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Articular cartilage is characterized by zonal organizations containing dual gradients of biochemical cues and mechanical cues. However, how biochemical gradient interacts with the mechanical gradient to drive the cartilage zonal development remains largely unknown. Here, we report the development of a dual-gradient hydrogel platform as a 3D niche to elucidate the relative contributions of biochemical and mechanical niche gradients in modulating zonal-specific chondrocyte responses and cartilage zonal organization. Chondroitin sulfate (CS), a major constituent of cartilage extracellular matrix, was chosen as the biochemical cue. Poly(ethylene glycol), a bioinert polymer, was used to create the stiffness gradient. Dual-gradient hydrogels upregulated cartilage marker expressions and increased chondrocyte proliferation and collagen deposition in a zonal-dependent manner. Hydrogels with CS gradient alone exhibited poor mechanical strength and degraded prematurely after 1 week of culture. While CS gradient alone did not support long-term culture, adding CS gradient to mechanical-gradient hydrogels substantially enhanced cell proliferation, glycosaminoglycan production, and collagen deposition compared to mechanicalgradient hydrogels alone. These results suggest that biochemical and mechanical gradient cues synergize to enhance cartilage zonal organization by chondrocytes in 3D. Together, our results validate the potential of dual-gradient hydrogels as a 3D cell niche for cartilage regeneration with zonal organization and may be used to recreate other tissue interfaces.

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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.