ECM derivatized alginate augmenting bio-functionalities of lyophilized mat for skin and liver wound treatment.

Peptides and molecular residues sourced from the fragmentation of the extracellular matrix (ECM) can exacerbate a plethora of cellular functions. We selected a natural ECM-derived complex peptide mixture to functionalize sodium alginate. Three alginate derivatives (sodium alginate conjugated with ECM) SALE-1, SALE-2, and SALE-3 were synthesized using the lowest (10 % w/w), moderate (50 % w/w), and highest (100 % w/w) concentrations of ECM. Thereafter, they were used to fabricate three groups of mat scaffolds EMAT-1 (ECM derivatized alginate thrombin-mat), EMAT-2, and EMAT-3, respectively by the freeze-drying process. To enhance the hemostatic activity, thrombin was loaded onto the scaffolds. Another group, AT, without any derivatized alginate was additionally included in order to comparative analysis. Physical characteristics revealed that the porous mat scaffold showed enhancement in degradation and swelling ability with the increase in ECM content. The higher cell proliferation, migration, and cell viability were noticed in the higher ECM-containing samples EMAT-2 and EMAT-3. In vivo studies using rodent hepatic and rabbit ear models were carried out to ensure the hemostatic ability of the scaffolds. EMAT-2 and EMAT-3 demonstrate excellent liver regeneration ability in rat models. Moreover, the rat cutaneous wound model depicted that EMAT-3 dramatically elevated the skin's healing ability, thereby rendering it an excellent candidate for future clinical application in wound healing.

Multi-functional dual-layer nanofibrous membrane for prevention of postoperative pancreatic leakage.

Postoperative pancreatic leakage due to pancreatitis in patients is a life-threatening surgical complication. The majority of commercial barriers are unable to meet the demands for pancreatic leakage due to poor adhesiveness, toxicity, and inability to degrade. In this study, we fabricated mitomycin-c and thrombin-loaded multifunctional dual-layer nanofibrous membrane with a combination of alginate, PCL, and gelatin to resolve the leakage due to suture line disruption, promote hemostasis, wound healing, and prevent postoperative tissue adhesion. Electrospinning was used to fabricate the dual-layer system. The study results demonstrated that high gelatin and alginate content in the inner layer decreased the fiber diameter and water contact angle, and crosslinking allowed the membrane to be more hydrophilic, making it highly biodegradable, and adhering firmly to the tissue surfaces. The results of in vitro biocompatibility and hemostatic assay revealed that the dual-layer had a higher cell proliferation and showed effective hemostatic properties. Moreover, the in vivo studies and in silico molecular simulation indicated that the dual layer was covered at the wound site, prevented suture disruption and leakage, inhibited hemorrhage, and reduced postoperative tissue adhesion. Finally, the study results proved that dual-layer multifunctional nanofibrous membrane has a promising therapeutic potential in preventing postoperative pancreatic leakage.

Small-diameter vascular graft composing of core-shell structured micro-nanofibers loaded with heparin and VEGF for endothelialization and prevention of neointimal hyperplasia.

Despite the significant progress made in recent years, clinical issues with small-diameter vascular grafts related to low mechanical strength, thrombosis, intimal hyperplasia, and insufficient endothelialization remain unresolved. This study aims to design and fabricate a core-shell fibrous small-diameter vascular graft by co-axial electrospinning process, which will mechanically and biologically meet the benchmarks for blood vessel replacement. The presented graft (PGHV) comprised polycaprolactone/gelatin (shell) loaded with heparin-VEGF and polycaprolactone (core). This study hypothesized that the shell structure of the fibers would allow rapid degradation to release heparin-VEGF, and the core would provide mechanical strength for long-term application. Physico-mechanical evaluation, in vitro biocompatibility, and hemocompatibility assays were performed to ensure safe in vivo applications. After 25 days, the PGHV group released 79.47 ± 1.54% of heparin and 86.25 ± 1.19% of VEGF, and degradation of the shell was observed but the core remained pristine. Both the control (PG) and PGHV groups demonstrated robust mechanical properties. The PGHV group showed excellent biocompatibility and hemocompatibility compared to the PG group. After four months of rat aorta implantation, PGHV exhibited smooth muscle cell regeneration and complete endothelialization with a patency rate of 100%. The novel core-shell structured graft could be pivotal in vascular tissue regeneration application.

