尿道细胞外基质修复尿道缺损的研究

目的 寻求一种较理想的尿道修复材料。方法 20只家兔尿道制成尿道细胞外基质(ECM)；另40只随机分3组：尿道ECM移植组(实验组)，对照组Ⅰ及对照组Ⅱ。实验组切除尿道1．O～1．5cm后用尿道ECM修复并于术后10d、3周、6周及24周行组织再生情况观察；另于术后10周、24周各取4只行膀胱尿道造影；24周时实验组及对照组Ⅰ各取4只行尿流动力学检测；24周实验组取4只行尿道镜检查。结果 缺损修复术后10d，基质中见单层上皮细胞且有血管长入ECM；3周时尿道ECM管腔已完全被上皮细胞覆盖；6周时可见平滑肌细胞再生，炎性细胞消失；24周后其组织结构与正常组织相比差异无统计学意义。膀胱尿道造影无尿液外渗，无梗阻及结石形成。尿流动力学检测结果实验组与对照组Ⅰ差异无统计学意义；尿道镜检查证实尿道黏膜完整光滑，尿道内径及其形态正常。结论 尿道细胞外基质是一种理想的尿道修复材料。     

应用异体脱细胞尿道基质修复尿道缺损

目的探讨应用同种脱细胞尿道基质修复尿道缺损的可行性。方法将14只雄性新西兰兔分为两组，切除实验组长约1.0～1．5cm的尿道，用相应长度脱细胞尿道基质修复；对照组行假手术。术后行尿道造影并取尿道标本作病理检查。结果12只实验兔的脱细胞基质移植物没有移位。除2例狭窄、2例尿瘘外，其余满意效果。病理检测示，术后3周尿道管腔上皮化，6个月基质中平滑肌及血管再生明显。结论同种脱细胞尿道基质材料可以修复兔尿道部分缺损。     

血管细胞外基质和尿道细胞外基质修复兔尿道缺损的比较研究

目的　寻求较理想的尿道修复材料。　方法　切取 10只家兔的腹主动脉和尿道各 3cm ,制备成血管细胞外基质 (VECM)和尿道细胞外基质 (UECM)。另 2 0只分别切除尿道 2 .5cm并随机分为VECM修复组和UECM修复组。两组均于修复术后 10d、3周、6周及 2 4周行组织再生情况研究 ;另于术后 10周、2 4周各取 2只行膀胱尿道造影 ;2 4周两组各取 2只行尿动力学检测和尿道镜检查。　结果　制备的VECM和UECM均为白色透明状 ,但VECM较UECM弹性和机械强度好。缺损修复术后10d ,基质中见单层上皮细胞且有血管长入ECM ,基质和受体尿道连接处有炎性细胞浸润 ;3周时基质管腔已完全被上皮细胞覆盖 ;6周时可见平滑肌细胞再生 ,炎性细胞消失 ;2 4周后其组织结构与正常尿道组织结构一致。VECM修复组和UECM修复组相比其组织再生过程无差异。尿动力学检测VECM修复组和UECM修复组的膀胱容量分别为 (30 .2± 1.6 )ml和 (32 .1± 1.4 )ml、尿道最高压分别为(15 .2 7± 1.36 )mmHg和 (14 .6 8± 1.6 5 )mmHg、尿道最低压分别为 (12 .4 9± 1.2 3)mmHg和 (11.96±0 .98)mmHg ,差异无统计学意义 (P >0 .0 5 )。膀胱尿道造影可见尿道壁完整光滑通畅 ,不能分辨移植区与正常组织 ,无尿液外渗 ,无梗阻及结石形成 ;尿道镜检查证实VECM修复组和U     

