Acellular Small Intestinal Submucosa Research Articles

Successful Use of Acellular Small Intestinal Submucosa Graft in Vaginal Reconstruction

PurposeVarious techniques for neovaginal construction have been employed in the pediatric and adult populations, including the use of intestinal segments, buccal mucosal grafts, and skin grafts. Small intestinal submucosa (SIS) extracellular matrix grafts have been described as a viable alternative, though prior experience is limited. Our purpose was to assess operative characteristics and patient outcomes with neovaginal construction using SIS grafts. MethodsThirteen patients underwent vaginoplasty with acellular porcine SIS grafts at our institution between 2018 and 2022. Operative and clinical data, postoperative mold management, vaginal dilating length, and complications were reviewed. ResultsAge at time of repair ranged from 13 to 30 years (median 19 years). Patient diagnosis included cloacal anomalies (n = 4), Mayer-Rokitansky-Küster-Hauser syndrome (n = 4), isolated vaginal atresia with or without a transverse vaginal septum (n = 4), and vaginal rhabdomyosarcoma requiring partial vaginectomy (n = 1). Following dissection of the neovaginal space, a silicon mold wrapped with SIS graft was placed with retention sutures and removed on postoperative day 7. Median (IQR) operative time was 171 (118–192) minutes, estimated blood loss was 10 (5–20) mL, and length of stay was 2 (1–3) days. The follow-up period ranged from 3 to 47 months (median 9 months). Two patients developed postoperative vaginal stenosis that resolved with dilation under anesthesia. Mean vaginal length on latest follow-up was 8.97 cm. All thirteen patients had successful engraftment and progressed to performing self-dilations or initiating intercourse to maintain patency. There were no cases of graft reaction or graft extrusion. ConclusionsWe conclude that acellular small intestinal submucosa grafts are effective and safe alternatives for mold coverage in neovaginal construction. Our experience demonstrates minimal perioperative morbidity, early mold removal, and progression to successful dilation with maintenance of a functional vaginal length. Future study on sexual outcomes, patient satisfaction, and comparison against alternative techniques has been initiated. Level of EvidenceIV. Type of StudyRetrospective Study.

Inductive Remodeling of Extracellular Matrix Scaffolds in the Temporomandibular Joint of Pigs.

The temporomandibular joint (TMJ) disc is a fibrocartilaginous tissue located between the condyle of the mandible and glenoid fossa and articular eminence of the temporal bone. Damage or derangement of the TMJ disc can require surgical removal (discectomy) to restore function. Removal of the TMJ disc, however, leaves the joint space vulnerable to condylar remodeling and degradation, potentially leading to long-term complications. No consistently effective clinical option exists for repair or replacement of the disc following discectomy. This study investigates the use of an acellular scaffold composed of extracellular matrix (ECM) derived from small intestinal submucosa (SIS) as a regenerative template for the TMJ disc in a porcine model. Acellular SIS ECM scaffolds were implanted following discectomy and allowed to remodel for 2, 4, 12, and 24 weeks postimplantation. Remodeling of the implanted device was assessed by longitudinal magnetic resonance imaging (MRI) over the course of 6 months, as well as gross morphologic, histologic, biochemical, and biomechanical analysis (tension and compression) of explanted tissues (disc and condyle) at the time of sacrifice. When the scaffold remained in the joint space, longitudinal MRI demonstrated that the scaffolds promoted new tissue formation within the joint space throughout the study period. The scaffolds were rapidly populated with host-derived cells and remodeled with formation of new, dense, aligned fibrocartilage resembling native tissue as early as 1 month postimplantation. De-novo formation of peripheral muscular and tendinous attachments resembling those in native tissue was also observed. The remodeled scaffolds approached native disc biochemical composition and compressive modulus, and possessed 50% of the tensile modulus within 3 months postimplantation. No degradation of the condylar surface was observed. These results suggest that this acellular bioscaffold fills a medical need for which there is currently no effective treatment and may represent a clinically relevant "off-the-shelf" implant for reconstruction of the TMJ disc.

