Nial A. Bullett

Adsorption of vitronectin, collagen and immunoglobulin-G to plasma polymer surfaces by enzyme linked immunosorbent assay (ELISA)

Polymeric functional thin films have been deposited from plasmas of allyl alcohol, acrylic acid, allylamine and octa-1,7-diene onto polystyrene microwells and aluminium foil, and analysed by X-ray photoelectron spectroscopy (XPS) and water contact angle measurement. The films were found to be conformal and pin-hole free. Advancing and receding water contact angles measured in air showed that the surfaces had a range of hydrophilicities. The adsorption of human vitronectin, human immunoglobulin G (IgG) and heat-denatured bovine type II collagen to the different plasma polymer surfaces from single solutions was compared by enzyme linked immunosorbent assay (ELISA). Results demonstrate that the adsorption of proteins depends not only on the chemistry of the surface, but also on the nature of the protein. Vitronectin adsorbed most extensively to the acrylic acid-deposited surface, while immunoglobulin G adsorbed more readily to the allylamine deposited surface. The functionalised surfaces performed poorly in terms of collagen binding, with much higher levels of adsorption to the hydrocarbon (octa-1,7-diene) plasma polymer, and the uncoated polystyrene control wells. The amount of adsorbed protein detected on a surface is often explained in terms of surface hydrophilicity/hydrophobicity. The results of this study show that adsorption of these proteins is not simply a matter of wettability, but relates more to the chemical functionality of the surface, which in turn affects a number of surface properties, including wettability, surface charge and pKa.

Adsorption of immunoglobulin G to plasma-co-polymer surfaces of acrylic acid and 1,7-octadiene

Functionalised thin films of copolymers of acrylic acid and 1,7-octadiene have been deposited by plasma-co-polymerisation to create a range of surfaces of well-defined chemistry, containing carboxyl functional groups. X-Ray photoelectron spectroscopy (XPS) has been used to characterise the deposits. Adsorption of immunoglobulin G from single solution has been studied by indirect, antibody detection (ELISA), and by direct, radio-labelling and fluorescent detection methods. In addition, XPS analysis of the surface adsorbed protein was also performed. Direct measurement techniques showed an apparent decrease in protein binding on carboxylic acid functionalised surfaces, whilst antibody detection showed an increase in binding with increased carboxylic acid content of the surface. This difference has been ascribed to orientational/conformational and/or packing differences of the proteins on the different surfaces, leading to an increased biological (“functional”) activity on the carboxylic acid functionalised surfaces. Adsorption of IgG from serum has also been probed by ELISA. An increase in IgG binding with surface carboxylic acid content was measured, indicating a similar effect when IgG is adsorbed from a complex protein mixture.

Simplifying the Delivery of Melanocytes and Keratinocytes for the Treatment of Vitiligo Using a Chemically Defined Carrier Dressing

Obtaining pigmentary function in autologous skin grafts is a current challenge for burn surgeons as is developing reliable robust grafting strategies for patients with vitiligo and piebaldism. In this paper, we present the development of a simple methodology for delivering cultured keratinocytes and melanocytes to the patient that is of low risk for the patient but also user friendly for the surgeon. In this study, we examined the ability of keratinocytes and melanocytes to transfer from potential cell carriers under different media conditions to an in vitro human wound bed model. The number of melanocytes transferred, their location within the neoepidermis, and their ability to pigment were evaluated as preclinical end points. Two inert substrates (polyvinyl chloride and silicone sheets) and three candidate plasma-polymerized coatings with controlled surface chemistry deposited on these substrates were explored. Two media for expansion of cells, Greens, currently used clinically (but which contains fetal calf serum), and a serum-free alternative, M2 (melanocyte medium), were explored. Reproducible transfer of physiologically relevant numbers of melanocytes capable of pigmentation from the coculture of melanocytes and keratinocytes was obtained using either Greens medium or M2 medium, and a silicone carrier pretreated with 20% carboxylic acid deposited by plasma polymerization.