Injury, Int. J. Care Injured 45S (2014) S49–S57

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Can we enhance fracture vascularity: What is the evidence?§ Ippokratis Pountos a, Michalis Panteli a, Elias Panagiotopoulos b, Elena Jones c, Peter V. Giannoudis a,* a

Academic Department of Trauma & Orthopaedics, School of Medicine, University of Leeds, UK Academic Department of Trauma & Orthopaedics, School of Medicine, University of Patras, Greece c LIMM, Section Musculoskeletal Disease, University of Leeds, UK b

A R T I C L E I N F O

A B S T R A C T

Keywords: Mesenchymal stem cells Bone healing Angiogenesis

Angiogenesis is a vital component of bone healing. The formation of the new blood vessels at the fracture site restores the hypoxia and nutrient deprivation found at the early stages after fracture whilst at a later stage facilitates osteogenesis by the activity of the osteoprogenitor cells. Emerging evidence suggests that there are certain molecules and gene therapies that could promote new blood vessel formation and as a consequence enhance the local bone healing response. This article summarizes the current in vivo evidence on therapeutic approaches aiming at the augmentation of the angiogenic signalling during bone repair. ß 2014 Published by Elsevier Ltd.

Introduction Bone is a dynamic tissue undergoing continues regeneration, i.e. osteoclastogenesis and osteogenesis, a feature responsible for the scarless healing after a fracture [1–3]. In this process, the formation of new blood vessels is vital and interdependent to bone formation facilitating primarily oxygen and nutrients delivery [4]. The availability of oxygen is essential for aerobic metabolism and the function of most cells but also for the activity of molecules like hydroxylases and cyclooxygenases, which play a vital role on collagen synthesis and the expression of growth factors [5–9]. Similarly, deprivation of nutrients results in poor cellular function, cell apoptosis and reduced regenerative capacity [10]. Still, the role of blood vessels is more complex than fulfilling solely bioenergetic functions as it has been shown for instance that blocking of the vascular invasion results in absence of ossification [11]. Equally, by blocking the osteogenesis, seen in the genetically modified animal models i.e. Runx2 / , a complete absence of vascular invasion is noted [12]. It has been also hypothesized that osteoprogenitor cells could use the vascular network as a scaffold to invade surrounding

§ No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article. No funds were received in support of this study. * Corresponding author at: Trauma & Orthopaedic Surgery, Academic Department of Trauma & Orthopaedics, Leeds General Infirmary, Clarendon Wing Level A, Great George Street, Leeds LS1 3EX, UK. Tel.: +44 113 3922750; fax: +44 113 3923290. E-mail address: [email protected] (P.V. Giannoudis).

http://dx.doi.org/10.1016/j.injury.2014.04.009 0020–1383/ß 2014 Published by Elsevier Ltd.

space during ossification [12]. This is further strengthened by the fact that osteogenesis follows vascularization [13]. It is still to be defined however, whether osteoprogenitor cells are in control of this process by pulling vessels to the surrounding soft tissue or whether interactions with other cells types like endothelial cells and osteoclasts are responsible for these observations. Overall, two distinct forms of new blood vessel formation exist the so called angiogenesis and vasculogenesis, both complementing each other in the restoration of mature circulation [14,15]. Angiogenesis occurs by intussusceptive microvascular growth where vessel sprouting occurs from an existing vessel [14,16]. In this process, resident endothelial cells adjacent to a mature vascular network are positioned in such a way to form a tubular structure which is reinforced by the presence and actions of other cell types like pericytes, fibroblasts and/or smooth muscle cells [14]. Vasculogenesis is the de novo formation of a vessel or vascular network [15,16]. It involves the recruitment and differentiation of endothelial cells from mesoderm-derived precursor cells (angioblasts) which are organized to form blood vessels [16]. Although vasculogenesis was thought to occur during embryogenesis, this process is recapitulated in several postnatal stages including bone and wound healing, myocardial or peripheral ischaemia, stroke and retinopathy [15–17]. Following injury, bone mounts a number of events aiming to restore its continuity and function. The initial hyperaemia following disruption of the existing blood vessels together with the vasodilatation, oedema and swelling lead to the formation of the fracture haematoma [1]. Immediately after, hypoxia follows associated with necrosis, intense osteoclast activity and the

