REVIEWThe role of vascular endothelial growthfactor inossificationYan-Qi Yang1, Ying-Ying Tan1,2, Ricky Wong1, Alex Wenden1, Lin-Kun Zhang1,3and A Bakr M Rabie4Osteogenesis and angiogenesis are two closely correlated processes during bone growth, development, remodelling and repair.Vascular endothelial growth factor (VEGF) is an essential mediator during the process of angiogenesis. Based on an extensive literaturesearch, which was carried out using the PubMed database and the keywords of osteogenesis, VEGF, endochondral ossification andintramembranous ossification, this manuscript reviews the role of VEGFin ossification, with emphasis onits effect inendochondral andintramembranous ossification. Osteogenesis and angiogenesis are closely correlated processes. VEGF acts as an essential mediatorduring these processes. It not only functions in bone angiogenesis but also in various aspects of bone development.International Journal of Oral Science (2012) 4, 6468; doi:10.1038/ijos.2012.33; published online 22 June 2012Keywords: endochondral ossification; intramembranous ossification; osteogenesis; vascular endothelial growth factorINTRODUCTIONBoneformationcanoccurthroughtwodistinct pathways, namely,endochondral ossification and intramembranous ossification.12Duringdevelopmentofsomeskullandfacialbonesorduringbonerepair,ifbonesegmentsarestabilized, mesenchymalprecursorcellsdifferentiate directly into bone-forming osteoblasts in a process calledintramembranousossification.Thisprocessof ossificationoccursinmany locations of the skeleton and includes formation of flat bones ofthe cranium, facial bones and addition to shafts of many other bones.Duringthisprocess,osteoblastslaydownbonematrixformingspi-cules. Initially, these spiculae lay down trabecular bone, which is even-tuallyreplacedwithlamellar.1Alternatively, duringdevelopmentoflong bones and vertebrae or bone repair in a biomechanically unstableenvironment, bone formation occurs via a cartilage intermediate in aprocess called endochondral ossification.3During endochondral ossi-fication, mesenchymal cells differentiatetochondrocytes. Vascularinvasionandchondrocytesproliferateinacoordinatedprocessthatlengthens the bone.2Osteogenesis and angiogenesis are two closely correlated processesduring bone growth, development, remodelling and repair.4Vascularendothelial growth factor (VEGF) is an essential mediator during theprocess of angiogenesis. Disruptionof asingleVEGFallelecausesembryonic lethality because of defective vasculogenesis, angiogenesisandlargevessel formation.56Postnatal ablationof VEGFleadstoabnormal organs development because of the reduced vascularisationand angiogenesis.7Except these effects on angiogenesis, VEGF worksin both processes of endochondral ossification and intramembranousossification812andactsasanessential mediatorduringthesepro-cesses. It is involved not only in bone angiogenesis, but also in variousaspects of bone development, including chondrocyte differentiation,osteoblast differentiation and osteoclast recruitment. This articlefocuses predominately on the role of VEGF in the above two ossifica-tion processes.MATERIALS AND METHODSA literature search was carried out using PubMed and the keywordsosteogenesis; VEGF; endochondral ossification; andintramembra-nousossification.ArticlesthatonlydescribedtheeffectofVEGFonangiogenesis were excluded. Atotal of 44 references were finallyincluded in this manuscript.VEGF AND ITS RECEPTORSVEGFisaspecificmitogenforvascularendothelialcells.Itwasfirstidentified as an endothelial-specific growth factor from bovine pitui-taryfollicularcellsbyFerraraandDavisSymth.13TheVEGFfamilyconsists of seven members, namely, placenta growth factor, VEGF-A,-B, -C, -D, -E and -F.1314They all share a common structure of eightcharacteristically spaced cysteine residues in a VEGF homologydomain.VEGF-Aisthemostabundantformandisthereforecom-monlyused instudiesinvestigatingthebiologicaleffectsof VEGF.15Thus,VEGFiscommonlyreferredtoasVEGF-A.16Usingthiscon-vention, the term VEGF mentioned in this review refers to VEGF-A.In the osteogenesisangiogenesiscoupling, hypoxia-induciblefac-torisoneofthekeyupstreamregulatorsofVEGF.Upregulationofhypoxia-induciblefactorcan