The Research Of Diagnostic Biomarkers And Assessment Of C5a Therapeutic Value In Periprosthetic Joint Infection

Objectives:(1)The first aim was to investigate the diagnostic value of albumin,globulin,albumin-to-globulin ratio(AGR),and fibrinogen in the diagnosis of periprosthetic joint infection(PJI).(2)The second aim was to explore the activation of the complement system in PJI and the diagnostic value of complement markers in PJI.(3)The third aim was to establish a low virulence PJI mouse model.(4)The fourth aim was to evaluate the therapeutic value of C5a in the treatment of PJI.Methods:(1)In this retrospective cohort study,from January 2017 to December 2019,patients admitted to our hospital who underwent revision THA or TKA were enrolled,including 31 cases of acute PJI,51 cases of chronic PJI,and 139 cases of aseptic failures.The 2013 criteria of the Musculoskeletal Infection Society(2013 MSIS)were used as the reference standard for the diagnosis of PJI.The preoperative blood routine examination,coagulation examination,and blood biochemistry were assessed.The receiver operating characteristic curve(ROC),sensitivity,and specificity were utilized to compare different biomarkers’diagnostic values.(2)In this prospectively designed study,40 patients who underwent revision THA or TKA were enrolled.Among them,20 patients were diagnosed as PJI according to the 2013 MSIS criteria,and 20 aseptic failure patients matching to PJI patients on age and sex were treated as a control group.Preoperative blood and intraoperative synovial fluid were collected,and blood routine examination,coagulation examination,PCT,IL-6,and complement associated factors were tested.The ROC,sensitivity,and specificity were utilized to compare different biomarkers’diagnostic values.(3)Distal femur and proximal tibia implants(a 3D-printed mouse side Ti-6Al-4V implant)were inserted into female C57BL/6J mice knees and then inoculated intra-articular with phosphate-buffered saline(PBS)or different colony-forming unit(CFU)(10~2,10~4,10~6,10~8)of Staphylococcus epidermis.Clinical outcomes,inflammatory biomarkers,X-ray imaging,micro-CT,scanning electron microscopy,histology,and bacterial loads were utilized to assess infection.(4)PJI mice models were constructed and divided into 3 groups,namely,group A(PJI model group),group B(C5a treatment group),and group C(PMX-53 inhibition group).Local injection of drugs was given to the right knee of every mouse every day after surgery for 14 days.Every mouse in Group A locally injected 50 ul 0.9%Na Cl solution,in group B locally injected 50 ul 10 n M C5a solution,and in group C locally injected 0.4 ug/ul 50 ul PMX-53 inhibitor solution.The infection severity of mice in each group was comprehensively evaluated by temperature,body weight,weight-bearing,blood routine test,ESR,inflammatory biomarkers,X-ray imaging,micro-CT,scanning electron microscopy,bacterial load,and pathology.The effects of the C5a/C5a R1 axis intervention on osteoblasts,osteoclasts,collagen fibers,and articular cartilage were investigated by PCR,immunohistochemistry,and special staining.Results:(1)Compared with the aseptic patients,the following biomarkers in PJI patients were significantly higher(P<0.05),including globulin,NLR,PVR,PLR,D-dimer,fibrinogen,FDP,ceruloplasmin,α1-antitrypsin,ESR,and CRP.On the contrary,the following biomarkers in PJI patients were significantly decreased(P<0.05),including albumin and AGR.The area under ROC curve(AUC),sensitivity and specificity of each biomarker were as follows:albumin(0.774,67.5%,77.54%),globulin(0.82,57.5%,89.86%),AGR(0.845,66.25%,93.48%),NLR(0.678,52.44%,81.3%),PVR(0.707,48.78%,86.33%),PLR(0.7,51.22%,80.58%),D-dimer(0.683,55.22%,75.83%),fibrinogen(0.832,78.48%,78.95%),FDP(0.664,38.81%,88.33%),ceruloplasmin(0.797,81.16%,72.41%),α1-antitrypsin(0.817,82.09%,71.55%),ESR(0.877,81.48%,85.07%)and CRP(0.909,83.95%,88.89%).Globulin,AGR,fibrinogen,α1-antitrypsin,and ESR showed good performance in PJI diagnosis,meanwhile,CRP showed excellent performance in PJI diagnosis.(2)Compared with the aseptic patients,the following biomarkers in PJI patients were significantly higher(P<0.05),including fibrinogen,IL-6,serum C1q,synovial C1q,serum C3b,synovial C3b,serum i C3b,synovial i C3b,serum C4b,synovia C4b,serum C5a,synovial C5a,ESR,CRP,synovial WBC count,and synovial PMN%.The AUC,sensitivity,and specificity of each biomarker were as follows:fibrinogen(0.739,75%,65%),IL-6(0.685,60%,75%),serum C1q(0.729,60%,75%),synovial C1q(0.824,100%,55%),serum C3b(0.785,60%,90%),synovial C3b(0.754,80%,60%),serum i C3b(0.733,50%,95%),synovial i C3b(0.778,100%,55%),serum C4b(0.695,50%,100%),synovial C4b(0.728,100%,45%),serum C5a(0.829,55%,100%),synovial C5a(0.954,95%,85%),ESR(0.79,90%,60%),CRP(0.95,85%,95%),synovial WBC count(0.892,80%,90%)and synovial PMN%(0.939,85%,100%).Synovial C1q,serum C5a,and synovial WBC count showed good performance in PJI diagnosis,meanwhile,CRP,synovial C5a,and synovial PMN%showed excellent performance in PJI diagnosis.(3)On the 14th day of postsurgery,the survival rate of the 10~8CFU group was 85.7%,and mice in the other groups all survived.The mouse infection rate of the 10~2CFU group was 66.7%,and that of the other infection groups was 100%.The clinical outcomes,inflammatory biomarkers,X-ray imaging,micro-CT,biofilm formation,histological findings,and bacterial loads showed the same trend.With increasing bacterial inoculation,the mouse bacterial loads and inflammation increased.The optical bacterial incubation was 10~6CFUs for S.epidermis to develop a mouse PJI model.(4)A comprehensive evaluation of body temperature,body weight,weight-bearing,clinical feature,WBC count,neutrophil count,ESR,SAA,X-ray score,bacterial biofilm,bacterial load,and pathological showed that local injection of C5a could significantly reduce joint infection,while local injection of PMX-53 could aggravate the joint infection.PCR and immunohistochemical results showed that the expressions of MPO and CD64 were significantly increased in group B and there were no significant differences in the expressions of osteocalcin,osterix,and Runx2 among three groups.There were no significant differences in TRAP staining and Masson staining among three groups.Safranin-fast green staining and toluidine blue staining showed that the area of articular cartilage in group B was significantly higher than that in group A.Conclusion:(1)Globulin,AGR,fibrinogen,andα1-antitrypsin are good diagnostic biomarkers of PJI.(2)Complement is widely activated in the pathogenesis of PJI,and synovial C5a is an accurate and reliable diagnostic biomarker of PJI.(3)This study successfully established a clinically representative low-virulence PJI mouse model.(4)Local injection of C5a can significantly reduce PJI infection,decrease bacterial load,and reduce biofilm formation.Blocking the C5a/C5a R1 axis will aggravate infection,increase the bacterial load,and increase biofilm formation.