The Effect Of Macrophage Phenotype Modulation In Murine Alveolar Bone On Orthodontic Tooth Movement

Objective:Understanding the mechanism of orthodontic tooth movement(OTM)and targeting the appropriate cell phenotype for intervention is of great significance to regulate orthodontic tooth movement.In this study,we established the murine OTM model to observe the effects of fucoidan on tooth movement rate,bone remodeling,and macrophage biological activity based on the major macrophage phenotypes and explore the preliminary mechanism.At the same time,we used advanced single cell sequencing technology to further refine the macrophage clusters in the murine alveolar bone,to select the cluster for regulation,and finally to observe the effects of macrophages on the OTM.Materials and Methods:The murine model of OTM was established.For the purpose of single cell sequencing and bioinformatics analysis,CD11b~+F4/80~+macrophages were isolated from the collected alveolar bone tissues around the first molars.At the same time,the murine OTM models were randomly divided into control group(TM)and fucoidan injection group(TM+FUC).After 0-,3-,5-,7-,and14-days movement,the OTM distance and periodontal bone density of the mice were observed by Micro-CT scanning.The periodontal bone remodeling state was observed by staining.The m RNA expression levels of factors such as TNF-α,i NOS,IL-1β,IL-6,and IL-10 in alveolar bone were analyzed by RT-PCR.The proportions of macrophages were identified by flow cytometry.Meanwhile,the bone marrow-derived macrophages of mice were cultured in vitro.Under a stable and pre-induced condition,fucoidan was administered to investigate its effect on macrophage phenotypes and the expression of the above-mentioned inflammatory factors.Western blot was employed to analyze the activation of STAT3 pathway in vivo and in vitro.STAT3 pathway specific inhibitor was utilized to observe its effect on macrophage immune response and biological activity,as well as the process of OTM.Results:Fucoidan significantly enhanced the stability of the teeth,increased the bone density around the teeth,and inhibited the inflammation in the alveolar tissue around the teeth.Fucoidan injection could regulate the polarization of macrophages in periodontal tissues.It could promote the polarization of restorative macrophages and inhibit proinflammatory macrophages both in vitro and vivo.In addition,fucoidan induced the restorative macrophages by activating STAT3 pathway in vitro and vivo.Single cell sequencing showed multiple clusters of macrophages in murine alveolar bone corresponding to different functions and the functions changed during OTM.Also,an important role played by Ccr2 cluster during the movement process was proved.Conclusion:Fucoidan could regulate the polarization of macrophages to restorative macrophages,thus affecting the process of OTM.OTM was observed to be slowed down by fucoidan via stabilizing the teeth and inhibition of required inflammatory process.Simultaneously,as confirmed by single cell sequencing,many clusters of macrophages in murine alveolar bone are attending,and their regulation might directly affect OTM process.Among those Ccr2 cluster was observed to play a remarkable role in OTM.