Effects of passage number and post-expansion aggregate culture on tissue engineered, self-assembled neocartilage.

unlabelledChondrocyte dedifferentiation presents a major barrier in engineering functional cartilage constructs. To mitigate the effects of dedifferentiation, this study employed a post-expansion aggregate culture step to enhance the chondrogenic phenotype of passaged articular chondrocytes (ACs) before their integration into self-assembled neocartilage constructs. The objective was twofold: (1) to explore how passage number (P2, P3, P4, P5, P6, and P7), with or without aggregate culture, affected construct properties; and (2) to determine the highest passage number that could form neocartilage with functional properties. Juvenile leporine ACs were passaged to P2-P7, with or without aggregate culture, and self-assembled into 5mm discs in non-adhesive agarose molds without using any exogenous scaffolds. Construct biochemical and biomechanical properties were assessed. With aggregate culture, neocartilage constructs had significantly higher collagen content, higher tensile properties, and flatter morphologies. These beneficial effects were most obvious at higher passage numbers. Specifically, collagen content, Young's modulus, and instantaneous compressive modulus in the P7, aggregate group were 53%, 116%, and 178% higher than those in the P7, non-aggregate group. Most interestingly, these extensively passaged P7 ACs (expansion factor of 85,000), which are typically highly dedifferentiated, were able to form constructs with properties similar to or higher than those formed by lower passage number cells. This study not only demonstrated that post-expansion aggregate culture could significantly improve the properties of self-assembled neocartilage, but also that chondrocytes of exceedingly high passage numbers, expanded using the methods in this study, could be used in cartilage engineering applications. STATEMENT OF SIGNIFICANCE: This work demonstrated that extensively passaged chondrocytes (up to passage 7 (P7); expansion factor of 85,000) could potentially be used for cartilage tissue engineering applications. Specifically, an aggregate culture step, employed after cell expansion and before cell integration into a neocartilage construct, was shown to enhance the ability of the chondrocytes to form neocartilage with better biochemical and biomechanical properties. The beneficial effects of this aggregate culture step was especially noticeable at the high passage numbers. Most interestingly, P7 chondrocytes, which are typically highly dedifferentiated, were able to form neocartilage with properties similar to or higher than those formed by lower passage number cells. The ability to obtain high chondrocyte yields with an enhanced chondrogenic potential could have a broad, beneficial impact in improving current therapies (e.g., using higher cell seeding densities for repair) or developing new strategies that require high cell numbers, such as a scaffold-free approach in forming engineered cartilage.