The Mechanism Of Osteoblast-osteoclast Crosstalk In Subcondylar Bone Remodeling Under The Overloaded Functional Orthopaedic Force

Objectives:Class II malocclusion is a common orthodontic disease with higher incidence in adults and adolescents.The clinical manifestations includes mandibular retraction,mouth leakage,shallow nasolabial sulcus,deepened mentolabial sulcus.Class II malocclusion affects the occlusal function and facial aesthetics of patients,and even harms the health of the respiratory system,digestive system and psychosocial health.Functional orthodontics is a widely accepted method for the treatment of Class II malocclusion by orthodontists at home and abroad.It mainly achieved through the mandibular repositioning to coordinate the sagittal difference of the upper and lower jaws,improve the facial profile,the normal occlusion and jaw relationship.Various clinical data have shown that proper mandibular advancement contributes to the adaptive remodeling of the condyle during the treatment of functional treatment,while excessive mandibular extension leads to condyle absorption and even iatrogenic temporomandibular joint disease.At present,the pathogenesis of TMD,especially the abnormal temporomandibular joint related to the orthodontic treatment,still needs further investigation.Bone resorption of osteoclasts and osteogenesis of osteoblasts regulates bone metabolism and bone remodeling in mandibular condyles during functional correction therapy.Osteoblasts and osteoclasts are the main cells in bone tissue and stress sensitive cells in the body.They are both the sensory cells of mechanical signals and the effector cells of stress stimulation,which play a central regulatory role in the process of bone metabolism.However,the mechanism of mechanical stretching on osteoblasts and osteoclasts and the mechanism of information interaction between the cells are still unknown.In this study,we established the overloading environment in vivo and in vitro to study the subcondylar cartilage bone remodeling and the signal interaction between osteoblasts and osteoclasts during the overload of functional orthopedic force,so as to optimize and guide the orthodontic clinical treatment.Meanwhile,we aimed to provide the new insights to further avoid the TMJ damage during overloaded functional orthopaedic force,to accelerate the process of functional orthopaedic and to expand the indications of functional orthopaedic.Materials and methods:The rat model of excessive mandibular extension was constructed.At the time points of 1 week,2 weeks,4 weeks and 8 weeks after the establishment of the model,the reconstruction of temporomandibular joint,the morphological and histological changes of subchondral bone and the expression of related factors in the experimental group and control group of rats were observed and evaluated by micro-CT and immunohistochemistry.The activation level and location of mTOR in the experimental group were determined by immunofluorescence double staining.An in vitro mechanical overstretch model was established to simulate functional orthopedic force overloading environment,and the effects of factors secreted by osteoblasts and activated osteoblasts on osteoclast activity were investigated.After mechanical stress,osteoblasts were collected and detected by WB,PCR and immunofluorescence localization to detect the changes of osteoblast related factors.The superfluid of osteoblast was collected and added to osteoclast induction medium.After osteoclast induced differentiation,mechanical stress was loaded in vitro,and MAPK family protein levels were collected and detected by WB and PCR.The superfluid of osteoclast was collected and added to osteoblast induction medium,and the differentiation level of osteoblast was detected by WB and PCR.Results:1.After the construction of the rat model of mandibular hyperextension,the condyle protrusion showed joint degeneration at 1 week,2 weeks,4 weeks and 8 weeks.Micro-ct showed that bone resorption occurred in the condyle at 1,2,4 and 8 weeks after mandibular hyperextension rat model construction.Compared with the control group,the horizontal diameter of the condyle in the experimental group decreased,and the shape was irregular.BMD,BV/TV,Tb.N and Tb.Th all decreased,while Tb.Sp increased.The condylar bone resorption was especially obvious in the 8-week group.2.Double immunofluorescence staining showed that,compared with the control group,the activation of mTOR in the experimental group was mainly distributed in the osteoblasts of the condyle in the model of excessive mandibular extension in rats.3.Immunohistochemical results showed that compared with the control group,the expression of RANKL and OPG in the subcondylar cartilage bone of the experimental group was higher.4.In vitro experiments,Western Blot and PCR results showed that mTOR signaling factor expression increased,RANKL expression increased,OPG expression decreased andRANKL/OPG decreased in osteoblasts after loading.In osteoblasts pretreated with mTOR inhibitor Rapamycin,mTOR expression was inhibited and the increased RANKL/OPG ratio was reduced.5.In vitro experiments,compared with the blank control group,the supernatant of osteoblasts in the stretched group significantly increased the number of trap staining positive cells.The effect of supernatant added with Rapamycin on the number of positive osteoclasts was not significantly different from that of the control group.6.In vitro experiments,results of Western Blot and PCR showed that the expressions of ERK,JNK and p38 in osteoclasts increased under overloaded stretch.7.In vitro experiments,compared with the blank control group,the supernatant of osteoclasts in the stretched group significantly increased the expression of Runx2,BMP-2and Osterix.ERK inhibitor can effectively inhibit the promotion of osteoclast supernatant on osteoblast related proteins.Conclusions:1.Overloaded functional orthopaedic force leads to bone resorption in the subchondral bone of the condyle in rats.2.The mTOR signaling factor in subcondylar cartilage bone of rats is activated,mainly distributed in osteoblasts when the functional orthopaedic force overloaded.3.The overloaded mechanical stretch promotes the activation of mTOR signaling factors in osteoblasts and promotes osteoclast differentiation through the downstream RANKL/OPG signaling pathway.4.The overloaded mechanical stretch activates the ERK signaling factor of osteoclasts and up-regulates the differentiation level of osteoblasts.