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OralMaxillofacialSurgClinNAm14(2002)ix Preface Emerging biomaterials and tissue engineering MichaelJ.Buckley,DMD,MS JohnC.Keller,PhD GuestEditors Inthefuture,thepracticeoforalandmaxillofacial emerging biomaterials and tissue-engineered ap- surgerywillbeshapedbythetoolswearegiven.The proaches to surgical treatment is exciting and will emerging fields of tissue engineering and biomate- help the advance of oral and maxillofacial surgery. rialsaretrulyexciting.Productsofthefuture,includ- Thefuture isbright. ingthosefromgeneticallyengineeredproteinsallthe way to tissue-engineered organs, are and will be in clinical trials in the very near future. Our specialty Michael J.Buckley,DMD, MS will benefit from the development of many of these University ofPittsburg biomaterials, and our future will be shaped by their SchoolofDental Medicine use. Our patients will benefit from us understanding 3501TerraceStreet and critically evaluating new products as they are Suite 440,Salk Hall brought to market. This issue contains articles au- Pittsburgh,PA15213, USA thoredbyadiversegroupofscientistsandclinicians representing a variety of fields. The articles have all JohnC.Keller,PhD been written by authors with great insight into their Dows Institute for DentalResearch particulartopic.Theyarewrittentobringtolightthe Dental Research, NY18,DSB present state of the art, with emphasis on future University of Iowa development of products in that arena. The use of IowaCity,IA52242, USA 1042-3699/02/$ – seefrontmatterD2002,ElsevierScience(USA).Allrightsreserved. PII:S1042-3699(02)00038-9 OralMaxillofacialSurgClinNAm14(2002)1–14 Bone morphogenetic proteins A review for cranial and maxillofacial surgery Kodi Azari, MDa, John S. Doctor, PhDb,c, Bruce A. Doll, DDS, PhDb,d, Jeffrey O. Hollinger, DDS, PhDb,* aDivisionofPlasticSurgery,UniversityofPittsburghMedicalCenter,Pittsburgh,PA,USA bBoneTissueEngineeringCenter,CarnegieMellonUniversity,125SmithHall,5000ForbesAvenue,Pittsburgh,PA15213,USA cDepartmentofBiologicalSciences,DuquesneUniversity,Pittsburgh,PA,USA dSchoolofDentalMedicine,UniversityofPittsburgh,Pittsburgh,PA,USA Along with vitamin D and the Iliazov callous bone tissue released stimulating factors that affect distractiontechnique,thediscoveryofbonemorpho- osteoblasts. By the 1920s it was hypothesized that a genetic proteins (BMPs) is deemed one of the three substance released from graft tissue resulted in the greatest scientific advances in the domain of bone differentiation of fibroblasts into bone and cartilage research of the twentieth century [105]. To date, a formingcells[79].Thenexthypothesis,proposedin Medlinesearchoftheavailableliteratureyieldsmore 1928, was that calcium materials contained in the than2100publicationsthatfocusinsomemanneron grafttissueweretheputativeagentsthatinducednew BMPs. In recent years the use of recombinant DNA bone formation [50]. As a means of managing vis- technology has led to the identification and molecu- ceraldefects,however,Neuhof[72]implantedfascia larcloningofbonemorphogenetic proteinsresulting alongtheurinarytractandnoticedlocalboneforma- in the production of quantities suitable for clinical tion. This finding suggested that extraskeletal ossi- applications [75,76,110–112]. This article serves as fication may be accomplished by means other than a review of the history, mechanisms of signaling, transplantingcalcium-containinggrafts. and synopsis of the experimental and clinically In some pathologic conditions, extraskeletal tis- relevant information available in the field of BMPs sues may ossify and exhibit all the features of bone, asitpertains to cranialandmaxillofacial reconstruc- includingmarrow[22].In1903,Binnie[8]described tive surgery. myositis ossificans traumatica, in which frequently repeated soft tissue trauma resulted in heterotopic ossification of muscle. Other causes of ectopic bone Historicalperspectives formationincludetumorsofchondrocyticandosteo- cytic