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Fabrication and Characterization of Porous Tubular Silk Fibroin Scaffolds

Article  in  Journal of Biomaterials Science Polymer Edition · September 2009

DOI: 10.1163/156856208X396056 · Source: PubMed

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Fabrication and Characterization

of Porous Tubular Silk Fibroin
Scaffolds
a b c d
Sijia Min , Xin Gao , Lin Liu , Li Tian , Liangjun Zhu
e f g
, Haiping Zhang & Juming Yao
a
College of Animal Sciences, Zhejiang University,
Kaixuan Road 268, Hangzhou 310029, P. R. China.
minsj@zju.edu.cn
b
College of Animal Sciences, Zhejiang University,
Kaixuan Road 268, Hangzhou 310029, P. R. China
c
The Key Laboratory of Advanced Textile Materials and
Manufacturing Technology of Ministry of Education,
College of Materials and Textile, Zhejiang Sci-Tech
University, Hangzhou 310018, P. R. China
d
College of Animal Sciences, Zhejiang University,
Kaixuan Road 268, Hangzhou 310029, P. R. China
e
College of Animal Sciences, Zhejiang University,
Kaixuan Road 268, Hangzhou 310029, P. R. China
f
College of Animal Sciences, Zhejiang University, Kaixuan
Road 268, Hangzhou 310029, P. R. China
g
The Key Laboratory of Advanced Textile Materials and
Manufacturing Technology of Ministry of Education,
College of Materials and Textile, Zhejiang Sci-Tech
University, Hangzhou 310018, P. R. China
Published online: 02 Apr 2012.

To cite this article: Sijia Min , Xin Gao , Lin Liu , Li Tian , Liangjun Zhu , Haiping Zhang
& Juming Yao (2009) Fabrication and Characterization of Porous Tubular Silk Fibroin
Scaffolds, Journal of Biomaterials Science, Polymer Edition, 20:13, 1961-1974, DOI:
10.1163/156856208X396056

To link to this article: http://dx.doi.org/10.1163/156856208X396056

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 Journal of Biomaterials Science 20 (2009) 1961–1974
www.brill.nl/jbs

Fabrication and Characterization of Porous Tubular Silk

Fibroin Scaffolds

Sijia Min a,∗ , Xin Gao a , Lin Liu b , Li Tian a , Liangjun Zhu a , Haiping Zhang a and
Juming Yao b
Downloaded by [Australian National University] at 20:02 22 January 2015

a
College of Animal Sciences, Zhejiang University, Kaixuan Road 268, Hangzhou 310029,
P. R. China
b
The Key Laboratory of Advanced Textile Materials and Manufacturing Technology of Ministry of
Education, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018,
P. R. China
Received 27 September 2008; accepted 28 November 2008

Abstract
Silk fibroin (SF) has been one of promising resources of biotechnology and biomedical materials due to
its unique properties. Here, different sizes of porous tubular scaffolds were fabricated from a SF aqueous
solution with the addition of poly(ethylene glycol diglycidyl ether) (PGDE). The scaffolds were generally
flexible and transparent at the wet state with a pore size of 81–128 µm and porosity of 90–96%, depending
on the concentrations of SF and PGDE. The mechanical properties measurement showed that the tubular SF
scaffolds had satisfying tensile and compression properties, especially the excellent deformation–recovery
ability. FT-IR spectra indicated that the SF in the tubular scaffolds was in a β-sheet structure, and no PGDE
characteristic band was observed, suggesting that the PGDE could be removed from the scaffolds by soaking
in deionized water. The cell compatibility of scaffolds was evaluated, and no obvious cytotoxicity to mouse
L-929 fibroblasts was detected.
© Koninklijke Brill NV, Leiden, 2009

Keywords
Silk fibroin, porous scaffold, tubular scaffold, polyethylene glycol diglycidyl ether (PGDE), biomaterial

1. Introduction

In the past decades, a variety of tubular scaffolds has been prepared as artificial
substitutes for vascular surgery, which is rapidly becoming the most promising
experimental approach for regenerating the native structural and functional prop-
erties of living tissues [1–7]. However, these scaffolds were only achieved for

* To whom correspondence should be addressed. Tel.: (86-571) 8697-1812; Fax: (86-571) 8697-1815;
e-mail: minsj@zju.edu.cn

