Urethral replacement: a comparison between small intestinal submucosa grafts and spontaneous regeneration

OBJECTIVE: To evaluate the use of commercially available single-layer small intestinal submucosa (SIS) for urethral replacement, both as an onlay and as a tube, in a rabbit model. MATERIALS AND METHODS: Thirty-six male rabbits were assigned to four experimental groups. Group 1 had the ventral wall of the penile urethra excised for 15 mm; in group 2 this created defect was patched with a SIS onlay graft; group 3 had complete excision of a 15 mm segment of the penile urethra; and in group 4, this created defect was replaced with a SIS tube graft. In all rabbits the urethra was stented for 2 weeks. A retrograde urethrogram was taken in all rabbits before death at 3, 6 and 12 weeks after surgery. The urethra was then exposed, examined carefully and excised for histopathological examination. RESULTS: In groups 1 and 2 the retrograde urethrograms were normal in 13 rabbits and there was relative narrowing in two rabbits in group 1 and three in group 2. In groups 3 and 4 all rabbits developed urethral fistulae or strictures. Histological examination of the urethra showed epithelial regeneration supported by smooth muscle backing in all rabbits in group 1, while rabbits in group 2 showed no regeneration of smooth muscle. By contrast, rabbits in groups 3 and 4 showed incomplete regeneration and progressive fibrosis. CONCLUSIONS: Single-layer SIS is not a suitable urethral substitute in this animal model. When used as an onlay, healing is inferior to spontaneous urethral regeneration, as SIS impedes smooth muscle cell regeneration. When used as a tube, there is complete scarring and urethral luminal occlusion.     

Endoscopic urethroplasty with unseeded small intestinal submucosa collagen matrix grafts: a pilot study

PURPOSE: We evaluated small intestinal submucosa (SIS) as a substitute for skin in endoscopic urethroplasty performed as treatment for inflammatory and iatrogenic strictures of the male bulbar urethra, and in the early treatment of bulbomembranous urethral injuries associated with recent pelvic fractures. Tissue integration and epithelialization of SIS in endoscopic urethroplasty were assessed, as was the long-term maintenance of urethral patency following this treatment. MATERIALS AND METHODS: Nine patients with bulbar urethral strictures defined by urethrography were enrolled in the study. Following optical urethrotomy the SIS grafts were tubularized over a purpose specific graft carrying balloon device and secured into the opened urethra as described for endoscopic urethroplasty. Patients were followed with urethroscopy and urethrography at regular intervals as per protocol or when symptoms arose. Failure was defined as the need for any further intervention. RESULTS: Two patients with short inflammatory strictures maintained urethral patency without any intervention at 1 and 2 years, respectively. Stricture recurrence developed in 6 patients within 3 months of surgery. Of these, 3 have undergone subsequent open urethroplasty, 2 are currently awaiting urethroplasty and 1 is maintaining urethral patency with regular self-dilatation. One patient was lost to followup. CONCLUSIONS: Endoscopic urethroplasty with unseeded SIS grafts was unsuccessful in this study.     

Functional restoration of rat bladder after subtotal cystectomy: in vivo cystometry and in vitro study of whole bladder

Functional restoration of the rat urinary bladder following subtotal cystectomy was studied via in vivo infusion cystometry and an in vitro whole bladder model. After the bladder had been separated from the prostate, subtotal cystectomy was achieved by ligating the bladder completely at a level just above the insertion of the ureters into the bladder. Bladder function was investigated immediately and 7, 14, and 28 days after surgery. Bladder weight was reduced to 17% that in sham-operated controls immediately after surgery, but recovered to 76% of that in controls 28 days after the operation. In vivo capacity also increased after surgery from 13% that of controls to 59% 28 days later. However, voiding pressure remained low (34% of control) 28 days later. An in vitro whole bladder study showed that the response to field stimulation decreased significantly on day 7, but had recovered considerably by day 28. The maximal response to bethanechol decreased significantly 7 days after surgery, but recovered thereafter. The response to phenylephrine increased significantly immediately after surgery, but gradually returnd to the control level. An in vitro volume-pressure study showed that passive complance of the cystectomized bladder decreased after surgery, but improved with time. The peak of the active pressure increase to field stimulation occurred at a low infusion volume immediately after surgery, but bladder capacity increased gradually until 28 days later, when the maximal active pressure was obtained. Our results suggest that restoration of the bladder following subtotal cystectomy may not derive simply from an expansion of the bladder wall. Functional alteration involving the bladder base was also observed.     

