Study On The Role And Mechanism Of Macrophage Induced By Orthodontically Driven Corticotomy

Objective:To study the role and mechanism of macrophages induced by corticotomy during accelerated orthodontic tooth movement in rodent models.Thus to provide new evidence for the regulation of macrophages in orthodontic tooth movement.Materials and Methods:Adult Wistar rats(male,220–240 g,8 weeks old)and C57BL/6 mice(male,25–28 g,8 weeks old)were randomly divided into tooth movement only(TM)or corticotomy-assisted tooth movement(CO+TM)groups.Animals in CO+TM group were operated corticotomy and the upper left first molar were ligated with Ni Ti coil springs to the upper incisors which were used as anchorage teeth,while in the TM group,only the molars were mesialized without any surgery.The rats in each group were sacrificed 3,5,7,14,21,28,and 42 days after force application for tissue collection.The mice in each group were sacrificed 5,7,14,and 21 days after force application.Micro-computed tomography was used to assess the distance of tooth movement and the bone mineral density(BMD)around the upper left first molars.Morphological changes of periodontal tissue and the expression TRAP~+/CD11b~+/CD68~+/CD86~+/CD163~+cells were determined by TRAP staining and immunohistochemistry.The relative mRNA expression of CD11b,CD68,CD86,CD163,TNF-?,IL-1?,CD206 and Arg-1 in the palatal gingiva of rats was respectively evaluated by RT-PCR.The proportions of CD11b~+,CD11b~+CD86~+and CD11b~+CD163~+cells in the palatal gingiva were evaluated by Flow cytometry.Macrophageinvolvementwasfurtherconfirmedbydepleting monocytes/macrophages with intravenous injection of liposome-encapsulated clodronate in a subgroup of mice.In vitro macrophage culture was used to assess molecular pathways.BMDMs were incubated with fresh tissue suspension from mouse palatal gingiva.The relative mRNA expression of TNF-?,IL-1?,CD206 and Arg-1 were respectively evaluated by RT-PCR.The protein change of p65 and STAT3 phosphorylation were tasted by Western blot.Results:1.Corticotomy increased the rate of OTM:The amount of accumulative tooth movement was significantly greater in the CO+TM group from day 5 to day 42 than that in the TM group at each equivalent time point.In both groups,the rate of tooth movement reached the highest by day 3,then decreased on days 5,7 and 14,before increasing again on day 21.Comparison of OTM rate between TM and CO+TM groups showed that it was significantly higher on day 21 in the CO+TM group.Bone mineral density(BMD)of the volume of interest(VOI)was measured from micro-CT scans.It increased with time in both groups and was significantly lower in the CO+TM group than in the TM group at multiple time points(days 3,7,14 and28).2.Corticotomy induced osteoclastogenesis and macrophage infiltration during OTM:Osteoclast activity was evaluated by immunohistochemical staining for tartrate-resistant acid activity(TRAP).Consistent between the two parameters(Oc.S/B.S and N.Oc/B.Pm),osteoclast density peaked at day 14 and was significantly higher in the CO+TM group than the TM group.Immunohistochemistry showed significantly greater positive area for CD11b~+macrophages in the CO+TM group than in the TM group from day 5 to day 42.The positive area for CD68~+macrophages was significantly higher in the CO+TM group than the TM group from day 5 to day 14.Consistent with protein expression,RT-PCR results showed significantly higher relative expression of CD11b and CD68mRNA in the CO+TM group than in the TM group from day 5 to day 21.These findings were further verified using flow cytometry:the proportion of CD11b~+cells was significantly greater in the CO+TM group than in the TM group from day 5 to day 21.3.Corticotomy induced macrophage polarization at different stages of OTM:Immunostaining against the M1-phenotype marker CD86 and the M2-phenotype marker CD163 revealed a significantly larger positive area for CD86~+macrophages on the compression side in the CO+TM group than in the TM group from day 5 to day 21.The positive area for CD163~+macrophages was significantly greater in the CO+TM group than in the TM group on days 5,7 and 21.Flow cytometry analysis revealed a greater proportion of CD11b~+CD86~+cells in the CO+TM group gingiva than in the TM group gingiva from day 5 to day 21,and a greater proportion of CD11b~+CD163~+cells in the CO+TM group than in the TM group on days 5,7,and21.These results were confirmed at the mRNA level using RT-PCR.Relative expression of the CD86 mRNA increased until day 14 in the CO+TM group,then declined slightly.In contrast,CD163 mRNA levels increased on day 5 and then fell until day 21 when it increased again in both groups.Levels of CD86 mRNA were higher in the CO+TM group than in the TM group during initial and middle periods of treatment(between day 5 and day 14),whereas expression of CD163 was higher in the CO+TM group than in the TM group during the OTM(between days 5 and 42).CD86 up-regulation correlated with high expression of IL-1?and TNF-?on day 5and day 14 in the CO+TM group.Expression of the M2-phenotype markers Arg-1and CD206 was significantly higher in the CO+TM group than in the TM group on day 5 and day 21.4.Corticotomy-induced OTM was decreased by macrophage depletion:After depleting macrophage by injecting liposome-encapsulated clodronate(LEC),which was confirmed by a 70%of decrease in circulating monocytes,we found that the number of CD11b~+F4/80~+macrophages in the gingiva also significantly decreased.Flow cytometry analysis further demonstrated that macrophage polarization was affected by LEC injection,showing that the upregulation of both CD86~+and CD206~+cells among CD11b~+F4/80~+cells induced by corticotomy was significantly repressed.More importantly,without macrophage depletion,significantly larger tooth movement was shown in the CO+TM group than in the TM group from day 5 to day21;while with macrophage depletion,the amount of OTM induced by corticotomy was significantly decreased.The rate of tooth movement(from day 5 to day 14)was significantly lower in mice receiving LEC injection than those receiving saline injection.5.Corticotomy induced macrophage polarization b y activating NF-?B and JAK–STAT pathways:BMDMs were incubated with fresh tissue suspension from mouse palatal gingiva.RT-PCR showed that levels of M1-phenotype markers TNF-?or IL-1?were significantly higher in the CO+TM group than in the TM group wit h the levels increase from day 5 to day 14 and then declined on day 21.Similarly,levels of M2-phenotype markers Arg-1 or CD206 were significantly higher in the CO+TM group than in the TM group;the levels in both groups increased initially,declined slightly until day 14,and then increased again.Western blotting showed that P65levels in BMDMs increased after incubation with tissue suspension from the CO+TM group between days 5 and 7,after which they began to decrease.Levels of phosphorylated STAT3 increased on day 5 and declined until day 21,after which they increased again.Conclusion:These data suggest that local alveolar corticotomy accelerates OTM by enhancing macrophage infiltration and regulation of their polarization at different OTM stages.We also revealed that these regulations may be NF-?B and JAK/STAT3pathways,respectively.