rprotein

Annals of Biomedical Engineering, Vol. 38, No. 3, March 2010 (Ó 2010) pp. 683–693
DOI: 10.1007/s10439-010-9935-3

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
1
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan; 2Department
of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea; 3Research Institute for Agriculture and Life
Sciences, 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 National
University, 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 roles INTRODUCTION
in tissue engineering because cellular growth and differenti-
ation, in the two-dimensional cell culture as well as in the Tissue engineering has attracted many scientists and
three-dimensional space of the developing organism, require medical doctors with a hope to treat patients in a mini-
ECM with which the cells can interact. Also, the develop-
mally invasive and less painful way because it can be used
ment of new synthetic ECMs is very important because
ECMs facilitate the localization and delivery of cells to the to restore, maintain, or enhance tissues and organs66 and
specific sites in the body. Therefore, the development of the engineered tissues could reduce or eliminate the need
synthetic ECMs to replace the natural ECMs is increasingly for organ replacement.20 Tissue engineering approaches
essential and promising in tissue engineering. Recombinant typically employ exogenous three-dimensional extracel-
genetic engineering method has enabled the synthesis of
lular matrix (ECM) to engineer new natural tissues from
protein-based polymers with precisely controlled functional-
ities for the development of new synthetic ECMs. In this natural cells.1 The exogenous ECMs are designed to
review, the design and construction of structure-based bring the desired cell types into contact in an appropriate
recombinant fusion proteins such as elastin-like polymers three-dimensional environment and also to provide
(ELPs) and silk-like polymers (SLPs), cell-bound growth mechanical support until the newly formed tissues are
factor-based recombinant fusion proteins such as basic
structurally stabilized.58 Design of exogenous ECMs for
fibroblast growth factor (bFGF) and epidermal growth
factor (EGF), hybrid system composed of recombinant the tissue engineering is to mimic the functions of the
protein and synthetic polymer, and E-cadherin-based fusion natural ECM molecules found in tissues because these
protein by recombinant genetic engineering were explained natural ECMs act as a scaffold to bring cells together in a
for application of the synthetic ECMs. Modulation of tissue, to control tissue structure, and to regulate cell
mechanical properties, stimuli-sensitivity, biodegradation
phenotype.62 Development of new artificial ECMs is
and cell recognition can be achieved through precise control
of sequence, length, hydrophobicity and cell binding domain very important because the ECMs facilitate localization
by recombinant genetic engineering. and delivery of cells to specific sites in the body, they
define and maintain a three-dimensional space for the
Keywords—Extracellular matrix, Tissue engineering, Elastin- formation of new tissues with appropriate structure and
like polymer, Silk-like polymer, E-cadherin. they guide development of new tissues with their
appropriate functions.7
Typically, artificial ECM constructs, which form the
backbone of most engineered tissues, are developed
using either natural polymers such as collagen, algi-
nate, chitosan, fibrin, hyaluronan, etc., or synthetic
polymers such as PLA, PGA, PLGA, PEG, PCL, etc.17
Address correspondence to Toshihiro Akaike, Graduate School The collagen, fibrin and hyaluronic acid among the
of Bioscience and Biotechnology, Tokyo Institute of Technology, natural polymers have been widely used as scaffold for
Yokohama 226-8501, Japan. Electronic mail: takaike@bio.titech.ac.jp
tissue engineering because their physiological proper-
Address correspondence to Chong-Su Cho, Department of Agri-
cultural Biotechnology, Seoul National University, Seoul 151-921, ties, biocompatibility and biodegradability are similar
Korea. Electronic mail: chocs@plaza.snu.ac.kr to natural tissues. However, the low mechanical
683
0090-6964/10/0300-0683/0 Ó 2010 Biomedical Engineering Society

