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Administration of growth factors for bone regeneration


Growth factors (GFs) such as BMPs, FGFs, VEGFs and IGFs have signicant impacts on osteoblast behavior, and thus have been widely utilized for bone tissue regeneration. Recently, securing biological stability for a sustainable and controllable release to the target tissue has been a challenge to practical applications. This challenge has been addressed to some degree with the development of appropriate carrier materials and delivery systems. This review highlights the importance and roles of those GFs, as well as their proper administration for targeting bone regeneration. Additionally, the invitro and invivo performance of those GFs with or without the use of carrier systems in the repair and regeneration of bone tissue is systematically addressed. Moreover, some recent advances in the utility of the GFs, such as using fusion technology, are also reviewed.
KEYWORDS: BMP n bone regeneration n carrier material n delivery system n FGF nfusion protein n growth factor n IGF n VEGF n wound healing

Growth factors (GFs) have been found in all tissues and are known to stimulate cellular growth, proliferation, migration and differentiation. Many GFs have been proposed for bone tissue engineering applications. GFs are usually stored in the extracellular matrix (ECM); however, they are released from the ECM after injury and then contribute to the bone repair process. During bone repair, cells of the bone environment, such as bone marrow stromal cells, endothelial cells, broblasts, inammatory cells and osteoblasts, produce GFs [1] . Among others, periosteum, a dense irregular connective tissue lining the outer surface of bone, is considered important as this contains a population of progenitor cells that directly differentiate into osteoblasts. Many GFs, including BMPs, VEGFs, FGFs, IGFs and PDGFs, have been engaged in directing and stimulating periosteal progenitors to osteoblastic differentiation during the bone-healing process[210] . The active roles of GFs with respect to the cells mentioned above have been comprehensively identied both invitro and invivo. GFs may act in an autocrine fashion and inuence cells of the same class or in a paracrine fashion and affect different cells. Accordingly, a variety of GFs have been widely used for bone tissue engineering. BMPs are stored within the ECM of mature bone [11] and their expression is also upregulated in the early phases of fracture repair [12] . Through previous studies, BMP-2, -4, -6 and -7 (also called OP-1) are widely known as osteoinductive factors [13,14] . Expression levels of the different BMPs
10.2217/RME.12.1 2012 Future Medicine Ltd

vary throughout the healing process, which causes up- or down-regulation of other BMP family members [14] . BMPs can inuence the expression of other GFs [13] . BMP-2 is involved in the expression of osteogenic markers via induction of the MAPK pathway [15] and osteogenic nuclear transcription factor Cbfa-1/Runx2 [13] . Thus, BMPs have been widely used in preclinical and clinical trials [16,17] . Among the FGFs, FGF-2 plays a role in the bone-remodeling process by maintaining the ne balance between boneforming osteoblasts and bone-resorbing osteoclasts [18] and supporting neovascularization, similar to VEGFs [13] . In addition, FGF-2 promotes osteogenic differentiation via downstream activation of Cbfa-1/Runx2 [19] . VEGFs themselves are powerful angiogenic factors that are responsible for mediating vascular responses in tissues [20,21] . They are particularly important for wound healing and are expressed in fracture sites where they promote new neovascularization and angiogenesis via recruitment of endothelial cells [13,22] . IGFs are also thought to have an important role in bone healing. IGF-I and -II are both expressed by cells of the musculoskeletal system and are highly expressed in bone fracture sites, although IGF-I has the most potent effect on bone metabolic activity [23] . IGFs induce expression and deposition of the typeI collagen matrix and also maintain the anabolic, bone-forming state by preventing expression of collagenase by osteoblasts [13,23] . Although the effects of GFs are pleiotropic, their short biological half-life, lack of long-term
Regen. Med. (2012) 7(3), 369385

Ye-Rang Yun1, JunHyeogJang2 EunyiJeon2, WonmoKang2, SujinLee2, Jong-Eun Won1,3, HaeWon Kim*1,3,4 &Ivan Wall3,5
Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, Korea 2 Department of Biochemistry, Inha University School of Medicine, Incheon 400-712, Korea 3 Department of Nanobiomedical Science & WCU Research Center, Dankook University Graduate School, Cheonan 330-714, Korea 4 Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, Korea 5 Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK *Author for correspondence: Tel.: +82 41 550 3081 Fax: +82 41 550 3085 kimhw@dku.edu Authors contributed equally
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stability and tissue selectivity, and possible toxicity and risk of tumor-promoting activity limit their practical therapeutic application and thus demand controllable and sustainable delivery [24] . Selection of an appropriate delivery system for GFs is essential to induce a specic biological effect. For example, locally administered solutions of FGF-2 did not promote bone regeneration in rabbit skull defects, whereas FGF-2 incorporated into gelatin hydrogels did [25] . Moreover, BMP in an insoluble bone matrix failed to induce bone growth around titanium implants because of unsuitable carrier properties [26] . Similarly, IGF-I failed to induce bone repair when delivered directly into the osteotomy via an osmotic pump [27,28] , but was successful when embedded in biodegradable microspheres to heal segmental long bone defects [29] . In the following sections, we will briey overview the requirements of biomaterial scaffolds that might be employed to deliver GFs and also explain the importance and roles of various GFs that have been used for bone regeneration based on in vitro and in vivo experimental results.