生物可降解性尿道内支架修复战伤性尿道狭窄的研究

目的建立战伤性尿道狭窄动物模型,探讨生物可降解性尿道内支架对其进行重建修复的可行性。方法将新西兰雄兔28只分为两组,实验组(n=20):以定位爆炸法建立尿道狭窄模型。一月后行逆行尿道造影、尿道镜检查,并切除狭窄段尿道,行病理组织学观察证实。后置入人工合成生物可降解尿道内支架,置入术后2、4、8、12周分别行逆行尿道造影、尿道镜检查以及尿流动力学检测。并在以上各时间点处死5只动物,取狭窄处尿道组织,观察组织学修复重建情况。对照组(n=8):于实验组爆炸处理后4周和支架置入12周,分别取对照组4只动物与实验组对比观察。结果实验组所有动物爆炸后4周在尿道球部狭窄形成稳定狭窄模型(狭窄段长5～10 mm,尿道腔缩窄50%以上)。尿道内支架置入后2周,组织学观察见黏膜上皮新生迹象,并有炎性细胞浸润;4周时上皮新生明显,炎性细胞消失;8周时出现尿道平滑肌细胞再生,12周时见损伤后尿道组织结构完全修复,与正常尿道组织比较差异无统计学意义(P>0.05)。。同时间点尿道镜检查证实尿道腔隙、黏膜形态结构无异于正常对照组。尿流动力学检测显示两组间差异无统计学意义(P>0.05)。。结论应用成功建立的战伤性尿道狭窄动物模型,证实生物可降解性尿道内支架能作为修复战伤性尿道狭窄的理想材料,具有损伤小,易操作,功能恢复快的特点。     

尿道上皮细胞/片状多孔丝素蛋白支架复合物修复兔尿道缺损的实验研究

目的探索用尿道上皮细胞/片状多孔丝素蛋白支架复合物修补长段尿道缺损的可行性及效果。方法取雄性新西兰兔(2.5kg)27只,随机分成3组:实验组:尿道上皮细胞/多孔丝素蛋白支架复合物组(n=9);对照组Ⅰ:假手术组(n=9);对照组Ⅱ:尿道缺损组(n=9)。三组共同饲养4周和16周后,分别对手术部位行病理学检查,8周后分别进行膀胱尿道造影、尿动力检测(尿道测压)和病理检测。结果 27只兔实验过程中无一例死亡。对照组Ⅰ术后4、8及16周,尿道组织结构无明显区别,尿道上皮细胞均匀覆盖,层数约4~5层,排列规则,未见炎症细胞。对照组Ⅱ术后4、8及16周瘢痕形成,组织质地坚韧,腔壁纤维化形成,可见大量的纤维组织及成纤维细胞,黏膜缺损:实验组术后4周时可见较多炎症细胞,材料表面细胞黏附,层数约6~8层,丝素蛋白复合体发生扭曲,术后8周片状多孔丝素蛋白支架降解成碎片状,组织均匀覆盖;术后16周尿道上皮细胞排列均匀,层数约4~5层,无明显炎症细胞浸润,可见较多血管,与对照组Ⅰ相似。术后8周,对照组Ⅰ膀胱尿道造影显示尿道黏膜连续性好,管腔光滑,管径均匀。对照组Ⅱ造影片显示尿道黏膜连续性的中断,管腔毛糙,尿道管径变细,呈现尿道狭窄的表现。实验组膀胱尿道造影提示尿道黏膜的连续性良好,管腔稍毛糙,管径与对照组Ⅰ基本一致。尿道测压:实验组术后8周手术部位的尿道阻力平均值(15.25±1.72)cmH_2O,对照组Ⅰ尿道阻力的平均值为(14.85±1.96)cmH_2O,两组对比无统计学差异(P0.05),对照组Ⅱ尿道阻力平均值为(27.83±3.71)cmH_2O,阻力明显增高,与实验组及对照组Ⅰ相比均有明显的统计学差异(P0.05)。结论尿道上皮细胞/多孔丝素蛋白支架复合物可以作为尿道修复的支架,具有促进尿道缺损修复的能力。     

