rprotein

Application of Recombinant Fusion Proteins for Tissue Engineering

MASATO NAGAOKA,1 HU-LIN JIANG,2,3,5 TAKASHI HOSHIBA,4 TOSHIHIRO AKAIKE,1 and CHONG-SU CHO2,3

1Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan; 2Departmentof Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea; 3Research Institute for Agriculture and LifeSciences, Seoul National University, Seoul 151-921, Korea; 4Biomaterials Center, National Institute for Materials Science,

Tsukuba 305-0044, Japan; and 5Brain Korea 21 Program for Veterinary Science, College of Veterinary Medicine, Seoul NationalUniversity, Seoul 151-742, Korea

(Received 9 May 2009; accepted 17 January 2010; published online 4 February 2010)

Associate Editor Julia E. Babensee oversaw the review of this article.

Abstract—Extracellular matrix (ECM) plays important rolesin tissue engineering because cellular growth and differenti-ation, in the two-dimensional cell culture as well as in thethree-dimensional space of the developing organism, requireECM with which the cells can interact. Also, the develop-ment of new synthetic ECMs is very important becauseECMs facilitate the localization and delivery of cells to thespecific sites in the body. Therefore, the development ofsynthetic ECMs to replace the natural ECMs is increasinglyessential and promising in tissue engineering. Recombinantgenetic engineering method has enabled the synthesis ofprotein-based polymers with precisely controlled functional-ities for the development of new synthetic ECMs. In thisreview, the design and construction of structure-basedrecombinant fusion proteins such as elastin-like polymers(ELPs) and silk-like polymers (SLPs), cell-bound growthfactor-based recombinant fusion proteins such as basicfibroblast growth factor (bFGF) and epidermal growthfactor (EGF), hybrid system composed of recombinantprotein and synthetic polymer, and E-cadherin-based fusionprotein by recombinant genetic engineering were explainedfor application of the synthetic ECMs. Modulation ofmechanical properties, stimuli-sensitivity, biodegradationand cell recognition can be achieved through precise controlof sequence, length, hydrophobicity and cell binding domainby recombinant genetic engineering.

Keywords—Extracellular matrix, Tissue engineering, Elastin-

like polymer, Silk-like polymer, E-cadherin.

INTRODUCTION

Tissue engineering has attracted many scientists andmedical doctors with a hope to treat patients in a mini-mally invasive and less painfulwaybecause it canbe usedto restore,maintain, or enhance tissues and organs66 andthe engineered tissues could reduce or eliminate the needfor organ replacement.20 Tissue engineering approachestypically employ exogenous three-dimensional extracel-lular matrix (ECM) to engineer new natural tissues fromnatural cells.1 The exogenous ECMs are designed tobring the desired cell types into contact in an appropriatethree-dimensional environment and also to providemechanical support until the newly formed tissues arestructurally stabilized.58 Design of exogenous ECMs forthe tissue engineering is to mimic the functions of thenatural ECM molecules found in tissues because thesenatural ECMsact as a scaffold to bring cells together in atissue, to control tissue structure, and to regulate cellphenotype.62 Development of new artificial ECMs isvery important because the ECMs facilitate localizationand delivery of cells to specific sites in the body, theydefine and maintain a three-dimensional space for theformation of new tissues with appropriate structure andthey guide development of new tissues with theirappropriate functions.7

Typically, artificial ECM constructs, which form thebackbone of most engineered tissues, are developedusing either natural polymers such as collagen, algi-nate, chitosan, fibrin, hyaluronan, etc., or syntheticpolymers such as PLA, PGA, PLGA, PEG, PCL, etc.17

The collagen, fibrin and hyaluronic acid among thenatural polymers have been widely used as scaffold fortissue engineering because their physiological proper-ties, biocompatibility and biodegradability are similarto natural tissues. However, the low mechanical

Address correspondence to Toshihiro Akaike, Graduate School

of Bioscience and Biotechnology, Tokyo Institute of Technology,

Yokohama 226-8501, Japan. Electronic mail: [email protected]

Address correspondence to Chong-Su Cho, Department of Agri-

cultural Biotechnology, Seoul National University, Seoul 151-921,

Korea. Electronic mail: [email protected]

Annals of Biomedical Engineering, Vol. 38, No. 3, March 2010 (� 2010) pp. 683–693

