>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 - 106 versus 7 - 107 cells/mL) and cultivated for up to 4 weeks under TGF-ß 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.
>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 - 106 versus 7 - 107 cells/mL) and cultivated for up to 4 weeks under TGF-ß 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.
