title: Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors: Orthopaedic Research Laboratory, Aarhus University Hospital, Aarhus, Denmark. foldager@ki.au.dk
authors: Casper B
authors: CB
authors: Foldager
authors: Anna B
authors: AB
authors: Nielsen
authors: Samir
authors: S
authors: Munir
authors: Michael
authors: M
authors: Ulrich-Vinther
authors: Kjeld
authors: K
authors: Søballe
authors: Cody
authors: C
authors: Bünger
authors: Martin
authors: M
authors: Lind
mesh_terms: D053674:Aggrecans / Q000096:biosynthesis; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D015687:Cell Hypoxia; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D024043:Collagen Type II / Q000096:biosynthesis; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D018613:Microscopy, Confocal; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000096:biosynthesis; D023822:Tissue Engineering
chemical_list: D053674:Aggrecans; D024043:Collagen Type II; D055758:SOX9 Transcription Factor