膀胱平滑肌细胞与小肠黏膜下层体外复合培养的实验研究

目的探讨体外快速培养犬膀胱平滑肌细胞的方法及观察膀胱平滑肌细胞在脱细胞小肠黏膜下层(small intestinal submucosa,SIS上的生长状况,为构建组织工程膀胱平滑肌组织提供实验依据。方法分别采用酶消化法和组织块培养法分离、获取和原代培养犬膀胱平滑肌细胞,倒置相差显微镜下观察细胞的生长情况,透射电镜观察细胞的超微结构,免疫组织化学染色进行细胞鉴定。将犬膀胱平滑肌细胞接种到SIS支架材料上,于复合培养5、7及9d取材,行苏木素染色、石蜡切片HE染色和扫描电镜观察膀胱平滑肌细胞在SIS上的生长状况。以细胞-SIS复合培养组为实验组,以膀胱平滑肌细胞为对照组,每组各设9孔,分别于接种后3、5及7d取材,酶消化后收集细胞并计数。结果酶消化法原代培养获取犬膀胱平滑肌细胞数量多,细胞生长速度快,形态良好,培养5d细胞在培养瓶底生长汇合。组织块培养法接种3d见长梭形的膀胱平滑肌细胞从植块边缘萌出,获取的细胞数量较少。透射电镜下见膀胱平滑肌细胞胞质中有特征性细肌丝和细胞膜的密斑。抗α-肌动蛋白免疫组织化学染色胞浆呈棕黄色阳性反应。膀胱平滑肌细胞在SIS表面能黏附、生长和增殖。体外复合培养5d后,膀胱平滑肌细胞铺满SIS表面,呈单层细胞结构。7、9d细胞形态与5d相似。实验组3、5及7d的细胞计数分别为(16.85±0.79)×105、(39.74±2.16)×105及(37.15±2.02)×105个,对照组分别为(19.43±0.54)×105、(34.50±1.85)×105及(33.07±1.31)×105个。两组5d细胞计数差异有统计学意义(P<0.05)。结论酶消化法原代培养膀胱平滑肌细胞可提供大量活性良好的种子细胞。SIS支持膀胱平滑肌细胞黏附和生长,可为构建组织工程膀胱平滑肌组织提供良好支架。     

异源性膀胱脱细胞基质移植修复兔膀胱缺损的局部和全身安全性评价北大核心CSCD

目的采用异源性膀胱脱细胞基质(bladder acellular matrix,BAM)移植修复兔膀胱缺损,检测组织再生和机体的组织反应情况,评价其生物安全性。方法将完整新鲜猪膀胱通过一系列物理、化学和酶消化法进行脱细胞处理,得到猪BAM,通过HE染色和扫描电镜观察其脱细胞效果和超微结构。将18只新西兰大白兔(体重2.5～3.0 kg)随机分为实验组(n=12)和对照组(n=6),行膀胱部分(约30%)切除术,实验组采用与切除面积等大的猪BAM替代修补,对照组直接行膀胱切口原位缝合。术后观察动物存活情况,分别于术后15 d,1、2、3、6个月行血常规及肾功能、电解质检测。术后1、2、3、6个月行尿流动力学检测最大膀胱容量、排尿点膀胱内压力以及膀胱顺应性;然后处死动物,大体观察膀胱再生情况,并取再生膀胱组织行HE染色及免疫组织化学染色;同时取周围脏器及淋巴组织,经大体观察及HE染色观察有无异常变化。结果制备的猪BAM完全去除了细胞成分,扫描电镜观察呈多孔样结构。实验动物均存活至取材各时间点,血常规检测示术后15 d两组白细胞计数升高,之后逐渐恢复至正常水平,两组间差异无统计学意义(P>0.05)。肾功能、电解质检测提示两组间差异无统计学意义(P>0.05),其中肌酐有上升趋势,但至术后6个月时仍维持在正常范围内。术后实验组最大膀胱容量及膀胱顺应性与对照组相比明显恢复,术后3、6个月两组比较差异有统计学意义(P<0.05);两组间各时间点排尿点膀胱内压力比较差异均无统计学意义(P>0.05)。组织学观察可见两组中均有尿路上皮层、平滑肌组织和血管的再生。两组术后1个月均可见慢性炎性细胞浸润,随后慢性炎性细胞明显减少(P<0.05),直至基本消退;术后3、6个月,两组间慢性炎性细胞浸润程度评分比较差异无统计学意义(P>0.05)。随着时间推移,两组α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达量明显增多(P<0.05),对照组各时间点α-SMA表达量明显高于实验组(P<0.05)。取检全身其他器官及区域淋巴结经大体观察及HE染色观察均无异常改变。结论采用猪BAM进行异种移植修补兔膀胱缺损未发现明显免疫排斥反应,具有相对安全性。     