origin and the uncommon connective tissue Inthelate1800s,Senn[93]implanteddemineral- diseasecalledmyositisossificansprogressiva,which, ized ox bone and iodoform to treat dog skull osteo- as the name implies, leads to progressive soft tissue myelitis and noted better than expected bony defect ossification andbonyskeletonmalformations [8,22]. healing. Later, Lexer [51] suggested that necrotic In1965,inanexperimenttorecalcifycorticalbone, MarshallUrist[99]madethekeydiscoverythatledto thehuntforfactorsresponsibleforboneformation.In thisclassicexperiment,demineralizedbonefragments were implanted subcutaneously or in intramuscular JohnS.DoctorwassupportedbytheNationalInstitutes ofHealthsabbaticalfellowshipF33AR08268. pouches in rats and rabbits. Several weeks after * Correspondingauthor. implantation new cartilage and bone appeared at the E-mailaddress:[email protected](J.O.Hollinger). implantationsites.Thisoccurrenceledtothehypoth- 1042-3699/02/$–seefrontmatterD2002,ElsevierScience(USA).Allrightsreserved. PII:S1042-3699(02)00011-0 2 K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 esis of bone formation by autoinduction, in which extracellularlyandintracellularly,and(3)thepotential graftedinducercellsinfluenceresidentprimitivecells for agonistic and antagonistic activities of BMPs to differentiate into osteoprogenitor or chondropro- dependingontheparticularcelltype.Theamountof genitorcells. crosstalk between the BMP signaling pathway and Urist’sdiscoveryledtoaseriesofinvestigationsto othergrowthfactorandhormonalsignalingpathways determine the putative agents in demineralized bone [46,48,65] must be evaluated as to avoid unwanted thatprovokeautoinduction.Theosteoinductiveactiv- sideeffectsfromBMP-basedtherapies. ity in bone matrix was found to be the result of a noncollagenous and water soluble substance coined TheBMPsaremembersofalargefamilyofsignaling BMP[100–102,104] proteins that are important during development and The next major advance was the purification of sharethesame basic structure BMPs and the isolation of 16, 18 and 30 Kda poly- peptidesfrombovinebone.Later,theseproteinswere The TGF-b superfamily contains several dozen namedBMP1-3[106].Inthelate1980s,recombinant members,includingtheBMPs,theactivins,mu¨llerian DNA technology was used to generate human com- inhibitingsubstance,andtheprototypicTGF-bs.More plementary DNA clones of BMP molecules [112]. thanadozenproteinsaregroupedintotheBMPfamily, Thesecloneswereinsertedintomammalianandnon- based on sequence similarity. The roles of BMPs mammaliancellstoproducerecombinantBMPs.Thus duringdevelopmentcanbeinferredfromtheirexpres- far more than 15 BMPs have been isolated and sioninvarioustissues[28,31,54].Geneticanalysesin reportedintheliterature[74]. mice demonstrate roles for BMPs in skeletogenesis andotheraspectsofdevelopment.Forexample,sev- eralmutationsthatperturbthefunctionofBMPgenes Mechanisms of BMPsignaling areknown,including,shortearandbrachypod.Short ear,whichresultsinabnormalgrowthandpatterning The BMPs are members of the transforming of skeletal structures and diminished repair of bone growth factor-b (TGF-b) family of growth and dif- fracturesinadults,iscausedbylesionsintheBMP-5 ferentiation factors. Although identified and named gene [44,45,57]. Mutations in GDF-5 (growth and because of their osteoinductive activity [14,110], the differentiationfactor-5,amemberoftheBMPfamily) BMPsplaydiverserolesduringembryonicandpost- resultinthebrachypodphenotypeinmiceandinthe embryonic development as signaling molecules in a autosomal recessive syndromes Hunter-Thompson wide range of tissues [30,31,40,94]. For example, in and Grebe-type chondrodysplasias in humans [24, addition to roles in skeletogenesis and bone forma- 95].Thesesyndromesarecharacterizedbytheshort- tion[83],BMPsareimplicatedinmesodermpattern- eningoftheappendicularskeletonandlossorabnor- ing, neurogenesis, and organogenesis [62,64,66,77, maldevelopmentofsomejoints,withlittleeffecton 