Silk Sericin-Functionalized Bacterial Cellulose as a Potential Wound

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Silk Sericin-Functionalized Bacterial Cellulose as a Potential Wound-Healing Biomaterial Lallepak LAMBONI, Ying Li, Ji an feng Liu, and Guang Yang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00995 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 30, 2016

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Silk Sericin-Functionalized Bacterial Cellulose as a Potential Wound-Healing Biomaterial Lallepak Lamboni,aYing Li,bJianfengLiu,cGuang Yanga* a

Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, P.R. China.

b

Department of Chemistry Institute for Advanced Study,Division of Biomedical Engineering, Hong Kong University of Science and Technology, Clear Water Bay , Kowloon, Hong Kong , China. c

The Key Lab of Molecular Biophysics of MOE, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China).

KEYWORDS: Bacterial cellulose, silk sericin, wound healing, tissue engineering, biomaterials. ABSTRACT: Bacterial cellulose (BC) is a polysaccharide known as a suitable matrix for proper wound healing. To improve this ability, BC was functionalized with silk sericin (SS) which has cytoprotective and mitogenic effects. The composites obtained by solution impregnation were stabilized by hydrogen bonds, and SS could be released in a controlled manner. The constructs were highly porous, with interconnected pores, allowing for high water uptake that varied with the SS concentration used for sample preparation. While SS did not disrupt the stability of the BC network, soluble SS diffusing from the composites did not influence keratinocyte growth but enhanced fibroblast proliferation, which would further optimize the wound healing process and improve extracellular matrix production, accelerating healing. Further, improved cell viability was observed upon the composites. Due to their attractive structure and properties, these BC-SS biomaterials represent potential candidates not only for wound dressing applications but also for tissue engineering.

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INTRODUCTION Wound dressing is a biomaterial designed to create a suitable micro environment for cell adhesion and proper proliferation in order to restore the physiological and structural properties of wounded skin.1,2 It controls cell behavior by regulating physical, chemical, and biological signals through its surface microarchitecture.1 Whether used as temporary coverage or meant for chronic applications, wound dressings are of capital importance especially for the treatment of large wounds, and may be critical, if not life-saving in certain cases such as extensive burns. In fact, according to the World Health Organization, over 300,000 people die of burn injuries, making skin wounds a major burden both socially and financially.3 This high rate is due in part to poor skin healing ability, additionally to the limitations of current treatments, mostly the organ donor limitation.4 Therefore, despite the numerous wound dressing methods available to date, there is still a pressing need for improved ones, as the former only meet part of the requirements for an ideal wound dressing.3,5 The ideal wound dressing as currently described represents an effective barrier to micro-organisms, which absorbs wound exudate and allows gas exchange, while keeping the necessary moisture at the wound interface without toxicity, allergenic responses, and can be removed from the wound with minimum pain. This should be made from readily available and affordable materials that require minimum processing.6,7 In this context, nanofibrous materials, which have attracted significant interest as scaffolds in current tissue engineering strategies aiming for the repair of various tissues including skin, have shown to improve biocompatibility.8,9 Due to their high porosity, they enable cell adhesion, growth, and differentiation, while further allowing for the delivery of bioactive molecules.9,10

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In recent years, bacterial cellulose (BC), a natural polysaccharide, has shown great potential as a biomaterial for tissue engineering applications including skin tissue repair.11 Because of its nanofibrillar structure, BC represents an adequate matrix for optimal wound healing.12 In fact, similarity with collagenous fibers facilitates the interaction between cells and BC fibers, improving its overall biocompatibility.13,14 It is biodegradable and offers good mechanical properties and high hydrophilicity, all of which contribute to its success in tissue engineering.1,11 Conversely, silk sericin (SS) is a natural hydrophilic protein and a byproduct of the silk industry which biomedical properties have been well elucidated.15,16 It has cytoprotective and mitogenic effects on mammalian cells, with regenerative abilities on mammalian tissues;17-19 particularly, its positive effects on fibroblasts and keratinocytes,20 major cell types in skin, has made it very attractive for the development of skin tissue repair materials.21 In addition to its potential for enhancing collagen production and accelerating wound healing,22,23 SS has shown antimicrobial properties necessary for a wound dressing.24,25 Nevertheless, due to its amorphous nature, SS only forms fragile materials unsuitable for biomedical applications. Interestingly, it has polar side chains with diverse functional groups (amine, hydroxyl, carboxyl groups) that allow interaction with other compounds through blending, cross-linking or copolymerization to yield improved biomaterials.26 This study aims at developing an improved wound dressing material which would accelerate wound healing, with BC as the niche for optimal wound healing, and SS as the bioactive molecule that regulates cell behavior and accelerates the healing process thereby reducing scar formation.27