© Koninklijke Brill NV, Leiden, 2009 DOI:10.1163/156856208X396056

 1962 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974

relatively large diameter arteries. The patency rates have been disappointing when
they are used as small-diameter (<6 mm) arterial substitutes such as for coronary
and the infragenicular vessels, because of the acute thrombogenicity of the graft,
anastomotic intimal hyperplasia, aneurysm formation, infection and progression of
atherosclerotic disease [6, 8]. To construct an artificial vessel, three basic elements
are generally required, i.e., a structural scaffold, cells and a nurturing environment.
The scaffold provides a temporary skeleton to support the growing tissue and pro-
vides the desired shape until the cells produce their own extracellular matrix. As a
natural polymeric material, collagen was widely reported to construct the artificial
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vessels in vitro [1, 9–11], in which the researchers have explored many routes to
overcome the poor mechanical strength of collagen-based artificial vessels. Also,
several synthetical polymers have been used as scaffolds for the construction of ar-
tificial vessels, e.g., poly(glycolic acid) (PGA) [3], polytetrafluoroethylene (PTFE)
[12], the co-polymers of PGA with polyhydroxyalkanoate (PHA) [13] or poly(L-
lactic acid) (PLLA) [14], modified PTFE [15], etc.
Silk proteins are usually produced within the specialized glands of silkworms
after biosynthesis in epithelial cells, followed by secretion into the lumen of these
glands, where the proteins are stored prior to spinning into fibers [16]. Bombyx
mori silk is the most extensively characterized silkworm silk, consisting of two fi-
broin threads adhered together with sericin gum resulting in a single thread about
10–25 µm in diameter [16, 17]. As a fibrous protein, silk fibroin (SF) fibers from
B. mori have been used as biomedical suture material for their outstanding me-
chanical properties, in contrast to the catalytic and molecular recognition functions
of globular proteins [16]. Also, researchers have investigated SF as promising re-
source of biotechnology and biomedical materials due to its other unique properties,
including excellent biocompatibility, favorable oxygen permeability and outstand-
ing biodegradability, the fact that the degradation product can be readily absorbed
by body with minimal inflammatory reaction, etc. [18, 19]. Recently, with the im-
provement of tissue engineering and the need for porous scaffolds, SF has been
explored for an extended variety of biomedical applications, including osteoblast
and fibroblast cell support matrices and for ligament tissue engineering [20, 21].
By dipping straight lengths of stainless steel wire into aqueous SF, the microtubes
were possibly prepared, where the addition of poly(ethylene oxide) (PEO) enabled
control of microtube porosity [7].
In this work, the porous tubular SF scaffolds with different sizes were fabri-
cated from SF solution with the addition of poly(ethylene glycol diglycidyl ether)
(PGDE). The properties, e.g., pore size and porosity, mechanical properties, as well
as the cell biocompatibility and enzymatic degradation of different tubular SF scaf-
folds were investigated.
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1963

2. Materials and Methods

2.1. Materials
B. mori silkworm cocoons were obtained from Huzhou Academy of Agricultural
Science (P. R. China). PGDE was purchased from Nagase (Japan). Protease XIV
was purchased from Sigma (USA). Other chemical agents used here were analytical
grade and purchased from Huipu Chemical Agents (P. R. China). The L-929 mouse
fibroblasts were kindly provided by Centers for Disease Control and Prevention of
Zhejiang Province (P. R. China). SF aqueous solution was obtained according to the
method described previously [22].
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2.2. Preparation of Tubular SF Scaffolds

B. mori silkworm cocoons were degummed twice with 0.5% (w/v) Na2 CO3 aque-
ous solution at 100◦ C for 30 min and washed thoroughly with distilled water to
remove silk sericin. The fibroin fibers were then dissolved in 9 M LiBr solution at
45◦ C for about 30 min, followed by the extensive dialysis against distilled water
for 3 days (MWCO 12 000) in order to obtain the regenerated SF aqueous solution.
In this work, different SF concentrations, determined by the Bradford protein assay
on a DU530 DNA/Protein analyzer (Beckman Coulter, USA), were used to prepare
the scaffolds.
To prepare the tubular SF scaffolds, the moulds with different sizes were pre-
pared firstly. Each mould was made from a polyethylene (PE) pipe and a PE film
wrapped PE rod, as illustrated in Fig. 1. The bottom of the mould was sealed with
paraffin wax. The SF aqueous solutions with different contents of PGDE were
poured into the moulds, i.e., the space between the pipe and the rod, and frozen
immediately at −20◦ C for 1 h. The samples were then thawed at ambient tempera-
ture, followed by taking off the wax, pipe, rod and film in turn. The obtained tubular
SF scaffolds were soaked by immersing in deionized water for 3 days to remove the
redundant PGDE, where the deionized water was replenished every 8 h. The cylin-
drical SF scaffolds were prepared with the same procedure, except that PE pipe was
only used as a mould.

