Role of angiogenesis in bone repair

Archives of Biochemistry and Biophysics xxx (2014) xxx–xxx

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Review

Role of angiogenesis in bone repair Uttara Saran a, Sara Gemini Piperni c, Suvro Chatterjee a,b,⇑ a

Vascular Biology Lab, AU-KBC Research Centre, Anna University, MIT Campus, Chennai, India Department of Biotechnology, Anna University, Chennai, India c Department of Applied Clinical Science and Biotechnology, University of L’ Aquila, L’ Aquila, Italy b

a r t i c l e

i n f o

Article history: Received 2 March 2014 and in revised form 1 July 2014 Available online xxxx Keywords: Angiogenesis Endothelial cells Endothelial progenitor cells VEGF Osteogenesis Osteoblasts

a b s t r a c t Bone vasculature plays a vital role in bone development, remodeling and homeostasis. New blood vessel formation is crucial during both primary bone development as well as fracture repair in adults. Both bone repair and bone remodeling involve the activation and complex interaction between angiogenic and osteogenic pathways. Interestingly studies have demonstrated that angiogenesis precedes the onset of osteogenesis. Indeed reduced or inadequate blood flow has been linked to impaired fracture healing and old age related low bone mass disorders such as osteoporosis. Similarly the slow penetration of host blood vessels in large engineered bone tissue grafts has been cited as one of the major hurdle still impeding current bone construction engineering strategies. This article reviews the current knowledge elaborating the importance of vascularization during bone healing and remodeling, and the current therapeutic strategies being adapted to promote and improve angiogenesis. Ó 2014 Published by Elsevier Inc.

Bone Circulation and Angiogenesis Vasculature is essential for embryonic skeletal development, bone growth and remodeling [1–4]. Apart from supplying the bone tissue with nutrients, growth factors, hormones, cytokines and chemokines as required; and removing waste products, bone vasculature acts as a communicative network between the bone and neighboring tissues [3,5]. Depending on their origin, bone development occurs via two distinct modes of ossification: Intramembranous (flat bones such as skull and clavicle) and Endochondral (load bearing bones) ossification respectively. Regardless of their differences, however vascularization is a prerequisite for both types of ossification [1,6,7]. In fact the development of the vascular network plays a vital role in directing limb morphogenesis during embryogenesis [3]. Vascularization is required for osteogenesis to occur, where vascular invasion promotes the chondroblast/osteoclast degradation of the hypertrophied cartilage core of primary bone which is eventually replaced by the bone marrow and bone (see Fig. 1C). Apart from playing vital roles during ossification process and bone remodeling [1,4,8], angiogenesis is the key component during bone repair (see Fig. 1). The formation of new blood vessels in ⇑ Corresponding author at: AU-KBC Research Centre, MIT Campus of Anna University, Chromepet, Chennai 600044, India. Fax: +91 44 2223 1034. E-mail address: [email protected] (S. Chatterjee).

the metabolically active regenerating callus is required for supplying nutrients, oxygen, growth factors, cytokines and osteoblast and osteoclast precursors [9]. Thus the close spatial and temporal association of bone formation with vascularization has been termed as ‘angiogenic–osteogenic coupling’ [1,8,10]. Anatomically vasculature in long bones is supplemented by four arteries namely the nutrient, metaphyseal, epiphyseal and periosteal arteries [11]. The largest of these is the nutrient artery which supplies more than 50% of total blood to the long bones [12]. These arteries enter the bone through their respective foramina and reach the medullary cavity, where they divide forming the cortical and marrow microcirculations [13]. In the cortical bone, the vessels feed the capillaries in the Haversian and Volkmann’s canals [14], while in the marrow, the capillaries drain into sinusoids [15]. These capillaries in turn unite to form a single venule that follows the arteriole route back to the perichondrial plexus [16]. One of the most important components of blood vessels is the endothelium, which plays an essential role in the maintenance of bone homeostasis not only by functioning as a highly permeable barrier but also secreting factors that recruit circulating cells particularly hematopoietic cells, to the bone site [3,17–19]. (See Fig. 1A). Adult bones continuously undergo remodeling in order to maintain their integrity and biomechanical stability [1,4]. Bone remodeling occurs in two important steps: osteoclast mediated bone resorption and osteoblast mediated new bone formation

