Sox9 expression of alginate-encapsulated chondrocytes is stimulated by low cell density

Recent research in tissue engineering for the treatment of cartilage defects have demonstrated that matrix-biomaterial, cell culture conditions, and cytokine-related factors influence the chondrogenic differentiation pattern, especially for the expression of matrix genes. However, little is known about the impact of cell seeding density in a three-dimensional environment on the key chondrogenic transcription factor Sox9. Here we investigated, whether the cell concentration of alginate encapsulated chondrocytes influences the Sox9 expression. Dedifferentiated passage-4 porcine chondrocytes were encapsulated in alginate beads at two different concentrations (4 X 10(6) versus 7 X 10(7) cells/mL) and cultivated for up to 4 weeks under TGF-beta Stimulation. The expression of Sox9, Collagen I, II, and X was assessed via quantitative RT-PCR and compared to those observed in the initial monolayer culture. Cellular viability, cell morphology, and the sulphated glycosaminoglycan-production were monitored. Interestingly Sox9 expression was significantly upregulated in the low-cell-density group, whereas no difference between high-cell-density and monolayer culture group could be observed. Furthermore, the cellular survival and the sulphated glycosaminoglycan production were higher in the low-cell-density group. Collagen I expression was downregulated in the low-cell-density group whereas it was upregulated in the high-cell-density one. Surprisingly, only the high-cell-density group showed the expression of Collagen II, although it appeared not significant. Collagen X expression was upregulated in the low-cell-density group. Taken together our data indicate that a low concentration of cell seeding in a three-dimensional environment is beneficial for the overall chondrogenic development. However, this article reveals discrepancies between Sox9 and the chondrogenic pathway in redifferentiating chondrocytes that should be addressed in further work. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 91A: 910-918, 2009