Whole transcriptome analysis for ossification of vertebral ligament using RNA interference and microbeads array

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Histological, immunohistochemical, and real-time RT-PCR analyses of the expression of cell signaling and transcriptional factors in human ossified vertebral ligament. We analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process in cultured ossified ligament cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexerce(R) FX-3000. The localization of these factors was examined in decalcified paraffin sections by immunohistochemistry. Controlled samples were harvested from non-ossified vertebral ligament of patients. Under resting conditions(no tensile strain), the mRNA levels ofβ-catenin, Runx2, Sox9 and Osteopontin in cultured ossified ligament cells were significantly higher than in the control cells. Application of cyclic tensile strain to ossified cells resulted in significant increases in mRNA expression levels ofβ-catenin, Runx2, Sox9, and Osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and Osteopontin. Immunoreactivity ofβ-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area. Our results indicated that cyclic tensile strain applied to ossified vertebral ligament cells activated their ossification through a process mediated by theβ-catenin signaling pathway.