Growth factor engineering for biomaterials - ACS Biomaterials Science

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Growth factor engineering for biomaterials Yoshihiro Ito ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01649 • Publication Date (Web): 20 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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definitively concluded because it was uncertain whether immobilization was complete. This was the result of a lack of progress in biomaterials science and evaluation parameters for biosignaling effects, which were unsuitable for measuring real activity of immobilized polypeptides.17-20 Early investigations of immobilized biosignals were reviewed by Venter in 1982.21 Once biosignaling polypeptides became recognized as GFs and important factors for the construction of non-serum-containing cell culture media, GF immobilization commenced a new era for biomaterial factors in 1991.22,23 Since this time, biomaterial studies have provided new tools for both fundamental investigation of GF mechanisms of action and the development of biomaterial applications. With regard to cell biology, in addition to basic cell signaling mechanisms such as paracrine, endocrine, and autocrine, the presence of juxtacrine or matricrine signaling by membrane-bound GFs has been revealed, thus supporting the notion of immobilized GFs as an “artificial juxtacrine” signal.24 2. Immobilization of growth factors 2.1.

Immobilized and soluble growth factors Tissue engineering comprises three components: cells, matrix, and soluble GFs,

as shown in Figure 1. The concept of GF immobilization involves the fusion of matrix and GF. As augmenting or controlling biological activities such as growth enhancement and differentiation, which are related to gene expression through nuclear signal transduction, is considered to be difficult on artificial matrices, studies using GF immobilization have opened a new field of biological materials. Figure 1 Accurate quantitative evaluation of precisely immobilized amounts of GF is required to compare the effects of an immobilized GF with that of its soluble form. To this end, photolithography methods have been developed to visualize the effect of immobilized GFs using micropatterning (Figure 2).25 For example, this method was used to demonstrate that immobilization of insulin,26 epidermal growth factor (EGF),27,28 and vascular endothelial growth factor (VEGF)29 enhanced cell proliferation only on immobilized areas. In addition, when the immobilized region is smaller than the size of an individual cell, only the contact area was partially activated by immobilized EGF.28 Notably, the capacity for repeated utilization of an immobilized GF further supports the benefits of immobilization, and is advantageous for deploying immobilization as a


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bioreactor with immobilized enzymes.23 Notably, GF-immobilized microcarriers did not affect the proliferation of anchorage-independent cells because of lack of cell adhesion, but remarkably promoted the proliferation of anchorage-dependent cells.30 Collectively, these results demonstrate the utility of GF immobilization. Figure 2 General tendencies of immobilized GFs compared with soluble GFs are illustrated in Figure 3. Direct comparison of the effects between soluble and immobilized GFs is difficult, because the former is three-dimensional (3D) while the latter is 2D. Therefore, the amount of GF present in medium is expressed on the x-axis. First, immobilization is considered to enrich local concentrations via direct contact with cell membranes; as such, an apparently lower amount of immobilized GF is sufficient for the same level of effect as its soluble form. Second, a multivalent effect of polymeric biosignaling molecules has been established, whereby opportunities for ligand-receptor interactions and dimerization of interacting complexes is increased.31-35 Third, and perhaps most importantly, growth is enhanced by immobilized GFs, which was not discussed in early investigations of immobilized biosignaling mechanisms. Enhanced growth has been explained by the inhibition of GF receptor downregulation caused by GF internalization.19,20 Notably, the interaction of cells with immobilized GFs requires their adhesion onto a substrate, which delays the triggering of downstream signaling compared with the direct interaction of soluble GFs; however, signaling continues for a longer period of time with immobilized GFs because of inhibited downregulation of GF receptor expression. Figure 3 Many researchers have reported the induction of prolonged intracellular signal transduction by immobilized GFs.1,7-15,36,37 Boucher et al. reported that cellular responses elicited by immobilized EGF were most likely caused by long-term activation of intracellular kinase pathways through the EGF receptor (EGFR).38 Functionally, Platt et al. showed that the response of primary human bone marrow-derived stromal cells to immobilized EGF was linked to long-term activation of EGFR.39 As a result, they showed that osteogenic differentiation of stromal cells was promoted.39 Notably, prolonged activation of cellular signal transduction systems was also elicited by immobilized insulin-like growth factor-1 (IGF-1).40-43 The effect of tethering has also been investigated for VEGF.29,44-49 Anchorage of



