Facile preparation of a strong chitosan-silk biocomposite film

Journal Pre-proof Facile preparation of a strong chitosan-silk biocomposite film Jiwei Huang, Jianzhong Qin, Peng Zhang, Xuanmo Chen, Xinran You, Feng Zhang, Baoqi Zuo, Mu Yao

PII:

S0144-8617(19)31183-X

DOI:

https://doi.org/10.1016/j.carbpol.2019.115515

Reference:

CARP 115515

To appear in:

Carbohydrate Polymers

Received Date:

10 July 2019

Revised Date:

20 October 2019

Accepted Date:

20 October 2019

Please cite this article as: Huang J, Qin J, Zhang P, Chen X, You X, Zhang F, Zuo B, Yao M, Facile preparation of a strong chitosan-silk biocomposite film, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115515

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Facile preparation of a strong chitosan-silk biocomposite film Jiwei Huanga,b,1, Jianzhong Qinc,1, Peng Zhangc, Xuanmo Chenb, Xinran Youd*, Feng Zhange,f*, Baoqi Zuoa, Mu Yaog a

Department of Textile Engineering, College of Textile and Clothing Engineering,

Soochow University, Suzhou, 215001, China b

Department of Biological and Chemical Engineering, Guangxi University of Science

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and Technology, Liuzhou, 545006, China c

Department of Orthopedics, The Second Affiliated Hospital of Soochow University,

Suzhou, 215004, China d

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Medical University, Suzhou, 215000, China

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Department of Medical Laboratory, The Affiliated Suzhou Hospital of Nanjing

Department of Immunology, School of Biology and Basic Medical Sciences, Soochow

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University, Suzhou, 215123, China f

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The Affiliated stomatological Hospital of Soochow University，Suzhou Stomatological

Hospital, Suzhou, 215005, China

Faculty of Textile & Material, Xi’an Polytechnic University, Xi’an 710048, China

*

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Corresponding authors: [email protected] (X. You); [email protected] (F.

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Zhang). 1

Co-first author with equal contribution to this work.

Highlights •

Mechanically robust chitosan-silk (CS) composite films were prepared.

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The CS composite films shows a specific nanostructure. 1

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The CS composite films displays a strong hydrogen bonding interaction.

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The CS composite films shows good cell biocompatibility.

Abstract Chitosan-silk biocomposite films with nanofibrous structures have been prepared by facile solution casting of chitosan and silk co-dissolved in formic acid. The morphology,

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structure and mechanical properties of the chitosan-silk biocomposite were characterized by SEM, FTIR, TG-DSC, and mechanical testing. The results

demonstrate that the prepared biocomposite films with a chitosan-silk ratio of 3:1 shows

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a high tensile strength of 97.8 MPa, a strain at break of 10.8% and a Young’s modulus of 3.5 GPa, indicating its high strength and elasticity. Also, the preliminary cell culture

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experiment demonstrated the ideal biocompatibility of chitosan-silk composite films.

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As a result the superior mechanical properties of this composite film can be attributed to the silk nanofibrils and chitosan self-assembled nanofibers, and the strong hydrogen

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bonding interaction between the silk nanofibril and chitosan nanofibers. The specific nanostructure, enhanced mechanical properties, and biocompatibility make the

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biocomposite films a promising material for applications in biomedical devices.

Keywords: Composite films; Chitosan; Silk; Mechanical properties 1. Introduction Silk, once an ancient and desired textile material for its beauty, has become a promising hi-tech material (Omenetto & Kaplan, 2010). Although native silk has unusual mechanical properties (Keten, Xu, Ihle, & Buehler, 2010), regenerated silk 2

films usually show poor strength and brittle performance(da Silva et al., 2017; Tu et al., 2019; C. Zhang et al., 2012), which greatly prevents its extensive application. The geometric nanofibril structure plays a critical role in defining the superior strength, extensibility and toughness of native silk(Giesa, Arslan, Pugno, & Buehler, 2011). However, the multi-scale fibrillar structure is completely destroyed during the silk dissolving process, resulting in the deterioration of the mechanical properties (Holland, Terry, Porter, & Vollrath, 2007). Researchers have prepared regenerated silk films with nanofibrous structures thrgouh self-assembly (C. Zhang et al., 2012) or extension (Yin,

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Chen, Porter, & Shao, 2010), and have achieved improvement in mechanical properties over other regenerated films. Recently, we reported the dissolution of silk in CaCl2formic acid while preserving its nanofibril structure, which signficantly improved the

mechanical performance of the silk nanofibers alone (F. Zhang et al., 2014), and within

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a film (F. Zhang et al., 2015).

