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。     

人包皮脱细胞基质组织工程化尿道的可行性研究

目的:为人包皮脱细胞基质作为尿道组织工程修复材料提供依据。方法:制备人包皮脱细胞基质,对其进行组织学观察、细胞毒性以及体内实验,检验其作为尿道材料的安全性和组织相容性。结果:制备的人包皮脱细胞基质,细胞去除完全,用原代包皮上皮细胞测定其细胞毒性,结果显示细胞相对增值率在75%～99%之间,细胞毒性为1级,符合国家标准,回植体内后,随着时间的延长,人包皮脱细胞基质与尿路上皮细胞结合完好,正常尿道复层结构层次逐步恢复。结论:人包皮脱细胞基质抗原性低,相容性好,可以作为组织工程尿道的支架材料。     

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.     

脂肪干细胞复合膀胱脱细胞基质促进膀胱再生的实验研究

目的 探讨脂肪干细胞(adipose-derived stem cell,ADSC)在兔的组织工程膀胱重建中的应用.方法 采用自体脂肪干细胞和膀胱脱细胞基质(bladder acellular matrix graft,BAMG)构建兔组织工程膀胱.应用酶消化法分离培养兔自体的ADSC,并进行流式细胞鉴定.对新鲜的兔膀胱组织进行脱细胞处理制得BAMG.将ADSC扩增后接种于BAMG表面并进行体外培养,获得组织工程膀胱.对24只雄性新西兰兔行40％ ～ 50％膀胱部分切除,分为实验组和对照组,每组12只.实验组采用自体培养的ADSC构建的组织工程膀胱对缺损膀胱进行修复,对照组采用单纯的BAMG进行修复.术后24周进行膀胱造影并取材观察组织修复再生情况. 结果 镜下观察ADSC贴壁后绝大多数为纺锤形,细胞之间以细丝拉网.流式细胞仪检测结果CD90、CD4、CD105、CD166、CD34表达均为阳性,CD106、CD45表达为阴性.ADSC种植于BAMG表面生长良好.修补24周后,对照组和实验组膀胱容量分别为术前的(69.33 ±5.05)％和(94.68±3.31)％.组织染色分析对照组显示上皮层正常,黏膜下纤维组织增生而肌层较薄;实验组则显示了与自体膀胱相似的正常的3层组织结构. 结论 采用ADSC和BAMG构建组织工程膀胱是理想的膀胱替代修补材料.     

脱细胞基质在尿道组织工程中的应用

脱细胞基质是经过物理化学方法 脱去组织中抗原成分且富含胶原的一种材料,在尿道修复和重建中,显示了极其广泛的应用前景.     

人脐动脉平滑肌细胞复合脱细胞基质构建海绵体平滑肌的动物实验研究

目的 探讨脐动脉平滑肌细胞(HUASMC)与阴茎海绵体脱细胞基质(ACCM)复合构建海绵体平滑肌的可行性.方法 以1%的Triton-X100与0.1%NH3H2O混合液对兔阴茎海绵体进行脱细胞处理,制备ACCM.采用贴块法分离、培养、扩增HUASMC.HUASMC以30×10<'6>/ml密度接种ACCM,细胞-ACCM体外复合10 d后将复合物植入9只5周龄BALB/C裸鼠背部皮下,术后10、20和40 d分别对移植物进行HE染色、免疫组织化学染色和器官浴槽实验,评价其裸鼠体内构建情况.结果 ACCM为白色圆柱状,镜下为富含胶原的疏松多孔结构,不含细胞成分.HUA-SMC与ACCM相容性良好,HUASMC在与ACCM接触部位充分伸展,并沿ACCM窦隙活跃生长.9只裸鼠均存活,植入部位无感染,无植入物排斥发生.随培育时间延长,裸鼠体内ACCM逐渐降解,植入的HUASMC分化形成结构良好、交错排列的平滑肌组织.器官浴槽实验显示,构建组织对去氧肾上腺素和电刺激均表现出收缩功能,去氧肾上腺素和电刺激所诱导的最大收缩力分别为(3.64±0.18)和(2.50±0.21)g.结论 HUASMC作为种子细胞与ACCM复合可构建出具有一定形态和功能的组织工程海绵体平滑肌.     

海绵体脱细胞基质和平滑肌细胞相容性的研究

目的探讨海绵体脱细胞基质（ACCM）与人脐动脉平滑肌细胞（HUASMCs）的相容性。方法以1％的Triton-X100与0．1％NH3H2O制备ACCM。异体肌肉内埋植实验评价ACCM的生物相容性。分离培养HUASMCs，噻唑蓝（MTT）法测定ACCM浸提液对HUASMCs增殖的影响。将3—5代的HUASMCs接种ACCM，共培养3、5、10d后观察HUASMCs与ACCM复合情况。结果ACCM无细胞残留，异体肌肉内埋植实验证实ACCM生物相容性良好。浸提液实验显示体外培养第4天和第6天，浸提液组A值明显高于对照组（P〈0．05）。HUASMCs能渗人ACCM内部，并分化形成平滑肌柬。结论ACCM与HUASMCs相容性良好，两者复合有望构建组织工程海绵体平滑肌。     

