Acellular 3D skin patch fortified with dermal fibroblast conditioned medium (DFCM) for immediate treatment of skin loss

Abstract

Skin substitutes is one of the prime treatment for skin loss. Large area of skin loss causes high mortality as well as morbidity. Having a skin substitute that is readily available would be the best treatment. However, majority of cell-based skin substitutes requires long production time, therefore long waiting time for patients. Secreted proteins from cells and tissues play vital roles in promoting wound healing. Ultimately, the aim was to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Dermal fibroblast provides a more vital role in wound healing, therefore the proteins secreted by fibroblasts was evaluated on its effect in in vitro and in vivo re epithelialisation. Fibroblasts isolated from human skin samples were cultured in vitro using the serum-free keratinocyte-specific medium (Epilife, KM1, or Define keratinocytes serum-free medium (DKSFM), KM2) and serum-free fibroblast-specific medium (FM) to collect DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. Mass spectrometry analysis identified 59, 46 and 58 secreted proteins in DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. Supplementation of DFCM-KM1 and DFCM-KM2 at a concentration of 200-400g/mL was shown to enhance keratinocytes attachment, proliferation, and migration at sub-confluence state. In contrast, supplementation of DFCM-FM at a concentration of 200-1600g/mL significantly improved keratinocytes wound healing rate, while 400-800g/mL were suitable for attachment and proliferation. Keratinocytes treated with different types of DFCM also expressed specific genes that are involved in wound healing process with 23 genes having higher fold regulation compared to control. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce any immune response and safe to be used allogenically. Implantation of a 3D skin patch, collagen hydrogel with or without DFCM on the dorsal of BALB/c mice with full thickness wound, demonstrated significantly faster healing rate compared to no treatment group after 7 days of implantation, and complete re-epithelialisation observed at day 17 in all groups. The histological analysis confirmed the structure and integrity of the regenerated skin with positive expression of CK14 in epidermal layer and collagen type I in the dermal layer. These findings highlighted the possibility of using fibroblast secretory factors together with collagen hydrogel as an acellular 3D skin patch allogeneically for immediate treatment of full thickness skin loss.