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Fig. 1. (A) Anatomical illustration of bone vasculature adapted from [http://www.anatomiahumana.ucv.cl/kine1/top2.html]. (B) Adult bone undergoes constant remodeling to maintain their structural integrity. This process consists of a osteoclast -mediated bone reabsorption and osteoblast-mediated new bone formation and mineralization phase. (C) Endochondral model of bone formation during embryo development. At first the hyaline cartilage substitutes as the new bone structure. The central hypertrophic chondrocytes start degenerating and the matrix surrounding them begins to calcify the future diaphyseal area forming the primary ossification center. Complete degeneration of the central area and calcification of the matrix results in formation of the secondary ossification center. Vascular penetration into the secondary structure initiates osteogenesis by supplementing the area with osteoprogenitor cells, chondroclasts, and hematopoietic cells, resulting in the formation of the mature bone. (D) Schematic representation of events mediated during bone fracture repair.

Fig. 2. Microenvironment during bone fracture repair.

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[13,20,21] (Fig. 1B). This process takes place in special vascularized structures called bone remodeling units (BRUs)1 [1,22]. The BRU facilitates the direct cell to cell interaction between osteoblast and osteoclast precursors, and secretion of receptor activator of NFjB ligand (RANKL) by osteoblast cells promotes osteoclast differentiation and activation [23–26]. Thus the BRC serves not only to integrate the bone remodeling process with the bone microvasculature but also to prompt the recruitment of osteoblast and osteoclast precursor cells and to signal molecules from circulation to the site of remodeling [27,28]. Studies have suggested that blood vessels development in bones and osteogenesis are coupled, which is indicative of the presence of a molecular crosstalk between endothelial and osteoblastic cells [29,30]. Recently Kusumbe et al. [31] identified a new capillary subtype present in the skeletal system processing distinct morphological, molecular and functional properties. They demonstrated that these blood vessels, were able to generate a distinct metabolic and molecular microenvironment, mediating growth of bone vasculature, maintaining perivascular osteoprogenitors and coupling angiogenesis to osteogenesis. Results also demonstrated that the abundance of these vessels and associated osteoprogenitors was greatly reduced in bones of aged animals [31]. Furthermore a second study by the same research group was able to establish a molecular connection among angiogenesis, angiocrine signals and osteogenesis [32]. They demonstrated that endothelial Notch activity plays a crucial role in promoting angiogenesis and osteogenesis in bones. The disruption of Notch signaling not only impairs bone vessels morphology and growth but also leads to decreased osteogenesis, chondrocyte defects, shortening of long bones, loss of trabeculae bone and reduced bone mass [32]. Bone remodeling in adults requires a precise balance between osteoclastic bone resorption and osteoblastic bone formation in order to maintain the integrity of the bone [1,33]. An imbalance between these two processes, i.e. increased bone resorption without adequate compensation in bone formation results in onset of osteoporosis [34], a syndrome frequently reported in post-menopausal women. The loss of bone mass and density results in weakening of bone strength, leading to an increase of the propensity to bone fractures. Burkhardt et al. [35] on examining histological samples of osteoporotic bones observed a reduced number of arterial capillaries and sinuses per unit area. Ding et al. [36] demonstrated a significant decrease in the expression of major angiogenic stimulator vascular endothelial growth factor (VEGF) in bones of ovariectomized mice. Zhao et al. [33] reported of an association between decreased bone vascularization and estrogen deficiency mediated bone loss. They showed that reduced expressions of hypoxia-inducible factor-a (HIF-a) and VEGF in bones of ovariectomized mice induced a decrease in bone vasculature as well as bone volume. Increased levels of cytokines - interleukin-1 (IL-1) and tumor necrosis factor (TNF) have also been suggested to cause the high osteoclast activity seen in post-menopausal osteoporosis. These cytokines have been suggested to induce precursor marrow cells to differentiate into osteoclasts and activate mature osteoclasts to resorb the bone [37].

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Abbreviations used: BRUs, bone remodeling units; RANKL, receptor activator of NF-

jB ligand; VEGF, vascular endothelial growth factor; HIF-a, hypoxia-inducible factora; IL-1, interleukin-1; TNF, tumor necrosis factor; EC, endothelial cell; MSCs, mesenchymal stem cells; PDGF, Platelet-Derived Growth Factor; TGF-b, Transforming Growth Factor b; FGF, Fibroblast Growth Factors; BMPs, Bone Morphogenetic Proteins; EPC, endothelial progenitor cells; CAM, chorioallantoic membrane; human osteoblast; HUVECs, human umbilical vein endothelial cells; rAAV, recombinant adeno-associated virus.

