Date of Award
Doctor of Philosophy (PhD)
The cellular mechanisms for loading-induced bone formation, from osteocyte mechano-sensation to osteoblast-directed bone formation, are not well understood. Elucidating these mechanisms and identifying any processes that are disrupted in aged mice can aide in the development of new anabolic drugs for treating diseases like osteoporosis. This thesis begins by investigating the genes expressed by osteocytes following loading at an early mechanosensitive (4-hr) timepoint, and later at a bone-forming (day 5) timepoint. We demonstrated increases in Ngf and Wnt1 in osteocyte-enriched intracortical bone by laser capture microdissection and microarray analysis. These results were important in demonstrating the presence of Ngf in osteocytes, and in suggesting the role of Wnt1 as a potential signaling molecule between osteocytes and osteoblasts which are currently unknown. Next, we investigated the effects of osteocyte-signaling on osteoblasts by studying the osteoblast differentiation stages at the bone surface and role of proliferation following loading-induced bone formation. We utilized lineage tracing with inducible Osx- and Dmp1-expressing reporter mice to evaluate the contribution of these cells to bone-forming surfaces at days 8 and 12 following the onset of loading. EdU was administered to mice via drinking water from the start of loading until sacrifice to identify any cells that arose via proliferation. Previously, it was shown that Osx-expressing cells are activated following loading to initiate bone formation 5 days following the onset of loading . We expanded these results by showing that Dmp1-expressing cells make up a large portion of the initial response to loading and become nearly depleted from the bone surface on days 8 and 12, especially in cases of rapid bone formation (higher stimulation). Our experiments evaluating bone formation at day 12 following the onset of loading also investigated age effects, comparing 5 mo and 22 mo mice. We demonstrated that similar levels of bone formation occurred at both ages, and similar mechanisms were utilized by young-adult and aged mice. Pre-existing Dmp1-expressing cells decreased at the bone surface in young-adult and aged mice to a similar degree. However, pre-existing Osx-expressing cells were not decreased with loading in aged mice, while a decrease occurred in young-adults. Thus, pre-existing preosteoblasts continue to sustain the bone formation response at day 12 in aged mice, but less differentiated cells perform this role in young-adult mice. Lastly, the proliferative response was shown to be dose dependent with more proliferation occurring in cases of more rapid bone formation, and no effect was found with age at day 12.
Matthew J. Silva
Roberto Civitelli, Farshid Guilak, Simon Tang, Erica Scheller,
Available for download on Tuesday, August 16, 2022