膀胱无细胞基质移植物重建兔阴茎白膜的动态观察(英文)

Dynamic observation on the bladder acellular matrix grafts for substituting albuginea penis in rabbits

背景:目前膀胱无细胞基质移植物已成功用于替代动物膀胱、尿道和修复尿道下裂,但膀胱无细胞基质移植物重建阴茎白膜有待观察。目的:采用同种异体膀胱无细胞移植物替代兔阴茎白膜,观察重建修复效果。设计:随机对照观察。单位:四川大学华西医学实验动物中心、华西组织工程实验室及贵阳医学院组织工程实验室。材料:选用50只雄性健康封闭新西兰兔,动物级别3级,体质量为2.6～3.0kg,均无包茎及阴茎发育不良,注射生理盐水后无阴茎弯曲,由四川大学华西实验动物中心提供。方法:实验于2005-12/2007-06在四川大学华西实验动物中心、四川大学组织工程实验室及贵阳医学院组织工程实验室完成。①取10只实验兔膀胱制备膀胱无细胞基质移植物。随机数字表法将40只新西兰兔分为对照组和膀胱无细胞基质移植物组,分别在阴茎背侧切除白膜10mm×5mm造成缺损,分别采用白膜原位缝合及膀胱无细胞基质移植物修复,每组20只。②分别于术后2,6,12及24周对2组动物进行阴茎海绵体内快速注射生理盐水诱导阴茎勃起,观察阴茎弯曲情况;于上述时间点分别处死实验兔,于术区取材进行苏木精-伊红、Masson染色观察修复部位组织结构变化;Stirus染色检测检测Ⅰ型和Ⅲ型胶原纤维阳性面积,免疫组化染色检测炎性标志因子诱导型一氧化氮合酶和促纤维化因子转化生长因子β1的表达。主要观察指标:①阴茎弯曲情况。②修复部位组织结构变化。③炎性标志因子诱导型一氧化氮合酶和促纤维化因子转化生长因子β1的表达。④Ⅰ型和Ⅲ型胶原纤维阳性面积。结果:纳入重建阴茎白膜术实验兔40只,2只死于麻醉药物过量,2只死于急性肠炎,其余36只均进入结果分析。①术后6周时弯曲发生率最高,对照组2例,膀胱无细胞基质移植物组1例;术后12周对照组及膀胱无细胞基质移植物组各1例发生弯曲;术后24周对照组1例发生弯曲,膀胱无细胞基质移植物组无弯曲发生。②修复部位组织结构变化:术后2周两组修复部位结构欠清,炎性细胞浸润明显,膀胱无细胞基质移植物组移植部位可见膀胱无细胞基质移植物内细胞生长,Masson染色显示术区白膜纤维纤细,排列欠规律。术后6周时白膜恢复完整性,膀胱无细胞基质移植物不能辨别,两组间无明显差异。术后24周移植部位白膜完整均匀,纤维恢复内环外纵排列,和正常白膜不能区别。③两组术后诱导型一氧化氮合酶及转化生长因子β1,2周表达最强,术后6周只有少数纤维细胞和血管内皮细胞有表达,术后12,24周仅有极少的血管内皮细胞表达诱导型一氧化氮合酶和转化生长因子β1,同时间点2组诱导型一氧化氮合酶和转化生长因子β1没有差别。④术后2周两组Ⅰ型和Ⅲ型胶原纤维共同存在,比例相当。以后Ⅰ型胶原纤维逐渐增加,而Ⅲ型胶原纤维逐渐减少,术后24周以Ⅰ型胶原纤维为主,Ⅲ型胶原纤维已不明显。结论:膀胱无细胞基质移植物修复新西兰兔阴茎白膜无明显的炎症反应和纤维化,是较为理想的阴茎白膜修复材料。

