inﬂammation and tooth movement: the role of cytokines...

Inflammation and Tooth Movement: The Roleof Cytokines, Chemokines, and GrowthFactorsIldeu Andrade Jr, Silvana R. A. Taddei, and Paulo E. A. Souza

When an orthodontic force is applied, the periodontal tissues express ex-tensive macroscopic and microscopic changes, leading to alterations in 5microenvironments: extracellular matrix, cell membrane, cytoskeleton, nu-clear protein matrix, and genome. Capability of adaptive reaction to appliedmechanical loading relies in the DNA of periodontal ligament (PDL) andalveolar bone cells. However, an inflammatory process is a precondition forthese modifications to occur, which will lead to orthodontic tooth move-ment (OTM). PDL’s vascularity and blood flow changes, as well as mechan-ical alterations in the cytoskeleton of PDL and bone cells, will result in localsynthesis and release of various key mediators, such as chemokines, cyto-kines, and growth factors. These molecules will induce many cellular re-sponses by various cell types in the periodontium, providing a favorablemicroenvironment for bone resorption or deposition and, consequently, forOTM. In these inflammation and tissue remodeling sites, cells may alsocommunicate with one another through the interaction of cytokines and otherrelated molecules. The aim of this review is to bring focus on the role of theseimportant local inflammatory mediators that are closely related to the mecha-notransduction involved in OTM. (Semin Orthod 2012;18:257-269.) © 2012Elsevier Inc. All rights reserved.

M echanotransduction induced by orth-odontic force occurs when external strain

induces mechanosensing (the cell senses struc-tural changes in the extracellular matrix, causedby external mechanical loading), transduction,

and cellular response in several paradental tis-sues. This process leads to vasculature and extra-cellular matrix remodeling in the periodontalligament (PDL), gingiva, and alveolar bone.This remodeling is facilitated by proliferation,differentiation, and apoptosis of local periodon-tal cells, bone cell precursors, and leukocytemigration from the microvascular compart-ment.1,2 In this context, an aseptic acute inflam-matory response is occurring in the early phaseof orthodontic tooth movement (OTM), fol-lowed by an aseptic and transitory chronic in-flammation. As orthodontic forces (continuous,interrupted, or intermittent) are not uniformthroughout the applied region, areas of tensionor compression are developed leading to variedinflammatory processes resulting in different tis-sue remodeling responses.1,3

This sequence of events leads to an increasein the number of monocytes and polymorpho-nuclear leukocytes, which exit from the micro-

Associate Professor, Department of Orthodontics, Faculty of Den-tistry, Pontifícia Universidade Católica de Minas Gerais (PUCMinas), Belo Horizonte, Minas Gerais, Brazil; Immunopharmacol-ogy, Department of Biochemistry and Immunology, Instituto deCiências Biológicas, Universidade Federal de Minas Gerais, BeloHorizonte, Minas Gerais, Brazil; Associate Professor, Department ofOral Pathology, Faculty of Dentistry, Pontifícia UniversidadeCatólica de Minas Gerais (PUC Minas), Belo Horizonte, MinasGerais, Brazil.

Address correspondence to Ildeu Andrade Jr, DDS, MS, PhD,Department of Orthodontics, Faculty of Dentistry, PontificalCatholic University of Minas Gerais (PUC Minas), Av. Dom JoséGaspar 500, CEP 31.270-901, Belo Horizonte, Minas Gerais,Brazil. E-mail: [email protected]

© 2012 Elsevier Inc. All rights reserved.1073-8746/12/1804-0$30.00/0http://dx.doi.org/10.1053/j.sodo.2012.06.004

257Seminars in Orthodontics, Vol 18, No 4 (December), 2012: pp 257-269

mailto:[email protected]

http://dx.doi.org/10.1053/j.sodo.2012.06.004

vasculature into the extravascular space.1 Thesemigratory cells produce various inflammatorymediators, which interact directly or indirectlywith the entire population of native paradentalcells. These molecules, such as chemokines, cy-tokines, and growth factors (GFs), mediate andmaintain the vascular and cellular changes in anautocrine or paracrine way, stimulating or inhib-iting cellular activity.1 Such mediators are be-lieved to activate tissue remodeling, character-ized by selective bone resorption or depositionin compression and tension sites of the PDL,respectively.1,3

This article reviews the current biomedicalliterature on inflammation in OTM. It seeks tosummarize the role of chemokines, cytokines,and GFs and to explore their clinical implica-tions in routine orthodontic practice. It does notpropose a complete picture (as the research is asyet incomplete in the current scenario), but ori-ents the reader to the role that these mediatorsplay in the inflammatory periodontal tissue re-actions, in response to orthodontic force appli-cation.

