Biomedical subjects

Heather Sheardown

Publications and source records attributed to Heather Sheardown.

16 recordsLinked to original sources

PubMed2006

Characterization of EGF coupling to aminated silicone rubber surfaces.

Tethering of growth factors to biomaterial substrates via a polyethylene glycol (PEG) spacer has been established as a means of controlling dosage and conformation of the protein at the material surface, while retaining biological activity. However, the extent of modification through a comparison of bound versus unbound protein has not generally been characterized. In this work, covalent tethering of epidermal growth factor (EGF) to allylamine plasma modified polydimethylsiloxane (PDMS) substrates is characterized to determine the nature of the bound growth factor and to optimize the conditions for the reaction. Tethering is achieved via conjugation of EGF with homobifunctional N-hydroxysuccinimide (NHS) ester of PEG-butanoic acid (SBA2-PEG) in solution, followed by exposure of the pegylated EGF to the aminated surfaces (solution first reaction). SDS-PAGE analysis indicates that a low ratio of EGF:PEG is required to maximize the yield of the EGF-PEG reaction; a relatively short reaction time is needed to limit hydrolysis of the NHS ester. With increasing amounts of PEG and a higher reaction time, a higher fraction of the EGF can be covalently tethered to the surfaces, as shown by binding of 125I-labeled EGF and subsequent washing with sodium dodecyl sulfate (SDS) to remove adsorbed protein. However, even under the optimal reaction conditions established by the SDS-PAGE analysis, higher molecular weight EGF-PEG complexes are observed by SDS-PAGE and matrix-assisted laser desorption/ionization (MALDI). The presence of these complexes, as well as unreacted growth factor, can lead to a surface of heterogeneous composition. While these surfaces were found to have biological activity, stimulating the adhesion and growth of corneal epithelial cells versus PDMS controls, further optimization of reaction conditions, including the use of a homobifunctional PEG linker and possibly separation of reaction species are required to achieve a uniformly active and well-defined biomaterial surface.

Adsorption↗

PubMed2006

Biofunctionalization of collagen for improved biological response: scaffolds for corneal tissue engineering.

Residual dendrimer amine groups were modified with incorporate COOH group containing biomolecules such as cell adhesion peptides into collagen scaffolds. YIGSR, as a model cell adhesion peptide, was incorporated into both the bulk structure of the gels and onto the gel surface. The effects of the peptide modified collagen gels on corneal epithelial cell behavior were examined with an aim of improving the potential of these materials as tissue-engineering scaffolds. YIGSR was first chemically attached to dendrimers and the YIGSR attached dendrimers were then used as collagen crosslinkers, incorporating the peptide into the bulk structure of the collagen gels. YIGSR was also attached to the surface of dendrimer crosslinked collagen gels through reaction with excess amine groups. The YIGSR modified dendrimers were characterized by H-NMR and MALDI mass spectra. The amount of YIGSR incorporated into collagen gels was determined by (125)I radiolabelling at maximum to be 3.1-3.4 x 10(-2)mg/mg collagen when reacted with the bulk and 88.9-95.6 microg/cm(2) when attached to the surface. The amount of YIGSR could be tuned by varying the amount of peptide reacted with the dendrimer or the amount of modified dendrimer used in the crosslinking reaction. It was found that YIGSR incorporation into the bulk and YIGSR modification of surface promoted the adhesion and proliferation of human corneal epithelial cells as well as neurite extension from dorsal root ganglia.

Amines↗

PubMed2006

Kinetics of in vitro lysozyme deposition on silicone hydrogel, PMMA, and FDA groups I, II, and IV contact lens materials.

We sought to compare the kinetics of in vitro lysozyme deposition on silicone hydrogel (SH), polymethyl methacrylate (PMMA), and FDA groups I, II, and IV contact lenses. Lenses were incubated in 125I-labeled lysozyme for time periods ranging from 1 hr to 28 days, and radioactive counts were determined. SH lenses and PMMA deposited less lysozyme than conventional hydrogel lenses (p < 0.05). Lysozyme accumulation on group IV lenses reached a maximum on the seventh day and then plateaued, whereas on groups I, II, and SH lenses, deposition continued to increase across all time periods, reiterating that kinetics of lysozyme deposition is highly material dependent.

