Small Intestinal Submucosa Scaffolds Research Articles

Reinforcing decellularized small intestine submucosa with cellulose acetate nanofibrous and silver nanoparticles as a scaffold for wound healing applications.

The formation of chronicwounds accounts for considerable costs in health care systems. Despite theseveral benefits of decellularized small intestinal submucosa (SIS) as anappropriate scaffold for different tissue regeneration, it has shortcomings such as lack of antibacterial features and inappropriate mechanical properties for skin tissue regeneration. We aimed to examine the efficacy and safety of decellularized SIS scaffold enhanced with cellulose acetate (CA) and silver (Ag) nanoparticles (NPs) for healing full-thickness wounds. The scaffolds wereprepared by decellularizing bovine SIS and electrospinning CA/Ag nanoparticles and characterized using a transmission electron microscope (TEM), scanning electronmicroscope (SEM), tensile testing, and X-ray diffraction. In vivo evaluations were performed using full-thickness excisions covered with sterile gauze as the control group, SIS, SIS/CA, and SIS/CA/Ag scaffolds on the dorsum of twenty male Wistar rats divided into four groups randomly with 21-days follow-up. All in vivo specimens underwent Masson's trichrome (MT) staining for evaluation of collagen deposition, transforming growth factor-β (TGF-β) immunohistochemistry (IHC), and Haematoxylin Eosin (H&E) staining. The IHC and MT data were analyzed with the ImageJ tool by measuring the stained area. The TEM results revealedthat Ag nanoparticles are successfully incorporated into CA nanofibers. Assessment of scaffoldshydrophilicity demonstrated that the contact angle of SIS/CA/Ag scaffold was the lowest. The in vivoresults indicated that the SIS/CA/Ag scaffold had the most significant wound closure. H&E staining of the invivo specimens showed the formation of epidermal layers in the SIS/CA/Ag group on day 21. The percentage of the stained area of MT and TGF-β IHC staining's was highest in the SIS/CA/Ag group. The decellularizedSIS/CA/Ag scaffolds provided the most significant wound closure compared toother groups and caused the formation of epidermal layers and skin appendages. Additionally, the collagen deposition and expression of TGF-β increased significantly in SIS/CA/Ag group.

The effect of Wharton’s jelly-derived stem cells seeded/boron-loaded acellular scaffolds on the healing of full-thickness burn wounds in the rat model

Burn wounds are the most destructive and complicated type of skin or underlying soft tissue injury that are exacerbated by a prolonged inflammatory response. Several cell-based therapeutic systems through the culturing of potent stem cells on modified scaffolds have been developed to direct the burn healing challenges. In this context, a new regenerative platform based on boron (B) enriched-acellular sheep small intestine submucosa (AOSIS) scaffold was designed and used as a carrier for mesenchymal stem cells derived from Wharton’s jelly (WJMSCs) aiming to promote the tissue healing in burn-induced rat models. hWJMSCs have been extracted from human extra-embryonic umbilical cord tissue. Thereafter, 96 third‐degree burned Wistar male rats were divided into 4 groups. The animals that did not receive any treatment were considered as group A (control). Then, group B was treated just by AOSIS scaffold, group C was received cell-seeded AOSIS scaffold (hWJMSCs-AOSIS), and group D was covered by boron enriched-cell-AOSIS scaffold (B/hWJMSCs-AOSIS). Inflammatory factors, histopathological parameters, and the expression levels of epitheliogenic and angiogenic proteins were assessed on 5, 14 and 21 d post-wounding. Application of the B/hWJMSCs-AOSIS on full-thickness skin-burned wounds significantly reduced the volume of neutrophils and lymphocytes at day 21 post-burning, whilst the number of fibroblasts and blood vessels enhanced at this time. In addition, molecular and histological analysis of wounds over time further verified that the addition of boron promoted wound healing, with decreased inflammatory factors, stimulated vascularization, accelerated re-epithelialization, and enhanced expression levels of epitheliogenic genes. In addition, the boron incorporation amplified wound closure via increasing collagen deposition and fibroblast volume and activity. Therefore, this newly fabricated hWJMSCs/B-loaded scaffold can be used as a promising system to accelerate burn wound reconstruction through inflammatory regulation and angiogenesis stimulation.

