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。     

脱细胞膀胱粘膜下筋膜修复尿道的实验研究

目的探索组织工程方法在尿道修复中的应用。方法将12只雄兔作为实验对象,切除部分尿道的全周粘膜,长度分1cm以内(Ⅰ组)和2cm以上(Ⅱ组)两组,将制备完成的同种家兔膀胱粘膜下脱细胞筋膜用可吸收线,缝合到尿道缺损处,术后2周、6周分别处死一批实验对象,通过尿道造影,大体观察,修复段组织切片HE染色光镜研究和免疫组化等来研究修复段尿道的解剖和组织学变化。结果Ⅰ组仅1只(1/6)发生尿瘘,其余尿道造影显示尿道基本通畅；而Ⅱ组4只(4/6)发生狭窄(不伴尿瘘),1只(1/6)发生尿瘘；术后2周两组植入筋膜依然存在；术后6周时,尿道植入筋膜已降解,Ⅰ组的修复段尿道已全部被宿主自身粘膜所覆盖,粘膜光洁完整,管腔无明显狭小；Ⅱ组的修复段尿道中间苍白增厚,致密坚硬且管腔狭小。结论单纯脱细胞膀胱粘膜下筋膜能修复短距离兔尿道缺损,而对长距离缺损修复效果不理想。     

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。     

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.     

表皮细胞作为种子细胞构建尿道的长期随访比较研究

目的将通过分离培养的表皮细胞与脱细胞膀胱黏膜下筋膜复合，修复缺损的尿道，观察表皮细胞表型在尿道环境中的转归。方法采用同种异体（家兔）的膀胱，经显微分离和脱细胞液处理，制成无细胞的生物支架（并经随机切片证实脱细胞效果），选择兔的小块包皮组织，消化收集分离出的表皮细胞，经过增殖、传代培养植入生物支架中，并加入Brdu标记物，将其卷成管状，回植入自体人工造成的尿道缺损，术后1、2、6及12个月分别测定治疗效果（每组各处死3只家兔／批），观察指标：尿道造影、大体外形、修复段尿道黏膜的HE染色、免疫组化和荧光标记等。结果术后实验对象伤口愈合正常，修复尿道的大体形态和尿道造影显示排尿通畅，无尿瘘和狭窄发生；HE染色和免疫组化显示，术后1月-6月，修复段尿道黏膜层次由单一向复层结构转变；而术后12月修复段尿道黏膜已基本显示为类似移行上皮细胞结构；术后角蛋白染色阳性；Brdu标记在术后1月清晰显示植入上皮细胞层存在，随后逐渐稀少。术后6及12月尿道黏膜结构中未见显影。结论作为种子细胞，表皮细胞可与胶原筋膜结合运用于尿道修复，修复效果良好，并随着时间延长向尿道原有细胞结构转变。     

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.     

脱细胞基质载体和表皮细胞结合构建尿道的实验研究

目的探索组织工程修复技术在尿道构建中的应用前景。方法采用同种异体家兔膀胱,经显微外科分离和脱细胞液处理,制成无细胞的生物支架,12只雄兔随机分为成实验和对照2组,剥离实验对象尿道黏膜2cm;实验组切取小块兔包皮组织,消化收集分离出的表皮细胞,经过增殖、传代培养,植入生物支架中,培养2周,并加入Brdu标记物,将其卷成管状,植入实验组人工尿道缺损区域;对照组单纯采用无细胞植入的生物支架修复尿道;术前和术后1、2、6个月每组各处死2只家兔/批,分别行尿道造影、大体外形、修复段尿道黏膜的HE染色、免疫组化和荧光标记。结果术后动物伤口愈合正常,排尿通畅,无尿瘘发生。修复尿道大体形态和尿道造影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1个月,HE和免疫组化显示,修复段尿道黏膜层次单一,缺乏复层和乳头结构。术后2个月基本恢复正常尿道结构,复层上皮结构形成,角蛋白染色阳性。术后6个月黏膜复层上皮结构更为丰富,角蛋白染色阳性;Brdu标记在术后1个月清晰显示植入上皮细胞层存在,术后2个月植入的原始上皮细胞显影稀少。术后6个月尿道黏膜结构中未见显影;而单纯生物支架修复组的实验对象则出现排尿变细,任何观察时段,均出...     

