Gradient nanofibrous chitosan/poly ɛ-caprolactone scaffolds as extracellular microenvironments for vascular tissue engineering

One of the major challenges of tissue-engineered small-diameter blood vessels is restenosis caused by thrombopoiesis. The goal of this study was to develop a 3D gradient heparinized nanofibrous scaffold, aiding endothelial cells lined on the lumen of blood vessel to prevent thrombosis. The vertical graded chitosan/poly ?-caprolactone (CS/PCL) nanofibrous vessel scaffolds were fabricated with chitosan and PCL by sequential quantity grading co-electrospinning. To mimic the natural blood vessel microenvironment, we used heparinization and immobilization of vascular endothelial growth factor (VEGF) in the gradient CS/PCL. The quantity of heparinized chitosan nanofibers increased gradually from the tunica adventitia to the lumen surfaces in the gradient CS/PCL wall of tissue engineered vessel. More heparin reacted to chitosan nanofiber in gradient CS/PCL than in uniform CS/PCL nanofibrous scaffolds. Antithrombogenic properties of the scaffolds were enhanced by the heparinization of these scaffolds, as shown by activated partial thromboplastin time and platelet adhesion assay. Compared to the uniform CS/PCL scaffold, the release of VEGF from the gradient CS/PCL was more stable and sustained, and the burst release of VEGF was reduced approximately 42.5% within the initial 12 h. The adhesion and proliferation of human umbilical vein endothelial cells (HUVEC) were enhanced on the gradient CS/PCL scaffold. Furthermore, HUVEC grew and formed an entire monolayer on the top side of the gradient CS/PCL scaffold. Therefore, use of vertical gradient heparinized CS/PCL nanofibrous scaffolds could provide an approach to create small-diameter blood vessel grafts with innate properties of mammalian vessels of anticoagulation and rapid induction of re-endothelialization.     

Development of a fibrin composite-coated poly(epsilon-caprolactone) scaffold for potential vascular tissue engineering applications

Poor cell adhesion, cytotoxicity of degradation products and lack of biological signals for cell growth, survival, and tissue generation are the limitations in the use of a biodegradable polymer scaffold for vascular tissue engineering. We have fabricated a hybrid scaffold by integrating physicochemical characteristics of poly(epsilon-caprolactone) (PCL) and biomimetic property of a composite of fibrin, fibronectin, gelatin, growth factors, and proteoglycans to improve EC growth on the scaffold. Solvent cast porous films of poly(epsilon-caprolactone) was prepared using PEG as a porogen. Porosity varied between 5 and 200 microm, and FTIR spectroscopy confirmed structural aspects of PCL. Films kept in PBS for 60 days showed tensile strength and elongation matching native blood vessel. Slow degradation of the scaffold was demonstrated by gravimetric analysis and molecular weight determination. Human umbilical vein endothelial cell (HUVEC) adhesion and proliferation on bare films were minimal. FTIR spectroscopy and environmental scanning electron microscopy (ESEM) of PCL-fibrin hybrid scaffold confirmed the presence of fibrin composite on PCL film. HUVEC was subsequently cultured on hybrid scaffold, and continuous EC lining was observed in 15 and 30 days of culture using ESEM. Results suggest that the new hybrid scaffold can be a suitable candidate for cardiovascular tissue engineering.     

Biological performances of collagen-based scaffolds for vascular tissue engineering

Collagen is widely used for biomedical applications and it could represent a valid alternative scaffold material for vascular tissue engineering. In this work, reconstituted collagen films were prepared from neutralized acid-soluble solutions for subsequent haemocompatibility and cell viability performance assays. First, haemoglobin-free, thrombelastography and platelet adhesion tests were performed in order to investigate the blood contact performance. Secondly, specimens were seeded with endothelial cells and smooth muscle cells, and cell viability tests were carried out by MTT and SEM. Results show that neutralized acid-soluble type I collagen films do not enhance blood coagulation, do not alter normal viscoelastic properties of blood and slightly activate platelet adhesion and aggregation. Cell culture shows that the samples are adequate substrates to support the adhesion and proliferation of endothelial and smooth muscle cells.     

Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering

The endothelialization of prosthetic scaffolds is considered to be an effective strategy to improve the effectiveness of small-diameter vascular grafts. We report the development of a nanofibrous scaffold that has a polymeric core and a shell mimicking mussel adhesive for enhanced attachment, proliferation, and phenotypic maintenance of human endothelial cells. Polycaprolactone (PCL) was chosen as a core material because of its good biodegradability and mechanical properties suitable for tissue engineering. PCL was electrospun into nanofibers with a diameter of approximately 700 nm and then coated with poly(dopamine) (PDA) to functionalize the surface of PCL nanofibers with numerous catechol moieties similar to mussel adhesives in nature. The formation of a PDA ad-layer was analyzed using multiple techniques, including scanning electron microscopy, Raman spectroscopy, and water contact angle measurements. When PDA-coated PCL nanofibers were compared to unmodified and gelatin-coated nanofibers, human umbilical vein endothelial cells (HUVECs) exhibited highly enhanced adhesion and viability, increased stress fiber formation, and positive expression of endothelial cell markers (e.g., PECAM-1 and vWF).     

In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing vascular grafts, the in vitro biocompatibility of poly(epsilon-caprolactone) (PCL), recently suggested for several clinical applications, was evaluated in this study using L929 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture on PCL films: adhesion, proliferation, morphology, LDH release and mitochondrial function. Since topography and other surface characteristics of materials play an essential part in cell adhesion, PCL membranes with either smooth or rough surface were prepared, characterized and used to carry out cell cultures. During short culture times, PCL produced a significant stimulation of mitochondrial activity evaluated by reduction of the MTT reagent. The results provide evidences of good adhesion, growth, viability, morphology and mitochondrial activity of cells on PCL films. Therefore, it can be concluded that PCL is a suitable and biocompatible material as a scaffold for vascular graft development.     

Effect of sustained heparin release from PCL/chitosan hybrid small-diameter vascular grafts on anti-thrombogenic property and endothelialization

Thrombus formation and subsequent occlusion are the main reasons for the failure of small-diameter vascular grafts. In this study, a hybrid small-diameter vascular graft was developed from synthetic polymer poly(ε-caprolactone) (PCL) and natural polymer chitosan (CS) by the co-electrospinning technique. Heparin was immobilized on the grafts through ionic bonding between heparin and CS fibers. The immobilization was relatively stable, and heparin could continuously release from the grafts for more than 1 month. Heparin functionalization evidently improved the hemocompatibility of the PCL/CS vascular grafts, which was illustrated by the reduced platelet adhesion and prolonged coagulation time (activated partial thromboplastin time, prothrombin time and thromboplastin time) as shown in the human plasma assay, and was further confirmed by the ex vivo arteriovenous shunt experiment. In vitro cell proliferation assay showed that heparin can promote the growth of human umbilical vein endothelial cells, while moderately inhibiting the proliferation of vascular smooth muscle cells, a main factor for neointimal hyperplasia. Implantation in rat abdominal aorta was performed for 1 month. Results indicate that sustained release of heparin provided optimal anti-thrombogenic effect by reducing thrombus formation and maintaining the patency. Furthermore, heparin functionalization also enhanced in situ endothelialization, thereby preventing the occurrence of restenosis. In conclusion, it provides a facile and useful technique for the development of heparinized medical devices, including vascular grafts.     