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EXPERIMENTAL SECTION Materials: Bombyx mori cocoons from Ningbo Industrial Co., Ltd. (China) were used. The bacterial strain Acetobacter xylinum (ATCC53582) was purchased from the American Type Culture Collection (ATCC), while the NIH-3T3 and HaCaT cell lines were obtained from China Infrastructure of Cell Line Resources. Yeast extract and peptone were provided from Beijing Shuangxuan Microbe Culture Medium Products Factory (China), with all other reagents of chemical grade except for poly (ethylene glycol) (PEG, Biosharp, China) purchased from Sigma. Dulbecco modified eagle medium (DMEM), fetal bovine serum (FBS), and trypsin-EDTA (TE) were products of Gibco. Penicillin / streptomycin mixture was obtained from Beyotime (China). Phosphate buffered saline (PBS) solution was supplied from Hyclone, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) powder from Sigma, and alamarBlue from Thermo Fisher Scientific. Fluorescein isothiocyanate labeled phalloidin (FITC-Phalloidin) was provided from Sigma, Hoechst 33342 from Invitrogen (Life Technologies), formaldehyde from Solarbio (Beijing, China) and triton X-100 from Amresco. The BCA (bicinchoninic acid) kit was purchased from Pierce, Thermoscientific. Preparation of bacterial cellulose films: BC was produced in Hestrin & Schramm (HS) medium from Acetobacter xylinum grown in static cultures at 30 °C. The HS medium was composed as follows: citric acid (0.15%, w/v), disodium phosphate (0.27%, w/v), yeast extract (0.5%, w/v),peptone (0.5%, w/v), and glucose (2%, w/v).28 The films were allowed to grow to a thickness of 2-3 mm after which they were harvested and subjected to cleaning steps to remove the bacteria and the adsorbed HS medium. They were washed 3 days in distilled water, then boiled in NaOH (1 wt%) for 40–45minutes, and subsequently washed in purified water to neutralize the pH. The clean BC membranes were autoclaved in purified 4

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water and stored at 4°C for further use. For different experimental purposes, various BC sizes were obtained from different sizes culture flasks or polystyrene cell culture vessels. Extraction of silk sericin from Bombyx mori cocoons and preparation of BC-SS composites. SS was extracted from Bombyx mori cocoons by a method described by Lee et al.29 with slight modification. The cocoons were cut into small pieces and boiled under pressure at 120 °C for 1 hour in purified water (1g of cocoon/10 mL water). Fibroin fibers were removed from the resulting soup by filtration followed by centrifugation. SS solution was then concentrated by dialysis against PEG solution, and lyophilized. The powder was stored at – 20 °C until use. The absorbance profile of the extract between 700 and 190 nm was scanned by UV spectroscopy (UV-1600 PC spectrophotometer, MAPADA, coupled to “Mwave –Professional 2” software), from a solution sample constituted as described further in this section. BC-SS composites were prepared by solution impregnation as depicted in Figure 1. To obtain SS solution, SS powder was re-suspended in purified water and dissolved by boiling for 20 min.30 The solution was further filtered through Minisart® high flow syringe filters (pore size 0.2 µm, Sartorius Stedim) for the removal of particulate impurities. Sterile filters were used when sterilization was required. Different constructs (BC-SS1:24, BC-SS2:24, BC-SS3:24) were thus fabricated, by varying SS concentration (1, 2 and 3 % (w/v), respectively).

