Vascularization of engineered musculoskeletal tissues

Vascularization of engineered musculoskeletal tissues

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J. Lim1, M. Chong1, Y. Liu1, A. Khademhosseini2,3,4, S.H. Teoh1 1Division of Bioengineering, Nanyang Technological University, Singapore; 2Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA; 3Biomaterials Innovation Research Center, Division of Biomedical Engineering, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Cambridge, MA, USA; 4Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, USA

11.1  Introduction Tissue-engineering strategies are believed to be the mainstay of tissue replacement in the future, and efforts to create reliable, reproducible off-the-shelf and customized substitutes are ongoing. Musculoskeletal tissues possess extensive vasculature in the form of nutrient arteries, periosteal vessels, and epiphyseal vessels. As such, the design of tissue-engineered products need to include sufficient vascularity. Current research has already established that adequate vascularization of the engineered graft is a key determinant of the clinical outcome of the implanted bone substitute (Liu, Lim, & Teoh, 2013). More specifically, the success of current clinically tested tissueengineered bone constructs is dependent on sufficient vascularization (see Chapter 5). To this end, this chapter aims to provide an overview of current strategies to engineer vascularized musculoskeletal tissues, and is organized along the central tenets of tissue-engineering: scaffolds, cells, growth factors (GFs), and bioreactors.

11.2  Lack of vascularization remains a bottleneck in tissue-engineering Mass transfer issues limit the size to which tissue can grow without the presence of adequate vascularization, with theoretical estimates suggesting 100–200 μm to be the maximum thickness for which sufficient nutrient and metabolite exchange may occur. Accordingly, the presence of an intricate intraosseous vasculature points toward the importance of blood vessels in maintaining the viability and metabolism of bone tissue (Santos & Reis, 2010). We have previously summarized the experimental evidence and clinical observations demonstrating the importance of vascularization in the field of bone-tissue-engineering (Liu, Lim, et al., 2013). In particular, avascularity has been suggested to be the cause of lack of callus formation or bone growth, as well as resorption at bone endings. Lafage-Proust, Prisby, Roche, and Vico (2010) go further Regenerative Engineering of Musculoskeletal Tissues and Interfaces. http://dx.doi.org/10.1016/B978-1-78242-301-0.00011-2 Copyright © 2015 Elsevier Ltd. All rights reserved.

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to say that “the bone vessel is at the center of the bone remodeling unit and hematopoietic stem cell niche.” On a similar note, significant research efforts in skeletal muscle tissue-engineering centers around providing sufficient vascularity to the engineered construct, to overcome diffusion limits (Bach et al., 2006). It follows that establishing viable vasculature is a key determinant in the clinical success of tissue-engineered grafts. Strategies that have been developed to promote graft vascularization may be organized along those targeting scaffold design, use of biological cues, use of mechanical cues, and /or use of chemical cues, respectively; these strategies shall be discussed in the ensuing sections of this chapter.

11.3  Biomaterial selection and microfabrication 11.3.1  Biomaterial selection and surface modification The basic requirements of scaffolds intended for use in musculoskeletal tissue-engineering include the ability to induce (1) cell adhesion, (2) proliferation, (3) migration, (4) differentiation, and (5) mineralization (McCullen, Chow, & Stevens, 2011; Nguyen et al., 2012). In addition to these, scaffolds must meet an additional requirement: to be able to support vascularization, particularly in clinical avascular non-unions (Tseng, Lee, & Reddi, 2008). Metals (Simon, Ricci, & Di Cesare, 1997; Zhang, Hamamura, & Yokota, 2008), ceramics (Hämmerle et al., 1997; Hollinger & Battistone, 1986), and polymers (Agrawal & Athanasiou, 1997; Hosseini et al., 2012; Hutmacher et al., 2001; Lam, Hutmacher, Schantz, Woodruff, & Teoh, 2009; Lim et al., 2013) have all been studied for musculoskeletal tissue-engineering applications, with polymers appearing to be a popular choice for scaffolds due to their ease of fabrication and versatility (McCullen et al., 2011). See Chapter 1 for a comprehensive discussion on biodegradable polymers as biomaterials for musculoskeletal tissue-engineering. The ability of various polymers to be tuned in terms of their physicochemical properties is particularly important to provide appropriate substrate stiffness for supporting vascularization and tissue regeneration. This will be discussed in greater detail later in hydrogel applications (Bae et al., 2014; Chen et al., 2012; Nikkhah, Edalat, Manoucheri, & Khademhosseini, 2012; Nikkhah, Eshak, et al., 2012; Seidi, Ramalingam, Elloumi-Hannachi, Ostrovidov, & Khademhosseini, 2011; Xiao et al., 2011). More relevant to bone tissue, it is apparent from a materials standpoint that one would be hard-pressed to identify an ideal candidate material from a single material class capable of meeting these myriad requirements. Consequently, composites (Bernstein et al., 2010; Rakovsky, Gotman, Rabkin, & Gutmanas, 2013; Yeo, Wong, Khoo, & Teoh, 2010) are increasingly employed to achieve a suitable blend of desirable characteristics. Specific to the problem of vascularization, scaffold porosity, pore size, and physicochemical properties should be optimized. It is generally well accepted that vascular infiltration is highly dependent on porosity (Hollister, 2005; Pilliar, Lee, & Maniatopoulos, 1986; Van Tienen et al., 2002), with some groups studying a range of porosities (Clark, Milbrandt, Hilt, & Puleo, 2014; DiRienzo et al., 2014; Hutmacher et al., 2001; Salgado, Coutinho, & Reis, 2004; Williams et al., 2005) to optimize vascular ingrowth. Through introduction of pores, Tabata et al. showed that vascularization into a poly(vinyl alcohol) (PVA) sponge could be achieved (Tabata, Miyao, Yamamoto, & Ikada, 1999), whereas

