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Proteomic Analysis of Silk Fibroin
Reveals Diverse Biological Function of
Different Degumming Processing
From Different Origin
Yaling Wang
1
,
2
, Yunyun Liang
1
, Jiacen Huang
3
, Yisheng Gao
1
, Zhixin Xu
1
, Xuejun Ni
4
,
Yumin Yang
1
*, Xiaoming Yang
1
,
3
* and Yahong Zhao
1
,
3
*
1
Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-innovation Center of Neuroregeneration, NMPA
Key Laboratory for Research and Evaluation of Tissue Engineering Technology Products, Nantong University, Nantong, China,
2
School of Pharmacy, Nantong University, Nantong, China,
3
School of Public Health, Nantong University, Nantong, China,
4
Affiliated Hospital of Nantong University, Nantong University, Nantong, China
Silk, as a kind of natural fibrin, has been prepared into various biomaterials due to its
excellent biocompatibility and mechanicalness. However, there are some
controversies on the biocompatibility of silk fibroin (SF), especially when it coexists
with sericin. In this study, two kinds of silk from Jiangsu and Zhejiang were degummed
with two concentrations of Na
2
CO
3
solution, respectively, to obtain four kinds of silk
fibroin. The effects of different degumming treatments on silk fibroin properties were
analyzed by means of color reaction, apparent viscosity measurement, and
transmission electron microscope and isobaric tags for relative and absolute
quantification analyses, and the effects of different silk fibroin membranes on the
growth of Schwann cells were evaluated. The results showed that the natural silk from
Zhejiang treated with 0.05% Na
2
CO
3
solution had a fuller structure, higher apparent
viscosity, and better protein composition. While SF obtained by degumming with 0.5%
Na
2
CO
3
solution was more beneficial to cell adhesion and proliferation due to the
thorough removal of sericin. This study may provide important theoretical and
experimental bases for the selection of biomaterials for fabricating artificial nerve grafts.
Keywords: silk, silk fibroin, silk sericin, degum, proteomic
INTRODUCTION
Peripheral nerve repair has been one of the difficult problems in the field of neuroscience, especially
the repair of long-distance serious injury. With the development of tissue engineering, artificial nerve
grafts are becoming more and more popular. It is expected to replace autologous nerve
transplantation (the “gold standard”at present) to repair long-distance injury because it can
make up for the shortage of autologous nerve transplantation, such as limited source, tissue size
and structure mismatch, long-term denervation, and secondary injury of the donor site (Martins
et al., 2013;Ring, 2013;Castillo-Galvan et al., 2014).
In the past few decades, a large number of natural or synthetic materials, such as chitosan (Wang
et al., 2005;Demina et al., 2017;Yu et al., 2017), silk fibroin (Baecker et al., 2017;Luo and Shao, 2017),
collagen (Sayanagi et al., 2020), and poly (lactic-co-glycolic acid) (Wang et al., 2014), have been used
to prepare artificial nerve grafts.
Edited by:
Diego Mantovani,
Laval University, Canada
Reviewed by:
Kunyu Zhang,
Johns Hopkins University,
United States
Renchuan You,
Wuhan Textile University, China
Mingzhong Li,
Soochow University, China
*Correspondence:
Yumin Yang
yangym@ntu.edu.cn
Xiaoming Yang
sammy@ntu.edu.cn
Yahong Zhao
Zhaoyh108@ntu.edu.cn
Specialty section:
This article was submitted to
Biomaterials,
a section of the journal
Frontiers in Bioengineering and
Biotechnology
Received: 15 September 2021
Accepted: 29 December 2021
Published: 07 February 2022
Citation:
Wang Y, Liang Y, Huang J, Gao Y,
Xu Z, Ni X, Yang Y, Yang X and Zhao Y
(2022) Proteomic Analysis of Silk
Fibroin Reveals Diverse Biological
Function of Different Degumming
Processing From Different Origin.
