Label-free multimodal profiling of collagen-matrix mineralization during nucleus pulposus osteogenesis to guide bioactive scaffold design for spinal fusion cell therapy

Osteogenic differentiation of nucleus pulposus cells (NPCs) offers a promising biological approach to cell-based spinal fusion, serving as a native alternative to bone marrow-derived mesenchymal cells (MSCs). However, clinical translation requires robust, non-destructive analytical methods to monitor matrix maturation and define biomechanical design targets. Here, we established a label-free, multimodal framework integrating Raman spectroscopy, two-dimensional correlation spectroscopy (2D-COS), X-ray diffraction (XRD), and nanoindentation to longitudinally track the in vitro osteogenesis of human NPCs from degenerated discs (Pfirrmann grades III-V). Raman analysis identified key spectral markers, with the phosphate-to-phenylalanine ratio progressively increasing from day 14 to 28 to track mineral integration. Crucially, Amide I deconvolution revealed an upshift of the central helix-associated band (1655 to 1665 cm^-1) and an increased Conformational Complexity Index (CCI), signifying the structural maturation and heterogeneity of the collagen-rich matrix. Furthermore, 2D-COS demonstrated a temporal sequence where collagen remodeling strictly precedes phosphate accumulation, followed by a terminal decline in bulk lipid metabolism. Full-range principal component analysis (PCA, 600-3100 cm^-1) unbiasedly resolved distinct phenotypic stages, identifying a major developmental transition at day 14 and stabilization by day 28. Nanoindentation confirmed the evolving matrix stabilized at an effective reduced modulus (E_r) of ~3.25 GPa, correlating strongly with Raman-derived mineral metrics (r = 0.737). Unlike the extreme rigidity of metallic implants, this ~3.25 GPa benchmark provides a biologically relevant compliance target for future scaffold design. Ultimately, this optical-mechanical framework shifts evaluation away from destructive assays, defining foundational parameters for bio-instructive collagen-matrix engineering.