Click chemistry reactions have become an important synthetic tool for fabrication of user-defined hydrogels consisting of poly(ethylene glycol) (PEG) and bioactive peptides/proteins for tissue engineering. In this work, novel approaches were developed for the creation of next-generation, bio-instructive PEG-based hydrogels using various sequential click reactions. The sequential click paradigm utilizes stoichiometric control of complimentary reactions, each leveraged for their own individual advantages, towards the goal of creating bio-instructive hydrogel platforms for stem cell and therapeutic protein delivery. Firstly, thiol-ene and tetrazine click reactions were explored. PEG microparticles were prepared off-stoichiometry using a photo-initiated thiol-ene click reaction resulting in microparticles with free norbornene groups. Subsequently, bioactive proteins alkaline phosphatase and glucose oxidase were functionalized with tetrazine groups for bioorthogonal conjugation to norbornene-bearing microparticles. This sequential click strategy allowed for scalable microparticle fabrication along with facile and dose-dependent protein conjugation without compromising bioactivity Next, microporous annealed particle (MAP) hydrogels, a new class of hydrogels fabricated from crosslinking hydrogel microparticles together, was investigated. Here, the sequential click platform was leveraged by assembling protein-functionalized microparticles into MAP hydrogels using a secondary tetrazine click reaction with a PEGdi-tetrazine crosslinker. This novel MAP hydrogel for tissue engineering was mechanically characterized, assessed for cellular compatibility, and demonstrated to be highly versatile by creating user-defined patterns with bioactive protein-functionalized microparticles. Next, protein modification strategies, specifically stochastic amine functionalization and site-selective functionalization, were compared to improve bioactivity of tethered growth factor bone morphogenetic protein-2 (BMP2). A bis-sulfone reagent, tetrazine-PEG-bis-sulfone, was successfully used to modify recombinant BMP2 with tetrazine groups selectively at C-terminal histidine-tags, which was further conjugated to MAP hydrogels. The bis-sulfone modification strategy resulted in tetrazineBMP2 functionalized MAP hydrogels that were more osteoinductive compared to all other MAP hydrogel counterparts and negative controls. This approach is promising for the delivery of sensitive, therapeutic proteins and can be extended to other biomaterial platforms. Lastly, the versatility of the sequential click approach was extended to other click reactions in order to overcome synthetic challenges associated with macromer-based hydrogel formation. Briefly, a thiol-maleimide Michael addition reaction was used to make PEG-peptide block co-polymers from short, linear components and subsequently assembled into hydrogels using a thiol-ene click reaction. This highly customizable platform was also coupled with a complimentary tetrazine click reaction for protein patterning. By leveraging the reactivity of norbornene, the utility of a tri-click system was demonstrated, thus opening the door to explore other unique combinations for creating user-defined hydrogel platforms.