VEGF to the ECM depends on its molecular weight and ability to anchor to endothelial cells, a feature conveyed by different signaling responses such as growth and migration.45 ECM-bound VEGF elicited prolonged receptor activation.46 Maia et al. demonstrated induction of prolonged intracellular bioactivity by VEGF-functionalized dextran compared with unmodified VEGF in endothelial cells.47 Moreover, a study using covalently immobilized VEGF showed that internalization of VEGF is not required for phosphorylation (activation) of VEGF receptor-2 in the cell membrane.48 Suzuki et al.49 reported that endothelial progenitor cells selectively adhered to VEGF-tethered surfaces and their differentiation was induced by arterial shear stress. Fibroblast growth factor (FGF)-2 immobilized on amine-modified nanofibrildeposited surfaces more potently elicited increases in cellular proliferation and viability compared with its soluble form through activation of FGF receptor and related signal transduction pathways.50 Arisaka et al.51 showed that heparin-functionalized surfaces clearly suppressed FGF-2 internalization within cells compared with soluble FGF-2. Heparinized surfaces with stably bound FGF-2 inhibited downregulation of FGF receptors and yielded continuous activation of downstream signal transduction. Schwab et al., who reported tethering of bone morphogenetic protein (BMP)-2, observed prolonged and elevated Smad signaling pathway activation compared with the same amount of soluble BMP-2.52 Additional results demonstrating that GF immobilization induces phenomena not observed with soluble GFs illustrate the importance of immobilization effects. In addition to quantitatively increasing effects such as growth enhancement, as described above, immobilized EGF induced qualitatively distinct effects from soluble EGF in some cells. Notably, immobilized EGF elicited not only mitogenic, but also morphological responses (rounding up) of primary hepatocytes.53,54 Moreover, cells cultured on tethered EGF reportedly exhibited directed migration with greater persistence compared with soluble EGF.55 In addition, cells incubated with soluble EGF demonstrated higher phosphorylation of extracellular signal-regulated kinase (ERK)1/2, while cells cultured on immobilized EGF exhibited higher expression of pPLC 1 (a member of phosphoinositide-specific phospholipase C), which was localized at the leading edge. This difference was considered to cause increased migratory activity. As another representative example, a qualitative switching phenomenon was reported. It is generally known that growth of the rat pheochromocytoma cell line PC12 is stimulated by soluble EGF, while its differentiation is stimulated by soluble nerve growth factor (NGF). The difference is explained by the activation period: EGF is short and NGF is long. Immobilized EGF stimulated PC12 differentiation through prolonged


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stimulation of intracellular signaling proteins mitogen-activated protein kinase (MAPK) and p38 (a member of the MAPK superfamily) in cells, as did soluble NGF (Figure 4A and 4B).

56

This switching between growth and differentiation stimulation is simply

explained by the length of activation period rather than the difference of GF. At the same time, it was demonstrated that GF immobilization can induce qualitatively different effects on cells. Figure 4 Apart from inhibiting the downregulation of GF receptor with immobilized GFs, EGF-immobilized nanoparticles (NPs) were shown to enhance the apoptotic efficacy of ligands (Figure 4C).57 The apoptotic efficacy of nano-conjugated EGF significantly depended on the size and morphology of NPs. Specifically, spherical NPs with an approximately 80-nm diameter induced apoptosis much more efficiently compared with smaller spherical NPs or rod-shaped NPs. Immobilization methods used in the studies described above progressed from chemical methods to protein engineering, as shown in Figure 5. Figure 5 2.2.