尿路上皮细胞和膀胱脱细胞基质的相容性研究

目的 研究尿路上皮细胞(UCs)和膀胱脱细胞基质(BAM)的生物相容性.方法 分离培养兔UCs,以1.0×105/cm2均匀接种于BAM上,观察细胞的粘附、生长及增殖.将复合物种植于兔背部皮下检测其组织相容性.结果 典型的UCs呈多角形的“铺路石样”结构,抗CK AE1/AE3抗体免疫组化阳性证实为上皮细胞;BAM内部未见细胞碎片,可见排列规则而紧密连接成网状的胶原纤维;细胞种植到支架上一周后,行HE染色检测显示膀胱上皮细胞在BAM上贴附生长良好;生长曲线显示,实验组和对照组生长曲线相似.结论 UCs在BAM表面生长良好,相容性较好,是组织工程良好的种子细胞.     

组织工程化膀胱平滑肌结构的体外构建

目的 利用组织工程方法在体外初步构建膀胱平滑肌结构.方法 置备膀胱脱细胞基质移植物(BAMG)并以HE和Masson染色进行支架材料评价.酶消化法获得膀胱平滑肌原代细胞,分别以免疫荧光和RT-PCR进行细胞鉴定.经过体外培养和扩增后,将P3代膀胱平滑肌细胞接种在BAMG上.结果 支架上的膀胱平滑肌细胞经过体外培养4周后形成多层结构,同时免疫组化检测α-Smooth Muscle Actin表达阳性.结论 运用组织工程方法能够在体外进行膀胱平滑肌结构的初步构建,为进一步膀胱组织工程修复奠定技术基础.     

组织工程化膀胱壁复层结构的体外构建

目的 探讨膀胱缺损组织工程修复方法.方法 机械法去除膀胱黏膜层、肌层和浆膜层,仅保留膀胱黏膜下层.经SDS、Trixon-100和三蒸水处理去除细胞结构,制备获得膀胱脱细胞基质移植物(BAMG)作为支架材料.取成年猪膀胱壁,机械法分离膀胱肌层和黏膜层.以酶消化法分别获取膀胱尿路上皮细胞和平滑肌细胞.常规细胞传代和扩增后,将膀胱平滑肌细胞和尿路上皮细胞分别接种在BAMG浆膜面和黏膜面.细胞-材料复合物体外培养4周并行组织学鉴定.结果 HE和Masson染色鉴定发现,BAMG以胶原成分为主,内部未见明显细胞残留.免疫荧光方法鉴定结果显示,膀胱尿路上皮细胞uroplakin Ⅱ阳性,膀胱平滑肌细胞α-平滑肌肌动蛋白阳性.体外培养4周后,BAMG浆膜面和黏膜面分别形成连续的复层结构尿路上皮层和膀胱平滑肌层,免疫组化显示平滑肌层α-平滑肌肌动蛋白表达阳性,尿路上皮层广谱角蛋白表达阳性.结论 以膀胱脱细胞材料作为支架,尿路上皮细胞和膀胱平滑肌细胞作为种子细胞,能够在体外构建形成包含膀胱黏膜层及平滑肌层的复层膀胱壁结构.细胞-材料复合物的体外构建将为体内膀胱缺损的组织工程修复提供实验依据.     

