Matrix Scaffold Research Articles

Formulating Customizable Extracellular Matrix Scaffolds From Decellularized Mouse Kidneys

Formulating Customizable Extracellular Matrix Scaffolds From Decellularized Mouse Kidneys

Human Hematopoietic Stem Cells Co-cultured in 3D with Stromal Support to Optimize Lentiviral Vector-mediated Gene Transduction.

HSC transplantation (HSCT) has emerged as a promising treatment option for hematological and immunological disorders. Unfortunately, many viral vectors are inefficient at transduction, limiting the number of cells available for gene therapy in cord blood HSC transplantation. Combining ex vivo expansion and genetic manipulation of cord blood cells is a potential gene therapy approach. We present a 3D co-culture method using a demineralized bone matrix scaffold to optimize lentiviral vector-mediated gene transduction. pLenti-III-miR-GFP-has-miR-124 was transduced into cord blood HSCs. Transduced CD34 + cells co-cultured on the stromal layer for 72h under cytokine-free conditions. We performed flow cytometry, colony assays, real-time polymerase chain reaction, and SEM morphological analysis. Seventy-two hours after transduction, when pLentiIII-miR-GFP-has-miR-124 and control vector-transduced expanded cord blood HSCs were compared to non-transduced expanded cord blood HSCs, the findings revealed 15 ± 3.04 and 55 ± 3.05-fold increases in miR-124 mRNA expression, respectively. Compared to a control culture on the same day, the expansion of CD34+, CD38-HSCs in 3D culture increased 544 ± 31.09 fold. This result demonstrated that the 3D-culture system could emerge as a novel approach to overcoming the current limitations of cord blood HSC transduction. In the future, this research could be applied in a therapeutic setting.

Decellularized Extracellular Matrix Scaffolds for Cardiovascular Tissue Engineering: Current Techniques and Challenges.

Cardiovascular diseases are the leading cause of global mortality. Over the past two decades, researchers have tried to provide novel solutions for end-stage heart failure to address cardiac transplantation hurdles such as donor organ shortage, chronic rejection, and life-long immunosuppression. Cardiac decellularized extracellular matrix (dECM) has been widely explored as a promising approach in tissue-regenerative medicine because of its remarkable similarity to the original tissue. Optimized decellularization protocols combining physical, chemical, and enzymatic agents have been developed to obtain the perfect balance between cell removal, ECM composition, and function maintenance. However, proper assessment of decellularized tissue composition is still needed before clinical translation. Recellularizing the acellular scaffold with organ-specific cells and evaluating the extent of cardiomyocyte repopulation is also challenging. This review aims to discuss the existing literature on decellularized cardiac scaffolds, especially on the advantages and methods of preparation, pointing out areas for improvement. Finally, an overview of the state of research regarding the application of cardiac dECM and future challenges in bioengineering a human heart suitable for transplantation is provided.

Exhale and Decell: Environmentally Sustainable Sterilization and Decellularization of Cartilage Grafts via Supercritical Carbon Dioxide

