Wang Et Al-2017-Journal of Biomedical Materials Research Part A

Evaluation of a series of silk fibroin protein-based nonwoven mats for
use as an anti-adhesion patch for wound management in robotic

surgery
Yu-Chi Wang3, Meng-Yi Bai*,, 1,2, Wan-Yuan Hsu1, Mu-Hsien Yu3
1
Graduate Institute of Biomedical Engineering,
National Taiwan University of Science and Technology, Taipei 10607, Taiwan (Republic of
China)
2
Adjunct appointment to the Department of Biomedical Engineering, National Defense
Medical Center, Taipei 114, Taiwan (Republic of China)
3
Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense
Medical Center, Taipei City, Taiwan
[*] Corresponding author: Meng-Yi Bai, Ph. D., TR-917, AAEON Building, no. 43 Keelung Road,
Section 4, Taipei 10607, Taiwan (Republic of China)
Tel; +886-2-2730-3743
Fax: +886-2-2730-3733
E-mail: mybai@mail.ntust.edu.tw
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/jbm.a.36225
This article is protected by copyright. All rights reserved.

Journal of Biomedical Materials Research: Part A Page 2 of 29
Abstract
A novel anti-adhesion nonwoven mat mainly composed of silk fibroin protein (SFP) was
fabricated via the single-spinneret electrospinning technique. A series of SFP-based electrospun
nonwoven mats containing additives of different synthetic polymer ratios, such as pure SFP,
SFP/poly(vinyl alcohol) (PVA), SFP/polyethylene glycol (PEG), and SFP/polyethylene oxide
(PEO) were produced and compared. All membranes were porous and had diameters of 324.02 ±
113.7, 308.86 ± 74.02, 366.22 ± 115.81, and 341.82 ± 119.42 nm, respectively. The average pore
size for each membrane was 1.132 ± 0.99, 0.811 ± 0.424, 0.975 ± 0.741, and 0.784 ± 0.497 µm2.
No nonwoven mats showed significant cytotoxicity toward fibroblast cells based on the results of
MTT assays. Surprisingly, for all groups of SFP-based nonwoven mats, nitrate formation was
reduced by up to 94.55 ± 14.50%, 92.16 ± 19.38%, 91.28 ± 28.375%, and 92.00 ± 12.64% in
lipopolysaccharide-induced RAW 264.7 macrophages model. Tissue anti-adhesion potential was
evaluated in an in vitro fibroblast cell adhesion model and in vivo wounded mice model. In vitro,
the mean cell anti-adhesion percentage of fibroblast cells changed over time in the following order:
PVA/SFP > SFP > PEG/SFP~PEO/SFP. In vivo, SFP and PVA/SFP-treated groups both showed
superior collagen regeneration and wound closure.
Keywords: nanotechnology; electrospinning; biomaterials; microfibers; nonwoven mat
2
John Wiley & Sons, Inc.
Page 3 of 29 Journal of Biomedical Materials Research: Part A
Introduction
Anti-adhesion to internal tissue is a major concern in the development of medical materials/or
device, such as wound dressing,1 peritoneal surgery barriers2, and artificial heart valves.3
Unwanted adhesion may have severe consequences, such as secondary damage when removing
the dressing, peritoneal adhesion, and undesired adhesion of clots on heart valves. Anti-adhesion
need is very important in wound management because wounds are among the most frequently
treated medical problems.4 Therefore, the design and preparation of anti-adhesion materials has
become increasingly important in the wound and tissue management fields.5
Numerous anti-adhesion products have been introduced into the commercial market involving
pharmacological inhibition6 and barrier prevention.7,8 However, none are composed of natural
materials except hyaluronic acid. Naturally derived materials have chemical compositions similar
to those of the tissues they are replacing or contacting. Therefore, they may be more biocompatible
with the surrounding tissue over time. For example, in the human history, silk and linen were the
most frequently used natural materials in east and middle Asia because of their abundant local
production. Because of its excellent biocompatibility, smoothness, hydrophilicity, and mechanical
strength, silk continues to be widely used. Because silk easily forms β-sheet secondary structures,
giving it a smooth surface, silk is a good material candidate as an anti-adhesion material. However,
previous anti-adhesion material studies mainly focused on synthetic polymers, such as
poly(L-lactic acid),9 polyethylene glycol (PEG),10 poly(D,L-lactic-co-glycolic acid),11
poly(ethylene oxide) (PEO),12 poly(vinyl alcohol) (PVA),13,14 and poly(lactide)-b-poly(ethylene
glycol);15 Recently, some studies have evaluated natural polymers, such as chitosan and sodium
alginate16 and hyaluronic acid17 Peritoneal adhesions are the most common complication after
abdominal surgery, often leading to chronic pelvic pain, infertility, and even bowel obstruction.
3
The best methods for preventing adhesion involve minimizing abdominal trauma, maintaining
hemostasis, avoiding infection, and applying tension sutures. Anti-adhesion patches reduce
adhesions after surgery, but silk fibroin protein is not commonly used as an anti-adhesion material.
Zhu et al. described the use of silkworm pupa in a rat cecal abrasion model.18 Vepari et al. used silk
fibroin protein film but PEGylated the surface of the film to improve its anti-adhesion properties.19
These studies revealed that raw silk fibroin protein may not a good anti-adhesion candidate except
when co-processed with other materials. However, they all used the same film type and material
processing procedures. This continuous film provides full surface contact with biological tissues,
enabling high adhesion to biological tissue. Additionally, continuous films retard water vapor
permeability and air transport. Based on these previous studies, we hypothesized that a porous
membrane composed of tiny silk fibroin protein fibers with excellent anti-adhesion ability
originating from its relatively low contact area and smooth fiber surface as a result of the formation
