title: Indian hedgehog signaling promotes chondrocyte differentiation in enchondral ossification in human cervical ossification of the posterior longitudinal ligament.
abstract: METHODS
  Histological, immunohistochemical, and immunoblot analyses of the expression of Indian hedgehog (Ihh) signaling in human cervical ossification of the posterior longitudinal ligament (OPLL).
  
  
  OBJECTIVE
  To 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.
  
  
  BACKGROUND
  In 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.
  
  
  METHODS
  Samples 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  Our results indicated that overexpression of Ihh signaling promotes abnormal chondrocyte differentiation in enchondral ossification and enhances bone formation in OPLL.
authors.affiliation: From the Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sugita
authors.forename: Takafumi
authors.initials: T
authors.lastname: Yayama
authors.forename: Kenzo
authors.initials: K
authors.lastname: Uchida
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kokubo
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nakajima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamagishi
authors.forename: Naoto
authors.initials: N
authors.lastname: Takeura
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Baba
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002454:Cell Differentiation; D002574:Cervical Vertebrae / Q000378:metabolism / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D005260:Female; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D046529:Microscopy, Electron, Transmission; D008875:Middle Aged; D017887:Ossification of Posterior Longitudinal Ligament / Q000378:metabolism* / Q000473:pathology; D010012:Osteogenesis; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D053823:Hedgehog Proteins; D044162:Parathyroid Hormone-Related Protein; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human