PURPOSE: Current decellularization techniques have important limitations, including obligatory use of harsh ionized substances or detergents that deplete bioactive cytokines and compromise the physical integrity of the resulting extracellular matrix (ECM) scaffold. Furthermore, the detergents used are increasingly recognized as harmful to the environment and are poised to be restricted from commercial use. Supercritical carbon dioxide (scCO2) is an appealing alternative, applying the principles behind supercritical fluid extraction and sterilization to the removal of immunogenic cellular components. We endeavor to evaluate the efficacy of scCO2 in both sterilization and decellularization of ovine costal cartilage xenograft and human costal cartilage allograft to develop a highly efficacious, environmentally sustainable, and biocompatible alternative to currently available decellularization strategies. METHODS AND MATERIALS: Xenograft preparation: Racks of lamb were purchased from a local butcher. Floating ribs were minced into ∼8 mm3 cubes or zested into flakes (1 mm × 1 mm). Allograft harvest: Human costal cartilage was obtained from patients undergoing microsurgical breast reconstruction necessitating partial rib resection and processed via mincing or zested as above. All samples were subjected to: 1) sterilization; 2) sterilization and decellularization; or 3) sterilization, decellularization, and a pretreatment wash. Sterilization: Samples were placed in a NovaSterilis ™ Nova2200 unit and subject to standard NovaSterilis ™ sterilization parameters. At least 3 samples with biologic indicators were placed within the packaging and processed with each run. Decellularization: Samples were subject to the standard parameters of the Nova2200 system as previously described by NovaSterilis ™ with an ethanol to scCO2 volume ratio of 1:3.3. Wash: Samples were serially soaked in saline and agitated with exposure to 16 mL 3% H2O2 and a 30-minute scCO2 run at 35° C and 1,436 psi. Histology: After treatment, H&E, DAPI, and safranin-O staining were performed. DNA Quantification: DNA content was quantified in unprocessed and decellularized graft samples using the DNeasy Blood & Tissue kit (Qiagen Inc.). RESULTS: Sterilization conditions were sufficient for a 6 log10 sterilization of B. atrophaeus. H&E staining revealed preservation of tissue architecture after both sterilization and decellularization in both allograft and xenograft cartilage. DAPI staining demonstrated depletion of nuclei in the decellularized zested samples, but persistence of visible nuclei in the minced samples. Safranin-O staining revealed immunogenic GAG depletion after decellularization. DNA content in minced and zested samples was 192.2 ng DNA/mg tissue and 321.6 ng DNA/mg tissue, respectively, while DNA content in sterilized and decellularized minced and zested samples were 24.8 ng DNA/mg tissue and 11.6 ng DNA/mg tissue, respectively (industry standard requirement for decellularization <50 ng DNA/mg tissue); after sterilization, decellularization, and the chemical wash, minced and zested samples contained 17.6 ng DNA/mg tissue and 4 ng DNA/mg tissue respectively. CONCLUSION: These preliminary data suggest that scCO2 sufficiently sterilizes and decellularizes cartilage derived from both human and animal sources, thereby supporting scCO2 as an efficacious, commercially appealing, and ecologically responsible alternative to current decellularization strategies. Biomechanical testing on treated samples, as well as the evaluation of scCO2 as a decellularization agent for human nasal septal cartilage are ongoing.

3D printing of bone scaffolds for treating infected mandible bone defects through adjustable dual-release of chlorhexidine and osteogenic peptide

Preventing implant-related bone infection during treating mandibular bone defect is still challenging as intractable bacterial infection in the irregular defects often leads to surgical failure and other fatal complications. Herein, we fabricate a multifunctional scaffold with controlled dual-stage drug release to achieve antibacterial and osteogenic therapy during infected bone reconstruction. Water-in-oil (w/o) β-tricalcium phosphate (TCP) nanoparticle/ poly(Lactic-co-glycolic acid) (PLGA)/dichloromethane composite emulsion inks containing the antibacterial chlorhexidine (CHX) loaded graphene oxide (GO) nanosheets and osteogenic peptide (p24) was produced through cryo-3D printing. A biphasic drug release profile is achieved via a rapid CHX release from CHX@GO on scaffold surface to kill the bacteria, and a sustained release of CHX and p24 from the scaffold matrix to prevent the recurrence of infection and induce bone regeneration. Interestingly, the GO nanosheet increased the antibacterial sensitivity through its direct physical contact which destroyed the bacteria membrane. Furthermore, the p24 and phosphate ions derived from dissociated TCP nanoparticles could induce the osteogenic differentiation of bone marrow derived stem cells. Eventually, the designed scaffolds effectively eliminated the bacteria, alleviated the accompanying inflammation, and improved bone regeneration in a rat model. The present study provides a facile strategy to treat bone defects with infection.

Novel metal matrix composites reinforced with Zr-based metallic glass lattices

Although various methods have been developed to manufacture metal matrix composites (MMCs), most of the currently employed methods use discontinued crystalline ceramic particles as reinforcements. Herein, we report a new approach of reinforcing metals using a three-dimensional metallic glass (MG) lattice. Specifically, the processing route involves additive manufacturing of a stainless-steel matrix scaffold containing a continuous lattice-structured cavity and subsequent high-pressure injection of a Zr-based glass-forming liquid into the scaffold. Our results demonstrate that the as-produced composites reinforced with 30 vol% MG in the form of a body-centered-cubic lattice possess excellent mechanical properties. Remarkably, the plasticity is higher than those of some particle-reinforced stainless-steel composites and most conventional ex situ MG matrix composites. The enhanced performances mainly arise from the well-designed structure, good combination of the hard MG and soft stainless steel, and their mutual reinforcement mechanism. This study not only reveals a new class of composites reinforced with lattice-structured MG that overcome the limitation of MG reinforcement, rendering it suitable for reinforcing high-melting-point materials, but also provides a novel design concept and fabrication approach that could be extended to other alloy systems for achieving improved mechanical properties.

