Journal of Investigative Medicine:
Symposium and Meeting Reports
Strategies for Vascularization of Polymer Scaffolds
Papavasiliou, Georgia PhD*; Cheng, Ming-Huei MD, MHA†; Brey, Eric M. PhD*‡
From the *Department of Biomedical Engineering, Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Chicago, IL;†Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China; and ‡Research Service, Hines Veterans Hospital, Chicago, IL.
Received May 5, 2010, and in revised form June 25, 2010.
Accepted for publication June 27, 2010.
Reprints: Eric M. Brey, PhD, Pritzker Institute of Biomedical Science and Engineering, Illinois Institute of Technology, Wishnick Hall, 3255 S Dearborn St, Chicago, IL 60616. E-mail: email@example.com.
Supported by funds from the National Science Foundation (0852048, 0731201, and 0854430), the Veterans Administration, the National Institutes of Health (R21HL094916 and RO1 DK 080897), Chang Gung Memorial Hospital (CRPG371861), and the Taiwan National Science Council (NSC96-2314-B-182A-075-MY2). This symposium was supported in part by a grant from the National Center for Research Resources (R13 RR023236).
There are no conflicts of interest in connection to the article.
Biocompatible, degradable polymer scaffolds combined with cells or biological signals are being investigated as alternatives to traditional options for tissue reconstruction and transplantation. These approaches are already in clinical use as engineered tissues that enhance wound healing and skin regeneration. The continued enhancement of these material strategies is highly dependent on the ability to promote rapid and stable neovascularization (new blood vessel formation) within the scaffold. Whereas neovascularization therapies have shown some promise for the treatment of ischemic tissues, vascularization of polymer scaffolds in tissue engineering strategies provides a unique challenge owing to the volume and the complexity of the tissues targeted. In this article, we examine recent advances in research focused on promoting neovascularization in polymer scaffolds for tissue engineering applications. These approaches include the use of growth factors, cells, and novel surgical approaches to both enhance and control the nature of the vascular networks formed. The continued development of these approaches may lead to new tissue engineering strategies for the generation of skin and other tissues or organs.
Wounds resulting from trauma, burn, or tumor resection remain some of the more challenging issues in tissue reconstruction. The reconstruction of these defects depends on the size of the defect, the donor site availability, and the health status of the patient. In a healthy adult, a wound less than 5 cm in its largest dimension can be healed by secondary intention. Healing can be enhanced through a variety of therapies, such as vacuum-assisted closure and/or hyperbaric oxygen treatment. If the defect is larger than 5 cm, then local or free flap transfer may be required for coverage of the defect. In a purely soft tissue defect with healthy soft tissue or muscle covering the wound bed, the wound can be easily treated with a skin graft. However, skin grafts have particular difficulty surviving when there is exposed bone and tendon. In addition, chronic wounds resulting from aging, diabetes, and peripheral artery occlusive disease are often refractory to conventional treatment.
New biologically active polymers that initiate or enhance wound healing are currently available potential alternative approaches for the treatment of large-volume or poorly healing wounds.1 In polymer design, engineers and scientists must consider the optimization of biomaterial properties to modulate important biological responses, including epithelialization, infection, and scar formation. In addition, the success of these strategies is highly dependent on whether the materials are able to promote rapid and stable neovascularization (new blood vessel formation) within the scaffold, typically before complete material degradation. There are a variety of approaches that have been explored for promoting neovascularization. Here we will describe some of the more common approaches and where they currently stand with respect to application for engineering skin, tissues, and organs.
Scaffold Delivery of Growth Factors
The most common approach for controlling neovascularization in biomaterial scaffolds is based on the incorporation of naturally occurring growth factors into the scaffolds. A complex temporal and spatial expression of multiple growth factors occurs in tissues regulating neovascularization in response to tissue ischemia, inflammation, or disease. The direct injection of growth factors into tissues has shown promise clinically for the treatment of ischemic diseases.2 For many tissue engineering applications, including skin, the scaffolds often need to permit extensive neovascularization before the bulk degradation of the material. This presents issues not encountered when promoting vascularization in ischemic tissues. The specific growth factors to use, their delivery kinetics, and spatial presentation must be optimized with material degradation to coordinate both vascularization and tissue regeneration.
