MR Spectroscopy of Local Metabolism in Engineered and Native Cartilage

工程软骨和天然软骨局部代谢的磁共振波谱

• 批准号: 7732354
• 负责人: Richard Spencer
• 金额: $ 17.23万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732354
• 关键词: Alginates; Area; Bathing; Bioenergetics; Bioreactors; Cartilage; Cartilage Diseases; Cartilage Matrix; Cell Respiration; Cell Survival; Characteristics; Chondrocytes; Citric Acid Cycle; Clinical; Collagen; Collection; Condition; Consumption; Data; Dense Connective Tissue; Deposition; Depth; Development; Diffusion; Electrodes; Energy Metabolism; Engineering; Equilibrium; Excision; Fiber; Fiber Optics; Fluorescence; Gel; Glucose; Glycolysis; Image; Implant; Liquid substance; Localized; Magnetic Resonance Spectroscopy; Maps; Measurement; Measures; Mechanics; Metabolic; Metabolism; Methods; Microdialysis; Microelectrodes; Modeling; Monitor; Natural regeneration; Nutrient; Oxygen; Oxygen measurement, partial pressure, arterial; Perfusion; Phase; Placement; Play; Positioning Attribute; Procedures; Production; Property; Rate; Reporting; Resolution; Role; Sampling; Sepharose; Site; Techniques; Testing; Thinking; Tissue Engineering; Tissue Viability; Tissues; Work; articular cartilage; base; extracellular; fluorophore; glucose uptake; improved; interest; repaired; research study; response; scaffold; sensor; solute

Mature articular cartilage is avascular and relies on diffusion of metabolites in order to maintain cellular metabolism and viability. As would be expected for dense connective tissue, the rate of diffusion within cartilage is modest, so that limited delivery of nutrients and removal of metabolic byproducts may play a substantial role in its highly restricted self-repair capacity. Although data are limited, there is evidence that chondrocytes rely heavily on glycolysis, rather than aerobic metabolism, for their bioenergetic demands. Chondrocyte viability in native tissue, and in culture is modulated by oxygen and glucose, as is matrix production itself. The tricarboxylic acid cycle is thought to contribute minimally to the direct energy demands of chondrocytes, but oxygen tension exerts a marked influence on glucose consumption. Lee et al. report a reduction in glycolysis of 40% in anoxic conditions based on lactate production and 25% based on glucose uptake. Lactate, the main by-product of chondrocyte energy metabolism, is an indirect measure of metabolic activity in cartilage. In addition, if production of lactate is greater than diffusive loss, lactate accumulation and the attendant lowering of extracellular pH limits matrix production and cell viability. The relationship of cell viability and matrix production to metabolic state, including substrate availability and byproduct removal, therefore places significant constraints on approaches to cartilage culture and tissue engineering. However, monitoring the levels of glucose, lactate, and oxygen in cartilage is difficult. Indirect, model-dependent, measures of metabolite diffusion rates and nutrient utilization have been performed. Moreover, because indirect measurements of metabolites are made via sampling bath concentrations, concentration profiles within the culture are not available. Therefore, results depend upon assumptions of metabolite mass balance and diffusion characteristics. Direct measurements of metabolites have also been performed, using microdialysis probes positioned at fixed positions within a chondrocyte-seeded gel. Probe placement permitted collection of metabolites as a function of depth into the culture. However, spatial resolution is limited in this procedure, sampling volumes are large and not well-defined, and the arrangement of measurement points is inflexible and determined by pre-defined probe placement. Finally this technique is not suitable for generating metabolite maps within tissue, as that would require many sampling sites. Thus, measurement of glucose, lactate, and oxygen within chondrocyte cultures or within cartilage remains an outstanding problem. Such measurements would permit study of the relationships among tissue viability, mechanical properties, metabolism, and matrix production. It would be of particular interest to characterize the metabolic response to alterations in the glucose and oxygen substrate levels in dynamic experiments. Finally, we note that while measurement of endogenous metabolite levels permits direct metabolic analyses, measurement of the transport of exogenous metabolites within and through cartilage permits assessment of their diffusion characteristics. It is also likely that clinical tissue engineering approaches to cartilage disease will use scaffold-based implants, with the choice of scaffold material having a significant impact on the production of matrix. Xu et al. report increased deposition of GAGs in alginate as compared to agarose in perfusion bioreactors, and increased deposition of collagen under perfused rather than static conditions. This indicates that the mobility of solutes within a construct plays a significant role in the properties of the developing tissue. Localized metabolite measurements would allow us to define relationships between metabolite diffusivity and tissue characteristics; much previous work in this area has relied upon destructive tests. As is the case with metabolites, measurement of oxygen concentrations in tissue is highly problematic. Classical Clark-type microelectrodes must be inserted directly into the tissue, offer limited spatial resolution (ca. 1 mm), consume oxygen during measurements and are sensitive to local flow and diffusion in the liquid phase of the tissue. Fiber optic sensors (optodes) measure dissolved oxygen through its quenching effect on the fluorescence of a fluorophore immobilized on the tip of the fiber. Optodes do not consume oxygen, are relatively insensitive to flow and diffusion effects and offer spatial resolution as high as 50 microns but must also physically penetrate the sample. Finally, as in the case of metabolite measurements with microdialysis probes, electrode-based techniques are not practical for constructing oxygen concentration maps. Previous work in the NMR Unit has led to the development and application of an EPR-based method for oxygen mapping in cartilage. While effective, this technique required availability of highly specialized EPR imaging apparatus. Therefore, the development of alternative, MR-based, oxygen mapping methods suitable for measurements in developing cartilage remains of interest.

