The Effect And Mechanism Of SPA On Osteolysis Mediated By Osteoclast During Bone Infection

Backgrounds and Objectives:Bone infection is a common and serious complication in the orthopedics field,which often leads to excessive bone destruction and non-union.Staphylococcus aureus(S.aureus)is a major pathogen causing bone infection,accounted for 65% to 70% of the pathogenic bacteria in traumatic osteomyelitis.SPA which widely presents in bacterial wall is one of the most important pathogenic components.It was found that SPA can be freely secreted into the extracellular environment and can widely bind to host defense proteins to alter the host immune response.Osteoclasts are derived from hematopoietic stem cell lineage,which is the only polykaryocytes with bone resorption function in the human skeletal system.It is the main cell involved in bone remodeling with osteoblasts,and the two of them regulate the dynamic balance of bone metabolism.In the state of infection,acute and chr onic inflammatory stimulation can change the dynamic balance and affect bone healing.Previous studies have demonstrated that SPA can accelerate apoptosis of osteoblast,suppress osteoblast proliferation and inhibit the process of bone formation and mineralization.In addition,osteoclast is associated with excessive bone loss,and the differentiation process is regulated by many factors.However,there are few reports about the effects of SPA on osteoclastogenesis and its molecular mechanism.Therefore,our research aimed: 1.To observe the effects of Staphylococcus aureus infection on bone mass and osteoclast in in traumatic osteomyelitis;2.To survey the effect of SPA on osteoclast differentiation,fusion and function;3.To explain the mechanism of SPA on osteoclastogenesis;in order to search for new targets for the prevention and treatment of such diseases.MethodsPart I:The effects of Staphylococcus aureus infection on bone mass and osteoclast in clinical specimens1.Clinical specimens which were diagnosed as chronic traumatic osteomyelitis were collected during surgery.2.Samples were fixed in 4% paraformaldehyde and then decalcified by 10%EDTA solution.After paraffin embedded,samples were prepared into 8?m thickness bone slices3.Masson,HE and TRAP staining were used to observe the bone mass and osteoclast.Part II: The effects of SPA on osteoclastogenesis in vitro1.RAW 264.7 cells were cultured with RANKL and M-CSF to build RANKL-induced osteoclastogenesis model.2.RAW264.7 cells were incubated with different dosages of SPA with or without RANKL and M-CSF respectively.CCK-8assay was used to evaluate the effects of SPA on cell proliferation to make sure the safe concentration of the experiment.3.TRAP stain was performed in order to observe osteoclast differentiation.The number of osteoclasts(nuclei ? 3)were counted.4.FAK staining was performed to investigate the effects of SPA on osteoclast fusion,and the number of osteoclasts and average nuclei were counted.5.Bone resorption assay was carried on to evaluate the effects of SPA on osteoclast bone resorption activity.6.Annexin-V/PI Staining were used to assess the effect of SPA on cell apoptosis during RANKL induced osteoclastogenesis.Part III: The mechanism of SPA promoted osteoclastogenesis1.The expression of m RNA associated with osteoclastogenesis were tested by RT-PCR.2.The expression of NFATC1,c-FOS and its upstream MAPK pathways related proteins were detected by western blot.3.The expression of NFATc1 and c-FOS were detected when MAPK pathways inhibitors were administeredResults:Part I:The thickness of bone trabecula was thinner in non-infection group compared with infection group.Meanwhile,the results of TRAP staining showed that OC surface/bone surface ratio was significantly increased.Part II:SPA promotes osteoclast differentiation,fusion,and enhances bone resorption activity.1.RAW264.7 cells were cultured with 50ng/m L RANKL and 50 ng/m L M-CSF for 3 days to induce osteoclast in vitro.2.The results of CCK-8 assay revealed that SPA concentration at 1000?g/m L significantly inhibited cell proliferation.In addition,the results of Annexin-V/PI Staining showed that SPA had no effect on both early and late apoptosis rate.3.According to the results of CCK8 assay,SPA dosages at 0.1?g/m L,1?g/m L,10?g/m L and 100?g/m L were selected for further study.The results of TRAP staining showed that the number of osteoclasts(nuclei?3 in the experimental group increased significantly compared with the control group.4.Quantification analysis of FAK staining showed that the number of osteoclasts and average nuclei of osteoclasts was significantly increased by SPA treatments.5.Statistical results of bone resorption areas showed that bone resorption activity was significantly increased by SPA treatment.Part III: q PCR results also revealed that osteoclastogenesis associated marker genes including CTSK,TRAP,CTR and DC-STAMP were significantly increased when SPA concentration was 10?g/m L or 100?g/m L.The results of western blot showed that the expression of p-ERK,p-JNK and p-P38 was increased compared with its non-phosphorylation levels by SPA treatment.At the same time,the expression of NFATc1 and c-FOS which were downstream of MAPK signaling were significantly increased.Meanwhile,the expression of NFATc1 and c-FOS were not increased when the cells was inhibited by ERK,JNK and P38 MAPKinhibitors.Conclusions:Staphylococcus aureus infection led to excessive bone loss and osteoclast activation.Our study also demonstrated that SPA treatment promotes osteoclast differentiation and fusion,and increases bone resorption activity in vitro.We showed that SPA promotes osteoclastogenesis through MAPK signaling activation.The results could be helpful to better understand the pathogenicity of S.aureus in bone infection,and it is meaningful to cure such diseases.