Isoforms of vascular endothelial growth factor (VEGF) have received significant attention as therapeutic agents and shown promise in clinical studies for the treatment of peripheral limb and myocardial ischemia. In these applications, the protein or DNA is typically delivered via multiple bolus injections into the diseased tissue. This can result in abnormally high levels that may stimulate vessels with abnormal structure, high permeability, and poor long-term stability.3,4 Addition of VEGF proteins into polymer scaffolds has been shown to enhance vascularization. The VEGF is typically incorporated in a form in which it diffuses from the scaffolds or is released as the polymer degrades, stimulating local vessels to sprout towards the implanted material. The proteins can also be covalently incorporated into polymer scaffolds. When attached to poly(ethylene glycol) (PEG) or fibrin-based materials, VEGF release is delayed, prolonging its biological function and reducing risk of ectopic effects.5,6 The attached VEGF had been shown to have improved activity and promote more extensive neovascularization relative to freely diffusing VEGF.6
A number of members of the fibroblast growth factor (FGF) family of proteins have also been investigated for their ability to stimulate vessel formation within polymer scaffolds. The most popular proteins of this family are FGF-1 (or acidic FGF) and FGF-2 (basic FGF), which are potent endothelial cell (EC) mitogens and are known to stimulate neovascularization. We have recently shown that the controlled delivery of FGF-1 from alginate microbeads results in a more persistent neovascularization response relative to bolus delivery.7-9 The incorporation of these beads into collagen scaffolds results in both an increase in initial vessel invasion into the collagen and a longer persistence of the vascular network formed.10 In addition, by delivering FGF-1 from alginate, the dose required to achieve vascularization is lower than what is required when the protein is suspended in the scaffold.9 Whereas the sustained delivery from alginate increases the lifetime of the vessels, measures of vessel maturity did not increase.
The sequential delivery of a protein that stimulates vessel sprouting and invasion followed by a factor involved in vessel stabilization can increase the stability or the maturity of the vessels formed. Whereas vessels stimulated by VEGF alone may regress, this regression can be inhibited by the delivery of angiopoietin-1, a vessel-stabilizing factor.11 The combination of platelet-derived growth factor-BB with FGF-2 increased arteriogenesis or collateralization in models of tissue ischemia.12 Multiple growth factor delivery has also been used to enhance scaffolds neovascularization, where VEGF followed by platelet-derived growth factor-BB leads to larger vessels and greater mural cell interactions.13 This research shows that controlled delivery of multiple growth factors may enhance neovascularization over a single factor. However, the delivery methods still need to be optimized. We have recently developed a technique for generating multilayer alginate microbeads that could be used for dual growth factor delivery with distinct release kinetics from the different alginate layers (Fig. 1).14
Growth Factor Gradients
Whereas the identity of growth factor and/or growth factor combinations is important in stimulating neovascularization, the spatial presentation of these factors plays a major role in this process. In studies in the postnatal retina, Gerhardt et al.15 showed that the migration of ECs on the tips of sprouting vessels was dependent on the gradient of VEGF, whereas proliferation was a function of its concentration. The approaches described in the previous section generate gradients due to the diffusion of the growth factor from the polymer scaffold and into the surrounding tissue. It may also be beneficial to generate scaffolds with controlled gradients of growth factors to accelerate and guide vessel invasion.
Barkefors et al.16 investigated EC migration in response to soluble gradients of VEGF and FGF-2 using a novel microfluidic chemotaxis chamber. An advantage of the microfluidic chemotaxis chamber as compared with more traditional chamber assays is that it allows for predictable gradients of various shapes and imaging of migratory cell paths using time-lapse microscopy. Gradients of VEGF resulted in a more potent chemotactic effect for ECs as compared with similar gradients of FGF-2. Further analysis of cell migration in different regions of the gradient indicated a maximal chemotactic response in exponential gradient as compared with linear gradient regions suggesting that the shape of the gradient dictates migratory response. These results suggest the complicated nature of the EC response to gradients of immobilized factors. The response depends on the specific growth factor and the shape of its concentration profile.
