Off-the-shelf acellular fetal skin scaffold as a novel alternative to buccal mucosa graft: the development and characterization of human tissue-engineered fetal matrix in rabbit model of hypospadiasis.

Abstract

In this study, we aimed to develop a novel alternative to buccal mucosal graft from the acellular human fetal skin to manage hypospadias in a rabbit model. We optimized the decellularization protocol to develop and characterize the human tissue-engineered fetal dermal matrix as an "off-the-shelf" natural biomaterial. Human fetal skin was obtained at 16-19weeks gestational age with respect to a signed informed consent from parents under the university ethical committee approval. The dissected full-thickness fetal skin tissues were placed into SDS and Triton X-100 in different dosages to achieve the optimum decellularization protocol. Histopathology of the acellular fetal matrix was assessed by Hematoxylin & Eosin (H&E) and DAPI staining to confirm the removal of all cell materials, Masson's trichrome staining for collagen evaluation, DNA quantification for confirmation of DNA content, and scanning electron microscopy (SEM) for evaluation of scaffold microstructure. Immunohistochemistry (IHC) staining was used to detect specific dermal markers, namely vimentin, type I collagen, cytokeratin (CK)19. The prepared dermal scaffolds were then grafted on the 8 rabbit models of hypospadias. The rabbits underwent evaluations at 1, 2, 3, and 6months postoperatively. H&E, Masson's trichrome, DAPI staining, and SEM confirmed the significant removal of cells; meanwhile, the ECM was completely preserved. At the time of biopsy, after 2, 4, and 6months, no evidence of inflammation, fibrosis, necrosis, or rejection was observed. The grafted dermal scaffolds appeared histologically and anatomically normal. It was observed that the scaffolds were recellularized by circulating CD 34 + bone marrow stem cells (BMSCs) inside the body, implicating the body as a natural bioreactor. The application of acellular fetal skin (AFS) is a safe and feasible method that can decrease surgical time in a complex hypospadias reconstruction. Moreover, AFS demonstrated excellent angiogenesis characteristics and migration of the stem cells to the scaffold observed during the course of treatment. Novel natural AFS scaffold without cell seeding is an excellent alternative to buccal mucosal graft; hence, it can overcome the limitations concerning the graft size and prevent the creation of wounds in oral mucosal tissue.