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Figure 1. Preparation of SS powder, BC pellicles, and BC-SS composites. (c = [SS] in % w/v, 1, 2, 3; A. xylinum = Acetobacter xylinum; HS = Hestrin and Schramm) Scanning electron microscope (SEM), Brunauer-Emmett-Teller (BET), and Fourier transform infrared (FTIR) analyses. SEM (NanoSEM 450) observation was carried out to examine the morphology of the constructs and confirm the incorporation of SS into the BC network. Cross-section imaging was performed after platinum coating (Precision etching coating system, Gatan, Model 682). Samples were prepared by freeze-drying (48 h freezing at – 20 °C, and 48 h lyophilization at – 50 °C), and cross-sections were achieved after steeping in liquid nitrogen. For SS sample, the lyophilized powder was gold-coated and observed similarly to the scaffolds. The microstructure of the membranes was further examined by BET analysis using Micromeritics ASAP 2020 (ASAP 2020 V4.00 (V4.00 H)). For this purpose, 6-well plates BC membranes were composited with SS and frozen-dried as indicated above. The membranes were weighed, placed in the automate, and subjected to a dehydrating phase where they were 6

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heated up to 120 °C to get rid of resident moisture. After cooling the samples back to ambient temperature, the surface area and porosity measurements where performed, using nitrogen as the adsorptive gas, with an equilibration time of 10 s. The chemical interaction between BC and SS was studied by FTIR (Vertex 70, Bruker). The samples were prepared by freeze-drying, and analyzed between 4000 and 450 cm-1 with the spectra obtained at a resolution of 4 cm-1 and a number of accumulated scans of 16. In vitro SS release from the samples: The physical integrity of the composites was assessed by measuring the release of SS at different time intervals while the never-dried samples were immersed in a known volume of PBS (1X, pH 7.4) containing sodium azide (0.02 wt%) to avoid contamination. For this purpose, BC was synthesized in 6-well cell culture plates (ø 34.8 mm; 9.5 cm2). The experiments were conducted at 37 °C over 7 time-points including 0, 1, 15, 30 min, 1, 2 and 3 days, respectively. The cumulated SS content at each time interval was determined by colorimetric test using the BCA protein assay kit. The absorbance was measured in a microplate reader (318C Microplate Reader). For each time-point, 3 replicates were used, and the experiment was repeated 3 times. Thermal and mechanical stability of the composites: The thermal behavior of the composites was determined by thermogrametric analysis (TGA) (Perkin Elmer Instruments). The samples were heated from room temperature to 600 °C, at a rate of 10 °C/min, under nitrogen atmosphere. A tensile test was performed on the samples in wet state, using the model CMT6503 of SANS Universal Testing Machines (SANS Test Machine Co. Ltd. Shenzhen, China). The test specimens, dumbbell-shaped, were cut out of the never-dried films using a mold of similar shape. For each sample, 3 specimens were tested. The tensile strength and Young’s modulus 7

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were thereafter determined. Water uptake ability: The swelling behavior of the composites in distilled water was studied by gravimetric method, as reported by Mandal et al.31, with slight modification. After freeze-drying, the dry weight (Wd) of each sample was measured. The sample was then placed in distilled water and left to swell at 37 °C in a closed container, after which the weight of the swollen sample (Ws) was measured. The swelling ratio was calculated according to the equation below: Swelling ratio = (Ws-Wd)/Wd The measurements were carried out on independent samples for different time intervals, 6, 12, 24, 48 and 72 h respectively (n = 3), using 24-well plates BC membranes (ø 15.6 mm; 1.9 cm2). Biocompatibility testing of the samples by use of extracts. To study the effect of the films on the cell behavior, the NIH-3T3 (mouse embryonic fibroblast cell line) and HaCaT (human skin keratinocytes) cell lines were used. Cells were routinely cultured in culture flasks in high glucose (4.5 g L-1) DMEM containing L-glutamin and pyruvate (110g L-1), supplemented with 10 % FBS (Gibco, USA) and 1 % penicillin / streptomycin, at 37 °C under an atmosphere of 5 % CO2. While fibroblasts were passaged at 70-80 % confluence using trypsin / EDTA, keratinocytes were sub-cultured at a confluence of 80-90 %. For both cell lines, culture media were changed every 2-3 days. For in vitro tests, cells from passages 6-9 were used. The cytotoxicity of the samples’ extracts was tested according to the international standard ISO 10993-5-2009 dedicated to in vitro evaluation of medical devices (Reference number. ISO 10993-5:2009(E)). Prior to extraction, un-modified BC and BC-SS samples were washed 3 times in PBS (1X, pH 7.4) and conditioned in culture media for 30 min at room temperature. 8