Growth factors and extracellular matrix molecules simultaneously regulate the migration of cells. Liu et al.17 created surface density gradients of both VEGF and fibronectin on gold substrates with varying gradient slopes generated by self-assembled monolayers using an electrochemical approach. To our knowledge, this was the first study to create surface density gradients of VEGF. Endothelial cells seeded on surface density gradients of fibronectin, VEGF, or both proteins exhibited increased cell displacement on gradient surfaces towards higher surface protein density as compared with surfaces with a uniform protein density. A 2-fold increase in cell migration was observed on VEGF gradient as compared with fibronectin gradient surfaces, and a further 2-fold increase in migratory response was observed on surfaces with combined gradients of both factors as compared with those with immobilized VEGF or fibronectin gradients alone.
Whereas the aforementioned studies have provided significant insight on EC haptotactic response on 2-dimensional (2D) surfaces, the creation of gradients within 3D polymeric scaffolds more accurately mimics the physiological environment. Hydrogels of PEG formed via photopolymerization have the potential to be used to create immobilized gradients of growth factors and cell adhesion sites. The advantage of this approach to engineer polymer scaffolds for tissue engineering applications is that both the physical properties and the incorporation of proteins and peptides can be controlled by polymerization conditions (eg, polymerization time, PEG macromer molecular weight, precursor concentration). Immobilized gradients of FGF-2 were incorporated into PEG-based hydrogels formed using a gradient maker.18 In this case, cells preferentially migrated up the gradient, but it is not clear whether the magnitude of the gradient influenced migratory behavior. In addition, although the hydrogel is 3D, the cells only migrated on the surface resulting in a 2D cell migratory response, and only smooth muscle cell migration was examined. Creation of 3D gradients within PEG hydrogels would be a better benefit for applications in regenerative medicine.
Microfluidic devices provide the versatility for multiparameter manipulation and the ability to image 3D cellular migration within scaffolds in response to local changes in their microenvironment.19-21 Studies have used these platforms to investigate the effects of gradients of growth factors.19 Endothelial cells were cultured in a channel in direct contact with collagen scaffolds localized in these microfluidic devices. Mass transport behavior is exploited to create gradients within the collagen by injection of VEGF in channels surrounding the collagen scaffolds. Endothelial cells in contact with the VEGF gradient rapidly migrated into the scaffold (up the gradient), whereas cells in contact with control channels in the absence of VEGF gradient exhibited significantly less migration.22
There have been significant efforts to enhance EC migration and neovascularization by incorporation of growth factors within polymer scaffolds. The biological response to these materials depends on a number of factors, including which growth factors are used and their temporal and spatial presentation. In addition, the optimal approach is likely to depend on the specific tissue to be regenerated as the microcirculation varies within every tissue and organ.23 The ability to generate tissue-specific vascular structures has received little attention.
Growth factor delivery alone may not stimulate vessel invasion rapidly enough to completely vascularize scaffolds used for the treatment of large-volume wounds.23 In these conditions, the central region of the scaffolds can become necrotic or cannot regenerate sufficiently. The addition of ECs either individually or organized into vascular structures before implantation may accelerate scaffold vascularization. If the cells can form network structures and inosculate with the host circulation, then, in theory, large volumes of tissues can rapidly establish a patent blood supply in a manner similar to transplanted organs and free tissue transfer.
