Anders Sellborn

Fibrinogen Adsorption and Conformational Change on Model Polymers: Novel Aspects of Mutual Molecular Rearrangement

By combining quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR), the organic mass, water content, and corresponding protein film structure of fibrinogen adsorbed to acrylic polymeric substrates with varying polymer chain flexibility was investigated. Albumin and immunoglobulin G were included as reference proteins. For fibrinogen, the QCM-D model resulted in decreased adsorbed mass with increased polymer chain flexibility. This stands in contrast to the SPR model, in which the adsorbed mass increased with increased polymer chain flexibility. As the QCM-D model includes the hydrodynamically coupled water, we propose that on the nonflexible polymer significant protein conformational change with water incorporation in the protein film takes place. Fibrinogen maintained a more native conformation on the flexible polymer, probably due to polymer chain rearrangement rather than protein conformational change. In comparison with immunoglobulin G and albumin, polymer chain flexibility had only minor impact on adsorbed mass and protein structure. Understanding the adsorption and corresponding conformational change of a protein together with the mutual rearrangement of the polymer chain upon adsorption not only has implications in biomaterial science but could also increase the efficacy of molecular imprinted polymers (MIPs).

Bacterial cellulose as a potential vascular graft: Mechanical characterization and constitutive model development

Bacterial cellulose (BC) is a polysaccharide produced by Acetobacter Xylinum bacteria with interesting properties for arterial grafting and vascular tissue engineering including high-burst pressure, high-water content, high crystallinity, and an ultrafine highly pure fibrous structure similar to that of collagen. Given that compliance mismatch is one of the main factors contributing to the development of intimal hyperplasia in vascular replacement conduits, an in depth investigation of support mechanical properties of BC is required to further supporting its use in cardiovascular-grafting applications. The aim of this study was to mechanically characterize BC and also study its potential to accommodate vascular cells. To achieve these aims, inflation tests and uniaxial tensile tests were carried out on BC samples. In addition, dynamic compliance tests were conducted on BC tubes, and the results were compared to that of arteries, saphenous vein, expanded polytetrafluoroethylene, and Dacron grafts. BC tubes exhibited a compliance response similar to human saphenous vein with a mean compliance value of 4.27 × 10(-2) % per millimeter of mercury over the pressure range of 30-120 mmHg. In addition, bovine smooth muscle cells and endothelial cells were cultured on BC samples, and histology and fluorescent imaging analysis were carried out showing good adherence and biocompatibility. Finally, a method to predict the mechanical behavior of BC grafts in situ was established, whereby a constitutive model for BC was determined and used to model the BC tubes under inflation using finite element analysis.

Methods for research on immune complement activation on modified sensor surfaces

Abstract We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a CH 3 end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.

An in vitro study of blood compatibility of vascular grafts made of bacterial cellulose in comparison with conventionally-used graft materials

In this study we analyzed the blood compatibility of bacterial cellulose (BC) as a new biosynthetic material for use as a vascular graft. As reference materials we used expanded polytetrafluoroethylene (ePTFE) and poly(ethylene terephthalate) (PET) vascular grafts. These materials are in clinical use today. Tubes with inner diameters of both 4 (not PET) and 6 mm were tested. Heparin-coated PVC tubes (hepPVC) were used as a negative control. Platelet consumption and thrombin-antithrombin complex (TAT) were used as parameters of coagulation and for complement activation, sC3a and sC5b-9 were used. The investigated parameters were measured after 1-h exposure to freshly drawn human blood supplemented with a low dose of heparin in a Chandler loop system. The results showed that BC exhibits no significant difference in platelet consumption, as compared with PET (6 mm), ePTFE and hepPVC. The PET material consumed more platelets than any of the other materials. The TAT generation for 4 mm tubes was not significantly different between BC and the other materials. For 6 mm tubes, however, differences were observed between hepPVC and PET (p < 0.0001); BC and hepPVC (p = 0.0016); ePTFE and PET (p < 0.0001); BC and ePTFE (p = 0.0029); BC and PET (p = 0.0141). Surprisingly, considering the low platelet consumption, the complement activation parameters (sC3a and sC5b-9) were much higher for BC, as compared with the other materials for both 4 and 6 mm tubes.

In vitro study of monocyte viability during the initial adhesion to albumin- and fibrinogen-coated surfaces

Surface adherent monocytes and macrophages play a central role in the inflammatory response to biomaterials. In the present study the adhesion, viability and apoptotic changes in material surface adherent monocytes during the first hours of cell-surface interactions in vitro were studied, using tissue culture polystyrene surfaces coated with human albumin and fibrinogen. Human peripheral blood monocytes were enriched by a two-step gradient centrifugation and resuspended (1 x 10(6)/ml) in RPMI with 10% fetal bovine serum. The cells were added to polystyrene surfaces coated with human fibrinogen or albumin and incubated in 37 degrees C (5% CO2, 100% humidity) for 30 min, 1, 2, 3 and 24 h. The adherent cells were stained for early apoptotic changes (exposed phosphatidylserine) and cell death using Annexin-V-fluorescein and propidium iodide staining, respectively. A bi-phasic adhesion was observed on the fibrinogen coated surface, having the highest number of adherent cells after 30 min and 24 h, while the cell number was markedly reduced after 1-3 h. The number of adherent cells on albumin was relatively low after all short time incubations but had reached a high level after 24 h. The number of adherent dead cells was highest after I h on both albumin (approximately 30%) and fibrinogen (approximately 15%). In the 24 h cultures, the viability of adherent cells was high on both surfaces (95-100%). Viable cells staining positive for early apoptotic changes could only be clearly observed on the albumin coated surface, after 30 min of cell-material surface interaction. Cell death, including apoptotic death, thus seems to play an important role during the initial interactions between monocytes and a foreign surface.

