An Experimental Study On The Construction Of Rabbit Condylar Resorption Animal Model And The Inhibitory Effect Of Kaempferide On Osteoclast

ObjectivesThe purpose of this study was to successfully build the condylar resorption animal model in growing rabbit through surgical induced temporomandibular joint(TMJ)anterior disc displacement(ADD)combined with bilaterial ovariectomy(OVX),and to preliminary explore the possible mechanism of osteoclasts on condylar resorption and the inhibitory effect of kaempferide on osteoclast.Methods1.The establishment and evaluation of rabbit condylar resorption animal model.(1)The influence of OVX and ADD on the development of mandible in growing rabbitsThe animal model of unilateral ADD and OVX was built in growing rabbits,the mandibular length and midline asymmetry was measured preoperation and 3 months post-operation.The serum 17β-estradiol was detected at 4 weeks,8 weeks and 12 weeks after surgical OVX using ELISA.(2)The influence of OVX and ADD on the condylar bone change and the expression of osteoclasts on the subchondral bone.Micro CT was used to testify the condylar bone change after surgical induced OVX and ADD,the bone loss of anterior and posterior part of the condyle were analyzed.The subchondral osteocytes apoptosis and expression of osteoclasts on the subchondral bone were also examined by TUNEL,hematoxylin-eosin(HE)staining and tartrate resistant acid phosphatase(TRAP)staining.2.The inhibitory effect of kaempferide on osteoclastIn vitro study,kaempferide(KF)was used to testify the inhibitory effect on osteoclast’s proliferation and differentiation,and the function of bone resorption.The specific protein and gene expression of osteoclast were also tested.In vivo study,the animal model of calvarial osteolysis was built and kaempferide was intraperitoneal injected and the calvarial bone change was examined by micro CT and histology examinations including HE staining and TRAP staining.Results1.The animal model of rabbit condylar resorption was built successfully(1)OVX+ADD significantly impaired the mandible growth.3 months postoperatively,in control group,no difference of mandibular length was found between left and right sides.In OVX group,the mandibular length increased evenly,and measurements revealed good facial symmetry with no significant difference of mandibular length between sides(p>0.05)and no difference was found between OVX group and control group.While in ADD group and OVX+ADD group,the mandibular length of the right side was significantly shorter than the left side(p<0.05),and in the above two groups,no difference was observed in the mandibular length of the left side compared to control group.The mandibular length of right side of OVX+ADD group was significantly shorter(p<0.05)than that of ADD group.The mandible in the control group and OVX group showed good symmetry with almost no midline deviation.In ADD and OVX+ADD group,the mandibular symphysis consistently deviated toward the operated side.The angles in the OVX+ADD group was larger than that in the ADD group.(2)The rabbit condylar resorption was more severe in OVX+ADD group.The bone volume(BV),trabecular number(Tb.N),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp)of OVX+ADD group were significantly decreased compared to CTRL group,and the posterior part of the condyle decreased more.HE staining also showed less trabecular bone of the posterior part of the condyle and the apoptosis of osteocytes was significantly higher than CTRL group.TRAP staining revealed more osteoclasts formation on the posterior part of the condyle in OVX+ADD group.(3)KF inhibited the osteoclast’s formation and function both in vitro and in vivo.In mouse bone marrow macrophages(BMMs),KF significantly inhibited the osteoclast formation and bone resorption at 12.5 μmol/L.However,KF does not affect the mature osteoclast F-actin ring formation.But when being co-treated with KF and anisomycin,BMMs differentiated into mature osteoclasts.At the molecular levels,the JNK phosphorylation was inhibited and the osteoclastogenesis-related specific gene expression including V-ATPase d2,TRAP,CTR,c-Fos and NFATc1 was markedly suppressed.In the mice with titanium particle induced calvarial osteolysis,the low dose of KF mildly reduced the resorption pits while in the high dose group,fewer scattered pits were observed on the surface of calvarium.Histological examination showed fewer osteoclasts formation in the KF group.Conclusions1.During growth period,ADD will impair mandible growth,leading to mandibular asymmetry while low level of serum estrogen will not affect mandible growth.The effect of OVX+ADD on mandibular development is more serious.2.The osteocytes apoptosis was more obvious and severe in the posterior part of the condyle,and much more TRAP positive osteoclasts were formed in this area,leading to condyle bone resorption of the posterior part more severe than the anterior part.Combined the results of the first two parts,we can conclude that we successfully built the animal model of condylar resorption.3.Kaempferide could inhibit the osteoclasts’ formation and function through downregulating the JNK signaling pathways in vitro and could rescue the calvarial osteolysis induced by Ti-particle in vivo.