A new ⁵¹Chromium assay for accurate measurement of cell attachment to demineralized and non‐demineralized dentine in vitro

Fibroblast‐like cells (RGF) derived from rat gingival explants were labelled with radioactive chromium (⁵¹Cr) and plated into Linbro 96‐well microtitre plates, each well of which contained one 200 μm‐thick slice of root. The root slices were removed at various times, washed, and counted in a gamma scintillation counter to determine the number of attached cells. The results obtained were expressed as counts per unit area of root, which could be converted to cell number per unit area. A linear correlation was established between the number of cells plated and the number of cells that attached in the range 0.6 to 10 × 10⁴ cells per well. Visual corroboration of this relationship was provided by examination of root slices using scanning electron microscopy. A significant proportion of cells was found by this assay not to be removed from the slices of tooth using standard trypsinization procedures. In the case of demineralized tissue, more cells remained adherent to the root slices than were removed. Consequently, trypsinization and counting procedures as they are normally employed no longer provide an acceptable assay for cell attachment to root surfaces. Cell attachment to root slices demineralized for 1/2 h at 37°C with ethylenediaminetetraacetic acid (EDTA) was compared with attachment to untreated roots. It was found that there was no significant difference in the number of cells attaching to undemineralized compared to demineralized roots over a 4 h period. Consequently, greater rates of migration and division as well as chemotaxis, rather than attachment, may account for the improved healing obtained with demineralized root surfaces as compared to undemineralized surfaces in experimental wounds of the periodontium.