Genetic differences in osteogenic differentiation potency in the thoracic ossification of the ligamentum flavum under cyclic mechanical stress

Mechanical stress and genetic factors play important roles in the occurrence of thoracic ossification of ligament flavum (TOLF), which can occur at one, two, or multiple levels of the spine. It is unclear whether single- and multiple-level TOLF differ in terms of osteogenic differentiation potency and osteogenesis-related gene expression under cyclic mechanical stress. This was addressed in the present study using patients with non-TOLF and single- and multiple-level TOLF (n=8 per group). Primary ligament cells were cultured and osteogenesis was induced by application of cyclic mechanical stress. Osteogenic differentiation was assessed by evaluating alkaline phosphatase (ALP) activity and the mRNA and protein expression of osteogenesis-related genes, including ALP, bone morphogenetic protein 2 (BMP2), Runt-related transcription factor-2 (Runx-2), osterix, osteopontin (OPN) and osteocalcin. The application of cyclic mechanical stress resulted in higher ALP activity in the multiple-level than in the single-level TOLF group, whereas no changes were observed in the non-TOLF group. The ALP, BMP2, OPN and osterix mRNA levels were higher in the multiple-level as compared to the single-level TOLF group, and the levels of all osteogenesis-related genes, apart from Runx2, were higher in the multiple-level as compared to the non-TOLF group. The osterix and ALP protein levels were higher in the multiple-level TOLF group than in the other 2 groups, and were increased with the longer duration of stress. These results highlight the differences in osteogenic differentiation potency between single- and multiple-level TOLF that may be related to the different pathogenesis and genetic background.