Review Article

Bone morphogenetic proteins: A powerful

osteoinductive compound with non-negligible

side effects and limitations

Ahmad Oryan1*

Soodeh Alidadi1

Ali Moshiri2

Amin Bigham-Sadegh3

1Department of Pathology, School of Veterinary Medicine, ShirazUniversity, Shiraz, Iran2Department of Clinical Sciences, Division of Surgery and Radiology,School of Veterinary Medicine, Shiraz University, Shiraz, Iran3Department of Surgery and Radiology, School of Veterinary Medicine,Shahrekord University, Shahrekord, Iran

Abstract

Healing and regeneration of large bone defects leading to

non-unions is a great concern in orthopedic surgery. Since

auto- and allografts have limitations, bone tissue engineering

and regenerative medicine (TERM) has attempted to solve this

issue. In TERM, healing promotive factors are necessary to

regulate the several important events during healing. An ideal

treatment strategy should provide osteoconduction, osteoin-

duction, osteogenesis, and osteointegration of the graft or bio-

materials within the healing bone. Since many materials have

osteoconductive properties, only a few biomaterials have

osteoinductive properties which are important for osteogene-

sis and osteointegration. Bone morphogenetic proteins (BMPs)

are potent inductors of the osteogenic and angiogenic activ-

ities during bone repair. The BMPs can regulate the produc-

tion and activity of some growth factors which are necessary

for the osteogenesis. Since the introduction of BMP, it has

added a valuable tool to the surgeon’s possibilities and is

most commonly used in bone defects. Despite significant evi-

dences suggesting their potential benefit on bone healing,

there are some evidences showing their side effects such as

ectopic bone formation, osteolysis and problems related to

cost effectiveness. Bone tissue engineering may create a local

environment, using the delivery systems, which enables BMPs

to carry out their activities and to lower cost and complication

rate associated with BMPs. This review represented the most

important concepts and evidences regarding the role of BMPs

on bone healing and regeneration from basic to clinical appli-

cation. The major advantages and disadvantages of such bio-

logic compounds together with the BMPs substitutes are also

discussed. VC 2014 BioFactors, 00(00):000–000, 2014

Keywords: bone morphogenetic protein; bone tissue engineering and

regenerative medicine; bone healing; delivery system; BMPs

substitutes

1. IntroductionInnate capacity of bone for regeneration and healing signifi-cantly reduces as size of the bone defect increases [1–3]. Sev-eral conditions such as bone loss, trauma, cyst or tumor resec-tion, bone diseases, osteoporosis and osteomyelitis mayproduce large bone defects [1,3]. In such situations, a bonegraft is often applied to improve and accelerate bone regener-ation. The iliac crest autologous graft (ICBG) is considered asthe gold standard owing to its osteogenic, osteoinductive,osteoconductive and osteointegrative properties [1,2]. How-ever, its drawbacks including the donor site morbidity, pain,

VC 2014 International Union of Biochemistry and Molecular BiologyVolume 00, Number 00, Month/Month 2014, Pages 00–00

*Address for correspondence: Ahmad Oryan, DVM, PhD, Professor ofComparative Pathology, Department of Pathology, School of VeterinaryMedicine, Shiraz University, Shiraz, Iran. Tel.: 198-7112286950; Fax: 98-7112286940; E-mail: Oryan@shirazu.ac.ir.Received 18 May 2014; accepted 26 July 2014DOI 10.1002/biof.1177Published online 00 Month 2014 in Wiley Online Library(wileyonlinelibrary.com)

BioFactors 1

limited availability, and further surgery limit its application[2,4–6]. Allogeneic and xenogeneic bone grafts are otheroptions but they have significant disadvantages composed ofan increased risk of disease transmission such as HIV andhepatitis, lack osteogenic property, lower osteoactivity thanautograft, donor incompatibility, and the possibility of graftrejection [2,6–8]. Bone tissue engineering as a new technol-ogy attempts to find a better solution to overcome these limi-tations and improve bone healing [7,9,10]. It triggers bonehealing via the employment of an osteogenic cell source suchas stem cells, an osteoinductive factor to promote healing likegrowth factors and an osteoconductive bioscaffold [2,3,11,12].Each of these can be used alone (monotherapy) or designedin combination with other components as poly-therapy[13,14]. One of the most important features that a tissue engi-neered bio-implant should exhibit is osteoinductivity usuallyprovided by growth factors [14–16]. Osteoinduction is the pro-cess of differentiation of the mesenchymal stem cells (MSCs)into osteoprogenitor cells and ultimately into osteoblasts toform new bone [14,15]. Among the osteoinductive agents,growth factors are the most important examples of the heal-ing promotive factors [2,17,18]. One of the most potentosteoinductive growth factors are multifunctional cytokinesbelonging to b-TGF superfamily, namely bone morphogeneticproteins (BMPs) [4,19,20]. BMPs exert significant inductiveeffects on different stages of bone healing process such as theinflammatory reaction, angiogenesis, the soft and hard callusformation, and bone remodeling [21,22]. Despite the signifi-cant positive effects of BMPs, their use is going to be limiteddue to several drawbacks including their rapid degradation,high costs, safety and efficacy concerns, need to high doses,osteolysis, ectopic bone formation, and soft tissue swelling[23,24]. Therefore, there is a considerable need for providinga suitable delivery system or vehicle for controlled and con-tinuous releasing of BMPs [24]. Collagen is the only approvedcarrier for this regard due to being natural and its good bio-degradability and biocompatibility [25,26]. However, due tolittle affinity of BMPs for collagen, they rapidly release fromthe carrier [1,27]. Although different carriers [27–30] andmethods for enhancing the affinity of the delivery systems[31,32] have been developed and examined for the delivery ofBMPs, this area of research is still a considerable challenge.On the other hand, given the disadvantages associated withBMPs, newer methods and strategies should be designed anddeveloped with the aiming to reduce their complications, orto substitute them with other osteoinductive agents such assimvastatin, strontium and nano- hydroxyapatite (nHA)[33–36]. In the present review, the most important conceptsregarding BMPs including their mechanisms of actions, appli-cation modalities, healing efficacy, and their advantages anddisadvantages have been discussed based on their basic toclinical applications. We finally introduced the BMPs substi-tutes and have provided some future directions for those whoare in close contact with bone regenerative medicine andreconstruction.

1.1. History of BMPsThe BMPs were discovered by Urist [37] when demineralizedbone matrix (DBM) implanted into ectopic sites in rats wasable to induce bone formation. This excellent discovery waspublished in Science, titled “Bone formation by auto-induction.” In the following years later, he named the pro-teins responsible for this effect “Bone morphogeneticproteins” [38]. Before the production of recombinant BMPsdeveloped, these proteins were obtained via their isolationfrom the bone. This procedure was difficult because onlyapproximately 1 to 2 mg of BMP was obtained from 1 kg ofcadaver bone [24]. Identification and cloning of differentBMPs have been carried out during the purification of theseproteins from bovine bone. Afterwards, using the recombi-nant gene technology, the recombinant human BMPs(rhBMPs) were synthesized [39]. The BMPs are low molecularweight non-collagenous glycoproteins belonging to the trans-forming growth factor-beta (TGF-b) superfamily [19,20].Other members of TGF-b superfamily consist of activins andinhibins [19]. To date, over 20 homodimeric or heterodimericmorphogenic proteins have been identified in human beingsand other species that they play a critical role in the develop-ment and function of many cell types in various tissues. How-ever, it should be highlighted that only a few members of thisfamily are truly osteogenic [19,39].

For the synthesis of BMPs, at first a large precursor mole-cule comprised of a poorly conserved amino (N)-terminal sig-nal peptide, a pro-region/domain and a highly conserved car-boxy (C)-terminal mature region is formed [40]. BMPs exceptfor BMP-15 contain the mature domain with seven cysteineresidues involving in intra- and inter-molecular disulfidebonds [41]. N-linked glycosylation site in the center of theTGF-b is shared by BMP-2, 4, 5, 6, 7, and 8, but absent inBMP-3 [42,43]. Additionally, it has been shown that themature domain of some BMPs such as BMP-12, 13 and 14 isnot N-glycosylated [40,44]. The mature region of BMP-15includes a 17 kDa band that has been reported to be O-linkedglycosylated. However, its physiological significance is stillunknown [41]. BMPs exhibit the classical TGF-b superfamilyarchitecture with covalently disulfide-linked (for BMP-15,non-covalently) dimeric structures containing a cystine knotmotif, the beta strands and the conserved a-helix [41,45].Dimeric molecules may be either homodimers, containing ofthe same subunits, or heterodimers, as both subunits are thesame [39]. Some of the BMPs such as BMP-2, 4, 6, 7, and 9contain a heparin binding domain that enables interactionswith extracellular matrix elements [20,46]. Changes in pHreduce bioactivity of the BMP-2 and therefore it should bereconstituted with carrier proteins to maintain its bioactivityprior to implantation [39]. Recombinant human BMP-2 wasfirst cloned and expressed in Chinese hamster ovary (CHO)cells in 1988 by Wozney et al. [47]. Currently, the clinicallyavailable rhBMPs are all derived from the mammalian cellcultures transfected by the BMP-gene. One of the major prob-lems regarding clinical application of such rhBMPs is their

BioFactors

2 Bone Morphogenetic Proteins in Bone Healing

high cost due to the necessity for high dosage. According to arandomized clinical trial study, production of the rhBMP-2from BMP-gene-transfected Escherichia coli (E. coli)(ErhBMP-2) has been shown to be a low cost strategy withhigh efficiency. In that study, the healing efficacy of theb-TCP/HA bone graft material containing ErhBMP-2 wasexamined in alveolar bone regeneration. ErhBMP-2-coatedbeta-three calcium phosphate/hydroxyapatite (b-TCP/HA)graft material was more effective than conventional b-TCP/HA alloplastic bone graft materials [48].

