Abstract
Introduction
Intraosseous transcutaneous amputation prostheses (ITAP) provide an alternative means of attaching artificial limbs for amputees. Conventional stump-socket devices are associated with soft tissue complications including; pressure sores and tissue necrosis. ITAP resolves these problems by attaching the exo-prosthesis transcutaneously to the skeleton. The aim of this study is to increase the attachment of dermal fibroblasts to titanium alloy in vitro. Fibronectin (Fn) and laminin 332 (Ln) enhance early cell growth and adhesion. We hypothesize that silanized dual coatings of fibronectin and laminin (SiFnLn) will be more durable when compared with adsorbed dual coating (AdFnLn), and will enhance early fibroblast growth and adhesion compared to single coatings.
Methods
The kinetics of dual single and dual protein coating attachment onto titanium alloy was quantified on silanized 10mm diameter discs using radiolabelled Fn (125I-Fn) and Ln (125I-Ln). Sixty discs were polished, sterilized and silanized. Coating durability was assessed when soaked in fetal calf serum (FCS) for 0, 1, 24, 48 and 72hrs. Data was compared to un-silanized Ti discs with the same coatings. Five thousand human dermal fibroblasts were seeded on discs (n = 6) of Ti polished alone (Pol), Ti with adsorbed fibronectin (AdFn), Ti with adsorbed laminin (AdLn), Ti adsorbed dual coating (AdFnLn), Ti silanized (Si), Ti silanized with fibronectin (SiFn), Ti silanized with laminin (SiLn), Ti silanized with a dual coating (SiFnLn) for 24hrs. In order to measure cell adhesion fibroblasts were fixed, vinculin stained using mouse vinculin antibody and alexa fluor. Axiovision Image Analysis software was used to measure cell area, vinculin focal adhesion markers per cell and per unit cell area. Data was analysed in SPSS and significance was assumed at the 0.05 level.
Results
Silanized dual coatings bonded to Ti alloy in significantly larger quantities compared with adsorbed coatings at all time points (all p values < 0.05). Fibroblasts cultured on dual coatings were significantly larger, produced more vinculin markers per cell, and per unit cell area compared with single coatings. Cells on SiFnLn were larger with more numerous vinculin markers per cell, and per unit cell area compared with AdFnLn (p<0.05).
Conclusion
This study has demonstrated that covalently bonding both fibronectin and laminin to Ti alloy provides a durable, dual coating that enhances early fibroblast growth and attachment compared with either protein coating alone in vitro. Our study showed that there is non-competitive binding of laminin on Ti surfaces in the presence of fibronectin. Dual coatings may be applied to the skin-penetrating region of transcutaneous devices to improve the skin seal and this may have positive implications for the development of ITAP.
