Modulation of Secondary Structure, Bioavailability, Immunomodulation, and Tissue Repair Outcomes Using Differential Processing of Silk Biomaterials.

The ability to modulate structure, physicochemical properties, and function makes naturally derived polypeptides attractive biomaterials for human health. Silk fibroin protein, derived from silkworm cocoons, has been explored in tissue engineering, wound healing, and drug delivery. Despite these advances, the influence of silk polypeptide processing on secondary structure, bioavailability, tissue responses, and healing outcomes is poorly understood. Here, we fabricated silk fibroin-indocyanine green (ICG) dye films and modulated their properties using different processing conditions, including laser-induced photothermal or methanol solvent treatments. Solid-state nuclear magnetic resonance (ssNMR), Fourier transform infrared (FT-IR) spectroscopy, and wide-angle X-ray scattering (WAXS) were employed to elucidate the structural attributes of these films. Secondary structure and dissolution-facilitated bioavailability of these differentially processed silk-ICG films were correlated with their immunomodulation and tissue response activities using a full-thickness wound model in immunocompetent mice. Although treatment with dissolution-resistant, β-sheet-rich, methanol-treated films accelerated early wound closure compared to saline-treated control mice, treatment with rapidly soluble, lower β-sheet-containing "as-prepared" films increased the deposition of granulation tissue in mice over time. As-prepared silk films exhibited an elevated immune response, characterized by the increased presence of neutrophils and pro-inflammatory and pro-repair macrophages in the wound compared to the less soluble silk films, potentially due to the ready bioavailability of silk in the tissue microenvironment. Our results indicate that processing methods influence secondary structure and dissolution-facilitated bioavailability of silk. It, in turn, determines immunomodulation activity and wound closure efficacy, thereby advancing our understanding and design of biomaterial-facilitated tissue repair.