DOI: 10.1007/s10439-010-9935-3

0090-6964/10/0300-0683/0 � 2010 Biomedical Engineering Society

683

properties, the risk of viral infection, the antigenicity,the instability of materials, deterioration and limitedversatility limit long-term implantation for the clinicalapplication. Therefore, the development of artificialECMs as alternatives to replace the natural polymerswill be useful in tissue engineering. It will be expectedthat development of new artificial ECMs for tissueengineering plays an important role in providing analternative to organ and tissue transplantation, both ofwhich suffer from a limitation of supply.17

This review summarizes the recent applications ofrecombinant fusion proteins for tissue engineering.This review mainly explains four recombinant ECMsbecause it is too broad to summarize the structure–function relationships of other artificial ECMs such ascollagen,31,41,44,74,84 fibrin,14,33 hyaluronic acid,9,13,85,86

glycosaminoglycans82 and proteoglycans70 for tissueengineering application although they can be preparedby recombinant genetic engineering technique andsome products are already commercial available.However, some literatures are cited for the readers.One is structure-based recombinant fusion proteinssuch as elastin- and silk-like polymers well character-ized for the most part. Second is cell-bound growthfactor-based recombinant fusion proteins. Third ishybrid system composed of recombinant protein andsynthetic polymer. Fourth is cadherin-based fusionprotein because it can be applicable for stem cell tissueengineering. Also, it can be expected that the devel-opment of recombinant fusion proteins will providethe promise of a safe, predictable and chemicallydefined source of ECMs for tissue engineering althougha prerequisite for the growth of applications based onrecombinant fusion proteins is the improvement of theproduction of larger amounts of recombinant proteinsand two-dimensional recombinant fusion proteins arealmost applied in many cell culture studies by far.

CHARACTERISTICS OF RECOMBINANT

FUSION PROTEINS

One of the parameters for the development of tissueengineering is to design and to produce materials foracting as an adequate scaffold in growing cells andtissues.18 The development of artificial ECMs startedwith the use of biocompatible and biodegradablechemically synthetic polymers, which showed low cellattachment and spreading capabilities of a rather non-specific nature5 although these polymers were soonimproved with more specific functionalities included intheir structure, specially peptide cell adhesive sequencesuch as RGD (arg-gly-asp) tripeptide found in variousnatural ECM molecules.27 The use of these sequencessignificantly enhanced the performances of those

ECMs. However, these artificial ECMs are still farfrom showing the efficiency and rich complexity andfunctionality of the natural ECMs.18

Recently, progress in recombinant DNA technologyhas allowed the synthesis of recombinant protein-basedpolymers with precisely defined molecular weights,compositions, sequences and stereochemistries.45 Suchprecise control over molecular structure of a polymerallows similarly fine control over its physicochemicalcharacteristics and biological fate.16 Also, it enablesconstruction of new tailor-made biomaterials with im-proved properties important for tissue engineering.

A recombinant protein-based polymer is a polymerconsisting of peptide sequence repeats, where eachrepeating unit can be composed of as few as two or asmany as hundreds of amino acid residues or withoutcontaining any repeating sequences, andmay recur froma few to hundreds of times.77 The key difference thatseparates genetically engineered polymers from poly(amino acid)s and polypeptides is that they are synthe-sized by recombinant techniques.22 The entire aminoacid sequence of recombinant polymers is controlled atthe DNA-level leading to polymers with preciselydefined, and potentially quite complex sequences andstructures.8 Therefore, recombinant polymers can bedesigned to incorporate a variety of functionalities, suchas responsiveness to microenvironmental stimuli, con-trolled biodegradation and presentation of informa-tional motifs for cellular interaction.22

CLASSES OF RECOMBINANT FUSION

PROTEINS

Mostly four recombinant fusion proteins have beenused in tissue engineering. One is structure-basedrecombinant fusion protein because several repeatingamino acid sequences or amino acid sequences withoutany repeating were constructed by recombinant tech-nique although elastin- and silk-like polymers aremainly covered in this review due to the limited appli-cations of other recombinant fusion proteins for tissueengineering. Second is cell-bound growth factor-basedrecombinant fusion proteins because cell-boundgrowth factor domain is mainly constructed byrecombinant technique. Third is hybrid system composedof recombinant fusion protein and synthetic polymer.Fourth is cadherin-based fusion protein because it canbe applicable for stem cell tissue engineering.