膀胱上皮细胞和膀胱脱细胞基质构建组织工程管状移植物行兔膀胱切除术后尿流改道

目的利用膀胱脱细胞基质(BAM)和膀胱上皮细胞构建组织工程管状移植物(TETG), 在兔模型上验证其进行尿流改道的可行性。方法选取雄性新西兰大白兔48只, 将其分为对照组和实验组, 各24只, 首先制作兔的BAM, 体外培养扩增兔膀胱上皮细胞, 观察上皮细胞在BAM上的生长情况。将复合物包绕尿管制作成长为4 cm、长径为0.8 cm的TETG, 使膀胱上皮细胞位于管腔内面, 将TETG移植到兔模型上验证其进行尿流改道的可行性。分别于尿流改道术后1、2、4、8周进行组织学、免疫组化检查, 术后12周进行静脉尿路造影(IVU)检查流出道的通畅情况。结果 BAM行HE检测染色显示未见细胞生长;膀胱上皮细胞种植到BAM上的生长曲线结果显示细胞生长良好。将TETG移植到动物体内进行尿流改道, 结果显示, 实验组均存活, 取材时发现移植物与盆腔脂肪轻度粘连, 未见明显尿瘘、狭窄及严重的肾积水。组织学检查揭示了有管腔上皮的生长。免疫组化检查也证实了管腔内上皮的再生情况, TETG移植到体内, 随时间增长, 内腔覆盖多层的成熟上皮细胞, 并可见新生血管形成。术后12周行IVU和膀胱镜检未见梗阻发生。相...     

犬膀胱平滑肌细胞的体外连续培养

目的通过体外培养不同代次的犬膀胱平滑肌细胞,比较其生物学特征,探讨多次传代后的平滑肌细胞作为种子细胞的可行性,为构建组织工程膀胱和尿道筛选种子细胞提供方法和依据。方法取体重10～12kg的12月龄雄性犬膀胱肌层,混合酶消化法获取平滑肌细胞,并于含10%小牛血清的培养基中培养,观察比较不同代次细胞的形态变化及生长、增殖过程,电镜下观察细胞超微结构,并通过免疫组织化学方法检测特征性蛋白的表达。结果体外培养的平滑肌细胞单层生长至亚融合状态后,倒置相差显微镜下细胞呈长梭形,并呈峰-谷样结构,可连续传至12代。生长曲线显示第7代以前的细胞具有相似的增殖特点,约每40小时为1次生长周期,透射电镜下可见平滑肌细胞特有的密体结构,平滑肌肌动蛋白免疫组织化学染色为阳性;第7代后细胞增殖逐渐迟滞,至第10代细胞内肌丝及密体结构消失。结论犬膀胱平滑肌细胞可在体外连续培养,通过适当的纯化方法可获得大量高纯度的平滑肌细胞。第7代以前的平滑肌细胞均可作为种子细胞,用于构建组织工程膀胱和尿道。     

膀胱出口梗阻后的膀胱功能保护

膀胱出口梗阻(Bladder outlet obstruction,BOO)可以引起膀胱结构和功能的改变,本文就BOO后引起的膀胱α1受体的变化、平滑肌肌球蛋白的变化和与缺血缺氧相关的变化等以及BOO后膀胱功能的药物保护作一综述。     

聚羟基乙酸作为支架材料体外构建复层膀胱壁结构的探讨

目的：探讨聚羟基乙酸（polyglycolic acid,PGA）作为支架材料并复合成年猪膀胱平滑肌细胞和尿路上皮细胞,体外构建复层膀胱壁结构的可行性。方法：采用PGA作为支架材料。并以聚乳酸（polylact acid,PLA）塑形。以酶消化法分别获得猪膀胱尿路上皮细胞和平滑肌细胞,并体外扩增。以MTT法分别检测P3代膀胱平滑肌细胞（SMC）和尿路上皮细胞（UC）的体外增殖能力。将P3代膀胱平滑肌细胞先接种在支架上,再接种尿路上皮细胞。将细胞一材料复合物体外培养1～2周并行组织学鉴定。结果：酶消化法获得的膀胱平滑肌细胞和尿路上皮细胞经过体外培养仍具有较强的体外增殖能力。细胞材料复合物体外培养1周后形成分层排列结构,包括黏膜上皮层和膀胱平滑肌层。免疫组织化学显示平滑肌层α-Smooth Muscle Actin表达阳性,黏膜上皮层广谱角蛋白表达阳性。而细胞材料复合物继续体外培养至2周,细胞从支架上大量脱落。结论：PGA适合作为复层膀胱壁结构体外构建的支架材料。采用PGA作为支架材料并复合膀胱平滑肌细胞和尿路上皮细胞,可以体外构建出类似复层膀胱壁结构。细胞材料复合物体外培养1周左右较适合进行体内回植修复。     