Considerations for carrier materials When designing appropriate delivery systems, the carrier materials need to satisfy some essential criteria. Specically, the materials should be biocompatible, noncytotoxic and nonimmuno genic to prevent any adverse effects on residing and recruited cells and neighboring tissues. Moreover, the chemistry of materials should be tailored to support cell adhesion and proliferation. Ultimately, the implanted carriers should be degraded by enzymes and/or circulating biological uid. Commonly used materials include natural polymers such as collagen, gelatin, brin, alginate, silk, hyaluronic acid and chitosan [30] , as well as synthetic polymers, including the a-hydroxy esters such as poly(lactic acid), poly(glycolic acid), poly(anhydride), poly(phosphazene), poly(propylene fumarate) and poly(ethylene glycol) [3145] . Although most GF delivery carriers have been produced from polymeric materials, inorganic materials are also used to promote bone formation during specic pharmacological intervention for musculoskeletal problems. Specically, calcium phosphates and silicate glasses have been processed to deliver GFs [4656] . BOX1 presents the carrier materials and matrix types used for the delivery of various GFs for bone regeneration. Those carrier compositions have been processed in the form of micro-/nano-particles, porous scaffolds, bers and hydrogels for specic
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applications. The GFs have either been incorporated within the structure of carriers or bound onto the surface. In either case, when the GFs were introduced through chemical bonds (covalently, ionically or afnity bindings), a more sustainable release could be achieved, possibly leading to potentiating the efcacy of GFs. When the carriers incorporate the GFs inside the structure, the chemical interaction between GFs and carriers and the degradation rate of the carriers will dominate the release prole of GFs, and the delivery kinetics is more tunable/controllable, allowing prolonged release. On the other hand, when introduced on the surface of carriers, the effects of GFs may occur more rapidly in a manner of direct interaction with the cells in the surroundings. Most of all, timed delivery of GFs to the site over certain periods at appropriate doses is the most critical issue that needs deep consideration in the design of delivery systems. In fact, the repair and regeneration process of bone is a highly complex and harmonized event involving various GFs. Therefore, many recent studies have focused on the delivery of dual/multiple GFs. These studies have been conducted in response to the nding that treatments with GF combinations exhibited additive or even synergistic effects on bone formation. Occasionally, administration of multiple GFs has been found to inhibit bone formation. For example, in a rat tibia fracture model, the combined application of IGF-I and TGF-b1 showed a synergistic effect in fracture healing [57] , whereas the combined administration of BMP-2 and FGF-1 with collagen sponge resulted in decreased bone formation in the same model [58] . These studies demonstrate that the combination of GFs should be carefully considered, and that adequate release kinetics might be critical for successful bone regeneration. For example, should dual GFs be released in combination or sequentially? Dual or multiple GF delivery regimes complicate the design of controlled-release devices, potentially requiring separated compartments to provide timely and sequential release.

BMP Among GFs, BMPs are known to play a critical role in bone regeneration. BMPs are multi functional GFs that belong to the TGF- b super family due to their striking similarities in structure and sequence [11] . Currently, there are 20 known BMP family members, which share some degree of similarity [59] . They are involved in a variety of functions in bone. Predominantly,
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Administration of growth factors for bone regeneration

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Box 1. Carrier materials for bone tissue regeneration.


Carrier materials Natural polymers: collagen, brin, alginate, silk, hyaluronic acid and chitosan Synthetic polymers: poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(caprolactone), poly(anhydride), poly(phospazene), poly(propylene fumarate) and poly(ethylene glycol) Inorganic materials: hydroxyapatite, tricalcium phosphate, calcium phosphate cement and bioactive glasses Matrix types: microparticles, nanoparticles, porous scaffolds, microbers, nanobers and hydrogels Growth factors: BMP-2, BMP-4, BMP-7, FGF-2, TGF- b1, TGF- b2, TGF- b3, VEGF, IGF-I, IGF-II, combinations of two growth factors and peptides

