血管组织工程支架材料的细胞相容性

背景：胶原与透明质酸均有利于组织培养中细胞的黏附、增殖和分化。 目的：观察血管内皮细胞、平滑肌细胞与胶原/透明质酸膜、明胶海绵的细胞相容性,并筛选最佳种植方法。 方法：将第3-5代兔血管平滑肌细胞种植在胶原/透明质酸膜(或明胶海绵)材料上,连续培养2周后将兔内皮细胞接种在平滑肌细胞-胶原/透明质酸膜(或明胶海绵)复合体上,并设置单纯平滑肌细胞与内皮细胞共同接种组。 结果与结论：①光镜和扫描电镜观察：细胞在两种材料上均随着培养时间,接种次数增加而生长加快,其中在胶原/透明质酸膜上的细胞生长更好,细胞连接更致密。②WST-1法检测：胶原/透明质酸膜组平滑肌细胞的黏附率及增殖率均高于明胶海绵组(P < 0.05),且细胞在材料上的生长随着接种次数的增加有不同程度提高。③³H-TDR掺入法检测DNA合成率：在胶原/透明质酸膜上的细胞DNA合成最高,明胶海绵上的较差。表明胶原/透明质酸膜具有较理想的细胞相容性,采用适当间隔、反复接种的方法可提高细胞的黏附和增殖。     

Enhanced functions of vascular cells on nanostructured Ti for improved stent applications

Vascular tissue possesses numerous nanostructured surface features, but most metallic vascular stents proposed to restore blood flow are smooth at the nanoscale. Thus, the objective of the present study was to determine in vitro vascular cell functions on nanostructured titanium (Ti) compared to conventional commercially pure (c.p.) Ti. Results of this study showed for the first time greater competitive adhesion of endothelial versus vascular smooth muscle cells on nanostructured Ti compared to conventional Ti after 4 hours. Moreover, when cultured separately, increased endothelial and vascular smooth muscle cell density was observed on nanostructured Ti compared to conventional c.p. Ti after 1, 3, and 5 days; endothelial cells formed confluent monolayers before vascular smooth muscle cells on nanostructured Ti. Results also showed greater total amounts of collagen and elastin synthesis by vascular cells when cultured on nanostructured Ti. Since a major mode of failure of conventional vascular stents is the overgrowth of smooth muscle cells compared to endothelial cells, these results suggest that while the functions of both types of vascular cells were promoted on nanostructured c.p. Ti, endothelial cell functions (of particular importance, cell density or confluence) were enhanced over that of vascular smooth muscle cells. Thus, the present in vitro study showed that vascular stents composed of nanometer c.p. Ti particles may invoke advantageous cellular responses for improved stent applications.     

In vitro functional testing of endothelial progenitor cells that overexpress thrombomodulin

This study investigated the augmentation of endothelial progenitor cell (EPC) thromboresistance by using gene therapy to overexpress thrombomodulin (TM), an endothelial cell membrane glycoprotein that has potent anti-coagulant properties. Late outgrowth EPCs were isolated from peripheral blood of patients with documented coronary artery disease and transfected with an adenoviral vector containing human TM. EPC transfection conditions for maximizing TM expression, transfection efficiency, and cell viability were employed. TM-overexpressing EPCs had a fivefold increase in the rate of activated protein C production over native EPCs and EPCs transfected with an adenoviral control vector expressing β-galactosidase (p<0.05). TM upregulation caused a significant threefold reduction in platelet adhesion compared to native EPCs, and a 12-fold reduction compared to collagen I-coated wells. Additionally, the clotting time of TM-transfected EPCs incubated with whole blood was significantly extended by 19% over native cells (p<0.05). These data indicate that TM-overexpression has the potential to improve the antithrombotic performance of patient-derived EPCs for endothelialization applications.     

Heparinized PCL/keratin mats for vascular tissue engineering scaffold with potential of catalytic nitric oxide generation

Abstract

Heparins are capable of improving blood compatibility, enhancing HUVEC viability, while inhibiting HUASMC proliferation. Combination of biodegradable poly(ε-caprolactone) (PCL) with keratin and heparins would provide an anticoagulant and endothelialization supporting environment for vascular tissue engineering. Herein, PCL and keratin were first coelectrospun and then covalently conjugated with heparins. The resulting mats were surface-characterized by ATR-FTIR, SEM, WCA, and XPS. Cell viability data showed that the heparinized PCL/keratin mats could motivate the adhesion and growth of HUVEC, while inhibit HUASMC proliferation. In addition, these mats could prolong blood clotting time and reduce platelet adhesion as well as no erythrolysis. Interestingly, these mats could catalyze the NO donor in blood to release NO, which could enhance endothelial cell growth, while decrease smooth muscle cell proliferation and platelet adhesion. In summary, the heparinized mats would be a good candidate as a scaffold for vascular tissue engineering. This study is novel in that we prepared a type of heparinized tissue scaffold that could catalyze the NO donor to release NO to regulate endothelialization without angiogenesis and thrombus formation.     

