Label-Free Evaluation of Osteogenic Differentiation Using Extended-Focus Raman Imaging

Periodontal ligament stem cell (PDLSC)-based therapy has emerged as a promising approach to regenerate damaged periodontal tissue in periodontitis treatments. To advance the application of this therapy, a nondestructive method to monitor osteogenic differentiation and evaluate the osteogenic abilities of PDLSCs is essential. In this study, we developed a rapid extended-focus Raman imaging technique for the label-free evaluation of cell differentiation. The developed technique enabled the high-throughput acquisition of Raman spectra of the whole cell volume, facilitating the monitoring of osteogenic differentiation of PDLSCs lineages with low and high osteogenic abilities. Using multivariate analysis of Raman spectra, we identified extracellular matrix (ECM) components represented by collagen type I as key contributors to the spectral differences between two cell populations. Combining Raman imaging and fluorescence observation, we confirmed the correlation between ECM deposition and hydroxyapatite (HA) generation, which indicated that ECM accumulation reflects the osteogenic ability of PDLSCs. These findings highlight the application of the extended-focus Raman imaging approach to realize the label-free assessment of PDLSC lineages with distinct osteogenic capabilities, paving the way to nondestructive cell screening in periodontal tissue regeneration treatment.