Poly(ether imide) membranes: studies on the effect of surface modification and protein pre-adsorption on endothelial cell adhesion, growth and function

Poly(ether imide) (PEI) membranes of which the surface was modified with carboxylic groups were tested in comparison to pure PEI and poly(ethylene terephtalate) (PET) for their ability to support attachment, growth and function of human umbilical vein endothelial cells (HUVEC) with respect to endothelization of the above materials. Flat sheet PEI membranes were modified by covalent binding of iminodiacetic acid (IDA) for different periods of time (1 to 30 min) to obtain surfaces with various content of carboxylic groups. In addition, fibronectin (FN) and fibrinogen (FNG) pre-adsorption on the various membranes were studied for their effect on HUVEC behaviour. The results show a decreased protein adsorption and HUVEC adhesion, growth and function in terms of prostacyclin production with an increase in carboxylic groups. Pre-adsorption of the membranes with FN or FNG promoted activity of HUVEC, which became superior to cells on PET. FN-coated membranes were found to be a better substrate for HUVEC adhesion and prostacyclin production, while on FNG-coated membranes cells grew better. Overall it can be concluded that PEI is a promising materials for endothelial cells immobilization as it is needed for improving the haemocompatibility of cardiovascular devices.     

On the tissue compatibility of poly(ether imide) membranes: an in vitro study on their interaction with human dermal fibroblasts and keratinocytes

Recently we have developed a novel type of membrane based on poly(ether imide) (PEI) which is considered for biomedical application. To improve its physical and biological performance it was modified by blending with poly(benzimidazole) (PBI). In the present study both membranes were characterized in terms of their physicochemical properties and in vitro tissue compatibility using human dermal fibroblasts and keratinocytes. The modified membrane (PEI*) was more hydrophilic, less porous and had an increased surface (zeta) potential. We further found that blending with PBI tends to promote cell contact, at least initially, as indicated by the improved overall cell morphology, adhesion and spreading of fibroblasts, and the development of focal adhesion complexes. The effects of fibronectin (FN) and serum coating were also beneficial when compared to pure PEI and tissue culture polystyrene (TCP), which correlates to a higher adsorption of both FN and vitronectin detected by ELISA. However, a clear tendency for homotypic cellular interaction particularly of keratinocytes was obtained in contact with membranes, which was much stronger pronounced on PEI*. Although the initial adhesion was greater on PEI*, a surprising decrease in cell growth was observed at later stages of incubation, which may be explained with the membrane-promoted cellular aggregation leading to an easier detachment from the substratum. Thus, membranes based on blends of PEI with PBI could provide a tissue compatible scaffold with lowered adhesive properties, which might be a useful tool for the transfer of cells, for example, to in vitro engineered tissue constructs.     

On the tissue compatibility of poly(ether imide) membranes: an in vitro study on their interaction with human dermal fibroblasts and keratinocytes

Recently we have developed a novel type of membrane based on poly(ether imide) (PEI) which is considered for biomedical application. To improve its physical and biological performance it was modified by blending with poly(benzimidazole) (PBI). In the present study both membranes were characterized in terms of their physicochemical properties and in vitro tissue compatibility using human dermal fibroblasts and keratinocytes. The modified membrane (PEI*) was more hydrophilic, less porous and had an increased surface (zeta) potential. We further found that blending with PBI tends to promote cell contact, at least initially, as indicated by the improved overall cell morphology, adhesion and spreading of fibroblasts, and the development of focal adhesion complexes. The effects of fibronectin (FN) and serum coating were also beneficial when compared to pure PEI and tissue culture polystyrene (TCP), which correlates to a higher adsorption of both FN and vitronectin detected by ELISA. However, a clear tendency for homotypic cellular interaction particularly of keratinocytes was obtained in contact with membranes, which was much stronger pronounced on PEI*. Although the initial adhesion was greater on PEI*, a surprising decrease in cell growth was observed at later stages of incubation, which may be explained with the membrane-promoted cellular aggregation leading to an easier detachment from the substratum. Thus, membranes based on blends of PEI with PBI could provide a tissue compatible scaffold with lowered adhesive properties, which might be a useful tool for the transfer of cells, for example, to in vitro engineered tissue constructs.     

