LIPUS Accelerates BMP-2Signaling Pathway During Rat Orthodontic Alveolar Bone Remodeling

Accelerating the alveolar bone remodeling and thus accelerating the velocity oforthodontic tooth movement is highly desirable by orthodontists and patients. Toothmovement is closely related to the response of applied orthodontic forces that causeremodeling of periodontal tissues, especially alveolar bone remodeling. Humanperiodontal ligament cells (hPDLCs) are considered as multipotential cells, and play animportant role in alveolar bone remodeling. Low-intensity pulsed ultrasound (LIPUS)stimulation has been reported to promote fracture healing, to treat bone nonunion and itcould accelerate bone maturation and remodeling during the consolidation stage ofdistraction osteogenesis. Bone morphogenetic proteins (BMPs) are multifunctional growthfactors, they not only result in proliferation, differentiation, apoptosis and other functionsof cells; but also participate in tissue regeneration and repair, particularly play a vital rolein bone remodeling. Among them, BMP-2is one of factors having the best effects ininducing bone formation. Hepatocyte growth factor (HGF) could stimulate osteoblastproliferation and differentiation, and the activation of HGF may activate Runt-relatedtranscription factor2(Runx2) during bone formation. Runx2is an osteoblastdifferentiation specific transcription factor which plays an important role in formation and differentiation of osteoblasts, and the formation and activation of osteoclasts. In addition,Runx2signaling pathways may be involved in increasing BMP-2expression duringLIPUS stimulation. The receptor activator of nuclear factor kappa-B ligand (RANKL) isone of the key factor in the regulation of osteoclast formation and may be involved inalevolar bone remodeling during orthodontic treatment.Objective:There is limited studies regarding the effects of LIPUS stimulation onHGF/Runx2/BMP-2signaling pathways and RANKL as well, This study was aimed toexamine these effects during orthodontic tooth movement in rats.Methods:1.Orthodontic appliances were placed on the rats between the homolateral first uppermolar and upper central incisor, and animals were treated daily LIPUS stimulation onupper first molar from the2ndday of orthodontic tooth movement. Determine the distanceof tooth movement after initiating tooth movement (0d,1d,3d,5d,7d, and14d).Meanwhile, the temperature rise of gingival surface of rat upper first molar was measuredat different time points. BMP-2immunoreactivity was observed in periodontium of upperfirst molar tension side in the LIPUS stimulation group and control group byimmunohistochemistry.2.Observed HE staining of rats periodontium of upper first molar pressure side (0d,3d,7d). By qRT-PCR and Western blot (WB), RANKL mRNA and protein expression ofperiodontium of upper first molar pressure side were detected (0d,3d,7d).3.Using qRT-PCR and WB to assay HGF, Runx2, BMP-2mRNA and proteinexpression of periodontium of upper first molar tension side on day0, day3and day7.4.We performed and identified the primary culture of Human periodontal ligamentcells (hPDLCs), and the cells were subjected to daily LIPUS stimulation. The BMP-2mRNA and protein expression in hPDLCs were detected by qRT-PCR and WB on day0,day1, day3, day5, day7and day14.5.hPDLCs were incubated with0ng/ml,5ng/ml,10ng/ml HGF at37℃for24h,BMP-2mRNA expression was detected by qRT-PCR, BMP-2protein amounts in cells for 48h were detected by WB. hPDLCs were transfected with Runx2siRNA and controlsiRNA for24h, and followed by LIPUS stimulation for5days. BMP-2mRNA andprotein amounts in hPDLCs were examined by qRT-PCR and WB.6.hPDLCs numbers were counted after LIPUS stimulation at day0, day1, day3, day5, day7, day14in test and control groups. The6-well plates of hPDLCs were cultured for14days at37℃,38℃,39℃in thermostatic incubator, and then BMP-2mRNA andprotein amounts in hPDLCs were detected by qRT-PCR and WB.Results:1.The amount of orthodontic tooth movement in test group was significant greatercompared to the control group on day5, day7and day14(P <0.05). Quantitative analysisof BMP-2immunohistochemistry results, the amounts of BMP-2positive cells in testgroup were significantly more than control group on day3, day5, day7and day14(P <0.05). The temperature of upper first molar gingival surface rose a little bit during LIPUSstimulation.2.The results of HE staining showed that compared with the control group, LIPUSstimulation increased number of osteoclasts on day3(P <0.05), while increasedsignificantly on day7(P <0.01). The relative RANKL mRNA and protein expressionlevels in rats maxillary first molar pressure periodontal tissue by LIPUS stimulation wereincreased on day3(P <0.01), while it had very significant increase on day7(P <0.001).3.The relative HGF, Runx2and BMP-2mRNA and protein expression levels in ratmaxillary first molar tension periodontal tissue by LIPUS stimulation were increased onday3(P <0.05), while they had significant increase on day7(P <0.01).4.The expression of BMP-2mRNA and protein amounts in hPDLCs weresignificantly increased as time gent on. Compared with control group, the test groupexpressed significantly more BMP-2mRNA and protein on3-14d in hPDLCs, andreached the peak level on day7, and then remained elevated through day14.5.Treatment of hPDLCs with5ng/ml HGF for24h increased BMP-2mRNA andprotein amounts (P <0.05), and with10ng/ml HGF for24h had significantly increased (P<0.01). Pretreatment of hPDLCs with Runx2siRNA reduced BMP-2mRNA and protein expression by LIPUS stimulation compared with control group, and the difference wasstatistically significant (P <0.05).6.There was no significant difference between test group and control group on day0,day1, day3, day5, day7, day14, so it meant LIPUS stimulation had no effects on theproliferation of hPDLCs (P>0.05). At the same time, the higher temperatures did notsignificantly affect BMP-2mRNA and protein expression (P>0.05).Conclusion:In summary, LIPUS stimulation promoted speed of tooth movement in theorthodontic rat model by increasing HGF/Runx2/BMP-2signaling pathway and theexpression of RANKL. Furthermore, we identified LIPUS stimulation could enhancedBMP-2mRNA and protein expression via Runx2expression in vivo and in vitro. Overall,these results confirm that the LIPUS stimulation could promote alveolar bone remodeling.Because LIPUS stimulation is safe and non-invasive and it is likely to become a newtherapy to accelerate orthodontic tooth movement.