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.     

Bladder reconstruction with adipose-derived stem cell-seeded bladder acellular matrix grafts improve morphology composition

Purpose  

To assess the feasibility of seeding adipose-derived stem cells (ADSCs) onto bladder acellular matrix grafts (BAMGs) for bladder reconstruction in a rabbit model.     

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.     

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。 ...     

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.     

Homologous bladder augmentation in dog with the bladder acellular matrix graft

OBJECTIVE: To determine the functional potential and antigenicity of the homologous bladder acellular matrix graft (BAMG) in a dog model. MATERIALS AND METHODS: Seven mongrel dogs underwent partial cystectomy (20-50%) and grafting with an equal-sized BAMG; two control animals underwent partial cystectomy (40%) only. The dogs were killed after 30 (one), 120 (one) and 210 days (five dogs). Blood samples were obtained before and at 1, 2, 4, 7, 14, 30, 90 and 210 days after surgery. The dogs underwent cystography, intravenous pyelography and ultrasonography before and after surgery, and on the day they were killed, with cystoscopy carried out just before death. The grafted tissue was assessed using routine and immunohistochemical techniques. RESULTS: All the dogs survived surgery; a complete blood cell count, chemical panel and white blood cell count showed no significant difference between the experimental and control animals. Cystography, cystoscopy and ultrasonography revealed no pathological changes in the upper urinary tract. After 7 months, the mean bladder capacity in the augmented dogs was significantly higher (P = 0.035) than in the controls (264 vs 172 mL). Histological evaluation showed an invasion of all bladder wall components during the first month; at 7 months, the morphological examination showed essentially complete regeneration. CONCLUSION: In this dog model, the potential of the BAMG as a bladder augmentation graft was confirmed, having minimal antigenicity with maximal acceptance. The reconstructed bladder matched the morphological and functional properties of the normal bladder.     

Functional improvement in spinal cord injury-induced neurogenic bladder by bladder augmentation using bladder acellular matrix graft in the rat

Spinal cord injury (SCI) rostral to the lumbosacral level causes bladder hyperreflexia and detrusor-sphincter dyssynergia (DSD), which are accompanied by bladder hypertrophy. We hypothesize that bladder augmentation using a bladder acellular matrix graft (BAMG) can improve the function of SCI-mediated neurogenic bladder. In female rats (n = 35), SCI was induced by transection of the spinal cord at the lower thoracic level. Eight weeks following spinalization, bladder augmentation using BAMG was performed after hemicystectomy of the hypertrophic bladder. Cystometrography was performed at 8 weeks after spinalization and again at 8 weeks after augmentation. Several urodynamic parameters were measured and the grafted bladder was histologically evaluated. Thirty one rats were alive 8 weeks after spinalization. Twenty two (71%) rats developed hyperreflexic bladders and nine (29%) rats had underactive bladders before bladder augmentation. Twenty six rats survived until 8 weeks after augmentation. Urodynamic parameters showed improvement in some bladder functions in both hyperreflexic and underactive bladders after augmentation. In addition, bladder compliance was increased in hyperreflexic bladders and decreased in underactive bladders. Bladder augmentation decreased bladder capacity in high-capacity rats and increased it in low-capacity rats. Histological evaluation showed complete regeneration of BAMG in SCI-induced neurogenic bladder at 8 weeks after augmentation. This is the first report suggesting that the voiding function in SCI-induced neurogenic bladder can be improved by augmentation using BAMG. Improved voiding function was accompanied by histological regeneration of BAMG.     

Reconstructive urethroplasty using porcine acellular matrix

Long tract urethral reconstruction nowadays still has no other resolution than two-stage techniques or graft and flap procedures that are neither simple nor trouble-free both for the patient and the surgeon. Tissue engineering simplifies this surgery using "porcine acellular matrix", obtained from small intestine submucosa. It is thin but strong, just ready for grafting, without rejection, because it is not immunogenic, being deprivated of cells. It serves as biological bridging of the reconstruction, promoting the generation of surrounding tissue in which it is completely transformed. We report the first use of porcine intestine submucosa in urethroplastic surgery.The up to date follow up is sixteen months with satisfactory urodynamic and subjective outcome.     

应用脱细胞异体真皮植入Bucks筋膜下加大阴茎

目的探讨一种加大阴茎的手术方法。方法将脱细胞异体真皮填充在阴茎Bucks筋膜与白膜之间加大阴茎。结果自2002年3月以来,我们在临床应用12例,术后自然状态下阴茎周径加大13～31cm,平均26cm,术后3个月有正常的性生活。1例因包扎过紧至阴茎皮肤部分坏死,经转移阴囊皮瓣修复愈合。结论该方法用于阴茎加大,创伤小、操作简便、效果确实,无不良反应。     

Different bladder defects reconstructed with bladder acellular matrix grafts in a rabbit model

Objective

To evaluate the potential use of the bladder acellular matrix graft (BAMG), two different bladder defects in the rabbit model were reconstructed.

