Alkylsilane-modified surfaces: inhibition of human macrophage adhesion and foreign body giant cell formation.

A homologous set of alkylsilane-modified glass surfaces with chain lengths ranging from methyl to octadecyl was prepared in order to examine the influence of alkyl surface chemistry on macrophage adhesion and foreign body giant cell (FBGC) formation. Contact angle and X-ray photoelectron spectroscopy analysis confirmed our silanation technique and indicated a consistent alkyl chain density independent of chain length. Human peripheral blood monocytes were isolated and cultured on these alkylsilane surfaces for a period of 10 days. The initial density of human monocytes was similar on all surfaces. Beyond day 0 the clean glass, methyl (DM and C1), propyl (C3), and hexyl (C6) surfaces maintained a high cell density and supported macrophage development. In contrast, long-term macrophage density was extremely low on the tetradecyl (C14) and octadecyl (C18) surfaces. When interleukin-4 was added to induce FBGC formation in vitro, the DM, C1, C3, and C6 surfaces supported high levels of macrophage fusion while clean glass strongly inhibited fusion. The C14 and C18 surfaces did not contain sufficient macrophages to support FBGC formation. Cage implant studies revealed that in vivo macrophage density and FBGC formation on clean glass and C6 surfaces was similar to in vitro data. In contrast to the monocyte culture results, the C18 cage implant samples supported significant FBGC formation, possibly as a result of different conditions within each experimental system. Radiotracer adsorption studies of eight human serum proteins identified the high concentration and tenacious hold of adsorbed von Willebrand factor as being possibly involved in the poor long-term macrophage density observed on C14 and C18.     

Effects of adsorbed proteins and surface chemistry on foreign body giant cell formation, tumor necrosis factor alpha release and procoagulant activity of monocytes

The adhesion and activation of monocytes and macrophages are thought to affect the foreign body response to implanted medical devices. However, these cells interact with devices indirectly, because of the prior adsorption of proteins. Therefore, we preadsorbed several "model" biomaterial surfaces with proteins and then measured foreign body giant cell (FBGC) formation, tumor necrosis factor alpha (TNFalpha) release, and procoagulant activity. The model surfaces were tissue culture polystyrene (TCPS), untreated polystyrene (PS), and Primaria, whereas the proteins used were albumin, fibronectin, fibrinogen, and immunoglobulin. FBGC formation, TNFalpha release, and procoagulant activity of monocytes were the highest for surfaces preadsorbed with IgG. FBGC formation was lower on surfaces with adsorbed fibrinogen and fibronectin than on uncoated surfaces. TNFalpha release and procoagulant activity of monocytes were similar on surface adsorbed with fibrinogen, fibronectin, or albumin. Monocyte activation was also affected by the surface chemistry of the substrates, because FBGC formation was the highest on PS and the lowest on TCPS. Monocyte procoagulant activity was the highest on Primaria. Adsorbed proteins and surface chemistry were found to have strong effects on FBGC formation, monocyte TNFalpha release, and procoagulant activity in vitro, providing support for the idea that these same variables could affect macrophage-mediated foreign body response to biomaterials in vivo.     

Co-adsorbed fibrinogen and von Willebrand factor augment platelet procoagulant activity and spreading

Previously we observed that platelets adherent to surfaces preadsorbed with blood plasma exhibited 1.3 to 2.4 times greater procoagulant activity than platelets on surfaces adsorbed with fibrinogen (Fg) only. These observations suggested that the adhesion proteins adsorbed from plasma may activate platelets in a cooperative, or synergistic manner. In the present study, polystyrene surfaces adsorbed with both Fg and vWF induced up to three times greater procoagulant activity than surfaces adsorbed with Fg or vWF only. The amounts of Fg and vWF adsorbed from binary mixtures that resulted in increased procoagulant activity were found to be similar to the amounts that adsorbed to PS from 100% plasma. The effect of adsorbed adhesion proteins on platelet spreading was also investigated. The proportion of fully spread platelets increased, depending on the adhesion protein preadsorbed to the surface, in the following order: vWF < Fg < Fn < (vWF + Fg) < Vn < plasma.     