The engineering of bioinspired materials with composite components is of great

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interest for replicating the unique mechanical properties of natural structures, such as nacre (Tang, Kotov, Magonov, & Ozturk, 2003), chiton tooth (Gordon & Joester, 2011),

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and the insect cuticle (Ortiz & Boyce, 2008). The common property of these structural materials is the nano-scale interaction between polysaccharides and silk-like protein

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(Benitez, Torres-Rendon, Poutanen, & Walther, 2013; Meyers, Chen, Lin, & Seki, 2008). Therefore, silk-chitosan or chitin composites have been widely explored(Hardy & Scheibel, 2010). However, these composites usually show poor mechanical

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properties likely resulting from the absence of a multi-scale structural hierarchy (H. Kweon, Ha, Um, & Park, 2001; Nerurkar et al., 2009; Niamsa, Srisuwan, Baimark,

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Phinyocheep, & Kittipoom, 2009). Recently, a novel chitosan-silk composite was engineered to resemble the chemical composition and laminar form of an insect cuticle, which exhibited mechanical parity with its natural counterpart (Fernandez & Ingber, 2012). In addition, a biomimetic chitin-silk composite was fabricated from chitin selfassembled nanofibers in a silk matrix, which also showed superior strength and toughness through mimicking the organic phase of the insect cuticle (J. Jin et al., 2013). In this study, we reported the fabrication of strong chitosan-silk composite films 3

with a specific nanostructure by a simple solution casting method. During the solution preparation process, the silk was dissolved into nanofibrils using a CaCl2-formic acid mixture (F. Zhang et al., 2014). The chitosan self-assembled into nanofibers, and then the silk nanofibrils and chitosan nanofibers were casted to form composite films. Compared with previously reported methods for fabricating chitosan-silk composite films, this method was simple, time-saving, and facile to be scaled up. The resulting composite film with a chitosan to silk ratio of 1:3 showed a high tensile strength of 97.8 MPa and modulus of 3.5 GPa, and a failure strain of 10.8%, which are equivalent to that

previously(Fernandez & Ingber, 2012; J. Jin et al., 2013).

2.1. Preparation of chitosan-silk biocomposites

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2. Materials and methods

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of a chitin nanofiber-silk biocomposite and chitosan-fibroin laminates reported

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Silk is purchased from Jiangsu silk industrial Co., Ltd., Nanjing, China. Low molecular weight chitosan (Brookfield viscosity 20.000 cps) was purchased from

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Sigma-Aldrich Chemistry (St. Louis. MO, USA). The degummed silk was dissolved in 4% w/v CaCl2-formic acid to form regenerated silk films as reported previously (F. Zhang et al., 2015). Then the regenerated silk film and chitosan in the designed weight

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ratios were dissolved in formic acid, generating a total concentration of 2% w/v. The resulting chitosan-silk biocomposite films were produced by casting the chitosan-silk

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solutions. The chitosan-silk weight ratios in the dry state were 3:1, 1:1, and 1:3, termed as CS31, CS11, and CS13. These biocomposite films were treated with 1% w/v sodium

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hydroxide to prevent chitosan dissolution and induce fibroin β-sheet transition, and then rinsed with deionized water. Figure 1 summarized the preparation and nanofibrillar structure of chitosan-silk biocomposite films. 2.2. Cell culture In this study, all experimental procedures involving animals were approved by the

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Animal Care and Use Committee of Soochow University. Bone marrow mesenchymal stem cells (BMSCs) were isolated as in our previous report (F. Zhang et al., 2015). . Briefly, BMSCs were derived from 5 week Sprague- Dawley rats (150-200g) by flushing femurs and tibias with low-glucose Dulbecco’s modified Eagle’s medium (LDMEM, Gibco, Carlsbad, CA, USA). The obtained cell suspension was centrifuged at 500 × g for 4 min. The resulting cell suspension was further purified by passing it

