Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension

Abstract

Objective: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. Methods: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O2 for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [3H]-proline incorporation and cell-viability by the trypan blue exclusion method. Results: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [3H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [3H]-proline incorporation. Viability was ∼90% for all alginate cultures. Conclusion: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures. © 2002 OsteoArthritis Research Society International.