1.2. Classification and Functions of BMPsBMPs perform fundamental activities in regard with the devel-opment of not only the musculoskeletal tissue (bone, cartilage,and tendon) but also of many other tissues such as teeth, nerv-ous system, eye, lung, heart, pancreas, liver, kidney, ovary,and testis [15,49]. They induce a consecutive cascade of eventsfor chondro/osteogenesis, composed of chemotaxis, prolifera-tion of MSCs and osteoprogenitor cells and differentiation,angiogenesis, and synthesis of extracellular matrix. Their reg-ulatory effects depend on the type of targeted cell, its differen-tiation status, the local concentration surrounding the ligandand interaction with other factors [20,50]. In bone tissue,BMPs are produced by MSCs, osteoprogenitor cells, chondro-cytes, osteoblasts, endothelial cells, and platelets within theextracellular matrix [20,51]. They are released during bonerepair and remodeling. After release of BMPs into the extracel-lular matrix, the matrix acts as a temporary storage for BMPs[19]. By comparing among the derived amino acid sequence ofBMPs present in osteoinductive extracts of bone, they can becategorized into four subclasses (Table 1) [15,19,20,49]. Thefirst subclass involves BMP-2 and BMP-4, the second subclassincludes BMP-5, BMP-6, BMP-7 (also known as osteogenicprotein-1, OP-1), and BMP-8 (OP-2) which are slightly largerproteins than the former subclass. BMP-9 and BMP-10 formthe third osteogenic subclass [19,39,50]. Finally, BMP-3 orosteogenin forms the fourth subclass that acts as BMP inhibi-tors [19]. The other members of the BMP family such as BMP-12 Growth and Differentiation Factor (GDF-7), BMP-13 (GDF-6), BMP-14 (GDF-5), and BMP-15 (GDF-9B) do not have osteo-genic activities. BMP-1 is not belonging to the TGF-b super-family and lacks any osteogenic property. However, this metal-loproteinase may prevent the action of BMP antagonists byproteolysis of their binding proteins. Therefore, BMP-1 maymodulate BMPs activities [50]. BMP-3 is the most abundantBMP in the demineralized bone, accounting for 65% of thetotal BMP stored in the bone matrix. BMP-3 plays an impor-tant role in fracture healing and mechanical loading of theskeleton as well as modulation of osteogenic BMPs [52,53].BMP-2, 4, 6, and 7 as well as BMP-9 are members of mainBMP family subclass that are responsible for inducing boneand cartilage regeneration and formation [54]; so that, it hasbeen shown that loss of both BMP-2 and BMP-4 leads to asevere failure in osteoblast differentiation [55]. BMP-2 pro-motes migration, proliferation, and differentiation of the osteo-

progenitors and production of extracellular matrix [56].Thereby, it is characterized as the most commonly usedgrowth factor for bone regeneration and the most promisingfactor for bone tissue engineering [39,57,58]. It is an essentialcomponent of the signaling pathway controlling fracture repair[58]. BMP-7 as an osteoinductive agent can be used for treat-ing resistant non-unions in the upper and lower limbs [59].BMP-9 is one of the most potent BMPs in inducing osteogenicdifferentiation of the MSCs and also preadipocytes via activat-ing BMP/Smad signaling pathway [60]. BMP-12 and BMP-14are unable to induce bone formation; instead, they induce theformation of cartilage and tendon [54]. Among all the BMPsubfamily members, BMP-2 is the most investigated member,not only because of being involved in almost all stages of boneregeneration process [61], but also for its promising resultswhen used in the clinical patients [39,58]. Moreover, rhBMP-2is one of the only two approved BMPs (and rhBMP-7) for clini-cal use in combination with absorbable bovine type I collagensponges in long bone fracture healing and spinal fusions [24].BMPs particularly the BMP-2 and BMP-7 have been applied inclinical use to enhance spinal fusion [62–65], for the alveolarridge and maxillary sinus augmentation [66], and for the treat-ment of long bone non-union fractures [67]. In 2002, theUnited State Food and Drug Administration (FDA) approvedthe administration of rhBMP-2 (Infuse, Medtronic) in the ante-rior lumbar interbody fusion and spinal fusion as a substituteto the iliac crest bone graft [68] and in 2004 for the open tibialfractures [69]. Since then, application of this growth factor inspinal fusion surgery has increased rapidly. In 2008, the FDAissued a public health notification of potentially life threatingcomplications associated with swelling of neck and throat afterapplication of the rhBMP-2 in the cervical spines [62,63]. Inthe recent years, members of the BMP family have receivedthe highest attention among potential factors for bone repairbecause of their ability to induce matrix synthesis and promoterepair in different connective tissues including bone [58,70].

1.3. BMPs and Bone HealingBMPs play a central and main role in the regulation of thethree major stages of fracture healing: the inflammatoryresponse, the chondrogenic phase, and the osteogenic phase[61,71,72]. The exact timing of the stimulatory effects of BMPson bone metabolism remains unknown. However, it is possibleto be initiated during the inflammatory stage [61]. Upon frac-ture, a hematoma forms surrounding the ridges of the fracturesite that activates an inflammatory reaction initiating the heal-ing process [2,73]. In order to repair the bone, the cellsinvolved in osteogenesis, chondrogenesis and angiogenesissuch as progenitor cells of the MSCs and endothelial cells mustbe present at the fracture site [74]. These processes are regu-lated by several local and systemic factors produced andreleased by cells of bone, bone marrow, blood vessels, perios-teum, and surrounding soft tissues [2]. After invasion of theosteoprogenitor cells into the hematoma, chondrogenesis takesplace so that the soft callus is formed [2,73]. Following the

Oryan et al. 3

production of the soft callus, osteoblasts start to mineralize thecartilaginous matrix and on the other hand, the calcifiedmatrix is resorbed by chondroclasts and subsequently a wovenbone or hard callus is replaced [2,73,74]. The woven bone orhard callus must be converted to lamellar bone during remod-eling phase. The woven bone is absorbed by the osteoclastsand lamellar bone is formed [73]. Bone remodeling consists ofstimulation of the preosteoclasts to differentiate into osteo-

clasts, osteoclastic resorption, preosteoblast migration to theresorption site, differentiation into osteoblasts and bone for-mation. The equilibrium between bone formation and boneresorption is regulated by the paracrine and autocrine growthfactors such as BMPs [21]. The importance of BMPs activityduring this stage is debated [74]. However, the BMPsexpressed in osteoclasts can initiate the remodeling phase ofbone healing, so that the osteoclastic BMPs, especially the

Types of BMPs, their tissue, and gene location and their activities

Type of BMP synonyms Tissue location Gene locus Functions in bone/cartilage

BMP-1 - 8p21 Not part of TGF-b superfamily

BMP-2 BMP-2A Bone, cartilage, teeth,

muscle, liver, heart, testis

20p12 Osteogenic, osteoinductive,

initiated bone regeneration

and healing, osteoblast

differentiation, chondrogenesis

BMP-3 osteogenin Bone, cartilage, teeth kidney, lung 14p22 Most abundant BMP in bone,

inhibition of osteogenesis,

BMP inhibitor

BMP-4 BMP-2B Bone, cartilage, teeth, muscle, kidney,

gut, uterus, liver, pancreas,

lung, heart, ovary, testis

14p22–23 Osteogenesis, chondogenesis

BMP-5 - Bone, cartilage, lung, kidney,

pancreas, heart

6p12.1 Cartilage development

BMP-6 Vgr-1 Cartilage, joints, heart, ovary, liver,

ureter, pancreas, epidermis

6p12.1 Osteogenic, osteoblast

differentiation,

enhance and accelerate

bone regeneration

BMP-7 OP-1 Bone, cartilage, lung, kidney,

synovium, liver, heart, ovary,

eye, testis, epidermis

20q13 Osteogenesis

BMP-8 OP-2, BMP-8B Bone, ovary, testis 1p35-p32 Osteoinduction, osteogenesis,

chondrogenesis

BMP-9 GDF-2 Liver, CNS - Induce oseogenic differentiation

of MSCs, chonrogenesis

BMP-10 - Heart 2p14 Not

BMP-11 GDF-11 CNS - Not

BMP-12 GDF-7, CDMP-3 Cartilage, tendon, CNS - Chondrogenesis, tendon healing

BMP-13 GDF-6, CDMP-2 Cartilage, tendon - Tendon healing

BMP-14 GDF-5, CDMP-1 Cartilage, tendon, eye chondrogenesis

BMP-15 GDF-9B Ovary Xp11.2 Oocyst development

BMP 5 bone morphogenetic protein; Vgr 5 vegetal related; OP 5 osteogenic protein; GDF 5 Growth and Differentiation Factor; CDMP 5 Cartilage-

derived morphogenetic protein; CNS 5 central nervous system.

TABLE 1

BioFactors

4 Bone Morphogenetic Proteins in Bone Healing

BMP-6, can stimulate the differentiation of the preosteoblaststo form a calcificable bone matrix [21]. Based on the study ofMarsell and Einhorn [74], BMP-2 and BMP-4 are produced bythe mesenchymal progenitor cells and thenafter, these cellsare differentiated into the chondrogenic cells. In their experi-ment, the highest expression level of the BMP-6 was seen inthe second phase and the expression of BMP-3, 4, and 5 alsoincreased at that stage. Osteogenesis phase occurs on days 14to 21 after injury. In this phase of healing, expression of theBMP-1, 2, 3, 4, 5, 6, 7, and 8 was high, and the expression ofBMP-3, 4, and 7 was at the maximum level. Expression of theBMP-7 occurred during days 14 and 21 and thereby, it playeda significant role in regulation of the osteogenic stage of frac-ture healing. Cho et al. [61] compared temporal expressionpatterns of several BMPs including BMP-2, 3, 4, 5, 6, 7, and 8as well as some other proteins during a 28-day period in amouse tibial fracture model. Briefly, BMP-2 was the earliestgene that was induced and it showed maximum expression onday 1 after fracture, during the period when the MSCs wererecruited to the fracture site to promote the chondrogenesis.The above findings represent the crucial role of the BMP-2 ininitiating the healing cascades. The second peak of the BMP-2expression was seen during the period of osteogenesis. TheBMP-3, 4, 7, and 8 were expressed within the restricted periodsince day 14 to day 21, at the time when resorption of the cal-cified cartilage is taking place and osteoblast recruitment andbone formation is maximal (osteogenic period). Expression ofthe BMP-5 and BMP-6 was observed between days 3 to 21 sug-gesting their stimulatory role in the chondrocyte maturation.BMP-6 as an autocrine factor can initiate chondrocytic matu-ration that overlaps with the role of BMP-2 [61]. They con-cluded that BMPs are actively involved in fracture healing,although they have distinct temporal expression patterns. Inanother study conducted by Yu et el. [71] it was showed thatall of the BMPs play important roles in various stages of heal-ing and regulate various cell types such as chondrocytes, peri-osteal cells and inflammatory cells in the granulation tissuewhich regenerates during the early stages of bone healing.Considering close link between angiogenesis and osteogenesis,it seems that BMPs can enhance angiogenesis by inducing vas-cular endothelial growth factor (VEGF) [71]. They believed thatBMP-2, 6, and 9 can induce osteogenic differentiation of theMSCs and osteogenesis in the early stages of healing. Indeed,BMP-2 was strongly expressed in chondrocytes and activatedin the early stages, where its role in recruiting precursor cellsis a key to initiate healing. Evidences suggest that BMP-2 maybe the most important BMP involved in bone healing and alarge number of studies carried out on this type of BMPs con-firmed this fact. Cottrell et al. [22] examined the effect ofrhBMP-2 on expression of the endogenous osteogenic growthfactors in rat femoral fracture model. Radiographic findingsdemonstrated that treatment with rhBMP-2 enhances callusformation and bridging time. On the other hand, they showedthat treatment with rhBMP-2 significantly increased the mRNAlevels of the BMP-2 on day 4, BMP-4 on days 2, 4, 8 and 10,

BMP-7 on day 2 and reduced the mRNA levels of the BMP-2on day 2 and 10 and BMP-6 on days 2, 10, 14 and 21 afterfracture. In addition to the direct effects on cell differentiation,they suggested that rhBMP-2 promotes bone healing by induc-ing expression of the endogenous osteogenic growth factorsthat could contribute to bone formation. Collectively, BMP-2seems to play a key role in fracture healing, especially at earlystages of fracture healing.

