Theses and Dissertations at Montana State University (MSU)
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Item Does bone-to-cartilage fluid transport exist and is it relevant to joint health?(Montana State University - Bozeman, College of Engineering, 2024) Hislop, Brady David; Chairperson, Graduate Committee: Ronald K. June II; This is a manuscript style paper that includes co-authored chapters.Osteoarthritis (OA) afflicts millions of people each year. The onset of OA has been associated with many factors including increased bone-cartilage fluid transport, yet a cure remains elusive. To implicate bone-cartilage fluid transport in the progression of OA, further studies are needed on fluid transport in health. Recent studies have challenged the assumption that no fluid transport occurs between bone and cartilage in healthy joints. However, many gaps remain in our understanding of bone-to-cartilage fluid transport, including 1) do fluid pressure gradients develop at the bone-cartilage interface, 2) do traumatic injuries impact subchondral bone stiffness, and synovial fluid metabolism 3) do larger molecules move from bone-to-cartilage and does cyclic loading enhance such movement, 4) what material properties influence bone-to-cartilage fluid transport 5) do distinct metabolism changes occur with osteoarthritis, evaluated using a novel clustering method. Our results showed the development of fluid pressure gradients at the osteochondral interface, and that cyclic compression enhances bone-cartilage fluid transport. Furthermore, our results showed that proteoglycan loss, and decreased subchondral bone stiffness increased bone-cartilage fluid transport. Finally, we showed that in the first week after traumatic joint injuries (e.g., ACL tears) subchondral bone volume decreases, and subchondral bone stiffness increases, while the synovial fluid metabolism shifts. In conclusion, we showed that osteochondral fluid transport is enhanced by cyclic compression for larger molecules than previously studied (3kDa dextran), and that material parameters changes associated with the progression of OA alter bone-cartilage fluid transport. These studies provide novel understanding of bone-to-cartilage fluid transport, leading us one step closer to understanding OA as a whole joint disease.Item Diffusion and diffusive exchange are sensitive to the structure of cartilage as measured by nuclear magnetic resonance(Montana State University - Bozeman, College of Engineering, 2017) Mailhiot, Sarah Elizabeth; Chairperson, Graduate Committee: Ronald K. June II; Nathan H. Williamson, Jennifer R. Brown, Joseph D. Seymour, Sarah L. Codd and Ronald K. June were co-authors of the article, 'T1-T2 correlation and biopolymer diffusion within human osteoarthritic cartilage measured with nuclear magnetic resonance' in the journal 'Applied magnetic resonance' which is contained within this thesis.; Sarah L. Codd, Jennifer R. Brown, Joseph D. Seymour and Ronald K. June were co-authors of the article, 'Pulsed gradient stimulated echo (PGSTE) NMR shows spatial dependence of fluid diffusion in human stage IV OA cartilage' submitted to the journal 'Magnetic resonance in medicine' which is contained within this thesis.; Fangrong Zong, James E. Maneval, Ronald K. June, Petrik Galvosas and Joseph D. Seymour were co-authors of the article, 'Quantifying NMR relaxation correlation and exchange in articular cartilage with time domain analysis' submitted to the journal 'Journal of magnetic resonance' which is contained within this thesis.; James E. Maneval, Ronald K. June and Joseph D. Seymour were co-authors of the article, 'Relaxation exchange in human OA cartilage impacts the observable T 2 relaxation rates' submitted to the journal 'Magnetic resonance in medicine' which is contained within this thesis.Osteoarthritis (OA) is the deterioration of the tissue on the surface of the articulating joints in mammals. OA is the progression loss of articular cartilage. OA affects 50% of people over age 65 and is the leading cause of workplace disability. There is no cure for OA and the state of the art treatment is joint replacement. One limitation for treating OA is the difficulty of diagnosing OA before tissue failure. Magnetic Resonance Imaging (MRI) is capable of detecting early pathologic changes to cartilage but challenges remain. The goal of this work is to evaluate how parameters, specifically relaxation and diffusion, used for creating imaging contrast in MRI are affected by disease in naturally occurring human osteoarthritis. Nuclear Magnetic Resonance (NMR) is utilized to measure the diffusion and magnetic relaxation in human OA cartilage samples. Diffusion Weighted Imaging (DWI) is a proposed imaging mechanism for diagnosing OA. The hypothesis is that fluid diffusion is faster in diseased tissue than in healthy tissue. We show that diffusion of fluid increases when cartilage is damaged by enzymes, such as during OA. We also show that the diffusion of fluid is donor specific in human OA cartilage. Diffusion of proteins in cartilage is also sensitive to enzyme degradation and donor as well as to the size and structure of the proteins in cartilage. These are complementary measures of the fluid and solid phase of cartilage. Relaxation weighted imaging is the most common way to image cartilage and is capable of measuring small structure changes due to OA. One limitation of this method is that reported relaxation rates vary between studies. We show that exchange, or motion of fluid, between the two sites of relaxation in cartilage alters the observed relaxation. Further, we show that the exchange rate is sensitive to donor and enzyme degradation. The results suggest that exchange rate is a sensitive measure of structure in cartilage and that relaxation should be cautiously interpreted when exchange occurs. Overall, this work shows that NMR and MRI are sensitive to the structure of cartilage and capable of detecting pathological damage to cartilage.Item New methods in computation of reaction fluxes from metabolomics data(Montana State University - Bozeman, College of Engineering, 2018) Salinas Duron, Daniel; Chairperson, Graduate Committee: Brendan MumeyChanges in cellular metabolism can be deduced from how they affect the measurable metabolites in cell samples. We provide methods to compute metabolic reaction rates from changes in measurable metabolites over time. The methods provided are intended to overcome technical challenges, such as the inapplicability of a steady state assumption, heterogeneity of samples from different donors, and the lack of targeted metabolomics data. Solutions to these challenges involve identifying metabolites constrained even under non-steady state, using components analysis to find the donor consensus, and using an integer linear program to solve a set cover variant designed to generate targeted data from untargeted data. The methods are applied on data derived from diseased articular cells. The results show that the reaction rates inferred from the incomplete data are biologically relevant, and that the minimal pathways captured ancillary processes that alternative approaches ignored. We conclude that, although the resulting rates and pathways are not conclusive, they provide useful guidance on experiments to pursue. On the experimental side, our findings have lead us to believe that osteoarthritic chondrocytes respond to compression by initiating protein synthesis, opening the possibility of physical therapy as a stimulus for cartilage regeneration.Item The interstitial fluid pressure response during stress-relaxation of articular cartilage due to viscosity and porous media effects: a computational study(Montana State University - Bozeman, College of Engineering, 2018) Paschke, Brandon James; Chairperson, Graduate Committee: Erick JohnsonArticular cartilage is a complex material made of several fluid and solid components. A model that fully describes the responses of cartilage is required to accurately create a cartilage replacement that can be used in cases of injury or disease. Modeling of articular cartilage has proven difficult and currently no constitutive law fully describes its solid and fluid responses. Many of the current models describe the interstitial fluid as inviscid, even though it is known that proteoglycan migration within cartilage causes a viscous response within interstitial fluid. The goal of this research was to create a viscous fluid porous media model that better captures the compressive resistance of cartilage created by migration of interstitial fluid during cartilage compression. Through the creation of this model it was possible to capture the experimental magnitudes of fluid pressure within cartilage during unconfined slow compression simulations. As part of this model, a porous media approximation was used, which demonstrates that small variations in the solid matrix, comprised of collagen fibers, can cause large variations in system response. Magnitudes of mean pressure values, after 150 seconds of compression, for the viscous fluid porous media model bound the values found in experimental testing. Limitations of the fluid model are that system relaxation isn't captured and the slope increase of pressures during compression for experiments don't match those of the fluid model. A main conclusion drawn from the model is that viscosity of