Biocompatible Materials↗

PubMed2006

Progress in the development of a corneal replacement: keratoprostheses and tissue-engineered corneas.

Rapid progress has been made in the past 5 years in the development of corneal replacements. Traditionally they are divided into two categories, keratoprostheses and tissue-engineered corneal equivalents, as replacement tissues are increasingly in demand worldwide. There are currently several different keratoprosthesis models in clinical use around the world. The most popular and most widely publicized is the AlphaCor model, which has enjoyed significant clinical success. However, improvements remain to be made, and the aim of most of the current research is to better understand the interactions between a synthetic material and the surrounding biology on a more fundamental level. This improved understanding will no doubt lead to improvements in current models and to the development of new models in the near future. While tissue-engineered corneal equivalents have been under investigation for considerably less time, there is growing evidence to suggest that a tissue-engineered corneal equivalent comprised of primarily natural materials will exist in the not too distant future. Research groups have reported strong in vitro and in vivo results. The strength of the collagen matrix and its ability to support cell infiltration have been the primary avenues of research. Various collagen crosslinking techniques have been used. Infiltration of three major cells of the cornea has been observed. Most importantly, the ability of these materials to support nerve ingrowth has been demonstrated. While challenges remain with both types of corneal replacements, the considerable progress in the recent past suggests that reliable implants for the treatment of a variety of corneal diseases will be available. This review will provide an overview of recent results, and will provide insight into the future of research on corneal replacements.

Animals↗

PubMed2005

In situ neutron reflectometry investigation of gold-chemisorbed PEO layers of varying chain density: relationship of layer structure to protein resistance.

In work reported previously [L.D. Unsworth, H. Sheardown, J.L. Brash, Langmuir 21 (2005) 1036] we investigated protein interactions with polyethylene oxide (PEO) layers formed by chemisorption of thiol-PEO on gold. It was shown that, as a function of surface chain density, protein adsorption passed through a minimum. In follow-on work reported here, neutron reflectometry (NR) was used to investigate the formation and properties (volume fraction and chain density) of such PEO layers in situ, i.e., in contact with water. Chain density was varied by varying solubility conditions (far from and near the cloud point) and chemisorption time. Neutron experiments were carried out using neutrons of de Broglie wavelength 2.37 A. Contrast matching techniques were used to improve sensitivity. Layers formed under high solubility conditions were found to have PEO volume fraction, layer thickness and chain density of 0.33, 28 A, and 0.56 chains/nm2, respectively, after 0.5 h chemisorption; and 0.31, 28.5 A, and 0.59 chains/nm2, respectively, after 11 h, suggesting that the layer is fully formed within 0.5 h. Both chain density and PEO volume fraction in the chemisorbed layers were significantly greater when the layers were formed under low solubility conditions. The PEO layers shown in our previous work to have maximum protein resistance were found to have a PEO volume fraction of approximately 40%. Moreover the limiting volume fraction in the PEO films formed under low solubility conditions was approximately 57%, a value similar to the solubility limit of PEO in aqueous solution, suggesting that local regions in the layers may be phase separated under these conditions. This may result in increased hydrophobicity and may explain why protein adsorption was found to increase on the layers of higher chain density.

Adsorption↗

PubMed2005

Polyethylene oxide surfaces of variable chain density by chemisorption of PEO-thiol on gold: adsorption of proteins from plasma studied by radiolabelling and immunoblotting.

