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

骨组织代表复杂的复合物，其包括成骨细胞、破骨细胞、骨细胞和在矿化胶原基质中空间组织的其它细胞类型。这三种主要骨细胞类型之间的相互作用调节骨重建和体内平衡。大多数关于骨代谢调节的研究是用动物模型或涉及永生化鼠细胞系的体外细胞培养模型进行的。本项目的目的是建立一个3D体外共培养模型，仅涉及原代人成骨细胞，破骨细胞和骨细胞，以获得这些细胞的串扰信息，具有更高的临床相关性和更好的分析方法的结构的可访问性。在该项目的第一个资助期，开发了不同的方法来产生稳定的原代人骨细胞培养物。在此基础上，进行了骨细胞与成骨细胞、骨细胞与破骨细胞的共培养研究。最后，建立了成骨细胞、破骨细胞和成骨细胞的稳定的三重培养物，其显示了各自细胞类型的典型形态学和基因表达特征，并允许对成骨细胞和成骨细胞进行单独分析。与单一培养物相比，我们可以证明成骨细胞的更成熟状态，破骨细胞标志物显著减少，骨细胞中骨钙素表达显著下调。在第二个资助期，我们希望通过a）不仅在基因上而且在蛋白质水平上研究骨标志物的表达，和B）通过分析构建物的矿化，通过缝隙连接的细胞-细胞相互作用和骨钙素的调节。此外，我们希望将三重培养物暴露于不同的生物活性分子和离子，以研究这些试剂对骨细胞相互作用的影响。这些研究对创新生物材料如生物玻璃、陶瓷或可吸收合金的测试特别有帮助。生物材料测试通常使用成骨细胞系进行，然而，从生物材料释放的生物活性化合物对体外骨模型的影响，包括三种主要的骨细胞类型，提供了更多的临床相关数据，并将有助于了解生物活性化合物对骨再生和体内平衡的影响。此外，我们想研究的影响，机械负荷和缺氧，这两种情况下，发生在自然骨组织中，对成骨细胞，骨细胞和破骨细胞在我们的体外骨模型的相互作用。

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.