Evaluation of small intestinal submucosa as scaffolds for intestinal tissue engineering

BACKGROUND: Biodegradable scaffolds have been used for regenerating the small intestine. The aim of this study was to evaluate the feasibility of small intestinal submucosa (SIS) as scaffolds for intestinal regeneration in a rat model. MATERIALS AND METHODS: A tubular one-ply or four-ply SIS was interposed between isolated jejunal segments in rats. The scaffolds were harvested at 2, 4, and 8 weeks after implantation, and the specimens were examined grossly and histologically. RESULTS: Significant contractions were observed in SIS scaffolds after implantation. The one-ply SIS contracted to 44% of its initial length at 2 weeks and continued to contract to 6% of its initial length at 8 weeks. The contraction of four-ply SIS scaffolds was less than that of the one-ply SIS, reaching 29% of its initial length at 8 weeks. Minimal epithelial and smooth muscular regeneration was observed in the SIS scaffolds after implantation. CONCLUSIONS: A significant shrinkage was observed in the SIS scaffolds after implantation. Although the four-ply SIS contracted less than the one-ply SIS, neither scaffold supported significant amount of intestinal regeneration.     

Bladder regeneration in a canine model using hyaluronic acid-poly(lactic-co-glycolic-acid) nanoparticle modified porcine small intestinal submucosa

What's known on the subject? and What does the study add? Urinary bladder tissue can be produced by several regenerative techniques although consistent regeneration remains elusive and no single protocol is superior to the others. Small intestinal submucosa has been used as substrate for regeneration in several models and allows for full‐thickness bladder regeneration. Nanotechnology has been applied to impart specific physical and biological properties onto biological substrates. Nanoparticles composed of PLGA can be modified by the addition of hyaluronic acid and can be added to SIS. This modified SIS demonstrates a potential improvement in biological activity as suggested by more consistent smooth muscle regeneration in a canine model of bladder regeneration. This study demonstrates both the feasibilty and potential of synthetically‐modified natural biomaterials.

OBJECTIVE

? To determine if hyaluronic acid (HA) can be incorporated into porcine small intestinal submucosa (SIS) through poly (lactide‐co‐glycolide‐acid) (PLGA) nanoparticles to improve the consistency of the naturally derived biomaterial and promote bladder tissue regeneration.

METHODS

? Beagle dogs were subjected to 40% partial cystectomy followed by bladder augmentation with commercial SIS or HA‐PLGA‐modified SIS. ? Urodynamic testing was performed before and after augmentation to assess bladder volume. ? A scoring system was created to evaluate gross and histological presentations of regenerative bladders.

RESULTS

? All dogs showed full‐thickness bladder regeneration. ? Histological assessment showed improved smooth muscle regeneration in the HA‐PLGA‐modified SIS group. ? For both groups of dogs, urodynamics and graft measurements showed an approximate 40% reduction in bladder capacity and graft size from pre‐augmentation to post‐regeneration measurements. ? Application of the scoring system and statistical analysis failed to show a significant difference between the groups.

CONCLUSIONS

? SIS can be modified through the addition of HA‐PLGA nanoparticles. The modified grafts showed evidence of improved smooth muscle regeneration on histological assessment, although this difference was not evident on a novel grading scale. ? The volume loss and graft shrinkage experienced are consistent with previous models of SIS bladder regeneration at the 10‐week time point. ? Additional research into the delivery of HA and the long‐term benefits of HA on bladder regeneration is needed to determine the full benefit of HA‐PLGA‐modified SIS. In addition, a more objective biochemical characterization will be needed to evaluate the quality of regeneration.     

