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Comparison of the in vitro and in vivo degradations of silk fibroin scaffolds from mulberry and
nonmulberry silkworms
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2015 Biomed. Mater. 10 015003
(http://iopscience.iop.org/1748-605X/10/1/015003)
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Biomed . Mater. 10 (2015) 0150 03
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REVISED
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ACCEPTED FOR PUBLICATION
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PUBLISHED
22 Decem ber 2014
1. Introduction
Silk fibroin (SF) is a promising biomaterial for tissue
engineering and regenerative medicine applications
due to its abundance, mechanical robustness,
biocompatibility, and tunable biodegradability [1–4].
Silks can be classified as mulberry and nonmulberry,
which are produced by domesticated Bombyx mori
(Bombycidae family) and wild silkworm species,
respectively. Bombyx mori silk-based biomaterials have
been extensively used for tissue engineering scaffolds
[5, 6], biomedical devices [7, 8] and for drug release [9].
Of the various wild silkworms, Antheraea pernyi and
Antheraea yamamai are relatively common species that
belong to the Saturniidae family (order Lepidoptera,
phylum Arthropoda). Antheraea pernyi is mass-
produced in northeast China for the production of silk
fiber, and Antheraea yamamai is primarily produced in
East Asia, such as in China, Japan and Korea [10, 11].
To date, nonmulberry silk has mainly been used in
high-quality clothing. Recently, the nonmulberry
silks (Antheraea pernyi, Antheraea yamamai and
Antheraea mylitta) have attracted considerable interest
for biomedical applications because they contain an
abundance of Arg–Gly–Asp (RGD) sequences, which
are known to function as integrin receptors [12–16].
Mulberry and nonmulberry silks are structurally
and functionally distinguishable. Bombyx mori SF (Bm-
SF) is composed of a heavy (H) chain and a light (L)
chain linked by a disulfide bond [2]. H-chains, L-chains
and a P25 glycoprotein are assembled in a 6 : 6 : 1 r atio in
Bombycidae [17]. The P25 protein is believed to play a
significant role in maintaining the integrity of the com-
plex, which is non-covalently linked to these chains [2].
R You et al
Printed in the UK
015003
BMM
© 2015 IOP Publishing Ltd
2015
10
Biomed. Mater.
BMM
1748-605X
10.1088/1748-6041/10/1/015003
00
00
Biomedical Materials
IOP
22
December
2014
Comparison of the in vitro and in vivo degradations of silk broin
scaffolds from mulberry and nonmulberry silkworms
Renchuan You1, Yamei Xu1, Yi L iu2, Xiufang Li1 and Mingzhong Li1
1 National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No 199 Ren’ai
Road, Industrial Park, Suzhou 215123, People’s Republic of China
2 Center of Burns and Plastic Surgery of CPLA, Lanzhou General Hospital of Lanzhou Command, 333 Binhe Road, Lanzhou 730050,
People’s Republic of China
E-mail: mzli@suda.edu.cn
Keywords: silk fibroin, Bombyx mori silks, nonmulberry silks, scaffolds, degradation
Abstract
Degradation behavior is very important in the field of silk-based biomaterials. Mulberry and
nonmulberry silk fibroins are structurally and functionally distinguishable; however, no studies
have examined the differences in the degradation behaviors of silk materials from various silkworm
species. In this study, Ca(NO3)2 was used as a uniform solvent to obtain regenerated mulberry
and nonmulberry (Antheraea pernyi and Antheraea yamamai) silk fibroin (SF) solutions, and
the degradation behaviors of various SF scaffolds were examined. In vitro and in vivo results
demonstrated that regenerated mulberry SF scaffolds exhibited significantly higher mass loss and
free amino acid content release than did nonmulberry SF scaffolds. The differences in the primary
structures and condensed structures between mulberry and nonmulberry SF contributed to the
significant difference in degradation rates, in which the characteristic (–Ala–)n repeats, compact
crystal structure and high α-helix and β-sheet contents make nonmulberry SF more resistant than
mulberry SF to enzymatic degradation. Moreover, the Antheraea pernyi and Antheraea yamamai
SFs possess similar primary structures and condensed structures, although a slight difference in
degradation was observed; this difference might depend on the differences in molecular weight
following the regeneration process. The results indicate that the original sources of SF significantly
influence the degradation rates of SF-based materials; therefore, the original sources of SF should be
fully considered for preparing tissue engineering scaffolds with matched degradation rates.
Paper
doi:10.1088/174 8- 60 41/10/1/015003
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Nonmulberry SF lacks both an L-chain and the P25 pro-
tein [18]. Moreover, the abundant (–Ala–)n polyp eptide
sequences in nonmulberry silks are more hydrophobic
than the (–Gly–Ala–)n repeats in mulberry silks [18].
The poly(–Ala–) β-sheets impart a higher binding
energy than do poly(–Gly–Ala–) β-sheets, which make
nonmulberry SF more resistant to dissolution in salt
solvents in comparison to mulberry SF [18–20]. Fur-
thermore, mulberry and nonmulberry SFs also exhibit
different bioactivities for cell adhesion, proliferation
and differentiation. The presence of abundant RGD
sequences in nonmulberry SF leads to enhanced cell
adhesion and proliferation [21]. A comparative study
on osteochondral repair demonstrated that Antheraea
mylitta SF scaffolds are more chondroinductive, whereas
mulberry SF scaffolds are more osteoinductive [22].