Cytokines and Tooth Movement

Cytokines are extracellular signaling proteins di-rectly involved in the bone remodeling and in-flammatory process during OTM, which act di-rectly or indirectly, to facilitate bone and PDLcells differentiation, activation, and apoptosis.1,3

Investigations of their mechanisms of actionhave identified their effector (proinflammatory)and suppressive (anti-inflammatory) functionsduring OTM.

The receptor activator of nuclear factor-!Bligand (RANKL) and macrophage colony-stimu-lating factor (M-CSF) expressed by osteoblast andapoptotic osteocyte are the most important proin-flammatory cytokines responsible for recruitment,differentiation, activation, and survival of oste-oclasts.4 These cytokines bind to their respectivereceptors, RANK and c-Fms, expressed in oste-oclast precursors and mature osteoclasts, to pro-duce these events through osteoclast–osteoblastcommunication.2,4 By contrast, osteoblasts also ex-press osteoprotegerin (OPG), a decoy receptor ofRANKL, which inhibits the RANK/RANKL inter-action, preventing osteoclastogenesis and acceler-ating mature osteoclast apoptosis.1,4

When subjected to continuous (0.5-3.0 g/cm2)or intermittent (2.0 or 5.0 g/cm2) mechanical–compressive force, PDL cells induce osteoclasto-genesis in vitro through downregulation of OPGexpression and upregulation of RANKL expres-sion, via prostaglandin E2 (PGE2) and interleukin(IL)-1" synthesis.5,6 In accordance, mice researchalso demonstrated that osteoclastogenesis ap-pears to be primarily regulated through M-CSFand RANKL signaling by PDL cells in the com-pression side in the first week of orthodonticforce application.7,8 In the compression sitesduring human OTM (250 g), the same standardof RANKL and OPG expression is observed ingingival crevicular fluid (GCF) after 24 hours.9

By contrast, in vitro cyclic tensile strain increasesOPG levels and decreases RANKL synthesis incultured osteoblasts and PDL cells in a forcemagnitude-dependent way.10

Furthermore, RANKL gene transfer or M-CSFadministration to the periodontal tissue inducesosteoclastogenesis and accelerates experimentalOTM in rodents,11 whereas local OPG genetransfer has opposite results.12 Therefore, localMCSF/RANKL or OPG treatment might be auseful tool for shortening the duration of orth-odontic treatment or improving orthodontic an-chorage, respectively, but their clinical applica-tions are still far off. Currently, alveolardecortication has been the most frequently ap-plied clinical alternative to accelerate orthodontictreatment in humans.13 Animal studies haveshown that alveolar corticotomy increases the ex-pression of M-CSF and RANKL in PDL, enhancingthe rate of OTM during the initial tooth-displace-ment phase.14 In addition, a previous study re-ported that OPG levels were greater thanRANKL levels under physiological conditions inhuman GCF.15 However, an increased RANKL/OPG ratio was observed in patients with severeroot resorption under orthodontic treatmentthat could be related to a greater bone resorp-tion. Therefore, a local OPG treatment might bea future tool to prevent or paralyze root resorp-tion during OTM.

Tumor necrosis factor (TNF)-# is anotherproinflammatory cytokine that has been investi-gated in OTM and is involved in bone resorptionand acute as well as chronic inflammation.TNF-# is produced primarily by activated mono-cytes and macrophages, but also by osteoblasts,epithelial cells, and endothelial cells.16 In vitro

258 Andrade Jr, Taddei, and Souza

studies have demonstrated that in bone, TNF-#can directly and indirectly induce osteoclasto-genesis by binding to its p55 receptor on oste-oclast precursors and by upregulating expres-sion of RANKL, M-CSF, and other chemokineson osteoblasts.17-19 TNF-# is also an apoptoticfactor for osteocytes, which could be the signalfor osteoclast recruitment to resorb bone in thePDL pressure side, at the same time inhibitingosteoblasts.20

The real role of TNF-# in bone resorption,upregulating and increasing the amount ofOTM, was shown in rodent models with TNF-#receptor impairment.21,22 A recent in vitro studysuggested that PDL fibroblasts secrete higherlevels of TNF-# at the PDL compression sidethan at the tension side.23 This imbalance leadsto RANKL expression by activating CD4! Tcells, thereby facilitating bone resorption duringOTM. Taken together, local TNF-# treatment orstimulation of cells that produce this proinflam-matory protein might be a future alternative toaccelerate OTM. Moreover, local TNF-#-anti-body injection might be a useful tool to improvethe anchorage site during OTM, as a previousstudy has demonstrated its clinical applicationby inhibiting bone resorption in rheumatoid ar-thritis.24