The mechanisms involved in the inhibition of protein adsorption by polyethylene oxide (PEO) are not completely understood, but it is believed that PEO chain length, chain density and chain conformation all play a role. In this work, surfaces formed by chemisorption of PEO-thiol to gold were investigated: the effects of PEO chain density, chain length (600, 750, 2000 and 5000 MW) and end-group (-OH, -OCH3) on protein adsorption from plasma are reported. Similar to previous single protein adsorption studies (L.D. Unsworth et al., Langmuir 2005;21:1036-41) it was found that, of the different surfaces investigated, PEO layers formed from solutions near the cloud point adsorbed the lowest amount of fibrinogen from plasma. Layers of hydroxyl-terminated PEO of MW 600 formed under these low solubility conditions showed almost complete suppression (versus controls) of the Vroman effect, with 20+/-1 ng/cm2 adsorbed fibrinogen at the Vroman peak and 6.7+/-0.6 ng/cm2 at higher plasma concentration. By comparison, Vroman peak adsorption was 70+/-20 and 50+/-3 ng/cm2, respectively, for 750-OCH3 and 2000-OCH3 layers formed under low solubility conditions; adsorption on these surfaces at higher plasma concentration was 16+/-9 and 12+/-3 ng/cm2. Thus in addition to the effect of solution conditions noted previously, the results of this study also suggest a chain end group effect which inhibits fibrinogen adsorption to, and/or facilitates displacement from, hydroxyl terminated PEO layers. Fibrinogen adsorption from plasma was not significantly different for surfaces prepared with PEO of molecular weight 750 and 2000 when the chain density was the same ( approximately 0.5 chains/nm2) supporting the conclusion that chain density may be the key property for suppression of protein adsorption. The proteins eluted from the surfaces after contact with plasma were investigated by SDS-PAGE and immunoblotting. A number of proteins were detected on the various surfaces including fibrinogen, albumin, C3 and apolipoprotein A-I. The blot responses were zero or weak for all four proteins of the contact system; some complement activation was observed on all of the surfaces studied.

Adsorption↗

PubMed2005

Protein resistance of surfaces prepared by sorption of end-thiolated poly(ethylene glycol) to gold: effect of surface chain density.

Nonspecific protein adsorption generally occurs at the biomaterial-tissue interface and usually has adverse consequences. Thus, surfaces that are protein-resistant are eagerly sought with the expectation that these materials will exhibit improved biocompatibility. Surfaces modified with end-tethered poly(ethylene oxide) (PEO) have been shown to be protein-resistant to some degree. Although the mechanisms are unclear, it has been suggested that chain length, chain density, and chain conformation are important factors. To investigate the effects of PEO chain density, we selected a model system based on the chemisorption of chain-end thiolated PEO to a gold substrate. Chain density was varied by varying PEO solubility (proximity to cloud point) and incubation time in the chemisorption solution. The adsorption of fibrinogen and lysozyme to these surfaces was investigated. It was found that for 750 and 2000 MW PEO layers, resistance to fibrinogen increased with chain density and was maximal at a density of approximately 0.5 chains/nm(2) (80% decrease in adsorption compared to unmodified gold). As PEO chain density increased beyond 0.5/nm(2) adsorption increased. For PEO of 5000 MW the optimal chain density was 0.27/nm(2) and gave only a 60% reduction in fibrinogen adsorption. It is suggested that, at high chain density, the chemisorbed PEO is dehydrated giving a surface that is no longer protein resistant. The PEO-modified surfaces were found also to be resistant to lysozyme adsorption with reductions similar to, if somewhat less than, those for fibrinogen. The fibrinogen to lysozyme molar ratios were within the expected range for close-packed layers of these proteins in their native conformation and were relatively insensitive to PEO chain density and MW. This may suggest that such adsorption as did occur, even at chain densities giving minimum adsorption, may have been on patches of unmodified gold.

Adsorption↗

PubMed2005

Chemisorption of thiolated poly(ethylene oxide) to gold: surface chain densities measured by ellipsometry and neutron reflectometry.