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Fig. 1. Angiogenic molecules and their effect on bone healing and angiogenesis [30–77].

formation of granulation tissue [1]. Subsequently, this hypoxic environment recovers mainly due to proliferation of blood vessels around the fracture zone [18]. New blood vessels appear on the second week after injury and mainly derive from the adjacent soft tissues but also the periosteum and medullary canal [18–21]. Later on, soft and hard callus develop which are associated with maturation of blood vessels followed by a final long-lasting remodelling stage [18]. Among the many factors that can influence the bone healing process, adequate blood supply is crucial and the lack of perfusion is closely related to delayed union or non-union [1,22–25]. Animal models of tibial bone healing with prior resection of the femoral artery resulted in delayed healing or non-union with decreased cell proliferation, increased apoptosis and fibrous tissue formation [26,27]. Similarly, the local inhibition of angiogenesis in animal models resulted in bone union arrest and development of atrophic non-union [28]. Clinical studies in patients with concomitant vascular injuries reported an impaired healing rate of 46% which is far higher than the 5–10% non-union rate seen for long bone fractures in general [2,29]. At a cellular level, a number of important molecules have been identified to be implicated in the physiological process of new blood vessel formation (Fig. 1) [30–77]. Vascular endothelial growth factor (VEGF) is a key angiogenic mediator which stimulates new vessel formation and increases blood flow [78]. It exerts a chemotactic and proliferative effect both on osteoblasts and endothelial cells [56,73]. Blocking VEGF signalling results in arrest of intramembranous bone formation and VEGF gene knockout animals die due to deficient endothelial cell development and lack of blood vessels [34,75,79]. Platelet-derived growth factor (PDGF) and Fibroblast Growth Factor (FGF) are involved in the regulation of migration, morphogenesis, proliferation and differentiation of osteoprogenitor cells, osteoblasts and endothelial cells [32,41,45,53,61]. The transforming growth factor beta (TGF-b) superfamily is strongly engaged in developmental angiogenesis but also regulates the vascular integrity [31,65].

TGF-b is involved in the regulation of endothelial cell functions and is required for the development and homeostasis of blood vessels and plays an irreplaceable role on the differentiation of the vascular smooth muscle cells in the blood vessels [31,54,65]. Animal models with deficient TGF-b receptors acquire vasculogenic defects resulting in haemorrhage and vascular leakage [49]. Insulin-like growth factor (IGF) promotes bone matrix formation, regulates the productions of other growth factors including the VEGF and is a potent angiogenic factor that can stimulate endothelial cell proliferation, migration and morphological differentiation via binding to IGF-1R [37,39,57,72]. Parathyroid hormone (PTH) promotes angiogenesis and upregulates the expression of VEGF been able to reverse the hypovascularity produced in radiation induced depleted osseous vascularity animal models [38,48,51,58]. Finally, angiopoietin, thrombin and erythropoietin play an important role in vessel formation, inhibit MSC apoptosis due to nutrient deprivation and enhance osteoblast differentiation and bone formation [50,55,60]. Erythropoietin upregulates the expression of VEGF, endothelial nitric oxide synthase, and inducible nitric oxide synthase [42,46]. Several authors have suggested that an adequate vascularity response enhances and up-regulates the fracture healing process and outcome [18,21]. Such observations, delineate the importance of new blood vessel formation during bone healing and triggers the hypothesis whether early angiogenesis promoting intervention could expedite the bone repair process and outcome. The aim of this systematic review therefore was to evaluate the effectiveness of in vivo interventions of manipulation of the local environment utilizing the above molecules in terms of blood vessel formation (neo-angiogenesis) and bone regeneration. Materials and methods We searched Medline for general keywords such as ‘angiogenesis’, ‘vasculogenesis’, ‘bone healing’ isolated or in combination to specific words including ‘fracture healing’, ‘growth factors’

