Bacteria/blood/material interactions. I. Injected and preseeded slime-forming Staphylococcus epidermidis in flowing blood with biomaterials.

Blood-material interactions were studied using in vitro recirculation with human blood, slime-forming Staphylococcus epidermidis, and cardiovascular materials. Staphylococcus epidermidis, under preseeded or injected conditions, adhered to nonsmooth materials and elevated plasma levels of fibrinopeptide A (FpA) and C3a in the presence of all materials. Increased white blood cell (WBC) and platelet adhesion and thrombospondin and platelet factor 4 (PF4) release were noted for respective materials in the presence of injected bacteria. Materials that adhered significant quantities of injected S. epidermidis exhibited low levels of adsorbed proteins. Materials with high levels of preseeded S. epidermidis showed high levels of adsorbed proteins. Adhesion of preseeded bacteria and blood plasma elevations of C3a and FpA were lowest on semicrystalline polymer substrates, intermediate on halogenated substrates, and highest on amorphous substrates. In the presence of injected bacteria, WBCs and platelets adhered at earlier recirculation times to amorphous substrates than to semicrystalline substrates.

Protein adsorption and macrophage activation on polydimethylsiloxane and silicone rubber.

Static and dynamic human blood adsorption studies on polydimethylsiloxane, PDMS, and silicone rubber show that these materials are similar, but not identical, in their protein adsorption behavior. Fibrinogen, immunoglobulin G, and albumin were the predominant proteins identified on the material surfaces with fibronectin, Hageman factor (factor XII), and factor VIII/vWF adsorbing at intermediate levels. While the protein adsorption characteristics for the two materials were similar, higher levels of the respective proteins were identified on silicone rubber compared to PDMS. Monocytes/macrophages incubated on PDMS, silicone rubber and low density polyethylene, LDPE, with or without protein adsorption produced variable levels of IL-1 beta, IL-6 and TNF-alpha dependent on the polymer and adsorbed protein. PDMS showed lower levels of the cytokines when compared to the polystyrene control and polyethylene. Protein preadsorption on the PDMS, polystyrene, and LDPE surfaces showed lower levels of cytokines when compared to the respective quantities produced with no protein adsorption suggesting a passivating effect by the protein adsorption phenomenon on monocyte/macrophage activation. Preadsorption of IgG, fibrinogen or fibronectin decreased the quantitative expression of IL-1 beta but increased the functional activity in the thymocyte proliferation assay indicating the presence of monocyte/macrophage activation products which either downregulated the activity of IL-1 beta or upregulated thymocyte proliferation in an independent fashion.

Attachment and proliferation of bovine aortic endothelial cells onto additive modified poly(ether urethane ureas).

To better understand endothelial cell interactions with poly(ether urethane urea) (PEUU) materials, and to assess bovine aortic endothelial cell attachment, films were incubated for 24 h with BAEC in media containing 5% fetal bovine serum. Other films were allowed to incubate for 4 more days in media containing 5% fetal bovine serum without cells to assess BAEC proliferation. The assay was performed on PEUU films modified with acrylate and methacrylate polymer and copolymer additives that spanned a wide range on the hydrophobicity/hydrophilicity scale. Tissue culture polystyrene (TCPS) was used as a control. The assay showed that PEUU films loaded with Methacrol 2138F [copoly(diisopropylaminoethyl methacrylate [DI-PAM]/decyl methacrylate [DM]) (3/1)] or with its hydrophilic component, DIPAM, in homopolymer form (i.e., h-DIPAM), significantly enhanced BAEC attachment (approximately 80% of TCPS values) and proliferation (approximately 80%) when compared to unloaded PEUU films (attachment 73%; proliferation, 47%) or to PEUU films loaded with the more hydrophobic acrylate or methacrylate polymer additives (attachment, 32-69%; proliferation, 18-57%). The assay also showed that PEUU films coated with homopoly(diisopropylaminoethyl acrylate) (h-DIPAA) significantly enhanced BAEC attachment and proliferation when compared to PEUU films coated with h-decyl acrylate (h-DA); films coated with the copolymer of these two acrylates (i.e., co-[DIPAA/DA] [3/1]) showed intermediate behavior. To explain the enhancement of BAEC interaction with films loaded with Methacrol 2138F or h-DIPAM, when compared to unmodified PEUU films or to PEUU films loaded with more hydrophobic acrylate and methacrylate polymer additives, it was assumed that the additives near the surface region of the solvent swollen PEUU matrix may have migrated to, or near to, the PEUU-air interface during film formation, creating an additive enriched PEUU surface region. It is suggested that, once at this surface region, dynamic reorientation in response to an aqueous medium ensured the additives were able significantly to influence protein adsorption, and concomitant endothelial cell behavior, but only if they interacted with aqueous media more favorably than the PEUU. The ability of Methacrol and h-DIPAM additives to enhance endothelial cell behavior is argued to be the result of increased hydrophilicity. This is the result of exposed, hydrogen-bonding DIPAM moieties and increased surface flexibility, which is itself due to the hydration of unhindered Methacrol chains, which may create an additive enriched PEUU-water interfacial zone.