Physical property studies of surfaces formed by chemisorption of polyethylene oxide (PEO) onto gold are reported. Such surfaces have potential as model materials for elucidation of the mechanism of resistance to protein adsorption by PEO surfaces. Thiolated monomethoxy poly(ethylene oxide) (PEO) was chemisorbed onto gold-coated silicon wafers under various conditions such that different surface chain densities were achieved. Chain density was varied by controlling PEO solubility (proximity to cloud-point conditions) as well as chemisorption time. Films prepared with PEO of molecular weight 750, 2000, and 5000 g/mol were studied. Chain densities determined in the dry state by ellipsometry were found to be in the range of 0.4-0.7, 0.33-0.58, and 0.12-0.30 chains/nm(2) for MW 750, 2000, and 5000 PEO, respectively. Chain density was found to decrease with increasing molecular weight and to increase as cloud-point conditions were approached. PEO-layer mass densities and chain densities were determined independently by neutron reflectometry. Under low-solubility conditions and for a 4-h chemisorption time, film mass and chain density values of 1.0 +/-0.3 g cm(-3) and 1.8 +/- 0.9 chains/nm(2) were found for MW 750 PEO, and 0.82 +/- 0.02 g cm(-3) and 0.23 +/- 0.07 chains/nm(2) for MW 5000 PEO. Ellipsometry data for these systems yielded graft densities of 0.63 +/- 0.13 and 0.30 +/- 0.02 chains/nm(2), respectively. Using the mass densities obtained from the neutron data in the ellipsometry calculations, chain densities of 0.6 +/- 0.3 and 0.25 +/- 0.02 chains/nm(2), respectively, were obtained for the MW 750 and 5000 films. The ellipsometry and neutron data for the MW 5000 system are thus in agreement within experimental error. In general, the chain-density values are much higher than those corresponding to layers of unperturbed random coil PEO ("mushrooms"), suggesting that the PEO layers are in the brush regime with the chains in an extended conformation.

Journal Article↗

PubMed2005

Protein repellant silicone surfaces by covalent immobilization of poly(ethylene oxide).

Polydimethylsiloxane elastomers were surface modified with passivating polyethylene oxide (PEO) polymers of different molecular weights, both monofunctional and bifunctional. Following the introduction of Si-H groups on the surfaces by acid-catalyzed equilibration in the presence of polymethylhydrosiloxane, the PEO was linked by platinum-catalyzed hydrosilylation. ATR-FTIR, X-ray photoelectron spectroscopy (XPS) and water contact angle results confirmed that the PEO was successfully grafted to the silicone rubber. Atomic force microscopy and XPS suggested that surface coverage with PEO was very high on the modified surfaces but not complete. The protein-resistant properties of the PEO-modified surfaces were demonstrated by measuring the adsorption of fibrinogen from both buffer and plasma. Fibrinogen adsorption from buffer to the PEO-modified surfaces was reduced by more than 90% compared with controls.

Adhesiveness↗

PubMed2005

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer.

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 microg/cm2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.

Adsorption↗

PubMed2005

Building in vitro models of organs.

Tissue-engineering techniques are being used to build in vitro models of organs as substitutes for human donor organs for transplantation as well as in vitro toxicology testing (as alternatives to use of animals). Tissue engineering involves the fabrication of scaffolds from materials that are biologically compatible to serve as cellular supports and microhabitats in order to reconstitute a desired tissue or organ. Three organ systems that are currently the foci of tissue engineering efforts for both transplantation and in vitro toxicology testing purposes are discussed. These are models of the cornea, nerves (peripheral nerves specifically), and cardiovascular components. In each of these organ systems, a variety of techniques and materials are being used to achieve the same end results. In general, models that are designed with consideration of the developmental and cellular biology of the target tissues or organs have tended to result in morphologically and physiologically accurate models. Many of the models, with further development and refinement, have the potential to be useful as functional substitute tissues and organs for transplantation or for in vitro toxicology testing.

Animals↗

PubMed2005

Surface properties of PEO-silicone composites: reducing protein adsorption.

Silicone-based polymers with reduced protein adsorption were successfully prepared by incorporating mono- or bifunctional poly(ethylene oxide) (PEO) derivatives, respectively, into PDMS during rubber formation using classic room temperature vulcanization chemistry. Characterization of the films by water contact-angle measurements and XPS showed that the PEO was present on the film surface, with greater amounts of PEO at the interface modified with monofunctional PEO. Scanning electron microscopy showed the PEO domains segregated into regular zigzag patterns on the PEO-modified surfaces. Significant reductions in the adsorption of fibrinogen, albumin and lysozyme were observed on both PEO-modified surfaces, although the monofunctional PEO surfaces performed much better in this regard. The reductions in protein adsorption were comparable for all three proteins on both surfaces, suggesting that molecular mass of the protein is not a significant factor in determining the magnitude of protein deposition. Western blot studies showed that the adsorption of proteins from plasma to the monofunctional PEO-modified surfaces was also significantly reduced and surprisingly selective, with very few bands noted relative to the control surfaces and those modified with bifunctional PEO.

