Control of hMSC Behavior and Hydrogel Mechanical Properties Via Crosslinker Structure

Synthetic hydrogels have garnered interest as extracellular matrix (ECM) mimics, yet still do not fully replicate the hierarchical nature of biological tissue, limiting their biological efficacy as stem cell culture scaffolds for tissue engineering and regenerative medicine. The native ECM is made up of hierarchical biopolymers with precise sequences and chain structures that yield high molecular rigidity. These structures result in stiff microenvironments at low polymer concentrations. Synthetic hydrogels, typically utilize changes in polymer concentration or crosslinking stoichiometry to modulate mechanics with flexible, disordered polymers, coupling hydrogel parameters. To decouple parameters like stiffness and network connectivity, we have developed two synthetic hydrogel systems using naturally-derived hyaluronic acid (HA) and polyethylene glycol (PEG) with non-natural poly(N-substituted glycines) (peptoids) as crosslinkers. Peptoids are biomimetic molecules with a large chemical diversity, tunable persistence length, and high degree of structural control, enabling hierarchical order in engineered materials. The PEG based network will allow for better comparison to existing hydrogel systems.

Three different peptoid sequences were synthesized for use as crosslinkers: 1) a chiral, helix inducing sequence (H), 2) an analog designed to disrupt helix formation (N), and 3) an unstructured peptoid (U). A peptide control was also synthesized (P). Crosslinking structure was found to have a marked effect on resulting hydrogel storage modulus, with the helical crosslinker resulting in the stiffest hydrogels. The non-helical peptoid and peptide crosslinker possess chirality, but are not helical, resulting in intermediate moduli values. Lastly, the unstructured peptoid, with no chirality, formed the softest. This trend is representative of the relaxed state modulus, but seeded cells experience the substrates in the swollen state. To probe this swollen modulus, nanoindentation was performed on each hydrogel, confirming the trend in stiffness holds in the swollen state. Upon seeding hMSCs, similar viability was observed on each substrate by MTT assay after one day of culture, when normalized to the peptide crosslinked control. Cell proliferation of hMSCs was measured via EdU. Softer substrates significantly increased proliferation after three days of culture. This was surprising as previous studies indicated the opposite trend.

In conclusion, hydrogel systems were developed in which the parameters of stiffness and network connectivity could be decoupled via the alteration of crosslinker secondary structure. It was determined that these hydrogel substrates are viable cell culture platforms for hMSCs and that crosslinker structure had a noticeable impact on cell behavior. This system provides more avenues to achieve mimicry of the natural ECM.