血管细胞外基质和膀胱平滑肌细胞的生物相容性研究

目的探讨血管细胞外基质(VECM)和膀胱平滑肌细胞的细胞相容性和组织相容性,为临床应用提供依据。方法制备兔VECM,分离培养兔膀胱平滑肌细胞,并以1×10~6个细胞/ml种植于VECM上,相差显微镜和扫描电镜观察细胞的黏附及生长。制备VECM和乳胶的提取液,分别作为实验组和阳性对照组,以培养基为阴性对照组,以无细胞的单纯培养基作为空白对照组。取2～4代对数生长期的膀胱平滑肌细胞,四甲基偶氮唑盐(MTT)法测定VECM浸提液对培养平滑肌细胞的细胞毒性；将VECM植入异体兔皮下,观察其组织相容性。结果兔膀胱平滑肌细胞能在体外培养扩增；接种1h后已有部分细胞黏附于VECM上,随时间推移,细胞逐渐增多,并伸展成梭形；培养第1、3、5天,实验组细胞增殖率分别为95．61%、98．34%、102．91%；阴性对照组为100．00%；阳性对照组分别为35．14%、38,95%、32．66%；实验组细胞增殖率明显高于阳性对照组,差异有统计学意义(P＜0．01),与阴性对照组比较差异无统计学意义(P＞0．05)。动物皮下埋植实验无排斥反应发生。结论VECM具有良好的生物相容性,是一种较好的泌尿道修复支架材料。     

Calcineurin mediates bladder smooth muscle hypertrophy after bladder outlet obstruction

PURPOSE: Bladder outlet obstruction (BOO) leads to compensatory bladder hypertrophy. However, the hypertrophy mechanism remains elusive. We report that calcineurin (Cn) is involved in bladder hypertrophy. MATERIALS AND METHODS: Partial BOO was surgically induced in 10-week-old female Wistar rats. The bladder was excised 2, 4 and 6 weeks following surgery in 9 rats each. Histological study was performed at each time point. Cn expression was examined by Western blot analysis. Myosin heavy chain expression was evaluated on gels stained with Coomassie blue. Primary cultured bladder smooth muscle cells were infected with recombinant adenoviruses encoding a constitutive active form of CnA (DeltaCnA), CnB and lacZ, and cell size was measured. RESULTS: In histological findings bladder smooth muscle hypertrophy was observed 2 and 4 weeks after surgery. However, the thickened muscles became thinner 6 weeks after BOO. CnA expression 2 weeks after BOO increased 3.2-fold compared with that of controls. Expression significantly decreased 4 and 6 weeks after surgery. In contrast, CnB expression was unchanged throughout hypertrophy development. Changes in myosin heavy chain expression correlated with changes in CnA. We observed significant hypertrophy in DeltaCnA and CnB over expressing smooth muscle cells. Moreover, FK506, which is a potent inhibitor of Cn, blocked hypertrophy in DeltaCnA and CnB over expressing smooth muscle cells. CONCLUSIONS: These data suggest that Cn has an important role in the induction of bladder smooth muscle hypertrophy.     

Mesenchymal cells infiltrating a bladder acellular matrix gradually lose smooth muscle characteristics in intraperitoneally regenerated urothelial lining tissue in rats