Chemical immobilization methods

2.2.1 Direct binding of growth factors to substrates Numerous GFs immobilized on various materials have been investigated for their effects. In addition to general GFs, cytokines (such as erythoropoetin58) and other types of biosignaling molecules (such as Shh,32 ephrin,33 Notch ligand,59 and Wnt60) have been immobilized and investigated for their effects on cells. Researchers have tried various immobilization strategies ranging from classical methods to new techniques. Guex et al.61 developed scaffolds with covalently bound VEGF using fibrous non-woven poly(epsilon-caprolactone) for immobilization of VEGF via the formation of stable amide bonds (standard carbodiimide chemistry) after surface treatment. Upon evaluating the mitogenic effect of immobilized VEGF on endothelial cells in vitro, they observed significant growth on immobilized surfaces. Princz and Sheardown immobilized EGF and heparin-binding EGF (HB-EGF) on dendrimercrosslinked collagen gels via carbodiimide chemistry, either during or following gel fabrication.62 Bioactivity of EGF and HB-EGF was enhanced following step-wise conjugation to gels, as verified by the proliferation of human corneal epithelial cells.




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Gradient formation is an important technique for quantitative investigation of immobilized GFs. Odedra et al. demonstrated VEGF gradient immobilization on porous collagen scaffolds using a flow method.69 Chen et al. developed a gradient surface of immobilized EGF by photolithography (Figure 6A).70 To photochemically immobilize NGF and semaphorin 3A (Sema3A), two known regulators of neural formation, on chitosan film, Wijekoon and Leipzig71 synthesized a compound carrying sulfhydryl and amine photoreactive ends. They found that in vitro outgrowth of embryonic chick dorsal root ganglia (DRG) was increased in response to the covalently tethered states compared with the adsorbed states of NGF and Sema3A. Subsequently, Joddar et al.72 prepared NGF and Sema3A gradient surfaces using the photolithographic method developed by Chen et al.70, and found that localized concentration gradients of these chemotropic factors directed axonal outgrowth from DRG in a gradient-dependent manner. Figure 6 In addition, concentration gradients of GFs were prepared for investigation of cell invasion in breast cancer, as such gradients are observed in the tumor microenvironment. To photochemically immobilize EGF gradients, Fisher et al. synthesized a hyaluronic acid-based hydrogel composed of matrix metalloproteinasecleavable peptides and multiphoton labile nitrodibenzofuran.73 They showed that cell invasion on pattern-immobilized EGF gradients depended on the level of EGFR expression in cells. Moreover, they used a gradient method to demonstrate that the EGFR inhibitor cetuximab decreased the invasiveness of moderate EGFR-expressing cells, but increased the invasiveness and number of high EGFR-expressing cells. 2.2.3.2 Plural immobilization To enhance the activity of immobilized GFs, immobilization on cell-adhesive matrix or co-immobilization with adhesion factors has commonly been performed (Figure 6B).74,75 The interaction of integrin and GF receptors is very important for the design of immobilized GFs. Fernandes-Cunha et al.76 reported photochemical immobilization of GFs with collagen in the presence of riboflavin under the irradiation of visible light (ca. 458 nm). Riboflavin was employed as a photosensitizer. EGF was immobilized onto collagen-coated surfaces or directly onto endogenous collagen from porcine corneas. Using co-immobilization, EGF was maintained on collagen surfaces for over a week. As a result, immobilized EGF enhanced in vitro proliferation and migration of corneal epithelial cells, and supported the maintenance of a normal cell phenotype.



