Single‐Cell Transcriptomic Analysis Reveals Biomechanical Loading‐Induced Imbalance in Bone and Fat, Leading to Ossification in Lumbar Intervertebral Disc Nucleus Pulposus Degeneration

ABSTRACT In this study, we explored the impact of different biomechanical loadings on lumbar spine motion segments, particularly concerning intervertebral disc degeneration (IVDD). We aimed to uncover the cellular milieu and mechanisms driving ossification in the nucleus pulposus (NP) during IVDD, a process whose underlying mechanisms have remained elusive. The study involved the examination of fresh NP tissue from the L3‐S1 segment of five individuals, either with IVDD or healthy. The analysis consisted of histopathological evaluation and single‐cell RNA sequencing. To further validate the impact of biomechanical loading on IVDD, particularly on the CITED4 + METRN + NP chondrocytes and the bone‐fat balance mechanism, a retrospective analysis was conducted using paraffin‐embedded NP samples from patients. A distinct subset of CITED4 + METRN+ chondrocytes in the degenerated NP that were influenced by biomechanical loading was identified. These cells were evaluated for their potential as diagnostic biomarkers. Pseudotemporal analysis indicated that inflammation and repair processes were integral to NP ossification. Notably, the L4/5 and L5/S1 segments with severe IVDD showed pronounced ossification and heightened lipogenic metabolism. Cell communication analysis sheds light on the roles of bone‐fat balance proteins and various ossification genes. Additionally, immunohistochemistry and immunofluorescence confirmed that biomechanical loading intensified IVDD by fostering osteogenic differentiation, mediated by macrophage migration inhibitory factor (MIF)‐regulated bone‐fat balance. This research reveals the microenvironmental factors of IVDD NP ossification under biomechanical loading, highlighting the role of bone‐fat imbalance. These insights significantly enhance the understanding of IVDD pathogenesis and pave the way for innovative therapeutic approaches.