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Protein-based signaling systems in tissue engineering
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Protein-based signaling systems in tissue engineering Tanyarut Boontheekul and David J Mooney Tissue engineering aims to replace damaged tissues or organs using either transplanted cells or host cells recruited to the target site. Protein signaling is crucial to regulate cell phenotype and thus engineered tissue structure and function. Biomaterial vehicles are being designed to incorporate and locally deliver various molecules involved in this signaling, including both growth factors and peptides that mimick whole proteins. Controlling the concentration, local duration and spatial distribution of these factors is key to their utility and efficacy. Recent advances have been made in the development of polymeric delivery systems intended to achieve this control. Addresses University of Michigan, 1011 North University Avenue, 5213 Dental Building, Ann Arbor, MI 48109-1078, USA e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:559–565 This review comes from a themed issue on Tissue and cell engineering Edited by Jeffrey Hubbell 0958-1669/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2003.08.004
Abbreviations BMP bone morphogenetic protein EC endothelial cell ECM extracellular matrix EGF epidermal growth factor FGF-2 fibroblast growth factor 2 NGF nerve growth factor PDGF platelet-derived growth factor PEG polyethylene glycol PGA poly(glycolide) PLA poly(L-lactide) PLGA poly(lactide-co-glycolide) PlGF placental growth factor TGF transforming growth factor VEGF vascular endothelial growth factor
Introduction Tissue development is regulated through the interplay of a variety of signals, including soluble signaling molecules, insoluble ligands, mechanical cues and cell–cell interactions [1]. Numerous peptides and proteins involved in this signaling possess a biological activity that marks them as potential therapeutics. Soluble growth factors and immobilized ligands can regulate the adhesion, migration, proliferation and differentiation of various cell types. These factors may be a key to success in tissue engineering, an emerging field combining engineering and life www.current-opinion.com
sciences with the goal of repairing damaged or lost tissues [2]. Tissue regeneration is affected by either transplanted or host cells, in concert with local environmental signals. One approach to direct the process of tissue formation is the incorporation of bioactive components, including growth factors and peptides mimicking the function of extracellular matrix (ECM) molecules, into biomaterials [3]. Advances in material engineering have led to new modes of presenting these molecules to control the cell response and new tissue development. Transplantation of cells that secrete the desired growth factors is also a promising strategy to deliver these bioactive proteins [4], as are approaches based on gene therapy [5]. However, these latter approaches will not be reviewed in this article. We review here recent advances in the development of delivery materials for two types of signals: growth factors and immobilized peptides (Figure 1). Growth factor delivery has advanced to the clinic and will be categorized by the specific therapeutic applications. Immobilized peptide presentation is not as clinically advanced and fundamental issues important to its ultimate clinical utility, including peptide biology and immobilization techniques, are discussed as well as potential therapeutic applications.
Growth factor delivery The development of appropriate delivery vehicles for growth factors will be crucial for their clinical utility. Growth factors, owing to their control of many biological processes, are finding wide-spread use in the regeneration of many tissue types. Typically, recombinant versions of the desired proteins are manufactured and delivered in solution form, either systemically or via direct injection into the tissue site of interest. However, growth factors typically have a short half-life once they are introduced into the body and are rapidly eliminated [6]. This is problematic, as the target cell population must often be exposed to factors throughout the entire course of repair, or at least for an extended period. To address this challenge, controlled delivery systems that incorporate the growth factors into polymeric biomaterials have been developed to prolong the tissue exposure time and to maintain growth factor stability. In addition to the duration of tissue exposure, the amount and timing can be crucial to the biological response. The release rate of growth factors from polymers is typically controlled by the diffusion of the factor or polymer degradation [7]. Among the most commonly used materials are synthetic polymers such as poly(L-lactide) (PLA), poly(glycolide) (PGA) and their copolymers poly(lactide-co-glycolide) Current Opinion in Biotechnology 2003, 14:559–565
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Figure 1
(a)
(b)
[Peptide]
[GF]
Distance
Distance
Current Opinion in Biotechnology
Delivery of two types of protein-based signaling (growth factors and immobilized peptides) is accomplished with distinct approaches. (a) Growth factors (red circles) are typically encapsulated within a polymer and subsequently released. The released growth factor diffuses through the surrounding tissue where it binds to receptors (green) on cells. This mechanism creates a concentration gradient of the factor that might direct cell movement. GF, growth factor. (b) By contrast, peptides (blue diamonds) mimicking ECM molecules are typically covalently coupled to the material and only cells in direct contact with the surface of the material can bind to the peptides with appropriate cell-surface receptors (green). This leads to a very localized regulation of cellular activity.