Tensile strength comparison of small intestinal submucosa body wall repair

BACKGROUND: Porcine small intestinal submucosa (SIS) has been studied for body wall repair. However, the best method to implant the biological material has not been investigated. The objective of this study was to compare tensile strengths achieved after healing when SIS was placed using three implant techniques (onlay, inlay, underlay) in a porcine model of abdominal wall defect. MATERIALS AND METHODS: Twenty female domestic pigs had three abdominal midline sites assigned to one of five test groups: SIS implantation using inlay, onlay, or underlay technique; sham surgery (sutured midline incision) or normal body-wall control. Full-thickness muscle/fascia midline abdominal defects (6 x 4 cm) were surgically created and then repaired using eight-layer SIS. Healing was evaluated at 1 and 4 post-operative months by tensile strength testing and histopathology. RESULTS: Hernias were not observed. Tensile strengths were not statistically different between the five test groups (P = 0.39) or between months 1 and 4 (P = 0.35). The caudal site was stronger than the cranial or middle sites in the 1 month group (P < 0.0001). Histologically, healing appeared to progress over time as the repair site showed remodeling towards an interlacing fibrous connective tissue pattern. CONCLUSIONS: No significant differences in tensile strength were found between implant techniques and were not statistically different from sham surgery and normal control tissue. This study suggested that SIS healing/remodeling provides sufficient tensile strength for the repair of ventral (anterior) abdominal wall defects when implanted using any of three common techniques.     

Morphologic study of three collagen materials for body wall repair

BACKGROUND: The search for ideal prostheses for body wall repair continues. Synthetic materials such as polypropylene mesh (PPM) are associated with healing complications. A porcine-derived collagen-based material (CBM), small intestinal submucosa (SIS), has been studied for body wall repair. Renal capsule matrix (RCM) and urinary bladder submucosa (UBS) are CBMs not previously evaluated in this application. This is the first implant study using RCM. MATERIALS AND METHODS: Full-thickness muscle/fascia ventral abdominal wall defects were repaired with SIS, RCM, UBS, and PPM in rats with omentum and omentectomy. A random complete block design was used to allot implant type to each of 96 rats. Healing was evaluated at 4 and 8 weeks. Adhesion tenacity and surface area were scored. Implant site dimensions were measured at implantation and necropsy. Inflammation, vascularization, and fibrosis were histopathologically scored. Data were compared by analysis of variance (P < 0.05). RESULTS: PPM produced a granulomatous foreign body response in contrast to the organized healing of CBM implants. CBM mean scores were lower than PPM scores for adhesion tenacity, surface area, and inflammation at each follow-up time for rats with omentums (P < 0.02). The CBMs had less tenacity and inflammation than PPM at each follow-up time in omentectomy groups (P < 0.008). Wound contraction was greater for PPM (P < 0.0001) for all rats. CONCLUSIONS: RCM and UBS were similar to SIS invoking reduced inflammation, adhesion, and contraction compared to PPM. The fibrotic response to PPM was unique and more intense compared to CBMs. These CBM implants appear morphologically acceptable and warrant continued investigation.     

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.     

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.     

小肠粘膜下层的制备及细胞相容性的实验研究

目的了解猪小肠粘膜下层(SIS)的细胞相容性,探讨用SIS为生长载体复合骨髓基质干细胞(BMSCs)构筑组织工程骨的可能性。方法用物理和化学方法处理猪小肠粘膜下层,将兔骨髓基质干细胞与SIS进行体外复合培养,分别进行组织学、相差显微镜和扫描电镜观察。结果经物理和化学处理的SIS纯度高,孔隙多,胶原纤维未受损;BMSCs在SIS材料上生长、粘附、增殖,并能长入材料的孔隙内,分泌大量的细胞外基质成分。结论SIS的细胞相容性良好,不影响BMSCs的形态,对细胞生长和功能表达无抑制作用,可以用作骨组织工程的支架材料。     

Morphologic evaluation of regenerated small bowel by small intestinal submucosa

Background/Purpose

Previous studies have shown small intestinal submucosa (SIS) can be used as biodegradable scaffolds in tissue engineering small intestine. The purpose of this study is to evaluate the regeneration of neointestine and its morphology using SIS.