Like TNF-#, IL-1 (alpha and beta) is a proin-flammatory cytokine that is highly expressed onthe PDL pressure side of humans and animalsand the adjacent alveolar bone in the earlystages of OTM.6,25-28 Its role in OTM has beenthe focus of previous human studies25,27,28 thatdemonstrated an increase in osteoclast activityand survival, while at the same time inducingbone marrow cells and osteoblasts to produceRANKL in the early phase of OTM.29,30 More-over, IL-1 can induce osteoclast formation di-rectly from osteoclast precursors under TNFstimulation in vitro.31

Other in vitro studies have also demonstratedthat IL-1 strongly promotes osteoclast formationby increasing M-CSF and PGE2 production anddecreased OPG production by osteoblasts.26,32

Under 24 hours of continuous compressiveforces in vitro (3.0 g/cm2), osteoblastic cellsrespond by expressing IL-1", IL-6, IL-11, TNF-#,and receptors for IL-1, IL-6, and IL-8, suggestingan osteoblastic autocrine mechanism induced bymechanical stress.33 Indeed, animal studies withabsence of IL-1" and/or TNF-# signaling dem-

onstrated impaired tooth movement,21,22 butthe mechanisms behind this finding remain un-known.

Polymorphisms in IL-1 gene, which deter-mines the degree of alteration in the amount ofcytokines secreted, have been studied in OTM.34

The allele 1 at the IL-1 gene, known to decreasethe production of IL-1 cytokine in vivo, signifi-cantly increases the risk of external apical rootresorption in patients during OTM. The clinicalimplication is that potential orthodontic pa-tients can be screened for the IL-1" genotype byanalyzing the DNA from a simple cheek swab ormouth wash taken during the initial examina-tion, to identify those who carry 2 copies of thehigh-risk allele. It would then be possible toinform patients about their predispositions be-fore starting treatment, and closely monitorthose at risk, by periodic radiographs. Moreover,a recent study has shown that IL-1" (!3954)genotype was associated with faster tooth move-ment in humans.35

In addition, IL-1 receptor antagonist (IL-1Ra)competitively blocks the interactions of IL-1 withIL-1 receptors, inhibiting its activity. IL-1Ra hasbeen used as a therapeutic tool in conditionsrelated to bone resorption, such as rheumatoidarthritis.36 Moreover, a decreased physiologicalIL-1Ra expression in GCF has been shown tocorrelate with faster OTM in humans.35 We spec-ulated that in the future, IL-1Ra can be clinicallyused to modulate the amount and side effects ofOTM.

Other cytokines, such as IL-6, IL-8, and IL-11,also stimulate alveolar bone resorption duringOTM by acting early in the inflammatory re-sponse.37-39 These cytokines can be enhancedby, or can act synergistically with, TNF-# andIL-1.40 By contrast, IL-11 can have anabolic ef-fects, alone or in association with bone morpho-genetic protein-2 (BMP-2), inducing osteoblasticdifferentiation in mouse mesenchymal cells.41

Different anti-inflammatory cytokines play in-hibitory effects, controlling inflammation andbone resorption. IL-18 and IL-10 are also ex-pressed in the PDL during OTM, and both inhibitosteoclastogenesis and bone resorption.42,43 Fur-thermore, IL-10 inhibits the production of IL-1,IL-6, and TNF-#, and its expression is higher inPDL tension than in compression sites.25,44

From a clinical standpoint, analysis of cyto-kine levels in GCF during OTM may, in the

259Inflammation and Tooth Movement

future, reveal the rate of OTM and determinethe optimum force level that should be appliedby orthodontic devices. Analysis of cytokine lev-els in GCF may also be helpful in monitoring thebiological activities in the periodontium duringthe retention period, which could provide infor-mation about possible relapse.

Chemokines and Tooth Movement

Chemokines belong to the superfamily of smallheparin-binding cytokines.45 The ability to in-duce cell migration is the common feature thatdistinguishes this group of cytokines.46 Structur-ally, the chemokines are classified in 4 subfami-lies based on the position of 2 highly conservedcysteine residues at the N-terminus: C, CC, CXC,and CX3C. To mediate their cellular effects,these molecules bind to selective 7-transmem-brane domain receptors, which are coupled toheterotrimeric G proteins, differentiating alsofrom other cytokines. The chemokine receptorsare named according to their ligand family, suchas CCR for receptors of CC ligands and CXCRfor CXC ligands.45 The chemokine system ispromiscuous or redundant, as different chemo-kines can bind to a given chemokine receptor,and a given chemokine may bind to differentchemokine receptors.45,46 However, binding ofchemokines to their respective receptors doesnot necessarily achieve the same functions invivo.46 Chemokines present different biologicaloutcomes in different tissues, which are con-trolled by geography and timing.45,46 They play acentral role in trafficking and homing of leuko-cytes, immune cells, and stromal cells, duringphysiological (homeostatic chemokines) and in-flammatory conditions (inflammatory chemo-kines).46 In addition, chemokines induce otherbiological processes, such as angiogenesis, cellproliferation, and apoptosis.45

In inflammatory sites, cellular recruitment be-gins when local cytokines, pathogens, GFs,chemokines themselves, and mechanical stresstrigger off the production of inflammatorychemokines by several cell types.45 These locallyproduced chemokines bind to the cell surface ofthe vascular endothelium and/or to the extra-cellular matrix, formatting gradients of chemo-kines, and, consequently, directing cell recruit-ment to inflammation sites.47 Therefore,recruited and activated macrophages, neutro-

phils, and lymphocytes will respond to the tissuefactors and secrete several mediators that will acttogether, leading to bone and PDL remodelingduring OTM.