Expression of leukocyte-endothelial cell adhesion molecules on monocyte adhesion to human endothelial cells on plasma treated PET and PTFE in vitro

We used a coculture model to evaluate the inflammatory potential of ammonia gas plasma modified PET and PTFE by flow cytometry and immunohistochemistry. In these studies, human endothelial cells from umbilical cord (HUVEC) and promonocytic U937 cells were used. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF-) were used as controls. U937 adhesion to endothelium on each surface was evaluated at day 1 and day 7. To further investigate the role of leukocyte–endothelial cell adhesion molecules (CAMs) in cell-to-cell interaction on material surfaces, the expression of the leukocyte–endothelial CAMs: ICAM-1, VCAM-1, PECAM-1, and E-selectin on HUVECs were evaluated after U937 cell adhesion. The results demonstrated that plasma treated PET (T-PET) and treated PTFE (T-PTFE) did not increase U937 cell adhesion compared to the negative control. Maximal adhesion of U937 cells to HUVEC was observed on TNF- stimulated endothelium with significant differences between day 1 and day 7, which is consistent with our prior observation that T-PET and T-PTFE did not cause HUVECs to increase the expression of adhesion molecules. After U937 cell adhesion, the expression of ICAM-1 and VCAM-1 of HUVECs were not different on T-PET and T-PTFE compared with the negative control. However, the expression of E-selectin was reduced on day 1, but not on day 7. The effects of plasma treated PET and PTFE on HUVEC adhesion and proliferation were also studied. On day 1 there were slight increases in the growth of HUVECs on both of T-PET and T-PTFE but this was not statistically significant. On day 7, the cell number increased significantly on the surfaces compared to the negative control. The results demonstrate that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and these surfaces do not exhibit a direct inflammatory effect in terms of monocyte adhesion and expression of leukocyte–endothelial CAMs. The monocyte adhesion to endothelial cells on surfaces can be used as a tool for the evaluation of material surface modification and further to study the mechanisms of cell-to-cell interactions in response to surfaces.     

Plasma-treated PET surfaces improve the biocompatibility of human endothelial cells

Failures of small internal diameter vascular grafts have been caused by the lack of a stable endothelial lining to form on their artificial surfaces. Polymer surfaces can be optimized by means of proper treatment to allow a homogeneous and uniform coverage in artificial prosthesis applications. Several solutions were studied to improve cell attachment and growth on artificial materials. In the present study, polyethyleneterephthalate (PET) surfaces were treated by plasma processes with oxygen and ammonia and also in the presence of a gas mixture to verify the effect of functional groups grafting onto the endothelial cell growth. Related surface chemical modifications were investigated by X-ray photoelectron spectroscopy (XPS). Then using cytotoxicity and cytocompatibility tests, the biocompatibility of the modified PET surfaces was assessed by studying the behavior of human umbilical vein endothelial cells (HUVEC). The results showed that plasma-treated PET samples have no toxic effect on HUVEC. The cytocompatibility tests revealed an increase in cell growth with incubation time and the presence of well-spread and flattened cells (SEM analyses). Thus it is reported that plasma treatments can improve PET biocompatibility to HUVEC.     

Mylar and Teflon-AF as cell culture substrates for studying endothelial cell adhesion

