| Purpose Ossification of the posterior longitudinal ligament(OPLL) is a common spinal disorder. The spinal cord and nerve tissue are compressed by ossified ligaments, resulting in neurological deficit or even diplegia. The incidence of OPLL is up to 20%~34% in people over 65 years old in Asian, In China, OPLL is one of the major spinal diseases which affects people’s health. There is still no special and efficient treatment for OPLL except operation. However, surgery often has high risks and severe complications such as spinal cord injury. It is very important to develop safe and effective drugs or other treatments for OPLL. Since OPLL was first reported, a lot of basic and clinical studies have been done. It was assumed that gene mutation, cytokines, hormone and cell signaling pathways were related to the mechanism of OPLL, such as BMPs, IGF, the protease A/C, SMAD and MAPK. The pathology of OPLL could be presented partly from those studies, but the exact mechanism remains unclear, further studies are needed.(1) Ligament differentiating into bone is the base of OPLL. It’s necessary to know the mechanism of translation between the cells and origination of the osteoblasts.(2) Connexin 43(Cx43) is a gap junction, which involves in the mentalism of bone. In our pre-study, we revealed that Cx43 in OPLL had a higher expression than non-OPLL, which might play an essential role in the occurrence of OPLL. Therefore, it is necessary to clarify the functions of Cx43 and molecular mechanisms in OPLL.(3) In recent years, many reports revealed that NF-κB/p65(p65) associated with inflammation was a key regulator in osteogenesis. OPLL is a type of heterotopic ossification(HO). There was no report about p65 involving in the development of OPLL. Further study is needed to verify that p65 really existed in OPLL, and verify the roles of inflammatory response in OPLL.Method Since December 2013 to August 2014, 22 patients with OPLL and 16 patients with non-OPLL underwent anterior cervical discectomy and fusion(ACDF) and anterior cervical corpectomy and fusion(ACCF). Tissues were obtained during operation and handled as following: the first part, samples were processed for hematoxylin and eosin staining(H&E staining) or immunohistochemistry to detected the protein of Cx43, p-p38 MAPK, p-erk1/2, p-JNK, p-p65 and the inflammatory cytokine of IL-1, IL-6 and TNF-α. Second part, samples were incubated to isolated ligament fibroblasts with tissue piece method. The monolayer cells which creeped out from the explants were passaged till cells spread to more than 80% of the bottom of the dish. Vimentin wree stained by immunofluorescence technique to identify the fibroblast. Flow cytometric analysis was used to analyze cell-surface marker. Cells from passages 3 were induced by osteogenic, adipogenic and chondrogenic mediums respectively. Cells were stained by ALP staining after inducntion, and Alizarin Red S, Oil Red O and Alcian blue staining were used to explore the cell multilineage differentiation. The expressions of genes BMP2, Runx2, ALP, PPARγ2, LPL, Sox9, COL2A1 and COL10A1 were analysed by Q-PCR. Cells also were implanted in NOD-mices in order to verify the osteogenisis in vivo. 3×105 cells per well were seeded into Flexcell plates and cultured. Subsequently, the cells were subjected to a cyclic tensile strain of 10% elongation at a frequency of 0.5 Hz by using a Flexcell 5000 Strain Unit(Flexcell) for 48 h. Control groups without cyclic strain. Q-PCR was used to detect the genes of ALP, COL I and OCN. The siRNA of Cx43 was designed and synthesized according to online software, and were transfected cells by cationic transfection reagent. Q-PCR and Western-blotting analysis was performed to clarify the knock-down efficiency of siRNAs. The OPLL and non-OPLL cells had been under mechanical stretch loading respectively for 0min, 15 min, 30 mn, 60 min and 90 min. Total proteins were collected accoding different times, and analyzed by Western-blotting to clarify the p-p38 MAPK, p-ERK1/2, p-JNK and p-p65. After special signal pathway inhibitor were used to inhibite the p38 MAPK, ERK1/2, JNK and p65 pathways, cells were loaded mechanical stretch for 48 h. Q-PCR was used to test the ALP, COL I and OCN. At the same time, IL-1β, IL-6 and TNF-α were checked by ELISA.Results The Cx43, p-p38 MAPK, p-ERK1/2, p-JNK, p-p65, IL-1β, IL-6 and TNF-α were highly expressed in non-ossified areas of the ligament tissues from OPLL patients but could barely be detected in the ligaments from non-OPLL patients by immunohistochemical analysis. In 2 weeks, few plastic-adherent nucleated cells climbed out around the ligmentous specimen. Cells bodies were slender, thin and spindle-shaped. About 3 weeks, cells climbed out rapidly with cluster appearance and reached to 80-90% subconfluent. Immunofluorescence staining of Vimentin showed green fluorescence in cytoplasm, which was the marker of fibroblasts. The cells were negative for hematopoietic markers such as CD34, CD45, but highly expressed typical mesenchymal associated markers CD29, CD44, CD90 and CD105. After cultured by induced mediums, cells could be show high multidifferentiated abilities in vitro. And in vivo study, new bone could be detected in the leg of NOD-mices two months after inplantation. Cx43 protein was much higher in OPLL cells than in non-OPLL. Cx43 was significantly down-regulated by using siRNA and the interference efficiency exceeded 70% according to Western-blotting and Q-PCR. Biomechanical stimulation can activated p38 MAPK, erk1/2, JNK and p65 phosphorylated, and these pathways were upregulated dramatically after 15 min, and came to peak at 30 min or 60 min both in OPLL and in non-OPLL, but the phosphorylation in OPLL groups were higher. After Cx43 gene was knocked-down, the phosphorylated ERK1/2, p38 MAPK, JNK and p65 were much lower following biomechanical loading in both OPLL cells and non-OPLL.OPLL cells were treated with Cx43/siRNA or inhibitors of the ERK1/2, p38 MAPK, JNK and p65 signaling pathways. Mechanical stress was loaded onto cells for 48 h prior to RNA extraction. Cx43 knocked-down almost completely blocked the osteoblastic differentiation of OPLL cells which were stimulated by mechanical loading, including the down-regulation of ALP, COL I, CON and Runx2. Compared with Cx43/siRNA, Cells were treated with ERK1/2, p38 MAPK and p65 inhibitor, which could partly diminished the osteogenic effect of mechanical stimulation, whereas inhibition of JNK pathway showed no significant difference(p<0.05). These results indicate that biomechanicalstimulated osteoblastic differentiation depends upon Cx43 expression levels, whereas the downstream molecular that mediated such osteogenic effects include other unidentified mechanisms. IL-1β, IL-6 and TNF-α were up-regulated stimulated by mechanical stress in OPLL, but no changed in non-OPLL. Cx43/siRNA or inhibitor of p65 could block the up-regulation stimulated by mechanical stress, statistical significance(p<0.05).Conclusion Posterior longitudinal ligament fibroblasts couid be isolated by tissue culture method and verified by immunofluorescence staining of Vimentin.(1) Cell surface markers, cell multilineage differentiation and osteogenesis capacity in vivo suggested mesenchymal stem cell characteristics.(2) Mechanical stress was a key role in the development of OPLL.(3) p38 MAPK, ERK1/2,and p65 pathways may be involved in the OPLL of the ossification process.(4) Cx43 hemichannels played an important role in mechanical-stress-induced OPLL by the transduction of mechanical signals, giving rise to the activation of the osteogenic signals ERK1/2, p38 MAPK and p65 in ligament fibroblasts.(5) Inflammatory cytokine may also be involved in the process of OPLL. |
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