In addition to leukocytes, the chemokinesprovide key signals for trafficking, differentia-tion, and activity of bone cells,17,48 playing animportant role in bone remodeling and boneinflammatory disease. In this context, some re-searchers have investigated how a given chemo-kine or chemokine receptor can regulate this pro-cess. Previous studies in vitro have demonstratedthat CC-chemokine ligand 3 (CCL3), CCL2,CCL5, and CXC-chemokine ligand (CXCL9)chemokines promote chemotaxis of osteoclastswhen binding to their respective CC receptors(CCR1, CCR2, CCR3, CCR5, and CXCR3), whichare expressed by osteoclast precursors.17,48,49,50

Others have shown that CCL5, CCL7, CCL2,CCL3, CXCL12, and IL-8 (CXCL8) promoteRANKL-induced differentiation of osteoclastprecursors.48,51-55 Chemokines also stimulate ac-tivity of osteoclasts, such as CCL2, CCL3, andIL-8,51,52,55 and prolong osteoclast survival, suchas CCL3 and CCL9 (ligands CCR1).48,51 More-over, RANKL induces osteoclast production ofCCL2, CCL3, and CCL5, which suggests an au-tocrine and paracrine signalization during oste-oclastogenesis, and an increase of bone resorp-tion.48,49

Chemokines can also induce recruitment,proliferation, and survival of osteoblasts. Osteo-blasts express chemokine receptors, such asCXCR1, CXCR3, CXCR4, CXCR5, CCR1, CCR3,CCR4, and CCR5.17 CCL5, a ligand of CCR1,CCR3, CCR5, and CCR4, can induce osteoblastrecruitment and avoid apoptosis of this cell.17

The chemokine CXCL10 induces osteoblast pro-liferation and release of alkaline phosphataseand "-acetylhexosaminidase, while CXCL12 andCXCL13 induce both proliferation and collagentype I mRNA expression in osteoblasts.56

The osteoblast– osteoclast communicationthrough RANK/RANKL/OPG system is essentialin the regulation of bone remodeling, as previ-ously described.4 This interaction between oste-oclast and osteoblast can also be mediated bychemokines through paracrine signalization. Ininflammation sites, this process is initiated whenthe proinflammatory cytokines IL-1 and TNF-#promote the production of CCL2, CCL3, andCCL5 by the osteoblast.46,48 The release of these


chemokines by osteoblasts can significantly con-tribute to recruitment and development of oste-oclasts at osteolysis sites, thereby exacerbatingbone loss.17,48 Furthermore, CCL3 is also indi-rectly involved with osteoclast differentiation, asthis chemokine stimulates increased expressionof RANKL by osteoblasts53 and induces cell–celladhesion between osteoclasts and osteoblasts.52

Because chemokines play an important rolein bone remodeling, several studies have inves-tigated the levels of these proteins in OTM.CCL2, CCL3, CCL5, IL-8 (CXCL8), and CXCL12expression were greatly increased in periodontaltissues of murines and humans submitted toorthodontic force.22,56-58 A recent study inWistar rats has demonstrated that IL-8 and CCL2may facilitate the process of root resorption after7 days of excessive mechanical loading (50 g).58

Although the expression of these chemokineshas been observed in periodontium submittedto orthodontic force, their functional role inOTM is yet to be known. The authors of thepresent article demonstrated that the axis TNF-#/p55 induces expression of CCL5 after 12hours of continuous force (10 g) in the PDL ofmice during OTM.22 As CCL5 is a ligand ofCCR5 receptor, we have investigated the roleof this chemokine receptor after the applica-tion of orthodontic force.56 The results demon-strated that the amount of tooth movement andthe number of osteoclasts were increased inCCR5-deficient mice (CCR5"/") after 12 days ofmechanical loading (10 g). The data also re-vealed that the mRNA levels of osteoclastogen-esis and osteoclast activity markers (Cathepsin K,RANKL, and matrix metalloproteinase-13(MMP-13)) were higher in CCR5"/" mice after 3 days.Moreover, osteoblast differentiation markers(runt-related transcription factor 2 [RUNX2]and osteocalcin [OCN]), and negative regula-tors of bone resorption (OPG and IL-10) weredecreased in CCR5"/" mice after 3 and 7 days.Taken together, these results suggested that di-minished osteoblast differentiation in CCR5"/"

led to a reduction of inhibitory signals for oste-oclasts, resulting in increased bone resorptionand greater OTM. Therefore, CCR5 might be adownregulator of bone resorption during OTM,as this receptor indirectly inhibits osteoclast re-cruitment and reduces this cell activity.56 Fur-ther studies are now required to confirm therole of other chemokines/chemokine receptors

involved in osteoclast and osteoblast recruit-ment, differentiation and activity during OTM,and their possible impact in clinical orthodon-tics.