Klotho functionalization on vascular graft for improved patency and endothelialization

The Klotho (KL) gene is related to aging. In this study, SKL (secreted KL) and heparin were cross-linked to the acellular small intestinal submucosa (SIS). Based on this, tissue-engineered bioactive small blood vessels were constructed. The goal of this study was to determine whether the release of SKL could improve the patency of small-diameter tissue-engineered blood vessels (TEVs) through promoting cell adhesion. The recombinant human SKL protein was generated from HEK293 cells with overexpression of SKL. Then the SIS membrane was cross-linked with heparin and SKL respectively, to prepare heparin group and SKL group artificial vascular grafts. SKL treatment promoted endothelial cells proliferation and upregulated the levels of Focal adhesion kinase (FAK) phosphorylation and Ras homolog gene family, member A (RhoA). SKL effectively enhanced the endothelial cells adhesion on the SIS membrane. In vivo evaluation of SKL modified SIS grafts in rabbits exhibited increased patency rate, endothelialization, and smooth muscle regeneration. In this study, SKL-modified SIS grafts can effectively improve patency of small-diameter TEVs through enhancing cell adhesion, and it is expected to exhibit an important effect in the construction of substitutes for coronary artery bypass grafting.

A biomimetic triple-layered biocomposite with effective multifunction for dura repair

Dura mater defect and subsequent cerebrospinal fluid (CSF) leakage usually appear in trauma or neurosurgical procedures and are followed by a series of serious complications and even death. The use of a qualified dura mater substitute with multifunction of leakage blockade, adhesion prevention, and dura reconstruction is one of the promising treatment methods. However, even though some products have been used in the clinic, none of the substitutes achieved the required multifunction. In this study, we aimed to design and fabricate a dura repair composite with the ideal multifunction. By biomimicking the structure and component of natural dura, we applied poly(L-lactic acid) (PLLA), chitosan (CS), gelatin, and acellular small intestinal submucosa (SIS) powders to successfully prepare a triple-layered composite. Then, a series of specific devices and techniques were developed to investigate the performance. The results revealed that satisfactory structural stability could be realized under good synergistic interactions among the components. In addition, all the findings suggested that the bionic triple-layered composite showed satisfactory multifunction of leakage blockade, adhesion prevention, antibacterial property, and dura reconstruction potential, and thus, it might be a promising candidate for dura repair. Statement of significanceDeveloping qualified dura mater substitutes with multifunction of leakage blockade, adhesion prevention, and dura reconstruction is crucial for treating dura mater defect and subsequent cerebrospinal fluid (CSF) leakage that appear in trauma or neurosurgical procedures. In this study, we designed and fabricated a triple-layered dura repair biocomposite with satisfactory structural stability and desired multifunction based on biomimicking of the structure and component of natural dura. Moreover, a series of specific devices and techniques were developed to investigate the relevant performance. Overall, the developed hydrogel electrospinning system exhibited excellent advantages in achieving multifunction and could be applied widely in the future to achieve multifunctional tissue repair materials.

Adipose-Derived Stem Cells Are an Efficient Treatment for Fistula-in-ano of Japanese Rabbit.

Fistula-in-ano (FIA, anal fistula) treatment remains a surgical challenge for coloproctologists. Adipose tissue-derived stem cells (ADSCs) are a new frontier in the treatment of FIA. In this study, we established a FIA model of Japanese rabbit and evaluated the effect of four treatments on fistula healing: ADSC transplantation, acellular small intestinal submucosa (ASIS), noncutting seton, and PBS as negative control. High-throughput RNA sequencing was also performed to investigate the anal tissue (normal and ADSC treatment group) expression profile of mRNA. Our data showed that ADSC treatment had the shortest time to promote fistula healing compared to the other treatments, and fistula filled with new adipose tissue and muscle cells without scar. Gene Ontology (GO) analysis of RNA-seq data showed that the differential genes are enriched in system development and animal organ development. Taken together, our findings demonstrate that ADSCs rapidly promote fistula healing through differentiation, a promising stem cell therapy for FIA. The rabbit is an effective animal model for evaluating FIA therapeutic options.

Development and Characterization of an Acellular Porcine Small Intestine Submucosa Scaffold for Use in Corneal Epithelium Tissue Engineering

ABSTRACTPurpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.

Stable subcutaneous cartilage regeneration of bone marrow stromal cells directed by chondrocyte sheet.

In vivo niche plays an important role in directing differentiation fate of stem cells. Due to lack of proper chondrogenic niche, stable cartilage regeneration of bone marrow stromal cells (BMSCs) in subcutaneous environments is always a great challenge. The current study demonstrated that chondrocyte sheet generated by high-density culture of chondrocytes in vitro could cearte a chondrogenic niche in subcutaneous environment and efficiently retain the chondrogenic phenotype of in vitro BMSC engineered cartilage (vitro-BEC). Furthermore, cell tracing results revealed that the regenerated cartilage mainly derived from the implanted vitro-BEC. The current study not only proposes a novel research model for microenvironment simulation but also provides a useful strategy for stable ectopic cartilage regeneration of stem cells.

