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Topical application of silk fibroin-based hydrogel in preventing hypertrophic scars
Colloids and Surfaces B: Biointerfaces 186 (2020) 110735
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Topical application of silk fibroin-based hydrogel in preventing hypertrophic scars
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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: Silk fibroin-based hydrogel Hypertrophic scar Collagen fibers α-Smooth muscle actin
Current medications for the treatment of hypertrophic scars suffer from bottlenecks of limited therapeutic efficacy and a slow recovery rate. Silk fibroin (SF) has gained attention for its ability to promote wound healing in burns and cutaneous wounds, but its therapeutic effects against hypertrophic scar have not been thoroughly investigated. We prepared SF-based hydrogels (SFHs) with various SF concentrations (1.5 %, 3 %, and 6 %) and characterized their physicochemical properties. Cell experiments showed that these SFHs had favorable biocompatibility 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 resulting 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 fibroblastderived 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,
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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 accelerated the application of many new materials and their composites for various applications, especially in the biomedical field. The development 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, biosensors, and bioimaging [16–19]. In abundant natural polymer materials, a variety of natural biomaterials such as chitosan, sodium alginate, 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: [email protected] (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.
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2.2. Preparation of SFH
advantageous features, including excellent biocompatibility, controllable 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 regeneration and repair, biosensor, and three-dimensional (3D) bioprinting, among others [28–31]. To date, many SF-based wound dressings such as membrane, nanofiber, and hydrogel have been fabricated [32–35], which have the capacity to promote wound healing and restore 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 nanoparticles 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 ultrasonic method (Fig. 1). Subsequently, its therapeutic effect on hypertrophic 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.
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 electrical 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 samples 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. 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 lithium 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 further purification.
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 volume 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 remaining aqueous volume was measured as V3. The calculation formula of porosity is as follows:
Fig. 1. Schematic illustration of SFH preparation and its application in inhibiting hypertrophic scars. 2
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P(%) = (V1 − V3)/(V2 − V3)×100%
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 thicknesses 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.
(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
2.8. Histological examination
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 compression, 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.
Pathological changes of hypertrophic scars in rabbit ears were examined 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 αsmooth muscle actin (α-SMA) in scar tissue. Glyceraldehyde-3-phosphate 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′.
2.6. 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 incubation 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 extraction 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 24well 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) following the manufacturer’s protocol.
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 distribution in the SFH samples can be observed from the aperture distribution 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 concentrations 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 absorption 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
2.7. In vivo inhibition of hypertrophic scar All animal experiments were conducted in compliance with institutional 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 previously 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 3
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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).
suggesting the formation of a small amount of Silk I (α-helix) structure [44–46]. These data collectively reveal that SFHs at different concentrations have a similar conformation, and have large amounts of Silk II (β-sheet) crystal structures and very few α-helices and random coil
20.7°. The higher the SF concentration, the sharper the diffraction peak, which indicated that the contents of Silk II structure increased with increasing SF concentration. Additionally, SFHs at different concentrations showed weak diffraction peaks at 9.7°, 24.3°, and 28.2°,
Fig. 3. Conformational analysis of SFHs. (A) FTIR. (B) XRD. 4
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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 intense 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).
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 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 spreadable gel. Furthermore, we found that SFH (6 %) had the highest hardness value (1142.0 ± 24.2 g), which was too hard to be evenly applied on the skin’s surface. These observations can be attributed to the fact that the higher the concentration of SF solution, the denser the network structure formed.
structures. 3.2. Porosity and swelling behavior As can be seen in Fig. 4A, the porosities of SFHs increased with decreasing SF concentration, and SFH (1.5 %) had the highest porosity among all SFHs. Fig. 4B shows that the swelling equilibrium times of SFH (1.5 %), SFH (3 %), and SFH (6 %) were 8, 6, and 3 h, respectively. Furthermore, we found that SFH (1.5 %) had the highest swelling ratio and could absorb 27-fold its own mass of water. In summary, the porosity and swelling of SFH decreased with an increase in SF concentration.