OBJECTIVE: To characterize serial long-term histological changes in mesenchymal cells infiltrating a collagen-based matrix, as in a hollow organ with differentiated urothelial lining created intraperitoneally by grafting cultured urothelial cells, mesenchymal cells with smooth-muscle immunohistochemical characteristics infiltrated into the scaffold, despite no mesenchymal cells being seeded into the scaffold before grafting. MATERIALS AND METHODS: To regenerate a urothelial lining tissue intraperitoneally, rat urothelial cells were cultured and seeded with the feeder-layer technique onto bladder acellular matrix (BAM). After 7 days of cultivation to attach urothelial cells on the BAM, the matrix was folded with the urothelial cells inside and grafted onto the mesentery of the previously partially cystectomized rat. RESULTS: The grafted urothelial cells on the BAM, which formed a monolayer before grafting, stratified into three to four layers as early as 4 days after grafting. Although the regenerated urothelium became thinner with time, there was urothelial stratification and a peculiar angular appearance on the apical surface of the regenerated urothelium even after 56 days. The mesenchymal cells infiltrating the BAM showed positive immunohistochemical staining to alpha-smooth muscle actin or desmin at 7 days. Subsequently, the number of actin- or desmin-positive cells gradually decreased with time. On transmission electron microscopy, the infiltrating mesenchymal cells were characterized as myofibroblasts at 7 days. Smooth muscle-like cells were identified at 14 and 28 days, and fibrocytes were the main population at 56 days. CONCLUSIONS: Although epithelial-mesenchymal interactions have been assumed to be one of the most critical factors in smooth-muscle development, mesenchymal cells infiltrating the scaffold in this intraperitoneal regeneration model gradually lost smooth muscle characteristics with time. These results suggest that interactions between cultured urothelial cells and infiltrating mesenchymal cells alone could not maintain the smooth muscle character of infiltrating mesenchymal cells.     

The effect of a cyclic uniaxial strain on urinary bladder cells

Purpose

Pre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.

Methods

Isolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.

Results

Dynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.

Conclusions

Mechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

     

前列腺增生并发尿潴留膀胱壁病理电镜观察

目的：观察前列腺增生症（ＢＰＨ）并发尿潴留膀胱逼尿肌功能变化。方法：对１０例ＢＰＨ并慢性尿潴留患者实施前列腺摘除术时取膀胱平滑肌组织进行透射电镜观察，同时用１例外伤性膀胱破裂及１例膀胱异物患者的膀胱平滑肌组织作对照。结果：ＢＰＨ并慢性尿潴留患者的膀胱平滑肌显示出下列病变，即平滑肌细胞肥大，排列紊乱，细胞间连接结构减少，其中以胞突连接为主，细胞内线粒体肿胀，胞膜有高电子密度沉积物，脱落细胞胞浆成分散     

In vitro comparative evaluation of recombinant growth factors for tissue engineering of bladder in patients with neurogenic bladder

Background

To compare the effects of various recombinant growth factors on bladder regeneration and angiogenesis for tissue engineering of bladder in patients with neurogenic bladder through in vitro cellular biological methods.

Materials and methods

Human bladder smooth muscle cells (HBSMCs) and human bladder urothelial cells (HBUCs) were cultured from patients with neurogenic bladder and used for comparative evaluations of various growth factors. Human umbilical vein endothelial cells (HUVECs) were also used. Eight potential growth factors, platelet-derived growth factor BB (PDGF-BB), platelet-derived growth factor CC (PDGF-CC), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), and transforming growth factor beta 1 (TGF-β1), were selected and their effects on the proliferation, migration, and wound healing of HBSMCs, HBUCs, and HUVECs were compared.

Results

PDGF-BB, PDGF-CC, bFGF, VEGF, IGF-1, or HGF enhanced the proliferation, migration, and wound healing of HBSMCs, whereas TGF-β1 inhibited their proliferation. Proliferation, migration, and wound healing of HBUCs and HUVECs were enhanced by bFGF, VEGF, EGF, IGF-1, or HGF, whereas inhibited by TGF-β1. PDGF-BB failed to enhance cell activity of HUVECs, whereas PDGF-CC could enhance their migration and wound healing. PDGF-BB, EGF, and VEGF were the most potent factors for stimulating the activities of HBSMCs, HBUCs, and HUVECs, respectively.

Conclusions

Our findings suggest the potential use of a combination of PDGF-BB, EGF, and VEGF for bladder regeneration and angiogenesis. The synergetic effects of the three growth factors on cell activities in a three-dimensional scaffold and an animal model with neurogenic bladder need to be further evaluated.     

