Poly(styrenesulfonate)/poly(allylamine) multilayers: a route to favor endothelial cell growth on expanded poly(tetrafluoroethylene) vascular grafts

Small-diameter synthetic vascular grafts of expanded poly(tetrafluoroethylene) (ePTFE) polymer concern one of the most common alternatives for the replacement of diseased vessels. However, high failure rates arise especially due to the lack of endothelial cells (ECs). EC seeding was developed to build a monolayer on the luminal surface. Because ECs show little or no adhesion on synthetic prostheses, it is necessary to promote their retention. On ePTFE surfaces we successfully deposited polyelectrolyte multilayer films (PMFs) consisting of poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) to obtain PEI-(PSS-PAH)3 films. EC adhesion and spreading on modified ePTFE were assessed by scanning electron and confocal microscopies. Cell viability was evaluated by Alamar Blue assay. After 7 days of culture, the ePTFE modified with PMF exhibited improvements of EC viability as compared to that of the controls (nonmodified ePTFE) or even ePTFE coated by a PAH monolayer (p < 0.05). Moreover, the spreading of ECs was largely enhanced compared to that of the same controls, resulting in a healthy confluent cell monolayer formation. Positive staining for the von Willebrand factor confirmed the EC phenotype. Promoting EC attachment and function on ePTFE modified with PMFs could become in the future a promising treatment for synthetic small-diameter vascular grafts.     

Multilayer rat hepatocyte aggregates formed on expanded polytetrafluoroethylene surface

Feasibility of using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE), for culturing hepatocytes on its surface was examined. Adult rat hepatocytes were attached to an ePTFE surface and cultured in a hormonally defined medium supplemented with or without fetal calf serum (FCS, 10%) or bovine serum albumin (BSA, 0.03–3%). When cultured in a FCS-suplemented medium, hepatocytes reorganized themselves into multilayer cell aggregates on an ePTFE surface. The morphological characteristics of hepatocytes were influenced by the modification of the ePTFE surface as well as the culture medium. Hepatocytes cultured on a polyvinylalcohol (PVA)-coated ePTFE surface formed many more multilayer cell aggregates than those cultured on an uncoated ePTFE surface. Such highly multilayered hepatocyte aggregates were also noted when the cells were cultivated in a BSA-supplemented medium. On the other hand, when cultured in a FCS- or BSA-free medium, hepatocytes formed cell monolayers on both PVA-coated and uncoated ePTFE surfaces as did the cells on a collagen-coated polystyrene surface. The hepatocytes in the aggregates exhibited high albumin expression capability and low DNA synthesis rate as compared with those in monolayer cultures. The multilayer hepatocyte aggregates, as immobilized on a PVA-coated ePTFE surface in a serum-supplemented medium, are shown to be not only morphologically, but functionally differentiated, and will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver. This revised version was published online in June 2006 with corrections to the Cover Date.     

The influence of exogenous fibronectin on blood granulocyte adherence to vascular endothelium in vitro

Fibronectin is a major cell surface and extracellular matrix glycoprotein. It binds to a variety of substrata and supports the attachment and spreading of a number of cell types. We have found that purified human plasma fibronectin can also support blood granulocyte adhesion to cultured human umbilical vein endothelial cells. This activity is protected by treatment of the fibronectin with a sulphhydryl-containing agent. The effect of granulocyte attachment was observed at fibronectin concentration of 100 ng/ml with maximum effect at a concentration of 10 μg/ml. The attached granulocytes retained a rounded appearance, compared with the flattening that occurs on attachment to plastic. Granulocytes attached poorly to cultured human vascular smooth muscle cells and no enhancement occurred when fibronectin was added. Immunofluorescence microscopy using monospecific rabbit anti-human fibronectin demonstrated that the sulphhydryl-treated fibronectin accumulated on the endothelial cell surface, forming aggregates on the apical surface by 3 h of continued incubation. Washed, cultured endothelial cells not exposed to fibronectin or exposed to untreated purified plasma fibronectin did not demonstrate an aggregation of cell-surface fibronectin.     

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the alpha(5)beta(1)-integrin complex, whereas ECs used either alpha(5)beta(1)- or alpha(v)beta(3)-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC alpha(5)beta(1)-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.     