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through a 23-gauge needle several times. Cells were cultured in a standard medium composed of L-DMEM with 10% Fetal Bovine Serum (FBS, Gibco, Carlsbad, CA, USA)

and 1% penicillin-streptomycin, and were replaced first at 24 h, then every 72 h. The

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adherent cells were washed twice consecutively in D-Hanks’ balanced salt solution

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(Gibco, Carlsbad, CA, USA). BMSCs at 80-90% confluence were dissociated with 0.25% trypsin-EDTA solution (Sigma, San Francisco, CA, USA) and subcultured at a ratio of 1:2.

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BMSCs at passage 5 (80% confluence) were used for the experiments. 1.0×104 cells

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were seeded on the chitosan-silk biocomposite films (3 repetitions for each group). The cell culture medium was composed of L-DMEM with 10% Fetal Bovine Serum and 1%

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penicillin–streptomycin, and was replaced first at 24 h, then every 72 h. BMSCs grown

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on films were observed every day using inverted phase contrast microscope. 2.3. Characterization The silk films were fractured in liquid nitrogen and observed with S-4800 scanning

electron microscope (SEM, Hitachi, Tokyo, Japan) at 3 kV. Fourier Transform Infrared (FTIR) spectra were obtained using a Magna spectrometer (NicoLET5700, America) in the spectral region of 400-4000 cm-1. XRD spectra were obtained at room temperature with a wide-angle X-ray 5

diffractometer (X’PERT PRO MPD, PANalytical Company, the Netherlands) with CuK radiation, operated at 40 kV and 30 mA. The swelling ratio of the composites was determined with the following equation: Swelling ratio (%) = (Ws-Wd)/Wd Where Ws and Wd are the weights of swollen the samples and dry samples, respectively. The samples were immersed in distilled water at 37 °C and measured at designed time points after removing the water at the film surface. All the tensile tests to measure Young’s modulus and the tensile strength are

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conducted with a universal testing machine (Instron 3365, Instron, Norwood, MA) (gauge length: 20 mm; cross-head speed: 10 mm/min) equipped with a 100 N capacity load cell at 25 ± 0.5 °C and 60 ± 5% relative humidity. For dry tests, samples were equilibrated for 24 h at 25 ± 0.5 °C and 60 ± 5% relative humidity. For wet tests, samples

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were hydrated in deionized water for 2 h prior to testing.

Thermogravimetry analysis was performed in a TG-DTA, PE-SⅡ (America) in the

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temperature range of 40 to 600 °C with a ramp rate of 10 °C min, and nitrogen flux of 50 mL/min.

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The cell morphology on the composite films was examined by confocal microscopy. After 3 and then 7 days, the cell-seeded films were washed once with PBS and fixed in

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4% paraformaldehyde (Sigma-Aldrich, St Louis, MO) for 30 min, followed by further washing. The cell-seeded films were permeabilized with 0.1% Triton X-100 for 5 min and incubated with FITC-phalloidin (Sigma-Aldrich, St. Louis, MO) for 20 min at room

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temperature, followed by washing with PBS and, finally, staining with DAPI (SigmaAldrich, St. Louis, MO) for 1 min. Representative fluorescence images of the stained

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samples were obtained using a confocal microscope (Olympus FV10 inverted microscope, Nagano, Japan) with excitation/emission at 358/462 nm and 494/518 nm. CCK-8 assay was used to detect the proliferation of BMSCs as in our previous report

(P. Zhang et al., 2019). After 1, 3, 5, and 7 days, the medium was aspirated, and the films were rinsed with PBS. Then 20 μl CCK-8 plus 500 μl Dulbecco’s modified Eagle medium (DMEM) was placed into each well and kept for 4 h at 37 °C. Subsequently, the 300 μl supernatant for each well was transferred to a new 96-well plate, and the 6

absorbance values were measured using microplate reader at 450 nm. 2.4. Statistical analysis All experiments were carried out in triplicate. The mean and standard deviation (SD) was calculated by the statistical significance of differences among each group examined by a one-way ANOVA. The significance was set at p﹤0.05. 3. Results and discussion

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3.1 Morphology

To produce the chitosan-silk biocomposite films, the regenerated silk film and

chitosan were first co-dissolved in formic acid (FA), and then dried on a polyethylene

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dish. The silk components in the biocomposite films were water insoluble. To prevent

dissolution of chitosan, the pure chitosan film and biocomposite film were neutralized

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in sodium hydroxide and rinsed with water. This simple fabrication process created a thin film that exhibited a specific nanostructure composed of silk nanofibrils and

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chitosan nanofibers, as shown in Figure 2.

