Fibrin-mediated endothelial cell adhesion to vascular biomaterials resists shear stress due to flow

In vitro endothelial cell (EC) seeding onto biomaterials for blood-contacting applications can improve the blood compatibility of materials. Adhesive proteins adsorbed from serum that is supplemented with the culture medium intercede the initial cell adhesion and subsequent spreading on material surface during culture. Nevertheless, physical and chemical properties of vascular biomaterial surface fluctuate widely between materials resulting in dissimilarity in protein adsorption characteristics. Thus, a variation is expected in cell adhesion, growth and the ability of cell to resist shear stress when tissue engineering on to vascular biomaterials is attempted. This study was carried out with an objective to determine the significance of a matrix coating on cell adhesion and shear stress resistance when cells are cultured on materials such as polytetrafluoroethylene (PTFE, Teflon) and polyethyleneterephthalate (Dacron), ultra high molecular weight polyethylene (UHMWPE) and titanium (Ti), that are used for prosthetic devices. The study illustrates the distinction of EC attachment and proliferation between uncoated and matrix-coated surfaces. The cell attachment and proliferation on uncoated UHMWPE and titanium surfaces were not significantly different from matrix-coated surfaces. However, shear stress resistance of the cells grown on composite coated surfaces appeared superior compared to the cells grown on uncoated surface. On uncoated vascular graft materials, the cell adhesion was not supported by serum alone and proliferation was scanty as compared to matrix-coated surface. Therefore, coating of implant devices with a composite of adhesive proteins and growth factors can improve EC attachment and resistance of the cells to the forces of flow.     

Human fibroblast attachment on fibrous parylene-C thin-film substrates

Although the polymeric form of parylene-C is used in many medical devices, the mechanistic nature of cellular attachment to polymeric parylene-C is not clear. We examined the effects of (i) substrate morphology, (ii) surface wettability and (iii) presence of serum proteins on fibroblast attachment. A physicochemical vapor deposition technique was implemented to deposit flat parylene-C substrates as well as fibrous substrates of three different morphologies: slanted columnar, chevronic and chiral. Flat parylene-C surfaces were moderately hydrophobic while fibrous substrates were superhydrophobic. Pretreatment with oxygen plasma changed the substrate surfaces from hydrophobic to superhydrophilic. The attachment efficiency of human fibroblast cells to the flat and three fibrous thin-film parylene-C substrates was investigated. Fibroblast attachment was better on fibrous substrates than on flat substrates, and oxygen plasma pretreatment facilitated fibroblast attachment on all four morphologies. Serum proteins also facilitated cell attachment on all substrates. The combination of oxygen plasma pre-treatment and serum proteins increased fibroblast adhesion in an additive manner on flat, but not on fibrous parylene-C substrates. The morphology of cell–substrate interactions differed between fibrous and flat parylene-C substrates.     

Monocyte adhesion and adhesion molecule expression on human endothelial cells on plasma-treated PET and PTFE in vitro

The aim of this study was to evaluate in vitro the inflammatory potential of endothelialized surfaces of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE) after ammonia gas plasma modification. HUVECs grown on polystyrene and HUVECs stimulated with tumor necrosis factor (TNF-alpha) were used as controls. At day 1 and day 7, surfaces were evaluated for U937 cells and HUVECs using flow cytometry and immunohistochemistry. Plasma-treated PET (T-PET) and treated PTFE (T-PTFE) increased U937 cell adhesion compared to the negative control but this was not statistically significant. Maximal adhesion of U937 cells to HUVEC was observed on TNF-alpha stimulated endothelium with significant differences between day 1 and day 7. There was a small increase in U937 cell adhesion to plasma-treated PET compared to PTFE on both day 1 and day 7, but this was not statistically significant. Immunohistochemical staining demonstrated two patterns of distribution for monocyte adhesion on materials. On T-PET the cells were positioned in clusters attached to HUVECs and on T-PTFE the cells were randomly distributed on HUVECs and material. 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, cell number increased significantly on all of 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. Plasma-treated PTFE enhances HUVECs growth and was less adhesive for monocytes as compared with treated PET. 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.     