Methods

A 2-cm tubular SIS graft from Sprague Dawley rat donors was interposed in the middle of a 6-cm ileal Thiry-Vella loop of Lewis rats, which was used to construct an ileostomy. The grafts were harvested at each of the time points ranging from 2 weeks to half a year after implantation, and native small intestine and grafts were investigated for morphology using histology and immunohistochemistry.

Results

At the early postoperative period, SIS grafts were colonized by numerous inflammatory cells. A mucosal epithelial layer began to line the luminal surface of the graft by 4 weeks, and by 12 weeks, the luminal surface was covered completely by a layer of neomucosa. Neomucosa with typical small bowel morphology was characterized by a columnar epithelial cell layer with goblet cells, Paneth cells, absorptive enterocytes, and enteroendocrine cells. Significant differences between neomucosa by 12 weeks and 24 weeks in the measurements of mucosal thickness, villus height, and crypt depth were found. The outer walls of SIS grafts were composed of distinct bundles of well-formed smooth muscle-like cells with some fibrovascular tissue.

Conclusions

This initial study suggests that tissue engineering neointestine using SIS can develop structural features of the normal intestine. Small intestinal submucosa might be a viable material in the creation of neointestine for patients suffering short bowel syndrome.     

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。     

小肠粘膜下层表面肝素化及肝素化后其抗凝血性能的研究

目的研究等离子体引发血管组织工程支架——小肠粘膜下层(SIS)表面肝素化及肝素化后SIS膜的抗凝血性能。方法利用等离子体引发技术在SIS膜表面结合肝素,通过体外凝血时间检测及循环血流下观察肝索化SIS管腔的长期通畅性,以评价SIS膜的血液相容性。结果肝素结合到低温等离子体引发的SIS膜表面。肝素化SIS膜表面水接触角降低,亲水性增强,表面自由能增大,其凝血酶原时间、部分凝血活酶时间及凝血酶时间明显延长,肝素化SIS小口径血管支架保持通畅达6周。结论SIS膜表面通过等离子体进行肝素固化处理后具有良好而持久的抗凝血性能,表现出良好的血液相容性。     

骨髓基质干细胞复合小肠黏膜下层体内异位成骨的研究

目的 观察骨髓间充质干细胞复合小肠黏膜下层构建组织工程骨膜的异位成骨能力.方法 将常规方法培养的BMSCs与SIS复合(M1组)及进行成骨诱导培养BMSCs与SIS复合(M2组)植入兔(n=8)背部肌袋内作为实验组,以单纯SIS作为阴性对照.术后4、8、12周观察其成骨情况.结果 BMSCs在SIS支架上黏附、生长良好.植入体内4、8、12周,M2、M1、SIS组成骨量分别为(25.08±0.79)%、(18.81±0.42)%和(13.98±1.86)%,各指标M2组高于M1或SIS组(P<0.05),M1组高于SIS组(P<0.05).随着时间的延长,两实验组成骨量逐渐增加(P<0.05),SIS组的成骨量变化不明显(P>0.05). 结论 BMSCs与SIS复合构建组织工程骨膜在体内有异位成骨作用,而且经过成骨诱导的组织工程骨膜成骨作用更加明显.     

Small intestinal submucosa (SIS) graft urethroplasty: short-term results

OBJECTIVE: We evaluated the use of small intestinal submucosa (SIS) graft in penile and bulbar urethroplasties. METHODS: From 2003 to 2004, 20 men (mean age, 41 yr) with anterior urethral strictures underwent urethroplasty using SIS (COOK) as an inlay or onlay patch graft. Stricture location was penile in 1 patient, bulbar in 16, and penile-bulbar in 3. Average stricture and graft lengths were 3 and 5.7 cm, respectively. A dorsal inlay graft was performed in 14 cases, ventral onlay graft in 1, and dorsal inlay plus ventral onlay in 5. Clinical outcome was considered successful if no postoperative procedure was needed. RESULTS: Mean follow-up period was 21 mo (range: 13-35 mo). Seventeen cases (85%) were successful and 3 (15%) were failures. No postoperative complications were related to the use of heterologous graft material, such as infection or rejection. Sixteen successes (94%) were bulbar repairs and one a penile-bulbar repair, with stricture and graft average lengths 2.6 and 5.35 cm, respectively. Cystoscopy at 3 mo revealed adequate calibre lumens, but SIS grafted areas were not completely replaced by urothelium. The three failures were penile and penile-bulbar urethral repairs with stricture and graft average lengths of 5.7 and 7.7 cm, respectively. Recurrences showed fibrous tissue involving the grafted area with extension into the penile and bulbar urethra. CONCLUSIONS: In our short-term results, SIS seems to be a versatile material that may have a role in select urethral reconstructions. Longer follow-up and further investigations in select patients are needed before widespread use is advocated.     

