Melt-electrowriting-enabled anisotropic scaffolds loaded with valve interstitial cells for heart valve tissue Engineering.

Tissue engineered heart valves (TEHVs) demonstrates the potential for tissue growth and remodel, offering particular benefit for pediatric patients. A significant challenge in designing functional TEHV lies in replicating the anisotropic mechanical properties of native valve leaflets. To establish a biomimetic TEHV model, we employed melt-electrowriting (MEW) technology to fabricate an anisotropic PCL scaffold. By integrating the anisotropic MEW-PCL scaffold with bioactive hydrogels (GelMA/ChsMA), we successfully crafted an elastic scaffold with tunable mechanical properties closely mirroring the structure and mechanical characteristics of natural heart valves. This scaffold not only supports the growth of valvular interstitial cells (VICs) within a 3D culture but also fosters the remodeling of extracellular matrix of VICs. The in vitro experiments demonstrated that the introduction of ChsMA improved the hemocompatibility and endothelialization of TEHV scaffold. The in vivo experiments revealed that, compared to their non-hydrogel counterparts, the PCL-GelMA/ChsMA scaffold, when implanted into SD rats, significantly suppressed immune reactions and calcification. In comparison with the PCL scaffold, the PCL-GelMA/ChsMA scaffold exhibited higher bioactivity and superior biocompatibility. The amalgamation of MEW technology and biomimetic design approaches provides a new paradigm for manufacturing scaffolds with highly controllable microstructures, biocompatibility, and anisotropic mechanical properties required for the fabrication of TEHVs.

Promotion of adhesion and proliferation of endothelial progenitor cells on decellularized valves by covalent incorporation of RGD peptide and VEGF.

Tissue engineered heart valve is a promising alternative to current heart valve surgery, for its capability of growth, repair, and remodeling. However, extensive development is needed to ensure tissue compatibility, durability and antithrombotic potential. This study aims to investigate the biological effects of multi-signal composite material of polyethyl glycol-cross-linked decellularized valve on adhesion and proliferation of endothelial progenitor cells. Group A to E was decellularized valve leaflets, composite material of polyethyl glycol-cross-linked decellularized valves leaflets, vascular endothelial growth factor-composite materials, Arg-Gly-Asp peptide-composite materials and multi-signal modified materials of polyethyl glycol-cross-linked decellularized valve leaflets, respectively. The endothelial progenitor cells were seeded for each group, cell adhesion and proliferation were detected and neo-endothelium antithrombotic function of the multi-signal composite materials was evaluated. At 2, 4, and 8 h after the seeding, the cell numbers and 3H-TdR incorporation in group D were the highest. At 2, 4, and 8 days after the seeding, the cell numbers and 3H-TdR incorporation were significantly higher in groups C, D, and E compared with groups A and B (P < 0.05) and cell numbers and the expression of t-PA and eons in the neo-endothelium were quite similar to those in the human umbilical vein endothelial cells at 2, 4, and 8 days after the seeding. The Arg-Gly-Asp- peptides (a sequential peptide composed of arginine (Arg), glycine (Gly) and aspartic acid (Asp)) and VEGF-conjugated onto the composite material of PEG-crosslinked decellularized valve leaflets synergistically promoted the adhesion and proliferation of endothelial progenitor cells on the composite material, which may help in tissue engineering of heart valves.

Chondroitin Sulfate Derivative Cross-Linking of Decellularized Heart Valve for the Improvement of Mechanical Properties, Hemocompatibility, and Endothelialization.

Tissue-engineered heart valve (TEHV) has emerged as a prospective alternative to conventional valve prostheses. The decellularized heart valve (DHV) represents a promising TEHV scaffold that preserves the natural three-dimensional structure and retains essential biological activity. However, the limited mechanical strength, fast degradation, poor hemocompatibility, and lack of endothelialization of DHV restrict its clinical use, which is necessary for ensuring its long-term durability. Herein, we used oxidized chondroitin sulfate (ChS), one of the main components of the extracellular matrix with various biological activities, to cross-link DHV to overcome the above problems. In addition, the ChS-adipic dihydrazide was used to react with residual aldehyde groups, thus preventing potential calcification. The results indicated notable enhancements in mechanical properties and resilience against elastase and collagenase degradation in vitro as well as the ability to withstand extended periods of storage without compromising the structural integrity of valve scaffolds. Additionally, the newly cross-linked valves exhibited favorable hemocompatibility in vitro and in vivo, thereby demonstrating exceptional biocompatibility. Furthermore, the scaffolds exhibited traits of gradual degradation and resistance to calcification through a rat subcutaneous implantation model. In the rat abdominal aorta implantation model, the scaffolds demonstrated favorable endothelialization, commendable patency, and a diminished pro-inflammatory response. As a result, the newly constructed DHV scaffold offers a compelling alternative to traditional valve prostheses, which potentially advances the field of TEHV.