Immobilization of human thrombomodulin onto PTFE

Human thrombomodulin (hTM) is an endothelial cell-surface glycoprotein and has effective anticoagulant properties. This protein was immobilized onto polytetrafluorethylene (PTFE) surfaces to create biomaterials with enhanced haemocompatibility. The PTFE surface was functionalized by CO2 plasma activation and subsequent vapour-phase graft polymerization of acrylic acid. Surface characterization after plasma treatment, grafting and hTM immobilization was achieved by attenuated total reflection-Fourier transform–infrared spectroscopy, X-ray photoelectron spectroscopy, zeta potential and wetting measurements. The activity of immobilized hTM was estimated using the protein C activation test.     

Protein, cell and bacterial fouling resistance of polypeptoid-modified surfaces: effect of side-chain chemistry

Peptidomimetic polymers consisting of poly-N-substituted glycine oligomers (polypeptoids) conjugated to biomimetic adhesive polypeptides were investigated as antifouling surface coatings. The polymers were immobilized onto TiO(2) surfaces via an anchoring peptide consisting of alternating residues of 3,4-dihydroxyphenylalanine (DOPA) and lysine. Three polypeptoid side-chain compositions were investigated for antifouling performance and stability toward enzymatic degradation. Ellipsometry and XPS analysis confirmed that purified polymers adsorbed strongly to TiO(2) surfaces, and the immobilized polymers were resistant to enzymatic degradation as demonstrated by mass spectrometry. All polypeptoid-modified surfaces exhibited significant reductions in adsorption of lysozyme, fibrinogen and serum proteins, and were resistant to 3T3 fibroblast cell attachment for up to seven days. Long-term in vitro cell attachment studies conducted for six weeks revealed the importance of polypeptoid side-chain composition, with a methoxyethyl side chain providing superior long-term fouling resistance compared to hydroxyethyl and hydroxypropyl side chains. Finally, attachment of both gram-positive and gram-negative bacteria for up to four days under continuous-flow conditions was significantly reduced on the polypeptoid-modified surfaces compared to unmodified TiO(2) surfaces. The results reveal the influence of polypeptoid side-chain chemistry on short-term and long-term protein, cell and bacterial fouling resistance.     

Ellipsometry analysis of conformational change of immobilized protein monolayer on plasma polymer surfaces

The conformational stability of surface immobilized protein monolayers is a key issue in applications requiring preservation of the protein bioactivity such as in biosensors and in vivo implants. Ellipsometry was used to detect conformational changes in a single monolayer of immobilized proteins on plasma polymer surfaces. The areal mass density of immobilized proteins was used to validate the data analysis in the protein denaturation analysis. We observed that the rate of conformation change was strongly dependent on the properties of the immobilized protein. Immobilized catalase showed a significantly slower denaturation rate than the immobilized horseradish peroxidase, indicating that the tetramer catalase is more stable than the immobilized monomer horseradish peroxidase at the surface/air interfaces. The ellipsometry results were in a good agreement with the enzyme activity analysis.     

Hyaluronic acid immobilization on the poly-allylamine coated nano-network TiO2 surface

Recently, biocompatibility report revealed that the TiO2 nano-network (TiO2 NT) structure has much higher cells colonization than the native TiO2 on Ti surface. In this study, we prepared the hyaluronic acid (HA) immobilized TiO2 NT layer by plasma surface modification and then evaluated biological behavior of MC3T3-E1 on the Ti, TiO2 NT and TiO2 NT/NH2/HA surface. The cell viability tests revealed slightly enhanced viability on the TiO2 NT/NH2/HA surfaces than on the untreated Ti surfaces.     

