El-Husseiny, M; (2015) Effects of Dual Coating Proteins in Intraosseous Transcutaneous Amputation Prosthesis (ITAP). Doctoral thesis , UCL (University College London).

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Abstract

Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative method of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including pressure sores, neuroma formation and tissue necrosis. ITAP overcomes these problems by attaching the articial limb transcutaneously to the skeleton. In order for ITAP to be successful, it requires an infection-resistant transcutaneous barrier at the skin-implant interface. Fibronectin (Fn) and Laminin 332 (Ln), are glycoproteins found abundantly in the extracellular matrix. Dual coating proteins 125 I-Fn + Ln and 125 I-Ln +Fn were covalently bonded to Ti6Al4V through silanization. The hypothesis tested was: silanized dual coating protein coatings with fibronectin and laminin, enhances both keratinocyte and fibroblast spreading and increases vinculin focal adhesion plaques on Ti6Al4V in vitro. Both remained stable when immersed in foetal calf serum compared with adsorbed dual coating proteins at all time points up to 72 hours (p<0.05). There was non-competitive binding of laminin on Ti6Al4V in the presence of fibronectin. Keratinocytes and fibroblasts were grown on Ti6Al4V surfaces with single coating Fn, Ln, and dual coating FnLn on adsorbed, silanized with passivation and silanized without passivation discs. Vinculin focal adhesion markers and cell size were quantified. Silanized dual coating proteins without passivation (SiFnLn-) produced the largest number of vinculin markers and biggest cell size at all time points upt to 24 hours (p<0.05). Hydroxyapatite (HA) is a naturally occurring osteoinductive mineral in the body. 125 I-Fn coated on HA discs was assessed for optimal time for loading, concentration and durability. Fibroblasts were grown on polished, HA and Fn coated HA discs. Vinculin markers and cell size were quantified. Fn coated HA discs increased fibroblast attachment compared to uncoated controls of Ti6Al4V discs and HA discs (p<0.05). My thesis demonstrated silanized without passivation dual coating proteins FnLn produced more viculin markers per cell unit and per cell area when compared to uncoated controls and single coating proteins on adsorbed and silanized, passivated discs. Further research is required to establish whether dual coating proteins will produce the same effect in vivo. This can be achieved by silanizing ITAP with dual coating FnLn and implanting them in animals. Histopathological analysis at the skin-implant interface would provide valuable information whether this biochemical and physical modification improve soft tissue integration to percutaneous implants.

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