Dual-layer macromolecular scaffold enabling oxygen, nitric oxide, and small extracellular vesicle delivery for...

Delayed and impaired wound healing remains a significant clinical challenge, often associated with hypoxia, excessive oxidative stress, insufficient angiogenesis, and impaired tissue repair. Current therapeutic approaches are often inadequate to address these complex and interrelated factors. In the present study, a dual-layer macromolecular scaffold incorporating small extracellular vesicles (sEVs), calcium peroxide (CP), and nitric oxide (NO) (GC-CP-NO-sEV) was developed using solvent casting and cryogelation techniques. The upper layer was designed to provide a controlled microenvironment through sustained release of oxygen and nitric oxide, while the lower layer functioned as a reservoir for sEVs to facilitate cellular infiltration and tissue regeneration. The physicochemical properties of the scaffold, including biodegradability, swelling behavior, and release kinetics, were systematically evaluated. Biocompatibility and proliferative effects were assessed using NIH/3T3 fibroblasts and HaCaT keratinocytes. Furthermore, in vivo wound-healing efficacy was investigated using a full-thickness excisional wound model in rats. The fabricated scaffold exhibited desirable biodegradability and generated non-cytotoxic degradation products. Sustained and controlled release of oxygen, nitric oxide, and sEVs was observed over an 8-day period. In vitro studies demonstrated excellent cytocompatibility and enhanced cellular proliferation. In vivo results revealed that the GC-CP-NO-sEV scaffold significantly improved wound closure and was associated with enhanced collagen deposition, improved tissue organization, elevated expression of wound-healing associated genes, and histological features consistent with advanced tissue remodeling. Collectively, these findings suggest that the dual-layer GC-CP-NO-sEV scaffold provides a multifunctional wound-healing microenvironment that supports multiple aspects of the repair process and represents a promising platform for wound management. However, because individual-component control groups were not included, the specific contributions of the scaffold, oxygen, NO, and sEVs to the observed effects could not be independently determined.