684 NAGAOKA et al.

properties, the risk of viral infection, the antigenicity, ECMs. However, these artificial ECMs are still far
the instability of materials, deterioration and limited from showing the efficiency and rich complexity and
versatility limit long-term implantation for the clinical functionality of the natural ECMs.18
application. Therefore, the development of artificial Recently, progress in recombinant DNA technology
ECMs as alternatives to replace the natural polymers has allowed the synthesis of recombinant protein-based
will be useful in tissue engineering. It will be expected polymers with precisely defined molecular weights,
that development of new artificial ECMs for tissue compositions, sequences and stereochemistries.45 Such
engineering plays an important role in providing an precise control over molecular structure of a polymer
alternative to organ and tissue transplantation, both of allows similarly fine control over its physicochemical
which suffer from a limitation of supply.17 characteristics and biological fate.16 Also, it enables
This review summarizes the recent applications of construction of new tailor-made biomaterials with im-
recombinant fusion proteins for tissue engineering. proved properties important for tissue engineering.
This review mainly explains four recombinant ECMs A recombinant protein-based polymer is a polymer
because it is too broad to summarize the structure– consisting of peptide sequence repeats, where each
function relationships of other artificial ECMs such as repeating unit can be composed of as few as two or as
collagen,31,41,44,74,84 fibrin,14,33 hyaluronic acid,9,13,85,86 many as hundreds of amino acid residues or without
glycosaminoglycans82 and proteoglycans70 for tissue containing any repeating sequences, and may recur from
engineering application although they can be prepared a few to hundreds of times.77 The key difference that
by recombinant genetic engineering technique and separates genetically engineered polymers from poly
some products are already commercial available. (amino acid)s and polypeptides is that they are synthe-
However, some literatures are cited for the readers. sized by recombinant techniques.22 The entire amino
One is structure-based recombinant fusion proteins acid sequence of recombinant polymers is controlled at
such as elastin- and silk-like polymers well character- the DNA-level leading to polymers with precisely
ized for the most part. Second is cell-bound growth defined, and potentially quite complex sequences and
factor-based recombinant fusion proteins. Third is structures.8 Therefore, recombinant polymers can be
hybrid system composed of recombinant protein and designed to incorporate a variety of functionalities, such
synthetic polymer. Fourth is cadherin-based fusion as responsiveness to microenvironmental stimuli, con-
protein because it can be applicable for stem cell tissue trolled biodegradation and presentation of informa-
engineering. Also, it can be expected that the devel- tional motifs for cellular interaction.22
opment of recombinant fusion proteins will provide
the promise of a safe, predictable and chemically
defined source of ECMs for tissue engineering although CLASSES OF RECOMBINANT FUSION
a prerequisite for the growth of applications based on PROTEINS
recombinant fusion proteins is the improvement of the
Mostly four recombinant fusion proteins have been
production of larger amounts of recombinant proteins
used in tissue engineering. One is structure-based
and two-dimensional recombinant fusion proteins are
recombinant fusion protein because several repeating
almost applied in many cell culture studies by far.
amino acid sequences or amino acid sequences without
any repeating were constructed by recombinant tech-
nique although elastin- and silk-like polymers are
CHARACTERISTICS OF RECOMBINANT mainly covered in this review due to the limited appli-
FUSION PROTEINS cations of other recombinant fusion proteins for tissue
engineering. Second is cell-bound growth factor-based
One of the parameters for the development of tissue
recombinant fusion proteins because cell-bound
engineering is to design and to produce materials for
growth factor domain is mainly constructed by
acting as an adequate scaffold in growing cells and
recombinant technique. Third is hybrid system composed
tissues.18 The development of artificial ECMs started
of recombinant fusion protein and synthetic polymer.
with the use of biocompatible and biodegradable
Fourth is cadherin-based fusion protein because it can
chemically synthetic polymers, which showed low cell
be applicable for stem cell tissue engineering.
attachment and spreading capabilities of a rather non-
specific nature5 although these polymers were soon
improved with more specific functionalities included in Structure-Based Recombinant Fusion Proteins
their structure, specially peptide cell adhesive sequence
Elastin-Like Polymers
such as RGD (arg-gly-asp) tripeptide found in various
natural ECM molecules.27 The use of these sequences Elastin is an ECM protein consisting of several
significantly enhanced the performances of those repeating amino acid sequences, including VPGZG,

