Method for selecting populations of rat articular chondrocytes that exhibit distinct growth and metabolic characteristics, and their responses to growth factors, PMA and vitamin D3.
Chondrocytes were released from articular cartilage fragments of 6-week-old Wistar rats by a 2-hour treatment with bacterial collagenase. The cells from one animal were seeded in a 25-cm2 culture flask at a density of 10(5) cells/cm2. After 1 h, the flask was gently shaken and the medium, containing nonadherent cells, was transferred to a new flask. The attached cells were incubated with 5 ml of fresh medium. This procedure was repeated after 3, 24, 48 and 96 h. Resulting cell populations were then analyzed. The earlier cells attached, the more rapidly they proliferated, and the less collagen and proteoglycan (PG) they produced. The cells that attached after 24 h grew much more slowly, piled up in many areas, exhibited strong alkaline phosphatase activity and calcified extracellular matrix (ECM). Differences in deoxyribonucleic acid (DNA) and protein/PG synthesis were also observed when these cell populations were challenged with growth factors and 12-myristate 13-acetate (PMA). Pretreatment of cells for 2 h with PMA strongly enhanced DNA and PG synthesis only in cultures containing insulin-like growth factor-1. Nonselected rat articular chondrocytes (AC) subcultured at least four times as monolayers still expressed mRNA specific for aggrecan and type II collagen, suggesting conservation of the chondrogenic phenotype. In conclusion, AC of young individuals seem to be heterogeneous with respect to their capacity to proliferate and synthesize ECM. By selecting and expanding in vitro the appropriate cell population, this method could be potentially useful for studies aimed at repairing damaged cartilage.
U606 INSERM, Lariboisiere Hospital, Paris, France.
Andrei
A
Panasyuk
Abdel-Majid
AM
Khatib
Gladys
G
Colantuoni
Maritza
M
Quintero
Abderrahim
A
Lomri
Dragoslav R
DR
Mitrovic
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000494:pharmacology*; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology*; D008297:Male; D011509:Proteoglycans / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D006133:Growth Substances; D011509:Proteoglycans; D002762:Cholecalciferol; D003094:Collagen; D000469:Alkaline Phosphatase; D013755:Tetradecanoylphorbol Acetate