Role And Mechanism Of Mechanical Stress On Spinal Ligament Cells Derived From Patients With Ossification Of The Posterior Longitudinal Ligament

Objective Ossification of the posterior longitudinal ligament (OPLL) is a pathological condition of spinal cord or nerve root compression caused by ectopic bone formation in the spinal ligament, which frequently occurs in cervical spine, and is seldom founded in thoracic spine and lumbar spine. The mechanism of OPLL development remains unclear. Some clinical studies reveal OPLL should develop after posterior cervical surgery in contrast with anterior cervical surgery. In addition, posterior longitudinal ligament fibroblasts should be osteogenic differentiation induced by the mechanical stress, such as gene expression promotion of alkaline phosphatase (ALP), collagen types I (COL I) and osteocalcin (OC). Therefore, it is very important to elucidate the development mechanism of the disease by exploring the role and mechanism of mechanical stress on spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament. ERK 1/2 is one of MAPK family, which phosphorylation and osteogenic differentiation of OPLL cells should change after its upstream protein MEK blocked. Therefore, we postulated ERK1/2 phosphorylation should play an important role in advancing the progression of OPLL. It is the phosphatidylinositol-3-kinase/ protein kinase B (PI3K/AKT) signaling pathway, which could promote cell proliferation, inhibition of apoptosis and cell differentiation. The signaling pathway could be activated by mechanical stress and it also plays an important role in the onset and progression of OPLL suffered with diabetes mellitus. Then, it is likely to be significant of AKT protein for OPLL formation.Methods Sixteen inpatients presenting with OPLL and sixteen NOPLL underwent anterior decompression between August 2009 and August 2010. Specimens of the posterior longitudinal ligaments which were extirpated carefully intraoperatively were put into an aseptic tubule filled with cold physiological saline and immediately transferred to the lab. All specimens were cultured with the modified"tissue fragment attachment-block"method. Hematoxylin eosin (HE) staining and immunostaining of vimentin were performed on the third passage cells. Fifth passage fibroblasts from OPLL and NOPLL patients were seeded on a BioFlex 6-well plate and incubated in DMEM supplemented with 1% FBS for 24 h. The Flexercell 4000 Tension Plus system (Flexercell International Corporation) was used to stretch the cells with mechanical stress of 10%, 0.5 Hz and respectively lasted 0h, 6h and 12 h. The expressions of RNA (ALP,COLI,OC and Runx2) and protein (ERK 1/2 and AKT) were detected. Then signaling pathway blocker, U0126 and LY294002, were used to clarify the mechanism.Results All specimens were cultured with the modified"tissue fragment attachment-block"method and the isolated cells could proliferate rapidly and well in vitro. Cells were observed around the tissue fragment after 7-10 days culture and exhibited a fibroblast-like, spindle-shaped or polygon-shaped appearance. Cytoplasm was stained into deep pink and nucleus was stained into light blue in HE staining. Vimentin immunostaining of cells exhibited spindle-shaped or polygon-shaped appearance with large, round and blue nucleus and green cytoplasm. Cells were identified as fibroblasts and non-fibroblasts not found. The expressions of ALP, COL I, OC and Runx2 of OPLL cells were positively regulated after stimulation for 12h compared to the resting cells. Meanwhile, the osteogenic genes expression significantly up-regulated compared to the resting cells. However, there were no significant changes observed in NOPLL cells. The osteogenic genes expressions were significantly down-regulated with the signaling pathway blocker compared with the group without signaling pathway blocker.Conclusions The modified"tissue fragment attachment-block"method had some advantages than the traditional one. Posterior longitudinal ligament cells exhibited spindle-shaped or polygon-shaped appearance and Vimentin immunostaining were positive. Mechanical stress could significantly promote the osteogenic genes expression and activate the MEK/ERK1/2 and PI3K/AKT pathway. Meanwhile, the osteogenic genes expressions of ALP,COL I,OC were significantly down-regulated with the signaling pathway blocker. Therefore, mechanical stress could induce osteogenic differentiation of spinal ligament cells derived from OPLL patients via these two signaling pathway, and ERK1/2 and AKT protein play important roles.