In summary, the chemokines interfere di-rectly and indirectly in osteoclast and osteoblastrecruitment, differentiation, and consequentbone resorption and formation. Therefore, thedevelopment of strategies that are capable to lo-cally and selectively modulate the chemokine path-ways might contribute to a better control of therate of OTM, as well as the stabilization of theorthodontic results. Drugs such as met-RANTESand P8A are currently used for blockage of specificchemokine receptors, leading to a diminishedbone resorption in rheumatoid arthritis.59,60 Inthe future, the local administration of these pres-ent and upcoming drugs could be useful to in-crease biological anchorage at specific sites andstability of the final results.

Growth Factors and Tooth Movement

GF are substances that bind to specific receptorson the surface of their target cells, stimulatingcell proliferation, migration, and differentia-tion. Moreover, they display important roles inhematopoiesis, the inflammatory process, angio-genesis, and tissue healing.61 GF may also actlocally to modulate bone remodeling, and con-sequently, OTM.27,61

Vascular endothelial growth factor (VEGF) isan essential mediator of angiogenesis and in-creased vascular permeability.61 As osteoblastand osteoclast express VEGF receptor-1, somestudies have investigated the effect of VEGFon bone remodeling under mechanical load-ing.62,63 In vitro studies have shown that PDLcells and apoptotic osteocytes increase VEGFproduction after compressive force applica-tion.62,63 VEGF can modulate the recruitment,differentiation, and activation of osteoclast pre-cursors, increasing bone resorption.64 More-over, VEGF can also indirectly induce bone re-sorption, as it promotes angiogenesis in vitro,allowing new capillaries to assist the recruitmentof osteoclast precursors to the bone surface closeto the resorption site.63 In accordance, VEGFenhances osteoclast recruitment and increasesthe rate of experimental OTM in vivo,65 whichare both inhibited by anti-VEGF polyclonal anti-body treatment.66 The expression of VEGF was


also detected in osteoblasts, osteocytes, and fi-broblasts in PDL tension sites after 10 days ofOTM in C57BL/6J mice.65 Taken together, it isreasonable to conclude that VEGF plays an im-portant role in bone remodeling at both com-pression and tension sites of the PDL duringOTM.

The transforming growth factor (TGF)-" su-perfamily (TGF-"1 to -"3) is another importantGF related to bone and PDL tissue remodelingduring OTM. Under mechanical loading, thecyclic tensile force upregulates TGF-" expres-sion in osteoblasts and also in PDL cells invitro.67 Furthermore, TGF-" stimulates OPGproduction and downregulates IL-6 expression,which inhibits the osteoclastogenesis-supportingactivity of these cells.67 In accordance, increasedlevels of OPG and TGF-"1 mRNA are observedin osteoblasts and other PDL cells in the tensionsites after 2 days of OTM in Wistar rats, simulta-neously with a decreased number of osteoclastsin this area.68 In humans, the level of TGF-"1 isenhanced in GCF and in PDL tissue a few daysafter continuous orthodontic force applicationfor rapid maxillary expansion and canine distalmovement (force of 2-2.5 N), respectively.27

However, there are no differences in the level ofthis GF between tension and pressure sites.25 Asit is clear that TGF-" plays an important role inthe tension site during OTM, further studiesshould now investigate the TGF-" effect on pres-sure sites after orthodontic force application be-cause it is an essential factor for RANKL-inducedosteoclastogenesis and, consequently, OTM.

Bone morphogenetic proteins (BMPs) are mul-tifunctional GFs that belong to the TGF-" super-family and play an important role in upregulatingvarious transcription factors involved in osteoblas-tic differentiation, and consequently, in bone for-mation.69 To date, more than 20 BMPs have beendiscovered, but BMP-2, BMP-6, BMP-7, and BMP-9seem to have the most potent osteogenic activ-ity.69,70 Studies have shown that under tensilestrain, human PDL cells in culture increaseBMP-2 and BMP-6 expression, suggesting thatthese BMPs might play an important role in PDLtensile sites during OTM.70,71 However, there isa lack of information on the actual role of BMPsin OTM.

