| BackgroundAs the main weight-bearing structure of human,lumbar spine are usually under complex mechanical status,e.g.,compression,extension,lateral bending or twisting.The injury and degeneration of lumbar is one of the most common diseases,which affects about 60%of adults and 30%of adolescents.It has been proved that low back pain is closely associated with lumbar spine degenerative diseases,and recent studies showed that Modic changes is a risk factor for low back pain.According to the signal changes of endplate region in MR images,Modic changes are subdivided into three types:Type ? Modic changes show increased signal intensity on T2-weighted images and decreased signal intensity on T1-weighted images,reflecting fissures and micro-fractures of the trabeculae.Type ? Modic changes demonstrate increased signal intensity on both T2 and T1-weighted images,representing damage and fatty degeneration.Type ? displays decreased signal intensity on both T2-and T1-weighted images,considered as sclerotic bone.The population prevalence of Modic changes is about 5.8%,and both the prevalence and affected area increase with age.Type ? Modic changes dominates(64.2%),followed by mixed Type ? and Type ?(18.1%),then simply Type ?(16%).The distribution of Modic changes in superior or inferior endplates are the same,and 77.9%of Modic changes present simultaneously in the superior and inferior endplates of a disc as pairs.There are many theories concerning the origin of Modic changes,and mechanical damage is the most classical one.Mechanically saying,the endplate of disc/vertebral is the weak point of spine,thus the load delivered by the disc may concentrate on the endplate region,leading to endplate degeneration.The stress concentration may also result in microfracture and fissure of the endplate,then part of the endplate may have ossification.The Modic changes reflects the process of acute edema and inflammation(Type I Modic changes)-chronic inflammation and restoration(Type ? Modic changes)-ossification(Type ? Modic changes).Insufficient nutrition supply might be another reason for Modic changes.Several researches have revealed that the serum level of Vitamin D is positively correlated with the prevalence of Modic changes,which can be explained that the serum level of Vitamin D is related with calcium metabolism.Blood vessel dysfunction caused by smoking and hyperlipidemia is also considered a risk factor of Modic.Besides,Propionibacterium acnes(P.acnes),although controversial,is considered to be a factor of Modic changes in recent years.Researchers have found that P.acnes infection is highly correlated with the prevalence of Modic changes,and a double blind randomized clinical trial showed that antibiotic treatment is more effective in those low back pain patients with Modic changes.The aim of the current research is trying to discuss the origin of Modic changes,investigating how low virulent bacterial infection(P.acnes)and ischemia of endplate will affect the disc and its endplate region with cell experiment and animal models.Radiological,molecular biological and histological methods are used to evaluate the results.ObjectiveTo investigate how low virulent bacterial infection and ischemia will affect the intervertebral disc and its adjacent endplate region,and try to reveal the potential damage mechanism and provide evidence for therapeutic strategy in the future.Method1.Separate the nucleus pulposus and vertebral endplate cells of rats for in vivo culture.Different stimulates will be applied to the cells(supernate of P.acnes,hypoxic or ischemia),the reaction of cells are investigated by gene expression analysis,cell viability test and live/dead staining.2.Build an open approach to expose the intervertebral disc,then inject P.acnes into the disc by fine needle to generate an intradisc infection rabbit model.Use radiological examination,genomic test,molecular biological test,histological analysis,etc.to evaluate the model.3.Build a percutaneous ssubchondral endplate puncture approach,inject P.acne or Bleomycin(vessel inhibitor)via this approach to establish corresponding animal models.Use radiological examination,genomic test,molecular biological test,histological analysis,etc.to evaluate the model.Results1.Changes caused by low virulent bacterial infection or hypoxic in nucleus pulposus and endplate cells.1.1 The supernate of low virulent bacteria can suppress nucleus pulposus and endplate cell growth.Cell culture results demonstrated that when the bacteria concentration of the supernate is higher than 4*106 CFU/ml nucleus pulposus death rate is higher than 20%;under this bacteria concentration,cell growth was significantly depressed.When the bacteria concentration of the supermate is higher than 2*106 CFU/ml,endplate:cell death rate is higher than 20%;under this bacteria concentration,cell growth was significantly depressed.1.2 The supernate of low virulent bacteria can stimulate inflammatory gene expression in nucleus pulposus and endplate cells.qPCR showed that the supernate of P.acnes can downregulate the expression of aggrecan and collagen,while upregulate the expression of matrix degenerative proteins,like MMPs and ADAMTSs,in NP and EP cells.1.3 The effect of Bleomycin on.EP cells.The viability of EP cells is not affected when cocultured with 62.5?g/ml Bleomycin.The expression of extracellular matrix,like collagen and aggrecan,are also not affected.1.4 Hypoxia can induce low viability of NP and EP cells.Cell culture showed that in hypoxia environment(5%CO2 and 3%O2),the proliferation rate of EP cells is decreased by about 40%,more glycolysis products are generated.The expression of extracellular