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‘osteoprogenitor cells’ and ‘mesenchymal stem cells’ from January 1980 to 15 March 2014. For paper selection, the initial inclusion criteria were studies publishing results on angiogenic molecules involved in bone healing in vivo both in humans and animal models. Exclusion criteria included publications of non-English literature or studies having incomplete documentation (methodology was not clearly described, reporting of results was blurred). Manuscripts that fulfilled the inclusion criteria were subjected to further review of their references list to ensure that no relevant study was left out from the final analysis. Relevant papers were reviewed and data including the studied molecules, methodology, model, results and conclusions were extracted and analyzed. Main outcome parameters analyzed were the effect of the molecule implanted on bone blood flow, the new blood vessel formation (neo-angiogenesis), torsional stiffness, and bone mineral content amongst others. Results Out of 478 studies initially screened, 60 met the inclusion criteria. Table 1 [38,80–138]. The vast majority of the studies involved small rodents while rabbits were frequently used as well. A great diversity of experimental approaches were analyzed including segmental and critical size defect in long bones, calvarial defects and models of induced non-unions or distraction osteogenesis. Briefly, the approaches used could be classified as local application of molecules at the fracture site or the application of genetically modified cells expressing such growth factors. It is of interest however, that the majority of the mentioned angiogenic factors possessed dual function: promotion of new blood vessel formation together with a direct effect on bone cells either inducing or upregulating the osteogenic differentiation. However, due to the experimental design limited conclusions could be made in terms of the most appropriate type of carrier to be used containing and releasing the tested molecule in a reliable, consistent and time dependent fashion; being biocompatible, biodegradable and ideally cost effective with minimal toxicity for the host. The most studied molecule was VEGF implanted in in vivo models for bone regeneration, Table 2 [88,91,104–106,110, 119,121,139]. Several carriers have been used including collagen gels, poly-lactide-co-glycolide (PLGA), anginate and gelatin

Table 1 Flow chart diagram of included studies [38,80–138].