Incorporating Heteroatom-Doped Graphene in Electrolyte for High-Performance Lithium-Sulfur Batteries

Despite dominating the current commercial energy storage landscape, Li-ion batteries are fast approaching their theoretical limit. Meanwhile, Lithium Sulphur (Li-S) batteries, owing to their ultrahigh theoretical energy density of about 2600 Whkg-1, low-cost, Earth-abundant, and environmentally friendly sulfur (S) cathode, are seen as promising replacements to realize energy densities beyond 500 Whkg-1. Despite these advantages, the large-scale implementation of Li-S technology has been stymied due to several issues, one of the most deleterious of them is the dissolution and shuttling of polysulfide intermediates during cycling resulting in severe self-discharge, lowered S utilization and coulombic efficiency. Over the past decades, substantial amount of research towards mitigating ‘polysulfide shuttling’ shuttling has been focused on cathodic modification strategies such as infiltration of S in porous carbon, metal oxide or conducting polymer scaffold matrix which retard the mobility and loss of active material. However, such simple confinement strategies have been shown to be lacking over long cycling due to the relatively weak intermolecular interactions between the host and polysulfide molecules. An effective solution comes in the form of engineering ‘sulfiphilic’ materials that adsorb polysulfide species. For example, surface modification of cathodes with rGO and functionalized carbon species have shown success in reducing the redox shuttling. However, these approaches require elaborate preparation and hence, are limited in their practical usage.To overcome this, we propose a facile approach to improve cell performance via incorporation of functionalized graphenic species in the Li-S electrolyte. In this work, we have probed the impact of using tailored single-layer, heteroatom-doped graphene as electrolyte additives. We have studied the impact of their morphology and functionalization on the electrochemical performance of the cell. Results show a marked improvement in the discharge capacity and a high capacity retention of 84% over 105 cycles at 0.2 C cycling rate. The GCD cycle analysis showed an improvement in first cycle discharge plateau suggesting improved S utilization which was further substantiated by extensive postmortem analysis and polysulfide adsorption testing. Further, the electrolyte-modified cells showed an impressive three-fold and two-fold improvement in capacity at 1C and 2C cycling rates, respectively.These results demonstrated that tailored heteroatom-doped graphene in the form of electrolyte additive is an effective strategy to improve electrochemical performance via enhanced polysulfide encapsulation, cell conductivity and anode stabilization. Finally, we will present the effect of heteroatom-doped graphenic species on mitigation of polysulfide shuttling and cell performance when they are incorporated in the gel electrolyte system.

Decellularized tympanic membrane scaffold with bone marrow mesenchymal stem cells for repairing tympanic membrane perforation.

Tympanic membrane perforation (TMP) is a common disease in otology, and few acellular techniques have been reported for repairing this condition. Decellularized extracellular matrix (ECM) scaffolds have been used in organ reconstruction. This study on tissue engineering aimed to develop a tympanic membrane (TM) scaffold prepared using detergent immersion and bone marrow mesenchymal stem cells (BMSCs) as repair materials to reconstruct the TM. General structure was observed that the decellularized TM scaffold with BMSCs retained the original intact anatomical ECM structure, with no cell residue, as observed using scanning electron microscopy (SEM), and exhibited low immunogenicity. Therefore, we seeded the decellularized TM scaffold with BMSCs for recellularization. Histology and eosin staining, SEM and immunofluorescence in vivo showed that the recellularized TM patch had a natural ultrastructure and was suitable for the migration and proliferation of BMSCs. The auditory brainstem response (ABR) evaluated after recellularized TM patch repair was slightly higher than that of the normal TM, but the difference was not significant. The synthetic ECM scaffold provides temporary physical support for the three-dimensional growth of cells during the tissue developmental stage. The scaffold stimulates cells to secrete their own ECM required for tissue regeneration. The recellularized TM patch shows potential as a natural, ultrastructure biological material for TM reconstruction.

Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application.

In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.

3D Bioprinted Patient-Specific Extracellular Matrix Scaffolds for Soft Tissue Defects.

Soft tissue injuries such as volumetric muscle loss (VML) are often too large to heal normally on their own, resulting in scar formation and functional deficits. Decellularized extracellular matrix (dECM) scaffolds placed into these wounds have shown the ability to modulate the immune response and drive constructive healing. This provides a potential solution for functional tissue regeneration, however, these acellular dECM scaffolds are challenging to fabricate into complex geometries. 3D bioprinting is uniquely positioned to address this, being able to create patient-specific scaffolds based on clinical 3D imaging data. Here, a process to use freeform reversible embedding of suspended hydrogels (FRESH) 3D bioprinting and computed tomography (CT) imaging to build large volume, patient-specific dECM patches (≈12×8×2cm) for implantation into canine VML wound models is developed. Quantitative analysis shows that these dECM patches are dimensionally accurate and conformally adapt to the surface of complex wounds. Finally, this approach is extendedto a human VML injury to demonstrate the fabrication of clinically relevant dECM scaffolds with precise control over fiber alignment and micro-architecture. Together these advancements represent a step towards an improved, clinically translatable, patient-specific treatment for soft tissue defects from trauma, tumor resection, and other surgical procedures.

Effect of hyaluronic acid on microscale deformations of collagen gels

As fibrous collagen is the most abundant protein in mammalian tissues, gels of collagen fibers have been extensively used as an extracellular matrix scaffold to study how cells sense and respond to cues from their microenvironment. Other components of native tissues, such as glycosaminoglycans like hyaluronic acid, can affect cell behavior in part by changing the mechanical properties of the collagen gel. Prior studies have quantified the effects of hyaluronic acid on the mechanical properties of collagen gels in experiments of uniform shear or compression at the macroscale. However, there remains a lack of experimental studies of how hyaluronic acid changes the mechanical properties of collagen gels at the scale of a cell. Here, we studied how addition of hyaluronic acid to gels of collagen fibers affects the local field of displacements in response to contractile loads applied on length scales similar to those of a contracting cell. Using spherical poly(N-isopropylacrylamide) particles, which contract when heated, we induced displacement in gels of collagen and collagen with hyaluronic acid. Displacement fields were quantified using a combination of confocal microscopy and digital image correlation. Results showed that hyaluronic acid suppressed the distance over which displacements propagated, suggesting that it caused the network to become more linear. Additionally, hyaluronic acid had no statistical effect on heterogeneity of the displacement fields, but it did make the gels more elastic by substantially reducing the magnitude of permanent deformations. Lastly, we examined the effect of hyaluronic acid on fiber remodeling due to localized forces and found that hyaluronic acid partially – but not fully – inhibited remodeling. This result is consistent with prior studies suggesting that fiber remodeling is associated with a phase transition resulting from an instability caused by nonlinearity of the collagen gel.

Maresin 1 enhances osteogenic potential of mesenchymal stem cells by modulating macrophage peroxisome proliferator-activated receptor-γ-mediated inflammation resolution

Inflammation resolution plays a significant role in attenuating bone injury aggravated by acute inflammation and maintaining bone homeostasis. Maresin 1 (MaR1), a specialized pro-resolving mediators (SPMs), is biosynthesised in macrophages (Mφs) that regulates acute inflammation. Strategies to accelerate the resolution of inflammation in bone repair include not only promoting vanish of acute inflammation, also improving osteogenic microenvironment. Here, previously prepared difunctional demineralized bone matrix (DBM) scaffold was used to study thoroughly the “cross-talk” between Mφs lipid metabolism and mesenchymal stem cells (MSCs) behaviors in vitro. The pro-resolving mechanism in Mφs treated with MaR1 was elaborated. Furthermore, the biological behaviors of MSCs in co-culture system were evaluated. The results indicated that MaR1 had an enhanced capability and performance in peroxisome proliferator-activated receptor-γ (PPAR-γ) activation, M2-type Mφs polarization, and lipid droplets (LDs) biogenesis in Mφs in vitro. The nuclear receptor PPAR-γ enhanced the anti-inflammatory proteins expression and the polarization of Mφs toward M2 subtype, thereby favoring the proliferation, migration, and osteogenesis of MSCs. Overall, the results verified that MaR1 facilitated MSCs behaviors by regulating PPAR-γ-mediated inflammatory response, which implied that PPAR-γ exhibited a significant role in the dialogue between MSCs behaviors and Mφs lipid metabolism.