Front. Bioeng. Biotechnol. 9:777320.
doi: 10.3389/fbioe.2021.777320
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org February 2022 | Volume 9 | Article 7773201
ORIGINAL RESEARCH
published: 07 February 2022
doi: 10.3389/fbioe.2021.777320
Among numerous biomaterials, silk, one of the earliest animal
proteins, was first used in the textile industry and as surgical
suture. Silk is a kind of natural high-molecular-protein polymer,
which is synthesized in the special glands of the epithelial cells of
silkworms, secreted into the cavity, and finally spun into fibers. It
mainly contains silk fibroin (SF) and sericin. SF is in the middle of
the silk, surrounded by sericin (Minoura et al., 1995). The content
of SF accounts for most of the silk, about 70–80%, and sericin
accounts for about 20–30%.
In vivo and in vitro studies have shown that SF without sericin
will not cause obvious inflammation (Wang et al., 2008;Yang
et al., 2007a). Therefore, SF-based biomaterials have been widely
used in bone, tendon, and nerve repair and other tissue
engineering fields due to their favorable biocompatibility,
robust mechanical properties, physicochemical properties, and
biological activities (Gotoh et al., 2004;Dal Pra et al., 2005;
Bhardwaj and Kundu, 2012;Guptaa et al., 2013). In terms of
sericin, it was not well investigated during the past decades and
was simply discarded as waste in traditional silk reeling industry
(Lamboni et al., 2015). Fortunately, in recent years, people have
gradually realized that sericin is a kind of polymer material with
specific biological properties (Yun et al., 2016). It facilitates cell
adhesion and can inhibit cell apoptosis, can promote cell
differentiation (Song et al., 2016), and has been used in the
fabrication of a variety of biomaterials, such as thin films
(Zhang et al., 2015), hydrogels (Siritientong et al., 2011;Tao
et al., 2019), and scaffolds (Dash et al., 2009).
Early studies have reported the biocompatibility of silk. It is
generally believed that sericin has certain immunogenicity
(Dewair et al., 1985;Wen et al., 1990), which is not conducive
to its application as a biomaterial in vivo. In recent years, with the
progress of science and technology and further research on
sericin, researchers believe that sericin itself will not cause a
strong immune rejection (Jiao et al., 2017). When sericin was co-
cultured with mouse macrophages (RAW264.7), the mRNA
expression levels of IL-1 βand TNF-αwere lower than those
in negative control group, which proved that sericin did not cause
an immune response (Panilaitis et al., 2003). Some people think
that only the co-existence of sericin and silk fibroin can produce
immunogenicity, but the two alone will not (Yang et al., 2007b;
Teuschl et al., 2014). Therefore, different silk and degumming
degrees will have different effects on the properties and
biocompatibility of silk fibroin.
In this paper, silk from Jiangsu and Zhejiang were degummed
with 0.05% Na
2
CO
3
and 0.5% Na
2
CO
3
, respectively, to obtain four
kinds of SF, and the antherea silk was taken as the control group. The
composition and structure differences of each sample and its
influence on cell growth were analyzed. The study is anticipated
to provide important theoretical and experimental bases for the
selection of biomaterials for artificial nerve graft preparation.
MATERIALS AND METHODS
Materials
Bombyx mori silk was purchased from Jiangsu and Zhejiang
(China). Dulbecco’s modified Eagle’s medium and fetal bovine
serum were obtained from Gibco (United States). Forskolin,
heregulin, cytosine arabinoside, and 488-labeled goat anti-
mouse IgG were obtained from Sigma-Aldrich (United States).
Rabbit anti-S100 beta monoclonal antibody was obtained from
Abcam. CCK-8 kit was purchased from Ribobio (Guangzhou,
China).
