Chemically grafted fibronectin for use in QCM-D cell studies.

Traditionally, fibronectin has been used as a physisorbed surface coating (physFN) in cell culture experiments due to its critical role in cell adhesion. However, because the resulting layer is thick, unstable, and of unpredictable uniformity, this method of fibronectin deposition is unsuitable for some types of research, including quartz crystal microbalance (QCM) experiments involving cells. Here, we present a new method for chemical immobilization of fibronectin onto silicon oxide surfaces, including QCM crystals pre-coated with silicon oxide. We characterize these chemically coated fibronectin surfaces (chemFN) as well as physFN ones using spectroscopic ellipsometry (SE), Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and contact angle measurements. A cell culture model demonstrates that cells on chemFN and physFN surfaces exhibit similar viability, structure, adhesion and metabolism. Finally, we perform QCM experiments using cells on both surfaces which demonstrate the superior suitability of chemFN coatings for QCM research, and provide real-time QCM-D data from cells subjected to an actin depolymerizing agent. Overall, our method of chemical immobilization of fibronectin yields great potential for furthering cellular experiments in which thin, stable and uniform coatings are desirable. As QCM research with cells has been rather limited in success thus far, we anticipate that this new technique will particularly benefit this experimental system by availing it to the much broader field of cell mechanics.

Hemocompatibility of Chitosan/poly(acrylic acid) Grafted Polyurethane Tubing.

The activation and adhesion of platelets or whole blood exposed to chitosan (CH) grafted surfaces is used to evaluate the hemocompatibility of biomaterials. The biomaterial surfaces are polyurethane (PU) tubes grafted with an inner poly(acrylic acid) (PAA) and an outer CH or quaternary ammonium modified CH (CH-Q) brush. The CH, CH-Q and PAA grafted layers were characterized by ellipsometry and fluorescence microscopy. Material wear tests demonstrate that CH (CH-Q) is stably grafted onto PU tubes upon exposure to saline solution for 7 days. Using quartz-crystal microbalances with dissipation (QCM-D), in-situ adsorption of blood plasma proteins on CH and CH-Q compared to a silicon oxide control was measured. The QCM-D results showed that the physically adsorbed plasma protein layer on CH-Q and CH surfaces is softer and more viscous than the protein layer on the SiO2 surface. The CH-Q layer thus has the weakest interaction with plasma proteins. Whole blood and platelet adhesion was reduced by ~92% on CH-Q, which showed the weakest interaction with plasma protein but more viscous adsorbed plasma protein layer, compared to SiO2. Last, to examine the biologic response of platelets and neutrophils to biomaterial surfaces, CH (CH-Q)/PAA, PAA and PU tubes were tested using a Chandler Loop apparatus as an ex vivo model and flow cytometry. The blood adhesion and biologic response results showed that CH and CH-Q reduced adhesion and activation of platelets and neutrophils and improved hemocompatibility relative to other surfaces (PU and PAA). Our studies demonstrated that the properties of physically adsorbed plasma protein layer on biomaterial surfaces correlates with blood coagulation on biomaterial surfaces.

Hyaluronan and dextran modified tubes resist cellular activation with blood contact.

This study was undertaken to evaluate the effects of thin film hyaluronic acid and dextran surface coatings to blunt cellular activation in a laboratory model of extracorporeal blood circulation. The inner lumen surface of polyurethane (PU) and poly(vinyl) chloride (PVC) tubing was grafted with hyaluronic acid and dextran. Surfaces were characterized for the presence of the grafted layer using ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Persistence of the surface layer was maintained for up to 5 days of continuous exposure to shear flow using a Chandler loop apparatus. The Chandler loop method was used to study human whole blood activation activity. Whole blood aggregometry and flow cytometry measures of CD18, CD62L, CD62P, Annexin V and myeloperoxidase performed on blood samples exposed to the tubing for up to three hours were complemented by scanning electron microscopy (SEM) analysis of adherent cells and state of activation. In these studies commercial hospital products and uncoated PVC and PU tubes were used as controls. We found that hyaluranized PU and PVC conferred the greatest resistance to blood activation and that dextranization of the PU and PU tubing also provided significant diminution of the bioresponses measured. Based on our findings, we suggest that surface coating with hyaluronic acid or dextran acts as a potent shield from blood cellular activation during forms of extracorporeal circulation.

Correlating macrophage morphology and cytokine production resulting from biomaterial contact.