Application of Recombinant Fusion Proteins for Tissue Engineering 685

VPGVG, APGVGV, VPGFGCGAG and VPGG.64 specific cellular responses and direct new tissue for-
The most renowned building block within this family is mation. The recombinant technique allows highly
the VPGZG, where Z can be any natural or modified specialized artificial ECM proteins to be obtained in
amino acid.76 Urry and co-workers performed a pio- good yield. This approach included RGD sequences,52
neer work for biomedical uses, and particularly in RGD and CS5 binding domains of fibronectin,26,42
tissue engineering78 because elastin-like polymers RGD binding domain of collagen11 and tenascin-C,32
(ELPs) showed an outstanding biocompatibility. heparin-binding domains,82 and RGD and heparin-
Interestingly, Nicol et al. reported that ELPs induced binding domains.83
little or no immunogenic response in vivo.51,79 Also,
their biodegradation proceeded by conventional met-
Silk-Like Polymers
abolic routes, yielding just natural amino acids.18
ELPs based on a repeating VPGVG are soluble in Silks are fibrous proteins composed of repeating
aqueous medium below their inverse transition tem- sequences of both crystalline and amorphous
perature (Tt). When this temperature is raised above domains.19 The silks are naturally produced by spiders
the Tt, they aggregate by hydrophobic self-assembly and Lepdopterai. The primary amino acid components
and undergo a phase transition.76 Reversibility of of silk proteins are glycine, alanine and other short
physical changes in temperature-sensitive ELPs was chain amino acids. Synthesis of silk-like polymers
applied for temporary functional scaffolds for tissue (SLPs) by recombinant technique is a powerful method
restoration.78 Urry et al.80 reported that apparently for varying properties, through appropriate choice of
natural tissue was generated with normal amounts of the different units, the number of units in each multi-
collagen and elastic fibers, and red blood cells in mers, the spacing between them, and the number of
arterioles were apparent as the darker bodies, indi- repeats of the multimer combination assembly.36 Also,
cating vascularization of the new tissue bodies, when SLPs can comprise chemically active sites, enzymatic
RGD-containing ELP was injected into guinea pig activity, receptor binding sites, and other functional-
subcutaneously. Recently, temperature-sensitive ELPs ities. Especially, biomedical applications offer the
were applied for cell sheet recovery because cell sheet highest potential for spider silk owing to the excellent
engineering is a powerful technique in tissue engineer- mechanical properties, biocompatibility and biodeg-
ing using poly (N-isopropylacrylamide) by Okano’s radation.2 Bini et al.6 included RGD cell-binding
group.67 The cell sheets were obtained at 20 °C after domains into the recombinant spider silk. The results
A549 cells were cultured on a culture dish coated with indicated that the recombinant spider silk with RGD
ELP containing GVGVP repeating units, His tag and encoded into the protein supported enhanced differ-
RGD sequence.46 entiation of human bone marrow-derived mesenchy-
Injectable biomaterials are of interest for the rapid mal stem cells (hMSCs) to osteogenic outcomes when
and in vivo formation of load bearing scaffolds for cultured to tissue culture plastic, suggestion of
tissue engineering.35 This system is very attractive for potential application in stem cell tissue engineering.
tissue engineering because cells and growth factors can Kaplan and coworkers focused carboxyl terminal
be homogeneously mixed with a liquid and injected domain of dentin matrix protein 1 to exploit the self-
into a defect site, followed by in situ formation of a assembly and physical properties of silk proteins with
hydrogel.39 Also, the rapid formation of an elastomeric controlled hydroxyapatite (HA) formation for bioma-
network can give the requisite mechanical provisional terial composites.28 The recombinant protein was
scaffold with cells and growth factors in a three- mineralized using simulated body fluids and induced
dimensional microenvironment.68 Several methods the formation of calcium-deficient carbonated HA,
such as chemical cross-linking, enzymatic cross- suggestion of potential application in bone tissue
linking, and physically cross-linked networks have engineering. While recombinant spider dragline silk
been reported to prepare ELP hydrogels.12,37,43,49,55,75 displays superior mechanical properties for scaffolds,
Recently, Chilkoti and coworkers reported that fibro- one of the technical hurdles is how to recapitulate these
blasts embedded in the ELP hydrogels cross-linked by properties in the laboratory on a scalable basis.81
organophosphorous cross-linker survived during the Asakura et al.4 reported a silk-like hybrid protein
cross-linking process and remained viable for at least consisting of polyalanine region of silk fibroin from
3 days in vitro, suggestion of controlling mechanical a wild silk worm, Samia cynthia ricini, and RDG
properties and potentially functional outcomes in tis- sequence derived from fibronectin. The obtained silk-
sue engineering by the ELPs.38 like hybrid protein film showed high cell adhesive and
A common goal to biomaterial design involves growth activities of kidney VERO cells when com-
creating biomimetic materials that resemble the ECM pared with those of collagen although strong acidic
protein components or short peptide sequences to elicit solvents were used to make the film.

