Roger E. Marchant

Design properties of hydrogel tissue-engineering scaffolds

This article summarizes the recent progress in the design and synthesis of hydrogels as tissue-engineering scaffolds. Hydrogels are attractive scaffolding materials owing to their highly swollen network structure, ability to encapsulate cells and bioactive molecules, and efficient mass transfer. Various polymers, including natural, synthetic and natural/synthetic hybrid polymers, have been used to make hydrogels via chemical or physical crosslinking. Recently, bioactive synthetic hydrogels have emerged as promising scaffolds because they can provide molecularly tailored biofunctions and adjustable mechanical properties, as well as an extracellular matrix-like microenvironment for cell growth and tissue formation. This article addresses various strategies that have been explored to design synthetic hydrogels with extracellular matrix-mimetic bioactive properties, such as cell adhesion, proteolytic degradation and growth factor-binding.

Biomimetic engineering of non-adhesive glycocalyx-like surfaces using oligosaccharide surfactant polymers

The external region of a cell membrane, known as the glycocalyx, is dominated by glycosylated molecules which direct specific interactions such as cell–cell recognition and contribute to the steric repulsion that prevents undesirable non-specific adhesion of other molecules and cells. Mimicking the non-adhesive properties of a glycocalyx provides a potential solution to the clinical problems, such as thrombosis, that are associated with implantable devices owing to non-specific adsorption of plasma proteins. Here we describe a biomimetic surface modification of graphite using oligosaccharide surfactant polymers, which, like a glycocalyx, provides a dense and confluent layer of oligosaccharides. The surfactant polymers consist of a flexible poly(vinyl amine) with dextran and alkanoyl side chains. We show that alkanoyl side chains assemble on graphite through hydrophobic interaction and epitaxial adsorption. This constrains the polymer backbone to lie parallel to the substrate, with solvated dextran side chains protruding into the aqueous phase, creating a glycocalyx-like coating. The resulting biomimetic surface is effective in suppressing protein adsorption from human plasma protein solution.

Effect of albumin coating on the in vitro blood compatibility of Dacron® arterial prostheses

A recirculating in vitro perfusion system was used to assess the effect of albumin precoating on the thrombogenicity of Dacron vascular grafts. A complete analysis of platelet activation was carried out, involving platelet count, release, adhesion and aggregation. Fibrin formation was assessed by measuring fibrinogen levels and fibrinopeptide A production; leucocyte interaction was analysed by measuring total leucocyte count as well as an analysis of cell adhesion to the surface by scanning electron microscopy. The platelet count decreased progressively with perfusion time for Dacron until by 30 min, it had declined to 69% +/- 2% of baseline. The platelet count did not, however, change significantly from baseline when albumin-coated Dacron was tested. Release of platelet factor 4 and beta-thromboglobulin at 180 min for Dacron was 37.8 +/- 29.8 times and 66.9 +/- 18.2 times baseline, respectively, while albumin coating caused significantly less (P less than 0.03) platelet release. Albumin coating diminished coagulation activation and fibrinopeptide A formation. The total leucocyte concentration decreased significantly for Dacron by 180 min, while that for albumin-coated Dacron did not change significantly from baseline levels. Albumin coating produced a film-like covering over the Dacron. For Dacron, there were numerous leucocytes and platelets adherent to the surface, whilst cellular deposition was minimal upon the albumin-coated surface. Thus, albumin coating improved the short-term blood compatibility of Dacron by all of the methods employed in this study.

Methionine 35 Oxidation Reduces Fibril Assembly of the Amyloid Aβ-(1–42) Peptide of Alzheimer's Disease

The major component of amyloid plaques in Alzheimers disease (AD) is Aβ, a small peptide that has high propensity to assemble as aggregated β-sheet structures. Using three well established techniques for studying amyloid structure, namely circular dichroism, thioflavin-T fluorescence, and atomic force microscopy, we demonstrate that oxidation of the Met-35 side chain to a methionine sulfoxide (Met-35ox) significantly hinders the rate of fibril formation for the 42-residue Aβ-(1–42) at physiological pH. Met-35ox also alters the characteristic Aβ fibril morphology and prevents formation of the protofibril, which is a key intermediate in β-amyloidosis and the associated neurotoxicity. The implications of these results for the biological function and role of Aβ with oxidative stress in AD are discussed.

Surface modification of liposomes for selective cell targeting in cardiovascular drug delivery.

Cardiovascular disease processes such as atherosclerosis, restenosis, and inflammation are typically localized to discrete regions of the vasculature, affording great opportunity for targeted pharmacological treatment. Liposomes are potentially advantageous targeted drug carriers for such intravascular applications. To facilitate their use as drug delivery vehicles, we have considered three components of liposome design: (i) identification of candidate cell surface receptors for targeting; (ii) identification of ligands that maintain binding specificity and affinity; and (iii) prevention of rapid nonspecific clearance of liposomes into the reticuloendothelial organs. In this report, we describe our work in developing liposomal surface modifications that address both targeting and clearance. An arginine-glycine-aspartic acid (RGD) containing peptide was used as a model ligand to target liposomes to the integrin GPIIb-IIIa on activated platelets. Additionally, oligodextran surfactants incorporated into liposomes provided insight into the effect of vesicle perturbations on liposome clearance, and the importance of molecular geometry in designing oligosaccharide surface modifications. Together these studies demonstrate the feasibility of using peptides to guide liposomes to desired receptors, and illustrate the influence of vesicle stability on liposome interactions in vivo. Furthermore, they underscore the importance of simultaneously considering both targeting specificity and vesicle longevity in the design of effective targeted drug delivery systems.

