Distinct differences between calvarial and long bone osteocytes in cell morphologies, gene expression and aging responses.

Osteocytes are the terminally differentiated bone cells resulted from bone formation. Although there are two distinct processes of bone formation, intramembranous and endochondral ossifications contributing to the formation of calvarial and long bones, it is not clear whether the distinct pathways determine the differences between calvaria and femoral cortical bone derived osteocytes. In the present study, we employed confocal structured illumination microscopy and mRNA-sequencing analysis to characterize the morphologic and transcriptomic expression of osteocytes from murine calvaria and mid-shaft femoral cortical bone. Structured illumination microscopy and geometric modelling showed round shaped and irregularly scattered calvarial osteocytes compared to spindle shaped and orderly arrayed cortical osteocytes. mRNA-sequencing analysis indicated different transcriptomic profiles between calvarial and cortical osteocytes and provided evidence that mechanical response of osteocytes may contribute to geometrical differences. Furthermore, transcriptomic analysis showed that these two groups of osteocytes come from distinct pathways with 121 ossification-related genes differentially expressed. Analysis of correlation between ossification and osteocyte geometries via a Venn diagram showed that several genes related to ossification, cytoskeleton organization and dendrite development were differentially expressed between calvarial and cortical osteocytes. Finally, we demonstrated that aging disrupted the organization of dendrites and cortical osteocytes but had no significant effects on calvarial osteocytes. Together, we conclude that calvarial and cortical osteocytes are different in various aspects, which is probably the consequence of their distinct pathways of ossification.