采用肌卫星细胞及异种无细胞基质构建组织工程膀胱的研究

目的探讨应用组织工程膀胱进行膀胱替代修复的可行性。方法采用自体肌卫星细胞（MDSC）和猪膀胱无细胞基质（PBAM）构建兔组织工程膀胱。应用Percoll非连续密度梯度离心法分离纯化培养兔MDSCs，采用去污剂洗涤法对新鲜猪膀胱进行脱细胞处理获得PBAM。MDSCs接种于PBAM表面并进行短暂体外培养，获得组织工程膀胱。雄性新西兰兔行膀胱大部分切除术，分别采用PBAM及构建的组织工程膀胱进行替代修补，每组6只，1、4、8、12周后进行膀胱造影并取材观察修复组织再生情况。结果非连续密度梯度离心法所得细胞纯度〉90％。酶消化法能有效去除膀胱中细胞成分，光镜及电镜观察无细胞成分残留，细胞毒性测定为1级，细胞相容性好。1周后所构建组织工程膀胱修补吻合区生长良好，12周后修补区组织结构基本接近正常膀胱。容积达到正常膀胱的80％，2组比较差异有统计学意义（P〈0．05）。结论采用MDSC及PBAM构建的组织工程膀胱是理想的膀胱替代修补材料。     

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。     

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.     

复合细胞的膀胱黏膜下层无细胞基质替代长段输尿管的实验研究

目的 评价复合尿路平滑肌细胞和移行上皮细胞的膀胱黏膜下层无细胞基质在大动物体内替代长段输尿管后的结构、功能状况.方法 取长风杂交白猪(不包括在本实验组内)膀胱黏膜下层,洗涤后获得无细胞基质,备用.实验猪10只,切取小块膀胱组织,分离出平滑肌细胞和移行上皮细胞,培养增殖后种植于膀胱黏膜下层无细胞基质,体外共培养.以细胞-无细胞基质复合物替代自体猪一侧中段输尿管,长约5.0～5.5 cm,术后1、2周,1、3、6个月各处死2只,大体观察、组织学和组织浴槽等方法观察替代段输尿管的结构,并进行功能评价.结果 移植替代1个月可见明显肌层形成,3、6个月时已形成良好的移行上皮层和平滑肌层.大体观察见替代段输尿管管腔存在,周围有明显的纤维组织形成.替代段以上输尿管和肾盂扩张.组织浴槽研究显示,替代段输尿管存在平滑肌肌条的一般体外特性.结论 以膀胱黏膜下层无细胞基质作为细胞载体的组织工程输尿管技术在大动物体内替代长段输尿管后,可以获得具有良好组织结构的肌性管道,但其周围的严重纤维化会影响替代段输尿管的功能.     

Urethral stricture repair with an off-the-shelf collagen matrix

PURPOSE: In select patients with urethral strictures in whom genital skin is insufficient alternative tissues are needed for urethral reconstruction. We explored the feasibility of using a bladder submucosa collagen based inert matrix as a free graft substitute for urethral stricture repair. MATERIALS AND METHODS: A total of 28 patients 22 to 61 years old with a diagnosis of urethral stricture underwent reconstructive surgery using a collagen based inert matrix for urethral repair. The inert collagen matrix was trimmed to size as needed for each patient and the neourethra was created by anastomosing the matrix in an onlay fashion to the urethral plate with continuous 6-zero absorbable sutures. The size of the created neourethra ranged from 1.5 to 16 cm. A voiding history, physical examination, retrograde urethrography, uroflowmetry and cystoscopic examinations were performed preoperatively and postoperatively. Random urethral biopsies were also performed. RESULTS: After a 36 to 48-month followup (mean 37) 24 of the 28 patients had a successful outcome. The remaining 4 patients had a slight caliber decrease at the anastomotic sites on urethrography. A subcoronal fistula developed in 1 patient which closed spontaneously 1 year after repair. Mean maximum urine flow rate increased from the preoperative value of 9 +/- 1.29 to 19.7 +/- 3.07 ml. per second postoperatively. Cystoscopic studies revealed adequate caliber conduits and normal appearing urethral tissues. Histological examination of the biopsy specimens showed the typical urethral stratified epithelium. CONCLUSIONS: Use of an off-the-shelf collagen inert matrix appears to be beneficial for patients with urethral strictures and obviates the need for obtaining an autologous graft, thus eliminating donor site morbidity.     

Urethral replacement with free urinary bladder mucosal transplant

An electro-resection was carried out in a subvesical blind-hole stenosis in a 5 month old male baby. Afterwards a 3 cm long urethral defect resulted in the penoscrotal junction. This was bridged with a free mucosal graft from the urinary bladder by the technique of Memmelaar and Hendren.     

Construction of cavernosum smooth muscle using umbilical artery smooth muscle cells seeded on acellular corporal collagen matrices

The objective of this study was to investigate the feasibility of tissue engineering of corpus cavernosal smooth muscle. Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting immunological reaction. Human umbilical artery smooth muscle cells (HUASMCs) were isolated from human umbilical arteries through explant techniques and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30 × 10⁶ cells/mL. Then, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyse the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues. The decellularization process successfully extracted all cellular components while preserving the original collagen fibers. The immunological reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro . Histological analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64 ± 0.18 g/100 mg and 2.50 ± 0.21 g/100 mg, respectively. Our study demonstrates that HUASMCs can be seeded on three-dimensional ACCM scaffolds and will develop tissues similar to that of the native corpus cavernosum smooth muscle.     

膀胱平滑肌细胞与膀胱脱细胞基质的生物相容性研究

目的:研究膀胱平滑肌细胞与膀胱脱细胞基质(BAM)的生物相容性。方法:分离培养兔膀胱平滑肌细胞,采用α-平滑肌肌动蛋白(α-SMA)抗体进行鉴定。将以1×10 5个/mL的单细胞悬液均匀接种于制备BAM支架上,通过与单独培养的膀胱平滑肌细胞作对照,绘制两组细胞生长曲线图,对比观察两条生长曲线的差异性。 ...