Quartz crystal microbalance study of endothelial cell number dependent differences in initial adhesion and steady-state behavior: evidence for cell-cell cooperativity in initial adhesion and spreading

The quartz crystal microbalance (QCM) technique has been applied to the real time monitoring of endothelial cell (EC) adhesion and spreading on the QCM gold surface. We previously showed that the measured QCM Deltaf and DeltaR shifts were due to cells adhering to the gold crystal surface, requiring proteolytic enzyme treatment to be removed from the surface, in order for the Deltaf and DeltaR shifts to return to zero. In the present report, we demonstrate the quantitative dependence and saturation of the measured Deltaf and DeltaR shifts on the number of firmly attached ECs as measured by electronic counting of the cells. We demonstrate through a light microscope simulation experiment that the different Deltaf and DeltaR regions of the QCM temporal response curve correspond to the incident ECs contacting the surface, followed by their adhesion and spreading, which reflect cellular mass distribution and cytoskeletal viscoelasticity changes. Also, we demonstrate that the dose response curve of Deltaf and DeltaR values versus attached EC number is more sensitive and possesses less scatter for the hydrophilically treated surface compared to the native gold surface of the QCM. For both surfaces, a Deltaf and DeltaR versus trypsinized, attached EC number plot 1 h post-seeding exhibits a sigmoid curve shape whereas a similar plot 24 h post-seeding exhibits a hyperbolic curve shape. This number dependence suggests cell-cell cooperativity in the initial cell adhesion and spreading processes. These QCM data and our interpretation are corroborated by differences in cell appearance and spreading behavior we observed for ECs in a light microscope fluorescence simulation experiment of the cell density effect. For a stably attached EC monolayer at 24 h post-addition, steady-state Deltaf and DeltaR values are higher and exhibit saturation behavior for both the hydrophilically treated gold surface as compared to the untreated surface. The steady-state 24 h Deltaf and DeltaR values of stably attached ECs are shifted from the 1 h attached ECs. The 24 h values are characteristic of a more energy-dissipative structure. This is consistent with the time-dependent elaboration of surface contacts in anchorage-dependent ECs via the attachment of intregrins to underlying extracellular matrix. It is also in agreement with the known energy dissipation function of the ECs that cover the interior of blood vessels and are exposed to continuous pulsatile blood flow.     

Use of trout serum to prepare primary attached monolayer cultures of hepatocytes from rainbow trout (Salmo gairdneri)

Summary The influence of trout serum on the attachment and spreading of isolated trout hepatocytes maintained in primary culture at different temperatures was evaluated. Hepatocytes were obtained from young rainbow trout (Salmo gairdneri) by collagenase dissociation and maintained in modified Leibowitz L15 medium at 10° or 27° C for 24 h in plastic dishes previously coated with type I bovine collagen. In the absence of serum, fewer than 10% of hepatocytes attached and none of them spread on the collagen substrate. Trout serum at concentrations as low as 1.25% in the medium resulted in a pronounced concentration-dependent increase in hepatocyte attachment, as determined by direct counts by phase contrast microscopy, or by percentage of lactate dehydrogenase activity attached to the dishes after washing away unattached cells. Attachment rates were greater at the lower temperature (10° C). Trout serum also substantially increased the proportion of attached hepatocytes that spread as monolayers on the collagen substrate, especially at 10° C. By comparison, fetal bovine serum had little influence on the attachment or spreading of trout hepatocytes. These studies demonstrate a simple inexpensive method for preparing attached monolayer trout hepatocyte cultures. This procedure may be useful in toxicologic or functional studies in which fish hepatocyte attachment is an operational requirement.     

Platelet thrombospondin mediates attachment and spreading of human melanoma cells

Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.     

The inhibitory effect of serum on cell attachment can be prevented by cobalt-protoporphyrin IX

The rate of attachment of chick embryo fibroblasts (CEF) incubated for 1 h at pH 6 in the absence of serum, was equal to about 90% with 2% of spreading cells. The addition of 1% horse serum (HS) decreased to about 30% the rate of attached cells but increased to about 30% the proportion of spreading cells. We found that 3 microM cobalt-protoporphyrin IX (CoPP) added for 3 min before the addition of 1% HS increased both the rate of cell attachment (about 60%) and the proportion of cell spreading (about 50%).     

