The Healing Effect of Biodegradable Scaffolds Treated with Bone-Marrow Obtained Mesenchymal Stem Cells on Major Tendon Damage in the Dog as a Model.

The present study aimed to evaluate the implantation of decellularized small intestinal submucosa- extracellular matrix )SIS-ECM( seeded with bone marrow mesenchymal stem cells (BM-MSCs) to repair full-thickness Achilles tendon defect. For this purpose, 20 healthy adult stray dogs aged 8-12 months old (15±3 kg of weight) were enrolled in this study under an aseptic environment and general anesthesia. A 1.5 cm-long segment-based resection was performed in the mid-substance of the Achilles tendon in the control group (n=10) that did not receive treatment. While, in the experimental group (n=10), regarding the defect of the tendon, the stumps were bridged with decellularized SIS seeded with BM-MSCs (5×106) cells implanted. Afterward, the stumps of the tendon were sutured using the modified Kessler technique (4-0) polypropylene thread. The biomechanical observations of the tendon defect showed an increase in the tensile strength in the experimental group, compared to the control animals. It should be mentioned that this difference was significant (P≤0.05). Histopathological observations of biopsies harvested after the 4th, 8th, and 12th weeks revealed that the implanted graft had seeded with MSCs enhanced high-quality cellular infiltration and the host tissue healing was improved. Similar to the normal tendon, a dense organized collagenous tissue with high cellularity and vascularity was observed due to the presence of the remodeled ECM. However, the arrangement of collagen-fiber-derived connective tissue appeared to be more dominant than that in the experimental group, with less adhesion in the 12th week post-treatment. These findings suggest that the BM-MSCs inoculated with SIS can be employed to repair a damaged Achilles tendon due to the fact that this combination enhances the regeneration of the affected tendon.

The clinical effectiveness of staple line reinforcement with different matrix used in surgery.

Staplers are widely used in clinics; however, complications such as bleeding and leakage remain a challenge for surgeons. To tackle this issue, buttress materials are recommended to reinforce the staple line. This Review provides a systematic summary of the characteristics and applications of the buttress materials. First, the physical and chemical properties of synthetic polymer materials and extracellular matrix used for the buttress materials are introduced, as well as their pros and cons in clinical applications. Second, we review the clinical effects of reinforcement mesh in pneumonectomy, sleeve gastrectomy, pancreatectomy, and colorectal resection. Based on the analysis of numerous research data, we believe that buttress materials play a crucial role in increasing staple line strength and reducing the probability of complications, such as bleeding and leakage. However, considering the requirements of bioactivity, degradability, and biosafety, non-crosslinked small intestinal submucosa (SIS) matrix material is the preferred candidate. It has high research and application value, but further studies are required to confirm this. The aim of this Review is to provide comprehensive guidance on the selection of materials for staple line reinforcement.

Enhancement of Tendon Repair Using Tendon-Derived Stem Cells in Small Intestinal Submucosa via M2 Macrophage Polarization.

(1) Background: Reconstruction of Achilles tendon defects and prevention of postoperative tendon adhesions were two serious clinical problems. In the treatment of Achilles tendon defects, decellularized matrix materials and mesenchymal stem cells (MSCs) were thought to address both problems. (2) Methods: In vitro, cell adhesion, proliferation, and tenogenic differentiation of tendon-derived stem cells (TDSCs) on small intestinal submucosa (SIS) were evaluated. RAW264.7 was induced by culture medium of TDSCs and TDSCs-SIS scaffold groups. A rat Achilles tendon defect model was used to assess effects on tendon regeneration and antiadhesion in vivo. (3) Results: SIS scaffold facilitated cell adhesion and tenogenic differentiation of TDSCs, while SIS hydrogel coating promoted proliferation of TDSCs. The expression of TGF-ß and ARG-1 in the TDSCs-SIS scaffold group were higher than that in the TDSCs group on day 3 and 7. In vivo, the tendon regeneration and antiadhesion capacity of the implanted TDSCs-SIS scaffold was significantly enhanced. The expression of CD163 was significantly highest in the TDSCs-SIS scaffold group; meanwhile, the expression of CD68 decreased more significantly in the TDSCs-SIS scaffold group than the other two groups. (4) Conclusion: This study showed that biologically prepared SIS scaffolds synergistically promote tendon regeneration with TDSCs and achieve antiadhesion through M2 polarization of macrophages.

