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.

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.

Adsorbed proteins on implanted biomedical devices mediate platelet and leukocyte adhesion. Radio frequency plasma deposited tetraglyme (CH3O(CH2CH2O)4CH3), which forms a PEO-like coating, has been shown to resist protein adsorption and monocyte adhesion in vitro. By using different plasma deposition powers (5-80 W), we produced a series of plasma-deposited tetraglyme surfaces that varied in surface chemistry as measured by electron spectroscopy for chemical analysis (ESCA) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Both fibrinogen and IgG adsorption were increased on surfaces made at high plasma power. Monocyte adhesion correlated linearly with the amount of adsorbed protein. To identify the surface chemical features that contributed to the nonfouling properties of plasma-deposited tetraglyme, multivariate analysis using partial least squares (PLS) regression was applied. A PLS calibration model based on deposited tetraglyme samples placed downstream in the plasma reactor successfully predicted fibrinogen adsorption to deposited tetraglyme samples placed midstream in the reactor. The model identified how each surface spectral variable from ESCA and ToF-SIMS contributed to protein adsorption. The fraction of carbon in ether carbon linkages as measured by ESCA and ToF-SIMS peaks at m/z 59 and 103 was higher on surfaces that exhibited ultralow fibrinogen adsorption (<10 ng/cm 2 ). The fraction of hydrocarbon-like carbons as measured by ESCA and low-mass ToF-SIMS peaks such as m/z 29 and 31 was greater on surfaces exhibiting high fibrinogen adsorption (>200 ng/cm 2 ). This study elucidated the surface chemical features of deposited tetraglyme that most affect its resistance to protein and cell uptake and provided guidelines for engineering improved nonfouling biomaterial surfaces.

Characterization of biomaterial surfaces requires analytical techniques that are capable of detecting a wide concentration range of adsorbed protein. This range includes detection of low amounts of adsorbed protein (<10 ng/cm2) that may be present on non-fouling biomaterials. X-ray Photoelectron Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) are surface sensitive techniques capable of detecting adsorbed proteins. We have investigated the lower limits of detection of both XPS and ToF-SIMS on four model substrates each presenting unique challenges for analysis by XPS and ToF-SIMS: mica, poly(tetrafluoroethylene), allyl amine plasma polymer and heptyl amine plasma polymer. The detection limit for XPS ranged from 10 ng/cm2 of fibrinogen (on mica) to 200 ng/cm2 (on allyl amine plasma polymers). The detection limit for ToF-SIMS ranged from 0.1 ng/cm2 of fibrinogen to 100 ng/cm2, depending on the substrate and data analysis. Optimal conditions provided detection limits between 0.1 ng/cm2 and 15 ng/cm2 on all of the substrates used in this study. While both techniques were shown to be effective in detecting protein, the sensitivity of both XPS and ToF-SIMS was shown to be dependent on substrate surface chemistry and the organization of the adsorbed protein film. This study specifically highlights the applicability of ToF-SIMS in the characterization of low level protein adsorption.

Further research is needed to determine if these immune responses are predictors of vaccine efficacy or markers of natural resistance to HIV-1 infection.