Physicochemical, in vitro and in vivo evaluation of VEGF loaded PCL-mPEG and PDGF loaded PCL-Chitosan dual layered vascular grafts.

The present study has attempted to evaluate the endothelialization and smooth muscle regeneration efficiency of a novel dual-layer small-diameter vascular graft. Two types of layers (PCL-mPEG-VEGF and PCL-Chitosan-PDGF) were fabricated to find out the best layer giving endothelialization support for the lumen and unique contractile function for outer layer of blood vessels. Platelet-derived growth factor (PDGF) and chitosan were immobilized onto PCL surface by aminolysis-based surface modification technique. Besides, Poly (ethylene glycol) methyl ether (mPEG) and vascular endothelial growth factor (VEGF) were directly blended with PCL. Morphological analysis of membranes ensured consistency of average fibers diameter with native extracellular matrix. A favorable interaction of PCL-mPEG-VEGF with cow pulmonary endothelial cells (CPAEs) and PCL-Chitosan-PDGF with rat bone marrow mesenchymal stem cells (RBMSCs) was obtained during in vitro study. Controlled growth factor release patterns were found from both layers. Further, PCL-mPEG-VEGF exhibited endothelial markers expression properties from RBMSCs. Up-regulation of SMCs markers expression was significantly ensured by the PCL-Chitosan-PDGF membrane. Thus, PCL-mPEG-VEGF and PCL-Chitosan-PDGF were preferred as inner and outer layers respectively of a finally prepared tubular hybrid tissue engineered small diameter vascular graft. Finally, the dual-layer vascular graft was implanted onto a rat abdominal aorta model for 2 months. The extracted samples exhibited the presence of endothelial marker (ICAM 1) in the inner layer and smooth muscle cell marker (αSMA) in the outer layer as well as substantial amount of collagen deposition was observed in the both layers.

Porosity controlled soya protein isolate-polyethylene oxide multifunctional dual membranes as smart wound dressings.

Multifunctional membranes S7P0.7, S7P3.0, and dual membranes composed of soya protein isolate (SPI) and polyethylene oxide (PEO) were produced for wound dressing applications. The internal structure of the membranes was confirmed by scanning electron microscopy (SEM) to be homogeneous and coarser with a porous-like network. S7P3.0 showed the tensile strength of 0.78 ± 0.04 MPa. In the absence of antibiotics, the dual membrane (combination of S7P0.7 and S7P3.0) exhibited potential antibacterial activity against Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria. Hemolysis quantitative data presented in the image demonstrates that all samples exhibited hemolysis levels below 5 %. Dual membrane showed 77.93 ± 9.5 % blood uptake which reflects its absorption capacity. The combination of S7P0.7 and S7P3.0 influenced the dual membrane's antibacterial, biocompatibility, and good hemolytic potentials. The dual membranes' promising histology features after implantation suggest they could be used as wound dressings.

Natural TEMPO oxidized cellulose nano fiber/alginate/dSECM hybrid aerogel with improved wound healing and hemostatic ability.

Natural biopolymers have attracted considerable attention in a variety of biomedical applications. Herein, tempo-oxidized-cellulose nanofibers (T) were incorporated into sodium alginate/chitosan (A/C) to reinforce the physicochemical properties and further modified with decellularized skin extracellular matrix (E). A unique ACTE aerogel was successfully prepared, and its nontoxic behavior was validated using mouse fibroblast L929 cells. In vitro hemolysis results revealed excellent platelet adhesion and fibrin network formation abilities of the obtained aerogel. A high speed of homeostasis was attained based on the quick clotting in <60 s. Skin regeneration in vivo experiments were conducted using the ACT1E0 and ACT1E10 groups. In comparison to ACT1E0 samples, ACT1E10 samples demonstrated enhanced skin wound healing with increased neo-epithelialization, increased collagen deposition, and extracellular matrix remodeling. ACT1E10 was found to be a promising aerogel for skin defect regeneration due to its improved wound-healing ability.