小肠粘膜下层(SIS)桥接周围神经缺损的实验研究

目的 应用小肠粘膜下层(SIS)桥接周围神经缺损并观察结果。方法 选取健康的SD大鼠30只，随机分成三组，每组10只。先造成坐骨神经10mm缺损，然后分别用SIS桥接、自体神经移植和旷置不做处理，即SIS桥接神经组、自体神经移植对照组和空白对照组。分别于术后6周和10周取材，通过病理组织检查、神经电生理检查和计算机图像分析来评价神经再生。结果 SIS桥接神经组可见有再生神经组织长过缺损，呈条索状连续样，且神经纤维多集中在SIS形成的桥接管周缘区域，而中心区域可见胶原组织和孔隙较多。从电生理检查和计算机图像分析结果来看，SIS桥接神经组比自体神经移植组略差。结论 SIS具有促进周围神经轴突再生的作用，有望成为自体神经的替代材料应用于周围神经缺损的修复。     

Porcine small intestinal submucosa graft for repair of anterior urethral strictures

OBJECTIVES: We evaluated porcine small intestinal submucosa (SIS) used in the treatment of inflammatory, iatrogenic, posttraumatic, and idiopathic strictures of bulbar and penile urethra. Midterm maintenance of urethral patency was assessed. METHODS: Fifty patients aged 45-73 yr with anterior urethral stricture underwent urethroplasty using a porcine SIS collagen-based matrix for urethral reconstruction. Stricture was localized in the bulbar urethra in 10 patients, the bulbopenile area in 31 cases, and in the distal penile urethra in nine patients. All patients received a four-layered SIS patch graft in an onlay fashion. A voiding history, retrograde and antegrade urethrography, and cystoscopy were performed preoperatively and postoperatively. Failure was defined as stricture confirmed on urethrogram. RESULTS: After a mean follow-up of 31.2 mo (range: 24-36 mo), the clinical, radiological, and cosmetic findings were excellent in 40 (80%) patients. Restricture developed in one of 10 bulbar, five of 31 bulbopenile, and four of nine penile strictures. These all occurred in the first 6 mo postoperatively. All patients with recurrences needed further therapy, but there has been no additional recurrence observed to date. No complications such as fistula, wound infection, UTI, or rejection were observed. CONCLUSIONS: Use of inert porcine SIS matrix appears to be beneficial for patients with bulbar and bulbopenile strictures. Midterm results are comparable to skin flaps and mucosal grafts.     

猪角朊细胞复合小肠黏膜下层体外培养实验

目的 探讨脱细胞小肠黏膜下层（SIS）作为组织工程皮肤支架的可行性.方法 将猪第二代角朊细胞接种于SIS上,接种后第11、13、15、17天通过直接染色、石蜡组织切片、HE染色、抗层粘连蛋白免疫组化染色、透射电镜等方法,观察角朊细胞在SIS上的生长情况.结果 猪角朊细胞接种在SIS上11d形态良好,部分区域形成2～3层细胞组成的复层结构,免疫组织化学染色显示在角朊细胞与SIS之间的细胞外基质中抗层粘连蛋白呈连续阳性表达.13、15、17 d细胞复层结构增多,细胞之间连接紧密,透射电镜下可见细胞内有张力微丝,细胞间有桥粒连接,角朊细胞与SIS之间可见基底膜形成.结论 在猪角朊细胞体外培养中,SIS是良好的细胞黏附支架.     

