The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces.

Monocytes and macrophages play critical roles in inflammatory responses to implanted biomaterials. Monocyte adhesion may lead to macrophage activation and the foreign body response. We report that surface chemistry, preadsorbed proteins, and adhesion time all play important roles during monocyte adhesion in vitro. The surface chemistry of tissue culture polystyrene (TCPS), polystyrene, Primaria, and ultra low attachment (ULA) used for adhesion studies was characterized by electron spectroscopy for chemical analysis. Fibrinogen adsorption measured by (125)I-labeled fibrinogen was the lowest on ULA, higher on TCPS, and the highest on polystyrene or Primaria. Monocyte adhesion on protein preadsorbed surfaces for 2 h or 1 day was measured with a lactate-dehydrogenase method. Monocyte adhesion decreased over time. The ability of preadsorbed proteins to modulate monocyte adhesion was surface dependent. Adhesion was the lowest on ULA, higher and similar on TCPS or polystyrene, and the highest on Primaria. Monocyte adhesion on plasma or fibrinogen adsorbed surfaces correlated positively and linearly to the amount of adsorbed fibrinogen. Preadsorbed fibronectin, immunoglobulin G, plasma, or serum also promoted adhesion compared with albumin preadsorbed or uncoated surfaces. Overall, biomaterial surface chemistry, the type and amount of adsorbed proteins, and adhesion time all affected monocyte adhesion in vitro.

Inhibition of monocyte adhesion and fibrinogen adsorption on glow discharge plasma deposited tetraethylene glycol dimethyl ether.

Monocytes and macrophages play important roles in host responses to implanted biomedical devices. Monocyte and macrophage interactions with biomaterial surfaces are thought to be mediated by adsorbed adhesive proteins such as fibrinogen and fibronectin. Non-fouling surfaces that minimize protein adsorption may therefore minimize monocyte adhesion, activation, and the foreign body response. Radio-frequency glow discharge plasma deposition (RF-GDPD) of tetraethylene glycol dimethyl ether (tetraglyme) was used to produce polyethylene oxide (PEO)-like coatings on a fluorinated ethylene-propylene (FEP) surface. Electron spectroscopy for chemical analysis (ESCA) and static time of flight secondary ion mass spectrometry (ToF-SIMS) were used to characterize the surface chemistry of tetraglyme coating. Fibrinogen adsorption to the tetraglyme surface was measured with 125I-labeled fibrinogen and ToF-SIMS. Adsorption of fibrinogen to plasma deposited tetraglyme was less than 10 ng cm(-2), a 20-fold decrease compared to untreated FEP or tissue culture polystyrene (TCPS). Monocyte adhesion to plasma deposited tetraglyme was significantly lower than adhesion to FEP or TCPS. In addition, when the surfaces were preadsorbed with fibrinogen, fibronectin, or blood plasma, monocyte adhesion to plasma deposited tetraglyme after 2 h or 1 day was much lower than adhesion to FEP. RF-GDPD tetraglyme coating provides a promising approach to make non-fouling biomaterials that can inhibit non-specific material-host interactions and reduce the foreign body response.

The role of fibronectin in platelet adhesion to plasma preadsorbed polystyrene.

Platelet adhesion to synthetic surfaces that come in contact with blood is mediated by the adsorption of adhesive plasma proteins, especially fibrinogen. However, the roles of other adhesive proteins, such as fibronectin, vitronectin, and von Willebrand factor in platelet adhesion are not yet clear. In this study, the role of fibronectin in platelet adhesion to surfaces was assessed using three approaches. First, platelet adhesion was measured on Immulon I preadsorbed with fibronectin-depleted plasma or fibronectin-depleted plasma replenished with increasing amount of fibronectin. Under these conditions, fibronectin adsorbed from plasma did not have any effect on platelet adhesion, while fibrinogen played a major role in mediating platelet adhesion. Since fibronectin might play a role in platelet adhesion to surfaces which adsorb little or no fibrinogen, we also used two other strategies to assess the potential role of fibronectin. One was to use platelets treated with a platelet activation inhibitor, prostaglandin E1, which prevents the activation of platelet fibrinogen receptor GP IIb/IIIa. The adhesion of prostaglandin E1-treated platelets to Immulon I preadsorbed with plasma was greatly decreased compared to that of untreated platelets, but was increased by the addition of supernormal concentrations of fibronectin to the plasma. This suggests that GP Ic/IIa, rather than GP IIb/IIIa, might be the platelet receptor which is responsible for platelet adhesion to surface-bound fibronectin. Finally, we studied the effect of fibronectin on platelet adhesion to surfaces preadsorbed with fibronectin-depleted afibrinogenemic plasma. We found that fibronectin re-addition to fibronectin-depleted afibrinogenemic plasma increased platelet adhesion. However, our most important finding was that fibronectin seems to play little or no role in mediating platelet adhesion to polystyrene surfaces preadsorbed with normal plasma.