BACKGROUND：At present, bladder acellular matrix grafts have been successfully used for substituting animal bladder and urinary canal, and for repairing hypospadia. However, reports on bladder acellular matrix grafts for substituting albuginea penis need to be investigated. OBJECTIVE： Allogeneic bladder acellular grafts were used for substituting albuginea penis of rabbits, in order to observe repairing results. DESIGN： A randomized controlled observation. SETTING： West China Medical Laboratory Animal Center and West China Laboratory of Tissue Engineering of Sichuan University as well as Laboratory of Tissue Engineering of Guiyang Medical College. MATERIALS： Fifty male healthy New Zealand Rabbits of grade 3, weighing 2.6-3.0 kg, without phimosis and penis dysplasia, and without presence of phallocampsis after normal saline being perfused, were provided by Huaxi Laboratory Animal Center of Sichuan University. METHODS： This study was performed at the West China Laboratory Animal Center and West China Laboratory of Tissue Engineering of Sichuan University as well as Laboratory of Tissue Engineering of Guiyang Medical College between December 2005 and June 2007. Bladders were taken from 10 experimental rabbits for preparing bladder acellular matrix grafts. The other 40 New Zealand rabbits were randomly divided into the control group, and the bladder acellular matrix grafts group, with 20 in each. An area of 10 mm×5 mm of albuginea penis was resected from dorsum penis of each rabbit. Suture in situ of albuginea penis and bladder acellular matrix grafting were conducted in rabbits of the control group and bladder acellular matrix grafts group, respectively. In the 2nd, 6th, 12th and 24th weeks postoperatively, each rabbit was intracavernously perfused normal saline for inducing penile erection, separately, in order to observe phallocampsis. At above-mentioned each time point, experimental animals were sacrificed. Sample was taken from surgical region for haematoxylin-eosin （HE） staining and Masson trichrome staining, in order to observe the changes of tissue and structure of surgical region. Types Ⅰand Ⅲ collagen fiber areas were detected by Stirus red staining, and the expressions of inducible nitric oxide synthase（iNOS） and transforming growth factor-β1 （TGF-β1） were detected by immunohistochemical staining. MAIN OUTCOME MEASURES： ①Phallocampsis status. ② Changes of tissue and structure of surgical region. ③iNOS and TGF-β1 expressions. ④TypeⅠand Ⅲ collagen fiber areas. RESULTS： Forty experimental rabbits were involved in the penile surgery, two of them died from overdose anesthesia, two died from chordapsus, so the remaining thirty-six rabbits were involved in the final analysis. In the 6th week postoperatively, phallocampsis reached its highest level, and 2 rabbits in the control group and 1 rabbit in the bladder acellular matrix grafts group presented phallocampsis. In the 12th week, every rabbit presented phallocampsis. In the 24th week, 1 rabbit in the control group but none in the bladder acellular matrix grafts group presented phallocampsis. In the 2nd week, the structure of surgical regions of each rabbit was poorly clear, with remarkable inflammatory infiltration. In the bladder acellular matrix grafts group, grafting regions presented cells ingrowing the bladder acellular matrix grafts. Masson trichrome staining results showed that in the surgical region, tunica albuginea fibers were thin and poorly arranged. In the 6th week, tunica albuginea recovered its integrity, and bladder acellular matrix grafts could not be distinguished. No significant difference existed between two groups. In the 24th week, tunica albuginea was even and complete in the sugical region, and fibers restored their arrangement of circular muscle in inner layer and longitudinal muscle in outer layer, without difference from normal tunica albuginea. iNOS and TGF-β1 expressions were the strongest in the 2nd week, and they were found in the fibrocytes and vascular endothelial cells in the 6th week, but a little in the 12th and 24th weeks postoperatively. There were no remarkable differences in iNOS and TGF-β1 expressions between two groups at the same time point. In the 2nd week, typesⅠand Ⅲ collagen fibers co-existed with equivalent proportion. Then, typeⅠcollagen fibers were gradually increased, while type Ⅲ collagen fibers were on the contrary. In the 24th week, typeⅠcollagen fibers took the main place and type Ⅲ collagen fibers were unremarkable. CONCLUSION： Bladder acellular matrix grafts have no remarkable inflammatory reactions and fibrosis in repairing tunica albuginea of New Zealand rabbits, so they are very ideal grafting materials for penile surgery.