Novel culture procedure permitting the synthesis of proteins by rat calvarial cells cultured on hydroxyapatite particles to be quantified.
A simple culture procedure and assay conditions are described which have permitted us to quantify the synthesis of proteins which are associated 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 but allows attachment and growth of cells on material particles embedded in it on glass coverslips. Cells were cultured for 27 d on hydroxyapatite particle-coated coverslips. Alkaline phosphatase, osteopontin and collagen type I were monitored in cell lysates from d 10 to d 20. After Western blotting, osteopontin and collagen type I were quantified using 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 comparison of cell behaviour on particulate substrata.
Sammons
R L
RL
School of Biological Sciences, University of Birmingham, UK.
el Haj
A J
AJ
Marquis
P M
PM
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D000818:Animals; D001672:Biocompatible Materials; D015153:Blotting, Western; D002448:Cell Adhesion; D002455:Cell Division; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D017886:Durapatite / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008163:Luminescent Measurements; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D010006:Osteoblasts / Q000166:cytology* / Q000648:ultrastructure; D053495:Osteopontin; D010521:Periosteum / Q000166:cytology; D010641:Phenotype; D051381:Rats; D017208:Rats, Wistar; D012795:Sialoglycoproteins / Q000096:biosynthesis*; D012886:Skull / Q000166:cytology; D013194:Staining and Labeling
D001672:Biocompatible Materials; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D003094:Collagen; D017886:Durapatite; D000469:Alkaline Phosphatase