冻干无细胞膀胱黏膜下基质修复兔尿道缺损

目的　探讨冻干无细胞膀胱黏膜下基质修复尿道缺损的效果。方法　应用反复冻融-酶法及冷冻干燥技术制备冻干无细胞人体膀胱黏膜下基质。 18只新西兰白兔建立尿道中段部分缺损模型 ,尿道缺损面积约 1 0cm× 0 5cm。其中 14只兔作为实验组 ,以冻干无细胞膀胱黏膜下基质修补尿道缺损 ,术后 1、2、3、4、8、12、2 4周分别取 2只行逆行性尿道造影 ,观察尿道情况 ,并采取尿道组织进行大体、组织学及超微结构观察 ;4只兔作为对照组 ,未采用任何材料修补尿道缺损 ,直接缝合尿道海绵体包膜、皮下组织及阴茎皮肤 ,术后 2、4周分别取 2只行逆行性尿道造影 ,采取尿道组织进行大体观察。结果　实验组 14只兔均未发现明显的尿道狭窄。冻干无细胞膀胱黏膜下基质组织相容性良好 ,移植后无细胞膀胱黏膜下基质内有细胞长入 ,新生血管形成 ,术后 2周无细胞膀胱黏膜下基质移植区完全上皮化。随着移植时间的延长 ,移植区胶原纤维排列由紊乱趋于规则。结论　冻干无细胞膀胱黏膜下基质能够诱导尿道黏膜细胞迁徙、生长和上皮化 ,初步认为可以作为尿道缺损修复材料。     

小肠黏膜下层修复尿道的实验研究

目的探讨小肠黏膜下层(small intestinal submucosa,SIS)在尿道修复重建中的应用价值.方法 24只日本雄性大耳白兔,随机分为A、B、C及D组(n=6).A、B组切除前尿道2.0 cm,A组,用管状SIS修复尿道缺损;B组将其断端与周围组织直接缝合作为对照.C、D组仅切除2.0 cm尿道前壁,保留一半尿道壁为底板,C组用片状SIS修复尿道缺损;D组将其残端与周围组织直接缝合作为对照.均于修复后6、12周行组织学观察;12周行尿道膀胱造影及尿动力学检查.结果术后6周,A、C组修复的尿道有单层上皮细胞覆盖,基层组织中可见SIS的微小碎片包裹,出现不规则紊乱的平滑肌细胞生长,A组较C组的炎性反应重,有白细胞及淋巴细胞浸润,C组出现新生血管.术后12周,C组的上皮组织及基层下组织与D组无明显差别,平滑肌排列规则,血管数目进一步增多,炎性反应消失,未见SIS组织;A组中仍可见少数SIS的微小碎片;B组1只、D组2只尿道自行修复,余可见尿道闭塞,大量结缔组织生长,炎性细胞浸润,无正常上皮结构.术后12周尿道膀胱造影,A、C组可见尿道完整、光滑,无尿液外渗、尿道憩室等形成;尿动力学检查示A、C组的膀胱容量、最大尿道压分别与术前比较,差异无统计学意义(P＞0.05),而B、D组不能置入测压管检测.结论 SIS可作为兔尿道修复重建的良好支架材料,片状SIS修复优于管状SIS修复.     

尿道细胞外基质的研制

目的 探讨尿道细胞外基质(ECM)的制备方法。方法 采用4因素3水平，9次实验的正交设计[L9(3^4)]。切取37只兔的尿道，27条按正交设计随机分为9组行尿道脱细胞处理，试验重复3次。采用HE染色及计算机图像分析对尿道基质进行残余细胞成分计数，统计分析获得最佳方法：0．4％胰蛋白酶、l％甲醛加0．2％戊二醛、40U／ml DNA酶(A3B2C3方案)。另l0条兔尿道制备成尿道ECM并用于同种异体尿道缺损修复实验；分别于修复术后l0d，3、6及24周取材观察缺损修复处组织再生情况。结果 各组脱细胞后残余细胞成分量均不相同，第7、9组未发现细胞残余成分；制备的尿道ECM经扫描电镜分析未发观细胞残片。缺损修复实验术后10d，基质中见单层上皮细胞，且有血管长入ECM中，但管径较小，基质和受体尿道连接处有炎性细胞浸润；3周时尿道ECM管腔已完全被上皮细胞覆盖；6周时可见平滑肌细胞再生，炎性细胞消失。24周后基本结构近似正常。结论 制备尿道细胞外基质中3个关键条件的最佳水平为A3B2C3，即在尿道脱细胞处理过程中采用0．4％胰蛋白酶、1％甲醛加0．2％戊二醛、40U／ml DNA酶。     

