In Vitro NMR Study of Magnetization Exchange at Low Field and Proteoglycan-Depletion at High Field in Articular Cartilage
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Magnetization exchange between different spin reservoirs, or spin groups, in cartilage tissue is an important aspect of the use of Magnetic Resonance Imaging (MRI) in the early detection of osteoarthritis (OA). A low field (Larmor frequency of 30 MHz) NMR study of relaxation times in bovine articular cartilage was undertaken with the aim of elucidating details about magnetization exchange in this tissue. A key element of successful multi-site exchange modeling is the availability of a sufficient number of apparent relaxation parameters to model the intrinsic multi- site exchange scenario. Two-Dimensional (2D) time domain NMR spin-lattice relaxation experiments in the laboratory frame (T1 experiments) and the rotating frame (T1ρ experiments) were performed in articular cartilage, which allows for effective extraction of relaxation parameters from the composite NMR response from the heterogeneous cartilage tissue. 2D inversion recovery T1 experiments using non-selective excitation and monitor pulses as well as selective excitation and non-selective monitor pulses were used. The 2D relaxation results for each of the above experiments were then analysed for exchange by comparing the experimentally observed parameters to the apparent parameters, calculated from a set of intrinsic parameters, which were adjusted until a reasonable match was realized. In this multi-experiment approach the exchange results from one experiment can be used to corroborate the exchange results from another experiment. In order to circumvent the considerable masking effect that the bulk water has on effective proton NMR characterization of cartilage macromolecular components, the above 2D relaxation-exchange analysis approach also was applied to bovine cartilage tissue in which H2O was replaced with D2O, as well as cartilage tissue in which the bulk water was frozen, so as to allow for the selective saturation of the ice proton magnetization. Combining relaxation times and magnetization exchange analyses results from the four cases, natural cartilage at 3oC and -10oC as well as deuterated cartilage at 3oC and -10oC, a 4-site exchange scenario involving water, proteoglycan (PG) and two collagen spin reservoirs, was arrived at for cartilage tissue. Approximate rates for magnetization exchange between macromolecule spins and water spins as well as for inter-macromolecule magnetization exchange are presented. In addition, the present results have clearly demonstrated that at -10oC, at which temperature the bulk water in the tissue is frozen, the collagen proton magnetization in the tissue exhibit a similar relaxation behavior as seen at 3oC; i.e., the collagen proton magnetizations do not appear to be appreciably affected by the freezing phase transition. Loss of PG is an important indicator of early OA. An ideal MRI scheme for OA detection can be envisioned to involve the direct detection of the degradation of PG with progression of the disease, while at the same time monitoring the collagen content, which is not significantly affected by early OA and can be used as internal reference. In this part of the thesis, first, high field (Larmor frequency of 500 MHz) Magic Angle Spinning (MAS) NMR was used to monitor the changes in the PG spectra of the articular cartilage with dehydration, and with PG-degradation (using trypsin). Second, articular cartilage samples were modified enzymatically to achieve different PG-depletion levels using the trypsin enzyme and then chemical shift imaging (CSI) was used to obtain the PG spectra and a static spectral experiment was performed to measure the broad collagen spectra. The ratio of the PG spectral area to that of the collagen spectral area defines PG content relative to collagen content. As the collagen content does not change appreciably for early OA, this approach provides a relative PG content, which is expected to be useful for in vivo OA detection.
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Ghada Khouqeer (2016). In Vitro NMR Study of Magnetization Exchange at Low Field and Proteoglycan-Depletion at High Field in Articular Cartilage. UWSpace. http://hdl.handle.net/10012/10573