• Daisuke Sugita, Takafumi Yayama, Kenzo Uchida, Yasuo Kokubo, Hideaki Nakajima, Atsushi Yamagishi, Naoto Takeura, and Hisatoshi Baba.
• From the Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui, Japan.
• Spine. 2013 Oct 15;38(22):E1388-96.

Study DesignHistological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL).ObjectiveTo examine the hypothesis that Ihh signaling in correlation with Sox9 and parathyroid-related peptide hormone (PTHrP) facilitates chondrocyte differentiation in enchondral ossification process in human cervical OPLL.Summary Of Background DataIn enchondral ossification, certain transcriptional factors regulate cell differentiation. OPLL is characterized by overexpression of these factors and disturbance of the normal cell differentiation process. Ihh signaling is essential for enchondral ossification, especially in chondrocyte hypertrophy.MethodsSamples of ossified ligaments were harvested from 45 patients who underwent anterior cervical decompressive surgery for symptomatic OPLL, and 6 control samples from patients with cervical spondylotic myelopathy/radiculopathy without OPLL. The harvested sections were stained with hematoxylin-eosin and toluidine blue, examined by transmission electron microscopy, and immunohistochemically stained for Ihh, PTHrP, Sox9, type X, XI collagen, and alkaline phosphatase. Immunoblot analysis was performed in cultured cells derived from the posterior longitudinal ligaments in the vicinity of the ossified plaque and examined for the expression of these factors.ResultsThe ossification front in OPLL contained chondrocytes at various differentiation stages, including proliferating chondrocytes in fibrocartilaginous area, hypertrophic chondrocytes around the calcification front, and apoptotic chondrocytes near the ossified area. Immunoreactivity for Ihh and Sox9 was evident in proliferating chondrocytes and was strongly positive for PTHrP in hypertrophic chondrocytes. Mesenchymal cells with blood vessel formation were positive for Ihh, PTHrP, and Sox9. Cultured cells from OPLL tissues expressed significantly higher levels of Ihh, PTHrP, and Sox9 than those in non-OPLL cells.ConclusionOur results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.

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