Bone Remodeling In Compressive Area During Orthodontic Tooth Movement Through EphrinB2/EphB4Pathway

The slow rate of alveolar bone remodeling is a challenge in adult orthodontictreatment, which involves in two aspects: one is the slow velocity of tooth movementresulting in the long duration of treatment, and the other is the relapse of toothposition after orthodontic treatment. To date, the solution is limited to the clinicalmethods, such as compromised treatment projects which could shorten the duration byminimising the distance of tooth movement, and long-term or lifetime retention toavoid relapse. All the above methods, however, cannot effectively solve the problemby regulating bone remodeling.Bone remodeling is a continual process which is necessary for skeletal growthand replaces damaged and aged bone[1]. The process of bone remodeling takes placein basic multicellular units(BMU) throughout the skeleton. It is traditionally thoughtof as a cycle, comprised of activation, resorption, reversal and formation phases, andis widely accepted as the way that the skeleton repairs itself. However, the precisemechanisms controlling the BMU are not well-understood. Osteoblasts andosteoclasts are the cytological basis of bone remodeling. Of paramount importance forbone remodeling is the RANK/RANKL/OPG signaling pathway[2,3]. Osteoblasts couldinduce osteoclast differentiation via RANKL/RANK pathway, leading to boneresorption. During the reversal phase, osteoclastic bone resorption is inhibited andosteoclasts undergo apoptosis. Osteoblasts are recruited to the site, leading to theformation phase.The coupling of bone resorption and bone formation is critical during the normalprocess of bone remodeling, and the dysregulation of this coupling results in thedevelopment of a range of pathological bone diseases. There is considerable evidenceto support the coupling of bone formation to bone resorption, however themechanisms are unclear. Several studies have implicated some factors, includingIGF-I and II, TGF-β, S1P and HGF involved in the coupling mechanism[4-7]. Morerecently, a new concept for the coupling has been proposed, involving bidirectional signaling between EphB4receptor on osteoblasts and ephrinB2on osteoclasts[8]. Forthe unique properties of the coupling process, that is,(i) the localized nature ofcoupling, which starts with resorption and is followed by bone formation, occurringonly at sites of prior resorption, and (ii) the cessation of bone resorption uponcommencement of bone formation. These suggest both local mechanism, and thenecessity for signaling to both osteoblasts to stimulate formation and to osteoclasts toinhibit formation, for which bidirectional signaling between osteoblasts andosteoclasts provides a novel and intriguing potential explanation.EphrinB2/EphB4signaling pathway on osteoclasts and osteoblasts plays animportant role in bone remodeling[8-11]. EphrinB2is the preferred ligand for theEphB4receptor. Interaction between ephrinB-and EphB-expressing cells results inbidirectional signal transduction. It is found that differentiating osteoclasts induceephrinB2expression and that the reverse signaling through ephrinB2suppressesosteoclast differentiation. Moreover, osteoblasts express EphB4receptor, and theforward signaling through EphB4into osteoblasts enhances osteoblast differentiation.The bidirectional activation of the ephrinB2/EphB4signaling pathway on osteoclastsand osteoblasts leads to the suppression of osteoclast differentiation with a concurrentstimulation of osteoblast differentiation and consequential bone formation. Thecommunication between ephrinB2and EphB4has recently been shown to be involvedin the stimulation of osteoblast differentiation within the osteoblast lineage[12,13].Alveolar bone remodeling is the biological basis of orthodontic toothmovement[14,15]. Orthodontic force causes the resorption and formation of alvelar bone.On the compressed side of an orthodontically moving tooth, bone resorption takesplace in response to the mechanical compressive force and hypoxia induced by thedecreased blood flow, resulting in tooth movement. Osteoblasts and osteoclasts aresensitive environment-to-genome-to-environment communicators, capable ofrestoring system homeostasis disturbed by orthodontic mechanics[16]. Under the loadof orthodontic forces, many molecular reactions occur in and around these cells.However, the mechanisms of these reactions have not been fully understood. Recently,it is found that what happened in compressive area is not only bone resorption, but theprocess of bone remodeling[17,18]. The profounder understanding of the underlyingmechanism will provide valuable information for the development of agentsregulating the activities of osteoblasts and osteoclasts to better control the bone remodeling in compressive area which is the rate-limiting step in orthodontic toothmovement[17].Mechanical loading and hypoxia are two important factors in regulating boneremodeling. Appropriate mechanical loading would promote bone remodeling[19].Osteoclasts and osteoblasts are both oxygen-sensitive cells. Hypoxia would facilitatethe formation of osteoclasts and bone