Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

人脐静脉内皮细胞在RGD肽聚酯材料表面的黏附稳定性研究

研究RGD肽对内皮细胞(Endothelialcell,EC)在生物材料表面黏附稳定性的影响。实验材料(聚酯)分为三组:RGD组(表面共价接枝人工合成的RGD三肽)、对照组(表面预衬纤维粘连蛋白)和空白组(表面未作任何处理),然后在三组材料表面种植体外培养的人脐静脉内皮细胞,并在定常流条件下观察比较RGD肽和纤维粘连蛋白对材料表面细胞黏附稳定性的影响。结果显示随着剪切力作用时间延长和剪切力加大,三组材料表面黏附的细胞脱落逐渐增多。空白组PET表面细胞脱落最为明显,8.19dyne/cm2作用4h后,材料表面细胞残留率仅为13.73%。PET表面结合RGD或纤维粘连蛋白后,细胞残留率明显增加,同样剪切力作用下细胞残留率分别为43.33%和40.75%,两组之间无显著性差异。由此得出结论,RGD可以提高EC在材料表面的黏附稳定性。本结果仅是一个体外实验的初步结果,需要进一步的体内实验加以证实。     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

Co-assembling peptides as defined matrices for endothelial cells

Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the β-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials' fibrillization, β-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.     

生物活性短肽RGD在PET表面接枝方法的研究

在高分子材料表面共价引入人工合成的精氨酰-甘氨酰-天冬氨酸三肽(Arg-Gly-Asp peptides，RGD)，以达到让内皮细胞与之特异结合并更加牢固的目的。实验用紫外辐照法将活性基团羧基(-COOH)接枝到聚对苯二甲酸乙二醇酯(Poly(ethylene terephtalate)，PET)膜的表面，将液相合成的RGD三肽耦合接枝到处理过的材料表面，光电子能谱对以羧基为活性基团的接肽反应结果进行分析，光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。内皮细胞生长实验结果表明，成功接枝的RGD序列对材料内皮细胞种植起到了促进作用。本实验成功运用紫外接枝与化学耦合，将生物活性短肽RGD接枝到膜表面，探索了一种新的接枝生物活性短肽的方法。     

Mechanisms underlying the attachment and spreading of human osteoblasts: From transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.     

Endothelial cell adhesion on polyurethanes containing covalently attached RGD-peptides.

Peptides based on cell-adhesive regions of fibronectin, Arg-Gly-Asp-Ser (RGDS), and vitronectin, Arg-Gly-Asp-Val (RGDV), were covalently bound to a polyurethane backbone via amide bonds. Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies were used to monitor the reactions. The amount of grafted peptide was determined by amino acid analysis. X-ray photoelectron spectroscopy (XPS) suggested the presence of the grafted peptide at the polymer-air interface in vacuo. Dynamic contact angle analysis showed that, in water, the peptide-grafted polyurethane surfaces were more polar than the underivatized polyurethane indicating enrichment of peptide groups at the surface. The attachment and spreading of human umbilical vein endothelial cells (HUVECs) on the underivatized and peptide-grafted polyurethanes was investigated. The GRGDSY- and GRGDVY-grafted substrates supported cell adhesion and spreading even without serum in the culture medium. The GRGDVY-grafted substrate supported a larger number of adherent cells and a higher extent of cell spreading than the GRGDSY-grafted substrate. These RGD-containing peptide-grafted polyurethane copolymers may be useful in providing an easily prepared cell-adhesive substrate for various biomaterial applications.     

