Preliminary Study On The Tissue Engineering Urethra

Tissue gngineering is a modern subject.It includes a scaffold that provides an architecture on which seeded cells can be organized and developed into the desired organ or tissue prior to implant. The scaffold provides an initial biomechanical profile for the replacement tissue until the cells produce an adequate accellular matrix. Tissue engineered organs can help people to resolve the proplem of lack of organs for transplantation.The aims of this experiment is to study the technique of culture of UEC , USMC and MSCs in vitro;and to explore the methods and feasibility of culture of Cells above on bladder acellular matrix(BAMG) in vitro as an experimental basis for urethra regeneration in vivo by tissue engineering.The study comprises of five parts:The first part is concerned of cultivation of rabbit normal UEC . The tissue samples were harvested on surgery of the New Zealand rabbit. The culture system for UEC growing is DMEM/F₁₂ added with BPE and rEGF. When cells grew we found that cells growed in the DMEM/F₁₂ system contained mainly UEC without frbroblasts and SMC. UEC climbed out of the explants and grew gradually to a monolayer for about 12 to 14 days. Immunocytochemistry coloration for pancytokeratin of the cells got positive result. Moreover transmission electric microscopy examination of the cells confirmed the derivation of UEC. After that, we observed the viability of the UEC obtained, and the cell growth curve and attachment efficiency showed good activities. Furthermore, the UEC was cryopreserved in liquid nitrogen for about 3 months and they gained the viability when they were thawed. All the results indicated that the DMEM/F₁₂ is anappropriate culture system for UEC growth and enough UEC can be produced in this way.In the second part of the study, we primarily cultivated rabbit ureter SMC under the condition of DMEM plus FBS and antibiotics. Samples were harvested according to aseptic principles. All the samples were digested by compound enzyme of 0.2% I collagenase and 2.5mg/ml dispase and 3% FBS, enough separate cells were harvested and were seeded. In order to identify the cells derivation, immumocytochemistry for anti a -actin of the cells was performed and positive result was received which made sure that the cell came from USMC. Then the cell growth curve, attachment efficiency we achieved demonstrated USMC were of normal vitality. We also cryopreserved some USMC and they recovered to the original growth state almost. After 6 to 7 passages the cells experienced senescence and lost viability. Anyway the USMC we obtained through the cultivation system are quite enough for further study such as tissue engineering of urinary organs.The third part is to test the potential of a kind of membrane made from BAMG extracted from rabbit bladder. We seeded the rabbit UEC or USMC harvested from the former parts in the BAMG membrane on one side alone, and seeded UEC on one side USMC on the other side of the membrane for co-culture. The membrane seeded with cells cultured in medium in a incubator for a couple of days. And histological analysis was applied to examine the cells growing in the membrane. We found that either kind of cells can grow in the membrane to a continuous layer, even more layers after a longer time. The double seeded membrane grew into a 'sandwich' complex with UEC layers on the top and SMC layers on the bottom side of the membrane. So we conclude that the BAMG membrane we used are useful for supporting human UEC and USMC growth, and primary cell-scaffold composite can be formed in this way.In the fourth part ,we used the method of density gradient centrifugation to isolate and culture rabbit MSCs under the culture condition of aDMEM. The higher purity of MSCs were obtained by the repeated removal of nonadherent cells .The rabbit MSCs in vitro cultured under this condition in our laboratory can be expanded efficiently. Through observing the cells morphology and assaying the cells surface antigen by flow cytometry (FCM), we found that the cells we cultured were positive for CD44 and negative for CD45 which were some characters of mesenchymal stem cells. The culture method used in this experiment is simple and reliable. Proliferative cultured cells grows well and posses the abilities of differentiation . They can not only be used in experimental investigation, but also meet a demand of tissue engineering as cell seeded.In the last part ,the MSCs were seeded onto BAMG The MSCs/ BAMG complex were cultured and were detected the proliferation condition during the several follow days by Scanning Electronic Microscopy. Results suggested that the MSCs came into proliferative phase, the cells morphology were normal. Their growth show that BAMG are scaffolds with good biocompatibility and feasible for urethra tissue engineering urethra.,Our study cultivated rabbit urinary cell including UEC and USMC and MSCs successfully in vitro which can be used for advanced studies on urinary tissue engineering. It offers a basis for future study of tissue engineering urethra.