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Figure 11.1  (a) Three-dimensionally fabricated PCL-TCP scaffolds facilitated the formation of microvascular networks in a murine model (red: host-derived murine CD31-positive vessels). (Images were adapted with permission from Liu, Y.C., Teoh, S.H., Chong, M.S.K., Lee, E.S.M., Mattar, C.N.Z., Randhawa, N.K., et al. (2012). Vasculogenic and osteogenesis-enhancing potential of human umbilical cord blood endothelial colony-forming cells. Stem Cells, 30, 1911–1924.) (b) Representative 3D confocal images displayed cytoskeletal organization of HUVECs within GelMA microconstructs. Scale bar represents 100 mm. (Images were adapted with permission from Nikkhah, M., Eshak, N., Zorlutuna, P., Annabi, N., Castello, M., Kim, K., et al. (2012). Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels. Biomaterials.) (c) Formation of lumens in GelMA microconstructs with ECFCs and MSCs in a heterotypic coculture. Scale bar represents 20 μm. (Images were adapted with permission from Chen, Y.C., Lin, R.Z., Qi, H., Yang, Y., Bae, H., Melero-Martin, J.M., et al. (2012). Functional human vascular network generated in photocrosslinkable gelatin methacrylate hydrogels. Advanced Functional Materials, 22, 2027–2039.)

Wray et al. showed that the incorporation of microchannels into a porous silk scaffold facilitated mature microvascular networks with endothelialized lumens (Wray, Tsioris, Gi, Omenetto, & Kaplan, 2013). In a similar fashion, Liu et al. have shown that threedimensional (3D)-printed polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds with well-defined, interconnected pore structures allow the formation of extensive microvascular networks in vivo following implantation in a murine model (Figure 11.1(a)) (Liu, Teoh, et al., 2012). These networks were found to promote survivability and differentiation capacity of implanted cellular grafts. Pore size and distribution may have a further role to play in determining vascularization events (Bai et al., 2010, 2011; Chiu et al., 2011; Klenke et al., 2008; Marshall, Barker, Sage, Hauch, & Ratner, 2004, p. 710). Klenke et al. studied the effect of pore size in biphasic calcium phosphate particles and concluded that pore sizes exceeding 140 μm had significantly higher functional capillary density as opposed to those smaller than 140 μm (Klenke et al., 2008). Chiu et al. attained a similar conclusion working with poly(ethylene glycol) (PEG) hydrogels, reporting that larger pores permitted mature vascularization (Chiu et al., 2011). These evidences demonstrate the importance of pore structures in vascularization, and the reliability of creating such intricate structures thus forms the focus of the next section.

11.3.2  Microfabrication for the generation of capillaries Traditionally, techniques such as low-pressure forming (Chung, Sugimoto, Koh, &Ameer, 2012), electrospinning (Dargaville et al., 2013; Hasan et al., 2014), fused-deposition modeling (Zhang, Teoh, Chong, et al., 2010), and salt leaching (Hu et al., 2013) have