(PLGA), in the physical forms of microspheres or sponges [8]. Hydrogel-forming materials, including collagen [9], alginate [10], polyethylene glycol (PEG) [11] and PLAPEG [12], are also frequently used. To address the instability of proteins immobilized within polymers, a variety of stabilization techniques have been developed [13,14]. Recent progress in developing delivery vehicles for growth factors in the three most clinically advanced applications are discussed in the following sections. Neovascularization
Neovascularization is crucial in treating many diseases (e.g. coronary artery disease) and in virtually all approaches to tissue engineering. A lack of vascularization leads to insufficient nutrient delivery and waste removal, cell death and limited tissue development and tissue loss. Angiogenesis, new blood vessel formation by sprouting from the sides and ends of pre-existing microvascular vessels, has been widely studied to determine the rules guiding blood vessel formation in the adult. Numerous
growth factors have been identified as regulators of this process (summarized in Table 1). Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, as it is a potent mitogen for endothelial cells (ECs) and induces EC migration and sprouting by upregulation of several endothelial integrin receptors [15]. There is a family of VEGF growth factors, VEGF-A through VEGF-E, and two isoforms of VEGF-A (VEGF121 and VEGF165) have progressed to clinical trials in the treatment of ischemic diseases [16]. Placental growth factor (PlGF), another VEGF family member, can also stimulate angiogenesis [17], but its potential side effects remain unclear. Various polymeric materials have been used for the controlled release of VEGF, including PGA, alginate and fibrin [8,10,18]. It has recently been noted that the VEGF release rate from polymers can be responsive to the local mechanical environment [10], and this may be crucial in applications involving a mechanically dynamic environment (e.g. heart tissue).
Table 1 Growth factors commonly used in therapeutic angiogenesis. Growth factor
Abbreviation
Relevant known activities
Vascular endothelial growth factor Basic fibroblast growth factor Platelet-derived growth factor Angiopoietin-1 Angiopoietin-2 Placental growth factor Transforming growth factor
VEGF bFGF-2 PDGF Ang-1 Ang-2 PlGF TGF
Migration, proliferation and survival of ECs Migration, proliferation and survival of ECs and many other cell types Promotes the maturation of blood vessels by the recruitment of smooth muscle cells Strengthens EC–smooth muscle cell interaction Weakens EC–smooth muscle cell interaction Stimulates angiogenesis Stabilizes new blood vessels by promoting matrix deposition
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Fibroblast growth factor 2 (FGF-2), another well-studied angiogenic factor, elicits diverse biological effects on numerous cell types, including ECs, and has been used in clinical trials to induce angiogenesis [19]. FGF-2 has been incorporated into various polymers, such as gelatin [20], collagen [21], chitosan [22] and PLA [23]. FGF-2 reversibly binds to heparin-like molecules and heparin sulfate proteoglycans. This can be exploited to increase the FGF binding capacity to polymeric delivery vehicles, resulting in a more gradual and sustained release of FGF and an enhancement of angiogenesis [21]. The dominant growth factor delivery approach in angiogenesis utilizes single growth factors, but this may not be the ideal approach to replicate this complex process. Angiogenesis results from a complicated series of interactions involving diverse cytokines, growth factors, cells and proteases, acting in a consecutive, concerted or synergistic manner [16]. Recent advances in polymeric delivery systems allow one to locally and controllably deliver multiple growth factors with controlled doses and rates of delivery. Delivery of VEGF and plateletderived growth factor (PDGF) at distinct rates and doses results in a dramatic increase in the maturity of engineered vessel networks [8]. Similarly, delivery of FGF– PDGF combinations synergistically induces stable vascular networks, whereas single growth factors are unable to maintain these newly formed vessels [24]. It must be noted that interactions between various growth factors may complicate interpretation of the results of their delivery, as PDGF and nerve growth factor (NGF) can raise VEGF secretion by cells [25,26]. Recent studies also illustrate interplay between VEGF, Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) in regulating angiogenesis [27]. Bone regeneration
A primary goal of growth factor delivery for bone tissue engineering is to accelerate the regeneration process and engineer bone in situations that normally lead to incomplete regeneration. Several growth factors that are intimately involved in bone formation and remodeling have been identified and are summarized in Table 2. The transforming growth factor (TGF) family of proteins have an essential role in bone formation through the
regulation of osteoprogenitor and osteoblast proliferation and differentiation [28]. A variety of materials have been used for TGF-b1 delivery, including PLGA-PEG and coral [28,29], and combined TGF-b1 and insulin-like growth factor (IGF) release from PLA carriers accelerates osteotomy healing [30]. However, the optimal conditions for TGF-b1 release during bone regeneration have yet to be defined. Bone morphogenetic proteins (BMP), members of the TGF superfamily, appear to act as differentiation factors, causing mesenchymal cells to differentiate into bone-forming cells [31]. Recombinant human BMP-2 and BMP-7 are currently in clinical use as osteoinductive agents [31]. Several carriers for BMP have been developed, including collagen [32], PLGA [33], PLA–p-dioxanone–PEG block copolymers [34], PLA-PEG [12] and PEG hydrogels [11]. These systems allow control over the BMP release rate and promote pre-osteoblast differentiation and mineralization in vitro and ectopic bone formation in vivo [11,12]. Other growth factors involved in bone regeneration include FGF and PDGF, and a variety of materials have been used for their delivery [35,36]. Wound healing
There is a significant need to enhance the healing of skin tissue resulting from ulcers, scars or burns. The therapeutic potential of several growth factors in wound healing (Table 3) has long been recognized [37]. PDGF is the first mediator to appear at the wound site and is active in all stages of the healing process. Delivery of PDGF by a carboxymethylcellulose-based gel (Regranex1) is employed for the treatment of diabetic foot ulcers and was the first growth factor system for tissue engineering to be approved by the Food and Drug Administration (FDA) [37]. Both epidermal growth factor (EGF) and TGF-b regulate keratinocyte phenotype [38], and therefore play an important role in the wound healing process. Sustained and localized EGF delivery substantially decreases the size of the wound [9,39], while released TGF-b inhibits epithelial cell proliferation [40]. FGF-2 sustained release also enhances skin regeneration [41].
