Label-Free Quantification of Microscopic Alignment in Engineered Tissue Scaffolds by Polarized Raman Spectroscopy

Monitoring of extracellular matrix (ECM) microstructureis essentialin studying structure-associated cellular processes, improving cellularfunction, and for ensuring sufficient mechanical integrity in engineeredtissues. This paper describes a novel method to study the microscalealignment of the matrix in engineered tissue scaffolds (ETS) thatare usually composed of a variety of biomacromolecules derived bycells. First, a trained loading function was derived from Raman spectraof 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(-1)) were detected with respect to the polarization angle. These changeswere mainly caused by the aligned matrix of many compounds withinthe tissue relative to the laser polarization, including proteins,lipids, and carbohydrates. Hence this trained function was appliedto quantify the alignment within ETS of various matrix componentsderived by cells. Furthermore, a simple metric called Amplitude AlignmentMetric (AAM) was derived to correlate the orientation dependence ofpolarized Raman spectra of ETS to the degree of matrix alignment.It was found that the AAM was significantly higher in anisotropicETS than isotropic ones. The PRS method revealed a lower p-value for distinguishing the alignment between these two types ofETS as compared to the microscopic method for detecting fluorescent-labeledprotein matrices at a similar microscopic scale. These results indicatethat the anisotropy of a complex matrix in engineered tissue can beassessed at the microscopic scale using a PRS-based simple metric,which is superior to the traditional microscopic method. This PRS-basedmethod can serve as a complementary tool for the design and assessmentof engineered tissues that mimic the native matrix organizationalmicrostructures.