Surface 200 nm

Cross-section

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1 µm

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Figure 1. Simplified scheme for the formation process of the biocomposite films made from silk nanofibrils and chitosan nanofibers. The material preparation starts with a codissolution of (a) silk and (b) chitosan in formic acid to give a transparent (c) 7

biocomposite film comprised of self-assembled chitosan nanofibers intertwined with partially dissolved (d) silk nanofibrils. (e) SEM image of a cross section of the CS31 biocomposite films showing the specific nanostructure.

The pure chitosan and silk films were made of ultrafine chitosan and silk nanofibers. These two kinds of nanofibers were intertwined in the biocomposite films. Silk nanofibrils were obtained by dissolving silk directly into nanofibrils using CaCl2FA, and then chitosan nanofibers were self-assembled from the chitosan/FA solution.

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With this method, chitosan-silk biocomposites with weight ratios of 3:1, 1:1 and 1:3 were easily prepared, suggesting a simple strategy to tune the biocomposite

composition. The chitosan-silk films were clear and transparent. Formic acid was a

commonly used solvent for preparing tissue engineering scaffolds, such as silk films (F.

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Zhang et al., 2015), silk nanofibers(Min et al., 2004), and chitin nanofibers(Singh &

Ray, 2000), which showed no or negligible toxicity caused by the possible residue of

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the solvent. The unique nanofiber structure had a potential ability to generate structural

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biocomposites with outstanding mechanical properties. Silk

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Chitosan

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CS31

1µm

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CS13

CS11

1µm

1µm

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Figure 2. SEM images of the cross section of the chitosan-silk biocomposite films showing the internal nanofibrous structure. 8

3.2 Swelling behavior Chitosan

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CS31 100

CS11

Silk

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0

5

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Exposure time /h

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CS13

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Swelling ratio/%

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Figure 3. Swelling kinetics of chitosan-silk composites in distilled water at 37 °C.

Considering the large effect of swelling features on mechanical performance (Liu & Kim, 2012), degradation rate (Cui, Qian, Liu, Zhao, & Wang, 2015), and possible drug

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release (Gil, Frankowski, Spontak, & Hudson, 2005), the swelling response of chitosansilk films of differing compositions in distilled water at 37 °C is presented in Figure 3.

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Overall, the chitosan-silk composite exhibited a rapid swelling process, reaching saturation after 2 h of exposure. This swelling behavior reflected the permeability of

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water within the composite films. The swelling ratios of chitosan and silk were about 140% and 60%, respectively. The swelling ratio of the chitosan-silk composite decreased with the increasing the content of silk, suggesting a composition dependence of swelling ratio (Gil et al., 2005). 3.3 Mechanical properties Interestingly, mechanical testing showed the significantly improved mechanical properties (Figure 4) of the chitosan-silk biocomposite compared to pure chitosan and 9

silk films, indicating the strong interaction between the silk nanofibril and chitosan nanofibers. The mechanical properties of the biocomposite films were closely related to the ratio of chitosan to silk. The biocomposite films exhibited a maximal modulus and strength at a 3:1 (w/w) ratio of chitosan: silk (Figures 4 b and c), whereas increasing the silk component further gave rise to a biocomposite that was mechanically closer to pure silk film. The CS3:1 biocomposite films showed the elastic modulus of 3.5 GPa, a strength of 97.8 MPa, and a strain of 10.8%. This unexpected increase in the modulus allowed the biocomposite film to resist external tension without deforming the internal

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structure.

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Figure 4. Mechanical properties of chitosan, silk, and their composite films in a dry state. (a) stress-strain curves, (b) elastic modulus, (c) tensile strength, and (d) ultimate

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strain of the biocomposite films. The Young’s modulus and strength for the chitosansilk biocomposite films are higher than what is predicted from the rule of mixtures, indicating a strong interaction between the two nanofibrils.