A new method for the preparation of biomedical hydrogels comprised of extracellular matrix and oligourethanes

This paper reports a new method to modify hydrogels derived from the acellular extracellular matrix (ECM) and consequently to improve their properties. The method is comprised of the combination of liquid precursors derived from hydrolyzed acellular small intestinal submucosa (hECM) and water-soluble oligourethanes that bear protected isocyanate groups, synthesized from poly(ethylene glycol) (PEG) and hexamethylene diisocyanate (HDI). The results demonstrate that the reactivity of oligourethanes, along with their water solubility, properly induce simultaneously the polymerization of type I collagen and its crosslinking. The polymerization rate and the gel network parameters such as fiber diameter, porosity, crosslinking degree, mechanics, swelling, in vitro degradation and cell proliferation, keep a direct relationship with the oligourethane concentration. Consequently, the hybrid hydrogels formulated with 15 wt.% of oligourethane exhibit enhanced storage modulus and degradation resistance, while maintaining the cell viability and impeding the fibroblast-induced contraction in comparison with the hECM hydrogels without oligourethanes. Therefore, this method is adequate to prepare novel hydrogels where the adjustment of the crosslinking degree controls the materials structure and their properties. This new method offers advantages for regulating the features of ECM-derived templates, thereby extending their possibilities for tissue engineering (TE) applications.

The use of bi-layer silk fibroin scaffolds and small intestinal submucosa matrices to support bladder tissue regeneration in a rat model of spinal cord injury

Adverse side-effects associated with enterocystoplasty for neurogenic bladder reconstruction have spawned the need for the development of alternative graft substitutes. Bi-layer silk fibroin (SF) scaffolds and small intestinal submucosa (SIS) matrices were investigated for their ability to support bladder tissue regeneration and function in a rat model of spinal cord injury (SCI). Bladder augmentation was performed with each scaffold configuration in SCI animals for 10 wk of implantation and compared to non-augmented control groups (normal and SCI alone). Animals subjected to SCI alone exhibited a 72% survival rate (13/18) while SCI rats receiving SIS and bi-layer SF scaffolds displayed respective survival rates of 83% (10/12) and 75% (9/12) over the course of the study period. Histological (Masson's trichrome analysis) and immunohistochemical (IHC) evaluations demonstrated both implant groups supported de novo formation of smooth muscle layers with contractile protein expression [α-smooth muscle actin (α-SMA) and SM22α] as well as maturation of multi-layer urothelia expressing cytokeratin (CK) and uroplakin 3A proteins. Histomorphometric analysis revealed bi-layer SF and SIS scaffolds respectively reconstituted 64% and 56% of the level of α-SMA+ smooth muscle bundles present in SCI-alone controls, while similar degrees of CK+ urothelium across all experimental groups were detected. Parallel evaluations showed similar degrees of vascular area and synaptophysin+ boutons in all regenerated tissues compared to SCI-alone controls. In addition, improvements in certain urodynamic parameters in SCI animals, such as decreased peak intravesical pressure, following implantation with both matrix configurations were also observed. The data presented in this study detail the ability of acellular SIS and bi-layer SF scaffolds to support formation of innervated, vascularized smooth muscle and urothelial tissues in a neurogenic bladder model.

Grafts of porcine small intestinal submucosa seeded with cultured homologous smooth muscle cells for bladder repair in dogs

BackgroundDue to numerous complications associated to gastrointestinal augmented cystoplasty, this study aimed to analyze the anatomic repair of the bladder of 10 female dogs using grafts of porcine small intestinal submucosa (SIS) seeded with cultured homologous smooth muscle cells, and compare them with the acellular SIS grafts.ResultsWe assessed the possible side effects and complications of each type of graft by clinical examination, abdominal ultrasound and laboratory findings. Anatomic repair of neoformed bladder was assessed by histological staining for H/E and Masson's Trichrome, analyzed with a Nikon Photomicroscope connected to the system of image analysis Image J.ConclusionsWe propose that SIS associated to homologous smooth cells can improve the quality of tissue repair, and consequently decrease the potential complications inherent to acellular SIS.

