Covalently immobilized biosignal molecule materials for tissue engineering

Abstract

Immobilization of biosignal molecules including growth factors and cytokines is important for developing biologically active materials which can contribute to tissue engineering as a component. The immobilization has more meanings than only immobilization of the enzyme in a bioreactor or ligand–receptor interactions, because the immobilized biosignal molecules work on cells which have very complex structures and functions. This review discusses recent progress in immobilization of biosignal molecules, including the mechanisms and design concepts.

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Yoshihiro Ito

Yoshihiro Ito received his Bachelor’s (1981) and Master’s (1983) degrees in Polymer Chemistry from Kyoto University and was awarded a Doctorate in Engineering from the same university in 1987. Since then he has held a number of posts at various institutions including Research Fellow of the Japan Society for the Promotion of Science (1987), Assistant (1988) and Associate (1996) Professor at Kyoto University, Research Fellow at the University of California, Irvine (1992–1993), Professor at the University of Tokushima (1999), and Project Leader at the Kanagawa Academy of Science and Technology (2002–2007). Now, he is Chief Scientist and Director of the Nano Medical Engineering Laboratory at the RIKEN Institute of Physical and Chemical Research (since 2004). His research focuses on biomaterials science, regenerative medical engineering, combinatorial bioengineering for the creation of functional polymers, and soft nanotechnology.

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1. Introduction

Current clinical technologies, especially donor transplants and artificial organs, have been excellent life-saving and life-extending therapies to treat patients who need to reconstitute diseased or devastated organs or tissues as a result of an accident, trauma, and cancer, or congenital structural anomalies. Although advances in surgical techniques, immuno-suppression, and postoperative care have improved survival and quality of life, there are still problems associated with the use of biological grafts, such as donor site morbidity, donor scarcity, and tissue rejection. A variety of synthetic and natural materials have been developed for the replacement of lost tissues, but the results have not been satisfactory. Tissue engineering has emerged as a promising alternative in which organs or tissue can be repaired, replaced, or regenerated.¹

In tissue engineering, a neotissue is generally generated from the cells on a bioresorbable scaffold, incorporating growth factors as shown in Fig. 1. It can consist of up to three components: cells, scaffold, and growth factors. Recent progress in cell biology revealed various types of stem cell. However, because of the scarcity of stem cells it is important to increase the number of stem cells for clinical utilization. Bioreactors to amplify the stem cells are required and, for the construction of bioreactors, some novel materials which enhance the growth or differentiation are desired.^2–6

Fig. 1 The principle of tissue engineering. This review focuses on the fusion, that is, immobilization of growth factor on the scaffold.

For the design of biomaterials, cells and proteins at the interface play an important role.^7,8 Especially for the development of such bioreactor materials or bioactive materials to enhance regeneration in the body, the utilization of biosignal proteins including growth factors and cytokines is reasonable. However, although there are many examples of material design to enhance cell adhesion, there are not so many investigations for regulation of various higher functionalities (gene expression) of cells including growth, differentiation, apoptosis, transformation and so on. In this review the possibilities for regulating the expression of cells by materials are discussed.

2. Immobilization of biosignal molecules

Fig. 2 shows the activation mechanism of biosignal molecules including growth factors and cytokines. First, the biosignal molecules interact with the cognate receptor and form a complex with it, resulting in autophosphorylation of the cytoplasmic domains of the receptors. The phosphorylation activates the intracellular signal transduction. On the other hand, the formed complexes are aggregated and the aggregates are internalized into cells. The internalization occurs by both clathrin-dependent and clathrin-independent mechanisms and leads to the recycling of receptors to the plasma membrane for resensitization or shuttling of receptors to lysosomes for degradatory down-regulation.

Fig. 2 Interaction of cells with soluble and immobilized growth factors.

If the signal transduction from biosignal molecules to cells is due to complex formation with the receptor, it is expected that the biosignal molecules immobilized on the material surface work. In addition, it is expected that the signal transduction by the immobilized molecules continues for a longer time than by the soluble molecules.

