NMR STUDIES OF PHYSIOLOGY AND BIOCHEMISTRY IN CELLS, ORGANS AND ANIMALS

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Summary of Work: We have begun a detailed exploration of cartilage growth and development in a hollow-fiber bioreactor specially designed for NMR studies. This system permits cells and the three-dimensional matrix which they elaborate to be studied longitudinally for several weeks in a non-invasive manner. Ultimately, we hope to define appropriate conditions for neocartilage development in osteoarthritic joints in vivo. Detailed correlations between histologic data and MRI results have been carried out in cartilage developing from chick sterna. MRI revealed the development of stromal layers between growth units of neocartilage centered about each hollow fiber. Density images show decreased mobile water content in these layers. Just outside the fiber walls, we find high proton density with relatively low mobility. Mobility increases with distance from the hollow fibers within the growth units, corresponding to differences in cell size and density. In magnetization transfer contrast images, we find that the lowest km values correspond to areas of high proteoglycan concentrations. These are prevalent in the mid-regions of the growth units. In contrast, the stromal layers and the regions around the fibers which are relatively proteoglycan-poor show the highest km values, potentially indicating greater collagen-water interactions. We are also using 31P NMR to gain insight into metabolic adaptations as chondrocytes mature. We have been able to establish the presence of phosphocreatine in this system, and have demonstrated a decrease in intracellular pH during early development of the tissue. This is consistent with the known tendency for developing chondrocyte-cartilage systems to become increasingly dependent on anaerobic metabolism. In addition, we are investigating the effects of biologic response modifiers on neocartilage development. Using MRI, we have found that matrix proliferation from human articular chondrocytes is accelerated by addition of IGF-1 + TGF- or IGF-1 + cTGF to the growth medium. Studies of the interactions of these growth factors and cytokines are ongoing. We have also utilized NMR spectroscopy to measure high-energy phosphate metabolites in muscle distal to experimental femoral artery resection in rats. We have found that over a period of weeks following femoral artery resection, 2 month old rats recover muscle metabolic reserve significantly more rapidly than 20 month old rats. This likely reflects loss of angiogenic potential with age. Further, because modulators of angiogenesis have great promise for treatment of arterial vascular disease, we are performing experiments involving application of vascular endothelial growth factor (VEGF) just prior to femoral artery resection. We found that, over a period of days to weeks, VEGF acted to normalize the pattern of high energy phosphate response to acute muscle stimulation and recovery from stimulation. This indicates an increase in the rate of development of perfusing vessels. Extensions of this work which are underway include variations in the timing and other central elements of delivery of VEGF therapy, either by transgenes or directly. We also plan to implement NMR imaging methods to look more directly at increased blood flow to the ischemic limb.