Adsorption↗

PubMed2004

Silicone elastomers for reduced protein adsorption.

Monofunctional poly(ethylene oxide) polymers of molecular weight (MW) 350, 750, and 2000, respectively, were modified with Si(OEt)3 groups. These polymers underwent classic condensation cure with hydroxy-terminated silicone polymers and Si(OEt)4 to give composites with poly(ethylene oxide) (PEO) rich surfaces under aqueous conditions, as shown by contact angle and XPS data. The hydrophobicity of the surfaces was considerably higher in air. The greatest PEO concentration was observed with relatively short chain polymers of MW 350. Silicone polymers bearing short chain PEO chains were also observed to be the most protein rejecting from either buffer (fibrinogen) (90%) or plasma (85%). The silicone/TES-MPEO formulation offers the advantage of a one step/one shot polymerization process that gives materials with a high protein rejection ability than can be cast as films, or molded into complex shapes. Covalently linked PEO films of a variety of chain lengths and total surface coverage can be readily accommodated.

Adsorption↗

PubMed2004

Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology.

A number of growth factors and their associated receptors, including epidermal growth factor, transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor and platelet-derived growth factor have been detected in the anterior segment of the eye. On binding to cellular receptors, these factors activate signalling cascades, which regulate functions including mitosis, differentiation, motility and apoptosis. Production of growth factors by corneal cells and their presence in the tear fluid and aqueous humour is essential for maintenance and renewal of normal tissue in the anterior eye and the prevention of undesirable immune or angiogenic reactions. Growth factors also play a vital role in corneal wound healing, mediating the proliferation of epithelial and stromal tissue and affecting the remodelling of the extracellular matrix (ECM). These functions depend on a complex interplay between growth factors of different types, the ECM, and regulatory mechanisms of the affected cells. Imbalances may lead to deficient wound healing and various ocular pathologies, including edema, neovascularization and glaucoma. Growth factors may be targeted in therapeutic ophthalmic applications, through exogenous application or selective inhibition, and may be used to elicit specific cellular responses to ophthalmic materials. A thorough understanding of the mechanism and function of growth factors and their actions in the complex environment of the anterior eye is required for these purposes. Growth factors, their function and mechanisms of action as well as the interplay between different growth factors based on recent in vitro and in vivo studies are presented.

Animals↗

PubMed2003

Cellular and nerve regeneration within a biosynthetic extracellular matrix for corneal transplantation.

Our objective was to determine whether key properties of extracellular matrix (ECM) macromolecules can be replicated within tissue-engineered biosynthetic matrices to influence cellular properties and behavior. To achieve this, hydrated collagen and N-isopropylacrylamide copolymer-based ECMs were fabricated and tested on a corneal model. The structural and immunological simplicity of the cornea and importance of its extensive innervation for optimal functioning makes it an ideal test model. In addition, corneal failure is a clinically significant problem. Matrices were therefore designed to have the optical clarity and the proper dimensions, curvature, and biomechanical properties for use as corneal tissue replacements in transplantation. In vitro studies demonstrated that grafting of the laminin adhesion pentapeptide motif, YIGSR, to the hydrogels promoted epithelial stratification and neurite in-growth. Implants into pigs' corneas demonstrated successful in vivo regeneration of host corneal epithelium, stroma, and nerves. In particular, functional nerves were observed to rapidly regenerate in implants. By comparison, nerve regeneration in allograft controls was too slow to be observed during the experimental period, consistent with the behavior of human cornea transplants. Other corneal substitutes have been produced and tested, but here we report an implantable matrix that performs as a physiologically functional tissue substitute and not simply as a prosthetic device. These biosynthetic ECM replacements should have applicability to many areas of tissue engineering and regenerative medicine, especially where nerve function is required.

Animals↗

PubMed2002

Artificial human corneas: scaffolds for transplantation and host regeneration.

PURPOSE: To review the development of artificial corneas (prostheses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. METHODS: Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. RESULTS: Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. CONCLUSIONS: Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

Animals↗