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Vascular induction and cell infiltration into peptide-modified

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bioactive silk fibroin hydrogels

Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
a a,b b c a
Yusuke Kambe, Akie Murakoshi, Hiroshi Urakawa, Yoshiharu Kimura and Tetsuji Yamaoka
In hydrogel-based soft tissue engineering, vascular induction into a hydrogel as well as long-term volume retention is
essential to maintain tissue shape and function without causing necrosis in the deeper part of the hydrogel. Silk fibroin (SF)
hydrogel shows a sufficiently high mechanical strength to maintain its shape during implantation for a month, but it has
not been well evaluated whether it has vascular-inducing bioactivity to achieve its replacement by vascularized tissue.
Here, we produced a vascular-inducing peptide (VIP) containing an endothelial cell (EC)-adhesive REDV and vascular
endothelial growth factor-mimic QK peptides to modify the SF hydrogel. In vitro experiments showed that the modification
of the SF hydrogel with VIP changed only biological properties of the hydrogel due to the bioactivity of VIP. Subcutaneous
implantation of SF hydrogels in rats revealed isotropic EC migration into the hydrogels, which was followed by infiltration
of macrophages and fibroblasts. Since these macrophages and fibroblasts appeared to degrade the SF network and to
produce collagen, respectively, SF hydrogels were replaced gradually by regenerated tissue. VIP accelerated cell infiltration
and doubled the formation of blood vessels in the regenerated tissue. These results suggest the potential of the VIP-
modified SF hydrogel as a material for soft tissue engineering applications.

1. Introduction
In soft tissue engineering, hydrogel implants have been used
as fillers that are expected to produce volume retention for a
long period (months to years).1 Particularly, for wrinkle
treatment and facial/breast reconstruction, hydrogels
composed of natural/synthetic materials, such as collagen,
hyaluronic acid, or silicon, are implanted to restore the
aesthetic contour.1,2 Recently, hydrogel injections into
infarcted cardiac tissues have been shown to improve cardiac
function in myocardial infarction models,3,4 whose mechanism
is believed to be based on Laplace’s Law; an increase in wall
thickness by injected hydrogels leads to a decrease in wall
stress, preventing infarct expansion and negative ventricular
remodeling.3-5 Thus, the mechanical strength of hydrogels to
retain their volume and shape is an important factor for
hydrogel-based soft tissue augmentation and regeneration.
Another essential factor is the replacement of the implanted
hydrogels by vascularized tissue. It is accepted that the
vascularization of regenerated tissues is critical for the
6,7
achievement of regenerative medicine and for the treatment
8
of ischemic tissue disorders. Local necrosis after the injection
®
of the bovine collagen gel Zyplast at the glabellar area, where
there are fewer and smaller blood vessels, was caused by
1,2,9
immature vascular networks in and around the injected gel.
Neovascularization and cell infiltration due to the degradation
of hydrogels injected into infarcted heart tissues play a key
3,4,10,11
role in the long-lasting enhancement of cardiac function
because nondegradable polyethylene glycol hydrogels retained
increased wall thickness, but showed no functional
10,11
improvement at several months post-injection. Therefore,
the vascular-inducing bioactivity, as well as the mechanical
strength, of hydrogels are required for hydrogel-based soft
tissue engineering in vivo.
Bombyx mori silk has been used for centuries in medicine as
a surgical suture, and, more recently, degummed B. mori silk
protein, called silk fibroin (SF), has been approved by the US
Food and Drug Administration for clinical use. After being
dissolved, SF can be processed into films, meshes, sponges,
and hydrogels for biomedical applications, including as
12-16
scaffolds for tissue engineering. The gelation of SF aqueous
solutions is triggered by physical stimulants such as
17-20
temperature, pH, vortexing, and sonication. Upon gelation,
the random-coil structures of SF molecules transform into β-
sheet structures, and then, the β-sheet structures aggregate to
21,22
form physical crosslinks that organize into hydrogels.
Hydrophobic amino acid sequences (GAGAGS/Y) in the SF
heavy-chain (FibH) play a central role in the gelation process.
The mechanical strength of SF hydrogels can be adjusted by SF
20,21,23
concentration and ionic additives. In particular, the

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compressive modulus of SF hydrogels ranges widely from by its subcutaneous implantation in rats. To clarify cell
21,23
several Pa to approximately 6 MPa. This pronounced infiltration into the SF hydrogel even after a long-term
mechanical strength is likely to enable an SF hydrogel to keep implantation (8 weeks), the hydrogel was formed in a
24
its shape for 3 months in vivo. In contrast, it is still unclear polylactic-acid (PLA) three

Journal of Materials Chemistry B Accepted Manuscript

whether SF hydrogels have sufficient vascular-inducing
bioactivity to achieve their replacement by vascularized tissue
after implantation. Although two sequences in the SF primary
25
structure were found to stimulate fibroblast adhesion, their
activity against endothelial cells (ECs) has not shown. Rather,
26-28
the following previous findings indicate that SF hydrogels
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lack vascular-inducing bioactivity: few ECs attached to a bare