Enhancing cartilage regeneration and repair through bioactive and biomechanical modification of 3D acellular dermal matrix.

Acellular dermal matrix (ADM) shows promise for cartilage regeneration and repair. However, an effective decellularization technique that removes cellular components while preserving the extracellular matrix, the transformation of 2D-ADM into a suitable 3D scaffold with porosity and the enhancement of bioactive and biomechanical properties in the 3D-ADM scaffold are yet to be fully addressed. In this study, we present an innovative decellularization method involving 0.125% trypsin and 0.5% SDS and a 1% Triton X-100 solution for preparing ADM and converting 2D-ADM into 3D-ADM scaffolds. These scaffolds exhibit favorable physicochemical properties, exceptional biocompatibility and significant potential for driving cartilage regeneration in vitro and in vivo. To further enhance the cartilage regeneration potential of 3D-ADM scaffolds, we incorporated porcine-derived small intestinal submucosa (SIS) for bioactivity and calcium sulfate hemihydrate (CSH) for biomechanical reinforcement. The resulting 3D-ADM+SIS scaffolds displayed heightened biological activity, while the 3D-ADM+CSH scaffolds notably bolstered biomechanical strength. Both scaffold types showed promise for cartilage regeneration and repair in vitro and in vivo, with considerable improvements observed in repairing cartilage defects within a rabbit articular cartilage model. In summary, this research introduces a versatile 3D-ADM scaffold with customizable bioactive and biomechanical properties, poised to revolutionize the field of cartilage regeneration.

A biomimetic in situ mineralization ECM composite scaffold to promote endogenous bone regeneration

Bone tissue engineering scaffolds constructed from single-component organic materials have inherent limitations. Inspired by the hierarchical structure of physiological natural bone hard tissues, our research explores the construction of organic-inorganic composite scaffold for bone regeneration. In this study, we used a natural and readily obtainable extracellular matrix (ECM) material, i.e., decellularized small intestinal submucosa (SIS), to build the organic component of a phosphorylated hydroxyapatite nanocrystal-containing composite scaffold (nHA@SIS). Guided by polymer-induced liquid-precursor theory, we introduced a soluble inorganic mineralization solution to achieve an inorganic component of nHA@SIS. Using in situ mineralization, we successfully formed inorganic component within SIS and constructed nHA@SIS composite scaffold. We analyzed the physicochemical properties and the osteogenic role of nHA@SIS via a series of in vitro and in vivo studies. Compared with SIS scaffold, the nHA@SIS possessed suitable physicochemical properties, maintained the excellent cell activity of SIS and better guided reorganization of the cell skeleton, thereby achieving superior osteoconductivity and maintaining osteoinductivity at the protein and gene levels. Furthermore, the rat cranial defect area in the nHA@SIS scaffold group was mostly repaired after 12 weeks of implantation, with a larger amount of higher-density new bone tissue being visible at the edge and center than SIS and blank control group. This significantly improved in vivo osteogenic ability indicated the great potential of nHA@SIS for bone tissue engineering applications.

Small intestinal submucosa promotes angiogenesis via the Hippo pathway to improve vaginal repair.