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Normal bone repair and microenvironment In the event of an injury, bones have the unique ability to heal by regenerating new bone sans development of fibrotic scars, a common phenomenon during soft tissue healing [1,5,38]. The development of fibrotic scars during bone repair results in critical-sized bone defects if left untreated and it would ultimately compromise the mechanical properties of the skeleton [5]. Hence adult bone repair mimics bone formation during organogenesis, consisting of a series of interdependent healing stages, where the newly regenerated bone is indistinguishable from the uninjured bone [5,38–40]. Following a fracture, the resulting trauma, together with compromised blood supply, disruption of oxygen supply and acute necrosis of the surrounding tissue create a hypoxic environment around the injured gap [41]. This hypoxic environment is an important physiological signal in bone repair as it regulates the production of key modulators by osteoblasts that in turn influence endothelial cell (EC) proliferation, direct cellular differentiation, and induce ECs to secrete osteogenic growth factors.[3,42,43]. The ensuing inflammation and re-vascularization represent the most crucial phase of bone repair [39]. The first responders at the injured site are platelets which bind to the exposed collagen of the sub-endothelium, initiating platelet aggregation and clotting. The blood clot or haematoma releases various signaling factors and angiogeneic growth factors which in turn activate the migration of inflammatory cells and repair cells such as fibroblasts, osteoblasts, stem cells and vascular endothelial cells [44–49]. The fracture haematoma also serves as the template for the formation of provisional vascular callus [41], removal of the haematoma has been shown to significantly attenuate bone repair, while its transplantation has been shown to induce new bone formation at the new site [48–51]. The recruited fibroblasts begin to lay down the stroma that help support vascular ingrowths while the responding macrophages remove tissue debris [52].The inflammatory cells also release growth factors and cytokines signals to recruit mesenchymal stem cells (MSCs), which then proliferate and differentiate into osteoprogenitor cells. Osteoprogenitor cells that lie close to the undamaged bone (and a ready supply of oxygen) differentiate into osteoblasts which then form osteoids that are subsequently mineralized to form a soft callus around the wounded area. This callus gradually undergoes endochondrial ossification bridging the fractured gap with woven bone. The healing bone then undergoes remodeling, getting restored to its original shape, structure and mechanical properties [52] (Figs. 1D and 2). The lack or inhibition of angiogenesis has been reported as one of the main reasons for improper fracture healing [3,4,13,53–55]. Hausman et al. [53] demonstrated that preventing angiogenesis in animal models resulted in the formation of fibrous tissue reminiscent of human atropic non-unions. Fang et al. showed that administration of anti-angiogenic drug prevents normal osteogenesis and results in fibrous nonunion [54]. Similarly Holstein et al. [55] observed that the treatment with immunosuppressant drug Rapamycin (which is known to have anti-angiogenic properties), inhibits neovascularization in the fracture callus and delays the healing process. Open fractures are considered to carry a greater risk of non-union as the resulting soft-tissue damage deprives the fracture of its normal blood supply. This, in turn, leads to diminished vascular callus ingrowth, increased bone necrosis and diminished resistance to infections [41]. Thus blood flow to the fracture site is essential for a proper healing to take place. Early vascularization is required for osteogenic repair of critical bone defects, as vessels provide nutritional support for the bone grafts [54,56,57]. Interestingly Sojo et al. [58] with the help of osteodistraction models, demonstrated that angiogenesis occurs predominantly before the onset of osteogenesis [3], and the newly formed

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blood vessels ensure steady transport of circulating osteoclast and osteoblast precursors to remodeling sites [59]. The fact that osteogenesis is a vascular dependent process suggests that the vascular endothelium itself regulates osteoprogenitor cell invasion into the fracture site [12,60,61]. In vitro studies have demonstrated that the endothelium separates the osteoclast precursors from the bone surface; these precursors would need to adhere and migrate through the endothelium to reach distant bone resorption sites [62]. For this process to occur precisely, it has been suggested that in cutting cones of cortical bone, EC may express a specific area code that enables the osteoclast precursors to access the bone in a highly time and space dependent manner [63]. The intercellular signaling between bone-forming and vessel-forming cells plays crucial roles in the development, remodeling and maintaining the integrity of bones [64]. Each cell type communicates via direct (gap junctions) or indirect cell contact and by secreting of diffusible factors, which in turn affect the growth and differentiation of both cell types [65,66] .