Structure-Based Recombinant Fusion Proteins

Elastin-Like Polymers

Elastin is an ECM protein consisting of severalrepeating amino acid sequences, including VPGZG,

NAGAOKA et al.684

VPGVG, APGVGV, VPGFGCGAG and VPGG.64

The most renowned building block within this family isthe VPGZG, where Z can be any natural or modifiedamino acid.76 Urry and co-workers performed a pio-neer work for biomedical uses, and particularly intissue engineering78 because elastin-like polymers(ELPs) showed an outstanding biocompatibility.Interestingly, Nicol et al. reported that ELPs inducedlittle or no immunogenic response in vivo.51,79 Also,their biodegradation proceeded by conventional met-abolic routes, yielding just natural amino acids.18

ELPs based on a repeating VPGVG are soluble inaqueous medium below their inverse transition tem-perature (Tt). When this temperature is raised abovethe Tt, they aggregate by hydrophobic self-assemblyand undergo a phase transition.76 Reversibility ofphysical changes in temperature-sensitive ELPs wasapplied for temporary functional scaffolds for tissuerestoration.78 Urry et al.80 reported that apparentlynatural tissue was generated with normal amounts ofcollagen and elastic fibers, and red blood cells inarterioles were apparent as the darker bodies, indi-cating vascularization of the new tissue bodies, whenRGD-containing ELP was injected into guinea pigsubcutaneously. Recently, temperature-sensitive ELPswere applied for cell sheet recovery because cell sheetengineering is a powerful technique in tissue engineer-ing using poly (N-isopropylacrylamide) by Okano’sgroup.67 The cell sheets were obtained at 20 �C afterA549 cells were cultured on a culture dish coated withELP containing GVGVP repeating units, His tag andRGD sequence.46

Injectable biomaterials are of interest for the rapidand in vivo formation of load bearing scaffolds fortissue engineering.35 This system is very attractive fortissue engineering because cells and growth factors canbe homogeneously mixed with a liquid and injectedinto a defect site, followed by in situ formation of ahydrogel.39 Also, the rapid formation of an elastomericnetwork can give the requisite mechanical provisionalscaffold with cells and growth factors in a three-dimensional microenvironment.68 Several methodssuch as chemical cross-linking, enzymatic cross-linking, and physically cross-linked networks havebeen reported to prepare ELP hydrogels.12,37,43,49,55,75

Recently, Chilkoti and coworkers reported that fibro-blasts embedded in the ELP hydrogels cross-linked byorganophosphorous cross-linker survived during thecross-linking process and remained viable for at least3 days in vitro, suggestion of controlling mechanicalproperties and potentially functional outcomes in tis-sue engineering by the ELPs.38

A common goal to biomaterial design involvescreating biomimetic materials that resemble the ECMprotein components or short peptide sequences to elicit

specific cellular responses and direct new tissue for-mation. The recombinant technique allows highlyspecialized artificial ECM proteins to be obtained ingood yield. This approach included RGD sequences,52

RGD and CS5 binding domains of fibronectin,26,42

RGD binding domain of collagen11 and tenascin-C,32

heparin-binding domains,82 and RGD and heparin-binding domains.83

Silk-Like Polymers

Silks are fibrous proteins composed of repeatingsequences of both crystalline and amorphousdomains.19 The silks are naturally produced by spidersand Lepdopterai. The primary amino acid componentsof silk proteins are glycine, alanine and other shortchain amino acids. Synthesis of silk-like polymers(SLPs) by recombinant technique is a powerful methodfor varying properties, through appropriate choice ofthe different units, the number of units in each multi-mers, the spacing between them, and the number ofrepeats of the multimer combination assembly.36 Also,SLPs can comprise chemically active sites, enzymaticactivity, receptor binding sites, and other functional-ities. Especially, biomedical applications offer thehighest potential for spider silk owing to the excellentmechanical properties, biocompatibility and biodeg-radation.2 Bini et al.6 included RGD cell-bindingdomains into the recombinant spider silk. The resultsindicated that the recombinant spider silk with RGDencoded into the protein supported enhanced differ-entiation of human bone marrow-derived mesenchy-mal stem cells (hMSCs) to osteogenic outcomes whencultured to tissue culture plastic, suggestion ofpotential application in stem cell tissue engineering.Kaplan and coworkers focused carboxyl terminaldomain of dentin matrix protein 1 to exploit the self-assembly and physical properties of silk proteins withcontrolled hydroxyapatite (HA) formation for bioma-terial composites.28 The recombinant protein wasmineralized using simulated body fluids and inducedthe formation of calcium-deficient carbonated HA,suggestion of potential application in bone tissueengineering. While recombinant spider dragline silkdisplays superior mechanical properties for scaffolds,one of the technical hurdles is how to recapitulate theseproperties in the laboratory on a scalable basis.81