Migration of human vascular endothelial and smooth muscle cells.

Migration of endothelial and smooth muscle cells was studied in vitro by measuring the increase in surface area at specific time intervals of confluent cell colonies advancing under agarose gels that contained both Morgan's medium 199 and variuos types of sera. First passage cultures of endothelial cells or 3 to 6 passage smooth muscle cells were plated into wells punched in agarose gels, at a seeding density of 50,000 cells per well. At zero time the size of the cell colonies was 35.4 sq. mm. +/- standard error 0.1. Irradiation (1500 rads) did not affect the expansion of the cell colonies although 3H-thymidine uptake was inhibited. Endothelial cells migrated under the agarose gels concentrically as contiguous sheets. When exposed to either 20 per cent platelet-poor plasma serum, platelet-rich plasma serum, or whole blood serum, the average increase in surface area was approximately 9 sq. mm. per day. In contrast, arterial smooth muscle cell colonies expanded with an increment of approximately 9 sq. mm. per day when exposed to 10 per cent platelet-poor plasma serum but 12 sq. mm. per day when exposed to 10 per cent platelet-rich plasma serum (p less than 0.001). Platelet factors also had stimulatory effects on the migration of venous smooth muscle cells. Cytochalasin B, dibutyryl cyclic AMP, and theophylline inhibited the migration of both endothelial and smooth muscle cells, but the latter responded more to the inhibitory effects of all three agents. It is concluded that in contrast to vascular smooth muscle, endothelial cells do not require platelet factors for migration and are less responsive to specific inhibitors affecting cell movement.     

Acrylic acid grafting and collagen immobilization on poly(ethylene terephthalate) surfaces for adherence and growth of human bladder smooth muscle cells

In tissue engineering, degradable or non-degradable polymer matrices can act as cell-carrier-scaffolds. Cell adhesion and growth on these scaffolds can be promoted by immobilizing extracellular matrix proteins. Therefore, in this study, polymer poly(ethylene terephthalate) (PET) films were surface modified by graft polymerization of acrylic acid, to subsequently allow collagen (types I and III) immobilization and human smooth muscle cell expansion. The surfaces of PET were activated by plasma, followed by acrylic acid graft polymerization, resulting in covalently bound brushes, containing an average of either 0.22+/-0.1 or 5.93+/-0.87 microg/cm2 of poly(acrylic acid) (PAA). Subsequent electrostatic adsorption of collagen gave a surface concentration of 4.96 and 17.2 microg/cm2, respectively, as determined using radiolabelled 125I collagen. Both PET films grafted with 0.22 microg/cm2 of PAA with or without adsorbed collagen were apt for smooth muscle cell adhesion and proliferation. However, films grafted with 5.93 microg/cm2 were not. PAA-grafted PET films, onto which serum proteins of the culture medium adsorbed spontaneously, proved to be better matrices than films on which collagen has been immobilized. It, therefore, can be speculated that other serum proteins are more important than collagen for the human smooth muscle cell adhesion and growth on surface-modified polymer matrices.     

内皮细胞条件培养液对平滑肌细胞合成胶原的影响

本实验采用胶原酶消化法分离、培养兔主动脉内皮细胞(EC)及平滑肌细胞(SMC),以[~3H]-脯氨酸掺入SMC合成的[~3H]-羟脯氨酸中的量作为测定胶原的指标,观察了兔主动脉内皮细胞条件培养液(EC-CM)对动脉SMC合成胶原的影响,结果发现融合的EC-CM能促进SMC的胶原合成,但SMC数无明显变化;1:2稀释的EC-CM促进SMC合成胶原的作用最大。可见EC通过促进SMC的胶原合成而参与了动脉粥样硬化过程。     

Controlled release from multilayer silk biomaterial coatings to modulate vascular cell responses

