| Backgroud:Osteoarthritis(OA)is a common disease in joint orthopedics.In recent years,with the increasing incidence of traffic accidents and sports injuries,the incidence of anterior cruciate ligaments(ACL)injury and post-traumatic osteoarthritis(PTOA)increased year by year.As a result,high incidence of OA,while the age of onset has gradually become younger,has become one of the main causes of adult disability.In the knee joint with untreated ACL injury,secondary meniscus and cartilage degeneration,and ultimately a traumatic osteoarthritis have been concluded.The most common treatment for patients with ACL injury is ACL reconstruction.However,after long-term follow-up,ACL reconstruction surgery can effectively restore the stability of the knee joint,but a considerable number of patients eventually develop traumatic osteoarthritis.The pathological mechanism of OA in these stabilized knee joints has not been clarified.Objective:The purpose of this study was to establish a knee OA animal model after ACL reconstruction,and observe subsequent histological degeneration of the meniscus,cartilage and subchondral bone,also detect the abundance of inflammation factors in the synovial fluid of animals.We want to verify that intra-articular inflammation can independently induce early OA.In addition,the inflammation-related proteins were screened by cartilage proteomics,and a specific protein was selected for in vitro experiments,in order to find new targets for early diagnosis and intervention of OA.Methods:1.Obtain the Micro CT tomographic data of normal knee joints of minipigs and sheep(two of the most common pre-clinical animal model species),and use the software of Mimics,Geomagic Studio 2014,Pro/Engineer,Nastran to establish the 3D geometric model of the knee joint,and validate the effectiveness of the model at the same time.Stress-load analysis of ACL with knee flexion 30° and 60°,is used to compare the biomechanical differences between the knee joints of minipigs and sheep.From a mechanical point of view,we try to find the most suitable model animal to simulate human ACL reconstruction surgery.2.Innovative use of ACL autologous transplantation in situ to establish OA minipig model,and verify the timeliness and stability of this model.Quantitative assessment of OA changes in minipig knee joints was performed by morphological score from early osteophyte formation,articular cartilage,and meniscus injury.The degree and trend of histological changes of OA were qualitatively detected by techniques such as Indian ink staining and Safranin O staining.The knee joint cartilage tissue was harvested after the knee X-ray film was taken at 1 month,2 months,3 months and 4 months after ACL reconstruction surgery.The expression of m RNA in cartilage tissue was quantified by quantitative real-time PCR.The abundance of inflammatory factors was quantified by luminex technique.Finally,multiple linear regression models were used to analyze the correlation between inflammatory factors and histological changes of OA after ACL reconstruction.3.Using TMT label proteomics,we detected differential proteins in cartilage tissue by tandem mass spectrometry.After screening,protein sequence clustering analysis,GO enrichment analysis,KEGG pathway enrichment analysis,and PPI network were performed for functional annotation and pathway prediction on the differential proteins.4.Select ANXA1,which is closely related to inflammatory response,as a target protein in differential proteins.The expression of ANXA1 protein in human knee OA tissue at different stages was verified by immunohistochemical staining and ELISA.At the same time,IL-1? induced inflammatory injury(OA-like changes)on mouse costal cartilage cells in vitro,which were transfected with ANXA1 sh RNA plasmid.Thereby artificially knocking down the endogenous expression of ANXA1 gene and observing its effect on chondrocyte apoptosis.Results:1.Through biomechanical analysis of 3D finite element,the stress differences of various structures in joints of minipigs and sheep under the bending moment of 30° and 60° were compared.Overall,tissue stress of the joint structure increases with increase of knee angle.However,under the same knee angle,the stress peaks of various structures of sheep are larger than that of minipigs,and the distribution of stress loads in ligaments is uneven.The stress of the ACL and medial collateral ligament of the sheep is much larger than that of the posterior cruciate ligament and the lateral collateral ligament.Conversely,the stress and change of the cartilage,meniscus,and ligaments of minipig are closer to the human body.2.(1)Successfully established the OA minipig model after ACL reconstruction,and observed that histological changes of OA appeared at 3 months after ACL reconstruction by X-ray,Indian ink staining,and Safranin O-green staining.At the time,the model proved to have good timeliness and reliability.(2)Significant OA histological changes with osteophyte formation were observed in IACLR group,accompanied by an increase expression in m RNA of MMP-1,13 and a decrease in both m RNA and protein expression of aggrecan.As a result,the gross morphology score of the IACLR group was significantly higher than that of the control group.In addition,the expression of inflammatory factors(such as IL-1?,4,6 and TNF-?)in synovial fluid of the IACLR group was significantly increased.Most importantly,a multivariate linear regression model analysis found a correlation between IL-1?,6 and TNF-? concentrations and OA morphological scores(The results of the operation group showed that the P values of IL-1?,6 and TNF-? were 0.023,0.006 and <0.001,respectively.The control group also obtained statistical results with predictive significance of IL-1?,6 and TNF-?,whose P values are <0.001,0.017 and 0.001,respectively).3.Using TMT quantitative proteomics technology,2950 proteins were identified in cartilage tissue of minipigs.491 differential proteins were screened according to the standard of multiple changes more than 1.2 times and P<0.05.Among them,there were 198 differential proteins up-regulated in IACLR group compared with the control group,and 293 differential proteins were down-regulated.After GO function and KEGG pathway enrichment analysis,the functions of these differential proteins are mainly binding,catalytic activity,structural molecule activity,transporter activity and molecular function regulation,etc.These differential proteins are mainly involved in the cellular process,metabolic process,biological regulation,regulation of the biological process,and cellular component organization or biogenesis,and other important biological processes.4.(1)By immunohistochemical staining and ELISA,it was found that the differential protein ANXA1 was expressed in cartilage tissue and synovial fluid specimens of human knee OA.Its expression level is reduced at early and middle OA specimens,while late OA increase.(2)Inflammatory damage induced by IL-1? agent,the mouse costal cartilage cells showed a state similar to OA chondrocyte damage.Through liposome transfection technology,we artificially knocked down the expression of ANXA1 gene in chondrocytes,and observed the phenomenon of early apoptosis reduction of chondrocytes by flow cytometry.Conclusion:Through 3D finite element analysis,we found that minipigs have advantages in simulating the distribution of stress loads in the knee joint.The OA model established by ACL reconstruction using minipigs has good stability and timeliness,and studies in this animal model indicate that inflammation is one of the independent causes of the onset of OA.Using TMT-labeled proteomics,491 differential proteins were screened in minipigOA cartilage tissue.Among them,the ANXA1 protein closely related to inflammation was selected as a new target,and the expression was verified in different stages of human OA tissue samples.Finally,in vitro,we found that ANXA1 protein has the effect of inhibiting chondrocyte apoptosis.Of course,the molecular mechanism needs further research to confirm.This topic is an international science and technology cooperation program of China "the role of alpha-2-macroglobulin in early diagnosis and treatment of osteoarthritis(No.: 2015DFA33050)". |
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