Silk Fibroin Scaffolds In Experimental Study Of Tissue Engineered Urethra Reconstruction

Long anterior urethral stricture often require urethra reconstructions thatincorporate substitutive materials to augment the stenotic segment. The typeof substitutive material used for the repair of urethral strictures remainsone of the most challenging problems of this procedure. At present, variousautologous tissues have been proposed for urethra reconstruction. However,harvesting these grafts can potentially cause donor site morbidity and canprolong the operation. Tissue engineering technology could address thesecomplications and limitations. This study firstly used the new type ofstretched electrospun silk protein materials as scaffolds to construct tissueengineering mucosa, repair canine urinary tract mucosa. The study assessedmucosa tissue engineering in the application of urethral reconstruction.Part1Preparation and detection of the stretched electrospun silk protein matricesObjection: We prepared stretched electrospun silk fibroin scaffold materialsfor urethral reconstruction of the of tissue engineering, and detected thestructure and mechanical strength of the scaffold. Methods and materials: Natural cocoons of Bombyx mori were degummed twicewith a0.5wt%Na2CO3aqueous solution at100°C for30min and rinsed withdeionized water to remove sericin. The degummed silk was dissolved in a9.0M LiBr aqueous solution. After being diluted, The SF aqueous solution wasconcentrated to33wt%in forced airflow. The concentration of the solutionwas determined using the weight method. Briefly, a prepared SF aqueoussolution was transferred to a2.5ml syringe capped with a6-G needle (ID=0.6mm) as a spinneret. The electrospinning was performed using a voltageof25kV, a flow rate of0.3ml/h, and grounded aluminum foil placed at adistance of9cm to collect random fibers. To improve the mechanicalproperties, the electrospun matrices were mechanically stretched in90vol%ethanol aqueous solutions at a stretch rate of0.1mm/s and stretch ratioof1.4×. The matrices were then immersed in the same90vol%ethanol solutionfor30min with a fixed length. The morphology of electrospun SF fibers wasobserved using a JSM-5600LV (JEOL Co, Japan) scanning electron microscope(SEM) at25°C and60%RH. The pore sizes of the matrices of each samplewere measured at five different locations using the KS400Image AnalysisSystem.Results： The SEM figure showed that the matrices formed a highlyinterconnected and porous structure. The size of the observed pores was40±5 μm. The breaking strength and breaking energy of the post-treated mats at1.4×draw ratio and0.1mm/s draw rate were8.6MPa and172.2J/kg,respectively. However, those of the as-spun mats were only1.8MPa and93.2J/kg, respectively.Conclutions: Stretched electrospun silk fibroin matrices were3D porousstructure, and its spatial structure and mechanical strength in line withurethral reconstruction repair needs.Part2Evaluation of stretched electrospun silk fibroin matrices seeded withurothelial cells for urethra reconstructionObjective: We investigated the feasibility of urethral reconstruction usingstretched electrospun silk fibroin matrices with urothelial cells.Materials and Methods: A novel electrospun silk fibroin matrix was prepared.Canine urothelial cells were isolated, expanded and seeded onto the materialfor1week to obtain a tissue-engineered graft. The tissue-engineered graftwas assessed using HE staining and scanning electron microscopy. A dorsalurethral mucosal defect was created in9female beagle dogs, In theexperimental group, tissue-engineered mucosa was used to repair urethramucosa defects in6dogs. No substitute was used in the3dogs of the controlgroup. Retrograde urethrography was performed at1,2and6months after grafting. The urethral grafts were analyzed grossly and histologically.Results: Urothelial cells grew on the material and showed goodbiocompatibility with the stretched silk fibroin matrices. Canines implantedwith tissue-engineered mucosa voided without difficulty. Retrogradeurethrography revealed no signs of stricture. Histological staining showedgradual epithelial cell development and stratified epithelial layers at1,2and6months. The canines in the control group showed difficulty in voiding.Retrograde urethrography showed urethra stricture. Histological stainingshowed that no or only one layer of epithelial cells developed. A severeinflammatory reaction was also observed in the control group.Conclusion: Stretched electrospun silk fibroin matrices have goodbiocompatibility with urothelial cells, which could prove to be a potentialmaterial for use in urethra reconstruction.Part3Tissue-engineered buccal mucosa using silk fibroin matrices for urethralreconstructionBackground: To investigated the feasibility of urethral reconstruction usingtissue engineered buccal mucosa with silk fibroin matrices in a canine model.Materials and Methods: Autologous oral keratinocytes and autologous fibroblasts were isolated, expanded and seeded onto silk fibroin matricesto obtain tissue engineered buccal mucosa. The tissue engineered buccalmucosa was assessed using hemotoxylin and eosin staining, and scanningelectron microscopy. A5cm urethral mucosal defect was created in10femalecanines. Urethroplasty was performed using tissue engineered buccal mucosain5canines in the experimental group. In control group, Urethroplasty wasperformed with silk fibroin matrices without cells in the other5canines.Retrograde urethrography was performed after6months grafting. The urethralgrafts were analyzed grossly and histologically.Results: Oral keratinocytes and fibroblasts had good biocompatibility withsilk fibroin matrices. Tissue engineered buccal mucosa could be constructedusing silk fibroin matrices. Canines implanted with tissue engineered mucosavoided without difficulty. Retrograde urethrography revealed no sign ofstricture. Histological staining performed that epithelial cell developedgradually, and showed stratified epithelial layers at6months. In controlgroup, canines voided difficultly. Retrograde urethrography showed urethrastricture. Histological staining showed that1-2layer epithelial cellsdeveloped.Conclusions: Tissue engineered buccal mucosa using silk fibroin matricescould be a potential material for urethra reconstruction.