Vaginal reconstruction has incorporated the use of gastrointestinal segments for decades, but the technique is inevitably associated with complications. Tissue-engineering techniques, however, have brought great hope for vaginal reconstruction. This study aimed to evaluate the utility of small intestinal submucosa (SIS) in reconstructing clinically significant large vaginal defects in a porcine model and to investigate the role of the Hippo pathway in the vascular remodeling process. The composition and mechanical properties of SIS were characterized. Full-thickness vaginal defects were established in 10 minipig donors, with 4 cm lengths removed and replaced by an equal sized SIS scaffolds. The neovaginas were subjected to macroscopic, histological, immunohistochemical and molecular evaluations at 4 and 12 weeks after the surgery. Four weeks after the operation, extracellular matrix reorganization and complete coverage the surface of the luminal matrix by vaginal epithelium were observed, accompanied by the formation of a microvascular network and the regeneration of smooth muscles, albeit disorderly arranged. Twelve weeks after implantation, enhacements were seen in the formation of the multi-layered squamous epithelium, angiogenesis, and large muscle bundle formation in the vagina. Aditionally, the expression levels of angiogenesis-related proteins, proliferation-related proteins and Hippo pathway-related proteins in the neovagina were significantly increased. These results indicate that SIS could be used to reconstruct large vaginal defects and that the vascular remodeling process is potentially regulated by the Hippo pathway.

Umbilical Cord Mesenchymal Stem Cells Seeded on Small Intestinal Submucosa to Repair the Uterine Wall Injuries.

Objective: The effectiveness of tissue engineering materials combining porcine small intestine submucosa (SIS) and umbilical cord mesenchymal stem cells (UC-MSCs) on uterine injury in female rat after full-thickness uterine resection was evaluated as a basis for clinical treatment of postoperative uterine injury. Methods: After complex culture with SIS and UC-MSCs, cell adhesion, growth, and proliferation were assessed. Before the implantation, a surgical procedure of bilateral full-thickness uterine resection (0.5-2.0 cm long and 0.3 cm wide) was performed to obtain the rat uterine injury model, while the sham-operated rats were used as controls. Hematoxylin-eosin (H&E) staining results and fertility of female rats in each group were assessed to determine the critical resection length of the full-thickness uterine resection. Then SIS or UC-MSCs-SIS were implanted into the female rats from the uterine injury group, followed by assessments of H&E staining, the expression of ki67, α-SMA, and leukemia inhibitory factor (LIF), and fertility to determine the effectiveness of SIS and UC-MSCs-SIS on uterine injury in female rat. Results: At 24, 48, and 72 h, the cells grew progressively on the SIS material. In the 1.5 cm and 2.0 cm groups, the pregnancy rate, proportion of the uterus supporting live embryo growth, number of live embryos, and proportion of live embryos were all significantly less than those in the 0.5 cm and sham-operated groups. In the 2.0 cm group, there was little tissue regeneration at the center of the injury and not conducive to subsequent assessment. The UC-MSCs-SIS and SIS groups were better on morphological development, cell proliferation, LIF expression, and fertility than the control group. Conclusions: UC-MSCs show good adhesion, growth, and proliferation on the SIS scaffold material. The optimal resection length in full-thickness uterine resection on female rat is 1.5 cm. UC-MSCs-SIS is the effective treatment for repairing a injury after the full-thickness resection of the uterus in this research. Impact Statement The acquired severe uterus injury is a serious condition, which prone to uterine adhesions. Postoperative endometrial repairment and prevention of intrauterine adhesion recurrence are two major clinical challenges. Fortunately, the development of tissue engineering technology makes repairing a uterine injury possible. There are two main contributions from this study. First, due to ethical requirements, it is difficult to assess the repairing effect on uterus by invasive experiments in a clinical practice. Therefore, we constructed a full-thickness uterine injury rat model, which allows us to assess the repairing effect of treatments after severe uterine injuries in vivo. Second, it explored the effect of using a combination of and umbilical cord mesenchymal stem cells and small intestine submucosa materials on improving uterine repairments, providing a potential possibility for a future clinical practice.

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-β 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.

CircPTP4A2-miR-330-5p-PDK2 Signaling Facilitates In Vivo Survival of HuMSCs on SF-SIS Scaffolds and Improves the Repair of Damaged Endometrium.