2.1. History of immobilized growth factor

Immobilization of insulin on a solid matrix was for the first time performed and reported by Cuatrecasas.⁹ He synthesized the conjugates to investigate the mechanism of insulin action. Although these conjugates facilitated extensive purification of solubilized insulin receptors by affinity chromatography , definitive conclusions regarding the effectiveness of immobilized insulin could not be drawn because of the possibility that the immobilization was incomplete.^10–15 Furthermore, given the marked time delay between the exposure of the cells to the immobilized insulin and the detection of signal transduction events, his study examining acute effects of insulin conjugates may not have allowed sufficient time for evaluation of activity. In addition, because his studies used porous agarose gel beads that were not accessible to the cells as the immobilizing support, it was difficult to evaluate quantitatively the relationship between the amount of immobilized insulin and biological activities.

After these works, in 1991 insulin was immobilized on a plain surface as a growth factor for investigation of its mitogenic (a long-term) effect.^16,17 Subsequently various types of biosignal molecules have been immobilized on materials to regulate cell functions. Table 1 summarizes reports on bioconjugate materials which were covalently immobilized with growth factors to regulate cellular functions.^18–109 Immobilized insulin and epidermal growth factor (EGF) enhanced the growth of cells.^16–65 Bone morphogenetic protein (BMP) –2 or –4 induced alkaline phosphatase activity, or calcium deposition, or regulated gene expression in cells.^73–77 The immobilized transforming growth factor-β1 (TGF-β1) induced collagen synthesis^86,89 or suppressed chondrocytes toward prehypertrophic chrondrocytes and osteolineage cells.⁸⁸ Notch ligands induced Notch activation in cells only in the immobilized state and it was suggested to expand human hematopoietic stem cell population.^93–95 Immobilization of leukemia inhibitory factor (LIF) kept the colony morphology, alkaline phosphatase activity, and stage-specific embryonic antigen-1 immunoreactivity of embryonic stem cells, which indicated an undifferentiated state.^96,97

Table 1 Biosignal molecules covalently immobilized on a matrix for cell culture

Growth factor	Substrate	Reference
Insulin	Surface-hydrolyzed poly(methyl methacrylate) (PMMA)	16–21
	Surface-treated glass or polyacrylamide	22
	Polyurethane	23
	Biodegradable polymer	24
	Poly(2-hydroxyethyl methacrylate) (poly(HEMA))	25
	Spacer, surface-treated PMMA with POE	26
	Polymer grafted with poly(acrylic acid)	27, 28
	Polyurethane with POE	29
	Biodegradable polymer + POE	30–32
	+ Adhesion factors (RGDS (Arg-Gly-Asp-Ser), pollyallylamine, collagen, etc.)	33–40
	+ Heparin	41
	Polystyrene + micropatterning	42, 43
	Poly(N-isopropylacrylamide)	44
	Poly(N-isopropylacrylamide) + RGDS	45–47
Epidermal growth factor (EGF)	Surface-modified glass	48–50
	Surface-hydrolyzed PMMA	51
	Surface-modifed PDMS	52, 53
	Polystyrene photo-immobilized (mircopatterned)	54–59
	Gene-engineered	60–66
Nerve growth factor (NGF)	Surface-modified glass	49, 67
	Poly(HEMA) grafted with PAA	68, 69
	Gelatin tricalcium phosphate crosslinked	70
	Micropatterned	71, 72
Bone morphogen protein (BMP)	Surface-modified titanium	73, 74
	Type I atelocollagen	75, 76
	Chitosan nanofiber	77
	Poly(lactide-co-glycolide)	78
Vasular endothelial growth factor (VEGF)	PAA-grafted polyethylene film	79
	Micropatterned	80
	Gene-engineered	81
Fibroblast growth factor (FGF)	Photoimmobilization	82
	Polymers	83
	+ Heparin	84, 85
Insulin growth factor-1 (IGF-1)	Photoimmobilization	82
Transforming growth factor-β1 (TGF-β1)	Collagen type I coated titanium	86
	PDMS	87
	Gelatin-hyaluronic acid-chondroitin-6-sulfate sponge	88
	+ POE	89
Hapatocyte growth factor (HGF)	Gene-engineered	90, 91
Notch ligand	Photo-immobilized	92
	Gene-engineered	93–95
Leukemia inhibitory factor (LIF)	Photo-immobilized	96
	Non-woven polyester fabrics	97
Stem cell growth factor (SCF)	Gene-engineered	98
Interleukin-2	Crosslinked	99
Interleukin-1	Gene-engineered	100
Tumor necrosis factor-α (TNF-α)	Photo-immoblized	67,101
Erythropoetin	Photo-immobilized with gelatin	102
Neurotrophin-3	Surface-modified glass	50
Transferrin	Surface-hydrolyzed PMMA	103
E-cadherin	Gene-engineered	104
Osteopontin	Poly(2-hydroxyethyl methacrylate)	105
	Collagen	106
P-selectin	Coated on polystyrene	107
CXCR3 ligand	Coated on polystyrene	108
Sonic hedgehog	Gene-engineered + interpenetrating polymer network	109

