The Role of Growth Factors on Acceleration of Bone Regeneration During Distraction Osteogenesis

Abstract

The distraction osteogenesis (DO) technique has been used worldwide to treat many complex orthopedic and craniofacial conditions. One limitation of this technique is the long time of fixator needs to be left in place until the bone is completely consolidated. Various biophysical, mechanical, and biological methods have been investigated to accelerate bone regeneration during DO. Several growth factors (GFs) are known to enhance bone regeneration such as bone morphogenic proteins, transforming growth factor beta, fibroblast growth factor, insulin growth factor, vascular endothelial growth factor, and platelet-derived growth factor. These GFs are known to stimulate cellular growth, proliferation, migration, and differentiation. In this review, an extensive overview of these GFs development and applications on acceleration of bone regeneration in the context of DO is discussed. Current challenges and alternative tissue engineering techniques to address the delivery and sustain release of these factors are also discussed. Finally, we highlighted our view regarding the remaining questions and future research directions.

Introduction

Distraction osteogenesis (DO) is a controlled surgical procedure that initiates a regenerative process and uses mechanical forces to stimulate endogenous biological responses of the injured tissues to create new bone.¹ Since the introduction of the DO concept by Codivilla and Ilizarov many complex orthopedic conditions, such as bone loss, nonunion, congenital short bone segments, and bone tumors, have been treated with satisfactory outcomes.² Currently, the DO technique has been used worldwide to achieve spontaneous bone regeneration and considered as a valuable tool that surgeons can keep in their armamentarium for complex reconstructive procedures. One limitation of this technique is the long period of time that the fixator is left in place until the bone is completely consolidated. This has unfavorable outcomes on patients such as increasing the risk of infection, financial burden, and a psychological negative impact.^3–5 The question remains, how can we accelerate bone regeneration in DO patients?

Previous animal experiments have found successful distraction is based on the stability of the fixator, rate and rhythm of the gradual distraction, and degree of damage to local soft tissue.^6–8 Although these studies have described the technical principles to achieve a successful outcome, the molecular mechanisms responsible for these processes were essentially undetermined until were primarily studied by Sato et al. and Rowe et al.^9,10

Application of various growth factors (GFs) is one potential approach of other various modalities to enhance bone regeneration in DO models (Fig. 1).^11–29

FIG. 1.

Illustrating different methods to accelerate bone regeneration during distraction osteogenesis. DO, distraction osteogenesis.

Several GFs are known to enhance bone regeneration such as the transforming growth factor beta (TGF-b1), fibroblast growth factor (FGF), insulin growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and bone morphogenic proteins (BMPs). These GFs are known to stimulate cellular growth, proliferation, migration, and differentiation.³⁰ They are released from the extracellular matrix after injury to contribute in the bone regeneration process. The interaction between various GFs and surface receptors on the target cells (adult mesenchymal stem cells, osteoblasts, fibroblasts, endothelial cells, and epidermal cells) activates production of proteins needed for the regenerative processes such as cellular proliferation, matrix formation, osteoid production, and collagen synthesis.³¹

While the basic biological functions of certain GFs and their endogenic roles in tissue development and regenerative process have relatively been well studied, the use of GFs in a tissue-engineering regime has recently gained significant interest. Interference with any of these GF pathways will determine whether or not to accelerate the bone regenerative process.

In this article, we will discuss the aforementioned GFs that play an important role on acceleration of the bone regeneration process in the DO model, current challenges, and the future research directions.

Bone Morphogenic Proteins

In 1965, Dr. Marshall Urtist has introduced the term of bone morphogeinc proteins. He demonstrated that certain substances (BMPs) in the demineralized bone matrix could induce ectopic bone formation when implanted in extraskeletal sites in rodents.³² Since then, more than 20 BMPs have been described in the literature.³³ It belongs to the TGF-B superfamily and has a critical role in stimulating bone regeneration leading to osteogenesis, chondrogensis, angiogenesis, and upregulation of other GFs, cytokines, and synthesis of the extracellular matrix.³⁴ They are also responsible for matrix formation and osteoblast formation and it is the only osteoconductive ones that play a role in the early differentiation process of undifferentiated mesenchymal cells.³⁵ Interestingly, Lou et al. have documented the ability of BMP-2 to stimulate mesenchymal cell proliferation in vitro under adenovirus-mediated BMP gene transfer.³⁶

BMP receptors (BMPRs) functioned as mediators for BMP signaling. These signals are transmitted by Smad proteins.³⁵ The action of BMPs is tempered by extracellular BMP-binding proteins such as noggin, chordin, dan family and twisted gastrulation, transmembrane BMP pseudoreceptors (Bambi), intracellular BMP antagonists (Smad 6/7), and intranuclear substance (c-SKI and Smurf1/2).^37,38 In addition, BMP-3 has been also identified as a BMPR antagonist that blocks the BMP activity.³⁹ Furthermore, other GFs can also interact with the BMP action. For instance, the hepatocyte growth factor (HGF) was found to inhibit the BMP-2 action via activation of ERK1/2, which subsequently suppresses the BMP-2 Smad axis.⁴⁰ In contrast, the epidermal growth factor (EGF) enhanced significantly the action of BMP-2 and BMP-7 in vitro and, therefore, promoted the osteoblast growth.⁴¹

BMPs are available in two forms, either heterodimers or homodimers. Previous reports have shown that heterodimer forms (BMP 2/7 and BMP 2/6) have a better effect on bone regeneration by 20 folds in vitro and 5 to 10 folds in vivo when compared to homodimers.⁴² Of 20 BMPs, BMP-2, −4, −6, −7, and −9 have the greatest osteoinductive potentials in vitro.^43,44

BMP expression and immunohistochemical localization of protein-signaling Smads have been investigated in the DO model. Sato et al. found high expression of BMP-2 and BMP-4 during the distraction phase in a DO rat model.⁹ In our laboratory, we have investigated the temporal and spatial expression of BMP 2, 4, and 7 proteins during DO of tibiae in a rabbit model using immunohistochemistry, and interestingly found intense staining for these BMPs in the latency period and it was maintained during the entire distraction phase and once the distraction has stopped, BMP (2, 4, and 7) expression gradually disappears.⁴⁵ Similar results were found when we investigated the mandibular DO in rabbits except that the expression of BMP 7 was limited during the distraction phase.⁴⁶ Furthermore, delayed bone regeneration in the DO model was observed in BMP-2 knocked out mice when compared to the control group.⁴⁷ This confirmed the critical role of endogenous BMPs in the DO bone regenerative process. We also investigated the expression of receptor-regulated Smad 1, 5, and 8, which showed a significant increase mainly in the distraction phase, while Smad 6 and 7 showed high expression in the consolidation phase.⁴⁸

Most of the recombinant BMP-2 and −7 applications in DO models have shown promising results from the animal data^49–54 (Table 1). However, the clinical use of systemic and local BMPs is limited. When injected systemically, the half-life of BMPs is short and rapidly metabolized by the liver,^55–57 while when injected locally its limitations could be explained by the short resident time in tissues and probably insufficient numbers of responding cells.^58,59 Therefore, large doses are required to reach an optimal biological response. This is associated with unknown side effects and huge costs.⁶⁰ Other limitations for using BMPs in humans include possible neoplastic risk, potential teratologic sequences, osteoclastic activation, and unknown consequences on skeletally immature patients.^61–63

Table 1.

Bone Morphogenic Protein Applications in Different Distraction Osteogenesis Animal Models

Author (date)	BMP subfamily	Model	Biological outcome
Yonezawa et al. (2006)⁴⁹	BMP-2	Mandibular DO in Rabbits	Promote bone regeneration
Cheung et al. (2006)⁵⁰	BMP-2	Mandibular DO in Rabbits	Promoted bone regeneration at both routine and rapid distraction rates.
Mandu-Hrit et al. (2006)⁵¹	BMP-7	Tibiae DO in Rabbits	Promote bone regeneration
Hamdy et al. (2003)⁵²	BMP-7	Tibiae DO in Rabbits	Failed to promote bone regeneration during the consolidation phase of DO. The authors suggest that it would be useful when applied early in the distraction phase.
Mizumoto et al. (2003)⁵³	BMP-7	Femur DO in Rats	Promote bone regeneration
Zheng et al. (2006)⁵⁴	BMP-2	Mandibular DO in Rabbits	Promoted bone regeneration at both routine and rapid distraction rates.

BMP, bone morphogenic protein; DO, distraction osteogenesis.