Asakura et al.4 reported a silk-like hybrid proteinconsisting of polyalanine region of silk fibroin froma wild silk worm, Samia cynthia ricini, and RDGsequence derived from fibronectin. The obtained silk-like hybrid protein film showed high cell adhesive andgrowth activities of kidney VERO cells when com-pared with those of collagen although strong acidicsolvents were used to make the film.

Application of Recombinant Fusion Proteins for Tissue Engineering 685

While recombinant silk proteins have been success-fully synthesized, their low aqueous solubility limitsbiomedical applications. To enhance the aqueous sol-ubility of recombinant silks, differentially chargedanalogues of block copolymers containing repeatingsequences from silk (GAGAGS) and elastin (GVGVP)were synthesized by recombinant technique byreplacing a valine residue with glutamic acid.21 Theblock copolymers showed potentials as injectable ure-thral bulking agents for the treatment of female stressurinary incontinence due to pH- and temperature-sensitive properties.50 The characteristics of structure-based recombinant ECMs are summarized in Table 1.

Cell-Bound Growth Factor-Based Recombinant FusionProteins

Fibroblast Growth Factor

Basic fibroblast growth factor (bFGF) is a potentmitogen and a modulator for fibroblasts and vascularendothelial cells69 and plays an important role in tissueregeneration and repair.72 The bFGF is found inabundance in tissues such as brain, kidney and carti-lage. However, there are limitations in application tosynthetic ECMs because it would diffuse rapidly evenadministered in topical side effects on the surroundingtissues87 and free bFGF molecules possess a shorterhalf-life.59 Hashi et al.24 constructed a fusion protein ofthe cell-binding domain of human fibronectin andhuman bFGF with both cell-adhesive activity andgrowth factor activity. The fusion protein adsorbed toculture dishes stimulated the growth of humanumbilical-vein endothelial cells (HUVECs) and theangiogenesis in chorioallantoic membranes of devel-oping chick embryos, suggestion of potential in neu-roregenerative medicine.

Andrades et al.3 constructed a fusion protein, whichcontained bFGF and collagen-binding domain (CBD),a decapeptide derived from vonWilleband’s factor. Therecombinant protein increased affinity for collagen andenhanced wound healing. Also, Zhao et al.87 introduced

two CBDs into the human bFGF to develop a collagen-based wound targeting repair system. The resultsshowed that the bFGF with the CBD derived fromcollagenase (C-bFGF) promoted vascularization at theimplanted sites more effectively than bFGF with theCBD derived from von Willeband’s factor (V-pFGF).Sheng et al.65 constructed a fusion protein consisted ofbFGF fused to the C-terminus of glutathione S-trans-ferase (GST). The GST-bFGF stimulated the growth ofHUVECs to the same extent as recombinant bFGF,whereas GST itself did not stimulate, suggesting thatbFGF retains its biological activity when fused to GSTalthough GST-bFGF must be supplemented withbovine serumbefore storage to retain biological activity.

Epidermal Growth Factor

Epidermal growth factor (EGF) is a 53-residue sin-gle chain polypeptide that plays important roles in tis-sue regeneration, stimulation of the initiation of DNAsynthesis, cell replication, and activation of RNA,acceleration of wound healing, enhancement of prolif-eration and keratinization of epithelial tissues.10 TheEGFs have been constructed to use for EGF fusionproteins consisted of EGF as the autocrine/paracrinepeptide signaling molecules and synthetic ECMs.

Kawase et al.34 constructed a fusion protein fromEGF and cell-binding domain of fibronectin. The fusionprotein exhibited both cell-adhesive activity and growthfactor activity, each ofwhichwas indistinguishable fromthat of the corresponding unfused-protein.