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On the other hand, in around 1990, in the field of cell biology, biologically natural “juxtacrine stimulation”, which indicates biological signal transduction in a nondiffusible manner to neighboring cells, was demonstrated for some membrane-anchored growth factors and lymphokines, including TGF-α, tumor necrosis factor α, colony-stimulating factor 1, c-kit ligand, and heparin-binding EGF.^110,111 Considering the biological system, Ito et al. named the effect induced by a chemically immobilized growth factor “artificial juxtacrine stimulation”.¹¹² Recently, some reviews for biomimetic approaches to biomaterials, which signal to cells via biologically active entities, have been published.^7,113

2.3. Interaction of immobilized growth factor

In order to demonstrate that the immobilized signal molecules work to transduce the signal to the cells, some works have been performed. Chen et al.²⁷ prepared three basic types of insulin conjugate and their effect on cell growth was investigated. The insulin–polyoxyethylene conjugate (Ins–POE), insulin–poly(acrylic acid) conjugate (Ins–PAA), and insulin–immobilized PAA-grafted polystyrene (Ins–PSt). Ins–POE and Ins–PAA were monovalent and multivalent water-soluble conjugates, respectively. On the other hand, Ins–PSt was a water-insoluble multivalent conjugate. The mitogenic activity of Ins–POE was lower than that of native insulin. This could be due to the inability of the insulin receptor to bind to the coupled insulin because of steric hindrance from the polyoxyethylene chain. With similar reasoning, poly(acrylic acid) should inhibit the binding of insulin to its receptor. However, Ins–PAA showed a slightly higher mitogenic activity compared to native insulin, presumably because Ins–PAA was multivalent and hence able to enhance receptor dimerization as well as aggregation of the insulin conjugate/receptor complex. On the other hand, Ins–PSt had an extremely high mitogenic effect being much higher than the other conjugates. Similar results were obtained using different types of matrix²⁸ and using the immobilized EGF⁵¹ as summarized in Fig. 3.

Fig. 3 Schematic comparison of the mitogenic effect of immobilized growth factor with that of nonimmobilized growth factor. Immobilized growth factor induces a greater effect than nonimmobilized growth factor. The smaller amount of immobilized growth factor is considered to induce the effect as a result of multivalency and high local concentration, as discussed in sections 3.1 and 3.3, respectively. The enhancement of the mitogenic effect is considered to be caused by inhibition of down-regulation and other factors as discussed in sections 3.2 and 3.3, respectively.

2.4. Certification of the interactions

As discussed previously,^10–15 in the case of immobilization of biosignal molecules on a solid, the completeness is very important. If there are some releases from the matrix, the effect may be due to some artifact. Therefore the amount of immobilized insulin was carefully measured by radioisotope labeling.²⁰ In addition, the immobilized growth factor was repeatedly used after each cell culture.^21–102 This repeated use confirms the stability of the immobilized molecules. In addition to these methods, there are some technologies to demonstrate the activity of immobilized biosignal molecules.¹⁴

Specific interaction of immobilized growth factor with the cognate receptor was examined by antibodys against immobilized growth factor. If the effect of the immobilized growth factor was inhibited by the antibody, the specific interactions were certified. This method has been employed by many researchers.^{14,19,80,110,111}

One of the technologies to certify the interactions, visualization of the signal transduction and spatial regulation of the cell functions, was performed by micropattern-immobilization using a photo-lithographic method.^{42,43,54–59,67,71,72,80,92,102} When the cell culture was on the micropattern-immobilized growth factor, the cell growth or differentiation was accelerated only in the biosignal molecule-immobilized areas. The immobilized biosignal molecules affected the cell functions including growth and differentiations without enhancing the adhesion.