Degumming of Silkworm Raw Silk
Fresh silkworm raw silk from different sources (Jiangsu and
Zhejiang) was weighed and put into a sodium carbonate
solution with different concentrations (0.05 and 0.5 wt%) in
proportion. The silk was boiled three times in Na
2
CO
3
solution (half an hour each time). The degummed silk was
thoroughly rinsed with Millipore water and air-dried on a
super clean platform to obtain refined silk, that is, silk fibroin
sample for standby. There are 4 samples in total: the raw silk from
Jiangsu degummed with 0.05 and 0.5% Na
2
CO
3
are sample 1 and
sample 2, respectively, while the raw silk from Zhejiang
degummed with 0.05 and 0.5% Na
2
CO
3
are sample 3 and
sample 4, respectively. The tussah silk degummed with 0.5%
Na
2
CO
3
is labeled as sample 5.
Dissolution of Silk Fibroin
The refined silk of silkworm was dissolved in a ternary solvent
system of CaCl
2
/H
2
O/C
2
H
5
OH (mole ratio, 1:8:2) at 75 ± 2°C and
then dialyzed against Millipore water in a cellulose tube
(molecular cutoff = 12,000–14,000) at room temperature for
3 days.
Degumming Degree Test
Picric acid–carmine staining was used to evaluate the degumming
degree of silk degummed with two kinds of Na
2
CO
3
solution
(Zhang et al., 2014). First, in preparing the staining solution,
carmine was dissolved in 25% ammonia, followed by adding
saturated picric acid aqueous solution and adjusting the pH to
8.0–9.0. Then, the refined bombyx silk samples were immersed in
the staining solution in test tubes, and the tubes were heated in
boiling water bath for 5 min. Lastly, the samples were thoroughly
rinsed with ddH
2
O and air-dried. Raw silk was taken as the
control.
Determination of Apparent Viscosity
The refined samples were dissolved in a tertiary solvent system of
CaCl
2
/H
2
O/C
2
H
5
OH (mole ratio, 1:8:2) and then kept in water
bath at 20°C for 2 h. Apparent viscosity was then measured with
NDJ-7 rotary viscometer.
Transmission Electron Microscopy
Observation
Samples under different degumming treatments were fixed in
pre-cooled 2.5% glutaraldehyde, post-fixed in 1% osmium acid,
dehydrated with gradient ethanol, embedded in EPON 812 epoxy
resin, and cut into slices of 70-nm thickness. The ultra-thin
sections were stained with lead citrate and uranium acetate,
followed by observation under a transmission electron
microscope (JEOL Ltd., Tokyo, Japan). In Photoshop 7.0, the
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Wang et al. Proteomic Analysis of Silk Fibroin
fiber diameter was measured by measuring tools. The diameter of
at least 60 fibers was measured from 10 photos taken in different
fields randomly. The average value and standard deviation of the
fiber diameter were calculated.
Isolation of Schwann Cells
In this study, all experimental procedures involving animals were
conducted as per the institutional animal care guidelines and
approved ethically by the administration committee of
experimental animals, Jiangsu Province, China.
Schwann cells (SCs) were harvested as described previously
(Chen et al., 2017). Briefly, the sciatic nerves and dorsal root
ganglia were isolated from neonatal Sprague–Dawley rats (1 to
2 days) to get primary rat Schwann cells. The obtained tissues
were triturated and enzymatically digested with 0.25% trypsin at
37°C for 30 min. Then, the mixture was centrifuged and re-
suspended in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), followed by
plating on poly-L-lysine pre-coated dishes. After incubation for
24 h, cytosine arabinoside was added to allow cell incubation for
another 24 h to remove fibroblasts. Next, the cells were cultured
in DMEM supplemented with 10% FBS, 2 mM forskolin, and
2 ng/ml heregulin to stimulate cell proliferation. When cells
covered 90% of the dish surface, they were further purified
with anti-Thy1 antibody (1:1,000, AbD Serotec, Raleigh, NC,
United States) and complement (Jackson Immuno, West Grove,
PA). Lastly, the purified SCs were cultured in DMEM with FBS
and growth factor until the cells were sufficient to seed on the SF
membrane.