Degradation behavior is very important in the
fields of biomaterials and regenerative medicine; ide-
ally, the degradation rate of scaffolds should match the
tissue regeneration rate [4]. SF, like most proteins, can
be catalytically hydrolyzed by proteinases in vitro and
in vivo [23–29], and the degradation products of SF are
soluble peptides and free amino acids, which are easily
metabolized and absorbed by the body [30]. The type of
enzyme plays a key role in the degradation of SF mate-
rials. α-Chymotrypsin can digest the less crystalline
regions of SF but does not degrade the β-sheet crystals,
leading to a weaker degradability [31], whereas protease
XIV and collagenase IA were found to be more aggressive
to regenerated mulberry and nonmulberry SF materi-
als [23, 24]. The degradation rates of SF-based materials
are correlated with the β-sheet content, in which a low
β-sheet content contributes to easier enzymatic degra-
dation [3, 4, 31–33]. Natural SF fibers contain abundant
β-sheet crystallites after natural spinning, whereas such
high β-sheet content appears impossible to achieve in
regenerated SF-based materials. Therefore, regenerated
SF materials, such as films, nanofibers and porous scaf-
folds, are more readily degraded compared with natural
SF fibers [24, 26, 28]. Moreover, the fabrication process
and pore structure of silk-based materials also affect the
degradation rate. The scaffold obtained using an aque-
ous process exhibited more rapid in vivo degradation
than hexafluoroisopropanol-derived scaffolds, and a
higher initial SF concentration and smaller pore size led
to lower levels of tissue ingrowth and degradation [27,
34]. However, none of these studies examined the differ-
ences in the degradation behaviors of SF materials from
different silkworm species in great detail. The structural
and physico-chemical diversity of polymers significantly
influence the degradation behaviors of polymers [35,
36]; therefore, it is of interest to compare the degradation
behaviors of mulberry and nonmulberry SF biomateri-
als. In this study, three races of SF were selected: Bm-SF
from mulberry silkworms and Antheraea pernyi (Ap-SF)
and Antheraea yamamai SF (Ay-SF) from nonmulberry
silkworms. Porous scaffolds derived from the three races
of silkworms were used to investigate the differences in
the in vitro and in vivo degradation behaviors.
2. Materials and methods
2.1. Preparation of regenerated SF scaffolds
Bombyx mori raw silks (Huzhou, Zhejiang, China) were
boiled three times in a 0.05% Na2CO3 solution for 30 min
to remove sericin. Antheraea pernyi raw silks (Dandong,
Liaoning, China) and Antheraea yamamai cocoons
(Changsha, Hunan, China) were boiled three times in
a 0.25% Na2CO3 solution for 30 min. After thoroughly
rinsing, the extracted fibers were air dried at 60 °C and
dissolved in molten Ca(NO3)2 at 105 °C ± 2 °C for 3 h. The
mixtures were dialyzed against distilled water (MWCO
9–12 kDa) in cellulose tubes for 4 d. The resulting
SF solution was stored at 4 °C after filtration. The SF
solutions derived from the three races of silkworms were
diluted to 1.5%, and then 2-morpholinoethanesulfonic
acid (MES), N-hydroxysuccinimide (NHS) and
1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide
hydrochloride (EDC) (Sigma-Aldrich) were added to
the SF solutions at 20%, 10% and 20% by SF weight,
respectively. The mixtures were poured into stainless
steel vessels and frozen at −40 °C for 6 h, followed by
lyophilization for 48 h using a Virtis Genesis 25-LE Freeze
Dryer to obtain porous scaffolds.
2.2. In vit ro enzymatic degradation
The SF scaffolds were first cut into samples with
approximately equivalent sizes (3 × 3 cm2). Then,
the scaffolds were weighed and incubated at 37 °C in a
phosphate-buffered saline solution (PBS; 0.05 M, pH
7.4) that contained 1.0 U ml−1 collagenase IA (from
Clostridium histolyticum, EC 3.4.24.3, Sigma-Aldrich).
The samples were incubated in enzyme solution (bath
ratio 1 : 50) for 1, 3, 6, 12 and 18 d under slow shaking,
and samples without enzyme but in PBS served
as controls. For each type of scaffold, at least three
samples were used to obtain statistically significant
data. The degradation solution was replaced with
fresh enzyme solution every 3 d. At the designated time
points, the degradation products and residues were
collected for analysis after rinsing and lyophilizing for
morphological observations and structural analyses.
To calculate the mass loss, the remaining scaffolds
were rinsed with deionized water and then dried at
105 °C to a constant weight, and the remaining ratio
was expressed as the percentage of retained dry weight
relative to the initial dry weight.
2.3. Mor phological observation and structural
analysis
The morphological changes of the SF scaffolds were
observed by scanning electron microscopy (SEM;
S-570, Hitachi, Japan) after gold sputtering. Fourier
transform infrared (FTIR) spectroscopy analysis
of the SF scaffolds was performed to determine the
conformational changes. The scaffolds were cut into
microparticles with a size of less than 40 μm, and then
the samples were prepared in KBr pellets. FTIR spectra
were recorded using a Nicolet 5700 spectrometer
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(Thermo Scientific, USA). To quantify the secondary
structures, Fourier self-deconvolution of the amide I
region (1595–1705 cm−1) was performed using Opus
6.5 software (Bruker, Germany), and the Fourier self-
deconvolution spectra were curve-fitted to measure
the relative areas of the amide I region components
[37]. Furthermore, x-ray diffraction (XRD) was used
to determine the crystal structures of the scaffolds using
an x-ray diffractometer (X′Pert-Pro MPD, PANalytical
B.V. Holland) with Cu Kα radiation at 40 kV and 30 mA
and with a scan rate of 0.6 min−1.