Protein adsorption and endothelial cell attachment and proliferation on PAPI-based additive modified poly(ether urethane ureas).

To better understand vascular interactions with poly(ether urethane urea) (PEUU) materials, protein adsorption, and endothelial cell attachment and proliferation assays were performed on a base PEUU formulation, on PEUU formulations loaded with hydrophobic and amphiphilic poly(methylene-[polyphenyl isocyanate]) (PAPI) based additives, and on PEUU formulations in which some of the polymer chains had been endcapped with either diisopropylaminoethyl (DIPAA) or decyl (DA) moieties. Protein adsorption experiments with PAPI-based additives showed that additive loaded PEUU formulations adsorbed significantly lower amounts of the studied proteins than did the unloaded PEUU. Protein adsorption to the DA and DIPAA endcapped PEUU films was found not to vary consistently from that of the unloaded PEUU film. Endothelial cell attachment and proliferation experiments with PAPI-DA and polyethylene glycol-PAPI-DA (PEG-PAPI-DA) loaded PEUU films showed that many of the films exhibited attachment and proliferation that was significantly enhanced compared to PEUU A' and that approached or equaled that of the tissue culture polystyrene control. Experiments with PAPI-DIPAA and PEG-PAPI-DIPAA loaded PEUU films exhibited attachment and proliferation data that was often below 10% of the tissue culture polystyrene control values. Experiments with the DA and DIPAA endcapped PEUU films showed endothelial cell attachment and proliferation that was statistically indistinguishable from the PEUU A' values. Contact angle analysis was carried out on the endcapped PEUU films, on the PAPI-based additive loaded PEUU films, and on PEUU A' using the sessile drop method. The advancing and receding contact angle behavior of the PAPI-based additive loaded PEUU films deviated markedly from the behavior of PEUU A', suggesting that the additives were present at the PEUU-water interface. The contact angle behavior of the endcapped PEUUs was similar to that of PEUU A', suggesting that the DA and DIPAA endcap moieties did not exist at the hydrated PEUU surface in appreciable quantities. To explain the differences in protein adsorption and endothelial cell behavior on the air side of additive loaded PEUUs when compared to the base PEUU, it was assumed that the additives near this region of the solvent swollen PEUU matrix may have migrated to, at, or near the PEUU-air interface during film formation, creating an additive enriched PEUU surface region.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein adsorption to poly(ether urethane ureas) modified with acrylate and methacrylate polymer and copolymer additives.

To understand better blood interactions with poly(ether urethane urea) (PEUU) materials, a radioimmunoassay and whole or diluted human plasma were used to characterize the presence of fibrinogen, immunoglobulin G, factor VIII/von Willebrand factor, Hageman factor (factor XII), and albumin on a PEUU formulation and on PEUU formulations modified with the amphiphilic additive Methacrol 2138F (co[diisopropylaminoethyl methacrylate (DIPAM)/decyl methacrylate] [3/1]), or with hydrophobic acrylate or methacrylate polymer or copolymer additives. The protein adsorption assay showed that PEUU films loaded or coated with Methacrol 2138F (Methacrol) or homopoly-DIPAM (h-DIPAM) adsorbed significantly lower amounts of the studied proteins than did either the base PEUU formulations or the PEUUs loaded with the more hydrophobic acrylate or methacrylate polymer additives. Experiments with Methacrol-loaded PEUUs, where the loading of Methacrol was varied from 0.25 wt% to 20.0 wt%, showed that the adsorption of each of the characterized proteins did not vary significantly throughout the Methacrol loading range, and that all Methacrol-loaded PEUU formulations adsorbed significantly lower amounts of the studied proteins than did the unloaded PEUU. Phase separation within the additive loaded PEUUs was characterized by scanning electron microscopy (SEM). The solubility parameters of the additives, as well as of the base PEUU, were calculated and used to interpret differences in phase separation of the additive modified PEUUs. The analysis showed that additives of lower solubility parameter phase-separated into fewer large microdroplets within the PEUU matrix. SEM analysis also showed that additive microdroplets were not present on the air side surface of loaded PEUUs. To explain the differences in protein adsorption to the air side of additive loaded PEUUs when compared to the base PEUU, it was assumed that the additives near this region of the solvent swollen PEUU matrix may have migrated to, at, or near the PEUU-air interface during film formation, creating an additive enriched PEUU surface region. Once at this surface region, it was suggested that dynamic surface reorientation in response to an aqueous medium ensured that the additives were able significantly to influence protein adsorption behavior only if they interacted with aqueous media more favorably than the PEUU.