Mitochondrial metabolism in the rat during bladder regeneration induced by small intestinal submucosa

OBJECTIVE: To assess mitochondrial metabolism of bladder tissue induced by small-intestinal submucosa (SIS), by comparing the mitochondrial enzyme metabolism in this tissue with that in normal bladder tissue and thus evaluate intracellular normality. MATERIAL AND METHODS: In all, 70 rats were grouped into healthy controls (10), surgical controls with a simple bladder incision (15) and rats treated by partial cystectomy with replacement by the SIS graft (45). At 1, 3 and 6 months the rats were killed, the enzymes of mitochondrial respiratory chain complexes assayed, and the respiration of permeabilized bladder fibres assessed using polarographic analysis. RESULTS: The enzyme activities of control and treated rats at 3 months were identical. The results from the polarographic analysis of respiration were also similar to that in normal tissue apart from a decrease in the number of mitochondria. Histologically, there was complete regeneration at 6 months. CONCLUSION: After a phase of inflammation the bladder regenerates after a patch is placed. The new tissue has the same enzymatic and histological features as normal bladder tissue.     

Regional variations in small intestinal submucosa evoke differences in inflammation with subsequent impact on tissue regeneration in the rat bladder augmentation model

OBJECTIVE

To examine the histological differences in the inflammatory response and regenerative outcomes of distal vs proximal porcine small intestinal submucosa (SIS) grafts in the rat bladder, as SIS from distal small intestine yields reliable and reproducible bladder regeneration, while SIS from proximal portions of small intestine does not provide similar results.

MATERIALS AND METHODS

In all, 30 Sprague‐Dawley rats underwent hemi‐cystectomy followed by anastomosis of a bladder patch of SIS prepared from either distal or proximal small intestine. After bladder harvest, immunohistochemistry was used to quantify mast cells, eosinophils, macrophages, and neutrophils (PMNs). Total cell count per unit area was compared across the time course in univariate and logistic regression modelling.

RESULTS

There were more eosinophils and mast cells in proximal SIS grafts, while there were more macrophages and PMNs in distal SIS grafts (all P < 0.05). Trichrome analysis showed increased collagen deposition in proximal SIS grafts and little smooth muscle regeneration. There was also significant graft contracture in proximal SIS grafts compared with distal SIS grafts (P < 0.05).

CONCLUSIONS

We conclude that the location of SIS origin may evoke different inflammatory responses, which results in altered bladder tissue regeneration.     

Rabbit urethra replacement with a defined biomatrix or small intestinal submucosa

OBJECTIVE: The evaluation of collagen-based biomatrix (SIS COOK((R))) in comparison to a biochemically reconstructed biomatrix for replacement of the urethra in a rabbit model as a preclinical model. MATERIAL AND METHODS: Rabbits underwent partial urethra replacement (resection of 0.5 to 1.0 cm segment of the urethra), which was replaced with 1 or 4 layers Small Intestinal Submucosa (SIS COOK) patch grafts or with a biochemically defined collagen biomatrix, partly sutured with unresolvable sutures for future reference. Six animals underwent a sham control operation. The grafts of regenerated urethras were harvested at 1, 3 and 9 months after implantation. Urethrography was performed pre-operatively and before sacrificing. The animals were evaluated macroscopically and by routine histology and immunohistochemistry. RESULTS: At 1 month after implantation, the biomatrices (1 layer, 4 layers and our biochemically defined biomatrix) were well distinguishable from the normal surrounding tissues and showed blood vessels at the periphery. Macroscopically, the unresolvable reference sutures were easy to find at all time points. At 3 months the graft was still distinguishable in the 4 layers SIS group. In the 1 layer and the defined biomatrix group a good regeneration of the urethra within the graft was seen with some central fibrosis. Histological and immunohistochemical evaluation showed urothelium regeneration on the 1 layer and on biochemically defined biomatrix with decreasing number of inflammatory cells from 1 month on. In the group treated with 4 layers SIS the urothelium was completely regenerated at 3 months. Histologically, the regeneration of muscle cells in the three biomatrices was comparable. The smooth muscle cells regenerated very slowly as 1 month after implantation no muscle cells were detectable within the grafts. At 3 months a few muscle cells were present in the graft, but cell density did not increase in the following 6 months. Strictures were not observed on control urethrography pre-operatively in the animals. In one case slight narrowing of the urethra on urethrography was seen, but apparently without causing voiding problems. One rabbit developed a fistula near the operation site. CONCLUSION: The biomatrices investigated are feasible scaffolds to repair urethral lesions. The results with our biochemically defined biomatrix are comparable to one layer Small Intestinal Submucosa. Almost no smooth muscle cells population was observed after nine months for the three biomatrices. We conclude that an improved molecularly defined biomatrix focussed on stimulation of smooth muscle cell growth may be necessary to obtain optimal cellular grafting results.     