3.4. Biocompatibility evaluation Biocompatibility is an important prerequisite for the application of biomaterials. Thus, we tested this property of SFHs in L929 cells. As presented in Fig. 5A, the results of the CCK-8 experiments showed that the cell viabilities of all treatment groups were higher than 85 %,
3.3. Mechanical properties The hardness values were determined using a texture tester. Fig. 4C shows that the hardness values of SFHs increased with increasing SF 5
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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).
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 hypertrophic scarring (11.5 % and 15.5 %, respectively) [47,48]. Additionally, we found that with the passage of time, scar thickness of the negative control group also decreased slowly.
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 biocompatibility 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 appearance 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 SFHtreated 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
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 6
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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).
groups were clearly reduced. Notably, compared with the SFH (1.5 %)-treated group, the SFH (3 %)-treated group had more uniform arrangement of collagen fibers, similar to normal skin. Eventually, SFH (3 %) achieved excellent treatment effects on hypertrophic scars by inhibiting the formation of collagen fibers in the scar, thereby reducing scar thickness. Furthermore, compared with ginsenoside Rb1on histopathology of hypertrophic scar tissue [49], SFH had more significant effects on improving the density and arrangement of collagen fibers in scar tissues.
inflammatory cells. In comparison with other treatment groups, the negative control group showed the highest density and the most disordered 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 decreased 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 fibroblasts and the amounts of collagen fibers. Collagen is mainly secreted by fibroblasts, which can aggregate into collagen fibrils and bond into collagen fibers. The densities of collagen fibers in the SFH-treated
3.7. Analysis of hypertrophic scar-related gene expression During the process of wound healing, the local inflammatory response can induce the differentiation of mesenchymal cells and promote the proliferation of fibroblasts [50,51]. Fibroblasts secrete fiber 7
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References
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 negative 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 between 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.
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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 reduced 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 competitiveness in the field of biomedicine. Further studies should be conducted to improve the SFH to develop low-cost SF by inhibiting hypertrophic 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 influence 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). 8
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Topical application of silk fibroin-based hydrogel in preventing hypertrophic scars
T
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: Silk fibroin-based hydrogel Hypertrophic scar Collagen fibers α-Smooth muscle actin
Current medications for the treatment of hypertrophic scars suffer from bottlenecks of limited therapeutic efficacy and a slow recovery rate. Silk fibroin (SF) has gained attention for its ability to promote wound healing in burns and cutaneous wounds, but its therapeutic effects against hypertrophic scar have not been thoroughly investigated. We prepared SF-based hydrogels (SFHs) with various SF concentrations (1.5 %, 3 %, and 6 %) and characterized their physicochemical properties. Cell experiments showed that these SFHs had favorable biocompatibility 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 resulting 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 fibroblastderived 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,
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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 accelerated the application of many new materials and their composites for various applications, especially in the biomedical field. The development 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, biosensors, and bioimaging [16–19]. In abundant natural polymer materials, a variety of natural biomaterials such as chitosan, sodium alginate, 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: [email protected] (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.
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2.2. Preparation of SFH
advantageous features, including excellent biocompatibility, controllable 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 regeneration and repair, biosensor, and three-dimensional (3D) bioprinting, among others [28–31]. To date, many SF-based wound dressings such as membrane, nanofiber, and hydrogel have been fabricated [32–35], which have the capacity to promote wound healing and restore 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 nanoparticles 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 ultrasonic method (Fig. 1). Subsequently, its therapeutic effect on hypertrophic 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.
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 electrical 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 samples 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. 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 lithium 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 further purification.
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 volume 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 remaining aqueous volume was measured as V3. The calculation formula of porosity is as follows:
Fig. 1. Schematic illustration of SFH preparation and its application in inhibiting hypertrophic scars. 2
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P(%) = (V1 − V3)/(V2 − V3)×100%
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 thicknesses 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.
(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
2.8. Histological examination
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 compression, 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.
