| BackgroundThe treatment of urethral defect is always a difficult problem for urology owing to congenital factor, trauma, inflammation or tumor. So far, as having been reported, the methods of clinical treatment are various. Most of treatments for repairing urethral defect are using other tissues, such as appendix, membrane of skin and mucous of bladder, mouth. However, these treatments would cause a damage to normal tissues, bring an extra physical injury and have more complications. From the above mentioned, we can see that the effects of these treatments are far from perfect.Tissue engineering shows a bright future for repairing urethral defect, and has gained a breakthrough. In the initial researches, urethral defect were repaired with synthesized cells scaffold in which urethral epitheliums and smooth muscle cells were seeded, but recently usually repaired with SIS (Small intestinal submucosa), BAMG (Bladder acellular matrix graft) or ECM (Extracellular matrix).The cells scaffolds which are mainly used in tissue engineering at present include synthesized cells scaffold, scaffold made with natural material and ECM. Though a great progress has been made in tissue engineering, there are still many problems, such as the shortage of perfect scaffold.The reasons are as follows: synthesized cells scaffold brings inflammation for its poor biocompatibility and it is expensive. As to the natural cells scaffold, for its rare material, limited processing ability and high cost, it is hard to get into industrialization. Therefore, how to get perfect scaffold with good biocompatibility, lower cost and abundant source is an important task on relevant researches. The researchers have been studying and exploring new cells scaffold, mainly in terms of raw materials, processing technique, surface modification, and so forth.Silk, a kind of macromolecule material, has taken great attention in recent years. It has been confirmed that silk fibroin, a key component of silk, has no stimulation, but avirulence, strong adherence, good physical and chemical character and biocompatibility. Silk fibroin has been used in biomedical field widely, such as making cells scaffold, artificial vessel, protective membrane for wounded skin and endocranium repairing material. To our knowledge, it has not been reported that silk fibroin film is used as cells scaffold in repairing urethral defect.ObjectivesTo estimate the effect and feasibility of repairing urethral defect with silk fibroin film.Methods1. Animal and material: 24 Male New Zealand rabbits (2.5—4.5Kg)(Southern medical university); Silk fibroin film:aperture 150～250μm,interval porosity 85% ~ 95%( presented by professor Min Si-Jia of Zhejiang University);Mouse Anti—Pan Cytokeratin (PCK) antibody (BOSTER BIOTECHNOLOGY CO. LTD.).2. 24 male New Zealand rabbits were divided into three groups randomly: experimental group(n=12),control group I (n=6) and control group II (n=6).3. Experimental method:3.1 Experimental group: Dissociated and cut off the cavernous body of urethra for 1.5 cm and then repaired the urethral defect with Silk fibroin film;3.2 Control group I : Sew up the cavernous body parcel film , subcutis and skin one by one layer after dissociating the urethra cavernous body;3.3 Control group II: Dissociated and cut off the cavernous body of urethra for 1.5 cm and then just Sew up urethra;4. Detecting method: Urethra reorganization and retrograde urethrography 4.1 The rabbits of the experimental group underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 2,4,8 and 16 weeks postoperatively were observed by histological technique (HE and VG staining) and immunohistochemistry.4.2 The rabbits of the control group I and control group II underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 8 and 16 weeks postoperatively were observed by histological technique (HE and VG staining) and immunohistochemistry respectively.Results1. The survival of the rabbits and the heal condition of operative incision during experiment: None of the 24 rabbits died during experiment. One rabbit of the control group II had urinary fistula 2 weeks after operation.2. The observation of the specimens of regenerative urethral with naked eye: The urethra of control group I had no cicatrices formation and stricture; Control group II had cicatrices formation and defect 8 weeks after operation,and the urethras of operation area were narrower than control group I 16 weeks postoperatively,also the organization was tenacious too; The Silk fibroin film of the experimental group fixed in the original place and had no obvious change 2 weeks after operation, the surface of the Silk fibroin film was covered by organization partly; 4 weeks, the Silk fibroin film partly degradated and the organization which covered Silk fibroin film increased;by 8 weeks, the surface of the Silk fibroin film was covered by organization uniformly and became fragments; 16 weeks , Silk fibroin film was difficult to identify and there was no obvious difference with control group I in operation area.3. Retrograde urethrography: 6 rabbits of control group I had no stricture or urinary fistula; Except for the rabbit who had urinary fistula 2 weeks after operation,all the other 5 rabbits of the control group II underwent retrograde urethrography,appearing stricture and rigid feeling; 12 rabbits of experimental group had no obvious stricture or urinary fistula. 2 weeks postoperatively,operation area appeared rough slightly. 8 weeks later after operation, there was no obvious difference from control group I;.4. Histological examination: Hematoxylin-eosin (HE) and Van Gieson (VG) staining.4.1 The urethral organization of control group I had no obvious difference between 8 weeks and 16 weeks after operation. About 4-5 layers urethral epitheliums covered uniformly and regularly,under the membrane many smooth muscle cells were visible.There was no inflammatory cell or fibrosis.4.2 In the control group II ,cicatrices formation emerged and large amounts of fibrous tissue and collagenoblasts could be seen in 8 weeks, in many areas there was no epithelium covered ; in 16 weeks much fibrous tissue still could be seen, epitheliums increased ,but arranged irregularly ,only a few smooth muscle cells were visible occasionally.4.3 In the experimental group,2 weeks after operation there was no epithelium covered obviously, only a few epitheliums were visible occasionally, in the Silk fibroin film there were large amount of inflammatory cells, containing neutrophil, lymphocyte, lymphomonocyte and eosinophile; 4 weeks, there were still many inflammatory cells and about 6-8 layers urethral epitheliums covered unregularly;8 weeks the urethral epitheliums arranged more uniformly than 4 weeks, gradually tending to be regular, about 5-7 layers.Some smooth muscle cells and small vessels were visible occasionally, the inflammation cells decreased, many collagenoblasts and collagen fibers were visible; 16 weeks,the urethral epitheliums arranged uniformly and gradually, about 4-5 layers,smooth muscle cells increased, collagenoblasts and collagen fibers decreased and arranged gradually, caliber of the blood vessels enhanced, there was no obvious difference from control group I .5 Immunohistochemistry examination : Dyeing with Mouse Anti—Pan Cytokeratin (PCK) antibody, the urethral epitheliums of control group I indicated positive, arranging uniformly; In the control group II ,in many areas there was no epithelium covered in 8 weeks.In 16 weeks epitheliums increased ,but arranged irregularly; The urethral epitheliums of experimental group indicatedpositive,gradually tending to be regular from 4 to 16 weeks.Conclusion:Silk fibroin film appears to promote the urethral defect repair,which induces the growth of urethra epitheliums and smooth muscle cells.
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