Regeneration of detrusor muscle after subtotal cystectomy in the rat: effects on contractile proteins and bladder mechanics.

The aim of the present study was to determine to what extent adult rats can produce new contracting bladder muscle and to see if such newly formed bladder tissue possesses characteristic mechanical properties or whether the ability to recover mechanically is so pronounced that the prehistory of the bladder is unimportant. Subtotal cystectomy was performed in adult female rats, leading to a pronounced decrease in total bladder weight. At 10 weeks, bladder weight had normalized. The histological appearance of such bladders was similar to that of the controls. Active and passive length-tension relations for the detrusor muscle were determined in controls and up to 10 weeks after surgery. Immediately after surgery active and passive forces showed a leftward shift and maximum active force decreased markedly. With time the length-tension curves shifted back to normal, but a decreased active force still remained at 10 weeks. Detrusor actin concentration and detrusor myosin/actin ratio were unaffected by the subtotal cystectomy. Intermediate filament protein/actin ratio showed a significant but transitory increase. We conclude that there is a remarkable recovery of detrusor muscle function after subtotal cystectomy, leading to a normalization of optimum length for active force and a net synthesis of contractile and cytoskeletal proteins. The ability to produce active force does, however, not fully recover.     

异体小肠粘膜下组织替代膀胱壁的研究

目的　探讨大面积异体小肠粘膜下组织 (SIS)作为膀胱壁替代物的价值。方法　将2 0头成年猪随机分为 2组 :A组 12头行大面积SIS(10cm× 10cm )替代膀胱壁术 ;B组 8头行大面积SIS替代膀胱壁和一侧输尿管种植术。动态观察SIS再生膀胱壁的移行上皮、平滑肌的再生情况和顺应性 ;输尿管种植后的愈合和吻合口情况。结果　再生膀胱壁具有与正常膀胱壁相似的外观、厚度和顺应性 ,组织学检查显示移行上皮和平滑肌的良好再生 ,所有种植输尿管的出口均被再生的膀胱壁所封闭 ,各组的平均最大膀胱容积术前 (2 88.3± 3 1.4)ml、术后 (2 83 .8± 3 7.1)ml;最大膀胱压力术前 (3 6.1± 9.3 )cmH2 O(1cmH2 O =0 .0 98kPa)、术后 (3 7.3± 9.5 )cmH2 O ,差异无显著性 (P >0 .0 5 )。结论　异体小肠粘膜下组织是一种较理想的膀胱部分替代和扩大材料。     