resorption[20].Based on studies mentioned above, we presented a scientific hypothesis thatcompressive force and hypoxia could regulate EphB4and ephrinB2expression inosteoblasts and osteoclasts, which might contribute to alveolar bone remodeling in thecompressive area during orthodontic tooth movement. In our study, we would focuson ephrinB2/EphB4pathway and investigate the mechanism in bone remodeling ofcompressive area in two ways: mechanical compressive force and hypoxia stimuli.The content of this research is divided into three parts:Experiment1: the expression of ephrinB2in compressive area ofperiodontal tissues during experimental tooth movement.Wistar rats were used as research subjects. A rat model of experimental toothmovement was conducted to examine the histologic changes and the expression ofephrinB2in compressive area of periodontal tissues during experimental toothmovement by means of histologic and immunohistochemical staining.Experiment2: the effects of compressive force on osteoblast proliferationand differentiation, as well as EphB4and ephrinB2expression in osteoblasts, andthe effects on osteoclast differentiation and ephrinB2expression in osteoclasts.RAW264.7cells stimulated with RANKL and ST2cells, used as precursor cellsof osteoclast and osteoblast respectively, were subjected to cyclic uniaxialcompressive force by the four-point bending system for1h,2hs and4hoursrespectively in vitro. The effects of compressive force on osteoblast proliferation wereexamined by MTT colorimetry. EphB4and ephrinB2gene expression, as well asosteogenic genes including Runx2and Sp7were examined in ST2cells byquantitative real-time PCR (qPCR). EphrinB2gene expression, as well asosteoclastogenic genes including NFATc1and CTR were examined in RAW264.7cells. Experiment3: the effects of hypoxia stimuli on osteoblast proliferation anddifferentiation, as well as EphB4and ephrinB2expression in osteoblasts, and theeffects on osteoclast differentiation and ephrinB2expression in osteoclasts.RAW264.7cells stimulated with RANKL and ST2cells, used as precursor cellsof osteoclast and osteoblast respectively, were subjected to hypoxia stimuli producedby Anaeropack system for1h,2hs and4hours respectively in vitro. The effects ofhypoxia stimuli on osteoblast proliferation were examined by MTT colorimetry.EphB4and ephrinB2gene expression, as well as osteogenic genes including Runx2and Col1were examined in ST2cells by qPCR. EphrinB2gene expression, as well asosteoclastogenic genes including NFATc1and Mmp9were examined in RAW264.7cells.Through the above3experiments, the results were as follows:Experiment1Histologic examination in experimental rats showed giant and multinucleatedosteoclasts and resorption lacunae on the bone surface in the compressive area ofalveolar bone. Immunohistochemistry revealed that ephrinB2was strongly andpositively expressed in osteoclasts in these areas.Experiment2MTT colorimetry revealed that the proliferation was decreased significantly incompressed ST2cells. QPCR showed that the mRNA levels of NFATc1, CTR andephrinB2were increased significantly in compressed RAW264.7cells, and theexpression of ephrinB2, EphB4, Sp7and Runx2was decreased significantly incompressed ST2cells.Experiment3MTT colorimetry revealed that the proliferation was decreased significantly inST2cells subjected to hypoxia stimuli. QPCR showed that the expression of NFATc1,Mmp9and ephrinB2was increased significantly in RAW264.7cells, and theexpression of ephrinB2, EphB4, Runx2and Col1was decreased significantly in ST2cells subjected to hypoxia stimuli.According to the results above, we can draw the following conclusions:1. There are many giant and multinucleated osteoclasts, and significantosteoclastic resorption in the compressive area during orthodontic tooth movement.EphrinB2was strongly and positively expressed in osteoclasts, which mightcontribute to alveolar bone remodeling in the compressive area during orthodontictooth movement. 2. Cyclic compressive force could directly stimulate osteoclast differentiation,and inhibit the proliferation and differentiation in osteoblasts. Futhermore, mechanicalcompressive force could regulate EphB4and ephrinB2expression in osteoblasts andosteoclasts. The applied mechanical loads could be converted to the biologicalresponse through ephrinB2/EphB4pathway in osteoblasts and osteoclasts, whichcould play a role in bone remodeling.3. Hypoxia stimuli could directly promote osteoclast differentiation, and inhibitthe proliferation and differentiation in osteoblasts. Futhermore, hypoxia stimuli couldregulate EphB4and ephrinB2expression in osteoblasts and osteoclasts. It could beephrinB2/EphB4pathway in osteoblasts and osteoclasts that linked hypoxia stimuli tothe biological response, which could contribute to bone remodeling in compressivearea during orthodontic tooth movement.4. The profounder understanding of the underlying mechanism will providevaluable information for the development of agents regulating the activities ofosteoblasts and osteoclasts to better control the bone remodeling in compressive areaduring orthodontic tooth movement, which could be of paramount significance.