To further investigate the interaction of immobilized EGF, CHO cells overexpressing EGF receptors were cultured on the plate immobilized with EGF in a narrow stripe pattern.⁵⁵ The contact area (stripes 2 µm in width) between the cells and the immobilized EGF was stained by an anti-phosphotyrosine antibody. Since free lateral diffusion and internalization of the bound EGF–EGF receptor complex were prohibited by immobilization of EGF, only signal proteins in the interaction regions were activated. This finding also indicates that the biological signal was transduced only to the cell that interacted with the immobilized EGF.

Recently Ichinose et al.⁴⁹ quantitatively evaluated the interaction. When the density of EGF was only slightly lower than that of the EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from being laterally diffused and internalized and kept their initial position. In addition, the immobilization made suitable targets for stable single molecule observation under total internal reflection fluorescence microscopy to study EGF signaling mechanisms, preventing lateral diffusion and internalization of EGF receptors. Ichinose et al. showed results of single molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF.

Shibata et al.⁶⁷ demonstrated that nerve growth factor (NGF)–receptor complexes had two distinct diffusive states, characterized as a mobile and an immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of the NGF–receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF–receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF–receptor complexes in the immobile phase indicating that signal transduction occurs during this phase. Thus, it was considered that NGF signaling was performed through a repetitive random process to induce formation of signaling complexes.

Previously the effect of immobilized growth factor was compared with the effect of adhesion factors including gelatin, collagen, and fibronection, or with albumin, immunoglobulin.¹⁸ Recently microarray systems have been employed to investigate the effect of immobilized molecules by some researchers.^{50,82,114,115} The recently developed systems also revealed the effectiveness of immobilized growth factors. At least the result that the immobilized biosignal molecules stimulate the cells has been confirmed by many methods.

3. Effect accompanying immobilization of growth factors

In addition to the fact that the immobilized growth factors interact with the cellular cognate receptors, it has been demonstrated that the immobilized growth factor had a higher or different effect from the soluble one as shown in Fig. 3, because the interaction is not only the ligand–receptor interaction. These effects of immobilized growth factor are considered to be due to the following mechanisms.²¹

3.1. Multivalency

The importance of mutivalency of immobilized biosignal molecules have been discussed by many researchers. This effect is the same as the high local concentration of immobilized proteins on material surfaces.^16,116 Recently Kiessling et al. considered synthetic multivalent ligands as probes of signal transduction.¹¹⁷ Multivalent ligands can bind avidly to multiple receptors on the cell surface, a process that is facilitated in the fluid lipid bilayer by the two dimensional diffusion of receptors. The multivalent ligands can activate signaling pathways if they can cluster signaling receptors (Fig. 4). They discussed the three major concepts that are critical for the application of multivalent ligands as probes of signal transduction: (1) signal transduction cascades are mediated by receptor–receptor interactions, and promoting receptor assembly is critical for signaling. (2) Multivalent ligands can interact with the target receptors through multiple binding modes. (3) The structure of a multivalent ligand will determine the favored binding modes. Thus, the structure can be optimized to elicit the desired biological response.

Fig. 4 Interactions of a multivalent ligand with a cell. The multivalent ligand enhances the formation of ligand–receptor complexes, and the interaction of activated ligand–receptor complexes, because of the high local concentration of ligands. In addition, the formed complexes are stabilized by the multivalent ligands.

In addition, prevention of lateral diffusion of the activated receptor or the receptor–ligand complex in the plane of the cell membrane must be taken into consideration. The prevention means the stabilization of the aggregates of complexes and thus leads to a long-lasting effect.