口腔黏膜细胞与膀胱黏膜下脱细胞基质复合物构建组织工程化尿道的实验研究

目的 探讨以兔口腔黏膜细胞与同种异体膀胱黏膜下脱细胞基质(BAMG)复合物构建组织工程化尿道的可行性.方法 新西兰雄性兔24只,距尿道外口2.0 cm剥离尿道黏膜(2.0 cm×0.8 cm)后,随机分实验组和对照组,每组12只.切取实验组兔口腔黏膜组织分离细胞,在有灭活的3T3细胞培养皿上进行培养扩增,将培养获得的第2代口腔黏膜细胞种植于BAMG(2.2 cm×1.0 cm)上,植入实验组兔尿道缺损区域;对照组单纯采用无细胞植入的BAMG修复尿道.分别于术后1、2、6个月观察动物排尿情况,行尿道造影,8 F尿管插管确定有无狭窄;随后处死实验兔,取修复段尿道黏膜组织行组织学检查.结果 细胞培养获得的口腔黏膜细胞形态均一,生长良好;组织形态学、扫描电镜观察见口腔黏膜细胞与BAMG具有良好的相容性.实验组兔术后1、2、6个月伤口愈合良好、排尿通畅,无尿瘘发生,组织学和尿道造影检查显示带细胞修复的尿道形态完整、清晰宽敞,无狭窄发生;术后6个月植入的口腔黏膜细胞仍然存在,并明显扩增.对照组兔则出现排尿困难、尿道狭窄,光镜下发现黏膜及黏膜下存在严重的炎症反应.结论 兔口腔黏膜细胞与同种异体BAMG复合后,可成功用于尿道缺损的修复,构建组织工程化尿道.     

脱细胞尿道细胞外基质修复尿道缺损的临床研究

目的 总结新型替代材料修复尿道缺损的临床经验.方法 采用组织工程学材料脱细胞尿道细胞外基质(ECM)修复男性长段尿道缺损8例.患者平均年龄56岁.病程3～60个月,平均16.7个月.其中陈旧性前尿道狭窄4例、球部尿道狭窄2例、狭窄段从前尿道延伸到后尿道2例.狭窄长度3～8 cm,平均5 cm.结果 8例随访3～21个月,平均12.5个月.术后6周拔除尿管2例,8周拔管6例.无狭窄、尿瘘,排尿通畅7例,1例排尿不畅者定期行尿道扩张.术后8周尿道造影显示尿道通畅,未见吻合口狭窄.结论 脱细胞尿道细胞外基质为机体提供了良好的供宿主组织爬行替代的框架,在修补后可有效地与宿主组织融合,修补后的尿道愈合良好.     

