CS nanofibers containing silk protein sericin as a wound dressing: In vitro and in vivo assessment

Journal Pre-proof Development of the PVA/CS nanofibers containing silk protein sericin as a wound dressing: In vitro and in vivo assessment

Hamid Reza Bakhsheshi-Rad, Ahmad Fauzi Ismail, Madzlan Aziz, Mohsen Akbari, Zhina Hadisi, Mahdi Omidi, Xiongbiao Chen PII:

S0141-8130(19)34965-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.139

Reference:

BIOMAC 14443

To appear in:

International Journal of Biological Macromolecules

Received date:

26 July 2019

Revised date:

12 January 2020

Accepted date:

13 January 2020

Please cite this article as: H.R. Bakhsheshi-Rad, A.F. Ismail, M. Aziz, et al., Development of the PVA/CS nanofibers containing silk protein sericin as a wound dressing: In vitro and in vivo assessment, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.139

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© 2020 Published by Elsevier.

Journal Pre-proof Development of the PVA/CS nanofibers containing silk protein sericin as a wound dressing: In vitro and in vivo assessment Hamid Reza Bakhsheshi-Rad1,2,*, Ahmad Fauzi Ismail2, Madzlan Aziz2, Mohsen Akbari3, Zhina Hadisi3, Mahdi Omidi1, and Xiongbiao Chen4 Advanced Materials Research Center, Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran 2 Advanced Membrane Technology Research Center (AMTEC), Universiti Teknologi Malaysia, 81310 Skudai, Johor Bahru, Johor, Malaysia 3 Laboratory for Innovations in MicroEngineering (LiME), Department of Mechanical Engineering, University of Victoria, Victoria, BC V8P 5C2, Canada 4 Department of Mechanical Engineering, College of Engineering, University of Saskatchewan, Saskatoon, SK, Canada

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Corresponding author: H.R. Bakhsheshi-Rad ([email protected]; [email protected])

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Abstract

Skin and soft tissue infections are major concerns with respect to wound repair. Recently, anti-

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bacterial wound dressings have been emerging as promising candidates to reduce infection, thus accelerating the wound healing process. This paper presents our work to develop and

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characterize poly(vinyl alcohol) (PVA)/ chitosan (CS)/ silk sericin (SS)/ tetracycline (TCN) porous nanofibers, with diameters varying from 305 to 425 nm, both in vitro and in vivo for

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potential applications as wound dressings. The fabricated nanofibers possess a considerable capacity to take up water through swelling (~325-650%). Sericin addition leads to increased

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hydrophilicity and elongation at break while decreasing fiber diameter and mechanical strength. Moreover, fibroblasts (L929) cultured on the nanofibers with low sericin content (PVA/CS/12SS) displayed greater proliferation compared to those on nanofibers without sericin (PVA/CS). Nanofibers loaded with high sericin and tetracycline content significantly inhibited the growth of Escherichia coli and Staphylococcus aureus. In vivo examination revealed that PVA/CS/2SSTCN nanofibers enhance wound healing, re-epithelialization, and collagen deposition compared to traditional gauze and nanofibers without sericin. The results of this study demonstrate that the PVA/CS/2SS-TCN nanofiber creates a promising alternative to traditional wound dressing materials. Keywords: Silk protein sericin; Biocompatibility; Antimicrobial performance; Wound dressing

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Journal Pre-proof 1. Introduction Physical damage to the skin is a very common injury that occurs over the course of a human’s lifespan. Developing novel wound dressing materials is an essential challenge in terms of current medical technological innovation [1, 2]. In new dressing materials manufactured in recent years, biocompatible hydrogels with water uptake absorption capacity are regarded as an appealing choice for functional applications in skin tissue engineering [3, 4]. An ideal wound dressing requires a number of properties, including an appropriate swelling ratio, oxygen permeability, wound exudate absorption, and preservation of moisture in the wound surroundings to enhance

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wound healing [5-7].

