title: Upregulated expression of connexin43 in spinal ligament fibroblasts derived from patients presenting ossification of the posterior longitudinal ligament.
abstract: METHODS
  A case-control study was conducted.
  
  
  OBJECTIVE
  To investigate different expressions of connexin43 (Cx43) between spinal ligament fibroblasts from patients with ossification of the posterior longitudinal ligament (OPLL) and non-OPLL patients and demonstrate knockdown of Cx43 protein expression by RNA interference inhibiting expression of osteoblast-specific genes in OPLL cells.
  
  
  BACKGROUND
  The OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant cellular signaling pathways remain unclear.
  
  
  METHODS
  Twenty patients presenting with OPLL and 18 non-OPLL patients underwent anterior decompression between January 2008 and June 2009. Specimens of the posterior longitudinal ligament were collected intraoperatively. Tissue fragment cell culture was performed. Inverted phase contrast microscopy and hematoxylin-eosin staining were used to observe cell morphology. The mouse antivimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence. The messenger RNA expression of osteoblast-specific genes of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of Cx43 was detected via Western blotting. And then, after 72 hours, when RNA interference against Cx43 was performed in OPLL cells, expression of the indexes mentioned earlier was compared again between the transfection group and the nontransfection group.
  
  
  RESULTS
  Cultivated cells were observed 7 to 10 days after cell culture. Hematoxylin-eosin staining showed fusiform and multiangular star morphologies, large and elliptical cell nuclei, and ill-defined cell appearances. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The messenger RNA expressions of OCN, ALP, and COL I and protein expressions of Cx43 from OPLL fibroblasts were greater than those from non-OPLL cells, and the difference was significant. Furthermore, knockdown of Cx43 protein expression inhibited the messenger RNA expressions of OCN, ALP, and COL I remarkably in the transfection group compared with the nontransfection group, 72 hours after RNA interference targeting Cx43 was performed in OPLL cells.
  
  
  CONCLUSIONS
  Tissue fragment culture of the cervical posterior longitudinal ligament provided a successful fibroblast culture, showing good adherence and subculture. The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, in which Cx43 played an important role.
authors.affiliation: Department of Orthopedics, Changzheng Hospital, Shanghai, China.
authors.forename: Hai-song
authors.initials: HS
authors.lastname: Yang
authors.forename: Xu-hua
authors.initials: XH
authors.lastname: Lu
authors.forename: De-yu
authors.initials: DY
authors.lastname: Chen
authors.forename: Wen
authors.initials: W
authors.lastname: Yuan
authors.forename: Li-li
authors.initials: LL
authors.lastname: Yang
authors.forename: Hai-long
authors.initials: HL
authors.lastname: He
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D016022:Case-Control Studies; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D017846:Longitudinal Ligaments / Q000378:metabolism / Q000473:pathology; D008858:Microscopy, Phase-Contrast; D017887:Ossification of Posterior Longitudinal Ligament / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010006:Osteoblasts / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation
chemical_list: D024042:Collagen Type I; D018031:Connexin 43; D015675:Osteocalcin; D000469:Alkaline Phosphatase