Pro-angiogenic signaling in bone wound micro-environment A plethora of pro-angiogenic factors such as the VEGF, PlateletDerived Growth Factor (PDGF), Transforming Growth Factor b (TGF-b), Fibroblast Growth Factors (FGF) and Bone Morphogenetic Proteins (BMPs) are involved in the bone repair cascade (see Fig. 2). Their role in initiating angiogenesis and/or regulating osteogenesis has been summarized in Table 1. Among these, VEGF, whose production is frequently stimulated by most osteoinductive factors [67–69], has been suggested to possibly act as a central mediator for the other secreted factors [70]. Various studies have reported that inhibiting VEGF blocks the angiogenic activity of FGF and BMP-2 [67,70] as well as BMP induction of primary osteoblast differentiation and bone formation. VEGF levels have been reported to be strongly elevated both locally in the facture hematoma as well as systemically in injured patients [40]. VEGF plays important roles in the mobilization and recruitment of endothelial progenitor cells (EPC), EC differentiation and proliferation [71] as well as the recruitment and survival of osteoblasts [45,47,49,72–74] and osteoclasts [47,75,76]. VEGF expression has also been reported to increase in direct response to hypoxia in osteoclasts [77–79]. The HIF pathway has been reported to directly increase VEGF expression [80], with Komatsu et al. [81] demonstrating delayed bone regeneration in their HIF-1a heterozygous mice models. Osteoblast cells also express components of HIF-1 and hypoxia can upregulate VEGF expression by these cells thereby promoting angiogenesis and osteogenesis [80]. VEGF has also been reported to cause upregulation of the receptor RANK in endothelial cells, enhancing their responsiveness to RANKL [82]. RANKL in turn has been shown to play an important role in maintaining EC survival via PI3K/Akt signal transduction pathway [83], and it has been shown to strongly stimulate angiogenesis [84].

Among the members of the VEGF family, VEGF-A is one of the most critical mediators of angiogenesis [1]. VEGF -C, -E and PIGF have also been reported to play a role in accelerating bone healing [41,47,85–87]. VEGF receptors (VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 and -2) are expressed in cells of the osteoblast lineage [47,74,88] and during mouse fracture repair [89]. The deletion of even a single copy of the VEGF gene induces early embryonic lethality owing to defective vascular development [29,90–93]. Inhibition of VEGF in vitro has been reported to decrease primary osteoblast differentiation [45], while its inhibition in vivo caused decreased blood vessel invasion, decreased osteoclastic bone remodeling, impaired callus mineralization and reduced trabecular bone healing [45,49,94–97]. Additionally mice only expressing freely diffusible isoform of VEGF were reported to have impaired endochondral bone formation [73,96,97]. Liu et al. [98] reported that conditional deletion of VEGF in osteoprogenitors induced a osteoporosis-like phenotype characterized by increased bone marrow adiposity and decreased bone mass. Conversely exogenous administration of VEGF has been reported to significantly promote angiogenesis and accelerate bone healing [40,45,46,70,89,99–102]. Similarly adenoviral delivery of VEGF was reported to increase bone formation in intact rabbit femurs, and stimulate angiogenesis and bone formation in rat femur defects [99]. FGF is another growth factor inducing angiogenesis [47,103], that has been reported to contribute significantly to bone development, remodeling and repair [104,105]. It is also a potent mitogenic factor for different cell types including fibroblasts, EC, mesenchymal cells and osteoblasts [106–108]. FGF is produced by various cells including EC, fibroblasts and osteoblasts. Osteoblasts secrete FGF in response to stimulation by prostaglandin and TGF-b [109,110] FGF has also been shown to induce osteoclastogenesis by regulating the production of osteoclast differentiation factors such as RANKL and COX-2 [111–113]. Collin-Osdoby et al. [103] upon implanting a bone chip in chick embryo chorioallantoic membrane (CAM) reported that FGF promoted osteoclast recruitment, formation, differentiation, and resorption activity on bone at sites of stimulated angiogenesis. Both TGF- b and PDGF are released by degranulating platelets during fracture healing. PDGF is said to be able to stimulate angiogenic and osteogenic pathways, the former either directly or via VEGF upregulation and the latter by exerting chemotactic and mitogenic signals for osteoblasts [114–120]. TGF- b is a potent chemotactic stimulator for the recruitment and proliferation of MSCs and, at later stages, it regulates the differentiation of osteoblasts [117,120,121]. Tang et al. [120] were the first to prove that TGF-b is an important chemotactic recruiter of osteoblast precursors to bone remodeling sites. The authors also reported perturbed bone remodeling in those mice lacking TGF-b. Interestingly VEGF expressed in haematopoietic and endothelial cells is found to be regulated by TGF-b, suggesting that TGF-b released by osteoclasts induces VEGF expression in cells within the BRC to help maintain vascular supply during bone remodeling [122,123]. Various studies have shown

