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

建立3D体外共培养模型以研究骨细胞、破骨细胞和成骨细胞之间的串扰

• 批准号: 281673234
• 负责人: Dr. Anne Bernhardt
• 金额: --
• 依托单位: Zentrum für Translationale Knochen-, Gelenk- und Weichgewebeforschung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/281673234?language=en
• 关键词: Establishment; 3D; vitro; co; culture

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.

骨组织代表一种复杂的复合材料，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他细胞类型。这三种主要骨细胞类型之间的相互作用调节骨重塑和体内平衡。大多数关于骨代谢调节的研究都是通过动物模型或涉及永生化小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仅涉及原代人成骨细胞、破骨细胞和骨细胞的 3D 体外共培养模型，以获得这些细胞的串扰信息，具有更高的临床相关性，并提高构建体对分析方法的可及性。在该项目的第一个资助期间，开发了不同的方法来产生稳定的原代人类骨细胞培养物。基于这些培养物，进行了骨细胞与成骨细胞以及骨细胞与破骨细胞的共培养研究。最后，建立了成骨细胞、破骨细胞和骨细胞的稳定三重培养物，显示了各自细胞类型的典型形态和基因表达特征，并允许对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处于更成熟的状态，破骨细胞标记物显着减少，骨细胞中骨钙素表达显着下调。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记物的表达，b）通过分析骨的矿化来扩展已建立的三重培养物的分析范围。 结构、通过间隙连接的细胞间相互作用以及骨钙素的调节。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。这些研究对于生物玻璃和陶瓷或可吸收合金等创新生物材料的测试特别有帮助。生物材料测试通常使用成骨细胞系进行，然而，生物材料释放的生物活性化合物对体外骨模型（包括三种主要骨细胞类型）的影响提供了更多临床相关数据，并将有助于了解生物活性化合物对骨再生和稳态的影响。此外，我们想要研究机械负荷和缺氧这两种发生在天然骨组织中的条件对体外骨模型中成骨细胞、骨细胞和破骨细胞相互作用的影响。