Protein adsorption onto poly(ether urethane ureas) containing Methacrol 2138F: a surface-active amphiphilic additive.

Surface characterization and protein adsorption studies were carried out on a series of additive dispersed and additive coated poly(ether urethane ureas), PEUUs, to characterize early events in the blood compatibility of these materials. A hypothesis that is based on surface hydrophilicity, surface flexibility, and adsorption media has been developed to understand the modulated adsorption of plasma proteins by PEUU additives. Electron spectroscopy for chemical analysis (ESCA) and contact angle analysis were performed on two PEUU formulation as well as on PEUU formulations modified with Methacrol 2138F (co[diisopropylaminoethyl methacrylate (DIPAM)/decyl methacrylate (DM)][3/1]) or acrylate or methacrylate polymer or copolymer analogs of Methacrol 2138F. Methacrol 2138F is a commercially used amphiphilic copolymethacrylate. ESCA showed that the PEUUs loaded with Methacrol 2138F or with its hydrophilic component, homopoly (DIPAM) (h-(DIPAM)), had a higher percentage of nitrogen at their surfaces than did the base PEUUs. Contact angle analysis also showed that the air side of PEUU formulations loaded with Methacrol 2138F were more hydrophobic than was the air side of base PEUUs when films were cast from dimethylacetamide. However, during contact angle testing, the air side of PEUU films loaded with Methacrol 2138F rapidly became more hydrophilic than did the air side of the base PEUU films. A radioimmunoassay and whole or diluted human plasma were also used to characterize the presence of the proteins fibrinogen, immunoglobulin G, factor VIII/von Willebrand factor, Hageman factor (factor XII), and albumin, on the surface of the same PEUUs as analyzed by ESCA and contact angle. The protein adsorption assay showed that PEUU films loaded or coated with Methacrol 2138F, with a copolyacrylate analog of Methacrol 2138F (co(diisopropylaminoethyl acrylate [DIPAA]/decyl acrylate [DA]) [3/1]), or with the hydrophilic polyacrylate or polymethacrylate component analogs of Methacrol 2138F (h-DIPAM or h-DIPAA) adsorbed significantly lower amounts of the proteins than did either the base PEUU formulations or the homopoly(decyl methacrylate) (h-DM) or homopoly(decyl acrylate) (h-DA) coated or loaded PEUUs.

In vivo leucocyte interactions on Pellethane surfaces.

In vivo leucocyte interactions of three Pellethane materials of varying hardness were qualitatively and quantitatively characterized using a cage implant system over a 21 d implantation period. Scanning electron microscopy (SEM) and cytochemical staining were utilized to observe the cellular events occurring at the leucocyte-biomaterial interface. Many of the quantitative assays performed, the intracellular alkaline phosphatase activity of exudate leucocytes, the intracellular acid phosphatase activity of adherent leucocytes, the density of adherent leucocytes and the foreign body giant cell network formation tendencies of adherent leucocytes, suggest increased cellular activation with increased Pellethane hardness. Qualitative SEM evaluation of Pellethane surfaces revealed a variety of cellular activities. These included macrophage adherence, cytoplasmic spreading and macrophage-macrophage membrane fusions to form foreign body giant cells. The foreign body giant cells exhibited nuclear reorganization and, when compared with adherent macrophages, they displayed an enhanced ability to fuse to neighbouring leucocytes, increased spreading of membrane processes over the polymer surface, the presence of large cytoplasmic vacuoles, and a lengthened duration of enzymatic activity. Contact angle analysis showed the Pellethane surfaces to be hydrophobic and of low hysteresis. The critical surface tension and the dispersive component of the total surface tension were found to increase with Pellethane hardness.