26
non-woven SF mesh in vitro; even coating an SF mesh with
collagen and fibronectin induced few blood vessels at 14 days
post-implantation in mice;27 and endothelial progenitor cells
failed to form angiogenic structures in SF hydrogels in vitro,
even after supplementation with vascular endothelial growth
factor (VEGF).28 Therefore, in order to make an SF hydrogel a
good candidate material for soft tissue augmentation and
regeneration, the vascular-inducing bioactivity of the hydrogel
needs to be enhanced.
To append such bioactivity to an SF hydrogel, herein, we
developed a vascular-inducing peptide (VIP) composed of
several functional peptides: FibH-derived peptide; cell-
adhesive REDV peptide; matrix metalloprotease (MMP)-
cleavable peptide; and VEGF-mimic peptide (from the N- to C-
terminal). The REDV peptide was derived from fibronectin and
binds to ECs selectively via integrin α4β1.29-31 This peptide
reportedly promotes EC adhesion32-34 and migration velocity34
when immobilized on scaffold materials. The VEGF-mimic
peptide (called QK) was based on the native α-helical receptor-
binding domain of VEGF and shown to activate VEGF receptors
(VEGFRs).35,36 Peptide-based nano-structures containing QK
enhanced blood perfusion in the ischemic area in model mice Fig. 1 (A) Amino acid sequences of VIP and VIP(MMP(-)).
for peripheral artery disease.37,38 In VIP, the REDV and QK Sequences of the FibH-derived peptide are italicized;
peptides were linked with the MMP-cleavable GPQG↓IWGQ sequences of the tandem repeat of EC-adhesive REDV are in
peptide (the ↓ symbol indicates the protease cleavage bold; sequences of the MMP-cleavable/uncleavable peptide
site).39,40 This cleavable peptide has been used widely to are underlined (the ↓ symbol indicates the protease cleavage
release bioactive peptides/proteins from base materials.41,42 site); and sequences of the VEGF mimic QK peptide are gray.
The FibH-derived peptide at the N-terminal of VIP contained a (B) SDS-PAGE analysis for the proteolytic cleavage of VIP.
repetitive GA sequence. Upon the gelation of an SF aqueous Novex® Sharp Protein Standard (Invitrogen, CA) was used as a
solution containing VIP, the FibH-derived peptide in VIP was marker (M). Prior to the analysis, 5 µM VIP (MW, 12.3 × 103) or
expected to be embedded in the aggregated β-sheet VIP(MMP(-)) (MW, 12.2 × 103) in PBS were incubated with
structures (i.e., physical crosslinks) of the SF hydrogel. various amounts of collagenase (0, 0.05, 0.1, 0.5, and 1 µg mL-
1
Therefore, our strategy to modify the SF hydrogel was as ). This treatment cleaved only VIP, resulting in the release of a
follows. Due to the presence of the FibH-derived peptide, VIP peptide whose MW was ~3 × 103 (indicated by the arrow).
is immobilized in the SF hydrogel during its gelation process,
and when the VIP-modified SF hydrogel is implanted in vivo, -dimensional (3-D) lattice. This in vivo system clarified that VIP
the MMP-cleavable peptide is cleaved proteolytically to accelerated EC infiltration into the SF hydrogel, resulting in the
release the QK peptide. The released VEGF-mimic QK peptide replacement of the hydrogel by highly vascularized tissue.
attracts ECs from the surrounding tissue to the SF hydrogel.
Then, using the REDV peptide as a foothold, the ECs infiltrate
the hydrogel to make the SF hydrogel rich in blood vessels. 2. Results and discussion
VIP was prepared as a fusion peptide by using transgenic 2.1. Production and in vitro bioactivity of VIP
Escherichia coli technology, and its bioactivity was shown in in
Using transgenic E. coli technology, the FibH-derived, EC-adhesive
vitro experiments. Since the immobilization of VIP in the SF
REDV, MMP-cleavable, and VEGF-mimic QK peptides were fused to
hydrogel was confirmed, the effects of VIP modification on the
form VIP. A subspecies of VIP designated as VIP(MMP(-)), containing
vascular-inducing bioactivity of the hydrogel were evaluated

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41
the MMP-uncleavable GDQGIAGF peptide instead of by the collagenase. This result indicates that the VEGF-mimic QK
GPQG↓IWGQ in VIP, was also produced. Their amino acid peptide at the C-terminal of VIP can be released proteolytically.
sequences are shown in Fig. 1A. The major protein after purification Human umbilical vein ECs (HUVECs) that adhered to VIP- or
with an Ni-chelate column had a molecular weight (MW) of ~15 × VIP(MMP(-))-coated glass substrates showed spread shapes with a
3

Journal of Materials Chemistry B Accepted Manuscript

10 (Fig. S1A, ESI†), corresponding to the theoretical MW of VIP
3 3
(12.3 × 10 ) and VIP(MMP(-)) (12.2 × 10 ). In addition, these
proteins contained recombinant protein-specific polyhistidine (Fig.
S1B, ESI†), suggesting that highly pure (>90%) VIP and VIP(MMP(-))
were obtained. Positively-charged amino acids in VIP and
VIP(MMP(-)), such as polyhistidine, might affect differences
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developed actin cytoskeleton and filopodia, while the cells on the
non-coated glass substrate formed polygonal shapes (Fig. 2A). More
HUVECs adhered to the VIP and VIP(MMP(-)) substrates, compared
to the glass substrate, within 90 min post-cell seeding. However, no
significant difference was detected between the VIP and VIP(MMP(-
)) groups (Fig. 2B). Therefore, VIP and VIP(MMP(-)) coated on the

between the apparent MW and theoretical MW.
After incubation with various concentrations of collagenase, VIP
and VIP(MMP(-)) solutions were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). As shown in
Fig. 1B, the collagenase cleaved VIP in a dose-dependent manner,
resulting in a fragment (MW, ~3 × 103); however, in contrast,
VIP(MMP(-)) was insensitive to the enzyme. Therefore, the MMP-
cleavable peptide (GPQG↓IWGQ) in VIP was shown to be cleaved
glass substrate enhanced EC adhesion. This result is likely to be
induced by REDV, which is found in both VIP and VIP(MMP(-)),
because the peptide is bound by integrin α4β1 to stimulate the
selective adhesion and spreading of ECs.29-34 The competitive
inhibition of HUVEC adhesion to the VIP and VIP(MMP(-)) surfaces
with soluble GREDVY pepƟdes (Fig. S2, ESI†) also supports the
enhancement of EC adhesion by REDV in VIP.
While incubation for one and a half day, HUVECs seeded onto the

Fig. 2 (A) Fluorescent staining of F-actin and nuclei in HUVECs on bare, VIP-coated, and VIP(MMP(-))-coated glass substrates at 90 min post-
cell seeding. Scale bar = 50 µm. (B) Density of HUVECs that adhered to bare, VIP-coated, and VIP(MMP(-))-coated glass substrates at 90 min
4 -2
post-cell seeding at 1.0 × 10 cells cm . Data are shown as mean ± standard error of the mean (SEM; n = 3). Asterisks indicate significant
differences from the bare glass (Glass) group, analyzed by one-way ANOVA followed by Tukey’s post hoc comparison (p < 0.05). (C)
Fluorescent staining of F-actin, VE-cadherin, and nuclei in HUVECs on bare, VIP-coated, and VIP(MMP(-))-coated glass substrates at 34 h
post-cell seeding. Scale bar = 50 µm. (D) Dose-dependent changes in HUVEC growth-promoting activity of VIP, VIP(MMP(-)), QK peptide,

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and VEGF. Higher absorbance means higher activity. Data are shown as mean ± SEM (n = 3). An asterisk indicates a significant difference
between the VIP(MMP(-)) and QK-VEGF groups, analyzed by two-way ANOVA followed by Tukey’s post hoc comparison (p < 0.05).
glass, VIP, and VIP(MMP(-)) substrates proliferated to form cell–cell synthesized by HUVECs might cleave VIP to release the QK peptide,
junctions mediated by VE-cadherin (Fig. 2C), which is a cell-specific which bound readily to the VEGFRs on the cells.
43