Endothelial cells mixed with Matrigel (BD Biosciences, Franklin Lakes, NJ), a solution of base ment membrane proteins, and seeded onto highly porous poly (l-lactic acid) (PLLA) scaffolds assemble into microvessels within 5 days after implantation.24 These capillarylike structures inosculated with the host circulation after 14 days and recruited host-derived support cells by 21 days. It is not clear if this approach would work for large scaffolds, as even the transplanted ECs are sensitive to hypoxic conditions within the scaffolds. However, the same research group showed that transfecting the cells to express an antiapoptotic protein could enhance the survival of the transplanted ECs.25
The use of adult ECs is hindered by cell availability and altered function of cells owing to comorbidities and age in the potential patient population.26,27 Endothelial progenitor cells or embryonic stem cells offer an alternative source of cells for therapeutic applications.28,29 Human embryonic stem cells can be induced to form ECs that assemble into networks in vitro and enhance patent vascular formation in PLLA/poly(lactic-co-glycolic acid) (PLGA) scaffolds when implanted in vivo.28 The addition of embryonic fibroblasts to the scaffolds improves network formation and stabilization of these vessels.29
The assembly of ECs into network structures is often enhanced because of the presence of other cells owing to either the production of soluble factors30 or stabilizing cell-EC interactions.31 The coimplantation of ECs and mesenchymal stem cells (MSCs) in fibronectin-type 1 collagen gel resulted in both a greater number of and longer-lasting vessels than ECs alone.31 The MSCs seemed to largely differentiate into mural cells that stabilized the vessels. The ECs still assembled into vascular networks without MSCs, but little perfusion was observed. The importance of coimplantation with other cells has also been shown with fibrin gels prevascularized with fibroblasts and ECs.32 In this case, the cells were induced to form networks before implantation. The vascularized gels had an increase in the number of perfused vessels and decreased the time required for perfusion relative to controls.
PATTERNING VASCULAR STRUCTURES
The techniques described previously rely on the ability of the ECs to self-assemble into networks, resulting in largely a single type of vessel with a limited control over spatial distribution. As an alternative to designing scaffolds with the appropriate signals to stimulate vascularization upon implantation in vivo, vascularized tissue constructs may be fabricated in vitro. In an effort to engineer polymer scaffolds that mimic the complexity and microarchitecture of biological tissues and result in the formation of functional vascular structures, numerous micropatterning techniques have been exploited such as microcontact printing, micromolding, photolithography, micromachining, and laser-guided writing.
Early efforts focused on EC patterning on rigid substrates such as gold,33 silicon, and Pyrex.34,35 Using microcontact printing, gold substrates with adhesive patterns of fibronectin of various geometries were created.33 Endothelial cell spreading and shape was found to be dependent on the size and geometry of the adhesive patterns. Endothelial cells cultured on single islands of fibronectin greater than 1500 μm2 entered the growth phase, whereas restriction of the cells to areas less than 500 μm2 resulted in cell apoptosis. These results suggested that local changes in cell growth and apoptosis, which are critical to the formation of tissue structures, may be generated by local changes in EC-extracellular matrix interactions. An extension of this study investigated whether defined patterned geometries resulted in cell differentiation.34 Endothelial cells cultured on 10-μm wide lines of fibronectin resulted in extensive cell-cell contacts and formed capillary tubular structures with a central lumen, whereas cells adhered to 30-μm wide lines of fibronectin resulted in EC monolayers. Thus, alterations in the geometry of cell spreading influence EC genetic programs, which may be switched between growth, apoptosis, and differentiation.
Micromachining techniques have also been used to create vascular structures. Kaihara et al formed vascular capillary networks with diameters as small as 10 μm that were etched into silicon and Pyrex surfaces that served as templates.35 Endothelial cells and hepatocytes cultured on these 2D branched structures lifted off as cell monolayers and were then implanted. Upon implantation, the fabricated structures resulted in the formation of vascularized hepatic tissue. Although these techniques have contributed significantly towards the creation of vascularized structures on rigid substrates, their application is limited to patterning in 2D with extension to 3D, resulting from replication of the 2D patterns in 3D.36
In an effort to extend 2D patterning technology to 3D application, natural and synthetic polymer systems have been used as substrates. A 3D printing process (3DP) was used to create a vascularized "miniliver" in vitro.37 Using this approach, 200-Km channels were created within biodegradable copolymer scaffolds of PLLA and PLGA. After 5 weeks in static culture, ECs attached and filled the channels. Mixed populations of hepatocytes and ECs seeded into these scaffolds demonstrated substantial reorganization and the formation of structures that seemed similar to sinusoids when cells bridged across channels. This scaffold system also demonstrated features of vessels and parenchymal space. Although this patterning technique was successful in creating hepatic sinusoids, it would be difficult to apply towards the creation of channels with smaller feature sizes (5-10 μm) similar to the dimensions of capillary networks. Another study focused on combining melt micromolding with thermal fusion bonding to create stacked, aligned, and interconnected multilayered microfluidic networks analogous to those of the microvasculature using PLGA biodegradable scaffolds.38 Channel widths ranged from 30 to 3000 μm with height dimensions of 35 μm. When these microfluidic networks were perfused, they showed no signs of leaks or occlusions.