3.2. Inhibition of down-regulation

Inhibition of the internalization process should be also taken into consideration as shown in Fig. 2. Generally cells decompose the biosignal molecules in the cells to reduce their stimulation and this is called down-regulation. The immobilization was considered to inhibit this down-regulation and as a result the stimulation continued for a long time without reduction.

To investigate the above hypothesis, the activation of cellular signaling proteins , insulin receptor β-subunits in the cells, or mitogen-activated protein kinase (MAPK) in the cells was measured in the presence of immobilized insulin or immobilized EGF (Fig. 5).^21,51 Native insulin or EGF rapidly activated the insulin or EGF receptor. However, the activation is usually transient. On the other hand, activation by the immobilized insulin or EGF continued to increase up to 12 h although some lag time was needed for adhesion of cells on the surface immobilized with insulin or EGF. These sustained activations of signaling proteins by the immobilized insulin or EGF should explain the high mitogenic effect of the immobilized growth factors.

Fig. 5 Time course of signal activation by immobilized and soluble growth factors. The difference induces different effects on PC12 cells.

In addition, this sustained activation provided another effect of immobilized biosignal molecules.⁵⁸ It is well known that growth of the rat phaeochromaocytoma cell line PC12 is stimulated by EGF and the differentiation is stimulated by NGF.¹¹⁸ However, the immobilized EGF stimulated PC12 differentiation.⁵⁸ The immobilized EGF caused a long-lasting stimulation of MAPK and a subfamily of the MAPK superfamily in cells, as did diffusible NGF. This switching between growth stimulation and differentiation was considered to be due to the duration of stimulus.⁵⁸

3.3. Other considerable mechanisms

In addition, the difference in stimulation sites between the cell–medium interface and cell–matrix interface, etc. should also be taken into consideration. The interaction of immobilized biosignal molecules is from the substrate and the local concentration is extremely high as shown in Fig. 6. The specific situation is considered to affect various functions.

Fig. 6 Immobilized growth factor, which is different from soluble growth factor, provides a different environment for cells.

In fact, the immobilization sometimes induced some specific effect on cell behavior in addition to the primary effect. Kuhl and Griffith-Cima⁴⁸ found that the rounding responses of primary rat hepatocytes on the surface immobilized with EGF were different to those on the non-immobilized. Ogiwara et al.⁶³ reported that the cytoskeleton of A431 cells adhering to immobilized EGF-Fc (Fc is a fragment of immunoglobulin) was filopodia whereas that of the cells adhering onto collagen in the presence of soluble EGF was lammellipodia. In addition, Ogiwara et al.⁶⁴ found that interaction between photo-immobilized EGF and the receptor in the cells was independent of Mg²⁺ although integrin-mediated cell adhesion to natural exracellular matrices is dependent on Mg²⁺. Phosphorylation of EGF receptors in A431 cells was induced by immobilized EGF the same as soluble EGF. DNA uptake of hepatocytes decreased with immobilized EGF whereas it increased with soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 d by immobilized EGF whereas they were not maintained by soluble EGF, indicating that immobilized EGF follows a different signal transduction pathway from soluble EGF. These differences with immobilized growth factors compared to the soluble ones should be due to the complex structure of interfaces surrounding the cultured cells.

Reddy et al.¹¹⁹ demonstrated an approach derived from understanding how the attenuation mechanisms including growth factor depletion and receptor down-regulation arise from ligand/receptor trafficking processes. A recombinant EGF mutant with reduced receptor binding affinity is a more potent mitogenic stimulus for fibroblasts than natural EGF because of its altered trafficking properties. Optimization of ligand binding parameters requires systemic integration of processes from the initial binding event to the final cellular response. Their experimental data showed that consideration of cellular trafficking processes is essential for an optimization effort. The immobilized growth factor may have a similar effect.

4. Design of immobilization

In order to efficiently derive the effect of immobilized biosignal molecules for material design, there are many strategies reported (Fig. 7).¹²⁰

Fig. 7 Material designs using immobilized growth factors. (a) Flexible spacer chains make the immobilized growth factor mobile like a soluble growth factor. (b) Micropattern-immobilization for growth factors induces micropattern formation of cells. (c) Co-immobilization of different types of ligands (growth factor) induced cross-talk of receptors. (d) Stimuli-responsive polymers add some functions to the growth factor immobilization.

