Colloids and Surfaces B: Biointerfaces 186 (2020) 110735

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Topical application of silk fibroin-based hydrogel in preventing T

hypertrophic scars
Zheng Li, Jiangbo Song, Jianfei Zhang, Kaige Hao, Lian Liu, Baiqing Wu, Xinyue Zheng, Bo Xiao,
Xiaoling Tong, Fangyin Dai*
State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, College of
Biotechnology, Southwest University, Chongqing 400716, China

A R T I C LE I N FO A B S T R A C T

Keywords: Current medications for the treatment of hypertrophic scars suffer from bottlenecks of limited therapeutic ef-
Silk fibroin-based hydrogel ficacy and a slow recovery rate. Silk fibroin (SF) has gained attention for its ability to promote wound healing in
Hypertrophic scar burns and cutaneous wounds, but its therapeutic effects against hypertrophic scar have not been thoroughly
Collagen fibers investigated. We prepared SF-based hydrogels (SFHs) with various SF concentrations (1.5 %, 3 %, and 6 %) and
α-Smooth muscle actin
characterized their physicochemical properties. Cell experiments showed that these SFHs had favorable bio-
compatibility in vitro. Further animal experiments in rabbits revealed that the SFH (3 %)-treated group achieved
scars on their ears that were thinner and significantly lighter in color compared with the negative control group.
Moreover, treatment with SFHs reduced the density and led to the orderly arrangement of collagen fibers. It was
found that the therapeutic effects of SFHs were attributed to the reduced expression levels of α-smooth muscle
actin. These results are the first to demonstrate that SFH can be exploited as an effective therapeutic agent for the
treatment of hypertrophic scars.

1. Introduction
Wound healing is a complex but coordinated process that occurs in
living organisms and requires the coordination of multiple types of
cells, growth factors, and extracellular matrix (ECM) [1–4]. However,
when an organism has a full-thickness dermal wound, granulation
tissue is formed and gradually matures into scar tissue, eventually re-
sulting in pathological scar [5,6]. Scars are an inevitable physiological
response during wound healing, whereas pathological scars are a type
of skin fibrosis disease caused by abnormal repair [7,8]. Hypertrophic
scarring, a type of pathological scar, often occurs after severe trauma,
burn, and surgery. It not only affects the beauty and function of the skin
but also causes trouble to patients. Thus, the treatment of hypertrophic
scars has become a critical issue in the field of biomedicine.
A hypertrophic scar is defined as a visible and elevated scar that
does not spread into surrounding tissues, which is characterized by the
proliferation of dermal tissue, with excessive deposition of fibroblast-
derived ECM proteins and especially collagen, over long periods and by
persistent inflammation and fibrosis [9,10]. It is worth noting that
hypertrophic scars reportedly occur in 39 %–68 % of patients after
surgery and in 33 %–91 % of patients after burn injury [11]. To date,
numerous methods have been described for the treatment of abnormal
scars, but an optimal treatment method has not been established [12].
Silicone-based products continue to be premier drug options in clinical
trials for preventing and treating hypertrophic scars [13]. Clinical
evidence has demonstrated that these silicone-based formulations can
improve the thickness and color of hypertrophic scars. However, these
effects are relatively slow and vary greatly among individuals, making
their therapeutic efficacy far from satisfactory [14,15]. Therefore, there
is an urgent need to develop alternative wound dressings for the
treatment of hypertrophic scars.
In recent years, remarkable progress in materials science has ac-
celerated the application of many new materials and their composites
for various applications, especially in the biomedical field. The devel-
opment of polymer materials which can be divided into synthetic and
natural polymer materials, is particularly important. These polymer
materials have been widely used in diverse biomedical fields such as
wound repair, drug delivery, tissue engineering, gene therapy, bio-
sensors, and bioimaging [16–19]. In abundant natural polymer mate-
rials, a variety of natural biomaterials such as chitosan, sodium algi-
nate, and silk fibroin (SF) have been used for wound repair [20–25].
Among them, SF has garnered great attention as it has various

⁎
Corresponding author.
E-mail address: fydai@swu.edu.cn (F. Dai).