Table 1 Factors influencing bone repair. Growth Factors

Osteogenic or Angiogenic effect

Function

Influence of/over other growth factor

References

VEGF

Both

Chemoattractant for osteoblasts, MSC and EC

TGF- b

Both

[29], [40], [45], [47], [49], [67], [71–76], [85,87], [90–97,73,98–101] [109], [110], [117], [120–123]

PDGF

Both

VEGF

[114–120]

FGF

Angiogenesis

VEGF

[47], [103], [106–108], [111–113]

BMP-2, 4, 7

Osteogenic, indirectly angiogenic

Chemoattractant for MSC, differentiation of osteoblasts Chemoattractant and mitogenic stimulation for osteoblasts Stimulated osteoblast migration, mitogenic factor for EC, MSC and osteoblasts differentiation of osteoblast-like cells; Chemoattractant for neighboring EC

Central mediator for other growth factors FGF, VEGF

VEGF-A

[44], [67,68], [70], [87], [129–131]

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that BMPs (BMP- 2, 4 and 7; osteogenic protein 1) released by osteoprogeniter cells, osteoblasts and bone extracellular matrix, promote bone repair by stimulating the proliferation and differentiation of MSC and osteoprogenitor cells [47,87,117,124–127]. Additionally BMP- 2, 4 and osteogenic protein 1 have been reported to induce angiogenesis via VEGF stimulation [68,70,128–130]. Peng et al. [70] reported that VEGF has greater effect on BMP-4 induced rather than on BMP-2 induced bone formation. BMPs may also induce bone formation by directly activating EC mediated angiogenesis [129,131]. Recent studies suggest that EC subjected to hypoxic, mechanical or VEGF treatments may have an osteogenic role through their production of BMP-2 and -4. Endothelial progenitor cells in bone repair EPCs are a population of cells that is found circulating in the bone marrow and peripheral blood, which has the ability to differentiate into mature EC. New blood vessels during fracture repair can be formed either via angiogenesis or vasculogenesis. The latter process occurs when new blood vessels form without a pre-existing vascular component and occurs via the differentiation of local or/and circulating EPCs [132–136]. EPC are comprised of a heterogeneous population composed of two sub-populations: early and late EPC [137]. Yoon et al. [138] showed that, despite the differences between the two EPC populations, they function synergistically during neovascularization. EPCs have greater proliferation capacity than mature EC and have been demonstrated to differentiate into EC in vitro and contributing to the formation of vascular networks [139,140]. EPCs are known to contribute to both angiogenesis and vasculogenesis at site of ischemic wounds. Distraction osteogenesis, a bone regeneration system presents an example of ischaemic regeneration [141,142]. Interestingly Smadja et al. observed selective expression of BMP-2 and -4 by late EPC [143]. Lee et al. [141] observed increased mobilization of EPC towards the site of fracture healing in rat distraction osteogenesis and mouse models. Atesok et al. [144] demonstrated that EPCs initiated neovascularization and new bone formation during fracture healing. Similarly, Rozen et al. [135] Masumoto et al. [145] reported that transplantation of autologous EPCs enhances angiogenesis and osteogenesis in critical-sized defects and delays fracture unions respectively. The mechanism by which EPC regenerates bone is still unknown, however the most likely explanation is that EPC mediated angiogenesis and vasculogenesis is indispensable to bone regeneration. Data from several studies suggest that EPC induces recruitment, proliferation and differentiation of skeletal progenitors via a paracrine mechanism [146–148]. Alternatively, on observing that EPC cultured in osteogenic conditions developed alizarin-red, von Kossa and osteocalcin positive nodules, Bick et al. [149] postulated that EPC may transform into osteogenic cells in an appropriate microenvironment. Current therapeutic tools in bone repair and their limitations The treatment for restoring bone function is often dependent on the type of orthopedic injury. Although bone fractures generally undergo repair via the process of callus formation [38], severe cases of bone damage (traumatic fractures, bone tumors etc) mostly require surgical reconstruction [52]. Current therapy involves bone grafting, a procedure that replaces missing bone with either autologous (bone material sourced from patients own body), allogenic (bone material sourced from a donor) or biomaterial implants [5,52]. The ideal requirements for bone grafts include osteoconductivity, biomechanical stability and minimal host immune response. Hence, to maximize clinical success, tissue engi-