2.4. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
The regenerated SF solutions and degradation
products at 3, 6 and 18 d were examined using SDS-
PAGE according to our previous report [4]. In brief,
the samples were run on polyacrylamide gel in running
buffer (0.25M Tris-HCl, 10% SDS, 0.5% bromophenol
blue, 50% glycerol and 5% 2-mercaptoethanol, pH
8.3). The stacking gel contained 5% acrylamide, 0.1%
ammonium persulfate and 0.1% SDS in 1.0M Tris-HCl
buffer (pH 6.8), and the separating gel contained 8–12%
acrylamide, 0.1% ammonium persulfate and 0.1% SDS
in 1.5M Tris-HCl buffer (pH 8.8). Pre-stained protein
served as the molecular weight (MW) marker.
2.5. Amino acid analysis
The free amino acids of the degradation products
were detected using an amino acid analyzer (L-8800,
Hitachi, Japan). An equal volume of 8% sulfosalicylic
acid solution was added to the degradation solution
and then centrifuged at 15,000 rpm for 15 min [4]. The
supernatant was diluted with 0.02N HCl and filtered
with 0.22 μm syringe filters, and then the filtrate was
analyzed using an amino acid analyzer. To measure
the amino acids contents in the SF scaffolds and
degradation residues, the samples were hydrolyzed in
6N HCl at 110 °C for 24 h and then analyzed using an
amino acid analyzer (L-8800, Hitachi, Japan).
2.6. In vivo degradation
All animal experiments were conducted in accordance
with the Management Ordinance of Experimental
Animal of China ([2001] No 545) and were approved
by the Jiangsu Province according to the experimental
animals management rules ([2008] No 26). The SF
scaffolds (approximately 20 × 20 mm2) were implanted
into the backs of SD rats (180–200 g, SPF grade, male),
with 5 rats in each group. Pentobarbital sodium
(30 mg kg−1 body weight) was administered prior to
surgery. Full-thickness wounds were created on the
upper back of each rat, and the scaffolds were implanted
as dermal substitutes into the defect sites, followed
by covering with thin split-thickness skin grafts.
The wounds were then closed with 6-0 silk sutures
and covered by Vaseline carbasus and dry carbasus.
Specimens were harvested at 28 d, and the harvested
samples were immediately fixed in 4% formaldehyde
in PBS at room temperature and embedded in paraffin
to cut tissue sections. The sections were stained with
hematoxylin and eosin (H & E) and were observed under
an optical microscope (Olympus BH-2, Japan). The
degradation ratio of the scaffolds was approximately
calculated using Image-Pro Plus 6.0 software (Media
Cybernetics Inc., USA). In a typical tissue, nuclei are
stained by hematoxylin and show dispersive blue dots,
whereas the cytoplasm and extracellular matrix have
varying degrees of pink staining. Silk fibroin that is
negatively charged will react with the positively charged
hematoxylin through electrostatic interactions,
resulting in a light blue–purple strip and sheet-like
staining. In H&E pictures, the blue–purple area was
highlighted by enhancing the contrast, and then the
highlighted area was captured as remaining scaffolds to
calculate the remaining area after filtering the cell nuclei
staining area. The degradation ratio was expressed as
the percentage of reduced area relative to the initial area
before implantation (
=n5
for each sample). Statistical
comparisons were performed using SPSS version 16.0
software (SPSS Inc., Chicago, Illinois). The deductive
statistics (t-test, ANONA) were conducted and the
data were expressed as mean ± standard deviation, and
<p0.05
was considered to be statistically significant.
3. Results
3.1. Molecular weight distribution (MWD) of the
regenerated SF
As shown in figure 1, the SDS-PAGE results
demonstrated that the SF solutions derived from the
different silkworms all have sequential bands from
Figure 1. SDS-PAGE of the SF from (a)
Bombyx mori, (b) Antheraea yamamai
and (c) Antheraea pernyi, where (M1)
is the molecular weight markers. The
concentration of the separating gel was 8%.
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approximately 30 to 300 kDa, indicating that the
SF had been dissolved into polypeptide mixtures.
However, the Bm-SF exhibited stronger bands in the
range from approximately 30 to 52 kDa compared
with nonmulberry SF (figure 1 (lane a)), whereas
stronger bands for Ay-SF (figure 1 (lane b)) and Ap-
SF (figure 1 (lane c)) appeared in the region above
72 kDa. This result indicated that the Bm-SF contained
more low-MW polypeptides (30–52 kDa) and less
high-MW polypeptides (larger than 72 kDa) than the
nonmulberry SF after the same regeneration process.
Moreover, the lane of Ap-SF (figure 1 (lane c)) was
stronger than that of Ay-SF (figure 1 (lane b)) in the
region above 100 kDa, suggesting that the regenerated
Ap-SF contained more high-MW polypeptides
(especially in the region above 200 kDa) than the Ay-SF.