84,109].Inthelastseveralyears,genetic,molecular, theaxialskeleton. and cellular approaches have provided important DirecteddisruptionsofBMPgenesinmicereveal detailsaboutthemechanismsofBMPsignaling.Much important functions in mesoderm induction and of our knowledge derives from studies on model organogenesis [20,53,109] but have been less organisms, including mouse, frog (Xenopus), nem- informative about the roles of BMPs during skeleto- atode(Caenhorabditis),andfruitfly(Drosophila).As genesis. For example, BMP-2 and BMP-4 knockout indicatedschematicallyinFig.1,extracellularBMPs mice die early in embryonic development, long bindandactivateamultimerictransmembranerecep- before development of the skeleton, because of tor complex. This ligand-activated receptor, serine/ defects in gastrulation [109,116]. BMP-7 knockout threonine kinase, phosphorylates members of the mice exhibit eye and kidney defects but only mild Smad family of signal transducers, which results in skeletal defects, perhaps because of the rebundant the direct translocation of Smads to the nucleus, function of other BMPs [53]. where they can modulate the expression of target Molecular analysis reveals that BMPs share the genes [58,113]. major hallmarks of all members of the TGF-b su- Thisdescriptiononlyhintsatthecomplexityinher- perfamily: initially to facilitate secretion, they are ent in the control of BMP signaling. If BMPs are to synthesized as large precursors that consist of an serveaseffectivetherapeuticagents[87,90],itisnec- amino-terminal signal sequence, a pro-domain and a essary to understand their action, including (1) their carboxy-terminal bioactive mature domain [13,111, roleinsignalingamultitude ofcelltypes,(2) mech- 112]. The amino-terminal signal sequence and pro- anisms by which BMP signaling can be modulated domain regions of the BMPs vary in size and K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 3 Fig.1.Signalingbybonemorphogeneticproteins(BMPs).ThebindingofanextracellularBMPdimericligandtothemultimeric BMPtransmembranereceptorcomplex.Formationoftheligand/receptorcomplexresultsintheactivationoftheserine/threonine kinaseofBMPreceptor-1(BMPR-I)andthephosphorylationofmembersoftheR-Smadfamilyofsignaltransducers(Smads1,5, or8).ThisphosphorylationcanbeantagonizedbytheactionofinhibitorySmad6(notshown).ThephosphorylatedR-Smadbinds withC-Smad4andtranslocatesintothenucleuswhereR-Smad/C-Smadcomplexdirectlyorindirectlyinteractswiththecore binding factor (CBF). The activated CBF translocates to the osteocalcin promoter region and activates Osteoblast-Specific Element-2(OSE-2).Thissequenceofeventsresultsinthetranscriptionoftheosteocalcingene.OsteocalcinmRNAistranslatedto osteocalcin protein on the ribosomes. P=phosphate. mRNA=messenger ribonucleic acid (Adapted from Schmitt J. Bone morphogeneticproteins:anupdateonbasicbiologyandclinicalrelevance.JOrthoRes1999;17:269–78;withpermission). sequence, whereas the mature domain shows greater During intracellular processing before proteolytic sequence similarity among family members (Fig. 2). cleavage and secretion, these mature domains form The mature domain is usually cleaved from the pro- eitherhomodimersmadeupoftwoofthesamefamily domain region by the subtilsin-like serine protease members or heterodimers made up of two different furin [6,18] to produce an active polypeptide of be- familymemberstoproducetheactivesignalingmol- tween110and140amino acids inlength. ecule. Depending on the expression of BMPs in a The mature region of BMPs is highly conserved particularcelltype,itispossibletoformcombinations amongthefamilymembersandincludessevennearly of various homodimers and heterodimers, each with invariant cysteines. Six of these are involved in the potentially differing activities. Overlapping expres- formation of intrachain disulfide bonds to make a sionofmurineBMPssupportsthiscontentionforthe rigidstructurecalleda‘‘cysteineknot’’;theseventhis combinatorial effects of homodimeric and