be induced not onlyby thedecrease ofoxygentension,1718butalsobyotherstimulus,suchasinsulin-likegrowth factor-1.1920Recent study indicated that changes of the levelofhypoxia-induciblefactorcanalterthelevelofVEGFsignificantlyand change the bone mass dramatically.21Thebiological effectsof VEGFaremediatedbyspecifictyrosinekinasereceptors(VEGFRs), i.e., Vascularendothelial growthfactorreceptor-1(VEGFR-1/Flt-1)andVascularendothelialgrowthfactorreceptor-2 (VEGFR-2/KDR). Both Flt-1 and KDR can be phosphory-latedontyrosineresidues, but showdifferent patternsof potentialintracellularsubstratesintheinvitroimmunecomplexkinaseassayandtheyalsomediatedifferentcellularresponses.KDR,ratherthanFlt-1, was found to be the major mediator of essential functions such as1Department of Orthodontics, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China;2Unit Pakar Ortodontik, Klinik Pergigian, Poliklinik Komuniti Mak Mandin,Malaysia;3Department of Orthodontics, Tianjin Stomatological Hospital of Nankai University, Tianjin, China and4Private PracticeCorrespondence: Dr YQ Yang, Department of Orthodontics, Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR, ChinaE-mail: yangyanq@hku.hkReceived 20 February 2012; accepted 26 April 2012International Journal of Oral Science (2012) 4, 64682012 WCSS. All rights reserved 1674-2818/12www.nature.com/ijoschemotaxis, mitogenesis and cytoskeletal reorganisations, whereas thefunctional significanceof theFlt-1remains tobedetermined.2223Inaddition, neuropilin-1andneuropilin-2arereceptorsforsema-phorins, but they alsohave beenshowntoserve as co-receptorsfor VEGF.24EFFECTS OF VEGF ON BONE CELLSBoneisadynamictissueinwhichboneresorptionandformation(bone remodelling) occur in a regulated manner under the influenceof systemichormones andlocal factors. Osteoclasts, themultinu-cleated giant cells that resorb bone, develop from haematopoietic cellsof the monocyte/macrophage lineage. The osteoblast is a type ofmononucleatecell,arisingfromosteoprogenitorcellslocatedintheperiosteum and the bone marrow that is responsible for bone forma-tion. Besides producing osteoid, which is composed mainly of type Icollagen, osteoblasts arealsoresponsiblefor mineralisationof theosteoidmatrix. Furthermore, osteoblastsandbonemarrowstromalcells support osteoclast development via the mechanism of cell-to-cellinteraction with osteoclast progenitors.EFFECTS OF VEGF ON OSTEOBLASTSVEGFhasbeenimplicatedinvariousaspectsofosteoblastfunction.Twostudies have shown adose-dependentchemoattractiveeffectofVEGFonprimary humanosteoblasts25andhumanmesenchymalprogenitor cells.26Inadditiontoits effect oncell migration, VEGFstimulates cellproliferationbyupto70%.26ItwasfoundthatVEGFdirectlypro-motes differentiation of primary human osteoblasts in vitro by increas-ingnoduleformationandalkalinephosphataseactivityinadose-dependent manner.27Alsoreportedisthat VEGFwasexpressedatlowlevelsatthebeginningofosteoblastdifferentiationandthatitsexpression was strongly increased only during terminal differentiationandreachedmaximumexpressionduringtheperiodofmineralisa-tion.28Thus,VEGFplaysanessentialroleintheregulationofboneremodelling by stimulating osteoblast differentiation.Mayr-Wohlfart et al. also revealed increased expression of Flt-1 andKDR on human osteoblasts.25An in vitro kinase assay failed to dem-onstrateactivationof KDRuponstimulationwithVEGF, whichisconsistentwiththeideathattheeffectofVEGFonprimaryhumanosteoblasts is mediated via Flt-1.25EFFECTS OF VEGF ON OSTEOCLASTSRecentdiscoveryofreceptoractivatornuclearfactorkappa b ligand(RANKL)RANKinteractionconfirms thehypothesis thatthere is adirect contact between osteoblasts and osteoclasts and osteoblasts playan essential role in osteoclast differentiation. Osteoblasts expressRANKL as a membrane-associated factor. Osteoclast precursors thatexpress RANK, a receptor for RANKL, recognize RANKL through thecellcell interaction and differentiateinto osteoclasts.29Yasuda et al.alsoidentifiedanotherkeyfactorforosteoclastogenesis,osteoprote-grin.30Osteoprotegrin is produced by osteoblasts or stromal cells andbinds to RANKL as a decoy receptor, therefore