they promote bone formation throughout development and after injury in adults [6062] . BMP signaling is mediated via BMP receptors (BMPRs). Activation of BMPRs leads to intracellular signaling transmitted by Smad proteins, thereby inducing the transcription of specic genes. Their function is primarily modulated by extracellular BMP-binding proteins such as noggin and chordin, which inhibit BMP signaling by blocking their interaction with BMPRs. Active BMPs are disulde-linked dimers and can be either heterodimers (two different family members) or homodimers (two identical family members). The composition of the dimer profoundly inuences bone cell function. Thus, heterodimers of BMP-2/7 or BMP-4/7 produce a greater morphogenic effect than homodimers of any of these factors [63] . Initially, BMPs were isolated from various cell types and then puried before usage; however, since the 1980s, BMPs have been produced using recombinant DNA technology and batch production methods. In early studies, highly puried BMPs were used invitro. For instance, BMP-2 and BMP-3 retain bioactivity in chondrocytes and osteoblastic cells [64] . The osteogenic activity of BMPs invitro has only been identied since the development of recombinant DNA technology, and there have been many reports of its activity [65,66] . Moreover, 14 different BMPs have been investigated in various cell types in recent studies. The results of these studies suggested an osteogenic hierarchical model in which BMP-2, -6 and -9 might play an important role in induction of osteoblast differentiation of mesenchymal stem cells, whereas most other BMPs stimulate osteogenesis in mature osteoblasts. Overall, the BMPs with greatest osteogenic capacity invitro are BMP-2, -4, -6, -7 and -9 [67] . The benecial effects of BMPs for repairing bone defects such as bone fracture, spinal fusion and osteoporosis have been demonstrated invivo in a variety of animal models [6883] . Of
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the BMPs, the therapeutic potential of recombinant human BMP-2 and -7 (rhBMP-2 and rhBMP7) has been identied in bone wound healing models, as well as in craniomaxillo facial, periodontal and dental diseases [60] . Various studies have reported the multiple functions of BMPs at different dosages for specic cell types such as osteoblasts, chondroblasts and stem cells [82] . For instance, elevated concentrations of BMPs accelerate bone growth [83] , whereas tooth ankylosis can be induced by BMP-2 in a dosedependent fashion following application for periodontal regeneration [84] . Contrary to other BMPs, BMP-3 inhibits BMP-2-induced osteogenic differentiation. Furthermore, BMP3knockout mice exhibited increased bone density, which indicates that BMP-3 is both an antagonist and inhibitor of BMP-induced osteogenesis invivo[85] . Gene transfer technologies have received a great deal of attention due to their potential to aid healing by permitting targeted delivery and sustained expression of BMPs within osseous lesions [86] . This and other examples of regenerative medicine approaches in numerous exvivo studies have demonstrated that BMPs show great promise for bone tissue regeneration strategies [81,87104] . A summary of the application of BMPs for bone tissue regeneration invitro, invivo and exvivo is presented in TAbLE1. For many years, the clinical use of BMPs was severely limited due to several problems. First, potential health risks, such as the transmission of slow viruses, which cause disease in a slow, progressive manner over a time-course spanning months to years and consequently remain undetected for long periods, or induction of malignancies have prevented straightforward translation to the clinic [105] . Second, the use of high doses not only raises safety concerns due to its mitogenic potential, but also greatly increases costs. For example, to induce bone regeneration, BMPs need to be administered at high doses that are greater than the levels at which they occur
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Table 1. Application of BMPs for bone tissue regeneration.


Subfamily
Invitro BMP-2 BMP-2, -3 14 BMPs BMP-2

Model
ROB-C26, ROB-C20 cells Chondrocyte, MC3T3-E1 cells C2C12, C3H10T1/2, TE-85 cells Mandible defects in rats Femoral defects in rabbits Pelvic defects in sheep Tibial defects in sheep Femoral defects in rats Femoral fracture in rats Osteotomy/ostectomy in horses Spine fusion in rats Mandible defects in rats Ulnar osteotomy in rabbits Cranial defects in mice Cranial defects in pigs Mandible defects in rats Femoral defects in rats

Biological effect
Stimulate osteoblastic maturation Promote osteoblastic differentiation BMP-2, -6 and -9 induce osteoblastic differentiation Promote mandibular distraction osteogenesis Enhance fracture healing Retard bone formation Enhance fracture healing Enhance healing of segmental bone defects Promote bone formation in fracture region Accelerate bone formation and osteotomy healing Enhance osteoblastic differentiation Repair bony defects in the craniofacial region Accelerate healing Repair healing bone defects Accelerate bone regeneration Accelerate bone regeneration Enhance healing of segmental femoral defects Induce heterotopic and orthotopic bone formation Repair segmental femoral defects Repair femoral defects Repair experimentally induced osteonecrosis Repair bone defects Induce bone formation Enhance spine fusion Induce bone formation Promote healing of osteoporotic bone fracture Accelerate bone healing Enhance cranial bone regeneration Repair segmental bone defects Enhance bone healing Enhance spine fusion

Ref.
[66] [64] [67] [68] [69,70] [71] [72] [7375] [76] [77] [78] [80] [81] [87] [88] [81] [89] [90] [91] [92] [93] [94,95] [96] [97] [98] [99] [100] [101] [102] [103] [104]

Invivo

BMP-2, -4 BMP-2, -6 BMP-2, -7 BMP-2, -9 BMP-6 Exvivo BMP-2

BMP-2, -7 BMP-4 BMP-7

Femoral defects in goats Tibial defects in goats Spine fusion in mice Spine fusion in rats Spine fusion in rabbits Osteoporotic rat model Osteotomy in horses Cranial defects in mice Femoral defects in rats Cranial defects in rabbits Spine fusion in rats

naturally in bone. Another concern was the lack of appropriate delivery systems required for the clinical use of BMPs. Recombinant BMPs have been shown to have a bone-inducing capacity, but require special carriers to maintain their activity at low doses [106] . The function of the carrier matrix is to immobilize the bone-inducing protein at a particular site for a sufcient amount of time to allow bone induction to occur. Studies using various carrier materials have demonstrated that combinations of carrier materials and BMPs enhance osteogenic activities. Various materials and fabricated forms are used as carriers. Natural polymers, including collagen, gelatin, brin, alginate, chitosan and silk, are widely employed as carriers, and the use of these compounds has resulted in signicant induction of osteogenesis [107113] . BMP-2-derived oligopeptide covalently coupled to alginate hydrogel was found to induce ectopic bone formation in rats [114] . Chitosan was also reported to be an effective carrier [115117] . Synthetic polymers, which primarily include a-hydroxy esters, such as poly(lactic acid),
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poly(glycolic acid) and their copolymers, have also been used as BMP carriers. The use of such compounds has been found to accelerate bone formation both invitro and invivo [45,109,118] . Because metallic implants are commonly used in bone, BMPs have been coated on the surface of metals. The rhBMP-2 coating on titanium scaffolds has been studied in various species and types of defects, including minipigs with tibia implants, aged sheep implanted below the tibial plateau, nonaged sheep subjected to alveolar segmental osteotomy, rabbits with femural and tibial implants and rats with subcutaneous implantations [119124] . In these studies, BMPcoated titanium scaffolds successfully facilitated bone regeneration, which generally occurred in a concentration-dependent manner. Moreover, the combined use of BMP with a second GF can have synergistic effects on bone bonding. For example, adult male dogs treated with titanium implants containing both rhTGFb2 and rhBMP-2 were found to have a greater implant xation strength than groups that received either type of GF alone [125] . In terms of the carrier,
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Administration of growth factors for bone regeneration