hetero- involvedintheformationofhomodimersandhetero- dimericBMPs[54].HeterodimersofXenopusBMP-4 dimersbyaninterchain disulfide bond[89]. and BMP-7 produced in vitro [27] are more potent 4 K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 Fig.2.ThepolypeptidestructureofBMPs. than homodimers in bone- and mesoderm-inducing internalizedanddegraded,follistatinmayacttoclear activities [2,96]. activin and perhaps, BMPs from the extracellular The pro-domain seems to be required for normal environment. Attention to these extracellular regula- synthesis and secretion of TGF-b superfamily mem- torsisimportantindesigningtherapiestodeliverand bers, and it can remain associated with the mature releaseBMPs. domaintoproduceaninactivelatentcomplexforsome membersofthefamily[86]butapparentlynotforthe BMP receptors areheteromericcomplexes BMPs.Otherproteinscanassociatewithextracellular withserine/threoninekinase activity BMPs with important implications for their biologic activity,asdescribedinthefollowingsection. Bone morphogenetic protein members interact withtheextracellulardomainofafamilyofcellsurface Severalextracellular proteins actto regulate type I and type II receptors to signal across the cell the activityof BMPs membrane and elicit a cellular response [41,58]. A complexofatypeIreceptorandatypeIIreceptoras- The extracellular environment seems to play an semblesthroughinteractionwithaBMPligand.For- important role in regulating the activity of BMPs mationof this ligand-receptor complex results in the [25]. In addition to interactions with extracellular phosphorylationofthetypeIreceptor’skinasedomain matrix components such as collagens and heparin by the serine/threonine kinase domain of the type II sulfate proteoglycans, BMPs interact specifically receptor.ThisactivationofthetypeIreceptor’sserine/ with several extracellular proteins. The protein nog- threoninekinaseresultsinthedownstreamphospho- gin binds BMP-2 and BMP-4 with highly affinity rylationoftargetsubstrates,includingtheSmadfamily and inhibits their association with BMP receptors ofsignaltransducingproteins(seeFig.1). (BMPR) [119]; noggin also binds BMP-7 with low In mammals, BMP type I receptors (BMPR-IA affinity. Another secreted protein, follistatin, origi- and BMPR-IB) and BMP type II receptors (BMPR- nallyidentifiedasahighaffinityantagonistofactivin II)areexpressedduringembryogenesisandinseveral [68],alsohasaffinityforBMP-4andBMP-7[23,37]. celllines[19,21,28,71].Evidenceforimportantroles StudiesofXenopusembryosalsoimplicatetheDAN of BMPRs during development comes from anal- family (cerebus, gremlin, DAN) as antagonists of ysis of defective BMPR-I receptors (loss-of-function BMP signaling by direct binding to BMPs, thereby experiments) [71,97] that block BMP signaling blocking ligand/receptor interactions [35]. Similar to and constitutively active receptors (gain-of-function noggin,theproteinchordinbindsBMP-2andBMP-4 experiments) [1] that elicit characteristics cell re- with high affinity and inhibits BMP/receptor inter- sponsesintheabsenceofBMPs.Importantforarole action [78]. In the case of chordin, members of the in osteogenesis, BMPRs are expressed during bone astacinfamilyofmetalloproteases(includingBMP-1 formation in skeletal development and in fracture and several tolloid-like proteases) cleave chordin, repair[19,38].Mutationsinthemousebrachydactyly therebyreleasingBMPstointeractwithBMPRs[91]. gene, which encodes BMPR-1B, indicate a role of Componentsoftheextracellularenvironmentactto BMPRsin skeletogenesis [7]. sequester or release BMPs (Fig. 3). Chordin/BMP complexesmayserveasanextracellularreservoirfor Smadsareessential componentsof BMPs, with the level of the reservoir depending on intracellular signalingbyBMPs the amounts of chordin, BMP, and chordin-specific BMP-1/tolloidproteases.Basedonobservationsinrat The discovery of Smads, the intracellular down- pituitary cells that follistatin/activin complexes are stream targets of BMPR-II kinase activity