Journal of Materials Chemistry B Accepted Manuscript

marker to be expressed by ECs. Culturing on the VIP and These results suggest that VIP had bioactivities derived from its
VIP(MMP(-)) substrates increased cell–cell junction maturation: the component peptides. Therefore, it is expected that, when
44
junctions formed a smooth line between the cells. This might immobilized in a scaffold, VIP enables the proteolytic release of
result from the developed actin cytoskeleton in HUVECs grown on VEGF-mimic QK from the scaffold, which stimulates the attraction
the VIP and VIP(MMP(-)) substrates since homophilic interactions of ECs in vivo. Subsequently, the REDV peptide in VIP is likely to
between VE-cadherins are strengthened by intracellular promote the adhesion and migration of ECs, resulting in the
43,44
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interactions involving actin cytoskeleton. formation of vascular structures with matured cell–cell junctions in
The dose-dependent effects of VIP and VIP(MMP(-)) on HUVEC the scaffold.
proliferation were evaluated by the WST assay (Fig. 2C), where
synthesized QK peptide and recombinant VEGF were used as 2.2. Physical and biological properties of SF hydrogels modified
positive controls. As additives to the medium, the four with VIP
peptides/proteins were shown to enhance HUVEC proliferation at a
concentration ranging from 1 to 1,000 nM. Although the effect of SF aqueous solutions with/without VIP were mixed with -1
a sodium
VIP(MMP(-)) was significantly lower than that of QK or VEGF, there citrate buffer (pH-1 3.0) (final concentrations, 20 mg mL SF, 0/10 µM
was no significant difference between VIP and QK or VEGF. Since [0/0.12 mg mL ] VIP, and 20 mM sodium citrate buffer) and
the α-helical structure of QK plays a critical role in its binding to incubated at 37°C overnight in a humidified atmosphere, resulting
VEGFRs, such as VEGFR-1 and VEGFR-2,35,36 collagenases in the formation of opaque SF or SF+VIP hydrogels (Fig. 3A).

Fig. 3 (A) Gelation of 20 mg mL-1 SF aqueous solution in the presence/absence of 20 mM sodium citrate buffer (pH 3.0) and 10 µM (0.12 mg
mL-1) VIP. Regardless of the presence/absence of VIP, turbid SF hydrogels were formed by the addition of the citrate buffer. (B) Release
behavior of TAMRA-VIP from SF hydrogels modified with TAMRA-VIP in PBS with (w/) or without (w/o) collagenase at 37°C. Curve fitting
was done using a single exponential association. Data are shown as mean ± SEM (n = 4). (C) Scanning electron micrographs of freeze-dried
SF hydrogels with (SF+VIP) or without (SF) VIP. Scale bar = 50 µm. No remarkable difference was observed. (D) Water content and
compression modulus of the SF and SF+VIP hydrogels. Data are shown as mean ± SEM (n = 3). No significant difference was detected by a
two-sided Student’s t test. (E) Time-dependent changes in the growth of HUVECs cultured on the SF hydrogels containing 0, 1, and 10 µM
VIP and Matrigel. Higher absorbance means higher number of cells. Data are shown as mean ± SEM (n = 4). An asterisk indicates a

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significant difference from the SF+VIP (10 µM) group, while sharps indicate significant differences from the Matrigel group, analyzed by
two-way ANOVA followed by Tukey’s post hoc comparison (*: p < 0.05; ##: p < 0.01; ###: p < 0.001).
Figure 3B shows the release behavior of VIP from the SF hydrogel target of the TAMRA labelling), might be released preferentially due
-1
in phosphate-buffered saline (PBS) with/without 1 µg mL to the cleavage of the GPQG↓IWGQ peptide by the enzyme.

Journal of Materials Chemistry B Accepted Manuscript

collagenase at 37°C, which was characterized by using a However, the final percentage of release in the presence of the
tetramethylrhodamine (TAMRA)-labelled VIP (TAMRA-VIP; 10 µM). collagenase was only ~19%. Thus, ~8% ([19 – 13] × 7/5) of the fed
The release of VIP stopped within 12 h and the final percentage of VIP was estimated to expose its bioactive part to the surface of the
release in pure PBS was ~13%. This value is obviously lower than SF hydrogel. The remaining ~79% of VIP (100 – [13 + 8]) might
3
the release of physically absorbed dextran (MW, 10 × 10 ; 50–60% embed its whole body in the physical crosslinks. In this experiment,
45 46
at 30 h and 70% at 7 days ), bone morphogenic protein-2 (MW, the SF hydrogels appeared not to be degraded even in the
47
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not shown; 75% at 1 week) , and recombinant human insulin (MW, collagenase soluƟon (Fig. S3, ESI†). This can be the reason why the
3 48
5.8 × 10 ; 30% at 10 days) from SF materials. By using the FibH- release of VIP saturated within a day. However, even VIP embedded
derived peptide, VIP was considered to be immobilized in the in the physical crosslinks is probably released with the degradation
aggregated β-sheet structures (i.e., physical crosslinks) of the SF of the SF hydrogel in vivo, since SF material degradation affects
hydrogel. It is inferred that, after immobilization, the bioactive part release behaviour.45
(REDV, the MMP-cleavable peptide, and QK) of VIP was exposed on As the physical properties of a scaffold reportedly influence cell
the surface of the SF hydrogel in wet conditions because the behavior in/on the scaffold, such as adhesion and migration,49-52 we
bioactive part is more hydrophilic than the FibH-derived peptide; then evaluated the effect of VIP modification on them. As shown in
the grand average of the hydropathicity values for the bioactive Fig. 3C, no remarkable difference was observed between the
part and FibH-derived peptide, calculated using the ProtParam tool microstructures of freeze-dried SF and SF+VIP hydrogels. Moreover,
(http://web.expasy.org/protparam/), was -1.197 and -0.222, there was no significant difference between the SF and SF+VIP
respectively. This theory is supported partly by the increased hydrogels with respect to water content and compression modulus;
release of VIP in the collagenase solution (Fig. 3B): the QK peptide, both hydrogels absorbed a huge amount of water (>95 wt%) and
which contains five of the seven primary amine groups in VIP (the showed a compression modulus of ~8 kPa (Fig. 3D).

Fig. 4 Photographs of H&E-stained implants. SF and SF+VIP hydrogels formed in PLA 3-D lattices were implanted s.c. in rats for 1,
2, 4, and 8 weeks. Matrigel was used as the positive control for cell infiltration. Cells infiltrated the SF and SF+VIP hydrogels

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isotropically (surrounded by a dotted-line). Inside these cells, SF networks were observed (asterisks), while blood vessels
(arrows) were shown outside them. Scale bar = 1 mm and 200 µm (a–d).
In contrast to physical properties, biological properties of the SF the biological properties of the hydrogel could be modulated by the
hydrogels changed by the modification with VIP, showing dose- VIP modification, due to the bioactivity of VIP.