Immobilized ligand signaling Insoluble ECM molecules clearly regulate local cellular activity, and many functions of the ECM can be mimicked by small peptide fragments of the entire molecules [42,43]. These fragments can be produced
Table 2 Growth factors commonly used in bone regeneration. Growth factor
Abbreviation
Relevant known activities
Transforming growth factor-b Bone morphogenetic protein Insulin-like growth factor Fibroblast growth factor-2 Platelet-derived growth factor
TGF-b BMP IGF-I FGF-2 PDGF
Proliferation and differentiation of bone-forming cells Differentiation of bone-forming cells Stimulates proliferation of osteoblasts and the synthesis of bone matrix Proliferation of osteoblasts Proliferation of osteoblasts
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Table 3 Growth factors commonly used in wound healing. Growth factor
Abbreviation
Relevant known activities
Platelet-derived growth factor Epidermal growth factor Transforming growth factor-b
PDGF EGF TGF-b
Fibroblast growth factor
FGF
Active in all stages of healing process Mitogenic for keratinocytes Promotes keratinocyte migration, ECM synthesis and remodeling, and differentiation of epithelial cells General stimulant for wound healing
synthetically and covalently coupled to synthetic polymers so as to present them in a solid-state form, upon which their activity often depends, and to infer biological activity to the synthetic materials. Cellular attachment to ECM molecules is crucial for the survival, growth and determination of a differentiated phenotype for anchorage-dependent cells. Many of these processes are mediated through ECM–integrin receptor binding. The integrin binding capability of ECM molecules has been mapped to specific oligopeptide sequences within ECM proteins, and these sequences include RGD, IKVAV and YIGSR (in single-letter amino acid code) [44,45]. Advantages of employing short peptides containing these signaling motifs to modulate cell function, rather than utilizing whole adhesive proteins (e.g. fibronectin), include the ease and reproducibility of synthesizing peptides, as compared with isolating ECM molecules from a natural source. Utilization of only a small fragment of an ECM molecule may also allow one to target an interaction with a specific cell population [3]. A limitation to this approach, however, is that the biological activity of short peptide sequences is often substantially lower than that of the complete protein, owing at least partially to the absence of complementary domains that are involved in integrin binding [46]. Utilizing larger ECM molecule fragments, produced recombinantly, may represent a robust approach to increase activity, while still offering advantages over using the entire molecule (e.g. reduced antigenicity) [47]. A key feature of natural ECM molecules is their susceptibility to celltriggered proteolysis, which permits cell invasion and subsequent remodeling of the matrix. Invasion and remodeling depend on the action of cell-secreted proteases that target specific sequences of the ECM molecules [48]. Recent developments indicate that it is possible to synthesize cell-remodelable synthetic materials by utilizing these sequences as cross-linking agents [42]. Immobilization techniques
Peptide immobilization on polymers can be achieved by simple adsorption, standard covalent chemical conjugation or enzymatic incorporation [49,50,51]. In situ peptide adsorption can be optimized through appropriate peptide design (e.g. inclusion of hydrophobic domains [49]). Peptides can also be covalently conjugated using Current Opinion in Biotechnology 2003, 14:559–565
carbodiimide chemistry, Michael-type addition or photochemical grafting [42,50,52]. There are many possible coupling techniques resulting from the variety of active chemical groups on peptides. The method of immobilization has an effect on both the receptor binding capacity and affinity [53]. Moreover, it has been demonstrated that peptides can be readily incorporated into fibrin matrices enzymatically [51]. To present the peptides with a high signal-to-noise ratio, the polymer chosen for peptide presentation should not mediate significant adsorption of contaminant proteins. The most commonly used polymer for low protein binding and cell adhesion resistance is poly(ethylene oxide). Poly(ethylene oxide) has hydrophilic and electrically neutral properties, and can be combined with other biomaterials to form block polymers with controlled structures [49]. Dextran-based gels and conjugates also have excellent biocompatibility and ability to limit cell adhesion, and have more surface-binding sites than PEG [54]. In addition, other hydrophilic polymers such as alginate can be employed, as alginate hydrogels discourage nonspecific protein adsorption [55]. The lack of chemically modifiable sites in synthetic aliphatic polyesters (e.g. PGA) makes it more complex to attach peptides; however, incorporation of monomer units containing appropriate functional groups into the polymer backbone can overcome this problem [56]. Alternatively, carboxylic acid groups can be generated on the surface by hydrolysis to allow for covalent attachment of ligands via amide bond formation [57]. Regulation of cell phenotype and tissue regeneration
Peptide-incorporating biomaterials (Table 4) have demonstrated control over the adhesion, proliferation and differentiation of various cell types, including fibroblasts, chondrocytes, osteoblasts, myoblasts, endothelial, smooth muscle and neuronal cells [42,49,54–56,58]. Several parameters involved in the immobilization process, including the type of peptide, peptide concentration, spacer arms, immobilization technique and spatial distribution of peptides, can regulate the cellular response [49,50,51,53]. The nanoscale organization of the peptide may also be a key to control cellular response, as intracellular signaling is typically only triggered when integrins are clustered in the cell membrane www.current-opinion.com
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Table 4 Immobilized ligands used in tissue engineering. Immobilized ligand
ECM molecule source
Application
RGD
Multiple ECM molecules, including fibronectin, vitronectin, laminin, collagen and thrombospondin Laminin Fibronectin Common MMP substrates, (e.g. collagen, fibronectin, laminin)
Enhance bone and cartilage tissue formation in vitro and in vivo Regulate neurite outgrowth in vitro and in vivo Promote myoblast adhesion, proliferation and differentiation Enhance endothelial cell adhesion and proliferation Regulate neurite outgrowth in vitro and in vivo Promote formation of focal contacts in pre-osteoblasts Encourage cell-mediated proteolytic degradation, remodeling and bone regeneration (with RGD and BMP-2 presentation) in vivo
IKVAV YIGSR RNIAEIIKDI Recombinant fibronectin fragment (FNIII7–10) Ac-GCRDGPQ-GIWGQDRCG
Sequences are given in single-letter amino acid code. MMP, matrix metalloproteinase.