The morphological and inflammatory responses of adherent macrophages are correlated to evaluate the biocompatibility of surfaces. Monocyte-derived macrophage (MDM), THP-1, and THP-1 cells expressing GFP-actin chimeric protein were seeded onto glass, polyurethane (PU), and glass surface modified with quaternary ammonium salt functionalized chitosan (CH-Q) and hyaluronic acid (HA). Using confocal microscopy, the surface area, volume and 3D shape factor of adherent macrophages was quantified. For comparison, functional consequences of cell-surface interactions that activate macrophages and thereby elicit secretion of a proinflammatory cytokine were evaluated. Using an enzyme linked immune sorbent assay, tumor necrosis factor-alpha (TNF-α) was measured. On glass, macrophages exhibited mainly an amoeboid shape, exhibited the largest surface area, volume, and 3D shape factor and produced the most TNF-α. On PU, macrophages displayed mainly a hemispherical shape, exhibited an intermediate volume, surface area and 3D shape factor, and produced moderate TNF-α. In contrast, on CH-Q and HA surfaces, macrophages were spherical, exhibited the smallest volume, surface area, and 3D shape factor, and produced the least TNF-α. These studies begin to validate the use of GFP-actin-modified MDM as a novel tool to correlate cell morphology with inflammatory cell response.

Human macrophage adhesion on polysaccharide patterned surfaces.

Despite many advances in designing biocompatible materials, inflammation remains a problem in medical devices and implants. We report two methods, microcontact printing and photodegradation by UV exposure, to pattern dextran and hyaluronic acid on glass, as well as demonstrate their utility for use as an anti-inflammatory biomaterial. The dextran/glass patterned surface can be further modified by grafting hyaluronic acid to glass, creating a binary polysaccharide patterned surface. We used two geometries, 90 µm squares and 22 µm stripes, to study the human macrophage (THP-1) adhesion on the patterned surfaces containing dextran, hyaluronic acid and the binary pattern. The results indicate that a majority of the macrophages are non-adherent on hyaluronic acid for three day culture. The ranking of surfaces according to macrophage adhesion is 3-aminopropyl triethoxysilane-modified glass culture dish, dextranized surfaces, glass, and hyaluronic acid-modified surfaces. On the binary pattern of dextran and hyaluronic acid, macrophages preferentially attach and adhere to the dextranized area. Patterned surfaces provide an excellent platform for mimicking the complexity of the glycocalyx and investigating the interface between this surface and cells. This binary polysaccharide pattern also offers a new route to address anti-inflammatory potential of surface coatings on biomaterials in a high through-put fashion.

The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.

CD47 is a transmembrane protein that is a marker of "self". CD47 binding to its cognate receptor in leukocytes and macrophages, signal-regulatory protein alpha (SIRPα), causes inhibition of inflammatory cell attachment. We hypothesized that immobilization of recombinant CD47 on polymeric surfaces would reduce inflammation. Recombinant CD47 was appended to polyvinyl chloride (PVC) or polyurethane (PU) surfaces via photoactivation chemistry. Cell culture studies showed that CD47 immobilization significantly reduced human neutrophil (HL-60) and human monocyte derived macrophage (MDM) (THP-1) attachment to PVC and PU respectively. A neutralizing antibody, directed against SIRPα, inhibited THP-1 and HL-60 binding to PU and PVC surfaces respectively. This antibody also increased the level of SIRPα tyrosine phosphorylation, thereby indicating a direct role for SIRPα mediated signaling in preventing inflammatory cell attachment. Studies using human blood in an ex vivo flow-loop showed that CD47 modified PVC tubing significantly reduced cell binding and neutrophil activation compared to unmodified tubing or poly-2-methoxy-ethylacrylate (PMEA) coated tubing. In ten-week rat subdermal implants, CD47 functionalized PU films showed a significant reduction in markers of MDM mediated oxidative degradation compared to unmodified PU. In conclusion, CD47 functionalized surfaces can resist inflammatory cell interactions both in vitro and in vivo.

Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study.

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.

Anomalous grazing incidence small-angle x-ray scattering studies of platinum nanoparticles formed by cluster deposition.

The size evolution of platinum nanoparticles formed on a SiO2/Si(111) substrate as a function of the level of surface coverage with deposited clusters has been investigated. The anisotropic shapes of sub-nanometer-size nanoparticles are changed to isotropic on the amorphous substrate as their sizes increased. Using anomalous grazing incidence small-angle x-ray scattering (AGISAXS), the scattering from nanoparticles on the surface of a substrate is well separated from that of surface roughness and fluorescence. We show that AGISAXS is a very effective method to subtract the background and can provide unbiased information about particle sizes of less than 1 nm.