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Biomedical subjects

T A Horbett

Publications and source records attributed to T A Horbett.

At least 55 records · Page 3Linked to original sources

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.

Acetone↗

Measurement of fibrinogen adsorption from blood plasma using 125I-fibrinogen and a direct ELISA technique.

A direct enzyme-linked immunosorbent assay (ELISA), using a polyclonal anti-fibrinogen conjugated to horseradish peroxidase, was used to detect fibrinogen adsorption from blood plasma to ten different materials. Adsorption was also measured with [125I]-fibrinogen. The materials studied included glass, Biomer, Immulon I, and a series of hydroxyethylmethacrylate (HEMA) and ethylmethacrylate (EMA) co-polymers. For all the materials studied, the results from the ELISA technique closely paralleled those obtained using [125I]-fibrinogen. The cross-reactivity of the antibody with proteins other than fibrinogen was generally small. Both experimental methods detected the presence of a maximum in fibrinogen adsorption (as a function of the plasma dilution) to the more hydrophobic materials. For all but two HEMA/EMA co-polymers, a linear correlation between the ELISA and [125I]-fibronogen measurements was indicated by inspection of cross plots as well as by a statistical test.

Adsorption↗

Postadsorptive transitions in fibrinogen: influence of polymer properties.

Changes in the fibrinogen molecule after its adsorption to biomaterials may be important in determining blood compatibility. Previously, postadsorptive transitions were detected by measuring the elutability of adsorbed proteins with surfactant solutions. The elutability decreased with increased residence time, suggesting that protein-surface interactions increased with residence time. In this study, we have examined the effects of polymer structure and composition, chain mobility, and hydrophobicity on the postadsorptive transitions of fibrinogen. Glassy, rigid polymers showed high fibrinogen adsorption, regardless of whether the polymer was hydrophilic or hydrophobic. However, the binding strength (as measured by elutability) was much lower on hydrophilic polymers and oxygen-containing hydrophobic polymers. Short-term transitions, requiring 2 h or less after adsorption, were observed only on hydrophobic polymers that contained no oxygen. More gradual transitions were observed on hydrophobic polymers containing oxygen, but only after a lag time of 1-4 h.

Adsorption↗

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.

Adsorption↗

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.

Adsorption↗

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.

Actins↗

Changes in the SDS elutability of fibrinogen adsorbed from plasma to polymers.

Baboon fibrinogen was adsorbed from diluted plasma solutions to glass, polyethylene, polystyrene, polydimethylsiloxane, poly(ethylene)terephthalate, and Biomer. Following adsorption, half of the samples were immediately placed in sodium dodecyl sulfate (SDS) solutions to elute the protein, while the others were stored in buffer for up to 5 days and then eluted in SDS. The elution was typically incomplete, but depended on the plasma concentration and the residence time. The elutability was generally lower for fibrinogen adsorbed from more diluted plasma, and substantially lower for samples on which the fibrinogen had resided for 5 days. The non-elutable portion of the protein layer formed rapidly on polystyrene, while on polyethylene elutability was high initially, followed by a gradual decrease.

Adsorption↗

Discrimination between platelet-mediated and coagulation-mediated mechanisms in a model of complex thrombus formation in vivo.

To study mechanisms of complex thrombus formation in vivo, and to compare the relative antithrombotic effects of anticoagulants and antiplatelet agents, a model was developed in baboons. Segments of collagen-coated tubing followed by two sequentially placed expansion chambers exhibiting disturbed flow patterns were exposed to native blood under laminar flow conditions. The device was incorporated for 1 hour into an exteriorized arteriovenous shunt in baboons under controlled blood flow (20 ml/min). Morphologic evaluation by scanning electron microscopy showed that thrombi associated with collagen were relatively rich in platelets but thrombi in the chambers were rich in fibrin and red cells. Deposition of indium 111-labeled platelets was continuously measured with a scintillation camera. Platelet deposition increased in a linear (collagen-coated segment) or exponential (chambers 1 and 2) fashion over time, with values after 40 minutes averaging 24.1 +/- 3.3 x 10(8) platelets (collagen segment), 16.7 +/- 3.4 x 10(8) platelets (chamber 1), and 8.4 +/- 2.4 x 10(8) platelets (chamber 2). Total fibrinogen deposition after 40 minutes was determined by using iodine 125-labeled baboon fibrinogen and averaged 0.58 +/- 0.14 mg in the collagen segment, 1.51 +/- 0.27 mg in chamber 1, and 0.95 +/- 0.25 mg in chamber 2. Plasma levels of beta-thromboglobulin (beta TG), platelet-factor 4 (PF4), and fibrinopeptide A (FPA) increased fourfold to fivefold after 60 minutes of blood exposure to the thrombotic device. Platelet deposition onto the collagen segment, chamber 1, and chamber 2 was linearly dependent on the circulating platelet count. Platelet accumulation in chamber 1 and chamber 2 was also dependent on the presence of the proximal collagen segment. An anticoagulating dose of standard heparin decreased platelet deposition in the chambers (p less than 0.05) but did not decrease deposition onto the collagen segment. Although beta TG and PF4 levels remained elevated after the administration of standard heparin, the elevation in plasma FPA was interrupted. Further evidence that the thrombotic process was dependent on platelets was provided by the finding that prostaglandin I2 at high concentration (35 ng/ml) decreased platelet deposition onto the collagen segment and in chambers 1 and 2, decreased beta TG and PF4 release, and reduced FPA formation. The combination of standard heparin and PGI2 produced the most potent inhibition of platelet thrombus formation and prevented the increases in plasma PF4, beta TG and FPA.

