Chondrogenic differentiation of human chondrocytes cultured in the absence of ascorbic acid

Bioreactor systems will likely play a key role in establishing regulatory compliant and costeffective production systems for manufacturing engineered tissue grafts for clinical applications. However, the automation of bioreactor systems could become considerably more complex and costly due to the requirements for additional storage and liquid handlingtechnologies if unstable supplements are added to the culture medium. Ascorbic acid (AA) isa bioactive supplement that is commonly presumed to be essential for the generation of engineered cartilage tissues. However, AA can be rapidly oxidized and degraded. In this work, we addressed whether human nasal chondrocytes can re-differentiate, undergochondrogenesis, and generate a cartilaginous extracellular matrix when cultured in the absence of AA. We found that when chondrocytes were cultured in 3D micromass pellets either with or without AA, there were no significant differences in their chondrogenic capacity in terms of gene expression or the amount of glycosaminoglycans. Moreover, 3D pellets cultured without AA contained abundant collagen type II and collagen type I extracellular matrix. While the amounts of collagen II and I were significantly lower (34% and 50% lower) than in pellets cultured with AA, collagen fibers had similar thicknesses and distributions for both groups, as shown by SEM imaging. Despite the reduced amounts of collagen, if engineered cartilage grafts can be generated with sufficient properties that meet defined quality criteria without the use of unstable supplements such as AA, bioreactor automation requirements can be greatly simplified, thereby facilitating the development of more compact, user-friendly, and cost-effective bioreactor-based manufacturing systems