|dc.description.abstract||All medical devices that pass through skin are plagued by infection and other problems at the skin interface. Such devices include intravenous lines, catheters, robotics, osseointegrated rods, bone anchored hearing aids, and tracheostomy tubes. A robust and functional skin interface could permit long-term infection-free implantion of these types of devices.
Failure of skin attachment to percutaneous devices results from skin avulsion, infection, and epidermal marsupialisation. Prevention of marsupialisation requires epidermal cell (keratinocyte) attachment and viable (living dynamic) subepidermal tissue integration into implant pores. To address these problems, a structure was designed, a “cap-scaffold,” to permit dynamic tissue integration aided by negative pressure wound therapy (NPWT). While the design and testing of a skin-material interface system for use with NPWT was the ultimate destination of this thesis, surface-optimisation to improve epidermal attachment was thought to be important. Hence, the majority of work reported herein preceded in vivo work and involved surface optimisation techniques and their measurement: a method of surface functionalisation with thiol groups and the conjugation of collagen and biotin to these groups with maleimide linkers; the novel use of conjugated collagen immunoassay as a method for assessing the efficacy of surface functionalisation; the novel finding that collagen 4 (C4) is more stable than collagen 1 (C1) in both adsorbed and covalently-bound forms; and the assessment of keratinocyte responses to collagen and laminin-332 (L332).
Thiol groups can undergo a large variety of chemical reactions. They are used commonly in solution phase to conjugate bioactive molecules. Previous research on solid substrates with continuous phase glow discharge polymerisation of thiol-containing monomers might have been compromised by oxidation. Thiol surface functionalisation via glow discharge polymerisation has been reported as requiring pulsing. Herein, continuous phase glow discharge polymerisation of allyl mercaptan (2-propene-1-thiol) was used to generate significant densities of thiol groups on a mixed macrodiol polyurethane and tantalum. Three general classes of chemistry are used to conjugate proteins to thiol groups, with maleimide linkers being used most commonly. Herein the pH specificity of maleimide reactions was used effectively to conjugate surface-bound thiol groups to amine groups in collagen. XPS demonstrated surface-bound thiol groups without evidence of oxidation, along with the subsequent presence of maleimide and collagen. Glow discharge reactor parameters were optimised by testing the resistance of bound collagen to degradation by 8 M urea. The nature of the chemical bonding of collagen to surface thiol groups was effectively assessed by colourimetric assay (ELISA) of residual collagen after incubation in 8 M urea over eight days and after incubation with keratinocytes over fifteen days. The facile creation of useable solid-supported thiol groups via continuous phase glow discharge polymerisation of allyl mercaptan opens a route for attaching a vast array of bioactive molecules.
Traditional methods of assessing surface functionalisation, including spectroscopy and chemical labelling, often involve significant error and conjecture about bonds. Proteins that improve cell attachment have specific pKa’s and optimum binding requirements that may differ from the conditions required for chemical labelling. The utility of collagen ELISA to optimise acetaldehyde glow discharge polymerisation (Aapp) reactor parameters was tested. Accurate stepwise increases in collagen conjugation strength were demonstrated by incubating specimens in 8 M urea for 5-8 days followed by ELISA to test for residual surface collagen. Surface modifications also were assessed by X-ray photoelectron spectroscopy (XPS). Results suggested that ELISA after bond-stressing with urea may suffice for optimising surface functionalisation and that traditional methods of analysis may be superfluous if protein conjugation is the aim of functionalisation.
C1 is used commonly to improve biological responses to implant surfaces. The stability of C1 was compared with C4 on a Elast-Eon™, both adsorbed and covalently bound via Aapp and reductive amination. Substrate specimens were incubated in solutions of C1 and C4. The strength of conjugation was tested by incubation in 8 M urea followed by ELISA to measure residual C1 and C4. L332 was superimposed via adsorption on C4-treated specimens. Keratinocytes were grown on untreated, C1-treated, C4-treated, and C4 + L332 treated specimens, followed by measurement of cell area, proliferation, and focal adhesion density. Adsorbed C4 was shown to be significantly more stable than C1 and covalent conjugation conferred even greater stability, with no degradation of C4 over twenty days in 8 M urea. Cell growth was similar for C1 and C4, with no additional benefit conferred by superimposition of L332. The greater resistance of C4 to degradation may be consequent to cysteine residues and disulphide bonds in its non-collagenous domains. The use of C4 on implants, rather than C1, may improve their long-term stability in tissues.
Following the completion of in vitro research and armed with the improved stability findings of covalently bound C4, thesis work progressed to pig studies. Six wounds were made on the backs of each of four 3-month old pigs. Four unmodified (no caps) scaffolds were implanted along with 20 cap-scaffolds. C4 was attached to 21 implants. NPWT then was applied. Structures were explanted and assessed histologically at day 7 and day 28. At day 28, there was close tissue apposition to scaffolds, without detectable reactions from defensive or interfering cells. Three cap-scaffolds explanted at day 28 showed likely attachment of epidermis to the cap or cap-scaffold junction, without deeper marsupialisation. NPWT appeared to facilitate dynamic integration with macroporous scaffolds, epidermis appeared to attach to caps in most full-thickness implanted cap-scaffolds, and there was no infection or inflammation at 28 days. This time span was insufficient to permit conclusions about long-term infection risk, but skin-interfaced devices implanted for 28 days could still be useful in clinical practice, e.g. a course of chemotherapy or dialysis. Implant numbers were insufficient to reveal effects of conjugated C4 on epidermal attachment in vivo. Given the improved epidermal attachment to implanted contact lenses optimised with C1 reported by others, the effects of conjugated C4 on skin-material interfacing warrants further investigation. While numbers were limited, qualitative results suggested that a robust skin-material interface system might be within reach using toric-shaped cap-scaffolds with NPWT.||en_US
|dc.subject||cell adhesion, collagen, glow discharge polymerisation, implant interface, percutaneous, pigs, scaffolds, surface analysis, surface modification, surgery||en_US