A multilayered silk fibroin protein coating system was employed as a drug carrier and delivery system to evaluate vascular cell responses to heparin, paclitaxel, and clopidogrel. The results demonstrated that the silk coating system was an effective system for drug-eluting coatings, such as for stent applications, based on its useful micromechanical properties and biological outcomes. Cell attachment and viability studies with human aortic endothelial cells (HAECs) and human coronary artery smooth muscle cells (HCASMCs) on the drug-incorporated silk coatings demonstrated that paclitaxel and clopidogrel inhibited smooth muscle cell (SMC) proliferation and retarded endothelial cell proliferation. Heparin-loaded silk multilayers promoted HAEC proliferation while inhibiting HCASMC proliferation, desired outcomes for the prevention of restenosis. The preservation of the phenotype of endothelial cells on silk and heparin-loaded silk coatings was confirmed with the presence of endothelial markers CD-31, CD-146, vWF and VE-Cadherin using immunocytochemistry assays. A preliminary in-vivo study in a porcine aorta showed integrity of the silk coatings after implantation and the reduction of platelet adhesion on the heparin-loaded silk coatings.     

The aortic intima. II. Repair of the aortic lining after mechanical denudation.

This article reports the ultrastructure of the aortic lining during the repair of mechanically denuded aortic intima in the rat. Three main features were observed: a) Although platelets form a pavement on the exposed components of the aortic intima, platelet thrombi do not form on the denuded surface. b) During the first weeks after injury, a temporary false endothelial lining is formed by modified intimal smooth muscle cells. While the modified smooth muscle cells do not constitute a continuous cell layer, they are like true endothelial cells in that platelets do not adhere to the cell membrane of either cell type. c) A continuous layer of true endothelial cells is formed within 2 months after the original injury. Even after reestablishment of a continuous endothelium, however, abnormalities persist in the form of incompletely formed intercellular junctions. This abnormal endothelium is associated with areas of intimal smooth muscle cell proliferation. These observations are compatible with two alternative interpretations of the role of endothelial injury in the intimal proliferation seen following injury to the vessel wall: a) persistent defects in the endothelium may result in proliferation of underlying arterial smooth muscle cells or b) the proliferation, in converse, may in some manner delay the healing process of the overlying endothelium.     

Heparin/collagen multilayer as a thromboresistant and endothelial favorable coating for intravascular stent

Endothelialization and antithrombogenicity are two key issues in stent implantation. The layer-by-layer (LbL) deposition of anticoagulant heparin and cell compatible collagen was explored to develop a multilayered coating with synergic property of antithrombogenicity and fast endothelialization. The quartz crystal microbalance with dissipation (QCM-D), UV spectrometer, spectroscopic ellipsometry, scanning electron microscopy, and confocal laser scanning microscopy investigations indicate that the LbL technique, which based on molecular assembly, provides an easy way to develop a smooth, homogenous, and stable coating onto stents. In vitro blood clotting time tests and the platelet adhesion tests show that the multilayer-modified stents present good hemocompatibility. In vitro endothelial cell (EC) culture results show that the multilayer-modified surfaces accelerate the adhesion and proliferation of ECs. These results illustrate that a stent surface coating with properties of antithrombogenicity and EC preference was obtained via heparin/collagen multilayer modification. This surface coating may have great potential in facilitating in situ endothelialization of blood contacting materials.     

Morphological and histological evaluations of 3D-layered blood vessel constructs prepared by hierarchical cell manipulation

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.     

Morphological and Histological Evaluations of 3D-Layered Blood Vessel Constructs Prepared by Hierarchical Cell Manipulation

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.     

Endothelial-smooth muscle interactions in vitro: effects of high pH, flowing medium and extracellular matrix

The interactions of cultured bovine aortic and human umbilical or saphenous vein endothelium with cultured fibroblasts or smooth muscle cells were studied using light microscopy, scanning electron microscopy, and a radioisotope adhesion assay. (1) Resuspended fibroblasts or smooth muscle cells readily 'overgrew' confluent endothelial monolayers under static culture conditions, but not when cultures were exposed to a continuously stirred medium. (2) Exposure of cultured endothelial or smooth muscle cells to a moderately alkaline environment alters the disposition of pericellular concanavalin. A positive extracellular material. This does not affect the initial adhesion of endothelial or smooth muscle cells, but does affect cell spreading. (3) Endothelial adhesion to cultured smooth muscle cells involves both adhesion and spreading. Recently subcultured or rapidly proliferating smooth muscle cells support initial adhesion, but not spreading. Spreading appears to require the establishment of a suitable extracellular matrix, and this is inhibited both by a flowing medium and by an alkaline extracellular environment.     

