Quantitative Analysis of the Proliferation and Differentiation of Rat Articular Chondrocytes in Alginate 3D Culture

Background: While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. Methods: Rat articular chondrocytes were released by enzymatic digestion, plated at 5 × 104 cells/cm2 and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-(A, 5- dimethylthiazolyl- 2-yl)-1, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. Results: Average population doubling time in alginate cultures (10.04 ± 0.9 days) tended to be significantly (P<0.05) higher than that in monolayer cultures (2.94 ± 0.3 days). The period of alginate culture could be subdivided into expansion phase (Days 0-40) during which proliferation appeared to be high and differentiation phase (Days 40-60) during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period (P<0.05). Conclusion: It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period.