A. Bantjes

Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities

The in vitro interaction of human endothelial cells (HEC) and polymers with different wettabilities in culture medium containing serum was investigated. Optimal adhesion of HEC generally occurred onto moderately wettable polymers. Within a series of cellulose type of polymers the cell adhesion increased with increasing contact angle of the polymer surfaces. Proliferation of HEC occurred when adhesion was followed by progressive flattening of the cells. Our results suggest that moderately wettable polymers exhibit a serum and/or cellular protein adsorption pattern that is favourable for growth of HEC.

Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers of hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) was found to be optimal on the moderately wettable copolymer (mol ratio 25 HEMA/75 MMA). Positively-charged copolymers of HEMA or MMA with trimethylaminoethyl methacrylate-HCl salt (TMAEMA-Cl), both with mol ratios of 85/15 and a negatively-charged copolymer of MMA with methacrylic acid (MAA), mol ratio 85/15, showed high numbers of adhering HEC. In CM, HEC adhered onto the three charged copolymers mentioned above, but neither onto the copolymer of HEMA and MAA (mol ratio 85/15) nor onto the HEMA/MMA co- and homopolymers. Complete cell spreading in CM was only observed on the positively-charged copolymers.

Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated polytetrafluoroethylene

From in vitro experiments it is known that human endothelial cells show poor adhesion to hydrophobic polymers. The hydrophobicity of vascular prostheses manufactured from Teflon or Dacron may be the reason why endothelialization of these grafts does not occur after implantation in humans. We modified films of polytetrafluoroethylene (Teflon) by nitrogen plasma and oxygen plasma treatments to make the surfaces more hydrophilic. Depending on the plasma exposure time, modified polytetrafluoroethylene surfaces showed water-contact angles of 15-58 degrees, versus 96 degrees for unmodified polytetrafluoroethylene. ESCA measurements revealed incorporation of both nitrogen- and oxygen-containing groups into the polytetrafluoroethylene surfaces, dependent on the plasma composition and exposure time. The thickness of the modified surface layer was approximately 1 nm. The adhesion of cultured human endothelial cells from 20% human serum-containing culture medium to modified polytetrafluoroethylene surfaces with contact angles of 20-45 degrees led to the formation of a monolayer of cells, which was similar to the one formed on tissue culture polystyrene, the reference surface. This was not the case when endothelial cells were seeded upon unmodified polytetrafluoroethylene. Surface-modified expanded polytetrafluoroethylene prosthesis material (GORE TEX soft tissue) also showed adhesion of endothelial cells comparable to cell adhesion to the reference surface. The amounts of serum proteins, including fibronectin, adsorbed from serum-containing medium to modified polytetrafluoroethylene surfaces were larger than those adsorbed to unmodified polytetrafluoroethylene. Moreover, the modified surfaces probably allow the exchange of adsorbed serum proteins with cellular fibronectin.

Preferential adsorption of high density lipoprotein from blood plasma onto biomaterial surfaces

In the present study a two step enzyme immuno assay (EIA) was used for the investigation of the adsorption of proteins and lipoproteins from solutions and from blood plasma onto polymer surfaces. It was found that only a small adsorption of the major blood proteins occurred from plasma. Evidence is presented that the reason for this adsorption behaviour is a preferential adsorption of high density lipoprotein (HDL).

Platelet deposition studies on copolyether urethanes modified with poly(ethylene oxide)

Pellethane 2363 80A films and tubings were chemically modified and the effect of these modifications on platelet deposition was studied. Grafting of high molecular weight poly(ethylene oxide) and graft polymerization of methoxy poly(ethylene glycol) 400 methacrylate resulted in surfaces with a good water wettability. The increased hydrophilicity of these modified surfaces could be demonstrated by contact angle measurements. The platelet deposition was investigated with tubings in a capillary flow system, using different types of perfusates. Platelet deposition from a buffer-containing perfusate on surfaces modified with either high molecular weight poly(ethylene oxide) or methoxy poly(ethylene glycol) 400 methacrylate was almost absent and less than on Pellethane 2363 80A. Using a citrated plasma-containing perfusate the amount of deposited platelets on Pellethane 2363 80A modified with high molecular weight poly(ethylene oxide) was low and about the same as on unmodified surfaces. However, a marked reduced platelet deposition compared to unmodified Pellethane 2363 80A was found when the platelets were activated by Ca2+ ionophore. The improved blood compatibility of the modified Pellethane 2363 80A tubings obviously indicates the favourable effect of the presence of grafted PEO on the surface.

