Effect of ascorbate and two different media on canine chondrocytes in three-dimensional culture

The objective of this study was to determine the effects of ascorbate and two different culture media on cell morphology and extracellular matrix formation of canine chondrocytes grown in a three-dimensional (3-D) culture system. Articular cartilage harvested from the humeral head of three adult canine cadavers was used to obtain chondrocytes for primary culture. Subcultured chondrocytes were seeded in a 3-D medium of RPMI-1640 (R), RPMI-1640 with 50 microg/mL ascorbate (RA), Ham's F-12 (F), or Ham's F-12 with 50 microg/mL ascorbate (FA) in agarose. Samples were harvested at 5, 10, 15, and 20 days of 3-D culture and analyzed for histologic appearance and proteoglycan staining, electron microscopic appearance, dimethylmethylene blue assay for glycosaminoglycan (GAG) content, and immunohistochemical staining for collagen type II production. Chondrocytes in all four groups maintained appropriate morphology and produced matrix over the entire study period. Chondrocytes from groups R and RA produced more GAG and collagen type II than did those from groups F and FA on days 10 (P = .00791) and 15 (P = .0173). Chondrocytes from group RA produced more GAG on days 5 (P = .0154) and 20 (P = .0044) than did those in groups R, F, and FA. With respect to matrix production, RPMI-1640 is superior to Ham's F-12 for 3-D culture of canine chondrocytes. The addition of ascorbate at 50 microg/mL to RPMI-1640 did have a positive effect on the production of GAG but had minimal effect on type II collagen production. Determining the most ideal in vitro microenvironment for canine chondrocytes grown in a 3-D culture system has important implications to the in vivo application of this technique.