Journal of Materials Chemistry B Accepted Manuscript

dependent effects on the growth behaviour of HUVECs seeded onto
the SF hydrogels (Fig. 3E). HUVECs on the SF hydrogel without VIP
proliferated from 1 to 3 days but, afterward, their growth was
saturated. On the other hand, the cells on the SF+VIP (1 µM)
hydrogel kept proliferating until 5 days, suggesting the stimulation
of cell growth by VIP. However, at a concentration of 10 µM, VIP
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2.3. In vivo behaviour of SF hydrogels modified with VIP
To clarify cell infiltration into the SF and SF+VIP (VIP concentration,
10 µM) hydrogels implanted subcutaneously (s.c.) in rats for a long
period, the gels were formed in a PLA 3-D lattice (6 × 6 × 3 mm3)
(Fig. S4, ESI†). The feed amount of VIP in the SF hydrogel was set at

immobilized in the SF hydrogel inhibited HUVEC proliferation, as is 10 µM in light of its concentration reduction by diffusion in vivo,
®
the case with BD Matrigel Basement Membrane Matrix (Matrigel), although HUVEC growth was inhibited on the SF+VIP (10 µM)
in which growth factors were not reduced. Since an overdose of hydrogel in vitro (Fig. 3E). Matrigel was also formed in the PLA 3-D
growth factors inhibits cell proliferation,53-55 the concentration of lattice and used as the positive control for cell infiltration. This is
VIP (especially QK) in the SF+VIP (10 µM) hydrogel and that of because Matrigel are used frequently to induce
growth factors in Matrigel were beyond the optimal range for cell neovascularization56-58 and shown to maintain its volume in vivo.58
growth in vitro. The hydrogels formed in the PLA 3-D lattice were implanted s.c. in
These results suggest that the modification with VIP had no rats for 1, 2, 4, and 8 weeks. At enucleation, no obvious differences
influence on the physical properties of the SF hydrogels, likely due among specimens were observed visually (Fig. S5, ESI†). Then, the
to the small amount of VIP (SF : VIP = 1 : 0.006 wt%). In contrast, implants were subjected

Fig. 5 Photographs of CD68 (monocyte/macrophage marker)-immunostained implants. SF and SF+VIP hydrogels formed in PLA 3-
D lattices were implanted s.c. in rats for 1, 2, 4, and 8 weeks. Matrigel was used as the positive control for cell infiltration. Round
cells (arrowheads) were observed at 1 week post-implantation. In contrast, trachychromatic, knurled cells (clear arrows) were

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shown at 2 and 4 weeks. These trachychromatic cells approached the center of the SF and SF+VIP hydrogels (surrounded by a
dotted-line), and SF networks remained inside them (asterisk). However, they disappeared at 8 weeks. Scale bar = 1 mm, 25 (a
and b), 200 (c), and 50 (d) µm.
to histology: hematoxylin and eosin (H&E) staining; and approaching cells were not observed; instead, collagen fibrils

Journal of Materials Chemistry B Accepted Manuscript

immunostained for CD68 (monocyte and macrophage marker), and blood vessels (Fig. 4c) were observed throughout the
prolyl 4-hydroxylase subunit β (P4HB; fibroblast marker), and CD31 whole area of the implants. These results suggest that the SF
(EC marker). hydrogel networks were degraded completely from 4 to 8
2.3.1. H&E staining. Photographs of H&E-stained implants are weeks and were replaced by regenerated tissue. The
shown in Fig. 4. From 1 to 8 weeks post-implantation, cells approaching cells infiltrated more deeply into the SF+VIP
infiltrated the hydrogels gradually. Besides, there appeared no hydrogels than into the SF hydrogels at 2 and 4 weeks post-
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encapsulation of the implants. Obviously, cell infiltration into implantation. Thus, the modification of SF hydrogels with VIP
Matrigel was the fastest; the cells reached the center of accelerated cell infiltration.
Matrigel within 2 weeks (Fig. 4d). Cells infiltrated the Matrigel 2.3.2. Immunostaining of CD68 (monocyte/macrophage marker).
anisotropically; however, in contrast, the outside-in approach Figure 5 exhibits photographs of CD68-immunostained
of cells appeared to be isotropic in the SF and SF+VIP implants. At 1 week post-implantation, positive cells with a
hydrogels (we call these cells “approaching cells;” surrounded round shape were observed inside the hydrogels (indicated by
by a dotted-line). There remained SF networks (indicated by arrowheads in Fig. 5a). These cells were likely to be
asterisks in Figs. 4a and 4b) inside the approaching cells. monocytes. In contrast, trachychromatic, knurled cells
Conversely, outside the approaching cells, SF was likely to be (indicated by clear arrows in Fig. 5b), which appeared to be
degraded and there were eosin-positive collagen fibrils and macrophages, infiltrated isotropically into the SF and SF+VIP
luminal structures with red blood cells located inside hydrogels from 2 to 4 weeks (surrounded by a dotted-line in
(indicated by arrows). These findings, particularly the collagen Fig. 5c). This behavior of the trachychromatic CD68-positive
deposition, were also supported by the results of van Gieson cells corresponded to that of the approaching cells shown in
staining (Fig. S6, ESI†). At 8 weeks post-implantation, the Fig. 4. Therefore, VIP showed an accelerating

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Fig. 6 Photographs of P4HB (fibroblast marker)-immunostained implants. SF and SF+VIP hydrogels formed in PLA 3-D lattices
were implanted s.c. in rats for 1, 2, 4, and 8 weeks. Matrigel was used as the positive control for cell infiltration. Trachychromatic
cells approached the center of the SF and SF+VIP hydrogels (surrounded by a dotted-line), while SF networks remained inside
(asterisks). Outside these cells, spread cells were observed (clear arrowheads). At 8 weeks, spread cells were observed