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The extracts were then obtained by incubating the scaffolds (BC and composites, 2-3 mm thick) at 37 °C in fresh complete culture media at the ratio of 1.25 cm2/mL. Before testing, cells were seeded at 10000 cells/ well directly into 96-well cell culture plate’s wells and cultured for 24 h. Thereafter, the culture media were fully replaced with the extracts, and the cells were incubated for further 1 and 3 days, respectively. Normal complete cell culture medium was used as control. The cell viability was measured by MTT assay. Briefly, the yellow MTT powder was solubilized in PBS (5 mg mL-1), microfilter-sterilized (Minisart® high flow syringe filters, pore size 0.2 µm, Sartorius Stedim), and used as indicated by the manufacturer. The MTT solution was aseptically added to ongoing cultures at a ratio of 1/10 relatively to the volume of culture media in individual wells, after replenishment of the latter. Cultures were put back to incubation for 4h, after which the MTT was converted to purple formazan crystals by mitochondrial dehydrogenases in viable cells. A syringe (1 mL) was used to gently remove the supernatant medium, as the crystals were formed on the bottom of the wells. Formazan was dissolved at 37 °C, using DMSO as solvent. The optical density (OD) in different wells was read at 492 nm, using a microplate reader (318C Microplate Reader). The cell viability which directly correlates with the absorbance of solubilized formazan solutions was expressed as the OD of representative wells, after blank corrections. The experiments were performed in triplicates. AlamarBlue assay: Fibroblast and keratinocyte cell proliferation on the constructs was quantified by alamarBlue assay, respectively. The test works on the principle that metabolizing cells reduce the blue oxidized form of the dye into a red form, which absorbance or fluorescence may be monitored. Cells were seeded onto the scaffolds (10000 cells/ scaffold) in 96-well plates, after confluence on culture flasks. Before cell seeding, the samples were prepared as described above. They were washed 3 times in PBS and incubated 9

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in culture media for 30 min at room temperature. They were then laid in new wells and kept in the cell incubator for about 10-15 min to level their temperature to 37 °C. The cell suspension was then seeded onto the scaffolds in a drop-wise manner, and put to incubation for 2 h to allow the cells to settle and attach to the membranes. Culture media were thereafter gently added into the wells, and the plates were incubated as routinely done. Empty polystyrene cell culture wells (designated as PS) were used as control, while 2 types of blanks, medium only and BC + medium (without SS modification) were employed. The culture was stopped at different time-points to assess the cell response to the scaffolds, with different specimens being used for the different time-points. For each sample type, the experiment was done in triplicate. For our scaffolds, the alamarBlue assay was performed as follows: At different time-points (1, 3 and 5 days), exhausted media from the cultures were replaced with fresh ones, and the cultures put back to incubation for at least 1h before addition of alamarBlue solution at a ratio of 1/10 relatively to the volume of culture media per well. The test plates were incubated for 3h in the cell incubator, after which aliquots of the supernatant mixture were taken for measurements. The absorbance was read at 570 nm with the reference wavelength of 630 nm in a microplate reader (318C Microplate Reader), and the percentage of reduced alamarBlue which reflects the cell growth was then calculated for each sample. Confocal microscopy: To visualize the cells onto the tested samples, the seeded cells were stained for actin filaments. Coverslips (CS) were used as controls. The cell-laden scaffolds and CS were washed in PBS and fixed with 4 % formaldehyde for 20 min, followed by a permeabilization step with 0.1 % triton X-100 after a 2nd washing step. They were then washed and stained for 20 min at room temperature with FITC-Phalloidin solution (15 µg mL-1) made up in DMSO and diluted in PBS. The constructs were washed again, and 10

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counterstained for 30 min at room temperature with Hoechst 33342 diluted in purified water (5 µg mL-1). The constructs then went through a final washing step with purified water, before being kept in PBS until mounting for microscopy. CS were mounted using mounting medium, while the cell-loaded scaffolds were mounted in PBS. Confocal images were captured using Ti Nikon Eclipse confocal microscope. STATISTICAL ANALYSIS For comparison among groups, One-way Analysis of Variance (ANOVA) test was used with Tukey's Multiple Comparison test for the post-hoc pair-wise comparisons, in OriginPro 8 software. Statistical significance was obtained at P