Study design: Histological, immunohistochemical, and real-time reverse transcription-polymerase chain reaction analyses of the expression of cell signaling and transcriptional factors in human ossification of ligamentum flavum (OLF).
Objective: To test the hypothesis that β-catenin plays a role in the ossification of OLF cells in response to cyclic tensile strain.
Summary of background data: Several studies have investigated the roles of biomechanical and metabolic factors in the development and progression of OLF, based on the importance of genetic and biological factors. The process of ossification includes enchondral ossification, although such pathology remains poorly defined.
Methods: Using real-time reverse transcription-polymerase chain reaction, we analyzed the mRNA expression levels of signaling factors known to be involved in the ossification process (β-catenin, Runx2, Sox9, and osteopontin) in cultured OLF cells subjected to cyclic tensile strain. Cyclic tensile strain was produced by Flexercell FX-3000 (Flexercell International, Hillsborough, NC), applied for 0, 6, 12, or 24 hours. The localization of these factors was examined in decalcified paraffin OLF sections by immunohistochemistry. Controlled samples were harvested from nonossified ligamentum flavum of patients who underwent thoracic posterior surgical procedures.
Results: Under resting conditions (no tensile strain), the mRNA levels of β-catenin, Runx2, Sox9, and osteopontin in cultured OLF cells were significantly higher than in the control non-OLF cells. Application of cyclic tensile strain to OLF cells resulted in significant increases in mRNA expression levels of β-catenin, Runx2, Sox9, and osteopontin at 24 hours. Hypertrophic chondrocytes present around the calcification front were immunopositive for Runx2 and osteopontin. Immunoreactivity of β-catenin and Sox9 was strongly present in premature chondrocytes in the fibrocartilage area.
Conclusion: Our results indicated that cyclic tensile strain applied to OLF cells activated their ossification through a process mediated by the β-catenin signaling pathway.