Interactions between osteocytes and osteoblasts in a novel 3D co-culture system [Abstract]

Introduction Osteocytes, the most abundant bone cell type, form an intercommunicating network within bone integrating hormonal, growth factor and mechanical stimuli to control osteoblast activity. The importance of this osteocyte-driven control is exemplified by human disorders where mutations in the osteocyte-derived protein sclerostin cause excessive bone mass. We have devised a co-culture system to investigate osteocyte/osteoblast interactions. Materials and Methods Osteocyte cell lines (MLO-Y4, MLOA5) were embedded within type I collagen gels with osteoblast cell lines (MG63/SaOS2) layered on top. Cell phenotypes were visually assessed by light microscopy, immunolocalisation and fluorescence microscopy. Gene expression was quantified by real time RT-PCR of RNA extracted separately from surface cells and gel-embedded cells within the same cocultures. Results Osteocytes formed a network of dendritic cells, maintained for at least 2 weeks, with extensive processes connecting neighbouring osteocytes and extending towards osteoblasts on the gel surface. Comparison of osteoblasts cultured on empty gels and those co-cultured with osteocytes revealed that osteocytes inhibit both invasion into the gel and expression of Type I collagen by osteoblasts. The osteocyte dendritic marker E11 was expressed by MLO-Y4 osteocytes. E11 was not expressed by MG63 osteoblasts on the surface of osteocyte-filled gels, but was expressed when MG63s were incorporated within the gel. Human primary osteoblasts embedded within collagen gels assume a dendritic morphology, downregulate Runx2 and upregulate osteocalcin mRNA expression, indicative of differentiation to an osteocyte-like phenotype. Discussion Osteocytes regulate osteoblast functions in this co-culture model and embedding osteoblasts within type I collagen induces differentiation to an osteocyte-like cell