| Summary: | Osteocytes are currently regarded as being pivotal in maintaining bone homeostasis. They differentiate from osteoblasts, are embedded in mineralised matrix and difficult to isolate. Current models for in vitro osteocyte studies are limited. Others have suggested that osteoblasts in 3-dimensional (3D) cultures differentiate to osteocytes. This study aimed to develop 3D cultures enabling differentiation of osteoblasts to osteocytes, which could be used for studies of osteocyte differentiation and responses to mechanical loading. Furthermore, the effects of external compounds on osteoblast differentiation in 3D were assessed. Mouse (MC-3T3, IDG-SW3) and human primary osteoblasts (hOBs) were maintained in type I collagen gels in either non-osteogenic or osteogenic media, and +/- compounds such as insulin-like growth factor-1 (IGF-1). Furthermore, mechanical loading (5 mins, 10 Hz, 2.5 N) was applied to 3D cultures and responses characterised. Cells were viable in collagen gels for 25 days, and expressed mRNA for mature osteocyte markers e.g. sclerostin in osteogenic medium. Furthermore, IGF-1 upregulated mRNA expression of osteocyte markers and other molecules (e.g. receptor activator of the nuclear factor kappa-β ligand - RANKL - 43-fold) in MC-3T3 cells, indicating modulation of cell differentiation and function. Osteocyte markers were expressed earlier in IDG-SW3 cells in 3D compared to published marker expression profiles in 2D monolayer cultures. Following mechanical loading, known mechanosensitive markers were modulated in IDG-SW3 cells in 3D, for example, RANKL and vascular endothelial growth factor (VEGF) up-regulated and sclerostin downregulated post-loading. This 3D model enables differentiation of osteoblasts to osteocytes in an environment akin to osteocytes in vivo. External compounds accelerated cell differentiation, and this was also accelerated in 3D compared to monolayer. Furthermore, the 3D model enabled osteocyte mechanical loading. This model can be used with human cells, will further our understanding of osteocyte differentiation, and inform on osteocyte function including their responses to mechanical loading.
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