One-Step Tunable Human Hair Keratin Gradient Hydrogel with Antibacterial Activity for Tissue Engineering

Gradient hydrogels are an emerging strategy in tissue engineering to mimic the heterogeneity in native tissues. Herein, the metal-thiolate complexation mechanism is exploited by allowing diffusion of silver ions (Ag⁺) through a cysteine-rich human hair keratin (HHK) solution to produce a novel gradient hydrogel. This one-step approach enables the combined benefits of a sustainable material with an established antimicrobial agent. Herein, the gelation kinetics, physical, mechanical, and biochemical properties of the gradient hydrogel are correlated over a range of thiol:Ag ratios. The presence of a porosity gradient within a single construct, along with dissimilar top and bottom surface morphologies, is shown. Disk diffusion tests against Staphylococcus aureus verified the antibacterial activity of this Ag-loaded hydrogel. Additionally, hydrogels at 1.25 thiol:Ag ratio supported > 95% viability and proliferation of human dermal fibroblasts (HDFs), comparable to collagen hydrogels. These HDFs produce fibronectin, collagen III, and express alpha-smooth muscle actin within the gradient hydrogels. In an in vivo full-thickness wound healing mouse model, the 1.25 hydrogel evoke minimal host tissue response, support reepithelialization more than the uniform Gelatin Methacrylate (GelMA) hydrogel, and promote the most collagen deposition. These findings demonstrate the practicality of metal-thiolate complexation in producing biomimetic gradient hydrogels for tissue regeneration.