in mam- K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 5 Fig. 3. BMPs interact specifically with extracellular proteins that act as antagonists to sequester BMPs from signaling. The inhibitoryactivitiesofdistincttypesofproteinsonextracellularBMPsaredepicted.Nogginfollistatin,chordin,andmembersofthe differentialscreening-selectedgeneabberativeinneuroblastoma(DAN)family(cerebus,gremlin,DAN)bindtomembersofthe TGF-bfamily,includingBMPs,andinhibittheirabilitytobindcognatereceptors.Theantagonisticactivityofchordinisitself inhibitedbyBMP1/tolloidproteases,whichacttodegradechordin,therebyreleasingBMPs. mals,derivesdirectlyfromgeneticanalysisofmuta- to BMPR-I and competes for binding by R-Smads tions that affect the signaling activity of the fruitfly [39].Smad6alsocaninhibitBMPsignalingbycom- BMP-2and-4-likeproteinknownasdecapentaplegic. petingforthebindingofphosphorylatedR-Smadsto WorkinDrosophilaontheMadgene(Mothersagainst Smad4[26]. decapentaplegic), which mediates decapentaplegic signaling [92] and on a series of sma genes in the R-Smad/C-Smad complexes interact with nematodeCaenorhabditis[88],ledtotheisolationof transcription factors in the nucleus sma/Mad-like genes, termed the Smads in mammals to modulate gene expression [4]. To date, genes that encode eight related mam- malian Smad proteins, Smad1 through Smad8, have Onceinthenucleus,R-Smad/C-Smad complexes been identified and characterized [16,29,41,113]. canbinddirectly,althoughwithlowaffinity,toDNA These proteins are grouped into three categories: in the regulatory regions of many BMP-responsive the receptor-regulated, pathway-restricted R-Smads genes[47,59].TheformationofstableDNA-binding (Smad1, 2, 3, 5, 8); the common mediator C-Smad complexes requires the interaction of Smad com- (Smad4);andtheinhibitoryI-Smad(Smad5,6).The plexeswith sequence-specificDNAbindingproteins R-SmadsareactivatedbyBMPR-Iphosphorylationof [41]. Several such DNA binding proteins have been threekeyserinesintheireffectordomaininaligand- identifiedforacitvin-specificSmads2and3,including specific fashion. Smad1, Smad5 and Smad8 are FAST-1,FAST-2,andAP-1[15,49,116].Inthecaseof involved in BMP signaling, whereas Smad2 and BMP Smads1, 5, and 8, several recent papers report Smad3 mediate TGF-b/activin signaling [4]. Smad4, SmadinteractionswithSIP1,theacutemyelogenous the C-Smad, is not a substrate for receptor kinases. leukemiaprotein,Hoxc-8,ahomeodomaincontaining Instead, it forms a heterodimeric complex with a transcriptionfactor,andp300/CBPandSTAT3,tran- phosphorylated R-Smad, and this complex translo- scriptionfactorsimportantinastrocytedifferentiation cates to the nucleus to modulate the expression of [59,70]. A particularly interesting result implicates a targetgenes(seeFig.1).R-Smadscontainamotifes- SmadinteractionwithRUNX2/CBFA1,acomponent sentialfortranslocationoftheR-Smad/C-Smadcom- of a transcription factor that is essential for proper plex intothe nucleus [114,117]. TheI-Smads inhibit bone development [118]. Gene knockouts of the signalingbybindingtotheintracellulardomainofthe RUNX2geneinmiceresultintheabsenceofmature receptor.Forexample,Smad6bindsinastablefashion osteoblastsandtheabsenceofboneformation.Muta- 6 K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 tionsoftheRUNX2geneinhumansareimplicatedin tractantgrowthfactorssuchasplatelet-derivedgrowth cleidocranial dysplasia,adominanthumanbonedis- factor(PDGF),TGF-b,fibroblastgrowthfactor(FGF), easecharacterizedbymultipleskeletalabnormalities. endothelial growth factor (EGF), insulin-like growth AtruncatedRUNX2proteinfromapatientwithclei- factor-1 (IGF-1), and platelet factor-4 (PF-4) [3,90]. docranialdysplasiafailstointeractwithandrespond Theearlyfracturemilieuischaracteristicallyanhypo- toSmads,therebyimplicatingimpairedSmadsignal- xic and acidic environment that is optimal for the inginthisgeneticdisease. activities of PMNs and tissue macrophages [32]. PMNsphagocytizebacteriaandmicrodebris,whereas larger sized materials