Materials and methods

Two groups of rabbits underwent partial bladder wall cystectomy (group?A, 30?C40%; group?B, 70?C60%) and reconstruction of the defects with an equally sized BAMG. After 4, 12, and 24?weeks, bladder cystographs were performed. Then the rabbits were killed after uneventful postoperative periods, and the grafts were harvested for H&;E staining and immunohistochemical staining.

Results

Two rabbits died on the postoperative days 3 and 6 in group?A due to urinary peritonitis. At 24?weeks, in group?A, the reconstructed bladders reached a mean volume of 94.39±0.54% of the precystectomy bladder capacity. Histologically, complete regeneration of smooth muscle and urothelium tissue was evident. Regenerated SMCs and urothelium stained positive for ??-smooth muscle actin and AE1/AE3. In group?B, the mean bladder volume was 64.5±3.19% of the precystectomy volume. Histologically, group?B was characterized by multilayered urothelium without organized muscle tissue.

Conclusion

The BAMG was an effective scaffold for bladder wall regeneration in the rabbit model. However, the use of BAMG reconstruction in larger bladder defects did not induce the same quality and quantity of bladder regeneration as the reconstruction of smaller bladder defects.     

Tunica repair with acellular bladder matrix maintains corporal tissue function

Penile conditions, such as Peyronie's disease or tumor resection may require surgical reconstruction of the tunica albuginea. Various materials have been proposed, as a biomaterial for tunica albuginea repair, however, little functional data are available. We examined the applicability and functional outcome of a collagen-based matrix derived from the bladder (acellular bladder matrix (ABM)), as a biomaterial for tunica repair. Biocompatibility testing was performed on the matrix, which included mitochondrial metabolic activity, cell viability and apoptosis. Approximately 50% of the dorsal penile tunica albuginea was replaced with the collagen-based matrix patch after surgical removal in 24 New Zealand White rabbits. Cavernosometry and cavernosography were performed. The animals were killed 1, 2 and 3 months after surgery for analyses. The matrix showed excellent biocompatibility. All animals implanted with the matrix survived without any noticeable untoward effects. There was no evidence of inflammation or infection at the time of retrieval. Cavernosometry of the implanted animals demonstrated normal intracavernosal pressures with visual erections. Cavernosography of the repaired corpora showed a normal anatomical configuration. Biomechanical analysis of the retrieved matrices demonstrated similar tensile strengths as native tunica. Histologically, there was only a minimal inflammatory response, which gradually decreased over time. These results show that ABM is biocompatible, durable and effective when used as a tunica substitute. The matrix may be useful as an off-the-shelf biomaterial for patients requiring tunica albuginea repair.     

Implant breast reconstruction using acellular dermal matrix

Thirteen breast reconstructions in 11 patients, averaging 58 years of age, underwent mastectomies. The technique uses a saline implant either totally or partially covered with a human acellular dermal matrix. The mean postoperative follow-up time was 14 months. Ninety percent of the patients were considered high risk; the thickness of the human acellular dermal matrix was an average of 1.3 mm, with an average area per breast of 121 cm. There were 12 successful breast reconstructions (92%) that provided stability, increased soft tissue padding, which allowed a greater resemblance to normal breast shape and decreased rippling and implant visibility. The graft was used in an onlay fashion or as an extension of the pectoralis major muscle that covers the implant. A representative histologic cross-section of well-integrated human acellular dermal matrix is presented.The use of a human acellular dermal matrix in breast reconstruction is an alternative protocol in high-risk patients, resulting in a minimal increase in operative time and a decrease in morbidity compared with more extensive procedures.     

Expression of VEGF and collagen using a latex biomembrane as bladder replacement in rabbits

Objective: To investigate the VEGF expression and collagen deposition using a latex biomembrane as bladder replacement in rabbits. Materials and Methods: After partial cystectomy, a patch of a non-vulcanized latex biomembrane (2 x 2 cm) was sewn to the bladder of rabbits with 5/0 monofilament polydioxanone sulfate sutures in a watertight manner. Groups of 5 animals were killed at 15, 45 and 90 days after surgery and the bladder was removed. Sections of 5μm were cut and stained with picrosirius-red in order to estimate the amount of extracellular matrix in the graft. To confirm the presence of VEGF in tissues, protein expression was determined by immunohistochemistry. Results: No death, urinary leakage or graft extrusion occurred in any group. All bladders showed a spherical shape. A progressive reduction in the amount of collagen occurred in the graft area and was negatively and linearly correlated with time (p < 0.001). VEGF expression was higher in grafted areas when compared to controls at 15 and 45 days after surgery and decreased with time (p < 0.001). Conclusion: The latex biomembrane as a matrix for partial bladder replacement in rabbits promotes temporary collagen deposition and stimulates the angiogenic process.