Establishment of a 3D in vitro co-culture model for the investigation of cross talk between osteocytes, osteoclasts and osteoblasts

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Bone tissue represents a complex composite comprising osteoblasts, osteoclasts, osteocytes and other cell types spatially organized in a mineralized collagen matrix. The cross talk between these three major bone cell types regulates bone remodeling and homeostasis. Most investigations on the regulation of bone metabolism are performed with animal models or in vitro cell culture models involving immortalized murine cell lines. The aim of the present project was to establish a 3D in vitro co-culture model involving solely primary human osteoblasts, osteoclasts and osteocytes to achieve information on the cross talk of these cells with higher clinical relevance and improved accessibility of the constructs to analytical methods. In the first funding period of the project, different approaches were developed to generate stable primary human osteocyte cultures. Based on these cultures, co-culture studies of osteocytes with osteoblasts, and osteocytes with osteoclasts were performed. Finally, stable tripel cultures of osteoblasts, osteoclasts and osteocytes were established showing typical morphological and gene expression characteristics for the respective cell types and allowing the separate analysis of osteocytes and osteoblasts. Compared to single cultures we could demonstrate a more mature state of osteoblasts, significantly decreased osteoclast markers and a significantly downregulated osteocalcin expression in osteocytes.In the second funding period, we want to extend the analytic range of the established tripel cultures by a) studying the expression of bone markers not only on gene, but also on protein level, and b) by analyzing the mineralization of the constructs, the cell-cell interaction via gap junctions and the regulation of osteocalcin. Furthermore, we want to expose the triple cultures to different bioactive molecules and ions to investigate the influence of these agents on the interaction of the bone cells. These investigations are especially helpful for the testing of innovative biomaterials like bioglasses and ceramics or resorbable alloys. Biomaterials testing is usually performed with osteoblast cell lines, however, the impact of bioactive compounds, released from biomaterials on the in vitro bone model , comprising the three major bone cell types provides more clinically relevant data, and will help to understand the influence of bioactive compounds on bone regeneration and homeostasis. Moreover, we want to study the effect of mechanical load and hypoxia, both conditions, which occur in natural bone tissue, on the interaction of osteoblasts, osteocytes and osteoclasts in our in vitro bone model.