Journal of Materials Chemistry B Accepted Manuscript

throughout the whole area of the implants. Scale bar = 1 mm, 200 (a), 100 (b), and 25 (c and d) µm.
effect on macrophage infiltration into SF hydrogels. The infiltrated the SF hydrogel, but they appeared not to break
trachychromatic cells seen at 2 and 4 weeks disappeared at 8 down the SF networks (Fig. 8a). Then, from 1 week onward,
weeks post-implantation, although round cells were observed ECs infiltrated the SF hydrogel isotropically to lead the
inside the implants (Fig. 5d). approaching cells, including macrophages and fibroblasts (Figs.
2.3.3. Immunostaining of P4HB (fibroblast marker). The time- 8b–8e). During this process, macrophages and fibroblasts were
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dependent infiltration of P4HB-positive cells into the hydrogels likely to degrade the SF networks and produce collagen,
(Fig. 6) was similar to that of CD68-positive cells (Fig. 5). resulting in the gradual replacement of the SF hydrogel by
Trachychromatic cells (surrounded by a dotted-line in Figs. 6a regenerated tissue. Additionally, luminal structures were
and 6b) approached the center of the SF or SF+VIP hydrogels in formed by ECs in the regenerated tissue, that is, outside the
synchrony with each other from 2 to 4 weeks, and this process approaching cells (Fig. 8e). Finally, by 8 weeks post-
was promoted by VIP. Hence, like macrophages, fibroblasts implantation, the SF hydrogel was degraded completely and
were likely to correspond to the approaching cells. P4HB- replaced by regenerated tissue (Fig. 8f). As there were few
positive, spread cells (indicated by clear arrowheads in Fig. 6c) trachychromatic CD68-positive cells in the implants at 8 weeks
were observed outside the trachychromatic approaching cells. (Fig. 5d), the hydrogels did not cause chronic inflammation. In
At 8 weeks, spread cells were observed throughout the whole contrast, there remained a number of P4HB-positive spread
area of the implants, as shown in Fig. 6e. cells (Fig. 6d), indicating these fibroblasts play a role in the
2.3.4. Immunostaining of CD31 (EC marker) and vascular area in homeostatic remodeling of the tissue. The blood vessels that
the implants. Photographs of CD31 immunostaining of the formed in the regenerated tissue were also likely to be
implants are shown in Fig. 7A. Similar to the results of CD68 involved in the maintenance of homeostasis, that is, they
and P4HB immunostaining, CD31-positive cells infiltrated provided oxygen and nutrients and removed waste products.
toward the center of the hydrogels from 1 to 4 weeks, which 2.3.6. Effects of VIP on the replacement of SF hydrogels by
was enhanced by VIP. In contrast to the CD68-positive cells, regenerated tissue. The modification of the SF hydrogel with
CD31-positive cells were not observed inside the SF and SF+VIP VIP enhanced cell infiltration, resulting in the accelerated
hydrogels at 1 week. Unlike macrophages and fibroblasts, ECs replacement of the hydrogel with tissue. This effect of VIP is
were not included in the approaching cells; rather, as shown in suggested to have resulted from its bioactivity, because
Figs. 7Aa and 7Ab, they entered the hydrogels to lead the modification of the SF hydrogel with VIP influenced not on its
approaching cells (surrounded by a dotted-line). Although physical properties but on biological properties (Fig. 3).
59
these leading ECs had an elongated shape, those outside the Considering that monocytes secrete several types of MMPs,
approaching cells formed luminal structures (indicated by VIP immobilized in the SF hydrogel could be cleaved at
arrows in Fig. 7Ab). This result corresponded well to the GPQG↓IWGQ to release the QK peptide from the hydrogel
findings from H&E staining (Fig. 4), suggesting the formation of within 1 week post-implantation. At this time, the released
blood vessels. There were a large number of luminal structures amount of QK might be around one-fifth part of the feed
formed by CD31-positive cells throughout the whole area of amount (Fig. 3B), which could be large enough to affect cell
6,7
the implants at 8 weeks post-implantation (indicated by behavior (Fig. 3E). Therefore, like VEGF, the released QK
arrows in Figs. 7Ac and 7Ad). peptide stimulated angiogenesis (i.e., EC infiltration into the SF
Figure 7B represents the time-dependent changes in the hydrogel). In addition, REDV immobilized in the SF hydrogel
area percentage of blood vessels (i.e., CD31-positve luminal possibly accelerated EC infiltration because the peptide
34
structures) in the implants, which was determined using CD31- reportedly increases the migration velocity of HUVECs.
immunostained figures. In all types of the hydrogels, vascular Therefore, it is suggested that, in the SF+VIP hydrogel, the
area tended to increase with the implantation period. infiltration of ECs that led the approaching cells (Fig. 8b) was
Particularly, there was a remarkable increase in the SF+VIP enhanced by the bioactivity of VIP. Furthermore, VIP doubled
group from 4 to 8 weeks, showing twice the size of the SF and the vascular area in the implant, which was, interestingly,
Matrigel groups at 8 weeks. Therefore, a vascular-inducing shown at 8 weeks post-implantation (Fig. 7B). This fact implies
function was appended to the SF hydrogel by its modification that the effect of VIP on vascular formation was still present
with VIP. even after the complete degradation of the SF+VIP hydrogel.
2.3.5. Process for the replacement of SF hydrogels by regenerated Although it is difficult to explain the precise mechanism
tissue. SF hydrogels implanted s.c. in rats were degraded and involved in the slow enhancement of vascular formation by VIP
replaced by regenerated tissue within 8 weeks. From time- from the results of this study alone, macrophages could be a
course histological evaluations of the SF hydrogel implants key factor for this phenomenon. Monocytes/macrophages are
(Figs. 4–7), the replacement mechanism was considered as reported to play an important role in remodeling the vascular
shown in Fig. 8 (note that this mechanism may not be the case network, such as reconstitution of the extracellular matrix,
for Matrigel). By 1 week post-implantation, monocytes had fusion of endothelial tip cells, and pruning of redundant

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60,61
vascular networks, although this is in the case of occurred from 4 to 8 weeks post-implantation, might change
developmental brain/retinal models. As the vascular remodeling process to increase the number of
62,63
monocytes/macrophages express VEGFR-1, VIP, vascular structures.
particularly the QK peptide released at the degradation of the In contrast to SF hydrogels, Matrigel allowed cell infiltration

Journal of Materials Chemistry B Accepted Manuscript

SF hydrogel, might affect the vascular remodeling activity of early after implantation (within 2 weeks), and the infiltration
macrophages. Then, the decrease of macrophages, which
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Fig. 7 (A) Photographs of CD31 (EC marker)-immunostained implants. SF and SF+VIP hydrogels formed in PLA 3-D lattices were
implanted s.c. in rats for 1, 2, 4, and 8 weeks. Matrigel was used as the positive control for cell infiltration. CD31-positive cells
infiltrated the SF and SF+VIP hydrogels (asterisks) isotropically. They appeared to lead “approaching cells” including

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Paper Journal of Materials Chemistry B

macrophages and fibroblasts (surrounded by dot-line) into the center of the hydrogels. There were luminal structures (arrows)
outside the approaching cells, and a number of these structures were observed throughout the whole area of the implants at 8
weeks post-implantation. Scale bar = 1 mm and 200 µm (a–d). (B) Time-dependent changes in the area percentage of vascular
structures in the s.c. implants. Data are shown as mean ± SEM (n = 4, except for data of the SF+VIP and Matrigel groups at 8