We can overcome these challenges by finding an alternative strategy such as intrinsically stimulate BMP expression or developing an appropriate delivery system will allow local sustained release of BMPs in low doses.

In addition, administration of more than one exogenous BMP with respect to its spatiotemporal expression might accelerate the bone regeneration process during DO. Up to date, various tissue-engineering techniques have been utilized to enhance BMP action during DO, including gene therapy, coadministration of the bioactive factor, and protein therapy^64–71 (Table 2).

Table 2.

Different Bone Morphogenic Protein Applications Accompanied with Delivery Systems in Distraction Osteogenesis Models

Author (date)	BMP subfamily	Model	Biological outcome
Lesaichot et al. (2011)⁶⁴	BMP-2 with ACS	Tibiae DO in Rabbits	Promote bone regeneration
Sailhan et al. (2010)⁷⁰	BMP-2 with ACS	Tibiae DO in Rabbits	Promote bone regeneration, rabbits that received high BMP-2/ACS doses developed premature consolidation.
Ashinoff et al. (2003)⁶⁵	BMP-2 (Adbmp-2)	Mandibular DO in Rats	Promote bone regeneration
Haidar et al. (2009)⁶⁶	BMP-7 with biodegradable core-shell nanoparticles	Tibiae DO in Rabbits	Promote bone regeneration at low doses of BMP-7
Long et al. (2011)⁶⁷	(Adv-BMP-2)	Mandibular DO in Rabbits	Promote bone regeneration at rapid distraction rate
Yang et al. (2011)⁶⁸	BMP-2 with TCP/HA	Femur DO in Rat	Promote bone regeneration
Zhang et al. (2012)⁶⁹	MSCs transfected with BMP-2/7	Irradiated mandibular DO in Rabbits	Accelerate bone regeneration
Zhu et al. (2011)⁷¹	BMP-2+Nell-1	Tibiae DO in Rabbits	Promote bone regeneration. Best results were found when BMP-2 was combined with Nell-1.

ACS, absorbable type I collagen sponge; Adbmp-2, adenovirus containing the gene for BMP; TCP/HA, tri-calcium phosphate/hydroxyapatite; MSCs, mesenchymal stem cells; Nell-1, NEL-like molecule-1.

Several reports have emphasized the role of inhibiting the BMP antagonist in bone repair as a potential method to accelerate bone formation^72–78 (Table 3). In the context of DO, we have previously reported that BMP antagonists such as of BMP-3, chordin, inhibin, and noggin showed significant expression during DO.⁷⁹ However, the role of inhibiting the BMP antagonist has not been investigated yet in the DO model.

Table 3.

Role of Extracellular, Intracellular, Intranuclear Bone Morphogenic Protein Antagonist on Bone Metabolism in Selected Studies

Author (date)	Purpose of the study	Model	Results
Kloen et al. (2012)⁷²	To document coexpression of BMPs, pSmad 1/5/8, and BMP-inhibitors.	Human fracture callus and human nonunions	Expression of BMP-inhibitors was stronger in nonunions than in fracture callus
Takayama et al. (2009)⁷³	Study the effect of Noggin silencing by transfection of small interfering RNA on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2	In vitro: C2C12 cells In vivo: mice	In vitro: acceleration of BMP-induced osteoblastic differentiation. In vivo: ectopic bone formation.
Kwong et al. (2008)⁷⁵	Study the effect of Chordin knockdown on osteogenic differentiation of human MSCs	In vitro: human MSCs	Significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral
Li et al. (2008)⁷⁶	The effect of sclerostin knockout (SOST) mice on bone formation	In vivo: mice	Increase of bone volume, formation, and strength
Ku et al. (2006)⁷⁷	Investigated the role of the transcriptional activator and Smad1/4 cofactor OAZ in regulating BMP signaling.	In vitro: pluripotent embryonal carcinoma cells	Removal of endogenous OAZ prevents the induction of Smad6 by BMP4 and extends the period of detection of phosphorylated Smad1 after BMP stimulation
Winkler et al. (2004)⁷⁴	Investigating the combined effect of Noggin–sclerostin complex as BMP antagonist	In vitro: rat osteosarcoma cell line ROS 17/2.8	Competitive complex that decrease BMP antagonist activity and can form a complex that allows BMP signaling to proceed
Zhao et al. (2004)⁷⁸	To determine the role of Smurf1 in osteoblast differentiation and bone formation	In vitro: 2T3 osteoblast precursor cells In vivo: transgenic mice	In vitro: Smurf1 overexpression inhibits BMP signaling and osteoblast differentiation In vivo: bone formation was significantly reduced during postnatal life

OAZ, smad-interacting transcription cofactor; ROS, rate osteosarcoma.

Transforming Growth Factor-B

TGF-Bs are 25-kDa proteins. Three isoforms have been found (TGF-b1, b2, and b3). TGF-Bs are potent osteoblast chemoattractants, have mitogenic effects on osteoblast precursors produced by osteoblasts in culture, and are expressed by osteoblasts and osteoblast precursors during endochondral ossification.⁸⁰ TGF-Bs play an integral part of collagen production and upregulate the expression of noncollagenous ECM proteins implicated in the regulation of bone turnover and mineralization.⁸¹ The most abundant GF of the bone matrix is TGF-b1, which has a wide range of cellular activities, including control of proliferation and expression of the differentiated phenotype of several cell types specific to bone, among them, mesenchymal precursor cells, chondrocytes, osteoblasts, and osteoclasts.⁸¹

The spatial and temporal expression of TGF-b1 were investigated in mandibular DO in the rat model, and demonstrated an increase of TGF-b1 expression at the end of the latency period and shortly after the initiation of distraction and remain elevated until 4 weeks of the consolidation period when compared to sham-operated animals.⁸⁰ Yeung et al. have also found strong expression of TGF-b1 at the osteoblast and osteocyte in the newly formed callus and remained elevated throughout the distraction phase.⁸²

The effect of TGF-b1 application has shown promising results in fracture healing and bone repair in various animal experiments.^83–86 However, few studies have reported the role of exogenous TGF-b1 in the DO model.^87,88 In our laboratory, TGF-b1 treatment had no detectable effect on the bone mineral density or histologically determined bone volume in the distraction gap. In addition, load to failure in uniaxial tension was lower in TGF-b1-treated rabbits.⁸⁷ In contrary, Ozakan et al. found that locally applied TGF-b1 improves the mineral density of the distraction gap and the load to failure, but no histomorphometric difference was detected between the treatment and control groups.⁸⁸ They have also noted downregulation of the TGF-b1 type 2 receptors in the TGF-b1-injected group. The authors have justified this discrepancy by the lack of a specific stain to the type 1 receptor in their experiment, which likely will show upregulation secondary to the exogenous TGF-B1 administration. However, further experiments are needed to draw a clear conclusion regarding the use of exogenous TGF-b1 in the DO models.

Insulin Growth Factor-I

The IGF plays an integral role in the cellular growth, metabolism, differentiation, and cell cycle progression.⁸⁹ The IGF-I is also a potent chemotactic factor for osteoblasts. Interestingly, when a mouse IGF-I was neutralized, delayed osteoblast monolayer wound healing and cellular polarizations were observed, but this effect reversed when the human IGF-I was added.⁹⁰ In addition, when a conditional deletion of the IGF-I was performed in mice, a significant decrease in the femoral size and bone formation was noted.⁹¹ Furthermore, osteoblast-specific knockout mice have also shown a decrease in cancellous bone volume, connectivity, and trabecular number, and an increase in trabecular spacing.⁹² These studies have established the critical role of the IGF-I in bone regeneration.

Schumacher et al. have investigated IGF-I expression in tibiae DO in rabbits and found that periosteal IGF-I increased after 2 weeks of distraction and ceased gradually when the distraction stopped.⁹³ Eingartner et al. have also found strong expression of the IGF-I in the distraction callus of 3 patients.⁹⁴ Furthermore, the administration of IGF-1 during mandibular DO in rabbits showed a significant increase in the osteoblastic activity in both rapid and slow distraction rates.⁹⁵ Bernstein et al. have investigated the local application of enriched polylactide membranes (IGF-I and TGF-b1) in tibiae DO in a sheep model, although no statistical significance was found, a higher relative bone area was observed when compared to groups treated with an uncoated membrane or packing of the osteotomy defect with cancellous bone.⁹⁶ Although these results are encouraging, further experiments are required to determine when and how the IGF-I should be given during DO procedures.