Animals↗

Cell adhesion to a series of hydrophilic-hydrophobic copolymers studied with a spinning disc apparatus.

Adhesion of cells to substrates strongly influences many of their functions and therefore plays an important role in a variety of processes, including growth, phagocytosis, hemostasis, and the response of tissue to implanted materials. In previous studies, the influence of substrate hydrophilicity on cell adhesion has not been separated from effects due to major differences in other properties of the substrate, such as charge, rigidity, and the specific chemical composition of the materials. In addition, very few careful studies of the force required for cell detachment from various substrates have been performed. In this study, 3T3 cell detachment from a chemically homologous series of copolymers based on hydroxyethylmethacrylate (HEMA) and ethylmethacrylate (EMA) was measured with a spinning-disc apparatus. The spinning-disc technique allowed measurements of cell detachment over a wide range of applied shear stress on each sample. Cell detachment did not occur until a critical value of shear stress was exceeded. The critical shear stress of detachment decreased linearly with increasing HEMA content, from 18 dynes/cm2 on poly-EMA to 0 on the polymers containing 83% or more HEMA. "Plating efficiency," calculated as the fraction of cells initially applied which remained after dip rinsing the surfaces, did not vary significantly among most of the copolymers. Dip rinsing, however, exposes the cells to only one, relatively low shear stress (estimated to be somewhat less than 3 dynes/cm2). The existence of a critical shear stress for 3T3 cell detachment suggests that cell adhesion to surfaces cannot be fully understood with single shear stress methods because cells may attach with a wide range of strengths which are either all above or all below the applied shear stress. The influence of surface hydrophilicity on cell adhesion and the variety of forces which may contribute to this phenomenon are discussed.

Animals↗

Analysis of in vitro enzymatic and oxidative degradation of polyurethanes.

In vitro biodegradation studies were performed to assess the long-term stability of poly(ether urethane) (PEU) implants. Three PEU's and one poly(ester urethane) were treated with enzymes characteristic of those released from inflammatory cells during the foreign body reaction. In addition, the effect of hydrogen peroxide was observed to examine oxidative degradation. Polymers were prepared as thin films on glass, gold, silver, and copper substrates to test the possibility of metal-catalyzed degradation. Molecular weights and polydispersities of the polymers were measured by gel permeation chromatography (GPC) before and after treatment. Changes in peak shape and location were also monitored. The results demonstrate that varying degrees of both enzymatic and oxidative degradation occurred.

Chymotrypsin↗

Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers.

The interaction of cells with solid surfaces is important in many settings, including the response of tissue to implanted materials. Protein adsorption to the surface plays a critical role in controlling cell interactions with surfaces. However, few comprehensive studies of both cell behavior and protein adsorption in complex protein mixtures (e.g., serum) have been done so the connection between these events is not well understood. In particular, methods to systematically perturb both protein adsorption and cell behavior in order to understand their relationship have been lacking. To induce changes in cell and protein behavior, the effects of serum dilution and substrate surface chemistry were studied. Surface chemistry was varied by using a series of polymers and copolymers of hydroxyethyl methacrylate (HEMA) and ethylmethacrylate (EMA) varying in their hydrophobic/hydrophilic balance. Large changes in cell spreading and fibronectin adsorption were observed when either serum concentration or polymer type was varied. The spreading of 3T3 cells in serum was found to be well correlated with the amount of fibronectin adsorption to the substrates. Attachment was not correlated with fibronectin adsorption, especially on glass preadsorbed with diluted serum. For 3T3 cells and perhaps other cells that have a receptor for a protein which is present in the medium, the amount of adsorption of this protein to the substrate appears to be a critical factor controlling cell interactions with the substrate.

Adsorption↗

Adsorption of proteins from artificial tear solutions to contact lens materials.

A series of polymers and copolymers of 2-hydroxyethyl methacrylate (HEMA) and methyl methacrylate (MMA) were synthesized in order to find surfaces that would adsorb minimal amounts of protein. The adsorption of albumin, lysozyme and immunoglobulin G from a three-way mixture of these proteins in isotonic buffered saline to the polymers was measured using 125I-labeled proteins. Apparently high protein uptake on copolymers rich in HEMA was found to be due to sorption of unbound 125I by the polymers. 125I sorption by the polymers was minimized by dialysis of the protein solution to remove unbound 125I iodide and inclusion of 0.01 M sodium iodide to block uptake of residual 125I iodide. Using these improved protocols, minimal total protein uptake was observed on copolymers containing 50% or more HEMA. The majority of adsorbed protein on all p(MMA-HEMA) polymers was albumin. Total protein uptake was greatest on pMMA. Commercial contact lenses composed of copolymers of HEMA and N-vinyl pyrrolidone (NVP) or acrylamide (AAm) adsorbed small amounts of all proteins whereas copolymers of methacrylic acid (MAAc) and HEMA adsorbed much larger quantities of lysozyme. These results indicate that protein uptake by contact lens materials varies greatly with polymer composition. Artifactually high "adsorption" can occur if precautions are not taken to prevent uptake of unbound 125I.

