The Mechanism Of Pathogenesis Of Ossification Of The Ligamentum Flavum Mediated By Osteopontin

1.Objective Ossification of the ligamentum flavum(OLF)can cause severe spinal nerve damage,as for the lack of effective drugs and other conservative treatments,laminectomy is the only effective treatment but with a limited degree of recovery in the spinal cord.Our previous studies have demonstrated that: osteopontin(OPN)and its receptor(CD44 and integrin)was found in the OLF tissues and OLF animal models can be induced by OPN successfully,pointing out that OPN palyes a critical role in the process of OLF.However,the underlying mechanisms remain elusive.This study aims to further elucidate the molecular mechanisms of osteopontin in the occurrence of OLF.2.Method The methods of Immunohistochemistry and molecular biology research are used to clarify the molecular mechanisms of osteopontin in the pathogenesis of ossification of the ligamentum flavum.In order to identify the OPN's role in the induction of osteogenic differentiation further,animal models of OLF were used to validate the mechanisms.The expression of OPN and its receptors in the ligament tissues and ossification tissues of human OLF.Grouping: Samples are divided into 2 groups including OLF group and NLF group.Each group involves 8 patients.Immunohistochemistry: After taking from laminectomy,the samples of human OLF were fixated in formaldehyde,decalcificated,dehydrated,embedded in paraffin and cut into slices successively;the slices were stained with antibodies against OPN,CD44,and integrin,then observed the stain results.Isolation,culture of LFCs and the ossification induction of OPN.Isolation and culture of the LFCs : LFCs were isolated and cultured with the methods tissue pieces inoculation in vitro and the left tissue pieces can be used repeatedly.The LFCs were passaged when they touched each other and the redundant cells were cryopreservated in liquid nitrogen for standby.The ossification Induction of OPN: The third generation of OLF-LFCs are cultured into 6-well plate.After cultured for 24 hours,the cells were under interference of 100 ng/ml OPN for 48 hours.We tested the fore-and after-changes of the expression of ossification markers: ALP and OCN.The testing and establishment of the molecular pathways involved in the OLF induced by OPNTesting the phosphorylation of the MAPK signaling pathway: The third generation of OLF-LFCs were cultured into 6-well plate and after 24 hours,the cells are under interference by 100 ng/ml OPN.Cellular proteins were extracted from OLF-LFCs for various periods of time and were then immunoblotted with antibodies against phospho-ERK1/2,phospho-JNK,and phospho-p38.signaling pathway blockade: Blockade of OLF-LFCs with ERK1/2 phosphorylation-selective inhibitor U0126 and P38 phosphorylation-selective inhibitor SB203580 selectively inhibited the phosphorylation of ERK1/2 and P 38 for 24 hours,respectively.Then the cells were under interference of 100 ng/ml OPN for 48 hours.The protein of ALP and osteocalcin were examed by Western blotting.SB203580 inhibition of P38 signaling molecules on the OPN induced OLF animal model.The treatment of animal model: At 4?,the OPN group(OPN-matrigel),the P38 block group(OPN-SB203580-matrigel)and the ERK block group(OPN-U0126-matrigel)were injected into the ligamentum flavum between the laminar.After 12 weeks,we tested the OLF situation of each group by Micro-CT.3.results The expression of OPN and its receptors in the ligament tissues and ossification tissues of human OLF.The expression of OPN and CD44 can be seen around the cartilage cells in all 8 ossification tissues of OLF,however the expression of integrin was negative;the expression of OPN,CD44 and integrin were all positive around the vascular endothelia cells.Isolation,culture of LFCs and the ossification induction of OPN.The isolated cells vary in size and with bright cytoplasm,spindle-shape and partly polygonal and oval.Primary cells from the tissue pieces were scattered in distribution early,then gradually growing intensively and as the tissue pieces for center radiating outward growth.When the cells attached each other,then presented a vortex growth.There are more polygon and oval cells in the OLF group than the NLF group and some small nodule distributions are observed in the OLF group.Compared with the NLF group,the expression of OPN are increased in the OLF group through the WB detection.Under the interference of OPN,the expression of the ossification markers,ALP and OCN,are increasing in the OLF-LFCs.the detection and establishment of the molecular pathway induced by osteopontin in the process of ossification of the ligamentum flavum.Cellular proteins were extracted from TOLF cells that were exposed to OPN for various periods of time and were then immunoblotted with antibodies against phospho-ERK1/2,phospho-JNK,and phospho-P38.The phosphorylation of P38,ERK1/2,and was stimulated by OPN but not JNK.Under the inhibition of SB203580 which is the phosphorylation inhibitor of P38,the expression of ossification marker of ALP and OCN was downregulated to the level of the control,even if induced by OPN.But the U0126,inhibitor of ERK1/2,can not inhibit the osteogenic differentiation induced by OPN.the inhibition of P38 signaling pathway by SB203580 on the animal model of ossification of ligamentum flavum induced by OPN.After 12 weeks,high density in the ligamentum flavum area was showed by Micro-CT examination in the OPN animal model group and ERK blocking animal model group all show,but the P38 block group have no high-density performance in the ligamentum flavum.4.conclusions 4.1 OPN and its receptor CD44 was expressed in the OLF regions but with no expression of its receptor integrin,and the OPN,CD44 and integrin were all expressed in the vascular endothelial cells area of no-ossification.4.2 LFCs can be isolated by tissue piece inoculation methods successfully and under the induction of OPN,the expression of ossification marker ALP increased in the OLF-LFCs.4.3 OPN can activate the MAPK signaling pathway by phosphorylating the ERK1 / 2 and P38 signaling molecules,but the phosphorylation of JNK of signaling molecules is not clear.Inhibiting the phosphorylation of ERK1 / 2 signaling molecules can not effectively block the OPN-induced cell ossification of ligamentum flavum,while inhibiting the phosphorylation of P38 signaling molecules can effectively block the OPN-induced cell ligamentum flavum ossification.4.4 Inhibiting P38 signaling pathway can inhibit the ossification process in the OPN-induced rabbit OLF models.