Schwann Cells Culture
The prepared SF membranes were sterilized with 75% alcohol for
30 min and rinsed extensively with sterilized phosphate-buffered
saline (PBS). Then, all samples were put in 24-well culture plates,
and cell suspension was added in. The seeding cell density was 1 ×
10
5
cells/well. At different times of culture, the morphological
changes of Schwann cells on these different SF membranes were
observed under an inverted light microscope.
CCK-8 Assay
CCK-8 kit was used to evaluate the viability and proliferation of
SCs on different samples after culturing for 1 and 3 days,
respectively. At different times, fresh DMEM medium with
CCK-8 reagent (V
medium
/V
CCK-8
= 10:1) was added to
replace the cell medium to allow cell incubation at 37°C for
4 h. Then, 150 μl of suspension was transferred to a 96-well plate.
The absorbance was measured at 450 nm by an ElX-800 micro-
ELISA reader (Bio-Tek Inc., United States).
Immunostaining of Schwann Cells
The morphology of Schwann cells on different samples was
examined by the immunostaining method. In brief, after
2 days of culture, cells were rinsed with PBS for three times
thoroughly; then, they were fixed in 4% paraformaldehyde at 4°C
for 4 h and stained with S100 (1:400) at 4°C for 24 h, followed by
further reaction with IgG-488 (1:400) at 37°C for 2 h.
Subsequently, the cells were incubated with Hoechst (final
concentration: 5 μg/ml) at room temperature for 15 min.
Finally, cell samples were observed under an immuno-
fluorescence microscope (Leica, Germany).
Calcein AM/PI Test
The viability of Schwann cells was evaluated with Calcein-AM/PI
Double Staining Kit (Invitrogen, L3224). Schwann cells were
seeded on four different silk fibroin membranes and control
plates at a density of 1 × 10
5
cells/well. After the SCs were
cultured for 1 and 3 days, DMEM with 10% FBS was discarded,
and the samples were rinsed with PBS. Then, calcein-AM and
propidium iodide (PI) were added, and the cells were incubated at
37°C for 30 min. Images were captured by an immuno-
fluorescence microscope.
Proteomic Quantitative Analysis of Different
Silk Fibroin Samples
To find out the different proteins between samples and select the
appropriate silk materials, we used isobaric tags for relative and
absolute quantification (iTRAQ) to analyze proteins in different
samples quantitatively (Zou et al., 2019). The phenol extraction
method was used for protein extraction, which can effectively
remove the small molecular interference in the sample. At the
same time, filter-aided proteome preparation enzymolysis
strategy was used for protein digestion. Then, the peptides of
each sample were labeled with iTRAQ, and the labeled samples
were graded by high-pH reversed-phase classification strategy.
Finally, data collection of samples obtained by classification was
carried out by the ultra-high-resolution mass spectrometer
Q-Exactive.
Statistical Analysis
The statistical significance was analyzed by GraphPad Prism 7.0
(GraphPad Software, Inc.). One-way ANOVA followed by
Tukey’spost-hoc test was used to compare individuals among
different groups of the same time. Two-way analysis of variance
(two-way ANOVA), followed by Sidak’s multiple-comparisons
test, was employed when comparing −ES and +ES groups of all
groups. Data were presented as mean ± SD. p<0.05 was
considered statistically significant.
RESULTS
Effect of Degumming Method on
Degumming Degree
Two kinds of Na
2
CO
3
solution (0.05 and 0.5 wt%) were chosen to
degum silk from different sources. All silk from different sources
in the two solutions were boiled for 0.5 h, and the test was
repeated three times. The degumming degree of two kinds of
Na
2
CO
3
solutions on the raw silk from different sources was
determined by the picric acid–carmine staining method. SF and
sericin have different absorbance capacity on picric acid and
carmine. Silk fibroin turns yellow in alkaline solution due to its
selective adsorption of picric acid. However, sericin has a strong
capacity to absorb both picric acid and carmine; red covers
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Wang et al. Proteomic Analysis of Silk Fibroin
yellow, so it appears red. Therefore, after dyeing and washing, the
yellow surface of raw silk indicates that sericin has been
completely removed; otherwise, it indicates that sericin has not
been completely removed. It can be seen from Figure 1 that, after
degumming with the Na
2
CO
3
solution, samples 1–4 are yellow
(Figures 1A,B,D,E), indicating that sericin has been removed,
FIGURE 1 | Optical images of dyed silkworm silk from different sources, degummed by Na
2
CO
3
with different concentration (A–F) and epivisal viscosity (G),*p<
0.05. Silk from Jiangsu degummed by 0.05% Na
2
CO
3
(A) and 0.5% Na
2
CO
3
(B); no degumming treatment (C). Silk from Zhejiang degummed by 0.05% Na
2
CO
3
(D)
and 0.5% Na
2
CO
3
(E); no degumming treatment (F).
