Department of Bioengineering, National University of Singapore, Singapore, Singapore.
Tissue Eng Part A. 2012 Sep;18(17-18):1902-11. doi: 10.1089/ten.TEA.2011.0580. Epub 2012 Aug 8.
Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.
生理状态下的骺软骨界面再生是一个巨大的挑战。本研究旨在探讨兔骨髓基质干细胞（rBMSCs）与兔成骨细胞在特殊设计的二维（2D）-三维（3D）共界面培养中的共培养，以体外构建中间骺软骨区域。通过独立培养支架上的软骨生成 rBMSC 和细胞培养板中的成骨细胞，随后将它们放置在接触和共培养来建立 2D-3D 共培养系统。作为对照，使用未与成骨细胞共培养的样本。通过实时聚合酶链反应定量检测成骨细胞对软骨生成 rBMSC 的调控作用。为了研究共培养对支架不同部位细胞的影响，将样本分为两部分，发现它们之间存在明显不同的基因表达模式。与对照组相比，软骨生成标志物基因，如 Collagen II 和 Aggrecan 的表达明显下调。然而，Sox-9 和 Collagen I 的表达增加。更重要的是，共培养系统中的软骨生成 rBMSC 通过表达钙化软骨带特异性细胞外基质标志物 Collagen X 和肥大软骨细胞标志物 MMP-13 形成骺软骨界面层，而在对照组中未观察到这些标志物。具体而言，只有与成骨细胞接触的软骨生成 rBMSC 层表达 Collagen X 和 MMP-13，表明共培养对界面形成有积极影响。生化分析、组织学结果和免疫组织化学染色进一步支持了这一观察结果。总之，本研究揭示了 2D-3D 界面共培养系统中成骨细胞的特定调控刺激可以诱导骺软骨界面的形成，用于骺软骨组织工程。