are removed by macrophages Roleof BMP afterbone injury that may develop into multinucleated giant cells. Macrophages perform myriad functions in the local When the bone, a living tissue, is injured, such woundsite,includingsynthesisofgrowthfactorsthat as by fracture (Fig. 4), an immediate inflammatory affect local cell activity, cell recruitment, chemoat- responseiselicited.Attheinjuredsitevascularendo- traction,andmitogenesis. thelial damage results in the activation of the com- By days 3 to 5 after bone injury, a local envi- plement cascade, platelet aggregation, andrelease of ronmentdevelopsthatincludesnewcapillarysprouts, itsalphagranulecontents.Thisplateletdegranulation fibroblasts,macrophages,andcollagenisotypes.Itis releases growth factors and triggers chemotactic sig- believed that the attachment of growth factors to nals. Polymorphonuclear leukocytes (PMNs), lym- collagensservestolocalize,protect,andarrangethese phocytes, blood monocytes and tissue macrophages factors for cellular interaction [80]. The collagenous areluredtothewoundbedandareactivatedtorelease portion of the healing wound renders a key instruc- basicfibroblastgrowthfactor(bFGF). tionalframeworktopositioncytokinesandBMPsto Theextravasatedbloodcollectionclots.Hematoma receptivecells.Primitiveundifferentiatedsystemically formationservesasahemostaticplugtolimitfurther circulating cells, along with osteoprogenitor cells of hemorrhage. The conductors of the cascade are the the periosteum and endosteum, attach to the granu- platelets,whichhavethedutyofhemostasisandme- lation tissuecollagen, which present signaling mole- diator signaling through the elaboration of chemoat- cules, including BMP. The cell-ligand interaction Fig.4.Sequenceofeventsfollowingbonefracture.Followingosseousfracture,plateletsdegranulate,ahematomaisformed,the immune system is activated at the wound site, and microdebris is removed by phagocytizing cells. By day 3–5 postinjury, neovascularization occurs while osteoprogenitor and mesenchymal stem cells localized at the wound site respond to environmentalfactors,includingBMPs,fibroblastgrowthfactor(FGF),transforminggrowthfactorb,insulin-likegrowthfactors (IGF), platelet-derived growth factor (PDGF). (Adapted from Schmitt J. Bone morphogenetic proteins: an update on basic biologyandclinicalrelevance.JOrthoRes1999;17:269–78;withpermission.) K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 7 promptsdifferentiationofthesecellsintoosteoblasts was either added as a paste of purified BMP and andchondrocytes[80,81].TheinfluenceofBMPson granular bone matrix or it was added to lyophilized celldifferentiationisofparticularinterestwithrespect cartilage or bone during rehydration. BMP paste toboneformation.Thecombinatorialfunctionsofcell probably consisted of several homologous proteins attachment, transduction, and cell-ligand interaction [14,112].Afterrigoroustestingforboneinductionina prompt cellular differentiation into specific pheno- mouse assay, Muthukumaran et al. [67] purified types to repair osseous wounds. Cellular armies in bovine BMP and delivered it to human skeletal the appropriate milieu of growth and differentiating defects. Sailer summarized the maxillofacial recon- factorssuchasTGF-b,FGF,VEGF,PDGF,andBMPs structionproceduresusingbovineBMPbetween1992 eventuateinfracturerepairby6to8weeksafterinjury and1995[52].Age,sex,surgicalsite,andpreexisting [32].Interestingly,therepairedbonestructureisindis- traumatic conditions did not hinder engraftment as tinguishablefromthepreinjurystructure.Theremod- judged by postsurgical clinical and radiographic eling of injured bone to the preinjury structure is examinationintheninecasespresented[52]. craftedcarefullybyosteoblastsandosteoclasts[103]. Theuseofnative,purifiedBMPislimitedbythe If,however,asufficientnumberofcellsarenotpresent availablesource,arduouspreparation,andlowyields. at the osseous injury site, they must be recruited, Small yields of BMP are obtained from kilogram