Establishment of a human 3D pancreatic adenocarcinoma model based on a patient-derived extracellular matrix scaffold

Pancreatic cancer is likely to become one of the leading causes of cancer-related death in many countries within the next decade. Surgery is the potentially curative treatment for pancreatic ductal adenocarcinoma (PDAC), although only 10%–20% of patients have a resectable disease after diagnosis. Despite recent advances in curative surgery the current prognosis ranges from 6% to 10% globally. One of the main issues at the pre-clinical level is the lacking of model which simultaneously reflects the tumour microenvironment (TME) at both structural and cellular levels. Here we describe an innovative tissue engineering approach applied to PDAC starting from decellularized human biopsies in order to generate an organotypic 3D in vitro model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence, mechanical analysis, and scanning electron microscopy. Mass spectrometry confirmed a different extracellular matrix (ECM) composition between decellularized healthy pancreas and PDAC by identifying a total of 110 non-redundant differently expressed proteins. Immunofluorescence analyses after 7 days of scaffold recellularization with PANC-1 and AsPC-1 pancreatic cell lines, were performed to assess the biocompatibility of 3D matrices to sustain engraftment, localization and infiltration. Finally, both PANC-1 and AsPC-1 cells cultured in 3D matrices showed a reduced response to treatment with FOLFIRINOX if compared to conventional bi-dimensional culture. Our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the pancreatic cancer microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of pancreatic cancer response to chemotherapy agents.

Human-derived decellularized extracellular matrix scaffold incorporating autologous bone marrow stem cells from patients with congenital heart disease for cardiac tissue engineering.

Stem cells are used as an alternative treatment option for patients with congenital heart disease (CHD) due to their regenerative potential, but they are subject to low retention rate in the injured myocardium. Also, the diseased microenvironment in the injured myocardium may not provide healthy cues for optimal stem cell function. In this study, we prepared a novel human-derived cardiac scaffold to improve the functional behaviors of stem cells. Decellularized extracellular matrix (ECM) scaffolds were fabricated by removing cells of human-derived cardiac appendage tissues. Then, bone marrow c-kit+ progenitor cells from patients with congenital heart disease were seeded on the cardiac ECM scaffolds. Cell adhesion, survival, proliferation and cardiac differentiation on human cardiac decellularized ECM scaffold were evaluated invitro. Label-free mass spectrometry was applied to analyze cardiac ECM proteins regulating cell behaviors. It was shown that cardiac ECM scaffolds promoted stem cell adhesion and proliferation. Importantly, bone marrow c-kit+ progenitor cells cultured on cardiac ECM scaffold for 14 days differentiated into cardiomyocyte-like cells without supplement with any inducible factors, as confirmed by the increased protein level of Gata4 and upregulated gene levels of Gata4, Nkx2.5, and cTnT. Proteomic analysis showed the proteins in cardiac ECM functioned in multiple biological activities, including regulation of cell proliferation, regulation of cell differentiation, and cardiovascular system development. The human-derived cardiac scaffold constructed in this study may help repair the damaged myocardium and hold great potential for tissue engineering application in pediatric patients with CHD.

The distribution of electrospun polylactic acid in polycaprolactone matrix controlled by traction rate and its effect on the foamed porous tissue engineering scaffolds

AbstractPolycaprolactone (PCL) matrix scaffolds have been extensively studied for tissue engineering applications, which were limited by poor cell adhesion and mechanical strength. Fiber reinforcement modification could be used to improve material properties and optimize material structure. In this study, polylactic acid (PLA) electrospun nanofibers were dispersed in PCL powder, and then the composites were extruded and blended at different traction speed. During the blending and extrusion process, the nanofibrous phase was uniformly dispersed in the matrix, and a kind of tissue engineering scaffold with high connectivity and network structure was prepared using supercritical carbon dioxide foaming. The effect of nanofibers on the foaming behavior of PCL matrix composites prepared at different traction rates was investigated, and the addition of nanofibers promoted the connectivity of the scaffold. Human umbilical vein endothelial cells (HUVECs) culture results showed that the high‐connectivity scaffold was biocompatible and could significantly promote the adhesion and growth of HUVECs. Therefore, fiber‐reinforced porous scaffolds have great application potential in the field of tissue engineering scaffolds.