Fibroblast Growth Factor

It is known to compromise 22 members. FGFs have multiple roles, including cell proliferation, migration, differentiation, and angiogenesis. These have been used for the regeneration of damaged tissues, including skin, blood vessel, muscle, adipose, tendon, ligament, cartilage, bone, tooth, and nerve. It functions through binding into the FGF receptors, which mainly stimulated through the RAS/MAP kinase pathway.⁹⁷ The FGF2 (bFGF) is recognized as a potent mitogen for a variety of mesenchymal cells.⁹⁸ The impact of locally applied bFGF injection under physiological conditions and osteoporotic-induced rabbits was investigated, the best results in terms of enhancing bone formation were found when a 400 mg or greater was given.^99,100 Kato et al. also found that bFGF injection will increase the volume and mineral content of newly formed bone in long bone defect in rabbits in a dose-depended manner with significant results seen at 100 mg or greater.¹⁰¹ Interestingly, the FGF-2 use in the fracture healing has been translated to the clinic. In a prospective randomized double-blind placebo-controlled multicenter trial that included 71 patients, local application of rhFGF2 hydrogel has proven to be effective in terms of accelerating fresh fracture healing.¹⁰²

The basic FGF plays an important role in neovascularization during DO and its expression found to be strongest from 0 to 7 days after the end of distraction in mandibular DO in a goat model,¹⁰³ while bFGF expression in long bone DO in the goat model was highest during the distraction phase.¹⁰⁴ Local applications of the bFGF in tibiae DO in rabbits showed significant enhancement in bone formation as early as 2 weeks after bFGF injection when compared with the control group.¹⁰⁵ Furthermore, the use of combined rhFGF-2 and b-tricalcium phosphate (b-TCP) have reduced the treatment period for DO and accelerated bone formation.¹⁰⁶ The basic FGF seems to be a promising agent. However, further experiments are required to establish the optimal time, dose, and delivery system of the bFGF during DO procedures.

Vascular Endothelial Growth Factor

The VEGF represents a critical rate-limiting step in the physiological angiogenesis.¹⁰⁷ The family of VEGF-related molecules includes seven mammalian members: VEGF-A, placenta growth factor, and VEGF-B, -C, -D, -E, and -F. The biological functions of VEGF are mediated by at least three corresponding receptors: VEGFR-1/Flt-1, VEGFR-2/Flk-1, VEGFR-3/Flt-4 and 2 coreceptors of neuropilin and heparan sulfate proteoglycans.¹⁰⁸ VEGF-A is most predominately involved in the bone regenerative process and several studies have demonstrated its role in the regulation of chondrocyte activities, chondroblast/osteoclast activities, and osteoblast activities.¹⁰⁸ Several ways have been described to be involved in VEGF-A upregulation and gene expression such as BMPs, hypoxia-induced factor (HIF), TGF-B1, tumor necrosis factor alpha, and hormones.¹⁰⁸

Local application of VEGF in severe soft tissue trauma with fracture and bone segment defects in rabbits has shown promising results in terms of enhancing bone regeneration.^109,110

VEGF mRNA expression has been investigated in femur DO in rats and found to be maximal during the active distraction phase.¹¹¹ However, in mandibular DO in goats, VEGF expression was highest from 0 to 7 days after the end of distraction.¹⁰³ Warren et al. have also found VEGF expression remains elevated throughout both distraction and consolidation phase in mandibular DO in rats.¹¹²

There is no doubt that VEGF-A plays a central role of angiogenesis during DO.¹¹³ Ohashi et al. have shown that better preservation of blood vessels during DO will lead to a better mineralization of the distracted callus.¹¹⁴ It has been also shown that the rapid distraction rate can lead to a decrease in the VEGF and other angiogenic GFs; thus, administration of these factors might improve bone healing at these rapid distraction rates.¹¹⁵ Surprisingly, Eckardt et al. did not find a significant difference in the bone mineral content, torsional stiffness, and histomorphometric parameters after local application of the VEGF in tibiae DO in rabbits.¹¹⁶ The authors have suggested that high endogenous VEGF concentrations are expected during DO; thus, additional delivery of the exogenous VEGF would have no effect due to optimal endogenous DO signaling. Wan et al. have demonstrated that activation of the HIF-1 alpha pathway stimulates bone regeneration in DO; they also emphasize the importance of the HIF-1 alpha pathway in angiogenesis and its critical role in VEGF stimulation.¹¹⁷ The authors suggested that the VEGF alone might be not sufficient to stimulate bone formation because HIFs are upstream of the entire angiogenic cascade and they would be expected to induce additional factors that are required for forming a new intact vascular network in the injured bone.

Platelet-Derived Growth Factor

The family of PDGF polypeptide GFs includes PDGF-A, B, C, and D, encoded by four genes located on different chromosomes. The PDGF has stimulatory effects as a potent mitogen and chemoattractant for various mesenchymal cells, including osteogenic cells.¹¹⁸ The PDGFs signal through two cell-surface receptors, PDGF-R alpha and PDGF-R beta, which are capable of forming homodimers and heterodimers.¹¹⁹ PDGF-BB is considered the universal PDGF isoform because of its ability to bind to all known PDGF receptor isotypes. The PDGF can modulate the bone regenerative process through other GFs, in which, it increases VEGF expression, enhances IGF-I signaling, and increases expression of some BMP antagonists.¹¹⁸

Local application of PDGF-BB was investigated in the context of fracture healing and has shown its ability to stimulate the bone regenerative process.^120,121 Moore et al. have investigated the role of PDGF-BB injections in femora DO in rats and found significant new bone formation in a dose-dependent manner at midconsolidation phase as well as union rate when compared to the control group.¹²² Numerous preclinical and clinical studies have proven the safety of recombinant human platelet-derived growth factor-BB applications.¹²³ One drawback is that the PDGF has a short half-life of ∼30 min when circulating in the blood; thus, it is crucial to be combined with an appropriate delivery system.¹¹⁸ However, although there is a paucity of data for using the PDGF in the DO context, it seems to be a promising candidate for future DO research and applications.

Platelet-Rich Plasma

Since the identification of platelet-secreted GFs, the use and development of platelet-rich plasma (PRP) gained significant interest.^124,125 PRP is an autologous source of various GFs such as PDGF, TGFb1-b2, FGF, VEGF, IGF, HGF, EGF, and platelet-activating factor-4.^126,127 These GFs are secreted secondary to the degranulation of alpha granules in the platelets. PRP also contain cell adhesion molecules (fibrin, fibronectin, and vitronectin), which act as a scaffold for osteoconduction.¹²⁸ In addition to that, PRP contain leukocytes, which provide an additional source of GFs.¹²⁴ The one major advantage of PRP is the autologous preparation, therefore, no risks about the transmission of diseases or immunogenic reactions.

PRP has been utilized in various domains, including skin ulcers, osteoporosis, bone regeneration tissue, and soft tissue healing.^129–132 The efficacy of PRP in bone healing has been debated. Robert Marx has argued that some studies did not show the efficacy of the PRP likely because they had either small sample size, used damaged platelet, and may have not activated the platelets.¹²⁸

The application of PRP has been studied in animal DO models. Kawasumi et al. have shown that cellular proliferation was enhanced in all PRP groups when cultured with bone marrow cells in a dose-dependent manner in the rat DO model.¹³³ Choi et al. have found that the proliferation of alveolar bone cells can be suppressed or stimulated by PRP.¹³⁴ Recently, Kitoh et al. have observed a shorter bone healing time when the expanded bone marrow cells and PRP were injected into the callus bone in 11 patients during limb lengthening.¹³⁵ Furthermore, Latalski et al. have echoed the same findings when the PRP was applied from autogenous sources alone to enhance bone healing during DO procedures.¹³⁶ Although these are promising results, future studies are required for dose optimization, methods, and rhythm of administration.

Considerations for Delivery Systems and Other Alternatives

There is no doubt that the mode of delivery will enhance the efficacy of using exogenous GFs.^137,138 The ideal delivery system has to be biocompatible, has predictable biodegradability, low immunogenicity and antigenicity while preserving the GF bioactivity. It should also have the ability to control and meter/tailor protein release at an effective dose, for the appropriate period of time while considering the defect of anatomical site, size and vascularity, malleability, ease of manufacture, safety, stability, sterility, availability and cost-effectiveness.¹³⁹

Most osteogenic and endogenous GFs are expressed during the early stages of the bone regenerative process. Therefore, it is critical to be delivered in the appropriate time.

