CELL ADHESION TO TITANIUM ALLOY: BIOPHYSICAL QUANTIFICATION

Abstract

Introduction: Cell adhesion to titanium alloy implants is important in osseointegration [1,2] and attachment of the soft tissues to skin penetrating implants e.g. external fixator pins and Intraosseous Transcutaneous Amputation Prostheses [3,4]. Cell adhesion can be assessed using cell area data and immunolocalisation of focal contact proteins e.g. vinculin; however no method of assessing biophysical attachment is performed routinely. Cell adhesion can be enhanced with adhesion proteins including fibronectin (Fn)[5]. We have previously shown that covalently binding Fn to titanium also increases cell adhesion, and produces a more robust protein coating [6]. However the strength of adhesion of cells to this coating has not been measured. Our hypothesis was that biophysical cell adhesion measured using novel radial flow apparatus would correlate with cell area and focal contact data and that covalently bound fibronectin substrates would increase cell adhesion compared with adsorbed and uncoated controls.

Method: Dermal fibroblasts were cultured for 1, 4, and 24 hours on 30mm and 10mm diameter polished titanium alloy discs (n = 6). Cells on 30mm discs were calcein stained and subjected to shear stress in a submerged, media filled, custom-made radial flow apparatus at 37¬C at 1.66ml/s for 15s. Cells were fixed in 10% formal saline and photographs were taken using a tangential light source. Fluorescent microscopy was performed at 2mm intervals along two perpendicular diameters. Using image analysis, the central cell free zone was measured and radial distance and shear stress calculated. Cells on 10mm discs were fixed, permeablised and vinculin stained (mouse vinculin antibody (1:200) 2hrs; FITC mouse antibody (1:100)1hr). Images were analyzed with a Zeiss microscope linked to image analysis software and the number of focal contacts were counted per cell area. The medians of the radial flow data were compared with data for cell area and focal contact production at the same time points using Spearman¡s regression correlation. This method was subsequently used to compare cell adhesion at one hour with adsorbed and covalently bound Fn substrates (10¥ìg/disc).

Results/Discussion: The shear strength of cells increased between 4 and 24hrs (p=0.002) on polished untreated control substrates. Attachment values (dynes/cm2) were 84.90 (73.98–97.19), 96.30 (91.66–100.89), and 136.69 (134.68–140.30) for 1, 4 and 24 hours respectively. At 1hr, covalently bound Fn (509.90 dynes/cm2 (490.55–528.49) significantly increased cell adhesion compared with adsorbed Fn(434.45 dynes/cm2(385.25–465.62)) and control substrates(p=0.002). There was significant correlation between shear stress and focal contacts/cell (1.00(p< 0.01)) and focal contacts/cell area (0.900(p=0.037)), but not cell area (0.600(p=0.285)).

Conclusion: Radial flow measurement is a useful direct method to quantify cell adhesion to orthopaedic implants and correlates well with other methods of measurement. Covalently bound Fn significantly increases biophysical cell attachment compared with adsorbed and uncoated controls.