Functional Oxidized Hyaluronic Acid Cross-Linked Decellularized Heart Valves for Improved Immunomodulation, Anti-Calcification, and Recellularization.

Tissue engineering heart valves (TEHVs) are expected to address the limitations of mechanical and bioprosthetic valves used in clinical practice. Decellularized heart valve (DHV) is an important scaffold of TEHVs due to its natural three-dimensional structure and bioactive extracellular matrix, but its mechanical properties and hemocompatibility are impaired. In this study, DHV is cross-linked with three different molecular weights of oxidized hyaluronic acid (OHA) by a Schiff base reaction and presented enhanced stability and hemocompatibility, which could be mediated by the molecular weight of OHA. Notably, DHV cross-linked with middle- and high-molecular-weight OHA could drive the macrophage polarization toward the M2 phenotype in vitro. Moreover, DHV cross-linked with middle-molecular-weight OHA scaffolds are further modified with RGD-PHSRN peptide (RPF-OHA/DHV) to block the residual aldehyde groups of the unreacted OHA. The results show that RPF-OHA/DHV not only exhibits anti-calcification properties, but also facilitates endothelial cell adhesion and proliferation in vitro. Furthermore, RPF-OHA/DHV shows excellent performance under an in vivo hemodynamic environment with favorable recellularization and immune regulation without calcification. The optimistic results demonstrate that OHA with different molecular weights has different cross-linking effects on DHV and that RPF-OHA/DHV scaffold with enhanced immune regulation, anti-calcification, and recellularization properties for clinical transformation.

Facile engineering of interactive double network hydrogels for heart valve regeneration.

Regenerative heart valve prostheses are essential for treating valvular heart disease, which requested interactive materials that can adapt to the tissue remodeling process. Such materials typically involves intricate designs with multiple active components, limiting their translational potential. This study introduces a facile method to engineer interactive materials for heart valve regeneration using 1,1'-thiocarbonyldiimidazole (TCDI) chemistry. TCDI crosslinking forms cleavable thiourea and thiocarbamate linkages which could gradually release H2S during degradation, therefore regulates the immune microenvironment and accelerates tissue remodeling. By employing this approach, a double network hydrogel was formed on decellularized heart valves (DHVs), showcasing robust anti-calcification and anti-thrombosis properties post fatigue testing. Post-implantation, the DHVs could adaptively degrade during recellularization, releasing H2S to further support tissue regeneration. Therefore, the comprehensive endothelial cell coverage and notable extracellular matrix remodeling could be clearly observed. This accessible and integrated strategy effectively overcomes various limitations of bioprosthetic valves, showing promise as an attractive approach for immune modulation of biomaterials.

Tissue engineering of heart valves: PEGylation of decellularized porcine aortic valve as a scaffold for in vitro recellularization.

BACKGROUND: Poly (ethylene glycol) (PEG) has attracted broad interest for tissue engineering applications. The aim of this study was to synthesize 4-arm -PEG-20kDa with the terminal group of diacrylate (4-arm-PEG-DA) and evaluate its dual functionality for decellularized porcine aortic valve (DAV) based on its mechanical and biological properties. METHODS: 4-arm-PEG-DA was synthesized by graft copolymerization of linear PEG 20,000 monomers, and characterized by IR1H NMR and 13C NMR; PEGylation of DAV was achieved by the Michael addition reaction between propylene acyl and thiol, its effect was tested by uniaxial planar tensile testing, hematoxylin and eosin (HE) and scanning electron microscopy (SEM). Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) peptides and vascular endothelial growth factor-165 (VEGF165) were conjugated onto DAV by branched PEG-DA (GRGDSPC-PEG-DAV-PEG-VEGF165). RESULTS: Mechanical testing confirmed that PEG-cross-linking significantly enhanced the tensile strength of DAV. Immunofluoresce confirmed the GRGDSPC peptides and VEGF165 were conjugated effectively onto DAV; the quantification of conjunction was completed roughly using spectrophotometry and ELISA. The human umbilical vein endothelial cells (HUVECs) grew and spread well on the GRGDSPC-PEG-DAV-PEG-VEGF165. CONCLUSIONS: Therefore, PEGylation of DAV not only can improve the tensile strength of DAV, and can also mediate the conjugation of bioactive molecule (VEGF165 and GRGDSPC peptides) on DAV, which might be suitable for further development of tissue engineered heart valve.