preventing interactionbetween RANKL with RANK. Thus, osteoclast differentiation is inhib-ited by osteoprotegrin.Min et al. have shown that VEGF can significantly increase both theexpression of RANK mRNA and surface protein in human microvas-cularendothelial cell lines.31Inaddition, theyrevealedthat VEGFmainly enhances RANK expression in endothelial cells through Flk-1/KDRprotein kinase CERKsignaling pathway, suggesting that VEGFplays animportant role in modulating the angiogenic action ofRANKLunder physiological or pathological conditions. Yaoet al.also found that the combination of VEGF and a low dose of colony-stimulating factor-1 can upregulate the RANKexpression in osteoclastprecursors that is neededfor osteoclastogenesis.32Nakagawaet al.suggested that VEGF is involved in osteoclastic recruitment, differen-tiationandenhancement of osteoclasticbone-resorbingactivityincultured rabbit mature osteoclasts.33EFFECTS OF VEGF ON ENDOCHONDRAL OSSIFICATIONEndochondral ossification (also referred to as intracartilaginous ossi-fication) plays a major role in bone formation. It is an essential processduring mandibular condylar growth, rudimentary formationandgrowth of long bones, and the healing of bone fractures. Unlike intra-menbranous ossification, cartilage, an avascular tissue is present andreplaced by bone during endochondral ossification.1Endochondral ossification occurs as chondrocytes undergo prolif-eration, hypertrophy, cell death and osteoblastic replacement. VEGFhas been indicated to serve as an important mediator during the pro-cess due to its ability to regulate blood vessel invasion (neovasculari-sation) into hypertrophic cartilage3436(Figure 1). The invadingblood vessels bring undifferentiated mesenchymal cells into themineralisation front and later differentiate into osteoblasts and engagein osteogenesis.34Also, a recent study by Bluteau et al. for the first timeshowedthat chondrocytes cansecretefour members of theVEGFfamily.37VEGF-A, -B, -Cand-Dweredetectedandupregulatedatthe mRNA and protein levels during chondrogenic differentiation ofprimarychondrocytes inthe ATDC5chondrogenic cell line. Thissuggests that these factors play an essential role during chondrogene-sis.Zelzeretal.providedfurtherinvivoevidencefortheimportantAngiogenesisBlood vesselinvasion orneovascularizationVEGFEndothelial cellGrowth factorscytokinesOsteoblastsIntramembranousossificationBoneHypertrophyChondrocyteEndochondralossificationMesenchymal cellFigure1 SchematicofeffectsofVEGFonangiogenesisandosteogenesis.VEGF impacts on endothelial cells and initiates the angiogenesis process whichrecruits original mesenchymal cells migrate to cartilage or subperioeteal connec-tive tissue through the neonatal blood vessels. During the process of endochon-dral ossification, themesenchymal cellsdifferentiateintochondrocytes, andundergochondrocyteshypertrophy. Then, theprocessof boneformationisinitiated. On the other hand, during the process of intramembranous ossification,themesenchymal cellswhichmigratetothesubperioeteal connectivetissuedifferentiate into osteoblasts directly and initiate the process of bone formation.VEGF upregulates the expression of growth factors and cytokines in endothelialcells and plays an important role during the process of intramembranousossi-fication. VEGF, vascular endothelial growth factor.The role of VEGF in ossificationYQ Yang et al65International Journal of Oral Scienceroleof VEGFinbloodvessel invasionintohypertrophiccartilageduringbonedevelopment.38Moreimportantly, theydescribedforthefirsttimeaconnectionbetweenVEGFandchondrocytesurvivalduring skeletal development.Duringmandibulargrowth,thecondyleundergoesendochondralossificationandthecondylarcartilageservesasatemplateforboneformation. Condylar growth involves a multistage process of cell dif-ferentiation, defined by molecules that are synthesized by cells in thecondyle.34VEGF is expressed at high levels in hypertrophic chondro-cytes in the mandibular condyle of growing and adult rats.34,39Maximumlevels of VEGF expressionprecedethe maximumlevelofnew bone formation in the condyle. This indicates a close correlationbetween vascularisation and