Nishi et al.54 constructed fusion proteins consistingof EGF or bFGF and CBD derived from clostridiumhistolyticum collagenase. The fusion proteins tightlybound to insoluble collagen and stimulated the growthof BALB/c3T3 fibroblasts as much as the unfusedcounterparts. Also, the CBDEGF remained at the sitesof injection for up to 10 days when injected subcuta-neously into nude mice whereas EGF itself was notdetectable at 24 h after injection, indicating that thefusion proteins are non-diffusible and long-standingin vivo. Also, Hayashi et al.25 constructed the fusion

TABLE 1. Characteristics of structure-based recombinant fusion proteins.

Protein Amino acid sequence Advantages Reference

ELP VPGZG No immunogenic response in vivo Nicol et al.51

VPGZG + RGD Vascularization of new tissue bodies Urry et al.80

GVGVP + RGD Cell sheet formation Mie et al.46

VGA or KVF In situ hydrogel formation Lim et al.39

SLP (ASAAAAAA)m(GPGQQ)n Improved mechanical properties Lazaris et al.36

Spider silk + RGD Enhancement of differentiation of hMSCs Bini et al.6

Spider silk + dentin Formation of calcium-deficient carbonated HA Huang et al.28

Silk fibroin + RGD Higher cell adhesive and growth activities of kidney

VERO cells

Asakura et al.4

SELP Silk + elastin Chondrogenesis of mesenchymal stem cells Haider et al.21

NAGAOKA et al.686

protein consisting of EGF and type III collagen. Thefusion protein was shown to hold the triple helicalconformation of collagen and the mitogenic activity ofEGF and the fusion protein can be immobilized ontissue culture dishes as a fibrous form, suggestion ofpotential in application to tissue engineering. Further-more, Ishikawa et al.30 reported that the fusion proteinconsisting of EGF and fibronectin collagen-bindingdomain induced granulation tissue formation in thewounds of kind limbs when applied with collagen gel.

Ogiwara et al.56 constructed a fusion protein con-sisting of EGF as a cell growth function and immu-noglobulin G (IgG) Fc region (EGF-Fc) to stablyadsorb to a cell culture surface. Mouse fibroblast Swiss3T3 cells adhered to EGF-Fc-coated surface, which issimilar to collagen-coated one whereas the cells did notadhere to IgG-coated surface as shown in Fig. 1. Also,phosphorylation of EGF receptors was induced by theimmobilized EGF-Fc as well as EGF itself, indicatingthat the immobilized EGF-Fc tranduced a signal to thecells through the phosphorylation of tyrosine residueson signal proteins. Furthermore, immobilized EGF-Fccontinued to activate MARK after 4 h, whereas theactivation of MARK in the cells cultured in the pres-ence of EGF itself rapidly decreased with time asshown in Fig. 2, suggesting that MARK activationinduced by the immobilized EGF-Fc in the cells iscontinuous without internalization of growth factorand the constructed EGF-Fc can be used as a syntheticECM. They also constructed recombinant photo-reactive EGF bearing p-azido phenylalanine at theC-terminal (HEGFP) to immobilize the HEGFP stablyto biomaterial surface.57 The results indicated thatalmost same amounts of A 431 cells adhered toHEGFP-immobilized surface when compared withcollagen-coated surface as shown in Fig. 3a. Also, as

FIGURE 1. Cellular adhesion to EGF-Fc. Swiss 3T3 cellsadhered to the EGF-Fc- and collagen-coated dishes after 6 hof incubation. The data represent the mean 6 SD of experi-ments (n 5 3). From Ogiwara et al.56

FIGURE 2. Sustained activation of MAPK. A431 cells wereseeded onto EGF-Fc- or collagen-coated PS dishes and cul-tured in DMEM containing 0.5% FBS for various periods oftime. Cells cultured in collagen-coated PS dishes were stim-ulated with or without 100 ng mL21 EGF. Lysates were sub-jected to Western blotting with anti-phospho-MAPK antibody.From Ogiwara et al.56