In vitro Studies on Binding and Release of Vascular Endothelial Growth Factor in Heparinized Bovine Pericardiums

Objective: To investigate binding and release of vascular endothelial growth factor (VEGF) and its effect on adhesion and proliferation of endothelial cells (Ecs)in acellular fresh specimens of bovine pericardiums,which were modified by heparinization.Methods:Cross-linked acellular fresh specimens of bovine pericardiums were heparinized by three methods:(1) heparinized N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) treated acellular tissue samples;(2) heparinized poly(ethyleneimine)(PEI) treated acellular tissue samples;(3) heparinized EDC-PEI treated acellular tissue samples.Controlled release of VEGF and its effect on adhesion and proliferation of Ecs was evaluated.Results:In the present study,binding and release of VEGF had better performance in heparinized EDC-PEI treated group,compared with heparinized EDC-alone treated group and heparinized PEI -alone group.We could observe enhanced ability to adhesion and proliferation via modest moisture and effective controlled binding and release of VEGF.Conclusion:Binding of VEGF in heparinized EDC treated group was stable,while Exeiease of VEGF in heparinized treated group was adjusted freely.Interestingly,controlled binding and release of VEGF could exert beneficial effect on adhesion and proliferation of Ecs in heparinized EDC-PEI treated group.     

Biocompatibility of Poly(epsilon-caprolactone) scaffold modified by chitosan--the fibroblasts proliferation in vitro

In this study, the surface of poly(epsilon-caprolactone) (PCL) scaffold was modified by chitosan (CS) in order to enhance its cell affinity and biocompatibility. It is demonstrated by scanning electronic microscopy (SEM) that when 0.5-2.0 wt% chitosan solutions are used to modify the PCL scaffold, the amount of adhesion of the fibroblasts on the chitosan-modified PCL scaffolds dramatically increase when compared to the control after 7 days cell culture. The results indicate that the chitosan-modified PCL scaffolds are more favorable for cell proliferation by improving the scaffold biocompatibility. The improvement may be helpful for the extensive applications of PCL scaffold in heart valve and blood vessel tissue engineering.     

The role of heparin binding surfaces in the direction of endothelial and smooth muscle cell fate and re-endothelialization

In this work, the effects of a heparin-functionalized coating on the growth behavior of vascular cells were studied. To retain its functionality, heparin was bound to a cationic plasma polymerized allylamine coating through electrostatic interaction. The heparin binding surface significantly inhibited human umbilical artery smooth muscle cell (HUASMC) adhesion and proliferation. In contrast, human umbilical vein endothelial cells (HUVECs) showed significant enhancement in cell adhesion, proliferation and migration, release of nitric oxide (NO) and secretion of prostaglandin I(2) (PGI(2)). The test of acute thrombogenicity assessed using human blood exhibited an excellent antithrombotic performance of the heparin grafted surface. The heparinized surface significantly promoted in?vivo re-endothelialization and effectively inhibited thrombosis formation. These observations form an important framework for further deciphering the biological functions of heparin. It is highlighted that these striking findings may serve as a guide for the design of multifunctional vascular devices.     

Preparation and characterization of electrospun PLGA/gelatin nanofibers as a drug delivery system by emulsion electrospinning

Novel biocompatible poly(lactide-co-glycolide) (PLGA) nanofiber mats with favorable biocompatibility and good mechanical strength were prepared, which could serve as an innovative type of tissue engineering scaffold or an ideal controllable drug delivery system. Both hydrophobic and hydrophilic drugs, Cefradine and 5-fluorouracil were successfully loaded into PLGA nanofiber mats by emulsion electrospinning. The natural bioactive protein gelatin (GE) was incorporated into the nanofiber mats to improve the surface properties of the materials for cell adhesion. Nanofibrous scaffolds were characterized by scanning electron microscopy, X-ray diffraction, differential scanning calorimetry, contact angle and tensile measurements. Emulsion electrospun fibers with GE had perfect hydrophilic and good mechanical property. The in vitro release test showed thedrugs released from emulsion electrospun fibers, which achieved lower burst release. The cells cytotoxicity experiment indicated that emulsion electrospun fibers were less toxic and tended to promote fibroblasts cells attachment and proliferation, which implied that the electrospun fibers had promising potential application in tissue engineering or drug delivery.     