Extensive research has been performed investigating various BMP delivery systems, including the use of natural polymers, synthetic polymers, inorganic materials, and composites.^140–144 However, each delivery system has limitations (Table 4).

Table 4.

Major Classes of Delivery Materials

Carrier material	Advantages	Disadvantages	Types
Natural polymers	Biocompatibility, biodegradability, and soluble in physiologic fluids with natural affinity for BMPs	Immunogenic, possible pathogen transmission and sterilization difficulties	Collagen (gel, nanofibers, scaffolds, and films), fibrin glue, alginate, and chitosan
Inorganic materials	Mimic the natural bone, degradable/nondegradable, osteoconductive, self-setting ability in vivo and have affinity for BMPs	Brittle, difficult mold and some CPC are exothermic	CPC, bioactive glasses, hydroxyapatite, hyaluronic acid, tricalcium phosphates, metals, ceramics, and calcium sulfate
Synthetic polymers	Outstanding chemical and mechanical properties, easy to process and sterilize, flexible to tailor and reproducible	Inflammatory reaction, localized pH drop due to by-product acidity and restricted biological function	Polymers of a-hydroxy esters such as PLA, PGA, and copolymers of these two monomers (PLGA)
Composites	According to combination from different material	Complex manufacturing procedures	e.g., collagen-HA and titanium-PLL

Modified and adapted from Haidar et al.¹⁴⁰

CPC, calcium phosphate cements.

In the past decade, intensive research was performed for the use of nanoparticle formulation in anticancer drugs, as many advantages were found in nanoparticles such as small size, long shelf life, ability to interact with drugs, longer stay in the circulation, and ability to cross the epithelial membranes.^145–147 These are attractable properties for tissue engineering, which can be utilized to develop a promising GF delivery system.^148,149 Recently in our laboratory, we have developed a novel hybrid nanoparticle core shell release-controlled delivery system, the core is composed of charged large unilamellar liposomes and the shell is constructed through the L-b-L self-assembly of alternating layers of sodium alginate and chitosan.¹⁵⁰ This was proven to be safe and biocompatible and also has the capability of releasing a range of BMP-7 concentrations up to 45 days.^151,152 Moreover, a single injection of nanoparticles loaded with low doses of BMP-7 have shown enhancement of bone regeneration in the DO model.⁶⁶

Gene therapy also is another attractable field to study. Although few studies have shown promising results when administrated adenovirus containing the gene of BMP-2 in the DO model,^65,67 further experiments are required to examine its safety and efficacy.

Administration of multiple GFs is also a potential approach to enhance the bone regenerative process during DO. Several studies have demonstrated promising results when dual GFs were administrated. Coadministration of BMP-2 and Nell-1 have shown promising results in a long DO animal model.⁷¹ Similarly, cotransfection with a combination of BMP-7 and IGF has promoted osteoblast cell differentiation in vitro.¹⁵³ However, Vanau et al. have reported decrease bone formation when BMP-2 and bFGF were coadministrated in a fracture-healing model.¹⁵⁴ Therefore, using this technique should consider mimicking the natural bone regeneration cascade to obtain the desired results.¹⁵⁵ It has been hypothesized that inserting a scaffold with a controllable multiple-gene delivery system can arrange the sequential expression of GFs, thus, manipulating cell proliferation, chemotactic properties, differentiation, and matrix synthesis.¹⁵⁶ The ultimate goal is to find what are the optimal GF combinations to accelerate bone regeneration during DO.

Moreover, several reports have proven the safety of administrating exogenous FGF-2 and PDGF-BB in human.^102,123 This should encourage scientists to conduct further experiments to examine its efficacy with regard to dosing, timing, and delivery system in the context of DO.

Conclusion

Most of the current osteogenic GF applications in DO have relatively satisfactory outcomes in vivo studies. However, there are some limitations to its use secondary to the lack of an optimal delivery system, controversial reports about its efficacy (e.g., TGF-b1), safety concerns, and lack of data that provide the full map of how these factors interact with each other during DO. Future research can be directed to identify the cross talk between various GFs during DO, investigate the role of endogenous stimulation of the GFs (e.g., inhibiting BMP antagonists), carefully administrate multiple GFs (e.g., EGF/BMP-2 or 7) or multiple BMPs (e.g., 2/7) mimicking the natural spatiotemporal expressions during DO, and optimizing the delivery systems to determine the appropriate time and dose during DO procedures.

Footnotes

Disclosure Statement

None of the authors received payments or services, either directly or indirectly (i.e., via his institution), from a third party in support of any aspect of this work.

References

Ai-Aql

Z.S.

, Alagl

A.S.

, Graves

D.T.

, Gerstenfeld

L.C.

, Einhorn

T.A.

Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res, 87:107. 2008.

Birch

J.G.

, Samchukov

M.L.

Use of the Ilizarov method to correct lower limb deformities in children and adolescents. J Am Acad Orthop Surg, 12:144. 2004.

Eldridge

J.C.

, Bell

D.F.

Problems with substantial limb lengthening. Orthop Clin North Am, 22:625. 1991.

Velazquez

R.J.

, Bell

D.F.

, Armstrong

P.F.

, Babyn

, Tibshirani

Complications of use of the Ilizarov technique in the correction of limb deformities in children. J Bone Joint Surg Am, 75:1148. 1993.

Garcia-Cimbrelo

, Olsen

, Ruiz-Yague

, Fernandez-Baillo

, Munuera-Martínez

Ilizarov technique. Results and difficulties. Clin Orthop Relat Res, 283:116. 1992.

Ilizarov

G.A.

The tension-stress effect on the genesis and growth of tissues. Part I. The influence of stability of fixation and soft-tissue preservation. Clin Orthop Relat Res, 238:249. 1989.

Ilizarov

G.A.

The tension-stress effect on the genesis, growth of tissues: Part I.I The influence of the rate and frequency of distraction. Clin Orthop Relat Res, 239:263. 1989.

Aronson

, Good

, Stewart

, Harrison

, Harp

Preliminary studies of mineralization during distraction osteogenesis. Clin Orthop Relat Res, 250:43. 1990.

Sato

, Yasui

, Nakase

, Kawahata

, Sugimoto

, Hirota

, Kitamura

, Nomura

, Ochi

Expression of bone matrix proteins mRNA during distraction osteogenesis. J Bone Miner Res, 13:1221. 1998.

10.

Rowe

N.M.

, Mehrara

B.J.

, Dudziak

M.E.

, Steinbrech

D.S.

, Mackool

R.J.

, Gittes

G.K.

, McCarthy

J.G.

, Longaker

M.T.

Rat mandibular distraction osteogenesis. Part I. Histologic and radiographic analysis. Plast Reconstr Surg, 102:2022. 1998.

11.

Mori

, Akagi

, Kikuyama

, Yasuda

, Hamanishi

Axial shortening during distraction osteogenesis leads to enhanced bone formation in a rabbit model through the HIF-1alpha/vascular endothelial growth factor system. J Orthop Res, 24:653. 2006.

12.

Gebauer

, Correll

Pulsed low intensity ultrasound: a new salvage procedure for delayed unions and nonunions after leg lengthening in children. J Pediatr Orthop, 25:750. 2005.

13.

Hou

W.W.

, Zhu

Z.L.

, Zhou

, Zhang

C.X.

, Yu

H.Y.

Involvement of Wnt activation in the micromechanical vibration-enhanced osteogenic response of osteoblasts. J Orthop Sci, 16:598. 2011.

14.

Hwang

S.J.

, Lublinsky

, Seo

Y.K.

, Kim

I.S.

, Judex

Extremely small-magnitude accelerations enhance bone regeneration: a preliminary study. Clin Orthop Relat Res, 467:1083. 2008.

15.

Lai

J.P.

, Wang

F.S.

, Hung

C.M.

, Wang

C.J.

, Huang

C.J.

, Kuo

Y.R.

Extracorporeal shock wave accelerates consolidation in distraction osteogenesis of the rat mandible. J Trauma, 69:1252. 2010.

16.

Kawamoto

, Kim

W.C.

, Tsuchida

, Tsuji

, Fujioka

, Horii

, Mikami

, Tokunaga

, Kubo

Effects of alternating current electrical stimulation on lengthening callus. J Pediatr Orthop B, 14:299. 2005.

17.

Cakarer

, Olgac

, Aksakalli

, Tang

, Keskin

Acceleration of consolidation period by thrombin peptide 508 in tibial distraction osteogenesis in rats. Br J Oral Maxillofac Surg, 48:633. 2010.