Figure 1. Illustration for the preparation of tubular SF scaffolds.

 1964 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974

For comparison, the SF solutions (4%, w/v) with PGDE (3%, v/v) and without
PGDE were poured into the polystyrene disks and kept at 60◦ C for 8 h and 3 days,
respectively, to obtain the SF gels. Here, the SF gel with PGDE was prepared by
the addition of 2% (w/v) NaCl. The SF gels with PGDE obtained were also soaked
in deionized water as described above.

2.3. Characterization

The morphology of tubular SF scaffolds was observed under a XL30-ESEM scan-

ning electron microscope (SEM; Philips, The Netherlands) with an accelerating
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voltage of 20 kV. The structure of SF samples was investigated by Fourier trans-

formed infrared spectroscopy (FT-IR). The samples were mixed with KBr in the
mass ratio of 1:20. FT-IR spectra were recorded on an ATR-8000 fourier transform
infrared instrument (Shimadzu, Japan).
The mechanical properties of tubular SF scaffolds were measured on an AG-IS
universal testing instrument (Shimadzu, Japan) at 22 ± 2◦ C. For the tensile testing,
the scaffolds with an outside diameter (od) of 8 mm and tube wall thickness (twt)
of 1 mm were used with an initial gage length of 25 mm and a cross-head speed of
2 mm/min. For the tensile–recovery testing, the samples with an initial gage length
of 25 mm were stretched to 30 mm with a cross-head speed of 2 mm/min, and
then returned at the same speed, which was repeated 100 times. Here, the tubular
scaffolds were too soft to apply the vertical compression. Therefore, for the com-
pression testing, cylindrical SF scaffolds (15 mm od) with an initial height of 14 mm
were used at a cross-head speed of 2 mm/min. For the compression–recovery test-
ing, cylindrical SF scaffolds with an initial height of 14 mm were compressed to
7 mm with a cross-head speed of 2 mm/min, and then returned at the same speed,
which was also repeated 100 times. The data reported are the mean of 5 examina-
tions.
Mapinfo 7.0 software (MapInfo, USA) was used to designate a square area ran-
domly on the SEM image of scaffolds, which contained ten integrated contiguous
pores. The measured area was then converted into the diameter of each pore, which
was regarded as the pore size. The pore sizes reported are the mean of 10 examina-
tions.
The porosity of scaffolds was measured using the liquid displacement method
[23]. A freeze-dried tubular scaffold was immersed in a known volume (V1 ) of
hexane in a graduated cylinder, which was then vacuumized at a vacuum degree of
0.05 MPa and 20 ± 2◦ C for 10 min. The total volume of hexane and the hexane-
impregnated scaffold was recorded as V2 . The hexane-impregnated scaffold was
removed from the cylinder and the residual hexane volume was recorded as V3 .
The total volume of the scaffold was V = (V2 − V1 ) + (V1 − V3 ) = V2 − V3 . Here,
(V2 − V1 ) is the volume of tubular scaffold and (V1 − V3 ) is the volume of hexane
within the scaffold. Thus, the porosity of the scaffold (ε) was obtained by ε(%) =
((V1 − V3 )/(V2 − V3 )) × 100. The data reported are the mean of 5 examinations.
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1965

