Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes

Background and purpose In vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments. Native chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. We compared monolayer culture to combined 3D and hypoxic culture using quantitative gene expression analysis. Methods Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were used for either monolayer or scaffold culture. The scaffolds were clinically available MPEG-PLGA scaffolds (ASEED). After harvesting of cells for baseline investigation, the remainder was divided into 3 groups for incubation in conditions of normoxia (21% oxygen), hypoxia (5% oxygen), or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2, and 6 days after the baseline time point, respectively. Quantitative RT-PCR was performed using assays for RNA encoding collagen types 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyde-3-phosphate dehydrogenase relative to 2 hypoxia-stable housekeeping genes. Results Sox9, aggrecan, and collagen type 2 RNA expression increased with reduced oxygen. On day 6, the expression of collagen type 2 and aggrecan RNA was higher in 3D culture than in monolayer culture. Interpretation Our findings suggest that there was a combined positive effect of 3D culture and hypoxia on cartilage-specific gene expression. The positive effects of 3D culture alone were not detected until day 6, suggesting that seeding of chondrocytes onto a scaffold for matrix-assisted chondrocyte implantation should be performed earlier than 2 days before implantation.