Patterns of vascular networks with channel widths varied from 3 mm (at the inlet and outlet) to capillary dimensions on the order of 45 μm with fixed 30-μm depth have been created within biodegradable cell adhesive elastomers of poly(glycerol sebacate).39 Vascular structures were formed by etching the patterns onto silicon wafers. These scaffolds contained inlet and outlet ports enabling perfusion. When these devices were perfused at physiological flow rates, they resulted in complete endothelialization after 14 days and remained stable for 4 weeks in culture illustrating the potential of this technique for inducing vascularization in engineered tissues.
Chrobak and Potter40 used micromolding to generate functional perfused microvascular cylindrical channels within collagen gels that demonstrated strong barrier properties after 5 days in culture and resisted leukocyte adhesion. Confluent EC monolayers resulted in the formation of channel diameters between 75 and 150 μm after maturation, with EC tube length spanning the entire collagen gel (5-7 mm). Cell-induced defects of these channels were minimized by optimizing the concentration of collagen and the temperature required for gel formation.
Other approaches to create microvascular patterns within scaffolds with high resolution have focused on laser-induced patterning. Laser-guided direct writing is a patterning technique used to confine multiple cell types in a laser beam and deposit them on various surfaces including biological gels with micrometer accuracy allowing for direct cell patterning within the engineered tissue scaffolds.41 This approach was used to pattern ECs on Matrigel in 2D and in 3D multilayers. Endothelial cells elongated and formed tubelike structures along the patterns; however, stabilization and maturation of these structures into capillaries was not observed. In a later study, Chen et al.42 used biological laser printing (BioLP) to directly deposit patterns of ECs onto Matrigel. Initial unconnected EC patterns resulted in interconnected patterns of different geometries suggesting that growth was achieved without surface modification and was solely a result of cell-cell interactions and cell differentiation.
Another patterning strategy that can be used to vascularize polymer scaffolds is the combination of photopolymerization of synthetic hydrogel precursors with noncontact photolithography. We used interfacial photopolymerization to produce microvascular patterns within multilayered PEG diacrylate hydrogels with feature sizes between 50 and 70 μm using vascular photomasks (Fig. 2, A and B).43 Interfacial photopolymerization was induced by the addition of the PEG-DA precursor solution onto a surface that was covalently immobilized with a photoinitiator (eosin Y) followed by exposure to visible light (λ = 514 nm). This resulted in the formation of a hydrogel that grew from the surface outward with thickness controlled solely as a function of polymerization conditions without the need of spacers or molds. The addition of a PEG aminoacrylate macromer to the hydrogel precursor resulted in the formation of a hydrogel containing pendant amines onto which the eosin was covalently attached, enabling the formation of covalently attached multilayers (Fig. 2, C and D). This approach could be exploited to allow formation of 3D multilayered structures with distinct pattern formation in each layer.