of β-sheet secondary structures could be a better design. The electrospinning (ES) technique can
be used to process the solution of silk fibroin protein containing other material mixing and produce
the porous membrane.
As serum protein is one of the important implications for evaluating the dressing adhesion to
wounds. Normally, reduced binding of serum proteins to the surface of the fiber dressing may help
reduce the adherence characteristics for this type of dressing, minimizing the second damage
during dressing changes.20 Based on the survey of many publications,21 we found that Antonella
Motta and co-workers have reported that both hydrophobicity and surface weaving structure could
influence the adsorption of key immunoproteins in serum to the silk fibroin protein. Basically, a
fabric and beta-sheet crystalline surface topography and protein structure is less favorable for the
adhesion of serum protein. In conjunction with our previous work that the electrospun SFP
4
nonwoven mats still maintain 76-77% of high beta-sheet crystalline structure,22 we believe that an
electrospun nonwoven mat composed of SFP-based fiber may have potential to strike a balance
between anti-adhesion and permeability.
Therefore, in this study, a locally sourced natural material, i.e., silk fibroin protein was
processed using the electrospinning technique to produce a porous nonwoven mat; its potential
ability in wound anti-adhesion was also evaluated. To determine the advantages of using silk
fibroin protein (SFP) and a series of water-soluble synthetic polymers commonly used as
anti-adhesion materials, such as PVA, PEG, and PEO, different combinations of starting materials
were co-electrospun. The use of natural-derived polymers or synthetic polymers has both
advantages and disadvantages; for instance, naturally- derived polymers have chemical
compositions similar to the biological tissues they are contacting and thus may be highly
biocompatible. However, the feasibility of obtaining large amounts of these materials for clinical
applications, their relatively low mechanical properties, and difficulty in ensuring pathogen
removal are major concern. Synthetic polymers can be easily mass-produced and sterilized under
harsh conditions. Additionally, their physical, chemical, and mechanical properties and molecular
weight can be tailored for specific applications.
The aim of this study was to find the optimum combination of the naturally derived polymer
SFP and synthetic polymer for fabrication of an anti-adhesion nonwoven mat. Criteria for mixing
these materials need to take into account the material availability, material cost, chemical and
physical properties of the nonwoven mat. Using a water-soluble polymer, we produced SFP-based
nonwoven mats with therapeutic potential. In contrast to previously described anti-adhesion
barriers, this anti-adhesion nonwoven mat exhibits both anti-adhesion and therapeutic potential
simultaneously.
5
Materials and Methods
Materials
Silkworm cocoon (Shitan Chuanmin Silkworm Farm, Miaoli, Taiwan) was used as the raw
material for harvesting SFP fibers and sericin. Lithium bromide (LiBr, ≥99%, Sigma- Aldrich, St.
Louis, MO, USA) and formic acid (FA, CH2O2, ≥98%, Sigma-Aldrich, St. Louis, MO, USA)
were used as received without purification to dissolve SFP. PVA (Mw 13,000–23,000, 98%,
Sigma-Aldrich, St. Louis, MO, USA), PEG (Mn 4,600, Sigma- Aldrich, St. Louis, MO, USA),
and PEO (Mv ~900,000, Sigma-Aldrich, St. Louis, MO, USA) were utilized to be the solubility
enhancer as needed. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (lyophilized
powder, Sigma-Aldrich, St. Louis, MO, USA) was used as purchased for anti-inflammatory tests.
A 1X phosphate-buffered saline (PBS) solution was prepared by diluting 1 mL of 10X PBS
(UniRegion Bio-Tech, Miaoli, Taiwan) with 9 mL of deionized water and stirring for 10–15 min.
Methods
Preparation of a series of SFP/water-soluble polymer electrospun nonwoven mats using the
ES technique
First, a 12 wt% SFP formic acid stock solution was prepared. Next, 8 wt%, 5 wt%, and 0.1
wt% of PVA, PEG, and PEO FA solutions were prepared. Five hundred microliters of 12 wt% SFP
formic acid stock solution was mixed with 500 µL of 8 wt%, 5 wt%, or 0.1 wt% of PVA, PEG, or
PEO FA solution to generate 1 mL of stock solution for ES, respectively. After pouring 1.0 mL of
this solution into a 3-mL syringe, the stock solution of SFP-based FA solution was fed from a
syringe pump (New Era Pump Systems, Inc., Syringe Pump NE-300, St. Farmingdale, NY, USA)
at a flow rate of 4–7 µL/min. In this experiment, a 20-gauge injection needle was used as a
spinneret head. An electrical field was established between the spinneret head and collection
6
substrate (with a positive voltage applied on the spinneret head assembly by a DC high-voltage
power supply) and electrically grounded on the collection substrate. The distance between the
spinneret head and collection substrate was 6–10 cm and the applied voltage range was 8–12 kV.
A U-shaped metallic frame was used as the collection substrate (see Figure S1 in supporting
information for detailed specification). After 10–15 min of the ES process, a piece of dried
SFP-based nonwoven mat was stretched across the gap of the metallic substrate. Depending on the
water-soluble polymer added to the stock solution, the chemical compositions of nonwoven mats
were fine-tuned to produce the pure SFP, PVA/SFP, PEG/SFP, and PEO/SFP nonwoven mats for
the following in vitro and in vivo tests. The fiber diameter and pore size were directly estimated
from the SEM image using NIH ImageJ software.
Robotic system
The Da Vinci SI model (Intuitive Inc, Sunnyvale, CA, USA) composed of a column was used