FIGURE 3. Cell adhesion to HEGFP: (a) A431 cells adheredto the HEGFP-immobilized and collagen-coated surfaces after30 min of incubation. The data represent means 6 SD ofexperiments (n 5 3); (b) A431 cells pretreated with 100 ng/mLsoluble EGF were seeded onto each surface. Adhesion ratioof the cells was measured after 30 min of incubation; (c) A431cells suspended in PBS (2) were seeded onto each surface.As a positive control, the cells suspended in DMEM contain-ing 0.5% FBS were seeded on collagen-coated surface. FromOgiwara et al.57


shown in Fig. 3b, the number of cells adhered ontoHEGFP-immobilized surface remarkably decreasedwhereas the number of cells adhered onto collagen-coated one were not much changed, when A 431 cellswere pretreated with EGF, indication of receptor-mediated adhesion. Furthermore, as shown in Fig. 3c,Mg2+ does not play a prominent role in the receptor-mediated interaction. Moreover, liver-specific func-tions, CYP1A2 and hepatocyte nuclear factor (HNF)-4a, were maintained as same level as hepatocytescultured on galactose-carrying polymer (PVLA) asshown in Fig. 4, suggesting that the HEGFP can beused as the synthetic ECM for liver tissue engineering.Elloumi et al.15 constructed a fusion protein consistingof RGD sequence as a cell adhesive function, EGF as acell growth function, and hydrophobic sequence as anefficient assembling function. The fusion protein coatedon hydrophobic surface retained both cell adhesiveactivity through the RGD sequence and cell growthactivity when A 549 cells were cultured, whereas EGFitself had no growth activity even though it wasattached to a solid surface to some extent, suggestingthat the hydrophobic sequence played roles of a celladhesion function and stabilization of protein structure.

Platelet-Derived Growth Factor

Dai and coworkers constructed a fusion proteinfrom platelet-derived growth factor (PDGF) andCBD.40 The fusion protein promoted the binding ofPDGF to collagen scaffold with enhanced vasculari-zation in vivo and caused more cells to proliferate onthe collagen gel than native PDGF, suggesting that thisprotein is effective for targeting tissue regeneration andwound repair. They also constructed a fusion proteinfrom nerve growth factor-b (NGF-b) and CBD.71

The fusion protein increased the expression level and

improved the bioactivity of fused NGF-b withenhancement of the nerve growth in vivo.

Hybrid Systems Composed of Recombinant FusionProtein and Synthetic Polymer

Hybrid materials were prepared to mimic the nat-ural ECM. It will be expected that they represent a newand versatile class of biomimetic materials with clinicalpromise in serving as implants because they promotewound healing and tissue regeneration.63 Halstenberget al.23 prepared hybrid system composed ofrecombinant protein containing an RGD integrin-binding motif, two plasmin degradation sites and aheparin-binding site, and PEG chains with terminalacrylate groups at the cysteine’s thiol groups, therebyrendering the protein covalently photo-cross-linkableto form a hydrogel. The results indicated that it hadspecific integrin-binding capability with heparin bind-ing and proteolytic penetration in 2D and 3D systems.Hubbell and coworkers also synthesized hybridhydrogels of consisting of recombinant protein havingthe activity of vascular endothelial growth factor(VEGF) to induce cell adhesion and to provide cell-mediated remodeling by cross-linking matrix metallo-proteinase (MMP), and reactive PEG.88 The resultsindicated that the hybrid hydrogel matrices atop thechick chorioallontoic membrane brought strong newblood vessel formation. When implanted subcutane-ously in rats, these hybrid hydrogel matrices werecompletely remodeled into native, vascularized tissue.Furthermore, they prepared hybrid hydrogels consist-ing of recombinant protein having cell adhesion motifRGD and degradation sites for plasmin and MMPs,and reactive PEG.60,61 The results showed that thehybrid hydrogels promoted specific cellular adhesionand exhibited degradability by the target enzymes.Also, the hybrid hydrogels promoted healing of bonedefects after treatment of rat calvarial defects, sug-gesting that the combination of recombinant genetictechnology and synthetic polymer emerges as a pow-erful for the development of artificial ECMs.

E-CADHERIN

E-cadherin is a member of the intercellular adhesionmolecules and E-cadherin-mediated adhesion is regu-lated by Ca2+-dependent homophilic interaction.73

E-cadherin is essential for tissue morphogenesis andmaintenance of organized solid tissues.53

Nagaoka et al.47 constructed a fusion proteinconsisting of E-cadherin extracellular domain andIgG Fc region (E-cad-Fc) to stably adsorb to a cellculture surface as shown in Fig. 5. They reported that

FIGURE 4. Liver-specific functions and DNA uptake ofhepatocytes: hepatocytes were seeded onto HEGFP-immobi-lized, collagen-coated or PVLA-coated surface and cultured inWE for 3 days. Cells cultured on collagen were stimulatedwith or without 100 ng/mL EGF. CYP1A2 and HNF-4a mRNAwere detected by RT-PCR analysis. From Ogiwara et al.57