3.2. Morphological changes
The porous scaffolds derived from the three silkworms
exhibited similar morphological structures, in
which the pore shapes are irregular polygons and
fusiform (figures 2(a)–(c-0)). The large pores have
approximately 100–500 μm long diameters and
approximately 50–200 μm short diameters, and the
diameters of the small pores were approximately 10–
100 μm. The pore diameters and pore wall thicknesses of
the three scaffolds were similar. After 6 d of incubation
in enzyme solution, the Bm-SF scaffold had completely
collapsed (figure 2(a-6)). In contrast, partial scaffold
integrity was retained in the Ap-SF and Ay-SF scaffolds,
and a shrunken pore structure could still be observed
(figures 2(b) and (c-6)). With further degradation,
the Bm-SF scaffolds degraded into powders and small
fragments after 18 d (figure 2(a-18)). However, there
were still many large sheet-like remains in the residual
Ap-SF and Ay-SF scaffolds (figures 2(b) and (c-18)).
The SEM results revealed that the Ap-SF and Ay-SF
scaffolds were capable of maintaining their structural
integrities for longer times in collagenase IA solution,
whereas the Bm-SF scaffold was more susceptible to
enzymatic degradation.
Figure 2. SEM images of the scaffolds after enzymatic degradation for 0, 6 and 18 d: (a) The Bm-SF scaffolds, (b) the Ap-SF scaffolds
and (c) the Ay-SF scaffolds. Scale bars: 500 μm.
Figure 3. Quantitative changes in the SF scaffolds
during degradation: (■) the Bm-SF scaffolds, (◆) the
Ap-SF scaffolds and (▲) the Ay-SF scaffolds.
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3.3. Mass loss
Figure 3 shows the mass loss of the scaffolds as a
function of the enzymatic degradation time. The
mass loss of the SF scaffolds significantly increased
with increasing enzymatic degradation time. After
3 d, the mass loss of the scaffolds showed significant
differences, and the residual weights of the Bm-SF,
Ap-SF and Ay-SF scaffolds were 51.32%, 72.98% and
70.27%, respectively. With further degradation for 18 d,
the residual masses of the Bm-SF, Ap-SF and Ay-SF
scaffolds were 2.95%, 43.8% and 37.14%, respectively.
This result demonstrated that the Bm-SF scaffold can
be almost completely degraded in 18 d when exposed
to collagenase IA and that the enzymatic degradability
of that regenerated Bm-SF scaffold was significantly
higher than that of nonmulberry SF scaffolds.
Moreover, the Ap-SF scaffold exhibited a slightly slower
degradation rate compared to the Ay-SF scaffold,
suggesting that the silkworm races of nonmulberry SF
influence the degradation behavior of the scaffolds.
3.4. Structural changes after in vitro degr adation
FTIR spectra were recorded to monitor conformational
transformations in the SF scaffolds. As shown in
figure 4(a), the SF scaffolds exhibited broad peaks at
1650 cm−1 (random coil, amide I) and at 1542 cm−1
(α-helix, amide II) before degradation, and weak
peaks appeared at 1521 cm−1 (β-sheet, amide II).
Moreover, the Bm-SF scaffolds exhibited a broad peak
at 1234 cm−1 (random coil, amide III), whereas weak
peaks at 1252 cm−1 (α-helix, amide III ) appeared in
the nonmulberry SF scaffolds. The results indicated
that the secondary structures of the regenerated
SF scaffolds were mainly random coil and α-helix
before degradation. After degradation, strong peaks
corresponding to the β-sheet structure appeared in
the residual SF scaffolds, whereas the peaks (amide III
region) corresponding to the random coil and α-helix
structures were weak. The residual Bm-SF scaffold
exhibited strong peaks at 1630 cm−1 (β-sheet, amide I)
and at 1530 cm−1 (β-sheet, amide II) (figure 4(a)(a-6)),
whereas strong peaks at 1630 cm−1 (β-sheet, amide I)
and 1521 cm−1 appeared for the residual nonmulberry
SF scaffolds (figure 4(a)(b),(c)-18). These results
demonstrated that the secondary structures of the
residual SF scaffolds were mainly β-sheet structures after
degradation. The fitting results for the amide I region
show the change in the secondary structure contents
in the SF scaffolds (table 1). The β-sheet contents of
the Bm-SF, Ap-SF and Ay-SF scaffolds were 25.6%,
32.3% and 31.7% before degradation, respectively.
This result indicated that the nonmulberry SF scaffolds
contained more β-sheet and α-helix structures than did
Figure 4. (a) FTIR spectra of (a) the Bm-SF scaffolds, (b) the Ap-SF scaffolds and (c) the Ay-SF scaffolds after degradation for 0,
6 and 18 d. (b) XRD data of (a) the Bm-SF scaffolds, (b) the Ap-SF scaffolds and (c) the Ay-SF scaffolds after degradation for 0, 6
and 18 d.
Table 1. The secondary structure contents of the SF scaffolds
derived from the three races of silkworms after degradation for 0, 6
and 18 d. * The secondary structure content of the residual Bm-SF
scaffold at 18 d was not examined because there was not a sufficient
amount of remains to collect.
SF scaffolds Conformation 0 d 6 d 18 d
Bm-SF β-sheet (%) 25.6 37.1 *
α-helix (%) 20.4 18.4 *
Random coil (%) 21.0 10.1 *
Turns (%) 28.2 27.5 *
Ap-SF β-sheet (%) 32.3 36.8 38.3
α-helix (%) 26.2 23.4 22.4
Random coil (%) 15.8 13.6 12.6
Turns (%) 22.0 21.6 20.6
Ay-SF β-sheet (%) 31.7 38.2 39.2
α-helix (%) 24.4 22.6 20.1
Random coil (%) 15.9 13.2 11.2
Turns (%) 25.1 23.9 21.9
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the mulberry SF scaffolds, and no significant difference
was observed between the Ap-SF and Ay-SF scaffolds.
