Skin substitutes are one of the main treatments for skin loss, and a skin substitute that is readily available would be the best treatment option. However, most cell-based skin substitutes require long production times, and therefore, patients endure long waiting times. The proteins secreted from the cells and tissues play vital roles in promoting wound healing. Thus, we aimed to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts from human skin samples were cultured using serum-free keratinocyte-specific media (KM1 or KM2) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. The acellular 3D skin patch was soft, semi-solid, and translucent. Collagen mixed with DFCM-KM1 and DFCM-KM2 showed higher protein release compared to collagen plus DFCM-FM. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce an immune response. The implantation of the 3D skin patch with or without DFCM on the dorsum of BALB/c mice demonstrated a significantly faster healing rate compared to the no-treatment group 7 days after implantation, and all groups had complete re-epithelialization at day 17. Histological analysis confirmed the structure and integrity of the regenerated skin, with positive expression of cytokeratin 14 and type I collagen in the epidermal and dermal layer, respectively. These findings highlight the possibility of using fibroblast secretory factors together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full-thickness skin loss.
The management of chronic nonhealing ulcers pose a great challenge because they are associated with morbidity and increased costs. This report presents the observations of standard management along with application of modified collagen with glycerin (MCG) in the periwound area for management of nonhealing wounds. This observational report included 50 patients (33 male, 17 female) aged 24 to 94 years having nonhealing wounds. All wounds were treated using standard treatment protocols (TIME concept), whereas the periwound severity was assessed using the Harikrishna Periwound Skin Classification (HPSC). All patients received once-daily application of MCG lotion directly in the periwound areas and compression bandaging until there was complete wound healing. Patient compliance was ensured by regular follow-up and counseling. All diabetic patients were counseled to ensure glycemic control during the entire follow-up period. The criteria used for wound healing were based on clinical observation, and proper epithelialization of the wound was the end point. The median age of the wounds was 12.0 weeks (95% CI = 8.00 - 58.08). Majority of the non-healing wounds were diabetic foot ulcers with age of wound between 4 weeks to 15 years. The median time to complete wound healing was 12.71 (95% CI = 10.00-16.67) weeks. Standard treatment protocol of TIME principle with periwound area assessment based on HPSC 2015 and treatment accordingly with topical application of MCG along with additional measures has shown complete healing of nonhealing wounds. However, further large-scale comparative studies are needed to substantiate these effects on a larger population.