NAGAOKA et al.688

hepatocytes adhered to the E-cad-Fc-coated surfacewere almost same as collagen-coated one and theadhesion was inhibited by pretreatment of hepatocyteswith anti-E-cadherin antibody, whereas E-cadherin-deficient Hepa 1-6 cells did not adhere to the E-cad-Fc-coated surface as shown in Fig. 6, suggesting that theadhesion of hepatocytes to E-cad-Fc is mediated byE-cadherin. Also, they reported that hepatocytesadhered to E-cad-Fc-coated surface showed differenti-ated phenotypes such as low DNA synthesis activity andmaintenance of tryptophan oxygenase expression as amarker gene of differentiated hepatocytes,29 suggestionof potential synthetic ECM in maintaining differentia-tion of hepatocytes for liver tissue engineering.

Also, Akaike and coworkers constructed E-cad-Fcfor culture of embryonic stem (ES) cells.48 Theyreported that EB 3 cells as the ES cell adhered to theE-cad-Fc-coated surface as similar with conventional

FIGURE 5. Construction and expression of E-cadherin-IgGFc fusion protein. (a) Generated segments of an extracellulardomain of mouse E-cadherin and an IgG Fc region weresubcloned into an eukaryotic expression vector pRC/CMV viaHindIII-NotI site and NotI-XbaI site respectively. CMV pro-moter, human cytomegalovirus immediate-early promoter/enhancer; BGH pA, bovine growth hormone polyadenylationsignal; Neor, neomycin resistance gene for selection; Ampr,ampicillin resistance gene. (b) Conditioned media fromtransfected CHO-K1 cells were analyzed by Western blotting.Fusion protein was detected by either anti-E-cadherin or anti-mouse IgG antibody. Arrowhead, mature protein; dottedarrow, precursor. (c) Cell lysate and conditioned media oftransfectant were analyzed to confirm the protein secretionsby Western blotting using anti-mouse IgG antibody. FromNagaoka et al.47

FIGURE 6. Adhesive activity to E-cad-Fc fusion protein. (a)Primary hepatocytes could adhere to E-cad-Fc-coated dish aswell as collagen-coated surface after 4 h. Inhibition assay wasperformed by incubating with neutralizing antibody (ECCD-1).BSA was a negative control. (b) Adhesion of mouse hepatomacell line Hepa 1-6 to collagen or E-cad-Fc was analyzed after3 h culture. (c) The expression levels of E-cadherin in twotypes of liver-derived cells were assessed by western blotting.The data represent the mean 6 SEM of experiments (n 5 3).From Nagaoka et al.47

FIGURE 7. Cell adhesion, morphology of ES cells on theE-cad-Fc fusion protein-immobilized surface. (a) ES cells (EB3)adhered to E-cad-Fc-coated dishes with equivalent efficiencyas to 0.1% gelatin-coated dishes after 3 h of incubation. (b) EScells (EB3) were cultured on E-cad-Fc-coated or fibronectin-coated dishes without serum. EGTA (5 mM) was added to theculture medium at 3 h after seeding (open bar). Detached cellswere removed and remaining cells were counted using alamarBlue reagent. *p < 0.05, §p < 0.001 vs. no treated condition(closed bar). (c, d) Morphological observation of ES cells (EB3)on the two different matrices. ES cells were cultured on poly-styrene surfaces coated with 0.1% (wt/vol) gelatin (c), or 10 mg/mL E-cad-Fc (d) in the presence of LIF for 2 days. High magni-fication images are shown in (c¢) and (d¢). From Nagaoka et al.48


gelatin-coated surface as shown in Fig. 7a. The cellsadhered even in serum-free condition with Ca2+-dependency as shown in Fig. 7b. Interestingly, theadhered cells remained separated from each other evenin the presence of leukemia inhibitory factor (LIF)with dendritic morphologies, whereas the cells adheredto gelatin-coated surface formed tightly aggregatedcolonies as shown in Figs. 7c and 7d, suggesting thatblocking of close contact among cells in the aggregatedcolonies may not generate a heterogeneous environ-ment within colonies, which potentially inhibit theproliferation of ES cells and the distribution of solublefactors. Also, the ES cells showed higher proliferationon the E-cad-Fc-coated surface than gelatin-coatedone as shown in Figs. 8a and 8b and the cells adheredto the E-cad-Fc-coated surface showed higher trans-fection efficiency than gelatin-coated one as shown inFig. 8c. Furthermore, the expression of Oct-3/4 as theundifferentiated marker of the ES cells was maintainedfor at least 3 days when cultured on E-cad-Fc-coated

dishes, suggesting that the E-cad-Fc will have a greatpotential as the artificial ECM for stem cell tissueengineering. The characteristics of growth factor-basedrecombinant ECMs are summarized in Table 2.