Building on these results, we constructed microchannels within PEG hydrogels using 2 photopolymerizable macromers, PEG-DA and PEG-PLLA-DA (Fig. 3).44 Hydrogel patterns of PEG-PLLA-DA could be formed within PEG hydrogels. Multiple layers could be created with distinct PEG-PLLA patterns. The PEG-PLLA serves as a sacrificial polymer that can be selectively degraded away after patterning owing to its greater susceptibility to hydrolysis. After completion of the multilayer process, the hydrogel was incubated in a high pH environment resulting in complete degradation of the PEG-PLLA-DA hydrogel patterns. Using this approach, multilayered interconnected channels were fabricated through hydrolytic degradation of PEG-PLLA-DA hydrogel-patterned regions within distinct layers. The channels can be functionalized with cell adhesion sequences to support EC growth to form capillarylike channels.44
The studies described previously have provided significant insight and developments into how cell interactions with polymer scaffolds can be designed to improve or accelerate vessel assembly. Patterning technologies are far from clinical application but have been shown to allow precise control over the generation of vascular structures and microchannels. Regardless, whether the cells are allowed to form networks on their own or are directly patterned into distinct regions, the source of the cells that would be used for future clinical applications is not clear. Studies are needed that address the capabilities of these specific cells to form networks and inosculate with the host vasculature.
Whether growth factors, cells, or combinations of the 2 are used to promote neovascularization in polymer scaffolds, the approaches can be very successful at generating microvascular networks in small-volume scaffolds. However, clinical application for the treatment of large defects requires a more complex vessel hierarchy within larger scaffold volumes. We have approached this issue by exploiting prefabrication approaches developed in reconstructive microsurgery.9,45-50 In these approaches, the scaffolds are implanted in a "donor" tissue location that would promote greater neovascularization than the defect location. After a period of prefabrication, the scaffolds with an extensive vascular network would then be transferred to the recipient site.
In one embodiment, scaffolds are implanted around a vascular pedicle composing a sizable artery and vein. The pedicle allows de novo vascularization of the scaffolds that could then be transferred to the recipient defect with or without microsurgical techniques. Using a rodent vascular pedicle model, we have shown that with the same FGF-1 delivery strategy from alginate beads, there is greater overall vascularization when the beads are implanted around a vascular pedicle model9 than other microcirculatory beads.7,8 In addition, these newly vascularized scaffolds, along with the pedicle, may be transferred together using microsurgical anastomoses to the recipient vessels. It may be difficult to translate the pedicle model to clinical application, but others have shown that larger volumes of vascularized tissues can be created by implantation of materials around microsurgically created vessel loops.51-56
We have also shown that the implantation of a cell-matrix-growth factor mixture into a highly vascularized donor location in the body can be used to guide fabrication of large volumes of vascularized tissues with complex 3D shape.47 The application of prefabricated or prelaminated flap has been widely applied in clinical cases.57-59 This approach was successfully applied clinically where the prefabricated tissue construct with neovascularization was easily transferred to the recipient location.48 The use of established surgical approaches to enhance vascularization in large-volume scaffolds has received little attention for applications in tissue engineering. When combined with a novel growth factor and/or cell strategy, surgical techniques may help optimize the volumes of scaffolds vascularized.
The ability to stimulate neovascularization within polymer scaffolds is critical to the success of tissue engineering strategies for the treatment of large wounds and engineering new tissues and organs. Although this issue has received significant attention in recent years, there remains a significant number of issues to overcome, including the following:
* Generation of new vessels in large, complex volumes of tissues. Research up to this point has focused on small polymer scaffolds, which do not approach the clinical volumes needed.
* Although numerous efforts have focused on strategies to incorporate growth factors or cells within a synthetic polymer, studies to date have not quantified how their 3D spatial presentation influences vessel assembly. These studies would significantly contribute to our understanding on how polymer scaffolds can be better designed to induce vascularization.
* Addressing fundamental alterations in neovascularization in targeted patient populations. Though not addressed in this article, many in the population targeted for these therapeutic approaches, including the elderly,27 people with diabetes,60 and those with other comorbidities,23 have a reduced capacity for vessel assembly. These issues need to be considered directly in the development of new therapeutic approaches.
The continued development of novel approaches to these issues will likely lead to new therapeutic approaches to applications for engineering skin and other tissues.
The authors thank Omaditya Khanna and Dr Monica Moya for their contributions to Figure 1 and Yu-Chieh Chiu to Figure 3.
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