For successful cartilage tissue engineering, the ability to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation is critical. The ability to accomplish this by using the abundant and easily sampled costal cartilage could provide a practical alternative to the use of articular cartilage and mesenchymal stem cells. Chondrocytes isolated from pig costal cartilage were expanded in either serum-free medium with FGF2 (SFM) or fetal bovine serum-containing medium (SCM), under either high (21%) or low (5%) oxygen conditions. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (p < 0.05). The effect of passage number on the differentiation status of the chondrocytes was analyzed by alkaline phosphatase (AP) staining and real-time PCR for known chondrocyte quality markers. AP staining indicated that chondrocytes grown in SCM had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (p < 0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (p < 0.05) in cells expanded in Low-SFM, indicating reduced de-differentiation. In vitro re-differentiation capacity was assessed after a 6-week induction, and chondrocytes grown in Low-SFM showed similar expression ratios of COL2/COL1 and ACAN/VCAN to native cartilage. Proteomic analysis of in vitro produced cartilage indicated that the Low-SFM condition most closely matched the proteomic profile of native costal and articular cartilage. In conclusion, Low-SFM culture conditions resulted in improved cell growth rates, reduced levels of de-differentiation during expansion, greater ability to re-differentiate into cartilage on induction, and an improved proteomic profile that resembles that of in vivo cartilage.
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