Bacterial nanocellulose as novel carrier for intestinal epithelial cells in drug delivery studies.

Synthetic cell carriers (A) represent common scaffold structures for the development of cell-based in vitro models of the human intestine but due to their low porosity or unwanted molecular adhesion effects, synthetic carriers can negatively affect cell function. Alternative scaffolds such as natural extracellular matrices (ECMs) (B) were shown to overcome some of the common drawbacks. However, their fabrication is time-consuming, less well standardized and not entirely conform to the 3R principle (replacement, reduction, refinement). Nowadays, biopolymers such as bacterial nanocellulose (BNC) (C) represent interesting scaffold materials for innovative tissue engineering concepts, as they can be generated in a faster and more standardized process workflow without the need for animal material. In this study, we demonstrate the BNC as suitable carrier for the development of Caco-2-based in vitro models of the human intestine. The BNC-based models exhibit organ-specific properties comprising typical cellular morphologies, characteristic protein expression profiles, representative ultrastructural features and the formation of a tight epithelial barrier. The proof of in vivo-like transport activities further validates the high quality of the BNC-based Caco-2 models. In summary, this illustrates the BNC as alternative bioscaffold of non-animal origin to develop functional organ models in vitro.

Decellularized bSIS-ECM as a Regenerative Biomaterial for Skin Wound Repair.

Tissue engineering-based regenerative applications can involve the use of stem cells for the treatment of non-healing wounds. Multipotent mesenchymal stem cells have become a focus of skin injury treatments along with many other injury types owing to their unprecedented advantages. However, there are certain limitations concerning the solo use of stem cells in skin wound repair. Natural bioactive extracellular matrix-based scaffolds have great potential for overcoming these limitations by supporting the regenerative activity and localization of stem cells. This chapter describes the use of bone marrow mesenchymal stem cells together with decellularized bovine small intestinal submucosa (SIS), for the treatment of a critical-sized full-thickness skin defect in a small animal model.

Decellularization of Bovine Small Intestinal Submucosa.

Decellularization technology promises to overcome some of the significant limitations in the regenerative medicine field by providing functional biocompatible grafts. The technique involves removal of the cells from the biological tissues or organs for further use in tissue engineering and clinical interventions. There are significant differences between decellularization protocols due to the intrinsic properties of different tissue types and purpose of use. This multistep, chemical-solution-based protocol is optimized for the preparation of decellularized bovine small intestinal submucosa (SIS).

Outcomes in patients undergoing robotic reconstructive uterovaginal anastomosis of congenital cervical and vaginal atresia.

OBJECTIVE: To introduce our experience of robotic surgery of reconstructive uterovaginal anastomosis and operative outcomes in congenital cervical and vaginal atresia patients. METHODS: Clinical observation and follow-up of four patients with congenital cervical and vaginal atresia who underwent robotic reconstruction of cervix and vagina by SIS (small intestinal submucosa, SIS) graft. RESULTS: Average patient age was 13.8 ± 2.2. Patients complained of severe periodic abdominal pain. Diagnosis was made according to clinical characteristics, physical examination, MRI and classified by ESHRE/ESGE system. All patients underwent reconstruction of cervix and vagina by uterovaginal anastomosis by SIS graft. Average operation time was 232.5 ± 89.2 min, average blood loss was 225.0 ± 95.7 mL. After surgery, all patients have regular menstruation without pain. Average follow up was 12 months, average vagina length was 8.9 ± 0.3 cm, average vagina width was 2.9 ± 0.1 cm. CONCLUSION: Robotic assisted reconstruction of cervix and vagina is feasible from our experience, enlarged cases and additional studies are required.

Small intestinal submucosa: A potential osteoconductive and osteoinductive biomaterial for bone tissue engineering.

