Centre for Translational Bone, Joint, and Soft Tissue Research, Technische Universität Dresden, Faculty of Medicine and University Hospital, 01307 Dresden, Germany.
Centre for Translational Bone, Joint, and Soft Tissue Research, Technische Universität Dresden, Faculty of Medicine and University Hospital, 01307 Dresden, Germany.
Eur J Cell Biol. 2022 Jun-Aug;101(3):151256. doi: 10.1016/j.ejcb.2022.151256. Epub 2022 Jul 8.
An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr as well as hypoxic cultivation (5% O content or chemically induced by Co) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr, hypoxic conditions or in the presence of 75 µM Co. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr free control. Hypoxic conditions induced by both 5% O or a Co treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.
体外骨三培养物涉及人原代成骨细胞、骨细胞和破骨细胞，可在天然骨组织模型系统中研究骨愈合因子、药物或生物材料。本研究分析了 Sr 以及缺氧培养（5%O 含量或化学诱导 Co）对骨细胞的影响。三种细胞类型在存在 100µM Sr、缺氧条件或存在 75µM Co 的情况下共同培养。培养后，将细胞类型分离并分别在 mRNA 和蛋白质水平上进行分析。在 Sr 的存在下，成骨细胞表现出 IBSP 表达下调和 ALP 活性刺激。成骨细胞基因标志物 PDPN、MEPE、RANKL、OPG、骨钙素的表达以及分泌的骨钙素量减少。与 Sr 自由对照相比，破骨细胞特异性酶 TRAP 和 CAII 的活性增强。由 5%O 或 Co 处理诱导的缺氧条件导致所有骨细胞的 DNA 含量降低，成骨细胞标志物 ALPL 和 IBSP 以及骨细胞标志物 PDPN、RANKL 和 OPG 的表达下调。此外，Co 诱导的缺氧降低了骨细胞中骨钙素的基因和蛋白质表达。Co 处理后，TRAP 基因表达和活性增加。本研究首次分析了 Sr 或缺氧对原代人骨细胞三培养物的影响。所研究的体外骨模型可能适合在生物材料和药物开发的早期阶段减少动物实验。
