Mechanically Induced M2 Macrophages Are Involved In Bone Remodeling Of The Midpalatal Suture During Palatal Expansion

Objective:Rapid palatal expansion(RPE)is the most common treatment for maxillary transverse deficiency(MTD)in adolescents.Although RPE is effective,there are still different degrees of relapse.Inadequate bone formation is the main factor leading to the relapse.Therefore,finding ways to promote bone formation during palatal expansion is the key to shorten the retention time and reduce the rate of relapse.The innate immune system’s main cell type,macrophages,is crucial for orthodontic bone remodeling.However,whether macrophages are involved in and influence mechanical force-mediated bone remodeling of the midpalatal suture is unknown.Therefore,the aim of this study was to investigate whether there are changes in the number and phenotype of macrophages during palatal expansion,and their effects on bone remodeling as well as the possible mechanisms.Methods:(1)Rats were randomly divided into the expansion group and the control group to construct the palatal expansion models.The changes in the width of the midpalatal suture,the status of bone remodeling in the midpalatal suture region,and the number and phenotypic changes of macrophages in both groups at 7 and 14 days were evaluated by micro-CT,HE staining,TRAP staining,and immunohistochemical staining for OCN,F4/80,iNOS,and CD206.(2)Rats were randomly divided into the depletion group and the control group,and the macrophages depletion models were constructed using clodronate liposomes.Macrophages depletion efficiency was detected by F4/80 immunohistochemical staining and flow cytometry.The changes in the width of the midpalatal suture and the status of bone remodeling in the midpalatal suture region at 7 and 14 days after palatal expansion was assessed by micro-CT,HE staining,TRAP staining,and immunohistochemical staining for ALP,OCN,OPG,and RANKL in both groups.(3)In vitro experiments were performed to culture RAW 264.7 macrophages and palatal osteoblasts.RAW 264.7 macrophages were induced to polarize toward the M2 phenotype using mechanical force,and M2 macrophage polarization was verified by qRT-PCR,ELISA,and immunofluorescence staining.ALP staining,alizarin red staining,qRT-PCR and western blot were used to detect the influence of M2 macrophages-derived conditioned medium on osteoblasts.Results:(1)At 7 and 14 days,the width of the midpalatal suture was significantly wider,bone remodeling was more active,and OCN and TRAP positive cells at the midpalatal suture were increased in the expansion group compared to the control group.New bone formation was also seen at the midpalatal suture of the rats in the expansion group at 14 days.In addition,the number of macrophages increased significantly with the increase of the expansion time and polarized to M2 phenotype,which was consistent with the trend of bone remodeling at the midpalatal suture during palatal expansion.(2)At 7 and 14 days,the midpalatal suture in the depletion group was noticeably broader than in the control group,and the function of osteoblasts in the midpalatal suture was weakened,the expression of ALP and OCN was decreased,and new bone formation was reduced at the late stage of palatal expansion.In addition,compared with the control group,the depletion group showed decreased OPG expression and increased RANKL expression,with the corresponding decrease in the RANKL/OPG ratio and the relative increase in osteoclasts.(3)In vitro,mechanical force successfully induced RAW264.7 macrophages to polarize toward M2 phenotype.Compared with M0 macrophages,osteoblasts induced by conditioned medium derived from the supernatant of M2 macrophages showed increased levels of ALP activity,mineralized nodule formation ability,and expression of osteogenesis-related genes and proteins including ALP and COL1,while the expression of osteoclastogenesis-related gene OPG was elevated,and the expression of RANKL as well as RANKL/OPG ratio were decreased.Conclusion:Macrophages in the local microenvironment during palatal expansion were recruited and polarized toward the M2 phenotype to participate in bone remodeling of the midpalatal suture,providing an idea to find ways to promote bone remodeling of the midpalatal suture during clinical palatal expansion from an immune perspective.