Surface Modification of Decellularized Heart Valve by the POSS-PEG Hybrid Hydrogel to Prepare a Composite Scaffold Material with Anticalcification Potential.

Tissue-engineered heart valves (TEHVs) are the most promising replacement for heart valve transplantation. Decellularized heart valve (DHV) is one of the most common scaffold materials for TEHVs. In actual clinical applications, the most widely used method for treating DHV is cross-linking it with glutaraldehyde, but this method could cause serious problems such as calcification. In this study, we introduced polyhedral oligomeric silsesquioxane (POSS) nanoparticles into a poly(ethylene glycol) (PEG) hydrogel to prepare a POSS-PEG hybrid hydrogel, and then coated them on the surface of DHV to prepare the composite scaffold. The chemical structures, microscopic morphologies, cell compatibilities, blood compatibilities, and anticalcification properties were further investigated. Experimental results showed that the composite scaffold had good blood compatibility and excellent cell compatibility and could promote cell adhesion and proliferation. In vivo and in vitro anticalcification experiments showed that the introduction of POSS nanoparticles could reduce the degree of calcification significantly and the composite scaffold had obvious anticalcification ability. The DHV surface-coated with the POSS-PEG hybrid hydrogel is an alternative scaffold material with anticalcification potential for an artificial heart valve, which provides an idea for the preparation of TEHVs.

Application of decellularized scaffold combined with loaded nanoparticles for heart valve tissue engineering in vitro.

The purpose of this study was to fabricate decelluarized valve scaffold modified with polyethylene glycol nanoparticles loaded with transforming growth factor-ß1 (TGF-ß1), by which to improve the extracellular matrix microenvironment for heart valve tissue engineering in vitro. Polyethylene glycol nanoparticles were obtained by an emulsion-crosslinking method, and their morphology was observed under a scanning electron microscope. Decelluarized valve scaffolds, prepared by using trypsinase and TritonX-100, were modified with nanoparticles by carbodiimide, and then TGF-ß1 was loaded into them by adsorption. The TGF-ß1 delivery of the fabricated scaffold was measured by asing enzyme-linked immunosorbent assay. Whether unseeded or reseeded with myofibroblast from rats, the morphologic, biochemical and biomechanical characteristics of hybrid scaffolds were tested and compared with decelluarized scaffolds under the same conditions. The enzyme-linked immunosorbent assay revealed a typical delivery of nanoparticles. The morphologic observations and biological data analysis indicated that fabricated scaffolds possessed advantageous biocompatibility and biomechanical property beyond decelluarized scaffolds. Altogether this study proved that it was feasible to fabricate the hybrid scaffold and effective to improve extracellular matrix microenvironment, which is beneficial for an application in heart valve tissue engineering.

Anticalcification Potential of POSS-PEG Hybrid Hydrogel as a Scaffold Material for the Development of Synthetic Heart Valve Leaflets.

Calcification of bioprosthetics is a primary challenge in the field of artificial heart valves and a main reason for biological heart valve prostheses failure. Recent advances in nanomaterial science have promoted the development of polymers with advantageous properties that are likely suitable for artificial heart valves. In this work, we developed a nanocomposite polymeric biomaterial POSS-PEG (polyhedral oligomeric silsesquioxane-polyethylene glycol) hybrid hydrogel, which not only has improved mechanical and surface properties but also excellent biocompatibility. The results of atomic force microscopy and in vivo animal experiments indicated that the content of POSS in the PEG matrix plays an important role on the surface and contributes to its biological properties, compared to the decellularized porcine aortic valve scaffold. Additionally, this modification leads to enhanced protection of the hydrogel from thrombosis. Furthermore, the introduction of POSS nanoparticles also gives the hydrogel a better calcification resistance efficacy, which was confirmed through in vitro tests and animal experiments. These findings indicate that POSS-PEG hybrid hydrogel is a potential material for functional heart valve prosthetics, and the use of POSS nanocomposites in artificial valves may offer potential long-term performance and durability advantages.