Polyethylene terephthalate membrane grafted with peptidomimetics: endothelial cell compatibility and retention under shear stress

The present work aimed to treat a polyethylene terephthalate (PET) surface to make the biomaterial more ‘attractive’ in terms of attachment and shear stress response to endothelial cells with a view to possible applications in vascular grafting. A surface wet-chemistry protocol was applied to graft track-etched PET membranes with RGD peptidomimetics based on the tyrosine template and active at the nano-level vs. isolated human α_vβ₃ receptor, which was monitored by X-ray photoelectron spectroscopy, contact angle measurement and atomic force microscopy for characterization. A primary culture of human saphenous vein endothelial cells was used before and after sterilization of the membranes (heat treatment or γ-ray irradiation) to test the benefit of grafting. The optimal surface concentrations of grafted molecules were around 50?pmol/cm². Compared to GRGDS, the peptidomimetics promoted cell attachment with similar or slightly better performances. Endothelialized grafted supports were further exposed to 2?h of shear stress mimicking arterial conditions. Cells were lost on non-grafted PET whereas cells on grafted polymers sterilized by γ-ray irradiation withstood forces with no significant difference in focal contacts. At the mRNA level, cells on functionalized PET were able to respond to shear stress with NFkB upregulation. Thus, grafting of peptidomimetics as ligands of the α_vβ₃ integrin could be a relevant strategy to improve the adhesion of human endothelial cells and to obtain an efficient endothelialized PET for the surgery of small-diameter vascular prostheses.     

The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces

Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution μ-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm²) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm² is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm² were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm². Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.     

Immobilization of biomacromolecules onto aminolyzed poly(L-lactic acid) toward acceleration of endothelium regeneration

By reaction of poly(L-lactic acid) (PLLA) membrane with 1,6-hexanediamine, free amino groups were introduced onto a PLLA surface, through which biocompatible macromolecules such as gelatin, chitosan, or collagen were covalently immobilized by employing glutaraldehyde as a coupling agent. The existence of free amino groups on the aminolyzed PLLA surface was verified quantitatively by the ninhydrin analysis method, which revealed that surface NH(2) density increased with 1,6-hexanediamine concentration or aminolyzing time. Scanning force microscopy measurements detected an increase in surface roughness after aminolysis. The culture of human umbilical vein endothelial cells (HUVECs) in vitro proved that the cell proliferation rate and cell activity of both aminolyzed and biomacromolecule-immobilized PLLAs were improved compared with control PLLA. Scanning electron microscopy observation showed more spreading and flat cell morphology after HUVECs were cultured for 4 days on either aminolyzed or biomacromolecule-immobilized PLLA membranes. Confluent cell layers were observed on the modified PLLA. Measurement of von Willebrand factor secreted by these HUVECs confirmed that endothelium function was maintained. Therefore, aminolysis and biomacromolecule immobilization are promising ways to accelerate endothelium regeneration, which is crucial for blood vessel tissue engineering.     

Influence de la densité de peptides RGD greffés en surface de polyéthylène téréphtalate sur l’attachement des MC3T3

The aim of this study was to evaluate the impact of different densities on MC3T3 cells attachment onto polyethylene terephthalate (PET) film surfaces. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions onto PET surface, coupling agent grafting and finally immobilization of peptides. The originality of this work consist, in one hand on quantifying RGD peptides densities grafted onto PET, and on the other hand on studying MC3T3 cells responses after seeding on such biomimetic surfaces. After each functionnalization step, modifications were validated by several physicochemical techniques: X-Ray Photoelectron Spectroscopy permitted to prove the grafting and high-resolution β-imager coupled with use of radiolabelled amino acids served in evaluation of peptides densities. Moreover, this last technique permit us to ensure stability of binding between peptides and polymer. The efficiency of this new route for biomimetic modification of PET surface was demonstrated by measuring the adhesion at 15 hours of osteoblast like cells. Study of cellular comportment was realized by means of focal contact proteins (vinculin, actin) immunostaining.     

Inhibitory effect of midkine-binding peptide on tumor proliferation and migration

Background: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. Methods: The midkine-binding peptides were panned by Ph.D.-7^™ Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. Results: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. Conclusion: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo.     