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neers primarily focus on fabricating bone constructs that possess similar properties (microstructure, mechanical strength) and functions of physiological bones. Recent studies have suggested the potential to use porous scaffold polymer implants (naturally derived or synthetic matrices) for bone reconstruction. Advantages of natural polymers like collagen include their biodegradability and biocompatibility [44], while synthetic polymers can be easily engineered and custom-designed to fit the anatomical defects of individual patients [150,151]. Despite current advances in bone tissue engineering strategies, therapeutic tools for bone reconstruction still face serious limitations including risks of infection, rejection, costs and limited supplies [152,153]. Drawbacks of autografts include the need for separate incisions at the harvesting site, increased operative period, blood loss and post-operative pain at the donor site [154], while allografts present delayed vascular penetration, delayed or incomplete graft incorporation, immune rejection and disease transmission [5,154]. In case of natural polymers, their applicative limitations include low mechanical strength and structural complexity resulting in varied scaffold matrices [5]. Limitations of synthetic polymers include lack of osteoconductivity [5] as well as risk of inducing inflammation and cytotoxicity [155–157]. Additionally the accumulation of high concentrations of nano or sub-mirco sized wear particles have been reported to incite immune response, ultimately causing loosening and failure of the implant. Another major hurdle in bone engineering strategies is the lack of vascularization within engineered bone constructs which results in poor implant integration and survival [5,158,159]. Slow penetration of host blood vessels into large engineered tissue grafts resulting in necrosis of the graft central region has been attributed as the main reason for the eventual failure of these implants [160]. Strategies for improving vascularization in Bone tissue engineering Biomaterial scaffolds in bone tissue engineering serve as templates for the establishment of the vascular system and bone-forming cell growth [160]. Lack of de novo tissue growth, insufficient nutrient supply and poor waste removal in 3D scaffolds are some of the limitations still hampering successful bone engineering and transplantation. The efficacy of the scaffolds for successful bone regeneration critically depends on their ability to induce and support vascular infiltration. Various studies have demonstrated that the application of a free-flap over the fractured area significantly improved fracture healing by providing a suitable microenvironment as well as a supply of undamaged well vascularized soft tissue, thereby highlighting the importance of vascularization during wound repair [134,161]. Thus optimal bone scaffolds, in addition to being able to withstand the forces exerted on the bone, also need to possess a sufficiently porous internal interconnecting microarchitecture that will promote cell growth [162]. Recent developments when designing bioengineered scaffolds to address this problem include ‘bioactive’ coating of implants with cells (osteoprogenitor cells) and/or incorporation pro-angiogenic factors in scaffold composites prior to transplantation. Incorporation pro-angiogenic factors in scaffolds The close association between angiogenesis and osteogenesis, makes angiogenic growth factors that are implicated in both neovascularization and endochondral ossification, important therapeutic agents for bone regeneration. The ability of pro-angiogenic factors like VEGF, FGF-2, BMP-2 and BMP-7 to accelerate fracture repair when administered exogenously is well established [45,46,67,70,89,99,100]. For example injecting FGF-2 into larger

