Experimental Study On Urethral Reconstruction Using Silk Fibroin Scaffold

Objective:To explore the method of building a stable urethral stricture model in New Zealand white rabbits.Methods:Twelve male New Zealand white rabbits were randomly divided into two groups, and each group had6rabbits with the average weight of2.84±0.15kg. After intravenous anesthesia by sodiμm pentobarbital, we made models by stripping1.0cm long urethral mucosa in Group B, then suturing the incision and fixing the F8catheter. And Group A was controlled. We did microsurgery for all models by10times Optical microscope. We observed the daily micturation condition after operation for both groups. We did the antegrade urethrography, urethral pressure profile (UPP) and histiocytes at two months postoperatively. We used antibiotics by peritoneal injection to prevent infection during operation and one week after operation.Results:One rabbit of Group B appeared shock during using antibiotics after anesthesia. It died after the external cardiac massage for fifteen minutes. After adding one rabbit in group B, all rabbits of two groups had completed the test. One week later the catheter were withdrawed by itself. We found the rabbits in group B were more irritable than those in group A after two months postoperatively.(1)CYSTOURJETHROGRAPHY:We found significant urethral stricture with narrow lumen and discontinuous mucosa in Group B. There was significant difference between Group A and Group B.(2)UPP:The urethral pressure on operative site in Group A was14.67±2.16cmH2O, and27.83±3.71cmH2O in Group B. There was significant statistical difference between two groups (p<0.01).(3)HISTIOCYTES:Urothelium was well-distributed covered without any inflammatory cells in Group A, which had3-4lays of the epithelial cells. And Urothelium was inequably covered with neutrophil and lymphocyte in Group B.Conclusion:We establish the way to build a stable, homogeneous and repetitive urethral stricture model of new Zealand rabbits by microsurgical technique, which can provide the foundation of laboratory animals to research all kinds of urethral stricture. Cystourethrogrraphy combined UPP and histiocytes is the reliable method to identify the urethral stricture. Objective:To research the cultivation and the amplification of urothelium cell in vitro and the growth of urothelium cell in the scaffold of silk fibroin, and to explore the aperture of scaffold and the experimental environment in which the urothelium cell was fit to grow.Methods:(1) The mucous membrane of urethra was derived from the male New Zealand white rabbits. The primary culturing of cell in vitro was done after the rinse, digestion, standing, filtration, centrifugate, float and cultivation of cell. After the transfer of cultivation, the cell was identified by contrast phase microscope and the immunohistochemical reaction of keratin antibody.(2) The scaffold of silk fibroin was divided into three groups by the aperture. Group A was the scaffold of silk fibroin which aperture was20μm. The aperture of scaffold was40u m in Group B and80μ m in Group C.(3) The urothelium cell was planted into the scaffold of silk fibroin of all groups under the same experimental environment. The cell growth in all groups was observed by scanning electron microscope after3days’ cultivation. And the metabolic products of the urothelium cell were detected by the spectrum analyzer to evaluate the cell growth in all groups.Results:(1) The mucous membrane of urethra was derived from the male New Zealand white rabbits successfully. The primary culturing and the subculturing of the cell in vitro were completed. The cultivated cell was identified to the urothelium cell by contrast phase microscope and the immunohistochemical reaction of keratin antibody.(2) After planting the urothelium cell into the scaffold of silk fibroin, we found that the metabolic products in Group A which aperture was20μ m were most of all three groups by the spectr μ m. And we also found that the urothelium cell could grow in the scaffold of silk fibroin in Group A by scanning electron microscope. The urothelium cell could not be found in thhe other two groups.Conclusion:We found that the urothelium cell which was derived from the mucous membrane of urethra in New Zealand white rabbits could be cultivated and amplified in vitro successfully. We also found that the urothelium cell could grow in the lamellar scaffold of silk fibroin which aperture was20u m. Objective:To research the degradation of the lamellar scaffold of silk fibroin in New Zealand white rabbits.Methods:(1) The18slices of silk fibroin which aperture was20u m were trimmed into square tablets (10*10*2mm). These square tablets of silk fibroin were implanted into the subcutaneous tissue of three New Zealand white rabbits. Each rabbit had six square tablets.