The establishment of murine blood outgrowth endothelial cells and observations relevant to gene therapy

Endothelial cells are an attractive vehicle for gene therapy because they may be used in an autologous fashion and may allow for direct exposure of the gene product into the intravascular space. To explore this future potential, a reproducible system was developed for the culture of murine blood outgrowth endothelial cells. These cells demonstrated acetylated low-density lipoprotein (LDL) incorporation, matrigel tube formation, and specific endothelial staining characteristics, namely P1H12, VeCAD, vascular cell adhesion molecule (VCAM), vWF, platelet endothelial cell adhesion molecule (PECAM-1), and vascular endothelial growth factor receptor-2 (VEGFR2). They were also negative for smooth muscle actin and monocytic markers CD11b, CD14, and CD16. Moreover, these cells were amendable to gene transfer with red fluorescent and green fluorescent expression vectors as well as human Factor VIII (hFVIII) while maintaining endothelial characteristics. Both source- and gene-introduced cells also manifested excellent proliferative potential. Furthermore, murine blood outgrowth endothelial cells (BOECs) demonstrated persistent in vivo seeding in the liver, lung, spleen, and bone morrow of recipient mice.     

Novel Approach for Endothelializing Vascular Devices: Understanding and Exploiting Elastin–Endothelial Interactions

Elastin is an essential component of arteries which provides structural integrity and instructs smooth muscle cells to adopt a quiescent state. Despite interaction of endothelial cells with elastin in the internal elastic lamina, the potential for exploiting this interaction therapeutically has not been explored in detail. In this study, we show that tropoelastin (a precursor of elastin) stimulates endothelial cell migration and adhesion more than smooth muscle cells. The biological activity of tropoelastin on endothelial cells is contained in the VGVAPG domain and in the carboxy-terminal 17-amino acids. We show that the effects of the carboxy-terminal 17 amino acids, but not those of VGVAPG, are mediated by integrin α_Vβ₃. We demonstrate that tropoelastin covalently linked to stainless steel disks promotes adhesion of endothelial progenitor cells and endothelial cells to the metal surfaces. The adherent cells on the tropoelastin-coated metal surfaces form monolayers that can withstand and respond to arterial shear stress. Because of the unique effects of tropoelastin on endothelial and smooth muscle cells, coating intravascular devices with tropoelastin may stimulate their endothelialization, inhibit smooth muscle hyperplasia, and improve device performance.     

Endothelial-smooth muscle interactions in vitro: effects of high pH,flowing medium and extracellular matrix.

The interactions of cultured bovine aortic and human umbilical or saphenous vein endothelium with cultured fibroblasts or smooth muscle cells were studied using light microscopy, scanning electron microscopy, and a radioisotope adhesion assay. (1) Resuspended fibroblasts or smooth muscle cells readily ''overgrew'' confluent endothelial monolayers under static culture conditions, but not when cultures were exposed to a continuously stirred medium. (2) Exposure of cultured endothelial or smooth muscle cells to a moderately alkaline environment alters the disposition of pericellular concanavalin. A positive extracellular material. This does not affect the initial adhesion of endothelial or smooth muscle cells, but does affect cell spreading. (3) Endothelial adhesion to cultured smooth muscle cells involves both adhesion and spreading. Recently subcultured or rapidly proliferating smooth muscle cells support initial adhesion, but not spreading. Spreading appears to require the establishment of a suitable extracellular matrix, and this is inhibited both by a flowing medium and by an alkaline extracellular environment.     

Shear stress responses of adult blood outgrowth endothelial cells seeded on bioartificial tissue

Human blood outgrowth endothelial cells (HBOECs) are expanded from circulating endothelial progenitor cells in peripheral blood and thus could provide a source of autologous endothelial cells for tissue-engineered vascular grafts. To examine the suitability of adult HBOECs for use in vascular tissue engineering, the shear stress responsiveness of these cells was examined on bioartificial tissue formed from dermal fibroblasts entrapped in tubular fibrin gels. HBOECs adhered to this surface, deposited collagen IV and laminin, and remained adherent when exposed to 15 dyn/cm(2) shear stress for 24 h. The shear stress responses of HBOECs were compared to human umbilical vein endothelial cells (HUVECs). As with HUVECs, HBOECs upregulated vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 when exposed to tumor necrosis factor (TNF)-α and shear stress decreased the expression of these adhesion molecules on TNF-α-activated monolayers. Nitric oxide production was elevated by shear stress, but did not vary between cell types. Both cell types decreased platelet adhesion to the bioartificial tissue, whereas pre-exposing the cells to flow decreased platelet adhesion further. These results illustrate the potential utility for HBOECs in vascular tissue engineering, as not only do the cells adhere to bioartificial tissue and remain adherent under physiological shear stress, they are also responsive to shear stress signaling.     

