الملخص
Objective To investigate the feasibility of urethral reconstruction by using stretched electrospun silk fibroin matrices.Methods Stretched electrospun silk fibroin matrix was prepared,and the structure of the material was assessed by electron microscopy.Canine urothelial cells were isolated,expanded and seeded onto the material for 1 week to obtain a tissue-engineered graft.The tissue-engineered graft was assessed using HE staining and electron microscopy scanning.A dorsal urethral mucosa defect was created in 9 female beagle dogs.In the experimental group,tissue-engineered mucosa was used to repair urethral mucosa defects in 6 dogs.No substitute was used in the 3 dogs of the control group.Retrograde urethrography was performed at 1,2 and 6 months after grafting.The urethral grafts were analyzed grossly and histologically.Results Electron microscopy scanning revealed that the material had a 3 dimensional porous structure.Urothelial cells grew on the material and showed good biocompatibility with the stretched silk fibroin matrices.Canines implanted with tissue-engineered mucosa voided without difficulty.Retrograde urethrography revealed no signs of stricture,and histological staining showed gradual epithelial cell development and stratified epithelial layers at 1,2 and 6 months.The canines in the control group showed difficulty in voiding.Retrograde urethrography showed urethral stricture,and histological staining showed that no or only one layer of epithelial cells developed.A severe inflammatory reaction was also observed in the control group.Conclusion Stretched electrospun silk fibroin matrices have good biocompatibility with urothelial cells,and could be a potential material for urethral reconstruction.
الملخص
An in-vitro system for the cultivation of urothelial cells has been developed. Urothelial cells were isolated from 30 tissue samples obtained from the urinary bladders of six adult New Zealand rabbits that underwent partial cystectomy. The cells from 15 samples were grown in M-199 culture media and that from the remaining 15 samples were grown over human placental membranes measuring 3 x 4 x 0.1 centimeter (cm) contained in the same media. Confluent cell monolayers covering the entire base of the plastic multiwells and the entire amniotic membranes were produced within 7 days respectively in all cases. Subsequent subcultures likewise achieved the same confluency within another 7 days. Morphological analysis of all cultures by inverted phase contrast microscopy revealed cells of epithelioid nature. Cells from the media were likewise concentrated by centrifugation and smeared over glass slides, heat-dried and fixed with absolute ethanol. The smears underwent Giemsa staining and were examined under light microscopy and confirmed the presence of urothelial cells. The cultured urothelial graft developed from this study is a potentially useful material for future in-vivo researches involving genito-urinary tract reconstructive surgeries.