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[91,93,104–106,110,119,121]. When VEGF was used alone, it promoted neovascularization with significant increases of blood vessel formation inducing a positive effect in the early phase of fracture healing with, greater bone coverage and increased bone mineral density (BMD) production [91,104,119]. Several studies have investigated the effect of a VEGF together with osteoprogenitor cells showing increased mineralization potential and integration of the construct into the host [93,105]. In addition to the osteoprogenitor cells, combination of VEGF with BMP-2 resulted in a synergistic effect with higher quality of regeneration and significantly higher levels of new endochondral bone matrix [106,110,121]. It should be noted that Kempen et al. found that VEGF implanted alone either ectopically or orthotopically in Sprague-Dawley rats was found unable to induce bone formation [110]. Equally to the above mentioned studies, research on VEGF gene transfer and transfected models reported favourable results in the induction of angiogenesis and bone healing [85,86,94,95,97, 100,107–109,114,115,122,124,131,134–136]. Katsube et al. studied the effect of VEGF gene transfer to augment surgical revascularization of necrotic bone [108]. Their results showed increased blood flow and vessel density one-week postoperatively. Rabbit bone marrow-derived mesenchymal stem cells (BMSCs) were cultured and infected with adeno-associated virus VEGF and BMP-7 and then implanted intramuscularly into the ischaemic limb. The use of DNA-VEGF (165) plasmid at bone defects could accelerate the formation of capillaries and the repair of bone defects [136]. The combination of VEGF and BMP-7 resulted in higher capillary growth and mean blood flow [135]. Similarly, Li et al. reported that VEGF gene therapy had significant angiogenic and osteogenic effects to enhance healing of a segmental defect in the long bone of rabbits [114]. Deproteinized bone grafting with VEGF gene transfer resulted in higher capillary regeneration and osteogenic potential. The utilization of fibroblast growth factor (FGF) has also shown to increase angiogenesis during fracture healing, Table 3 [84,90, 96,101–103,111,123,125,128,137]. FGF was found to accelerate vascularization, increase blood vessel volume enhancing fracture callus and the overall regeneration process [84,111,123,128,137]. FGF-2 applied on collagen sponge found to increase blood vessel volume and bone formation in critical-sized calvarial bone defects in mice [111]. Bland et al. however, failed to find any significant effect in animal tibial fractures injected with FGF and similar results were obtained by an earlier study on mandibular bone graft healing in the rabbits [83,90]. Similarly, Andreshak et al. did not find any difference in terms of healing and angiogenesis in tibial segmental defects in rats treated with coralline hydroxyapatite graft and Gelfoam with bFGF at 8 weeks after implantation despite their observation that increased vasculogenesis and development of hyaline cartilage occurred in 2 weeks [82]. Larsen et al. investigated the effect of FGF-2 and VEGF alone or in combination on blood flow and capillary density after application in rat’s femur. They reported higher capillary density in the FGF-2/VEGF group; however, the blood flow was the lowest of all studied groups [112]. On the contrary, in a rat model of revascularization of frozen allografts, FGF-2 was found to have no effect and when VEGF was compared with the VEGF/FGF-2 group no synergistic effect was identified [133]. Limited evidence exists on the direct angiogenic effect of BMPs in fracture healing in vivo. Donati et al. studied the effect of BMP-7 on the repair of a long bone critical size defect in a sheep model [87]. The results showed significantly higher penetration of newly formed vessels in the BMP-7 group together with faster callus formation and higher remodelling [87]. Similarly Ripamonti et al. presented a positive effect of BMP-7 on calvarial defects in vivo [126]. In a rabbit model of distraction osteogenesis, injection of

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Table 2 Studies presenting the effect of VEGF in treatment of bone defects in vivo. Author/year/ref.

Animal model

Scaffold

Osteoprogenitor cells

Eckardt et al., 2003 [88]

Rabbit model of distraction

No carrier used

–

osteogenesis

Murphy et al., 2004 [155]

Rat cranium critical defect

85:15 polylactide-coglycolide

–

Eckardt et al., 2005 [138]

Rabbit model of distraction osteogenesis

No carrier used

–

Kleinheinz et al., 2005 [156]

Mandibular defect in rabbits

Type-I collagen

–

Geiger et al., 2005 [94]

Critical size defects in rabbits

Gene-activated matrix

–

Eckardt et al., 2005 [157]

Atrophic non-union model in rabbits

Either autograft, hyaluronic acid or hyaluronic acid and VEGF

–

Kaigler et al., 2006 [104]

Irradiated calvarium of Fisher rats

Biodegradable PLGA (copolymer of D,L-lactide and glycolide) scaffold

–

Leach et al., 2006 [158]

Rat critical-sized defect model

Bioactive glass

–

Rabie et al., 2007 [124] Patel et al., 2008 [121]

Parietal bone defects in rabbits Rat cranial critical size defect

DBM Gelatin microparticles

– +/

BMP-2

Kempen et al., 2009 [110]

Sprague-Dawley rats orthotopic implantation

Poly(lactic-co-glycolic acid) microspheres surrounded with gelatin hydrogel

+/

BMP-2

Kanczler et al., 2010 [106]

Critical sized femural defect

Alginate scaffold

BM-MSCs + BMP-2

Gao et al., 2013 [93]

Femoral diaphysis defect in mice

Type I collagen gels

BM-MSCs

Farokhi et al., 2013 [91]

Rabbit

Silk/calcium phosphate/PLGA

–

Ozturk et al., 2013 [119]