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This paper describes the development of wound dressings composed of silk sericin (SS), a natural protein biomaterial and bioactive compound that does not require extra functionalization

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[8-12]. Silk sericin is a glycoprotein that comprises nearly 30% of the mass of silk cocoons and

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is generally a waste product of the textile industry [13]. Silk sericin has also been explored as a serum alternative on account of its bioactivity with respect to improving cell adhesion and

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promoting cell proliferation, biodegradability, and antibacterial activities [6, 14, 15]. However, neat sericin does not possess suitable properties due to its fragile nature [16]. Thus, it

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is crucial to improve the mechanical characteristics of sericin to broaden its potential applications [8, 16]. Blending with other polymers is one the best methods to enhance the

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mechanical characteristics of sericin [17]. Polyvinyl alcohol (PVA) is a synthetic polymer that possesses a variety of remarkable properties, such as biocompatibility, chemical stability,

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affordability, and outstanding film forming ability, and is often blended with natural polymers to increase their mechanical performance [16, 18, 19]. Chitosan (CS) is another appealing polymer as it possesses structural likeness to extracellular matrix (ECM) and anti-bacterial activity, and thus can assist in enhancing the effectiveness of ECM in skin tissue engineering [19-23]. Blending of sericin and PVA can improve the mechanical characteristics of sericin while preserving the swelling capacity of chitosan, hence expanding the utilization of sericin in biomedical fields as a drug carrier, antibacterial agent, and wound dressing [16, 24]. Gilotra [14] designed biopolymer nanofibers composed of PVA and SS with a wide range of porous structures for wound dressing and demonstrated greater attachment and proliferation of PVA/SS mats than neat PVA mats. Shi et al. [25] reported that the water uptake capability and swelling 2

Journal Pre-proof ratios of poly(γ-glutamic acid) hydrogel containing SS increased with SS concentration. Likewise, incorporation of SS into the hydrogel improved the adhesion and growth of L929 cells. A number of studies [20, 26, 27] demonstrate that nanofibers containing sericin have the ability to rebuild epidermal-dermal tissue, thus resulting in skin tissue regeneration and wound repair [20], and therefore the idea to prepare nanofibers containing sericin with enhanced biological properties, mechanical characteristics, and antibacterial performance for wound dressings is appealing [27]. Drug-incorporating biomaterials also provide a valuable opportunity to deliver medications to specific locations. Indeed, tetracycline (TCN) can be incorporated into nanofibers

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to impede bacterial infections and has the capacity to promote the body’s protection mechanisms

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to eliminate the bacteria that might result in infection [24, 28, 29]. Yang et al. [17] proposed a sericin-based nanocomposite hydrogel with substantial antibacterial performance for wound

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dressing, revealing that the macromolecular sericin within the nanocomposite attracts bacteria

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through charge interaction. Chao et al. [30] studied the antibacterial performance of sericin/PVAbased fibers and showed that the incorporation of tigecycline into the fibers leads to increased

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inhibition zones and significant antibacterial performance toward Escherichia coli and Bacillus subtilis. Therefore, the fabrication of nanofibers containing sericin with good mechanical and

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swelling properties, biocompatibility, and antibacterial performance has attracted considerable attention for wound dressing. In this study, PVA/CS/SS-TCN nanofibers were fabricated via

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electrospinning and subsequently examined both in vitro and in vivo for their potential in wound healing with the topical delivery of an antibiotic.

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2. Materials and Experiments

Sericin was prepared based on the method presented in the supporting information (S1). For preparation of the PVA/CS/SS-TCN solution, a mixture of PVA and SS powders was dissolved in hot water (80 °C) and stirred for 2 h. Chitosan (CS) powder was then dissolved in 90% acetic acid for 48 h. Sericin solutions with different concentrations (0, 1, 2, 3, and 5 w/v%) were blended with 10 w/v% PVA at a 1:1 ratio. Subsequently, 2 w/v% CS was added to the PVA/SS blend solutions at a ratio of 1:1 and stirred for 3 h, with the resulting solutions labeled 0 SS, 1 SS, 2SS, 3 SS, and 5 SS, respectively. The electrospinning (Fanavaran Nano-Meghyas) process was conducted at room temperature, a constant flow rate of 2.50 mL/h, and voltage of 15 kV. Aluminum (Al) foil was placed 20 cm from the needle tip to collect the electrospun fibers. Thereafter, the electrospun nanofibers were crosslinked using glutaraldehyde vapors for 12 h 3