SIS is an acellular, naturally occurring collagenous extracellular matrix (ECM) material with various bioactive factors, which broadly applied in tissue engineering in clinic. Several studies have applied SIS in bone tissue engineering to enhance bone regeneration in animal models. However, the mechanism was rarely investigated. The aim of the current study was to investigate the osteoconductivity and osteoinductivity of SIS scaffold to bone regeneration systematically and the potential mechanism. Our results showed that SIS scaffold with excellent biocompatibility was beneficial for cell attachment, proliferation, migration and osteogenic differentiation of various cells contributing to bone repair. In mouse calvarial defect model, bone regeneration was significantly enhanced in the defects implanted with SIS scaffolds, along with the up-regulation of BMP-2 and CD31 expression. Accordingly, ID-1, the downstream target gene of BMPs, was increased in BMSCs cultured on SIS scaffolds. The results of this study suggest that SIS scaffold is a potential osteoconductive and osteoinductive biomaterial which plays multiple roles to various cells during process of bone regeneration.

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical-sized full-thickness skin defect, alone and in combination with stem cells, in a small rodent model.

In this study, we initially described an efficient decellularization protocol for bovine-derived small intestinal submucosa (bSIS), involving freeze-thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm2 ) full-thickness skin defect. The wound models treated with either MSCs-seeded (1.5 × 106 cells/cm2 ) or non-seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs-seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non-seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.

Controlled delivery of icariin on small intestine submucosa for bone tissue engineering.

Small intestine submucosa (SIS) has been reported as an excellent biomaterial for tissue engineering because of its naturally occurring collagenous extracellular matrix property with growth factors. However, SIS from submucosal layer of intestine provides different microenvironment from bone tissue, which limits its application to bone regeneration. The object of this study was to improve osteoinductivity of SIS by controlled local delivery of icariin (Ic), a potent osteogenic compound. Sustained release of icariin from SIS scaffold was achieved for >30days and the loading of icariin on SIS scaffold was uniform as scanned by SEM. In vitro experiments revealed that expression of osteogenic differentiation markers (Alp, Bsp and Ocn) was increased after treatment of Ic-SIS scaffold, without significant cytotoxicity. In an in vivo mouse calvarial defect model, bone regeneration was enhanced by SIS implantation at 8weeks, compared to control defect. New bone formation was further improved by implantation with Ic-SIS (low and high) at both 4 and 8weeks. The results of this study suggest that SIS scaffold has the potential as an icariin delivery carrier for enhancement of bone regeneration.

In vivo assessment of two endothelialization approaches on bioprosthetic valves for the treatment of chronic deep venous insufficiency.

Chronic deep venous insufficiency is a debilitating disease with limited therapeutic interventions. A bioprosthetic venous valve could not only replace a diseased valve, but has the potential to fully integrate into the patient with a minimally invasive procedure. Previous work with valves constructed from small intestinal submucosa (SIS) showed improvements in patients' symptoms in clinical studies; however, substantial thickening of the implanted valve leaflets also occurred. As endothelial cells are key regulators of vascular homeostasis, their presence on the SIS valves may reduce the observed thickening. This work tested an off-the-shelf approach to capture circulating endothelial cells in vivo using biotinylated antikinase insert domain receptor antibodies in a suspended leaflet ovine model. The antibodies on SIS were oriented to promote cell capture and showed positive binding to endothelial cells in vitro; however, no differences were observed in leaflet thickness in vivo between antibody-modified and unmodified SIS. In an alternative approach, valves were pre-seeded with autologous endothelial cells and tested in vivo. Nearly all the implanted pre-seeded valves were patent and functioning; however, no statistical difference was observed in valve thickness with cell pre-seeding. Additional cell capture schemes or surface modifications should be examined to find an optimal method for encouraging SIS valve endothelialization to improve long-term valve function in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1610-1621, 2016.

Carbodimide cross-linked and biodegradation-controllable small intestinal submucosa sheets.

The small intestinal submucosa (SIS) is an acellular collagen-based matrix, primarily composed of fibrillar collagens (types I, II, and V). They enhance healing due to a minimal immune response. A good degradation rate is the degradation of materials equal to the rate of remodeling in the host. The SIS should apply a good degradation rate and cytocompatibility. In this study, a series of SIS with different degradation rates is obtained by treatment with Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Morphology, composition, degradable ratio, mechanical properties and cytocompatibility of the SIS are evaluated. We determined a 20 µm thickness and 60 µm pore size of the native SIS. The degradable ratio of the native SIS was approximately 90% in the presence of 0.25 mg/ml collagenase for 24 hours. The storage modulus of the native SIS was 388 MPa. The degradable ratio of the SIS decreased to 6% and the storage modulus increased to 777 MPa after being treated with 30 mM EDC for 24 hours. In cytocompatibility assay, cell numbers on the native SIS were similar as on the treated SIS due to the non-toxicity of the EDC treatment process. This SIS exhibited collagenase resistance, stronger mechanical strength and good cytocompatibility after the EDC treatment concluded. The cross-linked SIS could be utilized as a potential cell carrier for tissue engineering application.

Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-ß3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

Bladder Regeneration by Small Intestinal Submucosa with Release of Exogenous Growth Factors / 中国康复理论与实践

@#ObjectiveTo explore the release of exogenous growth factors from small intestinal submucosa (SIS) in bladder regeneration. MethodsThe release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from SIS in vitro were evaluated by ELISA and MTT method. The defected bladder walls of rats in experimental group were repaired with porcine small intestinal submuscosa. Partial bladder mucosa and smooth muscle of the rats in control groups were destroyed. At regular intervals, the VEGF and bFGF expression were observed by histological and immunohistochemical methods. ResultsThe concentration of bFGF and VEGF released in vitro from SIS in PBS solution were (121.8±2.683) ng/L and (93.8±3.033) ng/L respectively, and showed proliferation of vascular endothelial cell. In the SIS framework, the capillary and smooth muscle were observed followed histological evaluation. The weak expression of VEGF and bFGF in both experimental and control groups were found in the first week. Since the second week the VEGF and bFGF expression in experimental group began to increase with a peak in the 6th week, and began to decrease after 8 weeks. In the control group, the weak VEGF and bFGF expression were shown during the observation. ConclusionSIS functions as a carrier for exogenous growth factors release in rat bladder regeneration.

Bladder Regeneration by Small Intestinal Submucosa with Release of Exogenous Growth Factors / 中国康复理论与实践

@#ObjectiveTo explore the release of exogenous growth factors from small intestinal submucosa (SIS) in bladder regeneration. MethodsThe release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) from SIS in vitro were evaluated by ELISA and MTT method. The defected bladder walls of rats in experimental group were repaired with porcine small intestinal submuscosa. Partial bladder mucosa and smooth muscle of the rats in control groups were destroyed. At regular intervals, the VEGF and bFGF expression were observed by histological and immunohistochemical methods. ResultsThe concentration of bFGF and VEGF released in vitro from SIS in PBS solution were (121.8±2.683) ng/L and (93.8±3.033) ng/L respectively, and showed proliferation of vascular endothelial cell. In the SIS framework, the capillary and smooth muscle were observed followed histological evaluation. The weak expression of VEGF and bFGF in both experimental and control groups were found in the first week. Since the second week the VEGF and bFGF expression in experimental group began to increase with a peak in the 6th week, and began to decrease after 8 weeks. In the control group, the weak VEGF and bFGF expression were shown during the observation. ConclusionSIS functions as a carrier for exogenous growth factors release in rat bladder regeneration.

Study on antithrombogenicity and surface heparinization of small intestinal submucosa / 中华创伤骨科杂志

Objective To study surface heparinization of small intestinal submucosa(SIS)and an- tithrombegenicity of beparinized SIS films with plasma initiation technique for the engineering vascular scaffolds. Methods The SIS films were grafted with heparin by hypothermia plasma initiation technique.The blood com- patibility of the modified SIS films was assessed by observing blood coagulation time in vitro and the long term pa- tency of hepafinized SIS vascular scaffolds directly under the circulation of blood.Results The hypothermia plasma initiation method could attach heparin onto the SIS surface,The water contact angle of SIS films modified with heparin was decreased while the surface free energy and hydrophilicity increased.The prothrombin time(PT),ac- tivated partial thromboplastin time(APTT)and thrombin clotting Time(TT)of the SIS films modified with heparin were prolonged obviously.Small caliber engineering vascular scaffold made of heparinized SIS films kept the patency for six weeks.Conclusion Heparin can be attached to SIS films by hypothermia plasma initiation technique.The modified surfaces provide good and persistent antithrombogenicity,and possess potent blood compatibility,