https://doi.org/10.1016/j.colsurfb.2019.110735
Received 8 October 2019; Received in revised form 2 December 2019; Accepted 15 December 2019
Available online 16 December 2019
0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Z. Li, et al.
advantageous features, including excellent biocompatibility, con-
trollable biodegradation, and desirable mechanical properties [26,27].
Based on these characteristics, SF has also been widely used for the
preparation of sustained-release drug delivery systems, bone tissue re-
generation and repair, biosensor, and three-dimensional (3D) bio-
printing, among others [28–31]. To date, many SF-based wound dres-
sings such as membrane, nanofiber, and hydrogel have been fabricated
[32–35], which have the capacity to promote wound healing and re-
store the structures of wounded tissues. Among the various forms of SF
materials, SF-based hydrogel (SFH) has attracted growing attention. It
has the important physical property of being able to easily attach to the
skin surface without the need for biological adhesives. At the same
time, its gelation behavior, molecular structure, and high-water content
can keep skin hydrated [36]. The high affinity of SF to the skin enables
SFH to attach to the skin’s surface, which can enable coated nano-
particles to penetrate from the outermost layer of the stratum corneum
to the inner layer [37]. Moreover, the 3D network structure of SFH
facilitates the loading and stable slow release of drugs [38]. In addition,
SFH can be prepared quickly and easily by ultrasound, pH adjustment,
chemical crosslinking, and other methods [39].
To the best of our knowledge, SFH has not been applied to reduce
hypertrophic scars. In this study, SFH was facilely prepared by the ul-
trasonic method (Fig. 1). Subsequently, its therapeutic effect on hy-
pertrophic scars was studied based on a model of hypertrophic scars in
rabbit ears. Meanwhile, a traditional scar removal cream was selected
as the positive control. According to the therapeutic outcomes, we
show, for the first time, that SFH has excellent inhibitory effects on
hypertrophic scars.
2. Materials and methods
2.1. Materials
Bombyx mori cocoons were provided by the silkworm gene bank
(Southwest University, Chongqing, China). Mouse L929 fibroblastic
cells were obtained from the Stem Cell Bank, Chinese Academy of
Sciences (Shanghai, China). The dialysis bag (molecular weight cut-off
[MWCO] = 8–14 kDa) was purchased from Musheng Biotech Co., Ltd.
(Chongqing, China). Polyethylene glycol 20,000 (PEG 20,000) and li-
thium bromide (LiBr) were purchased from Sangon Biotech Co., Ltd.
(Shanghai, China). Buffered paraformaldehyde (4 %) was purchased
from Solarbio Science & Technology Co., Ltd. (Beijing, China). Scar
removal cream was supplied by Mili pharmaceutical Co., Ltd.
(Zhengzhou, China). All chemicals were used as received without fur-
ther purification.
Fig. 1. Schematic illustration of SFH preparation and its application in inhibiting hypertrophic scars.
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Colloids and Surfaces B: Biointerfaces 186 (2020) 110735
2.2. Preparation of SFH
B. mori cocoons were provided by the silkworm gene bank
(Southwest University). Silkworm cocoons were cut into small pieces
and boiled in 0.5 % (W/V) sodium carbonate solution for 30 min. Then
sericin was removed by rinsing with deionized water, and this process
was repeated twice. SF fibers were dried in an oven at 60 °C for 12 h.
Subsequently, the dried fibers were dissolved in LiBr solution (9.3 M)
and placed in a dialysis bag (MWCO = 8–14 kDa). After dialysis for 3
days, the dialysis bag was placed in PEG 20,000 solution (10 %, W/V)
and a concentrated SF solution was obtained. After dilution with water,
SF solutions with predetermined concentrations were attained. Finally,
SFH was prepared by the ultrasonic method. The SF solutions were
sonicated 15 times (3 s of ultrasonication, 1 s interval) at 60 W using an
ultrasonic cell crushing apparatus (Jingxin technology, Shanghai,
China). After ultrasound, SFH was formed.
2.3. Structural and morphological characterization
2.3.1. Morphological observation by scanning electron microscopy
SFH was freeze-dried in the LYOQUEST-55 lyophilizer (Telstar,
