Development and validation of a system for studying chondrocyte mechanotransduction with preliminary metabolomic results
Jutila, Aaron Arthur.
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Osteoarthritis (OA) is a degenerative disease currently affecting over 46 million Americans. OA is most commonly characterized by breakdown of articular cartilage within the joint resulting in abnormal loading, loss of motion, and pain. Articular cartilage is the tough, flexible, load-bearing material that allows for joints to articulate smoothly i.e. running with relative ease. Currently there is no cure for this disease and the exact causes remain relatively unknown. Chondrocytes are the only cell type found in cartilage and are responsible for all biological maintenance and repair. Previous studies have shown that chondrocytes respond to mechanical load by cellular mechanotransduction, the process by which cells convert mechanical stimuli into biochemical activity. The aim of this thesis is to study the effects that mechanical loads have on human chondrocyte metabolism to better understand OA. To study chondrocyte mechanotransduction it was vital to develop a machine that could simulate in vivo loading of chondrocytes within the human knee joint. A bioreactor was designed, built, and validated that can simulate physiological loading in a tissue culture environment. This bioreactor was then used to characterize the mechanical properties of a viscoelastic material (agarose) capable of maintaining viable 3-Dimensional cell cultures. Inside the body chondrocytes are surrounded by a pericellular matrix (PCM), which provides a unique stiffness much less than the stiffness of cartilage. The mechanical property tests performed on agarose allowed for an accurate representation of this cellular microenvironment. Agarose gel concentrations were found that can model both healthy PCM and OA PCM stiffness. Methods were then developed to encapsulate living chondrocytes within these physiologically stiff gels. Utilizing these newly developed gel constructs and custom built bioreactor, 3-Dimensional chondrocyte suspensions were subjected to dynamic compression simulating normal physiological loading i.e. walking. It was hypothesized that moderate dynamic loading would promote changing in the central metabolism pathways, such as glycolysis. To study this hypothesis, mass-spectrometry techniques were utilized to identify metabolites present in each sample, and if the amount of each metabolite changed due to dynamic compression. These results provide a robust foundation for understanding cellular mechanotransduction.