Few investigators think of bone as an endocrine gland, even after the discovery that osteocytes produce circulating fibroblast growth factor 23 that targets the kidney and potentially other organs. In fact, until the last few years, osteocytes were perceived by many as passive, metabolically inactive cells. However, exciting recent discoveries have shown that osteocytes encased within mineralized bone matrix are actually multifunctional cells with many key regulatory roles in bone and mineral homeostasis. In addition to serving as endocrine cells and regulators of phosphate homeostasis, these cells control bone remodeling through regulation of both osteoclasts and osteoblasts, are mechanosensory cells that coordinate adaptive responses of the skeleton to mechanical loading, and also serve as a manager of the bone's reservoir of calcium. Osteocytes must survive for decades within the bone matrix, making them one of the longest lived cells in the body. Viability and survival are therefore extremely important to ensure optimal function of the osteocyte network. As we continue to search for new therapeutics, in addition to the osteoclast and the osteoblast, the osteocyte should be considered in new strategies to prevent and treat bone disease.

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The central importance of osteocytes in regulating bone homeostasis is becoming increasingly apparent. However, the study of these cells has been restricted by the relative paucity of cell line models, especially those of human origin. Therefore, we investigated the extent to which SaOS2 human osteosarcoma cells can differentiate into osteocyte-like cells. During culture under the appropriate mineralising conditions, SaOS2 cells reproducibly synthesised a bone-like mineralised matrix and temporally expressed the mature osteocyte marker genes SOST, DMP1, PHEX and MEPE and down-regulated expression of RUNX2 and COL1A1. SaOS2 cells cultured in 3D collagen gels acquired a dendritic morphology, characteristic of osteocytes, with multiple interconnecting cell processes. These findings suggest that SaOS2 cells have the capacity to differentiate into mature osteocyte-like cells under mineralising conditions. PTH treatment of SaOS2 cells resulted in strong down-regulation of SOST mRNA expression at all time points tested. Interestingly, PTH treatment resulted in the up-regulation of RANKL mRNA expression only at earlier stages of differentiation. These findings suggest that the response to PTH is dependent on the differentiation stage of the osteoblast/osteocyte. Together, our results demonstrate that SaOS2 cells can be used as a human model to investigate responses to osteotropic stimuli throughout differentiation to a mature osteocyte-like stage.

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38.[Q1] The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.