Experimental non-union model, proximal tibiae defect in rabbits

Hydroxyapatite containing gelatin scaffold

–

BMP-7 resulted in an upregulation of VEGF expression without an increase in bone blood flow nor platelet endothelial cell adhesion molecule (PECAM) expression [116]. BMP-2 delivered in a hyaluronan gel carrier at a mid-tibial osteotomy model improved bone healing without any statistically significant difference in blood flow [89]. Similarly, BMP-2 application in a segmental femoral defect in rats did not result in any statistically significant difference in terms of vessel volume or vessel number in the newly formed bone [129]. Several authors have combined other angiogenic molecules with BMPs [99,118]. The results showed

Results  VEGF treatment increased the blood flow in bone of the distracted limb.  Failed to influence bone blood flow, torsional stiffness and bone mineral content.  Increased vascularization and enhanced mineralized tissue generation, but not significant osteoid formation.  VEGF treatment increased the blood flow in bone of the distracted limb.  No effect on bone blood flow, torsional stiffness, bone mineral content.  Increased number of blood vessels and improved bone volume.  At 6 weeks, 2–3 fold increase of the number of vessels.  At 12 weeks, the amount of vascularization decreased, whereas more new bone was detectable.  VEGF-treated osteotomies united whereas the carrier-treated osteotomies failed to unite.  The biomechanical properties of the groups treated with VEGF and autograft were identical.  There was no difference in bone blood flow.  Significant increases in blood vessel formation, greater bone coverage and increased BMD in VEGF scaffolds.  Enhanced angiogenesis and bone maturation.  Enhanced bone formation.  A synergistic effect of the dual delivery of VEGF and BMP-2 on bone formation was noted.  VEGF does not affect the amount of bone formation achieved by BMP-2 at 12 weeks.  VEGF did not induce bone formation, it did increase the formation of the supportive vascular network.  In orthotopic defects, no effect of VEGF on vascularization was found.  Alginate + BMP-2 + VEGF + MSCs group showed significant new endochondral bone matrix and quality of regeneration.  Rapidly mineralized and integrated into host.  New bone matrix formation with neovascularization in the angiogenic factors loaded scaffold after 10 weeks of implantation.  The local administration of VEGF on the graft had a positive effect in the early phase of fracture healing.

increased angiogenesis and bone regeneration. Local administration of VEGF and BMP-2 enhanced angiogenesis, osteoblastic activity and bone blood flow in a femoral diaphyseal bone transplantation model [118]. Samee et al. studied the effect of combination of BMP-2 and VEGF transfected human periosteumderived cells which were cultured and implanted to nude mice intramuscularly [140]. After implantation, ectopic bone was observed, however the combination of BMP-2 and VEGF formed significantly more bone at 4 weeks with more blood vessels. Enhanced osteogenic and angiogenesis was also reported by the

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Table 3 Animal studies presenting the effect of FGF in treatment of bone defects. Author/year/ref.

Animal/model

Model

Kigami et al., 2013 [111]

Critical-sized calvarial bone defects VEGFR2-luc transgenic mice

FGF-2 with an absorbable collagen sponge Starch/polycaprolactone (SPCL) scaffolds with VEGF + FGF-2

Zheng et al., 2011 [137]

Mice mandibular condyleshaped construct

Human BM-MSCs transfected with basic FGF seeded in a porous corral construct

Qu et al., 2011 [123]

Calvarial critical-sized defect model in rats

Guo et al., 2006 [96]

Critical-sized segmental bone defects in the radius in rabbits

BM-MSCs transfected with bFGF seeded in a nano-hydroxyapatite/ polyamide66 (n-HA/PA66) composite scaffold BM-MSCs transfected with bFGF on beta-TCP ceramic scaffold

Chen et al., 2004 [84]

Rabbit proximal tibial metaphyseal osteotomy

Injection of FGF-2, mixed with gelatin hydrogel

Iwakura et al., 2003 [101]

Sternal healing in dogs

Gelatin sheet incorporating FGF

Iwakura et al., 2001 [103]

