Department of Bioengineering, Stanford University, Palo Alto, California 94305, United States.
Department of Orthopaedic Surgery, Stanford University, Palo Alto, California 94305, United States.
ACS Biomater Sci Eng. 2023 Feb 13;9(2):831-843. doi: 10.1021/acsbiomaterials.2c00469. Epub 2023 Jan 11.
Cartilage tissue is characterized by zonal organization with gradual transitions of biochemical and mechanical cues from superficial to deep zones. We previously reported that 3D gradient hydrogels made of polyethylene glycol and chondroitin sulfate can induce zonal-specific responses of chondrocytes, resulting in zonal cartilage formation that mimics native tissues. While the role of cell-matrix interactions has been studied extensively, how cell-cell interactions across different zones influence cartilage zonal development remains unknown. The goal of this study is to harness gradient hydrogels as a tool to elucidate the role of cell-cell interactions in driving cartilage zonal development. When encapsulated in intact gradient hydrogels, chondrocytes exhibited strong zonal-specific responses that mimic native cartilage zonal organization. However, the separate culture of each zone of gradient hydrogels resulted in a significant decrease in cell proliferation and cartilage matrix deposition across all zones, while the trend of zonal dependence remains. Unexpectedly, mixing the coculture of all five zones of hydrogels in the same culture well largely abolished the zonal differences, with all zones behaving similarly to the softest zone. These results suggest that paracrine signal exchange among cells in different zones is essential in driving cartilage zonal development, and a spatial organization of zones is required for proper tissue zonal development. Intact, separate, or coculture groups resulted in distinct gene expression patterns in mechanosensing and cartilage-specific markers, suggesting that cell-cell interactions can also modulate mechanosensing. We further showed that 7 days of priming in intact gradient culture was sufficient to instruct the cells to complete the zonal development, and the separate or mixed coculture after 7 days of intact culture had minimal effects on cartilage formation. This study highlights the important role of cell-cell interactions in driving cartilage zonal development and validates gradient hydrogels as a useful tool to elucidate the role of cell-matrix and cell-cell interactions in driving zonal development during tissue morphogenesis and regeneration.
软骨组织的特征是具有区域化组织，其生化和力学线索从浅层到深层逐渐过渡。我们之前报道过，由聚乙二醇和硫酸软骨素组成的 3D 梯度水凝胶可以诱导软骨细胞产生区域特异性反应，从而形成模仿天然组织的区域特异性软骨。虽然细胞-基质相互作用的作用已经得到了广泛的研究，但细胞-细胞相互作用如何影响软骨的区域化发育仍不清楚。本研究的目的是利用梯度水凝胶作为一种工具，阐明细胞-细胞相互作用在驱动软骨区域化发育中的作用。当软骨细胞被包裹在完整的梯度水凝胶中时，它们表现出强烈的区域特异性反应，模仿天然软骨的区域化组织。然而，将梯度水凝胶的每个区域单独培养会导致所有区域的细胞增殖和软骨基质沉积显著减少，而区域依赖性的趋势仍然存在。出乎意料的是，将所有五个区域的水凝胶共培养物混合在同一个培养孔中，大大消除了区域差异，所有区域的行为都类似于最软的区域。这些结果表明，不同区域细胞之间的旁分泌信号交换对于驱动软骨区域化发育是必不可少的，并且区域的空间组织对于组织的正常区域化发育是必需的。完整的、单独的或共培养的分组导致了机械敏感和软骨特异性标志物的基因表达模式的显著差异，这表明细胞-细胞相互作用也可以调节机械敏感性。我们进一步表明，在完整的梯度培养中进行 7 天的启动足以指导细胞完成区域化发育，而在 7 天的完整培养后进行单独或混合的共培养对软骨形成的影响很小。本研究强调了细胞-细胞相互作用在驱动软骨区域化发育中的重要作用，并验证了梯度水凝胶作为一种有用的工具，可以阐明细胞-基质和细胞-细胞相互作用在组织形态发生和再生过程中驱动区域化发育的作用。