matrix is downregulated while more proteases that can degenerate extracellular matrix are synthesis.In NP cells,only the expression of HIF-la and ADAMTS-5 are upregulated by hypoxia.2.Intradisc low virulent bacterial infection in vivo can lead to MRI signal changes in disc and endplate region.2.1 Intradisc low virulent bacterial infection may lead to disc degeneration and endplate Modic changes in MRI.After exposure the intervertebral disc by open surgery via Wiltse approach,P.acnes were injected into the disc.Degenerative sign of the intervertebral disc(decreased signal intensity in T2 weighted MR images)can be found 2 weeks after surgery,and all the intervertebral discs presented degenerative symptoms at 9 months postoperatively.The endplate region of several segments injected with P.acnes presented Type ? Modic change after 3 months,and part of which turned into Type ? Modic changes after 6 months.2.2 Intradisc low virulent bacterial infection can induce matrix degeneration within the disc and inflammation in the endplate.Histological slices presented that the intradisc infection of P.acnes initiates the disintegration of disc extracellular matrix(aggrecan).At the same time,inflammatory cells can be found around the endplate region,which further promotes the degeneration of intervertebral disc.qPCR revealed that both the inflammatory cell factors(IL-1?,TNF-?,IFN-?,etc.)and matrix degenerative protease(MMPs and ADAMTSs)increased in the disc and the endplate region,suggesting a relatively severe inflammatory response stretching over the discs and their endplates was induced.2.3 Endplate subchondral bone absorption and remodeling caused by intradisc low virulent bacterial infectionNine months after intradisc injection of P,acnes,obviously bone structure remodeling can be found by Micro-CT.The trabeculae turned sparse,with bone marrow density and bone volume/total volume rate and trabeculae thickness decreased significantly.The bone surface/bone volume rate significantly increased.Furthermore,heterotopic ossification at several sites of the endplate region can be found by both Micro-CT and histological slices.3.The effects of low virulent bacterial infection or vessel depression at the endplate region.3.1 Low virulent bacterial infection at the endplate region generates Modic changes.By subchondral bone puncture and P.acnes injection at the endplate region,nearly one third of the injected segments presented increased signal intensity in both T1 and T2 weighted MR images,suggesting the occurrence of Type ? Modic changes.During the same period,none of the intervertebral discs adjacent to the injected endplates showed degenerative signs in MRI.3.2 Inflammatory injury of the endplate caused by local low virulent bacterial infection.Infected by P.acnes in the subchondral bone endplate region,the expression of inflammatory cell factors,such as IL-1?,TNF-? and IFN-?,are significantly upregulated in the endplate region,suggesting that an inflammatory-like responses are initiated within the endplate region.In the meantime,neighter obvious sign of inflammatory,nor evaluated inflammatory cell factors,can be found within the disc.3.3 Bone structure change caused by local low virulent bacterial infection.By histological slices and Micro-CT scanning,we found that compared to directly intradisc infection,subchondral bone region infection of P.acnes won't generate obvious signs of bone remodeling,but mild response of bone resorption can still be found.3.4 Radiological signs of ossification can be found when vessels are suppressed by Bleomycin.By subchondral bone puncture and Bleomycin injection at the endplate region,nearly one third of the endplate regions injected presented increased signal in T1 and T2 MR images,suggesting Type ? Modic changes,which refers to bone ossification.On the other side,no sign of intervertebral disc degeneration can be found in adjacent discs.3.5 Bone structure change caused by vessels suppressing.Micro-CT scanning of the injected site showed that the trabeculae number reduced a bit,while the bone mineral density and bone volume/total volume rate significantly increased all across the endplate region,suggesting large scale of ossification has occurred.qPCR also confirmed that the inflammatory cell factors,such as IL-1? and TNF-?,increased in this region.Conclusion1.In vivo,the supernate of low virulent bacteria can significantly restrain NP and EP cells' growth,activate multiple inflammatory cell factors like IL-1?,TNF-?,IFN-?,and upregulate the expression of their downstream genes,such as MMPs and ADAMTSs.Under hypoxia environment,the proliferation rate and metabolism level of NP and EP cells are decreased,the synthesis of extracellular matrix is reduced and degeneration is increased.Bleomycin,as a vessel inhibitor,does not have obvious cytotoxicity on NP and EP cells when used alone.2.Intradisc infection of low virulent bacteria may lead to time-dependent Modic changes and intervertebral disc degeneration after 3 months postoperatively,activate multiple inflammatory cell factors like IL-1?,TNF-?,IFN-?,and result in endplate subchondral bone absorption and remodeling.3.Infection of the subchondral endplate region by low virulent bacteria can lead to mild inflammatory-like response,presented as Type ? Modic changes at 6 months postoperatively,and sometimes generate endplate fissure,but the intervertebral disc maintains fine.On the other hand,using vessel inhibitor Bleomycin to treat the subchondral endplate region can lead to inflammatory response and calcification or ossification,which presented as Type ? Modic changes. |
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