Experimental Study On Urethral Reconstruction With Extracellular Matrix Of Human Umbilical Vein

Background and ObjectivesUrethral reconstruction is still the main therapy pathway for urethral stricture or defect caused by congenital malformation,acquired injury or other diseases.There are more than 300 operation methods for urethroplasty,but uerthral stricture and fistula are still common complications which explain the difficulty,complexity and challenge of urethroplasty.Patients often need to be operated again or for many times.Moreover, the repair for function and shape of penis is also difficult to urologists.Novel or improved methods continue to come forth to prevent urethral fistula and stricture in order to recover the function and shape of penis to the most extent.But no way is appropriate and generalized.The focus is on the stuff for urethral reconstruction.At present,biological materials for urethral reconstruction include:1 vesical or buccal mucosa grafts.The former can supply adequate amounts of tissue,but the later supply a limited amount.Both of them could bring secondary injuries,easily causing urethral fistula and stricture.2 prepuce of penis or skin free grafting.For lack of blood,this way has seldom been used now.3 ureter,artery,vein or appendix tissue.These ways are not the major methods at all times because of inconvenience to get them and bad treatment effect.4 Vascularized skin flaps from the prepuce,scrotum or penile shaft. These ways have the virtue of good blood circulation,but no adequate amounts of tissue.No tissue could be available for patients' lack of prepuce,with severe hypospadias,or urethral operation failure many times. With the rapid development of life science,material science and relative disciplines,a new intersect science named tissue engineering arises.Tissue engineering has been proposed as a strategy for urethral defect.In the studies of urologic tissue engineering the support material is very important for tissue or organ's reconstruction.As a supporting stuff,extracellular matrix has been paid more and more attenation to nowadays.ECM is a special natural biomaterial.It is a such low antigenicity substance as collagen protein,proteoglycans,or glucoprotein retained from organism via special decellularization,and can provide mechanical support and physical intensity for tissue,organ even the whole organism.Further study showed that ECM not only can provide mechanical support for organism,but also has profound influence on cell reproduction,adhere,differentiation,development,diffuseness,migration,ecesis anomaly,apoptosis etc.The theoretical basis of this material being used for tissue repair is reserving its natural extracellular matrix and biological characteristics, enabling it to no immunogenicity when antigenic cellular components are extracted.Many researchers have acquired significant achievements in urethral tissue engineering.ECM such as SIS,BECM and VECM were used to reconstruct the urethra.These studies indicated that urethral reconstruction with ECM was feasible. Therefore,based on the study of the forward researchers,we will use ECM of umbilical vein to repair the urethra,and observe its tissue regeneration capacity so as to seek an ideal material for urethral reconstruction.Materials and methodsUmbilical core was obtained from health woman of uterine-incision delivery. ECM of umbilical vein was prepared by use of hypotonic and hypertonic solutions, detergent and proteinase in a multistep process.Twenty-four male rabbits were divided into 3 groups:group A(n=12),group B(n=6),group C(n=6).In group A and B,a 2.0cm segment of urethra was resected.In group A,the resected urethra was replaced with ECM of human umbilical vein graft and in group B urethral defect was created as a control group.A 2.0cm segment of urethra was cut and then stitched up with 6-0 absorbable suture in group C.8~# silicone tubes were placed into the three groups of rabbits' urethras to play the role of support and urination.Every rabbit was injected with penicillin 8×10~6 u/d the first week after the operation.The silicone tube was removed in the tenth day after operation.Then the regenerative segment was studied with histological technique by hematoxylin-eosin straining and immunohistological straining after 2,4,8 and 16 weeks postoperatively.The retrograde urethrography and urodynamics were used to evaluate the function of the regenerative urethra at 16 weeks after operation.Results1 The ECM of human umbilical vein is achromatic color and has elastic behavior. Morphological analyses showed that the cells were removed effectively from umbilical veins,and the elastic fibers and collagen fibers were preserved.2 In group A,2 rabbits had infection which was controled after treatments,and urinary fistula occurred on two of them after the tube was removed.In group B,3 rabbits had urinary fistula,and in others urethral strictures and fibrous connective tissue were observed without normal epithelial structure.ln group C,all rabbits successfully recovered.3 In group A,epithelial cells grew from each side in ECM with infiltration of inflammatory cells 2 weeks after operation.However,these cells completely disappeared 4 weeks after operation and at the same time the luminal surface of matrix was completely covered by urothelium.At 8 weeks after operation, minimal extracellular matrix of human umbilical veins graft was observed in the host tissue,and irregular mussy smooth muscle cells were confirmed.At 16 weeks postoperatively,the amount of regenerative tissue was equivalent to the urethral tissue of group C with regular smooth muscle cells,increased vessels and no inflammatory reaction,and ECM of human umbilical veins disappeared in group A.4 The urethrography showed wide smooth urethra without urine exosmosis and urethral diverticulum in group A and C,meanwhile urodynamic evaluation didn't demonstrate significant statistical difference(P＞0.05) in the bladder volume and the maximum urethral pressure before and after operation between the two groups. Piezometer can't be placed in group B for detecting.Conclusions1 Hypotonic and hypertonic solutions,detergent and proteinase are ideal methods to prepare ECM from umbilical veins.2 ECM of umbilical vein can repair 2.0 cm long tubiform urethral defect. There are only two anastomosises when using it to reconstruct urethra.Good regeneration of mucosa occurred and the rate of urethral fistula and stricture is low after operation.3 Urethral reconstruction with ECM of umbilical vein permitted adequate materials, simple operation and few traumas.It is a good material to reconstruct urethra and worthy of further study.