[59]. Controllable nanoscale clustering of RGD peptides tunes cell adhesion [60] and can be used to study the effects of ligand density and clustering on cell adhesion and the strength of the adhesion [61]. An important advance over the past few years was the demonstration that peptide immobilization to materials can regulate in vivo events, and specifically enhance bone and nerve regeneration [50,51,58]. RGD-coupled alginate hydrogels can control osteoblast and chondrocyte differentiation in vitro and the formation of cartilaginous and bony tissues in vivo [50,58]. Strikingly, co-transplantation of both cell types together in this material leads to the formation of growing tissues that structurally and functionally resemble a growth plate [58]. The integration of RGD peptides and matrix metalloproteinase (MMP) substrates into a single material has been pursued to form a cell-responsive BMP-2 delivery vehicle, and this system can be used to regenerate bone tissue [42]. The proteolytic sensitivity of this system can be readily controlled to regulate cell infiltration and bone formation [43]. Further, fibrin modified with laminin-derived peptides has been shown to substantially enhance axon regeneration [51].
Summary and future directions Biomaterials are being increasingly designed to contain intrinsic bioactivity, through specific biological recognition mediated by released growth factors and immobilized peptides. Growth factor delivery has become a therapeutically powerful approach, and has been applied to neovascularization, bone regeneration and skin regeneration. Although certain growth factors (e.g. VEGF and BMP) have been widely studied and used in clinical trials or products, many additional growth factors (e.g. PlGF and NGF) and the various isoforms of the currently utilized growth factors have not been completely characterized. Many of these factors are likely to have therapeutic application in the future. Further, many growth factors have multiple actions and new therapeutic applications of the known growth factors are likely to emerge. A limitation of current growth factor delivery approaches www.current-opinion.com
is the focus on delivery of single growth factors, which may not be an ideal means to drive the highly regulated networks that lead to tissue regeneration. The development of multiple growth factor delivery systems that control the temporal availability of the factors has recently shown utility in angiogenesis [8], and this concept may be useful for other applications. Although less clinically advanced, peptide immobilization onto materials provides another approach to regulate cellular function in a very localized yet flexible manner. Recently, the utility of this system has been examined in the context of bone and nerve regeneration [51,58], and additional therapeutic applications will clearly emerge in the near future. Combining these multiple types of information into single biomaterials [42,55] will even more closely mimic the multifunctional nature of the native ECM and will be a powerful approach to regulate tissue regeneration and engineering.
Acknowledgements We wish to acknowledge financial support from the NIDCR/NIH to the laboratory of DM and a Royal King Anantamahidol Scholarship (Thailand) to TB.
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51. Schense JC, Bloch J, Aebischer P, Hubbell JA: Enzymatic incorporation of bioactive peptides into fibrin matrices enhances meurite extension. Nat Biotechnol 2000, 18:415-419. Factor XIIIa is used to covalently cross-link peptides into fibrin matrices, and these enzymatically incorporated matrices enhance neurite extension, depending on the type and concentration of peptides. 52. Chung TW, Lu YF, Wang SS, Lin YS, Chu SH: Growth of human endothelial cells on photochemically grafted Gly-Arg-Gly-Asp (GRGD) chitosans. Biomaterials 2002, 23:4803-4809. 53. Osmond RI, Kett WC, Skett SE, Coombe DR: Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization. Anal Biochem 2002, 310:199-207. 54. Massia SP, Stark J: Immobilized RGD peptides on surfacegrafted dextran promote biospecific cell attachment. J Biomed Mater Res 2001, 56:390-399. 55. Rowley JA, Sun Z, Goldman D, Mooney DJ: Biomaterials to spatially regulate cell fate. Adv Mater 2002, 14:886-889. 56. Quirk RA, Chan WC, Davies MC, Tendler SJ, Shakesheff KM: Poly(L-lysine)-GRGDS as a biomimetic surface modifier for poly(lactic acid). Biomaterials 2001, 22:865-872. 57. Lee KB, Yoon KR, Woo SI, Choi IS: Surface modification of poly(glycolic acid) PGA for biomedical applications. J Pharm Sci 2003, 92:933-937. 58. Alsberg E, Anderson KW, Albeiruti A, Rowley JA, Mooney DJ: Engineering growing tissues. Proc Natl Acad Sci USA 2002, 99:12025-12030. RGD-coupled alginate hydrogels have been utilized to cotransplant chondrocytes and osteoblasts, leading to the formation of complex tissues resembling growth plates. 59. Miyamoto S, Teramoto H, Coso OA, Gutkind JS, Burbelo PD, Akiyama SK, Yamada KM: Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J Cell Biol 1995, 131:791-805. 60. Irvine DJ, Mayes AM, Griffith LG: Nanoscale clustering of RGD peptides at surfaces using comb polymers 1. Synthesis and characterizatioin of comb thin films. Biomacromolecules 2001, 2:85-94. 61. Koo LY, Irvine DJ, Mayes AM, Lauffenburger DA, Griffith LG: Co-regulation of cell adhesion by nanoscale RGD organization and mechanical stimulus. J Cell Sci 2002, 115:1423-1433. The nanoscale organization of immobilized RGD peptides regulates cell adhesion and its strength.