FIGURE 2 | Transmission electron microscopy of silkworm silk from different sources, degummed by Na
2
CO
3
with different concentration. Silk from Jiangsu
degummed by 0.05% Na
2
CO
3
(A) and 0.5% Na
2
CO
3
(B); no degumming treatment (C). Silk from Zhejiang degummed by 0.05% Na
2
CO
3
(D) and 0.5% Na
2
CO
3
(E);no
degumming treatment (F). The parts circled in yellow in samples (C) and (F) are marked as the sericin layer. (G) Statistical graph of the fiberdiameterof(A–F).(A–F):bar=5μm.
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Wang et al. Proteomic Analysis of Silk Fibroin
while the control group without degumming can be clearly
observed to be red (Figures 1C,F). With the increase of
Na
2
CO
3
solution concentration, the samples appear pure
yellow (Figures 1B,E), showing that the degumming degree is
higher.
Effect of Silkworm Raw Materials on
Apparent Viscosity
The SF obtained by different degumming methods was dissolved in
ternary solution in the same proportion, and its apparent viscosity
was measured (Figure 1G). The viscosity of Zhejiang silk decreased
from 630 to 78 CP, while that of Jiangsu silk changed from 450 to 75
CP as the concentration of Na
2
CO
3
changed from 0.05 to 0.5%,
indicating that the high concentration of Na
2
CO
3
can catalyze the
degradationorhydrolysisofSF.Underthecatalysisofthehigh
concentration of Na
2
CO
3
, the molecular chain of SF becomes shorter,
the number of entangled nodes in the solution decreases, and the
friction resistance between molecules decreases, which leads to the
decrease of viscosity. Therefore, according to the viscosity change, the
concentration of Na
2
CO
3
should be reduced as much as possible to
reducethedamagetotheviscosityofSF.Atthesametime,wealso
FIGURE 3 | Morphology observation of Schwann cells on different samples for 24 and 48 h. Brightfield images (two columns to the left) and immunofluorescence
staining (column to the right, 48 h). (A) control, (B) JS-0.05, (C) JS-0.5, (D) ZJ-0.05, (E) ZJ-0.5. Scale bar, 50 μm.
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Wang et al. Proteomic Analysis of Silk Fibroin
found that the viscosity of the SF solution of Zhejiang silkworm was
higher than that of Jiangsu silkworm under two kinds of Na
2
CO
3
concentration.
The Effect of Degumming Method on the
Structure of Silk Fibroin
Silk from different sources was degummed with different
concentrations of Na
2
CO
3
solution. After degumming with
0.05% Na
2
CO
3
, the cross-section of the SF sample
demonstrates a homogeneous oval with complete structure,
smooth edge, and no attachment on the surface, indicating
that 0.05% Na
2
CO
3
can not only remove sericin on the
surface but also maintain the structure of SF. While treated
with 0.5% Na
2
CO
3
, the section of degummed SF presents oval
of different sizes and a long fusiform shape. It appears wrinkled
with incomplete edge, and the diameter gets smaller,
demonstrating that the high concentration of Na
2
CO
3
makes
sericin completely removed, but it also destroys the structure of
SF to some extent. Comparing the effect of source on the structure
of SF, despite the concentration of the Na
2
CO
3
solution, the
cross-section diameter of SF obtained from Zhejiang silkworms
was larger than that from Jiangsu silkworms, indicating that the
structure of Zhejiang silkworms was relatively full (Figure 2).
