The Role Of CD226 Molecule In The Distribution And Function Of Inflammatory Monocyte Affected By Orthodontic Stress

Orthodontic tooth movement is a complex process.It is the common result of a series of soft tissue and bone remodeling reactions of the periodontal ligament under orthodontic force.Normally,osteogenesis in tension side and osteoclastogenesis in pressure side.After orthodontic loading,the capillaries in the periodontal ligament on the pressure side of the orthodontic teeth are compressed,which results in the decrease of local blood flow,the decrease of local oxygen pressure and the change of hydrodynamics.As a result,the cells with sensory function（such as alveolar bone cells,mesenchymal stem cells,bone lining cells,etc.）express rapidly and secrete cytokines locally,promote the differentiation of local cells and stimulate periodontal cells at the same time.Local capillary endothelial cells in the periodontal ligament lead to vasodilation and permeability enhancement,and then recruit osteoclast precursor cells in the adjacent bone marrow.When the recruited osteoclast precursor cells reach the periodontal ligament,they express RANK under the stimulation of local environment.Combined with the RANKL expressed by osteoblast precursor cells in the periodontal ligament,they further promote the differentiation of local osteoclasts and induce bone remodeling.Under the same stimulation,the ability of osteoclast precursor cells to penetrate endothelial cells in the bleeding vessel becomes a key factor affecting orthodontic tooth movement.Inflammatory monocytes can penetrate the endothelium of the hemorrhagic duct to reach the site of action during inflammation and further differentiate into peripheral macrophages and osteoclasts.CD226 molecules are highly expressed on the surface of inflammatory monocytes,which play an important role in the process of inflammation penetrating the endothelium of the hemorrhagic duct,while CD226 molecules are relatively low on the surface of patrol monocytes that do not penetrate the endothelium of the hemorr This is the case.Therefore,this study infers that the expression of CD226 molecule can affect the recruitment process of osteoclast precursor cells after orthodontic tooth loading,thus affecting the tooth movement process.In this study,we successfully established the orthodontic tooth movement model in mice,taking CD226 molecule as the research center,to explore the proportion of inflammatory monocytes in peripheral blood and spleen and the expression of CD226molecule on the surface of inflammatory monocytes in mice after orthodontic stress,and to further confirm the effect of CD226 molecule on orthodontic tooth movement by using CD226 gene knockout mice.The experiment is divided into four parts:1.Establishment of orthodontic tooth movement model in miceObjective:To study the method of establishing orthodontic tooth movement model in mice.Methods:24 SPF-grade female C57BL mice aged 8-10 weeks were divided into three groups（1-day group,3-day group and 7-day group,each group was divided into experimental group and control group.）.In the control group,only the springs were bonded and no additional force was applied;In experimental groups nickel-titanium springs were bonded between the left first molar and incisor in each group,with a force of about 40 g.The maxillary bone of mice was taken out and the distance between the left first molar and second molar was observed under light microscope.After making paraffin section,HE staining was performed to observe the osteoclasts formation at the root of the tooth.Results:The orthodontic tooth movement model of mice was successfully established.Light microscopy showed that the distances between the first molar and the second molar on the left side of the maxilla increased with the prolongation of the loading time,and HE staining showed osteoclast formation in the periodontal ligament and bone remodeling.Conclusions:The orthodontic tooth movement model of mice was successfully established to provide the basis for the next experiment.2.Changes in the proportion of inflammatory monocytes and CD226 expression during orthodontic stressObjective:To establish orthodontic tooth movement model in mice and observe the changes of the proportion of inflammatory monocytes in spleen and peripheral blood and the expression level of CD226 molecule.Methods:Twenty-four SPF-grade female C57BL mice aged 8-10 weeks（without specific pathogens）were divided into three groups（1-day,3-day and 7-day）with nickel-titanium springs bonded between the first maxillary molar and the incisor in each group.The strength of nickel-titanium springs bonded between the first maxillary molar and the incisor in the experimental group.After the mice were killed,peripheral blood and spleen were collected for flow cytometry,decalcification of maxilla was collected,and then maxilla was made into paraffin sections for TRAP staining to observe osteoclasts formation in periodontal ligament.Results:With the prolongation of orthodontic force,osteoclast formation increased;the proportion of inflammatory monocytes in peripheral blood decreased on the 1st day;on the 3rd day,the proportion of inflammatory monocytes in peripheral blood and spleen increased,while the expression level of CD226 molecule decreased;on the 7th day,the proportion of inflammatory monocytes in peripheral blood increased compared with the control group,the expression level of CD226 on the surface of inflammatory monocytes in spleen and peripheral blood decreased.Conclusions:During orthodontics,the proportion of inflammatory monocytes in peripheral blood and spleen of mice and the expression level of CD226 molecule have dynamic changes.The expression level of CD226 molecule may affect the orthodontic tooth movement of mice.3.The effect of CD226 molecule on the function of inflammatory monocytes during orthodontic stress and its preliminary mechanismObjective:To establish orthodontic tooth movement model in mice and observe the changes of the proportion of inflammatory monocytes and CD226 expression in spleen and peripheral blood of cd226^-/-mice;To extract bone marrow from cd226^-/-mice and WT mice for osteoclast differentiation induction,and detect the effect of CD226 on osteoclast differentiation.Methods:Ten SPF-grade female C57BL WT(cd226^+/+)mice and cd226^-/-mice aged 8-10 weeks were used to establish orthodontic tooth movement models（3days,7days）.The left maxillary first molar and incisor of the two groups were bonded with nickel-titanium springs.After 3 days,the mice were killed to collect peripheral blood and spleen for flow cytometry;After 3 days and 7days,the maxillary bone was collected to observed under light microscope.Five female C57BL WT(CD226^+/+)mice and five CD226^-/-mice aged 8-10 weeks were selected.Bone marrow cells were extracted and cultured directly or with bovine bone slices.After 10 days of culture,the osteoclasts were stained with TRAP.The differentiation of osteoclasts was observed under light microscope.Results:cd226^-/-mice were successfully cultured to provide experimental animals for subsequent experiments.After 3 days of stress,there was no significant difference between KO mice and WT group in the distance of maxillary first molar movement.At 7days of stress,the distance of WT mice was more significant than KO mice,and there was statistical difference.on the 3rd day,the proportion of inflammatory monocytes in peripheral blood and spleen increased compared with the WT group;There was no significant difference in the number and volume of osteoclasts directly differentiated between WT group and KO group under light microscopy.However,compared with WT group,KO group produced smaller osteoclasts with fewer nuclei and less vacuoles in cytoplasm.Conclusions:The deletion of CD226 molecule affects the ratio of inflammatory monocytes in peripheral blood and spleen,the distance of the first molar movement was decreased in KO group during orthodontics;CD226 may affect osteoclast differentiation by influencing the fusion of osteoclasts.