Current Opinion in Biotechnology 2003, 14:559–565
Protein-based signaling systems in tissue engineering Tanyarut Boontheekul and David J Mooney Tissue engineering aims to replace damaged tissues or organs using either transplanted cells or host cells recruited to the target site. Protein signaling is crucial to regulate cell phenotype and thus engineered tissue structure and function. Biomaterial vehicles are being designed to incorporate and locally deliver various molecules involved in this signaling, including both growth factors and peptides that mimick whole proteins. Controlling the concentration, local duration and spatial distribution of these factors is key to their utility and efficacy. Recent advances have been made in the development of polymeric delivery systems intended to achieve this control. Addresses University of Michigan, 1011 North University Avenue, 5213 Dental Building, Ann Arbor, MI 48109-1078, USA e-mail: [email protected]
Current Opinion in Biotechnology 2003, 14:559–565 This review comes from a themed issue on Tissue and cell engineering Edited by Jeffrey Hubbell 0958-1669/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2003.08.004
Abbreviations BMP bone morphogenetic protein EC endothelial cell ECM extracellular matrix EGF epidermal growth factor FGF-2 fibroblast growth factor 2 NGF nerve growth factor PDGF platelet-derived growth factor PEG polyethylene glycol PGA poly(glycolide) PLA poly(L-lactide) PLGA poly(lactide-co-glycolide) PlGF placental growth factor TGF transforming growth factor VEGF vascular endothelial growth factor
Introduction Tissue development is regulated through the interplay of a variety of signals, including soluble signaling molecules, insoluble ligands, mechanical cues and cell–cell interactions [1]. Numerous peptides and proteins involved in this signaling possess a biological activity that marks them as potential therapeutics. Soluble growth factors and immobilized ligands can regulate the adhesion, migration, proliferation and differentiation of various cell types. These factors may be a key to success in tissue engineering, an emerging field combining engineering and life www.current-opinion.com
sciences with the goal of repairing damaged or lost tissues [2]. Tissue regeneration is affected by either transplanted or host cells, in concert with local environmental signals. One approach to direct the process of tissue formation is the incorporation of bioactive components, including growth factors and peptides mimicking the function of extracellular matrix (ECM) molecules, into biomaterials [3]. Advances in material engineering have led to new modes of presenting these molecules to control the cell response and new tissue development. Transplantation of cells that secrete the desired growth factors is also a promising strategy to deliver these bioactive proteins [4], as are approaches based on gene therapy [5]. However, these latter approaches will not be reviewed in this article. We review here recent advances in the development of delivery materials for two types of signals: growth factors and immobilized peptides (Figure 1). Growth factor delivery has advanced to the clinic and will be categorized by the specific therapeutic applications. Immobilized peptide presentation is not as clinically advanced and fundamental issues important to its ultimate clinical utility, including peptide biology and immobilization techniques, are discussed as well as potential therapeutic applications.
Growth factor delivery The development of appropriate delivery vehicles for growth factors will be crucial for their clinical utility. Growth factors, owing to their control of many biological processes, are finding wide-spread use in the regeneration of many tissue types. Typically, recombinant versions of the desired proteins are manufactured and delivered in solution form, either systemically or via direct injection into the tissue site of interest. However, growth factors typically have a short half-life once they are introduced into the body and are rapidly eliminated [6]. This is problematic, as the target cell population must often be exposed to factors throughout the entire course of repair, or at least for an extended period. To address this challenge, controlled delivery systems that incorporate the growth factors into polymeric biomaterials have been developed to prolong the tissue exposure time and to maintain growth factor stability. In addition to the duration of tissue exposure, the amount and timing can be crucial to the biological response. The release rate of growth factors from polymers is typically controlled by the diffusion of the factor or polymer degradation [7]. Among the most commonly used materials are synthetic polymers such as poly(L-lactide) (PLA), poly(glycolide) (PGA) and their copolymers poly(lactide-co-glycolide) Current Opinion in Biotechnology 2003, 14:559–565
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Figure 1
(a)
(b)
[Peptide]
[GF]
Distance
Distance
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Delivery of two types of protein-based signaling (growth factors and immobilized peptides) is accomplished with distinct approaches. (a) Growth factors (red circles) are typically encapsulated within a polymer and subsequently released. The released growth factor diffuses through the surrounding tissue where it binds to receptors (green) on cells. This mechanism creates a concentration gradient of the factor that might direct cell movement. GF, growth factor. (b) By contrast, peptides (blue diamonds) mimicking ECM molecules are typically covalently coupled to the material and only cells in direct contact with the surface of the material can bind to the peptides with appropriate cell-surface receptors (green). This leads to a very localized regulation of cellular activity.