686 NAGAOKA et al.

While recombinant silk proteins have been success- two CBDs into the human bFGF to develop a collagen-
fully synthesized, their low aqueous solubility limits based wound targeting repair system. The results
biomedical applications. To enhance the aqueous sol- showed that the bFGF with the CBD derived from
ubility of recombinant silks, differentially charged collagenase (C-bFGF) promoted vascularization at the
analogues of block copolymers containing repeating implanted sites more effectively than bFGF with the
sequences from silk (GAGAGS) and elastin (GVGVP) CBD derived from von Willeband’s factor (V-pFGF).
were synthesized by recombinant technique by Sheng et al.65 constructed a fusion protein consisted of
replacing a valine residue with glutamic acid.21 The bFGF fused to the C-terminus of glutathione S-trans-
block copolymers showed potentials as injectable ure- ferase (GST). The GST-bFGF stimulated the growth of
thral bulking agents for the treatment of female stress HUVECs to the same extent as recombinant bFGF,
urinary incontinence due to pH- and temperature- whereas GST itself did not stimulate, suggesting that
sensitive properties.50 The characteristics of structure- bFGF retains its biological activity when fused to GST
based recombinant ECMs are summarized in Table 1. although GST-bFGF must be supplemented with
bovine serum before storage to retain biological activity.
Cell-Bound Growth Factor-Based Recombinant Fusion
Epidermal Growth Factor
Proteins
Epidermal growth factor (EGF) is a 53-residue sin-
Fibroblast Growth Factor
gle chain polypeptide that plays important roles in tis-
Basic fibroblast growth factor (bFGF) is a potent sue regeneration, stimulation of the initiation of DNA
mitogen and a modulator for fibroblasts and vascular synthesis, cell replication, and activation of RNA,
endothelial cells69 and plays an important role in tissue acceleration of wound healing, enhancement of prolif-
regeneration and repair.72 The bFGF is found in eration and keratinization of epithelial tissues.10 The
abundance in tissues such as brain, kidney and carti- EGFs have been constructed to use for EGF fusion
lage. However, there are limitations in application to proteins consisted of EGF as the autocrine/paracrine
synthetic ECMs because it would diffuse rapidly even peptide signaling molecules and synthetic ECMs.
administered in topical side effects on the surrounding Kawase et al.34 constructed a fusion protein from
tissues87 and free bFGF molecules possess a shorter EGF and cell-binding domain of fibronectin. The fusion
half-life.59 Hashi et al.24 constructed a fusion protein of protein exhibited both cell-adhesive activity and growth
the cell-binding domain of human fibronectin and factor activity, each of which was indistinguishable from
human bFGF with both cell-adhesive activity and that of the corresponding unfused-protein.
growth factor activity. The fusion protein adsorbed to Nishi et al.54 constructed fusion proteins consisting
culture dishes stimulated the growth of human of EGF or bFGF and CBD derived from clostridium
umbilical-vein endothelial cells (HUVECs) and the histolyticum collagenase. The fusion proteins tightly
angiogenesis in chorioallantoic membranes of devel- bound to insoluble collagen and stimulated the growth
oping chick embryos, suggestion of potential in neu- of BALB/c3T3 fibroblasts as much as the unfused
roregenerative medicine. counterparts. Also, the CBDEGF remained at the sites
Andrades et al.3 constructed a fusion protein, which of injection for up to 10 days when injected subcuta-
contained bFGF and collagen-binding domain (CBD), neously into nude mice whereas EGF itself was not
a decapeptide derived from von Willeband’s factor. The detectable at 24 h after injection, indicating that the
recombinant protein increased affinity for collagen and fusion proteins are non-diffusible and long-standing
enhanced wound healing. Also, Zhao et al.87 introduced in 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 Asakura et al.4
VERO cells
SELP Silk + elastin Chondrogenesis of mesenchymal stem cells Haider et al.21