Insulin-like growth factors (IGFs) are in-volved in bone formation by inducing prolifera-tion, differentiation, and apoptosis of osteo-

blasts.72 The IGFs effect is regulated by growthhormone, parathyroid hormone, vitamin D3,corticosteroids, TGF-", IL-1, and platelet-de-rived growth factor. Studies have shown thatunder continuous tensile mechanical loading,rat tibiae osteocytes and calvaria osteoblasts in-crease IGF-I synthesis, which stimulates bone for-mation.73,74 In PDL tissues, IGF also acts as an-tiapoptotic and proliferative factor for fibroblastsand osteoblasts in vitro.75 Accordingly, an in vivostudy using Wistar rats demonstrated that 4hours of a continuous tensile force (0.1-0.5 N)applied to a tooth induces increased expressionof IGF-I and IGF-I receptor in PDL cells in ten-sion sites, but a decreased expression in com-pression sites.76 Therefore, a local increase ofIGF-I appears to provide a link between the me-chanical loading and tissue remodeling in thetensile site during OTM.

Fibroblast growth factors (FGFs) belong to afamily of 23 members that bind to 4 structurallyrelated high-affinity receptors.77 Among FGFs,FGF-2 can regulate bone remodeling by stimu-lating osteoblast-like cell proliferation and differ-entiation in vitro, and by increasing osteoclastformation and activity.78 An in vitro study dem-onstrated that compressive forces induce produc-tion of FGF-2 by human PDL cells, which stimu-lates RANKL expression.79 Moreover, there is anincreased expression of this GF in the compressionsite of PDL tissues after 1 day of mechanical load-ing in humans in vivo.39 The results suggest thatFGF-2 can be involved in bone resorption duringOTM.39,79 However, whether FGF-2 plays an im-portant role in the tensile site after mechanicalloading is yet to be elucidated. As FGF-6, FGF-8,FGF-9, FGF-18, and FGF-23 are also known asregulators of bone cell functions, they should betested following application of the orthodonticforce.39,77-79

A further GF showing increased levels in GCFafter the first 24 hours of OTM in humans is theepidermal growth factor (EGF).27 A recent studyhas shown that the administration of EGF–lipo-some in the mucosa adjacent to the tooth inHoltzman rats was able to stimulate the expres-sion of RANKL, leading to an increased numberof osteoclasts in the PDL compression sites, andconsequently, to enhancement of tooth move-ment under a continuous force (20 g).80 In ad-dition, the expression of both EGF and EGFR inosteoblasts and PDL fibroblasts is increased at


PDL tensile sites in rats and cats.80,81 Takentogether, these data suggest that EGF is involvedin remodeling of mineralized and nonmineral-ized extracellular matrix in both tensile andcompressive sites during OTM.

Taken together, the current knowledge in-dicates that mechanical loading stimulates lo-cal expression of many GF involved in boneand PDL remodeling in the early stages ofOTM in both tensile and compressive sites. Inthe future, local injection of a single GF or a

combination of multiple GFs in the periodon-tal tissues might modulate the rate of OTM orhelp to improve the stability of the orthodon-tic results.

Inflammation in OTM

When an orthodontic force is applied to atooth, immediate changes are observed inperiodontal tissues (Fig 1). Compression sites

Figure 1. In compression sites, the orthodontic force induces local hypoxia and mechanotransduction. Thelocal hypoxia increases the expression of interleukin (IL)-1", IL-6, IL-8, tumor necrosis factor (TNF)-#, andvascular endothelial growth factor (VEGF) in periodontal ligament (PDL) fibroblasts. The physical strain alsostimulates the PDL cells to produce growth factors, prostaglandin E2 (PGE2), and chemokines (mechanotrans-duction). Moreover, stressed PDL peripheral nerve fibers release vasoactive neurotransmitters, such as calcitoningene–related peptide and substance P. The cytokines IL-1", IL-6, IL-8, and TNF-# lead to increased expressionof adhesion molecules (VCAM-1 and ICAM-1) and chemokines, which in turn promote leukocyte adhesion andmigration. Furthermore, PGE2 and VEGF increase vascular flow and permeability, leading to plasma extravasa-tion and leukocyte diapedesis. These alterations trigger an acute inflammatory response, which is replaced by achronic process that allows leukocytes and osteoclast precursors to continue their migration into the strainedparadental tissues, modulating this remodeling process. (Color version of figure is available online.)


are characterized by tissue and cell damage,reduction in the number of patent capillaries,occlusion, and partial disintegration of bloodvessels, leading to ischemia and hypoxia.1

These alterations trigger an acute inflamma-tory response, featured by vasodilatation andmigration of leukocytes out of capillaries.1