SUMMARY AND FUTURE PERSPECTIVE

Precise control over molecular weight, composition,sequence and stereochemistry at the molecular level byrecombinant genetic engineering method has createdinterest in the artificial ECMs of biomaterials for tissueengineering. In this review, the design and construc-tion of ELPs, SLPs, bFGF, EGF, E-cadherin, andhybrid systems composed of recombinant protein andsynthetic polymer by recombinant technique wereexplained for the artificial ECMs. Modulation of thecell recognition is achieved through precise controls insequence and length. While some examples of theinfluence of sequence, length, hydrophobicity, and cell

FIGURE 8. ES cells show higher proliferation and higher transfection efficiency on the E-cad-Fc-coated surface. (a) The prolif-erative activity of ES cells on a gelatin- or E-cad-Fc-coated surface was evaluated. EB3 cells were seeded on gelatin-coated (opensquare) or E-cad-Fc-coated (filled square) dishes and the cell number was counted after staining with alamar Blue reagent. Thedata indicate means 6 SD of experiments (n 5 3). **p < 0.01 vs. gelatinized plates. (b) BrdU incorporation of EB3 cells undercolony-forming (on gelatin) or scattering conditions (on E-cad-Fc). Relative BrdU incorporation value was evaluated. The dataindicate means 6 SEM. §p < 0.001. (c) Transfection efficiency of ES3 cells cultured on gelatin- or E-cad-Fccoated surface. Relativeexpression of GFP was evaluated. The data indicate means 6 SEM. §p < 0.001 vs. gelatinized plates. From Nagaoka et al.48

TABLE 2. Characteristics of growth factor-based recombinant fusion proteins.

Protein Natural Fused domain Advantages Reference

FGF bFGF Fibronectin Stimulation of growth of HUVECs Hashi et al.24

bFGF Collagen Increased migration of fibroblasts Andrades et al.3

bFGF GST Stimulation of growth of HUVECs Sheng et al.65

EGF EGF Fibronectin Increased cell-adhesive activity of HB101 cells Kawase et al.34

EGF Collagen Stimulation of growth of BALB/c 3T3 fibroblasts

and nondiffusible

Hayashi et al.,25 Nishi et al.54

EGF Fibronectin Induction of granulation tissue formation in the wounds Ishikawa et al.30

EGF IgG Fc Longer activation of MARK of 3T3 fibroblasts Ogiwara et al.56

EGF p-Azido phenylalamine Stable immobilization of EGF Ogiwara et al.57

EGF RGD hydrophobic

sequences

Retention of cell adhesive activity and stabilization

of protein structure

Elloumi et al.15

PDGF PDGF Collagen Increased vascularization of collagen scaffold Lin et al.40

NGF NGF-b Collagen Enhancement of nerve growth Sun et al.71

NAGAOKA et al.690

binding domain on the cell recognition were reviewed inthis article, medical applications are still on the horizonand the full potential of this recombinant in tissueengineering has yet to materialize. The control ofmechanical properties, biodegradation, biorecognitionand checking of no immunogenic response can signifi-cantly expand the utility of these constructs in tissueengineering although a prerequisite for the growth ofapplications based on recombinant fusion proteins is theimprovement of the production of larger amounts ofrecombinant proteins. Also, there will be increasingdemands that 3D recombinant fusion proteins providebetter model systems for physiologic situations because3D fusion proteins induce more effectively cellularfunctions than 2Dones although 2D in vitro assay on 2Drecombinant fusionproteins are still applied inmany cellculture studies. Furthermore, future research will beaimed to the design of hybrid materials consisting ofintelligent recombinant proteins and other biomaterialswith high mechanical characteristics for hard tissues.

ACKNOWLEDGMENT

This work was supported by the funds provided byKorea Research Foundation (KRF) (E00244).

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