Immobilization of laccase and tyrosinase on untreated and plasma-treated cellulosic and polyamide membranes

Laccase and tyrosinase were immobilized by adsorption and covalent attachment onto microfiltration membranes made of cellulosic and polyamide material. Amine, hydroxyl and carboxylic functional groups for covalent attachment were generated by plasma polymerization of allylamine, allyl alcohol and acrylic acid using mild plasma parameters. Mass analysis of the modified membranes, surface tension and FTIR-ATR spectra were used to show the presence of stable plasma polymer on the membrane surface. It was shown that untreated and plasma treated cellulosic membranes were unsuitable for laccase and tyrosinase immobilization. Both, immobilization of laccase onto polyamide membrane modified with AlNH₂ and adsorption on the untreated membrane at pH 5.2 gave satisfactory and comparable results with better operational stability in 10 consecutive batch processes for covalently bound enzyme. In the case of tyrosinase, adsorption of the enzyme on the untreated PA at pH 7.0 was as effective as covalent binding onto PA-AlNH₂ (in pH 7.0). Operational stability was tested in the presence of diphenolic substrate, which exhibits strong suicide inactivation towards the enzyme. It was shown that immobilized tyrosinase seems to be exceptionally stable in the presence of diphenolic substrate.     

Collagen immobilization on polyethylene terephthalate surface after helium plasma treatment

An attractive alternative to add new functionalities such as biocompatibility due to the micro- and nano-scaled modification of polymer surfaces is offered by plasma processing. Many vital processes of tissue repair and growth following injuries depend on the rate of adsorption and self-assembling of the collagen molecules at the interfaces. Consequently, besides the amount of protein, it is necessary to investigate the form in which the collagen molecules are organizing on the polymer surface. In this study, direct current (DC) helium plasma treatment was used in order to obtain poly(ethylene terephthalate) (PET) films with different amounts of collagen and different shapes of aggregates formed from the collagen molecules. The immobilization of collagen on PET surface was confirmed by XPS measurements, an increase of the nitrogen content by increasing the plasma exposure time being recorded. The SEM and AFM measurements revealed the presence of grains and dendrites of collagen formed on the polymer surface. At 15 min plasma treatment time, the polymer surface after collagen immobilization has a homogenous topography. Usually, one can find fibrils, coil or dendrimers of collagen formed in buffer solutions and immobilized on different polymer surfaces. On the other hand, in this particular configuration, the combination of DC plasma and helium gas as a PET functionalization tool is an original one. As the collagen is not covalently immobilized on the surfaces, it may interact with the cell culture medium proteins, part of the collagen might being replaced by other serum proteins.     

稀土处理玻璃纤维填充PTFE复合材料的滑动磨损性能

研究了不同玻璃纤维表面处理对PTFE复合材料在干摩擦条件下滑动磨损性能的影响,并借助扫描电子显微镜(SEM)分析了磨损机理。结果表明:在干摩擦条件下,经表面处理玻璃纤维填充的PTFE复合材料的摩擦系数和摩擦表面温度比未经处理玻璃纤维填充的PTFE复合材料的低,且减磨性能优于未经处理的;而稀土处理玻璃纤维填充的PTFE复合材料的摩擦系数和摩擦表面温度最低,减磨性能最好;未经处理玻璃纤维填充的PTFE复合材料和偶联剂处理玻璃纤维填充的PTFE复合材料都发生了剧烈的粘着转移;偶联剂与稀土处理玻璃纤维填充的PTFE复合材料的磨损机理主要是明显的磨粒磨损;稀土处理玻璃纤维填充PTFE复合材料的磨损形式主要是粘着转移和轻微的磨粒磨损。     

Plasma treated polyethylene grafted with adhesive molecules for enhanced adhesion and growth of fibroblasts

The cell–material interface plays a crucial role in the interaction of cells with synthetic materials for biomedical use. The application of plasma for tailoring polymer surfaces is of abiding interest and holds a great promise in biomedicine. In this paper, we describe polyethylene (PE) surface tuning by Ar plasma irradiating and subsequent grafting of the chemically active PE surface with adhesive proteins or motives to support cell attachment. These simple modifications resulted in changed polymer surface hydrophilicity, roughness and morphology, which we thoroughly characterized. The effect of our modifications on adhesion and growth was tested in vitro using mouse embryonic fibroblasts (NIH 3T3 cell line). We demonstrate that the plasma treatment of PE had a positive effect on the adhesion, spreading, homogeneity of distribution and moderately on proliferation activity of NIH 3T3 cells. This effect was even more pronounced on PE coated with biomolecules.     