1.4. BMP Signaling PathwayAlthough multiple pathways are involved in the bone metabo-lism, the BMP/SMAD pathway has received the most attentionto date (Fig. 1). The BMP signaling cascade is initiated fromthe cell surface [55,75]. At first, most of the BMP ligands bindeither first to a BMPR-I receptor that then recruits BMPR-II orcooperatively to pre-form receptor complexes composed ofBMPR-I and BMPR-II [20,39]. Some of the BMPs such as BMP-7 at first bind to the type II receptor followed by recruitingand phosphorylation of the type I receptor [76]. Nevertheless,it is believed that the type II receptor does not actually bindthe ligand but stabilizes the type I receptor and acceleratesligand binding to the type I receptor [23,75,77]. After ligandbinding and activation of the BMPR-II that is constitutivelyactive, it catalyzes phosphorylation of the BMPR-I, which inturn, phosphorylates the Smad1, 5, and 8 [75,77]. BMP-7 bindsat first to the type II receptor (Act-II) and then trans-phosphorylates the type I receptor, Alk2 [76]. After releasingfrom the receptor, these activated R-Smads form a proteincomplex with a Co-Smad named Smad-4 which translocatestoward the nucleus. The phosphorylated R-Smads complexwith the Smad-4 enters into the nucleus to activate “runt-related transcription factor 2” (Runx2) and Osterix (Osx) genes[20,23,75]. Regulation of osteoblast differentiation and bonemetabolism induced by BMPs occurs upon expression of theRunx2 and Osx. Therefore, deletion of the Runx2 and Osxcauses loss of ossification [20]. In mammals, seven type Ireceptors have been identified termed activin receptor-likekinase 1 to 7 (Alk1- Alk7). Among these, BMPs preferably bindto the Alk1, 2, 3, and 6, while other members of TGF-b super-family such as TGF-b1, 2 and 3 and activins bind to the Alk5and Alk4, respectively [23]. Four type I (Alk1, BMPR-IA/Alk3,BMPR-IB/Alk6, and Alk2) and three type II serine/threoninekinase (BMPR-II, activin type II receptor (ACVR-II or Act-II),and ACVR-IIB or Act-IIB) BMP receptors are able to bind tothe BMPs [20,23]. Smad proteins play a crucial role in relayingthe BMP signal from the receptor to the target genes in thenucleus [39]. The Smad family includes eight members thatare divided into three groups: (1) the receptor-regulatedSmads (R-Smads) including Smad1, 2, 3, 5, and 8; (2) the com-mon mediator Smad (Co-Smad) such as Smad4; and (3) theinhibitory Smads (I-Smads) composed of Smad6 and Smad7.Among these, the Smad1, 5 and 8 are substrates for the BMPreceptors [23,78]. The Smad family more specifically Smad1, 5and 8 have been identified as the downstream effectors of thephosphorylated type I receptor [79]. The BMP pathway may be

Oryan et al. 5

inhibited via some natural extracellular proteins such as Nog-gin and Chordin and the inhibitory Smads including Smad6and Smad7 [20,70,79]. In addition to the Smad proteins, BMPscan transduce signals via a Smad-independent or non-Smadsignaling pathway via mitogen activated protein kinases(MAPKs) such as ERK, p38, and JNK, small GTPase and Aktpathways (Fig. 1) [75,77]. The p38/ERK MAPK pathway isrequired for the BMP-induced osteoblast differentiation andbone formation. The ERK1/2 MAPK and TGFb-activatedkinase-1 (TAK1) are important for regulating the Smad signal-ing [75,80]. TAK1 acts as a BMP agonist and synergizes with

the Smad1/5, while it also interacts with the R-Smads andinterferes with the R-Smads transactivation suppressing BMP-induced osteoblast differentiation [80]. Additionally, TAK1 isable to promote the Smad1/5/8 phosphorylation and thus itcan be an essential modulator for the canonical BMP-Smadpathway [81]. TGF-b signaling promotes osteoprogenitor pro-liferation, differentiation and commitment to the osteoblasticlineage via the selective mitogen activated protein kinases(MAPKs) and Smad2/3 pathways [75,77]. Nevertheless, it isunclear how BMPs promote bone differentiation or when andwhere the BMP signaling is active during bone regenerationprocess [55].

1.5. BMP Antagonists and RegulatorsDue to the importance of the BMPs in bone metabolism, fac-tors limiting the effects of BMPs play important roles in regula-tion of the bone metabolism. The activities of BMPs are time-dependent in a sequential cascade of events which result inangiogenesis, chondrogenesis, and subsequently osteogenesis[20]. The concentrations and actions of BMPs are locally regu-lated and tempered via several antagonists. Based on therecent investigations, the BMP signaling inhibitors play animportant role in bone healing and formation [78]. High BMPlevels stimulate the expression of these molecules that nega-tively affect fracture healing. These antagonists can act atthree levels including extracellular, the receptor or membraneand intracellular levels [75,78]. After the production andrelease of these antagonists by osteoblasts into the extracellu-lar matrix, they bind and form complexes with BMPs and thusprevent them from binding to their receptors. The mainextracellular antagonists of BMPs include Noggin, Chordin,Follistatin, Follistatin-like, BMP-3, Gremlin, and twisted gas-trulation (Tsg) [19,20,50,78,82]. Indeed, BMPs and their antag-onists moderate the fracture healing process. By a feedbackregulatory mechanism, the BMPs can also regulate the expres-sion of their antagonists [52]. Noggin is mainly expressed incells of mesenchymal origin such as osteoblasts and chondro-blasts and regulates osteoblast differentiation and bone forma-tion [52]. Although noggin inhibits several BMPs includingBMP-2, 4, 5, 7, 13, 14, currently, it is not clear why the BMP-3, 6, 9, 10, and 15 signaling is not affected [52,82]. Although,noggin can bind to the BMP-6, it does not decrease the activityof this BMP in osteoblast differentiation [82]. Noggin acts withthe BMP ligands at the extracellular region by binding tightlyto the BMPs and preventing them from binding to both type Iand type II receptors and thus blocking the BMP signaling[49,52,82].

Expression of Chordin in osteoblasts is little, and it isexpressed in chondrocytes and regulates their maturation[52,82]. Chordin is the antagonist of BMP-2, BMP-4, and BMP-7, and inhibits the BMP signaling via blocking the binding tothe BMP receptors [47]. BMP-3 is the antagonism of BMP-2and BMP-4 inducing osteogenesis [20,52]. BMP-3 inhibitsosteoblast differentiation and responsiveness to the BMP-2 viaantagonizing the BMP-2 signaling. BMP-3 can bind to the type

BMP-2 signaling pathways. Canonical Smad-

dependent pathway is initiated by binding of the

BMP ligands to a heteromeric complex of type I

(BMPR-I, Alk1, Alk2, Alk3, and Alk6) and type II

(BMPR-II, Act-II, and Act-IIB) transmembrane recep-

tors. Subsequently, the type II receptor phosphoryl-

ates and thus activates the type I receptor, which in

turn phosphorylates Smad1, 5, and 8 (R-Smads). The

phosphorylated R-Smads then form a complex with

Smad 4 (Co-Smad) and translocate into the nucleus

to modulate the transcription of target gene that reg-

ulate bone healing and regeneration. Smad 6 and

Smad 7 (I-Smads) can either prevent association of

R-Smads with Smad-4 or directly inactive type I

receptor and thereby inhibit R-Smads phosphoryla-

tion. Besides signaling via Smads, the BMP signal

can also be transduced via activation of p38

(MAPK14), ERK (MAPK1) and JNK (MAPK8) by a

complex composed of TAK1 (MAP3K7IP1), its activa-

tor called TAB1 (MAP3K7) and the X-linked inhibitor

of apoptosis protein (XIAP). MAPKs are transported

into the nucleus and activate transcriptional factors

initiating specific gene expression. On the other

hand, activation of PI3 kinase (PI3K) via the men-

tioned complex leads to transcriptional regulation by

Akt and non-transcriptional regulation (like direct reg-

ulation of cytoskeleton re-arrangement) by Rho

GTPase pathways.

FIG 1

BioFactors

6 Bone Morphogenetic Proteins in Bone Healing

II receptors such as Act-IIB and prevents the BMPs from ini-tiating signal transduction on target cells [83]. Thereby, itblocks the BMP-2-mediated differentiation of osteoprogenitorcells into osteoblasts [53]. Follistatin is an antagonist of theBMP-2, BMP-4, and BMP-7 that inhibits their effects throughbinding to the BMP receptors via BMPs, thus it forms a tri-meric complex with the BMPs and the BMP receptors [50,52].Another antagonist of BMPs is Tsg that binds to the Chordinand BMP-4, as a co-factor. They form a tertiary complextogether and therefore, inhibition of the BMP signaling isintensified through this complex. Moreover, Tsg can exert itsinhibitory effects on the BMP signaling by binding to the BMPsin the absence of Chordin [50,52]. Furthermore, the BMP sig-naling pathway can be negatively regulated by expression ofthe membrane pseudo-receptors (BMP and activin membranebound receptor, BAMBI), at receptor level [78]. BAMBI isstructurally similar to type I BMP receptors in the extracellulardomain, while it has no intracellular kinase domain. There-fore, BAMBI inhibits further signaling inside the cell by inter-fering with the formation of receptor complexes [20]. At intra-cellular level, the BMP signaling pathway is negativelyregulated by the activation of inhibitory Smads such as Smad6and Smad7, Smad8b, Smad ubiquitin regulatory factor(Smurf)-1 and Smurf-2 [20]. The signal inhibition by I-Smadsoccurs either through their interaction with the activated typeI BMP receptors, or through interfering with the Smad4 andthe formation of an inactive R-Smad/I-Smad complex. Gener-ally, Smad-6 prefers to inhibit the BMP signaling, whereasSmad7 can inhibit both the BMP and TGF-b signaling [23,50].For more clarification, Smad6 interferes specifically with theSmad1/5/8 pathway, while Smad7 is able to interfere with bothof the Smad1/5/8- mediated and Smad2/3-mediated signaltransduction [23]. Although Smad7 binds to the activatedBMPR-I to prevent R-Smads from becoming active, Smad6binds directly to the R-Smads competing with Smad4 to pre-vent R-Smads/Co-Smads complex formation [80]. Smurfsinhibit the BMP pathway by binding to R-Smads and promotingtheir degradation. In addition, Smurfs can bind to the BMPtype I receptors through I-Smads and thus induce degradationof the receptors [50]. Ehnert et al. [23] demonstrated thatrhTGF-b blocks rhBMP signaling in osteoblasts by reducing theexpression of factors required for the BMP signaling. Mean-while, Endolin (CD105), a transmembrane co-receptor, isinvolved in down-regulation of the BMPs [20]. Additionally,CRIM1 which is a transmembrane protein containing cysteine-rich repeats, similar to the chordin, regulates and depressesdelivery of the BMPs to the cell surface [20].