Ettelt et al.77 used a biotin-streptavidin coupling system to modify TiO2 surfaces with immobilized GFs to promote bone and soft tissue growth. As a result, they found that TiO2 with immobilized FGF-2 and heparin selectively increased the growth of fibroblasts, whereas immobilized BMP-2 promoted osteoblast proliferation. Furthermore, co-immobilization of GF and fibronectin yielded increased osteoblast adhesion compared with either protein alone. In the future, combined tethering of different types of ECM proteins will be useful to further influence cell-specific reactions of implants. Immobilization of plural GFs can also yield important enhancements of effects (Figure 6C). Chie and Radisic78 showed that VEGF and angiopoietin-1 covalently bound onto a porous collagen matrix enhanced vascularization. Mao et al.79 immobilized three reported GF replacements for serum [FGF-2, transforming growth factor-beta (TGF- , and platelet-derived growth factor (PDGF)] with photo-reactive gelatin for in vitro expansion of human mesenchymal stem cells (hMSCs) in serum-free culture medium. Use of multiple immobilized GFs supported cell growth in the absence of serum at the same level of serum-containing medium. In addition, immobilization significantly improved the stability of GF reagents, as the storage time of GFs was prolonged by immobilization at both 4°C and 37°C. Moreover, reutilization is an advantage of immobilization, as reagents can be repeatedly used without losing their biological activity on cells. Thus, multiple GF immobilization is a useful method for serum-free cell culture systems. For treatment of spinal cord injury (SCI), recovery of the central nervous system (CNS) is important. To promote the regeneration of new CNS tissue, Li et al.80 created a unique spinal cord bridge using a methacrylamide chitosan (MAC) hydrogel containing adult neural stem/progenitor cells. They incorporated recombinantly produced IFN- and PDGF-AA carrying biotin at the N-terminal. The resulting streptavidin-functionalization of MAC induced neuronal or oligodendroglial lineages in a model rat of SCI. 3. Design of binding growth factor Apart from chemical immobilization of GF proteins, protein engineering has been performed to augment the binding ability of GFs such that engineered GFs can be directly applied or used for surface modification of non-biological materials, as shown in Figure 7.

1-15

As biological binding targets, collagen, gelatin, fibrin, fibrinogen, cell

adhesion molecules (e.g. integrins or specific receptors), and cellulose have been reported. In addition, artificial substrates including organic and inorganic materials have been reported.1-15 Binding modes are classified into covalent and non-covalent bonding. Figure 8 summarizes the design of GF proteins with binding properties.


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Figures 7 and 8 3.1 Derivation from natural proteins Since the first report by Tuan et al.81, various types of chimeric proteins have been designed and prepared for non-covalent immobilization to add or enhance the binding affinity of GFs. The first example involves fusion of biologically active TGFto a collagen-binding domain (CBD) produced from Escherichia coli. Subsequently, Marino et al. identified a super-affinity peptide sequence to ECM in placenta growth factor.82 Kitajima et al.83 fused hepatocyte growth factor (HGF; an angiogenic factor) with a CBD polypeptide derived from fibronectin. The CBD-fused HGF, which was prepared using a baculovirus expression system, exhibited significantly increased collagen-binding affinity compared with unmodified HGF. As a result, CBD-HGF significantly promoted the proliferation of endothelial cells. Transplantation of collagenbased sponges bound with CBD-HGF in rats enhanced blood vessel formation 4–6 times as extensively as control sponges with native HGF after 7 days. Binding GFs have since been conjugated to various matrices for diverse applications.84-87 As HGF is a multipotent neural factor, Yamane et al.88 employed CBDHGF for the treatment of SCI. The bound HGF remained in the SCI model for 7 days even with only a single administration. This group later applied a visible-light-curable gelatin hydrogel with CBD-HGF,89and found that the combination more effectively promoted endogenous repair and recovery than the hydrogel containing unmodified HGF. 3.2 Artificially designed polypeptides 3.2.1 Non-covalent binding (known interaction) As an alternative to natural binding sequences, various artificially designed tags or peptide sequences have been conjugated to GFs.90,91 Instead of biotinylation, Yoo et al.92 employed biotin-like peptides (HPQ) to immobilize GFs. M13 phages were genetically modified to express HPQ and/or an integrin-binding peptide (RGD). The prepared phages, which formed nanofibrous matrices even after the modification of coat proteins, were immobilized with streptavidin-conjugated GFs such as FGF-2 and NGF. The results demonstrated synergistic effects of immobilized GFs and integrin-binding peptides. Boucher et al.37 used a coiled-coil interaction to prepare tethered EGF, which elicited enhanced growth of human corneal epithelial cells compared with physically adsorbed or soluble EGF. Murschel et al.93, who utilized tags to directly capture GFs on



