Early Alignment of Structural Proteins in Tissue Constructs Measured by Polarized Raman Spectroscopy

Structural protein alignment plays a vital role in determining the mechanical function and biological performance of engineered tissues. During tissue remodeling, alignment of fibrous components such as collagen and actin can evolve with cellular forces and geometric constraints. Previously, we developed a polarized Raman spectroscopy (PRS) technique that provides a nondestructive, label-free method to quantify protein alignment in tissues and hydrated constructs. Spectral data are analyzed using principal component analysis (PCA) to compute an amplitude alignment metric (AAM), reflecting molecular orientation. In this study, we employed PRS to quantify the spatiotemporal evolution of protein alignment in myoblast-seeded collagen constructs over a 5-day incubation period. Raman spectra were collected at seven polarization angles (0°-180°) across three defined regions-middle center (MC), middle edge (ME), and corner edge (CE)-on days 1, 2, and 5. PRS suggested a region-dependent temporal redistribution of alignment patterns, with early higher alignment at MC and later increased alignment at CE. On day 1, MC exhibited the highest alignment (AAM = 0.71 ± 0.38), while CE showed minimal organization (0.16 ± 0.23). By day 5, alignment was highest at CE (0.62 ± 0.28), while MC decreased (0.57 ± 0.36), indicating a spatial redistribution of matrix organization. There were significant main effects of region and region-by-time interaction, highlighting location- and time-dependent realignment. Within the CE region, alignment on days 2 and 5 was significantly higher than on day 1, consistent with delayed but sustained peripheral alignment. Qualitative immunofluorescence imaging provided complementary visualization of increasing fiber density and directional organization over time. Together, these findings show that PRS, with PCA-based spectral analysis, can detect early spatial and temporal changes in molecular anisotropy within myoblast-seeded collagen constructs. The observed region-dependent alignment changes are consistent with the combined influence of cellular contractility, boundary constraints, and matrix remodeling. Future studies with matched acellular controls and broader remodeling stages and construct conditions will help clarify matrix-associated contributions to PRS-derived alignment during engineered tissue maturation.