Activation of human neutrophils by organic polymer surfaces

By means of lucigenin-dependent chemiluminescence it has been demonstrated that films of organic polymers activate human neutrophils. The strongest response was always induced by polyvinyl chloride-D and the smallest by polyethylene. No significant differences were found between the number of adherent cells on the surfaces of the investigated materials. The stimulatory capacity of the organic polymers were changed after adsorption of some plasma proteins. These effects were not connected with similar alterations of cell attachment. A second stimulation by opsonized zymosan was only possible for neutrophils adherent to polyether urethane and polyvinyl chloride-D.     

Improved proliferation of human osteosarcoma cells on oxygen plasma treated polystyrene

Polystyrene (PS) foils were exposed to oxygen plasma in order to improve the ability of human osteosarcoma cells (HOS) proliferation. Plasma was created in an electrodeless radio-frequency (RF) discharge in pure oxygen. Plasma treatment time of 30 s allowed for saturation of the surface with oxygen rich functional groups. Incubation of proteins from cell culture media was studied versus incubation time for untreated and plasma treated PS by X-ray photoelectron spectroscopy (XPS). Then HOS cells were cultivated according to a standard procedure, applied on the PS substrates and incubated. The proliferation was studied qualitatively by optical microscopy, scanning electron microscopy (SEM) and quantitatively by MTT assay. All techniques revealed significant differences in HOS cells proliferation on untreated and oxygen plasma treated PS. The differences were most pronounced after 24 h of incubation when the cells have already stared to proliferate on plasma treated PS, while they were still in quiescence on untreated PS. The confluency was achieved till the 6th day on plasma treated PS, while on untreated PS such confluency has never been achieved. The cell viability assay revealed that HOS cells grew better on plasma treated PS for various incubation times. Similar trend was observed regarding the adhesion of proteins. They adhered better on plasma treated PS causing better environment for further cell proliferation. The results clearly show that oxygen plasma treatment allows for much better proliferation of HOS cells on plasma treated PS as compared to the untreated PS substrates.     

Surface modification of titanium by hydrothermal treatment in Mg-containing solution and early osteoblast responses

Surface modification on titanium was carried out in order to improve its bioactivity. Pure titanium was hydrothermally treated in distilled water and 0.1 M MgCl₂ solutions at 200°C for 24 h. Surface morphology, roughness, wettability and chemical composition were characterized before and after treatment. Bovine serum albumin was used as model to study protein adsorption. MC3T3-E1 cells were cultured and initial cell attachment, morphology, proliferation were evaluated. After hydrothermal treatment, nano-sized precipitations were observed and samples showed superhydrophilicity. Magnesium (Mg) was immobilized into titanium surface by hydrothermal treatment. Protein adsorption was significantly increased on Mg-containing samples. Cell attachment was improved and cell spreading was enhanced on Mg-containing samples compared with untreated or those treated in distilled water. Increased early cellular attachment on the MgTi surface resulted in subsequent increase of number of proliferated cells. Hydrothermal treatment in MgCl₂ solution was expected to be an effective method to fabricate titanium implant with good bioactivity.     

Tribological properties of PTFE composites filled with surface-treated carbon fiber

Carbon fibers (CF) were surface treated with concentrated HNO₃, silane coupling agent, rare earth solution and rare earth sol, respectively. The friction and wear behavior of polytetrafluoroethylene (PTFE) filled with differently surface treated CF were studied. The worn surfaces of CF/PTFE composites were then examined with scanning electron microscope (SEM). It is found that the wear volume loss of the rare earth sol treated CF filled PTFE composites reaches the lowest value when the modifier concentration is 10 wt%. It is only about 65% of the wear volume loss of the PTFE filled with untreated CF. Among all the treatments to CF, rare earth sol treatment is the most effective and the lowest friction and wear volume loss of CF/PTFE composite is exhibited.     

Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces

Immobilization of adhesive peptides interacting with cellular integrin receptors onto metallic implant surfaces represents a promising approach to improve osseointegration of implants into the surrounding tissue. In the present study, a functional dextran‐based coating system consisting of an amino titanate adhesion promoter with dendritic structure and a carboxymethyl dextran was established to bind an RGD‐containing adhesive peptide via a selective coupling methodology onto titanium surfaces. The three‐step reaction procedure was characterized by X‐ray photoelectron spectroscopy. In cell adhesion experiments it could be demonstrated that dextran coatings containing immobilized RGD promote attachment and spreading of fibroblast and pre‐osteoblastic cells compared to native as well as CMD‐coated titanium surfaces without RGD. The direct attachment of the RGD sequence to the metal surface via the amino titanate adhesion promoter did not increase pre‐osteoblastic cell spreading, whereas coupling of RGD to the polymeric carboxy­methyl dextran layer slightly enhanced spreading of the cells.     

Biocompatibility of TiO2 nanotubes fabricated on Ti using different surfactant additives in electrolyte

This study examined the in vitro cell-material interactions on four different types of titanium surfaces: a polished Ti surface, TiO₂ nanotube surfaces fabricated in a fluorinated glycerol solution (TN), fluorinated glycerol solution with 1 wt% anionic surfactant sodium dodecyl sulphate (TN-SDS), and fluorinated glycerol solution with 1 wt% cationic surfactant cetyl trimethyl ammonium bromide (TN-CTAB), respectively. The surfaces exhibited distinct surface morphologies and geometrical features. Surface energy calculation shows that TN surface enhances the hydrophilic character by significantly increasing the surface energy. The osteoblast cell growth behavior on the four different surfaces was examined using the MC3T3-E1 cell line for 1 day. When the anodized surfaces were compared for the cell-materials interaction, each of the surfaces showed different properties that affected the cell–material interactions. Proliferation of the cells was noticed with distinctive cell-to-cell attachment on the TN surfaces. Good cellular adhesion with extracellular matrix extensions between the cells was noticed in the TN samples. The TiO₂ nanotubes grown in the surfactant-assisted fluorinated electrolyte did not show significant cell growth on the surface and some cell death was observed. The cell adhesion, differentiation and alkaline phosphatase activity were more pronounced on the TN surface. The MTT assays also revealed an increase in living cell density and proliferation on the TN surfaces. Overall, a rough surface morphology and surface energy are important factors for better cell material interactions.     

A photoimmobilizable sulfobetaine-based polymer for a nonbiofouling surface

A novel photoreactive polymer containing sulfobetaine polar groups was prepared by copolymerization of two kinds of methacrylic acids with sulfobetaine and azidoaniline. The polymer was photoimmobilized on polyester and polystyrene surfaces. Its effects on surface modification were investigated from its interactions with water, proteins and cells. Polymer immobilization altered both of the plain surfaces to becoming hydrophilic in a similar range of static contact angles (12.5 ± 1.6° on polyester and 14.7 ± 2.2° on polystyrene). This suggests that the surfaces were covered with sulfobetaine polar groups. Micropattern immobilization was carried out on both polymers using a photomask. The formed pattern was identical to the photomask, showing that the polymer was formed in response to ultraviolet irradiation. Measurements using atomic force microscopy showed that the polymer was formed at a thickness of 550 nm, demonstrating that the polymer was cross-linked with itself and with the substrate molecules. Measurements using time-of-flight secondary ion mass spectrometry detected an abundance of sulfur-containing ions in the patterned polymer, confirming that sulfobetaine had been immobilized. Protein adsorption and mammalian cell adhesiveness were reduced markedly on the immobilized regions. The reduction of cell adhesiveness was concentration-dependent for the immobilized polymer on polyester surfaces. In conclusion, a novel sulfobetaine-containing polymer was immobilized photoreactively on conventional polymer surfaces and significantly reduced interactions with proteins and mammalian cells.     

Surface-modified poly(methyl methacrylate) capillary electrophoresis microchips for protein and peptide analysis

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.