Raman spectroscopy enables highly sensitive profiling of osteogenic mineralization in periodontal organoids

This study aimed to assess the capability of Raman spectroscopy to detect osteogenic differentiation in structurally intact organoids derived from human periodontal ligament stem cells (PDLSC) and to compare the mineral deposition capacity between PDLSC monolayers and organoids.

PDLSC organoids were generated using a scaffold-free cell sheet culture technique. Organoids and monolayers were subjected to osteogenic induction and/or inflammatory stimulation for 14 days. Samples were analysed by morphological assessment, weighing, histology, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Alizarin Red staining and Raman spectroscopy.

Osteogenic induction resulted in more than a threefold increase in organoid mass, partly due to enhanced cellularization confirmed by 4',6-diamidino-2-phenylindole (DAPI) staining. Histological analysis showed uniform cell distribution in both undifferentiated and differentiated organoids. Undifferentiated organoids exhibited higher baseline expression of osteogenesis-related genes (BMP2, MSX2, OP) compared with monolayers, suggesting inherent osteogenic priming. Calcium deposition increased by 2.2-fold in differentiated organoids and 1.3-fold in monolayers. Raman spectroscopy demonstrated superior sensitivity, revealing a 47.2-fold increase in the mineral-to-matrix (M:M) ratio in differentiated organoids versus a 3.7-fold increase in monolayers. Inflammatory stimulation did not significantly alter organoid mass, calcium deposition or M:M ratio during osteogenic induction.

Raman spectroscopy provides a precise, quantitative and non-destructive approach for assessing osteogenic differentiation in structurally intact periodontal organoids. These findings support its potential as a valuable tool for periodontal disease modelling and therapeutic screening.