Biodegradable PCL scaffolds with an interconnected spherical pore network for tissue engineering

A technique for producing controlled interconnected porous structures for application as a tissue engineering scaffold is presented in this article. The technique is based on the fabrication of a template of interconnected poly(ethyl methacrylate) (PEMA) microspheres, the introduction of a biodegradable polymer, poly-epsilon-caprolactone (PCL), and the elimination of the template by a selective solvent. A series of PCL scaffolds with a porosity of 70% and pore sizes up to 200 microm were produced and characterized (both thermally and mechanically). Human chondrocytes were cultured in monolayer on bulk PCL disks or seeded into porous PCL scaffolds. Cell adhesion, viability, proliferation, and proteoglycan (PG) synthesis were tested and compared with monolayer cultures on tissue-treated polystyrene or pellet cultures as reference controls. Cells cultured on PCL disks showed an adhesion similar to that of the polystyrene control (which allowed high levels of proliferation). Stained scaffold sections showed round-shaped chondrocyte aggregates embedded into porous PCL. PG production was similar to that of the pellet cultures and higher than that obtained with monolayer postconfluence cultures. This shows that the cells are capable of attaching themselves to PCL. Furthermore, in porous PCL, cells maintain the same phenotype as the chondrocytes within the native cartilage. These results suggest that PCL scaffolds may be a suitable candidate for chondrocyte culture.     

Autologous fibrin scaffolds cultured dermal fibroblasts and enriched with encapsulated bFGF for tissue engineering

Autologous fibrin scaffolds (AFSs) enriched with cells and specific growth factors represent a promising biocompatible scaffold for tissue engineering. Here, we analyzed the in vitro behavior of dermal fibroblasts (DFs) (cellular attachment, distribution, viability and proliferation, histological and immunohistochemical changes), comparing AFS with and without alginate microcapsules loaded with basic fibroblast growth factor (bFGF), to validate our scaffold in a future animal model in vivo. In all cases, DFs showed good adhesion and normal distribution, while in scaffolds with bFGF at 14 days, the cell counts detected in proliferation and viability assays were greatly improved, as was the proliferative state, and there was a decrease in muscle specific actin expression and collagen synthesis in comparison with the scaffolds without bFGF. In addition, the use of plasma without fibrinogen concentration methods, together with the maximum controlled release of bFGF at 14 days, favored cell proliferation. To conclude, we have been able to create an AFS enriched with fully functional DFs and release-controlled bFGF that could be used in multiple applications for tissue engineering.     

Shell-core bi-layered scaffolds for engineering of vascularized osteon-like structures

Bottom-up assembly of osteon-like structures into large tissue constructs represents a promising and practical strategy toward the formation of hierarchical cortical bone. Here, a unique two-step approach, i.e., the combination of electrospinning and twin screw extrusion (TSE) techniques was used to fabricate a microfilament/nanofiber shell–core scaffold that could precisely control the spatial distribution of different types of cells to form vascularized osteon-like structures. The scaffold contained a helical outer shell consisting of porous microfilament coils of polycaprolactone (PCL) and biphasic calcium phosphates (BCP) that wound around a hollow electrospun PCL nanofibrous tube (the core). The porous helical shell supported the formation of bone-like tissues, while the luminal surface of nanofibrous core enabled endothelialization to mimic the function of Haversian canal. Culture of mouse pre-osteoblasts (POBs, MC 3T3-E1) onto the coil shells revealed that coils with pitch sizes greater than 135 μm, in the presence of BCP, favored the proliferation and osteogenic differentiation of POBs. The luminal surface of PCL nanofibrous core supported the adhesion and spreading of mouse endothelial cells (ECs, MS-1) to form a continuous endothelial lining with the function similar to blood vessels. Taken together, the shell–core bi-layered scaffolds with porous, coil-like shell and nanofibrous tubular cores represent a new scaffolding technology base for the creation of osteon analogs.     