脱细胞尿道及其海绵体基质制备的实验研究

目的 探索脱细胞尿道及其海绵体基质的制备方法。方法取健康壮年兔完整尿道及其海绵体组织，以Triton-X100与NH3H2O联合提取法进行脱细胞处理。标本做HE染色，组织学观察分析脱细胞效果。结果脱细胞处理11天后，成功获得脱细胞及其海绵体基质，所得基质外观良好。HE染色观察无细胞存在。弹力纤维排列规整，间隙较大，结构无破坏。结论利用Triton-X100与NH3H2O联合提取法可成功制备完整无细胞尿道及其海绵体基质，为尿道再造修复提供崭新思路。     

异体真皮细胞外基质重建尿道的实验和临床研究

目的：寻求理想的尿道修复材料。方法: 2只犬真皮脱细胞处理后制成真皮细胞外基质框架;18只犬分为2组;异体真皮细胞外基质（ECM）移植组（实验组）15只,异体真皮移植组（对照组）3只,全麻下将尿道中段切除5cm,将同样长度真皮ECM或异体真皮缝成直径0．3cm管状替代缺损尿道,8F硅塑管留置4周,实验组分别于术后1,3,6,12,24周每次3只取材行光镜,电镜及免疫组化检查。结果：组织学显示：1周时移植物边缘腔内面有尿上皮,管壁可见少量肌束,6周时移植物区尿路上皮达4－5层,肌层分布均匀,24周后移植物已无法与宿主尿道辨别,对照组术后8－10d移植物坏死出现尿瘘,在实验基础上用于临床2例均获成功。结论ECM为理想的尿道修复材料。     

尿道细胞外基质在兔尿道重建中的应用

目的 评价尿道细胞外基质作为一种生物材料重建尿道的效果. 方法 切取新西兰兔的尿道制备尿道细胞外基质.手术切除实验组1～1.5 cm的尿道片段后用细胞外基质修复缺损,采用ELISA法检测术前、术后12、24及48 h血清TNFα的水平,评估术后兔的免疫反应状态.术后10d和3、6、24周取修复段尿道,行组织学观察并做尿道造影、尿道镜及尿流动力学检查. 结果 术后实验组血清TNFα水平较对照组略有升高,但无统计学意义(P>0.05).术后10 d,上皮细胞开始从边缘向细胞外基质移行并出现新生小血管;3周上皮细胞覆盖细胞外基质的整个管腔;6周出现排列不规则的平滑肌纤维;24周平滑肌数量明显增多,成束状排列.尿道造影、尿道镜及尿流动力学检测检查显示,尿道基质管壁光滑,排尿通畅. 结论 尿道细胞外基质是一种安全有效的尿道重建材料.     

Urethral replacement using epidermal cell-seeded tubular acellular bladder collagen matrix

OBJECTIVES: To investigate the feasibility of replacing urinary epithelium cells with foreskin epidermal cells to reconstruct engineered anterior urethra with an acellular collagen matrix. MATERIALS AND METHODS: Acellular collagen matrices were generated from allogeneic rabbit bladder submucosa. In nine rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and labelled with 5-bromo2'-deoxy-uridine (BrdU) before seeding onto a tubular acellular collagen matrix (1.5x1 cm). In male rabbits, a urethral mucosal defect was created, and urethroplasty performed with a tubular acellular collagen matrix seeded with epidermal cells (nine rabbits) or with a matrix with no cell seeding (nine rabbits; control group). Urethrography was done at 1, 2 and 6 months after grafting. The urethral grafts were harvested and analysed grossly and histologically. RESULTS: In the control group, gross views and urethrography revealed stricture of repaired defects at the different sample times. In the experimental group, a wide urethral calibre was maintained with no sign of strictures. Histology in the control group showed a single layer of epithelium cells with disorganized muscle fibre bundles in the submucosa layer at 1 month after grafting, and a transitional cell layer surrounded by disorganized muscle fibre bundles at 2 and at 6 months. Grafts seeded with epidermal cells formed a single-layer structure by 1 month, and at 2 and 6 months there were several layers of epidermal cells with abundant vessels in the submucosa. There was an evident margin between graft epidermal cells and host epithelium at 6 months. The implanted cells expressed keratin, shown by staining with anti-pancytokeratins. Immunofluorescence for BrdU confirmed the presence of implanted epidermal cells at 1 month after grafting; there were fewer positive cells at the implantation site at 2 months. At 6 months, there were several layers of epidermal cells with no signs of BrdU staining. CONCLUSIONS: Urethral reconstruction was better with an acellular collagen matrix seeded with epidermal cells than with the acellular collagen matrix alone. Foreskin epidermal cells seem adequate in replacing urethral epithelium cells for urethral reconstruction.     

Long-term study of male rabbit urethral mucosa reconstruction using epidermal cell

Aim： To investigate the transformation of characteristics of epidermal cells from foreskin which were used to reconstruct male rabbit anterior urethra in combination with acellular collagen matrices. Methods： In three rabbits, autologous foreskin epidermal cells were isolated, expanded in vitro, and seeded （inoculated） onto a tubular acellular collagen matrix, acquired from allogeneic rabbit bladder submucosa. A urethral mucosal defect was created, and urethral reconstruction was performed with the tubular acellular collagen matrix seeded with epidermal cells. Results： On gross examination at 12 months following the procedure, the mucosa of the urethral grafts appeared lubricous and smooth. Urethrography showed that a wide urethral caliber had been maintained without any sign of strictures. Histological examination showed a transitional cell layer in the graft without evidence of a margin between the graft and the host tissue at 12 months postoperatively. Conclusion： Epidermal cells seeded onto acellular collagen matrices can be successfully used to reconstruct urethras that have defects and are transformed to transitional epithelial cells.     

