Novel fluorescent probes for analysis of protein interactions under truly physiological conditions with real medical devices

Abstract

Protein adsorption under physiological conditions was recognised to be the key step in the modulation of biological responses between materials and an osseous environment. Many studies have shown variation of adsorption behaviour on medical materials but most experiments were performed under non-physiological idealised conditions with idealised samples. The aim of this thesis was to develop a method to analyse protein adsorption on real clinically relevant samples under physiologically relevant conditions. The use of fluorescent probes was identified as a methodology which would facilitate analysis under a range of conditions including fully competitive with real samples that required no specialised surface pre-treatment. Fluoresceinthiureidoaminocaproic acid (FTCA) and a sulforhodamine derivative (SR101), were identified as suitable for coupling to proteins. FTCA labelled bovine serum albumin (BSA) was initially used to validate the technique and found to have several advantages over commercially available total protein assays; including greater sensitivity and facilitate its use under competitive conditions. These experiments also confirmed sensitivity to temperature and test media as well as demonstrating that the technique could be used on idealised dense and real porous granular (as used clinically) samples. Conformational changes associated with protein-surface interactions were observed through variation in protein auto-fluorescence and confirmed with CD analysis, therefore, care in selection of appropriate experimental conditions and fluorophore probes was required. Additionally, labelling facilitated the visualisation in differences in the morphological habit of the surface adsorbed protein species. Investigation of differential response in protein exchange with hydroxyapatite (HA) and 0.8wt% silicon substituted hydroxyapatite (SA) with more biologically relevant proteins; such as bone morphogenetic protein-2 (BMP-2), fibronectin (FN) and osteopontin (OPN) individually and/or competitively in phosphate buffered saline (PBS) or minimum Eagles medium (MEM) supplemented with 10 % foetal bovine serum (FBS) demonstrated SA to have a greater capacity to adsorb selected osteogenic proteins under competitive conditions as compared to HA. Particularly interesting was the BMP-2 findings, which highlighted the role of media in promoting BMP-2 adsorption and the conformation sensitivity of traditional ELISA assays giving rise to unreliable results.