protein consisting of EGF and type III collagen. The
fusion protein was shown to hold the triple helical
conformation of collagen and the mitogenic activity of
EGF and the fusion protein can be immobilized on
tissue culture dishes as a fibrous form, suggestion of
potential in application to tissue engineering. Further-
more, Ishikawa et al.30 reported that the fusion protein
consisting of EGF and fibronectin collagen-binding FIGURE 2. Sustained activation of MAPK. A431 cells were
domain induced granulation tissue formation in the seeded onto EGF-Fc- or collagen-coated PS dishes and cul-
tured in DMEM containing 0.5% FBS for various periods of
wounds of kind limbs when applied with collagen gel. time. Cells cultured in collagen-coated PS dishes were stim-
Ogiwara et al.56 constructed a fusion protein con- ulated with or without 100 ng mL21 EGF. Lysates were sub-
sisting of EGF as a cell growth function and immu- jected to Western blotting with anti-phospho-MAPK antibody.
From Ogiwara et al.56
noglobulin G (IgG) Fc region (EGF-Fc) to stably
adsorb to a cell culture surface. Mouse fibroblast Swiss
3T3 cells adhered to EGF-Fc-coated surface, which is
similar to collagen-coated one whereas the cells did not
adhere to IgG-coated surface as shown in Fig. 1. Also,
phosphorylation of EGF receptors was induced by the
immobilized EGF-Fc as well as EGF itself, indicating
that the immobilized EGF-Fc tranduced a signal to the
cells through the phosphorylation of tyrosine residues
on signal proteins. Furthermore, immobilized EGF-Fc
continued to activate MARK after 4 h, whereas the
activation of MARK in the cells cultured in the pres-
ence of EGF itself rapidly decreased with time as
shown in Fig. 2, suggesting that MARK activation
induced by the immobilized EGF-Fc in the cells is
continuous without internalization of growth factor
and the constructed EGF-Fc can be used as a synthetic
ECM. They also constructed recombinant photo-
reactive EGF bearing p-azido phenylalanine at the
C-terminal (HEGFP) to immobilize the HEGFP stably
to biomaterial surface.57 The results indicated that
almost same amounts of A 431 cells adhered to
HEGFP-immobilized surface when compared with
collagen-coated surface as shown in Fig. 3a. Also, as

FIGURE 3. Cell adhesion to HEGFP: (a) A431 cells adhered
to the HEGFP-immobilized and collagen-coated surfaces after
30 min of incubation. The data represent means 6 SD of
experiments (n 5 3); (b) A431 cells pretreated with 100 ng/mL
soluble EGF were seeded onto each surface. Adhesion ratio
of the cells was measured after 30 min of incubation; (c) A431
FIGURE 1. Cellular adhesion to EGF-Fc. Swiss 3T3 cells cells suspended in PBS (2) were seeded onto each surface.
adhered to the EGF-Fc- and collagen-coated dishes after 6 h As a positive control, the cells suspended in DMEM contain-
of incubation. The data represent the mean 6 SD of experi- ing 0.5% FBS were seeded on collagen-coated surface. From
ments (n 5 3). From Ogiwara et al.56 Ogiwara et al.57