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combining different materials can improve the delivery potential [48,126,127] . For example, a novel chitosan/g-PGA composite scaffold for the controlled release of rhBMP-2 has been established and can release larger amounts of the GF in a sustained and controlled manner. Some BMPs have been investigated in both animal studies and human clinical trials [128132] . This has led to several commercially available BMP-based products that will be discussed later in this review.

FGF Originally detected as substances in the brain and pituitary gland, FGFs isolated from these areas were found to promote the growth of broblasts [301] . FGFs are representative GFs that have potential effects on the repair and regeneration of tissues [133] . The FGF family comprises at least 22 different monomeric proteins with molecular weights of 1618 kDa. These compounds bear 55% amino acid sequence homology and are encoded by distinct genes [134] . The 22 members of the FGF family mediate their cellular responses by binding to and activating different isoforms of their receptors encoded by the four receptor tyrosine kinases (i.e., FGFR1, FGFR2, FGFR3 and FGFR4) [135] . FGFs mediate their effects via activation of signaling pathways, including R AS/MAPK, PI3K/AKT and PLC g, among which the RAS/MAPK pathway is known to be predominant [136] . FGFs have various biological functions that occur both invivo and invitro, including roles in mitogenesis, cellular migration, differentiation, angiogenesis and wound healing [133] . Application of FGFs for regenerative medicine may be suitable for promoting regeneration in a wide range of tissues, including skin, blood vessels, muscle, adipose, tendons/ligaments, cartilage, teeth, nerves and bone. Indeed, FGFs are key regulators of bone development [137] , and bone regeneration can be achieved by using bone replacement material that comprises one or more polypeptides having the biological action of FGFs in a porous matrix [132] . FGF pathways can mediate the effects of FGF signaling on osteoblast gene regulation. These pathways can stimulate proliferation of osteogenic cells and chondrocytes and promote cell differentiation and angiogenesis [138] . A brief summary of the roles of different FGF family members in bone growth is presented in FIGURE1. Among the 22 members of the FGF ligand family, FGF-2, -9 and -18 in particular play important roles during bone development. Knockout
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models have revealed that FGF-2-/- adult mice have decreased skeletal bone mass. Moreover, FGF-9-/- embryonic mice exhibit delayed initiation of chondrogenesis. Finally, FGF-18 -/embryonic mice with reportedly smaller cranial vaults and widened cranial sutures have been described [137] . Similar to BMPs, the release effect of FGFs from various carrier materials has been investigated to identify appropriate delivery strategies that regulate the sustained and targeted release of FGFs. FGF-1 and -2 have primarily been used for such studies. For example, in calvarial defects in rats, FGF-1 administered via a coated agarose gel implant was found to promote osteogenesis for cranioplasty [139] . FGF-2 delivered in a hyaluronan or gelatin hydrogel has also been reported to accelerate bone healing invivo [140] . In addition, the osteoinductive effect of hydroxyapatite implants coated with FGF-2 was greatly enhanced compared with uncoated control implants in a rabbit model [141] . Other FGF-binding scaffolds for bone regeneration have also been reported. Collagens with perlecan domain I improved binding of FGF-2, and perlecan domain I-coated electrospun collagen bers were more effective than heparin bovine serum albumin collagen bers at binding FGF-2. Because FGF-2 regulates cell growth, differentiation, migration and survival, the
Preosteoblast Proliferation FGF-2, FGF-4, FGF-9, FGF-18

Osteoblast

Osteoid Matrix

Differentiation FGF-2, FGF-4, FGF-8, FGF-9, FGF-18 Mineralization FGF-2, FGF-9, FGF-18 Osteocyte

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Protects against cell apoptosis FGF-2

Figure 1. FGF functions for bone growth.

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ability to effectively bind FGF-2 to the matrix surface is a key improvement in the fabrication of scaffolds for successful tissue engineering [142] . When titanium nonwoven fabrics with FGF-2 delivery systems of gelatinhydroxyapatite hydrogel microspheres were implanted in rabbit skull defects, bone regeneration was signicantly enhanced compared wth cases without the FGF-2 delivery system or simply with combined systems of FGF-2 in titaniumhydroxyapatite, suggesting the importance of a proper delivery system [143,144] . The negatively charged gelatin protein structure may help the ionic bonding with oppositely charged FGF-2, and consequently sustained release from the system invivo condition.