(PLGA), in the physical forms of microspheres or sponges [8]. Hydrogel-forming materials, including collagen [9], alginate [10], polyethylene glycol (PEG) [11] and PLAPEG [12], are also frequently used. To address the instability of proteins immobilized within polymers, a variety of stabilization techniques have been developed [13,14]. Recent progress in developing delivery vehicles for growth factors in the three most clinically advanced applications are discussed in the following sections. Neovascularization
Neovascularization is crucial in treating many diseases (e.g. coronary artery disease) and in virtually all approaches to tissue engineering. A lack of vascularization leads to insufficient nutrient delivery and waste removal, cell death and limited tissue development and tissue loss. Angiogenesis, new blood vessel formation by sprouting from the sides and ends of pre-existing microvascular vessels, has been widely studied to determine the rules guiding blood vessel formation in the adult. Numerous
growth factors have been identified as regulators of this process (summarized in Table 1). Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, as it is a potent mitogen for endothelial cells (ECs) and induces EC migration and sprouting by upregulation of several endothelial integrin receptors [15]. There is a family of VEGF growth factors, VEGF-A through VEGF-E, and two isoforms of VEGF-A (VEGF121 and VEGF165) have progressed to clinical trials in the treatment of ischemic diseases [16]. Placental growth factor (PlGF), another VEGF family member, can also stimulate angiogenesis [17], but its potential side effects remain unclear. Various polymeric materials have been used for the controlled release of VEGF, including PGA, alginate and fibrin [8,10,18]. It has recently been noted that the VEGF release rate from polymers can be responsive to the local mechanical environment [10], and this may be crucial in applications involving a mechanically dynamic environment (e.g. heart tissue).
Table 1 Growth factors commonly used in therapeutic angiogenesis. Growth factor
Abbreviation
Relevant known activities
Vascular endothelial growth factor Basic fibroblast growth factor Platelet-derived growth factor Angiopoietin-1 Angiopoietin-2 Placental growth factor Transforming growth factor
VEGF bFGF-2 PDGF Ang-1 Ang-2 PlGF TGF
Migration, proliferation and survival of ECs Migration, proliferation and survival of ECs and many other cell types Promotes the maturation of blood vessels by the recruitment of smooth muscle cells Strengthens EC–smooth muscle cell interaction Weakens EC–smooth muscle cell interaction Stimulates angiogenesis Stabilizes new blood vessels by promoting matrix deposition
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Fibroblast growth factor 2 (FGF-2), another well-studied angiogenic factor, elicits diverse biological effects on numerous cell types, including ECs, and has been used in clinical trials to induce angiogenesis [19]. FGF-2 has been incorporated into various polymers, such as gelatin [20], collagen [21], chitosan [22] and PLA [23]. FGF-2 reversibly binds to heparin-like molecules and heparin sulfate proteoglycans. This can be exploited to increase the FGF binding capacity to polymeric delivery vehicles, resulting in a more gradual and sustained release of FGF and an enhancement of angiogenesis [21]. The dominant growth factor delivery approach in angiogenesis utilizes single growth factors, but this may not be the ideal approach to replicate this complex process. Angiogenesis results from a complicated series of interactions involving diverse cytokines, growth factors, cells and proteases, acting in a consecutive, concerted or synergistic manner [16]. Recent advances in polymeric delivery systems allow one to locally and controllably deliver multiple growth factors with controlled doses and rates of delivery. Delivery of VEGF and plateletderived growth factor (PDGF) at distinct rates and doses results in a dramatic increase in the maturity of engineered vessel networks [8]. Similarly, delivery of FGF– PDGF combinations synergistically induces stable vascular networks, whereas single growth factors are unable to maintain these newly formed vessels [24]. It must be noted that interactions between various growth factors may complicate interpretation of the results of their delivery, as PDGF and nerve growth factor (NGF) can raise VEGF secretion by cells [25,26]. Recent studies also illustrate interplay between VEGF, Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) in regulating angiogenesis [27]. Bone regeneration
A primary goal of growth factor delivery for bone tissue engineering is to accelerate the regeneration process and engineer bone in situations that normally lead to incomplete regeneration. Several growth factors that are intimately involved in bone formation and remodeling have been identified and are summarized in Table 2. The transforming growth factor (TGF) family of proteins have an essential role in bone formation through the
regulation of osteoprogenitor and osteoblast proliferation and differentiation [28]. A variety of materials have been used for TGF-b1 delivery, including PLGA-PEG and coral [28,29], and combined TGF-b1 and insulin-like growth factor (IGF) release from PLA carriers accelerates osteotomy healing [30]. However, the optimal conditions for TGF-b1 release during bone regeneration have yet to be defined. Bone morphogenetic proteins (BMP), members of the TGF superfamily, appear to act as differentiation factors, causing mesenchymal cells to differentiate into bone-forming cells [31]. Recombinant human BMP-2 and BMP-7 are currently in clinical use as osteoinductive agents [31]. Several carriers for BMP have been developed, including collagen [32], PLGA [33], PLA–p-dioxanone–PEG block copolymers [34], PLA-PEG [12] and PEG hydrogels [11]. These systems allow control over the BMP release rate and promote pre-osteoblast differentiation and mineralization in vitro and ectopic bone formation in vivo [11,12]. Other growth factors involved in bone regeneration include FGF and PDGF, and a variety of materials have been used for their delivery [35,36]. Wound healing