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animal models was reported to significantly increase the callus area, bone mineral content, and biomechanical strength [164–167]. In contrast, Tomanek et al. [168] demonstrated that bolus injection of VEGF induced inappropriate neovascularization in avascular areas while FGF injections initially enhanced blood vessel growth, but this effect was transient and disappeared in later stages. The main advantage of incorporating or entrapping bioactive factors within biodegradable scaffolds is that this method facilitates their slow release over a longer duration, thereby prolonging their effect in aiding implant integration with host cells. Both Formiga et al. [160] and Kaigler et al. [169] reported that incorporation and sustained release of VEGF from scaffolds enhanced neovascularization and bone regeneration. Similarly other studies also observed increased neovascularization following implantation of 45S5 bioactive glass coated VEGF-releasing biodegradable scaffolds in their respective animal models [170–172]. Furthermore Jabbarzadeh et al. [173] showed enhanced and uniformed growth of EC on implanted VEGF incorporated scaffolds. Similarly both Vogelin et al. [161] and Chu et al. [174] reported that load bearing BMP-2 scaffolds enhanced bone regeneration and maintenance of bone length in critical sized defects. Solorio et al. [175] demonstrated that encapsulating BMP2 in gelatin microparticles induced MSC mediated bone formation. While Chen et al. [163] reported that treatment with gelatin hydrogel incorporated with FGF2 increased bone mineral density and cancellous bone, at 4 and 8 weeks post injury respectively. Similarly, both Moya et al. [176] and Uriel et al. [177] observed increased vessel invasion and sustained vascular network formation in collagen scaffolds impregnated with FGF micro-beads. Improved mechanical strength as well as increased callus density and volume was also reported following local administration of PDGF on collagen gels [178]. As bone regeneration involves interactions between multiple growth factors and cytokines [179], the delivery and isolated action of a single growth factor is not enough to emulate the complex process of bone regeneration. Researchers are now investigating the benefits of delivering a combination of growth factors. Numerous studies have shown the synergistic effects of increased bone formation in vivo following the dual delivery of BMP-2/7 and TGF-b, in contrast to the negligible bone formation following individual delivery of either growth factor [180–183]. Theoretically the ideal combination of growth factors should simultaneously stimulate both angiogenesis and osteogenesis enhancing vascularization in the tissue constructs. In order to facilitate this process researchers are now looking at delivering VEGF, the central molecule in angiogenesis along with other growth factors. Richardson et al. [184] found that sustained dual delivery of both VEGF and PDGF resulted in highly dense and well established vessels; Other studies have reported that the combined delivery of VEGF and BMP-2 promoted both blood vessel formation as well as osteogenesis [185– 187]. Due to their effect on osteogenic differentiation, when strategizing delivery of growth factor combinations it is important to carefully consider not only the correct combinations but also the time of their delivery i.e. simultaneous or sequential. This point was emphasized by two studies demonstrating that the early release of BMP-2 followed by BMP-7 suppressed rat MSC proliferation and increased osteogenic differentiation [188,189]. Determining the correct dosage of growth factor to be delivered by the impregnated scaffold is another critical aspect that must be considered when designing these delivery scaffolds. Experimental data has shown that treatment with high doses of VEGF and FGF resulted in malformed, leaky blood vessels with poor long-term stability [190–192]. Furthermore it is imperative to consider that treatment with angiogenic growth factors can exacerbate pathological effects of angiogenesis including tumor development, atheroscleosis, and proliferative retinopathies [193].

‘Biocoating’ of scaffolds Generating vascularized engineered bone tissue constructs by culturing MSCs or co-culturing ECs and bone cells in scaffolds presents another approach to simultaneously promote osteogenesis and vascularization [194–196]. ECs are well established to play a key role in angiogenesis, thus enhancing EC migration into the matrix to develop vascular beds is critical for the survival of implanted bone constructs. Schechner et al. [197] observed that in vivo implantation of primitive vascular networks derived from ECs remained immature and did not survive. However Koike et al. [198] reported that co-culturing of ECs and perivascular cell precursors in engineered constructs enhanced the development of long lasting stable microvessels in vivo. Similarly Levenberg et al. [199] showed that tri-culturing of ECs, myoblasts and embryonic fibroblasts lead to the development of higher density vascular networks when compared to co-cultured controls. Interestingly increased VEGF levels was identified as the factor responsible for the increased vessel density observed [199]. Holder et al. [200] following seeding aortic ECs in porous matrices reported organized/ unorganized EC within matrix as well as increased capillaries and lymphatic like structures. Apart from angiogenesis, EC has recently been reported to stimulate osteogenesis thereby enhancing bone regeneration [201,202]. It is well known that cellular interaction between osteoblasts and ECs is essential in bone formation. This was confirmed by Wang et al. [203] who reported increased cell number and VEGF expression in co-cultures of human osteoblastlike cells (HOBs) and human umbilical vein endothelial cells (HUVECs), but no increase in HOBs monocultures. Other studies have demonstrated that co-culturing MSCs and ECs results in vascularized bone formation, as ECs promotes MSCs osteogenesis and accelerated local vascularization [204,205]. Other powerful candidate cell types for bone regeneration include EPCs and MSCs [206], with the former being critical regulators of the angiogenic switch that differentiate into ECs while the latter promotes bone regeneration by differentiating into osteoblasts [45,144,207–211]. Numerous studies have shown that in vitro pre-vascularization of bone grafts with EPC is a promising strategy in improving implant survival. Lee et al. observed increased EPC mobilization towards the bone injury site in both their mice and rat models [142]. Additionally EPCs seeded grafts have been shown to significantly improve in vivo vascularization when compared to MSCs seeded and control grafts respectively [61,142,206–208,212–215]. Although the formation of a primitive vascular plexus was observed with MSCs seeded grafts, highest amounts of vascularization was seen when EPCs and MSCs were co-cultured together suggesting a synergistic effect [56,207,208, 212,213,215,216]. It is possible that as EPC and MSC contribute to bone regeneration via different but complementary pathways, their co-transplantation on tissue-engineered bone constructs facilitates osteogenesis due to better vascularization of the graft. Other studies have shown that implanting BMP2 modified MSCs scaffolds promoted bone regeneration [217–219], even if these strategies were unable to successfully heal the defects due to insufficient blood supply [220,221]. He et al. [2] attempted to resolve this problem by combining BMP2 modified MSCs and EPCs on their scaffolds to promote vascularized bone regeneration. They observed increased blood vessel density within groups with EPCs as opposed to groups without EPCs, which showed reduced vascularization and bone formation. Transplanted EPCs have been reported to stimulate vascularization by releasing VEGF to recruit host EPCs and MSCs migration into the repair site, allowing for greater availability of nutrients and molecular factors involved in the bone healing process [2,56,142]. Thus these experimental data suggest that initial neovascularization by EPCs is vital for the