Enhanced wound-healing capability with inherent antimicrobial activities of usnic acid incorporated poly(ε-caprolactone)/decellularized extracellular matrix nanofibrous scaffold

An extracellular matrix-mimicking, biodegradable tissue-engineered skin substitute with improved antibacterial, antibiofilm, and wound healing capabilities is essential in skin tissue regeneration applications. The purpose of this study was to develop a novel biodegradable composite nanofibrous poly(ε-caprolactone) (PCL)/decellularized extracellular matrix (dECM) scaffolds loaded with usnic acid (UA); (PEU), where UA is employed as an antibacterial agent as well as a wound-healing accelerator. The architecture and fiber structure of the scaffolds were examined using scanning electron microscopy, and the results revealed that the average diameters decreased as the dECM content increased. The chemical composition, changes in the crystalline structure, homogeneity, and thermal stability of the nanofiber scaffolds with different material compositions were determined using Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis, respectively. The composite nanofibrous scaffolds exhibited strong antibacterial activity against various bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and Cutibactrium acnes, and fungal pathogens (such as Candida albicans). Additionally, the composite nanofibrous scaffolds exhibited biofilm inhibition properties against Klebsiella pneumoniae and Pseudomonas aeruginosa. An evaluation of the appearance of in vivo full-thickness excisional wounds treated with the composite nanofiber scaffolds, as well as a histological analysis of the wounds 21days after surgery, revealed that treatment with nanofibrous PEU scaffolds enhanced wound healing. This study reveals that the proposed composite nanofibrous PEU scaffold has substantial potential for treating infectious full-thickness wounds.

Decellularized small intestine submucosa device for temporomandibular joint meniscus repair: Acute timepoint safety study.

Temporomandibular joint (TMJ) Meniscus removal is an option for the patient to regain full range of motion if the disc is irreversibly damaged or unable to be reduced. However, this procedure leaves the joint vulnerable to condylar remodeling and degeneration. We have shown that extracellular matrix (ECM) scaffolds remodel into a tissue with near native TMJ meniscus in previous studies. The next step towards clinical translation is to manufacture the ECM scaffold as a device under good manufacturing practices (GMP) and test it in a pre-clinical animal study under good laboratory practices (GLP). The primary objective of this study was to evaluate the in-vivo histopathological response to a Prototype GMP manufactured device made of decellularized porcine small intestinal submucosa (SIS), by observing for signs of surrounding tissue reaction to the device that are indicative of an adverse host response in comparison to an empty control at 21 days post-surgical implantation in a canine TMJ meniscus removal and implant model in a GLP setting. The conclusive findings were that the ECM device is safe for placement in the TMJ. After 21 days post implantation, histology of tissue surrounding the device and draining lymph nodes showed that the Prototype GMP device had no negative effects compared to the empty site (as evaluated by the board-certified veterinary pathologist). Furthermore, there was a lack of negative findings for clinical pathology (hematology and clinical chemistry), mortality, and body weight/weight change. Future studies will go to one year after implantation to show that the remodel device remains as a viable tissue with near native mechanical properties.

Reduced Graphene Oxide-Extracellular Matrix Scaffolds as a Multifunctional and Highly Biocompatible Nanocomposite for Wound Healing: Insights into Characterization and Electroconductive Potential.