18.

Fujio

, Yamamoto

, Ando

, Shohara

, Kinoshita

, Kaneko

, Hibi

, Ueda

Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors. Bone, 49:693. 2011.

19.

Yamane

, Okano

, Kishimoto

, Hagino

Effect of ED-71 on modeling of bone in distraction osteogenesis. Bone, 24:187. 1999.

20.

Namazi

Enhancing bone healing during distraction osteogenesis with platelet-rich plasma: a novel molecular mechanism. Injury, 43:1225. 2012.

21.

Shao

, Liu

, Peng

, Liu

, Xu

, Liu

Transplantation of osteoblast-like cells to the distracted callus in the rabbit mandible. Plast Reconstr Surg, 119:500. 2007.

22.

Xue

, Peng

, Yuan

, Wang

, Zhang

, Liu

, Fan

, Wang

, Xu

, Ting

, Zhang

, Lu

NELL1 promotes high-quality bone regeneration in rat femoral distraction osteogenesis model. Bone, 48:485. 2011.

23.

Perrien

D.S.

, Nicks

K.M.

, Liu

, Akel

N.S.

, Bacon

A.W.

, Skinner

R.A.

, Swain

F.L.

, Aronson

, Suva

L.J.

, Gaddy

Inhibin A enhances bone formation during distraction osteogenesis. J Orthop Res, 30:288. 2012.

24.

Song

H.R.

, Oh

C.W.

, Kyung

H.S.

, Park

I.H.

, Kim

P.T.

, Baek

S.H.

, Kim

S.J.

, Lee

S.T.

Injected calcium sulfate for consolidation of distraction osteogenesis in rabbit tibia. J Pediatr Orthop B, 13:170. 2004.

25.

Sen

, Gunes

, Erdem

, Koseoglu

R.D.

, Filiz

N.O.

Effects of calcitonin and alendronate on distraction osteogenesis. Int Orthop, 30:272. 2006.

26.

Abbaspour

, Takahashi

, Sairyo

, Takata

, Yukata

, Inui

, Yasui

Optimal increase in bone mass by continuous local infusion of alendronate during distraction osteogenesis in rabbits. Bone, 44:917. 2009.

27.

Kurklu

, Yildiz

, Kose

, Yurttas

, Karacalioglu

, Serdar

, Deveci

Effect of alpha-tocopherol on bone formation during distraction osteogenesis: a rabbit model. J Orthop Traumatol, 12:153. 2011.

28.

Jiang

, Song

, Ye

, Wang

, Song

, Yang

, Hu

Effect of intermittent administration of adiponectin on bone regeneration following mandibular osteodistraction in rabbits. J Orthop Res, 29:1081. 2011.

29.

Claes

, Augat

, Schorlemmer

, Konrads

, Ignatius

, Ehrnthaller

Temporary distraction and compression of a diaphyseal osteotomy accelerates bone healing. J Orthop Res, 6:772. 2008.

30.

Cillo

J.E.

Jr. , Gassner

, Koepsel

R.R.

, Buckley

M.J.

Growth factor and cytokine gene expression in mechanically strained human osteoblast-like cells: implications for distraction osteogenesis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 90:147. 2000.

31.

Pape

H.C.

, Marcucio

, Humphrey

, Colnot

, Knobe

, Harvey

E.J.

Trauma-induced inflammation and fracture healing. J Orthop Trauma, 24:522. 2010.

32.

Urist

M.R.

Bone: formation by autoinduction. Science, 150:893. 1965.

33.

Ducy

, Karsenty

The family of bone morphogenetic proteins. Kidney Int, 57:14. 2000.

34.

Sakou

Bone morphogenetic proteins: from basic studies to clinical approaches. Bone, 22:591. 1998.

35.

Miyazono

, Kamiya

, Morikawa

Bone morphogenetic protein receptors and signal transduction. J Biochem, 147:35. 2010.

36.

Lou J

, Merkel

, Manske

Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J Orthop Res, 17:43. 1999

37.

Gazzerro

, Canalis

Bone morphogenetic proteins and their antagonists. Rev Endocr Metab Disord, 7:51. 2006.

38.

Tsialogiannis

, Polyzois

, Oak Tang

, Pavlou

, Tsiridis

, Heliotis

, Tsiridis

Targeting bone morphogenetic protein antagonists: in vitro and in vivo evidence of their role in bone metabolism. Expert Opin Ther Targets, 13:123. 2009.

39.

Rosen

Harnessing the parathyroid hormone, Wnt, and bone morphogenetic protein signaling cascades for successful bone tissue engineering. Tissue Eng Part B Rev, 17:475. 2011.

40.

Kawasaki

, Niki

, Miyamoto

, Horiuchi

, Matsumoto

, Aizawa

, Toyama

The effect of timing in the administration of hepatocyte growth factor to modulate BMP-2-induced osteoblast differentiation. Biomaterials, 31:1191. 2010.

41.

Laflamme

, Curt

, Rouabhia

Epidermal growth factor and bone morphogenetic proteins upregulate osteoblast proliferation and osteoblastic markers and inhibit bone nodule formation. Arch Oral Biol, 55:689. 2010

42.

Israel

D.I.

, Nove

, Kerns

K.M.

, Kaufman

R.J.

, Rosen

, Cox

K.A.

, Wozney

J.M.

Heterodimeric bone morphogenetic proteins show enhanced activity in vitro and in vivo. Growth Factors, 13:291. 1996.

43.

Abe

Function of BMPs and BMP antagonists in adult bone. Skelet Dev Remodel Health Dis Aging, 1068:41. 2006.

44.

Cheng

, Jiang

, Phillips

F.M.

, Haydon

R.C.

, Peng

, Zhou

, Luu

H.H.

, An

, Breyer

, Vanichakarn

, Szatkowski

J.P.

, Park

J.Y.

, He

T.C.

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs)

J Bone Joint Surg Am, 85-A:1544. 2003.

45.

Rauch

, Lauzier

, Croteau

, Travers

, Glorieux

F.H.

, Hamdy

Temporal and spatial expression of bone morphogenetic protein-2, −4, and −7 during distraction osteogenesis in rabbits. Bone, 27:453. 2000.

46.

Campisi

, Hamdy

R.C.

, Lauzier

, Amako

, Rauch

, Lessard

M.L.

Expression of bone morphogenetic proteins during mandibular distraction osteogenesis. Plast Reconstr Surg, 111:201. 2003

47.

Alam

, St-Arnaud

, Lauzier

, Rosen

, Hamdy

R.C.

Are endogenous BMPs necessary for bone healing during distraction osteogenesis?

Clin Orthop Relat Res, 467:3190. 2009.

48.

Haque

, Mandu-Hrit

, Rauch

, Lauzier

, Tabrizian

, Hamdy

R.C.

Immunohistochemical localization of bone morphogenetic protein-signaling Smads during long-bone distraction osteogenesis. J Histochem Cytochem, 54:407. 2006.

49.

Yonezawa

, Harada

, Ikebe

, Shinohara

, Enomoto

Effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on bone consolidation on distraction osteogenesis: a preliminary study in rabbit mandibles. J Craniomaxillofac Surg, 34:270. 2006.

50.

Cheung

L.K.

, Zheng

L.W.

Effect of recombinant human bone morphogenetic protein-2 on mandibular distraction at different rates in an experimental model. J Craniofac Surg, 17:100. 2006.

51.

Mandu-Hrit

, Haque

, Lauzier

, Kotsiopriftis

, Rauch

, Tabrizian

, Henderson

, Hamdy

R.C.

Early injection of OP-1 during distraction osteogenesis accelerates new bone formation in rabbits. Growth Factors, 24:172. 2006.

52.

Hamdy

R.C.

, Amako

, Beckman

, Kawaguchi

, Rauch

, Lauzier

, Steffen

Effects of osteogenic protein-1 on distraction osteogenesis in rabbits. Bone, 33:248. 2003.

53.

Mizumoto

, Moseley

, Drews

, Cooper

V.N.

3rd , Reddi

A.H.

Acceleration of regenerate ossification during distraction osteogenesis with recombinant human bone morphogenetic protein-7. J Bone Joint Surg Am, 85-A:124. 2003.

54.

Zheng

L.W.

, Chenug

L.K.

Effect of recombinant human bone morphogenetic protein-2 on mandibular distraction at different rates in a rabbit model. Tissue Eng, 12:3. 2006.

55.

Hollinger

J.O.

, Uludag

, Winn

S.R.