Small-diameter polyurethane vascular graft with high strength and excellent compliance.

In this study, a polyurethane vascular graft with excellent strength and compliance for clinical application was designed and fabricated by preparing three small-diameter vascular graft layers via the textile techniques of wet spinning and knitting. The polyurethane filament that was fabricated by wet spinning formed the inner layer. The polyurethane tubular fabric was used as the middle layer. The outer layer was prepared by spraying polyurethane solution. The three layers of the polyurethane vascular graft have uniform wall thickness, high strength, excellent compliance, and good puncture resistance compared with clinical poly(ethylene terephthalate) (PET) and expanded polytetrafluoroethylene (ePTFE) vascular graft. Therefore, these layers can have potential clinical applications in the replacement of the conventional artificial vascular graft prepared from PET and ePTFE.

Effect of poly (lactic acid) porous membrane prepared via phase inversion induced by water droplets on 3T3 cell behavior.

Phase inversion induced by water droplets has garnered attention in the field of polymer science as a novel method for preparing porous membranes. This study investigates the effect of the porous structure of poly (lactic acid) (PLA) membranes prepared through phase inversion induced by water droplets at four different temperatures (25, 50, 75, and 100 °C) on the morphology and proliferation of 3T3 cells. The surface properties of the PLA porous membrane, including pore size, pore size distribution, surface roughness, surface hydrophilicity, and cytocompatibility with 3T3 cells, were evaluated. The results indicated that the synthesized PLA membrane had two surfaces with different structures. The upper surface in contact with the water droplets during preparation contained uniformly distributed micropores, whereas the bottom surface was smooth and composed of small particles in contacted with the mold. The upper surface showed high cytocompatibility with 3T3 cells, and the 3T3 cells migrated and grew within the pores at 25 °C. In contrast, the bottom surface exhibited low biocompatibility with the 3T3 cells. Our study has wide-ranging implications and will improve the fabrication and implementation of 3D cultured scaffolds with excellent cytocompatibility.

Nanostructured Non-Newtonian Drug Delivery Barrier Prevents Postoperative Intrapericardial Adhesions.

With the increasing volume of cardiovascular surgeries and the rising adoption rate of new methodologies that serve as a bridge to cardiac transplantation and that require multiple surgical interventions, the formation of postoperative intrapericardial adhesions has become a challenging problem that limits future surgical procedures, causes serious complications, and increases medical costs. To prevent this pathology, we developed a nanotechnology-based self-healing drug delivery hydrogel barrier composed of silicate nanodisks and polyethylene glycol with the ability to coat the epicardial surface of the heart without friction and locally deliver dexamethasone, an anti-inflammatory drug. After the fabrication of the hydrogel, mechanical characterization and responses to shear, strain, and recovery were analyzed, confirming its shear-thinning and self-healing properties. This behavior allowed its facile injection (5.75 ± 0.15 to 22.01 ± 0.95 N) and subsequent mechanical recovery. The encapsulation of dexamethasone within the hydrogel system was confirmed by 1H NMR, and controlled release for 5 days was observed. In vitro, limited cellular adhesion to the hydrogel surface was achieved, and its anti-inflammatory properties were confirmed, as downregulation of ICAM-1 and VCAM-1 was observed in TNF-α activated endothelial cells. In vivo, 1 week after administration of the hydrogel to a rabbit model of intrapericardial injury, superior efficacy was observed when compared to a commercial adhesion barrier, as histological and immunohistochemical examination revealed reduced adhesion formation and minimal immune infiltration of CD3+ lymphocytes and CD68+ macrophages, as well as NF-κß downregulation. We presented a novel nanostructured drug delivery hydrogel system with unique mechanical and biological properties that act synergistically to prevent cellular infiltration while providing local immunomodulation to protect the intrapericardial space after a surgical intervention.