There is a significant need to enhance the healing of skin tissue resulting from ulcers, scars or burns. The therapeutic potential of several growth factors in wound healing (Table 3) has long been recognized [37]. PDGF is the first mediator to appear at the wound site and is active in all stages of the healing process. Delivery of PDGF by a carboxymethylcellulose-based gel (Regranex1) is employed for the treatment of diabetic foot ulcers and was the first growth factor system for tissue engineering to be approved by the Food and Drug Administration (FDA) [37]. Both epidermal growth factor (EGF) and TGF-b regulate keratinocyte phenotype [38], and therefore play an important role in the wound healing process. Sustained and localized EGF delivery substantially decreases the size of the wound [9,39], while released TGF-b inhibits epithelial cell proliferation [40]. FGF-2 sustained release also enhances skin regeneration [41].
Immobilized ligand signaling Insoluble ECM molecules clearly regulate local cellular activity, and many functions of the ECM can be mimicked by small peptide fragments of the entire molecules [42,43]. These fragments can be produced
Table 2 Growth factors commonly used in bone regeneration. Growth factor
Abbreviation
Relevant known activities
Transforming growth factor-b Bone morphogenetic protein Insulin-like growth factor Fibroblast growth factor-2 Platelet-derived growth factor
TGF-b BMP IGF-I FGF-2 PDGF
Proliferation and differentiation of bone-forming cells Differentiation of bone-forming cells Stimulates proliferation of osteoblasts and the synthesis of bone matrix Proliferation of osteoblasts Proliferation of osteoblasts
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Table 3 Growth factors commonly used in wound healing. Growth factor
Abbreviation
Relevant known activities
Platelet-derived growth factor Epidermal growth factor Transforming growth factor-b
PDGF EGF TGF-b
Fibroblast growth factor
FGF
Active in all stages of healing process Mitogenic for keratinocytes Promotes keratinocyte migration, ECM synthesis and remodeling, and differentiation of epithelial cells General stimulant for wound healing
synthetically and covalently coupled to synthetic polymers so as to present them in a solid-state form, upon which their activity often depends, and to infer biological activity to the synthetic materials. Cellular attachment to ECM molecules is crucial for the survival, growth and determination of a differentiated phenotype for anchorage-dependent cells. Many of these processes are mediated through ECM–integrin receptor binding. The integrin binding capability of ECM molecules has been mapped to specific oligopeptide sequences within ECM proteins, and these sequences include RGD, IKVAV and YIGSR (in single-letter amino acid code) [44,45]. Advantages of employing short peptides containing these signaling motifs to modulate cell function, rather than utilizing whole adhesive proteins (e.g. fibronectin), include the ease and reproducibility of synthesizing peptides, as compared with isolating ECM molecules from a natural source. Utilization of only a small fragment of an ECM molecule may also allow one to target an interaction with a specific cell population [3]. A limitation to this approach, however, is that the biological activity of short peptide sequences is often substantially lower than that of the complete protein, owing at least partially to the absence of complementary domains that are involved in integrin binding [46]. Utilizing larger ECM molecule fragments, produced recombinantly, may represent a robust approach to increase activity, while still offering advantages over using the entire molecule (e.g. reduced antigenicity) [47]. A key feature of natural ECM molecules is their susceptibility to celltriggered proteolysis, which permits cell invasion and subsequent remodeling of the matrix. Invasion and remodeling depend on the action of cell-secreted proteases that target specific sequences of the ECM molecules [48]. Recent developments indicate that it is possible to synthesize cell-remodelable synthetic materials by utilizing these sequences as cross-linking agents [42]. Immobilization techniques
Peptide immobilization on polymers can be achieved by simple adsorption, standard covalent chemical conjugation or enzymatic incorporation [49,50,51]. In situ peptide adsorption can be optimized through appropriate peptide design (e.g. inclusion of hydrophobic domains [49]). Peptides can also be covalently conjugated using Current Opinion in Biotechnology 2003, 14:559–565
carbodiimide chemistry, Michael-type addition or photochemical grafting [42,50,52]. There are many possible coupling techniques resulting from the variety of active chemical groups on peptides. The method of immobilization has an effect on both the receptor binding capacity and affinity [53]. Moreover, it has been demonstrated that peptides can be readily incorporated into fibrin matrices enzymatically [51]. To present the peptides with a high signal-to-noise ratio, the polymer chosen for peptide presentation should not mediate significant adsorption of contaminant proteins. The most commonly used polymer for low protein binding and cell adhesion resistance is poly(ethylene oxide). Poly(ethylene oxide) has hydrophilic and electrically neutral properties, and can be combined with other biomaterials to form block polymers with controlled structures [49]. Dextran-based gels and conjugates also have excellent biocompatibility and ability to limit cell adhesion, and have more surface-binding sites than PEG [54]. In addition, other hydrophilic polymers such as alginate can be employed, as alginate hydrogels discourage nonspecific protein adsorption [55]. The lack of chemically modifiable sites in synthetic aliphatic polyesters (e.g. PGA) makes it more complex to attach peptides; however, incorporation of monomer units containing appropriate functional groups into the polymer backbone can overcome this problem [56]. Alternatively, carboxylic acid groups can be generated on the surface by hydrolysis to allow for covalent attachment of ligands via amide bond formation [57]. Regulation of cell phenotype and tissue regeneration