Morphology of Schwann Cells
Schwann cells play an important role in the formation of myelin
sheath during peripheral nerve regeneration. Therefore, in this
study, the adhesion, survival, and growth of Schwann cells on
different silk fibroin membranes were studied to choose a suitable
FIGURE 4 | Viability of Schwann cells on different samples for 24 and 48 h. (a) Images of calcein-AM/propidium iodide (PI) double-stainin g: (A) control, (B) JS-0.05,
(C) JS-0.5, (D) ZJ-0.05, and (E) ZJ-0.5. Scale bar, 50 μm. Green fluorescence indicates live cells stained with calcein-AM, and red fluorescence indicates dead cells
stained with PI. Scale bar, 200 μm. (b) CCK-8 test of Schwann cells.
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Wang et al. Proteomic Analysis of Silk Fibroin
SF to prepare peripheral nerve grafts which can better promote
peripheral nerve regeneration.
Figure 3 shows the morphology observation of SCs on different
samples for 24 and 48 h, respectively. After 1 day of culture, there
were certain amounts of cell on all samples, but the number of cells
on JS-0.5 and ZJ-0.5 was more than that on JS-0.05 and ZJ-0.05,
and some cells on JS-0.05 and ZJ-0.05 shrank into a circle,
indicating that cells could grow on all samples, but samples JS-
0.5 and ZJ-0.5 were more conducive to cell adhesion. It may be that
a small amount of residual sericin in JS-0.05 and ZJ-0.05 affected
the interaction between cells and samples, resulting in inferior
number and the morphology of cells on JS-0.05 and ZJ-0.05. Two
days later, the cell density on sample JS-0.5 and ZJ-0.5 increased,
indicating good cell growth and proliferation, and the number of
cells on samples JS-0.05 and ZJ-0.05 increased, too, but not as
much as that of JS-0.5 and ZJ-0.5, indicating that JS-0.05 and ZJ-
0.05 can also support cell growth and promote cell proliferation,
but the cell growth was slower than that of JS-0.5 and ZJ-0.5. The
results of immunofluorescence staining tells us that, on samples JS-
0.5 and ZJ-0.5, SC cells exhibited better adhesion and spreading
and displayed a spindle-like shape with filopodia at both ends,
while on sample JS-0.05 and ZJ-0.05 the cell bodies were spindle or
round, which is consistent with the brightfield images. Moreover,
the morphology and quantity of the cells on ZJ-SF were better than
those on JS-SF.
Survival and Proliferation of Schwann Cells
on Different Silk Fibroin Membranes
The viability and proliferation of Schwann cells on all samples
were evaluated by calcein-AM/PI double staining and CCK-8 test,
respectively. The results are shown in Figure 4. It was found that
Schwann cells can grow on all samples, and almost all cells are
green (Figure 4A), indicating that all samples can support cell
survival and growth. Both staining test and CCK-8 test
(Figure 4B) showed that the number of cells increased over
time, which further proved that all the samples exhibited no
cytotoxicity and supported cell proliferation. However, the
number of cells on the sample made of silk fibroin degummed
with 0.05% Na
2
CO
3
was relatively larger, as the thoroughly
degummed silk fibroin membrane was more conducive to cell
adhesion. In addition, under the same degumming conditions,
compared with JS-SF, the number of cells on the ZJ-SF samples is
relatively more in 1 or 3 days, and the morphology of the cells was
better.
Proteome-Wide Identification and
Classification of Different Samples
In order to find out the reasons for the differences of cell adhesion and
growth on different products, we analyzed the protein of samples and
found that there were 55 kinds of proteins which were classified
according to their function. Figure 5 shows the correlation analysis of
each sample. It can be seen that the correlation between sample JS-
0.05 and sample ZJ-0.05 is very high, as well as that between sample
JS-0.5 and ZJ-0.5, indicating that the silk fibroin composition of
different sources treated with the same degumming method is similar.