Substrate ifluences human epidermal melanocyte attachment and sperading in vitro

Summary Previous culture systems for melanocytes have employed serum-supplemented medium and uncoated plastic dishes, prohibiting examination of possible substrate influences on cellular morphology and function. We now report, using a sensitive serum-free system and a quantitative procedure for evaluating cellular morphology, that modification of the plating surface affects human epidermal melanocyte attachment rate and subsequent morphology in vitro. Melanocytes attach and spread more rapidly on surfaces coated with fibronectin or Type I/III collagen or on surfaces previously conditioned by human keratinocytes, dermal fibroblasts, melanocytes, or melanoma cells than do melanocytes on untreated control surfaces. Type IV collagen and laminin, although minimally beneficial for cell attachment, do support a characteristics melanocyte morphology that differs from that seen either on the other coated surfaces or on uncoated plastic controls. Addition of fetal bovine serum at the time of inoculation has no appreciable effect on attachment but markedly improves cell spreading on untreated surfaces, while addition of nerve growth factor with or without serum to this system fails to affect cell attachment or spreading. Our data establish that human epidermal melanocytes are indeed capable of responding morphologically to substrate signals. The ability of several biochemically unrelated surfaces to enhance melanocyte attachment rate and spreading suggests that melanocytes have surface receptors with a variety of specificities. This work is relevant to the development of improved culture systems for melanocytes in vitro and to understanding melanocyte behavior in vivo. This work was supported by the USDA Agricultural Research Service, by a grant from Cheesebrough-Ponds, Inc., and by a Dermatology Foundation Fellowship (Dr. Yaar).     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

The effectiveness of antibiotic activity of penicillin attached to expanded poly(tetrafluoroethylene) (ePTFE) surfaces: a quantitative assessment

Recent studies identified and established a platform of polymer surface modifications allowing the attachment of penicillin (PEN) to expanded poly(tetrafluoroethylene) (ePTFE) surfaces. The effectiveness of this approach was accomplished by creating surfaces with chemically attached PEN that prevent the proliferation of microbes. In this study, quantitative assessments of PEN effectiveness attached to ePTFE were conducted. Using variable-angle attenuated total reflectance (ATR-FTIR) spectroscopy, the volume concentration changes of PEN were determined as a function of depth from the ePTFE surface. At depths ranging from 0.2 to 1.2 mum from the surface, PEN concentration levels decrease from 8.85 to 3.33 mug/m3. Assessments of concentration levels of the colony forming units (CFUs) of Staphylococcus aureus bacteria as a function of contact time with the penicillin-polyethylene glycol spacer separated by maleic anhydride ePTFE (PEN-PEG-MA-ePTFE) surfaces showed profound effectiveness of PEN in preventing microbial proliferation. Hydrolytic stability of PEN-PEG-MA-ePTFE surfaces revealed that even with a 32% loss of PEN due to the cleavage of the ester linkages between PEN and PEG spacer, antimicrobial activity is still maintained.     

A novel technique for loading of paclitaxel-PLGA nanoparticles onto ePTFE vascular grafts

The major cause of hemodialysis vascular access dysfunction (HVAD) is the occurrence of stenosis followed by thrombosis at venous anastomosis sites due to the aggressive development of venous neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective in inhibiting hyperplasia without causing systemic side effects. We have previously demonstrated that paclitaxel-coated expanded poly(tetrafluoroethylene) (ePTFE) grafts, by a dipping method, could prevent neointimal hyperplasia and stenosis of arteriovenous (AV) hemodialysis grafts, especially at the graft-venous anastomoses; however, large quntities of initial burst release have remained a problem. To achieve controlled drug release, paclitaxel (Ptx)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Ptx-PLGA-NPs) were prepared by the emulsion-solvent evaporation method and then transferred to the luminal surface and inner part of ePTFE vascular grafts through our micro tube pumping and spin penetration techniques. Scanning electron microscope (SEM) images of various stages of Ptx-PLGA-NPs unequivocally showed that micro tube pumping followed by spin penetration effectively transferred Ptx-PLGA-NPs to the inner part, as well as the luminal surface, of an ePTFE graft. In addition, the in vitro release profiles of paclitaxel demonstrated that this new system achieved controlled drug delivery with a reduced initial burst release. These results suggest that loading of Ptx-PLGA-NPs to the luminal surface and the inner part of an ePTFE graft is a promising strategy to ultimately inhibit the development of venous neointimal hyperplasia.     