2.4. Cell Culture

The L-929 mouse fibroblasts were cultured in Dulbecco’s modified Eagle medium
(DMEM) high glucose, supplemented with 10% fetal bovine serum (FBS) and an-
tibiotics (100 U/ml penicillin and 100 U/ml streptomycin). The cells were digested
using 0.25% (w/v) trypsin for 3–5 min, centrifuged at 800 × g for 5 min, and re-
suspended in the medium. 100 µl/well of mixture with 4% (w/v) SF and 3% (v/v)
PGDE was poured into the 24-well plate followed by freezing at −20◦ C for 4 h.
The samples were then thawed at ambient temperature and soaked by immersing
in deionized water for 3 days to remove the redundant PGDE, where the deionized
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water was replenished every 8 h. The sample surfaces were scraped with a knife in
order to allow the cell transfer into the scaffolds, followed by sterilization in 60 Co
γ irradiation (25 kGy). After immersion in 1 ml of culture medium for 24 h, 100 µl
cell suspension (about 6 × 105 cells/ml) was added into each well. The scaffold/cell
constructs were incubated at 37◦ C for 2 h to allow the cells to attach to them. Then
the scaffold/cell constructs were turned over and 100 µl of cell suspension at the
same cell density were added in each well again. After another 2 h of incubation,
1 ml culture medium was added. The scaffold/cell constructs were cultured in a hu-
midified incubator at 37◦ C with 5% CO2 . Culture medium was replenished every
2 days.
After cultivation, the scaffold/cells constructs were fixed with 2.5% (v/v) glu-
taraldehyde overnight at 4◦ C and washed with PBS solution (pH 7.4) for 3 times.
The samples were then fixed with 1% (v/v) osmic acid for 2 h and washed with
PBS solution (pH 7.4), followed by dehydrating with the aqueous ethanol, i.e., 50%
(v/v), 70% (v/v), 80% (v/v), 90% (v/v) and 95% (v/v) gradually for 15 min, and
lastly with 100% ethanol twice for 20 min. The dehydrated scaffold/cells constructs
were treated with a mixture of ethanol and isoamyl acetate (1:1, v/v) for 30 min,
followed by treating with isoamyl acetate for 2 h. The constructs were dried on a
K850 critical point dryer (Emitech, UK) and the morphology was observed on a
XL30-ESEM scanning electron microscopy (SEM) (Philips) with an accelerating
voltage of 10 kV.

2.5. In Vitro Enzymatic Degradation

The in vitro degradation experiments were carried out using protease XIV
(5.9 U/mg). The tubular SF scaffolds (od 8 mm, twt 1 mm) with the length of
10 mm were immersed in 3 ml PBS buffer solution (pH 7.4) containing antibiotics
(100 U/ml penicillin, 100 U/ml streptomycin) and protease XIV at 37◦ C. Here, dif-
ferent concentrations of protease XIV, i.e., 0 U, 1 U and 2.5 U in 3 ml PBS buffer
solution were used for comparison. The solution was replenished every 2 days. The
scaffolds immersed for different times were removed from the enzyme solution and
dried at 105◦ C to constant weight. After cooling, the scaffolds were weighed and the
mass residues were calculated. The data reported are the mean of 3 examinations.
 1966 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974

(A) (B)

Figure 2. Images of tubular SF scaffolds (4% (w/v) SF, 3% (v/v) PGDE). (A) (a) outside diameter
Downloaded by [Australian National University] at 20:02 22 January 2015

(od) of 8 mm and tube wall thickness (twt) of 1 mm; (b) 5 mm od and 0.8 mm twt; (c) 4 mm od and
0.8 mm twt. (B) Scaffolds (5 mm od, 0.8 mm twt) in the (a) wet state and (b) dry state, respectively.

3. Results and Discussion

3.1. Morphology of Tubular SF Scaffolds
Figure 2A shows the different sizes of tubular SF scaffolds prepared in this work.
The scaffolds are generally flexible and transparent in the wet state, as seen in
Fig. 2B, while they are a little stiff in the dry state. Figure 3 shows the SEM images
of a tubular SF scaffold (8 mm od, 1 mm twt) prepared from 4% (w/v) of SF solu-
tion with the addition of 3% (v/v) PGDE, indicating a porous structure. The pores
at both outside and inside surfaces of tubular scaffold are mostly fine and close in
texture, which become loose and are interconnected with each other in the scaffold
as seen from the SEM image (Fig. 3f) of the outside surface after scraping with a
knife.
The pore size and porosity are important features in the scaffolds. Generally,
a pore size of 100 µm in diameter is required for tissue in-growth based on the
cell size and migration [21, 24], and the porosity needs to be more than 30% to
achieve interconnection, providing sufficient opportunity for cell migration and ex-
pansion while maintaining transport interconnection [25]. In this work, the tubular
SF scaffolds could be easily fabricated by freezing the SF/PGDE solution between
−5◦ C and −40◦ C within a short period followed by thawing at ambient tempera-
ture. The concentrations of SF and PGDE should be the key factors for the texture
of scaffolds. Figure 4 shows the SEM images of tubular SF scaffolds prepared from
different SF and PGDE concentrations followed by freezing at −20◦ C. All of the
scaffolds showed the loose and interconnected pores. Table 1 summarizes pore size
and porosity of different scaffolds. Lower concentration of SF led to the larger pore
size and porosity. The pore size ranged from 81 µm to 128 µm, and the poros-
ity was 90–96% for the prepared scaffolds in this work. It is noteworthy that the
concentration of PGDE had the significant influence on the pore size, i.e., higher
concentration of PGDE led to the smaller pore size, but minor influence on the
porosity. Here, the fabrication of tubular scaffold was failed at lower concentrations
of SF and PGDE, i.e., 2.5% (w/v) SF and 1.0% (v/v) PGDE.
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1967
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Figure 3. SEM images of tubular SF scaffolds (8 mm od, 1 mm twt) (4% (w/v) SF, 3% (v/v) PGDE).
(a, b) Transverse sections, scale bars 2 µm and 1 µm, respectively; (c) vertical section, scale bar 1 µm;
(d) outside surface, scale bar 200 µm; (e) inside surface, scale bar 200 µm; (f) outside surface after
scraping with a knife, scale bar 200 µm.

