Osteoarthritis has been estimated to affect 13.5% of the adult U.S. population. Cartilage lesions range from small focal defects to large defects extending the entire articulating surface. While reparative techniques (microfracture, auto/allografts, autologous chondrocyte implantation, etc.) can treat the former, total or unicompartmental knee arthroplasty is the only solution for the latter. Given the limited lifespan of these prosthetics, a solution to repair and regenerate large cartilage lessions is needed. Tissue engineering offers a promising approach. In particular, the selfassembling process was shown to generate neocartilage with functional biomechanical properties near native tissue values, albeit the engineered constructs are up to 5 mm in diameter. If a sufficiently large number of cells can be obtained, this technology could be scaled-up to engineer large, anatomically shaped constructs with potential in repairing large defects, such as a unicondylar surface.

Toward this goal, the global objective of this thesis was to scale-up the engineering process, both in terms of articular chondrocyte expansion, as well as self-assembly, to fabricate neocartilage of clinically relevant dimensions. First, using a 5 mm dia. construct model, culture conditions appropriate for the self-assembly of passaged articular chondrocytes were determined. Specifically, the highest cell seeding density (so as to generate the thickest neocartilage) and the highest cell passage number (so as to maximize cell expansion yields) that could be used to generate neocartilage without compromised properties were determined. In a parallel effort, novel chemical stimuli (digoxin and ATP) were explored to enhance neocartilage properties. Subsequently, using the identified optimized culture conditions, a new protocol to fabricate large (> 8 cm²) neocartilage constructs was established. This then paved the way toward establishing the tissue engineering strategy for fabricating large, shape-specific osteochondral constructs.

Results from these studies demonstrated that passaged chondrocytes can be self-assembled into large neocartilage, alone or as an osteochondral construct. A critical seeding density of 2 million cells per 5 mm dia. was determined to be the highest that can form functionally viable neocartilage, possessing properties on the high end of what had been previously achieved;​ specifically, neocartilage with Young’s modulus of 4 MPa and collagen/wet weight of 7%. Above this density, construct properties were drastically inferior. Under specific culture conditions, it was also found that young rabbit articular chondrocytes can be expanded up to passage 7 (expansion factor of 85,000) and form self-assembled neocartilage without losing functional biomechanical properties. Being able to expand articular chondrocytes to such high numbers overcomes challenges in fabricating large constructs. In a parallel study, digoxin and ATP were shown to enhance neocartilage collagen content up to 2-fold and Young’s modulus up to 5-fold over controls.

Significant scale-up of the self-assembling process, from constructs 0.4 cm² to > 8 cm² (23-fold scale-up) did not adversely affect neocartilage properties;​ this was only possible when employing new culture strategies, specifically use of static mechanical loading and a chemical stimuli regimen (cytochalasin D, TGF-β1, chondroitinase ABC, and lysyl oxidase-like 2 cocktail). Using these scale-up strategies, in combination with custom 3D fabrication techniques, it was possible to fabricate an osteochondral construct with the shape and size of a sheep femoral condyle and with robust interfacial properties between the two phases. Culturing strategies were developed the osseous phase to be on par with native cartilage. These constructs can potentially be used to repair large cartilage lesions, representing critical steps toward engineering a bioprosthesis for unicompartmental reconstruction.