Journal Pre-proof (The optimized parameters presented in supporting information S2). Tetracycline was loaded to the nanofibers by the diffusion method by soaking the nanofibers in 0, 0.1, 0.2, 0.3 and 0.5 g of TCN dissolved in 10 mL of distilled water labeled PVA/CS, PVA/CS/1SS-TCN, PVA/CS/2SSTCN, PVA/CS/3SS-TCN, and PVA/CS/5SS-TCN, respectively. A schematic presentation of this method is shown in Fig. 1. Water contact angle was determined via video-based optical contact angle equipment (VCA Optima, AST Products Inc.) using ~1 μL water droplets at room temperature. Further measurements were conducted on 20 × 20 mm electrospun nanofiber mats. To determine the

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swellability, the weight of dried and wet nanofiber mats was compared at specific time points.

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Dried nanofibers were weighed and soaked in PBS (pH 7.4) at 37 °C. Weights of the wet nanofibers were recorded at specific time points until saturation was achieved. Swelling ratio (S)

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was measured using the following relationship:

(1)

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S (%) = (Ws−Wd) / Wd × 100,

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where Wd and Ws are the dry and wet weight of the electrospun nanofiber mats, respectively. Water vapor transmission rate (WVTR) of the nanofiber was determined based on the Ref.

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[31,32]. In this context, the nanofiber were cut into small pieces (disk form) and subsequently mounted on the mouth of bottles (diameter is 35 mm) containing 15 ml purified water.

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Consequently, the bottles were weighted (Wi) and afterward placed into in the incubator at 37 °C with humidity of 80%. WVTR was attained using the following equation: WVTR (g/m2/day) = (Wi−Wf )/A

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(2)

where A is the effective exposure area, and Wi and Wf are the initial and final weight of bottles, respectively. The tensile strength and elongation of electrospun nanofibers (20 mm in length × 10 mm in width) was determined using a Universal Testing Machine (Instron-5569) at a displacement rate of 10 mm/min with a 5 N load cell at room temperature. For antibacterial assessment, Gram-positive Staphylococcus aureus (ATCC 12600) and Gramnegative E. coli (ATCC 9637) were employed in disc diffusion testing and to determine the number of colony forming units (CFUs), with gentamicin used as a control according to [33].

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Journal Pre-proof The antibacterial effect of the samples incorporating different amounts of sericin was determined by evaluating the inhibition area (IA). To assess cell attachment on the fiber layers, an L929 fibroblast cell line at a concentration of 2×104 cells/mL was seeded on sterilized SS-containing nanofibers and cultured for 3 d, after which the cell scaffold constructs were stained with DAPI (4΄,6-diamidino-2-phenylindole, blue fluorescence in live cells) and then subjected to fluorescence image analysis. All specimens were sterilized using ultraviolet radiation for at least 2 h before the cell experiment. In addition, the morphology of the attached cells was examined utilizing scanning electron microscope (SEM)

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imaging. The cell toxicity of the samples was evaluated utilizing the MTT technique as per a

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previous study; cells were seeded at a concentration of 104 cells/mL in a 24-well plate and allowed to grow for various time periods at 37 ºC [33]. The entire experiment was performed in

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at least triplicate to ensure reproducibility of the results. An average of three measurements was

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considered for each sample.

For in vivo studies, BALB/c mice (male, 20–30 g) were purchased from the Pasteur Institute

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(Tehran, Iran) and acclimatized for 7 d before experiments. Each mouse was subjected to two second-degree burn injuries induced by applying an 85 °C hot plate (0.5 cm diameter) for 10 s.

