Gradient-based hydrogels : for mimicking cartilage zonal organization and high-throughput screening of cell-niche interactions
Keywords: Bioengineering.

Articular cartilage injury is one of the leading causes for disability. Native articular cartilage is characterized by gradients of biochemical and mechanical cues, which is critical for rendering the unique mechanical properties of articular cartilage. Most tissue engineering strategies developed to date only allow regenerate cartilage with homogeneous biochemical and physical properties. As such, there remains a critical need to develop novel strategies to regenerate cartilage with biomimetic zonal organization. To address this unmet need, my thesis work focuses on developing tissue-scale gradient hydrogels as 3D scaffold to guide cartilage regeneration with biomimetic zonal organization. Unlike conventional methods, our gradient hydrogel platform allows rapid and homogeneous cell encapsulation with high cell viability and facilitates tunable biochemical and mechanical gradients. Using this gradient hydrogel platform as a tool, we first examined the effects of single vs. dual gradient niche cues in driving chondrocyte-based cartilage formation in 3D. Our results showed stiffness gradient alone was effective in inducing zonal-dependent cartilage formation. Inhibiting mechanosensing using blebbistatin abolished such stiffness-dependent tissue zonal development, confirming the zonal response is dependent on the stiffness gradient from hydrogels. Using dual gradient hydrogels containing chondroitin sulfate gradient and stiffness gradient, I further demonstrate that these two gradient cues synergize to enhance zonal-dependent cartilage formation by encapsulated chondrocytes in 3D. In addition to modulate cell-matrix interactions, we also harnessed gradient hydrogels as an in vitro model to uncover the role of cell-cell interactions between zones in driving cartilage zonal development, which remains unknown due to lack of suitable research tools. Our results showed inter-zonal cell-cell interactions can directly alter mechanosensing of chondrocytes encapsulated in 3D stiffness gradient hydrogels. Specifically, removing inter-zonal paracrine signaling using separate culture downregulated mechanosensing gene expression and reduced cartilage formation by cells in all zones, yet still maintained a stiffness-dependent trend. Unexpectedly, co-culture all zones together but disrupting their native spatial positions minimized inter-zonal differences in cartilage formation. Together, our findings suggest that tissue zonal organization is driven not only by insoluble matrix gradients, but also by inter-zonal cell-cell interactions. While the present thesis focuses on recreating cartilage zonal organization to demonstrate proof-of principle, the concepts arise from such studies can be broadly applicable to recreate other tissue types. In the second part of this thesis, we have demonstrated that such 3D gradient hydrogels can be used for high-throughput screening of stem cell-niche interactions. Also, we expanded the application of such gradient hydrogels as a biomimetic cancer cell niche with stiffness and biochemical cues in a range that close to the brain tissue, which could help optimize design for 3D brain cancer models with more physiological relevance to accelerate the discovery of novel therapeutics for treating brain cancer. In summary, our developed gradient-based hydrogel platform helps elucidate how cells respond to mechanical and biochemical properties of their niche, which can be harnessed to accelerate functional tissue formation in vitro that recapitulates native tissue organization. Furthermore, such hydrogel platform can also be used to better understand the interplay between cell-matrix and cell-cell interactions that drive tissue formation in 3D, which can be broadly applicable to other tissue engineering applications.