Correlation between corneal epithelial cell outgrowth and monoclonal antibody binding to the cell binding domain of adsorbed fibronectin.

The ability of corneal epithelial cells to attach, spread, and migrate on synthetic surfaces is largely determined by the characteristics of the adsorbed protein layer. In previous studies we have described an in vitro model for quantitating epithelial cell outgrowth from explanted corneal buttons onto synthetic materials (Pettit et al., Invest. Ophthalmol. Vis. Sci., 31, 2269 [1990]). We have also described the role of fibronectin (fn) adsorption and binding strength on epithelial cell outgrowth (Pettit et al., J. Biomed. Mater. Res., 26, 1259 [1992]). In the current study we have used a monoclonal antibody against the RGD cell binding domain of fn (mAb 3E3) to further characterize the role of adsorbed fn in promoting epithelial cell outgrowth. Ten materials of diverse chemical and physical properties were adsorbed with fn (0.1 mg/ml) or mixtures of fn and albumin (concentrations totaling 0.1 mg/ml) and tested for antibody recognition of the cell binding domain. The surface density of bound anti-cell binding domain antibody varied from a low of 0.66 +/- 0.11 for fluorinated ethylene propylene copolymer (FEP) to a high of 1.90 +/- 0.26 for tissue culture polystyrene dish substrates (units are OD at 450 nm measured in the ELISA technique normalized to polyethylene). A general increase in cell outgrowth areas was noted, with increases in recognizable cell binding domain. However, several exceptions to this trend were noted as well (e.g., low cell outgrowth but high antibody recognizability for glass). These results suggest that, although the number of cell binding domains exposed on adsorbed fn molecules may influence cell outgrowth, other characteristics of the adsorbed protein, such as the binding strength to the underlying substrate, may be equally important in characterizing epithelial cell-substrate interactions.

Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. Student Research Award in the Doctoral Degree Candidate Category, Fourth World Biomaterials Congress (18th annual meeting of the Society for Biomaterials), Berlin, Germany, April 24-28, 1992.

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. Circular corneal buttons (5 mm) were punched from excised rabbit corneas and placed onto bare substrates or substrates preadsorbed with fibronectin (fn), albumin, or binary mixtures of both fn and albumin. Cell outgrowth areas were measured after culturing the buttons for 4 days in serum-free medium. Fibronectin adsorption to the materials was measured from pure and binary solutions with 125I-radiolabeled fibronectin. A parameter thought to be related to the binding strength of fn to polymeric substrates was measured in parallel experiments by partial elution of the adsorbed fn by 3% sodium dodecyl sulfate (SDS). Following pure solution fibronectin adsorption a range of outgrowth areas was measured (from 0.86 +/- 0.03 cm2 for glass to 1.49 +/- 0.03 cm2 for TCPS). On all of the materials tested cell outgrowth areas increased following fn preadsorption and decreased following albumin preadsorption relative to bare surfaces (p less than 0.05). Following preadsorption with binary protein mixtures cell outgrowth areas increased with fibronectin adsorption, however, the outgrowth areas were not determined solely by the concentration of fn adsorbed onto the surfaces. This result suggested that the biological efficiency of the adsorbed fibronectin was substrate-dependent. When the cell outgrowth data were cross-plotted against fn retention following SDS elution, the outgrowth areas were found to increase along with increases in fn retention. Based on these data we suggest that epithelial cell outgrowth may be partially governed by the tightness of binding between the fn molecules and the underlying substrate.

Glow discharge plasma deposition of tetraethylene glycol dimethyl ether for fouling-resistant biomaterial surfaces.

