(411a) Tunable Fibrillar Collagen Hydrogels Strengthened By Bioorthogonal Covalent Crosslinks

The extracellular matrix (ECM) consists of a network of macromolecules that are organized in a tissue-specific manner, providing support to the constituent cells and regulating several cellular processes including adhesion, proliferation, and migration. The geometry and arrangement of the ECM components themselves influence how cells behave. Collagens are the most prevalent proteins in the ECM. Type I collagen, which accounts for 90% of collagens in the human body, possesses a characteristic fibrillar structure that guides cell elongation and migration, and thereby tissue development. This intrinsic biocompatibility makes collagen-based hydrogels a promising candidate for tissue engineering and fabrication of implantable medical devices. However, their utility is limited by their poor mechanical properties, tendency to deform against cellular contraction, and rapid enzymatic degradation in vivo.

To address these shortcomings and improve the usability of collagen hydrogels, we have developed a material system that employs strain-promoted azide-alkyne cycloaddition (SPAAC) chemistry to covalently crosslink the collagen fibrils within a hydrogel. Whereas typical crosslinking strategies may require cytotoxic catalysts or produce cytotoxic byproducts, SPAAC chemistry allows for a bioorthogonal crosslinking mechanism. We achieve this by modifying the collagen through conjugation with azide groups, which are subsequently crosslinked with a multi-armed polyethylene glycol small molecule modified with strained alkynes. This bioconjugation reaction can be performed either on solubilized collagen protein or on pre-assembled collagen fibers. We hypothesized that by changing the phase in which the collagen was modified, we could tune the resulting SPAAC network to be either amorphous or fibrous. Using second harmonic generation imaging, we found that blending unmodified collagen with azide-modified collagen produces gels that are mechanically stronger without altering the characteristics of the fibrillar network, including fibril length and mesh size. Furthermore, by encapsulating human corneal mesenchymal stromal cells (cMSCs) into the collagen hydrogels, we found that these blends demonstrate improved resistance against cellular contraction, while still permitting cells to spread and elongate. Blends with an amorphous SPAAC-crosslinked network around the collagen fibrils are exceptionally resistant to deformation and enzymatic degradation, whereas those containing collagen with fibrillar SPAAC crosslinking allow for more rapid cell spreading. Thus, our system provides a collagen hydrogel that can be fine-tuned to achieve structural and cell-responsive properties appropriate to distinct tissue engineering applications.