Mechanical and Aerospace Engineering, University of Florida, Gainesville, Florida 32611, United States.
J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, Gainesville, Florida 32611, United States.
ACS Biomater Sci Eng. 2023 Jun 12;9(6):3206-3218. doi: 10.1021/acsbiomaterials.3c00242. Epub 2023 May 12.
Monitoring of extracellular matrix (ECM) microstructure is essential in studying structure-associated cellular processes, improving cellular function, and for ensuring sufficient mechanical integrity in engineered tissues. This paper describes a novel method to study the microscale alignment of the matrix in engineered tissue scaffolds (ETS) that are usually composed of a variety of biomacromolecules derived by cells. First, a trained loading function was derived from Raman spectra of highly aligned native tissue via principal component analysis (PCA), where prominent changes associated with specific Raman bands (e.g., 1444, 1465, 1605, 1627-1660, and 1665-1689 cm) were detected with respect to the polarization angle. These changes were mainly caused by the aligned matrix of many compounds within the tissue relative to the laser polarization, including proteins, lipids, and carbohydrates. Hence this trained function was applied to quantify the alignment within ETS of various matrix components derived by cells. Furthermore, a simple metric called Amplitude Alignment Metric (AAM) was derived to correlate the orientation dependence of polarized Raman spectra of ETS to the degree of matrix alignment. It was found that the AAM was significantly higher in anisotropic ETS than isotropic ones. The PRS method revealed a lower -value for distinguishing the alignment between these two types of ETS as compared to the microscopic method for detecting fluorescent-labeled protein matrices at a similar microscopic scale. These results indicate that the anisotropy of a complex matrix in engineered tissue can be assessed at the microscopic scale using a PRS-based simple metric, which is superior to the traditional microscopic method. This PRS-based method can serve as a complementary tool for the design and assessment of engineered tissues that mimic the native matrix organizational microstructures.
监测细胞外基质（ECM）的微观结构对于研究与结构相关的细胞过程、改善细胞功能以及确保工程组织具有足够的机械完整性至关重要。本文描述了一种研究通常由细胞衍生的各种生物大分子组成的工程组织支架（ETS）中基质微尺度排列的新方法。首先，通过主成分分析（PCA）从高度排列的天然组织的拉曼光谱中得出一个经过训练的加载函数，其中与特定拉曼带（例如 1444、1465、1605、1627-1660 和 1665-1689 cm）有关的明显变化相对于偏振角进行了检测。这些变化主要是由组织内许多化合物的排列基质相对于激光偏振引起的，包括蛋白质、脂质和碳水化合物。因此，该训练函数用于量化细胞衍生的各种基质成分在 ETS 中的排列。此外，还衍生出一种称为振幅对准度量（AAM）的简单度量标准，以将 ETS 的偏振拉曼光谱的取向依赖性与基质对准程度相关联。结果发现，各向异性 ETS 的 AAM 明显高于各向同性 ETS。与在类似微观尺度下检测荧光标记蛋白基质的微观方法相比，PRS 方法在区分这两种类型的 ETS 的对准方面的 PRS 值较低。这些结果表明，使用基于 PRS 的简单度量标准可以在微观尺度上评估工程组织中复杂基质的各向异性，这优于传统的微观方法。这种基于 PRS 的方法可以作为设计和评估模仿天然基质组织微观结构的工程组织的补充工具。