组织工程补片膀胱扩大术治疗神经源性膀胱的疗效分析

目的 探讨使用小肠黏膜下层(small intestinal submucosa,SIS)组织工程材料补片行膀胱扩大术治疗神经源性膀胱的可行性和有效性. 方法 2011年1月至2014年3月收治14例神经源性膀胱患者,男10例,女性4例.年龄14～65岁,平均29岁.脊髓发育不良8例,脊髓损伤6例.尿动力学检查:最大膀胱测压容量平均为(150.1±64.2) ml,膀胱顺应性平均为(5.2±3.9)ml/cmH2O(1 cmH2O=0.098 kPa),最大逼尿肌压力平均为(44.1±29.2) cmH2O.14例均接受SIS组织工程补片膀胱扩大术,术中将补片锁边缝合至纵向剖开的膀胱浆肌层,达到扩大膀胱的目的,其中7例同期行输尿管抗反流再植术.对术后并发症、影像尿动力学检查参数、尿路核磁水成像及肾功能进行观察评价. 结果 本组14例手术均顺利完成,手术时间平均为120 min.患者术后无代谢紊乱.复查肌酐水平均正常.术后随访6～48个月,平均24个月.尿动力学检查术后6、12、24个月最大膀胱测压容量分别为(274.9±88.7)、(322.5± 144.4)、(279.9± 157.9) ml,与术前比较差异有统计学意义(P＜0.05);术后12、24个月最大逼尿肌压力为(20.1±9.8)、(20.2± 19.1) cmH2O,与术前比较差异有统计学意义(P＜0.05);术后24个月膀胱顺应性为(26.1±29.4) ml/cmH2O,与术前比较差异有统计学意义(P＜0.05).术后1个月,2例出现膀胱吻合口尿外渗,更换导尿管引流通畅后愈合.术后3个月,1例出现膀胱结石,行经尿道取石术后未再复发.术后12个月,4例出现膀胱输尿管反流,其中2例予膀胱逼尿肌A型肉毒素注射术,术后留置尿管3个月复查反流消失;2例保留导尿,口服琥珀酸索利那新(5 mg,2次/d)和酒石酸托特罗定(4 mg,1次/d)6个月后,1例反流消失,1例仍存在反流.结论 SIS组织工程补片用于膀胱扩大术可达到有效增加膀胱容量的目的.生物工程补片的使用为治疗神经源性膀胱提供了新的选择.     

A comparison of engineered urinary bladder and intestinal smooth muscle for urinary bladder wall replacement in a rabbit model

Background/Purpose

The small intestine is the most common resource for bladder augmentation. Little is known whether intestinal smooth muscle cells (SMCs) may be engineered into bladder tissue. We investigated the phenotypic and functional characteristics of engineered bladder and intestinal SMCs as bladder wall replacement in a rabbit model.

Methods

One month after an initial 70% partial cystectomy, 3 autoaugmentation surgeries were performed, including traditional autoaugmentation (TA, n = 6), TA using engineered bladder SMCs (TA + B, n = 6), and TA using intestinal SMCs (TA + I, n = 6). All were followed up by bladder volume measurement and retrieved on the first, third, and sixth month. The grafts and the native bladder wall were evaluated with immunocytochemistry and electrical field stimulation (EFS). Statistical analysis was performed using analysis of variance.

Results

Both the TA + I and TA + B groups showed significant and similar bladder capacity increment in all time points. The engineered muscle cells demonstrated the typical “contraction-relaxation” response to supramaximal EFS. There were no statistical differences in both the TA + I and TA + B groups in contractility force.

Conclusion

Engineered SMCs derived from urinary bladder and small intestine could retain their phenotype after implantation in vivo. Both exhibited a similar degree of contractility to EFS. These results suggest that there are no phenotypic or functional differences between muscle cells obtained from the 2 different organs. Both have the potential to be engineered into normal bladder tissues.     

Evaluation of small intestinal submucosa grafts for meniscal regeneration in a clinically relevant posterior meniscectomy model in dogs