The glow discharge plasma deposition (GDPD) of tetraethylene glycol dimethyl ether is introduced as a novel method for obtaining surfaces that are resistant to protein adsorption and cellular attachment. Analysis of films by x-ray photoelectron spectroscopy and several biological assays indicate the formation of a fouling-resistant, PEO-like surface on several substrata (e.g., glass, polytetrafluoroethylene, polyethylene). Adsorption of 125I-radiolabelled proteins (fibrinogen, albumin and IgG) from buffer and plasma was very low (typically less than 20 ng/cm2) when compared to the untreated substrata, which exhibited much higher levels of protein adsorption. Not all coated substrata adsorbed equal amounts of protein (e.g., coated glass samples typically adsorbed more protein than coated polyethylene or coated polytetrafluoroethylene samples), suggesting that the substratum used may affect the amount of protein adsorbed. Measurement of dynamic platelet adhesion, using epifluorescent video microscopy, and endothelial cell attachment further demonstrates the short-term nonadhesiveness of these surfaces.

Adsorption of baboon fibrinogen and the adhesion of platelets to a thin film polymer deposited by radio-frequency glow discharge of allylamine.

Platelet adhesion under static and flow conditions from a washed platelet suspension containing albumin to a polymer deposited by radio-frequency glow discharge of allylamine vapour on a poly(ethylene terephthalate) substrate was measured. Electron spectroscopy for chemical analysis was used to characterize the surface. Fibrinogen adsorption from a series of dilute plasma solutions to radio-frequency glow discharge/allylamine, measured using 125I radiolabelled baboon fibrinogen, increased with decreasing plasma dilution to a level much higher than that previously observed on polyurethanes. Elutability by sodium dodecyl sulphate of fibrinogen adsorbed from dilute plasma also increased with increasing plasma concentration, but fibrinogen preadsorbed from plasma became non-elutable when surfaces were stored in buffer for 5 d before contact with sodium dodecyl sulphate. Platelet adhesion to substrates which had been pre-adsorbed with dilute plasma was measured using baboon platelets radiolabelled with 111In. Adhesion greatly decreased as the plasma concentration used for preadsorption increased, suggesting that non-specific platelet binding to the bare surface occurs when protein coverage is incomplete. Non-specific platelet binding was inhibited to varying degrees by preadsorption of different proteins to the surface. Platelet adhesion to surfaces preadsorbed with dilute (1.0%) baboon and human plasmas lacking fibrinogen (i.e. serum, heat-defibrinogenated plasma and congenitally afibrinogenemic plasma) was diminished compared with normal plasma. Addition of exogenous fibrinogen to the deficient plasma partially restored platelet adhesion to normal levels. Adhesion to surfaces preadsorbed with human plasma deficient in von Willebrand factor was comparable to that observed with normal plasma. The plasma preadsorption studies with fibrinogen deficient media suggested that adsorbed fibrinogen is necessary for platelet adhesion to the radio-frequency glow discharge/allylamine substrate at high protein coverage. However, since adhesion was greatly reduced when the plasma preadsorbed substrate was stored in buffer before platelet contact, the conformation of adsorbed fibrinogen is also important in mediating platelet adhesion to radio-frequency glow discharge.

Protein adsorption to poly(ethylene oxide) surfaces.

Surfaces containing poly(ethylene oxide) (PEO) are interesting biomaterials because they exhibit low degrees of protein adsorption and cell adhesion. In this study different molecular weight PEO molecules were covalently attached to poly(ethylene terephthalate) (PET) films using cyanuric chloride chemistry. Prior to the PEO immobilization, amino groups were introduced onto the PET films by exposing them to an allylamine plasma glow discharge. The amino groups on the PET film were next activated with cyanuric chloride and then reacted with bis-amino PEO. The samples were characterized by scanning electron microscopy, water contact angle measurements, gravimetric analysis, and electron spectroscopy for chemical analysis (ESCA). The adsorption of 125I-labeled baboon fibrinogen and bovine serum albumin was studied from buffer solutions. Gravimetric analysis indicated that the films grafted with the low-molecular-weight PEO contained many more PEO molecules than the surfaces grafted with higher-molecular-weight PEO. The high-molecular-weight PEO surfaces, however, exhibited greater wettability (lower water contact angles) and less protein adsorption than the low-molecular-weight PEO surfaces. Adsorption of albumin and fibrinogen to the PEO surfaces decreased with increasing PEO molecular weight up to 3500. A further increase in molecular weight resulted in only slight decreases in protein adsorption. Protein adsorption studies as a function of buffer ionic strength suggest that there may be an ionic interaction between the protein and the allylamine surface. The trends in protein adsorption together with the water contact angle results and the gravimetric analysis suggest that a kind of "cooperative" water structuring around the larger PEO molecules may create an "excluded volume" of the hydrated polymer coils. This may be an important factor contributing to the observed low protein adsorption behavior.