The textured and opaque nature of Dacron and ePTFE has prevented the use of these fabrics in conventional cell culture techniques normally employed to optimize cell attachment and retention. This lack of optimization has led, in part, to the poor performance of endothelialization strategies for improving vascular graft patency. Here we show that thin, transparent films of Mylar and Teflon-AF are viable in vitro cell culture mimics of Dacron and ePTFE vascular graft materials, particularly for the study of protein mediated endothelial cell (EC) attachment, spreading and adhesion. Glass substrates were used as controls. X-ray photoelectron spectroscopy (XPS) and contact angle analysis showed that Mylar and Teflon-AF have surface chemistries that closely match Dacron and ePTFE. (125)I radiolabeling was used to quantify fibronectin (FN) adsorption, and FN and biotinylated-BSA "dual ligand" co-adsorption onto glass, Mylar and Teflon-AF substrates. Native human umbilical vein endothelial cells (HUVEC) and streptavidin-incubated biotinylated-HUVEC (SA-b-HUVEC) spreading was measured using phase contrast microscopy. Cell retention and adhesion was determined using phase contrast microscopy under laminar flow. All surfaces lacking protein pre-treatment, regardless of surface type, showed the lowest degree of cell spreading and retention. Dual ligand treated Mylar films showed significantly greater SA-b-HUVEC spreading up to 2 h, but were similar to HUVEC on FN treated Mylar at longer times; whereas SA-b-HUVEC spreading on dual ligand treated Teflon-AF was never significantly different from HUVEC on FN treated Teflon-AF at any time point. SA-b-HUVEC retention was significantly greater on dual ligand treated Mylar compared to HUVEC on FN treated Mylar over the entire range of shear stresses tested (3.54-28.3 dynes/cm(2)); whereas SA-b-HUVEC retention to dual ligand and HUVEC retention to FN treated Teflon-AF gave similar results at each shear stress, with only the mid-range of stresses showing significant difference in cell retention to Teflon-AF.     

Oxidized laminin‐1 induces increased monocyte attachment and expression of ICAM‐1 in endothelial cells

Atherosclerosis has been associated with increased oxidative stress and monocyte recruitment by endothelial cells. Sub‐endothelial basement membrane proteins, such as laminins that play a central role in cell adhesion, are exposed to reactive oxygen species. In the present study monocyte attachment on human umbilical cord vein endothelial cells (HUVEC) that were preattached to oxidized or native laminin, was investigated. Intracellular cell adhesion molecule‐1 (ICAM‐1) expression by HUVEC was estimated by an enzyme‐linked immunosorbent assay. HUVEC attachment to oxidized or native laminin‐1 was examined using the Hemacolor kit. Anti‐alphaL, anti‐alphaM, anti‐alpha2 and anti‐beta2 integrin subunit antibodies were used in order to further investigate the above phenomena. HUVEC that were preattached to oxidized laminin expressed higher levels of ICAM‐1 and monocytes attached at a higher degree to these cells as compared to HUVEC that were preattached to native laminin. Incubation of monocytes with monoclonal antibodies against the alphaM and beta2 integrin subunits equalized the above mentioned differences. Moreover, HUVEC attached to oxidized laminin at a higher degree as compared to native laminin. This difference was equalized after incubation with the antibody against the alpha2 integrin subunit. These results indicate a modified interaction between HUVEC and the basement membranes in cases where laminin is oxidatively modified. This modified interaction results in increased ICAM‐1 expression by endothelial cells and consequently increased monocyte recruitment capacity.     

The adhesive properties of endothelial cells on endovascular stent coated by substrates of poly-l-lysine and fibronectin

Optimizing endothelial cell growth and adhesion on the surface of metallic stents implanted in the vascular system is a fundamental issue in understanding and improving their long-term biocompatibility. The ability of the endothelial cell to attach and adhere to the luminal stent surface as well as the capacity to withstand the significant shear stress associated with blood flow are important determinants. The adhesive characteristics of human umbilical vein endothelial cellsectin (HUVEC) on stent surfaces coated with either Poly-L-Lysine (PLL) or fibron (FN) were compared with uncoated controls. Increasing concentrations of PLL and FN were measured using a micropipette aspiration system. The adhesivenamic properties of HUVECs under static flow conditions were compared to a dy environment on endovascular stents using a parallel-plate-flow chamber. A scanning electron microscope picture was used to measure the number and the adhesive cell ratio as well as the percentage of surface coverage of stent by endothelial cells. The adhesive forces of HUVECs on foreign surfaces coated with PLL and FN were higher compared to uncoated surfaces, and were dependent on incr ing concentrations. These coatings resulted in significant increase of the adhesive force of HUVECs. The influence of substrates on the adhesion of the endothelial cell monolayer under static or dynamic flow conditions was highly significant compared with controls (p<0.01). No significant differences were observed between PLL and FN substrates. Both PLL and FN coated surfaces can significantly increase the adhesion and growth of HUVECs on metallic stent surfaces.     