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been used to fabricate scaffolds with microarchitecture. However, these methods typically result in random structures, ceding control over the pore size and consequently more stochastic outcomes. The Khademhosseini team has been actively developing various microfabrication techniques (Bae et al., 2014; Mihaila et al., 2013; Ostrovidov, Seidi, Ahadian, Ramalingam, & Khademhosseini, 2013, pp. 52–79; Ramalingam, Jabbari, Ramakrishna, & Khademhosseini, 2013; Schukur, Zorlutuna, Cha, Bae, & Khademhosseini, 2013; Shin et al., 2013; Zhang et al., 2013) for various tissueengineering applications, including that of facilitating vascularization within scaffolds. By conjugating the well-investigated arginine-glycine-aspartic acid (RGD) peptide in PEG hydrogels (Gobin & West, 2002), endothelial cell (EC) differentiation from embryonic stem cells was accelerated by three to five days, potentiating its use for tissue-engineering applications, including that of creating a prevascularized construct. Hyperbranched polyesters functionalized with acrylate groups (Zhang et al., 2013) were shown to be capable of various pore sizes with tunable mechanical properties. In a separate in vivo study, Bai et al. were able to induce vascularization of TCP scaffolds with micropores, and concluded that pore sizes beyond 400 μm resulted in better vascularization following radiological assessment (Bai et al., 2011). Micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical topographies were investigated for 3D endothelial cord formation by Nikkah et al. (Nikkhah, Eshak, et al., 2012). Results suggested that micron-sized topographies were able to align and organize human umbilical vein ECs (HUVECs), forming cord structures that were circular and epithelial in cross-section. Interestingly, 100 μm constructs provided the optimal microenvironment to form stable structures (Figure 11.1(b)). GelMA hydrogels fabricated by varying the degrees of methacrylation resulted in controllable compressive moduli and pore sizes, which appeared to play a role in determining the number of branches and branch points and the average length of capillaries formed (Chen et al., 2012). These results potentiate the application of GelMA hydrogels in the biomedical field, and in the construction of complex engineering tissues (Figure 11.1(c)). The aim of such microfabrication techniques is focused on guiding the direction of cellular growth and migration, and has been shown useful for targeted vascular infiltration. The next section will focus on the use of biological components, namely cells and soluble factors.

11.4  Cells and growth factors 11.4.1  Vasculogenic and angiogenic cells in coculture models In the traditional approach to engineering musculoskeletal tissue, bone marrow-derived mesenchymal stem cells (bMSCs) were used in monoculture systems. Although highly mineralized bony constructs can be generated in vitro using such techniques, concerns have similarly emerged over the lack of vasculature, and consequently, compromised viability and functional engraftment of such constructs post implantation. To address this need, cocultures involving the inclusion of vasculogenic/angiogenic cells were thus studied. A variety of vasculogenic/angiogenic cells spanning across various sources

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have been identified: cells from an endothelial lineage, such as endothelial progenitor cells (EPCs), ECs, and pericytes, all of which have high potential for vessel formation, have been the subject of widespread investigation. By introducing a heterogeneous cellular population that is native to physiology, heterotypic interactions between endogenous cell populations are encouraged, stimulating paracrine and autocrine interactions that subsequently lead to increased vascular infiltration, as well as de novo vessel formation. Consequently, a gradual shift in research focus to heterocellular cocultures has been observed, with some of the most impactful work focusing on heterogeneous interactions between endogenous cells (Aguirre, Planell, & Engel, 2010; Chen et al., 2012; Fuchs, Ghanaati, et al., 2009; Fuchs, Jiang, et al., 2009; Grellier, Bordenave, & Amédée, 2009; Kirkpatrick, Fuchs, & Unger, 2011; Liu, Chan, & Teoh, 2012; Liu, Lim, et al., 2013; Liu, Teoh, et al., 2013; Rouwkema, Rivron, & van Blitterswijk, 2008; Santos, Unger, Sousa, Reis, & Kirkpatrick, 2009). Our discussion continues with an overview of vasculogenic/angiongenic cells (Table 11.1) used to support vascularization strategies, with a focus on endothelial type cells, and introducing other cell types that reportedly are able to stimulate angiogenesis. We begin by discussing one of the most widely used ECs at present: circulating EPCs. EPCs were first described by Asahara et al. in 1997 (Asahara et al., 1997), in which hemopoietic progenitors were prospectively isolated from human peripheral blood (PB), and shown capable of generating endothelial progeny. More strikingly, these cells were shown able to rescue tissue ischemia in a murine model, suggesting the utility of EPCs as a highly potent angiogenic and vasculogenic cell source (Asahara et al., 1997; Carano & Filvaroff, 2003; Hankenson, Dishowitz, Gray, & Schenker, 2011; Kalka et al., 2000; Kocher et al., 2001; Lee et al., 2008, 2010; Li & Wang, 2013; Lin, Weisdorf, Solovey, & Hebbel, 2000; Matsumoto et al., 2008; Murohara et al., 2000; Papathanasopoulos & Giannoudis, 2008; Peichev et al., 2000; Reyes et al., 2002). EPCs have since been isolated from a variety of sources (Hill et al., 2003), including circulating bone marrow, umbilical cord blood, and even placenta (Patel et al., 2013). Despite significant debate on the exact identity of these cells, EPC populations have been studied extensively for bone regeneration. Matsumoto et al. (2006) reported in 2006 that human PB CD34 + cells played an important role Table 11.1  Various

sources of endothelial cells for the stimulating

angiogenesis Endothelial cell type

References

Human embryonic ECs Human umbilical vein endothelial cells (HUVECs)

