes. We concluded that osteocytes are mostly studied in 2D-monolayer culture, but that 3D in vitro models of osteocyte-like cells and primary osteocytes have been established as well. 3D-osteocyte models mimic the native environment of osteocytes and show superior osteocyte morphology and behavior, enabling the development of human disease models. Osteocytes cultured in their native matrix resemble osteocytes in their in vivo situation most closely. The native matrix of osteocytes affects their mechanoresponsiveness. To study the role of the matrix surrounding the osteocytes in their mechanoresponsiveness, we developed, in chapter 3, a 3D-mechanical loading model of human cortical bone containing osteocytes in their native matrix. Using our model, mechanical loading can be reliably applied to osteocytes in their native matrix. Mechanically loaded osteocytes in their native matrix maintained their viability, and slightly upregulated SOST expression with increasing mechanical loading magnitude up to 8000 µɛ. This demonstrated that osteocytes in our model showed responsiveness to mechanical loading, indicating that our model is suitable for further studies on the role of healthy or diseased native matrix on osteocyte mechanoresponsiveness. Moreover, osteocytes in their native matrix responded to 1,25-dihydroxyvitamin D3, which indicates that our model can be used for studies on the role of small molecules in bone metabolism, and for drug screening. The mechanism of osteocyte-regulated mechanical adaptation of bone is not fully understood. More insight into target gene expression in osteocytes in their native matrix in response to mechanical loading is necessary. In chapter 4, we identified mechanosensitive genes by mapping the response of osteocytes in their native matrix to mechanical loading using RNA sequencing. Forty-seven new differentially expressed genes by mechanical loading were discovered in osteocytes in their native bone matrix. Eleven of these genes were related to bone metabolism. The functional aspects of these genes will be further explored, since they might play a role in the mechanical adaptation of bone. Our findings will be helpful to unravel the possible role of osteocytes in metabolic bone diseases, thereby providing new insight in the pathogenesis of these diseases, and facilitating potential gene targeted therapy. The inherited bone disorder osteogenesis imperfecta (OI) is characterized by bone fragility. Most OI patients have mutations leading to defected type I collagen, which is the main protein component of the extracellular matrix of bone. Osteocytes play an important role in bone homeostasis, but whether and how the function of osteocytes is affected in OI is unknown. In chapter 5, we studied whether abnormalities in OI bone matrix affect the osteocyte response to mechanical loading. Isolated non-OI and OI osteocytes cultured in monolayer showed similar mechanoresponsiveness, indicating that after removal of the altered bone matrix, isolated OI osteocytes retained their normal ability to respond to mechanical loading. This demonstrates that intrinsic mechanosensing by osteocytes is unaffected in OI. Moreover, osteocytes in OI bone matrix exhibited an altered response to mechanical loading compared to osteocytes in non-OI bone matrix. Abnormalities in OI bone matrix, i.e. hypermineralized collagenous matrix, may affect osteocyte mechanoresponsiveness, which could be partly responsible for the bone fragility in OI patients. This study provided new insight in the pathogenesis of OI, which may contribute to the development of new strategies for OI treatment in the future.