Journal of Materials Chemistry B Accepted Manuscript

weeks (n = 3)). An asterisk indicates a significant difference between the SF+VIP and Matrigel groups, analyzed by two-way
ANOVA followed by Tukey’s post hoc comparison (p < 0.05).
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Fig. 8 Time-course events after the implantation of SF hydrogel s.c.. First, monocytes enter the hydrogel (a), and then endothelial
cells infiltrate gradually toward the center of the hydrogel to lead macrophages and fibroblasts (b–e). Macrophages degrade the
SF networks and fibroblasts produce collagen, resulting in the gradual replacement of the SF hydrogel by regenerated tissue (d–
f). Outside these cells, vascular structures are formed by ECs (e). Finally, the SF networks are degraded completely and their
replacement by regenerated tissue is achieved (f). There are a few macrophages, but a number of fibroblasts. Blood vessels
supply oxygen and nutrients and remove waste products. Fibroblasts and blood vessels play a role in the maintenance of tissue
homeostasis.

pattern was anisotropic, showing nodular structures at 4
weeks. This could be the result of by the heterogeneity of
Matrigel because it consists of a tumor-derived extracellular
matrix with various growth factors and is not fully defined.56
Another possibility is that Matrigel was replaced by
hypertrophic scar-like tissue, which exhibits nodular structures
composed of fibroblastic cells, small vessels, and randomly
organized collagen fibers,64 although these characteristic
structures were not observed at 8 weeks post-implantation.
Despite the fact that Matrigel is used frequently to stimulate
56-58
cell infiltration and angiogenesis, the vascular structures in
the regenerated tissues that had displaced the SF+VIP hydrogel
were significantly richer than when using Matrigel (Fig. 7B).
Since Matrigel contains a considerable amount of growth
factors, vascular permeability might be promoted excessively,
resulting in destabilized vascular structures. Thus, accelerated
cell infiltration into hydrogels does not always lead to
enhanced angiogenesis. Zisch et al.41 implanted polyethylene
glycol-based hydrogels that were formed in a porous
polyurethane disc s.c. in rats. They revealed that degradation
is critical and that an angiogenic stimulus (e.g., VEGF) is
important for the replacement of hydrogels by vascularized
tissues. The biodegradation of reconstituted SF 3-D scaffolds is
adjustable from weeks to years without causing chronic
65
inflammation, and this slow degradation of SF-based
materials is considered to enable optimal healing in