VEGF VEGFs are important GFs that stimulate the growth of new blood vessels. With molecular weights ranging from 34 to 42 kDa, VEGFs are secreted by various human and rodent tumor cell lines. The VEGF family contains VEGF-A, VEGF-B, PlGF, VEGF-C and VEGF-D [145] . Different isoforms of VEGF can be generated through alternative splicing, including VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206. Among these, the major forms (i.e.,VEGF121, VEGF165 and VEGF189) are secreted by most cell types [146] . VEGFs have various functions. The primary function of VEGFs is related to both vasculogenesis and angiogenesis. Accordingly, VEGFs are known as powerful angiogenic factors that stimulate proliferation and migration of endothelial cells, resulting in the formation of tubular blood vessels. Moreover, VEGFs play a crucial role in bone formation and healing through recruitment, survival and activity of bone forming cells [147] . The process of angiogenesis involves new vessel formation from a pre-existing vascular network. These newly formed vessels are essential for transporting nutrients and oxygen, migration of cells and preservation of the appropriate metabolic microenvironment during bone regeneration and bone repair [148] . Therefore, the activation of angiogenic factors using VEGFs leads to coordinated angiogenesis and bone regeneration [149] . Additionally, VEGFs were found to enhance the migration of osteoprogenitor cells [150] and stimulate migration and differentiation of primary human osteoblasts [151,152] . Furthermore, the expression of VEGF165 by osteoblasts was actively upregulated during osteogenesis [153] . These results suggest that VEGFs can enhance
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bone formation. Interestingly, VEGF in combination with BMP-2 enhanced BMP-2-induced bone formation, further indicating that additive or synergistic effects can be achieved using combinations of GFs for bone regeneration. VEGF can also act as a mediator of various other osteoinductive factors, including TGFb1, IGF and FGF-2, which in turn control the expression of VEGF [154] . Therefore, the combined application of VEGFs with other GFs could synergistically enhance bone formation and fracture healing. VEGFs not only enhance the formation of bone directly, but also indirectly via the induction of angiogenesis at the fracture site, enabling the delivery of additional circulating progenitor cells [154] . TAbLE2 shows a summary of the application of VEGFs for bone tissue regeneration invitro [146,149,151,152,155158] and invivo[150,157,159161] . Due to the rapid diffusion of GFs away from the injury site after injection, delivery of VEGF was performed using poly(lactic-co-glycolic acid) (PLGA) biopolymer carriers [162] . In a rat calvarial defect model, signicant improvements in neovasculatrization, bone coverage and bone mineral density were observed after implantation of VEGF scaffolds compared with PLGA control scaffolds [162] . In another study, VEGF coated onto the surface of bioactive glass resulted in additive bone healing effects within the rat critical- sized defect [163] . Moreover, PLGA scaffolds seeded with human bone marrow stromal cells containing combinations of condensed plasmid DNA encoding BMP-4 and VEGF promoted new bone formation [164] .

IGF IGFs are polypeptides with high sequence similarity to insulin. There are two IGF ligands (IGF-I and IGF-II) and two cell-surface receptors (IGF1R and IGF2R). IGF-I has been used for the treatment of injuries in various tissues. IGF-I generally functions by stimulating cell proliferation and survival and inhibiting apoptosis [165] . IGF-I is the most abundant GF present in skeletal tissues [166] . It is an important regulator of skeletal growth and development [167] , as well as a regulator of osteoblastic function [166] . In addition, IGF-I stimulates the proliferation and differentiation of osteoblastic precursor cells [168] . This osteoblastic function is mediated through the PI3K pathway by stabilizing b -catenin, thereby enhancing Wnt-dependent activity [169] . In a monolayer model of wound healing, IGF-I secreted from osteoblasts directed
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Table 2. Application of VEGFs for bone tissue regeneration.


Cell/animal type
Invitro Endothelial cells

Biological effect

Ref.
[146] [149] [155] [156] [157] [146] [158] [157] [159] [150,160,161]

Proliferation, sprouting, migration and tube formation Osteoblasts Migration, proliferation and survival Chondrocytes Conversion of cartilage into bone and osteoblasts Muscle-derived stem cells Differentiation and proliferation (angiogenesis) Mouse osteoprogenitor cells Ossication and angiogenesis Enhancement of bone formation and healing Bone formation Mouse Rabbit Resorption of cartilage Blood vessel formation, ossication and new bone (callus) maturation Bone healing and increased bone density