In vitro construction of tissue-engineered cardiac tissue sheet by small intestinal submucosa with bone mesenchymal stem cells

Objective To explore the feasibility of in vitro construction of tissue-engineered cardiac sheet by small intestinal submucosa(SIS) with bone mesenchymal stem cells(BMSCs). Methods BMSCs separated from rat bone marrow were induced by 5-azacytidine(5-Aza) for 3 weeks,and were seeded on the serosal side of acellular SIS scaffold.The tissue-engineered cardiac tissue sheet was established in vitro after co-culture in dynamic culture system for 2 weeks,and its properties were evaluated by histological,ultrastructural and immunohistochemical examinations. Results After co-culture for 2 weeks,HE staining revealed that the seeded cells were not restricted in the upper area of SIS,forming multiple uniform layers on SIS,and some cells penetrated and migrated into the porous matrix.Scanning electronic microscopy revealed that BMSCs adhered,proliferated and migrated well on SIS,and secreted a large amount of extracellular matrix.Immunohistochemical staining demonstrated that the cells on SIS were cardiomyocyte-like cells with the expression of α-actin,cTnⅠ and connexin-43. Conclusion The engineered cardiac tissue sheet has been successfully constructed by SIS with 5-Aza-induced BMSCs in vitro,and SIS is an ideal bio-scaffold material for cardiac tissue engineering.

The Artificial Conduit for Urinary Diversion in Rats: A Preliminary Study

Objectives Small intestinal submucosa forms a scaffold for tubular construction. The aim of this study was to build the artificial conduit using small intestinal submucosa (SIS) and 3T3 fibroblasts for urinary diversion in rats. Materials and methods 3T3 fibroblasts were multiplied to a total of 10 9. Two groups consisted of three Wistar rats each. The left ureters were separated from the bladder and anastomosed to the proximal end of the tubular scaffold. No splitting of the ureteral junction or drainage was done. The distal end of the scaffold was implanted into a previously performed channel in the abdominal wall. Cell-seeded grafts were used in the first group and acellular SIS scaffolds in the second group. Rats were sacrificed after 2 and 4 weeks. X-ray pyelography was performed. Hematoxylin and eosin staining was prepared from conduit cross sections. Results All animals survived the observation. An inflammatory reaction was observed within the peritoneal cavity in both groups. It was difficult to dissect the adhesions in the cell-seeded group. The ureteral-conduit anastomoses were tight in five cases, except there was leakage and pseudocyst formation after 14 days in one cell-seeded graft. No ureterohydronephrosis was observed in two acellular conduits after 14 or 30 days, and in one case of a cell-seeded graft. A neovascularisation process was observed in the acellular conduit after a month. Multilayered epithelium covered the conduit lumen near the anastomosis at the distal end of acellular conduit, a small islet-forming epithelial layer was observed after a month. Conclusions 3T3 fibroblasts cannot serve as a “feeder layer” for ureteral augmentation. It seems that there is no need to split the ureteral-conduit junction. An SIS scaffold was used for tubular construction for urinary diversion in an animal model.

Prevention of device-related tissue damage during percutaneous deployment of tissue-engineered heart valves

Endovascular application of pulmonary heart valves has been recently introduced clinically. A tissue-engineering approach was pursued to overcome the current limitations of bovine jugular vein valves (degeneration and limited longevity). However, deployment of the delicate tissue-engineered valves resulted in severe tissue damage. Therefore the objective of this study was to prevent tissue damage during the folding and deployment maneuver. Porcine pulmonary heart valves, small intestinal submucosa, and ovine carotid arteries were obtained from a slaughterhouse. After dissection and antimicrobial incubation, the valves were trimmed (removal of sinus and most of the muscular ring) to fit into the deployment catheter. The inside (in-stent group, n = 6) or outside (out-stent group, n = 6) of a nitinol stent was covered by an acellular small intestinal submucosa, and the valves were sutured into the stent. The valves were folded, tested for placement in the deployment catheter, and decellularized enzymatically. Myofibroblasts were obtained from carotid artery segments and seeded onto the scaffolds. The seeded constructs were placed in a dynamic bioreactor system and cultured for 16 consecutive days. After endothelial cell seeding, the constructs were folded, deployed, and processed for histology and surface electron microscopy. The valves opened and closed competently throughout the entire dynamic culture. Surface electron microscopy revealed an almost completely preserved tissue in the in-stent group. Stents covered with small intestinal submucosa on the outside, however, showed severe damage. This study demonstrates that small intestinal submucosa covering of the inside of a pulmonary valved stent can prevent stent strut-related tissue damage.