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。     

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.     

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.     

Comparison of partial urethral replacement with acellular matrix versus spontaneous urethral regeneration in a canine model

OBJECTIVE: To determine whether acellular matrix could be used for partial urethral replacement and to compare regeneration over acellular matrix versus normal spontaneous urethral regeneration. MATERIALS AND METHODS: The study included 21 male mongrel dogs in which a 3-cm segment including half of the urethral circumference was excised. In 13 dogs (study group), the defect was covered by acellular matrix of the same length and width obtained from female mongrel dogs and prepared to have complete cell lysis with keeping of the fiber framework. In 8 dogs (control group), the urethral defect was not covered by any urethral tissue. In both groups, an 8F feeding tube was kept inside the urethra for a mean duration of 2 weeks. In the study group, dogs were sacrificed at 1 week, 2 weeks, 3 weeks and then one dog every month for 10 months. In the control group, one dog was sacrificed every month for 8 months. RESULTS: All dogs survived the procedure. In the study group, 10 dogs underwent urethrogram; 8 were normal, 1 had diverticulum and 1 had relative narrowing. In the control group, 6 dogs underwent urethrogram; 5 were normal and 1 showed relative narrowing. Histopathological examination of the study group showed gradual regeneration over the acellular matrix with normal appearance at 20 weeks. In the control group, normal healing was observed at 2 months and thereafter. CONCLUSION: Regeneration of all components of the urethra can occur gradually over acellular matrix and is complete at 20 weeks. Regeneration of a urethral defect 3-cm long including half of the urethral lumen is possible with or without acellular matrix.     

Tunica albuginea acellular matrix graft for treatment of Peyronie's disease--an experimental study in dogs

OBJECTIVE: To study the value of an acellular matrix graft of the tunica albuginea for reconstruction of the penis in cases of severe Peyronie's disease. MATERIAL AND METHODS: In nine mongrel dogs, an acellular matrix graft of the tunica albuginea was used to cover a 30 x 10 mm2 tunical defect. Equal numbers of animals were sacrificed at 1, 3 and 6 months after surgery. Before death, an erection was induced by means of papaverine injection and cavernosography was performed. After death the penis was prepared for histopathological study. RESULTS: All animals survived the surgery and none developed haematoma, wound infections or dehiscence. All dogs developed a straight, rigid erection. Cavernosography showed patent corpora cavernosa in all animals. The papaverine injection and cavernosographic results did not change over time. Inspection of the graft site and measurement of its length and width showed healing with no contracture. Histologically, the regenerated matrix appeared thicker than the neighbouring tunica albuginea in the 1-month group; otherwise the appearance was normal. Gradual orientation of the fibrocytes, capillaries and collagen fibres was demonstrated at 1 month and was complete at 3 and 6 months. Comparison between an implanted tunica at 6 months and a control tunica from a normal dog showed no significant histological difference. CONCLUSION: A homologous acellular matrix graft of the tunica albuginea may be an alternative treatment for severe cases of Peyronie's disease.     

Experimental defect in rabbit urethra repaired with acellular aortic matrix

Urethral reconstruction following failed hypospadias repair or post-traumatic chronic stricture requires adequate amounts of tissue. Many surgical techniques utilizing different types of biological tissues have been attempted: (a) vascularized skin flaps from the prepuce, scrotum or penile shaft; (b) full-thickness free skin grafts; (c) vesical or buccal mucosa grafts; (d) ureter; artery; vein and appendix tissue. More recently, biodegradable polymers have also been used as delivery vehicles of urothelial cells in animals. It has been demonstrated that the implant of an acellular tissue matrix in the bladder can guide the regeneration of urothelium, blood vessels, smooth muscle and nerves. The aim of this study was to create an experimental model of urethral defect, and then repair it by implanting homologous acellular aortic grafts as urethral substitutes. An acellular matrix was obtained by detergent enzymatic treatment of rabbit thoracic aorta. The growth of urethral epithelium was verified in vitro, and homologous acellular vessels were then implanted in rabbits, bridging a previous surgical urethral defect. The outcome of reconstructive surgery was evaluated histologically at 10 days, 3 weeks, 3 and 12 months. As the time after surgery increased, the neourothelium became less thick, signs of inflammatory response disappeared, and the orientation of collagen fibrils and smooth muscle fascicles resembled that of a normal urethra. The implants displayed abundant vascularization, and the luminal surface started to become irregular. Acellular blood vessels may represent a promising approach to urethral defect therapy for different reasons: (a) unlimited availability, (b) readily obtainable in different lengths and gauges, (c) the potential for being organized as tissue bank, and (d) that just one simple surgical procedure is needed. Nevertheless, before this technique can be applied in humans, it must be tested in more species and animals. Received: 25 March 1999 / Accepted: 25 June 1999