Platelet adhesion to polystyrene-based surfaces preadsorbed with plasmas selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor

Four plasma proteins have been shown to be able to mediate platelet adhesion to synthetic materials when they are adsorbed as purified proteins: fibrinogen (Fg), fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWF). Among them, Fg is thought to play a leading role in mediating platelet adhesion to plasma-preadsorbed biomaterials, but this has been established for only three types of materials so far in our laboratory. Furthermore, the role of Fn, Vn, and vWF in mediating platelet adhesion to plasma-preadsorbed surfaces is still unclear. The aim of the current study was to assess the importance of Fg, Fn, Vn, and vWF in mediating platelet adhesion to a series of polystyrene-based surfaces. The strategy applied in the present investigation was to compare platelet adhesion to surfaces preadsorbed with normal plasma, plasma selectively depleted in Fn or Vn or both Fn and Vn, plasma from donors who were genetically deficient in vWF, and serum. Few platelets adhered to the surfaces preadsorbed with serum, whereas depletion of Fn, Vn, or vWF from plasma did not decrease platelet adhesion significantly. Replenishment of exogenous Fg to serum before protein adsorption restored platelet adhesion to the surfaces, suggesting that Fg was the major plasma protein that mediated platelet adhesion. Also, we found that a surface density of adsorbed Fg far below the amount that usually adsorbs to synthetic surfaces was sufficient to support full-scale platelet adhesion.     

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.     

Protein and surface effects on monocyte and macrophage adhesion, maturation, and survival

Cell adhesion and maturation can be affected by the protein adsorption profile on the surface of an implanted biomaterial. In this study we have investigated how surface chemistry and adsorbed proteins can modulate monocyte and macrophage adhesion, IL-13-induced foreign-body giant cell formation, and apoptosis in vitro. Compared to a dimethylsilane-modified surface (DM), a surface modified with RGD peptides had no effect on adhesion density, foreign-body giant cell (FBGC) formation, or apoptosis in nondepleted serum conditions. The depletion of specific adhesive proteins affected adhesion, FBGC formation, and apo- ptosis. While the depletion of fibronectin and vitronectin had no overall effect compared to nondepleted serum conditions, the depletion of IgG from serum caused a significant decrease in initial adherent cell density [1000 +/- 200 compared to 2460 +/- 590 (p = 0.02)], a significant decrease in FBGC formation [2% compared to 17% (p = 0.02)], and a significant increase in the level of apoptosis [57% compared to 32% (p = 0.01)] on DM. The lowered initial adherent cell density on DM was not observed on the RGD surface, indicating that the RGD surface promotes increased initial adhesion. However, the RGD surface does not affect FBGC formation (i.e., macrophage fusion) or levels of apoptosis, which remained comparable to those on the DM surfaces at days 7 and 10.     

Vitronectin is a critical protein adhesion substrate for IL-4-induced foreign body giant cell formation

An in vitro system of interleukin (IL)-4-induced foreign body giant cell (FBGC) formation was utilized to define the adhesion protein substrate(s) that promotes this aspect of the foreign body reaction on biomedical polymers. Human monocytes were cultured on cell culture polystyrene surfaces that had been pre-adsorbed with a synthetic arginine-glycine-aspartate peptide previously found to support optimal FBGC formation, or with various concentrations of potential physiological protein substrates, i.e. complement C3bi, collagen types I or IV, fibrinogen, plasma fibronectin, fibroblast fibronectin, laminin, thrombospondin, vitronectin, or von Willebrand factor. Cultures were evaluated on days 0 (1.5 h), 3, and 7 by May-Grünwald/Giemsa staining. Initial monocyte adhesion occurred on all adsorbed proteins. However, by day 7 of culture, only vitronectin was striking in its ability to support significant macrophage adhesion, development, and fusion leading to FBGC formation. Vitronectin supported high degrees of FBGC formation at an absorption concentration between 5 and 25 microg/mL. These findings suggest that adsorbed vitronectin is critical in the collective events that support and promote FBGC formation on biomedical polymers, and that the propensity for vitronectin adsorption may underlie the material surface chemistry dependency of FBGC formation.     

