Single-cell transcriptome profiling reveals the heterogeneity of ossification of the posterior longitudinal ligament (OPLL) and its immune microenvironment

<h4>Background</h4>Ossification of the posterior longitudinal ligament (OPLL) is a heterotopic ossification process of the posterior longitudinal ligament (PLL) that can compress the spinal cord and nerve roots, yet its cellular heterogeneity and pathogenesis remain unclear.<h4>Methods</h4>We performed single-cell RNA sequencing (scRNA-seq) with integrative computational analyses and histological validation on 4,683 cells from surgically resected cervical OPLL lesions obtained from human patients.<h4>Results</h4>We identified 15 major cell subsets, including chondrocyte-lineage populations-progenitor/proliferative fibrocartilage chondrocytes (ProFCs), pre-hypertrophic chondrocytes (preHTCs), and hypertrophic chondrocytes (HTCs)-as well as osteoblasts, endothelial cells, and diverse immune cells. Pseudotime analysis suggested an inferred trajectory from cartilage progenitor-like cells through inflammatory/reactive and hypertrophic states toward osteogenic programs, with progressive activation of ossification, extracellular-matrix, and inflammation-related pathways. Cell-cell communication analyses showed coordinated immune-stromal crosstalk, with FGF signaling enriched in earlier pseudotime states and SPP1-CD44 signaling enriched in later states. SPP1 signals originated predominantly from HTCs, whereas macrophages were prominently involved in the broader inflammatory niche and immune-stromal communication within OPLL lesions. Consistently, SPP1/CD44-positive cells were enriched in OPLL tissues and further increased by IL-1β stimulation in vitro and in an Enpp1-driven OPLL mouse model.<h4>Conclusions</h4>This exploratory single-cell atlas suggests that an inflammatory niche may couple chondrocyte hypertrophy with osteogenic activation and highlights candidate immune-stromal pathways for future biomarker and therapeutic exploration in OPLL.