Overcoming Challenges in Engineering Large, Scaffold-Free Neocartilage with Functional Properties.

Although numerous cartilage engineering methods have been described, few report generation of constructs greater than 4 cm2, which is the typical lesion size considered for cell-based therapies. Furthermore, current cell-based therapies only target focal lesions, while treatment of large nonisolated lesions remains an area of great demand. The objective of this study was to scale up fabrication of self-assembled neocartilage from standard sizes of 0.2 cm2 to greater than 8 cm2. Passaged sheep articular chondrocytes were self-assembled into 5 or 25-mm-diameter scaffoldless neocartilage constructs. The 25-mm-diameter constructs grew up to 9.3 cm2 (areal scale-up of 23) and possessed properties similar to those of the 5-mm-diameter constructs; unfortunately, these large constructs were deformed and are unusable as a potential implant. A novel neocartilage fabrication strategy-employing mechanical confinement, a minute deadweight, and chemical stimulation (cytochalasin D, TGF-β1, chondroitinase-ABC, and lysyl oxidase-like 2 protein)-was found to successfully generate large (25-mm diameter) constructs with flat, homogeneous morphologies. Chemical stimulation increased collagen content and tensile Young's modulus 140% and 240% in the 25-mm-diameter constructs and 30% and 70% in the 5-mm-diameter constructs, respectively. This study not only demonstrated that exceedingly large self-assembled neocartilage can be generated with the appropriate combination of mechanical and chemical stimuli but also that its properties were maintained or even enhanced.