(2) Six square tablets of silk fibroin were taken out of the subcutaneous tissue of three rabbits at2,4,8weeks after implanted. The size of each square tablet was measured. And the reaction between the square tablet of silk fibroin and host was observed.Results:(1) SIZE—The square tablets of silk fibroin had the original shape two weeks after implanted. The mean volμme of square tablets was156±9.30mm3, There was significant statistical difference between the preoperative and postoperative volμ me of square tablet (p=0.014). The square tablets of silk fibroin lost the former shape, and showed irregular form four weeks after implanted. The mean volume of square tablets was38.33±14.56mm3. There was significant statistical difference between the preoperative and postoperative volume of square tablet (p<0.001). The last six square tablets of silk fibroin showed irregular form eight weeks after implanted. The mean volume of square tablets was7.00±2.00mm3.There was significant statistical difference between the preoperative and postoperative volume of square tablet (p<0.001).(2) FORM—The square tablets of silk fibroin had the original shape two weeks after implanted. These square tablets had no obvious adhesions with surrounding tissue and were separated easily. The square tablets of silk fibroin lost the former shape, and showed irregular form four weeks after implanted. These square tablets had obvious adhesions with surrounding tissue and were separated easily, however. The last six square tablets of silk fibroin showed irregular form eight weeks after implanted. The size of square tablet was much smaller than that at four weeks. These square tablets had obvious adhesions with surrounding tissue and were separated difficultly.Conclusion:The square tablets metabolized about96.32%in vivo for eight weeks. There was some local adhesions and dissolution. Objective:To research the repairing long-segmental urethral stricture and deletion by the lamellar scaffold of silk fibroin.Methods:(1)Twenty-four male New Zealand white rabbits were randomly divided into four groups, and each group had6rabbits with the average weight of2.87±0.16kg. Group A was controlled. Group B was the group of urethral stricture in which the mucous membrane of urethra was divested and the wound surface was healed by itself without any repairing. Group C was the group of the scaffold of silk fibroin with which the deletion of urethra was repaired after the mucous membrane of urethra stripped. Group D was the group of the compound of cell and scaffold with which the deletion of urethra was repaired after the mucous membrane of urethra stripped.(2)After raising for2months, the rabbits in all groups underwent the antegrade urethrography, urethral pressure profile (UPP) and histiocytes at two months postoperatively to observe the effect of repairing by the the lamellar scaffold of silk fibroin or the lamellar compound of cell and scaffold.Results:One rabbit of Group B appeared shock during using antibiotics after anesthesia. It died after the external cardiac massage for fifteen minutes. After adding one rabbit, rabbits of Group B had completed the test. One rabbit in Group C died at six weeks postoperatively. One scaffold was prolapsed in Group D at four weeks and another one died at five weeks postoperatively.(1) CYSTOURETHROGRAPHY:We found significant urethral stricture with narrow1μmen and discontinuous mucosa in Group B. There was significant difference between Group A and Group B. We also found that the continuity and diameter of urethra in Group C and D was better than those in Group B and was worse than those in Group A. However, Between Group C and Group D, was there no significant difference in vision.(2)UPP:The urethral pressure on operative site in Group A was14.67±2.16cmH2O, and27.83±3.71cmH20,18.00±1.87cmH2O and18.00±1.41cmH2O in Group B, C and D, respectively. There was significant statistical difference between three groups and Group A, respectively (p<0.05). There was significant statistical difference between the latter two groups and Group B, respectively (p<0.01). However, there was no difference between Group C and D. The graph of UPP showed that there was much more closed waveform of high pressure at the operative site in Group B, worse than that in Group C and D. There was the gentle waveform of low pressure in Group A. And there was similar waveform between Group C and D.(3)HISTIOCYTES:Urothelium was well-distributed covered without any inflammatory cells in Group A, which had3-4lays of the epithelial cells. And Urothelium was inequably covered with neutrophil and lymphocyte in Group B. In Group C and D urothelium was covered by4-5lays with less inflammatory cells, which was no difference between Group C and D.Conclusion:Using the scaffold of silk fibroin which acted as the replaced material of urethral deletion wound improve the urethral stricture through the comparative study in four groups. However, there was no difference between by the scaffold of silk fibroin alone and by urothelium-scaffold compound in the effect of repairing the urethral deletion. The inflammatory function was not obvious in the repairing segment of urethra.