A Novel Click-by-Click Preparation Strategy of In Situ Porous Hydrogels to Spatially Control Both Physical and Biochemical Signals
The development of advanced functional biomaterials to interact with biological systems has a wide range of biomedical applications, from the delivery of bioactive molecules and cell adhesion mediators to mimicking living tissue to treat diseases. Integration of heterogenous cues within biomaterials can be used to stimulate tissue regeneration by recapturing the complexity of native tissue to direct cellular actions. Biomaterial scaffolds can be used as structural constructs, providing the cells with mechanical support, and as depots, providing cells with the biochemical signals necessary to stimulate growth and guide tissue regeneration. The presentation of physical and biochemical cues within the body is diverse and heterogeneous, and controlling the topography, mechanical strength, and the bound versus soluble presentation of growth factors within an engineered scaffold is key to further our understanding of how native tissue directs cellular processes in either the diseased or healthy states. However, hydrogel scaffolds where the physical or chemical cues cannot be controlled are still used and engineered hydrogel platforms where these cues can be controlled have lost their injectability, rendering them unsuitable for some in vivo biomedical applications. A novel bottom-up approach to synthesizing nanoporous and macroporous hydrogel biomaterials to control physical and biochemical signals homogenously or heterogeneously while maintaining injectability was developed. First, a strategy to control the presentation of physical and biochemical cues was developed. In this approach, the selection of the polymeric backbone and how they were functionalized with reactive moieties was key to controlling the microstructure, mechanics, and protein presentation of the hydrogel scaffold. Although tuning the reaction speed of the thiol-maleimide Michael-type addition reaction altered the homogeneity of the hydrogel microstructure and thereby cell spreading, pivoting to the thiol-norbornene radially mediated step growth reaction allowed for the necessary spatiotemporal control of these properties. The incorporation of heparin into the hyaluronic acid hydrogel platform allowed for the tuning of the bound and soluble protein presentation, release profile, and cellular response. Next, a strategy to control the physical and biochemical cues in an injectable hydrogel platform was developed by implementing the concept of annealing microgels in situ. Utilizing the inverse electron demand Diels-Alder tetrazine-norbornene click reaction to functionalize the microgels post-fabrication and anneal the microgels led to a highly versatile injectable platform were the bulk scaffold’s stiffness, strength and biochemical composition could be independently tuned from its building blocks. Finally, by controlling how the microgels were loaded into a syringe, the presentation of microgels were successfully layered in a single injection, in vitro and in vivo. Transitioning to heterogeneous biomaterials as a synthetic mimic to native tissue will allow us to evaluate how the presentation of cellular cues impacts tissue regeneration and ultimately improve the design approach.