Abstract
A non-invasive technique for labelling S phase osteoblasts in vitro following immunolabelling of their focal adhesions is proposed. Quantification of cell adhesion area in the S phase (where the cells are most spread) of the cell cycle is then possible with a scanning electron microscope (SEM).
Primary calvarial osteoblasts (isolated by migration) were cultured on plastic and implant quality metal discs. S-phase cells were labelled by a pulse of 3H thymidine in the culture medium for 30 min. Cells were cultured for a further 2h in normal media before being processed for immunogold labelling of vinculin. Briefly, cells were permeabilised and fixed in 4% paraformaldehyde. Non specific binding sites were blocked for 30 min. Cells were incubated with mouse anti vinculin for 1h before rinsing and blocking with 5% goat serum for 30 min. Secondary incubation was with goat anti mouse 5nm gold conjugate for 2h. After rinsing, cells were permanently fixed with 2.5% glutaraldehyde. For SEM visualisation, the gold label was enhanced with gold enhance solutions. Postfixation and staining was performed with osmium tetroxide. Samples were dehydrated and critically point dried. The discs were carbon coated and covered with a thin layer of photographic emulsion in a dark room and left in a light tight box at 4°C for 7 days before developing the emulsion.
Backscattered electron imaging with the SEM revealed silver grains on the nuclei of S-phase cells, produced by the interaction of radioactive emissions, from the labelled DNA, and the photographic emulsion. Immunolabelled focal adhesions were also observed at higher magnifications on the same cells.
This combination of autoradiography and high resolution SEM removes cell cycle variability, which has been a problem with previous in vitro adhesion studies. This method will be applied to quantify osteoblast cell adhesion to various implant materials to evaluate cell/implant interactions.