4.1. Spacer insertion and surface stiffness

As shown in Fig. 2, after the ligand complexes with the receptor, the complexes are generally considered to aggregate in the cell membrane to transmit the signal to the nuclei. Therefore, the insertion of the spacer between the surface and the biosignal molecules is useful for enhancement of diffusion of the complex in the membrane. Some studies using the spacer chains were performed and the increase in the activity is reported.^{26,29–32,48} However, possible implications of steric hindrance in spacing growth factor from the substrate and the possible impact on growth factor binding to its cell surface receptor should be taken into consideration. In fact, the POE conjugation reduced the biological activity of insulin.²⁷

The insertion of a spacer arm is also related to the surface stiffness. It is well known that the cells sense and react to extracellular stiffness as revealed by recent experiments with soft elastic substrates.^121,122 Therefore, immobilization was performed on polymeric materials having different water contents and the effect of immobilized growth factor was discussed. The increase in flexibility of immobilized growth factor increased the biological activity.

Iwamoto and Mekada¹¹¹ discussed the juxtacrine mechanism according to the flexibility of membrane-anchored growth factor, heparin-binding epidermal growth factor (HB-EGF). Stable transfectants of mouse L cells expressing precursor HB-EGF (proHB-EGF) were fixed with formalin to prevent the release of soluble HB-EGF (sHB-EGF), and then the fixed donor cells were cultured with EP170.7 cells, an EGF receptor–ligand dependent cell line. Under these conditions, growth stimulation of EP170.7 cells was observed and this EP170.7 cell growth was found to be dependent on the amount of proHB-EGF expressed on the donor cells. On the other hand, in order to examine the activities of proHB-EGF, a modified coculture system was reported, in which an intact monkey kidney cell line expressing proHB-EGF was incubated with EGF receptor-expressing 32D cells. Under these conditions, proHB-EGF was shown to have a growth-inhibitory activity and to induce apoptosis of the recipient cells, while sHB-EGF stimulated cell growth. They consider that the fixation by formalin perturbed the aggregation of ligand/receptor complexes in the cell membrane.

4.2. Micropatterning

Biomaterials for cell patterning have been used to regulate cellular processes, such as proliferation and differentiation, through cellular adhesion.^{120,123–132} As cells adhere to micropatterned substrates, they align themselves to the shape of the underlying adhesion region. This shape change induces changes in cytoskeleton features and has been shown to influence apoptosis and proliferation.¹³³ Gene expression and protein synthesis were also altered by changing the nuclear shape.¹³⁴ It has been determined that cell shape can also control stem cell differentiation¹³⁵ and that the tissue form itself can feed back to regulate patterns of proliferation through micromechanical forces.¹³⁶ Controlling the cellular microenvironment through micropatterning may be used for directing cell fate for tissue engineering applications.

Moreover, inside the body, cells lie in contact or in close proximity to other cell types in a tightly controlled architecture.¹²⁵ Tissue engineering constructs, which aim to reproduce the architecture and geometry of tissues, will benefit from methods of controlling cell–cell interactions within these tissues. Patterned cocultures are a useful tool for tissue engineering constructs and for studying cell–cell interactions in vitro because they can be used to control the degree of homotypic and heterotypic cell–cell contact. Pioneering work in this area was performed by studying the interaction of hepatocytes and nonparenchymal fibroblasts in cocultures.¹³⁷ Recently developed methods are based on thermally reversible polymers,¹³⁸ layer-by-layer deposition of ionic polymers,¹³⁹ microfluidic deposition,¹⁴⁰ and molding of hydrogels.¹⁴¹

As mentioned in section 3.1, the micropatterning is also useful for investigating the effect of immobilized growth factors. If no signal transduction is observed on non-immobilized growth factor, it is possible to conclude that there is no release of immobilized growth factor to act on the cells.^112,120

In addition to the certification method, micropatterning has been employed for regulation of cellular morphology and tissue formation. Ito et al.⁸⁰ demonstrate the micropatterning of blood endothelial cells to form vessels. Gomez et al.⁷² reported axon extension in neurons on the surface covalently immobilized with NGF and microtopography was introduced in the form of microchannels. When the two surface stimuli were presented in combination, a synergistic increase in axon length was detected, which could be a result of faster polarization triggered by topography plus enhanced growth from NGF.