Acrylates↗

Complement activation by hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Certain biomaterials, including nylon oxygenator and cellulosic dialysis membranes, are potent activators of human complement. In this study, the effect of polymers containing 2-hydroxyethyl methacrylate (HEMA) on the human complement system was investigated. Copolymers of HEMA with ethylmethacrylate (EMA) varying from 100 to 40% HEMA in the monomer made by radiation initiation were used to coat glass discs. These were equilibrated with human plasma in vitro and the degree of complement activation was quantitated by C3a radio immunoassay. Significant activation was caused by copolymers made from monomers containing 60% or greater HEMA. A direct relationship between the amount of activation and the percentage of HEMA was found. The degree of activation by poly HEMA, when corrected for surface area, was quite similar to that observed for dialysis and oxygenator membranes. Similar observations were made when solid casts of crosslinked HEMA/N-vinylpyrrolidone (NVP) copolymer gels were tested, but the magnitude of activation was much greater. The results are significant because complement activation may play an important role in the response to foreign surfaces, in both extravascular and intravascular settings. A new concept of molecular biocompatibility is proposed in which surfaces eliciting molecular transformations in any of the biological defense systems are deemed nonbiocompatible. By this criterion, the hydrogel poly-HEMA, which has so frequently been thought of as biocompatible, is considered to be a molecularly non-biocompatible material.

Biocompatible Materials↗

The effects of surface chemistry and coagulation factors on fibrinogen adsorption from plasma.

Fibrinogen adsorption from plasma exhibits an unusual displacement phenomenon (the Vroman effect) in that decreases in adsorption occur after longer contact time or as the plasma concentration increases. Fibrinogen adsorption and the displacement effect were found to depend markedly on the nature of the surface, the time and temperature of adsorption, and to a lesser extent on certain contact activation factors. Displacement still occurred from plasmas lacking kininogens, factor IX, and prekallikrein, and from plasma that had been treated with BaSO4 to remove factors II, VII, IX, and X. Furthermore, displacement was also observed from fibrinogen solutions to which either albumin or hemoglobin had been added. In addition, competitive adsorption of binary protein mixtures was also shown to depend strongly on surface type. It therefore appears that fibrinogen adsorption from plasma is subject to similar if not identical competitive processes that occur in simpler protein mixtures. The final adsorption then reflects the influence of all the proteins in plasma, each competing for the limited number of adsorption sites according to the fundamental physical properties of surface activity and mass concentration.

Adsorption↗

von Willebrand Factor/Factor VIII adsorption to surfaces from human plasma.

The adsorption of human von Willebrand factor/factor VIII (VWF/VIII) from citrated plasma to a series of polymeric surfaces and glass was measured with both prelabelled 125I VWF/VIII and an 125I antibody to VWF/VIII. Adsorption to the various surfaces differed but was generally quite low (0.24 to 6.5 ng/cm2). Results from both techniques agreed well. The relationship between VWF/VIII adsorption and the thrombogenicity of the surfaces was examined but no clear trend could be discerned. Further studies will therefore be required to determine whether the variation in VWF/VIII adsorption influences the thrombogenicity of the surfaces.

Adsorption↗

Mass action effects on competitive adsorption of fibrinogen from hemoglobin solutions and from plasma.

The strong effect of protein adsorption on blood and tissue compatibility is well known. Little is presently known about the mechanisms which control the composition of the adsorbed protein layer which forms upon exposure of surfaces to mixtures of proteins. Reexamination of the ability of hemoglobin to inhibit the adsorption of 125I fibrinogen to polyethylene revealed that the inhibition was strongly dependent on the fibrinogen concentration. These results suggested that fibrinogen adsorption from more complex mixtures such as plasma should also be strongly dependent on total concentration. Fibrinogen adsorption from plasma was found to be maximal at intermediate plasma concentrations, and was reduced at both low and high plasma concentrations. The plasma concentration at which this maximum occurred was 10% for polytetrafluoroethylene, 1% for polyethylene, and 0.1% for glass. The unusual concentration dependence is attributed to mass action effects on the competitive adsorption of proteins, specifically that competitive effectiveness is expected to increase as unoccupied surface adsorptive sites become less frequent. Analogous effects of adsorption time on competitive adsorption are also predicted due to the changing concentration at the interface as the buffer is gradually replaced by protein solution. These mass action effects are very similar to previous qualitative observations by Vroman and are therefore dubbed the " Vroman effect".

Adsorption↗