Pathological changes of hypertrophic scars in rabbit ears were examined 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 αsmooth muscle actin (α-SMA) in scar tissue. Glyceraldehyde-3-phosphate 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′.
2.6. 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 incubation 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 extraction 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 24well 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) following the manufacturer’s protocol.
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 distribution in the SFH samples can be observed from the aperture distribution 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 concentrations 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 absorption 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
2.7. In vivo inhibition of hypertrophic scar All animal experiments were conducted in compliance with institutional 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 previously 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 3
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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).
suggesting the formation of a small amount of Silk I (α-helix) structure [44–46]. These data collectively reveal that SFHs at different concentrations have a similar conformation, and have large amounts of Silk II (β-sheet) crystal structures and very few α-helices and random coil
20.7°. The higher the SF concentration, the sharper the diffraction peak, which indicated that the contents of Silk II structure increased with increasing SF concentration. Additionally, SFHs at different concentrations showed weak diffraction peaks at 9.7°, 24.3°, and 28.2°,
Fig. 3. Conformational analysis of SFHs. (A) FTIR. (B) XRD. 4
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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 intense 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).
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 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 spreadable gel. Furthermore, we found that SFH (6 %) had the highest hardness value (1142.0 ± 24.2 g), which was too hard to be evenly applied on the skin’s surface. These observations can be attributed to the fact that the higher the concentration of SF solution, the denser the network structure formed.
structures. 3.2. Porosity and swelling behavior As can be seen in Fig. 4A, the porosities of SFHs increased with decreasing SF concentration, and SFH (1.5 %) had the highest porosity among all SFHs. Fig. 4B shows that the swelling equilibrium times of SFH (1.5 %), SFH (3 %), and SFH (6 %) were 8, 6, and 3 h, respectively. Furthermore, we found that SFH (1.5 %) had the highest swelling ratio and could absorb 27-fold its own mass of water. In summary, the porosity and swelling of SFH decreased with an increase in SF concentration.
3.4. Biocompatibility evaluation Biocompatibility is an important prerequisite for the application of biomaterials. Thus, we tested this property of SFHs in L929 cells. As presented in Fig. 5A, the results of the CCK-8 experiments showed that the cell viabilities of all treatment groups were higher than 85 %,
3.3. Mechanical properties The hardness values were determined using a texture tester. Fig. 4C shows that the hardness values of SFHs increased with increasing SF 5
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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).
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 hypertrophic scarring (11.5 % and 15.5 %, respectively) [47,48]. Additionally, we found that with the passage of time, scar thickness of the negative control group also decreased slowly.
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 biocompatibility 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 appearance 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 SFHtreated 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
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 6
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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).
groups were clearly reduced. Notably, compared with the SFH (1.5 %)-treated group, the SFH (3 %)-treated group had more uniform arrangement of collagen fibers, similar to normal skin. Eventually, SFH (3 %) achieved excellent treatment effects on hypertrophic scars by inhibiting the formation of collagen fibers in the scar, thereby reducing scar thickness. Furthermore, compared with ginsenoside Rb1on histopathology of hypertrophic scar tissue [49], SFH had more significant effects on improving the density and arrangement of collagen fibers in scar tissues.
inflammatory cells. In comparison with other treatment groups, the negative control group showed the highest density and the most disordered 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 decreased 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 fibroblasts and the amounts of collagen fibers. Collagen is mainly secreted by fibroblasts, which can aggregate into collagen fibrils and bond into collagen fibers. The densities of collagen fibers in the SFH-treated
3.7. Analysis of hypertrophic scar-related gene expression During the process of wound healing, the local inflammatory response can induce the differentiation of mesenchymal cells and promote the proliferation of fibroblasts [50,51]. Fibroblasts secrete fiber 7
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References
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 negative 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 between 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.
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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 reduced 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 competitiveness in the field of biomedicine. Further studies should be conducted to improve the SFH to develop low-cost SF by inhibiting hypertrophic 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 influence 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). 8
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