In this study, we evaluated the regulatory functions of ascorbic acid (AA) on cartilage matrix metabolism and the mechanisms of AA and dehydroascorbate (DHA) transport in human chondrocytes, to discern the physiologically relevant pathways of vitamin C transport in cartilage.

We evaluated the regulation of cartilage matrix components by different AA forms. Transcription of type II collagen, prolyl 4-hydroxylase, and aggrecan increased in response to the anti-scorbutic forms of AA and was stereospecific to the L-forms. Collagen and aggrecan protein synthesis also increased in response to the anti-scorbutic forms but only in the absence of acidity. This stereoselective regulation was a manifestation of the specific transport of the Lforms of AA by sodium dependent vitamin C transporter 2 (SVCT2).

Next, we characterized the transport of AA and DHA in human chondrocytes. Chondrocytes were robust concentrators of AA, capable of transporting and storing AA 960-fold over the concentration in the extracellular milieu. Chondrocyte transport of AA was sodium-dependent, stereoselective for the L-forms, and inhibited by sulfinpyrazone. When SVCT2 transcripts were suppressed with small interfering RNAs (siRNAs), the active transport component of AA was abolished.

DHA transport in human chondrocytes was glucose-sensitive, cytochalasin B inhibitable, and upregulated by low oxygen tension. Based on RNA interference (RNAi) results, glucose transporter 1 (GLUT1), at least in part, mediated DHA uptake, while GLUT3 had a minimal effect on DHA transport. We analyzed the Asc content of synovial fluid from patients with osteoarthritis (OA) and found that DHA constituted 8% of total ascorbic acid (Asc) in OA joints, as compared to 80% of total Asc reported for rheumatoid arthritis (RA) joints.

We provide the first evidence that SVCT2 mediates the active and concentrative transport of AA in human chondrocytes and that these cells also transport DHA via the GLUTs. Furthermore, under physiologic conditions in the joint, DHA would be expected to contribute 26% of the total intracellular Asc in OA chondrocytes, 39% of the total Asc in non-arthritic chondrocytes, and 94% of the total Asc in chondrocytes from RA patients. These results demonstrate that both AA and DHA are physiologically relevant sources of Asc for chondrocytes.