Competitive adsorption of vitronectin with albumin, fibrinogen, and fibronectin on polymeric biomaterials.

Vitronectin (VN) was competitively adsorbed with human serum albumin (HSA), fibrinogen (FGN), and fibronectin (FN) from binary component mixtures in order to compare the relative affinities of these proteins for various polymer materials. Competitive adsorption was monitored by incubating radiolabeled protein solutions inside 0.125-in. i.d. tubing of the polymers, flushing with buffer, and measuring the adherent radioactivity. Adsorption experiments at equal mass concentrations of the competing proteins revealed that VN comprises at least 75% by weight of the adsorbed protein when competitively adsorbed with HSA and approximately 50% by weight when competitively adsorbed with FGN and FN on all surfaces except a poly(ethylene oxide)-based polyurethane where it comprised closer to 80 wt%. When VN was competitively adsorbed in the presence of increasing amounts of HSA, FGN, and FN, the amount of VN adsorbed on a weight basis was diminished the most by FGN. HSA had the least inhibitory effect at low bulk concentrations and FN had the weakest effect at higher bulk concentration levels. When HSA, FGN, and FN were competitively adsorbed in the presence of increasing amounts of VN, VN diminished their adsorption on a weight basis in the order: HSA greater than FN greater than FGN.     

Effects of surface-coupled polyethylene oxide on human macrophage adhesion and foreign body giant cell formation in vitro.

Surface immobilized polyethylene oxide (PEO) has been shown to efficiently reduce protein adsorption and cellular adhesion, resulting in a biologically passive surface. To explore the in vitro effects of surface immobilized PEO on the human inflammatory cells, macrophages, and foreign body giant cells (FBGCs), we developed a diisocyanate-based method for coupling PEO to amine-modified glass, a surface previously shown to enhance macrophage adhesion and FBGC formation. Contact angle analysis and X-ray photoelectron spectroscopy confirmed the presence of PEO molecules bound to the surface and revealed that PEO molecular weight significantly influenced the efficiency of PEO coupling. We used a 10-day human monocyte culture protocol to demonstrate that the presence of surface coupled PEO molecules does not significantly decrease initial monocyte density or monocyte-derived macrophage density after 3 days. However, PEO-coupled surfaces significantly reduced long-term monocyte-derived macrophage density and virtually eliminated interleukin-4-induced FBGC formation observed at day 10. The cellular response to these PEO-coupled surfaces was related to the molecular weight of the PEO chains, which was varied between 200 Da and 18.5 kDa. These results suggest that an optimized PEO surface treatment may be effective in reducing inflammatory cell adhesion and possible degradation during the inflammatory response to an implanted biomedical device.     

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.     

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.     

A comparison of the adsorption of three adhesive proteins to biomaterial surfaces.

The adsorption of three cell adhesive proteins with known thrombogenic activity [fibrinogen (FGN), fibronectin (FN), and vitronectin (VN)] was quantified from mono-component protein solutions, from a quaternary-component protein solution, and from plasma and diluted plasma in order to compare their potential for adsorption to polymeric substrates from solutions of varying complexity. The surfaces studied included polyethylene (PE), silicone rubber (SR), Teflon-FEP (FEP), and two polyetherurethanes: one with a poly(tetramethylene oxide) soft segment (PTMO-PU) and one with a poly(ethylene oxide) soft segment (PEO-PU). The adsorption of these proteins from single-component solutions followed the Freundlich isotherm and the adhesive proteins showed similar trends in Freundlich parameters for surfaces of similar surface wettability. Adsorption from a quaternary-component solution composed of physiological molar ratios of the three proteins and human serum albumin (HSA) revealed a significant enrichment of adsorbed vitronectin as determined from ratios of the adsorbed surface fraction of each protein to its respective bulk fraction. The other proteins' adsorption was enriched to a lesser extent in the decreasing order of FGN greater than FN greater than HSA for all surfaces. The relative enrichment of VN from plasma was also high as compared with its bulk concentration, whereas the enrichment of FGN, FN, and HSA was much lower and of approximately the same magnitude. Compared with the three other proteins, VN showed a resistance to displacement from the polymer substrates as either the plasma concentration was increased or the length of contact with plasma and diluted plasma was increased.     