in this work for demonstration of produced nonwoven mats and control sample, which holds the
robotic arms and surgeon’s console. The column had four arms that hold the instruments are move
to the surgical field. The surgeon sits at the console that away from column had a stereoscopic
image. The surgeon controls the robotic arms using tow hand and foot. The surgeon’s hand can
control the instrument include bipolar forceps and monopolar cautery spatula.
Statistical analysis
All data were expressed as the means ± standard deviation. Student’s t test was used to
compare results between experimental groups and control group. P < 0.05 between the
experimental groups and control group was considered statistically significant. SigmaPlot v10.0
(Systat Software, Inc., San Jose, CA, USA) was used for statistical analysis.
7
Results and discussion
Surface morphology and porosity of produced SFP, PVA/SFP, PEG/SFP, and PEO/SFP
nonwoven mats
The single-nozzle ES technique was successfully used to fabricate a series of SFP-based
nonwoven mats composed of submicron-fibers with varying compositions. Figure 1 shows the
SEM images of SFP (fiber diameter: 340 ± 113.7 nm), PVA/SFP (fiber diameter: 308.9 ± 74 nm),
PEG/SFP (fiber diameter: 366.2 ± 115.8 nm), and PEO/SFP (fiber diameter: 341.8 ± 119.4 nm)
nonwoven mats. The silk fibroin protein used in this study had a high molecular weight of
approximately ＞245 kDa, limiting its potential application in drug delivery when using this
nonwoven mat as an anti-adhesion patch.23 However, in some cases, these two functions are
required simultaneously, such as in wound management. Thus, a series of water-soluble polymers
was screened and used as a carrier excipient for co-spinning with SFP. This combination of
synthetic and natural-derived polymers achieved both aqueous solubility and biocompatibility. In
addition, utilization of the ES technique aided in forming these polymers into tiny fibers, greatly
enhancing the permeability and porosity. Additionally, the electrospun nonwoven mat possessed
pores much smaller than those in conventional cotton gauze. For instance, the estimated pore sizes
shown in Figure 1a, 1c, 1e, and 1g are 1.13 ± 0.99, 0.81 ± 0.42, 0.98 ± 0.74, and 0.78 ± 0.50 µm2,
respectively, revealing a remarkable reduction in pore size after incorporation of water-soluble
polymer into the electrospun nonwoven mats. Notably, the pore size was much smaller than most
microorganisms, which are typically several micrometers. Thus, use of this nonwoven mat for
interior or exterior wound management can greatly limit pathogen invasion.
Characterization of the produced SFP, PVA/SFP, PEG/SFP, and PEO/SFP nonwoven mats
by FTIR
8
For all biomaterials, because the surface is the first point of contact with the biological system,
understanding surface properties and composition is very important. Herein, FT-IR equipped with
an ATR accessory was used to characterize a series of SFP-based nonwoven mats. As shown in
Figure 2b, SFP nonwoven mat showed two prominent peaks at 1650 and 1536 cm-1, which were
attributed to amide I and amide II stretching in the silk fibroin protein chain. In contrast, the amide
I and amide II stretching mode in the PVA/SFP nonwoven mat was shifted to lower frequencies of
1623 and 1514 cm-1 because of the formation of hydrogen bonds between the amide bond
(-CONH-) of the protein and hydroxy bond (-OH) of PVA. In addition, the characteristic peak of
PVA, i.e., -C-O-, was observed at a lower frequency of 1071 cm-1 as compared to the normal PVA
peak (-C-O-, vibration at 1087 cm-1). These results indicate that the surface of the generated
PVA/SFP nonwoven mat contained the PVA blend and that this combination showed potential to
be used as a barrier against foreign invasion and therapeutic agent to promote recovery when
needed.
In vitro cytotoxicity and anti-inflammatory effects of a series of SFP-based nonwoven mats
on 3T3 fibroblast cells and LPS-induced inflammatory RAW 264.7 macrophage cells
The extract method was used to perform the MTT assay to determine the cytotoxicity of a series of
SFP-based nonwoven mats. Figure 3 shows the results of the MTT assay of a series of nonwoven
mats, including SFP, PVA/SFP (5/5), PEG/SFP (5/5), and PEO/SFP (5/5) nonwoven mats. Because
fibroblast cells play a critical role in wound healing, they were used as an in vitro cell model to test
the cytotoxicity of substances leached from the nonwoven mats. As shown in Figure 3, the pure
SFP nonwoven mat showed no cytotoxicity towards fibroblast cells compared to that in the control
group (medium only). In addition, all other water-soluble polymer/SFP nonwoven mats showed
cell viability above 80%, indicating no remarkable cytotoxicity towards fibroblast cells. Student t
9
test revealed no significant difference between the experimental groups and the control group.
Although the produced nonwoven mats were designed for using as anti-adhesion patches on
wound, however, inflammatory conditions are frequently seen and accompanying by the creation
of wound. The silk fibroin peptide derived from silkworm Bombyx mori has been reported to show
an anti-inflammatory effect alone or strengthen the effect of Tat-SOD in a mouse edema model. 21
Therefore, an LPS-induced inflammatory RAW 264.7 macrophage cell model was used to
evaluate the anti-inflammatory effects of the SFP-based nonwoven mats. Figure 4 shows the
results of the Griess test of inflammatory RAW cells after treatment with a series of nonwoven
mats. Figure 4a shows a calibration line established using standard nitrite reagents and was used to
determine the nitrite concentration. Figure 4b shows the results of the cytotoxicity test of using
different concentrations of LPS reagent to determine the optimum dosage of LPS that could