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The mice were then randomly divided in three groups (n=6 per group) as follows: burns without treatment, burns treated with nanofibers without SS, and burns treated with nanofibers containing

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SS. At 7 d post-injury, mice were euthanized and wounds from the different treatment groups were collected for histological (haemotoxylin and eosin (H&E) and Masson trichrome (MT))

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staining. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Tehran [16]. Results are reported as mean ± standard error (SE), analyzed using SigmaPlot software with p values of < 0.05 (*) to reveal significant differences between all data. 3. Results and Discussion SEM images and plots indicating the diameter distribution of the PVA/CS and PVA/CS/SS nanofibers are depicted in Fig. 2. The SEM images show that nanofibers are beadless, homogeneous, and smooth, confirming that the sericin was properly incorporated inside the fibers and that the average diameter marginally decreased as the amount of the sericin increased. The fibers produced in this investigation were arbitrarily oriented with interconnecting pores, 5

Journal Pre-proof which enhances the exchange/mass transfer of oxygen, fluids, and nutrients [20]. This could be associated with the decrease in the viscosity of the electrospinning solution as the amount of sericin increased, resulting in fewer interactions between polymer molecules via hydrogen bonding [29]. The diameter of the PVA/CS/SS nanofibers decreased with increasing sericin concentration as follows (Fig. 2f): 0 SS (425 ± 34 nm), 1 SS (397 ± 29 nm), 2 SS (371 ± 27 nm), 3 SS (328 ± 27 nm), and 5 SS (305 ± 26 nm). The range in fiber diameters attained was fairly comparable to that of collagen fibrils (10-300 nm) present in native tissue [14], implying that these nanofibers will be able to support cell growth.

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Wettability of the wound dressing is an essential component for identifying its appropriateness

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pertaining to biomedical applications. The fiber mats should have outstanding hydrophilicity in order to be employed as wound dressings [34]. Contact angle was measured to evaluate the

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hydrophilicity of various nanofibers scaffolds (Fig. 3a). The hydrophilicity of the PVA/CS/SS

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nanofibers increased with increasing sericin concentration, which should enhance the attachment of human fibroblast cells on the surface of the nanofibers [27]. The surface of the nanofibers

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with and without sericin had contact angles of 24 ± 2.0 (PVA/CS/5SS) and 42 ± 3.1° (PVA/CS), respectively (Fig. 3b). SS and CS polymers both possess hydrophilic characteristics due to

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interacting functional groups, including OH, COOH, and NH2, which generates a suitable template to be employed for manufacturing skin repair materials [35].

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Swelling is an essential factor for wound dressing nanofibers as it influences the incorporation of antibacterial agents and drugs. The pattern of swelling ratio of the PVA/CS/SS nanofibers is

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depicted in Fig. 3c. The swelling ratio rises nearly linearly with increasing sericin content up to 5 w/v%. Sericin is very hydrophilic as it features a number of polar chemical groups [25]. Incorporating sericin into the nanofibers resulted in swelling ratios of ~325-650%, which is greater than PVA/CS (325%) and implies the nanofibers containing sericin have outstanding swellability. High hydrophilicity and swellability of PVA/CS/SS nanofibers will assist in further absorption of fluid infiltrating the wound. PVA/CS/5SS nanofibers had a substantially greater swelling capacity (650 ± 30%) than other nanofibers. This high swelling ratio could be due to the formation of a more flexible network due to intra-polymer chain reactions, increasing the flexibility and number of hydrophilic groups on the nanofibers containing sericin [35-37]. Furthermore, the greater swelling ratio is attributed to the higher hydrophilicity and water

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Journal Pre-proof absorption capabilities of the free -COOH groups and free -NH2 in the nanofibers containing sericin compared to only the free -COOH groups in nanofibers without SS [25]. The capability of a wound dressing control the water loss is often identified through the water vapor transmission rate (WVTR). The perfect WVTR value needs to be made up between 2000 and 2500 g/m2/day to assist in the spread, proliferation and attachment of epidermal cells along with the water vapor exchanges [5,31,32]. The WVTR of the PVA/CS/SS nanofibers increased with increasing sericin concentration as follows (Fig. 3d): 0 SS (1872 g/m2/day), 1 SS (2125 g/m2/day), 2 SS (2203 g/m2/day), 3 SS (2296 g/m2/day), and 5 SS (2387 g/m2/day). The obtained