Supramolecular assemblies of adsorbed collagen affect the adhesion of endothelial cells

The behavior of endothelial cells (HUVECs) in contact with thin collagen films presenting different supramolecular organizations was investigated. Collagen was adsorbed on polystyrene (PS) and plasma-oxidized PS (PSox) in conditions ensuring the formation of continuous layers presenting an increasing density of fibrillar structures. Discontinuous collagen layers were also prepared on PS by adsorption followed by dewetting. The morphology of the obtained collagen films was checked by using atomic force microscopy. HUVECs adhesion was evaluated in terms of cell number, cell area, cell shape, and actin structure after 4 h of contact with the prepared collagen layers. In the presence of serum, no adhesion was observed on PS, whereas a substantial adhesion was found on PSox. This is explained by the competition for adsorption, which turns in favor of adhesive proteins secreted by the cells on the hydrophilic PSox, but turns in favor of serum albumin on the hydrophobic PS. The progressive coating of PS by smooth collagen films increased cell adhesion and spreading. However, cell spreading and cytoskeleton organization were adversely affected by the appearance of a high density of collagen fibrillar structures. This latter trend was similarly observed on PSox. On the other hand, HUVECs spreading and cytoskeleton organization were clearly enhanced on discontinuous collagen layers compared with continuous ones. A possible explanation for these observations lies in the modification of exposure and/or spatial distribution of recognition sequences due to spontaneous collagen self-assembly on fibril formation or to collagen aggregation on dewetting.     

Vascular biosynthesis of nitric oxide: effect on hemostasis and fibrinolysis

Nitric oxide (NO) is a multifunctional effector molecule that plays a central role in the regulation of vascular homeostasis. NO is synthesized from L-arginine by a family of enzymes called NO synthases. The principal source of NO in the vascular system of healthy mammals is the constitutively expressed NO synthase in endothelial cells. The basal endothelial formation of NO can be increased by receptor-dependent agonists (i.e., bradykinin) in a calcium-calmodulin-dependent manner, and also by physical forces (i.e., shear stress), predominantly without changes in the intracellular concentration of free calcium. Nitric oxide can diffuse toward the blood vessel wall where the major target is the smooth muscle cell. NO regulates vascular tone, and the free radical is also a potent inhibitor of smooth muscle cell proliferation, migration and synthesis of extracellular matrix proteins. NO can also diffuse toward the lumen of the blood vessel where it helps maintain blood fluidity. NO inhibits platelets' and leucocytes' adhesion to endothelial cells. In addition, NO inhibits platelet aggregation and facilitates the dissolution of small platelet aggregates. However, the regulatory action of NO on blood cells is most likely limited to the luminal surface of endothelial cells since NO is rapidly scavenged by hemoglobin in erythrocytes and inactivated by oxygen-derived radicals such as superoxide anions. NO can also affect the fibrinolytic activity by regulating the release of tissue-type plasminogen activator and plasminogen activator inhibitor-1. The crucial role of vascular NO in the control of blood fluidity has been demonstrated by the regulation of the bleeding time in humans.     

Smooth muscle cell adhesion to tissue engineering scaffolds

Synthetic polyesters of lactic and glycolic acid, and the extracellular matrix molecule collagen are among the most widely-utilized scaffolding materials in tissue engineering. However, the mechanism of cell adhesion to these tissue engineering scaffolds has not been extensively studied. In this paper, the mechanism of adhesion of smooth muscle cells to these materials was investigated. Vitronectin was found to be the predominant matrix protein adsorbed from serum-containing medium onto polyglycolic acid, poly(lactic co-glycolic) acid, and collagen two-dimensional films and three-dimensional scaffolds. Fibronectin adsorbed to both materials as well, although to a much lower density. Smooth muscle cell adhesion was mediated through specific integrin receptors interacting with these adsorbed proteins, as evidenced by both immunostaining and blocking studies. The receptors involved in adhesion included the alpha(v)beta5 to vitronectin, the alpha5beta1 to fibronectin and the alpha2beta1 to collagen I. Identification of the specific receptors used to adhere to these polymers clarifies why smooth muscle tissue development differs on these scaffolds, and may allow one to design tissue formation by controlling the surface chemistry of tissue engineering scaffolds.