After 6 d of degradation, the β-sheet contents increased
to 37.1%, 36.8% and 38.2%, respectively. With further
degradation for 18 d, the β-sheet contents of the Ap-
SF and Ay-SF scaffolds increased to 38.3% and 39.2%,
respectively. Furthermore, XRD was performed to
determine crystal structure changes in the SF scaffolds
(figure 4(b)). The Bm-SF and nonmulberry SF scaffolds
showed a diffraction peak at approximately 21.1°,
which indicated that the regenerated SF scaffolds
were primarily amorphous before degradation
(figure 4(b)(a-0)). However, the nonmulberry SF
scaffolds (figure 4(b)(b), (c)-0) showed significant
peaks at 11.8° (silk I) and at 22.2° (an overlapped peak
of silk I and silk II) and a weak peak at 31.6° (silk II),
demonstrating that abundant silk I crystal structures
and minor silk II crystal structures formed in the
nonmulberry SF scaffolds. A primary peak at 21.1°
(silk II) and a weak peak at 24.1° (silk II) appeared in
the residual Bm-SF scaffold (figure 4(b)(a-6)) after 6 d,
and significant peaks at 16.7° (silk II), 20.0° (silk II) and
24.1° (silk II) appeared in the residual nonmulberry
SF scaffolds (figure 4(b)(b), (c)-18) after 18 d. These
results indicated that the amorphous regions were
preferentially degraded by collagenase IA in both
mulberry and nonmulberry SF scaffolds, leading to an
increase in β-sheet contents after degradation.
3.5. MWD of the degradat ion products
SDS-PAGE was used to determine the molecular weights
of the degradation products at 3, 6 and 18 d (figure 5).
After 3 d of degradation, all samples showed sequential
bands at 10–50 kDa (figure 5(a)). However, a significant
band at 10–15 kDa appeared in lane a, whereas strong
bands at 27 and 40 kDa appeared in lanes (b) and
(c). These results indicated that the SF scaffolds were
degraded into polypeptides by collagenase IA and that the
Bm-SF scaffold was more readily degraded into low-MW
polypeptides. After degradation for 6 d, the bands at less
than 15 kDa strengthened and high-MW polypeptides
were further released into the enzyme solution
(figure 5(b)). Bands at 25 and 40 kDa appeared in Bm-
SF, and a band at 90 kDa appeared in the nonmulberry
SF. With further degradation, bands at 15 and 27 kDa
were observed in Bm-SF and nonmulberry SF after 18 d
(figure 5(c)), respectively. Moreover, the sequential band
at 10–50 kDa weakened compared to the degradation
products at 6 d, suggesting that the polypeptides released
from the scaffolds were further degraded into lower MW
polypeptides and free amino acids.
3.6. Amino acid analysis of the degradation
products and remains
The amino acid compositions of various races of SF were
examined. The amino acid composition of the residual
Bm-SF scaffold at 18 d was not examined because
the amount of collected remains was not sufficient
for conducting amino acid analysis. The amino acid
composition of SF consists primarily of Gly, Ala and
Ser. As shown in table 2, the Gly, Ala and Ser contents
account for approximately 84% of the total amino acid
content in Bm-SF, and the content in nonmulberry
SF is approximately 81.5%. However, the Gly and Ala
contents in the scaffolds are significantly different. The
Bm-SF contains 43.41% Gly and 30.2% Ala, whereas the
nonmulberry SF contains approximately 28.5% Gly and
42.5% Ala. The abundant Gly–X repeats in mulberry
silk contribute to the higher content of Gly, whereas the
abundant (–Ala–)n repeats in the nonmulberry silks
Figure 5. SDS-PAGE of the degradation products from (a) the Bm-SF scaffolds, (b) the Ap-SF scaffolds and (c) the Ay-SF scaffolds.
(M) The molecular weight marker. (a) 3 d, (b) 6 d and (c) 18 d. Concentrations of the separating gel: (a) 10%, (b), (c) 12%.
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result in a higher Ala content [38, 39]. Furthermore,
amino acids with large side chains and polar groups are
mainly present in the amorphous regions [4, 38, 39].
After degradation, the contents of Gly, Ala and Ser
increased. In contrast, the amounts of amino acids
with large side chains and polar groups, including Asp,
Glu, His, Arg, Lys, Thr, Cys, Tyr, Phe and Pro, decreased
after degradation. The content of these amino acids
decreased from 10.60%, 15.66% and 15.42% to 10.33%,
13.56% and 14.13% in Bm-SF, Ap-SF and Ay-SF,
respectively. This result was consistent with the FTIR
and XRD results, demonstrating that the amorphous
regions of SF were readily degraded. The SF scaffolds
were degraded into soluble peptides and free amino
acids. As shown in table 3, the content of free amino acids
released from the Bm-SF scaffold was 1764.15 μg ml −1
after 6 d of degradation, which was significantly higher
than the values of 29.30 and 31.60 μg ml−1 for the
Ap-SF and Ay-SF scaffolds. This result was consistent
with the mass loss examination, indicating that the Bm-
SF scaffold was more rapidly degraded by collagenase
IA. Moreover, compared to the Bm-SF scaffold, almost
no free Ala could be detected in the degradation
products of the nonmulberry SF scaffolds, indicating
that the polyalanine segments are strongly resistant to
enzymatic attack. Due to rapid degradation, the content
of free amino acids released during the degradation of
the Bm-SF scaffold was lower than that released from
the nonmulberry SF scaffold at 18 d.