Considering the possible applications of these composites in a biomedical field, the state of the mechanical properties when wet is one of the important factors in determining the usefulness of the composite. The chitosan-silk composites exhibited a 10

significantly lower Young’s modulus, strength, and much higher elongation due to the water plasticization than as compared with their dry state, as shown in Figure 5. Compared with a chitosan film, a silk film typically had a higher Young’s modulus. The addition of 25% chitosan content to silk was effective for the improvement of the Young’s modulus, and no significant change was achieved by further increasing chitosan content, likely due to the significant increase of the swell ratio (Figure 3). The silk film and chitosan-silk composite films showed a similar strength but were higher than that of a pure chitosan film. This reported strength of the composite films appeared 17

. In addition, the elongation at break was

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to be sufficient for practical applications

150% and 230% for silk and chitosan film, respectively. The chitosan film showed

excellent elongation, and the elongation of the composite films increased with increasing chitosan content. The composite film containing 75% chitosan exhibited as

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high as 260% elongation at breaking due to the high swelling ratio up to 140%.

Figure 5. Mechanical properties of chitosan, silk, and their composite films in a wet state: (a) elastic modulus, (b) tensile strength, and (c) ultimate strain of the biocomposite films. The Young’s modulus and strength for the chitosan-silk films are higher than what is predicted from the rule of mixtures, indicating a strong interaction between the two nanofibrils.

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To visually confirm the interactions between chitosan and silk, the CS3:1 films without sodium hydroxide neutralization were swelled in water to separate the constituents of chitosan and silk by using their different swelling behavior (Silva et al., 2008). As shown in Figure 6, the swollen biocomposite films exhibited a complex structure composed of phase separated chitosan nanofibers and silk lamellar-like layers (H. J. Jin et al., 2005). The chitosan nanofibers closely adhered to the silk layer, indicating the chitosan nanofibers interacted with, or were embedded in the silk components (J. Jin et al., 2013). These biocomposites replicated the component phase

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of an insect cuticle and an exoskeleton of a crustacean, thus superior mechanical properties were achieved (Nerurkar et al., 2009). In general, the significantly enhanced

strength and flexibility in the dry and wet state also endowed the biocomposite films with the potential application in high-technology directions, such as flexible electronic

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displays or biomedical materials (C. Zhang et al., 2012).

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Figure 6. Cross-sectional SEM images of the swollen CS31 films showing the interface

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between the chitosan and silk components.

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3.4 FTIR analysis

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FTIR analysis was carried out to study the secondary structure of the biocomposite films. Figure 7 showed the FTIR spectra of silk-chitosan biocomposites with different compositions. The pure chitosan film showed characteristic absorption bands at 1650 cm-1, 1581 cm-1, 1409 cm-1, 1150 cm-1, and 1060 cm-1 all assigned to the polysaccharide structure (Gu, Xie, Huang, Li, & Yu, 2013), which was consistent with the incomplete deacetylation of chitin (Fernandez & Ingber, 2012). The pure silk fibroin film showed absorption bands at 1618 cm-1 (amide I), 1515 cm-1 (amide II), and 1260 cm-1 (amide 12

III) attributed to the β-sheet conformation (Silva et al., 2012). As expected, the biocomposites exhibited the presence of both the polysaccharide structure of the chitosan and the amide I and amide II bands from the silk fibroin. Nevertheless, there was an obvious displacement of the amide I and amide II which was not expected from the simple sum of the chitosan and silk spectra. The displacement of amide I and amide II to higher wavenumbers with increased chitosan content suggested that hydrogen bonding occurred between silk and chitosan (J. Jin et al., 2013). This hydrogen bonding interaction between chitosan or chitin and silk had been reported in previous literature

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(Bhardwaj & Kundu, 2011; Fernandez & Ingber, 2012; J. Jin et al., 2013; Niamsa et al., 2009). From the FTIR results, it was confirmed that silk in the biocomposites was in

the β-sheet conformation (Silk II), and a strong hydrogen bonding interaction between

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chitosan and silk formed. (a)

