输尿管无细胞基质移植物的制备和评价

Construction and evaluation of acellular matrix for ureter tissue engineering

背景:与小肠黏膜下层等材料相比,脱细胞血管具有天然管状结构,与输尿管形态结构相近,替代输尿管时仅需端端吻合即可,手术操作简单,血管外壁光滑,获取及制备方法简便等优点。目的:拟应用同种颈动脉血管无细胞基质体外构建输尿管。设计、时间及地点:观察性实验,于2006-09/2008-06在上海交通大学医学院附属新华医院动物实验中心完成。材料:长风杂交白猪由上海松联实验动物公司提供。上海交通大学医学院实验动物中心提供的健康成年大鼠8只,用于血管无细胞基质的动物毒性实验。方法:剥去猪颈动脉血管外膜,PBS冲洗若干遍,然后将血管置于pH7.1的PBS中4℃下振荡清洗,0.5%十二烷基硫酸钠振荡24h,后使用双蒸水4℃下反复振荡洗涤1周,每日换双蒸水2次。对于解剖过程中肌肉残留相对稍多的血管,在双蒸水洗涤前以混合消化液37℃下振荡消化约2h,再行双蒸水洗涤。制备的无细胞基质置于青、链霉素溶液中,4℃保存,完成脱细胞支架制备。主要观察指标:光镜及电镜观察脱细胞后管壁无细胞基质片的主要成分。将同种异体来源内皮祖细胞培养增殖后植入血管无细胞基质,观察细胞生长情况,并进行血管无细胞基质动物毒性实验。拉力实验了解血管无细胞基质材料的收缩性能。结果:颈动脉血管无细胞基质中已无细胞成分,无细胞基质主要由胶原成分组成。扫描电镜未见该材料表面存在细胞及细胞碎片,同时发现该无细胞基质存在孔隙样结构。体外同种异体来源的内皮祖细胞培养增殖后植入血管无细胞基质,细胞黏附于无细胞基质。血管无细胞基质按毒性分级属于无毒级。拉力实验说明该无细胞基质具有一定的韧性和牵张性。结论:采用胰酶和十二烷基硫酸钠制备的颈动脉血管无细胞基质材料无细胞残留,具有一定的韧性和牵张性,种植于其中的种子细胞具备一定的生长能力。

BACKGROUND：Compared to small-intestine submucosa,acellular vascular grafts have natural tubic structure,which is similar to ureter.When it is used as replacement for ureter,end-to-end anastomosis is used.It is characterized by simple operation,smooth vessel wall,collection and preparation method.OBJECTIVE：To prepare acellular vascular matrix as scaffold in tissue-engineered ureter in vitro.DESIGN,TIME AND SETTING：The observational experiment was performed at the Animal Experimental Center of Xinhua Hospital Affiliated to School of Medicine,Shanghai Jiao Tong University from September 2006 to June 2008.MATERIALS：Swines were supplied by Shanghai Song Lian experimental animals company.Eight healthy adult rats were supplied by Animal Experimental Center Affiliated to School of Medicine,Shanghai Jiao Tong University for the animal toxicity study of acellular vascular matrix.METHODS：Swine carotid artery membrana was removed and placed in phosphate-buffed saline（PBS,pH 7.1）.The tissue was stirred at 4 ℃ with 0.5% sodium dodecylsulfate for 24 hours.Then the tissue was treated by double distilled water for 1 week at 4 ℃.Double distilled water was changed twice every day.Vessel with muscle were digested in mixed digestive juice at 37 ℃ for 2 hours before washing.The acellular matrix was stored in penicillin and streptomycin solution at 4 ℃.MAIN OUTCOME MEASURES：The components of acellular scaffold was investigated by optical and electron microscopes.Allogenic endothelial progenitor cells following proliferation were transplanted into acellular vascular matrix to observe cell growth.Animal toxicity study of acellular vascular matrix was performed.Tensile force study was employed to understand the contractility of acellular vascular matrix.RESULTS：Acellular vascular matrix was without cell component.Acellular vascular matrix was mainly composed of collagen.Under scanning electron microscope,cells and cell debris were not found on the matrix.Acellular vascular matrix showed pore-like structure.In vitro allogenic endothelial progenitor cells following proliferation were transplanted into acellular vascular matrix.Cells adhered to the acellular vascular matrix.The acellular vascular matrix was not toxic.Tensile force test showed that the acellular vascular matrix had a certain tenacity and stretch.CONCLUSION：Trypsin and sodium dodecylsulfate-prepared acellular vascular matrix is without cell residual,with a certain tenacity and stretch.Seed cells on the acellular vascular matrix possess growth ability.