3.7. In vivo degradation assessment
Macroscopic observations revealed that there was no
empyema or infection in the wound in the backs of the
SD rats for SF scaffolds from various races, and the skin
morphology and architecture were fully developed in all
groups after 4 weeks. The scaffolds were well tolerated
Table 2. The amino acid compositions of the various SF scaffolds and degradation remains. ND*: not determined.
Amino acids
The amino acid compositions of SF scaffolds (mol%)
Bm-SF Ap-SF Ay-SF
0 d 6 d 0 d 18 d 0 d 18 d
Gly 43.41 ± 0.01 43.80 ± 0.00 28.34 ± 0.12 28.44 ± 0.02 28.66 ± 0.05 28.62 ± 0.04
Ala 30.20 ± 0.05 30.21 ± 0.04 42.54 ± 0.04 44.47 ± 0.00 42.80 ± 0.10 44.51 ± 0.02
Ser 10.36 ± 0.02 10.53 ± 0.01 10.17 ± 0.35 10.37 ± 0.03 10.06 ± 0.06 10.07 ± 0.01
Asp 1.66 ± 0.01 1.60 ± 0.01 5.23 ± 0.03 5.10 ± 0.01 5.49 ± 0.01 5.38 ± 0.02
Glu 1.57 ± 0.03 1.52 ± 0.01 1.04 ± 0.57 0.37 ± 0.01 0.64 ± 0.01 0.32 ± 0.00
His 0.19 ± 0.01 0.17 ± 0.01 0.80 ± 0.01 0.67 ± 0.00 0.94 ± 0.01 0.83 ± 0.01
Arg 0.46 ± 0.00 0.43 ± 0.01 2.59 ± 0.01 2.62 ± 0.00 2.64 ± 0.02 2.82 ± 0.02
Lys 0.22 ± 0.00 0.24 ± 0.00 0.07 ± 0.00 0.06 ± 0.00 0.03 ± 0.01 0.03 ± 0.00
Thr 0.89 ± 0.01 0.87 ± 0.01 0.37 ± 0.01 0.19 ± 0.01 0.31 ± 0.01 0.16 ± 0.00
Cys 0.01 ± 0.00 0.02 ± 0.00 0.59 ± 0.12 0.05 ± 0.00 0.72 ± 0.01 0.31 ± 0.06
Tyr 4.64 ± 0.01 4.54 ± 0.05 4.53 ± 0.03 4.28 ± 0.01 4.34 ± 0.03 4.10 ± 0.04
Phe 0.67 ± 0.00 0.65 ± 0.00 0.25 ± 0.01 0.22 ± 0.01 0.13 ± 0.01 0.12 ± 0.00
Pro 0.29 ± 0.00 0.29 ± 0.01 0.19 ± 0.01 ND* 0.18 ± 0.03 0.06 ± 0.09
Val 1.88 ± 0.01 1.81 ± 0.00 0.66 ± 0.03 0.38 ± 0.01 0.62 ± 0.01 0.38 ± 0.01
Ile 0.57 ± 0.01 0.54 ± 0.01 0.30 ± 0.01 0.18 ± 0.00 0.21 ± 0.014 0.11 ± 0.00
Leu 0.35 ± 0.01 0.31 ± 0.01 0.22 ± 0.00 ND* 0.14 ± 0.01 ND*
NH32.62 ± 0.00 2.46 ± 0.00 2.09 ± 0.01 2.61 ± 0.00 2.08 ± 0.00 2.17 ± 0.01
Total 100 100 100 100 100 100
Table 3. The free amino acid contents of the degradation products from various SF scaffolds. ND*: not determined.
Amino acids
The free amino acid contents of degradation products (μg ml−1)
Bm-SF Ap-SF Ay-SF
6 d 18 d 6 d 18 d 6 d 18 d
Gly 913.40 ± 3.11 0.75 ± 0.21 1.60 ± 0.00 0.90 ± 0.00 12.60 ± 0.00 ND*
Ala 494.55 ± 1.35 1.25 ± 0.35 0.85 ± 0.07 ND* 0.45 ± 0.64 ND*
Ser 10.00 ± 0.28 2.75 ± 0.48 16.95 ± 0.07 15.95 ± 0.50 5.95 ± 0.50 15.05 ± 0.35
Asp 1.20 ± 0.14 0.55 ± 0.78 1.95 ± 0.07 1.85 ± 0.35 1.25 ± 0.07 1.75 ± 0.35
Thr 13.50 ± 0.00 1.50 ± 0.71 2.95 ± 0.07 2.75 ± 0.21 4.25 ± 0.21 2.70 ± 0.42
Cys 23.50 ± 0.14 2.90 ± 1.70 2.25 ± 0.07 1.75 ± 0.35 5.10 ± 0.42 7.15 ± 1.06
Tyr 267.25 ± 0.78 6.85 ± 0.97 2.75 ± 0.07 1.25 ± 0.21 2.00 ± 0.28 9.65 ± 0.35
Phe 40.75 ± 0.50 5.35 ± 0.57 ND* ND* ND* 6.40 ± 0.71
Total 1764.15 21.90 29.30 24.45 31.60 42.70
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Biomed . Mater. 10 (2015) 0150 03
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by the host animals, and no abnormal conditions were
observed during the four weeks of implantation. H & E
staining images showed that new tissue had grown into
the interior of the scaffolds (figure 6) and that most of
the SF scaffolds had been degraded. Compared to the
nonmulberry SF scaffold, the Bm-SF scaffold completely
degraded into fragments (figure 6(a)) and the new tissue
formation was more obvious. Furthermore, the changes
after degradation were approximately calculated (figure
6(d)). The degradation ratio of the Bm-SF scaffold
was about 82%, whereas the degradation ratios of the
Ap-SF and Ay-SF scaffolds were about 51% and 58%,
respectively. No significant difference was found between
the Ay-SF and Ap-SF scaffolds. The present study does
not provide sufficiently accurate calculation method to
show the slight in vivo degradation difference between
the Ay-SF and Ap-SF scaffolds. However, the degradation
ratio of the Bm-SF scaffold was significantly higher than
that of the nonmulberry SF scaffold after four weeks
implantation. The results demonstrated that the in vivo
degradation rate of the Bm-SF scaffold was significantly
higher than that of the nonmulberry SF scaffold, which
was consistent with the in vitro degradation results.