688 NAGAOKA et al.

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

Hybrid Systems Composed of Recombinant Fusion
Protein and Synthetic Polymer
Hybrid materials were prepared to mimic the nat-
ural ECM. It will be expected that they represent a new
and versatile class of biomimetic materials with clinical
promise in serving as implants because they promote
wound healing and tissue regeneration.63 Halstenberg
et al.23 prepared hybrid system composed of
recombinant protein containing an RGD integrin-
FIGURE 4. Liver-specific functions and DNA uptake of binding motif, two plasmin degradation sites and a
hepatocytes: hepatocytes were seeded onto HEGFP-immobi- heparin-binding site, and PEG chains with terminal
lized, collagen-coated or PVLA-coated surface and cultured in
WE for 3 days. Cells cultured on collagen were stimulated acrylate groups at the cysteine’s thiol groups, thereby
with or without 100 ng/mL EGF. CYP1A2 and HNF-4a mRNA rendering the protein covalently photo-cross-linkable
were detected by RT-PCR analysis. From Ogiwara et al.57 to form a hydrogel. The results indicated that it had
specific integrin-binding capability with heparin bind-
shown in Fig. 3b, the number of cells adhered onto
ing and proteolytic penetration in 2D and 3D systems.
HEGFP-immobilized surface remarkably decreased
Hubbell and coworkers also synthesized hybrid
whereas the number of cells adhered onto collagen-
hydrogels of consisting of recombinant protein having
coated one were not much changed, when A 431 cells
the activity of vascular endothelial growth factor
were pretreated with EGF, indication of receptor-
(VEGF) to induce cell adhesion and to provide cell-
mediated adhesion. Furthermore, as shown in Fig. 3c,
mediated remodeling by cross-linking matrix metallo-
Mg2+ does not play a prominent role in the receptor-
proteinase (MMP), and reactive PEG.88 The results
mediated interaction. Moreover, liver-specific func-
indicated that the hybrid hydrogel matrices atop the
tions, CYP1A2 and hepatocyte nuclear factor (HNF)-
chick chorioallontoic membrane brought strong new
4a, were maintained as same level as hepatocytes
blood vessel formation. When implanted subcutane-
cultured on galactose-carrying polymer (PVLA) as
ously in rats, these hybrid hydrogel matrices were
shown in Fig. 4, suggesting that the HEGFP can be
completely remodeled into native, vascularized tissue.
used as the synthetic ECM for liver tissue engineering.
Furthermore, they prepared hybrid hydrogels consist-
Elloumi et al.15 constructed a fusion protein consisting
ing of recombinant protein having cell adhesion motif
of RGD sequence as a cell adhesive function, EGF as a
RGD and degradation sites for plasmin and MMPs,
cell growth function, and hydrophobic sequence as an
and reactive PEG.60,61 The results showed that the
efficient assembling function. The fusion protein coated
hybrid hydrogels promoted specific cellular adhesion
on hydrophobic surface retained both cell adhesive
and exhibited degradability by the target enzymes.
activity through the RGD sequence and cell growth
Also, the hybrid hydrogels promoted healing of bone
activity when A 549 cells were cultured, whereas EGF
defects after treatment of rat calvarial defects, sug-
itself had no growth activity even though it was
gesting that the combination of recombinant genetic
attached to a solid surface to some extent, suggesting
technology and synthetic polymer emerges as a pow-
that the hydrophobic sequence played roles of a cell
erful for the development of artificial ECMs.
adhesion function and stabilization of protein structure.

Platelet-Derived Growth Factor
E-CADHERIN
Dai and coworkers constructed a fusion protein
from platelet-derived growth factor (PDGF) and E-cadherin is a member of the intercellular adhesion
CBD.40 The fusion protein promoted the binding of molecules and E-cadherin-mediated adhesion is regu-
PDGF to collagen scaffold with enhanced vasculari- lated by Ca2+-dependent homophilic interaction.73
zation in vivo and caused more cells to proliferate on E-cadherin is essential for tissue morphogenesis and
the collagen gel than native PDGF, suggesting that this maintenance of organized solid tissues.53
protein is effective for targeting tissue regeneration and Nagaoka et al.47 constructed a fusion protein
wound repair. They also constructed a fusion protein consisting of E-cadherin extracellular domain and
from nerve growth factor-b (NGF-b) and CBD.71 IgG Fc region (E-cad-Fc) to stably adsorb to a cell
The fusion protein increased the expression level and culture surface as shown in Fig. 5. They reported that


FIGURE 5. Construction and expression of E-cadherin-IgG
Fc fusion protein. (a) Generated segments of an extracellular
domain of mouse E-cadherin and an IgG Fc region were
subcloned into an eukaryotic expression vector pRC/CMV via
HindIII-NotI site and NotI-XbaI site respectively. CMV pro-
moter, human cytomegalovirus immediate-early promoter/
enhancer; BGH pA, bovine growth hormone polyadenylation
signal; Neor, neomycin resistance gene for selection; Ampr,
ampicillin resistance gene. (b) Conditioned media from
transfected 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; dotted
arrow, precursor. (c) Cell lysate and conditioned media of
transfectant were analyzed to confirm the protein secretions
by Western blotting using anti-mouse IgG antibody. From
Nagaoka et al.47

