A Quantitative Method of Measuring Cell-Substrate Adhesion Areas

Variability in measurements of the \u27cell adhesion strength\u27 of fibroblasts to substrates using mechanical disruption techniques causes difficulty in determining precisely the position, in the cytoskeleton-focal adhesion-matrix -substrate interface, where failure has occurred. In the present study, a quantitative in vitro procedure for measuring the total area and percentage of fibroblast adhesion to biomaterials, using the scanning electron microscope (SEM), is described. The amount of adhesion ofL929 and Balb/c3T3 fibroblasts to discs of stainless steel, commercially pure titanium, and polyethylene terepthalate (Thermanox) was quantified. Cells were fixed, stained with heavy metals, dehydrated and embedded in resin. The resin blocks were removed from the substrate and sputter coated. The samples were examined in a field emission SEM using backscattered electron imaging. Demonstration that the cells had been removed with their adhesion sites, was by immunocytochemical labelling of vinculin within the focal adhesions of the embedded ~lis. Quantification of cell adhesion was performed by measuring the total area of each cell (imaged at 15 kV) and the area of their adhesion sites (imaged at 4kV) using an image analysis system. The in vitro results show (under static conditions, with the materials, roughnesses and cell types used) that roughness does not affect the total area of adhesion. The procedure could be applied to any cells that form focal adhesions, such as osteoblasts and may be developed to assess connective tissue adhesion to implant surfaces from in vivo experiments