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Date of Award
Doctor of Philosophy (PhD)
Osteoarthritis (OA) is a debilitating joint disease that is primarily characterized by the degeneration of articular cartilage, the soft connective tissue that covers articulating bone surfaces in diarthrodial joints. While there are a number of risk factors for developing OA, the progression of this disease is mediated in part by pro-inflammatory cytokines from both the synovium and chondrocytes, the resident cells of articular cartilage. These cytokines, specifically interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-α), induce aberrant expression of catabolic and degradative enzymes and inflammatory cytokines in OA, which promotes degradation of engineered tissues as well as native articular cartilage. This loss of the homeostatic balance in chondrocytes results in a challenging environment for cartilage repair and regeneration. In the first aim of this dissertation, we show that the combination of gene therapy and tissue engineering can be applied for the development of an artificial gene circuit in murine induced pluripotent stem cells (miPSC). This system is based on a NF-κB responsive synthetic promoter, which drives expression of a therapeutic transgene (interleukin-1 receptor antagonist, IL-1Ra) and is packaged into a lentiviral vector (NRE-IL1Ra), and results in inflammation driven, self-regulating delivery of biologic drugs. miPSCs were transduced with NRE-IL1Ra in monolayer or through biomaterial-mediated transduction and were either maintained in monolayer or differentiated into cartilage constructs. For both of these delivery methods, stimulation with various doses of IL-1 resulted in production of high levels of IL-1Ra, which inhibited inflammatory signaling and protected tissue-engineered cartilage from proteoglycan degradation. In the second aim of this dissertation, we determined novel therapeutic targets in the response of tissue-engineered cartilage to inflammatory cytokines. MicroRNAs (miRNAs) are important regulatory molecules that have been implicated in many diseases, including OA. However, the specific roles and regulatory networks of miRNAs in response to IL-1 and TNF-α have not been elucidated. We performed miRNA and mRNA sequencing to determine the temporal and dynamic responses of miRNAs and genes to IL-1 and TNF-α. We found genes and miRNAs that were differentially expressed with both cytokines, and also those that were unique to IL-1 or TNF-α. Through integration of miRNA and mRNA sequencing data sets, we created networks of miRNA-mRNA interactions and identified hub miRNAs miR-17-5p, miR-20a-5p, and miR-29b-3p. Delivery of miR-17-5p and miR-20a-5p mimics in combination decreased degradative enzyme activity levels and expression of inflammation-related genes in cytokine treated cells. The work presented in this dissertation improves our understanding of the mechanisms driving inflammatory responses in OA and provides novel strategies and therapeutic targets to prevent inflammation-driven degradation of native and tissue-engineered cartilage.
Yousef Abu-Amer, Nathaniel Huebsch, Audrey McAlinden, Muhammad Farooq Rai,
Available for download on Wednesday, April 20, 2022