Many aspects of the process of in vitro differentiation of chondrocytes in three-dimensional (3D) scaffolds need to be further investigated. Chitosan scaffolds were produced by freeze-drying 3% w/v 90% DDA chitosan gels. The effect of the cell seeding concentration was evaluated by culturing human adult chondrocytes in chitosan scaffolds After the first passage, cells were seeded into chitosan scaffolds with a diameter of 8 mm. The final cell seeding concentration per cm3 of chitosan scaffold was: Group A, 3 × 106;Group B, 6 × 106; Group C, 12 × 106; and Group D, 25 × 106 cells. After 14 and 28 days in 3D culture, the constructs were assesed for collagen, glucosaminoglycans and DNA content. The mechanical properties of the constructs were determined using a dynamic oscillatory shear test. The histological aspect of the constructs was evaluated using the Bern score. The collagen and GAG concentration increased, varying the cell seeding concentration. There was a significant increase in proteoglycan and hydroxyproline production between groups C and D. The sulphated GAG content increased significantly in the group D as compared to the other groups. The mechanical properties of the different constructs increased over time, from 9.6 G?/kPa at 14 days of 3D culture to 14.6 G?/kPa at 28 days under the same culture conditions. In this study we were able to determine that concentrations of 12–25 million cells/cm2 are needed to increase the matrix production and mechanical properties of human adult chondrocytes under static conditions. Copyright © 2008 John Wiley & Sons, Ltd.
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