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WVTR values are in the range of an excellent dressing, implying our fabricated nanofiber might

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result in generate an good environment contain perfect moisture level for exudative wounds

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without having too much dehydration and appropriate as wound dressing material [3,30,32]. Mechanical performance is essential because the matrix must possess the appropriate mechanical

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strength to assist in the healing of tissue, thereby resulting in skin repair [25]. Fig. 4a shows the stress-strain curves for the PVA/CS/SS nanofibers. The tensile strength (Fig. 4b) of the

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nanofibers diminished with increasing sericin content while the strain at break tended to increase (Fig. 4c). This trend indicates that the addition of sericin decreases the mechanical strength but

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simultaneously increases the flexibility of the nanofibers. PVA/CS/SS nanofibers with higher amounts of sericin demonstrate a longer plastic region compared to nanofibers without SS. In

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this regard, the blending of sericin and PVA-based polymer resulted in the creation of a

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hydrogen bonds network among the hydroxyl groups of PVA and the polar amino acid residues of sericin, which considerably improved the mechanical characteristics [16]. Visualizing cell morphology was used as a primary method to evaluate the performance of the fibers in terms of cell adhesion and proliferation. The SEM images in Fig. 5a shows that L929 cells could successfully proliferate on PVA/CS/SS nanofibers with a low sericin content. Moreover, nanofibers containing low amounts of sericin exhibited considerably greater proliferation of fibroblasts compared to nanofibers without sericin (PVA/CS) after 7 d of incubation. In this context, nanofibers with sericin exhibit a spread-out morphology of L929 cells together with a greater density, which additionally confirms the superior characteristics of sericin in terms of supporting cell proliferation. This can be attributed to the hydrophilic nature of sericin supporting the attachment and proliferation of cells [14]. This enhancement could 7

Journal Pre-proof potentially accelerate skin repair by sustaining the surrounding moisture and assisting in cell migration. Fluorescence imaging revealed the extent of cell adhesion on sericin-containing nanofibers after 3 d (Fig. 5b). PVA/CS/SS nanofibers with low amounts of SS supported cell adhesion and viability to a greater extent than nanofibers with higher amounts or without sericin (PVA/CS). The favorable influence of the incorporation of a minimal concentration of sericin demonstrates its great potential to enhance cell viability. An MTT assay was conducted to further assess the biocompatibility of the nanofibers, which is essential for their potential use in clinical treatments

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[25]. To determine the cytocompatibility of the PVA/CS/SS nanofibers, we evaluated the

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influence of the nanofibers on L929 fibroblast cell viability. Fig. 5c depicts L929 fibroblasts in direct contact with the nanofibers in culture media as employed to measure the viability of cells

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and mimic a wound dressing. The MTT assay demonstrated in the presence of SS-containing

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nanofibers a cell viability in the range of 75–102% after 3 d and 86–118% after 7 d (Fig. 5d). This trend indicates that the cell viability of the sericin-containing nanofibers increased with

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incubation time and that the fibers were effective in supporting cell proliferation. The cell viability of the nanofibers with a low sericin content was greater than without sericin (PVA/CS)

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or with a high sericin content (PVA/CS/5SS). These results indicate that the nanofibers with low sericin content are biocompatible and exhibit less cytotoxicity than the nanofibers with high

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sericin amount [30]. This result concurs with previous work that indicates sericin does not lead to inflammatory reactions but instead accelerates the skin repair process [20]. In this context, other

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studies [20] indicate it is safe to employ the silk protein sericin for the development of biomaterials. However, another study [38,39] reports that sericin concentrations above a certain amount lead to some cytotoxic effects. This is most likely because more unbound sericin was released into the culture medium during the early days of the incubation. Likewise, the result of other research demonstrate [40] that gelatin films containing high amounts of sericin presented low cell viability after a few days of culturing which may owing to leaching of sericin molecules in preliminary stages from Gel with high SS (5-7.5 wt.%). To evaluate the possible application of the drug loaded PVA/CS/SS as a wound dressing, TCN was incorporated PVA/CS/SS for the further studies. The TCN drug release model presented in Fig. 6a, while drug release pattern could be split up into two phases: rapid and steady (Fig. 6b). The rapid stage lasted for approximately 12 h, which 8