Sustained release emphasizing recombinant human bone morphogenetic protein-2. Adv Drug Deliv Rev, 31:303. 1998.

56.

Bouxsein

M.L.

, Turek

T.G.

, Blake

C.A.

, D'Augusta

, Li

, Stevens

, Seeherman

H.J.

, Wozney

J.M.

Recombinant human bone morphogenetic protein-2 accelerates healing in a rabbit ulnar osteotomy model. J Bone Joint Surg Am, 83-A:1219. 2001.

57.

Kirker-Head

C.A.

Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliv Rev, 15:181. 2000.

58.

Wozney

J.M.

Overview of bone morphogenetic proteins. Spine (Phila Pa 1976), 27:S2. 2002.

59.

Bishop

G.B.

, Einhorn

T.A.

Current and future clinical applications of bone morphogenetic proteins in orthopaedic trauma surgery. Int Orthop, 31:721. 2007.

60.

Rengachary

S.S.

Bone morphogenetic proteins: basic concepts. Neurosurg Focus, 13:e2. 2002.

61.

Dohin

, Dahan-Oliel

, Fassier

, Hamdy

Enhancement of difficult nonunion in children with osteogenic protein-1 (OP-1): early experience. Clin Orthop Relat Res, 467:3230. 2009.

62.

Hsu

M.Y.

, Rovinsky

, Penmatcha

, Herlyn

, Muirhead

Bone morphogenetic proteins in melanoma: angel or devil?

Cancer Metastasis Rev, 24:251. 2005.

63.

Gautschi

O.P.

, Frey

S.P.

, Zellweger

Bone morphogenetic proteins in clinical applications. ANZ J Surg, 77:626. 2007.

64.

Lesaichot

, Leperlier

, Viateau

, Richarme

, Petite

, Sailhan

The influence of Bone Morphogenic Protein-2 on the consolidation phase in a distraction osteogenesis model. Injury, 42:1460. 2011.

65.

Ashinoff

R.L.

, Cetrulo

C.L.

Jr. , Galiano

R.D.

, Dobryansky

, Bhatt

K.A.

, Ceradini

D.J.

, Michaels

5th , McCarthy

J.G.

, Gurtner

G.C.

Bone morphogenic protein-2 gene therapy for mandibular distraction osteogenesis. Ann Plast Surg, 52:585. 2004.

66.

Haidar

Z.S.

, Tabrizian

, Hamdy

R.C.

A hybrid rhOP-1 delivery system enhances new bone regeneration and consolidation in a rabbit model of distraction osteogenesis. Growth Factors, 28:44. 2010.

67.

Long

, Li

, Du

H.M.

, Liu

, Zheng

X.H.

, Lin

Y.F.

, Wang

, Jing

, Tang

, Chen

W.H.

, Tian

W.D.

Effects of bone morphogenetic protein 2 gene therapy on new bone formation during mandibular distraction osteogenesis at rapid rate in rabbits. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 112:50. 2011.

68.

Yang

J.H.

, Kim

H.J.

, Kim

S.E.

, Yun

Y.P.

, Bae

J.H.

, Kim

S.J.

, Choi

K.H.

, Song

H.R.

The effect of bone morphogenic protein-2-coated tri-calcium phosphate/hydroxyapatite on new bone formation in a rat model of femoral distraction osteogenesis. Cytotherapy, 3:315. 2012

69.

Zhang

W.B.

, Zhang

L.W.

, Chua

D.T.

, Cheung

L.K.

Treatment of irradiated mandibles with mesenchymal stem cells transfected with bone morphogenetic protein 2/7. J Oral Maxillofac Surg, 70:1711. 2012.

70.

Sailhan

, Gleyzolle

, Parot

, Guerini

, Viguier

Rh-BMP-2 in distraction osteogenesis: dose effect and premature consolidation. Injury, 41:680. 2010.

71.

Zhu

, Song

, Jiang

, Zhou

, Hu

Combined effects of recombinant human BMP-2 and Nell-1 on bone regeneration in rapid distraction osteogenesis of rabbit tibia. Injury, 42:1467. 2011.

72.

Kloen

, Lauzier

, Hamdy

R.C.

Co-expression of BMPs and BMP-inhibitors in human fractures and non-unions. Bone, 51:59. 2012.

73.

Takayama

, Suzuki

, Manaka

, Taguchi

, Hashimoto

, Imai

, Wakitani

, Takaoka

RNA interference for noggin enhances the biological activity of bone morphogenetic proteins in vivo and in vitro. J Bone Miner Metab, 27:402. 2009.

74.

Winkler

D.G.

, Yu

, Geoghegan

J.C.

, Ojala

E.W.

, Skonier

J.E.

, Shpektor

, Sutherland

M.K.

, Latham

J.A.

Noggin and sclerostin bone morphogenetic protein antagonists form a mutually inhibitory complex. J Biol Chem, 279:36293. 2004.

75.

Kwong

F.N.

, Richardson

S.M.

, Evans

C.H.

Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells. Arthritis Res Ther, 10:R65. 2008.

76.

, Ominsky

M.S.

, Niu

Q.T.

, Sun

, Daugherty

, D'Agostin

, Kurahara

, Gao

, Cao

, Gong

, Asuncion

, Barrero

, Warmington

, Dwyer

, Stolina

, Morony

, Sarosi

, Kostenuik

P.J.

, Lacey

D.L.

, Simonet

W.S.

, Ke

H.Z.

, Paszty

Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res, 23:860. 2008.

77.

, Howard

, Ni

, Lagna

, Hata

OAZ regulates bone morphogenetic protein signaling through Smad6 activation. J Biol Chem, 281:5277. 2006

78.

Zhao

, Qiao

, Harris

S.E.

, Oyajobi

B.O.

, Mundy

G.R.

, Chen

Smurf1 inhibits osteoblast differentiation and bone formation in vitro and in vivo. J Biol Chem, 279:12854. 2004.

79.

Haque

, Hamade

, Alam

, Kotsiopriftis

, Lauzier

, St-Arnaud

, Hamdy

R.C.

Characterizing the BMP pathway in a wild type mouse model of distraction osteogenesis. Bone, 42:1144. 2008.

80.

Mehrara

B.J.

, Rowe

N.M.

, Steinbrech

D.S.

, Dudziak

M.E.

, Saadeh

P.B.

, McCarthy

J.G.

, Gittes

G.K.

, Longaker

M.T.

Rat mandibular distraction osteogenesis: II. Molecular analysis of transforming growth factor beta-1 and osteocalcin gene expression. Plast Reconstr Surg, 103:536. 1999.

81.

Joyce

M.E.

, Roberts

A.B.

, Sporn

M.B.

, Bolander

M.E.

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur. J Cell Biol, 110:2195. 1990.

82.

Yeung

H.Y.

, Lee

K.M.

, Fung

K.P.

, Leung

K.S.

Sustained expression of transforming growth factor-beta1 by distraction during distraction osteogenesis. Life Sci, 71:67. 2002.

83.

Mundy

G.R.

, Bonewald

L.F.

Role of TGF beta in bone remodeling. Ann N Y Acad Sci, 593:91. 1990.

84.

Nielsen

H.M.

, Andreaseen

T.T.

, Ledet

, Oxlund

Local injection of TGF-beta increases the strength of tibial fractures in the rat. Acta Orthop Scand, 65:37. 1994.

85.

Lind

, Schumacker

, Søballe

, Keller

, Melsen

, Bünger

Transforming growth factor-beta enhances fracture healing in rabbit tibiae. Acta Orthop Scand, 64:553. 1993.

86.

Peterson

D.R.

, Glancy

T.P.

, Bacon-Clarke

A study ofdelivery timing and duration on the transforming growth factor beta 1 induced healing ofcritical size long bone defects. J Bone Miner Res, 12:S304. 1997.

87.

Rauch

, Lauzier

, Travers

, Glorieux

, Hamdy

Effects of locally applied transforming growth factor-beta1 on distraction osteogenesis in a rabbit limb-lengthening model. Bone, 26:619. 2000.

88.

Ozkan

, Eralp

, Kocaoglu

, Ahishali

, Bilgic

, Mutlu

, Turker

, Ozkan

F.U.

, Sahin

, Guven

The effect of transforming growth factor beta1 (TGF-beta1) on the regenerate bone in distraction osteogenesis. Growth Factors, 25:101. 2007.

89.

Yakar

, Courtland

H.W.

, Clemmons

IGF-1 and bone: new discoveries from mouse models. J Bone Miner Res, 25:2543. 2010.