Invivo

the migration of osteoblasts, promoted cell spreading and regulated cell polarization [170] . Similarly, in developing fracture calluses, IGF-I and its receptors are widely expressed in periosteal cells, mesenchymal cells, proliferating chondrocytes and osteoblasts, suggesting that it plays a coordinated role among several cell types during fracture healing [171] . There is invivo evidence that serum IGF-I concentrations are positively correlated with skeletal mass. Increases in serum IGF-I levels during growth lead to enhancement of all bone phenotypic traits and may play a protective role later on during aging. Such increases also emphasize the contribution of serum IGF-I to both cortical size and bone density [172] . By contrast, locally produced skeletal IGF-I plays a greater role in trabecular bone integrity. This is demonstrated in transgenic mice expressing IGF-I in osteoblasts and in conditional IGF-I receptor-null mice, which display decreased osteoblast numbers and function, causing reduced bone formation and trabecular bone volume [173] . Global deletion of IGF-I in mice resulted in postnatal growth retardation [174] ; similarly, in IGF-I heterozygous mice, reduction of serum IGF-I levels resulted in a change of body weight and reduced cortical bone mineral density [175] . Other studies in mice with reduced serum IGF-I levels also showed signicant decreases in transverse bone growth. Reductions in serum IGF-I during postnatal growth are extremely important markers of a potential compromise in bone robustness and increased fracture risk during adulthood and aging. IGF-I also induces VEGF expression in skeletal cells, and so may enhance the direct and indirect positive effects of VEGF on endochondral bone formation and osteoblastic differentiation and function [176] . Only a few invivo studies investigated the use of intra-articular injections of IGF-I in mice,
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dogs and rabbits. These studies all demonstrated little therapeutic effect of the periodic use of IGF-I alone for cartilage and subchondral bone repair in osteoarthritic joints [176] . The effect of IGF release from carrier materials has also been investigated. When IGF-I was coated onto a dextran-co-gelatin, periodontal tissue regeneration was enhanced [177] . In another study, IGF-I entrapped in PLGA micro particles also enhanced new bone formation [178] . Moreover, the combined delivery of TGF-b and IGF-I within gelatin hydrogels to promote bone formation was evaluated, and the combined implantation of TGF-b and IGF-I was shown to have greater bone healing potential than IGF-I treatment alone [179] . The long-term effects of combined TGF-b and IGF-I on fracture healing have also been determined, and while enhanced bone regeneration was evident in a rat closed tibial fracture model for up to 6weeks, by 12weeks, no difference in bone regeneration status remained in comparison with control fractures [180] . However, when IGF-I and TGF-b were coated onto titanium with poly(l-lactide acid), they failed to induce ectopic bone formation in ovine muscle [181] . Nevertheless, IGF-I and IGF-II delivered within collagen gels were found to enhance the repair of facial osseous defects[182] .

Other GFs HGF HGF has been shown to stimulate both osteoblast proliferation and osteoclast chemotactic migration [183] . In bone tissue, HGF is produced by osteoclasts and induces responses in both osteoblasts and osteoclasts. In osteoblasts, HGF induces DNA synthesis, whereas in osteoclasts, in addition to DNA synthesis, HGF also stimulates chemotactic-oriented migration, as previously observed in epithelial and endothelial cells [184] . Cell migration is important in the
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early steps of bone resorption, when osteoclast precursors are chemotacticly attracted to the bone matrix. In these cells, HGF also stimulates DNA synthesis, thereby controlling both migration and proliferation of osteoclasts at the sites of bone resorption [185] . HGF reportedly exerts benecial effects on bone formation at the boneimplant interface of orthopedic prostheses [184] . However, HGF has occasionally been found to inhibit BMP-2-induced bone formation through activation of ERK1/2 when the cells are treated with both HGF and BMP-2 simultaneously [186] . Furthermore, HGF was shown to inhibit the BMP-2-induced expression of Runx2 [187] , which, along with osterix, is an essential transcription factor for osteogenic differentiation [188] . Treatment with HGF prior to BMP-2 was shown to promote cell proliferation without disturbing BMP-2-induced osteoblast differentiation. Thus, HGF treatment can potentially be benecial for increasing cell numbers, but the timing of HGF treatment is considered to be critical and should be carefully determined for successful bone formation induced by BMPs [186] . HGF readily adsorbs to the bone mineral surface, maintains integrity (i.e., does not degrade) over time and enhances osteoblast differentiation. These characteristics make HGF a promising potential candidate for GF coating of bone implant/scaffold surfaces [183] . EGF EGF plays an important role in osteoblast growth and development [189] . EGF alone promotes osteoblast proliferation, and the combination of EGF with BMP-2 and -7 signicantly upregulates this proliferation [190] . The effect of EGF on osteoblast growth may occur through specic receptors that are expressed by the osteoblasts. EGF delivered either alone or in combination with BMP-2 and -7 also induced expression of ALP and OC proteins, but in spite of this, there is an overall net negative effect on bone mineralization due to inhibition of nodule formation [190] . Moreover, EGF combined with calcitriol was able to stimulate osteoblast differentiation, characterized by increased synthesis of both ALP and OC [189] . EGFR signaling is also involved in bone resorption. EGF has the ability to strongly stimulate bone resorption in cultured fetal rat long bones, newborn mouse calvarial cultures and long-term human marrow cultures, suggesting that this GF regulates osteoclastogenesis and bone resorption [191] .
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MGF Many splice variants of the IGF-I gene can be produced that have different functions [192] , and one splice alternative variant is MGF [193] . MGF acts locally as a tissue repair factor that responds to changes in either physiological conditions or environmental stimuli [193] . The MGF splice variant of IGF-I is produced when cells experience significant physical trauma such as mechanical overload, severe temperature uctuations or hypoxia, among others [192] . The presence of a unique E domain (a 48-bp insert between exons 5 and 6 introduced during splicing) in the C-terminal region of MGF (called MGF-Ct24E) functionally distinguishes MGF from other IGF-I isoforms [194] . Recently, MGF and its E peptide have attracted much attention because of their reported woundhealing role [195] . MGF and MGF-Ct24E were found to improve osteoblast proliferation [192] . Particularly, MGF-Ct24E increased proliferation activity compared with IGF-I. To date, the study of MGF using carrier materials has not been investigated, although MGF is a candidate for targeting bone tissue regeneration. Therefore, further study on MGF is needed. PDGF PDGF is produced by skeletal cells. These cells express three pdgf genes (i.e., pdgfa, pdgfb and pdgfc), indicating that PDGF may act as an autocrine regulator of skeletal cell function. PDGF is present in bone matrix and is secreted by platelets at the site of fracture during early tissue repair [196] . It is a potent mitogen that facilitates wound healing and stimulates bone repair by expanding osteoblastic precursor cells [197,198] . However, additional signals are required to induce the differentiation of these cells towards mature osteoblasts [166] . The angiogenic effects of PDGF, which are similar to the effects of VEGF, may also be favorable to osseous wound repair. PDGFs have a similar structure to VEGF, and PDGF-BB enhances FGF-2-stimulated VEGF release [199] . These additional factors can accelerate and enhance the bone-induction effects of PDGF, promoting greater bone volume, and thus enabling earlier implant placement and loading [197] . Collagen gels loaded with PDGF-BB were found to enhance their bone-regenerative effect on tibial osteotomies in rabbits [200] . Furthermore, PDGF-BB within a chitosan/ tricalcium phosphate sponge carrier was also shown to have bone-regenerative effects [201] . Taken together, these studies suggest that
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delivery of PDGF within carriers can enhance bone regeneration strategies.