FIGURE 7. Cell adhesion, morphology of ES cells on the
E-cad-Fc fusion protein-immobilized surface. (a) ES cells (EB3)
adhered to E-cad-Fc-coated dishes with equivalent efficiency
as to 0.1% gelatin-coated dishes after 3 h of incubation. (b) ES
cells (EB3) were cultured on E-cad-Fc-coated or fibronectin-
coated dishes without serum. EGTA (5 mM) was added to the
culture medium at 3 h after seeding (open bar). Detached cells
were removed and remaining cells were counted using alamar
Blue 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

hepatocytes adhered to the E-cad-Fc-coated surface
were almost same as collagen-coated one and the
adhesion was inhibited by pretreatment of hepatocytes
with 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 the
adhesion of hepatocytes to E-cad-Fc is mediated by
E-cadherin. Also, they reported that hepatocytes
adhered to E-cad-Fc-coated surface showed differenti-
ated phenotypes such as low DNA synthesis activity and
FIGURE 6. Adhesive activity to E-cad-Fc fusion protein. (a) maintenance of tryptophan oxygenase expression as a
Primary hepatocytes could adhere to E-cad-Fc-coated dish as marker gene of differentiated hepatocytes,29 suggestion
well as collagen-coated surface after 4 h. Inhibition assay was
performed by incubating with neutralizing antibody (ECCD-1). of potential synthetic ECM in maintaining differentia-
BSA was a negative control. (b) Adhesion of mouse hepatoma tion of hepatocytes for liver tissue engineering.
cell line Hepa 1-6 to collagen or E-cad-Fc was analyzed after Also, Akaike and coworkers constructed E-cad-Fc
3 h culture. (c) The expression levels of E-cadherin in two
types of liver-derived cells were assessed by western blotting. for culture of embryonic stem (ES) cells.48 They
The data represent the mean 6 SEM of experiments (n 5 3). reported that EB 3 cells as the ES cell adhered to the
From Nagaoka et al.47 E-cad-Fc-coated surface as similar with conventional

690 NAGAOKA et al.

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 (open
square) or E-cad-Fc-coated (filled square) dishes and the cell number was counted after staining with alamar Blue reagent. The
data indicate means 6 SD of experiments (n 5 3). **p < 0.01 vs. gelatinized plates. (b) BrdU incorporation of EB3 cells under
colony-forming (on gelatin) or scattering conditions (on E-cad-Fc). Relative BrdU incorporation value was evaluated. The data
indicate means 6 SEM. §p < 0.001. (c) Transfection efficiency of ES3 cells cultured on gelatin- or E-cad-Fccoated surface. Relative
expression 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 Hayashi et al.,25 Nishi et al.54
and nondiffusible
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 Retention of cell adhesive activity and stabilization Elloumi et al.15
sequences of protein structure
PDGF PDGF Collagen Increased vascularization of collagen scaffold Lin et al.40
NGF NGF-b Collagen Enhancement of nerve growth Sun et al.71

gelatin-coated surface as shown in Fig. 7a. The cells dishes, suggesting that the E-cad-Fc will have a great
adhered even in serum-free condition with Ca2+- potential as the artificial ECM for stem cell tissue
dependency as shown in Fig. 7b. Interestingly, the engineering. The characteristics of growth factor-based
adhered cells remained separated from each other even recombinant ECMs are summarized in Table 2.
in the presence of leukemia inhibitory factor (LIF)
with dendritic morphologies, whereas the cells adhered
to gelatin-coated surface formed tightly aggregated SUMMARY AND FUTURE PERSPECTIVE
colonies as shown in Figs. 7c and 7d, suggesting that
blocking of close contact among cells in the aggregated Precise control over molecular weight, composition,
colonies may not generate a heterogeneous environ- sequence and stereochemistry at the molecular level by
ment within colonies, which potentially inhibit the recombinant genetic engineering method has created
proliferation of ES cells and the distribution of soluble interest in the artificial ECMs of biomaterials for tissue
factors. Also, the ES cells showed higher proliferation engineering. In this review, the design and construc-
on the E-cad-Fc-coated surface than gelatin-coated tion of ELPs, SLPs, bFGF, EGF, E-cadherin, and
one as shown in Figs. 8a and 8b and the cells adhered hybrid systems composed of recombinant protein and
to the E-cad-Fc-coated surface showed higher trans- synthetic polymer by recombinant technique were
fection efficiency than gelatin-coated one as shown in explained for the artificial ECMs. Modulation of the
Fig. 8c. Furthermore, the expression of Oct-3/4 as the cell recognition is achieved through precise controls in
undifferentiated marker of the ES cells was maintained sequence and length. While some examples of the
for at least 3 days when cultured on E-cad-Fc-coated influence of sequence, length, hydrophobicity, and cell