90.

Nakasaki

, Yoshioka

, Miyamoto

, Sasaki

, Yoshikawa

, Itoh

IGF-I secreted by osteoblasts acts as a potent chemotactic factor for osteoblasts. Bone, 43:868. 2008.

91.

Govoni

K.E.

, Wergedal

J.E.

, Florin

, Angel

, Baylink

D.J.

, Mohan

Conditional deletion of insulin-like growth factor-I in collagen type 1alpha2-expressing cells results in postnatal lethality and a dramatic reduction in bone accretion. Endocrinology, 148:5706. 2007.

92.

Zhang

, Xuan

, Bouxsein

M.L.

, von Stechow

, Akeno

, Faugere

M.C.

, Malluche

, Zhao

, Rosen

C.J.

, Efstratiadis

, Clemens

T.L.

Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem, 277:44005. 2002.

93.

Schumacher

, Albrechtsen

, Keller

, Flyvbjerg

, Hvid

Periosteal insulin-like growth factor I and bone formation. Changes during tibial lengthening in rabbits. Acta Orthop Scand, 67:237. 1996.

94.

Eingartner

, Coerper

, Fritz

, Gaissmaier

, Koveker

, Weise

Growth factors in distraction osteogenesis. Immuno-histological pattern of TGF-beta1 and IGF-I in human callus induced by distraction osteogenesis. Int Orthop, 23:253. 1999.

95.

Stewart

K.J.

, Weyand

, van't Hof

R.J.

, White

S.A.

, Lvoff

G.O.

, Maffulli

, Poole

M.D.

A quantitative analysis of the effect of insulin-like growth factor-1 infusion during mandibular distraction osteogenesis in rabbits. Br J Plast Surg, 52:343. 1999.

96.

Bernstein

, Mayr

H.O.

, Hube

Can bone healing in distraction osteogenesis be accelerated by local application of IGF-1 and TGF-beta1?

J Biomed Mater Res B Appl Biomater, 92:215. 2010.

97.

Yun

Y.R.

, Won

J.E.

, Jeon

, Lee

, Kang

, Jo

, Jang

J.H.

, Shin

U.S.

, Kim

H.W.

Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng, 2010:218142. 2010.

98.

Rifkin

D.B.

, Moscatelli

Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol, 109:1. 1989.

99.

Nakamura

, Kurokawa

, Kato

, Okazaki

, Mamada

, Hanada

, Hiyama

, Aoyama

, Nakamura

, Tamura

Local application of basic fibroblast growth factor into the bone increases bone mass at the applied site in rabbits. Arch Orthop Trauma Surg, 115:344. 1996.

100.

Nakamura

, Kawaguchi

, Aoyama

, Hanada

, Hiyama

, Awa

, Tamura

, Kurokawa

Stimulation of bone formation by intraosseous application of recombinant basic fibroblast growth factor in normal and ovariectomized rabbits. J Orthop Res, 15:307. 1997.

101.

Kato

, Kawaguchi

, Hanada

, Aoyama

, Hiyama

, Nakamura

, Kuzutani

, Tamura

, Kurokawa

, Nakamura

Single local injection of recombinant fibroblast growth factor-2 stimulates healing of segmental bone defects in rabbits. J Orthop Res, 16:654. 1998.

102.

Kawaguchi

, Oka

, Jingushi

, Izumi

, Fukunaga

, Sato

, Matsushita

, Nakamura

TESK Group. A local application of recombinant human fibroblast growth factor 2 for tibial shaft fractures: a randomized, placebo-controlled trial. J Bone Miner Res, 25:2735. 2010.

103.

, Zou

, Li

, Chen

, Wang

, Gao

Temporospatial expression of vascular endothelial growth factor and basic fibroblast growth factor during mandibular distraction osteogenesis. J Craniomaxillofac Surg, 31:238. 2003.

104.

Yeung

H.Y.

, Lee

S.K.

, Fung

K.P.

, Leung

K.S.

Expression of basic fibroblast growth factor during distraction osteogenesis. Clin Orthop Relat Res, 385:219. 2001.

105.

Okazaki

, Kurokawa

, Nakamura

, Matsushita

, Mamada

, Kawaguchi

Stimulation of bone formation by recombinant fibroblast growth factor-2 in callotasis bone lengthening of rabbits. Calcif Tissue Int, 64:542. 1999.

106.

Higuchi

, Kabasawa

, Sato

, Kikuchi

, Aoki

, Ohya

, Maruoka

, Omura

Effect of recombinant human fibroblast growth factor-2 on bone formation in rabbit mandibular distraction models using beta-tricalcium phosphate. Congenit Anom (Kyoto), 50:95. 2010.

107.

Ferrara

, Gerber

H.P.

, LeCouter

The biology of VEGF and its receptors. Nat Med, 9:669. 2003.

108.

Dai

, Rabie

A.B.

VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res, 86:937. 2007.

109.

Ochman

, Frey

, Raschke

M.J.

, Deventer

J.N.

, Meffert

R.H.

Local application of VEGF compensates callus deficiency after acute soft tissue trauma—results using a limb-shortening distraction procedure in rabbit tibia. J Orthop Res, 29:1093. 2011.

110.

Kleinheinz

, Stratmann

, Joos

, Wiesmann

H.P.

VEGF-activated angiogenesis during bone regeneration. J Oral Maxillofac Surg, 63:1310. 2005.

111.

Pacicca

D.M.

, Patel

, Lee

, Salisbury

, Lehmann

, Carvalho

, Gerstenfeld

L.C.

, Einhorn

T.A.

Expression of angiogenic factors during distraction osteogenesis. Bone, 33:889. 2003.

112.

Warren

S.M.

, Mehrara

B.J.

, Steinbrech

D.S.

, Paccione

M.F.

, Greenwald

J.A.

, Spector

J.A.

, Longaker

M.T.

Rat mandibular distraction osteogenesis: part III. Gradual distraction versus acute lengthening. Plast Reconstr Surg, 107:441. 2001.

113.

Carvalho

R.S.

, Einhorn

, Lehmann

, Edgar

, Al-Yamani

, Apazidis

, Pacicca

, Clemens

T.L.

, Gerstenfeld

L.C.

The role of angiogenesis in a murine tibial model of distraction osteogenesis. Bone, 34:849. 2004.

114.

Ohashi

, Ohinshi

, Kageyama

, Fukuda

, Tsuchiya

, Imai

, Matsuyama

, Nakamura

Effect of vascularity on canine distracted tibial callus consolidation. Clin Orthop Relat Res, 438:253. 2005.

115.

Schiller

J.R.

, Moore

D.C.

, Ehrlich

M.G.

Increased lengthening rate decreases expression of fibroblast growth factor 2, platelet-derived growth factor, vascular endothelial growth factor, and CD31 in a rat model of distraction osteogenesis. J Pediatr Orthop, 27:961. 2007.

116.

Eckardt

, Bundgaard

K.G.

, Christensen

K.S.

, Lind

, Hansen

E.S.

, Hvid

Effects of locally applied vascular endothelial growth factor (VEGF) and VEGF-inhibitor to the rabbit tibia during distraction osteogenesis. J Orthop Res, 21:335. 2003.

117.

Wan

, Gilbert

S.R.

, Wang

, Cao

, Shen

, Ramaswamy

, Jacobsen

K.A.

, Alaql

Z.S.

, Eberhardt

A.W.

, Gerstenfeld

L.C.

, Einhorn

T.A.

, Deng

, Clemens

T.L.

Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration. Proc Natl Acad Sci U S A, 105:686. 2008.

118.

Hollinger

J.O.

, Hart

C.E.

, Hirsch

S.N.

, Lynch

, Friedlaender

G.E.

Recombinant human platelet-derived growth factor: biology and clinical applications. J Bone Joint Surg Am, 90:48. 2008.

119.

Seifert

R.A.

, Hart

C.E.

, Phillips

P.E.

, Forstrom

J.W.

, Ross

, Murray

M.J.

, Bowen-Pope

D.F.

Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem, 264:8771. 1989.

120.

Hollinger

J.O.

, Onikepe

A.O.

, MacKrell

, Einhorn

, Bradica

, Lynch

, Hart

C.E.

Accelerated fracture healing in the geriatric, osteoporotic rat with recombinant human platelet-derived growth factor-BB and an injectable beta-tricalcium phosphate/collagen matrix. J Orthop Res, 26:83. 2008.

121.

Nash

T.J.

, Howlett

C.R.