Fusion proteins & peptides Fusion proteins are formed by combining sequences from two or more genes that originally coded for those individual proteins. The single protein product contains functional characteristics of each individual protein. This is made possible by the fact that a particular protein function is often specically attributable to a single domain of that protein. Therefore, the whole protein does not have to be fused, but only the sequences containing the functional domains are of interest. Fusion proteins have been widely used for identifying and purifying proteins, (e.g., using hexa-His peptide [6Histag], fusing a GST protein or FLAG peptide). In addition, fusion proteins can have toxins or antibodies attached to them, offering the opportunity to target specic cell types, such as tumor cells. Because of their wide range of uses, fusion proteins are being widely investigated. FGFs are effective GFs for fusion with a variety of proteins. For example, FGF-1 conjugated with heparan sulfate proteoglycan was constructed to protect FGF-1 from proteolytic degradation [202] . In addition, fusion of FGF-2 and OC has been investigated [144] . The FGF-2mOC product has advantages for bone repair and regeneration owing to the synergistic effects of FGF and OC. The hydroxyapatite (HA)-binding capacity of FGF-2mOC protein is signicantly greater than that of native FGF-2, due to the high afnity of OC to HA. Furthermore, fusion of FGF-2 and mOC in a HA surface-immobilized state results in a signicant increase in cell proliferation and differentiation of MC3T3-E1 osteoblastic cells. These properties, which are not exhibited by native FGF-2, may offer a novel strategy to potentiate the therapeutic effect of FGF-2 in bone repair and regeneration. In particular, collagen-binding domain (CBD)-based GF fusion proteins have been found to be useful for combining GFs with collagen carriers to injured tissues in some studies. For instance, fusion protein delivery of VEGF has been investigated when Ishikawa etal. established the bronectin CBD (FNCBD)VEGF121 [203] . FNCBDVEGF121 stably maintained an optimally high and local concentration of VEGF within the collagen matrix, stimulating both endothelial cells and endothelial progenitor cells insitu, thereby supplying a vascular regeneration niche. The same group also established a collagen-binding GF
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consisting of EGF and FNCBD, designated FNCBDEGF [204] . FNCBDEGF is a biologically active fusion protein that was able to stably bind to collagen materials and exert its GF activity even after collagen binding. Thus, in an injured artery, infused FNCBDEGF remained bound to collagen exposed in the injured tissues, even after blood circulation was restored. Injection of the fusion protein into the rat hindlimbs was found to be effective for direct administration to muscular tissue. The effects of a HGF fusion protein exhibiting collagen-binding activity (CBDHGF) were studied to determine the re-endothelialization and neointimal formation in a balloon-injured rat carotid artery [205] . CBDHGF accelerated re-endothelialization and neointimal formation invivo compared with both the negative control and HGF-treated groups. TGF-b1 was also engineered to generate a fusion protein bearing a CBD to selectively target typeI collagen and ensure the slow and localized release of the GF [206] . Taken together, CBD fusion protein is a useful vehicle to deliver vascular GFs to injured arteries. Genetically engineered fusion proteins with specic binding domains for the ECM are becoming particularly attractive for bone targeting.

Regulator-approved products: towards the clinic There are still relatively few recombinant GF products on the market for bone regeneration purposes. Of fundamental importance for the delivery of biologics, such as recombinant GFs, is that they are safe to use and do not compromise the wellbeing of the patient. The second main requirement is that they actually demonstrate efcacy in the context of accelerated tissue regeneration responses, yielding substantial improvement in regeneration capacity. The US FDA has approved two BMP therapeutic products for clinical application. Both BMP-2 and -7 are available commercially for clinical use as the active components of the products Infuse (Medtronic Sofamor Danek; MN, USA) and OP-1 (Stryker Biotechnology; MA, USA). These BMPs have been rigorously investigated in both animal studies and human trials [128132] , with many successful results. Specifically, BMP-2 (Infuse) has been approved for the treatment of fusion of the lumbar spine in patients with degenerative disc disease. It has also been approved for certain oral and maxillofacial uses and has been demon strated to be highly effective in the treatment of
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tibial fracture [130] . BMP-7 has been approved as an alternative to autografting in recalcitrant long bone nonunions [207] and in compromised patients requiring revision posterolateral lumbar spinal fusion. However, recent evidence suggests that the safety prole of BMP treatment is not as high as was originally thought, and patients treated with BMP-2 for spinal fusion have an increased risk of cancer [208] . Therefore these
Executive summary

treatments should only be used with signicant caution. PDGF has been used to stimulate bone regeneration in dental procedures. Being incorporated into a calcium phosphate matrix, it has been demon strated to provide safe and effective stimulation of bone regeneration in patients with severe periodontal bone loss [209] . It is available under the trade name GEM21S (Osteohealth Company,