successfully induce cell inflammation without harming the cell. When the concentration of LPS
was falling between 0.5–2.5 µg/mL, no significant cytotoxicity was observed, and cell viability in
all test groups was well-above 80%. To successfully induce cell inflammation and reduce LPS
toxicity, 1.0 µg/mL of LPS reagent was determined to be the optimum dosage for treatment in
LPS-induced inflammatory RAW 264.7 macrophage cells. Figure 4c shows that the nitrite
concentration of LPS-induced RAW 264.7 macrophage cells reached 25 µΜ by using 1.0 µg/mL of
LPS treatment, which was remarkably higher than that in the control group (medium-treated only
group). In contrast, all SFP-based groups showed significantly reduced nitrite formation compared
to that in the control group, indicating that the SFP-based nonwoven mats suppressed
inflammation in macrophage cells. A plausible mechanism for the anti-inflammatory ability of the
SFP-based nonwoven mats is as follows: cells treated with LPS triggered inflammation symptoms,
which commonly generate large amounts of reactive oxygen species. These reactive oxygen
10
species induce the expression of various pro-inflammatory genes and activate numerous signal
transduction pathways, such as tumor necrosis factor-α, interleukin (IL)-1, and IL-6. However,
Kim et al. reported that silk fibroin peptide suppressed the increase in the levels of the
pro-inflammatory cytokines cyclooxygenase-2, IL-6, IL-1β, and tumor necrosis factor-α.24 The
low-molecular weight protein in SFP-based nonwoven mats may have been released from the
matrix, as these small molecules typically have better water-solubility.
In vitro anti-adhesion test of SFP-based nonwoven mats on 3T3 fibroblast cells
The main goal of this study was to develop an anti-adhesion patch for wound management. Thus,
an in vitro anti-adhesion test using skin fibroblast cells was conducted to evaluate the
anti-adhesion performance of the SFP-based nonwoven mats. Figure 5a–e shows the optical
images of the DAPI-stained fibroblast cells attached to the PCL, SFP, PVA/SFP, PEG/SFP, and
PEO/SFP nonwoven mats. PCL nonwoven mats were used as the control material for comparison,
as these mats showed good fibroblast cell adhesion in our previous study.25 Densely attached
fibroblast cells were detected on the PCL nonwoven mats and PEG/SFP and PEO/SFP mats. In
contrast, much fewer and scattered attached fibroblast cells were observed on SFP and PVA/SFP
nonwoven mats (Figure 5b and 5c). In addition, the time-dependent trend in the statistical results
of these in vitro cell anti-adhesion test is shown in Figure 5f. After 3-days of seeding, SFP and
PVA/SFP nonwoven mats maintained the 1st and 2nd lowest cell adhesion percentages compared to
that in the PCL control group and this anti-adhesion effect could last as long as 5 days. In addition,
a decreasing trend in cell adhesion was observed within 3 days of application and persisted for as
long as 5 days, revealing that the SFP and PVA/SFP nonwoven mats were the most suitable
candidates for use as anti-adhesion patches in wound management.
In vitro drug release profile of PVA/SFP nonwoven mats
11
As described above, we expected that with the assistance of a water-soluble polymer, the
produced SFP-based nonwoven mats may have a therapeutic function, as silk fibroin protein is not
water-soluble. Regarding the therapeutic function in water environment, in vitro cytotoxicity and
anti-inflammatory assays of the SFP-based nonwoven mats, the PVA/SFP nonwoven mats showed
the most promising results and struck a balance between these requirements. Therefore, an in vitro
drug release profile test of PVA/SFP nonwoven mats was conducted. Figure 6a shows the UV-vis
absorption spectrum of PVA, revealing two prominent peaks at 283 and 323 nm. Figure 6b shows
the concentration-dependent calibration line based on optical density acquired at 323 nm (most
intense absorption band of PVA). Figure 6c shows the release profile of PVA from PVA/SFP
nonwoven mats, which indicates that nearly 100% of PVA release was achieved at day 5 of the test
and the cumulative release amount of PVA was up to 0.06 µg/mL (approximately 0.06 ppm). This
result was set as the foundation for subsequent in vivo animal studies.
In vivo macroscopic evaluation of wound-healing and anti-adhesion ability of SFP-based
nonwoven mats using trauma mouse model
As wound adhesion is one of the most common side effects causing secondary damage when
removing dressing during wound management, an anti-adhesive patch is needed. Additionally,
anti-adhesion patches are frequently used in abdominal surgery to prevent post-surgery peritoneal
adhesion in clinical practice, such as the hyaluronic acid patch following minimally invasive
surgery such as da Vinci surgery. Therefore, a trauma mouse model was used to evaluate the
putative wound-healing and anti-adhesion ability potential of a series of SFP-based nonwoven
mats (see Figure_S2 for a typical image of wound bound up by dressing in animal study).26 Figure
7 shows time-dependent images of the wound healing course for each group of treated animals. As
12
shown in Figure 7, full skin removal was conducted on the dorsal area of mice to create
approximately 1.2 × 1.2 cm clean wounds on day 0. All wound areas were covered with a layer of
nonwoven mats and changed every 5 days based on the results of in vitro cytotoxicity,
anti-inflammatory, and drug release profile studies. Images recorded on days 0, 5, 10, 15 and 20 of
post-wounding revealed gradual closure of the wound area in all four groups of animals (Figure
7a). Notably, there was no significant difference in wound recovery and skin hair regeneration, as
no therapeutic agent was contained in the nonwoven mats. Figure 7b shows the statistical analysis