Several agents have the ability to positively affect the BMPpathway either directly or indirectly, including statins. Statinssuch as lovastatin and simvastatin (hydrophobic statins), prav-astatin, rosuvastatin (hydrophilic statins), atorvastatin, pitavas-tatin, and fluvastatin are widely used for reducing cardiovas-cular diseases and lowering cholesterol. It has been shownthat statins increase the expression of BMP-2 in bone cells[84–86]. Pravastatin is unable to stimulate the BMP-2 expres-

sion and therefore, it cannot induce new bone formation.Indeed, all statins except pravastatin can stimulate BMP-2 pro-moter activity [84,86,87]. Relaxin (Rln) is a polypeptide hor-mone belonging to the insulin superfamily that plays a signifi-cant role in angiogenesis, and collagen turn-over [88]. Rlnreceptors have been detected on the osteoblasts and osteo-clasts and Rln stimulate osteoclast differentiation from itshematopoietic precursors [87–89]. An investigation carried outby Moon et al. [88] has examined the effects of Rln on osteo-blast differentiation and bone formation induced by BMP-2.Rln synergistically enhanced the BMP-2-induced Smad phos-phorylation and Runx-2 expression. In addition, retinoic acids,derivatives of vitamin A, have showed synergetic effects withthe BMP-9 and BMP-2 in inducing osteogenic differentiationfor the MSCs and preadipocytes [60].

1.6. Delivering BMPsNew bone formation may be achieved by direct application ofBMPs alone. However, this approach requires application oflarge doses of BMPs, since they have a short systemic half-lifeof about 7 to 16 min in bloodstream and undergo rapid degra-dation by proteinases after administration [90]. Indeed, thesefactors have short retention at the defect site that may fail toachieve signaling. Thus, such clinical applications use the dos-age much higher than the effective dose to compensate fastdegradation of the proteins [91]. On the other hand, singleapplication of BMPs seems to stimulate macrophages, lympho-cytes and plasma cells, and activate moderate production ofthe anti-BMP antibodies [24]. In humans, the physiologicalconcentrations of the BMPs are estimated to be about 2 ng/g ofbone, while such concentrations of BMPs are sufficient to exerttheir activities, compared to the levels of mg used in most clin-ical trials [24]. Therefore, application of specific carriers sup-plemented with BMPs can improve their osteogenic activities[24,92]. The carriers for the growth factors delivery shouldretain the growth factors at the defect site and avoid their sys-temic diffusion, maintain their local concentrations andrelease at the implantation site, since bone healing efficiencyis correlated with the prolonged presence of the BMPs at theimplantation site [20,24,93]. Moreover, the carrier shouldmaintain the structural conformation and biological propertiesof the incorporated growth factor during its releasing period.With a constant and prolonged release of the growth factorfrom the delivery carriers, the growth factor can act more effi-caciously [1,94]. On the other hand, because the rhBMPs areproduced in a liquid form that easily dissolve and subsequentlyinactivate in vivo, their clinical application requires the pres-ence of a carrier vehicle that allow a high concentrationcapacity and time-controlled release [94]. Furthermore, thecarrier may have osteoconductive property that allows cellinfiltration and ingrowth of new bone and blood vessels[24,92]. An optimal carrier or delivery system should be three-dimensional and consist of a highly porous network of inter-connected pores to promote cell ingrowth, adhesion, prolifera-tion, and differentiation [24,58,92]. The carrier should be

Oryan et al. 7

biodegradable while protecting BMPs from degradation. Onthe other hand, it should be biocompatible but stimulate aminimal inflammatory reaction. The ideal delivery systemshould be transformed from liquid to solid in situ, presentadhesion for cell ligands, contain affinity sites for growth fac-tor binding, permit the integration of the newly formed bonewith native surrounding tissues, and fill the defect [92,95,96].Additionally, it should be non-toxic, non-allergic and do notinduce any adverse reactions in the body [20]. Meanwhile,appropriate mechanical properties are the essential and fun-damental requirements of an ideal scaffold [97]. Moreover,when using as a delivery system in animal and human studies,it should be confirmed that it is non-carcinogenic and non-toxic. Finally, it should be easily sterilized, stable and cost-effective [98,99]. Nonetheless, none of the available carrierspossess all the mentioned features to be considered as idealmaterial [96].

However, collagen, the main organic constituent of boneand the most abundant protein in the body, is the only carrierapproved for clinical application of BMPs [24,100]. Despite thepoor biomechanical strength of the collagen, its biocompatibil-ity, biodegradability, and low immunogenicity are desired[20,24,25,101,102]. For its preparation, the bovine type Iabsorbable collagen sponges (ACS) are soaked in the proteinbefore implantation [24]. Unfortunately, most growth factorshave little natural affinity for collagen. Protein deliverythrough a collagen sponge in the presence of BMP results forup to 8 days, locally [91]. BMPs rapidly liberate from the colla-gen sponge resulting in a high initial burst release (30%),while a prolonged or sustained release of BMPs is critical andessential for their osteoinductive actions [1,103]. Thus, by slowand sustained release of BMPs from the carrier and theirretention at the target site, it palliates most of the problemsassociated with the application of BMPs [24]. The main con-cern regarding the delivery of BMPs is their retention to opti-mize their osteogenic potential at the injury site. The criticalfactor is the system delivery used that affects BMPs retention[99]. In order to overcome this problem, Hannink et al. [32]fabricated a carrier-based delivery system with a localizedsustained release. Heparin, a sulfated polysaccharide, pos-sesses a good binding affinity with many biologically importantproteins such as BMPs. It covalently attaches to a cross-linkedcollagen coated TCP/HA bone substitute and load with BMP-2.Therefore, it incorporates into biomaterials to immobilizeBMPs through its growth factor binding domain. This heparin-containing delivery system revealed the effectiveness of hepa-rin in the controlled released of BMP-2. In fact, binding ofBMPs to heparin stabilizes these factors and protects themfrom proteolytic degradation. Furthermore, half-life of theBMPs has been shown to be prolonged by binding to the hepa-rin. On the other hand, heparin could also enhance the osteo-blastic differentiation induced by BMPs, and thus induce boneformation [32]. Additionally, rapid and high initial burstrelease of BMP-2 with titanium-based implants has also beenprevented via heparin-based delivery systems by Lee et al.

[31]. In another study, Lee and colleagues [1] evaluated thebone regeneration via BMP-2 signaling using the fibronectin-like peptide amphiphile nanofibers with a strong potential tobind heparin sulfate chains in the pores of an absorbable col-lagen scaffold in a rat femoral bone defect model. Theyshowed that a hybrid biomaterial containing collagen scaffoldwith supramolecular nanofibers promoted bone regenerationand amplified the regenerative capacity of the BMP-2. More-over, the degradation rate of the collagen scaffold was reducedby cross-linking treatment [104]. The methods of combiningBMP-2 with the carrier are mostly physical like static adsorp-tion, or physically embedding BMP-2. Hence, the affinity ofthese delivery systems is reduced, and is inadequate for sus-tained controlled release of BMP-2. Many researchers haveused different types of materials covalently functionalized withBMP-2 [31,32,104–106].

Several types of carriers have been designed and investi-gated to facilitate delivery of BMPs. These carriers are mainlygelatin [30], chitosan [28,105], b-tricalcium phosphate[29,107], hydroxyapatite (HA) [32], polylactide/polyglycolideacid (PLGA) [108], alginate [27], hyaluronic acid [25], andfibrin [109]. Lopiz-Morales et al. [110] used alginate as a car-rier material because of its biocompatibility, and its gelationproperties. In fact, alginate can fill any shape of defect andalso it can incorporate with various agents such as BMPs inorder to be applied as a delivery vehicle. Currently, chitosanthat is a natural biomaterial has received considerable atten-tion and interest in the field of tissue engineering, because ofits properties including enzymatic biodegradability, non-toxicity, and biocompatibility [68,111].

The release of a growth factor can be either diffusion-controlled, solvent-controlled, chemical reaction-controlled, ora combination of these mechanisms [99]. While BMP is physi-cally immobilized in a carrier matrix and released by degrada-tion of the carrier through a chemical-controlled fashion,release of BMP within the pores of a porous scaffold is basedon a diffusion-controlled mechanism [90,92]. The rate of BMPrelease relies on its molecular weight, its conformation, and itssolubility [24]. Gene therapy-based strategies have also beenintroduced to improve BMPs delivery and their effectiveness atthe target site [49,93]. This technology provides the gene forthe protein and results in a higher and more constant level ofBMPs for a sustained time period [112]. To include the BMPsgene into the target cell, a delivery vehicle or viral or non-viralvector is needed [49]. Some viruses including retroviruses andespecially the adenoviruses can be used as carriers of BMPcDNA in bone tissue engineering [93]. Adenoviral vectors caninfect a number of cells, thereby they lead to expression of theBMPs protein. Therefore, they may be considered as a suitablevector for bone regeneration [49,113]. Adenoviral vectors donot incorporate their DNA into the host chromosomal DNA athigh frequency, so that they are not responsible in producinginsertional mutagenesis. In one study, the effect of delayedpercutaneous injection of adenoviral vectors containing codinggenes for BMP-2 and BMP-6 on the healing of large

BioFactors

8 Bone Morphogenetic Proteins in Bone Healing

osteochondral defects in a femoral condyle of pony was eval-uated. Such strategy supported osteochondral regeneration,but was unable to provide long-time quality osteochondralrepair [49].