Journal Pre-proof could possibly be due to the release of TCN from the PVA/CS/SS surface; in the steady phase, TCN was gradually released from the PVA/CS/SS nanofibers. An ideal wound dressing would present a sustainable drug release capability to minimize the need for regular replacement of the dressing, hence lessening the risk of exposure of the wound to bacteria [1,18,29,30]. The raising amount of TCN from 1 to 5 wt.% results in an enhance in TCN generating, implying release rate to elevate in a content level‐ dependent fashion, where PVA/CS/5SS-TCN revealed more rapidly release in comparison with PVA/CS/1SS-TCN. This might be owing to the fact the diameter of PVA/CS/5SS-TCN nanofibers is evidently smaller in comparison with PVA/CS/1SS-TCN, which

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reduced the TCN diffusion distance from the matrix of nanofiber to the release medium.

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Inhibition area was measured to determine the antibacterial performance of neat PVA and PVA/CS/SS-TCN toward Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria. The

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results indicate PVA itself fails to form an inhibition area and does not affect bacterial

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expansion. However, PVA/CS and PVA/CS/SS-TCN nanofibers formed clear inhibition areas and substantially inhibited bacterial expansion (Fig. 7a). The diameter of the inhibition area

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increased with SS-TCN concentration (Fig. 7b), implying that increasing SS-TCN augments antibacterial performance and drastically inhibits the growth of E. coli and S. aureus. The

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PVA/CS/SS-TCN nanofibers reduced bacterial expansion in vitro, and this notable action towards each type of bacteria would likely aid in reducing secondary infection. The broad-

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spectrum antimicrobial performance towards E. coli and S. aureus could be due to the particular structures and compositions of their cell membranes [27, 37]; the E. coli cell membrane has a

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more complex structure that includes numerous polysaccharides, peptidoglycan, and proteins. Fig. 7c presents the percent reduction in S. aureus and E. coli for the nanofibers with different amounts of SS-TCN (0 to 5 wt%). With the combination of SS and TCN, significant bacterial reduction was noted for both bacteria and this decrease diminished with increasing SS-TCN concentration in the nanofibers. Again, no antibacterial activity was noted for PVA fibers without SS-TCN. A feasible antibacterial mechanism for the nanofibers containing SS-TCN is illustrated in Fig. 7d. In this respect, numerous antibacterial mechanisms regarding the nanofibers containing sericin and drug have been proposed. Although the mechanism behind the sericin anit-bacterial activity may have not been well elucidated or documented, the authors believe the mechanism behind be more complicated than the explanation of using the cell surface charges alone [38-40]. 9

Journal Pre-proof Some other examinations [24, 28] suggested that the synergistic influence of sericin and antibiotic TCN together enhance the antimicrobial performance of the nanofibers. In this view, TCN offers an outstanding bactericidal performance toward a number of microorganisms including E. coli and S. aureus bacteria and is regularly employed for clinical infection treatment [24,41,42]. While, sericin with low molecular weight might easily infiltrate the bacteria cells and combine with anion components in bacterial cells, which leads to an impairment of the cell integrity [27]. Similarly, it was also depicted [43,44] that sericin presents antioxidant performance owing to its free radical scavenging capacity. However, in this study the dominant

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mechanism of antibacterial performance for the SS-TCN containing nanofibers is attributed to

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the release of 76% of the total TCN concentration from the nanofibers within 24 h, which appears to be adequate to prevent the bacterial growth throughout its penetration onto the agar. In

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this context, it was suggested [20] that the presence of a great amount of sericin in nanofibers escalates the release of TCN from the nanofibers and hence, they experience a significantly

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better probability of a more effective TCN delivery. Bacteriostatic effects of TCN are attributed