3.2. Mechanical Properties of SF Scaffolds

Table 2 shows the tensile mechanical properties of different tubular scaffolds. The
results indicated that the mechanical properties of scaffolds generally improved
with the increase of SF concentration, and the scaffolds prepared at the PGDE con-
centration of 3.0% (v/v) showed more favorable properties compared to the other
two cases. Table 3 compares the tensile mechanical properties of tubular SF scaf-
folds after different sterilizations, showing that no significant change occurred on
both strength and elongation after a dose of 25 kGy radiation sterilization, except
for a slight increase of modulus. However, a significant increase of modulus and
decrease of elongation were observed after steam sterilization, i.e., autoclaving at
121◦ C for 20 min.
Figure 5 compares the compression curves of different cylindrical SF scaffolds
(15 mm od). Generally, a decrease in the porosity led to an increase in the compres-
sion modulus, and the pore size of different scaffolds had some, but only a minor
influence on the compression modulus compared to the porosity. In this work, all
of the scaffolds could be compressed from the original length of 14 mm to around
 1968 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974
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Figure 4. SEM images of tubular SF scaffolds (8 mm od, 1 mm twt) prepared from (a) 2.5% (w/v) SF,
3% (v/v) PGDE, (b) 4% (w/v) SF, 3% (v/v) PGDE, (c) 6% (w/v) SF, 3% (v/v) PGDE, (d) 4% (w/v)
SF, 1% (v/v) PGDE, (e) 4% (w/v) SF, 3% (v/v) PGDE and (f) 4% (w/v) SF, 5% (v/v) PGDE. Scale
bars = 200 µm.

Table 1.
Pore size and porosity of different tubular SF scaffolds

PGDE SF (%, w/v)

(%, v/v)
2.5 4.0 6.0
Pore size Porosity Pore size Porosity Pore size Porosity
(µm) (%) (µm) (%) (µm) (%)

1.0 – – 128.4 ± 21.7 94.0 ± 1.8 106.6 ± 16.1 93.9 ± 2.1

3.0 96.7 ± 9.8 95.7 ± 1.2 81.8 ± 9.1 93.7 ± 1.1 79.6 ± 10.0 90.2 ± 1.3
5.0 88.4 ± 12.9 95.5 ± 1.1 81.1 ± 20.5 93.2 ± 0.9 53.5 ± 10.0 90.0 ± 1.5

1 mm without any fracture, which could return to the original size after soaking in
the water. Figure 6 shows the last 5 cycles of tensile–recovery curve of tubular SF
scaffold (4% (w/v) SF, 3% (v/v) PGDE) (8 mm od, 1 mm twt), indicating that the
scaffold had a high tensile–recovery ability.
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1969

Table 2.
Tensile mechanical properties of different tubular SF scaffolds

PGDE SF (%, w/v)

(%,
2.5 4.0 6.0
v/v)
Modu- Strength Elonga- Modu- Strength Elonga- Modu- Strength Elonga-
lus (kPa) tion lus (kPa) tion lus (kPa) tion
(kPa) (%) (kPa) (%) (kPa) (%)

