Hyaluronic acid-based hydrogels with independently tunable mechanical and bioactive signaling features.

Extracellular matrix provides critical signaling context to resident cells through mechanical and bioactive properties. To realize the potential of tissue engineering and regenerative medicine, biomaterials should allow for the independent control of these features. This study investigates a hydrogel system based on thiol-modified hyaluronic acid (HA-S) and polyethylene glycol diacrylate (PEGDA). The mechanical properties of HAS-PEGDA are dictated by two cytocompatible crosslinking reactions that occur at distinct time points: a rapid, Michael-type nucleophilic addition reaction between HA-thiols and PEG-acrylates and a prolonged maturation of disulfide crosslinks from remaining thiols. It is hypothesized that these reactions would enable the independent tuning of the mechanical and bioactive features of HAS-PEGDA. Rheological studies confirmed that initial gelation reached completion by 1 day, at which point the shear modulus was proportional to the concentration of PEGDA. Over time, the shear modulus evolved dramatically, and final stiffness depended on the availability of HA-thiols. The addition of PEG-monoacrylate (PEGMA) after the initial gelation occupied a percentage of remaining thiols to prevent disulfide crosslinking, decreasing the steady-state stiffness in a dose-dependent manner. A fraction of the PEGMA was then replaced with acrylated peptide ligands to introduce specific bioactivity to the otherwise non-cell-adhesive network. The degree of latent stiffening was controlled by the total amount of peptide-PEGMA, while adhesivity was tuned with the balance of bioactive and inactive peptides. The functional effects of the tunable mechanical and bioadhesive ligand properties were confirmed with assays of cell adhesion and morphology.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers-Danlos syndrome-associated substitutions.

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2ß1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2ß1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2ß1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.