Understanding the multiscale basis of solute transport in the cartilage endplate

项目摘要 腰痛是导致残疾的主要原因，并与椎间盘退变有关。现有的治疗方法 椎间盘退变本质上是外科手术，因此，对非手术替代疗法的需求尚未得到满足。 椎间盘内治疗看起来很有希望，但它需要很高的营养供应。营养素和代谢物进入和 离开髓核必须穿过软骨终板(CEP)，其通透性取决于基质 生化成分(基质数量)和结构，例如，矿物质沉积增加， 蛋白多糖或胶原蛋白限制了可供溶质通过的物理孔隙空间，而稠密和 与完整的CEP相比，受损的CEP基质可能会损害营养物质的运输。事实上，一位高级 我们实验室的研究表明，CEP中较高的AGE含量与较低的溶质吸收有关 矩阵的数量。尽管对基质数量和质量如何影响散装CEP运输有一般了解 性质，与CEP基质成分的多尺度组织有关的运输知识是 缺乏。这些差距激发了我的主要假设，即1)较高的葡萄糖摄入量与 跨多个长度尺度的结构特征，包括更大的孔隙网络连通性和更高的 基质组织的程度，以及2)低CEP运输特性与较高的年龄相关，独立于 矩阵数量。这些假设将通过以下两个目标得到检验。在目标1中，我将发现 跨毫米到纳米的CEP矩阵的结构组织，以及这些层次结构是如何 影响溶质运移性质。总共126个人类CEP组织样本(来自15具身体)将被 收获后，根据养分运输的顺序(高、中、低吸收)将其分为三组。 然后，这些将与生化成分、组织水合作用和块状材料属性(孔隙率)相关 和厚度)。将测量局部材料属性(微米和纳米孔隙率以及孔大小/分布) 并与其他可量化的数据进行比较，如孔连通性、孔尺寸分布和 胶原/基质各向异性。将获得一系列2D数据集，对其进行分割和重建以量化 孔隙面积/体积和3D。这些结构结果将与生化成分、水合作用、 和孔隙度。在目标2中，我将确定由体外效应改变的纳米结构和生化变化。 核糖化试验。先前的研究表明，带负电荷的SGAG会吸引水，而水的含量会增加 AGEs与组织水合呈负相关。我将开发一种体外核糖化分析和葡萄糖 摄取试验测量CEP样品(对照和孵化)AGE含量的变化。最后， 得到的数据将与基质电荷分布、纳米孔和胶原D-周期的变化有关 间距。这些研究将阐明和表征层级结构和基质质量的影响 CEP组织对溶质转运的修饰。

PROJECT ABSTRACT Low back pain is the leading cause of disability and linked to disc degeneration. Existing treatments for disc degeneration are surgical in nature, and thus, there is an unmet need for non-surgical alternatives. Intradiscal therapy looks promising but it requires high nutrition supply. Nutrients and metabolites entering and exiting the nucleus pulposus must penetrate the cartilage endplate (CEP) and its permeability depends on matrix biochemical composition (matrix quantity) and structure, for example, increased deposition of mineral, proteoglycans, or collagen limits the physical pore space available for solutes to pass, while a dense and damaged CEP matrix from degenerated discs might impair nutrient transport than an intact CEP. Indeed, a prior study from our lab showed that higher AGE content in the CEP associates with lower solute uptake for a given amount of matrix. Despite a general understanding of how matrix quantity and quality impact bulk CEP transport properties, knowledge about transport in relation to the multiscale organization of the CEP matrix constituents is lacking. These gaps motivate my main hypotheses that 1) higher glucose uptake is positively correlated with structural characteristics across multiple length scales, including greater pore network connectivity and a higher degree of matrix organization, and 2) low CEP transport properties correlate with higher AGEs, independent of matrix quantity. These hypotheses will be tested through the following two aims. In Aim 1, I will discover the structural organization of the CEP matrix across millimeter to nanometer, and how these hierarchical structure affects solute transport properties. A total of 126 samples of human CEP tissue (from 15 cadavers) will be harvested and then ordered as three groups by the rank of nutrient transport (high, middle, and low uptake). Then, these will be related to biochemical compositions, tissue hydration, and bulk material properties (porosity and thickness). Local material properties (micro- and nano- porosity and pore size/distribution) will be measured and compared with the other quanfiable data, such as pore connectivity, pore size distribution, and collagen/matrix anisotropy. A series of 2D dataset will be obtained, segmented, and reconstructed to quantify pore area/volume and in 3D. These structural outcomes will be related to biochemical composition, hydration, and porosity. In Aim 2, I will identify nanostructural and biochemical changes altered by the effect of in-vitro ribosylation assay. Previous studies showed that negatively charged sGAG attracts water, while increased levels of AGEs negatively correlated to tissue hydration. I will develop an in-vitro ribosylation assay and a glucose uptake assay to measure the variation AGE contents (control vs. incubated) of the CEP samples. Lastly, the obtained data will be related to the changes of matrix charge distribution, nanoporosity, and collagen D-period spacing. These studies will clarify and characterize the effect of hierarchical structure and matrix quality modification of CEP tissue on solute transport.