一氧化氮合成酶在膀胱癌中的表达及意义

目的　了解三种类型一氧化氮合成酶 (NOS)在膀胱癌中的表达情况 ,探讨其与肿瘤发生发展的关系。　方法　对 2 5例开放手术切除的膀胱癌组织标本进行三种NOS免疫组织化学染色 ,自动图像分析仪对染色程度分级 ;6例肾移植供体正常膀胱组织标本作为对照。　结果　诱导型NOS(iNOS)在肿瘤上皮细胞呈阳性表达 ,在正常膀胱移行细胞不表达或仅微弱表达 (P <0 .0 5)。内皮细胞型NOS (eNOS)在肿瘤基质毛细血管内皮细胞中的表达阳性率 1 0 0 % ,高于对照组中的 67%。两组标本中神经型NOS(nNOS)表达部位及强度相似。统计学分析显示iNOS和eNOS表达与肿瘤分级、分期无明显相关性 ,P均 >0 .0 5。　结论　iNOS及eNOS在膀胱癌的高表达可能与肿瘤的发生发展有关。     

Inhibition of pressure induced bladder smooth muscle cell hyperplasia using CRM197

PURPOSE: In vivo the effects of sustained hydrostatic pressure on the bladder wall and its components are evident under physiological and pathological conditions. We previously reported that exposure of bladder smooth muscle cells to 20 and 40 cm. H2O hydrostatic pressure for as little as 1 hour resulted in the up-regulation of heparin binding epidermal growth factor messenger RNA in a time dependent fashion as well as in activation of the heparin binding epidermal growth factor growth factor gene. In our current study we investigated the use of CRM197 as an agent for blocking undesirable cellular level events, such as smooth muscle cell hyperplasia, eliminating the irreversible alterations in bladder and kidney function that result from chronic and/or severe bladder outlet obstruction. MATERIALS AND METHODS: Control and experimental neonatal ovine smooth muscle cells were exposed to 0.3 pressure and 8.5 cm. H2O, respectively, for 7 days. We evaluated the mitogenic activity of the supernatant medium from bladder smooth muscle cells exposed to 8.5 cm. H2O for 5 days (conditioned medium) before and after the addition of 0.1 mg./ml. CRM197. Bladder smooth muscle cell apoptosis was also assessed after CRM197 exposure. Statistical analysis was performed using the Student t test with p <0.05 considered significant. RESULTS: Exposing bladder smooth muscle cells to sustained 8.5 cm. H2O hydrostatic pressure for 7 days resulted in increased cell proliferation. Conditioned medium contained mitogenic activity, which was ablated after CRM197 was added. No direct toxic effect of CRM197 on bladder smooth muscle cell growth was appreciated (no apoptosis). CONCLUSIONS: We demonstrated a proliferative response of neonatal bladder smooth muscle cells after exposure to sustained hydrostatic pressure. This response was partially due to the release of heparin binding epidermal growth factor and was blocked by adding CRM197. These data support the potential use of CRM197 in drug targeted therapy for diseases involving bladder outlet obstruction.     

Development of a seeded scaffold in the great omentum: feasibility of an in vivo bioreactor for bladder tissue engineering

OBJECTIVES: Tissue engineering is very promising in bladder reconstruction. However, one of the main problems is to limit the development of ischaemic fibrosis during tissue maturation. We describe a model using the omentum as an in vivo bioreactor for a previously seeded scaffold. METHODS: Bladder biopsies were taken from five female pigs, from which both urothelial and smooth muscle cells cultures were made. These cultured cells were used to seed a sphere-shaped small intestinal submucosa (SIS) matrix, which was transferred into the omentum after 3 wk of cell growth. The grafts were harvested 3 wk later and histologic, immunohistochemical, and functional studies were performed. RESULTS: We obtained a highly vascularized tissue-engineered construct that contracted in response to acetylcholine stimulation. The wall thickness was 4mm, on average. Histologic and immunostaining analysis of the construct confirmed the presence of a multilayer urothelium on the luminal aspect and deeper fascicles organised tissue composed of differentiated smooth muscle cells and mature fibroblasts without evidence of inflammation or necrosis. Large- and small-diameter vessels were clearly identified histologically in the tissue obtained. CONCLUSION: The omentum permitted in vivo maturation of seeded scaffolds with the development of a dense vascularisation that is anticipated to prevent fibrosis and loss of contractility. This in vivo maturation into the omentum could be the first step before in situ implantation of the construct.