4.3. Co-immobilization

In order to enhance the effect of immobilized growth factors, other macromolecules have been co-immobilized with them. Biological or physico-chemical enhancement of cell adhesion increased the mitogenic effect of immobilized growth factors.^{33–40,45,46} For biological enhancement of cell adhesion, cell adhesion factors including collagen, fibronectin, gelatin, and the core RGDS peptide were co-immobilized. On the co-immobilized surface both adhesion and growth of cells were remarkably enhanced. For physico-chemical enhancement of cell adhesion, cationic polymers such as poly(allyl amine) and polylysine were employed.

In addition, recently various types of stimuli-responsive materials were developed and cells were manipulated on these materials.^142–145 Chen et al.⁴⁴ immobilized insulin with a thermo-responsive polymer, poly(N-isopropylacryamide). They observed growth enhancement by immobilized insulin and harvested the cells by lowering the temperature. Coimmobilization with insulin and RGDS peptide was reported by Hatakeyama et al.^45–47 recently.

4.4. Protein engineering for immobilization

Recently many types of gene-engineered proteins for immobilization have been reported. Nishi et al.⁶⁰ constructed fusion proteins consisting of a growth factor moiety and the collagen-binding domain (CBD) of collagenase, which acted as an anchor to the collagen fibrils. They chose EGF and basic fibroblastgrowth factor (bFGF) as parts of the fusion proteins (collagen-binding EGF, CBEGF; collagen-binding bFGF, CB-FGF). As a result, CBEGF, when injected subcutaneously into nude mice, remained at the sites of injection for up to 10 d, whereas EGF was not detectable 24 h after injection. Although CBEGF did not exert a growth-promoting effect in vivo, CBFGF, but not bFGF, strongly stimulated the DNA synthesis in stromal cells at 5 d and 7 d after injection. These results indicate that CBD may be used as an anchoring unit to produce fusion proteins nondiffusible and long-lasting in vivo.

Hayashi et al.⁶¹ developed a recombinant technology to confer mitogenic activity on type III collagen by fusing it to EGF at the collagen's N-terminus. The fusion protein was shown to hold the triple helical conformation of collagen and the mitogenic activity of EGF. It was also demonstrated that the chimeric protein can be immobilized on tissue culture dishes as a fibrous form and in collagen fibrils without abolishing the original mitogenic activity of EGF.

Collagen-binding or fibrin-binding growth factors consisting of EGF, HGF, and VEGF and the binding domains in fibronectin were reported as shown in Fig. 8.^62,90 The fusion protein bound to gelatin and fibrillar collagen sponges and substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF or HGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. On the other hand, Elloumi et al.⁶⁵ reported a novel protein consisting of a RGD (Arg-Gly-Asp) sequence functioning as a cell adhesive function, EGF as a cell growth function, and a hydrophobic sequence as an efficient assembling function in one molecule. The protein coated on an unmodified hydrophobic surface of a cell culture plate (through the hydrophobic moiety) retained both cell adhesive activity (through the RGD sequence) and cell growth activity (through the EGF moiety).

Fig. 8 Gene-engineered growth factors (EGF, VEGF, HGF, etc.) containing collagen-binding domain (CBD) found in fibronectin.

Recently Kim et al.¹⁰⁰ prepared a fusion protein of a recombinant human interleukin-1 (IL-1) receptor antagonist and an elastin-like peptide (IL-1ra-ELP) and found that the immobilized IL-1ra-ELP modulates the inflammatory profile of lipopolysaccharide (LPS)-stimulated cultured human monocytes. Specifically LPS-stimulated THP-1 monocytes that were exposed to either soluble or immobilized IL-1ra-ELP did not differentiate, but showed attenuated expression of pro-inflammatory cytokines, and had enhanced production of anti-inflammatory and pro wound-healing cytokines. The extent of signaling by immobilized and soluble fusion proteins were similar in magnitude, indicating roughly equivalent bioactivity and that cultured monocytes are clearly signaled by the immobilized IL-1ra-ELP.