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.     

Intracellular signaling involved in macrophage adhesion and FBGC formation as mediated by ligand-substrate interaction

Fibronectin and RGD- and/or PHSRN-containing oligopeptides were preadsorbed onto physicochemically distinct substrata: polyethyleneglycol-based networks or tissue culture polystyrene (TCPS). The role of selected signaling kinases (namely protein tyrosine kinases, protein serine/threonine kinases, PI3-kinase, Src, and MAPK) in the adhesion of human primary blood-derived macrophages and the formation of foreign-body giant cells (FBGC) on these modified substrata was investigated. The involvement of individual intracellular signaling molecules in mediating macrophage adhesion dynamically varied with the culture time, substrate, and ligand. For example, fibronectin on TCPS or networks involved similar signaling events for macrophage adhesion; however, fibronectin and G(3)RGDG(6)PHSRNG, but not peptides with other RGD and/or PHSRN orientations, mediated similar signaling events for macrophage adhesion on TCPS but mediated different signaling events on networks. Depending on the substrate, a specific molecule (i.e., Src, protein kinase C) within the protein tyrosine kinase or protein serine/threonine kinase family was either an antagonist or agonist in mediating FBGC formation.     

Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.     

The correlation between the adsorption of adhesive proteins and cell behaviour on hydroxyl-methyl mixed self-assembled monolayers

The objective of this study was to compare the biological effects of two key cell-adhesive proteins, fibronectin (FN) and vitronectin (VN), upon adsorption onto molecularly-designed model surfaces. Single-component and mixed self-assembled monolayers (SAMs) of alkanethiols on gold with OH and CH(3) terminal groups were prepared at 100%, 65%, 36% and 0% of OH at the surface, to generate a range of surfaces with a simple chemistry and a wettability gradient. FN and VN were adsorbed under non-competitive (single-protein solutions) and competitive (multi-protein solutions) conditions, and compared at different levels: adsorbed amount (radiolabelling), elution, functional presentation of cell-binding domains (ELISA), and role in mediating cell adhesion (antibody-based assay). The observed trends were related to mesenchymal stem cell response in terms of adhesion and overall cell morphology. Under non-competitive conditions, adsorption of both proteins increased with surface hydrophobicity. The presence of competitive proteins significantly decreased the adsorbed amounts, although both proteins were still detected in all SAMs. Adsorption of FN followed a trend similar to that of non-competitive conditions, while adsorption of VN was higher on 100%OH-SAMs. Concerning elution, retention of adsorbed VN was always higher than that of FN. For both proteins, functional presentation of cell-binding domains was more effective on the more hydrophilic 100%OH-SAMs. This fact, coupled to the ability of this type of SAMs to selectively recruit and retain VN in the presence of competitive serum proteins, seems to correlate with the better cell response observed on these surfaces, as compared with hydrophobic 0%OH(100%CH(3))-SAMs.     

Volumetric interpretation of protein adsorption: ion-exchange adsorbent capacity, protein pI, and interaction energetics

