Enhanced Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in Ossification of the Posterior Longitudinal Ligament Through Activation of the BMP2-Smad1/5/8 Pathway.

Ossification of the posterior longitudinal ligament (OPLL) is characterized by ectopic OPLL. To date, the specific molecular pathogenesis of OPLL has not been clearly elucidated. In this study, bone marrow-derived mesenchymal stem cells obtained from healthy donors (HD-MSCs) and patients with OPLL (OPLL-MSCs) were cultured in osteogenic differentiation medium for 21 days. The osteogenic differentiation capacity was determined by alizarin red S (ARS) and alkaline phosphatase (ALP) assays. Gene expression levels of osteoblastic markers were measured by quantitative reverse transcription-polymerase chain reaction. Protein levels of related genes and the activation of related signaling pathways were measured by western blotting. LDN193189 was used to inhibit the Smad1/5/8 pathway, and small interfering RNA was used to regulate BMP2 expression. Our results showed that the OPLL-MSCs had stronger ARS staining and ALP activity and higher expression of RUNX2, Osterix, and OCN than the HD-MSCs. During osteogenic differentiation, the Smad1/5/8 pathway was overactivated in the OPLL-MSCs, and LDN193189 inhibition reversed the enhanced osteogenic ability of these cells. Besides, BMP2 was upregulated in the OPLL-MSCs. After BMP2 knockdown, the abnormal osteogenic differentiation of OPLL-MSCs was rescued. Thus, abnormal activation of the BMP2-Smad1/5/8 pathway induces enhanced osteogenic differentiation of OPLL-MSCs compared with HD-MSCs. These findings reveal a mechanism of pathological osteogenesis in OPLL and provide a new perspective on inhibiting pathological osteogenesis by regulating BMP2.