Viral types of the vectors are considered as the most effi-cient approach available for delivering the BMP gene to thetarget cell [49,93]. However, these vectors could stimulateimmune reactions and thus inhibit transgene expression [112].On the other hand, the gene sequence of some viral vectorsmight integrate within the genome of the host cells, and there-fore may lead to an uncontrolled dissemination or even malig-nant transformations [93]. Therefore, these drawbacks limitthe use of the viral vectors in the field of bone tissue engineer-ing for bone healing [112]. For this purpose, non-viral genedelivery uses several techniques for transfection includingexposing the target to naked DNA, liposomes, and methodslike electroporation. These methods have some advantagessuch as minimal immunogenicity and therefore may be saferthan viral methods. Moreover, it is easier to produce non-viralvectors compared to the viral vectors [114]. Nevertheless,effectiveness of these methods to deliver the desired gene iscurrently under the debate [93,112]. BMP-2 delivery in a con-ventional collagen scaffold requires a high dose to provide apromising outcome, whereas such dosage may result in seri-ous side effects [90]. Therefore, the researchers and orthope-dic surgeons should attempt to find clinically acceptable strat-egies reducing the required dose of BMP-2 by improving thedelivery systems and optimizing the preclinical testing of thenew approaches.

1.7. In Vitro StudiesSeveral in vitro studies have shown the role of BMPs using dif-ferent delivery systems in bone repair and regeneration[58,90,110,115,116]. However, these studies are often consid-ered as quality control examinations and their results may notbe reliable enough for generalizing to animal experimentaland human clinical practices. Herein, some of these in vitroinvestigations are summarized in Table 2. In a study, Sharmaet al. [99] evaluated the release of BMP-2, derived from E. coliexpression system, from bi-resorbable and osteoconductivea-tricalcium phosphate/poly (lactic-co-glycolic acid) (a-TCP/PLGA) nanocomposite, at in vitro level. BMP-2 was successfullyadsorbed onto the surface of the nanocomposite and themajority of the adsorbed BMP-2 released from the carrier dur-ing 2 h. The early rapid release of BMP-2 could promote thedifferentiation of mesenchymal cells into osteoblasts followedby proliferation of osteoblasts through the nanocompositeitself. In another study, Chen et al. [123] evaluated the effectof PRP-released growth factors and microsphere-encapsulatedBMP-2 within a poly(lactide-co-glycolide) cube scaffold on theproliferation and osteoblast differentiation of human adipose-derived stem cells (hADSCs) in vitro. They showed that thesynergistic delivery of PRP-released growth factors and BMP-2effectively stimulate the proliferation of hADSCs and their dif-ferentiation into osteoblasts. Hence, they suggested that sus-

tained delivery of BMP-2 in a combination with PRP to a targetsite may be useful in bone regeneration. In another study,Zhang et al. [56] determined the effects of BMP-2 and VEGFon bone marrow stem cells (BMSCs) in bone regeneration.They showed that both the VEGF and BMPs stimulate the che-motaxis of BMSCs. They revealed that with facilitating themobilization of stem cells and subsequently the differentiationof them into the osteogenic and endothelial cells by BMP-2 andVEGF, respectively, their localized release from the porous silkprotein scaffold can promote bone regeneration.

1.8. In Vivo Experimental Animal StudiesThe animal studies are a bridge between in vitro and clinicalstudies, and thus they should be taken into consideration. Theresults of the in vivo studies are more reliable to judge and toconclude because they are conducted in different species andexamined using standard methodologies. Nonetheless, the ana-tomical and physiologic differences between animals andhuman beings are an important issue that should be consid-ered when generalizing the results of animal studies for clini-cal application. Several animal studies have investigated theefficacy of BMPs delivered by different biomaterials and scaf-folds. The summary of the recent in vivo animal studiesregarding the effectiveness of BMPs on the healing and regen-eration of the bony tissue has been provided in Table 2. b-TCPas an osteoconductive bioceramic material has been widelyused for bone regeneration and repair and is chemically simi-lar to the apatite composition in bone tissue [124]. To obtainboth the osteoconductivity and osteoinductivity essential for atissue engineered construct by taking advantage of the favor-able osteoconductivity of b-TCP and the osteoinductivity ofBMPs, the application of b-TCP as a vehicle for BMPs and theproduction of b-TCP/BMPs composite materials have beendeveloped with the hope that the mixture might be helpful forimproving and accelerating bone regeneration [125,126]. Soh-ier et al. [125] investigated the efficiency of BMP-2 deliveredby macroporous beta tricalcium phosphate (b-TCP) scaffolds.The scaffolds loaded with 15 and 30 mg of BMP-2, wereimplanted into the femoral defects and the back muscles ofrabbits, respectively. Bone was formed within the BMP-2-loaded scaffold pores, both in the back muscles and bonedefects independent of the implant site effect. The results oftheir study indicated the efficacy and suitability of b-TCP scaf-folds as BMP-2 carriers for bone regeneration. The naturalbased fibrin scaffold plays an important role in hemostasis andbone healing and has a considerable value for use in tissueengineering [127].

One of the initial events during the bone healing process isthe blood clot formation, and the fibronectin-heparin complexof the clot enhances binding and bioavailability of the endoge-nous growth factors. Fibrin scaffold can mimic this blood coag-ulation process [110,127]. Autologous fibrin avoids the poten-tial risk of foreign body reactions or infections and thereby, itis an immunecompatible and safe scaffold [127]. Due to highcell binding capacity of fibrin, it can provide a suitable

Oryan et al. 9

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BioFactors

10 Bone Morphogenetic Proteins in Bone Healing

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tin

do

gs

Rh

BM

P-2

/AC

Sca

nce

led

the

bo

ne

rem

od

elin

g

inh

ibit

ion

of

zole

dro

nic

acid

incre

ase

db

on

efi

ll

an

db

on

ere

mo

de

lin

g

[10

3]

CP

Sa

nd

rhB

MP

-2C

hit

osa

nS

ca

ffo

ldR

ab

bit

tib

ial

bo

ne

de

fect

Ind

uce

dm

ore

bo

ne

form

ati

on

insca

ffo

lds

wit

hb

oth

CP

Sa

nd

rhB

MP

-2th

an

tho

se

wit

ho

ut

CP

Sa

nd

rhB

MM

P-2

TA

BL

E2

Oryan et al. 11

(Co

nti

nu

ed

)

Re

fere

nce

Gro

wth

facto

rC

arr

ier

ma

teri

al

Fo

rmu

lati

on

of

ma

teri

al

Stu

dy

mo

de

lM

ain

ou

tco

me

s

[12

2]

BM

P-2

Ap

ati

te-c

oa

ted

co

lla

ge

n

Sp

on

ge

Lu

mb

ar

po

sto

late

ral

fusio

nin

rab

bit

s

En

ha

nce

bo

ne

reg

en

era

tio

n,

ten

sile

str

en

gth

an

d

fusio

nra

tea

fte

ra

6-w

ee

kd

ura

tio

n

[10

8]

Erh

BM

P-2

PL

GA

Me

mb

ran

eR

at

ca

lva

ria

ld

efe

ct

Su

ita

ble

ca

rrie

r,str

on

g

oste

oin

du

cti

vit

yw

ith

co

mp

lete

rep

air

[30

]M

SC

sa

nd

BM

P-2

Ge

lati

n/b

-TC

PS

po

ng

eE

qu

ine

the

thir

d

me

tata

rsa

l

bo

ne

de

fect

Pro

mo

ted

bo

ne

de

ge

ne

rati

on

[10

7]

Erh

BM

P-2

Au

tog

en

ou

sIC

BG

co

mp

are

dw

ith

b-T

CP

Inte

rbo

dy

ca

ge

An

teri

or

ce

rvic

al

dis

ce

cto

my

an

dfu

sio

n

Incre

ase

dce

rvic

al

fusio

n,

ne

wb

on

ea

rea

,a

nd

ma

teri

al

de

gra

da

tio

n

rate

an

dre

du

ce

d

resid

ua

lm

ate

ria

la

rea

Rh

BM

P-2

5re

co

mb

ina

nt

hu

ma

nb

on

em

orp

ho

ge

ne

tic

pro

tein

-2;

IGF

-I5

insu

lin

-lik

eg

row

thfa

cto

r-I;

TC

P5

tric

alc

ium

ph

osp

ha

te;

HA

5h

yd

rox

ya

pa

tite

;P

EI-

PE

G5

po

lye

thy

len

imin

e-

po

ly(e

thy

len

eg

lyco

l);

PU

R5

po

lyu

reth

an

e;

bF

GF

5b

asic

fib

rob

last

Gro

wth

facto

r;P

LG

A/P

CL

/nH

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po

lyla

cti

de

/po

lyg

lyco

lid

ea

cid

/po

lyca

pro

lac-

ton

e/n

an

o-h

yd

rox

ya

pa

tite

;P

LC

L/C

ol/

nH

A5

po

lyca

pro

lacto

ne

/co

lla

ge

n/n

an

o-h

yd

rox

ya

pa

tite

;C

RM

5co

mp

ressio

n-r

esis

tan

tm

atr

ix;

OP

5o

ste

og

en

icp

rote

in;

S-N

P/G

52

-N,6

-

O-s

ulf

ate

dch

ito

sa

nb

ase

dn

an

op

art

icle

/ge

lati

nsp

on

ge

;A

SC

s5

ad

ipo

se

-de

riv

ed

ste

mce

lls;

Ti5

tita

niu

m;

AC

S5

ab

so

rba

ble

co

lla

ge

nsp

on

ge

;C

PS

5ca

lciu

mp

ho

sp

ha

te

sa

lts;

Erh

BM

P-2

5E

sch

eri

ch

iaco

lire

co

mb

ina

nt

hu

ma

nb

on

em

orp

ho

ge

ne

tic

pro

tein

-2;

PL

GA

5p

oly

lacti

de

/po

lyg

lyco

lid

ea

cid

;M

SC

s5

me

se

nch

ym

al

ste

mce

lls;;

ICB

G5

ilia

ccre

st

bo

ne

gra

ft.