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completion of bone regeneration at later phases [61,142,144,207, 212,213]. However, the therapeutic application of EPC transplantation is mainly limited due to the scarcity of these progenitor cells [208]. Various studies have proposed to resolve this issue by genetically modifying transplanted EPCs and MSCs with factors like BMP2 and VEGF, which are essential to osteo-endothelial communication [166,128,221]. It is well known that BMP2, apart from stimulating MSC to differentiate into osteoblasts also increases VEGF expression which in turn stimulates EPC differentiation into endothelial cells and vascularization [222]. Song et al. [223] demonstrated that co-culturing VEGF-modified EPCs and BMP2-modified MSCs accelerated osteogenesis and bone formation by promoting vascularization in vivo. Similarly, He et al. [2] demonstrated that transfecting BMP2 into EPCs and MSCs significantly improved blood vessel and ectopic bone formation in defects when compared to control groups. Furthermore other studies have shown that VEGF up-regulates Id1 gene expression which in turn positively influences EPC functioning, while the loss of Id1 resulted in complete loss of EPC populations and subsequent prevention of neovascularization [224,225]. Gene therapy The application of gene therapy as a mean to deliver growth factors for the clinical management of orthopedic disorders is another promising area in the field of bone tissue engineering [226]. The transference of genetic material can be performed by either in vivo or ex vivo gene-transfer procedure, implemented via viral (transfection) or non-viral (transduction) vectors [227,228]. The in vivo technique where the genetic material is directly transferred to the host is generally the easier of the two methods, whereas the indirect ex vivo technique involves in vitro harvesting and genetic modification of cells before their transfer back to the host. However the former raises safety concerns, while the latter is deemed safer as it allows for both the selection of greatest expressing cells as well as the screening of cells depicting abnormal behavior prior to re-implantation [226]. Schechner et al. [197] observed that the addition of anti-apoptotic Bcl2 gene into the EC resulted in the survival, formation and differentiation of the primitive structures into arteries, veins and capillaries. Similarly Yang et al. [228] showed that retroviral-mediated transduction of hTERT in EC resulted in the development of microvascular structures on implantation. Tarkka et al. [99] reported that adenoviral vector delivery of VEGF in mouse femur defects induced angiogenesis, improved bone healing and bone mineral content. While Ito et al. [229] showed increased remodeling and vascularization in post-implanted allografts coated with recombinant adeno-associated virus (rAAV) encoding RANKL or VEGF. Gene therapy has also been used to promote fracture repair through expression of BMP-2 [2,222,230,231] and -4 [232] in animal models. Additionally Lieberman et al. [233] reported that the endogenous BMP production by transfected cells placed on fracture site was more efficient than exogenous delivery of the recombinant protein. Although gene therapy poses as a promising strategy, issues relating to oncogenic risk associated with genetic manipulation, biosafety and efficacy of the procedure need to be resolved before human trials can take place. Conclusions In conclusion, while much of the current strategies for fabricating functional vascularized bone grafts are still in their infancy, experimental data offers great potential for their future application in treatment of bone repair. However, in order to accelerate our progress in the field it is imperative to obtain a better understanding of bone wound microenvironment, which is the home of

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