Bioengineered skin constructs based on mesenchymal stromal cells and acellular dermal matrix exposed to inflammatory microenvironment releasing growth factors involved in skin repair
Authors: Correa-Araujo Luz , Prieto-Abello Leonardo , Lara-Bertrand Adriana , Medina-Solano Martha , Guerrero Linda , Camacho Bernardo , Silva-Cote Ingrid 
Published: October 2023

Abstract
Background
Skin tissue engineering is a rapidly evolving field of research that effectively combines stem cells and biological scaffolds to replace damaged tissues. Human Wharton’s jelly mesenchymal stromal cells (hWJ-MSCs) are essential to generate tissue constructs, due to their potent immunomodulatory effects and release of paracrine factors for tissue repair. Here, we investigated whether hWJ-MSC grown on human acellular dermal matrix (hADM) scaffolds and exposed to a proinflammatory environment maintain their ability to produce in vitro growth factors involved in skin injury repair and promote in vivo wound healing.

Methods
We developed a novel method involving physicochemical and enzymatic treatment of cadaveric human skin to obtain hADM scaffold. Subsequently, skin bioengineered constructs were generated by seeding hWJ-MSCs on the hADM scaffold (construct 1) and coating it with human platelet lysate clot (hPL) (construct 2). Either construct 1 or 2 were then incubated with proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α) for 12, 24, 48, 72 and 96 h. Supernatants from treated and untreated constructs and hWJ-MSCs on tissue culture plate (TCP) were collected, and concentration of the following growth factors, bFGF, EGF, HGF, PDGF, VEGF and Angiopoietin-I, was determined by immunoassay. We also asked whether hWJ-MSCs in the construct 1 have potential toward epithelial differentiation after being cultured in an epithelial induction stimulus using an air–liquid system. Immunostaining was used to analyze the synthesis of epithelial markers such as filaggrin, involucrin, plakoglobin and the mesenchymal marker vimentin. Finally, we evaluated the in vivo potential of hADM and construct 1 in a porcine full-thickness excisional wound model.

Results
We obtained and characterized the hADM and confirmed the viability of hWJ-MSCs on the scaffold. In both constructs without proinflammatory treatment, we reported high bFGF production. In contrast, the levels of other growth factors were similar to the control (hWJ-MSC/TCP) with or without proinflammatory treatment. Except for PDGF in the stimulated group. These results indicated that the hADM scaffold maintained or enhanced the production of these bioactive molecules by hWJ-MSCs. On the other hand, increased expression of filaggrin, involucrin, and plakoglobin and decreased expression of vimentin were observed in constructs cultured in an air–liquid system. In vivo experiments demonstrated the potential of both hADM and hADM/hWJ-MSCs constructs to repair skin wounds with the formation of stratified epithelium, basement membrane and dermal papillae, improving the appearance of the repaired tissue.

Conclusions
hADM is viable to fabricate a tissue construct with hWJ-MSCs able to promote the in vitro synthesis of growth factors and differentiation of these cells toward epithelial lineage, as well as, promote in a full-thickness skin injury the new tissue formation. These results indicate that hADM 3D architecture and its natural composition improved or maintained the cell function supporting the potential therapeutic use of this matrix or the construct for wound repair and providing an effective tissue engineering strategy for skin repair.