Body wall repair using small intestinal submucosa seeded with cells

Background/Purpose: Prosthetic repair of large ventral abdominal wall defects has been associated with high complication rates. This study was aimed at applying tissue engineering to body wall replacement.Methods: Syngeneic Lewis rats underwent harvest of skeletal muscle specimens. Once expanded in vitro, skeletal muscle cells or fibroblasts were suspended in a collagen gel. All animals underwent creation of a 2.5- × 3-cm abdominal wall defect. The defect was repaired with the cell-seeded gel placed in between 2 pieces of small intestinal submucosa (SIS). The control group was repaired by SIS with acellular gel. Animals were killed at different time-points for histologic and mechanical examination. Statistical analysis was by analysis of variance (ANOVA).Results: Abdominal wall hernia was present in 6 of 24 fibroblast-seeded constructs (25%), 5 of 21 skeletal muscle cell-seeded constructs (23.9%), and 16 of 21 acellular grafts (76.2%), respectively (P < .05). At harvest, cell-seeded constructs were thicker with better cellular infiltration, whereas acellular grafts were thin, low in cell density, and poor in mechanical resistance.Conclusions: Unlike acellular collagen matrices, engineered cellular constructs have better cell infiltration and mechanical performance. Tissue engineering may be a viable alternative for body-wall replacement.     

Dorsal onlay augmentation urethroplasty with small intestinal submucosa: Modified Barbagli technique for strictures of the bulbar urethra

AIM: To present the results from one clinic's experience of using small intestinal submucosa (SIS) in augmentation urethroplasty for management of strictures of the bulbar urethra. METHODS: Urethral surgery was performed in nine men with strictures 4-6 cm. All of the patients were evaluated by history, physical examination, retrograde urethrogram, and uroflowmetry. Four layers of SIS were soaked in saline or Ringer's solution for 15 minutes at 37 degrees C, and the inner surface of the patch was gently fenestrated with a thin scalpel. The patch was spread-fixed onto the tunica albuginea. The mucosa was sutured to the submucosal graft first at 2-3 mm inwards from the SIS margins, then the spongiosum tissue was attached to the margins with interrupted absorbable sutures. RESULTS: Of the nine patients who underwent augmentation urethroplasty using SIS, only one had re-stricture at 6 months due to urethral infection. At 18 months after the surgery the uroflowmetry of the other eight patients was 20-21 mL/s. In terms of complications, six patients reported having post-micturition dribbling, and seven patients reported lack of morning erections for 35-69 days after surgery. CONCLUSIONS: Using SIS is a safe procedure; however, long-term follow-up is needed to substantiate the good short-term results.     

The bolster technique utilising small intestinal submucosa wound matrix: A novel approach to wound treatment

Managing acute wounds with soft tissue loss can be very challenging for both patients and physicians. Successful wound healing depends on several factors including exudate control, prevention of infection, and moisture balance. In this case series, we describe a novel combination treatment method utilising small intestinal submucosa wound matrix (SISWM) with the bolster technique as a way of assisting the integration of collagen‐based wound treatment products into the base of complex wounds with the intent of restoring a dysfunctional extracellular matrix. In case 1, a 44‐year‐old female presented with an acute wound resulting from a spider bite to the posterior aspect of the right knee. In case 2, a 12‐year‐old male sustained multiple injuries to his right foot from an all‐terrain vehicle accident. In case 3, an 80‐year‐old female on anticoagulants sustained an avulsion injury to her left lower leg. In case 4, a 41‐year‐old female sustained a severe complex avulsion injury to the dorsal left forearm sustained from a dog bite. All patients were successfully treated with SISWM and the bolster technique, and their wounds healed completely within 6 weeks. The bolster technique, when combined with an SISWM, is a novel method designed to enable the SISWM to impart its wound healing properties to these complex traumatic wounds. This case series presents treating clinicians with a different treatment methodology to assist the patient in achieving a successful outcome.     

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.