organic substrates via coiled-coil interactions, efficiently adsorbed a tagged form of VEGF by poly(allylamine)-functionalized polystyrene or poly(ethylene terephthalate) film through electrostatic and hydrophobic interactions. The adsorbed VEGF maintained its biological activity and was gradually released over several days. Addi et al.94 extended the coiled-coil capture of GFs to FGF-2, and immobilized EGF and FGF-2 on collagenbased biomaterials using two high-affinity interactions: a coiled-coil interaction and CBD-gelatin interaction. They designed a chimeric protein carrying a coil-forming peptide and CBD derived from fibronectin. Chimeric proteins were prepared and the tethered GFs maintained their biological activities. Protein-based nanoparticles were developed by Kobatake’s group using genetically engineered elastin-like polypeptides (ELPs).95 They fused ELP with poly(aspartic acid) tails (ELP-D) and displayed EGF on the resulting ELP-D nanoparticles. Peptide sequences capable of forming a coiled-coil structure were designed and used to couple ELP-D with single-chain VEGF, thus facilitating non-covalent tethering. In addition, they developed another GF-tethered ECM comprising peptide motif repeats derived from elastin and the cell-adhesive RGD peptide.96 To conjugate FGF-2 onto the ECM, an acidic amino acid-rich sequence consisting of five repeats of four aspartic acids and a serine (DDDDS) was introduced at the C-terminus of the ECM protein. As FGF-2 has a highly basic amino acid domain, FGF-2 was non-covalently bound to the modified ECM protein by electrostatic interactions. Cells adhered well to the FGF-2-tethered ECM and grew without the addition of soluble FGF-2. Thatikonda et al.97 proposed a gene-level strategy to covalently conjugate FGF2 to a spider silk protein. The designed and prepared FGF-2 fusion silk protein selfassembled into silk-like fibers or was used for coating surfaces. The coated silk-FGF-2 bound its cognate receptor and enhanced cell adhesion. Primary human endothelial cells were successful cultured on the coated matrix or fibers and integrated without GF supplementation. 3.2.2 Covalent binding (known interaction) Genetically modified GFs have also been employed for covalent immobilization. To realize and optimize an antiapoptotic coating for vascular applications, EGF was combined with chondroitin sulfate. Riahi and Murschel98 designed and prepared a chimeric EGF in which the N-terminal was fused with a cysteine or coil-forming peptide. In the former case, the chimeric EGF was covalently tethered through the thiol group. In the latter case, it was non-covalently immobilized on the surface through coiled–coil interactions. Upon comparing the interactions of immobilized EGFs, its receptor, and the


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(Figure 9 below). The selected CB-VEGF exhibited high collagen-binding affinity and promoted cell proliferation on collagen. As a result, it enhanced angiogenesis in skin wounds and infarcted myocardium in vivo. Figure 9