TA

BL

E2

BioFactors

12 Bone Morphogenetic Proteins in Bone Healing

environment for adhesion, migration and proliferation of cellsand serves as a good natural reservoir and delivery system forgrowth factors such as BMPs [110,127]. Fibrin is also a biode-gradable and biocompatible scaffold and allows using a lowerdose of growth factors for tissue engineering purposes [110].In another study, the in vitro and in vivo effectiveness of anabsorbable collagen sponge (ACS) with 72 mg rhBMP-2 (BMPC)and fibrin matrix with 10 mg rhBMP-2 (BMPF) were comparedwith the ACS alone, fibrin alone, and empty groups. BMP-2release was significantly higher in the BMPF group than theBMPC group. The bone union of femoral defects and the bonevolume were higher in the BMPC and BMPF groups than thecontrols. Interestingly, fibrin matrix even with a seven-foldlower concentration of BMP-2 provided equivalent results withcollagen sponge. According to their results, it seems fibrinmatrix could be an excellent carrier for BMP-2 [110]. Keratinis an intermediate filament protein that is usually derived fromhuman hair [128]. Keratin extract is processed as reduced andoxidized forms termed kerateine and keratose, respectively[129]. Keratin-based biomaterials are readily-accessible, inex-pensive, easy to handle, biocompatible and biodegradable withnon-toxic byproducts, highly integrate with the host tissues,can be sterilized by gamma ray, and tolerate cellular and vas-cular infiltration. For these reasons, they can be suggested asa promising BMP delivery system in tissue engineering andregenerative medicine [128,129]. In a recent investigation, deGuzman et al. [129] used keratose scaffold for BMP-2 deliveryto facilitate bone regeneration of the femoral bone defects inmice. They obtained keratose biomaterial from oxidization ofhuman hairs by peracetic acid and successive extraction ofsoluble keratin proteins in Tris base and deionized water andsterilized by gamma ray (25 kGy). Keratose scaffold with anegative charge was bounded to positively-charged BMP-2through ionic/electrostatic interaction for providing localizedand controlled BMP-2 delivery during its degradation. In vitroanalysis showed that BMP-2 release correlates with degrada-tion of the keratose scaffold. In vivo, they showed that treat-ment with keratose causes deposition of more bone outgrowththan the control. Nonetheless, keratose was associated withreduced and suppressed formation of adipose tissues withinthe gap, thus it may indirectly enhance bone regeneration. Col-lectively, they indicated that a keratin-based biomaterial as adelivery system can extend the applications of BMP-2 for bonerepair and regeneration [130]. Jun et al. [94] fabricated asilica xerogel-chitosan hybrid for incorporating the BMP-2 ona porous HA scaffold. They evaluated the biological propertiesof the hybrid coating incorporated with the BMP-2, in terms ofthe release behavior of BMP-2 and also its in vivo performanceon calvarial defects in rabbits. The BMP-2 loaded hybrids sig-nificantly enhanced new bone formation in comparison to thepure porous HA scaffolds without BMP-2. Indeed, incorpora-tion of the BMP-2 into the porous scaffold promoted itsosteoinductive properties. They introduced the silica xerogel-chitosan hybrid as a promising candidate for improving osteo-genic properties of the HA scaffold with the constant and pro-

longed release of BMP-2. In another study, the effectiveness ofthiolated chitosan (Thio-CS) was evaluated as delivering sys-tem for BMP-2 and ectopic bone formation induction at thedorsum of mice. They used type I collagen gel (Col-gel) as acontrol for BMP-2 delivery. They showed Thio-CS scaffoldmight be useful in delivering BMP-2 with promising boneregeneration. The BMP-2 released from Thio-CS inducedectopic bone formation to a much greater extent than thatreleased from Col-gel and the control. They suggested theThio-CS scaffold as a biocompatible synthetic polymer in deliv-ering BMP-2 in bone regeneration strategies [68]. Rahmanet al. [92] investigated the potential of the composite poly (D,L-lactic acid-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG)scaffolds to deliver BMP-2 in a sustained and controlled man-ner and their osteogenic capability in a mouse calvarial defectmodel. Approximately 70% of the BMP-2 loaded into these sin-tered polymer scaffolds was released. The released BMP-2was active and induced osteogenesis in cell culture. A 55%and 31% increase in new bone mass was seen for PLGA/PEGscaffolds loaded with BMP-2 and for PLGA/PG without BMP-2,respectively, in comparison to the empty defect control. Theseresults revealed the potential of the PLGA/PG scaffolds in sus-tained delivery of the BMP-2 for bone regeneration. Most ofthe animal studies have revealed the osteoinductive activitiesof the BMPs [27,103,105,106,108]. However, the efficacy andreliability of these results obtained from the animal studies isin doubt and controversial in using in human practices[69,130,131].

1.9. Human Clinical StudiesThere are a considerable number of clinical studies in thefield of rhBMP-2 application in patients associated with open[69] or closed [97] long bone fractures, maxillofacial defects[132], joint arthrodesis [133], and in particular spinal fusionincluding lumbar interbody fusion (LIF) [134], transforaminallumbar interbody fusion (TLIF) [135], posterolateral lumbarfusion (PLF) [136], and anterior cervical discectomy andfusion (ACDF) [130]. A number of human studies regardingthe application of BMPs in bone defects have been presentedin Table 3.

In a prospective controlled study, Zimmermann et al. [142]compared the efficiency of BMP-7 with autogenous bone graftfor treatment of non-unions of the tibial shaft. During about 7years, 82 patients with delayed union of tibial shaft fractureafter primary stabilization were treated with autologous bonegrafting. Then, 26 cases with failure of the graft were treatedwith local implantation of BMP-7 covered by collagen spongeand complete radiological followed up was performed for atleast 1 year. Of the 26 patients, bone consolidation wasobserved after 4 months in 24 cases and only two patientsneeded revision surgery. They showed that BMP-7 has signifi-cantly higher healing capacity than the autograft alone.Papanna et al. [59] studied the safety and efficacy of localimplantation of BMP-7 for treating the resistant non-unions inthe upper and lower limb. Fifty-two patients (30 males and 22

Oryan et al. 13

Eff

ecti

ve

ne

ss

of

the

ap

plica

tio

ns

of

BM

PS

inclin

ica

lstu

die

s

Re

fere

nce

Stu

dy

typ

eN

o.

pa

tie

nts

Gro

wth

facto

ru

se

dB

on

ed

efe

ct

mo

de

lC

arr

ier

use

dM

ain

resu

lts

Do

se

of

gro

wth

facto

r

[69

]R

an

do

miz

ed

tria

l2

77

pa

tie

nts

Rh

BM

P-2

Acu

teo

pe

nti

bia

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actu

re

tre

ate

dw

ith

rea

me

d

intr

am

ed

ulla

ryn

ail

fix

ati

on

Ab

so

rba

ble

co

lla

ge

n

sp

on

ge

imp

lan

t

Wa

sn

ot

sig

nifi

ca

ntl

y

acce

lera

ted

by

the

rhB

MP

-2/A

CS

imp

lan

t

Imp

lan

tco

nta

inin

g

1.5

mg

/mL

[13

7]

Me

ta-a

na

lysis

of

ten

ran

do

miz

ed

co

ntr

olle

d

tria

ls

1,3

42

pa

tie

nts

Rh

BM

P-2

Lu

mb

ar

fusio

nA

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,co

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are

d

toIC

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Rh

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wa

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pe

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r

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for

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iev

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n

su

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an

da

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reo

pe

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on

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–4

0m

gin

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e

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d1

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g

info

ur

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die

s

[13

0]

Mu

ltic

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ter,

ran

do

miz

ed

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ed

tria

l

46

3p

ati

en

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hB

MP

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pin

al

art

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de

sis

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as

asso

cia

ted

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n

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sk

of

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r

tha

na

uto

ge

no

us

bo

ne

gra

ft

Hig

hd

ose

of

40

mg

rhB

MP

-2

[13

8]

Ra

nd

om

ize

d,

co

ntr

olle

d

cli

nic

al

tria

l

29

pa

tie

nts

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BM

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ple

ss

ex

tra

cti

on

of

tee

th

wit

hb

ucca

ld

eh

isce

nce

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ers

us

CS

alo

ne

Re

ge

ne

rate

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st

bu

cca

lp

late

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d

red

uce

dre

ma

inin

g

bu

cca

ld

eh

isce

nce

rad

iog

rap

hic

ally

an

d

clin

ica

lly

No

tsh

ow

n

[13

1]

Re

tro

sp

ecti

ve

rev

iew

50

9p

ati

en

tsR

hB

MP

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pin

al

de

form

ity

,

sp

on

dy

lolisth

esis

an

d

de

ge

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ea

se

No

tsh

ow

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pro

ve

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n,

bu

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ith

so

me

co

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lica

tio

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su

ch

as

se

rom

a,

an

d

ecto

pic

bo

ne

form

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on

An

av

era

ge

of

7.3

mg

(ra

ng

ing

2–1

2m

g)

pe

rd

isk

[66

]R

an

do

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ed

,co

ntr

olle

d,

pa

ralle

l-g

rou

p,

op

en

lab

el

clin

ica

ltr

ial

24

pa

tie

nts

Rh

BM

P-2

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op

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an

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or

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AC

So

ra

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no

us

ma

nd

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lar

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om

ola

rb

on

eg

raft

Incre

ase

dra

dio

gra

ph

ic

ho

rizo

nta

lb

on

e

ga

inw

ith

rhB

MP

-2

1.5

mg

/mL

[13

6]

Mu

ltic

en

ter,

ran

do

miz

ed

co

ntr

oll

ed

tria

l

19

7p

ati

en

tsR

hB

MP

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oste

rola

tera

lin

str

um

en

ted

lum

ba

rfu

sio

n

AC

Sco

mp

are

dto

ilia

ccre

st

au

tog

raft

Imp

rov

ed

rad

iog

rap

hic

al,

bu

tn

oclin

ica

l,

fusio

nra

tein

co

mp

ari

so

nto

the

use

of

au

tog

raft

No

tsp

ecifi

c

[97

]D

ou

ble

-bli

nd

,ra

nd

om

ize

d,

co

ntr

oll

ed

ph

ase

-II/III

tria

l

36

9p

ati

en

tsR

hB

MP

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cu

teclo

se

dti

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l

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ph

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re

Inje

cta

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CP

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ed

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sig

nifi

ca

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y

the

tim

eo

ffr

actu

re

un

ion

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dp

ain

-fre

e

full

we

igh

t-b

ea

rin

g

2.0

mg

/mL

(rh

BM

P/C

PM

)

TA

BL

E3

(Co

nti

nu

ed

)

Re

fere

nce

Stu

dy

typ

eN

o.

pa

tie

nts

Gro

wth

facto

ru

se

dB

on

ed

efe

ct

mo

de

lC

arr

ier

use

dM

ain

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lts

Do

se

of

gro

wth

facto

r

[13

2]

Un

sp

on

so

red

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do

miz

ed

op

en

-la

be

lclin

ica

ltr

ial

20

pa

tie

nts

Rh

BM

P-2

La

rge

ve

rtic

al

de

fects

of

ma

xilla

Aco

mp

osit

eg

raft

of

ace

llu

lar

CS

,P

RP

an

d(C

CF

DA

B)

