| Osteocytes comprise 90--95% of all bone cells and function as the mechanosensors within bone. Individual osteocytes are embedded in lacunae, intimately connected to the surrounding bone matrix, making them ideally situated to sense the strain caused by the deformation of loaded bone; however, this also makes the cells difficult to observe in vivo. When studying cells in vitro it is important to consider the interactions between cells, their environment, and the mechanical stimuli that they receive. The studies presented here sought to study the contributions of each of these components by deconstructing the in vivo condition and developing models and performing experiments in which each could be studied as an independent variable. Through the use of finite element modeling, I have illustrated how strains are transmitted to the embedded osteocyte and how variations in the perilacunar matrix influence these strains. I have shown that the strains experienced by individual osteocytes in vitro when exposed to fluid flow and substrate stretching are correlated to biological responses. I have shown that by utilizing a rest-inserted loading methodology MLO-Y4 cells experienced changes in gene expression and cytoskeletal organization. I have established a protocol to isolate primary osteocytes that results in a higher yield and purity than any previously published methods and enables the isolation of osteocytes from the long bones of skeletally mature and old animals. Finally, I have illustrated the importance of seeding cells in a manner in which they are allowed to infiltrate the scaffold completely and how strains are transmitted to cells seeded on scaffolds in varying configurations. My future plans include applying the methods presented in these studies to primary osteocytes isolated from osteoporotic bone in an attempt to better understand the disease and investigate potential treatments. |
