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

工作总结：我们已经开始了软骨生长的详细探索 核磁共振专用中空纤维生物反应器的研制 问题研究 该系统允许细胞和三维矩阵， 他们详细阐述了几个星期的纵向研究， 非侵入性方式。 最终，我们希望确定适当的条件， 用于体内骨关节炎关节中的新软骨发育。 详细 组织学数据和MRI结果之间的相关性已经进行 在鸡胸骨的软骨发育中。 核磁共振成像显示 新生软骨生长单位之间的基质层， 中空纤维 密度图像显示，这些区域中的移动的水含量减少， 层次。就在纤维壁外，我们发现高质子密度， 流动性相对较低。 移动性随着距离的增加而增加 生长单位内的中空纤维，对应于 细胞大小和密度。 在磁化传递对比图像中，我们发现 最低的km值对应于高蛋白聚糖的区域， 浓度的 这些在增长的中部地区普遍存在， 单位 相反，基质层和纤维周围的区域 相对蛋白聚糖贫乏的显示最高的Km值， 这可能表明胶原蛋白-水的相互作用更强。 我们也 使用31 P NMR深入了解软骨细胞的代谢适应性 成熟 我们已经能够确定磷酸肌酸的存在 这一系统，并已证明细胞内pH值的下降， 组织的早期发育。 这与已知的 软骨细胞-软骨系统的发展趋势是 越来越依赖于无氧代谢。 此外，我们在 研究生物反应调节剂对新生软骨的影响 发展 使用MRI，我们发现， 通过加入IGF-1 + TGF-β 1促进人关节软骨细胞的生长。 或IGF-1 + cTGF加入到生长培养基中。 研究了 这些生长因子和细胞因子仍在持续。 我们还利用核磁共振光谱来测量高能磷酸盐 实验性股动脉切除术远端肌肉中的代谢物 大鼠我们发现，在股动脉栓塞后的几周内， 切除后2月龄大鼠肌肉代谢储备明显恢复 比20个月大的老鼠更快。 这可能反映了 血管生成潜力。 此外，由于调节剂的 血管生成对于动脉血管的治疗具有很大的前景 疾病，我们正在进行实验，涉及应用血管 血管内皮生长因子（VEGF）。 我们发现，在几天到几周的时间里，VEGF起到了恢复正常的作用。 高能磷酸盐对急性肌肉刺激的反应模式， 从刺激中恢复。 这表明， 灌注血管的发育。 这项工作的延伸， 正在进行的包括时间和其他核心要素的变化， 通过转基因或直接递送VEGF疗法。 我们还计划 利用核磁共振成像方法， 流向缺血肢体

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.