Human acellular amniotic membrane loaded with hair follicle-derived cells promotes wound repair and appendage-like structure formation.

Background: Deep skin injuries often heal with scarring and lack skin appendage regeneration, posing a significant clinical challenge. This study aimed to develop an hAAM-based combinatorial strategy for co-delivery of human hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) to full-thickness skin wounds, and to evaluate its effects on wound repair and appendage-like structure formation. Methods: hAAM was prepared and characterized. HFSCs and DPCs were isolated from human hair follicles and identified. The composite construct (hAAM + HFSCs + DPCs) was applied to full-thickness skin wounds in nude mice. Wound healing rates were monitored. Histological and immunohistochemical analyses were performed to assess re-epithelialization, collagen deposition, the presence of CD31-positive vascular structures, cell proliferation, fibrosis-related markers, and morphological features suggestive of appendage-like structures. Results: The hAAM scaffold co-loaded with HFSCs and DPCs was associated with accelerated wound closure, tissue repair, and altered expression of fibrosis-associated markers, including TGF-β and α-SMA, together with altered collagen deposition. The treatment was also associated with the appearance of PPAR-γ-positive structures with morphological features resembling sebaceous glands, as well as epithelial bud-like structures morphologically suggestive of early follicular development, in a subset of specimens. Although these morphological observations are preliminary and require further characterization with additional lineage-specific markers, they represent preliminary morphological observations of appendage-like structures. Conclusion: The hAAM scaffold co-loaded with HFSCs and DPCs was associated with accelerated wound closure, tissue repair, and altered extracellular matrix remodeling in a murine full-thickness wound model. Structures with morphological features resembling sebaceous glands and epithelial bud-like structures suggestive of early follicular development were observed in some samples; however, their cellular identity, maturity, and functionality remain to be determined. Further lineage-specific characterization and long-term functional studies are required to establish whether this strategy can promote appendage-like structure formation and functional skin restoration.