Rl. Sammons et al., NOVEL CULTURE PROCEDURE PERMITTING THE SYNTHESIS OF PROTEINS BY RAT CALVARIAL CELLS CULTURED ON HYDROXYAPATITE PARTICLES TO BE QUANTIFIED, Biomaterials, 15(7), 1994, pp. 536-542
A simple culture procedure and assay conditions are described which ha
ve permitted us to quantify the synthesis of proteins which are associ
ated with an osteoblastic phenotype, by rat calvarial periosteal cells
grown on particulate materials. The main feature of the method is the
use of an adhesive which does not permit cells to attach to itself bu
t allows attachment and growth of cells on material particles embedded
in it on glass coverslips. Cells were cultured for 27 d on hydroxyapa
tite particle-coated coverslips. Alkaline phosphatase, osteopontin and
collagen type I were monitored in cell lysates from d 10 to d 20. Aft
er Western blotting, osteopontin and collagen type I were quantified u
sing specific antisera and enhanced chemiluminescence. Maximum levels
coincided with peak alkaline phosphatase activity, after 10 and 17 d.
The procedures described will be generally applicable to the compariso
n of cell behaviour on particulate substrata.