results of wound closure measurement for all groups of tested animals. All experimental groups
showed similar natural recovery behavior to the control groups and the wounds in both
experimental groups were fully closed in all animals by 20 days. Adhesion grades assessed using
Nair’s macroscopic adhesion classification were grouped into four categories (0, 1 and 2, 3, and 4)
(see Table S1). In the positive control group (PCL nonwoven mats), 100% of mice developed
gross adhesions, including 50% showing mild to moderate adhesion and 50% showing severe to
very severe adhesion. In the 100% SFP nonwoven mat group, 33% of mice developed grades 1 and
2 adhesions and 16.7% of mice developed grades 3 and 4 adhesions compared to 50% and 50% in
the positive control group, respectively. In contrast, the PVA/SFP (5/5) group showed a large
reduction in the rates of adhesions and up to 66.7% of mice showed no adhesion (see Table S1 and
S2 for all gross adhesion percentages and classification index).
The highest nonoccurrence of gross adhesions was detected in the PVA/SFP (5/5) group
(66.7%), followed by the 100% SFP (50%), negative control (16.7%), and positive control (0)
groups. Additionally, comparison of the gross adhesion percentages in the SFP, PVA/SFP groups
and that of the positive control group revealed p-values of 0.005 and 0.003, respectively,
13
suggesting that there was a significant decrease in gross adhesion formation in SFP-based
nonwoven mats, particularly for the 50% PVA addition. Figure 8 shows the optical micrographs
for pathological evaluation on days 5,10, 15, and 20 in mice during dressing change with
Tegaderm®, PCL nonwoven mats, 100% SFP nonwoven mats, and PVA/SFP nonwoven mats.
The positive and negative control groups showed severe wound adhesion and hair adhesion (red
arrowheads indicate wound adhesion or second damage condition, black arrowheads indicate hair
adhesion condition). Notably, adhesion was prominent and severe at an early stage of wound
healing (within 10 days of treatment) in both control groups, but the same symptoms were
moderate or absent in the two SFP-based nonwoven mat-treated groups.
Masson’s trichrome staining for morphological evaluation of wound recovery
The dermal maturation of the regenerative tissues was also investigated based on histological
sections. All newly regenerative tissues taken from sacrificed mice at the endpoint of the study
(day 20) were evaluated. Figure S3 shows the Masson trichrome staining images for all four
wound mice model groups. Figure S3a–d show the results of the negative and positive control
groups using TegadermR and PCL nonwoven mats, respectively, which reveal fully regenerated
normal skin but with loosely packed collagen fibril bundles in the dermis layer. However, the
tissues taken from the 100% SFP and PVA/SFP nonwoven mat groups revealed not only
three-layered structures but also compact packing of collagen fibril bundles. Statistical results of
all histological biopsies (Table S3, n = 6 for each group) revealed that the 100% SFP and PVA/SFP
nonwoven mat-treated groups showed relatively low porosity in the dermis layer of the
regenerated skin (35.69 ± 3.87 and 37.22 ± 5.34, respectively) compared to those of the positive
and negative control-treated groups (43.70 ± 6.75 and 41.34 ± 5.71, respectively).
14
This result supports those of previous studies showing that silk fibroin protein is suitable for
keratinocyte and fibroblasts cell adhesion and dispersion within the matrix.27 Thus, the observed
more compact packing of collagen fibril bundles in the regenerated dermis layer may be attributed
to the addition of silk fibroin protein in the nonwoven mats which promoted compact packing of
collagen fibril bundles in SFP-based nonwoven mat-treated groups.
In vitro comparison of PVA/SFP nonwoven mats and hyaluronic acid-based antiadhesive
barriers for intraperitoneal anti-adhesion prevention using a da Vinci surgical system
Synthetic mesh is currently used in state of the art incisional abdominal surgery. The meshes
are placed either extraperitoneally or intraperitoneally using an onlay mesh technique with
minimally invasive surgical tools. Although hyaluronic acid-based anti-adhesive barriers have
been approved by the FDA to prevent and reduce adhesion in abdominal cavity surgery, some
limitation remained, preventing the application of these materials in laparoscopic surgery. For
instance, hyaluronic acid-based antiadhesive films are brittle and rolling up after contacting within
the moist body tissue. In this study, we evaluated combinations of naturally-derived polymer SFP
and synthetic polymer to fabricate anti-adhesion nonwoven mats to overcome the limitations of
hyaluronic acid-based antiadhesive film. PVA/SFP nonwoven mats and hyaluronic acid-based
anti-adhesive barriers were compared in microinvasive surgery, i.e., the da Vinci surgical system,
to evaluate their potential use and handling in microinvasive surgery. Figure S4 shows time-course
in situ images of using the da Vinci surgical system to perform full-sheet placement and cigar
placement. As shown in Figure S4a and S4b (see supplementary video for more details), both
hyaluronic acid-based antiadhesive barriers and our PVA/SFP nonwoven mats were fully
extended as flat sheets in the onlay mesh technique. Figure S4c and S4d show the roll-up
15
properties of the film. Because of the brittleness of hyaluronic acid-based antiadhesive barriers,
these barriers cannot fully roll up and easily bounce back to a flat sheet. However, PVA/SFP
nonwoven mats exhibit flexible properties and can fully conform to a curve topography. Another
necessary technique in laparoscopic surgery is the cigar technique, which is used to pass the
barriers through the port and incision. Figure S4e and Figure S4f show the preparation step for