Terrassa, Spain). The dried samples were mounted on sample shelves
with conductive adhesive and coated with gold to improve their elec-
trical conductivity. The morphologies of SFH were observed by the
TM4000 desktop scanning electron microscope (Hitachi, Tokyo, Japan)
using an accelerating voltage of 10 kV. Fifty pores in each sample were
selected to analyze the pore diameter distribution in SFH with Nano
measure software.
2.3.2. Fourier transform infrared and X-ray diffraction
The freeze-dried SFH samples were ground into powder, mixed with
KBr, and pressed into disks. Infrared spectra were measured using the
Nicolet IS10 FTIR spectrophotometer (Thermo Fisher Scientific,
Waltham, MA, USA). X-ray diffraction (XRD) patterns of various sam-
ples were analyzed by the BRUCKER D8 X-ray diffractometer
(BRUCKER, Ettlingen, Germany) at a voltage of 40 kV and a current of
40 mA using CuKα radiation. The scanning scope of 2θ ranged from 5°
to 45°.
2.4. Porosity and swelling behavior
The porosity of SFH was measured by the liquid substitution theory.
Briefly, the dried SFH was immersed in anhydrous ethanol with a vo-
lume of V1, and the total volume of solution containing dried SFH and
water was V2. After incubation for 1 h, SFH was removed and the re-
maining aqueous volume was measured as V3. The calculation formula
of porosity is as follows:
Z. Li, et al.
P(%) = (V1 − V3)/(V2 − V3)×100% (1)
The swelling property of SFH was evaluated by the gravimetric
method in phosphate-buffered saline (PBS). In brief, freeze-dried SFH
with a volume of 1 cm3 was weighed (Wd) and immersed in 10 mL PBS
solution (pH 7.4). The samples were removed from the solution at
predetermined time points and their weights were measured (Ws). The
swelling rate (SR) is calculated as follows:
SR(%) = (Ws − Wd)/Wd × 100%. (2)
2.5. Mechanical property
After ultrasonication, SF solution was immediately transferred to
24-well cell culture plate. Cylindrical SFH samples with a height of
15 mm and a diameter of 15.6 mm was prepared. The mechanical
properties of the SFH samples were tested by the TA.XT Plus Texture
Analyzer (Stable Micro Systems, Surrey, UK). The test mode was com-
pression, the pre-test rate was 2 mm s−1, the test rate was 1 mm s−1,
the puncture distance was 10 mm, the probe model was P/0.5, and the
trigger force was 5 g.
2.6. In vitro cytotoxicity
The cytotoxicity of SFH was detected in L929 mouse fibroblast cells
(Chinese Academy of Sciences Stem Cell Bank) by the extraction
method. Initially, L929 cells were seeded in 96-well plates at a density
of 5 × 103 cells/well and incubated overnight. The freeze-dried SFH
was sterilized by ultraviolet irradiation for 30 min. According to the
extraction conditions in the ISO 10993-12, the sterilized freeze-dried
SFH samples were placed in sterile 6-cell plates and immersed in sterile
Dulbecco’s modified Eagle’s medium (DMEM) with fetal bovine serum
(Gibco, Gaithersburg, MA, USA) in proportion (0.1 g mL−1). After in-
cubation at 37 °C for 24 h, the medium was removed, after which 10 μL
extraction solution was added to each well and the wells without ex-
traction solution were used as negative controls. After incubation for
different time periods (24, 48, and 72 h), cell viability was evaluated
using Cell Counting Kit-8 (YEASEN Biotechnology, Shanghai, China)
according to the manufacturer’s protocol. L929 cells were seeded in 24-
well plates and incubated overnight. Then 50 μL SFH extract was added
to each well and the same amount of DMEM with serum was added as
the control. After 72 h of incubation, the cell morphology was observed
and photographed using the IX73 inverted fluorescence microscope
(Olympus, Tokyo, Japan). Cell viability was examined using the
Calcein-AM/PI Double Stain Kit (US Everbright, Soochow, China) fol-
lowing the manufacturer’s protocol.
2.7. In vivo inhibition of hypertrophic scar
All animal experiments were conducted in compliance with in-
stitutional ethical use protocols, and were approved by the National
Center of Animal Science Experimental Teaching at the College of
Animal Science and Technology at Southwest University of China, in
accordance with the college’s “Guide for the Care and Use of Laboratory
Animals.” The New Zealand rabbits were from the College of Animal