8
binding domain on the cell recognition were reviewed in Cappello, J., and K. Park (Eds.). Controlled Drug Deliv-
this article, medical applications are still on the horizon ery: Challenges and Strategies. Washington DC: American
Chemical Society, 1997, p. 439.
and the full potential of this recombinant in tissue 9
Chong, B. F., L. M. Blank, R. McLaughlin, and L. K.
engineering has yet to materialize. The control of Nielsen. Microbial hyaluronic acid production. Appl.
mechanical properties, biodegradation, biorecognition Microbiol. Biotechnol. 66:341–351, 2005.
10
and checking of no immunogenic response can signifi- Cohen, S. The epidermal growth factor (EGF). Cancer
cantly expand the utility of these constructs in tissue 51:1787–1791, 1983.
11
Cutler, S. M., and A. J. Garcia. Engineering cell adhesive
engineering although a prerequisite for the growth of
surfaces that direct integrin alpha5beta1 binding using a
applications based on recombinant fusion proteins is the recombinant fragment of fibronectin. Biomaterials 24:
improvement of the production of larger amounts of 1759–1770, 2003.
12
recombinant proteins. Also, there will be increasing Di Zio, K., and D. A. Tirrell. Mechanical properties of
demands that 3D recombinant fusion proteins provide artificial protein matrices engineered for control of cell and
tissue behavior. Macromolecules 36:1553–1558, 2003.
better model systems for physiologic situations because 13
Duan, X. J., H. X. Niu, W. S. Tan, and X. Zhang.
3D fusion proteins induce more effectively cellular Mechanism analysis of effect of oxygen on molecular
functions than 2D ones although 2D in vitro assay on 2D weight of hyaluronic acid produced by Streptococcus zoo-
recombinant fusion proteins are still applied in many cell epidemicus. J. Microbiol. Biotechnol. 19:299–306, 2009.
14
culture studies. Furthermore, future research will be Ehrbar, M., S. C. Rizzi, R. Hlushchuk, V. Djonov, A. H.
Zisch, J. A. Hubbell, F. E. Weber, and M. P. Lutolf.
aimed to the design of hybrid materials consisting of
Enzymatic formation of modular cell-instructive fibrin
intelligent recombinant proteins and other biomaterials analogs for tissue engineering. Biomaterials 28:3856–3866,
with high mechanical characteristics for hard tissues. 2007.
15
Elloumi, I., R. Kobayashi, H. Funabashi, M. Mie, and
E. Kobatake. Construction of epidermal growth factor
fusion protein with cell adhesive activity. Biomaterials
27:3451–3458, 2006.
ACKNOWLEDGMENT 16
Ghandehari, H., and J. Cappello. Genetic engineering of
protein-based polymers: potential in controlled drug
This work was supported by the funds provided by
delivery. Pharm. Res. 15:813–815, 1998.
Korea Research Foundation (KRF) (E00244). 17
Ghosh, K., and D. E. Ingber. Micromechanical control of
cell and tissue development: implications for tissue engi-
neering. Adv. Drug Deliv. Rev. 59:1306–1318, 2007.
18
Girotti, A., J. Reguera, J. C. Rodriguez-Cabello, F. J.
Arias, M. Alonso, and A. M. Testera. Design and bio-
production of a recombinant multi(bio)functional elastin-
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