Percutaneous Vertebroplasty and Facet Blocking for Treating Back Pain Caused by Osteoporotic Vertebral Compression Fracture.

Methods: Clinical and radiological data of 204 patients were reviewed. The patients were divided into Group A (PVP alone) and Group B (PVP and FB combined therapy) according to treatments. Back pain was evaluated with Visual Analog Scale (VAS) and Oswestry Disability Index (ODI). The operation, fluoroscopic exposure time, and bone cement leakage were recorded. The χ 2 test, Student's t-test, and repeated measures analysis of variance were used to compare the differences between the two groups. Results: There were 125 patients in Group A and 79 patients in Group B. Their baseline characteristics were similar (P > 0.05). The mean VAS scores of Group A and Group B were 7.03 and 7.21 at admission, 4.7 and 3.2 at 1 day after operation, 4.0 and 3.0 at 3 months, and 2.2 and 2.2 at 12 months after operation, respectively. The mean ODI scores of Group A and Group B were 30.9 and 29.8 at admission, 17.6 and 17.7 at 3 months, and 10.5 and 10.9 at 12 months after operation, respectively. The mean operation time and fluoroscopic exposure time of Group A (35.6 minutes and 7.2 seconds, respectively) was significantly shorter than that of Group B (45.7 minutes and 11.7 seconds, respectively, P < 0.01). The incidence of bone cement leakage and new fractures after operation did not have statistically significant difference between groups. Conclusion: PVP and FB combined therapy could provide better pain relief than PVP alone in short term after operation in patients with OVCFs associated back pains.

A riboflavin-ultraviolet light A-crosslinked decellularized heart valve for improved biomechanical properties, stability, and biocompatibility.

Tissue-engineered heart valves are a promising alternative to current valve substitutes. As the main scaffold of tissue-engineered heart valves, the decellularized heart valve (DHV) has problems such as biomechanical property damage and rapid degradation. In this study, we applied a photo-crosslinking reaction induced by riboflavin and ultraviolet light A (UVA) in the DHV for improving its biomechanical properties and stability. The results showed that the biomechanical properties of the DHV significantly improved following riboflavin-UVA (R-UVA) crosslinking. Moreover, the R-UVA-crosslinked DHV (R-UV-DHV) showed better resistance to enzymatic degradation in vitro, with significantly higher thermal denaturation temperature compared to that of the untreated DHV, indicating that the stability of the R-UV-DHV improved. Histological staining and scanning electron microscopy showed that the leaflet ultrastructure was preserved better after R-UVA crosslinking compared to a glutaraldehyde-crosslinked DHV. In addition, we found that the R-UV-DHV exhibited excellent human umbilical vein endothelial cell adhesion and cells could readily grow on its surface. In an in vitro anti-calcification experiment, the R-UV-DHV demonstrated non-calcifying properties in a simulated body fluid. Furthermore, the R-UV-DHV showed characteristics of slow degradation, non-calcification, and reduced pro-inflammatory response through a rat subcutaneous implantation model. As a result, R-UVA can effectively crosslink the DHV and the R-UV-DHV possessed satisfactory biocompatibility. R-UVA crosslinking can be a new approach for improving the performance of the DHV to prepare a better scaffold for tissue-engineered valves.

Effect of temperature on the thermal property and crystallization behavior of poly (lactic acid) porous membrane prepared via phase separation induced by water microdroplets.

Poly (lactic acid) (PLA)-based porous membrane were fabricated through phase separation induced by water microdroplets at different ambient temperature to unravel the relationship between the physical properties (including thermal properties and crystallization) and preparation temperature. Cross-sectional scanning electron micrographs revealed that the thickness of the membrane decreases with increasing temperature between 25 °C and 100 °C. In the bilayer structure, each layer has a different morphology. Differential scanning calorimetry (DSC) and X-ray diffraction studies indicate that the preparation temperature influences the ratio between imperfect and perfect crystals in the membrane, leading to a bimodal melting peak in the DSC thermogram. The change in the initial decomposition temperature in the thermogravimetric analysis curve is weak, suggesting a negligible effect of the preparation temperature on the thermal stability of the membranes. Thus, PLA porous membranes can be prepared with better crystallinity by controlling the ambient temperature during the phase separation induced by water microdroplets.

Fabrication of a novel hybrid heart valve leaflet for tissue engineering: an in vitro study.