The sample Antheraea pernyi silk is different from the other samples
andisclosesttosampleZJ-0.5,whichtellsusthattheprotein
composition of Zhejiang silk treated with 0.5% is the most similar
to that of A. pernyi silk.
Both A. pernyi silk fibroin and mulberry silk fibroin are fibrous
proteins, and the category of amino acids is almost identical.
However, there are obvious differences in quantity (Kim et al.,
2012). The amino acid residues in the silk fibroin of A. pernyi are
large, and the side chain contains rich active groups, including
aspartic acid (ASP) and arginine (Arg), which can form a special
RGD tripeptide sequence with glycine. Thus, A. pernyi silk is
FIGURE 5 | Correlation heat map of different samples. S1: JS-0.05, S2:
JS-0.5, S3: ZJ-0.05, S4: ZJ-0.5, S5: Antheraea pernyi silk.
FIGURE 6 | Classification of proteins in silk fibroin.
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Wang et al. Proteomic Analysis of Silk Fibroin
favorable to cell adhesion (Kopp et al., 2020), and its elongation
and elasticity are higher than that of silkworm silk. Therefore, in
our quantitative analysis, tussah silk was selected as the control.
Through iTRAQ analysis, the silk with similar properties as
tussah silk was determined so as to find the suitable silk
source and degumming treatment method. According to the
above-mentioned results, sample ZJ-0.5 is similar to sample A.
pernyi silk, that is, the silk sample from Zhejiang Province being
degummed in 0.5% Na
2
CO
3
. Previous cell experiments show that
sample ZJ-0.5 is the best for cell adhesion, viability, or
proliferation, which is well explained by the sample protein
clustering here.
The 55 proteins identified in the samples could be classified
into several categories based on their annotated molecular
function: extracellular (16), binding (13), antimicrobial (1),
fibroin (8), cell adhesion (2), integral of membrane (11),
sericin (1), cyclosketen (2), and so on (Figure 6). It can be
found that, except silk fibroin and sericin, other proteins are
closely related to cell growth, which is one of the reasons that silk
fibroin has good biological activity and is widely used in the
biomedical field.
Comparison of the Relative Abundance of
Proteins in the Samples
To compare protein abundances across different samples, we
used scale function to realize data standardization. Figure 7
shows that the content of silk fibroin in samples JS-0.5 and ZJ-
0.5 is higher, while the sericin content is lower, which is
consistent with the degumming treatment method. Samples
JS-0.5 and ZJ-0.5 were treated with 0.5% Na
2
CO
3
,andsericin
is removed more thoroughly. Cell adhesion and DNA
polymerase are also higher in samples JS-0.5 and ZJ-0.5,
and there is more toxin-activity protein in samples 1 and 3,
explaining why the cells on samples JS-0.5 and ZJ-0.5 survived
more and grew better. However, the contents of cytoskeleton,
binding, and extracellular proteins in samples JS-0.05 and ZJ-
0.05 are higher than those in samples JS-0.5 and ZJ-0.5,
indicating that a high concentration of Na
2
CO
3
can
effectively remove sericin but also make silk fibroin lose
some useful proteins. Therefore, the content of each
protein in every sample is different. They play various roles
in the process of cell adhesion and proliferation. We need to
consider their effects on cell growth comprehensively,
retaining as much natural matrix components as possible
on the premise of thoroughly removing sericin.
DISCUSSION
A high concentration of Na
2
CO
3
solution can remove sericin
more thoroughly, but it also destroys the structure of SF to a
certain extent, resulting in the incomplete edge of SF and the
decrease in cross-sectional diameter. At the same time, SF is
degraded to a great extent and more peptide bonds, hydrogen
bonds, and other secondary bonds are destroyed, forming shorter
protein molecular chains and making the structure of SF loose
and extended. Therefore, the viscosity of SF degummed with a
high concentration of sodium carbonate is smaller.
