Centre for Translational Bone, Joint and Soft Tissue Research, Technische Universitaet und Universitaetsklinikum Dresden, Dresden, Germany.
Tissue Eng Part A. 2019 Oct;25(19-20):1347-1355. doi: 10.1089/ten.TEA.2018.0338. Epub 2019 Jun 14.
Osteocytes play a key role in orchestrating bone homeostasis and turnover and, therefore, investigations with osteocytes are of high relevance for biomaterial and drug testing in future. In this study, collagen type I gels and collagen gels modified with biomimetically mineralized collagen were tested as three-dimensional (3D) environment for the maintenance of the osteocytic phenotype of primary human osteocytes. After cultivation in different collagen gels, cells were analyzed microscopically for the osteocytic phenotype and gene expression of osteocyte marker genes osteocalcin, podoplanin ()/, phosphate regulating endopeptidase homolog, X-linked (), matrix extracellular phosphoglycoprotein (), dentin matrix protein 1 (), and sclerostin (). Directly after isolation from bone tissue the cells expressed all examined osteocyte markers. After 7 days of 3D cultivation in collagen gels the osteocytic marker genes and were upregulated and the other marker genes still expressed. Modification of collagen gels with biomimetically mineralized collagen and strontium-doped mineralized collagen prevented the cell-seeded gels from shrinking. Osteocyte morphology was not affected by the gel modification. However, the isolation of RNA from the mineralized gel variants was heavily impaired. Alternatively, the osteocytic differentiation of human osteoblasts in the different collagen gels was examined. Primary human osteoblasts were embedded into the gels and cultivated under osteogenic stimulation. After 14 days of cultivation, embedded osteoblasts showed osteocyte-like morphology and positive staining for DMP-1. Early osteocyte marker genes, such as and , were expressed while the expression of the osteoblast marker gene alkaline phosphatase () increased. This upregulation was partly prevented by modification of collagen gels with mineralized collagen. Impact Statement This research focuses on the three-dimensional cultivation of primary human osteocytes instead of rodent osteocyte cell lines. Stable cultures of these regulating cells provide the opportunity to establish co- and triple cultures with osteoblasts and osteoclasts to analyze the cross talk between these cell species and to establish bone models for the testing of bioactive molecules, growth factors, drugs, and biomaterials.
成骨细胞在协调骨稳态和更新中起着关键作用，因此，对成骨细胞的研究对于未来生物材料和药物测试具有重要意义。在这项研究中，我们将Ⅰ型胶原凝胶和仿生矿化胶原修饰的胶原凝胶作为维持原代人成骨细胞成骨细胞表型的三维（3D）环境进行了测试。在不同胶原凝胶中培养后，通过显微镜分析细胞的成骨细胞表型和骨钙素、Podoplanin（）/、磷酸调节内肽酶同源物、X 连锁（）、细胞外基质磷蛋白聚糖（）、牙本质基质蛋白 1（）和硬骨素（）等成骨细胞标记基因的表达。细胞从骨组织中直接分离出来时表达了所有检测到的成骨细胞标记物。在 3D 胶原凝胶培养 7 天后，上调了 和 等成骨细胞标记基因，其他标记基因仍有表达。用仿生矿化胶原和锶掺杂矿化胶原修饰胶原凝胶可防止细胞接种凝胶收缩。矿化凝胶变体对 RNA 的提取有严重影响。成骨细胞形态不受凝胶修饰的影响。然而，替代方法是研究不同胶原凝胶中人类成骨细胞的成骨细胞分化。将原代人成骨细胞嵌入凝胶中，并在成骨刺激下培养。培养 14 天后，嵌入的成骨细胞表现出成骨细胞样形态，并且 DMP-1 染色呈阳性。早期成骨细胞标记基因，如 和 ，表达上调，而碱性磷酸酶（）的成骨细胞标记基因表达增加。这种上调被矿化胶原修饰的胶原凝胶部分阻止。研究意义 本研究侧重于原代人成骨细胞的三维培养，而不是鼠源性成骨细胞系。这些调节细胞的稳定培养为与成骨细胞和破骨细胞进行共培养和三重培养提供了机会，以分析这些细胞之间的串扰，并建立用于测试生物活性分子、生长因子、药物和生物材料的 骨模型。