The objective of this study was to fabricate biomatrix/polymer hybrid heart valve leaflet scaffolds using an electrospinning technique and seeded by mesenchymal stem cells. Mesenchymal stem cells were obtained from rats. Porcine aortic heart valve leaflets were decellularized, coated with basic fibroblast growth factor/chitosan/poly-4-hydroxybutyrate using an electrospinning technique, reseeded, and cultured over a time period of 14 days. Controls were reseeded and cultured over an equivalent time period. Specimens were examined biochemically, histologically, and mechanically. Recellularization of the hybrid heart valve leaflet scaffolds was significantly improved compared to controls. Biochemical and mechanical analysis revealed a significant increase of cell mass, 4-hydroxyproline, collagen, and strength in the hybrid heart valve leaflets compared to controls. This is the first attempt in tissue-engineered heart valves to fabricate hybrid heart valve leaflets using mesenchymal stem cells combined with a slow release technique and an electrospinning technique.

Fabrication of a novel hybrid scaffold for tissue engineered heart valve.

The aim of this study was to fabricate biomatrix/polymer hybrid scaffolds using an electrospinning technique. Then tissue engineered heart valves were engineered by seeding mesenchymal stromal cells (MSCs) onto the scaffolds. The effects of the hybrid scaffolds on the proliferation of seed cells, formation of extracellular matrix and mechanical properties of tissue engineered heart valves were investigated. MSCs were obtained from rats. Porcine aortic heart valves were decellularized, coated with poly(3-hydroxybutyrate-co-4-hydroxybutyrate) using an electrospinning technique, and reseeded and cultured over a time period of 14 days. In control group, the decellularized valve scaffolds were reseeded and cultured over an equivalent time period. Specimens of each group were examined histologically (hematoxylin-eosin [HE] staining, immunohistostaining, and scanning electron microscopy), biochemically (DNA and 4-hydroxyproline) and mechanically. The results showed that recellularization was comparable to the specimens of hybrid scaffolds and controls. The specimens of hybrid scaffolds and controls revealed comparable amounts of cell mass and 4-hydroxyproline (P>0.05). However, the specimens of hybrid scaffolds showed a significant increase in mechanical strength, compared to the controls (P<0.05). This study demonstrated the superiority of the hybrid scaffolds to increase the mechanical strength of tissue engineered heart valves. And compared to the decellularized valve scaffolds, the hybrid scaffolds showed similar effects on the proliferation of MSCs and formation of extracellular matrix. It was believed that the hybrid scaffolds could be used for the construction of tissue engineered heart valves.

[Controlled release of siRNA nanoparticles loaded in a novel external stent prepared by emulsion electrospinning attenuates neointima hyperplasia in vein grafts].

OBJECTIVE: To evaluate a strategy of using TF siRNA loaded in a novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles as a therapy for vein graft disease. METHODS: Hybrid ultrafine fibrous membranes consisting of PLGA/Chitosan nanoparticles were fabricated via a specially designed electrospinning setup. After soaking in chloroform to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined. The water uptake of the hybrid ultrafine fibrous membranes was investigated by incubation in phosphate buffer solution. Right jugular vein-carotid artery interposition grafting models in Sprague-Dawley rats were randomly divided into five groups:Group A (external stent consisting of PLGA/CS-TFsiRNA nanoparticles), Group B (external stent consisting of PLGA/CS-Stealth(TM) RNAi negative control nanoparticles), Group C (external stent consisting of PLGA/CS blank nanoparticles), Group D (external stent consisting of PLGA), Group E (without perivenous external stent). BLOCK-iT(TM) Fluorescent Oligo was used to confirm its stability and successful transfer into the vein graft wall. The vein grafts were harvested at 1, 3, 7, 14, 28 d after operation, respectively. The TF protein expression of vein grafts was analyzed by Western blot and immunochemistry at 1, 3, 7 d after operation, respectively. The expression of proliferating cell nuclear antigen (PCNA) was identified by immunochemistry methods. The thickness of neointima at 28 d was calculated by computer imaging analysis system. RESULTS: The PLGA and CS amount in PLGA/Chitosan nanoparticles membranes could be well controlled by adjusting the flow rate for electrospinning of PLGA and chitosan nanoparticles, respectively. Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid membranes exhibited good water absorption properties. BLOCK-iT(TM) Fluorescent Oligo could be detected in the graft wall even 12 days after operation. The expression of TF protein in Group A was significantly less than that in control groups at 3 d after operation (P < 0.05, 0.40 +/- 0.03 vs 0.75 +/- 0.01, 0.75 +/- 0.05, 0.77 +/- 0.07) and at 7 d after operation (P < 0.05, 0.30 +/- 0.03 vs 0.84 +/- 0.05, 0.86 +/- 0.06, 0.85 +/- 0.06). The expression of PCNA in Group A decreased significantly in comparison with control groups at 14 d after operation (P < 0.01, 13.0% +/- 2.6% vs 25.0% +/- 2.8%, 24.2% +/- 3.9%, 24.0% +/- 4.1%, 44.8% +/- 3.7%). The thickness of neointima at 28 d after grafting in Group A was significantly less than the untreated group (P < 0.01, 18.8 microm +/- 2.9 microm vs 38.7 microm +/- 5.0 microm, 37.3 microm +/- 3.6 microm, 37.2 microm +/- 2.6 microm, 67.5 microm +/- 4.8 microm). CONCLUSION: The novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles inhibits early neointima formation in rat vein grafts. This strategy may be a practicable and promising form of gene delivery against vein graft failure.