Science (Southwest University). New Zealand white rabbits (2.5 kg/
rabbit, 10 weeks of age, 50:50 ratio of males to females) were used to
establish a rabbit model of hypertrophic scarring according to a pre-
viously published protocol [40,41]. Briefly, after disinfection of the skin
on the ventral side of the rabbit ears, the anesthetic Zoletil® (Virbac,
Shanghai, China) was intramuscularly injected (10 mg kg−1). In the
middle ventral flank of each rabbit ear, five full-thickness skin defects
(5 mm in diameter) were made along the long axis. The full-layer skin
was excised, and the pericardium was scraped while the cartilage was
retained. Each defect was > 1 cm apart. The defects healed completely
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Colloids and Surfaces B: Biointerfaces 186 (2020) 110735
after 3 weeks, forming hypertrophic scars. After wound healing, 12
rabbits were randomly divided into a negative control group, SFH (1.5
%)-treated group, SFH (3 %)-treated group, and scar removal group.
There were 3 rabbits, 6 ears, and 30 wounds in each group. Each group
received different treatments once a day for 56 days. The scar thick-
nesses were measured by spiral micrometer on days 35 and 56 after
medication, and the morphology and color of hypertrophic scar in
rabbit ears were observed. At the end of the experiment, rabbits were
euthanized and scar tissues were excised. Each scar tissue was cut into
two parts: one was fixed in paraformaldehyde solution (4 %, v/v), and
the remaining tissue was stored at −80 °C.
2.8. Histological examination
Pathological changes of hypertrophic scars in rabbit ears were ex-
amined by hematoxylin and eosin (H&E) staining. These pathologic
sections were observed and photographed using the BX63 microscope
(Olympus).
2.9. Quantitative real-time PCR
Quantitative real-time PCR (qPCR) was used to detect the relative
expression levels of transforming growth factor-β1 (TGF-β1) and α-
smooth muscle actin (α-SMA) in scar tissue. Glyceraldehyde-3-phos-
phate dehydrogenase (GAPDH) was selected as the reference gene in
the experiment. The assay was performed using the Real-Time PCR
system (Bio-Rad, CA, USA). The primers used for qPCR were as follows:
(1) GAPDH: F 5′-TGGTGAAGGTCGGAGTGAAC-3′, R 5′-ATGTAGTGGA
GGTCAATGAATGG-3′;(2) TGF-β1: F 5′-AGAGAAGAACTGCTGTG
TGC-3′, R 5′-GTCCAGGCTCCAGATGTAGG-3′; and (3) α-SMA: F
5′-TGTTCCAGCCCTCCTTCATC-3′, R 5′-CCCTGAGAGCACATTGTT
AGC-3′.
2.10. Statistical analysis
Statistical analysis was performed using SPSS version 22. The data
were analyzed statistically using the Student’s t-test. P < 0.05 was
considered statistically significant. The data are expressed as the
mean ± standard error of the mean (SEM).
3. Results and discussion
3.1. Structural and morphological characterization
The scanning electron micrograph in Fig. 2 shows that all SFH
samples had porous structures, and the detailed porosity data are
shown in Fig. 2J. As can be seen from the diagrams, the higher SF
concentration resulted in more compact pore structure formed by SF
molecules, and the pore size gradually decreased with an increase in SF
concentration. The approximate normal distribution of pore size dis-
tribution in the SFH samples can be observed from the aperture dis-
tribution histograms.
Since the conformation of SF chain determines its solubility and
morphological structure, we investigated the conformation of SFHs at
different concentrations. As shown in Fig. 3A, SFHs at different con-
centrations exhibited absorption peaks at 1627, 1520, 1230 cm−1,
which corresponded to amides I, II, and III, respectively. According to
previous reports [42,43], the absorption peak in amide I at
1645–1655 cm−1 is a random coil, 1656–1662 cm−1 is an α-helix, and
1620–1630 cm−1 is a β-sheet. Similarly, the absorption peak in the
amide II located at 1515–1530 cm−1 is a β-sheet. However, the ab-
sorption peak of amide III located at 1230–1240 cm−1 is a random coil.
Therefore, these characteristic absorption peaks reveal the formation of
mainly β-sheets and a few random coils in the SFH. In addition, the
conformation of SFH was also confirmed by XRD. As seen in Fig. 3B,
SFHs at various concentrations presented strong diffraction peaks at
Z. Li, et al. Colloids and Surfaces B: Biointerfaces 186 (2020) 110735