Sternal healing in diabetic rats

Gelatin sheet incorporating FGF

Iwakura et al., 2000 [102]

Sternal healing in rats

Gelatin sheet incorporating FGF

Radomsky et al., 1999 [125]

Human primate fracture model

Andreshak et al., 1997 [82]

Tibial segmental defect in rats

Injection of Viscous gel containing hyaluronan and FGF-2 Coralline hydroxyapatite graft and Gelfoam with FGF

Bland et al., 1995 [83]

Rabbit tibial fractures

Eppley et al., 1988 [90]

Mandibular bone graft healing in the rabbits

Santos et al., 2013 [128]

Results

3 micrograms of either FGF-1 or FGF-2 injected around fractures FGF administered through a subcutaneous osmotic pumps

delivery of osteoprogenitor cells transduced with BMP-2, VEGF and angiopoietin-1 adenoviral vectors [99]. BMP-2 at suboptimal doses together with platelet rich plasma found to result in increased numbers of blood vessels, bone mineral density and bone mineral content [120]. A sequential angiogenic and osteogenic growth factor release i.e. VEGF release followed by BMP-2 release showed to enhance bone regeneration [110]. Finally, by blocking the BMP-2 inhibitor noggin, Levi et al. demonstrated significantly higher number of vessel formation in vivo [113]. Thrombin peptides (TP) have been reported to be effective in enhancing the fracture healing response through both the upregulation of osteogenesis and blood vessel formation. TP508 treatment was associated with an up-regulation of early response elements, inflammatory mediators, and genes related to angiogenesis [127]. Similarly, Wang et al. found that a single injection of thrombin peptide (TP508) in a closed rat femoral fracture model, accelerated the fracture repair and significantly increased the number on blood vessels in the callus [132]. In distraction osteogenesis performed in tibiae of rabbits over a period of 6 days TP508 has been found to up-regulate the levels of Runx2 and osteopontin expressed preosteoblasts, osteoblasts, osteocytes of newly formed bone and blood vessel cells [81]. Collectively, these studies suggest that TP508 peptide can accelerate bone repair by initiating a cascade of events that lead to an increased rate of tissue revascularization and regeneration [127]. Limited data exist on the in vivo angiogenic effect of the remaining molecules, despite the well documented upregulation of osteogenesis. Liposome carrying PDGF and IGF-I have been found to promote trabecular bone formation and VEGF expression inducing a higher number of blood vessels [80]. Similar results were reported by Ferreira et al. with the use of EGF, TBF-B and

 Increased blood vessel and bone formation.  The release of VEGF and FGF-2 from the constructs enhanced the expression of VEGFR2 and other important mediators in neovascularization (VEGF and VEGFR1).  Bone formation and collagen type I and type II deposition noted.  Neovascularization was observed around newly formed bone tissue.  Accelerated vascularization and bone regeneration on these composite scaffolds.

 Increases osteogenic properties of MSCs, enhanced capillary regeneration providing a rich blood supply.  Bone mineral density and the cancellous bone area in the healing region of the fracture site were significantly larger  Complete healing of the sternum with marked angiogenesis compared to the control group.  Marked angiogenesis and healing in the FGF group.  Blood flow significantly increased and marked angiogenesis noted around the sternum.  Enhanced callus size, periosteal reaction, vascularity, and cellularity.  At 2 weeks increased development of normal hyaline cartilage and vasculogenesis.  No difference between the groups at 8 weeks.  No effect.  No difference detected between the stimulated and non-stimulated grafts.