Physico-biological evaluation of 3D printed dECM/TOCN/alginate hydrogel based scaffolds for cartilage tissue regeneration.

Cartilage damage is the leading cause of osteoarthritis (OA), especially in an aging society. Mimicking the native cartilage microenvironment for chondrogenic differentiation along with constructing a stable and controlled architectural scaffold is considerably challenging. In this study, three-dimensional (3D) printed scaffolds using tempo-oxidized cellulose nanofiber (TOCN), decellularized extracellular matrix (dECM), and sodium alginate (SA) were fabricated for cartilage tissue regeneration. We prepared three groups (dECM80, dECM50, dECM20) of 3D printable hydrogels with different ratios of TOCN and dECM where SA concentration remained the same. Two-step crosslinking was performed with CaCl2 solution to achieve the highly stable 3D printed scaffolds. Finally, the fundamental physical characterizations showed that increasing the ratio of TOCN with dECM significantly improved the viscoelastic behaviour, stability, mechanical properties, and printability of the scaffolds. Based on the results, the 3D printed dECM50 scaffolds with controlled and identical pore sizes increased the whole-layer integrity and nutrient supply in each layer of the scaffold. Furthermore, evaluation of in vitro and in vivo biocompatibility of the scaffolds with rBMSCs indicated that dECM50 scaffolds provided a suitable microenvironment for cell proliferation and promoted chondrogenesis by remarkably expressing the cartilage-specific markers. This study demonstrates that 3D printed dECM50 scaffolds provide a favourable and promising microenvironment for cartilage tissue regeneration.

Fabrication and characterization of cellulose nano crystal and soya modified injectable brushite bone cement for enhanced bone regeneration.

An inventive, cellulose nano crystal (CNC) and isolated soya flour (SPI) laden brushite-based injectable bone substitute (IBS) material has been developed in the present research. The purpose of the study was to discover the physical, mechanical, in-vitro biological, and in-vivo bone forming ability of the prepared IBS. The incomparable abilities of CNC together with SPI resulted in enhanced biocompatibility, mechanical strength, and biodegradability, which together with its exclusive properties, sort it ideal for bone restoration. The CNC/SPI laden composites showed suitable mechanical strength of ~10.5 MPa for BM23 composite. The in- vitro cytocompatibility of the prepared samples were evaluated by osteoblast type MC3T3-E1 cells via MTT assay. Protein absorption and mineralization behavior of BRCNC2.0 was around (1.7 and 2.3)-fold higher than that of BR, respectively. In vivo performance was also found appreciable with ~(31.33 ± 2.04) % BV/TV. Incorporation of SPI resulted in enhanced bone formation at the central zone of the defect, while unmodified samples resulted in bone formation only at the peripheral zone. The findings of the current study proposed that CNC/SPI laden, brushite based injectable bone substitute might be proficient for bone regeneration ability.

In-vitro and in-vivo biocompatibility of dECM-alginate as a promising candidate in cell delivery for kidney regeneration.

In this study, kidney decellularized extracellular matrix (dECM) and alginate (ALG) hybrid injectable hydrogel, with the purpose of delivering progenitor cells for tissue engineering, were prepared by using a physical crosslinking method in a CaCl2 solution with high porosity for the exchange of nutrition and waste. In addition, the physical appearance and surface morphology of the hydrogel were investigated using optical and scanning electron microscopy, respectively. The functional groups of the dECM/ALG xerogels was examined via Fourier transform infrared spectroscopy. The biocompatibility of dECM/ALG xerogels was examined in-vitro using renal progenitor cells obtained from adult rat kidney. Enhanced biocompatibility and significant hemostatic behavior was noticed. Furthermore, the in-vivo biocompatibility of dECM/ALG hydrogel with progenitor cells was determined in the deep renal cortex for 7 and 21 days, in order to assess the foreign body reaction and inflammatory response. Early-stage glomerulus-like structure and dense linear cell network-like phenomenon were noticed. Loading of progenitor cells together with hydrogel enhances the cell density obviously due to cell migration from host and form a pattern. The desired early stage in-vivo response to progenitor cell-laden dECM/ALG hydrogel plays a potential role in kidney regeneration long term.