Curative effect and histocompatibility evaluation of reconstruction of traumatic defect of rabbit urethra using extracellular matrix

Objective： To investigate the curative effect and histocompatibility of reconstruction of traumatic urethral defect of rabbit using urethral extracellular matrix （ECM）.
Methods： Urethral ECM was obtained by excision of the urethra in 20 donor rabbits. In experimental group, 20 rabbits were resected a 1.0 cm-1.5 cm segment of the urethra and artificially made a model of traumatic urethral defect, then reconstructed by the urethral extracellular matrix of the same length. The rabbit immunity response was assessed by lymphocyte transformation test and serum TNF- α level. The reconstructed urethral segments were stained with hematoxylin-eosin and Van Gieson stain and observed by histological examination postoperatively. The urethrography, urethroscopy and urodynamic examinations were performed.
Results： There was no significant difference in stimulative index of lymphocyte transformation between ECM group and control group. The serum TNF- α levels of ECM group slightly rose, but the increase was not significant as compared with control group. On postoperative day 10, epithelial cell had migrated from each side and small vessels were found in the extracellular matrix. In the 3rd week, several layers of urothelium covered the whole surface of the matrix tube. In the 6th week, the disorganized arrangements of smooth muscle fibers were firstly observed by Van Gieson staining. In the 24th week, the smooth muscle cells increased and the matrix tube appeared fairly similar to normal urethral wall components. The urethroscopy and urodynamic evaluation revealed that the surface of reconstructed urethra was smooth and emiction was unobstructed.
Conclusion： The urethral extracellular matrix might be an ideal and safe biomaterial for the reconstruction of urethral traumatic defect.     

Urethral replacement: a comparison between small intestinal submucosa grafts and spontaneous regeneration

OBJECTIVE: To evaluate the use of commercially available single-layer small intestinal submucosa (SIS) for urethral replacement, both as an onlay and as a tube, in a rabbit model. MATERIALS AND METHODS: Thirty-six male rabbits were assigned to four experimental groups. Group 1 had the ventral wall of the penile urethra excised for 15 mm; in group 2 this created defect was patched with a SIS onlay graft; group 3 had complete excision of a 15 mm segment of the penile urethra; and in group 4, this created defect was replaced with a SIS tube graft. In all rabbits the urethra was stented for 2 weeks. A retrograde urethrogram was taken in all rabbits before death at 3, 6 and 12 weeks after surgery. The urethra was then exposed, examined carefully and excised for histopathological examination. RESULTS: In groups 1 and 2 the retrograde urethrograms were normal in 13 rabbits and there was relative narrowing in two rabbits in group 1 and three in group 2. In groups 3 and 4 all rabbits developed urethral fistulae or strictures. Histological examination of the urethra showed epithelial regeneration supported by smooth muscle backing in all rabbits in group 1, while rabbits in group 2 showed no regeneration of smooth muscle. By contrast, rabbits in groups 3 and 4 showed incomplete regeneration and progressive fibrosis. CONCLUSIONS: Single-layer SIS is not a suitable urethral substitute in this animal model. When used as an onlay, healing is inferior to spontaneous urethral regeneration, as SIS impedes smooth muscle cell regeneration. When used as a tube, there is complete scarring and urethral luminal occlusion.     

Urethral Reconstruction Using Bone Marrow Mesenchymal Stem Cell– and Smooth Muscle Cell–Seeded Bladder Acellular Matrix

Background

Congenital or acquired abnormalities may lead to a urethral defect that often requires surgical reconstruction. The traditional methods often lead to complications, including urethrocutaneous fistulae and strictures. In this study, we proposed to construct a tissue-engineered sheet graft (TESG) using a bone marrow mesenchymal stem cell (BMSC)– and smooth muscle cell (SMC)–seeded bladder acellular matrix (BAM) for urethral reconstruction.

Methods

Rabbit BMSCs and SMCs were isolated, expanded, and identified in vitro before seeding into BAM as the experimental group, whereas BAM-only was the control group. The graft was used to construct TESG for implantation into the rabbit omentum for 2 weeks before urethral reconstruction. We divided 24 male rabbits into four experimental groups six each, and six other were the control group. Histological analysis was performed at 2 weeks, 4 weeks, 8 weeks, and 16 weeks postoperatively. Retrograde urethrography was performed at 16 weeks postoperatively.

