Evaluation of the Regenerative Potential of Osteoarthritic Chondrocytes for Cartilage Tissue Engineering

Abstract

Focal defects in articular cartilage and other traumatic joint injuries trigger destructive pathways including inflammation, oxidative stress and increased expression of catabolic enzymes that often lead to post-traumatic osteoarthritis (PTOA). These pathways promote phenotypic changes characteristic of chondrocyte hypertrophy and senescence1. Senescence and hypertrophy are major phenotypic shifts seen in aging and OA and share similar markers such as expression of metalloproteinases (MMPs) that drive matrix degradation as well as an altered response growth factors that have been shown to be anabolic in healthy chondrocytes1,2,3,4. The term “chondrosenescence” was recently coined to describe this phenotype which is driven by inflammation, joint trauma, aging and other mechanical and chemical stimuli5. Accordingly, using aged or OA chondrocytes, which exhibit features of chondrosenescence, in clinically approved treatments for focal defect repair will yield subpar production of structural macromolecules and, therefore, subpar cartilage regeneration. Understanding the process of chondrosenescence will improve cartilage tissue engineered from these cells. This thesis focuses on the role of RUNX2 (Runt-related transcription factor 2), a transcription factor (TF) that drives chondrocyte hypertrophy during endochondral ossification. Specifically, we suppress RUNX2 in OA cells and investigate its role in the development of the chondrosenescent phenotype. We investigated the role of RUNX2 in two ways. First we defined the differences between young, healthy chondrocytes and aged/OA chondrocytes when regenerated in vitro. We found that OA cells have regeneration potential and produce cartilage matrix, limited by the upregulation of RUNX2 through multiple pathways. Next, we investigate the matrix production of OA cells when RUNX2 is suppressed using our previously developed cell regulation gene circuit. We found that high levels of RUNX2 suppression increases chondrogenic gene expression and increases cartilage matrix production. Finally, we developed an in vitro model of chondrosenescence to investigate that lead to the development of this phenotype. We show that our model leads to chondrosenescence and that multiple pathways are responsible for this development. Overall, this dissertation explores methodologies to enhance cartilage matrix production in aged/OA chondrocytes including suppressing RUNX2 and investigating other potential targets for suppression.