Development of a reinforced porcine elastin composite vascular scaffold

Elastin, a principal structural component of native arteries, has distinct biological and mechanical advantages when used as a biomaterial; however, its low ultimate tensile strength has limited its use as an arterial conduit. We have developed a scaffold, consisting of a purified elastin tubular conduit strengthened with fibrin bonded layers of acellular small intestinal submucosa (aSIS) for potential use as a small diameter vascular graft. The addition of aSIS increased the ultimate tensile strength of the elastin conduits nine-fold. Burst pressures for the elastin composite vascular scaffold (1,396 +/- 309 mmHg) were significantly higher than pure elastin conduits (162 +/- 36 mmHg) and comparable to native saphenous veins. The average suture pullout strength of the elastin composite vascular scaffolds was 14.612 +/- 3.677 N, significantly higher than the pure elastin conduit (0.402 +/- 0.098 N), but comparable to native porcine carotid arteries (13.994 +/- 4.344 N). Cyclic circumferential strain testing indicated that the composite scaffolds were capable of withstanding physiological loading conditions for at least 83 h. Implantation of the elastin composites as carotid interposition grafts in swine demonstrated its superiority to clinically acceptable ePTFE with significantly longer average patency times of 5.23 h compared to 4.15 h. We have developed a biologically based elastin scaffold with suitable mechanical properties and low thrombogenicity for in vivo implantation, and with the potential for cellular repopulation and host integration reestablishing an appropriate elastic artery.

Diaphragmatic reconstruction with autologous tendon engineered from mesenchymal amniocytes

Purpose This study examined the effects of amniocyte-based engineered tendons on partial diaphragmatic replacement. Methods Ovine mesenchymal amniocytes were labeled with green fluorescent protein (GFP), expanded, and seeded into a collagen hydrogel. Composite grafts (20 to 25 cm 2) based on acellular dermis (group I), or acellular small intestinal submucosa (group II) received either a cell-seeded or an acellular hydrogel within their layers. Newborn lambs (n = 20) underwent partial diaphragmatic replacement with either an acellular or a cellular autologous construct from either group. At 3 to 12 months’ postoperatively, implants were subjected to multiple analyses. Results Diaphragmatic hernia recurrence was significantly higher in animals with acellular grafts (5 of 5) then in animals with cellular ones (1 of 4) in group I ( P < .05) but not in group II (3 of 6 and 4 of 5, respectively). Cellular grafts had higher modular (5.27 ± 1.98 v. 1.27 ± 0.38 MPa) and ultimate (1.94 ± 0.70 v. 0.29 ± 0.05 MPa) tensile strength than acellular implants in group I (P < .05), but not in group II. Quantitative analyses showed no differences in extracellular matrix components between cellular and acellular implants in either group. All cellular implants showed GFP-positive cells. Conclusions Diaphragmatic repair with an autologous tendon engineered from mesenchymal amniocytes leads to improved mechanical and functional outcomes when compared with an equivalent acellular bioprosthetic repair, depending on scaffold composition. The amniotic fluid may be a preferred cell source for engineered diaphragmatic reconstruction.

Fibrochondrogenesis of free intraarticular small intestinal submucosa scaffolds.

Naturally occurring biomaterials, such as small intestine submucosa (SIS), are attractive as potential scaffolds for engineering various tissue types. The aim of this study was to determine whether acellular SIS scaffolds can support cell attachment and ingrowth in a diarthroadial joint without significant intraarticular hemorrhage. Disks of porcine SIS were arthoscopically implanted freely within a randomized knee joint of 21 dogs and harvested 1, 2, 3, and 6 weeks postoperatively. Harvested disks were assessed for gross and histologic appearance, cellular infiltration, and immunoreactivity of collagenase and collagen types I and II. Knee synovium and synovial fluid were also evaluated. All disks were thickened and opacified at harvest. Eleven disks (52%) had adhered to intraarticular tissues and cellular infiltration into the disks was positively correlated with tissue adherence. Further, tissue adherence was positively correlated with duration of intraarticular implantation. Five disks (24%) contained focal areas of homogeneous extracellular matrix. A trend toward more collagenase immunoreactivity was noted in the 3-week disks. Collagen type I was present in remaining SIS and extracellular matrix associated with infiltrated cells. Placed freely within a joint, acellular SIS disks underwent cellular and extracellular matrix modification resulting in fibrocartilage-like tissue. Utilization of SIS as a scaffold for intraarticular tissue-engineering applications is supported as cytoconductivity, appropriate residence time, and absence of untoward effects of implantation are desirable criteria for a tissue-engineering biomaterial.