Since the residual sericin around the core SF is considered to
be the source of silk-related undesirable immune reactions, it is
important to develop silk purification procedures by removing
FIGURE 7 | Ratio of representative proteins in different samples. R1: JS-0.05, R2: JS-0.5, R3: ZJ-0.05, R4: ZJ-0.5, R5: Antheraea pernyi silk.
Frontiers in Bioengineering and Biotechnology | www.frontiersin.org February 2022 | Volume 9 | Article 7773208
Wang et al. Proteomic Analysis of Silk Fibroin
sericin thoroughly and retaining SF. In this study, Na
2
CO
3
solution with two different concentrations was chosen to
remove sericin.
As a kind of glial cells, Schwann cells can not only guide the
growth of regenerated axons to establish a precise innervation
(Xu et al., 2013) but also secrete various neurotrophic factors, cell
adhesion molecules, that are conducive to nerve regeneration
(Guenard et al., 1992). Therefore, it is significant to figure out the
growth of cells on silk fibroin scaffold. For this reason, we chose
two different sources of silk and used different degumming
methods to obtain different SF, and then SCs were co-cultured
with membranes prepared with these SF.
On the SF-0.5% (SF treated with 0.5% Na
2
CO
3
) membrane,
the cell survival rate was high and the cells took the standard
spindle shape, showing a good growth situation. This is because
SF obtained by degumming with 0.5% Na
2
CO
3
was purer, and
there was no inflammatory reaction caused by residual sericin.
Therefore, SF-0.5 promotes cell adhesion and supports cell
proliferation.
In order to further explore the effect of different SF samples on
cell survival and growth, we used iTRAQ technology to analyze
the protein composition of different samples and took A. pernyi
silk fibroin as a reference standard. A. pernyi silk contains a
special RGD tripeptide sequence which is beneficial to cell
adhesion and gives A. pernyi silk better elongation and
elasticity. The iTRAQ results showed that the composition of
ZJ-0.5 is the closest to that of A. pernyi silk. Therefore, ZJ-0.5
should be the best choice to support cell adhesion, proliferation,
and growth, which is consistent with the results of the cell
experiment.
CONCLUSION
In this paper, silk fibroin from different sources were treated
with different degumming methods, their morphology,
viscosity, and protein composition were analyzed, and silk
fibroin membranes were prepared to co-culture with SC cells.
The results indicated that the natural silk from Zhejiang
Province that was treated with 0.05% Na
2
CO
3
solution had a
fuller structure, higher apparent viscosity, and better protein
composition, while SF obtained by degumming with 0.5%
Na
2
CO
3
solution was more beneficial to cell adhesion and
proliferation due to the thorough removal of sericin.
Moreover, the different growth status of cells on different
samples was explained by proteomic analysis. Overall, this
study may offer an important basis for the construction of
nerve conduit with suitable biomaterials.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be
directed to the corresponding authors.
ETHICS STATEMENT
The animal study was reviewed and approved by the Laboratory
Animal Center of Nantong University.
AUTHOR CONTRIBUTIONS
YW contributed to conceptualization and writing—review. YL
contributed to conceptualization, methodology, and
investigation. YG contributed to analysis and revision. XN
contributed to methodology and investigation. JH contributed
to investigation and writing—review and editing. ZX contributed
to formal analysis and investigation. XY contributed to
investigation. YY contributed to conceptualization and
supervision. YZ contributed to conceptualization, supervision,
and writing—review and editing.
FUNDING
This study was supported by the Priority Academic Program
Development of Jiangsu Higher Education Institution (no.
21KJA430011), the National Key Research and Development
Program of China (no. 2018YFC1105600), the Key Program of
NSFC (no. 31830028) and the Undergraduate Innovation
Training Programs of Nantong University (202010304031Z).
ACKNOWLEDGMENTS
The authors thank Professor Guicai Li for assistance in
manuscript preparation.
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