The Research Of Ossification Of Posterior Longitudinal Ligament Affected By Herniated Cerbical Intervertebral Disk

BackgroundOssification of the posterior longitudinal ligament(OPLL) is a pathological ectopic ossification of thisligament at the cervical and thoracic spine, causing myeloradiculopathy as a result of chronic pressure on thespinal cord and nerve roots. However, the exact pathogenesis and natural history of OPLL remain unclear. OPLL and herniated IVD are important causes of cervical stenosis. In clinical work, we find that OPLL often coexists with cervical disk hernia. But there has been no research analyzing the association between degenerated IVDand OPLL.ObjectiveIn vitro, to investigate the role and effect of cytokines secreted by nucleus pulposus cells on longitudinal ligament cell proliferation, cytotoxicity and osteogenic differentiation. To investigate the effect of herniated cervical intervertebral disk(IVD) on ossification of posterior longitudinal ligamen(OPLL). To provide a new theoretical basis for the diagnosis and treatment of ossification of posterior longitudinal ligament.Material and methods1. OPLL Cells CulturePosterior longitudinal ligamen were obtained from 10 patients during surgery for cervical vertebral canal stenosis combined with OPLL. The posterior longitudinal ligamen ossified was noted on preoperative magnetic resonance imaging and was also visualized directly during surgery. The longitudinal ligamen tissue specimens were washed with saline to remove blood and cut to move ossified ligamen.Posterior longitudinal ligamen(PLL) cells were harvested by tissue fragment cell culture and passaged when they reached approximately 80-90%confluency. The cells were used at passage 3 for all experiments.2. Nucleus Pulposus Cells CultureHerniated IVD tissues were obtained from a patient diagnosed degenerative disc disease during surgery. Nucleus pulposus (NP) cells were isolated by enzymatic digestion of nucleus pulposus tissue and passaged when they reached approximately 80-90%confluency. The cells were used at passage 3 for all experiments.3. Experimental groupingExperimental group:OPLL cells were co-cultured with NP cells in transwell permeable supports, OPLL cells were cultured in lower compartment, NP cells were cultured in upper compartment. The materials of membrane we used is polyester(PET), and the pore size in the membrane is 0.4 um. In control group:OPLL cells were cutured in ordinary 6-well plates.4. Enzyme-linked Immunosorbent Assay (ELISA)After incubation for 48 hours. Spernatants were collected form the OPLL+NP cells group, OPLL cells group and NP cells group. IL-1 a, IL-6, TNF-a, PGE2 from culture supernatants were quantified using a ELISA kits according to manufacturer’s instructions. The colorimetric reactions were analysed at 450 nm on a Varioskan flash multifunction plate reader.5. MTT Assay for CytotoxicityAfter incubation for 48 hours, MTT reagent was added to each well and incubated for 4 hours in 5%CO2 at 37.1℃ with humidity. Then the supernatant was removed and DMSO was added to each well to dissolve the formazan crystals. OD490 was measured in a Varioskan flash multifunction plate reader.6. EdU Assay for Cellular ProliferationCell proliferation of OP11 cells in each well was assessed using the EdU kit. After incubation for 48 hours, supernatants were removed, and culture medium contained EdU(1/2500 vol/vol) was added to each well. OPLL cells continued incubating for 4 hours, then OPLL cells were washed by PBS and fixed by 4%paraformaldehyde. DNA was stained by Apollo and Hoechst Staining successively. Keeping visual flied unchanged, green fluorescence and ultraviolet fluorescence were used to stimulate OPLL cells with fluorescence microscope, then we calculated ceil proliferation ratio.7. RT-PCRAnalysisFirst, total RNA was extracted using the Trizol. Second, total R.NA was reverse transcribed into cDNA using reverse transcription kit. Third, RT-PCR was performed. To normalize types I, XI collagen and osteocalcin genes expression, (3-actin was used as an internal control. The cycle threshold (Ct) data were obtained, and the relative expression levels results were analysed using the 2-△ △ Ct method.8. Von Kossa and Alkaline Phosphatase StainVon kossa staining was used for determining mineralization. OPLL cells were stained by cell Von Kossa staining kit according to manufacturer’s instructions. For alkaline phosphatase staining, OPLL cells were stained using alkaline phosphatase staining kit according to manufacturer’s instructions.9. Statistical AnalysisData were analysed using SPSS version 11.5. Mean values were calculated and are presented with an error bar representing x ± s. t test was used to evaluate data. Statistical significance was accepted at P< 0.05 for all analyses.Results1. CytotoxicityCell viability evaluated by MTT assays showed that OPLL cells co-cultures with NP cells showed nocytotoxicity compared with the control. Each culture demonstrated uniform increasesin optical density in the MTT assay. In the exexperimental group,the OD450 was 0.28±0.09, whereas,OD45o was 0.25±0.06 in the control group(p< 0.05).2. Cellular proliferationOPLL cells co-cultures with NP cells increased cellular proliferation compared with the control group. In the experimental group, the cellular proliferation rate(number of red nucleus/total number of nucleus) was 14.30±2.67%%approximately within 4 hours incubation, the cell proliferation rate was 2.21+0.64%in the control group(P < 0.05).3. RT-PCRExperimental data suggest that expression of types I, and XI collagen and osteocalcin mRNA increase in the experimental group compared to the Control group. Upregulation of types I, XI collagen and osteocalcin mRNA imply osteogenic collagen synthesis(p<0.05).4. Von Kossa and Alkaline Phosphatase StainOPLL cells co-cultures with degenerated NP cellsexhibited positive stains for alkaline phosphataseand Von Kossa stains, confirming expression of osteogenicphenotype and bone nodule formation, whereas control cultures show negative stains.5. CytokinesExperimental results demonstrate that the amount of IL-la, TNF-a and PGE2 significantly increased in the OPLL+NP cells group compared with OPLL cells group and NP cells group. IL-6 was not detected in all groups. (P< 0.05)Conclusions In vitro, nucleus pulposus(NP) cells can not only promote the proliferation of OPLL cells, but also improve osteogenic differentiation of OPLL cells without obvious cytotoxicity. The cytokines secreted by the nucleus pulposus play an important role in the ossification of the posterior longitudinal ligament. Collectively, herniated cervical intervertebral disk provide an important pathomechanism in ossification of the posterior longitudinal ligamen through inflammatory cytokines.