4. Discussion
Degradation behavior is a fundamental subject in
the field of silk-based biomaterials. The degradation
behaviors of silk materials have been investigated in
numerous in vitro and animal models as shown in
table 4. The results have showed that the regeneration
process, molecular weight and β-sheet crystal content
affected the degradability of regenerated SF materials
(table 4). Moreover, the processing methods, pore
structure and morphological features influenced the
degradation rate of SF-based materials (table 4). In this
study, the degr adation behaviors of SF scaffolds derived
from various races of silkworms were investigated.
Natural silk fibers must be regenerated and processed
into diverse biomaterial morphologies, such as films,
nanofibers and porous scaffolds, for various biomedical
applications. However, the poly(–Ala–) β-sheets make
nonmulberry SFs more resistant to dissolution in salt
solvents. Although there are many solvents for the
regeneration of mulberry SF, few salt solvents have
been reported for nonmulberry SF, namely Ca(NO3)2
and LiSCN [19, 20]. Here, we used Ca(NO3)2 as a
uniform solvent to obtain regenerated mulberry and
nonmulberry SF solutions.
The SDS-PAGE results indicated that the SF scaf-
folds were initially degraded into polypeptides. More-
over, the amino acid analysis and the FTIR and XRD
results demonstrated that the amorphous regions in the
SF scaffolds were more readily degraded by collagenase
IA, leading to an increase in the content of β-sheets after
degradation. Our data revealed significant differences
Figure 6. (a)–(c) H & E staining of the SF scaffolds after subcutaneous implantation in rats for 28 d for (a) the Bm-SF scaffolds,
(b) the Ap-SF scaffolds and (c) the Ay-SF scaffolds. The black arrows mark the residual scaffolds. Scale bars: 100 μm. (d) The
degradation percentages of the SF scaffolds for (a) the Bm-SF scaffolds, (b) the Ap-SF scaffolds, and (c) the Ay-SF scaffolds
(
<
p
**0
.0
1
, error bars indicate SD).
9
R You et al
Biomed . Mater. 10 (2015) 0150 03
IOP Publishing
in the degradation rates of mulberr y and nonmulberry
SFs. After enzymatic degradation for 18 d, the remain-
ing masses of the Ap-SF and Ay-SF scaffolds were 43.8%
and 37.14%, respectively. However, the Bm-SF scaffold
was almost completely degraded; the remaining mass
was only 2.95%. Furthermore, the in vivo degradation
level in the SF scaffolds exhibited the same trend as the
in vitro degradation level. After subcutaneous trans-
plantation for 28 d in SD rats, the degradation ratio of
the Bm-SF scaffold was about 82%, whereas the deg-
radation ratios of the Ap-SF and Ay-SF scaffolds were
about 51% and 58%, respectively. The results obtained
from in vitro and in vivo degradations indicated that
the regenerated nonmulberry SF was more resistant to
biodegradation.
The degradation rates of polymers are significantly
influenced by the chemical composition and crystal
structure of the polymer [35, 36]. The SFs from mul-
berry and nonmulberry are structurally distinguish-
able. The amino acid analysis revealed significant
differences in amino acid composition between the
mulberry and nonmulberry SF, especially for Gly and
Ala (table 2). The crystalline domains in the in mul-
berry silk consist of Gly–X repeats, with X being Ala,
Ser, Thr and Val [39]. In contrast to the (–Gly–Ala–)n
repeats of mulberry silks, the nonmulberry silks con-
tain abundant (–Ala–)n polypeptide sequences [38].
The abundant (–Ala–)n repeats in nonmulberry silks
are more hydrophobic than the (–Gly–Ala–)n sequences
in mulberry silks [18], which may be more resistant to
enzymatic attack [24, 41]. Moreover, the amorphous
segments in nonmulberry SF (11–22 amino acid resi-
due linkers) are shorter than the nonrepetitive seg-
ments of mulberry SF (42–44 residue linkers [39, 42,
43]), which may allow the formation of more compact
α-helix or β-sheet crystals, thereby leading to enzyme
penetration resistance. The amino acid analysis data
confirmed that Ala was minimally released from the
nonmulberry SF scaffolds, indicating that the polyala-
nine crystals are highly resistant to enzymatic attack.