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24,48,66,67
vivo. Therefore, the appropriate inflammation
produced by both the moderate biodegradation and
angiogenic effect of the SF+VIP hydrogel is inferred to
contribute to its replacement by highly vascularized tissue.
2.4. Advantages of SF hydrogels modified with VIP
Two of the advantages of the present modification to alter the
biological property of SF hydrogels are the ease of the modification
procedure and the maintenance of the bioactivity of the modifier.
SF biomaterials can be chemically modified with bioactive
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28,68,69 70
molecules; however, as mentioned by Sato et al., its
procedure is often accompanied by technical difficulties and high
manufacturing costs. Although recent advances in transgenic
silkworm technology have enabled the production of recombinant
55,70-72
SF protein fused to bioactive peptides/proteins, the amount
of the recombinant SF protein against SF remains low (at most
70,73
30% ). In contrast, owing to the FibH-derived peptide, our
modification procedure is simple (just add VIP to SF solutions prior
to gelation) and its modification yield is likely to rely on the amount
of VIP added. Additionally, because the bioactive part (from REDV
to QK) of VIP consists of 33 amino acids, such a small peptide does
not require a complex tertiary structure for bioactivity, as Van Hove
42
et al. mentioned previously. In fact, more than 85% of fed VIP was
immobilized in the SF hydrogel to show the expected bioactivity:
the stimulation of cell infiltration and vascular induction. However,
this modification had an insignificant effect on the characteristics of
the SF hydrogel, such as mechanical strength and monthly
biodegradation. These results suggest the promise of SF+VIP
hydrogels in soft tissue augmentation and treatment of myocardial
infarction. Since several gelation triggers make SF hydrogel
20,48,67
injectable, the authors are currently investigating the
potential of SF+VIP injectable hydrogels for the treatment of
myocardial infarction.
3. Experimental
3.1. Vector construction
An expression plasmid for VIP was constructed as follows, and the
sequences of the primers (Sigma-Aldrich, MO) are shown in Table
S1 (ESI†). Two oligonucleoƟde sets (REDV2-MMP(+)-5 and REDV2-
MMP(+)-3, with a NotI-compatible overhang at the 5′ terminal; and
QK-5 and QK-3, with a SacI-compatible overhang at the 3′ terminal)
were annealed, respectively, to prepare two oligonucleotide
cassettes containing a compatible overhang. These cassettes were
ligated with NotI- and SacI-digested pBluescript II KS(+) vector
(Agilent Technologies, CA) and the resulting plasmid was designated
as pBS[REDV2-MMP(+)-QK]. Restriction sites were added to the
cDNA of a FibH-derived peptide (5′ BamHI/3′ NotI) by polymerase
chain reaction (PCR) with primer sets (FibH-5 and FibH-3). The
resulting PCR product was inserted into the BamHI–NotI sites of
pBS[REDV2-MMP(+)-QK] to prepare pBS[FibH-REDV2-MMP(+)-QK].
To obtain an expression vector for VIP, the expression cassette in
pBS[FibH-REDV2-MMP(+)-QK] was digested with BamHI and SacI,
and finally, the resulting fragment was cloned between the BamHI
and SacI sites of the pTrcHisA vector (Invitrogen, CA). This
expression plasmid was designated as pTrcHis[FibH-REDV2-
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MMP(+)-QK]. The construct was verified by restriction digest and
DNA sequencing before expression.
3.2. Recombinant production of VIP
Journal of Materials Chemistry B Accepted Manuscript
E. coli strain BL21 competent cells, transformed with the
pTrcHis[FibH-REDV2-MMP(+)-QK] vector, were grown at 37°C in LB
-1
medium containing 150 µg mL ampicillin to an optical density of
0.5–0.6 at 600 nm. After 3-h induction with 1 mM isopropyl-
thiogalactoside, the cells were pelleted by centrifugation, washed
with PBS (pH 7.4; Gibco, MA), and stored at -80°C until use. A
subspecies of VIP designated as VIP(MMP(-)), containing the MMP-
41
uncleavable GDQGIAGF peptide instead of GPQG↓IWGQ in VIP,
was produced in E. coli strain BL21, transformed with the
pTrcHis[FibH-REDV2-MMP(-)]-QK] vector. This expression plasmid
was constructed using the same protocol for preparing
pTrcHis[FibH-REDV2-MMP(+)-QK] but REDV2-MMP(+)-5 and REDV2-
MMP(+)-3 were replaced with REDV2-MMP(-)-5 and REDV2-MMP(-
)-3, respectively. VIP or VIP(MMP(-)) were extracted from the
transgenic E. coli cell pellets and purified with an Ni-chelate column
(HisTrap FF crude; GE Healthcare, UK) as shown in Fig. S1 (ESI†).
After dialysis against PBS, VIP and VIP(MMP(-)) were sterilized by
filtering through a polyethersulfone membrane with 0.22-µm pores.
The purity of VIP or VIP(MMP(-)) was >90%, which was estimated by
SDS-PAGE band intensity analysis.
3.3. Cleavage analysis of VIP
Different concentration collagenase solutions (collagenase L; Nitta
Gelatin, Japan) were added to solutions of VIP or VIP(MMP(-)) in
PBS (5 µM VIP/VIP(MMP(-)) and 0, 0.05, 0.1, 0.5, or 1 µg mL
-1
collagenase). Following incubation at 37°C for 21 h, the protein
composition of the solutions was analyzed by SDS-PAGE on a Mini-
PROTEAN Peptide gel (10–20%; Bio-Rad Laboratories, CA) under
reducing conditions. Separated peptides were fixed and visualized
with an EzStain AQua solution (Atto, Japan).
3.4. In vitro EC adhesion assay
VIP/PBS or VIP(MMP(-))/PBS (10 µM) were poured into 24-well
glass-based plates (Asahi Glass, Japan) and 35-mm glass-based
dishes (diameter of glass part, 12 mm; Asahi Glass), which were
incubated at room temperature (RT) for 30 min to make the
peptides adsorb to the glass surface. After the removel of the
excess solution, the peptide surfaces on the plates/dishes were
dried in air at 50°C for more than 12 h. Then, the surfaces were
treated with 80 vol% methanol at RT for 30 min to render the
peptides water insoluble by changing the FibH-derived peptide from
74
a random conformation to a β-sheet conformation. After drying in
air at 50°C for more than 12 h, the peptide surfaces were sterilized
by 70 wt% ethanol treatment for 20 min and rinsed twice with
autoclaved ultrapure water and twice with PBS.
HUVECs (passage number, 3–5; KE-4109; Kurabo, Japan) were
seeded onto the peptide-coated plates at a concentration of 1.0 ×
4 -2 TM
10 cells cm in basal medium (EBM -2; Lonza, Switzerland)
supplemented with ascorbic acid, hydrocortisone, heparin, and
TM TM
gentamicin/amphotericin-B from an EGM -2 SingleQuots Kit
(Lonza). Following a 90-min incubation at 37°C in a humidified
atmosphere of 95% air and 5% CO2, the number of cells that had
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adhered to the peptide surfaces was determined using a lactate
75
dehydrogenase assay as described previously. The adherence of
HUVECs cultured on non-coated glass-based plates was measured
as a control. Three different wells were used (n = 3). F-actin and
nuclei of HUVECs adhered to the peptide-coated dishes were
71
stained at 90 min post-cell seeding as described previously.
Stained HUVECs were observed with a confocal laser scanning
microscope (IX-81 and FV1000-D; Olympus, Japan).
TM
HUVECs on the peptide-coated dishes were cultured with EBM -
TM TM
2 containing all supplements from the EGM -2 SingleQuots Kit
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except for VEGF for 34 h. Then, F-actin, VE-cadherin, and nuclei of
71
the cells were immunostained as described previously, where a
rabbit anti-VE-cadherin polyclonal antibody (ab33168; Abcam, UK)
®
and Alexa Fluor 488 conjugated anti-rabbit IgG (H+L), F(ab’2)
fragment (4412; Cell Signaling Technology, MA) were used as
primary and secondary antibodies to stain VE-cadherin,
respectively.
3.5. In vitro EC proliferation assay
HUVECs were seeded in 96-well tissue culture polystyrene plates
3
(Asahi Glass, Japan) at a concentration of 2.0 × 10 cells well-1 and
TM TM
cultured with EBM -2 containing all supplements from the EGM -
TM
2 SingleQuots Kit at 37°C in a humidified atmosphere of 95% air
and 5% CO2. After incubation for 4 h, the medium was replaced
TM TM
with EBM -2 containing all supplements from the EGM -2
TM
SingleQuots Kit except for VEGF, and different amounts of VIP,
35
VIP(MMP(-)), synthesized QK peptide (Ac-KLTWQELYQLKYKGI-NH2;
TM
purity >95%; Scrum, Japan), or VEGF from the EGM -2
TM
SingleQuots Kit. After culturing for 2 days, the cells were
incubated with a WST-1 reagent (Roche Diagnostics, Germany) for 2
h, followed by absorbance measurement at 450 nm using a plate
TM
reader (Varioskan ; Thermo Fisher Scientific) Absorbance at 650
nm was used as a reference. Three different wells were measured
for each culture condition (n = 3).
3.6. Formation of SF hydrogels with/without VIP
An SF aqueous solution was prepared from degummed silk fibers of
76
B. mori cocoons as described previously. The solution was
