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...     

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.     

膀胱粘膜尿道成形术后尿道护理的改进

膀胱粘膜尿道成形术后尿道护理的改进福建省泉州市第一医院郭丽芬，苏仲宁，王丽华尿瘘和尿道口狭窄是先天性尿道下裂尿道成形术后最常见的并发症，多因尿道内分泌物不能及时排出引起感染所致。因此，成形术后尿道的护理对手术成败至关重要。我院自１９８６～１９９３年，...     

膀胱粘膜尿道成形术治疗复杂性尿道下裂

目的对膀胱粘膜尿道成形术治疗复杂性尿道下裂的适应证及手术方法进行讨论，以重新认识其应用价值。方法总结2002～2004年经多次手术失败的复杂性尿道下裂7例，平均尿道缺损5、7cm，采用改进后的膀胱粘膜尿道成形术Ⅰ划尿道成形的资料。结果术后均获得较为满意的疗效，阴茎伸直满意，外形好，尿线粗，开口近似正常位置。2例术后尿瘘，6个月后再次修补获成功，手术一次成功率71.4％。结论对多次手术失败、阴茎疤痕严重、局部取材困难、尿道缺损过长的复杂尿道下裂的病例，采用膀胱粘膜尿道成形术Ⅰ期尿道成形仍是目前合理、有效的手术方法。     

膀胱粘膜移植治疗长段尿道狭窄

本文报告作者１９８４年１０月至１９９３年１０月为１２例长段尿道狭窄（５例为前尿道狭窄，７例为后尿道狭窄）患者施行膀胱粘膜尿道一期成形术。治愈１０例，好转２例。随访半年至９年无特殊。     

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。     

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.     

组织工程尿道粘膜的体外构建

目的:利用组织工程方法在体外构建形成尿道粘膜结构。方法:酶消化法获得猪膀胱尿路上皮细胞(UC),以免疫荧光和RT-PCR方法进行UC鉴定。制备膀胱脱细胞基质移植物(BAMG)作为支架材料,以苏木精-伊红染色、Masson染色、免疫组化和扫描电镜进行评价。经过体外培养和扩增后,将P3代UC接种在BAMG上。结果:BAMG支架上的UC经过体外培养1周后即可形成沿基膜排列的复层上皮结构。扫描电镜显示细胞-材料复合良好,免疫组化检测支架上的UC广谱角蛋白表达阳性。结论:运用组织工程方法能够在体外快速进行尿道粘膜上皮结构的初步构建,为进一步临床修复诸如尿道下裂、尿道狭窄等尿道缺损奠定技术基础。     

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     

冻干脱细胞膀胱黏膜下基质在尿道下裂修复中的临床应用

目的在动物实验的基础上,将异体脱细胞膀胱黏膜下基质用于修复阴茎型尿道下裂患者,以初步了解其临床疗效。方法我们采用反复冻融—酶法获得了同种异体人脱细胞膀胱黏膜下基质,于2004年将其试用于3例尿道下裂患者的治疗。结果一期手术2例,尿线粗、直,无尿瘘,无散射;术后1年随访,阴茎无下弯,无尿痛、尿瘘,尿线粗,尿流测定仪及尿道镜检查未见异常。二期手术1例,远端尿道未见狭窄及闭锁。结论同种异体脱细胞膀胱黏膜下基质是一种有前途的较为理想的尿道组织修复替代材料,远期效果还有待继续进行严密仔细的观察。     

Morphologic study of small intestinal submucosa as a body wall repair device

BACKGROUND: The extracellular matrix (ECM) derived from porcine small intestinal submucosa (SIS) has been used as a constructive scaffold for tissue repair in both preclinical animal studies and human clinical trials. Quantitative characterization of the host tissue response to this xenogeneic scaffold material has been lacking. MATERIALS AND METHODS: The morphologic response to a multilaminate form of the SIS-ECM was evaluated in a chronic, 2-year study of body wall repair in two separate species: the dog and the rat. Morphologic response to the SIS-ECM was compared to that for three other commonly used bioscaffold materials including Marlex mesh, Dexon, and Perigard. Quantitative measurements were made of tissue consistency, polymorphonuclear cell response, mononuclear cell response, tissue organization, and vascularity at five time points after surgical implantation: 1 week, 1, 3, and 6 months, and 2 years. RESULTS: All bioscaffold materials functioned well as a repair device for large ventral abdominal wall defects created in these two animal models. The SIS-ECM bioscaffold showed a greater number of polymorphonuclear leukocytes at the 1-week time point and a greater degree of graft site tissue organization after 3 months compared to the other three scaffold materials. There was no evidence for local infection or other detrimental local pathology to any of the graft materials at any time point. CONCLUSIONS: Like Marlex, Dexon, and Perigard, the SIS-ECM is an effective bioscaffold for long-term repair of body wall defects. Unlike the other scaffold materials, the resorbable SIS-ECM scaffold was replaced by well-organized host tissues including differentiated skeletal muscle.     