Fig. 2. Morphology observation by scanning electron microscopy and pore size distribution analysis of lyophilized SFHs at different concentrations. (A, D, G) 1.5 %
SFH. (B, E, H) 3 % SFH. (C, F, I) 6 % SFH. (J) Pore size analysis of SFHs (n = 50).

20.7°. The higher the SF concentration, the sharper the diffraction peak, suggesting the formation of a small amount of Silk I (α-helix) structure
which indicated that the contents of Silk II structure increased with [44–46]. These data collectively reveal that SFHs at different con-
increasing SF concentration. Additionally, SFHs at different con- centrations have a similar conformation, and have large amounts of Silk
centrations showed weak diffraction peaks at 9.7°, 24.3°, and 28.2°, II (β-sheet) crystal structures and very few α-helices and random coil

Fig. 3. Conformational analysis of SFHs. (A) FTIR. (B) XRD.

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Z. Li, et al. Colloids and Surfaces B: Biointerfaces 186 (2020) 110735

Fig. 4. Physical properties of SFHs. (A) Porosity of SFHs measured by the liquid substitution method. (B) Swelling behavior of SFHs evaluated by the gravimetric
method. (C) Mechanical properties of SFHs characterized by the hardness test. The error bar represents mean ± SEM (*P < 0.01, **P < 0.001, ***P < 0.0001).

Fig. 5. Cytotoxicity evaluation of the SFHs. (A) CCK-8 assay of

L929 cells treated with SFHs. (B) Growth observations of L929
cells treated with SFHs: (a,a”) blank control, (b, b’) 1.5 % SFH,
(c, c’) 3 % SFH, (d, d’) 6 % SFH. (C) Calcein AM/EthD-I Double
Stain Kit assay of L929 cells upon treatment with SFHs: (a)
blank control, (b) 1.5 % SFH, (c) 3 % SFH, and (d) 6 % SFH.
Live cells are stained by Calcein AM dye, and produce an in-
tense uniform green fluorescence (ex/em ∼488 nm nm/
∼530 nm). Dead cells are stained with EthD-I dye and emit
bright red fluorescence (ex/em ∼488 nm/∼610 nm). The
error bar represents the mean ± SEM. Vs. the control group,
N.S. means no significant difference (For interpretation of the
references to colour in this figure legend, the reader is referred
to the web version of this article).

structures. concentration. SFH (1.5 %) had a hardness value of 63.7 ± 2.6 g,

which could be easily broken by touching gently with the hand, and
formed a sticky and applicable hydrogel. In the context of SFH (3 %), its
3.2. Porosity and swelling behavior
hardness value was 278.0 ± 3.3 g, which was much higher than that of
SFH (1.5 %). However, this hydrogel could be mashed to form a
As can be seen in Fig. 4A, the porosities of SFHs increased with
spreadable gel. Furthermore, we found that SFH (6 %) had the highest
decreasing SF concentration, and SFH (1.5 %) had the highest porosity
hardness value (1142.0 ± 24.2 g), which was too hard to be evenly
among all SFHs. Fig. 4B shows that the swelling equilibrium times of
applied on the skin’s surface. These observations can be attributed to
SFH (1.5 %), SFH (3 %), and SFH (6 %) were 8, 6, and 3 h, respectively.
the fact that the higher the concentration of SF solution, the denser the
Furthermore, we found that SFH (1.5 %) had the highest swelling ratio
network structure formed.
and could absorb 27-fold its own mass of water. In summary, the por-
osity and swelling of SFH decreased with an increase in SF concentra-
tion.
3.4. Biocompatibility evaluation

3.3. Mechanical properties Biocompatibility is an important prerequisite for the application of

biomaterials. Thus, we tested this property of SFHs in L929 cells. As
The hardness values were determined using a texture tester. Fig. 4C presented in Fig. 5A, the results of the CCK-8 experiments showed that
shows that the hardness values of SFHs increased with increasing SF the cell viabilities of all treatment groups were higher than 85 %,