stimulatedtoproliferate,andacteduponbytheappro- quantities of dry bovine bone. The preparation priateadmixtureofgrowthfactors.Atthefracturesite, includes defatting, demineralization, and chromato- fragments of the ubiquitous attachment factor fibro- graphic steps—a time-consuming process for yields nectinanddegradationproductsfromtheextracellular under 1:50,000. Relative ratios of BMPs within the matrix stimulate the transformation of monocytes of preparation vary and confound assignment of osteo- osteoclasts[32].Macrophagesatthelocalwoundsite genic potential for each BMP. Despite the apparent elaborate bFGF and VEGF, thus inducing neovascu- usefulness of cross-species applications, concern for larizationtofurnishtransportpathwaysforadditional antigenicity encourages use of human recombinant cellstoreplenishthoselosttoinjury[36]. BMPs. The availability of large amounts of highly The clinical relevance of BMPs in the regenera- purified,efficientlyproduced,predictablyconstituted tionandrepair of injuredbonerequires aresponsive recombinant proteins facilitates incorporation into regionalcellpopulationatthewoundsite[82].Cells tissue-engineeredvehicles[107].Whatfollowsisthe mustbecapableofrespondingtotheBMPsignalor description of several different biodegradable com- signals,andsufficientquantityandformsofbiologi- posite bone graft materials that are in clinical or cally active BMPs must be attendant to produce the preclinical use for the treatment of skeletal defects sought outcome, (e.g., to regenerate the form and inorthopedic,maxillofacial,andperiodontalsurgery. function of bone). BMPs and their receptors are the executives inthis remarkable process[87,90]. Recent studies reveal increased expression of Animal studies BMP-2,-4and-7intheprimitivemesenchymaland osteoprogenitor cells, fibroblasts, and proliferating Theposteriormaxillaisachallengingareainwhich chondrocytespresentatthefracturesite[9,69,73].In toplacedentalimplantssuccessfullybecauseofpoor aratmodel,mesenchymalcellsthathadmigratedinto bone quality, poor vascularity, and close approxi- thefracturegapandhadbeguntoproliferateshowed mationtothemaxillarysinus.Remainingboneoften increasedexpressionofBMP-2and-4[9].Inasimilar necessitates sinus augmentation. Prior attempts to rat fracture healing model, Onishi et al. [73] con- increasethebonethicknessusedxenograft,allograft, firmedthatBMP-2,-4,and-7werepresentinnewly andautograftmaterials.Thesematerialshaveprovided formedtrabecularboneandmultinucleatedosteoclast- therapeutic success but did not result in a consistent likecells.Inconclusion,severalfindingssuggestthat andpredictablesinusaugmentationinallapplications. BMP-2,-4,and-7worktopromotefracturehealing McAllisteretal.[61]examinedthreedosesofrecom- andboneregeneration[87]. binant human osteogenic protein-1 (rhOP-1; also known as BMP-7), 0.25, 0.6, and 2.5 mg OP-1 per gram of collagen matrix, natural bone mineral, or Bonemorphogenetic proteinapplication collagenmatrixalone(control)placedinthemaxillary tocorrectcraniofacial defects sinus of adult chimpanzees. Based on histologic, tomographic,andclinicalexamination,rhOP-1treat- CompositegraftsthatcontainpurifiednativeBMP ment groups had greater amounts of bone formation wereappliedinskeletaldefects bySailer [85].BMP relative to the control group at 7.5 months. Bone 8 K.Azarietal./OralMaxillofacialSurgClinNAm14(2002)1–14 mineral density and the rate of mineralization were onstrated favorable trabeculation patterns in the enhancedinthetreatmentgroups. resectedareas[10].Table1summarizesotheranimal In a related study [56], Margolin et al. demon- experiments of interest to the cranial and maxillofa- strated short-term histologic and radiographic bone cialreconstructive surgeon. healing after sinus grafting with anorganic bovine bone mineral (Bio-Oss) in the chimpanzee sinus. At 18months,radiographicandhistologicevaluationof Humanstudiesusingbonemorphogeneticprotein the chimpanzee sinus grafts demonstrated mainte- formaxillofacial