Large meniscal defects are a common problem for which treatment options are limited. Successful meniscal regeneration has been achieved by using grafts of small intestinal submucosa in posterior, vascular meniscal defects in a dog model. This study investigates the long-term effects of a tibial tunnel fixation technique and a clinically based meniscectomy defect on meniscal regeneration using this model. Eight mongrel dogs underwent medial arthrotomy and partial meniscectomy. The dogs were divided into groups based on defect treatment: small intestinal submucosa (n = 4) or meniscectomy (n = 4). Dogs were scored for lameness by subjective scoring postoperatively, sacrificed at 6 months, and assessed for articular cartilage damage, gross and histologic appearance of the operated meniscus, amount of new tissue in the defect, and relative compressive stiffness of articular cartilage. Dogs in the meniscectomy group were significantly (P = .002) more lame than dogs treated with small intestinal submucosa. Small intestinal submucosa-treated joints had significantly (P = .01) less articular cartilage damage than meniscectomy joints. Small intestinal submucosa meniscal implants resulted in production of meniscal-like replacement tissue, which was consistently superior to meniscectomy in amount, type, and integration of new tissue, chondroprotection, and limb function during the study period. Small intestinal submucosa implants may be useful for treatment of large posterior vascular meniscal defects in humans. The tibial tunnel technique used for fixation may have clinical advantages and therefore warrants further investigation.     

小肠黏膜下层移植修复海绵体神经损伤的实验研究

目的:研究小肠黏膜下层(SIS)移植替代损伤的双侧海绵体神经(CN)恢复大鼠的勃起功能。方法:制备SIS,建立动物模型,分为CN损伤组、假手术组、SIS移植组,分别给予切断双侧的CN、仅游离CN以及SIS移植修复损伤的CN。术后3个月进行阿朴吗啡试验,了解阴茎勃起情况。取中、后段阴茎海绵体组织,进行nNOS免疫组化染色,记录nNOS阳性神经纤维的数目。结果:阿朴吗啡试验:30 min内SIS移植组72.73%(8/11)的大鼠出现阴茎勃起,平均勃起(1.07±0.89)次;CN损伤组勃起率和勃起次数均为0;假手术组则为90.91%(10/11)和(2.19±1.17)次。无论是勃起率还是勃起次数,SIS移植组均显著高于CN损伤组(P<0.01),但仍然比假手术组低(P<0.05)。nNOS神经纤维数目:SIS移植组为(70.36±10.09)条,CN损伤组为(22.09±4.76)条,差异有统计学意义(P<0.01),但二者均低于假手术组[(90.81±5.69)条,P<0.01]。结论:SIS作为移植物修复损伤的大鼠CN损伤,有利于恢复CN损伤所致的勃起功能障碍。     

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder

OBJECTIVE: To assess the potential use of bone marrow stromal cell (BMSC)-seeded biodegradable scaffold for bladder regeneration in a canine model, by characterizing BMSCs and comparing them to bladder smooth muscle cells (SMCs) by immunohistochemistry, growth capability, and contractility. MATERIALS AND METHODS: Bone marrow was taken by direct needle aspiration from the femurs of five beagle dogs for the in vitro study. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and cultivated in medium 199 with 10% fetal bovine serum. BMSCs were characterized by cell proliferation, in vitro contractility, immunohistochemical analysis, and the growth pattern on small intestinal submucosa (SIS) scaffolds compared to bladder SMC cultures from the same dogs. Another six dogs had a hemicystectomy and bladder augmentation with BMSC-seeded (two), bladder cells including urothelial cells plus SMC-seeded SIS (two) and unseeded SIS scaffolds (two). The six dogs were followed for 10 weeks after augmentation. RESULTS: In vitro BMSCs had a significant contractile response to calcium-ionophore, with a mean (sem) 36 (2)%, relative contraction (P < 0.01), which was similar to bladder SMCs but markedly different from fibroblasts. BMSCs also expressed alpha-smooth muscle actin by immunohistochemical staining and Western blotting, but did not express desmin or myosin. In vivo, both BMSC-seeded and bladder cell-seeded SIS grafts had solid smooth-muscle bundle formation throughout the graft. CONCLUSIONS: BMSCs had a similar cell proliferation, histological appearance and contractile phenotype as primary cultured bladder SMCs. SIS supported three-dimensional growth of BMSCs in vitro, and BMSC-seeded SIS scaffold promoted bladder regeneration in a canine model. BMSCs may serve as an alternative cell source in urological tissue engineering.     

Experimental assessment of small intestinal submucosa as a small bowel graft in a rat model

Background/Purpose:

Small intestinal submucosa (SIS) is an extracellular matrix used in tissue engineering. The purpose of this study is to evaluate the feasibility of using SIS as a scafford for small bowel regeneration in a rat model.