Background Growth factors promote cell responses and are important mediators of wound healing after injury. They hold promise for use in tissue engineering and for exogenous stimulation of intrinsic regenerative responses if delivered to the injury site using appropriate carrier materials. Considerations for carrier materials Materials that will be implanted should be biocompatible, nontoxic and nonimmunogenic, and ideally will degrade over time by the action of enzymes or circulating body uid. Polymer materials that are generally used are either natural (i.e., collagen) or synthetic (i.e., poly[lactic acid] or poly[glycolic acid]), andin the context of bone regeneration, inorganic materials such as calcium phosphate, which supports bone cell responses, are oftenused. BMP Most BMPs stimulate bone regeneration (BMP-3 being a notable exception), and delivery of BMPs using synthetic polymers has proven successful in accelerating bone regeneration in animal models. FGF FGF is a key regulator of bone development and has broad effects on repair and regeneration responses, including cell proliferation, migration, differentiation and angiogenesis. VEGF VEGF is an important growth factor that mediates angiogenic and vasculogenic responses, which are helpful for production of bone tissue as it requires a vascular supply. VEGF in combination with BMP2 enhances the osteogenic effects of BMP2. IGF IGF is an important regulator of skeletal growth and development and of osteoblast function, particularly proliferation and differentiation. Delivery using a variety of carrier materials in animal models of osteoid defects has yielded favorable regenerative repsonses. Other factors HGF HGF stimulates osteoblast proliferation, osteoclast proliferation and chemotaxis to regulate bone turnover, but inhibits BMP-2-induced bone formation. EGF EGF stimulates osteoblast proliferation and has a synergistic effect in combination with BMP-2 or -7. MGF MGF is an IGF splice variant that stimulates osteoblast proliferation and is a general positive regulator of wound healing. PDGF PDGF is a potent inducer of proliferation, but in itself is not capable of directing osteoblastic precursors towards a mature phenotype. Fusion proteins Fusion proteins that are engineered to contain functionally active components of two or more individual proteins have proven effective at enhancing bone regeneration. Notably, fusion proteins of FGF-2 combined with OC have been successfully employed to enhance cell responses on hydroxyapatite-coated scaffolds due to the dual impact of mitogenic FGF-2 and maturation-inducing OC. Regulator-approved products: towards the clinic Several recombinant protein products are currently available on the market for bone-regeneration therapy. Specically, products containing BMP-2, BMP-7 or PDGF as the active ingredient were successful in several clinical trials. However, in light of recent evidence, regulator concern still exists regarding the safety of recombinant protein products that have mitogenic capacity. Therefore, it is necessary to proceed with caution in the development of recombinant growth factors and delivery methods for bone regeneration.

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NY, USA) and meets the International Standard for biocompatibility of medical devices [401] . To date, no severe adverse effects have been reported, and it is therefore very promising as a treatment strategy. Broadly speaking, orthopedic and oral surgery have beneted greatly from commercially available GFs in the last few years. Positive patient responses have been reported in many clinical trials. However, recently reported safety concerns highlight the continued requirement to carry out more research and understand the global system effects of GFs on the patient. It also highlights the need to understand precisely the dosages that will elicit positive effects without compromising safety and the need to use biomaterials that will carefully control and regulate GF release in a precise and localized manner.

bone regeneration. Future strategies to restore functions in difcult-to-heal bone fractures will depend on carefully controlled, targeted delivery of osteogenic GFs to re-establish the healthy tissue architecture. In addition, administration of multiple GFs in combination is expected to be required to maximize the potential of GFs. Many strategies are being explored for producing carrier systems that will ensure targeted delivery in a sustained and controlled manner, including both synthetic and natural degradable biomaterials. A biotechnological approach using fusion proteins that preserve the active domains of the GFs to stimulate regenerative cells, as well as containing matrix-specic binding motifs to be effectively loaded onto delivery systems, offers an exciting addition to the current arsenal of GF-based therapy in bone regeneration.
Financial & competing interests disclosure
This work was supported by the Priority Research Centers Program (grant no.: 2009-0093829) and World Class University program (grant no.: R31-10069) through the National Research Foundation funded by the Ministry of Education, Science and Technology. The authors have no other relevant afliations or nancial involvement with any organization or entity with a nancial interest in or nancial conict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
endothelial growth factor receptors. Mol. Biol. Rep. 38(2), 14431450 (2011). chondrogenesis invitro. Osteoarth. Cartil. 11(1), 5564 (2003).
11

Future perspective GFs are generally stored in the ECM of bone tissue. GFs, including BMPs, FGFs, VEGFs and IGFs, are released after injury and contribute to bone repair, and have consequently been proposed for use in bone regeneration applications. There is considerable potential to use these biofactors for regenerative medicine approaches that not only manage fracture healing, but also actively enhance the regenerative process. Indeed, BMPs have already been tested clinically and recombinant proteins are available on the market to enhance
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