Thomas A. Horbett

Chapter 13 Principles underlying the role of adsorbed plasma proteins in blood interactions with foreign materials

Abstract The influence of adsorbed proteins on platelet interactions with biomaterials and the principles underlying protein adsorption to biomaterials from blood are reviewed. These principles are: 1) the monolayer model; 2) differences in the intrinsic affinity and bulk concentration of the proteins that are the driving forces determining the composition of the adsorbed layer from plasma; 3) the contribution of surface properties to selective adsorption; and 4) variations in the biological reactivity of the adsorbed proteins. Schematic representations of data from single, binary, and multiple protein mixtures are used to illustrate the principles involved. Physicochemical aspects of protein adsorption are briefly summarized. The author concludes that fundamental progress in designing new and better biomaterials will ensue from a deeper understanding of the role of adsorbed proteins in determining platelet interactions with biomaterials.

Human plasma fibrinogen adsorption and platelet adhesion to polystyrene

The purpose of this study was to further investigate the role of fibrinogen adsorbed from plasma in mediating platelet adhesion to polymeric biomaterials. Polystyrene was used as a model hydrophobic polymer; i.e., we expected that the role of fibrinogen in platelet adhesion to polystyrene would be representative of other hydrophobic polymers. Platelet adhesion was compared to both the amount and conformation of adsorbed fibrinogen. The strategy was to compare platelet adhesion to surfaces preadsorbed with normal, afibrinogenemic, and fibrinogen-replenished afibrinogenemic plasmas. Platelet adhesion was determined by the lactate dehydrogenase (LDH) method, which was found to be closely correlated with adhesion of 111In-labeled platelets. Fibrinogen adsorption from afibrinogenemic plasma to polystyrene (Immulon I(R)) was low and <10 ng/cm2. Platelet adhesion was absent on surfaces preadsorbed with afibrinogenemic plasma when the residual fibrinogen was low enough (<60 microg/mL). Platelet adhesion was restored on polystyrene preadsorbed with fibrinogen-replenished afibrinogenemic plasma. Addition of even small, subnormal concentrations of fibrinogen to afibrinogenemic plasma greatly increased platelet adhesion. In addition, surface-bound fibrinogens ability to mediate platelet adhesion was different, depending on the plasma concentration from which fibrinogen was adsorbed. These differences correlated with changes in the binding of a monoclonal antibody that binds to the Aalpha chain RGDS (572-575), suggesting alteration in the conformation or orientation of the adsorbed fibrinogen. Platelet adhesion to polystyrene preadsorbed with blood plasma thus appears to be a strongly bivariate function of adsorbed fibrinogen, responsive to both low amounts and altered states of the adsorbed molecule.

PEO-like plasma polymerized tetraglyme surface interactions with leukocytes and proteins: in vitro and in vivo studies

Polyethylene oxide (PEO) surfaces reduce non-specific protein and cell interactions with implanted biomaterials and may improve their biocompatibility. PEO-like polymerized tetraglyme surfaces were made by glow discharge plasma deposition onto fluorinated ethylene propylene copolymer (FEP) substrates and were shown to adsorb less than 10 ng/cm2 of fibrinogen in vitro. The ability of the polymerized tetraglyme surfaces to resist leukocyte adhesion was studied in vitro and in vivo. Polymerized tetraglyme and FEP were implanted subcutaneously in mice and removed after 1 day or 4 weeks. Histological analysis showed a similar degree of fibrous encapsulation around all of the 4-week implants. Darkly stained wells were present in the fibrous tissues at the tissue-material interface of both FEP and tetraglyme. Scanning electron micrographs showed that in vivo macrophage adhesion to polymerized tetraglyme was much higher than to FEP. After 2-hour contact with heparinized whole blood, polymorphonuclear leukocyte (PMN) adhesion to polymerized tetraglyme was much higher than to FEP, while platelet adhesion to polymerized tetraglyme was lower than to FEP. When PMNs isolated from blood were suspended in 10% autologous plasma, cell adhesion to polymerized tetraglyme was higher than to FEP; however when the cells were suspended in heat inactivated serum, cell adhesion to FEP was higher than to polymerized tetraglyme. The surface chemistry of polymerized tetraglyme did not change after 2-hour blood contact, but displayed nitrogen functional groups after 1-day implantation and became slightly degraded after 4-week implantation. The surface chemistry of FEP did not change significantly after blood contact or implantation. Loosely bound proteins such as fibrinogen on polymerized tetraglyme may contribute to the adhesion of PMNs and macrophages and ultimately to fibrous encapsulation (the foreign body response) around the implants.

