Application Of Silk Fibroin Film For Repairing Maledog Urethral Defect

BackgroundReconstruction of urethral defect has always been 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 membrane of skin,appendix, mucous of bladder and mouth and so on.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.It's the most important thing to seek a new way to solve the problem.Tissue engineering shows a bright future for repairing urethral defect,and has gained a breakthrough.Court Replaced a urethral segment with a silicone elastomer tube in the maledog in 1971,it was a new beginning thought the experiment failed.Italiano observed that using hyaluronic acid to replace the defective urethra section in the rabbits would help the defetive urethra to regenerate the construction well.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).Sievert indicated homologous acellular matrix graft will help for urethral reconstruction in the rabbit.FuQiang used acellular matrix succeed in repairing the lcm defective section,but failed in repairing the 2-3cm defective section.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:①synthesized cells scaffold brings inflammation for its poor biocompatibility;②as to the natural cells scaffold,for its rare material,limited processing ability,it also hard to get into industrialization,all the above lead to a expensive price;③the mechanical properties,such as ductibility and retractility can't suit to the clinical treatment.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 on.As a kind of macromolecule material,silk has been taken more and more 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.Liu Chunxiao and Lin Yangyang reported that using silk fibroin can repair the 1.5cm defect in rabbit urethra,it's unclear that the length of urethral defect can be repair only by silk fibroin.ObjectivesTo estimate the effect of different length of urethral defect repaired with Silk fibroin filmMethods1.0)Animal and material:14 maledogs(10.0—12.9Kg)(GUANGDONG ZHAOQING LABORATORY ANIMALS RESEARCH CENTER);Silk fibroin film:aperture 150～250μm,interval porosity 65%～95%(presented by professor Min Si-Jia of Zhejiang University);Mouse Anti—Pan Cytokeratin(PCK) antibody (BOSTER BIOTECHNOLOGY CO.LTD.).2.0) 14 Male maledogs were divided into three groups randomly:experimental groupⅠ(n=6),experimental groupⅡ(n=6) and control group(n=2).3.0) Experimental method:3.1) In the experimental groupⅠwith a defect of 1.5cm while groupⅡwith a defect of 3.0cm in the urethra in each maledog,all of them were repaired with Silk fibroin film;3.2) Control group of 2 maledogs underwent a sham operation;4.0) Detecting method:Urethra reorganization and retrograde urethrography4.1) The maledogs of the control group underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 6 and 12 weeks postoperatively were observed by histological technique(HE and VG staining) and immunohistochemistry respectively.4.2) The maledogs of the experimental group underwent retrograde urethrography and then the specimens of regenerative urethral obtained at 6 and 12 weeks postoperatively were observed by histological technique(HE and VG staining) and immunohistochemistry.Results1.The survival of the maledogs and the heal condition of operative incision during experiment:None of the 14 maledogs died during experiment.2.The observation of the specimens of regenerative urethral with naked eye: The control group had no cicatrices formation and stricture and there were no obvious difference between 6 weeks and 12 weeks after the operation.The experimental groupⅠwith 1.5cm defective section had a little cicatrices formation or stricture 6 weeks post operation,12 weeks after operation tend to be better than 6 weeks;fistula or strictures were observed in all of the animals in experimental groupⅡwith 3.0cm defective section,and there were no obvious difference between 6 weeks and 12 weeks after operation.The Silk fibroin film of the experimental group fixed in the original place and 6 weeks later,the Silk fibroin film partly degradated and the surface of the Silk fibroin film was covered by a little organization uniformly;12 weeks,Silk fibroin film was difficult to identify and there was no obvious difference with control group.3.Retrograde urethrography:All the experimental groupsⅡhad stricture or urinary fistula while the all control groups and the experimental groupⅠdidn't have.4.Histological examination:Hematoxylin-eosin(HE) and Van Gieson(VG) staining.4.1 The urethral organization of control group had no obvious difference between 6 weeks and 12 weeks after operation.About 4-5 layers urethral epitheliums covered uniformly and regularly,There was no inflammatory cell or fibrosis under the membrane and many smooth muscle cells were observed..4.2 In the experimental groupⅠof the 1.5cm defective section there were large amount of inflammatory cells 6 weeks after operation,containing neutrophil, lymphocyte,lymphomonocyte and eosinophile,and besides there were 6-8 layers of urethral epitheliums covered unregularly,some smooth muscle cells and small vessels were visible occasionally,many collagenoblasts and collagen fibers were observed;12 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.4.3 In experimental groupⅡof the 3.0cm defective section,6 weeks after operation there were large amount of inflammatory cells,containing neutrophil, lymphocyte,lymphomonocyte and eosinophile,and besides little urethral epitheliums,smooth muscle cells and small vessels were visible occasionally,many collagenoblasts and collagen fibers were visible;12 weeks latter,the urethral epitheliums,smooth muscle and small vessels didn't increased,collagenoblasts and collagen fibers remaind the same as 6 weeks ago.There were no obvious difference between 6 and 12 weeks after operation.5 Immunohistochemistry examination:Dyeing with Mouse Anti—Pan Cytokeratin(PCK) antibody,the urethral epitheliums of control group indicated positive,arranging uniformly,no obvious difference between 6 and 12 weeks;In the experimental groupⅠof the 1.5cm defective section indicated positive and the arrangement of the epitheliums tend to be more and more regularly from 6 weeks to 12 weeks.In experimental groupⅡof the 3.0cm defective section indicated seldom areas were covered by epithelium after 6 weeks.12 weeks after the operation,the epitheliums didn't increased obviously.Conclusion:Silk fibroin film appears to promote the urethral defect repair,which induces the growth of urethra epitheliums and smooth muscle cells.It can repair short length defect within 1.5cm,while it is unpredictable to repair defect longer than 3.0cm only by the materials.