Acoustics of blood plasma on solid surfaces

We have quantified surface associated coagulation of human blood plasma with a recently developed methodological system consisting of a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), a method that measures the weight of adsorbed molecules on surfaces as a function of frequency shifts of a quartz crystal. Further, it measures the damping energy (i.e. viscoelasticity) of the adsorbed layer. Four different surfaces where studied: Heparin (Hep) surface as an active inhibitor of clot formation, titanium (Ti) surfaces that are known to activate the intrinsic pathway, polystyrene (PS) surfaces and poly(urethane urea) (PUUR) surfaces. The experiments were initiated by applying citrated human plasma at the sensor surfaces; calcium was then added to initiate coagulation. The Hep surfaces showed no apparent indication of clot formation during one hour of incubation at room temperature. However, on Ti surfaces we observed an early and rapid change in both frequency shift and viscoelastic properties of the coagulating plasma. We inhibited the intrinsic pathway activation by using corn trypsin inhibitor (CTI), which is specific for factor FXIIa in the bulk phase, which prolonged the coagulation times for all non-heparinized surfaces. We have also found a peculiar initial plasma protein interaction phenomenon on Ti surfaces. The described methodology would be very efficient for basic studies of surface associated coagulation and as a screening method for new biomaterials.

Self-Assembled Arrays of Dendrimer–Gold-Nanoparticle Hybrids for Functional Cell Studies†

Engineered surfaces with nanoscale features of gold on silicon or glass have recently been used to improve the understanding of adhesion-mediated environmental sensing of cells. Often such surfaces present a cell-binding ligand, such as arginine–glycine–aspartic acid (RGD) peptide motifs, at controlled intramolecular distances on an inert background surface such as polyethylene glycol (PEG). The adhesion mechanism of macromolecular ligands in which direct interaction with cells is nonspecific is not known and the cell response is dictated by the type and the concentration of proteins adsorbed from solution. Dendrimers may increase the availability and multivalency of cell-interacting ligands as a consequence of their branched shape and inherently high concentration of end groups. It is therefore interesting to examine the eventual effect of the macromolecular architecture on the cell viability by the controlled reduction of ligands on a surface. Herein, we demonstrate the fabrication of selfassembledmacromolecular hybrid arrays in which the relative position of two anionic macromolecules of different architectures—a carboxy-functionalized dendrimer and a linear polymer—is straightforwardly controlled on a PEG surface. We also show how the interaction of primary human endothelial cells with these surfaces is modulated by the molecular spacing and how protein binding to the macromolecular arrays can be evaluated by using a standard surface plasmon resonance (SPR) technique. Self-assembled, short-range-ordered Au nanoparticle (NP) arrays were used as a versatile template to arrange polymeric entities at the nanometer scale (Figure 1a). This

Molecular Understanding of Endothelial Cell and Blood Interactions with Bacterial Cellulose: Novel Opportunities for Artificial Blood Vessels

Cardiovascular disease (CVD) is the main cause of death or invalidism in high-income countries today. Moreover, worldwide demographic changes are aiding CVD’s rapid progression towards the number one killer in middleand low-income countries. The World Health Organisation estimates that if current trends are allowed to continue, about 20 million people will die from CVD by 2015. This group of disorders, which affect the heart and blood vessels, includes coronary heart disease, cerebrovascular disease and peripherial arterial disease, deep vein thrombosis and pulmonary embolism. The main cause of these acute life-threatening conditions is atherosclerosis. Atherosclerotic plaques and restenosis can result in severe occlusions of peripheral and coronary arteries. Current treatments include drug therapy and bypass surgery, and depend on the severity of the disease. All treatments require molecular understanding of the processes that govern atherosclerosis. This is especially important when introducing artificial graft materials in vivo. Generally, the first choice for vascular replacement graft material is the patient’s own vessels, i.e., autologous vessels. If these are in shortage supply or do not exhibit sufficient quality due to, e.g., other diseases or previous surgery, artificial alternatives become necessary. Today, clinics use biomaterials such as expanded polytetrafluorethylene (ePTFE) and polyethylene terephtalate fibre (Dacron®) as prosthetic grafts for reconstructive vascular surgery. However, their performance is dismal in small diameter vessels (>6 mm) like coronary arteries and peripheral arteries below the knee, resulting in early thrombosis and intimal hyperplasia. Therefore, about 10% of patients with CVD are left untreated due to the lack of replacement material for small vessels. Considering the large number of patients who need replacement vessels, the substantial demand for alternative small-caliber grafts is urgent, driving scientists to search for and develop new materials. Recently, this has even led to the use of completely biological vessels. However, the growth of such requires months, rendering them unsuitable for acute situations such as heart infarction, which demand a substitute vessel immediately.