3.4 Generally adhesive growth factors In addition to specific binding of the GF, an “all around” adhesive GF is desired. To achieve this, engineers have looked to the many known underwater adhesive proteins.104 For example, mussels secrete 3,4-dihydroxyphenylalanine (DOPA)-rich proteins, which are adhesive to various materials.105-109 Salivary statherin, which contains phosphorylated serine residues in its active region, is an adhesive protein that binds to hydroxyapatite (HA) in teeth.110 Key amino acids (e.g. DOPA and phosphorylated serine) are usually incorporated into the protein by post-translational modification. Generally adhesive GFs can be designed by incorporating these peptides with non-natural amino acids (Figure 10). Using this approach, a wide range of organic and inorganic materials can be modified with various GFs. Figure 10 In the case of short peptides, it is possible to directly incorporate the abovementioned non-coding amino acids by a solid-phase synthetic method. Kang et al.40 used such a method to prepare a 54-residue peptide carrying phosphorylated serine residues. EGF conformation was not significantly changed by the conjugation of this binding peptide sequence. As a result, the binding affinity to HA and Ti was significantly increased, and the EGF construct maintained its mitogenic activity. The EGF-modified HA significantly promoted the proliferation of cells on the Ti surface. High molecular weight proteins are difficult to prepare using the solid-phase method. Therefore, bioorthogonal approaches are required to incorporate non-coding or non-natural amino acids.111,112 Sakuragi et al.113 designed a BMP-4 derivative incorporating phosphorylated serine residues. For ligation, sortase A was employed (Figure 11A). Both the recombinant BMP-4 and a peptide carrying recognition sites were designed for enzymatic ligation of the terminal amino acids of the polypeptide and peptide. The prepared BMP-4 derivative bound HA more efficiently than BMP-4 carrying canonical serine residues and elicited increased osteogenic activity of MSCs compared


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with cells cultured on HA alone. Figure 11 Zhang et al.41 used tyrosinase to incorporate DOPA into IGF-1 by adding a tyrosine-containing peptide sequence onto IGF-1 using recombinant DNA technology and enzymatic treatment (Figure 11B). As tyrosinase treatment induces hydroxylation of tyrosine residues in the resulting chimeric proteins, there is a concern that this treatment generally reduces its biological activity. However, it is known that the tyrosine residues in IGF-1 do not contribute to its activity, therefore the tyrosinase enzyme was used for modification. The modified IGF-1 exhibited strong binding affinity to Ti, as expected, and significantly promoted cell proliferation on the modified surface. 5. Growth factors with other physical factors Cellular microenvironments involve biochemical factors, ECM, and interactions with neighboring cells. Homeostasis of cellular microenvironments is dynamically maintained under physical conditions. Incorporation of GF signaling molecules onto materials, which involves combining biochemical factors and ECM, for the development of new biomaterials began in the 1990s. More recent research has investigated combinations of biochemical factors (such as GFs) with physical factors such as mechanotransduction.2,114-117 Specifically, understanding how to apply engineered interfaces with GFs and the resulting synergistic interactions of affected signaling pathways has become increasingly important. Indeed, by controlling surface properties such as hydrophilicity, stiffness, elasticity, mobility, geometrical patterns, and gradients of physical cues, immobilized GFs can be exploited to specifically induce cellular responses, such as growth or differentiation, from a solid phase by triggering specific signaling pathways. 5.1. Surface hydrophilicity Early work concerning the combination of GF and surface properties was reported by Ito et al.118 As a GF, insulin was immobilized on different hydrophilic/hydrophobic surfaces and the resulting effects on cell growth were investigated. The copolymer surface was tuned by altering compositions of hydrophilic and hydrophobic monomers. Without immobilization of insulin, too hydrophilic or hydrophobic surfaces reduced cell activities. Although a small amount of immobilized insulin did not affect cell behavior on different copolymer compositions, large amounts of immobilized insulin masked the influence of