成熟的关节软骨是血管的，依赖代谢物的扩散，以维持细胞代谢和生存能力。 正如致密结缔组织所预期的那样，软骨内的扩散速率是适度的，因此养分有限和代谢副产品的去除可能在其高度限制的自我修复能力中起着重要作用。 尽管数据有限，但有证据表明软骨细胞严重依赖糖酵解而不是有氧代谢，因为它们的生物能量需求。 天然组织中的软骨细胞生存力，并且培养中的氧气和葡萄糖也可以调节基质的产生本身。 三羧酸周期被认为对软骨细胞的直接能量需求的贡献最小，但是氧张力对葡萄糖消耗产生了显着影响。 Lee等。报告说，基于乳酸产生的缺氧条件下40％的糖酵解降低，基于葡萄糖摄取的25％。 乳酸是软骨细胞能量代谢的主要副产品，是软骨中代谢活性的间接度量。 另外，如果乳酸的产生大于扩散损失，则乳酸积累以及伴随的细胞外pH限制限制基质生产和细胞活力。 细胞活力和基质产生与代谢状态的关系，包括底物的可用性和副产品去除，因此对软骨培养和组织工程的方法施加了重大限制。 但是，难以监测软骨中葡萄糖，乳酸和氧气的水平。 已经进行了间接，模型依赖性的代谢产物扩散率和养分利用率的度量。 此外，由于代谢物的间接测量是通过抽样浴浓度进行的，因此培养物中的浓度分布不可用。 因此，结果取决于代谢物质量平衡和扩散特征的假设。 还使用位于软骨细胞种子凝胶中固定位置的微透析探针进行了直接测量代谢物。 探针放置允许收集代谢产物，这是深度的函数。 但是，在此过程中，空间分辨率受到限制，采样量很大且不定义，并且测量点的排列是不灵活的，并由预定的探针放置确定。 最后，该技术不适合在组织中产生代谢物图，因为这需要许多采样位点。 因此，在软骨细胞培养物或软骨内的葡萄糖，乳酸和氧的测量仍然是一个鲜明的问题。 这种测量将允许研究组织生存能力，机械性能，代谢和基质产生之间的关系。 表征动态实验中葡萄糖和氧底物水平改变的代谢反应将特别感兴趣。 最后，我们注意到，尽管测量内源性代谢产物水平允许直接代谢分析，但测量了在软骨内和通过软骨内部和通过软骨内外代谢物的运输测量，以评估其扩散特征。 软骨疾病的临床组织工程方法也很可能会使用基于支架的植入物，而选择脚手架材料对基质的产生产生了重大影响。 Xu等。与灌注生物反应器中的琼脂糖相比，报告增加了藻酸盐中插孔的沉积，并在灌注而非静态条件下增加了胶原蛋白的沉积。 这表明构建体内溶质的迁移率在发育组织的性质中起着重要作用。 局部代谢物测量将使我们能够定义代谢物扩散率和组织特征之间的关系。该领域的许多以前的工作都依赖于破坏性测试。 与代谢产物一样，组织中氧浓度的测量是有问题的。 经典的克拉克型微电极必须直接插入组织中，提供有限的空间分辨率（约1毫米），在测量过程中消耗氧气，并且对局部流动和组织液相中的局部流动敏感。光纤传感器（OPTODES）通过其对纤维尖端上荧光团荧光的荧光作用测量溶解氧。 Optodes不消耗氧气，对流动和扩散效应相对不敏感，并提供高达50微米的空间分辨率，但也必须物理地穿透样品。 最后，与微透析探针的代谢物测量一样，基于电极的技术对于构建氧浓度图是不切实际的。 NMR单元的先前工作导致了基于EPR的方法在软骨中开发和应用。 虽然有效，但该技术需要提供高度专业化的EPR成像设备。 因此，开发适合开发软骨的测量的替代性，基于M的氧映射方法。