, Martin

, Steele

, Johnson

K.A.

, Hicklin

D.J.

Effect of platelet-derived growth factor on tibial osteotomies in rabbits. Bone, 15:203. 1994.

122.

Moore

D.C.

, Ehrlich

M.G.

, McAllister

S.C.

, Machan

J.T.

, Hart

C.E.

, Voigt

, Lesieur-Brooks

A.M.

, Weber

E.W.

Recombinant human platelet-derived growth factor-BB augmentation of new-bone formation in a rat model of distraction osteogenesis. J Bone Joint Surg Am, 91:1973. 2009.

123.

Solchaga

L.A.

, Hee

C.K.

, Roach

, Snel

L.B.

Safety of recombinant human platelet-derived growth factor-BB in Augment(®) Bone Graft. J Tissue Eng, 3:2041731412442668. 2012.

124.

Malhotra

, Pelletier

M.H.

, Yu

, Walsh

W.R.

Can platelet-rich plasma (PRP) improve bone healing? A comparison between the theory and experimental outcomes. Arch Orthop Trauma Surg, 133:153. 2013.

125.

Marx

R.E.

, Carlson

E.R.

, Eichstaedt

R.M.

, Schimmele

S.R.

, Strauss

J.E.

, Georgeff

K.R.

Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 85:638. 1998.

126.

Kanno

, Takahashi

, Tsujisawa

, Ariyoshi

, Nishihara

Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation. J Oral Maxillofac Surg, 63:362. 2005.

127.

Weibrich

, Kleis

W.K.

, Hafner

, Hitzler

W.E.

Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg, 30:97. 2002.

128.

Marx

R.E.

Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg, 62:489. 2004.

129.

Cervelli

, Gentile

, Scioli

M.G.

, Grimaldi

, Casciani

C.U.

, Spagnoli

L.G.

, Orlandi

Application of platelet-rich plasma in plastic surgery: clinical and in vitro evaluation. Tissue Eng Part C Methods, 15:625. 2009.

130.

Hokugo

, Ozeki

, Kawakami

, Sugimoto

, Mushimoto

, Morita

, Tabata

Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel. Tissue Eng, 11:1224. 2005.

131.

de Vos

R.J.

, van Veldhoven

P.L.

, Moen

M.H.

, Weir

, Tol

J.L.

, Maffulli

Autologous growth factor injections in chronic tendinopathy: a systematic review. Br Med Bull, 95:63. 2010.

132.

Liu

H.Y.

, Wu

A.T.

, Tsai

C.Y.

, Chou

K.R.

, Zeng

, Wang

M.F.

, Chang

W.C.

, Hwang

S.M.

, Su

C.H.

, Deng

W.P.

The balance between adipogenesis and osteogenesis in bone regeneration by platelet-rich plasma for age-related osteoporosis. Biomaterials, 32:6773. 2011.

133.

Kawasumi

, Kitoh

, Siwicka

K.A.

, Ishiguro

The effect of the platelet concentration in platelet-rich plasma gel on the regeneration of bone. J Bone Joint Surg Br, 90:966. 2008.

134.

Choi

B.H.

, Zhu

S.J.

, Kim

B.Y.

, Huh

J.Y.

, Lee

S.H.

, Jung

J.H.

Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg, 34:420. 2005.

135.

Kitoh

, Kitakoji

, Tsuchiya

, Katoh

, Ishiguro

Distraction osteogenesis of the lower extremity in patients with achondroplasia/hypochondroplasia treated with transplantation of culture-expanded bone marrow cells and platelet-rich plasma. J Pediatr Orthop B, 27:629. 2007.

136.

Latalski

, Elbatrawy

, Thabet

A.M.

, Gregosiewicz

, Raganowicz

, Fatyga

Enhancing bone healing during distraction osteogenesis with platelet-rich plasma. Injury, 42:821. 2011.

137.

Boerckel

J.D.

, Kolambkar

Y.M.

, Dupont

K.M.

, Uhrig

B.A.

, Phelps

E.A.

, Stevens

H.Y.

, García

A.J.

, Guldberg

R.E.

Effects of protein dose and delivery system on BMP-mediated bone regeneration. Biomaterials, 32:5241. 2011.

138.

W.G.

, Kang

S.W.

, Yang

H.S.

, Bhang

S.H.

, Lee

S.H.

, Park

J.H.

, Kim

B.S.

The efficacy of bone morphogenetic protein-2 depends on its mode of delivery. Artif Organs, 34:1150. 2010.

139.

Haidar

Z.S.

, Hamdy

R.C.

, Tabrizian

Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part A: current challenges in BMP delivery. Biotechnol Lett, 31:1817. 2009.

140.

Haidar

Z.S.

, Hamdy

R.C.

, Tabrizian

Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: delivery systems for BMPs in orthopaedic and craniofacial tissue engineering. Biotechnol Lett, 31:1825. 2009.

141.

Yamamoto

, Takahashi

, Tabata

Enhanced bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein-2 from a biodegradable hydrogel. Tissue Eng, 12:1305. 2006.

142.

Barnes

, Boden

S.D.

, Louis-Ugbo

, Tomak

P.R.

, Park

J.S.

, Park

M.S.

, Minamide

Lower dose of rhBMP-2 achieves spine fusion when combined with an osteoconductive bulking agent in non-human primates. Spine (Phila Pa 1976), 30:1127. 2005.

143.

Saito

, Murakami

, Takahashi

, Horiuchi

, Ota

, Kato

, Okada

, Nozaki

, Takaoka

Synthetic biodegradable polymers as drug delivery systems for bone morphogenetic proteins. Adv Drug Deliv Rev, 57:1037. 2005.

144.

Wei

, Jin

, Giannobile

W.V.

, Ma

P.X.

The enhancement of osteogenesis by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomaterials, 28:2087. 2007.

145.

Zhang

, Feng

S.S.

The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles. Biomaterials, 27:4025. 2006.

146.

Dong

, Feng

S.S.

Methoxy poly(ethylene glycol)-poly(lactide) (MPEG-PLA) nanoparticles for controlled delivery of anticancer drugs. Biomaterials, 25:2843. 2004.

147.

Gref

, Minamatake

, Peracchia

M.T.

, Trubetskoy

, Torchilin

, Langer

Biodegradable long-circulating polymeric nanospheres. Science, 263:1600. 1994.

148.

Jeon

, Song

S.J.

, Yang

H.S.

, Bhang

S.H.

, Kang

S.W.

, Sung

M.A.

, Lee

J.H.

, Kim

B.S.

Long-term delivery enhances in vivo osteogenic efficacy of bone morphogenetic protein-2 compared to short-term delivery. Biochem Biophys Res Commun, 369:774. 2008.

149.

Yilgor

, Hasrici

, Hasirci

Sequential BMP-2/BMP-7 delivery from polyester nanocapsules. J Biomed Mater Res A, 93:528. 2010.

150.

Haidar

Z.S.

, Hamdy

R.C.

, Tabrizian

Protein release kinetics for core-shell hybrid nanoparticles based on the layer-by-layer assembly of alginate and chitosan on liposomes. Biomaterials, 9:1207. 2008.

151.

Haidar

Z.S.

, Azari

, Hamdy

R.C.

, Tabrizian

Modulated release of OP-1 and enhanced preosteoblast differentiation using a core-shell nanoparticulate system. J Biomed Mater Res A, 91:919. 2009.

152.

Haidar

Z.S.

, Hamdy

R.C.

, Tabrizian

Biocompatibility and safety of a hybrid core-shell nanoparticulate OP-1 delivery system intramuscularly administered in rats. Biomaterials, 31:2746. 2010.

153.

Yeh

L.C.

, Lee

J.C.

Co-transfection with the osteogenic protein (OP)-1 gene and the insulin-like growth factor (IGF)-I gene enhanced osteoblastic cell differentiation. Biochim Biophys Acta, 1763:57. 2006.

154.

Vonau

R.L.

, Bostrom

M.P.

, Aspenberg

, Sams

A.E.

Combination of growth factors inhibits bone ingrowth in the bone harvest chamber. Clin Orthop Relat Res, 386:243. 2001.

155.

Chen

F.M.

, Zhang

, Wu

Z.F.

Toward delivery of multiple growth factors in tissue engineering. Biomaterials, 31:6279. 2010.

156.

Liu

, Xu

, Li

, Ma

Temporally controlled multiple-gene delivery in scaffolds: a promising strategy to enhance bone regeneration. Med Hypotheses, 76:173. 2011.