Background Human umbilical cord mesenchymal stem cells- (HuMSCs-) based therapy has shown promising results in the treatment of intrauterine adhesions (IUA). In this study, we aimed to construct a HuMSCs-seeded silk fibroin small-intestinal submucosa (SF-SIS) scaffold and evaluate its ability to repair the damaged endometrium in an IUA mouse model. Methods To identify the functional effect of HuMSCs-SF-SIS scaffolds on the repair of damaged endometrium, a mouse IUA model was established. Uterine morphology and fibrosis were evaluated by hematoxylin-eosin staining and Masson staining. CircRNA sequencing, real-time PCR, and RNA fluorescence in situ hybridization were used to screen and verify the potential circRNAs involved in the repair of damaged endometrium by HuMSCs. Real-time integrated cellular measurement of oxygen consumption rate was performed using the Seahorse XF24 Extracellular Flux Analyzer. The potential downstream miRNAs and proteins of circRNAs were analyzed by dual-luciferase reporter assay and western blot. Results HuMSCs-SF-SIS not only increased the number of glands but also reduced the ulcer area in the IUA model. circPTP4A2 was elevated in the HuMSCs seeded on the SF-SIS scaffolds and was targeted by miR-330-5p-PDK2. It also stabilized the mitochondrial metabolism of HuMSCs. Moreover, miR-330-5p was found to inhibit PDK2 expression through the 3′ UTR target region. A rescue experiment further showed that circPTP4A2-miR-330-5p-PDK2 signaling was critical to HuMSCs-SF-SIS in decreasing the fibrosis area and increasing the number of glands in the IUA model. Conclusion We demonstrated that circPTP4A2 was elevated in HuMSCs-seeded on SF-SIS scaffolds and stabilized the mitochondrial metabolism through miR-330-5p-PDK2 signaling, which contributes to endometrial repair progression. These findings demonstrate that HuMSCs-seeded SF-SIS scaffolds have potential for the treatment of IUA.

Independent effects of structural optimization and resveratrol functionalization on extracellular matrix scaffolds for bone regeneration

Due to their natural biological activity and low immunogenicity, decellularized extracellular matrix (ECM) materials have aroused interest as potential scaffold materials in tissue engineering. Decellularized small intestinal submucosa (SIS) is one ECM biomaterial that can be easily sourced. In the present study, we tested whether the osteogenesis of SIS scaffolds was enhanced via structural optimization and resveratrol (RSV) functionalization and explored the independent effects of these modifications. We obtained SIS scaffolds with different pore structures by controlling the preparation concentration. The group with superior osteogenic properties was further RSV-functionalized via covalent immobilization. We conducted a series of in vitro and in vivo studies to explore the effects of these two optimization strategies on the osteogenic properties of SIS scaffolds. The results showed that pore structure and RSV functionalization significantly affected the osteogenic properties of SIS scaffolds. With a fabrication concentration of 1%, the SIS scaffolds had superior osteogenic properties. Through covalent coupling, RSV was successfully grafted onto SIS scaffolds, where it was slowly released. The most significant improvements in osteogenic properties were obtained with a coupling concentration of 1%. Furthermore, in in vivo experiments, vascular and new bone tissue formation was enhanced with RSV/SIS scaffolds compared with SIS scaffolds and the blank control group at 4 weeks after implantation. These findings indicate that the RSV/SIS scaffolds obtained via dual optimization strategies show promise as biomaterials in bone tissue engineering.

BMSCs and Osteoblast-Engineered ECM Synergetically Promotes Osteogenesis and Angiogenesis in an Ectopic Bone Formation Model.