bone formation.3536Gene therapyexploredinthecondylarareabyRabieandco-workers, hasshownthatrecombinantadeno-associatedvirus-mediatedVEGFisaneffi-cient delivery systemto induce mandibular condylar growth911. Herewe see a direct cause and effect. This exogenous VEGF leads to signifi-cant condylar growth.3536,4042Data fromCortina-Ram rez and Chimal-Monroy suggest differentialeffects of VEGF on different joints during development.41They evalu-ated VEGF effects on joints by implanting VEGF beads in the presump-tive limb joints of chick embryos. The wrist and elbow showed partialand complete fusions. They found that VEGF inhibited joint formationwhen it was applied after transforming growth factor-beta.The spheno-occipital synchondrosis is animportant growth center ofthe craniofacial skeleton. It forms an important link between the cranialvault and facial skeleton and can influence the positions of the maxillaandmandible.42Itundergoesendochondral ossificationandcontri-buteslargelytotheexpansionoftheossificationcentresandgrowthof thecranial baseduringthepostnatal period. Lei et al. identifiedfactors that regulate endochondral ossification in the spheno-occipitalsynchondrosis through their experiment on mice, in which they foundthatmechanical stressappliedtothespheno-occipital synchondrosiselicits core-binding factor subunit alpha-1 expression and subsequentlyupregulates the expression of VEGF.43Both factors play an importantrole in growth of the spheno-occipital synchondrosis.As hypertrophic chondrocytes are avascular, these cells are a poten-tial source of VEGF within the growth plate. To determine the role ofVEGFinendochondral bone formation, Gerber et al. inactivatedVEGF through the systemic administration of a soluble receptor chi-meric protein (Flt-(13)-IgG) to 24-day-old mice.44Blood vessel inva-sionwasalmostcompletelysuppressed,concomitantwithimpairedtrabecular bone formation and expansion of the hypertrophic chon-drocytezone. Recruitment and/ordifferentiationof chondroclasts,whichexpressgelatinaseB/matrixmetalloproteinase-9, andresorp-tion of terminal chondrocytes decreased. Although proliferation,differentiation and maturation of chondrocytes were apparentlynormal,resorptionwasinhibited.Cessationoftheanti-VEGFtreat-ment was followed by capillary invasion, restoration of bone growth,resorptionof the hypertrophic cartilage andnormalisationof thegrowth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growthplate morphogenesis and triggers cartilage remodelling. Thus, VEGF isa potential coordinator of chondrocyte death, chondroclast function,extracellularmatrixremodelling, angiogenesis andboneformationinthe growth plate. More investigates are needed to prove thishypothesis.Healing of fractures is dependent on vascularisation of bone, whichis in turn promoted by VEGF. In skeletogenesis, which is tightly linkedtoangiogenesis, VEGFpromotesthevascularisationof thegrowthplate and transformation of cartilage to bone. Street et al. determinedwhether VEGF is required for bone repair by inhibiting VEGF activityduring secondary bone healing via a cartilage intermediate/endochon-dral ossificationinanovel mousemodel.27Femoral fractureswereused as models of endochondral ossification. Fracture repair occurredinaseriesofevents, involvinganinitial inflammatoryphase, asoftcallus phase, ahardcallus phase andaremodellingphase. VEGFinhibitioninmicebyFlt-IgG,asolubleVEGFreceptorblocker,dis-rupted repair of femoral fractures and impaired new bone formation.Their results provide evidence that VEGF activity is essential for con-versionofsoftcartilaginouscallustoahardbonycallusandmine-ralisationinresponsetoboneinjury. Geiger et al. basedontheirexperiment onrabbits, showedthat VEGF-gene-activatedmatrix,led to a significant increase in vascularisation and bone regenerationinlargesegmentaldefects.45Thus, VEGF-gene-activatedmatrixcanserve as an appropriate tool to promote angiogenesis, osteogenesis andbone healing. Li et al. demonstrated enhanced healing of a segmentaldefect inthelongboneof rabbitsusingthecell-basedVEGFgenetransferwithout viralvectors.12Theirfindingsprovedthatthisdeli-verymethodcanbeeffectiveinmanagement