Surface-dependent Conformations of Human Fibrinogen Observed by Atomic Force Microscopy under Aqueous Conditions

Conformational differences in human fibrinogen under aqueous conditions on hydrophobic, positively charged and negatively charged surfaces, were examined by atomic force microscopy (AFM). Hydrophobic and positively charged surfaces were prepared by depositing octadecyltrichlorosilane (OTS) and 3-aminopropyltriethoxysilane (APTES) respectively on cleaned glass coverslips forming self-assembled monolayers. The negatively charged surface was prepared by freshly cleaving muscovite mica. AFM operated in fluid tapping mode with an ultrasharp carbon spike probe was used to obtain the molecular scale images. Fibrinogen displayed a characteristic trinodular structure on all three surfaces. although additional U-shaped conformations were observed on mica. In its native hydrated state, fibrinogen is well represented by three connected ellipsoids in close proximity. Quantitative dimensional analysis, which yielded structural information in three dimensions, indicates that surface-dependent structural deformation or spreading of fibrinogen increases according to the order: mica < APTES < OTS. Molecular length, and D and E domain widths of fibrinogen are increased, while the corresponding heights are decreased. The results provide direct evidence that material surface properties affect the conformational state of interacting fibrinogen.

Atomic force microscopy for characterization of the biomaterial interface

The molecular processes that occur at the interface of an implanted biomaterial determines the host response, including phenomena such as protein adsorption, conformational changes, and subsequent interactions with cellular components. Until recently, such processes could not be observed directly. Over the past decade, atomic force microscopy (AFM) has provided mechanistic insights into the molecular level interactions that occur at the biomaterial interface. Several unique operational modes have been developed which utilize intermittent contact with the sample and decrease applied shear forces. These dynamic modes also can be used to study the role of different structural components on biomaterial micromechanical properties. Force detection techniques allow molecular level studies of individual receptor-ligand binding events, and force mapping for determining structure/function relationships. Advancements in tip manufacturing, image processing techniques, the use of model surfaces and labeling all have contributed to the advancement of the AFM as a state-of-the-art research instrument. In this report, we examine the applicability of the AFM to the study of biomaterials and cell/molecular interactions.

Degradation of a poly(ether urethane urea) elastomer: infra-red and XPS studies

The potential for a poly(ether urethane urea) (PEUU) elastomer to undergo degradation, under the conditions preyalent in the biological environment, was investigated using an in vitro model system. The effect of exposing an unstabilized PEUU to an aqueous environment containing the proteolytic enzyme, papain, for one month was examined by Fourier transform infra-red spectroscopy, X-ray photoelectron spectroscopy and chromatographic methods. Evidence of degradation was observed in both enzyme- and water-treated PEUU, but was restricted to the surface regions of the polymer. Analysis of methanol extracts from polymer samples revealed evidence for the degradation of ether linkages, which was independent of the enzyme, whereas the degradation of urethane and urea groups, indicated by the detection of a primary aromatic amine degradation product, depended on the presence of the proteolytic enzyme.

Affinity Manipulation of Surface-conjugated RGD-peptide to Modulate Binding of Liposomes to Activated Platelets

Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD motifs that can selectively target and bind integrin GPIIb-IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high affinity cyclic RGD motif to modulate the GPIIb-IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb-IIIa. The platelet-binding of RGD-modified liposomes was studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands.

Bacterial surface properties of clinically isolated Staphylococcus epidermidis strains determine adhesion on polyethylene

The role of surface physiochemical properties of Staphylococcus epidermidis strains in adhesion to polyethylene (PE) was investigated under physiological flow conditions in phosphate buffered saline (PBS) and 1% platelet poor plasma (PPP). Four clinically isolated strains were divided into two groups: low and high relative hydrophobicity, and the F1198 and RP62A strains showing significantly greater hydrophobicity than the F21 and F1018 strains. In PBS, adhesion of all S. epidermidis strains was shear dependent from 0 to 15 dyn/cm2, after which adhesion becomes shear independent. Strains with higher surface hydrophobicity showed higher adhesion to PE, demonstrating the influence of bacterial surface hydrophobicity in nonspecific adhesion. Bacterial adhesion correlated well with bacterial surface hydrophobicity at low shear stresses (0-8 dyn/cm2). In 1% PPP, adhesion of all strains dramatically decreased and we found no correlation between bacterial surface hydrophobicity and adhesion. The presence of plasma proteins reduced nonspecific adhesion. S. epidermidis surface charge did not correlate with bacterial adhesion in either test media. The results suggested that S. epidermidis surface hydrophobicity may mediate nonspecific adhesion to PE at low shear stresses in protein-free media.