Bone mesenchymal stem cells (BMSCs) have been extensively used in bone tissue engineering because of their potential to differentiate into multiple cells, secrete paracrine factors, and attenuate immune responses. Biomaterials are essential for the residence and activities of BMSCs after implantation in vivo. Recently, extracellular matrix (ECM) modification with a favorable regenerative microenvironment has been demonstrated to be a promising approach for cellular activities and bone regeneration. The aim of the present study was to evaluate the effects of BMSCs combined with cell-engineered ECM scaffolds on osteogenesis and angiogenesis in vivo. The ECM scaffolds were generated by osteoblasts on the small intestinal submucosa (SIS) under treatment with calcium (Ca)-enriched medium and icariin (Ic) after decellularization. In a mouse ectopic bone formation model, the SIS scaffolds were demonstrated to reduce the immune response, and lower the levels of immune cells compared with those in the sham group. Ca/Ic-ECM modification inhibited the degradation of the SIS scaffolds in vivo. The generated Ca/Ic-SIS scaffolds ectopically promoted osteogenesis according to the results of micro-CT and histological staining. Moreover, BMSCs on Ca/Ic-SIS further increased the bone volume percentage (BV/TV) and bone density. Moreover, angiogenesis was also enhanced by the Ca/Ic-SIS scaffolds, resulting in the highest levels of neovascularization according to the data ofCD31 staining. In conclusion, osteoblast-engineered ECM under directional induction is a promising strategy to modify biomaterials for osteogenesis and angiogenesis. BMSCs synergetically improve the properties of ECM constructs, which may contribute to the repair of large bone defects.

Establishment of the SIS scaffold-based 3D model of human peritoneum for studying the dissemination of ovarian cancer

Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer.

A Novel Bionic Extracellular Matrix Polymer Scaffold Enhanced by Calcium Silicate for Bone Tissue Engineering.

A novel porous calcium silicate (CS)-enhanced small intestinal submucosa (SIS) scaffold was prepared by freeze-drying to mimic the natural extracellular matrix environment for bone tissue engineering. The micro-morphology, physicochemical properties, biological characteristics, and effects on osteogenic differentiation in vitro were explored; the effects on promoting bone formation in vivo were evaluated. The composite scaffold had an ideal three-dimensional porous structure. The amount of calcium silicate played a significant role in improving mechanical properties and promoting osteogenic differentiation. The SIS/2CS scaffold promoted proliferation and osteogenic differentiation in human bone marrow mesenchymal stem cells; it also significantly increased osteogenesis in vivo. This novel composite polymer scaffold has potential applications in bone tissue engineering.

Evaluation of Bi-Layer Silk Fibroin Grafts for Penile Tunica Albuginea Repair in a Rabbit Corporoplasty Model.

The use of autologous tissue grafts for tunica albuginea repair in Peyronie’s disease and congenital chordee is often restricted by limited tissue availability and donor site morbidity, therefore new biomaterial options are needed. In this study, bi-layer silk fibroin (BLSF) scaffolds were investigated to support functional tissue regeneration of tunica albuginea in a rabbit corporoplasty model. Eighteen adult male, New Zealand white rabbits were randomized to nonsurgical controls (NSC, N = 3), or subjected to corporoplasty with BLSF grafts (N = 5); decellularized small intestinal submucosa (SIS) matrices (N = 5); or autologous tunica vaginalis (TV) flaps (N = 5). End-point evaluations were cavernosography, cavernosometry, histological, immunohistochemical, and histomorphometric assessments. Maximum intracorporal pressures (ICP) following papaverine-induced erection were similar between all groups. Eighty percent of rabbits repaired with BLSF scaffolds or TV flaps achieved full rigid erections, compared to 40% of SIS reconstructed animals. Five-minute peak erections were maintained in 60% of BLSF rabbits, compared to 20% of SIS and TV flap reconstructed rabbits. Graft perforation occurred in 60% of TV group at maximum ICP compared to 20% of BLSF cohort. Neotissues supported by SIS and BLSF scaffolds were composed of collagen type I and elastin fibers similar to NSC. SIS and TV flaps showed significantly elevated levels of corporal fibrosis relative to NSC with a corresponding decrease in corporal smooth muscle cells expressing contractile proteins. BLSF biomaterials represent emerging platforms for corporoplasty and produce superior functional and histological outcomes in comparison to TV flaps and SIS matrices for tunica albuginea repair.