Nitric oxide-releasing polyurethane-PEG copolymer containing the YIGSR peptide promotes endothelialization with decreased platelet adhesion

Thrombosis and intimal hyperplasia are the principal causes of small-diameter vascular graft failure. To improve the long-term patency of polyurethane vascular grafts, we have incorporated both poly(ethylene glycol) and a diazeniumdiolate nitric oxide (NO) donor into the backbone of polyurethane to improve thromboresistance. Additionally, we have incorporated the laminin-derived cell adhesive peptide sequence YIGSR to encourage endothelial cell adhesion and migration, while NO release encourages endothelial cell proliferation. NO production by polyurethane films under physiological conditions demonstrated biphasic release, in which an initial burst of 70% of the incorporated NO was released within 2 days, followed by sustained release over 2 months. Endothelial cell proliferation in the presence of the NO-releasing material was increased as compared to control polyurethane, and platelet adhesion to polyethylene glycol-containing polyurethane was decreased significantly with the addition of the NO donor.     

Axially aligned 3D nanofibrous grafts of PLA–PCL for small diameter cardiovascular applications

Axially aligned nanofibrous matrices were evaluated as small diameter cardiovascular grafts. Grafts were prepared using the poly(L-lactic acid) (PLA) and poly(ε-caprolactone) (PCL) physical blends in the ratios of 75:25 and 25:75 with the dimension of (40?×?0.2?×?4) millimeter by electrospinning using dynamic collector (1500 RPM). Hydrophobicity and tensile stress were significantly higher in PLA–PCL (75:25), whereas tensile strain and fiber density were significantly higher in PLA–PCL (25:75). Properties such as anastomatic strength porosity, average pore size, degradation with retained fiber orientation, and thromboresistivity were comparable between blends. Human umbilical vascular endothelial cells (HUVEC) adhesion on the scaffolds was observed within 24 h. Cell viability and proliferation were rationally influenced by the aligned nanofibers. Gene expression reveals the grafts thromboresistivity, elasticity, and aided neovascularization. Thus, these scaffolds could be an ideal candidate for small diameter blood vessel engineering.     

Structural characterization and cell response evaluation of electrospun PCL membranes: micrometric versus submicrometric fibers

Electrospinning is a valuable technique to fabricate fibrous scaffolds for tissue engineering. The typical nonwoven architecture allows cell adhesion and proliferation, and supports diffusion of nutrients and waste products. Poly(epsilon-caprolactone) (PCL) electrospun membranes were produced starting from 14% w/v solutions in (a) mixture 1:1 tetrahydrofuran and N,N-dimethylformamide and (b) chloroform. Matrices made up of randomly arranged uniform fibers free of beads were obtained. The average fiber diameters were (a) 0.8 +/- 0.2 microm and (b) 3.6 +/- 0.8 microm. PCL matrices showed the following tensile mechanical properties: tensile modulus (a) 5.0 +/- 0.7 MPa (b) 6.4 +/- 0.2 MPa, yield stress (a) 0.55 +/- 0.06 MPa (b) 0.43 +/- 0.02 MPa, and ultimate tensile stress (a) 1.7 +/- 0.2 MPa and (b) 0.8 +/- 0.1 MPa. The ultimate strain ranged between 300% and 400%. Cytotoxicity of electrospun membranes was continuously evaluated by means of electric cell-substrate impedance sensing technique using human umbilical vein endothelial cells (HUVEC). PCL matrices resulted free of toxic amounts of contaminants and/or process by-products. In vitro studies performed by culturing HUVEC on micrometric and submicrometric fibrous mats showed that both structures supported cell adhesion and spreading. However, cells cultured on the micrometric network showed higher vitality and improved interaction with the polymeric fibers, suggesting an increased ability to promote cell colonization.     