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to the infiltration of TCN into the cell membrane cytoplasm and amalgamation within the cells which leads to the prevention of protein synthesis [24, 28] and also subsequent disruption of the

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regular functions of bacteria and their ultimate death. Fig. 8 shows the H&E and MT staining results for tissues post injury from wounds treated with dressings made from the different fiber groups. The in vivo experiment results indicate a smaller

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number of macrophages and fewer inflammatory cells in the blood vessels of skin tissues treated

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with PVA/CS/2SS-TCN wound dressings; this is in contrast to the severe inflammatory response including infiltration of macrophages and neutrophils observed in the gauze-treated group. The H&E staining images show that the PVA/CS/2SS-TCN nanofiber obviously improves healing compared to the traditional clinical grade gauze and PVA/CS, as there is an obvious epithelialization along with many newly formed blood vessels. Images of thicker and freshly emerging epidermal layers in tissues treated with PVA/CS/2SS-TCN nanofiber indicate that healing began earlier in these animals and should lead to full repair of the skin tissue. The reconstruction of the impaired tissue with compact and thick collagen fibers was likewise noticed in the MT staining. The presence of hair follicle cells together with sebaceous glands in the tissue treated with PVA/CS/2SS-TCN nanofiber demonstrates the potential of these fibers to support the wound healing process. In the images associated with gauze or PVA/CS fiber 10

Journal Pre-proof dressing treatments, the thickness of the epidermal layer was less, irregular collagen was evident, and the regeneration of hair follicles was low. The MT staining shows that the collagen fibers in the PVA/CS/2SS-TCN nanofiber groups formed well-organized bundles and were denser and thicker. However, the control (gauze) group and PVA/CS group displayed the least amount of collagen fibers and mainly loose collagen. Animals in the PVA/CS/2SS-TCN nanofiber demonstrated almost full coverage of the wound by the end of the treatment procedure, implying the electrospun nanofibers are able to support healing in the wound area.

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4. Conclusions In this study, sericin-tetracycline (SS-TCN) was successfully incorporated into poly(vinyl

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alcohol)/chitosan (PVA/CS) nanofibers using electrospinning technology. The novel nanofibers

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containing sericin were continuous, with a uniform diameter distribution between 305 and 425 nm. Furthermore, increased SS content led to an increase in the swelling capacity and flexibility

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but a decline in mechanical strength. In vitro cell investigations demonstrated that nanofibers with low sericin content display considerably greater adhesion and higher proliferation potential

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for L929 cells in comparison to nanofibers without sericin (PVA/CS). In addition, the nanofibers loaded with SS-TCN demonstrated excellent bactericidal performance towards both Gram-

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positive and Gram-negative bacteria, with better reduction at more elevated concentrations. In vivo results indicated that the application of the PVA/CS/2SS-TCN nanofiber results in skin

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repair along with re-epithelialization and formation of compact collagen fibrils; this further

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confirms the benefits of employing the PVA/CS/2SS-TCN nanofiber over traditional clinical gauze for wound dressing applications.

Acknowledgements The authors acknowledge support from the Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Foundation for Innovation (CFI), and Universiti Teknologi Malaysia (UTM) for this research.

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Journal Pre-proof Figure Captions Fig. 1. A schematic representation of the preparation and characterization of PVA/CS/SS-TCN electrospun nanofibers. Fig. 2. SEM images and diameter distribution of PVA/CS/SS nanofibers with sericin concentrations of (a) 0, (b) 1, (c) 2, (d) 3, and (e) 5 w/v% and (f) variation in fiber diameter with varying w/v% sericin concentration. Fig. 3. (a) Images of water contact angle, (b) comparison of water contact angle for varying concentrations of sericin (SS), (c) swelling behaviour and (d) WVTR of PVA/CS/SS nanofibers

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with various concentrations of SS (*p < 0.05).

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Fig. 4. Graphical presentation of (a) stress-strain curves, (b) tensile strength, and (c) strain at break of PVA/CS/SS nanofibers with various sericin concentrations.