copolymer composition and, as a result, equally enhanced cell growth. 5.2 Surface rigidity Cells are known to respond to the stiffness of their environment. Engler et al. reported that osteoblastic differentiation of cells was induced on stiff substrates over 25 kPa, but not on soft ones less than 10 kPa.119 Moreover, Couzier et al.120 showed that cells on stiff membranes respond similarly to BMP-2, regardless of it being bound to the surface or soluble in culture media, whereas cells were only responsive to matrix-tethered BMP-2, not diffusible BMP-2, on soft membranes. Toda et al.121 immobilized ephrinB2 onto different elasticity (1 to 70 kPa) of polyacrylamide (PAAm) hydrogels and the effect on cultured hMSCs was investigated. hMSCs on PAAm hydrogels tethered with ephrinB2 exhibited a cuboidal shape, while spherical shapes were primarily observed on nonconjugated PAAm, independent of hydrogel elasticity. In addition, Runx2 expression was promoted by immobilization of ephrinB2, with high expression observed on hydrogels with 3.6 and 5.9 kPa elasticity. Notably, use of these hydrogels significantly downregulated RhoA activity. 5.3 Surface geometry The ECM has unique structures in the nano- and micrometer range, and thus presents a variety of defined 3D geometries and topologies.122 Kawazoe et al. summarized geometric control of cells by photolithography, as well as subsequent regulation of cell behaviors (adhesion, growth, and differentiation).123 Cell spreading was regulated by the micropatterning of poly(vinyl alcohol) on polystyrene, with high spreading eliciting osteogenic differentiation, and low spreading promoting adipogenic differentiation of hMSCs. The shape of single cells was also regulated at the micro-scale level by the same method, and subsequent effects on hMSC differentiation were investigated. Precise control of cell protrusion was achieved by geometric patterning and the resulting cytoskeletal reorganization or changes in orientation were investigated in detail. 5.4 Surface mobility The mobility of an immobilized biosignal affects the function of contacting cells. Indeed, Zhou et al.124 demonstrated a correlation between the biological activity and membrane fluidity of chemically fixed substrates. Specifically, they developed chemically fixed murine embryonic fibroblasts (MEFs) as replacement for feeder cells to maintain the undifferentiated state of murine induced pluripotent stem cells (iPSCs).125 Chemical fixation is useful for ready-use and maintenance of MEFs. Their results


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demonstrated that the condition of chemically fixed feeder cells determined the membrane fluidity and stemness of contacting cells,125,126 as increased concentrations of fixation agent reduced membrane fluidity. For contacting iPSCs to maintain similar expression levels of pluripotency markers as observed on conventional feeder cells (mitomycin C-treated MEFs), a certain level of membrane fluidity was required. Thus, the mobility of immobilized biosignals is important for signal transduction of the contacting cell through interactions between cells and the immobilized biosignal. 5.5 Physical stimulations In addition to surface properties, many physical stimuli (e.g., mechanical127,128, electrical129,130, and ultrasonic131) have also been investigated. Lee et al. immobilized NGF on electrically conductive substrates to enhance neurite outgrowth using electrical stimulation.130 Considering that physical stimuli are already applied for medical treatments, understanding the use of physical stimulation in combination with immobilized biosignals may lead to new therapeutic devices. Indeed, the combination of GFs and physical stimuli should spawn an entirely new research field. 6. Future outlooks Engineered biomaterial systems to regulate cell fate are important for a broad range of applications in tissue engineering, regenerative medicine, cancer research, and drug development. As such, GF engineering is a key factor in biomaterial systems for the development of various medical treatments. Indeed, the development of advanced biological systems that make efficient use of GFs will be achieved in combination with key (physically and dynamically tunable) material properties. Considering that combinations of physical and regenerative therapies have not been thoroughly investigated, the present trend should significantly contribute to this interdisciplinary field. Moreover, as various strategies have already been developed for GF engineering, cooperative signaling by GFs and physical properties of the microenvironment have potential applications in various medical fields. Acknowledgements This work was financially supported by KAKENNHI from JSPS (15H01810, 16F16059, 16K12902, and 15F15054). We thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.