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Z. Li, et al.
Fig. 6. Therapeutic efficacies of SFHs on hypertrophic scarring. (A) Observation of hypertrophic scars in the rabbit ear. (B) Scar hyperplasia index. The error bar
represents the mean ± SEM. Vs. the control group, N.S. means no significant difference (*P < 0.05, **P < 0.01).
indicating that SFHs with different concentrations had no cytotoxicity.
Furthermore, we observed the cell morphology of different treatment
groups. At 36 h after incubating L929 cells with SFHs, all cells showed
normal fusiform and there was no significant difference in cell density
(Fig. 5B). In addition, all SFH extract-treated cells emitted obvious
green fluorescent signals, and only a small amount of dead cells showed
red fluorescence (Fig. 5C). These observations were consistent with the
experimental results of the CCK-8 assay, confirming the excellent bio-
compatibility of the SFHs.
3.5. Therapeutic effects of SFHs on hypertrophic scars
As demonstrated above, SFH (6 %) had a high hardness and was not
easy to apply on the skin’s surface. Therefore, only SFH (1.5 %) and SFH
(3 %) were selected for animal experiments. We observed the scar ap-
pearance and further measured the scar hyperplasia index on days 0,
35, and 56. As displayed in Fig. 6, the fresh scar appeared red and stiff
on day 0. After 35 days of treatment, the color of all the SFH-treated
wound surfaces became lighter than that on day 0. In contrast, the scar
color of the negative control group was deeper than that of the SFH-
treated and scar removal cream-treated groups. Moreover, the index of
scar hyperplasia decreased in all groups, especially in the scar removal
cream-treated group. After 56 days of treatment, the wound color of all
treatment groups became obviously lighter and was closely similar to
the surrounding normal skin color. However, the wound color of the
negative control group was slightly darker than that of the SFH-treated
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Colloids and Surfaces B: Biointerfaces 186 (2020) 110735
groups and the scar removal cream-treated group. At the same time, the
texture of scar tissue softened in the SFH-treated groups. Furthermore,
statistics on the scar hyperplasia index of each group showed that the
SFH (3 %)-treated group and the scar removal cream-treated group had
a significantly lower scar hyperplasia index than the negative control
group. The scar hyperplasia index of the SFH (1.5 %)-treated group was
also lower than that of the negative control group, but there was no
significant difference between these two groups. As mentioned above,
SFH (1.5 %) and SFH (3 %) showed therapeutic effects on hypertrophic
scars, which could whiten the color of the scars and reduce the wound
thicknesses, but only SFH (3 %) had statistically more significant
therapeutic effects. More importantly, the average scar hyperplasia
index in the SFH (3 %)-treated group was reduced by 16.6 % compared
with the control group. This was more significant than the previously
reported effect of artesunate and bacterial cellulose on inhibiting hy-
pertrophic scarring (11.5 % and 15.5 %, respectively) [47,48]. Ad-
ditionally, we found that with the passage of time, scar thickness of the
negative control group also decreased slowly.
3.6. Histological analysis
Inspired by the therapeutic effects of SFHs on hyperplastic scars, we
further analyzed the pathology of scar tissues by using H&E staining. As
observed in Fig. 7, the negative control group showed that the dermis of
hypertrophic scar was significantly thickened and contained a large
amount of dense, thick, and disordered collagen fibers and
Z. Li, et al. Colloids and Surfaces B: Biointerfaces 186 (2020) 110735

Fig. 7. Histological evaluation of the therapeutic effects on hypertrophic scars on day 56. (A) Blank control. (B) 1.5 % SFH. (C) 3 % SFH. (D) Scar removal cream.

Fig. 8. Expression level of TGF-β1 and α-SMA on day 56. (A). TGF-β1. (B) α-SMA The error bar represents mean ± SEM. Vs. the control group, N.S. means no
significant difference (*P < 0.05, **P < 0.0001).