BMP-4 [92,117]. Recombinant parathyroid hormone has been found to enhance arteriogenesis which was mediated through decreased angiopoietin-2 expression [38]. In addition, erythropoietin has significantly enhanced the outcomes of BMP2-induced cranial bone regeneration in part through its actions on osteoclastogenesis and angiogenesis [130]. In another model of femoral segmental bone defect model in mice, Holstein et al. presented that intraperitoneal erythropoietin (EPO) injections stimulated bone formation, cell proliferation and VEGF-mediated angiogenesis resulting in a higher number of blood vessels [98]. Discussion Therapeutic angiogenesis has been proposed by a number of authors for the management of complicated fracture healing cases, non-unions and distraction osteogenesis associated with an impaired healing response. During the angiogenic cascade several molecules are involved and their upregulation is coupled to the osteogenesis but there are various other factors that could influence this process. Nowadays, it is well accepted that excessive movement at the fracture site, for instance, leads to the inhibition of vascularization and formation of fibrocartilage [141]. On the contrary, mechanical stability and micromovement is associated with angiogenesis [142]. In addition, other factors like the personality of the fracture, associated soft-tissue injuries and patient’s comorbidities and habits could equally influence angiogenesis and healing [143]. Smoking for example is highly associated with disruption of angiogenesis [144]. Therefore, these factors should be clearly analyzed and be addressed appropriately before any attempt for reconstructive surgery is contemplated.

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Once the risk factors that influence angiogenesis and bone healing are addressed, strategies to restore/enhance local angiogenesis can be utilized. Such strategies could be divided into 3 categories. The first category involves the implantation of a growth factor locally. Such approach would result in a diffuse dose dumping of the molecule with high levels locally only for a limited amount of time. The second strategy involves the loading of such molecules on a scaffold, which in theory facilitate prolonged controlled release of the molecule. Several carriers have been used in animal studies [101,123,137]. However, very limited data exist on the release kinetics and the elution levels of the molecules released. This requirement is further reinforced by observations showing arrest of osteogenesis, vascular leakage, fibromatosis and hypotension in cases where high doses of VEGF or FGF were used [145,146]. In addition, when cells are loaded in such constructs, the amount of bone formed could be proportionate to the culture period, whereas the type of culturing may have a positive effect on the expression of osteogenic markers but not on the quantity of bone formation [146]. Therefore, presenting the final outcome of such approaches in animals is important. Nonetheless, before any clinical application is contemplated, patient safety should be ensured. A third strategy involves the local delivery of cells carrying foreign nucleic acids sustaining the transgene expression even after host cells replicate [147]. Such techniques are able to enhance or inhibit specific gene expression in cells, and to produce recombinant proteins [147]. It is not fully elucidated as yet, whether there is a genetic defect or suboptimal cellular production of angiogenic growth factors in cases of delayed union or nonunion. Therefore, uncontrolled upregulation of angiogenic factors in humans is at the moment unpredictable. Furthermore, bone healing is a complex process demanding production of molecules and tissue interfaces to repair the injured structures and this will require identification and controlled delivery of complex signals in both time and space. Our view regarding manipulation of the genetic programming of the cells involved in bone healing is that further research is required to demonstrate their safety and efficacy prior to any potential future clinical application. Several authors have highlighted potential safety issues with the use of growth factors in bone healing and regeneration strategies. Osseous overgrowth, immune reactions and local toxicity have been reported [148]. In addition, recent data raised concerns regarding potential carcinogenic effect with the application of such molecules [149–151]. Two recent studies reported an increased, but not statistically significant, risk for either cancer in general or pancreatic cancer in patients who received rhBMP [150,151]. Significantly increased risk of cancer was reported by Fu et al. in patients who received BMP-2 for spinal fusion [149]. Although the cancer types were heterogeneous and the cancer incidence was low, nevertheless, concerns exist in potential application of such molecules in clinical applications. VEGF, FGF and the majority of the angiogenesis promoting molecules are involved in oncogenesis and constitute a target for cancer medication. Noteworthy, their molecular pathway in bone healing has not been yet fully elucidated [152]. Despite the work that has been done up to now, no robust recommendations can be made with respect to which molecule could be used in the clinical setting. BMP-2 and BMP-7 are already approved for clinical applications (open tibial fractures and tibial non-unions respectively) and their results have been favourable. However, both molecules are considered strong osteogenic differentiation inducers rather than solely angiogenic promoters. Possibly, a dual approach with the combination of BMPs and VEGF or any other angiogenic molecule could be ideal in this respect. This theory is further strengthened by the finding of Garcia et al. who reported adequate vascularization in cases of non-union but deficient stimulation of osteogenesis [153]. It is of note however,