of clinical situationswherevascularityis compromisedandneovascularisationis tobeencouraged, suchasfractureswithbonelosswithoutthecomplica-tions regarding viral vectors.Peng et al. showed interaction between angiogenic and osteogenicfactorsinboneformationandbonehealing.46Theydiscoveredthatthe combination of VEGF and bone morphogenetic protein 4 is able torecruit more mesenchymal stem cells to enhance cell survival and toinducecartilageformationintheearlystagesofendochondralboneformation. Further, the beneficial effect of VEGF on bone healing wasfound to be dependent on the ratio of VEGF to bone morphogeneticprotein 4. Table 1 summarizes the effects of VEGF on endochondralossification.EFFECTS OF VEGF ON INTRAMEMBRANOUS OSSIFICATIONIntramembranous ossification is a process whereby the formation ofbone tissue occurs directly from connective tissue without a prelimi-nary cartilage stage. A good example occurs in the glenoid fossa whichisformedwhenmesenchymal cellsdirectlydifferentiateintoosteo-blasts before ultimately forming bone.1Experimental studies on ratshaveshownthat mechanical straincausedbyforwardmandibularpositioningstimulatesthebonecellsin thesubperiostealconnectivetissue of the glenoid fossa to secrete VEGF. During natural growth andforwardmandibular positioning, VEGFexpressionandnewboneformationwerethehighest intheposterior regionof theglenoidfossa.3536The identification of the temporal sequence of the cellularresponse revealed that the mesenchymal cells in the posterior region oftheglenoidfossawereorientedinthedirectionofthepull.47VEGFenhancesneovascularisation,whichinturnincreasesthenumberofmesenchymal cellsintheperivascularconnectivetissue(Figure1).VEGF also stimulates the vascular endothelial cells to secrete growthfactors and cytokines that influence the differentiation of mesenchy-mal cells to enter the osteogenic pathway and engage in osteogenesis48(Figure1). Furthermore, thegreatest amount of VEGFexpressionprecedesthegreatestincreaseinnewboneformation. Therefore, ithas been concluded that an increase of VEGF is spatially and tempo-rallyrelatedtotheamountofnewboneformationintheposteriorglenoid fossa.3536Bonerepairisamultistepprocesswhichinvolvesmigration,pro-liferation, differentiationandactivationofseveralcellstypes. Studyfrom Tatsuyama et al.49on fracture repair in bone indicates that thisThe role of VEGF in ossificationYQ Yang et al66International Journal of Oral Scienceprocess is complex, occurringthroughseveral steps andinvolvingvarious growth factors including VEGF. Bone growth and bone repairaresomehowregulatedinasimilarmannerandthus,VEGFshouldhave a similar effect during bone repair.8Repair of large bony defectsin the craniofacial region represent a major challenge for the surgeon,since autogenous graft material from patients may not be available insufficient amounts. It has been shown that addition of VEGF to demi-neralizedintramembranous bone matrix (DBMIM) improves thequalityandquantityofnewlyformedboneinthegraftedsite, thusindicatingthatVEGF1DBMIMisagoodgraftmaterial. It isbotheasilyobtainable and could eliminatethe needto harvest bone fromthe patient. Its future application, however, will require further clinicalevidence.ResultsfromStreetetal. onmicealsoshowtheroleofVEGFinintramembranous ossification.27They have shown direct bone repairthrough intramembranous ossification in a novel mouse model (tibialcortical bone defects). Inhibition of VEGF by Flt-IgGtreatment, just asin the femoral fracture model, had distinct effects on bone healing ofcortical bonedefects. Thepersistenceofunresorbed, unmineralizedfracture haematoma at 7 days and the persistence of woven bone at 14days indicate remodelling defects in Flt-IgG-treated mice. They foundthat the effects of Flt-IgGon intramembranous bone formation(cortical defects were more prominent at the earlier (7 days) than later(14 days) time point) were due to the fact that intramembranous ossi-fication does not involve a cartilage intermediate. Thus, they suggestedthat aslow releaseformulation of VEGF,applied locallyat thesite ofbonedamage, canbeaneffectivetherapytopromotehumanbonerepair.Another example of the role of VEGF in