inflammatory cells. In comparison with other treatment groups, the
negative control group showed the highest density and the most dis-
ordered arrangement of collagen fibers. Interestingly, we found that the
density of collagen fibers in the SFH (1.5 %)-treated group decreased,
but the arrangement of collagen fibers still showed obvious vortices.
Furthermore, it was found that the SFH (3 %)-treated group exhibited
less density of collagen fibers and more orderly collagenous fibers
compared with the SFH (1.5 %)-treated group. In the scar removal
cream-treated group, the density of collagen fibers significantly de-
creased and there were large gaps between collagen fiber bundles with
regular arrangement. In addition, the hypertrophic scar tissues from
various groups were characterized by the proliferation profiles of fi-
broblasts and the amounts of collagen fibers. Collagen is mainly se-
creted by fibroblasts, which can aggregate into collagen fibrils and bond
into collagen fibers. The densities of collagen fibers in the SFH-treated
groups were clearly reduced. Notably, compared with the SFH (1.5
%)-treated group, the SFH (3 %)-treated group had more uniform ar-
rangement of collagen fibers, similar to normal skin. Eventually, SFH (3
%) achieved excellent treatment effects on hypertrophic scars by in-
hibiting the formation of collagen fibers in the scar, thereby reducing
scar thickness. Furthermore, compared with ginsenoside Rb1on histo-
pathology of hypertrophic scar tissue [49], SFH had more significant
effects on improving the density and arrangement of collagen fibers in
scar tissues.
3.7. Analysis of hypertrophic scar-related gene expression
During the process of wound healing, the local inflammatory re-
sponse can induce the differentiation of mesenchymal cells and pro-
mote the proliferation of fibroblasts [50,51]. Fibroblasts secrete fiber

7
Z. Li, et al.
connexin and collagen and a large amount of collagen deposits in the
ECM when granulation tissue matures eventually form a hypertrophic
scar [52]. When the TGF-β1/Smad3 signaling pathway is activated, the
secretion levels of TGF-β1 increase, leading to the increased expression
of collagen I and reduced secretion of collagen II and collagen III. As a
result, scar hyperplasia is formed [53]. α-SMA is the main protein in the
intracellular contractile system, which can lead to scar contracture by
driving changes in collagen fiber position. In the later formation stage
of scars, the expression levels of α-SMA in tissues are upregulated.
Meanwhile, the expression of α-SMA in scar tissue is significantly higher
than that in normal tissue, which is related to scar hyperplasia and
contracture [54]. In this study, to explore the molecular mechanism of
therapeutic effects of SFHs against hypertrophic scar, the mRNA levels
of TGF-β1 and α-SMA in scar tissues were studied by qPCR after 56 days
of medication. As shown in Fig. 8, there was no significant difference in
the expression level of TGF-β1 among all groups. However, after
treatment for 56 days, the mRNA expression levels of α-SMA were
significantly decreased in all treatment groups compared with the ne-
gative control group. Among them, there was a significant difference
when the SFH (1.5 %)-treated group was compared with the negative
control group, and an extremely significant difference was found be-
tween the scar removal cream-treated and negative control groups.
Furthermore, the SFH (3 %)-treated group had the lowest expression of
α-SMA. These results indicate that SFHs may inhibit scar hyperplasia by
inhibiting α-SMA expression.
4. Conclusions
This study is the first to explore the therapeutic effects of SFH on
hypertrophic scars. SFH not only whitened the scar color but also re-
duced its thickness. Furthermore, the therapeutic outcomes with SFH (3
%) were much better than those with SFH (1.5 %). There is still a lot of
room for improvement in the therapeutic effect of SFH. SF has extensive
supply sources, easy accessibility, low cost, excellent biocompatibility,
and controllable degradation, which contribute to its strong competi-
tiveness in the field of biomedicine. Further studies should be con-
ducted to improve the SFH to develop low-cost SF by inhibiting hy-
pertrophic scar in the future. It was found that the inhibition of SFH on
hypertrophic scar could be induced by downregulating the expression
levels of α-SMA. These results show that SFH has a favorable inhibitory
effect on hypertrophic scar, which expands the application scope of SF.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Authors’ contributions
Zheng Li conceived and performed the study. Jiangbo and Jianfei
Zhang helped design the study. Kaige Hao, Lian Liu, and Xinyue Zheng
helped perform the materials related experiments. Baiqing Wu helped
perform the animal experiments. Bo Xiao, Xiaoling Tong and Fangyin
Dai reviewed and edited the manuscript. All authors read and approved
the manuscript.
Acknowledgments
The research was supported by the National Natural Science
Foundation of China (Nos. 31830094 and 31472153), the Hi-Tech
Research and Development 863 Program of China Grant (No.
2013AA102507), the Project funded by Chongqing Special Postdoctoral
Science Foundation (No. XmT2018058), and Funds of China
Agriculture Research System (No. CARS-18-ZJ0102).
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Colloids and Surfaces B: Biointerfaces 186 (2020) 110735
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