Cobalt Chloride-simulated Hypoxia Promoted Ossification of the Posterior Longitudinal Ligament Through the HIF1A–BMP4 Axis

Background: The ossification of the posterior longitudinal ligament (OPLL) is a progressive spinal disorder predominantly observed in Asian populations. Unfortunately, there is a limited availability of conservative therapies to impede the progression of OPLL. This study investigates the effects of cobalt chloride (CoCl2), which simulates an in vitro hypoxic microenvironment, on OPLL and explores its potential mechanisms, aiming to enhance our understanding of the pathogenesis of OPLL. Methods: Ligament cells were extracted from patients with OPLL and normal posterior longitudinal ligament (PLL) tissues, confirming their mesenchymal stem cell (MSC)-like properties. To simulate hypoxia, cells were treated with varying concentrations of CoCl2, and the effects on hypoxia-inducible factor 1-alpha (HIF1A) expression and osteogenic differentiation were assessed. Real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and Western blotting were used to quantify HIF1A and bone morphogenetic protein 4 (BMP4) expression. Immunohistochemistry was performed to visualize HIF1A in tissue samples. Osteogenic differentiation was evaluated through Alizarin Red S staining and alkaline phosphatase (ALP) assays, with optical density (OD) values measured using a microplate reader. Transcriptome sequencing was conducted to identify differentially expressed genes following CoCl2 treatment. Results: We found that HIF1A was differentially expressed in the OPLL. Both PLL cells and OPLL cells exhibited mesenchymal stem cell properties; however, OPLL cells demonstrated a greater capacity for osteogenic differentiation (p < 0.05). Following stimulation with cobalt chloride, the expression of HIF1A increased, which correlated with an enhanced osteogenic differentiation ability in PLL cells. Biosignature analysis revealed that HIF1A plays a regulatory role in BMP4 expression. Notably, BMP4 was downregulated, and the degree of osteogenic differentiation decreased upon inhibition of HIF1A with siRNA in OPLL cells. Conclusions: Primary cells derived from normal PLL and OPLL exhibited MSC-like properties and demonstrated the capacity for osteogenic, adipogenic, and chondrogenic differentiation, with OPLL cells showing a greater propensity for osteogenic differentiation. This study reports the potential involvement of HIF1A in the development of OPLL and investigates the regulatory role of the HIF1A-BMP4 axis in this process.