Physico-mechanical and biological evaluation of heparin/VEGF-loaded electrospun polycaprolactone/decellularized rat aorta extracellular matrix for small-diameter vascular grafts.

Although the continuous development of small-diameter vascular grafts (SDVGs) (D < 5 mm) continues, most vascular grafts are made from synthetic polymers, which lead to serious complications from arteriosclerosis, thrombosis, and vascular ischemia. Here, to address these shortcomings, we combine synthetic polymers with natural decellularized small-diameter vessels and loaded with growth factor. We fabricated vascular grafts by electrospinning polycaprolactone (PCL) to decellularized rat aorta matrix (ECM) followed by heparin and vascular endothelial growth factor (VEGF) loading. In- vitro studies showed that PCL/ECM/VEGF vascular grafts, showed excellent hemocompatibility and biocompatibility properties. The vascular grafts implanted into the rat aorta revealed that the PCL/ECM/VEGF grafts promotes endothelial cells and smooth-muscle cells infiltration with a rate of FLK-1, ICAM1, and a-SMA distribution higher than that of the PCL and PCL/ECM vascular grafts at 2 weeks and 4 weeks after implantation. The PCL/ECM/VEGF vascular graft should be considered for potential small-diameter vascular grafts in clinical fields.

Tailored alginate/PCL-gelatin-ß-TCP membrane for guided bone regeneration.

Membranes prepared for guided bone regeneration (GBR) signify valued resources, inhibiting fibrosis and assisting bone regenration. However, existing membranes lack bone regenerative capacity or adequate degradation profile. An alginate-casted polycaprolactone-gelatin-ß-tricalcium phosphate dual membrane was fabricated by electrospinning and casting processes to enhance new bone formation under a GBR process. Porous membranes were synthesized with suitable hydrophilicity, swelling, and degradation behavior to confirm the compatibility of the product in the body. Furthermore, osteoblast-type cell toxicity and cell adhesion results showed that the electrospun membrane offered compatible environment to cells while the alginate sheet was found capable enough to supress the cellular attachment, but was a non-toxic material. Post-implantation, thein-vivooutcomes of the dual-layered membrane, showed appreciable bone formation. Significantly, osteoid islands had fused in the membrane group by eight weeks. The infiltration of fibrous tissues was blocked by the alginate membrane, and the ingrowth of new bone was enhanced. Immunocytochemical analysis indicated that the dual membrane could direct more proteins which control mineralization and convene osteoconductive properties of tissue-engineered bone grafts.

Autologous stromal vascular fraction-loaded hyaluronic acid/gelatin-biphasic calcium phosphate scaffold for bone tissue regeneration.

Bone defect augmentation with synthetic materials is crucial due to the unavoidable limitations of auto- and allografting. Although there are different promising synthetic materials for filling bone defects, the functionalization of these materials with cells is still challenging due to the lack of ideal cell sources. Here, we used stromal vascular fraction (SVF) heterogeneous cells that could be obtained from autologous adipose tissue to functionalize hyaluronic acid/gelatin-biphasic calcium phosphate (HyA-Gel/BCP) scaffolds for bone regeneration. The SVF cells were isolated, and the cellular composition and osteogenic differentiation potential were analyzed. Then, they were cultured on HyA-Gel/BCP scaffolds for in vitro characterization. An In vivo evaluation of the autologous SVF-loaded HyA-Gel/BCP scaffolds was performed using a rat skull critical-size defect model. The results showed that the SVF was successfully isolated and contained different types of cells, including mesenchymal stem like-cells with osteogenic differentiation ability. Also, the SVF cells could be cultured and expanded on the HyA-Gel/BCP scaffolds without affecting their viability. In vivo implantation of autologous SVF-loaded HyA-Gel/BCP scaffolds showed excellent bone regeneration compared to unloaded HyA-Gel/BCP scaffolds. Thus, autologous SVF-loaded HyA-Gel/BCP scaffolds could be a promising transplantable bone grafting material for bone tissue engineering.

