Composite PU/SF hybrid tubular scaffolds for fabrication of engineered neovagina in-vivo explants in a 3D-culture perfusion bioreactor

Background: Vaginal reconstruction is the choice medical approach in many congenital abnormalities, injuries, or cancers. Reconstructive techniques applying non-vaginal tissues can be associated with more complications. In special situations such as dilatation failure or large defects, surgical vaginoplasty is recommended. In this regard the main challenge is the lack of sufficient native tissue to reconstruct the organ.Objective: Our aim is development of a tubular electrospun hybrid scaffold to fabricate engineered neovagina for these patients.Materials and Methods: In this study, hybrid fibrous scaffolds of elastomeric polyurethane (PU) and silk fibroin (SF) with various mass ratios were fabricated by electrospinning. We expanded primary cultured human vaginal epithelial cells (HVECs) onto these scaffolds at a density of 1×105 cells/ml. Chemical and physical properties of scaffolds were evaluated using scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), contact angle measurement, biodegradation test and tensile strength analysis. The toxicity and biocompatibility of each scaffold was evaluated by the MTT assay using HVECs. Cell homing and proliferation was evaluated on the scaffolds by SEM and Hematoxylin & Eosin staining. Epithelial origin of the cell-seeded scaffolds characterized with RT-PCR analyses. PU/SF hybrid scaffolds were optimized to obtain the best imitator of normal vagina. Tubular structures of the optimized PU/SF nanofiber was fabricated by electrospinning with a novel designed rotating collector. The cell-seeded scaffolds were placed in the designed 3D perfusion bioreactor to prepare in vivo explants.Results: SEM micrographs of electrospun scaffolds showed that the mass ratio of the PU/SF hybrid highly influenced the fibers morphology due to variations in conductivity and viscosity. By increasing the SF proportion, the fibers had lower diameters and higher uniformity. Surprisingly, ultrafine nanofibers and nanowebs had been widely distributed among the usual fibers. These nanowebs effectively increased the surface area and were favorable for cell attachment and spreading. The PU/SF scaffolds significantly promoted epithelial cell homing and proliferation compared to PU scaffold alone (p<0.05). RT-PCR analysis of growing cells on the tubular scaffolds confirmed the expression of epithelial cell surface markers such as cytokeratin19. Optimized PU/SF hybrid scaffold (60/40 ratio) with the most similar mechanical characteristics to normal vagina, was applied to produce tubular scaffolds. The cell-seeded tubular scaffolds, cultured in our designed perfusion bioreactor, had the same favorite properties as earlier 2D constructs. All of the results clarified that PU/SF 60/40 tubular scaffolds meet the required specifications for vaginal tissue engineering. Conclusion: Results showed that the electrospun PU/SF 60/40 tubular scaffold possess proper biocompatibility and capability to promote vaginal tissue regeneration. 3D culture technology may be pursued further experiments in order to achieve engineered neovaginal tissues for the clinical applications.

Keywords:

PU/SF tubular scaffolds , Electrospinning , 3D culture bioreactor , Tissue engineering , Neovagina