Effects of adhesive proteins on endothelialization of PC crosslinking yak pericardial materials--an experimental study]

To investigate the conditions of endothelialization of biomaterials, we examined in vitro the growth properties of endothelial cells on PC treated yak pericardial materials. After rinsed in normal saline (0.9%), the surface of PC crosslinking yak pericardial materials was precoated with three kinds of adhesive proteins--Laminin (La), Fibronectin(FN) and Collagen-I(CL-I). Ednothelial cells(EC) from the canine were seeded on the precoated materials, and on the uncoated materials as control. The result showed that in the La and FN groups the seeded EC survived and grew on the patches, while in the CL-I group and uncoated group the seeded EC on and around the patches did not continue to live. Conclusively, in this experiment, PC yak pericardial materials had cytotoxicity; the cytotoxicity of PC crosslinking yak pericardial materials could be decreased by La and FN precoating; La and FN promoted the adhesion and growth of EC on the PC yak pericardial materials patches; the materials met the needs of endothelialization in vitro, prominently in the La group; there was significant difference in endothelialization between La and FN groups (P < 0.05); CL-I had no part in promoting EC adhesion and growth.     

Adhesion and penetration properties of human lymphocytes acting on allogeneic vascular endothelial cells.

Lymphocyte infiltration through vascular endothelium is one important step in the course of graft rejection. To investigate this process more exactly we established a monolayer invasion assay which enabled us to discriminate between adherent and penetrated cells. Detailed studies of adhesion and penetration kinetics of peripheral blood lymphocytes (PBL) acting on allogeneic human umbilical vein endothelial cells (HUVEC) were carried out by combined phase contrast and reflection interference contrast microscopy. Between 30 and 35% of all PBL attached to HUVEC after 4 hr. Out of these less than 10% penetrated. When HUVEC were prestimulated for 2 hr by interferon (IFN)-alpha,-beta,-gamma or interleukin (IL)-1, PBL adhesion in the early phase of cellular attachment to endothelial cells was accelerated. Overall adhesion however did not increase. Long-term pretreatment of HUVEC for 72 hr with IFN-gamma or IL-1 also modified PBL-HUVEC interactions. However, a 72-hr pretreatment with IFN-alpha or -beta did not influence lymphocyte binding behaviour. PBL penetration was not only accelerated but also enhanced by IFN-alpha,-beta,-gamma, irrespective of whether HUVEC were prestimulated for 2 hr or PBL and cytokines were added simultaneously to HUVEC. On the other hand IL-1 was not able to enhance the amount of penetrated cells but only accelerated the infiltration process. Up-regulation or de novo expression of the adhesion molecules ICAM-1 (intercellular adhesion molecule), ELAM-1 (endothelial leucocyte adhesion molecule) and VCAM-1 (vascular cell adhesion molecule) did not parallel PBL binding kinetics. Therefore an ICAM-, ELAM- and VCAM-independent modulation in the early phase of lymphocyte attachment to endothelium seems likely. The lymphocyte cytoskeleton may have a role in this process.     

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。     

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.     