Endothelial cell growth on oxygen-containing films deposited by radio-frequency plasmas: the role of surface carbonyl groups.

Polystyrene substrates were modified by radio-frequency plasma deposition from mixtures of various organic vapors (acetone, methane, methanol, and formic acid) and oxygen. The resulting surfaces exhibited a wide range of surface oxygen concentrations, as measured by electron spectroscopy for chemical analysis (ESCA). The surface hydroxyl, carboxyl, and carbonyl groups were derivatized with trifluoroacetic anhydride, trifluoroethanol, or hydrazine, respectively, and their concentrations subsequently determined by ESCA. The growth of bovine aortic endothelial cells was found to increase with the surface carbonyl concentration but did not appear to correlate with the hydroxyl or carboxyl concentrations.

Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth.

Polystyrene and poly(ethylene terephthalate) substrates were modified by radiofrequency plasma deposition with organic vapors comprised of carbon, oxygen, and hydrogen (acetone, methanol, glutaraldehyde, formic acid, allyl alcohol, and ethylene oxide). The treatments resulted in the deposition of a film at least 100 A thick containing up to 26% atomic oxygen at the surface. A high oxygen incorporation was observed for vapors with a large oxygen-to-carbon ratio. Bovine aortic endothelial cell growth measured on acetone, methanol, and glutaraldehyde films was linearly correlated with the oxygen content of the treated surfaces. Nitrogen was incorporated in the surface by blending nitrogen gas into the organic vapor used for plasma deposition. The resulting nitrogen-containing substrates exhibited a high affinity for serum fibronectin but a moderate cell growth.

Quantitation of rabbit corneal epithelial cell outgrowth on polymeric substrates in vitro.

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. To study the effects of polymer surface properties on corneal epithelial cell outgrowth, a quantitative in vitro cell outgrowth assay was done. Polymers with systematic variations in hydroxyl content were used as outgrowth substrates. These polymers were characterized by electron spectroscopy for chemical analysis for elemental surface composition and by captive air-bubble contact angle for surface wettability. Circular corneal buttons were punched from excised rabbit corneas, placed onto these substrates, and incubated in a hormonally enriched culture medium. Outgrowth of the epithelial cells was allowed to proceed onto the substrates for 4 days. The outgrowth areas were measured, and an outgrowth index was calculated for each substrate by comparing it with tissue culture polystyrene substrate. The highest outgrowth generally occurred on substrates with intermediate wettabilities (captive air-bubble contact angles of approximately 45-75 degree); it was less on substrates of lower or higher wettabilities. Protein coatings of albumin, immunoglobulin G (IgG), fibronectin, and culture medium were found to lower the wettabilities of native substrates. Albumin and IgG precoating were shown to reduce epithelial cell outgrowth; fibronectin precoating was shown to improve outgrowth on most substrates. These results suggest that epithelial cell outgrowth is influenced by both substrate and protein interactions.

Plasma gas discharge deposited fluorocarbon polymers exhibit reduced elutability of adsorbed albumin and fibrinogen.