The development of novel regenerative technologies based on the implementation of natural extracellular matrix (ECM), or individual components of ECM combined with multifunctional nanomaterials such as graphene oxide and reduced graphene oxide, has demonstrated remarkable results in wound healing and tissue engineering. However, the synthesis of these nanocomposites involves great challenges related to maintaining the biocompatibility with a simultaneous improvement in their functionalities. Based on that, in this research we developed novel nanoengineered ECM-scaffolds formed by mixing small intestinal submucosa (SIS) with graphene oxide (GO)/reduced graphene oxide (rGO) to improve electrical conductivity while maintaining remarkable biocompatibility. For this, decellularized SIS was combined with GO to form the scaffold precursor for subsequent lyophilization, chemically crosslinking and in situ reduction. The obtained GO and rGO were characterized via Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray diffraction (XRD), electrical conductivity testing and atomic force microscopy (AFM). The results confirm the suitable synthesis of GO, the effective reduction to rGO and the significant increase in the electrical conductivity (more than four orders of magnitude higher than bare GO). In addition, the graphene oxide/reduced graphene oxide-SIS scaffolds were characterized via Raman spectroscopy, FTIR, TGA, SEM, porosity assay (higher than 97.5% in all cases) and protein secondary structural analysis. Moreover, the biocompatibility of scaffolds was studied by standardized assays of hemolysis activity (less than 0.5%), platelet activation and deposition, and cell viability in Vero, HaCat and HFF-1 cells (higher than 90% for all evaluated cell lines on the different scaffolds). The obtained results confirm the remarkable biocompatibility, as supported by high hemocompatibility, low cytotoxicity and no negative impact on platelet activation and deposition. Finally, structural characteristics such as pore size and interconnectivity as well as superior cell attachment abilities also corroborated the potential of the developed nanoengineered ECM-scaffolds as a multifunctional nanoplatform for application in regenerative medicine and tissue engineering.

SMAR1 inhibits proliferation, EMT and Warburg effect of bladder cancer cells by suppressing the activity of the Wnt/β-catenin signaling pathway

ABSTRACT This study aimed to investigate the effects of scaffold matrix attachment region binding protein 1 (SMAR1) on the development of bladder cancer (BCa). SMAR1 expression in paired tumor and corresponding adjacent normal tissues from 55 BCa patients was detected by quantitative reverse transcription-polymerase chain reaction. BCa cells were transfected to regulate SMAR1 expression. BCa cells were treated with XAV-939, LiCl and 2-deoxyglucose. The effect of SMAR1 on the viability, proliferation, migration, invasion and Warburg effect of BCa cells was researched by counting kit-8, colony formation assay, Transwell and aerobic glycolysis assays. Western blot was performed to detect protein expression. BCa cell growth in vivo was recorded in nude mice. Immunohistochemical staining was performed for clinical and xenografted tumor tissue specimens. SMAR1 expression was down-regulated in BCa patients, associating with worse prognoses. SMAR1 knockdown enhanced the viability, proliferation, migration, invasion, EMT and Warburg effect of BCa cells. The opposite effect was found in the SMAR1 overexpression BCa cells. XAV-939 treatment reversed the elevation of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect in Bca cells post-SMAR1 knockdown. LiCl treatment abrogated the inhibition of β-catenin, c-Myc and Cyclin D1 proteins expression and Warburg effect proteins due to SMAR1 overexpression in BCa cells. SMAR1 overexpression inhibited the growth of BCa cells in vivo. SMAR1 might suppress the Wnt/β-catenin signaling pathway activity to inhibit the progression of BCa. It might be an effective treatment target for BCa.

ToxR is a c-di-GMP binding protein that modulates surface-associated behaviour in Pseudomonas aeruginosa

Pseudomonas aeruginosa uses multiple protein regulators that work in tandem to control the production of a wide range of virulence factors and facilitate rapid adaptation to diverse environmental conditions. In this opportunistic pathogen, ToxR was known to positively regulate the production of the major virulence factor exotoxin A and now, through analysis of genetic changes between two sublines of P. aeruginosa PAO1 and functional complementation of swarming, we have identified a previously unknown role of ToxR in surface-associated motility in P. aeruginosa. Further analysis revealed that ToxR had an impact on swarming motility by regulating the Rhl quorum sensing system and subsequent production of rhamnolipid surfactants. Additionally, ToxR was found to tightly bind cyclic diguanylate (c-di-GMP) and negatively affect traits controlled by this second messenger including reducing biofilm formation and the expression of Psl and Pel exopolysaccharides, necessary for attachment and sessile communities matrix scaffolding, in P. aeruginosa. Moreover, a link between the post-transcriptional regulator RsmA and toxR expression via the alternative sigma factor PvdS, induced under iron-limiting conditions, is established. This study reveals the importance of ToxR in a sophisticated regulation of free-living and biofilm-associated lifestyles, appropriate for establishing acute or chronic P. aeruginosa infections.