Photo-initiated grafting of gelatin/N-maleic acyl-chitosan to enhance endothelial cell adhesion,proliferation and function on PLA surface

Vascular graft surface properties significantly affect adhesion, growth and function of endothelial cells (ECs). The bulk degradation property of poly(lactic acid) (PLA) makes it possible for it to be replaced by cellular materials and PLA is desirable as a scaffold material for vascular grafts. However, PLA has an unfavorable surface property for EC adhesion and proliferation due to the lack of a selective cell adhesion motif. Photo-initiated surface-grafting polymerization is a promising method for immobilizing certain biomacromolecules on material surfaces without compromising bulk properties. N-Maleic acyl-chitosan (NMCS) is a novel biocompatible amphiphilic derivative of chitosan with double bonds and can be initiated by ultraviolet light. In this study, gelatin was complexed with NMCS via hydrophobic interaction, and gel/NMCS complex thus formed was then grafted on the PLA surface to improve EC biocompatibility. X-ray photoelectron and Fourier transform infrared spectroscopy, and water contact angle measurement confirmed immobilization of the gel/NMCS complex on PLA surface. Moreover, the gel/NMCS modified PLA enhanced human umbilical vein endothelial cell (HUVEC) spreading and flattening, and promoted the expression of more structured CD31 and vWF compared to unmodified PLA film. Compared to the unmodified PLA surface, the HUVECs on the modified PLA surface had elevated uptake of acetylated low-density lipoprotein, and maintained the ability to modulate metabolic activity upon exposure to shear stress at 5 dyn cm^?2 by up-regulating nitric oxide and prostacyclin production. Cell retention was 1.6 times higher on the gel/NMCS–PLA surface, demonstrating its improved potential for hemocompatibility. These results indicate that photo-initiated surface-grafting of the biomimetic gel/NMCS complex is an effective method to modify material surfaces as vascular grafts.     

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.     

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.     

Surface modification of poly(hydroxybutyrate) films to control cell-matrix adhesion

Tailoring surface properties of degradable polymer scaffolds is key to progress in various tissue engineering strategies. Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thin films were modified by low pressure ammonia plasma, low pressure water vapour plasma, or immersion in a sodium hydroxide solution to elaborate means to control the cell-matrix adhesion of human umbilical cord vein endothelial cells grown on these materials. Fibronectin (FN) heteroexchange and cell adhesion were correlated to the physicochemical characteristics of the modified polymer surfaces which were investigated by X-ray photoelectron spectroscopy (XPS), scanning force microscopy (SFM), electrokinetic measurements, and contact angle measurements. All treatments increased the hydrophilicity of the polymer samples, which could be accounted to newly created amine or carboxyl functionalities for ammonia plasma or water vapour plasma treatments, respectively, and ester hydrolysis for treatments with alkaline aqueous solutions. Main features of cell adhesion and FN reorganisation-evaluated after 1h and after 5 days-could be attributed to the anchorage strength of pre-coated FN layers at the polymer surface, which was, in turn found to be triggered by the type of modification applied. In line with earlier studies referring to different materials cell adhesion and matrix reorganisation were shown to be sensitively controlled through the physicochemical profile of poly(hydroxybutyrate) surfaces.     

登革病毒感染对人血管内皮细胞PGIS表达及PGI2分泌的影响

目的：研究登革病毒感染对人血管内皮细胞分泌血管活性物质前列环素（PGI2）的影响,以了解登革出血热/登革休克综合征（DHF/DSS）的发病机制。方法：用登革病毒Ⅱ型感染人脐静脉内皮细胞（HUVEC）,于感染后6、12、24、48、72、96h,分别收集病毒感染的HUVEC,用RT-PCR检测细胞内前列环素合酶（PGIS） mRNA的水平;分别收集病毒感染的上清液,用放射免疫检测法测定PGI2的含量。结果：登革病毒感染可使PGIS mRNA的水平增高,导致HUVEC分泌PGI2的量明显升高。在病毒感染后48、72、96h,与对照组比较HUVEC表达PGISmRNA的水平和HUVEC分泌PGI2的量明显升高（P〈0.05）。结论：DV2感染可显著上调HUVEC中PGIS mRNA转录及PGI2的分泌,导致登革病毒所致血管内皮细胞的功能障碍,可能与DHF/DSS的发病机制有关。     

Vascular graft endothelialization: comparative analysis of canine and human endothelial cell migration on natural biomaterials