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Fig. 5. (a) SEM images, (b) DAPI staining, (c), wound infection model, and (d) cell viability on PVA/CS/SS nanofiber with various sericin concentrations (*p < 0.05).

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Fig. 6. (a) drug release model and (b) TCN release profile of the PVA/CS/SS nanofiber scaffolds

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(*p < 0.05).

Fig. 7. (a) Images of inhibition zones, (b) measurements of growth inhibition zones, (c) percent

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bacterial inhibition for E. coli and S. aureus for PVA/CS/TCN nanofibers with various sericin concentrations, and (d) schematic illustration of the antimicrobial mechanism of the nanofibers containing sericin. Note: asterisks represent for statistically significant differences in comparison

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to the control group (p < 0.05).

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Fig. 8. Images of H&E and MT staining of wound sites 7 d after treatment with gauze, PVA/CS, and PVA/CS/2SS-TCN (H: hair follicles; BV: blood vessels; EP: epithelialization; IF: inflammatory cells; C: collagen; LC: loose collagen; SG: sebaceous glands; R: rupture; E: newly generated epidermis; scale bar=200 µm).

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Cell proliferation

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Fig. 1. A schematic representation of the preparation and characterization of PVA/CS/SS-TCN electrospun nanofibers.

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Fig. 2. SEM images and diameter distribution of PVA/CS/SS nanofibers with sericin concentrations of (a) 0, (b) 1, (c) 2, (d) 3, and (e) 5 w/v% and (f) variation in fiber diameter with varying w/v% sericin concentration.

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Fig. 3. (a) Images of water contact angle, (b) comparison of water contact angle for varying concentrations of sericin (SS), (c) swelling behaviour and (d) WVTR of PVA/CS/SS nanofibers with various concentrations of SS (*p < 0.05).

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Fig. 4. Graphical presentation of (a) stress-strain curves, (b) tensile strength, and (c) strain at break of PVA/CS/SS nanofibers with various sericin concentrations.

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Fig. 5. (a) SEM images, (b) DAPI staining, (c), wound infection model, and (d) cell viability on PVA/CS/SS nanofiber with various sericin concentrations (*p < 0.05).

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Fig. 6. (a) drug release model and (b) TCN release profile of the PVA/CS/SS nanofiber scaffolds (*p < 0.05).

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PVA/CS/SS-TCN nanofibers

Fig. 7. (a) Images of inhibition zones, (b) measurements of growth inhibition zones, (c) percent bacterial inhibition for E. coli and S. aureus for PVA/CS/SS-TCN nanofibers with various SS-TCN concentrations, and (d) schematic illustration of the antimicrobial mechanism of the nanofibers containing SS-TCN. Note: asterisks represent for statistically significant differences in comparison to the control group (p < 0.05).

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Fig. 8. Images of H&E and MT staining of wound sites 7 d after treatment with gauze, PVA/CS, and PVA/CS/2SS-TCN (H: hair follicles; BV: blood vessels; EP: epithelialization; IF: inflammatory cells; C: collagen; LC: loose collagen; SG: sebaceous glands; R: rupture; E: newly generated epidermis; scale bar=200 µm).

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Graphical abstract

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Authorship contribution H.R. Bakhsheshi-Rad: Conceptualization, Data curation, Writing - original draft, Formal analysis. A.F. Ismail: Supervision, Visualization, Writing - review & editing. M. Aziz: Supervision, Visualization, Writing - review & editing. Z. Hadisi: Conceptualization, Methodology, Data curation, Formal analysis.

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M. Akbari: Supervision, Visualization, Writing - review & editing.

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M. Omidi: Supervision, Visualization, Writing - review & editing.

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X.B. Chen: Supervision, Visualization, Writing - review & editing.

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Journal Pre-proof Highlights Functional PVA/CS/TCN/SS nanofibers were prepared using the electrospinning method. Incoportion of sericin into nanofiber results in superior cytocompatibility than PVA/CS/TCN. Antibacterial performance of nanofiber mats increases with increasing sericin content.

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Antibacterial mechanism of the nanofiber-contaning sericin is proposed.

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