[Impact of immobilized RGD peptides on cell attachment of decellularized valve scaffolds].

This is a comparative study on three groups. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were surface modified with one of arginine -glycine -aspartic acid (RGD) containing peptides by covalent bond(the treated group). After rat aortic myofibroblast seeding, MTT test showed that more cells of the treated group attached on the valve scaffolds coupled with RGD peptides when compared with the cells of the coated group and untreated group. Moreover, correlatioins of attachment with attaching time and peptide concentrations were observed. Light and electron microscopy and cell count also confirmed the findings. Therefore, immobilizing the RGD peptides on the decellularized valve scaffolds is effective for improving cell attachment, which is helpful to constructing tissue engineering heart valve.

Role of TGF-ß1 Signaling in Heart Valve Calcification Induced by Abnormal Mechanical Stimulation in a Tissue Engineering Model.

A tissue engineering model of heart valve calcification induced in a bioreactor was established to evaluate the calcification induced by abnormal mechanical stimulation and explore the underlying molecular mechanisms. Polyethylene glycol (PEG)-modified decellularized porcine aortic leaflets seeded with human valve interstitial cells (huVICs) were mounted on a Ti-Ni alloy frame to fabricate two-leaflet and threeleaflet tissue engineered valves. The two-leaflet model valves were exposed to abnormal pulsatile flow stimulation with null (group A), low (1000 mL/min, group B), medium (2000 mL/min, group C), and high velocity (3000 mL/min, group D) for 14 days. Morphology and calcification were assessed by von Kossa staining, alkaline phosphatase (ALP) content, and Runx2 immunostaining. Leaflet calcification and mRNA and protein expression of transforming growth factor (TGF)-ß1, bone morphogenetic protein 2 (BMP2), Smad1, and MSX2 were measured at different time points. ALP content was examined in two-leaflet valves seeded with BMP2 shRNA plasmid-infected huVICs and exposed to the same stimulation conditions. The results showed that during 14 days of flow stimulation, huVICs on the leaflet surface proliferated to generate normal monolayer coverage in groups A, B, and C. Under mechanical stimulation, huVICs showed a parallel growth pattern in the direction of the fluid flow, but huVICs exhibited disordered growth in the high-velocity flow environment. von Kossa staining, ALP measurement, and immunohistochemical staining for Runx2 confirmed the lack of obvious calcification in group A and significant calcification in group D. Expression levels of TGF-ß1, BMP2, and MSX2 mRNA and protein were increased under fluid stimulation. ALP production by BMP2 shRNA plasmid-infected huVICs on model leaflets was significantly reduced. In conclusion, abnormal mechanical stimulation in a bioreactor induced calcification in the tissue engineering valve model. The extent of calcification correlated positively with the flow velocity, as did the mRNA and protein levels of TGF-ß1, BMP2, and MSX2. These findings indicate that TGF-ß1/BMP2 signaling is involved in valve calcification induced by abnormal mechanical stimulation.