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

Abstract

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 10⁵ 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.

Morphology and Function of Ovine Articular Cartilage Chondrocytes in 3-D Hydrogel Culture

“…After 10 weeks, we found a low proliferation rate together with an increased cell density and predominantly FRM matrix deposition. This situation complies with the fi ndings by Panasyuk et al [2004]: highly proliferating chondrocytes produce less matrix and vice versa. But it is not yet understood by which mechanisms ECM and proliferation are mutually regulated [Qi and Scully, 2003].…”

“…It remains to be shown whether this simplifi cation is due to species differences (sheep vs. rabbit/human/canine) or not [Giannoni et al, 2005]. The heterogeneity of articular chondrocytes may also play a role in cell survival, proliferation and matrix synthesis [Panasyuk et al, 2004]. In addition, mitotic activity, seeded cell density and ECM composition may play important roles inasmuch as mutual infl uences are not fully understood yet [Paige et al, 1995;Gagne et al, 2000;Qi and Scully, 2003].…”