Canines are typically used as the standard preclinical model to gauge the success of vascular graft materials. However, canines spontaneously re-endothelialize vascular grafts, whereas humans do not, even after years. This raises questions of why there are differences in vascular healing between humans and other species and whether the canine is the appropriate preclinical model. In the present study we evaluated human and canine endothelial cell (EC) migration on the novel cross-linked collagen biomaterial PhotoFix(TM) pericardium. We compared in vitro migration of these cells on PhotoFix alone and on PhotoFix adsorbed with various growth factors (aFGF and bFGF) and adhesion proteins (fibronectin, collagen IV, vitronectin, and laminin). We also compared human and canine ECs in terms of their morphologies and prostacyclin production. We found that human umbilical vein ECs (HUVECs) and canine ECs (CECs) migrated well on PhotoFix, suggesting that this biomaterial may be a good vascular graft candidate. Both cell types responded similarly to different growth factors and adhesive proteins, but HUVEC migration was consistently higher than that for CECs. This suggested that human in vivo graft re-endothelialization is likely not hindered by poor endothelial migration but is hindered by other cellular or graft properties.     

Effect of cytokines on the expression of cell adhesion molecule and on the adhesion of melanoma cells to endothelial cells.

We examined the role of cell adhesion molecules (CAM) by which tumor cells bind to the endothelial cells using human umbilical vein endothelial cells (HUVEC) and cultured melanoma cells. Endothelial cells from human umbilical veins were isolated and examined for CAM expression and its modulation by tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6) or interferon-gamma (IFN-gamma). The expression of intercellular adhesion molecule 1 (ICAM-1) on HUVEC was increased by TNF-alpha, IL-1 and IFN-gamma when measured by ELISA or flow cytometric (FACS) analysis. IL-6 did not increase ICAM-1 expression on HUVEC. Two melanoma cell lines, Malme-3M and SK-Mel-28, showed increased expression of ICAM-1 after treatment with TNF-alpha, IL-1 and IFN-gamma in FACS analysis. IFN-gamma induced increased expression of HLA-DR only in SK-Mel-28 melanoma cells, not in Malme-3M melanoma cells. Neither HUVEC nor melanoma cells expressed lymphocyte function-associated antigen 1 (LFA-1) in either the basal (i.e., cytokine untreated) condition or the cytokine treated condition. Melanoma cells showed minimal increment in adhesion to TNF-alpha or IL-1 treated HUVEC than to cytokine untreated HUVEC. HUVEC and melanoma cells did not express LFA-1 and increased ICAM-1 expression by TNF-alpha, IL-1 and IFN-gamma treatment in FACS analysis did not coincide with minimal increase of melanoma cells adhesion to cytokine treated HUVEC. These results suggest that adhesion between melanoma cells and HUVEC is probably mediated by molecular interaction other than ICAM-1/LFA-1.     

轻度修饰低密度脂蛋白对血管内皮细胞粘附功能影响及其机理初探

目的:观察轻度修饰LDL(MM-LDL)对培养人脐静脉内皮细胞(HUVEC)与人类单核细胞系U937粘附功能及其表面粘附分子表达的影响。方法:利用计数法观察经MM-LDL作用的HUVEC与U937细胞的粘附率;用ELISA方法检测MM-LDL作用后HUVEC膜表面粘附分子血管细胞粘附分子-1(VCAM-1)、细胞间粘附分子-1(ICAM-1)及P选择素(P-selectin)的表达。结果:MM-LDL(75mg/L)作用HUVEC4h后,其对U937细胞粘附率明显增加(P<0.01),HUVEC膜表面未见VCAM-1,ICAM-1,P-selectin表达上调,作为阳性对照重组肿瘤坏死因子α(rTNF_α)5.0μg/L可显著诱导以上3种粘附分子表达。延长MM-LDL与HUVEC作用时间至18h可诱导产生P-selectin表达,对VCAM-1表达无影响。结论:MM-LDL诱导的HUVEC与U937粘附不是通过ICAM-1、VCAM-1介导的,P-selectin可能起一定的作用。