Granular hydrogels or packed microgels have gained increasing interest in recent years owing to their high tunability compared with “bulk hydrogels”. Although granular hydrogels have been utilized for the assessment of cancer cell invasion, the heterogeneous stiffness increase in the tumor microenvironment (TME) due to the accumulation of extracellular matrix and how the mechanical properties of TME and accumulation of extracellular matrix impact cell fate has not been well studied. We have previously synthesized norbornene and carbohydrazide dually modified gelatin (i.e., GelNB-CH) for dynamic tuning gel stiffness. In this study, we sought to create granular hydrogels using GelNB-CH and sequentially use orthogonal click chemistry to form hyaluronic acid and stiffness gradients through diffusion and hydrazone/tetrazine click reactions. GelNB-CH microgel was created through water-in-oil emulsification and crosslinked with PEG-tetra-thiol (PEG4SH) through thiol-ene reaction. The GelNB-CH microgels were packed, transferred to a cylindrical mold, and either deposited without annealing or annealed with PEG-tetrazine through tetrazine-norbornene click chemistry. As a comparison, GelNB-CH bulk gel was made using the same gel precursor in the same cylindrical mold, but without the additional step of emulsification. Aldehyde-bearing oxidized HA (oHA) solution was labeled with green fluorescent dye and added on top of the microgels to enable diffusion and additional hydrazone bonds for immobilization of HA. GelNB-CH microgels were successfully crosslinked through UV light induced polymerization (size: ~100 to ~300 μm). oHA gradient was successfully created through diffusion and hydrazide/hydrazone click reaction between the aldehyde on oHA and the carbohydrazide on GelNB-CH. Specifically, the gradient distance is the shortest (~1 mm) in bulk gel, whereas gradient distance was increased in annealed microgels, and reaches the longest distance in non-annealed microgels (~4 mm), due to the most permeable interstitial space for diffusion. Future work will be focused on different physicochemical gradient and cell response to the HA gradient.

Orthogonal click chemistry to generate physicochemical gradients in granular hydrogels

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