This process is initiated when local hypoxiaincreases the expression of IL-1", IL-6, IL-8,TNF-#, and VEGF in PDL fibroblasts.82 At thesame time, the physical strain stimulates thePDL production of these cytokines, as well asof GFs and chemokines, through a processcalled mechanotransduction.2 In this context,IL-1 and TNF-# activate microvascular endo-thelial cells, leading to increased expression ofadhesion molecules (VCAM-1 and ICAM-1),inducing local chemokine expression, whichin turn promotes leukocyte adhesion and mi-gration.83 Moreover, stressed PDL peripheralnerve fibers release vasoactive neurotransmit-ters, such as calcitonin gene–related peptideand substance P.1 These neuropeptides, to-gether with VEGF and PGE2, increase vascularflow and permeability, leading to plasma ex-travasation and leukocyte diapedesis.61 Theserecruited leukocytes interact directly or indi-rectly with the entire population of native pa-radental cells, increasing the production ofspecific chemokines, cytokines, and GFs in-volved in bone resorption. In this way, theacute phase of inflammation is replaced by achronic process that allows leukocytes and os-teoclast precursors to continue their migra-tion into the strained paradental tissues, mod-ulating this remodeling process.1

Bone Resorption

Under mechanical loading, fibroblasts, osteo-blasts, and other PDL cells, located at the pres-sure site (Fig 2), release signaling molecules,such as PGE2, IL-1, IL-6, TNF-#, and IL-11.25,26

Among these mediators, IL-1 and TNF-# stimu-late osteoblasts to produce chemokines, such asCCL3, CCL2, and CCL5.17,26,48,56,57,83 Thesechemotactic proteins, joined by others, such asCXCL12, and cytokines (RANKL and TNF-#)induce chemotactic recruitment of osteoclastprecursors to osteolysis sites, where these cells dif-ferentiate into mature osteoclasts through osteo-blast–osteoclast communications.17,48,49,54 For the

differentiation to occur, PGE2 and cytokines, suchas IL-1, IL-6, IL-8, and TNF-#, stimulate, directly orindirectly, osteoblast/stromal cells to produce themain regulators of osteoclast differentiation: M-CSF and RANKL.3-6 This process is achievedwhen M-CSF and RANKL bind to their respec-tive specific receptors, c-Fms and RANK, whichare both expressed on osteoclast precursors.However, osteoclastogenesis can be downregu-lated when OPG, a RANKL decoy receptor pro-duced by osteoblastic and PDL cells, binds toRANKL, inhibiting the RANK/RANKL interac-tion.4 Therefore, OPG level is decreased in com-pression sites during OTM, enhancing osteoclas-togenesis in this area.25

The mechanical loading-induced hypoxiapromotes the expression of hypoxia induciblefactor 1-#, which increases the expression ofRANKL by human PDL fibroblasts at pressuresites, enhancing osteoclastogenesis.84 In addi-tion to osteoblast and PDL cells, damaged osteo-cytes are also a source of RANKL and M-CSF. Inthis context, orthodontic force causes mi-crodamages in alveolar bone near PDL pressuresites that compromise osteocyte integrity, physi-cally damaging the cells by oxidative stress, or bydisruption of the blood flow and/or fluid flow inthe lacunar–canalicular system.63 This damagedtissue, together with local TNF-# and IL-1, caninduce osteocyte apoptosis, which initiates boneresorption close to the microdamaged sites, as itupregulates the expression of RANKL, VEGF,and M-CSF and, consequently, modulates theosteoclast precursor recruitment and differenti-ation.63 Moreover, VEGF also indirectly inducesbone resorption, as it promotes angiogenesis,allowing new capillaries to augment osteoclastprecursors recruitment to the bone surface closeto the resorption sites.63

Not only RANKL, but also other cytokines(IL-", TNF-#, IL-6, IL-11), GFs (FGF-2, EGF),and chemokines (CCL2, CCL3, CCL5, CCL7,CCL9, IL-8) can, directly or indirectly, increaseosteoclast differentiation, survival, and activ-ity.17,31,48,78,80 Moreover, in an amplified loop,CCL3 increases the expression of RANKL byosteoblasts.53 In parallel, RANKL induces pro-duction of CCL2, CCL3, and CCL5 by oste-oclasts, suggesting an autocrine and paracrinesignalization during osteoclastogenesis, and anincreased bone resorption.48,49


Bone Formation

In orthodontics, bone formation (Fig 3) begins40-48 hours after force application in the PDLtension sites.85 Osteocytes participate in the pro-cess of osteogenesis, being acutely sensitive andresponsive to applied tensile orthodontic forces.Their cellular projections facilitate communica-tions with neighboring osteocytes, as well as withalveolar bone surface-lining cells and bone mar-row cavity cells. Osteoblasts, which maintain di-rect contact with osteocytes, respond to thesesignals and initiate bone apposition.86