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Figure legends Figure 1 Three components of tissue engineering. Growth factor (GF) immobilization involves the combination of a matrix (scaffold) and growth factor. Figure 2 The effect of immobilized growth factors (GFs) was conveniently visualized by micropattern immobilization of a GF using optical microscopy. (A) Insulin was immobilized according to the photomask and growth of insulin-dependent cells was observed. However, only cells on immobilized regions responded and significantly grew. Reproduced with permission from ref 25. Copyright 1999 John Wiley. (B) Epidermal growth factor (EGF) was immobilized according to the photomask (size of immobilized regions was smaller than the size of cells), and activated signal transduction proteins in the part of the cell contacting immobilized regions were observed by staining. Partial activation corresponding to immobilized regions was confirmed. Reproduced with permission from ref 27. Copyright 1997 American Chemical Society. Figure 3 Schematic illustration of comparison between soluble and immobilized growth factors (GFs). (A) High local concentration. A lower amount of immobilized GF is sufficient to produce the same effect as a soluble GF because of a high local concentration. (B) Multivalent effect. By increasing the collision frequency between ligand and receptor, enhanced receptor dimerization rendered immobilized GFs more effective than soluble GFs. (C) Inhibition of downregulation. The saturation effect of an immobilized GF is higher than that of a soluble GF because of inhibited downregulation. Whereas the soluble GF is internalized after complex formation with its cognate receptor, as shown on top, the immobilized GF is not internalized, thus allowing signal transduction to continue for a longer period of time, as shown on bottom. Figure 4 (A) Schematic illustration of the time course of signal transduction induced by immobilized and soluble GFs. Although an immobilized GF exhibits lag time for interaction with its cellular receptor because cells must adhere to the substrateimmobilized GF, signal transduction continues for a long time without internalization. As a result, long-term activation by epidermal growth factor (EGF) was similar to that of nerve growth factor (NGF). (B) According to this mechanism, PC12 cells form neurites



on EGF-immobilized regions, although soluble EGF induces growth, not neurite formation. (C) Soluble and immobilized EGF induced different responses by cells. Soluble EGF enhanced growth, whereas EGF immobilized on nanoparticles induced apoptosis. Figure 5 Classification of immobilization methods described in this article. Figure 6 Strategies to enhance the effect of immobilized growth factors (GFs). (A) Gradient immobilization of GFs permits the visualization of surface density-dependent effects of immobilized GFs. (B) Co-immobilization of GFs with adhesion factors synergistically enhances the effect of immobilized GFs by collaborative stimulation of integrin and GF-specific receptors. (C) Co-immobilization of different GFs synergistically enhances the effect of immobilized GFs by multiplying the stimulation of specific GF receptors. Figure 7 Application of binding growth factors (GFs) prepared by engineering. One involves direct injection, while the other uses surface modification of artificial organs. Reproduced with permission from ref 6. Copyright 2014 Elsevier. Figure 8 Design strategies for the binding of growth factor (GF). Binding peptide sequences extracted from natural binding proteins or artificially designed peptides are joined with GF sequences. Figure 9 Design strategy for the binding of growth factor (GF) by molecular evolutionary engineering. If the pre-in vitro selected peptide is conjugated to the GF, it induces a conformational change of the peptide that reduces its binding affinity, as shown above. However, as shown below, this situation can be avoided by using in vitro selection after the joining of random sequences with GFs prior to conjugation. This is called a direct or “all-in-one” in vitro selection (molecular evolutionary engineering) method. Reproduced with permission from ref 102. Copyright 1997 Elsevier. Figure 10 Preparation of generally adhesive growth factors using a bioinspired approach of incorporating adhesion active regions of underwater adhesion proteins, which have non-coding (natural) amino acids. Figure 11 Bioorthogonal synthesis using sortase (A) and tyrosinse (B). In the case of (A),


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non-natural amino acid-containing proteins can be prepared without modification of other parts. In the case of (B), as tyrosinase modifies every tyrosine, tyrosine residues in the growth factor (GF) are required for non-contribution of biological activity. Figure 1



Figure 2


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Figure 7



Figure 8


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Figure 11



"For Table of Contents Use Only"

Cell

Matrix

Growth Factor Chemical Conjugation Protein Engineering Molecular Evolutionary Engineering Bioorthogonal Approaches

Growth factor engineering for biomaterials Yoshihiro Ito


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Figure 1 254x190mm (96 x 96 DPI)





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Figure 10 254x190mm (96 x 96 DPI)


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Figure 11 254x190mm (96 x 96 DPI)


Growth factor engineering for biomaterials - ACS Biomaterials Science

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