intramembranous ossifica-tion is observed in distraction osteogenesis, a surgical therapy used inthe treatment of skeletal injuries and deformities and in the correctionof limblengthabnormalities.50Boneformationduringdistractionosteogenesis occurs primarily through an intramembranous process.Investigatorsobservedclosecorrelationbetweenoptimalangiogenicresponseandthe rateof distractionandspeculatedthat it is thischaracteristicthatdrivesboneformationthroughtheintramembra-nous pathway.51Toelucidatethefunctional roleof VEGFsignalingduringboneformation in distraction osteogensis, Jacobsen et al. used two blockingantibodiesMF-1(anti-Flt-1)andDC101(anti-KDR)tospecificallydisrupt the activities of KDR and Flt-1.52The results showed that bothKDRandFlt-1playimportantrolesinneovascularisationandboneformationduringdistractionosteogenesis. Table2summarizestheeffects of VEGF on intramembranous ossification.Table 1 Effects of VEGF on endochondral ossificationMethods Materials Effects ReferencesIn vivo Mouse femur Stimulated capillary invasion and bone growth 44Rat mandibular condyle Blood vessel invasion ( neovascularisation) into hypertrophic cartilage was regulated 34Rat mandibular condyle. Promoted neovascularisation. Close correlation between vascularisation and bone formation35Mouse calvarial defects Synergistic effect between VEGF and BMP4 occurred and recruited more mesenchymal stemcells to enhance cell survival and to induce cartilage formation46Mouse femur fractures healing model Conversion of soft cartilaginous callus to a hard bony callus and mineralisation was induced 27Mouse embryos VEGF increased blood vessel invasion into hypertrophic cartilage during bone development 38Rabbit bone defects VEGF-GAM (gene-activated matrix) increased vascularisation and bone regeneration 45Rat mandibular condyle Mandibular condyle increased in size 11Limb joints of chick embryos Differential effects on different joints: partial fusion on wrist joint; complete fusion on elbowjoint; inhibited joint formation when applied after TGF-b41Cranial base synchondroses of mice Cbfa1 and VEGF promoted growth of the spheno-occipital synchondrosis 43Rabbit tibial fracture defects Healing of segmental defect in the long bone of rabbits were enhanced 12In vitro ATDC5 chondrogenic cell line Stimulated chondrogenic differentiation of primary chondrocytes 37BMP4, bone morphogenetic protein 4; Cbfa1, core-binding factor subunit alpha-1; GAM, gene-activated matrix; TGF-b, transforming growth factor beta; VEGF, vascularendothelial growth factor.Table 2 Effects of VEGF on intramembranous ossificationMethods Materials Effects ReferencesIn vivo Rat tibial fracture defects VEGF enhanced fracture bone repair 49Tibial cortical bone defects Improved direct bone repair 27Rat glenoid fossa Maximum level of VEGF expression and new bone formation in the posterior region of the glenoidfossa during natural growth and forward mandibular positioning. Greatest amount of VEGFexpression precedes the greatest increase in new bone formation36Rat glenoid fossa Secretion of growth factors and cytokines by vascular endothelial cells was enhanced 48Rabbit parietal bone defects Combination of VEGF and DBMIM is a good graft material 8Mouse tibia distractionosteogenesisBoth KDR and Flt-1 play important roles in neovascularisation and bone formation duringdistraction osteogenesis52In vitro Distraction osteogenesis Optimal angiogenic response and rate of distraction are closely related 51DBMIM, demineralized intramembranous bone matrix; Flt-1, vascular endothelial growth factor receptor-1; KDR, vascular endothelial growth factor receptor-2; VEGF,vascular endothelial growth factor.The role of VEGF in ossificationYQ Yang et al67International Journal of Oral ScienceCONCLUSIONThe evidence from a large amount of research has shown conclusivelythat VEGF is able to promote ossification by either inducing neovas-cularisationorbydirectlyaffectingbonecells. 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Toviewacopyof thislicense, visit http://creativecommons.org/licenses/by-nc-nd/3.0The role of VEGF in ossificationYQ Yang et al68International Journal of Oral Science