Effect Of Connexin43 Gap Junction In The Development Of OPLL Induced By Mechanical Stress

Objective Ossification of the posterior longitudinal ligament(OPLL) is a pathological condition causing ectopic bone formation in the cervical spinal ligament and is a common disease in Japan and throughout Asia. Although the mechanism of OPLL development remains unclear, genetic factors and local factors have been proposed and partly confirmed. OPLL can compress the spinal cord and its roots, leading to various degrees of neurological symptoms from discomfort to severe myelopathy. However, it is known that the presence of OPLL does not always indicate the presence of cervical myelopathy. OPLL is a chronic and progressive disease, and clinical symptoms may only occur after the ossified ligaments develop to a certain degree. Although some studies have investigated the osteogenesis of fibroblasts from ligaments of OPLL and the role of mechanical stress in OPLL development, the mechanism of cellular signaling transduction remains unclear. Gap junctions are pores or channels that span the cellular membrane and allow molecules less than 1 kD to pass from one cell to another, providing bilateral communication between cellular cytoplasms. These junctions are formed by two hexameric hemichannels, composed of 6 protein subunits, termed connexins. There are more than 20 identified mammalian connexins, and the most abundant connexin family member present in bone cells is connexin43 (Cx43). This mode of cell-cell communication is of particular importance in the skeleton, where a large variety of systemic and locally generated signals are transduced into biological signals and transmitted to cells at specific locations to permit bone formation. Mineralization in Cx43 null osteoblasts was reported to be defective, and osteoblast responses to anabolic signals were flawed. Thus, we hypothesized that the mRNA and protein expression of the Cx43 gap junction in spinal ligament fibroblasts derived from OPLL patients may be up-regulated and play a key role in the signal transmission from one cell to another, thereby promoting the development of OPLL. To explore this possibility, we evaluated the different expressions of osteoblastic genes (osteocalcin (OCN), alkaline phosphatase (ALP) and type I collagen (COL I)) and the Cx43 gap junction via RT-PCR in cells cultured from spinal ligament specimens from OPLL and non-OPLL patients. Mechanical stress was loaded on OPLL ligament fibroblasts, and the above mentioned gene expressions were compared before the application of mechanical stress and again at 12 h and 24 h after stress loading. Additionally, we performed siRNA interfering targeting Cx43 gap junction and again evaluated the above mentioned gene expressions.Methods Twenty-four patients presenting with OPLL and 20 non-OPLL patients underwent anterior decompression between December 2008 and December 2009. Specimens of the posterior longitudinal ligaments were collected intraoperatively. Enzymatic digestion culture was performed on four OPLL ligament specimens, and tissue fragment culture was performed on the remaining 40 specimens. Inverted phase contrast microscopy and hematoxylin eosin (HE) staining were used to observe cell morphology. The mouse anti-vimentin antibody was used to identify the cultured cells via immunocytochemistry and immunofluorescence (ICC/IF). Fibroblasts from OPLL patients were preloaded with mechanical stress using a Flexercell 4000 Tension Plus system. The mRNA expressions of osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COL I) and Cx43 were detected in OPLL and non-OPLL cultures, at pre-stress and at 12 and 24 h after stress loading by semi-quantitative RT-PCR. The protein expression of the Cx43 gap junction was also detected via Western blotting. Small interfering RNA (siRNA) targeting the Cx43 gap junction was transfected into the fibroblasts derived from the OPLL patients using Lipofectamine 2000 Reagent. The expressions of the indexes mentioned above were compared between the negative control group and the transfection group, and between groups before and after stress loading.Results No adherent cells were found in the four specimens of the enzymatic digestion culture; however, in the remaining 40 specimens that underwent tissue fragment culture, cultivated cells were observed 7-10 days after culture. Inverted phase contrast microscopy revealed a more hypertrophic cell appearance and cell matrix in the OPLL group. However, the cells were more orderly in the non-OPLL group. HE staining showed fusiform and multi-angular star morphologies, large and elliptical cell nuclei and ill-defined cell appearances. ICC/IF exhibited positive results of vimentin staining. The expressions of OCN, ALP, COL I and Cx43 from OPLL ligament fibroblasts were greater than those from non-OPLL cells as measured by RT-PCR and Western blotting. The same results were obtained after 12 h and 24 h of mechanical stress loading as compared with cells without stress. Specific siRNA that targeted the Cx43 gap junction reduced the mRNA expressions of OCN, ALP and COL I remarkably. The expressions of the osteoblastic genes did not up-regulate after 72 h of transfection in OPLL group, even with mechanical stress loading. 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, and applied mechanical stress may aid in the progression of OPLL. In these processes, the Cx43 gap junction played an important role.