Biological and synthetic prostheses in gynecologic microsurgery]

Biological prostheses of human umbilical vein and synthetic prostheses of ePTFE were comparatively studied in the microsurgical anastomosis of the uterine horns in twenty rats. Patency rate after 30 days was 60% for umbilical vein and 75% for the ePTFE prostheses. Gross and histological examination of umbilical vein graft specimens showed minimal alteration with a smooth intimal surface. On the contrary tissue covering the inner surface from the adjacent ends of the uterine horns was noted in ePTFE prostheses.     

Studies on cell adhesion and recognition. II. The kinetics of cell adhesion and cell spreading on surfaces coated with carbohydrate- reactive proteins (glycosidases and lectins) and fibronectin

The kinetics of cell attachment and cell spreading on the coated surfaces of two classes of carbohydrate-reactive proteins, enzymes and lectins, have been compared with those on fibronectin-coated surfaces with the following results: (a) A remarkable similarity between the kinetics of cell attachment to fibronectin-coated and glycosidase- coated surfaces was found. In contrast, cell attachment kinetics induced by lectin- and galactose oxidase-coated surfaces, in general, were strikingly different from those on fibronectin and glycosidase surfaces. The distinction between fibronectin- or glycosidase- and lectin- or galactose oxidase (an enzyme with lectin-type characteristics)-coated surfaces was further supported by the finding that cytochalasin B and EDTA inhibited cell attachment to fibronectin- and glycosidase-coated surfaces but not lectin-coated surfaces. (b) Fibronectin, if labeled and added to a cell suspension, showed only low or negligible interaction with the cell surface. However, fibronectin absorbed on plastic surfaces showed a high cell-attaching activity. It is assumed that fibronectin coated on plastic surfaces may form polyvalent attachment sites in contrast to its lower valency in aqueous solution. (c) Various inhibitors of cell attachment to both fibronectin- , galactose oxidase-, and lectin-coated surfaces were effective only during the first few minutes of the adhesion assay, after which time the attached cells became insensitive to the inhibitors. It is suggested that the initial specific recognition on either lectin-type or fibronectin-type surfaces is followed by an active cell-dependent attachment process. The primary role of the adhesion surface is to stimulate the cell-dependent attachment response. (d) Cells attached on tetravalent concanavalin A (Con A) spread very rapidly and quantitatively, whereas divalent succinyl Con A and monovalent Con A were effective stimulators of cell attachment but not cell spreading. Cross-linking of succinyl Con A restored the cell spreading activity. Tetravalent Con A surfaces specifically bind soluble glycoproteins, whereas succinyl Con A has a greatly reduced ability to bind the same glycoproteins. These results suggest that cross-linking of cell surface glycoproteins by the multivalent adhesive surface may trigger the cellular reaction leading to cell spreading.     

The serum dependence of baby hamster kidney cell attachment to a substratum

Normal attachment and spreading of baby hamster kidney cells onto a non-living substratum requires the presence of a specific serum component adsorbed to the substratum surface and Ca²⁺ ions in the medium. In the absence of the adsorbed serum factor or Ca²⁺ ions cells attach but do not spread. Thus, although the initial rate of BHK cell attachment is faster in serum-free medium than serum-containing medium, no cell spreading occurs in serum-free medium. Adsorption of serum onto the substratum results in a lag phase in the time course of cell attachment which can be eliminated by blocking the negatively charged groups of the serum components adsorbed to the substratum surface; blocking positively charged groups or free sulfhydryl groups of the adsorbed serum components is without effect. The requirement for serum components can be substituted for by adsorbing molecules such as concanavalin A or polycationic ferritin to the substratum surface; however, only ConA results in normal morphology of cell spreading. The data are discussed in terms of a non-electrostatic direct cell-substratum binding model of cell attachment.     

Digital image analysis of growth and starvation responses of a surface-colonizing Acinetobacter sp.