1.0 – – – 285 ± 45 41 ± 24 58 ± 21 369 ± 32 34 ± 11 37 ± 8

3.0 356 ± 60 28 ± 2 37 ± 5 563 ± 27 69 ± 9 56 ± 8 1085 ± 48 172 ± 3 120 ± 1
Downloaded by [Australian National University] at 20:02 22 January 2015

5.0 152 ± 62 14 ± 4 31 ± 1 373 ± 3 58 ± 9 60 ± 5 564 ± 32 73 ± 2 67 ± 8

Table 3.
Tensile mechanical properties of tubular SF scaffolds after sterilization

Modulus (kPa) Strength (kPa) Elongation (%)

Controla 587 ± 15 70 ± 9 54 ± 7
Radiation sterilizationb 622 ± 13 75 ± 9 52 ± 6
Steam sterilizationc 934 ± 26 80 ± 15 30 ± 8
a Tubular SF scaffolds (4% (w/v) SF, 3% (v/v) PGDE) (8 mm od, 1 mm twt).
b Scaffolds irradiated at a dose of 25 kGy.
c Scaffolds autoclaved at 121◦ C for 20 min.

Figure 5. Compression curves of cylindrical SF scaffolds (15 mm od) prepared from (a) 2.5% (w/v)
SF, 3% (v/v) PGDE, (b) 2.5% (w/v) SF, 5% (v/v) PGDE, (c) 4% (w/v) SF, 1% (v/v) PGDE, (d) 4%
(w/v) SF, 3% (v/v) PGDE, (e) 4% (w/v) SF, 5% (v/v) PGDE, (f) 6% (w/v) SF, 1% (v/v) PGDE, (g)
6% (w/v) SF, 3% (v/v) PGDE and (h) 6% (w/v) SF, 5% (v/v) PGDE.
 1970 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974
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Figure 6. Tensile recovery curve of tubular SF scaffolds (8 mm od, 1 mm twt) (4% (w/v) SF, 3% (v/v)
PGDE). Here, the last 5 of 100 cycles are shown.

3.3. FT-IR Spectrum of Tubular SF Scaffolds

Figure 7 shows the FT-IR spectra of tubular SF scaffolds and two kinds of SF gels
for comparison. The conformationally sensitive bands for SF could be detected
in the FT-IR spectra at amide-I, -II and -III modes. Here, all the spectra showed
the characteristic absorption bands of SF in a β-sheet structure, i.e., 1627 cm−1
with the presence of a shoulder at 1701 cm−1 (amide I), 1524 cm−1 (amide II),
1238 cm−1 (amide III) [26]. Generally, there are two crystalline forms, silk I and
silk II, reported for SF on the basis of extensive investigations from X-ray fiber
diffraction, electron diffraction and NMR spectroscopy [17]. The silk I form is the
SF structure in the solid state before spinning, where the molecular conformation
is repeated β-turn type II. The silk II form is the structure of SF after spinning,
i.e., silk fiber, where the molecular conformation is a heterogeneous anti-parallel
β-sheet. Therefore, the β-sheet structure is essential for SF, which to a large extent
provides the silk fiber with outstanding mechanical properties. Here, the absorption
band at 1104 cm−1 (indicated as arrows in the expanded FT-IR spectral figure) in
Fig. 7a and 7c is noticeable, which is assigned to the N–CH3 and C–CH3 stretch-
ing vibration modes in the SF molecules. Interestingly, a broad but not clear band
was detected in Fig. 7b, since the 1104 cm−1 band was overlapped with the char-
acteristic ether stretching band of PGDE, which was reported at 1100 cm−1 [27],
probably suggesting that PGDE is only the solidifier for SF at a lower temperature,
e.g., −20◦ C for the preparation of a tubular SF scaffold, and does not react with it,
because it could be removed after soaking in deionized water for 3 days. However,
PGDE is a cross-linker agent at a higher temperature, e.g., 60◦ C, for the preparation
of SF gel.
3.4. In Vitro Cultivation and Enzymatic Degradation of SF Scaffolds
The proliferation of mouse L-929 fibroblasts on the SF scaffolds is shown in Fig. 8.
No significant cell growth was observed within 3 days of cultivation. The cells
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1971
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Figure 7. FT-IR spectra of (a) SF gel prepared from 4% (w/v) SF solution, (b) SF gel prepared from
4% (w/v) SF solution with the addition of 3% (v/v) PGDE and 2% (w/v) NaCl after washing and
(c) tubular SF scaffolds (4% (w/v) SF, 3% (v/v) PGDE).

