Cell–Cell Interactions Enhance Cartilage Zonal Development in 3D Gradient Hydrogels

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.