(326d) Mimicking Biopolymer Structure in Synthetic Hydrogels for Model Extracellular Matrices

The native extracellular matrix (ECM) is composed of hierarchically structured biopolymers containing precise monomer sequences and chain shapes with high molecular rigidity (e.g., persistence lengths) to yield stiff hydrogels at relatively low polymer contents. This molecular rigidity is typically absent in synthetic hydrogel systems, which rely instead on increasing polymer content or crosslinking density to control bulk mechanics. To better mimic the hierarchical order of biopolymer networks in an artificial ECM, we incorporated crosslinkers with different persistence lengths into a synthetic hydrogel. Specifically, we developed a synthetic hydrogel system using commercially available hyaluronic acid (HA) macromers with non-natural, sequence-defined poly(N-substituted glycines) (peptoids) as crosslinkers. Peptoids are of particular interest due to their ability to form polyproline-type helices upon incorporation of bulky, chiral monomers into their sequence. Since these helical peptoids have been shown to have greater persistence lengths, we synthesized three peptoid sequences for study: one helical, one non-helical but chemically similar, and one entirely unstructured.

Varying the peptoid crosslinker structure enabled control over bulk hydrogel mechanics, with the more rigid crosslinkers leading to an increase in hydrogel elasticity. Additionally, we found that crosslinker structure dictated the scaling behavior of mechanical properties with crosslinker length: increasing helical crosslinker length further increased elasticity, while increasing length of unstructured crosslinkers led to a decrease in elasticity. Leveraging the ability to decouple elasticity from network connectivity, we next evaluated each hydrogel as a substrate for human mesenchymal stromal cell (hMSC) culture and found that all crosslinker formulations resulted in high cell viability. Furthermore, the cells spread more on stiffer substrates, with the most molecularly rigid substrates leading to cell areas and morphologies similar to those cultured on tissue culture plastic. Lastly, we determined how each hydrogel affected Indoleamine 2,3-dioxygenase (IDO) production, which is a measure of the immunosuppressive capacity of the hMSCs. Excitingly, we found that softer hydrogels upregulated IDO production. These results demonstrate the potential utility of this hydrogel platform in hMSC culture, where mechanics and network connectivity can be decoupled using crosslinker structure. Further studies investigating the impact of molecular rigidity on cell-matrix interactions will facilitate the design of more biomimetic artificial ECMs.