then began to grow rapidly, reaching a maximum density of 5.15 × 105 cells/ml
after 11 days. Further cultivation led to the decline of cell density. Figure 9 shows
the morphology of L-929 on the surface and inside of scaffolds cultivated for 5
and 10 days, respectively. The fibroblasts attached to the surface of scaffolds were
flattened and extensively spread out, and their filopodia could be observed clearly.
On the inside of the scaffolds, the fibroblasts attached on the pore walls or spread
in the pores with filopodia as the bridges.
 1972 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974
Downloaded by [Australian National University] at 20:02 22 January 2015

Figure 8. In vitro proliferation of mouse L-929 fibroblasts on SF scaffolds (4% (w/v) SF, 3% (v/v)
PGDE).

Figure 9. Morphology of mouse L-929 fibroblasts on the SF scaffolds (4% (w/v) SF, 3% (v/v) PGDE).
(a) Surface, 5 days; (b) surface, 10 days; (c) inside, 5 days; (d) inside, 10 days.

Figure 10 compares the in vitro degradation behavior of tubular SF scaffolds

under different conditions. The tubular SF scaffolds did not show any degradation
within 20 days in PBS buffer solution without protease XIV. With 1 U of protease
XIV, the scaffolds degraded gradually with time. Generally, lower concentrations of
SF and/or PGDE led to higher degradation rates of scaffolds at a constant amount of
protease XIV. However, the protease concentration was more effective to the initial
stage of scaffold degradation. The scaffolds (4% (w/v) SF, 3% (v/v) PGDE) rapidly
degraded with 2.5 U protease XIV (within the initial 5 days), and the mass residues
were 53% and 42% after 5 days and 10 days treatment, while the mass residues for
 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974 1973
Downloaded by [Australian National University] at 20:02 22 January 2015

Figure 10. In vitro enzymatic degradation of tubular SF scaffolds (8 mm od, 1 mm twt). (×) without
enzyme; (Q) 4% (w/v) SF, 5% (v/v) PGDE, 1 U enzyme; (2) 4% (w/v) SF, 3% (v/v) PGDE, 1 U
enzyme; (F) 2.5% (w/v) SF, 3% (v/v) PGDE, 1 U enzyme; (!) 4% (w/v) SF, 3% (v/v) PGDE,
2.5 U enzyme.

the same scaffolds was 87% and 69% with 1.0 U protease concentration. However,
further treatment in the protease XIV buffer solution did not show any significant
degradation of scaffolds at 2.5 U, while the scaffolds still degraded gradually at
1.0 U of protease. After 20 days of degradation treatment, the mass residues were
37% and 43%, respectively, for the scaffolds (4% (w/v) SF, 3% (v/v) PGDE) treated
with 2.5 U and 1 U of protease XIV.

4. Conclusion
Porous tubular SF scaffolds with an outside diameter of 4–8 mm and tube wall
thickness of 0.8–1 mm were fabricated from SF solution by the addition of PGDE.
The pore size of the tubular scaffolds ranged from 81 µm to 128 µm, and the poros-
ity was from 90–96%, depending on the concentrations of SF and PGDE, where the
pores both on the outside and inside surface of the tubular scaffold are mostly fine
and close in texture, which become loose and are interconnected with each other
in the scaffolds. The FT-IR spectra showed that the scaffolds had a β-sheet struc-
ture, providing the scaffolds with satisfying mechanical properties, especially the
high ability of deformation–recovery. The PGDE in the scaffolds may be only the
solidifier for SF and does not react with it, because it could be removed by soaking
in deionized water. Thus, the scaffolds showed excellent biocompatibility to mouse
L-929 fibroblasts, suggesting that the tubular SF scaffolds prepared in this work
may be an ideal candidate for the temporary skeleton to support the growing tissue,
providing the desired shape until the cells produce their own extracellular matrix.
 1974 S. Min et al. / Journal of Biomaterials Science 20 (2009) 1961–1974

Acknowledgements
This work was financially supported by Special Funds of Cooperation between
Hangzhou City and Zhejiang University, Key Program of Science and Technol-
ogy Plan of Zhejiang Province (2007C2118), Program for New Century Excellent
Talents in University (NCET070763) and National Natural Science Foundation of
China (Nos 30870628 and 10672145).

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