As another binding method, fusion with immunoglobulin G Fc region,⁶³p-azido phenylalanine,⁶⁴histidine tag,⁶⁶cysteine-containing tag⁸¹ and cellulose-binding domain⁹⁸ were employed. These gene engineering methods contributed oriented immobilization of biosignal molecules for efficient interaction with receptors to induce cellular signal transduction.

5. In vivo applications

Covalent immobilization of growth factor onto a substrate has been used mainly for cell culture. However, some in vivo experiments have also been performed for tissue engineering. Ohyama et al.⁸⁵ prepared bFGF-immobilized platinum microcoils and performed coil embolization of aneurysms constructed using a canine common carotid artery via the endovascular approach. The percentage of occlusion at the aneurysm orifice in animals treated with bFGF-immobilized coils was significantly greater than with unmodified coils. Liu et al.⁷⁸ immobilized BMP-2 on a poly(lactide-co-glycolide) scaffold and created bilateral, full-thickness cranial defects in rabbits to investigate the osteogenic effect of cultured mesenchymal stromal cells on bone regeneration in vivo. Histomorphometry and histology demonstrated that the BMP-2 conjugate enhanced bone formation after surgery. Nishi et al.⁶⁰ prepared collagen-binding EGF and injected it subcutaneously into nude mice. It remained at the sites of injection for up to 10 d, whereas EGF was not detectable 24 h after injection. In addition, collagen-binding FGF strongly stimulated the DNA synthesis in stromal cells at five and seven days after injection. Kitajima et al.⁹⁰ prepared collagen-binding HGF and its angiogenic activity in rat tissues was examined by subcutaneously implanting collagen sponges containing the binding HGF. Blood vessel formation in the sponges after seven days was four to six times more extensive when compared with the control sponges without any sample. Implanted sponges with native HGF did not show significant any difference from the controls. Recently, Ohkawara et al.⁹¹ studied the re-endothelialization and neointimal formation in balloon-injured rat carotid arteries in the presence of HGF and binding HGF. The left common carotid artery of male Sprague Dawley rats was injured with an inflated balloon catheter, and treated with binding HGF, native HGF, or saline (control) for 15 min. Rats were injected with Evans blue and sacrificed after 14 d. The length of the re-endothelialized area was significantly longer in binding HGF-treated rats than in control or HGF-treated rats. Neointimal formation was significantly greater in binding HGF-treated rats than in others.

In tissue engineering, design of scaffold has been the main target. Growth factors are usually physically mixed for utilization. However, to effectively utilize the growth factors, modification is favored. Covalent or noncovalent immobilization regulates the diffusion of growth factors to maintain the effects and provide specific microenvironments to regulate cellular responses with matrices.^146–150 In addition, the immobilization of growth factor is important for geometrical control of complex tissue formation of different types of cells within or near the scaffolds.

6. Future perspective

The field of tissue engineering has created a need for biomaterials that are capable of providing biofunctional and structural support for living cells outside the body. Most of the commonly used biomaterials in tissue engineering are designed based on their physico-chemical properties, thus achieving precise control over mechanical strength, compliance, porosity, and degradation kinetics. Biofunctional signals are added to the scaffold by tethering, immobilizing, or supplementing biofunctional macromolecules, such as growth factors, directly to the scaffold material. The challenge in tissue engineering remains to find the correct balance between the biofunctional and the physical properties of the scaffold materials for each application.¹⁵¹ Communication between cells and extracellular environment using the engineered scaffold should be correctly regulated. For this purpose the surface biolization by immobilization of growth factor is a powerful tool for constructing elaborate intelligent biofuntional materials. Control of surface function with the immobilization is important for design. It will be also important to prepare biomaterials mimicking the growth factorproteins for mass production of biofunctional materials without using the native proteins .

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