Upregulated Expression of Connexin43 in Spinal Ligament Fibroblasts Derived From Patients Presenting Ossification of the Posterior Longitudinal Ligament

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Abstract

Study Design.

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.

Summary of Background Data.

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.

Conclusion.

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.

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