Abstract
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.
Highlights
Background and purposeIn vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments
Cartilage biopsies were collected from the intercondylar groove in the distal femur
Cells were used for either monolayer or scaffold culture
Summary
Cartilage samples Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 healthy patients undergoing anterior cruciate ligament reconstruction. Following trypsinization (with 1.25% trypsin and 5 mM EDTA), the cells were divided to undergo monolayer or scaffold seeding. Scaffolds with cells were vortexed in 1 mL of TRIzol Reagent (Invitrogen, Taastrup, Denmark) and total RNA was extracted according to the manufacturer’s instructions. It was dissolved in nuclease-free water (Ambion, Cambridgeshire, UK). Data analysis was performed using 7500 Fast System Sequence Detection software version 1.3 (Applied Biosystems, Naerum, Denmark). The data were analyzed using repeated-measurements ANOVA taking into account that the cells from each patient were used on both surfaces, at all 3 time points, and in either one or two of the three oxygen tensions. All p-values less than 0.05 were considered significant
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