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Figure 7. Molecular analysis of biocomposite films using FTIR. (a) FTIR spectra of

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chitosan, CS31, CS11, CS13, and silk, FTIR peaks for (b) amide I (1617 cm−1) and (c) amide II (1512 cm−1 ) in biocomposite films. Note that, the 1617 cm−1 and 1512 cm−1

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peaks of silk decreased with chitosan content. In addition, there were a clear shifts in the absorption peaks of amide I and amide II bands of the biocomposite films relative to that of pure silk. The shift of above amide bands strongly suggested the interaction between chitosan and silk which was probably due to the hydrogen bonds (Sionkowska & Planecka, 2013).

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3.5 X-ray diffraction (XRD) analysis XRD analysis was performed to further evaluate the crystal structure of the biocomposite films. Figure 8 showed the XRD spectra of the silk-chitosan biocomposite with different compositions. The pure chitosan showed two main diffraction peaks at 2θ = 10.0° and 19.8° in the diffraction pattern corresponding to the reflection of the crystalline structure of chitosan, indicating the high degree of crystalline morphology (Chen, Chen, & Lai, 2012; Kumar, Dutta, & Dutta, 2009; K. Zhang, Geissler, Fischer, Brendler, & Baucker, 2012). On the other hand, the three

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diffraction peaks centered at 2θ = 9.5 °, 20.5 ° and 24.5 ° are indicative of mainly silk

II crystal structures of the pure silk film (Liang et al., 2013). The new diffraction peaks or peak shifts were not observed in the biocomposite, indicating the weak interactions

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within the crystal structure of silk and chitosan. The above FTIR analysis confirmed

the strong hydrogen bonding interaction between chitosan and silk in the biocomposite.

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We hypothesize that the hydrogen-bonded interaction mainly occurred between the silk nanofibril and chitosan nanofibers. The silk nanofibril and chitosan nanofibers

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possessed a large specific surface area that could maximize the degree of hydrogen bonding between the two polymers. Furthermore, the two polymers did not mediate the crystallization process for each other and thereby decreased the mechanical

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properties(Fernandez & Ingber, 2012). Therefore, the unique mechanical properties of silk-chitosan biocomposites were due to strong hydrogen-bonded inter-nanofibrillar

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interactions as well as the independent crystalline structure.

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Figure 8. XRD patterns of chitosan, CS31, CS11, CS13, and silk. In chitosan, two main characteristic peaks at 10.0° and 19.8° indicated that the chitosan components are

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mainly in a crystalline structure. In silk, the three diffraction peaks at 9.5°, 20.5° and 24.5° correspond to a silk II (β-sheet) secondary structure. Note that, for silk-chitosan

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biocomposite films, the silk and chitosan diffraction peaks do not show any obvious

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shifts, indicating the negligible interaction within the crystal forms of silk and chitosan.

3.6 Thermogravimetric analysis (TGA)

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The thermograms of chitosan, silk and their biocomposites are shown in Figure 9a.

Overall, the films showed a thermal weight loss as the temperature increased. The process of decreasing weight could be divided into three distinct regions. For region I, the initial weight loss that took place with increasing temperature was due to water evaporation. Compared to the chitosan film, the silk film showed higher weight loss. For region II, after the water loss was complete, the rate of weight loss increased continuously with the increase in temperature, and it reached a maximum at around 15

300 °C, as shown in the DTG curves (Figure 9b). The weight loss showed a nearly linear relationship in a range of 260-390 °C due to polymer decomposition. In region III, the weight loss increased continuously with the increase in temperature, but with a slow decomposition rate where a maximum weight loss at 750 °C was attained. Finally, the pure silk and chitosan films showed the lowest and highest weight loss, and the

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weight loss of the biocomposite was between the two (Chen et al., 2012).

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Figure 9. (a) TGA thermograms, (b) DTG curves and (c) DSC curves of chitosan, CS31,

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CS11, CS13, and silk films.