Results

All experimental rabbits survived to they were humanly killed. At 8 weeks, there was no difference between the graft and the normal urethra with no severe shrinkage. At 8 and 16 weeks after TESG grafting in vivo, multilayer urothelium covered the graft, neovascularization was visible within the center of TESG, and organized smooth muscle bundles were present. Retrograde urethrography failed to demonstrate diverticula formation or urethral stricture. Three control rabbits died within 4 weeks postoperatively. Autopsy showed their urethras to be almost completely blocked whereas another three hosts displays urethral strictures.

Conclusion

A TESG was constructed using a BMSC- and SMC-seeded BAM for urethral reconstruction.     

异种膀胱无细胞基质替代尿道的研究

目的探讨异种膀胱无细胞基质(ACM)管状替代尿道的可行性。方法19只成年雄性新西兰白兔分成3组：A组3只，为假手术对照组；B组10只，切除一段1．0cm尿道；C组6只，切除一段3．5～4．0cm尿道，之后应用已经事先制备好的异种膀胱ACM制成相当长度的管状替代被切除的尿道。术后1、2、4、8、16周动态观察替代尿道的尿道上皮、平滑肌和血管的再生情况。结果所有实验动物在术后7d拔除尿管后都恢复了自主排尿，没有排斥、尿瘘、感染等并发症发生。组织学检查显示实验组术后2周尿道上皮再生良好，4周完全覆盖尿道内腔，术后8周平滑肌见于近吻合口处，平滑肌生长缓慢，观察期内未能覆盖全长尿道。尿道造影未见明显尿道狭窄和憩室。结论异种膀胱ACM是一种良好的尿道修复和替代的材料。     

Rabbit urethra replacement with a defined biomatrix or small intestinal submucosa

OBJECTIVE: The evaluation of collagen-based biomatrix (SIS COOK((R))) in comparison to a biochemically reconstructed biomatrix for replacement of the urethra in a rabbit model as a preclinical model. MATERIAL AND METHODS: Rabbits underwent partial urethra replacement (resection of 0.5 to 1.0 cm segment of the urethra), which was replaced with 1 or 4 layers Small Intestinal Submucosa (SIS COOK) patch grafts or with a biochemically defined collagen biomatrix, partly sutured with unresolvable sutures for future reference. Six animals underwent a sham control operation. The grafts of regenerated urethras were harvested at 1, 3 and 9 months after implantation. Urethrography was performed pre-operatively and before sacrificing. The animals were evaluated macroscopically and by routine histology and immunohistochemistry. RESULTS: At 1 month after implantation, the biomatrices (1 layer, 4 layers and our biochemically defined biomatrix) were well distinguishable from the normal surrounding tissues and showed blood vessels at the periphery. Macroscopically, the unresolvable reference sutures were easy to find at all time points. At 3 months the graft was still distinguishable in the 4 layers SIS group. In the 1 layer and the defined biomatrix group a good regeneration of the urethra within the graft was seen with some central fibrosis. Histological and immunohistochemical evaluation showed urothelium regeneration on the 1 layer and on biochemically defined biomatrix with decreasing number of inflammatory cells from 1 month on. In the group treated with 4 layers SIS the urothelium was completely regenerated at 3 months. Histologically, the regeneration of muscle cells in the three biomatrices was comparable. The smooth muscle cells regenerated very slowly as 1 month after implantation no muscle cells were detectable within the grafts. At 3 months a few muscle cells were present in the graft, but cell density did not increase in the following 6 months. Strictures were not observed on control urethrography pre-operatively in the animals. In one case slight narrowing of the urethra on urethrography was seen, but apparently without causing voiding problems. One rabbit developed a fistula near the operation site. CONCLUSION: The biomatrices investigated are feasible scaffolds to repair urethral lesions. The results with our biochemically defined biomatrix are comparable to one layer Small Intestinal Submucosa. Almost no smooth muscle cells population was observed after nine months for the three biomatrices. We conclude that an improved molecularly defined biomatrix focussed on stimulation of smooth muscle cell growth may be necessary to obtain optimal cellular grafting results.