interstitial fluid plays a large role in creating compressive resistance within articular cartilage. Another takeaway is that the porous media approximation greatly impacts the magnitude of fluid pressurization, which creates a need to accurately represent the solid matrix within cartilage.Item In vitro and in vivo systems mechanobiology of osteoarthritic chondrocytes(Montana State University - Bozeman, College of Engineering, 2015) Zignego, Donald Lee; Chairperson, Graduate Committee: Ronald K. June II; Aaron A. Jutila, Martin K. Gelbke and Daniel M. Gannon were co-authors, and Ronald K. June was a corresponding author of the article, 'The mechanical microenviroment of high concentration agarose for applying deformation to primary chondrocytes' in the journal 'Journal of biomechanics' which is contained within this thesis.; Aaron A. Jutila was a main author, Bradley K. Hwang, Jonathan K. Hilmer, Timothy Hamerly, Cody A. Minor and Seth T. Walk were co-authors, and Ronald K. June was a corresponding author of the article, 'Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements' in the journal 'Archives of biochemistry and biophysics' which is contained within this thesis.; Carley N. McCutchen, Jonathan K. Hilmer were co-authors, and Ronald K. June was a corresponding author of the article, 'Mechanotransduction in primary human osteoarthritic chondrocytes is mediated by metabolism of energy, lipids, and amino acids' submitted to the journal 'Arthritis and rheumatology' which is contained within this thesis.; Jonathan K. Hilmer was a co-author, and Ronald K. June was a corresponding author of the article, 'Shotgun phosphoproteomics identifies activation of vimentin, ankyrin, vam6/vpS39-like protein in primary human osteoarthritic chondrocytes after mechanical stimulation' submitted to the journal 'eLife' which is contained within this thesis.; Sarah E. Mailhiot, Timothy Hamerly, Edward E. Schmidt were co-authors, and Ronald K. June was a corresponding author of the article, 'Alterations in joint metabolomics following surgical destabilization and exercise in a novel cartilage reporter mouse model' submitted to the journal 'Annals of biomedical engineering' which is contained within this thesis.All cells are subjected to and respond to mechanical forces, but the underlying processes linking the mechanical stimuli to biological responses are poorly understood. In the joints of the body (e.g. the knee, hip, etc...) articular cartilage serves as a low friction, load bearing material and is subjected to near-constant mechanical loading. Through excessive loading of the joint, usually caused by obesity or injury, the protective articular cartilage begins to diminish, leading to the progression of osteoarthritis (OA). Osteoarthritis is the most common joint disorder in the world and is characterized by the deterioration of articular cartilage. Determining the link between cartilage deterioration and mechanical loading is one motivation that drove this research. Articular cartilage is composed of a dense extracellular matrix (ECM), a less-stiff pericelluar matrix (PCM), and highly specialized cells called chondrocytes. As the sole cell type in cartilage, chondrocytes are responsible for the healthy turnover of the ECM by creating, maintaining, and repairing the matrix. Multiple lines of evidence suggest chondrocytes can transduce mechanical stimuli into biological signals. The hypothesis for this research is that physiologically pertinent loading of chondrocytes results in a specific set of bio-signals resulting in matrix synthesis. To test this hypothesis, two unbiased, large-scale metabolomic and phosphoproteomic datasets were generated by modeling physiological compressive loading on 3D-embedded chondrocytes. To assess loading-induced changes in metabolites (e.g. small molecules representing the functional state of the cell) and proteome-wide patterns of post-translational modifications (i.e. phosphorylation), chondrocytes were encapsulated in physiologically stiff agarose, compressively loaded in tissue culture, and analyzed via liquid chromatography -- mass spectrometry (LC-MS). The results helped identify global and local biological patterns in the chondrocytes which are a direct result from mechanical loading. In addition, a novel mouse model that expresses cartilage specific bioluminescence was used to assess loading induced changes in vivo. The results from the mouse model allowed for in vivo validation and integration of the in vitro results from the metabolomic and phosphoproteomic results. To my knowledge, such research has never been done, and considerably expands the scientific knowledge of chondrocyte mechanotransduction.