Study On The Prevention Effect Of Semaphorins On Prosthesis Osteolysis

PurposeTotal hip arthroplasty(THA)is a safe and effective way for the treatment of severely degraded,post-traumatic and other end-stage hip diseases.However,with THA generally spreading to a younger population,the need for revision surgery is increased.The probability of THA revision surgery in USA from 1990 to 2002 increased from 0.95 in ten thousandths to 1.52 in ten thousandths.The aseptic loosening accompanied by osteolysis is the main reason for the failure of THA.Osteolysis and aseptic loosening is a result of the reaction of the tissue surrounding prosthesis for the prosthetic wear particles.Prosthetic wear particles can stimulate immune cells around the prosthesis to express pro-inflammatory cytokines,pro-osteoclastogenic cytokines and other substances to improve OCs activity,and simultaneously inhibit the OBs activity.Thus wear particles disrupt the bone homeostasis,resulting in occurrence of bone dissolution,further leading to loosening of the prosthesis.Different materials of hip prosthesis stimulate the surrounding tissues to different extents.For wear debris particles generated around the prosthesis,the titanium particle is a common ingredient.OCs can be activated as well as proliferation of OBs inhibited by the titanium particle whose diameter is less than 10?m,inducing the aseptic loosening of joint prosthesis.Semaphorins commonly exist in vertebrates,whose family contains more than twenty members divided into eight classes.More and more studies have found that a plurality of members of the family plays an important role in functions of OCs and OBs and other associated cells.Wherein Semaphorin7A(Sema-7A)exerts its important action in bone homeostasis by promoting osteoclast proliferation and osteoblast apoptosis.This study was designed to investigate that whether Sema7 A can inhibit the activation of OCs induced by titanium particles,further explore the molecular mechanisms of Sema7 A intervention for the differentiation,proliferation and activation of OCs induce by titanium particles,as well as discuss its intervention of apoptosis in OBs induced by titanium particles,revealing its potential value in prevention of bone dissolving around the prosthesis,which can provide new ideas for the treatment of dissolved bone around the prosthesis.MethodsThe mRNA expression of Sema-7A,plexin-C1 and ?1-integrin subunits in the osteoblast precursor cells(MC3T3-E1),Bone sialoprotein(BSP)and andosteocalcin(OC),which are the osteogenesis cell markers,were determined through Semi-quantitative RT-PCR.The expression levels of phosphorylated the ERK-1 and ERK-2 protein in MC3T3-E1 cells were measured using western-blot.The effects of sema-7A on the migration of osteoblast were verified by a scratch test.The mRNA expression of Sema-7A,its receptor plexin-C1 and ?1-integrin subunits in the osteoclast precursor cells(RAW264.7),and receptor activation of nuclear factor NF-kB(RANK),the osteoclast marker,were detected through Semi-quantitative RT-PCR.TRAP staining was used to observe the expression of the osteoclasts at different stages under a fluorescence microscope.The protein was extracted to access the expression and distribution of Sema-7A using western-blotting.Q-PCR and ELISA was performed to measure the expression levels of mRNA and protein of various inflammatory cytokines including the interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor ?(TNF-?).Western-blot was used to detect the expression levels of the osteoclast markers(RANK,MMP-9)and phosphorylated P38 protein.MTT was used to determine the osteoclast proliferation.Immunohistochemistry was used to observe the percentages of the MMP-9 and RANK positive cells.TRAP staining was used to observe and quantity the number of mature osteoclasts.Apoptosis of MC3T3-E1 cells were evaluated by flow cytometery.The mRNA expression of BSP,OC and collagen type I was measured with Q-PCR.The protein expression of BSP,OC and collagen type I was detected by Western blotting.The degree of osteoblast mineralization was assayed by Alizarin Red calcium nodule staining.ResultsDuring osteoblast differentiation in MC3T3-E1 cells,the expression of Sema-7A mRNA presents bimodal.Sema-7A induced an increase in ERK1/2 phosphorylation in a time-dependent manner.The scratch test showed that Sema-7A treatment group heals fast than ERK-1 and ERK-2 inhibitor group,demonstrating Sema-7A mediates the migration of MC3T3-E1 cells through MAPK pathway.Sema-7A ? plexinC1 and ?1-integrin has low expression during osteoblast differentiation.However,they highly expressed when immature osteoclasts integrate,suggesting that Sema-7A plays a role in osteoclasts' s integration process.The titanium particle can increase osteoclast peptide cytokine expression and induce proliferation of the number of mature osteoclasts.After transfection Sema-7A siRNA can reverse the process,resulting in up-regulating the expression of inflammatory cytokines and reducing the number of mature osteoclasts accordingly,which indicated that osteoclast differentiation and proliferation processes induced by titanium particles was related to the protein expression of Sema-7A.The expression levels of IL-1?,IL-6 and TNF-? increased,and MMP-9 and RANKL receptor activator protein levels decreased using inhibition of p38 MAPK pathway with SB203580.Sema-7A regulates the osteoclast proliferation and secretion of inflammatory cytokines through p38 MAPK signaling pathwayThe results suggested that in group 1,the expression of BSP,OC and collagentype I reduced significantly,and in group 2 the expression increased compared with standard control group(P<0.05).The results of flow cytometry suggested that in group 1,the osteoblast apoptosis rate was significantly higher than the other groups(P<0.05),and apoptosis rate in group 2 was lower than the standard control group(P<0.05).The results of Alizarin red staining showed that there was no significant mineralized nodules in group 1 and the formation of mineralized nodule occurred in group 2.Further results showed that overexpression of Sema-7A can lead to an increase in the expression of phosphorylated ERK1 and ERK2,on the contrary after interference of Sema-7A,and the expression of BSP,OC and collagen type I reduced due to the inhibition of ERK1/2 activity.These results demonstrate that Sema-7A may have a certain inhibitory effect on the differentiation of mouse MC3T3-E1 osteoblasts that inhibited by titanium particles.ConclusionSema-7A can mediate the migration of osteoblast,enhance the inhibitory effects on the osteoblast differentiation,and is associated with MAPK signaling pathways.Sema-7A can inhibit osteoblast mineralization and promote apoptosis.Furthermore,Sema-7A may be involved in the regulation of promoting the integration of osteoclast.Further studies have shown that Sema-7A has the effects on the mature osteoclasts proliferation induce by the titanium particles,the process also is associated with p38 MAPK signaling pathways.Sema-7A regulates the osteoclast proliferation and secretion of inflammatory cytokines through p38 MAPK signaling pathwayWe confirmed that the osteolysis induced by titanium particles was blocked from the cell level experiment,thus providing a feasible way for the clinical treatment toward particles-induced osteolysis using biotechnology.