that the mixing ratio between the molecules could influence their synergistic effect [154–158]. The elution kinetics of the growth factors and scaffold constructs should be elucidated in future research. Basic science research expounding the receptors and cellular functions governing the angiogenesis together with the complex mechanism and feedback loops found during bone healing would be of paramount importance. Further research on the recently proposed safety issues governing some of the molecules presented in this study is still to be clarified. Conclusion Basic science research has presented a strong interdependent link between angiogenesis and bone healing. The utilization of several angiogenic molecules in a variety of therapeutic approaches in vivo has shown their potential in bone regeneration. More attention should be drawn on the release kinetics of such molecules as well as their safety and efficacy in potential future applications. On this note, clinical data are limited. Through further basic science research elucidating the complex parameters involved in bone healing, such approaches could serve as future therapeutic alternatives in cases where bone regeneration is desirable. Conflict of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References [1] Pountos I, Georgouli T, Calori GM, Giannoudis PV. Do nonsteroidal antiinflammatory drugs affect bone healing?. A critical analysis. Scientific World J 2012;2012:606404. [2] Pountos I, Georgouli T, Kontakis G, Giannoudis PV. Efficacy of minimally invasive techniques for enhancement of fracture healing: evidence today. Int Orthop 2010;34:3–12. [3] Pneumaticos SG, Panteli M, Triantafyllopoulos GK, Papakostidis C, Giannoudis PV. Management and outcome of diaphyseal aseptic non-unions of the lower limb: a systematic review. Surgeon 2013. pii: S1479-666X(13)00138-8 (Epub ahead of print). [4] Liu Y, Cao L, Ray S, Thormann U, Hillengass J, Delorme S, et al. Osteoporosis influences osteogenic but not angiogenic response during bone defect healing in a rat model. Injury 2013;44:923–9. [5] Fong GH. Regulation of angiogenesis by oxygen sensing mechanisms. J Mol Med (Berl) 2009;87:549–60. [6] Kivirikko KI, Prockop DJ. Enzymatic hydroxylation of proline and lysine in protocollagen. Proc Natl Acad Sci U S A 1967;57:782–9. [7] Muzylak M, Price JS, Horton MA. Hypoxia induces giant osteoclast formation and extensive bone resorption in the cat. Calcif Tissue Int 2006;79:301–9. [8] Nicolaije C, Koedam M, van Leeuwen JP. Decreased oxygen tension lowers reactive oxygen species and apoptosis and inhibits osteoblast matrix mineralization through changes in early osteoblast differentiation. J Cell Physiol 2012;227:1309–18. [9] Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002;109: 1405–15. [10] Hoff P, Maschmeyer P, Gaber T, Schutze T, Raue T, Schmidt-Bleek K, et al. Human immune cells’ behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model. Cell Mol Immunol 2013;10:151–8. [11] Colnot C, Lu C, Hu D, Helms JA. Distinguishing the contributions of the perichondrium, cartilage, and vascular endothelium to skeletal development. Dev Biol 2004;269:55–69. [12] Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell 2010; 19:329–44. [13] Clarkin CE, Gerstenfeld LC. VEGF and bone cell signalling: an essential vessel for communication? Cell Biochem Funct 2013;31:1–11. [14] Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–60. [15] Tepper OM, Capla JM, Galiano RD, Ceradini DJ, Callaghan MJ, Kleinman ME, et al. Adult vasculogenesis occurs through in situ recruitment, proliferation, and tubulization of circulating bone marrow-derived cells. Blood 2005; 105:1068–77.

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