Levenberg et al. (2005) Levenberg et al. (2005) Rouwkema et al. (2006, 2008) Verseijden et al. (2009) Liu, Teoh, et al. (2012), Liu, Teoh, et al. (2013), Liu, Chan, et al. (2012) Groeber et al. (2013)

Endothelial progenitor cells (EPCs) Human dermal microvascular ECs

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in creating a favorable microenvironment for stimulating both angiogenesis and osteogenesis, and contributed significantly to fracture healing postinjection. Our group and others have demonstrated that by coculturing MSCs with umbilical cord blood-derived endothelial colony-forming cells (UCB-ECFC; a subpopulation of blood-derived outgrowth cells containing EPC), extensive endothelial networks could be formed in vitro. Following implantation in mice, the thus-engineered bone grafts were further seen to be highly perfused by a chimeric vascular network containing ECs of both human and murine origin (Liu, Teoh, et al., 2012). Additionally, both angiogenic and osteogenic gene expression levels were significantly higher in UCB-ECFC/MSC coculture than in either monoculture, providing further evidence of critical cross talk during tissue morphogenesis and supporting the use of such cocultures. In a similar study, a differential analysis was performed between the PB-derived EPCs and bone marrow-derived EPCs when EPCs were cocultured with MSCs. The results demonstrated superiority of the PB-derived EPCs in terms of their synergy with MSCs for enhanced osteogenesis and angiogenesis (Amini, Laurencin, & Nukavarapu, 2012; Nukavarapu & Amini, 2011). Presently, many ongoing clinical trials involve the use of EPCs for the treatment of various diseases, suggesting the potential of EPCs for future clinical application in the field of musculoskeletal regeneration (Table 11.2) (ClinicalTrials.gov). More commonly, however, mature ECs have been studied for similar applications. Common sources include umbilical cords (Kachgal, Carrion, Janson, & Putnam, 2012; Villars et al., 2002), dermal tissue, or clinically harvested vessels, including the Table 11.2 

Current ongoing clinical trials in relation to the usage of EPCs for regenerative medicine Region

Country/Number of trials

East Asia

China/3 Japan/1 Taiwan/2 Austria/4 France/7 Germany/2 Italy/5 Latvia/1 Netherlands/1 Spain/2 Switzerland/1 Israel/1 Canada (all states)/2 United States of America (all states)/18 Brazil/1 Malaysia/1 Singapore/1

Europe

Middle East North America South America Southeast Asia

Data retrieved from clinicaltrials.gov, correct as of January 2014.

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saphenous vein (Hendrickx, Vranckx, & Luttun, 2010). Criswell et al. have recently showed that the interplay between ECs and muscle progenitor cells (MPCs) resulted in enhanced vascularization when implanted in vivo in a subcutaneous mouse model (Criswell et al., 2013). One of the potential mechanisms elucidated was the secretion of vascular endothelial growth factor (VEGF), which had a two-fold effect on promoting MPC migration and inhibiting apoptosis, and also that of encouraging angiogenesis (Yuen, Du, Chan, Silva, & Mooney, 2010). Few studies have, however, compared the efficacy of EPCs against ECs as vasculogenic agents, and the ideal conditions for coculture remain to be elucidated.