Improving endothelial cell adhesion to vascular graft surfaces: Clinical need and strategies

Synthetic vascular grafts do not spontaneously endothelialize in humans and require some form of anticoagulation to maintain patency. Preseeding synthetic graft materials such as expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate (PET) with endothelial cells (EC) has been examined in various in vitro and in vivo models. Although various studies provide encouraging results, clinical trials for EC seeding on synthetic grafts have not been equally successful. This paper provides a brief review of the various reports on EC seeding in animal and clinical studies. We discuss the inefficiencies associated with the EC seeding process and examine plasma protein treatment of the graft surfaces as a viable option for improving EC attachment, retention and spreading. As an alternative to exsisting therapies we present data on a heterogeneous ligand treatment of fibronectin (Fn) and avidin-biotin for enhanced human umbilical vein endothelial cell (HUVEC) adhesion to ePTFE graft surfaces. Control consisted of HUVECs seeded on Fn treated ePTFE graft surfaces. Functionality of HUVECs was assessed by measuring prostacyclin production of cells on both homogeneous and heterogeneous ligand treated surfaces. Laminar flow studies with a variable width flow chamber and scanning electron microscopy were used to measure initial cell retention and observe initial cell spreading on ePTFE surfaces, respectively. HUVEC retention on heterogeneous ligand treated graft surface was significantly (p < 0.001) higher compared to homogeneous ligand treated surfaces for shear stress in the range of 10-30 dyn cm^-2. HUVEC showed more cellular spreading on the heterogeneous ligand treated surface after seeding for 1-2 h. In vivo experimentation was performed in immune deficient (nude) rats by replacing a section of both the femoral arteries with 8 mm long, 1 mm internal diameter denucleated ePTFE grafts treated with homogeneous and heterogeneous ligands respectively. Both grafts were seeded with similar cell density for 15 min prior to implantation. EC attachment and retention was measured by staining EC with hematoxylin and counting the cells before and after flow using light microscopy. The results indicate that a heterogeneous ligand treatment of graft surfaces using avidin-biotin and Fn-integrin attachment mechanisms increase cell seeding efficiency, initial cell retention and cellular spreading.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

In vitro study of cell-promoting multiple-armed peptides

The purpose of this study was to compare the effectiveness of several linear and branch cell-binding peptides to promote cell growth in prosthetic vascular grafts. In this in vitro study, the peptides were covalently immobilized onto expanded polytetrafluoroethylene (ePTFE) vascular grafts. Cell-growth properties were studied using primary human umbilical vein endothelial cells (HUVECs) and primary human umbilical artery smooth muscle cells (HUASMCs). Linear peptides (P15 and P15') and multiple-armed peptides (MAP4-I and MAP4-II) were covalently bonded onto ePTFE grafts by an atmospheric plasma coating method. X-ray photoelectron spectroscopy and amino acid analysis were used to analyze the surface characteristics of the peptide-coated samples. Cell adhesion, proliferation, and morphology were evaluated by culturing HUVECs and HUASMCs onto the surfaces of different samples: ePTFE control, chemically activated ePTFE, P15-coated ePTFE, and MAP4-coated ePTFE. The cell culture experiments were repeated several times to obtain statistically reliable cell-growth data. Cell-growth data were statistically analyzed by the two-way statistical analysis of variance. The study showed that multiple-armed MAP4 peptides were significantly more effective in promoting endothelial cells than the structurally similar linear P15 peptides. There were 800% more HUVECs proliferated on the MAP4-coated ePTFE samples compared with the ePTFE control. MAP4 peptides were 80% more effective for promoting HUVECs than P15 peptides. In contrast, MAP4 peptides were significantly less effective for promoting HUASMCs than HUVECs. There were only about 100% more HUASMCs proliferated on the MAP4-coated ePTFE samples compared with the ePTFE control. MAP4 and P15 peptides had similar cell-promoting characteristics for SMCs.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Effects of plasma treated PET and PTFE on expression of adhesion molecules by human endothelial cells in vitro