Re

ge

ne

rate

db

on

ew

ith

less

mo

rbid

ity

,e

qu

al

co

st,

an

dm

ore

via

ble

ne

wb

on

efo

rma

tio

nb

ut

wit

hm

ore

ed

em

ath

an

au

tog

en

ou

sg

raft

1.0

5m

g

rhB

MP

/AC

S

[13

4]

Mu

ltic

en

ter

clin

ica

lstu

dy

32

1p

ati

en

tsR

hB

MP

-2

ve

rsu

s

(Oste

oA

MP

)

Lu

mb

ar

or

tra

nsfo

ram

ina

l

inte

rbo

dy

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n

AC

SR

ed

uce

dfu

sio

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me

an

d

co

mp

lica

tio

nw

ith

Oste

oA

MP

co

mp

are

d

wit

hrh

BM

P-2

Av

era

ge

of

3.0

7m

go

f

rhB

MP

-2

[13

9]

Re

tro

sp

ecti

ve

clin

ica

lca

se

se

rie

sa

ta

sin

gle

insti

tuti

on

(20

07

–2

01

0)

57

3p

ati

en

tsR

hB

MP

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LIF

Co

lla

ge

n-s

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sp

on

ge

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mp

tom

ati

ce

cto

pic

bo

ne

form

ati

on

,v

ert

eb

ral

oste

oly

sis

an

d

pse

ud

art

hro

sis

we

re

co

mp

lica

tio

ns

12

mg

(la

rge

kit

),

or

4.2

mg

(sm

all

kit

)

[13

3]

Re

tro

sp

ecti

ve

co

ho

rtstu

dy

82

pa

tie

nts

Rh

BM

P-2

An

kle

art

hro

de

sis

Co

lla

ge

nsp

on

ge

Incre

ase

db

on

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rid

gin

g,

ach

iev

ed

su

cce

ssfu

l

un

ion

wit

ho

ut

furt

he

r

op

era

tio

n

1.5

mg

/mL

of

rhB

MP

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[14

0]

Re

tro

sp

ecti

ve

rev

iew

11

pa

tie

nts

Rh

BM

P-2

L5

-S1

art

hro

de

sis

in

Lo

ng

se

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tfu

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n

for

ne

uro

mu

scu

lar

sp

ina

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lio

sis

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SD

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ase

dco

mp

lica

tio

n

rate

an

db

loo

dlo

ss

An

av

era

ge

of

14

.2m

g

rhB

MP

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[14

1]

Ra

nd

om

ize

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ntr

olle

d

cli

nic

al

tria

l

69

pa

tie

nts

Rh

BM

P-2

To

oth

ex

tra

cti

on

Inje

cta

ble

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el

No

an

ticip

ate

da

dv

ers

e

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en

ts,

no

sig

nifi

ca

nt

imm

un

ere

acti

on

s,

sa

fe

an

de

asy

tou

se

0.0

5m

g/m

L

of

rhB

MP

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BM

ge

l

[13

5]

Pro

sp

ecti

ve

,ra

nd

om

ize

d,

co

ntr

oll

ed

tria

l

52

pa

tie

nts

Rh

BM

P-2

TL

IFA

CS

co

mp

are

dto

Sil

ica

te-s

ub

sti

tute

d

ca

lciu

mp

ho

sp

ha

te

Re

du

ce

dra

teo

f

art

hro

de

sis

wa

s

asso

cia

ted

wit

h

Sil

ica

te-s

ub

sti

tute

d

ca

lciu

mp

ho

sp

ha

te

4.2

mg

of

rhB

MP

-2

Rh

BM

P-2

5re

co

mb

ina

nt

hu

ma

nb

on

em

orp

ho

ge

ne

tic

pro

tein

-2;

AC

S5

ab

so

rba

ble

co

lla

ge

nsp

on

ge

;IC

BG

5ilia

ccre

st

bo

ne

gra

ft;

CR

M5

co

mp

ressio

n-r

esis

tan

tm

atr

ix;

CS

5co

lla

ge

nsp

on

ge

;

CP

M5

ca

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females) were treated by a bovine bone-derived collagen pastecontaining BMP-7. Based on the clinical and radiological find-ings, union reached to 94% during a time period of 5.6months. Therefore, it was revealed that BMP-7 is able to beused as an efficient adjunctive treatment for resistant non-unions in limbs. In a double-blind, randomized, controlledtrial, the efficiency of rhBMP-2 and an injectable calciumphosphate matrix (CPM) formulation was evaluated in 369patients with acute closed tibial diaphyseal fractures treatedwith reamed intramedullary nail fixation. In that study, thetime of fracture union and pain were not significantly reducedby 2.0 mg/mL rhBMP/CPM in comparison to the standard carealone including fracture fixation within 72 h as undreamedintramedullary nail fixation [97]. In a meta-analysis ofindividual-participant data by Simmonds et al. [64], the safetyand effectiveness of rhBMP-2 were evaluated. They analyzedand compared a number of randomized, controlled trialsregarding application of rhBMP-2 versus iliac crest bone graft(ICBG) in spine fusion surgery for degenerative disc diseaseand related conditions. By 24 months, pain, adverse eventsand radiographic fusion were better in those treated withrhBMP-2 than those treated with ICBG. However, at or shortlyafter surgery, pain and cancer risk were more common inthose treated with rhBMP-2 compared with those treated withICBG. In another meta-analysis and systematic review [143],the effectiveness and harms of rhBMP-2 in spinal fusion wasassessed in 13 randomized, controlled trials (RCTs) and 31cohort studies. For lumbar spine fusion, rhBMP-2 and ICBGwere similar in fusion, overall success. For anterior lumbarinterbody fusion, the rhBMP-2 was associated with increasedrisk for retrograde ejaculation and urogenital problems. Foranterior cervical spine fusion, the rhBMP-2 was associatedwith increased dysphagia and wound complications. Moreover,the cancer risk with the rhBMP-2 was a little higher than theICBG. Therefore, these studies did not prove the effectivenessand safety of the rhBMP-2 compared to the ICBG in spinalfusion. Based on the recent trends, concerns regarding BMPsinclude increased risk of heterotopic bone formation, radiculi-tis, osteolysis and retrograde ejaculation as well as risk of can-cers such as melanoma, pancreatic, prostatic and thyroidtumors [64,130,131,134,143]. Although BMPs can promotebone formation, their clinical efficacy is controversial. It isunclear why the impressive and convincing results seen invitro and in animal models are difficult to reproduce in theclinical studies.

1.10. Side Effects and Disadvantages of BMPsDespite the positive effects of BMPs especially the rhBMP-2 onbone healing such as elimination of the risk of autograft har-vesting and osteoinductivity, their application especially in spinefusion, may associated with surgical site infection, wound com-plication, ectopic bone formation, local bone resorption, pseu-doarthrosis, local edema and erythema, osteolysis, compart-ment syndrome and nerve injury [20,23,63,131,143,144].Meanwhile, in some instances there is resistance to BMP ther-

apy since the systemic increase in TGF-b interfering with BMPsignaling has been shown [23]. It is believed that some of thesedrawbacks may be due to the inductive effects of rhBMP-2 onthe inflammatory host reactions [24,63]. Another critical com-plication related to the application of rhBMP-2 includes inflam-matory vessel fibrosis and scarring resulting in the life-threatening vascular injury [63]. The complications associatedwith the treatment using rhBMP-2 may depend on the type andlocation of the fracture and the surgical approach [145]. Inaddition, it has been shown that application of BMP is associ-ated with significantly higher costs compared to procedureswithout BMP. The hospital costs for operations involving BMP isabout $15,000 more than those procedures perform withoutBMP. Therefore, it is a general agreement that these high costsmust be decreased or prevented [144]. In Nationwide InpatientSample (NIS) retrospective cohort examination, the impact ofBMPs on use of autograft, rates of operative treatment for lum-bar pseudoarthrosis, and hospital charges has been investigatedin 46,452 patients from 2002 to 2008. They showed that appli-cation of BMPs, added more than 900 million dollar to hospitalcharges. The findings included overall decrease in rates of revi-sion fusion procedures. In addition, they reported that introduc-tion of BMP did not correlate with decrease in the use of auto-graft bone harvest. Over the past few years, there has been acontroversy surrounding the promotion and use of rhBMP-2 forspinal fusion. A number of studies have revealed rhBMP-2 hasshown outcomes similar to those of autologous iliac crest bonegraft (AICBG) [4,130,134,136]. Nonetheless, there is no clearevidence of a clinically important difference between rhBMP-2and AICBG in inducing spinal fusion. On the other hand, bothoptions are associated with similar complication rates whenused as graft material in anterior lumbar interbody fusion orpostolateral fusion including neurologic complication, retro-grade ejaculation and ectopic bone formation as well as cancerrisk in patients receiving rhBMP-2. The prevalence of thereported adverse events and complications related to the use ofrhBMP-2 has raised many ethical and legal concerns for sur-geons. In addition, the cost of rhBMP-2 has required the identi-fication of a viable alternative [4,134].

