Articular cartilage, the thin avascular connective tissue lining the ends of articulating joints that allows for load support and smooth articulation, is a highly resilient tissue that can withstand decades of strenuous use before showing signs of breakdown. However, when cartilage breaks down, either gradually because of idiopathic, age-related changes, or rapidly following a traumatic joint injury, the progression of a viscous cycle of tissue degeneration invariably leads to the severe disease state called osteoarthritis (OA). OA is characterized by excessive joint inflammation, stiffness, pain, and immobility. Currently, the clinical outlook for OA patient is bleak, as existing therapeutic strategies only address secondary symptoms such as pain and inflammation. Ultimately, because de novo articular cartilage restoration is effectively impossible, due to the tissue’s poor capacity for repair, the only cure for OA at present is joint replacement surgery.
Recently, strategies founded in tissue engineering & regeneration (TER) have arisen as potential solutions for cartilage’s poor intrinsic repair capacities; however, prior TER approaches have shown limited efficacy with regards to in vivo longevity, with even the most promising clinical approaches resulting in the generation of fibrocartilage, which lacks the biomechanical and tribological properties of native hyaline cartilage, and typically only moderately postpone the time until total joint replacement is necessary. More recently, advancements in TER have highlighted the importance of leveraging directed chondrocyte calcium (Ca2+) signaling to enhance biosynthetic outcomes and maturation of engineered cartilage tissues ex vivo. However, such approaches have lacked the necessary specificity by which they invoke Ca2+ signaling processes to truly dissect its role in cartilage and chondrocyte biology. Therefore, this dissertation sought to determine the efficacy of a novel chemogenetic signaling platform, the hM3Dq DREADD (designer receptor exclusively activated by designer drugs), to enable synthetic, targeted, and precise intracellular calcium [Ca2+]i signal transduction capable of uncovering the role(s) of Ca2+ signaling in chondrogenesis, chondrocyte physiology, health, and metabolism, and as a potential tool for enhancing cartilage engineering.
Over the last decade, DREADDs have transformed the study of neuroscience and have been applied to a small number of non-neuronal cell types, but they have never been utilized in the context of musculoskeletal cells and tissues. Therefore, the first part of this dissertation (Chapter 2) focuses on establishing the use of the hM3Dq DREADD as a tool to precisely and synthetically activate Gαq-GPCR-mediated calcium signals in the chondrocyte-like ATDC5 cell line. Through transient transfection techniques, we demonstrate that hM3Dq can be expressed in these cells and that they can activate [Ca2+]i transients in response to the inert compound clozapine N-oxide (CNO) in a dose-dependent manner. hM3Dq-mediated Ca2+ activations were on par with currently employed synthetic and mechanical Ca2+ activators, GSK101 and hypo-osmotic shock. These activations could be repeated over the course of a few days without inducing cytotoxic effects through apoptosis or mitochondrial dysfunction. The findings from Chapter 2 established the successful first use of DREADDs and hM3Dq to synthetically regulate [Ca2+]i signaling in a chondrocyte-like cells.
The second part of this dissertation (Chapters 3-5) sought to improve upon the techniques employed in Chapter 2 by focusing on generating a stable ADTC5 cell line expressing a fluorescently-tagged hM3Dq protein to identify the temporal dynamics of calcium signaling in these cells and investigate the effects that CNO concentration and stimulation frequency have on gene and protein outcomes during long-term 2D culture. By leveraging lentiviral transduction techniques, we generated a stable ATDC5-hM3Dq-mCherry cell line that could permit co-localization of hM3Dq expression and [Ca2+]i fluorescence during live-cell imaging and enable the ability to conduct long-term culture experiments. The findings from Chapter 3 demonstrated the temperature- and concentration-dependent nature of CNO-evoked [Ca2+]i signaling and identified the presence of robust, cell-autonomous hM3Dq-evoked Ca2+ oscillations that were reliant on ER Ca2+ store release and refilling kinetics. In Chapters 4 & 5 we shed light on the role that [Ca2+]i activation patterns and Ca2+ oscillations play in chondrocyte metabolism in vitro. The findings from these chapters demonstrated the robust, and safe cell- and tissue-level responses to CNO-driven Gαq and [Ca2+]i activation patterns, which robustly drove cartilage-like neotissue formation and chondrogenic gene expression profiles, and modulated intra/extracellular organization and alignment through Ca2+-dependent processes.
The last part of this dissertation (Chapter 6) focuses on the effects that hM3Dq-mediated Ca2+ signaling dynamics have on tissue development and maturation in 3- dimensional (3D) culture models to determine the translatability of the outcomes observed in the previous chapters. Exploring the chondrogenic potential of cells cultured in 3D pellets and cell-laden hydrogels, we found that hM3Dq-mediated Ca2+ activations could drive stimulation frequency- and CNO concentration-dependent chondrogenic tissue-level responses that mimic what has been reported during embryonic endochondral ossification. The findings from Chapter 6 demonstrated the potential for utilizing hM3Dq and DREADDs, along with other targeted, synthetic Ca2+ signaling platforms, to potentially improve a number of aspects of cartilage tissue engineering and regeneration by overcoming specific barriers to TER success, while also providing a novel toolkit for dissecting precisely how Ca2+ and GPCR signaling regulate chondrocyte and chondroprogenitor physiology in vitro and in vivo.
Overall, this dissertation reveals the importance of regulating [Ca2+ ]i signaling dynamics in chondrocyte and cartilage biology and how leveraging targeted, synthetic Ca2+ signaling platforms, like DREADDs, might be leveraged to improve upon and synergize with current cartilage tissue engineering techniques to accelerate and enhance the development of functional engineered cartilage tissues. Additionally, this work establishes the ability to extend our knowledge of GPCR signaling in chondrocyte differentiation and cartilage development and perturb the pathway mechanisms more in-depth to develop a better working understanding behind cartilage and other musculoskeletal diseases and design and implement strategies to improving joint health.
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