curving the membrane into a cigar shape. Because of the brittleness of hyaluronic acid-based
anti-adhesive barriers, they could not form a cylinder and the membrane cracked when the da
Vinci endowrist instrument was applied. Figure S4g Figure S4h show the successful insertion of
cigar-shaped PVA/SFP nonwoven mats into the catheter. In summary, the PVA/SFP nonwoven
mats exhibited superior flexibility and durability compared to the hyaluronic acid-based barrier, as
well as ease of handling and tolerance of the endowrist instrument grasp.
Conclusions
A series of SFP-based nonwoven mats composed of a blend of synthetic water-soluble
polymers was fabricated and their bio-safety, anti-inflammatory ability toward fibroblasts and
macrophages were compared. First, all non-woven mats were composed of submicron-fibers with
diameters ranging from 308 to 366 nm and showed no cytotoxicity towards skin cells such as
fibroblast cells. Additionally, this series of nonwoven mats suppressed inflammatory factor
secretion, including nitrite. The mechanism of this effect requires further investigation, but may
involve potent release of silk fibroin peptide, which has been shown to have anti-inflammatory
effects in a mouse model.19 Second, co-incorporation of a water-soluble polymer, particularly
PVA/SFP nonwoven mats, markedly improved the anti-adhesion ability of SFP alone.
(Comparisons of gross adhesion percentage in mice model revealed 66.7% zero adhesion and 0%
severe adhesion in the PVA/SFP nonwoven mats group, but a lower zero adhesion occurrence rate
16
and higher severe adhesion occurrence rate were observed in the 100% SFP nonwoven mat group).
Based on these in vitro and in vivo results, PVA/SFP nonwoven mats outperformed the other
groups of SFP-based nonwoven mats. This is because of the optimum balance in the blend ratio
between SPF and PVA. SFP is natural and shows good flexibility and mechanical properties,
promoting the biocompatibility and anti-inflammatory effects. Additionally, the blend of
water-soluble PVA conferred drug-release ability and better anti-adhesion performance, which
may be attributed to the dissolution of PVA when the film contracted with the body fluid. Finally,
compared to the frequently used hyaluronic acid-based antiadhesive barriers, PVA/SFP nonwoven
mats showed flexible and stretchable features with high conformity and could be used with the
onlay and cigar techniques in microinvasive surgery. Therefore, the PVA/SFP nonwoven mats
with a 50%: 50% blend ratio shows potential as an anti-adhesion membrane for wound
management of internal or topical wounds.
Acknowledgments
M.Y. Bai would like to thank the National Science Council of Taiwan for financial support
under contract no. (MOST 104-2221-E-011-086-), funding from MOST (106-2622-E-011-002
-CC2), and in part by the National Taiwan University of Science and Technology–Tri-Service
General Hospital Joint Research Program, Taiwan (NTUST-TSGH-105-03). M.Y. Bai authored
the manuscript and prepared all figures. M.Y. Bai and W. Y. Hsu designed and performed all
experimental procedures, including setup of the electrospinning system, materials preparation, and
cell studies. Y. C. Wang and M. H. Yu performed the da Vinci surgical operation and provided
medical materials, professional consultation.
Conflict of Interest
The authors have no conflict of interest to declare.
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19
Figure Legends
Figure 1. SEM images and statistical bar charts of the fibrous diameter of (A,B) 100% SFP, 7500x,
(C,D) PVA/SFP, 7500x。(E,F) PEG/SFP, 7500x, and (G, H) PEO/SFP, 7500x.
Figure 2. Fourier-transform infrared (FT-IR) spectra of (A) pure PVA powder, (B) 100% SFP
powder, and (C) PVA/SFP (5/5) nonwoven mats.
Figure 3. Cytotoxicity assays of SFP-based nonwoven mats toward 3T3 fibroblast cells. The cell
viability in each group was expressed as the mean ± SD (n = 3/group) compared to the control
group. p values <0.05 were considered statistically significant with respect to the control group.
Figure 4. Nitrite assays of SFP-based nonwoven mats toward LPS-induced inflammatory 264.7
macrophage cells. (A) Calibration line of nitrite, (B) cytotoxicity results of LPS solution with
different concentrations. (C) Nitrite level of each group after treatment with nonwoven mats. The
results were expressed as the mean ± SD (n = 3/group) as compared to the control group. Asterisk
in figure indicates p < 0.001.
Figure 5. (A–E) Optical images of the density of fibroblast cells attached to PCL, SFP, PVA/SFP,
PEG/SFP, and PEO/SFP nonwoven mats, respectively, after 24 h culture. Nuclei of attached cells
were stained with DAPI. (F) Statistical results of adhered cell numbers compared to the control
group (i.e. PCL nonwoven mats).
Figure 6. In vitro drug-release profiles of PVA/SFP nonwoven mats soaked in PBS at pH 7.4 and
37°C . (A) UV-vis absorption wavelength of PVA, (B) calibration line of PVA solution with known
concentrations, (C) 9 day cumulative release curve of PVA/SFP nonwoven mats.
Figure 7. In vivo wounded animal model studies: (A) representative images of relationship
between type of dressing and wound healing on days 0, 5, 10, 15, and 20. (B) Statistical results of
20
measured area of wound remained as percentage of area of wound measured on day 0 (mean ± SD,
n = 6/group).
Figure 8. Representative optical micrographs for pathological evaluation at days 5, 10, 15, and 20
on mice during the period of dressing change with Tegaderm®, PCL nonwoven mat, 100% SFP
nonwoven mat, and PVA/SFP nonwoven mat treatment.
21
Figure 1. SEM images and statistical bar charts of the fibrous diameter of (A,B) 100% SFP, 7500x, (C,D)
PVA/SFP, 7500x。(E,F) PEG/SFP, 7500x, and (G, H) PEO/SFP, 7500x.
281x352mm (300 x 300 DPI)