Adsorption of lysozyme (Lys), human serum albumin (HSA), and immunoglobulin G (IgG) to anion- and cation-exchange resins is dominated by electrostatic interactions between protein and adsorbent. The solution-depletion method of measuring adsorption shows, however, that these proteins do not irreversibly adsorb to ion-exchange surfaces, even when the charge disparity between adsorbent and protein inferred from protein pI is large. Net-positively-charged Lys (pI=11) and net-negatively-charged HSA (pI=5.5) adsorb so strongly to sulfopropyl sepharose (SP; a negatively-charged, strong cation-exchange resin, -0.22 mmol/mL exchange capacity) that both resist displacement by net-neutral IgG (pI=7.0) in simultaneous adsorption competition experiments. By contrast, IgG readily displaces both Lys and HSA adsorbed either to quaternary ammonium sepharose (Q; a positively-charged, strong anion exchanger, +0.22 mmol/mL exchange capacity) or to octadecyl sepharose (ODS; a neutral hydrophobic resin, 0 mmol/mL exchange capacity). Thus it is concluded that adsorption results do not sensibly correlate with protein pI and that pI is actually a rather poor predictor of affinity for ion-exchange surfaces. Adsorption of Lys, HSA, and IgG to ion-exchange resins from stagnant solution leads to adsorbed multi-layers, into or onto which IgG adsorbs in adsorption competition experiments. Comparison of adsorption to ion-exchange resins and neutral ODS leads to the conclusion that the apparent standard free-energy of adsorption Delta Gads( degrees ) of Lys, HSA, and IgG is not large in comparison to thermal energy due to energy-compensating interactions between water, protein, and ion-exchange surfaces that leaves a small net Delta Gads( degrees ). Thus water is found to control protein adsorption to a full range of substratum types spanning hydrophobic (poorly water wettable) surfaces, hydrophilic surfaces bearing relatively-weak Lewis acid/base functionalities that wet with (hydrogen bond to) water but do not exhibit ion-exchange properties, and surfaces with strong Lewis acid/base functional groups that exhibit ion-exchange properties in the conventional chemistry sense of ion-exchange.     

Adhesion of human peripheral lymphocytes on biomaterials preadsorbed with fibronectin and vitronectin

The adhesion of human peripheral lymphocytes (HPL) was studied after preadsorption of fibronectin (FN) and vitronectin (VN) on hydrophilic glass and hydrophobic octadecyl glass. The adhesion of HPL was shown to be dependent not only on the wettability but also on the protein preadsorbed. Vitronectin expressed not only a higher extent of adhesion under static conditions but also a stronger interaction with HPL, indicated by the low detachment under shear stress. The flow experiments also demonstrated that FN adsorbed on octadecyl glass may undergo conformational changes because HPL could be easily removed. Scanning electron microscopy revealed that HPL on both FN-and VN-coated glass spread well whereas particularly on FN-coated octadecyl glass less cell spreading was observed; moreover, some round cells were detected. The typing of adherent HPL by immunofluorescence microscopy showed that on FN- and VN-coated glass about 70% of all HPL were T-cells (CD 3+). However, on octadecyl glass, particularly on VN, a smaller percentage of CD 3+cell was observed. The testing for the β₁ integrin-the receptor for FN and the α_v integrin-the receptor for VN demonstrated that about 70% of all cells on FN-coated glass were positive for the β₁ integrin. On VN-coated glass, however, only 5% of HPL were positive for the β₁ integrin. Although on VN a high adhesion and strong binding of HPL was observed, no presence of the α_vintegrin was detected.     

Adsorbed IgG: a potent adhesive substrate for human macrophages

Previous reports from our laboratory have demonstrated qualitatively that preabsorbed IgG can enhance long-term macrophage adhesion in vitro. This investigation further characterizes and quantifies the biological effect of adsorbed human IgG on human macrophages and probes the potential mechanisms. Ten-day human monocyte/macrophage cultures on Plastek M (PM), a normally poor cellular substrate for macrophages, confirmed the ability of preabsorbed IgG to dramatically enhance long-term macrophage adhesion. An adsorption solution concentration of 200 microg/mL of IgG was necessary to provide a consistent, optimal cellular response. (125)I adsorption studies indicated Langmuir-style IgG adsorption at low concentrations; however, no adsorption maximum was observed. Additional adsorption analysis revealed that the IgG fragments Fab, F(ab')(2), and Fc adsorb at levels only 20-40% that of the whole molecule. Despite the lower adsorption levels, both preabsorbed Fab and F(ab')(2) were shown to be as effective as whole molecule IgG at enhancing long-term macrophage adhesion. Surprisingly, the preabsorbed Fc fragment demonstrated no IgG-like activity, thereby eliminating the possibility of an Fc receptor-based mechanism. Other possible mechanisms, such as macrophage lectins, novel macrophage Fab receptors, and complement activation by adsorbed IgG and IgG fragments, are discussed.