Surface growth of an Acinetobacter sp. cultivated under several nutrient regimens was examined by using continuous-flow slide culture, phase-contrast microscopy, scanning confocal laser microscopy, and computer image analysis. Irrigation of attached coccoid stationary-phase Acinetobacter sp. cells with high-nutrient medium resulted in a transition from coccoid to bacillar morphology. Digital image analysis revealed that this transition was biphasic. During phase I, both the length and the width of cells increased. In contrast, cell width remained constant during phase II, while both cell length and cell area increased at a rate greater than in phase I. Cells were capable of growth and division without morphological transition when irrigated with a low-nutrient medium. Rod-shaped cells reverted to cocci by reduction-division when irrigated with starvation medium. This resulted in conservation of cell area (biomass) with an increase in cell number. In addition, the changes in cell morphology were accompanied by changes in the stability of cell attachment. During phase I, coccoid cells remained firmly attached. Following transition in high-nutrient medium, bacillar cells displayed detachment, transient attachment, and drifting behaviors, resulting in a spreading colonization pattern. In contrast, cells irrigated with a low-nutrient medium remained firmly attached to the surface and eventually formed tightly packed microcolonies. It is hypothesized that the coccoid and bacillar Acinetobacter sp. morphotypes and associated behavior represent specialized physiological adaptations for attachment and colonization in low-nutrient systems (coccoid morphotype) or dispersion under high-nutrient conditions (bacillar morphotype).     

Dextran T 500--induction of spreading in Ehrlich ascites tumour cells on glass surface

The experiments were carried out in order to find factors which could induce attachment and spreading of Ehrlich ascites tumour (EAT) cells on solid substrata. In normal culture media, serum-free as well as serum-containing, these cells did not spread and very weakly attached onto glass. It was found that after coating the cell surfaces with dextran T 500 the EAT cells strongly attached and spread extensively on glass. This spreading could be inhibited or reversed by washing out the dextran or adding calf serum. Dextran T 500 caused rapid spreading also in chick embryo fibroblasts and mouse lymphocytes. Some aspects of these results in connection with contemporary views concerning the processes of cell attachment and spreading are discussed.     

RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion

A novel class of surface-active copolymers is described, designed to protect surfaces from nonspecific protein adsorption while still inducing specific cell attachment and spreading. A graft copolymer was synthesized, containing poly-(L-lysine) (PLL) as the backbone and substrate binding and poly(ethylene glycol) (PEG) as protein adsorption-resistant pendant side chains. A fraction of the grafted PEG was pendantly functionalized by covalent conjugation to the peptide motif RGD to induce cell binding. The graft copolymer spontaneously adsorbs from dilute aqueous solution onto negatively charged surfaces. The performance of RGD-modified PLL-g-PEG copolymers was analyzed in protein adsorption and cell culture assays. These coatings efficiently blocked the adsorption of serum proteins to Nb(2)O(5) and tissue culture polystyrene while specifically supporting attachment and spreading of human dermal fibroblasts. This surface functionalization technology is expected to be valuable in both the biomaterial and biosensor fields, because different signals can easily be combined, and sterilization and application are straightforward and cost-effective.     

Enhancement of cell attachment and tissue integration by a IKVAV containing multi-domain peptide

Laminin contains a number of cell binding motifs including IKVAV and some that bind heparin. We developed a multi-domain synthetic peptide, LA2, which combines IKVAV sequences with a heparin-binding domain with the goal of improving cell attachment to otherwise non-adherent substrates. LA2 was used to coat polystyrene, ethyl vinyl acetate (EVA), expanded polytetrafluoroethylene (ePTFE), polycarbonate, titanium and stainless steel. In cell attachment studies, LA2 dramatically increased cell attachment to polystyrene and EVA compared to uncoated counterparts or those coated with SIKVAV. Similar increases were observed on ePTFE and titanium. On polystyrene, LA2 enhanced the attachment of endothelial cells, smooth muscle cells, epithelial cells, myoblasts, and osteoblast progenitor cells. Following adhesion, the cells underwent proliferation to form confluent monolayers with phenotypic morphologies. Using osteoblast progenitor cells (MC3T3 cells) grown on LA2/polystyrene, the cells exhibited an increased production of a differentiation marker, alkaline phosphatase. In vivo, LA2 improved tissue integration into ePTFE when implanted subcutaneously in rats. After 2 weeks, cells had penetrated deep into the LA2 coated ePTFE implant whereas little cell penetration was found in uncoated grafts. The implant sites exhibited little inflammation or other untoward effects. The results indicated that the LA2 peptide improved cell adhesion and tissue integration and might be useful in a number of tissue engineering applications.