The adsorption and subsequent detergent elutability of fibrinogen and albumin were measured on various treated and untreated polymer films in order to determine whether the relative adsorption of these proteins was responsible for the enhanced thromboresistance of Dacron vascular grafts treated with tetrafluoroethylene in a radio frequency glow discharge (RFGD) apparatus. Fluorocarbon-coated surfaces varying in the relative proportions of CF, CF2, and CF3 groups and in the ratio of fluorine to carbon were prepared by RFGD treatment of poly(ethylene terephthalate) (PET) films with tetrafluoroethylene or perfluoropropane. The adsorption of fibrinogen and albumin to these fluorocarbon-coated surfaces was comparable to the adsorption of the proteins to polytetrafluoroethylene (PTFE) and PET. However, the elutability of fibrinogen and albumin from the RFGD fluorocarbon surfaces with sodium dodecyl sulfate was much lower than that from PTFE or PET. Other RFGD treatments of PET, such as ethylene deposition or argon etching, did not reduce the extent of albumin elutability as dramatically as did the RFGD fluorocarbon treatments. The strong albumin binding to RFGD fluorocarbon surfaces may be exploited clinically to enhance the retention of albumin preadsorbed to blood-contacting surfaces to render them thromboresistant.

Rat peritoneal macrophage adhesion to hydroxyethyl methacrylate-ethyl methacrylate copolymers and hydroxystyrene-styrene copolymers.

Macrophage adhesion to a wide variety of substrates has been measured, but no systematic study of the influence of specific substrate chemical properties on adhesion is available. These studies were conducted using two series of materials, copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) and copolymers of hydroxystyrene and styrene, to determine the effect of a single chemical property, polar character, on adhesion. Rat peritoneal macrophages were allowed to contact polymer substrates for periods ranging from 1 to 240 min before being subjected to a shear stress of 60-120 dynes/cm2 in a thin-channel flow cell. Percentage adhesion was calculated from the number of cells that remained adherent to the substrate after 30 s of applied shear stress. Macrophages remained adherent to 100% EMA and all hydroxystyrene-styrene copolymer surfaces after only 1 min of contact. In copolymers of the HEMA-EMA series, the time required to attain peak adhesion levels increased with increasing substrate hydrophilicity (increasing HEMA content). Cells did not attach to the 20% EMA/80% HEMA copolymer and the 100% HEMA polymer. The results demonstrate that there is a time delay between contact and adhesion of the cells to surfaces of increasing hydrophilicity within the HEMA-EMA series and no time delay with the hydroxystyrene-styrene series. The time delay is thought to be a function of the excluded volume provided by polymers that are able to undergo significant chain rotation and or swelling in the solvent, water. Small excluded volumes present in copolymers of high EMA content and all hydroxystyrene-styrene copolymers offer little or no resistance to formation of adhesive bonds by macrophages, whereas copolymers with large excluded volumes (high HEMA content) prevent contact and/or adhesion. A mechanism based on the net excluded volumes of both the cell and substrate surface macromolecule is proposed to explain this phenomenon.

The reactivity of alpha-chymotrypsin immobilized on radiation-grafted hydrogel surfaces.

The enzymatic activity of alpha-chymotrypsin (CT), immobilized on hydrogel-coated polymer film supports, has been investigated. The support was prepared by radiation-graft copolymerization of 2-hydroxyethyl methacrylate (HEMA) and methacrylic acid (MAAc) on silicone rubber films. The enzyme was covalently coupled to the carboxylic group of MAAc via the N-hydroxysuccinimide (NHS) ester active intermediate. Increasing MAAc contents of the hydrogel resulted in increased attachment of CT. The integrity of the CT active site after attachment was assessed by an active site titration with diisopropyl fluorophosphate (DFP). As the MAAc content of the hydrogel was increased, an increasing fraction of the attached CT retained its activity to DFP. A greater fraction of CT was active towards DFP when adsorbed than when coupled. The rates of hydrolysis of some synthetic model substrates by the immobilized CT were also measured. The negative charge on the hydrogel had a large effect on the rates of these hydrolyses. The pH optimum for the hydrolysis of N-acetyl-L-tyrosine ethyl ester (ATEE) by immobilized CT was higher than that of free CT. Increasing MAAc content of the hydrogel resulted in larger shifts in the pH optimum. The maximum rates of ATEE hydroylsis per mg CT declined sharply with increasing MAAc content of the hydrogel. This is probably related to the increasing repulsive force between the ATEE (negatively charged above congruent to pH 9.5) and the hydrogel with increasing MAAc content. The activity of immobilized CT to ATEE is small compared to that of free CT, partly due to this charge effect. Conversely, the rate of hydrolysis of BAEE, a positively charged substrate, by immobilized CT at pH 11, is almost fourfold greater than that by free CT at its pH optimum.