Deposition of cellular fibronectin and desorption of human serum albumin during adhesion and spreading of human endothelial cells on polymers

More insight into the mechanism of adhesion of human endothelial cells (HEC) on to polymeric surfaces may lead to the development of improved small-diameter vascular grafts. HEC suspended in 20% human serum-containing culture medium adhere and spread well on moderately water-wettable polymers such as tissue culture polystyrene (TCPS). Earlier it was demonstrated that during adhesion and spreading of HEC on TCPS, cellular fibronectin is deposited on to this surface. It was postulated that fibronectin deposition is accompanied by desorption of adsorbed serum proteins, e.g. human serum albumin (HSA). The amounts of adsorbed (cellular) fibronectin and HSA on TCPS surfaces pretreated for 1 h with solutions of human serum (ranging from 0.01%–20%), were determined after incubation of these surfaces for 6 h with HEC in culture medium and after incubation with culture medium without cells. Protein adsorption was determined by means of a two-step enzyme-immunoassay (EIA). HEC adhesion and spreading on TCPS resulted in a significant deposition of fibronectin irrespective of the serum concentration in the solution used for the pretreatment of TCPS. The deposition of cellular fibronectin on to TCPS, pretreated with human serum, was accompanied by displacement of adsorbed HSA. Desorption of HSA from TCPS was only detectable with the EIA at serum concentrations ranging from 0.01%–1%. Using131-l-labelled HSA as tracer protein; it could, however, be demonstrated that HSA was also displaced from TCPS, pretreated with solutions of higher serum concentrations. Pretreatment of the hydrophobic vascular graft material PET (poly(ethylene terephthalate); Dacron) and of FEP (fluoroethylenepropylene copolymer; a Teflon-like polymer) with a solution containing 20% human serum resulted in a reduced adhesion of HEC compared to uncoated surfaces. We suggest that this may be caused by a poor displacement of adsorbed serum proteins from these hydrophobic surfaces by cellular fibronectin. This may explain why HEC normally fail to adhere on to prosthetic surfaces.

Small diameter blood vessel prostheses from blends of polyethylene oxide and polypropylene oxide

Studies on the relationship between blood platelet adhesion and type and amount of polyether segments in copolyetherurethanes report a reduced platelet adhesion with increasing polyether content. We therefore assumed that combinations of polyethylene oxide (PEO) and polypropylene oxide (PPOX) might give materials with a good blood compatibility. Water-soluble PEO was attached to PPOX by u.v.-initiated crosslinking. Films were tested for hydrophilicity, mechanical properties, protein adsorption and blood compatibility. The hydrophilicity was determined by swelling experiments. A compromise between hydrophilicity (PEO) and mechanical strength (PPOX) was met at a swelling of 0.5 (PPOX/PEO ratio: 90/10). In protein adsorption studies only small amounts of adsorbed proteins were found. Three blood material interaction in vitro tests gave good results: a low platelet adhesion and kallikrein generation and a high APTT value. Porous tubings (inner diameter 1.3 mm) were fabricated, by spinning from solution, for implantation in the abdominal aorta of rats. Stress-strain diagrams were comparable to those reported for natural blood vessels.

Platelet deposition in a capillary perfusion model: quantitative and morphological aspects

The capillary perfusion model according to Cazenave and co-workers was characterized by investigating the effects of protein precoating, perfusion time and shear rate on platelet deposition using 111Indium labelling of human platelets and scanning electron microscopy (SEM). Compared with uncoated polyethylene, platelet deposition was increased after precoating with purified human von Willebrand factor, fibrinogen or fibronectin, and decreased by preadsorbed immunoglobulin G, albumin or whole plasma. Platelet aggregates were observed on immunoglobulin G-coated polyethylene, whereas all other surfaces showed single adherent platelets. Complete platelet spreading was only observed after precoating with fibronectin. The quantitative data concerning platelet deposition were evaluated by using the convective-diffusion theory. Our results indicate the applicability of this perfusion model for the in vitro testing of biomaterials.

Deposition of endothelial fibronectin on polymeric surfaces

Cellular fibronectin is deposited on tissue culture polystyrene during the adhesion and spreading of cultured human endothelial cells (HEC). Following the seeding of HEC upon this polymer, larger amounts of fibronectin are deposited as both cell density and incubation time increase. Our results indicate that the ability to deposit cellular fibronectin onto a polymeric surface is a condition for the spreading and proliferation of HEC.

POLY(VINYL ALCOHOL)-HEPARIN HYDROGELS AS SENSOR CATHETER MEMBRANES

Poly(vinyl alcohol)-heparin hydrogels with varying water content were synthesized for use as sensor catheter membranes. Films were cast from aqueous mixtures of poly(vinyl alcohol) (PVA), a photosensitive cross-linker p-diazonium diphenyl amine polymer (PA), glutaraldehyde (GA) and heparin. After drying, the films were cross-linked by successive UV irradiation and heat treatment. To get an indication about the cross-linking density of the networks, the water content of the hydrogels was measured after equilibration in water. Hydrogels from PVA, PA, GA and heparin, with a water content of 35-95%, could be obtained if the components were dissolved in saline instead of water. The release of heparin from PVA-heparin or PVA-PA-heparin hydrogels was studied using different receiving phases. The cumulative amount of released heparin appeared to be dependent on the initial water content of the hydrogels and the composition of the receiving phase. For the PVA-PA-heparin hydrogels as well as the PVA-heparin hydrogels the cumulative amount of released heparin in water was about six times higher than in a Tris buffer. Using Tris buffer as receiving phase PVA-PA-heparin hydrogels with water contents of 53, 61 or 71% released heparin for at least 3 wk. The cumulative amount of released heparin increased with initial water content of these hydrogels. Recalcification times (RCT) of plasma exposed to PVA-PA-heparin hydrogels (water content 53%), which released heparin at a low rate (2 micrograms/cm2 per day), were markedly prolonged compared with the RCT values for PVA-PA hydrogels without heparin.(ABSTRACT TRUNCATED AT 250 WORDS)