11.4.2  Prevascularization strategies The objective of prevascularization is to create vascular networks that may quickly anastomose to the host circulatory system following implantation, and many studies have already shown that this may be done (Hutton & Grayson, 2014; Lesman et al., 2011; Singh, Wu, & Dunn, 2011; Unger et al., 2010). In a landmark study by Levenberg et al. in 2005 (Levenberg et al., 2005), cocultured ECs, myoblasts, and embryonic fibroblasts stimulated extensive prevascular-like networks over one month. The formation of prevascular networks was primarily reasoned as due to the elevated expression levels of VEGF. Importantly, subsequent in vivo evaluation in various models resulted in improved vascularization, blood perfusion, and viability of the tissue-engineered prevascular constructs at the site of implantation. Just a year later, Rouwkema et al. described the formation of prevascular networks after coculturing hMSCs and HUVECs (98:2%) for 10 days, suggesting a higher efficiency of prevascular network formation. In addition, the authors noted that the addition of 2% HUVECs was sufficiently able to stimulate the formation of prevascular networks, although the formation of a lumen was not observed, indicating that the vessel structures were not mature. Despite this, the vascular network was maintained after implantation into nude mice, with histological analysis pointing toward the formation of lumen (Rouwkema, Boer, & Blitterswijk, 2006). Amini et al. developed oxygen tension-controlled scaffolds that support bone- and vessel-forming cell survival throughout the scaffold pore structure to support vascularized bone regeneration in large areas for the repair and regeneration of critical-sized bone defects (Amini & Nukavarapu, 2014; Amini, Adams, Laurencin, & Nukavarapu, 2012). Taken together, this is indicative that a prevascularized strategy may help augment current vascularization strategies in musculoskeletal tissue-engineering. Kirkpatrick et al. (2011) recently published a concise and informative review on coculture systems for vascularization, and in it elucidated an important mechanism of bidirectional stimulation for osteogenesis and angiogenesis, citing the potential effect of VEGF in this process. Interestingly, it was also mentioned that endothelial monocultures did not provide measurable levels of VEGF secretion, whereas EC-osteoblast coculture systems actually produced significantly higher amounts of measurable VEGF (Santos et al., 2009). From there, additional amounts of GFs may initiate a refractory state of angiogenesis, inhibiting angiogenic activity rather than promoting (Santos et al., 2009). Apart from the bidirectional stimulatory effect and reciprocal signaling mechanisms, Verseijden et al. also proposed the possibility of cell–cell contact (Verseijden et al., 2009) as a

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potential mechanism for stimulating angiogenesis. They observed that conditioned media from bone marrow MSCs did not stimulate endothelial outgrowth, whereas adipose-derived stem cells (ASCs) did. Interestingly, when either of them was cocultured together with HUVECs, both coculture models developed prevascularized networks, suggesting that other than soluble trophic factors as earlier mentioned, direct cell–cell contact may have played a part in stimulating endothelial outgrowth into prevascular-like networks. In summary, the prevascular networks may be readily generated in vitro through the application of heterocellular cocultures, with the provision of permissive conditions of appropriate mechanical support. Soluble factors were found key in this process, and we now turn our attention to discuss the use of angiogenic GFs to promote vascularization.

11.4.3  Use of angiogenic growth factors In the preceding section, we discussed the stimulatory effects of soluble trophic factors consequently producing prevascular-like networks, mentioning on more than one occasion that VEGF was one of these factors. Here, we discuss the direct incorporation of this and other soluble trophic factors, otherwise known as growth factors (GFs), as an alternative strategy for generating vascularized musculoskeletal tissues. GFs are a known, potent source of biomolecules that can stimulate vascular growth, with VEGF and members of the FGF family understood to promote EC migration and proliferation, whereas platelet-derived growth factor (PDGF), TGF-β1, and Angiopoietins play a role in the recruitment of pericytes for the formation of stable vasculature (Harris, Rutledge, Cheng, Blanchette, & Jabbarzadeh, 2013). It was also mentioned that angiogenesis is an intimate interplay between both pro- and antiangiogenic factors, and the identification of some of these factors have been detailed in Ballara, Miotla, and Paleolog (1999). A search of the literature over the past few years indicated that VEGF and fibroblast growth factor (FGF), along with combinatorial approaches with Angiopoietins, have been widely studied. Aside from the absolute concentration and proportion, spatiotemporal considerations may also be critical, leading to suggestions that GF delivery within engineered tissues should be timed (Guldberg, 2009). This idea was supported by clinical studies in 2003 (Masquelet, 2003) and 2009 (Biau, Pannier, Masquelet, & Glorion, 2009) using two-stage procedures for the treatment of bone defects. In one case (Biau et al., 2009), a 12-year-old child with Ewing’s sarcoma underwent a two-stage procedure: initial removal of the tumor followed by the implantation of a cement spacer stabilized with a locked intramedullary nail; then autologous bone grafting seven months later. Results showed that osseointegration was permitted, with radiographic evidence suggesting integration with host bone and cortical reconstruction. Among the panel of GFs, VEGF is a commonly studied GF due to the propensity for both MPCs and osteoblasts/MSCs (Lafage-Proust et al., 2010) to secrete VEGF, and importantly, have receptors for VEGF. As such, the secretion of VEGF has a two-fold function: to encourage angiogenesis, and to stimulate musculoskeletal progenitor cell differentiation, or otherwise known as osteogenic/myogenic reciprocity between these two cell populations (Matsumoto et al., 2008). A critical limitation of VEGF is its very short half-life of 90 min (Lazarous et al., 1996) in vivo, because sustained expression

