Development and Validation of a System for Studying Chondrocyte Mechanotransduction With Preliminary Metabolomic Results

Links

URL: https://scholarworks.montana.edu/handle/1/8760

Abstract

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, massspectrometry 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 mechanotransductio

Cited Works (13)

Year	Entry
1999	Herberhold C, Faber S, Stammberger T, Steinlechner M, Putz R, Englmeier K, Reiser M, Eckstein F. In situ measurement of articular cartilage deformation in intact femoropatellar joints under static loading. J Biomech. December 1999;32(12):1287-1295.
2005	Alexopoulos LG, Setton LA, Guilak F. The biomechanical role of the chondrocyte pericellular matrix in articular cartilage. Acta Biomater. May 2005;1(3):317-325.
2003	Huang C-Y, Soltz MA, Kopacz M, Mow VC, Ateshian GA. Experimental verification of the roles of intrinsic matrix viscoelasticity and tension-compression nonlinearity in the biphasic response of cartilage. J Biomech Eng. February 2003;125(1):84-93.
2005	Alexopoulos LG, Williams GM, Upton ML, Setton LA, Guilak F. Osteoarthritic changes in the biphasic mechanical properties of the chondrocyte pericellular matrix in articular cartilage. J Biomech. March 2005;38(3):509-517.
2011	Guilak F. Biomechanical factors in osteoarthritis. Best Pract Res Clin Rheumatol. December 2011;25(6):815-823.
2003	Ng L, Grodzinsky AJ, Patwari P, Sandy J, Plaas A, Ortiz C. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J Struct Biol. September 2003;143(3):242-257.
2001	Kurz B, Jin M, Patwari P, Cheng DM, Lark MW, Grodzinsky AJ. Biosynthetic response and mechanical properties of articular cartilage after injurious compression. J Orthop Res. 2001;19(6):1140-1146.
2000	Loening AM, James IE, Levenston ME, Badger AM, Frank EH, Kurz B, Nuttall ME, Hung H-H, Blake SM, Grodzinsky AJ, Lark MW. Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis. Arch Biochem Biophys. September 15, 2000;381(2):205-212.
1995	Kim Y-J, Bonassar LJ, Grodzinsky AJ. The role of cartilage streaming potential, fluid flow and pressure in the stimulation of chondrocyte biosynthesis during dynamic compression. J Biomech. September 1995;28(9):1055-1066.
2001	Jin M, Frank EH, Quinn TM, Hunziker EB, Grodzinsky AJ. Tissue shear deformation stimulates proteoglycan and protein biosynthesis in bovine cartilage explants. Arch Biochem Biophys. November 1, 2001;395(1):41-48.
2006	Knight MM, Toyoda T, Lee DA, Bader DL. Mechanical compression and hydrostatic pressure induce reversible changes in actin cytoskeletal organisation in chondrocytes in agarose. J Biomech. 2006;39(8):1547-1551.
2009	Fox AJS, Bedi A, Rodeo SA. The basic science of articular cartilage: structure, composition, and function. Sports Health. November–December 2009;1(6):461-468.
2000	Lee DA, Knight MM, Bolton JF, Idowu BD, Kayser MV, Bader DL. Chondrocyte deformation within compressed agarose constructs at the cellular and sub-cellular levels. J Biomech. January 2000;33(1):81-95.

Cited By (2)

Year	Entry
2015	Zignego DL. In Vitro and in Vivo Systems Mechanobiology of Osteoarthritic Chondrocytes [PhD thesis]. Bozeman, MT: Montana State University; July 2015.
2024	Myers EP. Characterization and Manufacture of an Agarose Hydrogel System for Loading Embedded Chondrocytes [PhD thesis]. Bozeman, MT: Montana State University; December 2024.