Furthermore, the high β-sheet content leads to a more
crystalline structure that increases the resistance to
enzymatic degradation [3, 4, 31–33]. The (–Ala–)n
repeats facilitate the formation of an α-helix structure
in nonmulberry silks, and the α-helix structure would
be capable of being converted into a β-sheet structure
through thermal and chemical induction [38]. The
chemical crosslinking process is able to induce the con-
formational transition due to the rearrangement of
chains to form covalent bonds [44]. The XRD results
showed that more silk I crystal structures formed in the
nonmulberry SF scaffolds, and the analysis of the FTIR
spectra also confirmed that the nonmulberry SF scaf-
folds contained more β-sheet and α-helix structures
than did the mulberry SF scaffolds. The more crystalline
structure, particularly the increased content of β-sheet
crystals, can enhance the resistance of silk materials to
enzymatic degradation. Therefore, the higher content
of α-helix and β-sheet structures provided the nonmul-
berry SF scaffolds with greater resistance to enzymatic
degradation. In conclusion, the characteristic (–Ala–)n
repeats, compact crystal structure and higher β-sheet
content make nonmulberry SF more resistant to enzy-
matic degradation. On the other hand, the MW of SF
can influence the degradation rate of scaffolds, in which
a higher MW imparts more resistance to enzymatic
degradation [4]. Although all three regenerated SFs
can be obtained using Ca(NO3)2 as a solvent, the MW
level showed some differences between the mulberry
and nonmulberry SFs, in which the regenerated Bm-SF
contained more low-MW polypeptides (30–52 kDa).
The lower MW level would facilitate degradation of the
Bm-SF scaffold, and the slight degradation differences
between the Ap-SF and Ay-SF scaffolds supported the
influence of the MW level on the degradation rate. The
gene sequence similarity between Ap-SF and Ay-SF was
Table 4. Factors influencing the degradation behavior of silk fibroin materials.
Influence factors Degradation model Degradation properties References
Regeneration process In vitro using α-chymotrypsin / col-
lagenase IA, F / protease XIV, XXI
Degradation of regenerated SF materials
is more rapid than natural SF fibers due to
the disruption of β-sheet crystallites after
regeneration process
[24, 26]
Molecular weight • In vitro using collagenase IA
• Subcutaneous implantation in rats
Higher MW SF imparted more resistance
to enzymatic degradation
[4]
Condensed
structures
(β-sheet crystals)
In vitro using α-chymotrypsin /
collagenase IA / protease XIV
• Low β-sheet content contributes to
easier enzymatic degradation
• Regulating β-sheet crystal content could
control degradation rate
[3, 31–33]
Processing methods • In vitro using Protease XIV
• Cartilaginous implantation in rabbits
• Intramuscular / subcutaneous implan-
tation in rats
The degradation rate of aqueous-derived
scaffolds was more rapid than that of
HFIP-derived scaffolds
[27, 34]
Pore structure Intramuscular / subcutaneous implanta-
tion in rats
Smaller pore size led to lower levels of tis-
sue ingrowth and degradation
[27]
Morphological
properties
• In vitro using Protease XIV
• Cartilaginous implantation in rabbits
High surface area, porous morphology
increased degradation rate
[34, 40]
10
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Biomed . Mater. 10 (2015) 0150 03
IOP Publishing
quite high, showing 72% identity [42], and the Ap-SF
and Ay-SF scaffolds also showed similar secondary
structure contents (table 1). Therefore, the slight differ-
ence in degradation rate between the Ap-SF and Ay-SF
scaffolds should depend on the MW of the regenerated
SF. The regenerated Ap-SF contained more high-MW
polypeptides compared to the regenerated Ay-SF, espe-
cially in the region above 200 kDa, leading to a slightly
slower degradation rate in the Ap-SF scaffold.
5. Conclusion
The degradation behaviors of regenerated SFs obtained
from different original sources of silkworms were
presented. The results obtained through in vitro and in
vivo degradations revealed significant differences in the
degradation rates of mulberry and nonmulberry SFs.
The significant differences in the primary structures
and condensed structures of SF contributed to the
differences in degradation behaviors. The abundant
(–Ala–)n repeats and high α-helix and β-sheet contents
in the nonmulberry silks provided strong resistance to
enzymatic degradation. Consequently, the regenerated
mulberry SF scaffolds exhibited a significantly rapid
degradation rate compared to the nonmulberry SF
scaffold. Moreover, the difference in the degradation
behaviors of the Ap-SF and Ay-SF scaffolds might
depend on the MW of the nonmulberry SF solutions
after the regeneration process. These results
demonstrate that the original sources of SF significantly
influence the degradation rates of SF-based materials,
indicating that the original sources of SF are crucial in
controlling the degradation rate of tissue engineering
scaffolds.
Acknowledgments
This work was supported by the National Nature
Science Foundation of China (31370968), the Key
Program in Medical Science Research of Military (No
BWS11C061), Nature Science Foundation of Jiangsu
Province (BK20131177), College Natural Science
Research Project of Jiangsu Province (12KJA430003),
Priority Academic Program Development of Jiangsu
Higher Education Institutions and Jiangsu Province
Ordinary Universities and Colleges Graduate Scientific
and Innovation Plan (KYLX_1243).
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