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is necessary for up to four weeks to establish mature vasculature (Dor et al., 2002; Helmrich et al., 2013; Ozawa et al., 2004; Tafuro et al., 2009). As such, there has been rising interest in developing different platforms and strategies for the sustained delivery of VEGF (Figure 11.2). Helmrich et al. (2013) described the augmentation of bMSCs to constitutively express VEGF to improve angiogenesis and bone formation in vivo. In an attempt to mimic clinical cellular transplantation, the isolated bMSCs from human donors were aggressively expanded in vitro (Passage 7, 30–35 population doublings) and subsequently engineered to overexpress VEGF (efficiency of 90% (Helmrich et al., 2011)). These modified bMSCs were then implanted into rats and shown to induce the formation of extensive microvascular networks with morphology akin to that of stable capillaries (Figure 11.3(a)–(c)). They also observed the formation of a few arterioles of larger diameter, demonstrating further that a hierarchical vascular bed could be formed (Figure 11.3(c), white arrows). Silva and Mooney (2010) provided their opinion on VEGF temporal and spatial effects, using a previously described method (Silva & Mooney, 2007) to produce an injectable alginate hydrogel system for the sustained delivery of VEGF over 30 days. Implantation of this system in ischemic murine models led to a robust and potent angiogenic effect (Figure 11.3(d)–(f)). In an alternative strategy, Leslie-Barbick, Saik, Gould, Dickinson, and West (2011) developed a synthetic amino peptide that has similar biological activity to that of VEGF, while having a lower likelihood of triggering immune response. Additionally, it allows customization to achieve a biomimetic matrix system.

Figure 11.2  General interest in the sustained delivery of angiogenic growth factors (information collected as of Scopus, January 2014).

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Figure 11.3  Immunofluorescence staining for the endothelium: CD31 (green), pericytes: NG2 (red) and smooth muscle cells: smooth muscle actin (blue) in naive bMSCs (a), control CD8-bMSCs (b) and NG2-positive pericytes (c). Overexpression of VEGF in NG2-positive pericytes resulted in the formation of hierarchical vascular bed. Scale bar represents 50 μm. (Images adapted with permission from Helmrich, U., Di Maggio, N., Güven, S., Groppa, E., Melly, L., Largo, R.D., et al. (2013). Osteogenic graft vascularization and bone resorption by VEGF-expressing human mesenchymal progenitors. Biomaterials, 34, 5025–5035.) VEGF-expression led to significant recovery of blood flow in a hindlimb ischemia murine model (d–f). (Images adapted with permission from Silva, E.A., Mooney, D.J. (2010). Effects of VEGF temporal and spatial presentation on angiogenesis. Biomaterials, 31, 1235–1241.)

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In vivo, ischemic environments are known to express potent signals to stimulate angiogenesis. In 1998, it was reported that the hypoxia-inducible factor (HIF) pathway may stimulate angiogenesis from a hierarchically higher position (Carmeliet et al., 1998). Clear upregulation of vessel formation were seen in the HIF-1α groups, with corresponding expression of VEGF as validated by polymerase chain reaction (PCR) results. Vascular density was also found to be higher in the HIF-1α positive groups. However, HIF-1α is prone to degradation under normoxic conditions, making in vitro studies challenging. A small molecule inhibitor known as prolyl hydroxylase (PHD) has been shown to inhibit the degradation of HIF-1α, consequently activating the translocation of HIF-1α to the cell nucleus for the activation of target genes, one of them being VEGF. By incorporating PHD inhibitors in various concentrations in a murine model, higher expression of HIF-1α was recorded, bringing along with it higher three- to seven-fold improvement in VEGF expression, and capillary sprouting (Shen et al., 2009). In a study by Borselli et al. (2010), they combined the delivery of VEGF and insulin growth factor-1 (IGF-1), to achieve parallel regeneration of skeletal muscle, angiogenesis, and re-innervation, after observing that individual delivery of either factor alone separately led to angiogenesis or myogenesis. In summary, the biological effects of cells and GFs in stimulating vessel formation have been described in this section, with proposed mechanisms justifying the use of these components either individually, or incorporated in scaffolds. Musculoskeletal tissues are constantly exposed to varying levels of stress and fluid shear, provoking the interest of the scientific community in the area of various forms of mechanical stimulation on vessel formation.

