Ceragenin-Loaded Tri-Layered Skin Substitute Composed of Natural and Synthetic Biopolymers for Burn Wound Healing

Burn wounds present significant clinical challenges due to high infection risk, delayed healing, and extensive tissue damage. The development of biomimetic skin substitutes capable of simultaneously supporting tissue regeneration and preventing infection remains a critical need in burn wound management. In this study, a novel ceragenin (CSA-44)-loaded tri-layered skin substitute was developed to mimic the epidermis, dermis, and hypodermis and address both wound healing and infection prevention simultaneously. The substitute comprised a poly(ε-caprolactone) (PCL) film as the upper layer, polyvinyl alcohol (PVA)/sodium alginate (SA)-PCL nanofibers as the middle layer, and a CSA-44-loaded PVA/Gelatin (Gel) hydrogel as the bottom layer. The tri-layered scaffold exhibited a hierarchical porous architecture, high swelling capacity (557.75% ± 52.87%), controlled degradation behavior, and a water vapor transmission rate of 2514.92 ± 63.41 g/m²/day, indicating suitability for maintaining a moist wound environment. Drug release studies demonstrated controlled CSA-44 delivery, with 92.26% ± 6.90% cumulative release after 180 min. The scaffold exhibited strong antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA), with complete inhibition observed within minutes. Cytocompatibility studies using human keratinocyte (HaCaT) cells demonstrated that the ceragenin-loaded scaffold maintained acceptable cell viability within the tested concentration range. In addition, co-culture experiments with HaCaT and human umbilical vein endothelial (HUVEC) cells revealed enhanced endothelial tube formation, suggesting a favorable microenvironment for angiogenic signaling. The results suggested that the ceragenin-loaded tri-layered skin substitute holds promise as a multifunctional biomaterial for burn wound management by combining antimicrobial efficacy and tissue regeneration capability.