reconstruction nance of radiographic bone height and bone mineral density [60]. Anorganic bone was replaced by vital Human studies using recombinant BMP are lim- bone.Thecontrolsexhibitedwidevariationinparam- ited.Elevenpatientswereevaluatedradiographically eters insufficient for implant placement. Direct com- foroverallboneheightchangesafterrhBMP-2place- parisonsofrecombinantgraftsandallograftswerenot ment in the maxillary sinus [11]. A collagen sponge included inthese studies. (ACS,absorbablecollagenhemostaticagent)vehicle Usingasimilarcollagen-typeIsponge,either0.2 was usedto deliver the rhBMP-2 (doserange 1.77– or0.8mg/mLrhBMP-2wasaddedtobilateralhemi- 3.4 mg). A dose to bone formation analysis was not mandibulectomies in adult male Macaca fasicularis performed.Immunologicevaluationrevealednoanti- monkeys.Fourmonthsafterengraftment,endosseous body titers to rhBMP-2 or human type-1 collagen. implants were placed in a two-stage procedure. The One patient, however, developed an antibody to implants were uncovered and restored to function bovine type-1 collagen yet remained asymptomatic after 4 additional months. Ridges were regenerated and yielded ‘‘positive bone formation.’’ The vehicle completely on both doses. Histomorphometry dem- wasconsideredeasytousewhenform,cohesiveness, Table1 Experimentalcraniomaxillofacialapplicationsofbonemorphogeneticproteins:animalstudies Author/year Factor Species Defect Protocol Results Barbozaetal rhBMP-2 Dog ClassIII Alveolarridgeaugmentation Clinicallyrelevantridge 2000[5] alveolar withrhBMP-2/absorbable augmentationwhenHAwas collagenspongewithor addedtorhBMP-2and withoutHA absorbablecollagensponge Breitbartetal BMP-7 Rabbit Calvarium Rabbitperiostealcellswith BMP-7additionenhanced 1999[12] orwithouttransfected bonerepair humanBMP-7addedto critical-sizeddefects Khourietal BMP-3 Rat Calvarium 1500radirradiatedcalvarial Significantlygreaterhealing 1996[42] defectcoveredwithmuscle withBMP-3 flapwithorwithoutBMP-3 Mardenetal rhBMP-2 Rat Calvarium Criticalsizeddefectswere AdditionofrhBMP-2was 1994[55] treatedwithrhBMP-2inan superiortoICBMand insolublecollagenousbone DBMalone matrix(ICBM)orwith demineralizedmatrix(DBM) Toriumietal rhBMP-2 Dog Mandible 3cmdefectswereplate By10weeksastiff,noncom- 1991[98] stabilizedandtreatedwith pressible,mineralizedbone inactivedogbonematrix formedacrossthedefects, carrierwithorwithout allowingplateremovaland rhBMP-2 chewingasoliddiet Khourietal BMP-3(osteogenin) Rat Thighadductormuscleflaps Flapstreatedwithosteogenin 1991[43] wereplacedinsidebivalved wereentirelytransformedinto siliconerubbermolds, cancellousbonethatmatched injectedwithosteogenin,and theexactshapeofthemold; coatedwithdemineralized successfullygeneratedperfused bonematrix boneintheshapesoffemoral headsandmandibles Abbreviation:HA,hydroxyapatite. K . A z a r i e t Table2 a Maxillofacialapplicationsofbonemorphogeneticproteins:humanstudies l./ O Author/year Sample Factor/dose Controls Defect Protocol Results ra l Boyneetal 11patients rhBMP-21.77–3.4mg None Insufficientsinusridge ACSsoakedinrhBMP-2placed Immunologicresponseunremarkable;three M a 1997[11] forimplantplacement ipnatmieanxtsillaaprpyrosixniumsa,tbeiloyp6symfoivneths pwaotiveenntsbhoandesduebtescetqeudenintivmarpilaabnltepalmacoeumnetnt, xillofa aftersurgery cia Howelletal 12patients rhBMP-2,meandose0.83mg None Sixridgeaugmentations, ACSsoakedinrhBMP-2,assessment Healinguneventful,minimalclinically lS u 1997[34] (ridgeaugmentation)and sixextraction +graft at4monthsaftergraftplacement evidentridgeaugmentation rg 0.27mg(extractionsites) C Cochranetal 12patients Follow-upofstudy2 None Sixgraftedextraction Implantswereplacedandrestored, Follow-upforstudy2,histologicanalysis lin N 2000[17] sites,fourgraftedridge assessmentat2yearsafter ofgraftsitesimilarsurroundingnativebone A augmentationsites graftplacement m 1 Abbreviations:ACS,absorbablecollaqenhemostaticagent(Heliostat);rhBMP-2,recombinanthumanbmp-2. 4 (2 0 0 2 ) 1 – 1 4 9