11.5  Bioreactor conditioning of engineered musculoskeletal tissue 11.5.1  Effect of mechanical (ambulatory, uniaxial straining) stimulation on angiogenesis The role of mechanical stimulation on musculoskeletal tissue regeneration is broadly covered in Chapter 4. The basis of mechanical stimulation on the musculoskeletal system was recently explained by Migliaccio, Wannenes, Lenzi, and Guidetti (2013). The effects of mechanical stimulation of vessel formation both in vitro and in vivo have been of interest to the scientific community over the last 25 years (McCormick et al., 2001; Sampath, Kukielka, Smith, Eskin, & McIntire, 1995; Tzima, del Pozo, Shattil, Chien, & Schwartz, 2001), with early evidence suggesting that fluid shear stress has a direct effect on vascular ECs (Resnick et al., 1993), leading to an upregulation of gene transcription factors that promote PDGF, a known angiogenic factor mentioned earlier (this will be discussed in greater detail). Several studies on imposed mechanical stimulation, such as cyclic strains on vasculature formation or neovascularization, followed (Liu, Ensenat, Wang, Schafer, & Durante, 2003; Sipkema, van der Linden, Westerhof, & Yin, 2003; Upchurch, Leopold, Welch, & Loscalzo, 1998). Matsumoto et al. showed that unidirectional

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stretching of HUVECs regulated EC migration, proliferation, and differentiation into immature vessels (Matsumoto et al., 2007). A modeling study by Geris et al. found good agreement with experimental results of enhanced angiogenesis and osteogenesis when bone-implant loading was altered (Geris et al., 2010). Groothuis et al. reported that hematomas that were exposed to mechanical stimulation had a higher incidence of endothelial tube formation, which was a consequence of VEGF production (Groothuis et al., 2010). With increasing evidence that mechanical strain plays a key role in stimulating angiogenesis, specific levels of cyclic strains began to be reported, such as that of 6% cyclic uniaxial strains in two-dimensional (2D) and 8% in 3D cultures of ECs (Yung, Chae, Buehler, Hunter, & Mooney, 2009). Strain of 2.5% was also reportedly able to increase production of proangiogenic factors, although a disruption in endothelial networks was also mentioned. Guldberg and team then hypothesized that vessel formation was dependent on the timely introduction of mechanical loading, which was studied in a rat model of large bone defect. Importantly, they reported that delayed mechanical stimulation resulted in the formation and quantity of larger blood vessels in the defect region (Figure 11.4(a)–(e)) (Boerckel, Uhrig, Willett, Huebsch, & Guldberg, 2011). F



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Figure 11.4  Early loading (compliant plates) impeded angiogenesis and vascular connectivity (a–e). Images reprinted with permission from Boerckel, J.D., Uhrig, B.A., Willett, N.J., Huebsch, N., Guldberg, R.E. (2011). Mechanical regulation of vascular growth and tissue regeneration in vivo. Proceedings of the National Academy of Sciences, 108, E674–E80.

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11.5.2  Mechanical influences We have previously discussed the role of mechanical forces like cyclic straining in vessel formation, and briefly mentioned fluid shear. Here we focus on how fluid shear has been able to induce vessel formation in engineered musculoskeletal tissues. The impact of fluid shear on vessel formation has been described many years ago (Baiguera & Ribatti, 2013; Li, 2005; McCormick et al., 2001; Sampath et al., 1995; Tzima et al., 2001). More recently, the field has been employing the understanding of fluid shear on vessel formation to use in bioreactor cultures. Recently, Nishi et al. employed the rotating-wall vessel (RWV) bioreactor for the coculture of MSC-derived ECs and MSCs for the generation of bone scaffolds. The low-shear conditions provided by the RWV bioreactor were sufficient to induce vascular-like structures (Nishi, Matsumoto, Dong, & Uemura, 2013). We have also contributed to the study of vessel formation in engineered bone tissue with the development of the biaxial bioreactor (Liu, Chan, et al., 2012; Liu, Teoh, et al., 2013; Zhang, Teoh, Chong, et al., 2010; Zhang, Teoh, Teo, et al., 2010). Through fluid shear stimulation and intimate interactions between MSCs and EPCs, Liu et al. have shown that a prevascularized bone construct with early neovasculogenesis could be achieved (Liu, Teoh, et al., 2013). Groeber et al. recently developed a bioreactor system that is capable of generating a pulsatile flow similar to the physiological vascular system. Fourteen days of culture with human dermal microvascular ECs led to the expression of specific endothelial markers like von Willebrand factor, suggesting the possibility of generating functional vasculatures for tissue-engineering applications (Groeber, Kahlig, Loff, Walles, & Hansmann, 2013). As presented, fluid shear forces have been shown to be able to induce vascularization from EC colonies. A noteworthy point in the evaluation of fluid shear stresses on vessel formation using bioreactors led to the discovery of hypoxia-induced angiogenesis.