Peptide-incorporating biomaterials (Table 4) have demonstrated control over the adhesion, proliferation and differentiation of various cell types, including fibroblasts, chondrocytes, osteoblasts, myoblasts, endothelial, smooth muscle and neuronal cells [42,49,54–56,58]. Several parameters involved in the immobilization process, including the type of peptide, peptide concentration, spacer arms, immobilization technique and spatial distribution of peptides, can regulate the cellular response [49,50,51,53]. The nanoscale organization of the peptide may also be a key to control cellular response, as intracellular signaling is typically only triggered when integrins are clustered in the cell membrane www.current-opinion.com
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Table 4 Immobilized ligands used in tissue engineering. Immobilized ligand
ECM molecule source
Application
RGD
Multiple ECM molecules, including fibronectin, vitronectin, laminin, collagen and thrombospondin Laminin Fibronectin Common MMP substrates, (e.g. collagen, fibronectin, laminin)
Enhance bone and cartilage tissue formation in vitro and in vivo Regulate neurite outgrowth in vitro and in vivo Promote myoblast adhesion, proliferation and differentiation Enhance endothelial cell adhesion and proliferation Regulate neurite outgrowth in vitro and in vivo Promote formation of focal contacts in pre-osteoblasts Encourage cell-mediated proteolytic degradation, remodeling and bone regeneration (with RGD and BMP-2 presentation) in vivo
IKVAV YIGSR RNIAEIIKDI Recombinant fibronectin fragment (FNIII7–10) Ac-GCRDGPQ-GIWGQDRCG
Sequences are given in single-letter amino acid code. MMP, matrix metalloproteinase.
[59]. Controllable nanoscale clustering of RGD peptides tunes cell adhesion [60] and can be used to study the effects of ligand density and clustering on cell adhesion and the strength of the adhesion [61]. An important advance over the past few years was the demonstration that peptide immobilization to materials can regulate in vivo events, and specifically enhance bone and nerve regeneration [50,51,58]. RGD-coupled alginate hydrogels can control osteoblast and chondrocyte differentiation in vitro and the formation of cartilaginous and bony tissues in vivo [50,58]. Strikingly, co-transplantation of both cell types together in this material leads to the formation of growing tissues that structurally and functionally resemble a growth plate [58]. The integration of RGD peptides and matrix metalloproteinase (MMP) substrates into a single material has been pursued to form a cell-responsive BMP-2 delivery vehicle, and this system can be used to regenerate bone tissue [42]. The proteolytic sensitivity of this system can be readily controlled to regulate cell infiltration and bone formation [43]. Further, fibrin modified with laminin-derived peptides has been shown to substantially enhance axon regeneration [51].
Summary and future directions Biomaterials are being increasingly designed to contain intrinsic bioactivity, through specific biological recognition mediated by released growth factors and immobilized peptides. Growth factor delivery has become a therapeutically powerful approach, and has been applied to neovascularization, bone regeneration and skin regeneration. Although certain growth factors (e.g. VEGF and BMP) have been widely studied and used in clinical trials or products, many additional growth factors (e.g. PlGF and NGF) and the various isoforms of the currently utilized growth factors have not been completely characterized. Many of these factors are likely to have therapeutic application in the future. Further, many growth factors have multiple actions and new therapeutic applications of the known growth factors are likely to emerge. A limitation of current growth factor delivery approaches www.current-opinion.com
is the focus on delivery of single growth factors, which may not be an ideal means to drive the highly regulated networks that lead to tissue regeneration. The development of multiple growth factor delivery systems that control the temporal availability of the factors has recently shown utility in angiogenesis [8], and this concept may be useful for other applications. Although less clinically advanced, peptide immobilization onto materials provides another approach to regulate cellular function in a very localized yet flexible manner. Recently, the utility of this system has been examined in the context of bone and nerve regeneration [51,58], and additional therapeutic applications will clearly emerge in the near future. Combining these multiple types of information into single biomaterials [42,55] will even more closely mimic the multifunctional nature of the native ECM and will be a powerful approach to regulate tissue regeneration and engineering.
Acknowledgements We wish to acknowledge financial support from the NIDCR/NIH to the laboratory of DM and a Royal King Anantamahidol Scholarship (Thailand) to TB.
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