Figure 2. Fourier-transform infrared (FT-IR) spectra of (A) pure PVA powder, (B) 100% SFP powder, and (C)
PVA/SFP (5/5) nonwoven mats.
164x168mm (300 x 300 DPI)

Figure 3. Cytotoxicity assays of SFP-based nonwoven mats toward 3T3 fibroblast cells. The cell viability in
each group was expressed as the mean ± SD (n = 3/group) compared to the control group. p values <0.05
were considered statistically significant with respect to the control group.
259x388mm (300 x 300 DPI)

Figure 4. Nitrite assays of SFP-based nonwoven mats toward LPS-induced inflammatory 264.7 macrophage
cells. (A) Calibration line of nitrite, (B) cytotoxicity results of LPS solution with different concentrations. (C)
Nitrite level of each group after treatment with nonwoven mats. The results were expressed as the mean ±
SD (n = 3/group) as compared to the control group. Asterisk in figure indicates p < 0.001.
411x962mm (300 x 300 DPI)

Figure 5. (A–E) Optical images of the density of fibroblast cells attached to PCL, SFP, PVA/SFP, PEG/SFP,
and PEO/SFP nonwoven mats, respectively, after 24 h culture. Nuclei of attached cells were stained with
DAPI. (F) Statistical results of adhered cell numbers compared to the control group (i.e. PCL nonwoven
mats).
220x336mm (300 x 300 DPI)