3.7 Differential scanning calorimetry (DSC)

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The DSC thermograms of silk, chitosan and their biocomposites are shown in

Figure 9c. The main exothermic and endothermic peaks were observed for pure chitosan and silk, respectively. The strong endothermic peak of silk located at 319 °C was due to polymer decomposition (Fan, Zhang, Liu, & Zuo, 2014), and shifted to 323 °C and 329 °C when 25% and 50% chitosan was present. On the other hand, the exothermic peak of the pure chitosan film at 296 °C was attributed to the thermal degradation of chitosan (Kumar et al., 2009). Interestingly, the DSC curves of the silk17

chitosan biocomposite with a 1:3 ratio showed an exothermic peak similar to that of pure chitosan, and the silk-chitosan biocomposites with 1:1 and 3:1 ratios showed an endothermic peak similar to that of silk. In particular, the decomposition temperature of the silk component in the biocomposites increased from 319 °C to 329 °C with the increasing chitosan content, indicating the formation of intermolecular interactions between the two components (H. Y. Kweon, Um, & Park, 2001).

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3.8 Biocompatibility To evaluate the cytocompatibility of the biocomposite films, the attachment and

proliferation of rat bone marrow-derived mesenchymal stem cells (BMSCs) were

characterized. As shown in Figure 10(A), the morphology of the attached cells on the

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biocomposite films were observed by confocal laser microscopy. The biocomposite films supported the BMSCs adhesion and spreading as previously reported (Ding et al.,

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2017). Generally, most BMSCs presented in a dipolar and tripolar shape, and the cell proliferation was also directly observed in Figure 10(A). Cell proliferation experimental

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results further revealed a continued cell proliferation during the cell culturing period, as shown in Figure 10(B). Compared to the chitosan film, cell proliferation on the silk film was higher, in agreement with previous reports (Bhardwaj & Kundu, 2012; Ding

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et al., 2017). However, significantly improved cell proliferation was achieved on the silk-chitosan biocomposite films and was optimized on the CS31 films at day 7, which

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was likely due to the proper content ratio of silk and chitosan mimicking the extracellular matrix (ECM) composition (Dunne, Iyyanki, Hubenak, & Mathur, 2014;

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Lu, Zhang, Hu, & Kaplan, 2010). The character of native ECM was a network with multifibrillar collagens embedded in glycosaminoglycan (K. H. Zhang, Yu, & Mo, 2011). The chitosan-silk composite with the 3:1 ratio could be processed into a 3D scaffold, aiming to regulate cell behavior, such as cell morphologies, long-term cell proliferation, and cell differentiation (Li et al., 2017). Therefore, the preliminary results suggested that the silk-chitosan biocomposite films supported BMSCs attachment, growth, and proliferation, and tuning the silk and chitosan content was beneficial for 18

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improving material biocompatibility.

Figure 10. Confocal laser microscopy images of BMSCs on chitosan-silk biocomposite films at days 3 and 5 (A). BMSCs proliferation on silk/chitosan composite films (B). The cell proliferation on the CS31 biocomposite film was significantly higher than that on other films at day 7 (*p < 0.05). The data is represented as the average ± standard deviation. TRITC labeled phalloidin (green) for F-actin, DAPI (blue) for nuclei. 19

4. Conclusions In conclusion, we have demonstrated that biocomposite films composed of chitosan and silk are easily prepared from formic acid. CaCl2-formic acid mixture firstly dissolved silk into nanofibrils which interacted with chitosan nanofibers, generating composite films with a specific nanostructure. The composites are characterized by strong interfacial bonds between the interfaces of chitosan nanofibers and silk nanofibrils. The nanofibril structures and the interfibrillar interactions gave the

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biocomposite films significantly improved mechanical properties with respect to the mechanical properties of the individual components. The silk-chitosan biocomposites supported BMSC adhesion and proliferation in vitro, indicating desireable bicompatibility. In total, based on the outstanding strength and flexibility, the simple

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process, low cost, as well as good biocompatibility, the chitosan-silk biocomposite was

valuable for biomedical applications, such as tissue engineering scaffolds, drug delivery,

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and wound dressing.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China

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(51763001, 51403142), Suzhou Planning Project of Science and Technology (SYS201732, SYS2018052, SYS2019005), Pre-Research Project of the Second Affiliated Hospital of Soochow University (SDFEYBS1801), and a project funded by

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the Priority Academic Program Development of Jiangsu Higher Education Institutions

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(PAPD).

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