1.11. BMPs Substitutes and Future DirectionsAfter an initial promising start, concerns regarding safety andcost-effectiveness of BMPs are raising. These problems impli-cate that BMP application may not be the final solution in thechallenging field of non-union and delayed union treatment.The question remains how to improve the efficacy or osteoin-ductivity of rhBMP-2 for successful application especially inthe patients undergoing spinal fusion and other bone defectsor to reduce the dosages required [19]. In other words,because treatment of the bone defects with rhBMP-2 has somedisadvantages, the attempt for an alternative treatment strat-egy is still required. A solution may be inhibition of the BMPantagonists to decrease the need for high doses of BMPs andto prevent their complications. In an attempt, Bae et al. [146]lowered the required dose of rhBMP-2 to exert its actions and

BioFactors

16 Bone Morphogenetic Proteins in Bone Healing

enhanced its performance in spinal fusion by the addition ofbone marrow aspirate (BMA) to 0.006 mg/mL rhBMP-absorbable collagen sponges. A study was carried out to inves-tigate improving the osteogenic effects of BMP-2 by transientnon-viral gene silencing of Chordin and Noggin in human adi-pose tissue-derived stromal cells (hASCs) [36]. For this pur-pose, hASCs were tranfected with short interfering ribonucleicacid (siRNA), using a commercial liposomal transfection rea-gent. Osteogenic differentiation of hASCs has been determinedby matrix mineralization and alkaline phosphatase (ALP). Incontrast with Noggin, ALP activity of hASCs supplemented tosiRNA against Chordin without BMP-2 was increased. In com-bination with BMP-2 (100 ng/mL), silencing both the Chordinand Noggin strongly improved the ALP activity compared tothe BMP-2 alone. Matrix mineralization started earlier ingroups receiving siRNA against Chordin and Noggin. Collec-tively, Chordin silencing was more successful than Nogginsilencing in increasing the BMP-2 effects on osteogenic differ-entiation. In a study by Roh et al. [134], they applied osteoallogeneic morphogenetic protein (OsteoAMPVR ) that is a com-mercially available allograft-derived growth factor rich inosteoinductive, angiogenic, and mitogenic proteins such asBMP-2, BMP-7, TGF-b1, FGF, VEGF and ANG1, within bonemarrow cells. They offered OsteoAMP as a substitute torhBMP-2. The radiographic fusion in the patients receivingOsteoAMP was 59.7%, 93.3% and 98.9% at 6, 12, and 18months, respectively in comparison to 39.3%, 83.5%, and90.1% in the patients receiving rhBMP-2. Additionally, totaltime for fusion for OsteoAMP was about 40% shorter than thatof rhBMP-2. The average cost of 3.07 mg (2.05 mL) rhBMP-2used inside the interbody device was 2,523.52 US dollar, whilethat of 2.5 mL OsteoAMP used inside the spinal spacer was649.20 US dollar, so the osteoAMP arm was 80.5% less expen-sive than the rhBMP-2 arm per patient. Regarding the compli-cation observed related to two groups based on x-rays, radio-logical, and CT assays, ectopic bone formation and osteolysiswere 24.2% versus 5.3%, and 10.5% versus 5.3%, for rhBMP-2versus OsteoAMP groups, respectively. Collectively, their studyshowed that OsteoAMP can be considered as a cost-effectiveand viable alternative to rhBMP-2. Moreover, small-moleculeBMP-signaling activators have been recently discovered andtheir in vitro and/or in vivo effects on osteogenesis in the pres-ence of low dosages of exogenous rhBMPs have been reported[70,147]. It is believed that application of these small mole-cules can significantly reduce the dosages of exogenousrhBMPs required or eliminate the need for exogenous rhBMPs,thus significantly reduce the overall treatment cost [147].Wong et al. [147] demonstrated that a single dose of the small-molecule BMP activator, SVAK-12, could accelerate fracturehealing in a rat femoral fracture model without the need forexogenous rhBMPs. In addition, it is believed that icariin flavo-noid could be candidate as a substitute or as an assistant forBMPs, due to being cheaper and safer than BMPs. It possessesan osteoinductive potential, due to its activities of inducingosteogenesis, chondrogenesis, and angiogenesis [148]. Icariin

exerts its osteogenic effects via the induction of BMP-2 synthe-sis and BMP-2/Smad4 signal transduction pathway [70]. Plate-let concentrates including platelet rich plasma (PRP), platelet-rich fibrin (PRF), concentrated growth factor (CGF), and PRPgel (PRP plus thrombin and calcium chloride (CaCl2) to formglue) have been used for bone healing and repair [149–152].Particularly PRP similar to BMPs have been demonstrated tohave many therapeutic effects [153,154] due to its growth fac-tors including platelet-derived growth factor (PDGF), trans-forming growth factor beta (TGF-b), fibroblast growth factor(FGF), insulin-like growth factor 1 (IGF-1), insulin-like growthfactor 2 (IGF-2), vascular endothelial growth factor (VEGF),epidermal growth factor (EGF), Interleukin 8 (IL-8), keratino-cyte growth factor (KGF), and connective tissue growth factor(CTGF) [153,154]. However, BMPs still present a considerablyhigher cost as compared with the PRP. In addition to theseadvantages of PRP, for example PRP gel can be used in deliv-ering of BMPs. In this condition, PRP gel can be alternativelyused as a controlled release system for BMPs, while its effec-tiveness in improving new bone formation remains unclear[151]. On the other hand, the autologous nature of PRPinvolves a minimum risk of immune reactions and transmis-sion of infectious and contagious disease. For these reasons, itmay be an option to substitute the BMPs. Despite the highpotential for applicability, it is difficult and there is still a needfor the standardization and fabrication a high quality PRP[152,154]. In addition, demineralized bone matrix (DBM) has asmall quantity of BMPs that provide osteoinduction capabilities[152,155]. Moreover, it is also osteoconductive because theorganic portions of bone remain after demineralization [155].DBM does not stimulate immune response, because of thedestruction of the angiogenic surface structure during demin-eralization by acid. Nonetheless, there is still no clinical evi-dence to support its application as an optimal graft material inreconstructive medicine and orthopedic surgery [156]. It ishoped that in the near future, ongoing evaluations woulddetermine the true position of this adjunct. Alternatively, sim-vastatin as a member of statins with osteoinductive activityhas been shown to increase the expression of BMP-2 mRNAand VEGF and by these mechanisms it was shown that simva-statin can improve the bone regeneration [33]. Oral adminis-tration of simvastatin and its early extensive metabolism in theliver reduce bioavailability of this drug (about 2 h) [33,157]. Inorder to enhance both the circulating and local concentrationof simvastatin in promoting bone formation, an appropriatedelivery system should be developed [157]. Recently, Assafet al. [33] evaluated the efficacy of simvastatin in combinationwith PLGA in stimulating the regeneration of rat calvarialdefects. PLGA is a biocompatible, biodegradable, and non-toxicsynthetic polymer that has been extensively used for bonehealing and drug delivery. The application of such scaffoldcontaining simvastatin significantly increased bone formationand therefore, the osteoinductive property of simvastatin wasconfirmed. Furthermore, biphasic calcium phosphate (BCP)based ceramic biomaterials including HA and b-TCP exhibit

Oryan et al. 17

good biocompatibility, bioactivity, biodegradability and osteo-conductivity but no osteoinductivity, and thus, they need anosteoinductive biomolecule. Nano-hydroxyapatite has osteoin-ductive activity, can not only improve the biocompatibility, bio-activity, and osteointegration of the biomaterial, but also canpromote the adhesion, proliferation and osteogenic differentia-tion and improve mineral deposition [34]. In a study, porousBCP ceramic scaffolds were coated with nHA and then theosteogenic differentiation of rabbit BMSCs seeded on thesescaffolds was studied. It was demonstrated that coating thescaffold with nHA can increase the osteoinductive potential ofBCP ceramics making this biomaterial more suitable thanuncoated type for application in bone tissue engineering [34].In another study, Wang et al. [158] evaluated the applicationof the nHA/chitosan/PLGA (nHA/CS/PLGA) scaffold seeded withhuman umbilical cord mesenchymal stem cells (hUCMSCs) inbone tissue engineering. They compared the cell capability todifferentiate into osteoblasts associated with the nHA/CS/PLGA,nHA/PLGA, CS/PLGA, and PLGA scaffolds. Among these, thenHA/CS/PLGA scaffolds were the most suitable choice for theadhesion, proliferation, and osteogenic differentiation ofhUCMSCs both in vitro and in vivo. Another possible option asan alternative for BMPs is strontium (Sr), a trace element,exhibiting an osteoinductive activity. Strontium has been incor-porated into some biomaterials including calcium phosphatesand bioactive glasses to enhance bone formation [35,159]. Fur-thermore, Sr can increase osteoblast attachment, mineraliza-tion, osteointegration, bone strength, bone growth and differ-entiation of MSCs into bone lineage and reduce boneresorption [35,159]. Polytherapy using the implantation of sev-eral components may be regarded as a new and promisingstrategy in order to enhance the healing process [14,160]. Inthis approach, the biomaterials may provide a certain propertyfor fabricating an appropriate tissue engineered construct.The best construct is the one that possesses all osteogenic,osteoinductive, osteoconductive, and osteointegrative charac-teristics [14]. Calori et al. [160] compared polytherapyapproach by the simultaneous implantation of MSCs, rhBMP-7and autologous bone graft as the scaffold versus monotherapywith implantation of bone autograft, in the treatment of fore-arm non-unions. They suggested polytherapy approach as aneffective treatment method for such patients. Three dimen-sional printing (3D printing) of the scaffolds as a new approachin the tissue engineering technology holds great promise forfabricating bone graft substitute with increased performance[14,161]. This technology could be combined with controlledrelease of bioactive substances such as growth factors, andbioactive molecules with the aiming to heal and regeneratebone tissue. Shim et al. [162] developed a delivery system forrhBMP-2 encapsulated in collagen or gelatin solutions withslow mode in three-dimensional printing polycaprolactone(PCL)/PLGA scaffolds for bone formation in rabbit diaphysealdefect. They showed that a burst release of rhBMP-2 from thePCL/PLGA/gelatin scaffold did not induce the osteogenic differ-entiation of human nasal inferior turbinate-derived mesenchy-

mal stromal cells (hTMSCs) in vitro. However, in the in vivoanimal experiments, micro-computed tomography (micro-CT)and histological investigations confirmed that PCL/PLGA/colla-gen/rhBMP-2 scaffolds with a long-term delivery mode showedbetter bone healing quality at both weeks 4 and 8 afterimplantation without inflammatory response. Moreover, pluri-potent (embryonic and fetal) or multipotent (amniotic, adult)stem cells, especially the MSCs that have the potential to dif-ferentiate into bone tissue cell types such as osteoblasts can beconsidered as an attractive option [11,163,164]. So that, it ispossible to use stem cells with osteoinductive and osteoconduc-tive agents to fabricate a promising tissue-engineered con-struct with the aiming to improve and accelerate healing ofbone defects, which is important particularly in non-unionfractures [30,165].

Collectively, although BMPs are remarkable osteoinductiveagents, they have limited availability and are expensive.Regarding the limitations of exogenous BMPs extensively dis-cussed in this review, such compounds may not be suitableosteoinductive agents for clinical application. The future inves-tigations should focus on the BMPs substitutes having osteoin-ductive property, which affect the expression or function of theendogenous BMPs.

2. ConclusionIn conclusion, it seems BMPs have some beneficial effects onfracture healing. Most of the in vitro studies have showndesired but unreliable results. Despite the somewhat promis-ing results obtained from the in vivo animal and human clini-cal studies, there were some complications that should be con-sidered. Indeed, there are many controversies in application ofBMPs in the healing of different bone defects and non-unions.It appears that it still needs more studies to offer BMPs as apromising and effective therapeutic modality in orthopedicsurgery and regenerative medicine. These findings are impor-tant in guiding the orthopedic surgeons to make a more reli-able decision. They should carefully weigh the demonstratedand potential benefits and harms as well as the costs whenconsidering the adoption and use of these technologies inimproving bone regeneration and repair.

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