Local urethral recurrence after radical cystectomy and orthotopic bladder substitution in women: a prospective study

PURPOSE: We prospectively studied the pathological outcome and incidence of urethral recurrence after radical cystectomy and orthotopic bladder substitution in women. MATERIALS AND METHODS: Between January 1995 and December 2001, 145 women with a mean age of 50 +/- 8.5 years underwent standard radical cystectomy and orthotopic substitution for bladder cancer. Histopathological examination of the cystectomy specimens was assessed by a single pathologist. Clinically evident pelvic lymphadenopathy, bladder neck and/or vaginal wall involvement, or positive intraoperative frozen section from the urethra were considered contraindications. RESULTS: One patient died postoperatively of a massive pulmonary embolism. Followup was 12 to 97 months (mean 36, median 55.8). Pathological stage was P1, P2 (superficial muscle invasion), P3a (deep muscle invasion), P3b (perivesical fat infiltration) and P4a in 12, 29, 56, 44 and 4 patients, respectively. Grade was G1 in 61 patients, G2 in 62 and G3 in 22. Lymph nodes were positive in 28 cases and negative in 117. Histopathology of the trigone revealed carcinoma in situ in 11 cases and squamous metaplasia in 7, while results were free of disease in the remainder. At followup isolated urethral recurrence developed in 2 patients (1.4%), in whom definitive pathological findings showed P3a N1 (positive iliac lymph nodes) M0, grade 2 squamous cell carcinoma in 1 and P3b N0M0 tumor associated with trigonal carcinoma in situ in 1. Local pelvic recurrence developed in 18 patients, distant metastasis developed in 6 and the 2 conditions developed in 10. Oncological failure positively correlated with high stage, high grade and positive lymph nodes. CONCLUSIONS: The rate of urethral recurrence after radical cystectomy and orthotopic bladder substitution in women is low and acceptable and, thus, it justifies the continued performance of this type of diversion. Proper selection of cases is mandatory. Close followup for oncological failure in this group of patients after cystectomy is necessary.     

Assessment of bladder and urethral sphincter function before and after radical retropubic prostatectomy

PURPOSE: In a prospective study we analyzed the effects of radical retropubic prostatectomy (RRP) on detrusor and urethral sphincter function by comparing urodynamic status preoperatively with that during longitudinal followup. MATERIALS AND METHODS: A total of 49 consecutive patients underwent urodynamics with pressure flow studies and Valsalva leak point pressure measurements 3 to 7 days before RRP (baseline), and then 1 and 8 months after surgery. We assessed bladder compliance, detrusor overactivity, detrusor contractility and intrinsic sphincter deficiency (ISD). RESULTS: There was no significant change in detrusor overactivity at 1 and 8 months of followup. Decreased bladder compliance was observed in 20.4% of patients at baseline, and in 38.7% and 30.6% at 1 and 8 months, respectively. De novo decreased compliance was detected in 18.4% and 10.2% of patients at the same points. Impaired bladder compliance was comparable to that before surgery in 20% of cases. Impaired detrusor contractility was detected in 42.8% of patients at baseline, and in 61.2% (p <0.05) and 42.8% at 1 and 8 months, respectively. De novo hypocontractility was observed in 28.6% and 10.2% of patients at 1 and 8 months, respectively. A strong association between detrusor overactivity and ISD was observed at 1 and 8 months (p <0.01). CONCLUSIONS: Following RRP detrusor hypocontractility and decreased bladder compliance represent de novo transient dysfunction probably due to bladder denervation and an established condition not influenced by the operation. The strong association between overactivity and ISD suggests that stress urinary incontinence increases urethral afferent nerve activity and induces involuntary detrusor contractions.     

Osteochondral repair using an acellular dermal matrix—pilot in vivo study in a rabbit osteochondral defect model

The aim of this pilot project was to introduce a novel use of acellular dermal matrix (ADM) in combination with infrapatellar fat pad mesenchymal stromal cells (IPFP‐MSCs) to effect repair in a rabbit osteochondral defect model. ADM, in a range of surgical procedures, has been shown to promote remodelling of tissue at the site of implantation. Rabbit‐derived ADM (rabADM) was prepared from the skin of donor rabbits. Autologous IPFP‐MSCs were obtained at the time of knee surgery. Osteochondral defects (4 mm cartilage outer/2 mm central bone defect) were drilled into distal femoral condyles of 12 New Zealand White rabbits. Treatments groups: (i) defect only; (ii) rabADM alone; (iii) IPFP‐MSCs alone; and (iv) rabADM with IPFP‐MSCs. Condyles were harvested at 12 weeks, and analyzed using histology, immunohistochemistry (types I and II collagen) and histomorphometry to evaluate osteochondral repair. The rabADM only group achieved the highest ratio of type II to non‐type II collagen (77.3%) using areal measures (similar to normal cartilage), which indicated a higher quality of cartilage repair. The addition of IPFP‐MSCs, with or without rabADM, formed a fibrous collagen cap above the lesion site not seen with rabADM alone. Macroscopically, there was no joint erosion, inflammation, swelling or deformity, and all animals maintained full range of motion. Conclusions: RabADM alone resulted in neocartilage formation similar to native cartilage. IPFP‐MSCs limited osteochondral repair and contributed to fibrosis, even in combination with the rabADM. Further studies using ADM for osteochondral repair are warranted in a more appropriate pre‐clinical model of osteochondral repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1919–1928, 2018.