Primary Murine Osteoblast Cultures Contain Macrophages that Enhance Osteoblast Mineralisation

Delineation of the phenotype and functional capacity of osteoblasts (OBs) has been widely studied using primary OBs harvested via enzymatic digestion of neonatal rodent calvaria (calvarial OBs). Previous studies have suggested heterogeneity within this cell preparation and reported immune functions that aOre not traditionally performed by mesenchymal cells. These immune functions have been attributed to the OB, however, the heterogeneous nature of the culture has not been considered. We have demonstrated that macrophages are a significant population in standard calvarial OBs using cells isolated from Mac- Green mice (macrophages express an eGFP transgene driven by a myeloid restricted promoter). Microarray analysis of differentiating calvarial OB cultures (day 5, 14 and 21) confirmed a large number of macrophage-associated genes at all time points. Clustering analysis using microarray datasets representing nearly all tissues and cell lineages (symatlas.gnf.org) linked the gene expression profiles of calvarial OBs to macrophages and osteoclasts. Immunocytochemistry and flow cytometry confirmed that calvarial OB preparations co-isolated a population of F4/80+ macrophages that persist and expand during OB differentiation in vitro. Multiple passaging did not eliminate macrophages from these cultures. Bone explant cultures were also examined as an alternative approach to generating primary OBs and were similarly shown to contain F4/80+ macrophages. Given these observations, and our recent data demonstrating that macrophages are intercalated within bone lining tissues, we hypothesised that macrophages and OBs cooperate in the control of bone metabolism. To delineate the cooperative and distinct functional roles of macrophages and OBs, we used magnetic-assisted cell sorting to generate a population of highly enriched calvarial OBs by removing haematopoietic cells. The majority of cells removed via this method expressed the F4/80 macrophage marker. Strikingly, macrophage removal significantly decreased both osteocalcin mRNA expression levels and in vitro mineralisation (von Kossa) in enriched OB differentiation cultures. The presence of a persistent population of macrophages within primary OB cultures raises the possibility that our existing understanding of in vitro OB biology has been influenced by the contribution of this cell population. Our data provide evidence that macrophages regulate OB function and specifically enhance mineralisation in vitro.