Methods:

A 2-cm length tubular SIS graft from donor Sprague Dawley rats was interposed with bilateral anastomosis in the median tract of an isolated ileal loop of Lewis rats used to construct an ileostomy. The grafts were harvested and analyzed at each of the time-points ranging from 2 weeks to 24 weeks after operation using histology and immunohistochemistry.

Results:

Macroscopic examination found no adhesion in the surrounding area of neointestine by 24 weeks, and no stenosis was visible. The shrinkage of neointestine was indicated from 20% to 40%. Histologic and immunohistochemical evaluation showed that SIS grafts were colonized by numerous inflammation cells by 2 weeks. Neovascularization was evident, but the luminal surface was not epithelized. By 4 weeks, transitional mucosal epithelial layer began to line the luminal surface of the graft, and nearly 70% luminal surface of the graft had been covered by mucosal epithelium at 8 weeks. By 12 weeks, the luminal surface was covered completely by a mucosal layer with distinct bundles of smooth muscle cells in the neointestine. At 24 weeks, the neointestine wall showed 3 layers of mucosa, smooth muscle, and serosa.

Conclusions:

The preliminary study suggested that SIS allow rapid regeneration of mucosa and smooth muscle and might be a viable material for the creation of neointestine.     

Penile enhancement using a porcine small intestinal submucosa graft in a rat model

Several biodegradable materials have been experimented for penile enhancement, but none show the potential for clinical use. This study was designed to use porcine small intestinal submucosa (SIS) augmenting the normal tunica albuginea to increase the functional girth of the rat penis. In all, 20 adult male Sprague-Dawley rats constituted the study population. The animals were divided into two groups: group 1 consisted of the control (n=10) and group 2 (n=10) consisted of rats that underwent penile enhancement by a longitudinal I-shaped incision of the tunica albuginea on both sides, and the dissection of the plane between tunica albuginea and cavernosal tissue was carried out (n=10). The incision was then patched with a 3 x 10 mm2 piece of SIS, using a 6/0 nylon suture material. The penile length and mid-circumference were then measured using a Vernier Caliper before and 2 months after surgery. All rat penises underwent histological examination using Masson's trichome and Verhoff's van Giesen's stain for collagen and elastic fibers. The penile length, mid-circumference and degree of fibrosis score were expressed as mean+/-s.e. (standard error) and analyzed using a Wilcoxon rank-sum test. A statistical significance was accepted at P-value < or =0.05. Our results showed similar preoperative penile length and circumference in both groups. However, 2 months after the surgery, the mean penile circumference of the SIS group has grown significantly larger than the control group, while the mean penile length remained unchanged. The histological study of the rat penises revealed minimal amounts of fibrosis under the graft, and the elastic fibers of the graft showed orientation in a circular manner. In conclusion, SIS appears promising for material use in a penile enhancement.     

小肠粘膜下层与雪旺细胞生物相容性的研究

目的研究猪小肠粘膜下层(SIS)与体外培养的鼠雪旺细胞(SCs)的生物相容性。方法在体外将分离培养的SD乳鼠SCs接种于制备好的猪SIS上进行复合培养,通过相差显微镜和扫描电镜观察不同时间段SCs在SIS上的黏附、生长与增殖情况,用酶联免疫吸附测定和逆转录聚合酶链反应方法检测SCs分泌神经生长因子-β和脑源性神经生长因子的情况。对照组为接种于未平铺SIS的孔中正常培养的SCs。结果培养3～5 d后,相差显微镜下见SIS边缘SCs较为密集,黏附良好,多呈长梭形生长;扫描电镜观察见SIS表面SCs增殖黏附良好,胞体突起显著,呈端对端连接或成束排列,细胞表面可见蛋白颗粒分泌。酶联免疫吸附测定和逆转录聚合酶链反应方法检测发现,SCs与SIS复合培养后,神经生长因子-β和脑源性神经生长因子分泌良好,与对照组的SCs差异无统计学意义。结论猪SIS与鼠SCs有良好的生物相容性,有望用作支架,供SCs黏附生长,构建组织工程神经,用于修复周围神经缺损。