11.5.3  Chemical influences Hypoxia, medically defined, is a condition in which tissues or organs are deprived of normal oxygen supplies. Clinically, it provides important information on the development of vessels, or angiogenesis. The early 2000s saw a sudden surge in interest, and it was generally understood then that, by creating a hypoxic region within a construct, endothelial tip cells are directed toward the low-oxygen region, leading to the commonly known phenomenon of sprouting (Germain, Monnot, Muller, & Eichmann, 2010). When MSC–EPC coculture was conducted in static conditions in a 3D scaffold, significant sprouting of EPCs leading to the formation of vascular-like networks was observed. The lack of nutrient transfer and oxygen transfer could have naturally created an oxygen gradient that ultimately led to the formation of these vascular-like networks, although a continued persistence in a hypoxic microenvironment eventually led to a compromise in terms of cellular viability, particularly in the center of the scaffold (Liu, Teoh, et al., 2013). Lu et al. also reported on the enhanced vascularization in vivo with treatment of hypoxia (13% oxygen) (Lu et al., 2013). In skeletal cells, hypoxia (2–5% oxygen) has been shown to influence the expression of various osteogenic markers (Genetos et al., 2010; Steinbrech et al., 2000; Tseng, Yang,

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Lai, & Tang, 2010), but more importantly, VEGF expression (Steinbrech et al., 1999) also increased. The increased expression of VEGF may promote more angiogenesis. In skeletal muscle engineering (Deveci, Marshall, & Egginton, 2002; Hudlicka, Milkiewicz, Cotter, & Brown, 2002; Li et al., 2011; Lundby, Calbet, & Robach, 2009; Singh, Wu, & Dunn, 2013; Wagner, 2001) and behind the ear (bone tissue-engineering) (Götz, Reichert, Canullo, Jäger, & Heinemann, 2012; Portal-Núñez, Lozano, & Esbrit, 2012; Street et al., 2005; Zhou et al., 2013), the effect of hypoxia has been widely studied in recent years. Gotz et al. studied the effect of hypoxia in explanted human tissue, and noted the co-localization of VEGF with hypoxia-inducible factor-1 (HIF-1), thereby postulating that hypoxia plays a role in bone repair (Götz et al., 2012). At a molecular level, hypoxia was found to be capable of inducing osteogenesis and angiogenesis through the extracellular regulated kinase (ERK)1/2 and/or p38 pathways (Zhou et al., 2013). Specifically, oxygen does not exist solely as a metabolic substance; at low concentrations, oxygen acts as a signaling molecule for directing both angiogenesis and osteogenesis (Zhou et al., 2013).

11.6  Conclusion and future perspectives 11.6.1  Summary of vascularization strategies In summary, this chapter has discussed vascularization of engineered muscoskeletal tissue by centering on the important arms of tissue-engineering: scaffolds, cells, GFs, and bioreactor technology. From the perspective of scaffolds, some characteristics that have continued influence over scaffold design include those of porosity, pore size, and tunability in terms of mechanical properties and surface functionalities. As part of a tissue-engineered construct, astute selection of cell sources remains an important feature that determines the eventual biological functionality. Coculture systems have thus been developed, focusing on paracrine signaling effects in precisely designed microenvironments created using scaffolds. In the event in which an enhanced effect is needed, the incorporation of relevant biomolecules like GFs form the next important aspect. By inducing earlier angiogenesis and ensuring cell survival within 3D constructs through the generation of prevascularized tissues, engineered musculoskeletal tissues may be made functional. Currently, bioreactor technology may be able to aid in the creation of engineered, autogenous tissue/organs as replacements for patients. As such, bioreactors have been designed to mimic physiological conditions, and one way would be the generation of low-shear conditions for stimulating angiogenesis. Accordingly, hypoxia and varying oxygen concentrations within the body also regulate the secretion of soluble, angiogenic factors that stimulate endothelial outgrowth. This has also been translated into ex vivo cultures in an attempt to both understand, and enhance current tissue-engineering strategies.

11.6.2  Future perspectives It appears that tissue-engineering will become the mainstay of future regeneration strategies due to the ever-declining numbers for autologous transplants. As such, it is

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imaginable that scaffold design and manufacturing techniques will continue to evolve. New, clinically relevant fabrication techniques that are solvent-free may be developed, paving the way for novel biomaterials with appropriate and controllable microarchitectures. These techniques may also be combined in a single step with the incorporation of GFs and/or other biomolecules for stimulating vascularization. Cell selection continues to be a key challenge in this field, and efforts may be directed toward the identification of specific cell types for specific regenerative niches. Bioreactor designs will continue to evolve and incorporate more physiological conditions, with the aim of attaining perfect biomimicry to exalt the biological and functional performance of engineered musculoskeletal constructs.

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