Human-Derived Scaffold Components and Stem Cells Creating Immunocompatible Dermal Tissue Ensuing Regulated Nonfibrotic Cellular Phenotypes

Regeneration of large-sized acute and chronic wounds provoked by severe burns and diabetes is a major concern worldwide. The availability of immunocompatible matrix with a wide range of regenerative medical applications, more specifically, for nonhealing chronic wounds is an unmet clinical need. Extrapolating the in vitro tissue engineering knowledge for in vivo guided wound regeneration could be a meaningful approach. This study aimed to develop a completely human-derived and minimally immune-responsive scaffold comprising of acellular amniotic membrane (AM), fibrin (FIB) and hyaluronic acid (HA), termed AMFIBHA. The potential for in vivo guidance of skin regeneration was validated through in vitro dermal tissue assembly on the combination scaffold by growing human fibroblasts, differentiated from human adipose tissue-derived mesenchymal stem cells (hADMSCs). An effective method was standardized for obtaining decellularized amnion (dAM) for assuring better immunocompatibility. The biochemical stability of dAM upon plasma sterilization (pdAM) confirms its suitability for both in vitro and in vivo tissue engineering. The problem of poor handling characteristics was solved by combining the dried dAM with fibrin derived from a clinically used fibrin sealant kit. An additional constituent HA, derived from human umbilical cord tissue, imparts the required water absorption and retention property for better cell migration and growth. Post sterilization, the combination scaffold AMFIBHA demonstrated hemo-/cytocompatibility, confirming the absence of detergent residuals. Upon long-term (20 days/40 days) culture of hADMSC-derived fibroblasts, the suppleness of generated tissue was established by demonstrating regulated deposition of collagen, elastin, and glycosaminoglycans using both qualitative and quantitative measurements. Regulated expressions of transforming growth factors-beta 1 (TGF-beta 1) & TGF-beta 3, alpha smooth muscle actin (alpha-SMA), fibrillin-1, collagen subtypes, and elastin suggest nonfibrotic fibroblast phenotype, which could be an effect of microenvironment endowed by the AM, FIB, and HA. In burn wound model experiments, immune response to cellular AM was prominent as compared to untreated/sham control wounds and decellularized AM-treated and AMFIBHA-treated wounds, ensuring biocompatibility. Wound regeneration with complete epithelialization, angiogenesis, development of rete pegs, and other skin appendages were dearly visualized in 28 days after treating large-sized (4 x 4 cm(2)), debrided, full-thickness third-degree burn wounds, indicating guided wound regeneration potential of AMFIBHA dermal substitute.