Figure 6. In vitro drug-release profiles of PVA/SFP nonwoven mats soaked in PBS at pH 7.4 and 37°C . (A)
UV-vis absorption wavelength of PVA, (B) calibration line of PVA solution with known concentrations, (C) 9
day cumulative release curve of PVA/SFP nonwoven mats.
325x703mm (300 x 300 DPI)

Figure 7. In vivo wounded animal model studies: (A) representative images of relationship between type of
dressing and wound healing on days 0, 5, 10, 15, and 20. (B) Statistical results of measured area of wound
remained as percentage of area of wound measured on day 0 (mean ± SD, n = 6/group).
187x318mm (300 x 300 DPI)

Figure 8. Representative optical micrographs for pathological evaluation at days 5, 10, 15, and 20 on mice
during the period of dressing change with Tegaderm®, PCL nonwoven mat, 100% SFP nonwoven mat, and
PVA/SFP nonwoven mat treatment.
156x116mm (300 x 300 DPI)


Wang Et Al-2017-Journal of Biomedical Materials Research Part A

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Wang Et Al-2017-Journal of Biomedical Materials Research Part A

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Evaluation of a series of silk fibroin protein-based nonwoven mats for

use as an anti-adhesion patch for wound management in robotic

Yu-Chi Wang3, Meng-Yi Bai*,, 1,2, Wan-Yuan Hsu1, Mu-Hsien Yu3

This article is protected by copyright. All rights reserved.

fabricated via the single-spinneret electrospinning technique. A series of SFP-based electrospun

SFP/poly(vinyl alcohol) (PVA), SFP/polyethylene glycol (PEG), and SFP/polyethylene oxide

lipopolysaccharide-induced RAW 264.7 macrophages model. Tissue anti-adhesion potential was

superior collagen regeneration and wound closure.

Keywords: nanotechnology; electrospinning; biomaterials; microfibers; nonwoven mat

Anti-adhesion to internal tissue is a major concern in the development of medical materials/or

become increasingly important in the wound and tissue management fields.5

production. Because of its excellent biocompatibility, smoothness, hydrophilicity, and mechanical

previous anti-adhesion material studies mainly focused on synthetic polymers, such as

poly(L-lactic acid),9 polyethylene glycol (PEG),10 poly(D,L-lactic-co-glycolic acid),11

poly(ethylene oxide) (PEO),12 poly(vinyl alcohol) (PVA),13,14 and poly(lactide)-b-poly(ethylene

the porous membrane.

between anti-adhesion and permeability.

weight can be tailored for specific applications.

nonwoven mats with therapeutic potential. In contrast to previously described anti-adhesion

Materials and Methods

enhancer as needed. Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (lyophilized

A 1X phosphate-buffered saline (PBS) solution was prepared by diluting 1 mL of 10X PBS

Preparation of a series of SFP/water-soluble polymer electrospun nonwoven mats using the

from the SEM image using NIH ImageJ software.

Results and discussion

The single-nozzle ES technique was successfully used to fabricate a series of SFP-based

required simultaneously, such as in wound management. Thus, a series of water-soluble polymers

respectively, revealing a remarkable reduction in pore size after incorporation of water-soluble

interior or exterior wound management can greatly limit pathogen invasion.

In vitro cytotoxicity and anti-inflammatory effects of a series of SFP-based nonwoven mats

matrix, as these small molecules typically have better water-solubility.

In vitro anti-adhesion test of SFP-based nonwoven mats on 3T3 fibroblast cells

candidates for use as anti-adhesion patches in wound management.

In vitro drug release profile of PVA/SFP nonwoven mats

In vivo macroscopic evaluation of wound-healing and anti-adhesion ability of SFP-based

nonwoven mats using trauma mouse model

removing dressing during wound management, an anti-adhesive patch is needed. Additionally,

putative wound-healing and anti-adhesion ability potential of a series of SFP-based nonwoven

S2 for all gross adhesion percentages and classification index).

moderate or absent in the two SFP-based nonwoven mat-treated groups.

Masson’s trichrome staining for morphological evaluation of wound recovery

collagen fibril bundles in SFP-based nonwoven mat-treated groups.

In vitro comparison of PVA/SFP nonwoven mats and hyaluronic acid-based antiadhesive

barriers for intraperitoneal anti-adhesion prevention using a da Vinci surgical system

well as ease of handling and tolerance of the endowrist instrument grasp.

A series of SFP-based nonwoven mats composed of a blend of synthetic water-soluble

effects in a mouse model.19 Second, co-incorporation of a water-soluble polymer, particularly

promoting the biocompatibility and anti-inflammatory effects. Additionally, the blend of

management of internal or topical wounds.

The authors have no conflict of interest to declare.

1. Xu H, Ma L, Shi H, Gao C, Han C. Chitosan–hyaluronic acid hybrid film as a novel wound

(C,D) PVA/SFP, 7500x。(E,F) PEG/SFP, 7500x, and (G, H) PEO/SFP, 7500x.

powder, and (C) PVA/SFP (5/5) nonwoven mats.

in figure indicates p < 0.001.

group (i.e. PCL nonwoven mats).

concentrations, (C) 9 day cumulative release curve of PVA/SFP nonwoven mats.

nonwoven mat, and PVA/SFP nonwoven mat treatment.

281x352mm (300 x 300 DPI)

John Wiley & Sons, Inc.

164x168mm (300 x 300 DPI)

John Wiley & Sons, Inc.

259x388mm (300 x 300 DPI)

John Wiley & Sons, Inc.