Ro Hyunuk, Park Jungha, Yang Kisuk, Kim Jiyong, Yim Hyun-Gu, Jung Giyoung, Lee Hyukjin, Cho Seung-Woo, Hwang Nathaniel S
School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul, 151-742, Republic of Korea.
Department of Biotechnology, Yonsei University, Seoul, 120-749, Republic of Korea.
Cell Tissue Res. 2015 Oct;362(1):115-26. doi: 10.1007/s00441-015-2195-7. Epub 2015 May 9.
Transient cartilage and a mineralizing microenvironment play pivotal roles in mesenchymal cell ossification during bone formation. In order to recreate these microenvironmental cues, C3H10T1/2 murine mesenchymal stem cells (MSCs) were exposed to chondrocyte-conditioned medium (CM) and seeded onto three-dimensional mineralized scaffolds for bone regeneration. Expansion of C3H10T1/2 cells with CM resulted in enhanced expression levels of chondrogenic markers such as aggrecan, type II collagen, type X collagen, and Sox9, rather than of osteogenic genes. Interestingly, CM expansion led to reduced expression levels of osteogenic genes such as alkaline phosphatase (ALP), type I collagen, osteocalcin, and Runx2. However, CM-expanded C3H10T1/2 cells showed enhanced osteogenic differentiation as indicated by increased ALP and Alizarin Red S staining upon osteogenic factor exposure. In vivo, CM-expanded C3H10T1/2 mesenchymal cells were seeded onto mineralized scaffolds (fabricated with polydopamine and coated with simulated body fluids) and implanted into critical-sized calvarial-defect mouse models. After 8 weeks of implantation, mouse skulls were collected, and bone tissue regeneration was evaluated by micro-computed tumography and Masson's trichrome staining. In accordance with the in vitro analysis, CM-expanded C3H10T1/2 cells gave enhanced bone mineral deposition. Thus, chondrocyte-conditioned factors and a mineralized microenvironment stimulate the bone formation of MSCs.
短暂性软骨和矿化微环境在骨形成过程中的间充质细胞骨化中起关键作用。为了重现这些微环境线索，将C3H10T1/2小鼠间充质干细胞（MSCs）暴露于软骨细胞条件培养基（CM）中，并接种到三维矿化支架上用于骨再生。用CM扩增C3H10T1/2细胞导致软骨生成标志物如聚集蛋白聚糖、II型胶原、X型胶原和Sox9的表达水平升高，而不是成骨基因的表达水平升高。有趣的是，CM扩增导致成骨基因如碱性磷酸酶（ALP）、I型胶原、骨钙素和Runx2的表达水平降低。然而，CM扩增的C3H10T1/2细胞在暴露于成骨因子后，碱性磷酸酶和茜素红S染色增加，表明其成骨分化增强。在体内，将CM扩增的C3H10T1/2间充质细胞接种到矿化支架（用聚多巴胺制备并涂覆模拟体液）上，并植入临界大小的颅骨缺损小鼠模型中。植入8周后，收集小鼠颅骨，并通过显微计算机断层扫描和Masson三色染色评估骨组织再生。与体外分析一致，CM扩增的C3H10T1/2细胞骨矿物质沉积增加。因此，软骨细胞条件因子和矿化微环境刺激了间充质干细胞的骨形成。
