Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture.

中文翻译：

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在胶原蛋白凝胶中原代人类成骨细胞和成熟人类破骨细胞的三维共培养。

破骨细胞是用于骨重塑的关键细胞，其活性由位于骨基质内部的骨细胞协调。体外因此，破骨细胞和破骨细胞的共培养有利于分析这些细胞物种之间的串扰。在这项研究中，通过多步分离过程从人骨中分离出原代骨细胞，并嵌入到三维胶原蛋白凝胶中。成熟的人破骨细胞是通过分化人外周血单个核细胞（PBMC）而产生的。测试了不同表面的破骨细胞形成：悬浮培养皿，胶原蛋白凝胶和正常的组织培养聚苯乙烯。从表面脱离后，破骨细胞显示出典型的形态和破骨细胞标志物的基因表达。在胶原上分化的破骨细胞表现出最高的破骨细胞标志物表达。成熟破骨细胞与骨细胞的共培养是在Transwell系统中与骨细胞一起进行的，包埋在多孔聚对苯二甲酸乙二酯膜的顶侧的胶原蛋白凝胶和基底侧的破骨细胞中，从而可以对破骨细胞和破骨细胞进行单独的基因表达分析。共培养7天后，两种细胞均显示出典型的形态，破骨细胞为多核巨细胞，破骨细胞为树突状延伸的星形细胞。此外，破骨细胞标志物抗酒石酸酸性磷酸酶，碳酸酐酶II和组织蛋白酶K在单一和共培养中均在基因表达和蛋白质水平上被检测到。骨细胞在单一和共培养中均显示出典型的骨细胞标记物E11，硬化蛋白，牙本质基质蛋白1，骨钙素和核因子-κ配体的受体激活剂的基因表达。