Fabrication and characterization of chitosan/OGP coated porous poly(ε-caprolactone) scaffold for bone tissue engineering

As one of the stimulators on bone formation, osteogenic growth peptide (OGP) improves both proliferation and differentiation of the bone cells in vitro and in vivo. The aim of this work was the preparation of three dimensional porous poly(ε-caprolactone) (PCL) scaffold with high porosity, well interpore connectivity, and then its surface was modified by using chitosan (CS)/OGP coating for application in bone regeneration. In present study, the properties of porous PCL and CS/OGP coated PCL scaffold, including the microstructure, water absorption, porosity, hydrophilicity, mechanical properties, and biocompatibility in vitro were investigated. Results showed that the PCL and CS/OGP-PCL scaffold with an interconnected network structure have a porosity of more than 91.5, 80.8%, respectively. The CS/OGP-PCL scaffold exhibited better hydrophilicity and mechanical properties than that of uncoated PCL scaffold. Moreover, the results of cell culture test showed that CS/OGP coating could stimulate the proliferation and growth of osteoblast cells on CS/OGP-PCL scaffold. These finding suggested that the surface modification could be a effective method on enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering application and the developed porous CS/OGP-PCL scaffold should be considered as alternative biomaterials for bone regeneration.     

Embroidered and Surface Modified Polycaprolactone-Co-Lactide Scaffolds as Bone Substitute: <Emphasis Type="Italic">In Vitro</Emphasis> Characterization

The aim of this study was to evaluate an embroidered polycaprolactone-co-lactide (trade name PCL) scaffold for the application in bone tissue engineering. The surface of the PCL scaffolds was hydrolyzed with NaOH and coated with collagen I (coll I) and chondroitin sulfate (CS). It was investigated if a change of the surface properties and the application of coll I and CS could promote cell adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSC). The porosity (80%) and pore size (0.2–1 mm) of the scaffold could be controlled by embroidery technique and should be suitable for bone ingrowth. The treatment with NaOH made the polymer surface more hydrophilic (water contact angle dropped to 25%), enhanced the coll I adsorption (up to 15%) and the cell attachment (two times). The coll I coated scaffold improved cell attachment and proliferation (three times). CS, as part of the artificial matrix, could induce the osteogenic differentiation of hMSC without other differentiation additives. The investigated scaffolds could act not just as temporary matrix for cell migration, proliferation, and differentiation in bone tissue engineering but also have a great potential as bioartificial bone substitute.     

子痫前期血清通过PLCγ1-IP3R信息途径促进脐动脉平滑肌细胞内钙离子聚集

目的探讨子痫前期血清对脐动脉血管平滑肌细胞内钙离子浓度变化的影响以及磷脂酶C-γ1（PLC-γ1）-三磷酸肌醇受体（IP3R）信息途径对其的调控作用。方法体外建立内皮细胞与平滑肌细胞共培养体系,子痫前期血清处理共培养细胞,MTT检测平滑肌细胞活力,Western blot检测PLc—γ1（磷酸化）、三磷酸肌醇受体（IP3R）活性,Fluo-3／AM检测平滑肌细胞内钙离子浓度,PLC-γ1siRNA沉默平滑肌细胞PLC-γ1表达后,观察子痫前期血清对共培养平滑肌细胞IP3R及细胞内钙离子浓度变化的影响。结果子痫前期血清可促进共培养平滑肌细胞PLC-γ1、IP3R磷酸化及钙离子浓度,平滑肌细胞PLC-γ1沉默可逆转子痫前期血清引起的共培养平滑肌细胞内IP3R活性及细胞内钙离子增加。结论子痫前期血清通过PLC—γ1-IP3R信息通路促进脐动脉平滑肌细胞内钙离子增加,PLC-γ1-IP3R激活引起平滑肌细胞内钙离子增加可能在子痫前期血管平滑肌细胞病理过程中起重要作用。     

仿生构建具有一氧化氮自催化生成功能的人工血管材料

目的 利用有机硒催化一氧化氮(No)供体释放NO的能力设计一种新型人工血管支架材料。方法固载有机硒催化剂的聚乙烯亚胺( SePEI)作为聚阳离子,与聚阴离子聚谷氨酸(PGA)在静电纺丝得到的纳米纤维支架聚己内酯(PCL)表面层层自组装,用紫外和原子吸收进行了定性和定量的表征层层自组装结构;在还原型谷胱甘肽(GSH)的存...