autoclaved and SF concentration was determined by a bicinchoninic
acid assay using an SF standard. SF aqueous solutions with/without
VIP were mixed with a sodium citrate buffer (pH 3.0) (final
-1 -1
concentrations, 20 mg mL SF, 0/10 µM [0/0.12 mg mL ] VIP, and
20 mM sodium citrate buffer) and incubated at 37°C overnight in a
humidified atmosphere, resulting in the formation of opaque SF or
SF+VIP hydrogels.
3.7. Release of VIP from SF hydrogel
The solvent of purified VIP was replaced from PBS to 150 mM
sodium carbonate buffer (pH 9.0) by ultrafiltration, and the
-1
concentration of VIP was adjusted to 3 mg mL . Fifteen microliters
-1
of TAMRA-isothiocyanate (10 mg mL in dimethyl sulfoxide;
Thermo Fisher Scientific) were added to the VIP solution, which was
then stirred at RT for 1 h. TAMRA-VIP was purified and its solvent
was replaced to PBS by using a PD-10 column (GE Healthcare) in
accordance with the manufacturer’s protocol.
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SF aqueous solutions with/without TAMRA-VIP were poured into
a silicon rubber mold (inner diameter, 8 mm; height, 5 mm) and
then mixed with a sodium citrate buffer (pH 3.0) (final
-1
concentrations, 20 mg mL SF, 0/10 µM TAMRA-VIP, and 20 mM
Journal of Materials Chemistry B Accepted Manuscript
sodium citrate buffer). They were incubated at 37°C overnight in a
humidified atmosphere to form hydrogels. Each hydrogel was
moved carefully to a sample tube and 200 µL PBS with/without 1 µg
-1
mL collagenase were added. After incubation at 37°C for 1, 3, 6, 9,
12, 24, 48, 72, 120, and 168 h, the PBS was collected and replaced.
The collected solution was centrifuged at 15,000 × g at 4°C for 5 min
and the 576-nm fluorescence at 557-nm excitation of the
supernatant was measured by using a plate reader. The fluorescent
intensity of the TAMRA-VIP-free samples was subtracted as the
autofluorescence of SF and/or PBS. Cumulative release percentage
was determined based on the feed amount of TAMRA-VIP in the
SF+TAMRA-VIP hydrogels. Four different hydrogels were used (n =
4).
3.8. Scanning electron microscopy
SF and SF+VIP hydrogels were immersed extensively in deionized
water and frozen at -80°C. Specimens were freeze-dried, coated
with gold by a vacuum vapor deposition, and imaged using a
scanning electron microscope (JCM-5700; JEOL, Japan).
3.9. Water content
The water content of the SF and SF+VIP hydrogels was measured by
weighing wet and dry hydrogels. The hydrogels were submerged in
deionized water at RT for 1 h, and excess water was removed using
a tissue. The weight of wet hydrogels (Ww) was measured and then
the hydrogels were freeze-dried. The weight of freeze-dried
hydrogels (Wd) was also measured and the water content of the
gels (%Water) was calculated using the following equation:
%Water = 100 × (Ww – Wd ) × Ww
-1
In this measurement, three different samples were used (n = 3).
3.10. Compressive modulus
The compressive properties of SF and SF+VIP hydrogels were
measured using a compression tester (MCR301; Anton Paar,
Austria), equipped with an 8-mm diameter load plate, at 1% strain
-1
sec head speed at RT. The samples were completely wetted in
PBS, and excess solution was removed using a tissue before
measurement. The compressive modulus of the hydrogels was
determined from the initial slope of the stress/strain curves at a
strain of 0.5–2.5%. Three different hydrogels were tested (n = 3).
3.11. EC proliferation on SF hydrogels
SF hydrogels containing 0, 1, and 10 µM VIP and BD Matrigel
®
Basement Membrane Matrix (Matrigel; 356234; Corning, NY) were
formed in a 96-well tissue culture polystyrene plate and washed
three-times with PBS. HUVECs (passage number, 5) were seeded
4 -2
onto the hydrogels a concentration of 1.0 × 10 cells cm and
TM TM
cultured with EBM -2 containing all supplements from the EGM -
2 SingleQuots
TM
Kit except for VEGF at 37°C in a humidified
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atmosphere of 95% air and 5% CO2. After culturing for 1, 3, 5, and 7
days, an assay with the WST-1 reagent was performed as described
in Section 3.5. Four different hydrogels were measured (n = 4).
3.12. Subcutaneous implantation of hydrogels in rats
SF and SF+VIP hydrogels were formed in a PLA 3-D lattice (6 × 6 × 3
3
mm ). Matrigel was also formed in the lattice as the positive
control. Male Sprague Dawley rats (8–9 weeks old; Japan SLC,
-1
Japan) were anesthetized with 50–80 mg kg pentobarbital. Under
general anesthesia, four subcutaneous pockets were made on the
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back of each rat using surgical scissors, and a hydrogel formed in a
3-D lattice was placed into the pocket. Then, the pocket was
sutured with 3–0 silk (Ethicon, NJ) and the animals were allowed to
recover. Four different hydrogels were implanted (n = 4).
At 1, 2, 4, and 8 weeks post-operation, the implants with
surrounding subcutaneous tissues were extracted under general
anesthesia. Hydrogels/tissues in the extracted PLA 3-D lattice were
dimidiated in the middle of the lattice using a scalpel. One of them
was fixed in 10% neutral-buffered formalin and the other was
frozen in O.C.T compound (TissueTek; Sakura Finetek Japan, Japan)
in liquid nitrogen. All animal experiments in this study were
conducted in accordance with the animal experiment guidelines of
the NCVC Research Institute (permit number, 15051). All efforts
were made to minimize any pain and suffering felt by the animals.
3.13. Histological staining of implants
The implants were cut into 6-µm-thick sections and subjected to
histology: formalin-fixed sections were subjected to H&E staining
and immunostained for CD68 and P4HB; and cryo-sections were
immunostained for CD31. Mayer’s hematoxylin and eosin Y
solutions (Wako Pure Chemical Industries, Japan) were used for
H&E staining. For immunostaining, a mouse anti-rat CD68
monoclonal antibody (MCA341R; AbD Serotec, UK), mouse anti-rat
P4HB monoclonal antibody (AF5110-1; Acris Antibodies, CA), and
mouse anti-rat CD31 monoclonal antibody (550300; BD Biosciences,
NJ) were used as primary antibodies. A horseradish peroxidase-
conjugated polymer reagent (414191; Nichirei, Japan) and a 3,3′-
diaminobenzidine chromogen kit (K3467; Dako, Denmark) were
used to detect the antigens. Images of the stained samples were
acquired using a digital microscope (Coolscope II; Nikon, Japan). In
the CD31-immunostained images, luminal structures were
identified manually, and their area was quantified by image analysis
using ImageJ (http://imagej.nih.gov/ij/). The area of
hydrogels/tissues in the PLA lattice was also measured to calculate
the area percentage of blood vessels in the hydrogels/tissues.
3.14. Statistical analysis
Data from the in vitro HUVEC adhesion assay were analyzed with
one-way analysis of variance (ANOVA) followed by Tukey’s post hoc
comparison. Two-way ANOVA followed by Tukey’s post hoc
comparison was used to analyze data from the in vitro HUVEC
proliferation assays and the area percentage of luminal structures.
For the analysis of water content and compressive modulus of SF
and SF-VIP hydrogels, a two-sided Student’s t test was used. A value
of p < 0.05 was considered significant.
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4. Conclusions
VIP, composed of multi-bioactive peptides (the FibH-derived,
REDV, MMP-cleavable, and QK peptides), was produced
recombinantly in order to append vascular-inducing bioactivity
Journal of Materials Chemistry B Accepted Manuscript
to SF physical hydrogels. The subcutaneous implantation of SF
hydrogels in rats revealed that ECs infiltrated the hydrogels to
lead macrophages and fibroblasts, resulting in the replacement
of SF networks by regenerated tissue. This cell infiltration
process was accelerated by the bioactivity of VIP. Moreover,
the SF+VIP hydrogel was replaced by highly vascularized tissue,
showing an area percentage of blood vessels that was twice as
large as with unmodified SF hydrogel and Matrigel. In addition
to the inherent features of SF hydrogels, such as mechanical
strength and slow biodegradation, the vascular-inducing
bioactivity induced by peptide modification described in this
study may make SF hydrogels a useful scaffold for soft tissue
augmentation and ischemia tissue regeneration.
Conflict of interest
There are no conflict of interest to declare.
Acknowledgements
This work was supported financially in part by a Japan Society
for the Promotion of Science (JSPS) Grant-in-Aid for JSPS
Fellows (25-10369), JSPS Grant-in-Aid for Scientific Research(C)
(17K01402), and Intramural Research Fund of NCVC (28-2-1).
The authors thank Dr. Kyoko Shioya and the staff of the
Laboratory of Animal Experiments and Medicine Management,
NCVC, for helping us to care for the rats.
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