Effects of hypoxia on Achilles tendon repair using adipose tissue-derived mesenchymal stem cells seeded small intestinal submucosa

BackgroundThe study was performed to evaluate the feasibility of utilizing small intestinal submucosa (SIS) scaffolds seeded with adipose-derived mesenchymal stem cells (ADMSCs) for engineered tendon repairing rat Achilles tendon defects and to compare the effects of preconditioning treatments (hypoxic vs. normoxic) on the tendon healing.MethodsFifty SD rats were randomized into five groups. Group A received sham operation (blank control). In other groups, the Achilles tendon was resected and filled with the original tendon (Group B, autograft), cell-free SIS (Group C), or SIS seeded with ADMSCs preconditioned under normoxic conditions (Group D) or hypoxic conditions (Group E). Samples were collected 4 weeks after operation and analyzed by histology, immunohistochemistry, and tensile testing.ResultsHistologically, compared with Groups C and D, Group E showed a significant improvement in extracellular matrix production and a higher compactness of collagen fibers. Group E also exhibited a significantly higher peak tensile load than Groups D and C. Additionally, Group D had a significantly higher peak load than Group C. Immunohistochemically, Group E exhibited a significantly higher percentage of MKX + cells than Group D. The proportion of ADMSCs simultaneously positive for both MKX and CM-Dil observed from Group E was also greater than that in Group D.ConclusionsIn this animal model, the engineered tendon grafts created by seeding ADMSCs on SIS were superior to cell-free SIS. The hypoxic precondition further improved the expression of tendon-related genes in the seeded cells and increased the rupture load after grafting in the Achilles tendon defects.

The effects of bone marrow stem and progenitor cell seeding on urinary bladder tissue regeneration

Complications associated with urinary bladder augmentation provide the motivation to delineate alternative bladder tissue regenerative engineering strategies. We describe the results of varying the proportion of bone marrow (BM) mesenchymal stem cells (MSCs) to CD34 + hematopoietic stem/progenitor cells (HSPCs) co-seeded onto synthetic POC [poly(1,8 octamethylene citrate)] or small intestinal submucosa (SIS) scaffolds and their contribution to bladder tissue regeneration. Human BM MSCs and CD34 + HSPCs were co-seeded onto POC or SIS scaffolds at cell ratios of 50 K CD34 + HSPCs/15 K MSCs (CD34-50/MSC15); 50 K CD34 + HSPCs/30 K MSCs (CD34-50/MSC30); 100 K CD34 + HSPCs/15 K MSCs (CD34-100/MSC15); and 100 K CD34 + HSPCs/30 K MSCs (CD34-100/MSC30), in male (M/POC; M/SIS; n = 6/cell seeded scaffold) and female (F/POC; F/SIS; n = 6/cell seeded scaffold) nude rats (n = 96 total animals). Explanted scaffold/composite augmented bladder tissue underwent quantitative morphometrics following histological staining taking into account the presence (S+) or absence (S−) of bladder stones. Urodynamic studies were also performed. Regarding regenerated tissue vascularization, an upward shift was detected for some higher seeded density groups including the CD34-100/MSC30 groups [F/POC S− CD34-100/MSC30 230.5 ± 12.4; F/POC S+ CD34-100/MSC30 245.6 ± 23.4; F/SIS S+ CD34-100/MSC30 278.1; F/SIS S− CD34-100/MSC30 187.4 ± 8.1; (vessels/mm2)]. Similarly, a potential trend toward increased levels of percent muscle (≥ 45% muscle) with higher seeding densities was observed for F/POC S− [CD34-50/MSC30 48.8 ± 2.2; CD34-100/MSC15 53.9 ± 2.8; CD34-100/MSC30 50.7 ± 1.7] and for F/SIS S− [CD34-100/MSC15 47.1 ± 1.6; CD34-100/MSC30 51.2 ± 2.3]. As a potential trend, higher MSC/CD34 + HSPCs cell seeding densities generally tended to increase levels of tissue vascularization and aided with bladder muscle growth. Data suggest that increasing cell seeding density has the potential to enhance bladder tissue regeneration in our model.

Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model.

Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.

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.

Biomimetic Human Tissue Model for Long-Term Study of Neisseria gonorrhoeae Infection.

Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.

Characterization of different biodegradable scaffolds in tissue engineering.

The aim of the present study was to compare the characteristics of acellular dermal matrix (ADM), small intestinal submucosa (SIS) and Bio-Gide scaffolds with acellular vascular matrix (ACVM)-0.25% human-like collagen I (HLC-I) scaffold in tissue engineering blood vessels. The ACVM-0.25% HLC-I scaffold was prepared and compared with ADM, SIS and Bio-Gide scaffolds via hematoxylin and eosin (H&E) staining, Masson staining and scanning electron microscope (SEM) observations. Primary human gingival fibroblasts (HGFs) were cultured and identified. Then, the experiment was established via the seeding of HGFs on different scaffolds for 1, 4 and 7 days. The compatibility of four different scaffolds with HGFs was evaluated by H&E staining, SEM observation and Cell Counting Kit-8 assay. Then, a series of experiments were conducted to evaluate water absorption capacities, mechanical abilities, the ultra-microstructure and the cytotoxicity of the four scaffolds. The ACVM-0.25% HLC-I scaffold was revealed to exhibit the best cell proliferation and good cell architecture. ADM and Bio-Gide scaffolds exhibited good mechanical stability but cell proliferation was reduced when compared with the ACVM-0.25% HLC-I scaffold. In addition, SIS scaffolds exhibited the worst cell proliferation. The ACVM-0.25% HLC-I scaffold exhibited the best water absorption, followed by the SIS and Bio-Gide scaffolds, and then the ADM scaffold. In conclusion, the ACVM-0.25% HLC-I scaffold has good mechanical properties as a tissue engineering scaffold and the present results suggest that it has better biological characterization when compared with other scaffold types.

Biomimetic Composite Scaffold Containing Small Intestinal Submucosa and Mesoporous Bioactive Glass Exhibits High Osteogenic and Angiogenic Capacity.

Biomaterials with excellent osteogenic and angiogenic activities are desirable to repair massive bone defects. Decellularized matrix from porcine small intestinal submucosa (SIS) has attracted particular attention for tissue regeneration because it has strong angiogenic effects and retains plentiful bioactive components. However, it has inferior osteoinductivity and osteoconductivity. In this study, we developed porous composite of SIS combined with mesoporous bioactive glass (SIS/MBG) with the goal of improving the mechanical and biological properties. SIS/MBG scaffolds showed uniform interconnected macropores (∼150 μm), high porosity (∼76%), and enhanced compressive strength (∼0.87 MPa). The proliferation and osteogenic gene expression (Runx2, ALP, Ocn, and Col-Iα) of rat bone marrow stromal cells (rBMSCs) as well as the proliferation, angiogenic gene expression (VEGF, bFGF, and KDR), and tube formation capacity of human umbilical vein endothelial cells (HUVECs) in SIS/MBG scaffolds were significantly upregulated compared with nonmesoporous bioactive glass (BG)-modified SIS (SIS/BG) and SIS-only scaffolds. Western blot analysis revealed that SIS/MBG induced rBMSCs to osteogenic differentiation through the activation of Wnt/β-Catenin signaling pathway, and SIS/MBG enhanced angiogenic activity of HUVEC through the activation of PI3k/Akt pathways. The in vivo results demonstrated that SIS/MBG scaffolds significantly enhanced new bone formation and neovascularization simultaneously in critical-sized rat calvarial defects as compared with SIS/BG and SIS. Collectively, the osteostimulative and angiostimulative biomimetic composite scaffold SIS/MBG represents an exciting biomaterial option for bone regeneration.