The aim of this study was to evaluate the expression of adhesion molecules on the surface of human endothelial cells in response to the systematic variation in materials properties by the ammonia plasma modification of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE). These adhesion molecules act as mediators of cell adhesion, play a role in the modulation of cell adhesion on biomaterials and therefore condition the response of tissues to implants. First and second passage human umbilical vein endothelial cells (HUVECs) were cultured on plasma treated and untreated PET and PTFE. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-alpha) were used as controls. After 1 day and 7 days, the expression of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), Integrin alphavbeta3, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin and L-selectin were evaluated using flow cytometry and immunohistochemistry. There was a slight increase in positive cell numbers expressing the adhesion molecules ICAM-1 and VCAM-1 on plasma treated PET and PTFE. A significant increase in E-selectin positive cells on untreated PTFE was demonstrated after 7 days. Stimulation with TNF-alpha demonstrated a significant increase in the proportion of ICAM-1. VCAM-1 and E-selectin positive cells. Almost all cells expressed PECAM-1 and integrin alphavbeta3, on both materials and controls but did not express P- and L-selectin on any surface. When second passage cells were used, the expression of the adhesion molecules ICAM-1 and VCAM-1 was markedly increased on all surfaces but not with TNF-alpha. These significant differences were not observed in other adhesion molecules. These results were supported by immunohistochemical studies. The effects of plasma treated PET and PTFE on cell adhesion and proliferation was also studied. There was a 1.3-fold increase in cell numbers adhered on ammonia plasma treated PET compared to untreated PET and a 5.5-fold increase in cell numbers on treated PTFE compared to untreated PTFE after 1 day. This is significantly different when analysed statistically. After 7 days, cell number increased significantly on all surfaces compared to 1 day, except for untreated PTFE which conversely reduced by 41%. Cell number on the surface of untreated PET was no different to treated PET on days 1 and 7 when second passage cells were used. The study has shown that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and slightly upregulates the expression of adhesion molecules. This surface modification should promote colonisation of an artificial vascular prosthesis by endothelial cells and make it less vulnerable to immune system cells of the recipient. In addition, it should be considered which passage of cells is used due to the different adhesion features of different passages of HUVECs on untreated PET.     

RGD肽与生物材料的内皮化

生物材料的表面内皮化可以明显改善材料的血液相容性,促进各种生物材料,尤其是心血管移植物的临床应用,种植内皮细胞在生物材料表面的贴附及生长状况是决定内皮化能否成功的关键因素,RGD是许多粘附蛋白所共有的高度保守氨基酸序列,这一序列在介导细胞粘附,迁移及生长方面起重要作用,我们综述了RGD与整合素受体的相互作用,RGD生物活性的影响因素以及它在生物材料内皮化方向的应用。     

Procoagulant phenotype of endothelial cells after coculture with biomaterial-treated blood cells

Understanding endothelial cell (EC)/blood/biomaterial interactions is crucial for the advancement of cardiovascular devices that often fail because of the lack of nonthrombogenic biomaterials. To begin to assess these interactions, a static EC/blood cell/biomaterial model was used. Isolated blood cells were pretreated with model biomaterial beads with different surface chemistries: polystyrene (PS), and PS beads grafted with 3-kDa polyethylene glycol (PEG) with either a hydroxyl (PS-PEG-OH) or amine (PS-PEG-NH(2)) terminal group at 5.4 or 54 x 10(4) beads/mL. Biomaterial-treated monocytes, neutrophils, or platelets were applied to human umbilical vein ECs (HUVECs) for 5 or 24 h of static coculture, and the resultant procoagulant HUVEC phenotype was characterized using several methods. Flow cytometry was used to assess surface expression of tissue factor (TF), adenosine triphosphate diphosphohydrolase, phosphatidylserine, and thrombomodulin, a functional TF assay was used to assess TF activity, and a plasma recalcification assay examined clotting times on HUVECs. Static coculture of HUVEC with biomaterial-treated neutrophils induced a procoagulant phenotype as exemplified by upregulation of TF expression and total functional activity, and downregulation of adenosine triphosphate diphosphohydrolase and thrombomodulin expression. The plasma recalcification assay demonstrated that HUVECs cocultured with biomaterial-treated monocytes significantly shortened clotting times, with some effect of similarly treated neutrophils.