Fibroblast cell derived extracellular matrix containing electrospun scaffold as a hybrid biomaterial to promote in vitro endothelial cell expansion and functionalization.

Biomaterials to be used for vascular tissue engineering must allow attachment, proliferation, and functionalization of vasoactive cells especially endothelial cells. In this study, decellularized L929 fibroblast cell-derived ECM containing electrospun scaffolds were fabricated and their biological response was investigated using rat glomerulus endothelial cells (rGECs). The L929 cells were grown for one week to get cell sheets on PCL membranes followed by decellularization of whole cell sheet-PCL membrane (PCL-ECM) using sodium dodecyl sulfate (SDS)/triton X-100 (TX) or freeze/thaw (F/T)/Deoxyribonuclease cycle to yield the corresponding mechanically stable scaffold. The nucleic acids and structural proteins quantification were performed on various membranes before and after decellularization process. Seeded rGECs on PCL, PCL-ECM (SDS/TX) and PCL-ECM (F/T) membranes were investigated through immunofluorescence and cell proliferation assay. The bio-macromolecules contents on decellularized scaffolds showed diverse outcome because of different decellularization methods used. The hydrophilic PCL-ECM (F/T) scaffold showed the best result by ensuring stability, good cytocompatibility, and interconnections among endothelial cells as was further confirmed by endothelial gene expression analysis. In short, the outcomes of this study may pave the way for the construction of new cell-derived ECM based vascular tissue engineering scaffolds as well as for the development of in vitro models to study endothelial cell function.

Controlled release of Mitomycin C from modified cellulose based thermo-gel prevents post-operative de novo peritoneal adhesion.

The complications from surgery associated peritoneal adhesion can be alleviated by combination of physical isolation and pharmaceutical treatment. This work aims to develop thermo-sensitive hydrogel barrier by combining mitomycin C (MMC) with modified tempo oxidized nanocellulose (cTOCN) through EDC/NHS-chemical conjugation followed by integration with methyl cellulose (MC). The MMC was successfully combined with cTOCN and ensured controlled release of MMC from hydrogel throughout 14 days. Amount of MC (1.5, 2.5, 3.5% w/v) was proportional to gelation time and inversely proportional to degradation of hydrogel. The optimized hydrogel (C2.5T1M0.2) needed only 30 s for thermoreversible sol-gel (4℃-37℃) phenomenon and did not show in vitro fibroblast cells toxicity as well as ensured complete adhesion prevention efficacy, reperitonealization in rat side wall-cecal abrasion model. Overall, the developed C2.5T1M0.2 thermo-gel advances state-of-the-art in view of cytocompatibility, mechanical stability, optimum degradation, good injectability, sustain drug release from surgical sites, and satisfactory de novo anti-adhesion capacity.

The E3 ubiquitin ligase, NEDD4L (NEDD4-2) regulates bestrophin-1 (BEST1) by ubiquitin-dependent proteolysis.

The bestrophin family comprises well-known Ca2+-activated chloride channels (CaCC) that are expressed in a variety tissues including the brain, eye, gastrointestinal tract, and muscle tissues. Among the family members, bestrophin-1 (BEST1) is known to exist mainly in retinal pigment epithelium cells, but we recently reported that BEST1 mediates Ca2+-activated Cl- currents in hippocampal astrocytes. Despite its critical roles in physiological processes, including tonic γ-aminobutyric acid release and glutamate transport, the mechanisms that regulate BEST1 are poorly understood. In this study, we identified NEDD4L (NEDD4-2), an E3 ubiquitin ligase, as a binding partner of BEST1. A series of deletion constructs revealed that the WW3-4 domains of NEDD4L were important for interaction with BEST1. We observed that BEST1 underwent ubiquitin-dependent proteolysis and found that the conserved lysine370 residue in the C-terminus of BEST1 was important for its ubiquitination. Finally, we demonstrated that NEDD4L inhibited whole cell currents mediated by BEST1 but not by the BEST1(K370R) mutant. Collectively, our data demonstrated that NEDD4L played a critical role in regulating the surface expression of BEST1 by promoting its internalization and degradation.

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes.

TWIK-1 is a member of the two-pore domain K(+) (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoid-induced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.