Moreover, stretched PDL fiber bundles stim-ulate cell replication.1 Stem-cells (pericytes), mi-grated from blood vessel walls, and mesenchy-

mal stem-cells differentiate into preosteoblasticcells 10 hours after force application.86 Chemo-kines, cytokines, and GFs are directly involved inthis process. CCL3, CCL5, CXCL10, CXCL12,and CXCL13 induce osteoblast precursor re-cruitment, proliferation, differentiation, andsurvival.17,53,57,86 Local osteoblasts and osteo-cytes express GFs, such as TGF-" and IGF-1, thatpromote osteoblast precursors proliferation anddifferentiation, and mineralization of new boneby mature osteoblasts.73,74,85 In addition, BMP,EGF, and IL-11 upregulate osteoblast differenti-ation and function.41,69,81 In PDL tension sites,osteoblasts and PDL fibroblasts express VEGF,which stimulates angiogenesis, an important

Figure 2. In compression site, fibroblasts, osteoblasts, and other PDL cells release PGE2, IL-1, IL-6, TNF-#, andIL-11, under mechanical loading. Among these mediators, IL-1 and TNF-# stimulate osteoblasts to producechemokines, such as CCL3, CCL2, and CCL5. Together with CXCL12, as well as the cytokines receptor activatorfor nuclear factor-!B ligand (RANKL) and TNF-#, they induce chemotactic recruitment of osteoclast precursorsto osteolysis sites, where these cells differentiate into mature osteoclasts through osteoblast–osteoclast commu-nications. PGE2 and cytokines (IL-1, IL-6, IL-8, and TNF-#) stimulate osteoblast/stromal cells to producemacrophage colony-stimulating factor and RANKL, which bind to their receptors c-Fms and RANK expressed onosteoclast precursors, respectively. Damaged osteocytes are also a source of RANKL and macrophage colony-stimulating factor. Other cytokines (IL-", TNF-#, IL-6, IL-11), growth factors (fibroblast growth factor-2,epidermal growth factor), and chemokines (CCL2, CCL3, CCL5, CCL7, CCL9, IL-8) can increase osteoclastdifferentiation, survival, and activity. However, osteoclastogenesis can be downregulated when osteoprotegerinbinds to RANKL, inhibiting the RANK/RANKL interaction. (Color version of figure is available online.)


process in new bone formation.61,65 Moreover,anti-inflammatory cytokines involved in boneformation, such as IL-10 and OPG, are producedby osteoblasts and inhibit osteoclastogenesis.4,44

To maintain the integrity of the PDL apparatussimultaneously with bone formation, TGF-" andIGF-1 stimulate proliferation and differentiationof osteoblasts and PDL cells, as well as collagensynthesis.68,72,75

Conclusions

Chemokines, cytokines, and GFs are the mainmolecules involved in bone cell recruitment, ac-

tivation, proliferation, differentiation, and sur-vival. These molecules stimulate PDL and bonecells to orchestrate an inflammatory response,followed by osteoclastogenesis and bone resorp-tion in compression sites, and by osteoblast andnew bone formation at PDL tension sites. Theresearch trend is now directed toward elucidat-ing the molecular mechanisms involved in theseevents. Although several studies have investi-gated chemokines, cytokines, and GFs in OTM,it is difficult to establish a global vision thatillustrates the specific role and the exact mo-ment that these molecules participate in PDL

Figure 3. Osteoblast differentiation and bone formation occur in the tension site during orthodontic toothmovement. Stretched PDL cells stimulate cell replication by releasing chemokines, cytokines, and growth factors.Stem-cells (pericytes), migrated from blood vessel walls, and mesenchymal stem-cells differentiate into preos-teoblastic cells after force application. The chemokines, CCL3, CCL5, CXCL10, CXCL12, and CXCL13, induceosteoblast precursors recruitment, proliferation, differentiation, and survival. Local osteoblasts and osteocytesexpress growth factors (transforming growth factor-" and insulin-like growth factor-1) that promote proliferationand differentiation of osteoblast precursors, as well as mineralization of new bone by mature osteoblasts. Inaddition, bone morphogenetic protein, epidermal growth factor, and IL-11 upregulate osteoblast differentiationand function. Osteoblasts and PDL fibroblasts express VEGF, which stimulates angiogenesis. Meanwhile, trans-forming growth factor-" and insulin-like growth factor-1 stimulate proliferation and differentiation of osteoblastsand PDL cells, as well as collagen synthesis. (Color version of figure is available online.)


and bone remodeling. Different experimentalconditions, such as animal species, time andforce magnitude, appliance design, and meth-ods to detect the expression of these molecules,restrain the general comprehension of the cel-lular and molecular mechanisms involved inOTM. Nevertheless, current knowledge raisesthe possibility of local administration of sub-stances that are able to act on specific cytokines,chemokines, and GF. These molecules can mod-ulate the outcome of the application of orth-odontic force, accelerating OTM, enhancing bi-ological anchorage at specific sites, possiblydecreasing the rebound effect, and assisting inthe prevention of root resorption.

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