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.

Changes in adsorbed fibrinogen and albumin interactions with polymers indicated by decreases in detergent elutability

Abstract The adsorption of proteins to polymers is typically irreversible. Even the use of detergents does not elute all the adsorbed protein from all polymers. The fundamental reasons for such apparently tight protein binding are not well understood, so a study of several aspects of elution was undertaken. A method for examining the interaction of proteins at the protein/polymer interface using SDS elutability as a measure of the protein-surface interaction strength was developed. The effects of polymer type, elution agent, elution conditions, protein type, protein concentration, sample age, and storage temperature on elutability were examined. The results show that protein elutability from polymers decreases slowly over a period of days at 4°C but proceeds much more rapidly at elevated temperatures. The results indicate that protein denaturation may be responsible for both the initial incomplete elution and the decreases in SDS elutability of fibrinogen and albumin from polymers with time.

The role of adsorbed proteins in animal cell adhesion

Abstract Animal cell adhesion to extracellular matrices is a fundamental aspect of their behavior that derives from the organization of cells into large ensembles in the formation of specialized tissues and organs. Most cells in animals are adherent throughout their lives, e.g. fibroblasts and myoblasts. Even those that are not normally adherent such as the platelets and white cells in blood must undergo an adhesion step to perform their ultimate biological function. Thus, platelets adhere to matrices and each other when activated in order to stem blood flow, and neutrophils and monocytes must adhere to endothelial cells prior to their passage out of the blood to sites of inflammation and infection. Adhesion of animal cells to extracellular matrices is mediated by membrane bound receptor proteins, the integrins, that bind specifically to specialized adhesion proteins such as fibronectin, vitronectin, and fibrinogen. This paper briefly reviews the integrin receptor-extracellular adhesion protein system as well as the three known cell-cell adhesion receptor systems. Examples illustrating how the roles of the integrins and the adhesion proteins in cell attachment have been denned are presented. The adsorption behavior of the adhesion proteins fibrinogen, fibronectin, and vitronectin onto synthetic substrates, and changes in their biologic activity induced by adsorption on different surfaces, are reviewed. The concept of “substrate activation” of the adhesion proteins is also described. Some of the technological innovations based on studies of cell adhesion are presented. Finally, how the properties of the adsorbing surface might be varied in order to modulate the influence of the adsorbed proteins in cell adhesion is discussed.

Glucose sensitive membranes for controlled delivery of insulin: Insulin transport studies☆

Abstract Glucose sensitive membranes which increase their permeability in the presence of glucose have been developed. The potential of these membranes to deliver insulin at rates controlled by the external glucose concentration is assessed in this study. In order to perform meaningful membrane transport measurements of 125 I-labelled insulin, it has been found necessary to thoroughly remove unbound 125 I. Substantial errors in the apparent permeation rate will be obtained if this unbound material is not completely removed. Membranes that have insulin permeation rates that are great enough to achieve estimated physiologic insulin requirements must have a macroporous as opposed to homogenous structure. Macroporous membranes containing amine groups and entrapped glucose oxidase have been found to alter insulin permeability in response to external glucose concentration.

The effect of adsorbed fibrinogen, fibronectin, von Willebrand factor and vitronectin on the procoagulant state of adherent platelets.

Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the effect of adsorbed adhesion proteins (fibrinogen (Fg), fibronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn < vWF = Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg < Fn < vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas deficient in or depleted of von Willebrand factor (de-vWF), fibronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and fibronectin (de-VnFn) resulted in varied platelet adhesion, but little difference in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.

Hydrophilic-hydrophobic copolymers as cell substrates: Effect on 3T3 cell growth rates☆

Cell-substrate interactions are important in many processes, including biocompatibility, marine biofouling, cell growth in culture, and cell separation. The influence of surface properties such as polarity, hydrophobic/hydrophilic or acid/base balance on cell interactions is poorly understood, however. In this study 2-hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) in various volume ratios were copolymerized and spun cast onto glass to produce a chemically homologous series of surfaces varying principally in hydrophilicity. Growth, spreading, and attachment of mouse 3T3 cells were measured with time-lapse photomicroscopy, visualization of the nucleolus, and [3H]thymidine labeling, respectively. Fibronectin adsorption from serum to the copolymers was also measured, using 125I-labeled fibronectin. On all polymers made with monomers containing 50% or more EMA, cell numbers increased logarithmically with time at the same rate observed for glass (21-hr doubling time). Cells on 40% EMA polymers remained virtually constant in number while decreases in cell number were observed on all polymers made with 30% or less EMA. Attachment measured after either 2 or 20 hr was not well correlated with growth behavior since significant attachment occurred on polymers on which growth had not occurred. Spreading at 2 hr but especially after 24 hr was very similar to the variation in growth. The kinetics of attachment and spreading varied markedly among the copolymers. Fibronectin adsorption varied from 0.1 to 1.5 ng/cm2 on the copolymers and was similar to the variation in 2-hr spreading. Fibronectin adsorption from the serum and fibronectin exudation by the cells both appear to be important in determining 3T3 cell interactions with the HEMA-EMA copolymers. However, the relative influence of these two processes on cell behavior probably varies greatly among the copolymers. The interfacial properties of the copolymers which could account for a variation in the ability to support cell growth are discussed.

Theoretical and experimental studies of glucose sensitive membranes

A mathematical model has been developed to describe the steady-state behavior of two types of glucose sensitive membranes. Both membranes are synthetic hydrogels containing immobilized glucose oxidase enzyme (GluOx). The formation of gluconic acid from glucose and oxygen, catalyzed by GluOx, is the key to the functioning of either type of membrane since it causes a pH decrease within the membrane. In amine group-containing membranes, the pH decrease enhances the swelling (and permeability) of the gels, allowing control of insulin delivery in response to glucose concentrations. In membranes containing immobilized pH indicator dyes the pH decrease causes a color change, thus providing the basis for a glucose sensor. The models predictions of the response of amine-containing membranes to glucose show that the pH decrease is often limited by O2 depletion and that the occurrence of O2 depletion is strongly influenced by enzyme loading and membrane thickness. At a given thickness, an optimal enzyme loading exists which results in the maximum response to glucose over a given range of glucose concentrations. The influence of buffer and amine concentrations, amine pK, solute diffusivities, and flowrate past the membrane also have been examined. For polyacrylamide membranes containing phenol red (but no amines), substrate turnover rates, oxygen depletion, and the pH decrease within the membrane have been calculated using the model and found to agree qualitatively with experimentally determined values of these parameters. Quantitative agreement is lacking, however. In particular, the model predicts that as GluOx loading is increased (all other parameters, including glucose concentration, held constant) the pH decrease should asymptotically approach a maximum value. However, it is found experimentally that as GluOx loading is increased, the measured pH decrease increases progressively over the full range of GluOx concentrations that we have studied. Possible reasons for the quantitative disagreements between simulated and observed results will be discussed. Implications of the model with respect to optimizing membrane designs will be presented. One finding of particular interest is that a glucose sensor using immobilized GluOx will achieve a maximum response at sub-physiological concentrations of glucose, and not respond to higher glucose concentrations, unless the enzyme loading is made sufficiently low.