MoS2 Composite Nanofibers on Growth

Growth Behavior of Bone Marrow Mesenchymal Stem Cells. Shuyi Wu,. #†. Jieda Wang,. #†. Lin Jin, ... investigation about the response of MoS2 compo...
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Effects of Polyacrylonitrile/MoS2 Composite Nanofibers on Growth Behavior of Bone Marrow Mesenchymal Stem Cells Shuyi Wu, Jieda Wang, Lin Jin, Yan Li, and Zhenling Wang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00188 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on January 9, 2018

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Effects of Polyacrylonitrile/MoS2 Composite Nanofibers on Growth Behavior of Bone Marrow Mesenchymal Stem Cells Shuyi Wu, #† Jieda Wang, #† Lin Jin, ‡, §* Yan Li, †* Zhenling Wang‡, †

⊥*

Department of Prosthodontics, Guanghua School of Stomatology, Hospital of

Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, 510055, P. R. China ‡

International Joint Research Laboratory for Biomedical Nanomaterials of Henan,

Zhoukou Normal University, Zhoukou 466001, P. R. China ⊥

Henan Key Laboratory of Rare Earth Functional Materials; The Key Laboratory of

Rare Earth Functional Materials and Applications, Zhoukou Normal University, Zhoukou, 466001, P. R.China §

Key Laboratory of Polymeric Composite & Functional Materials of Ministry of

Education *Corresponding author. E-mail: [email protected]; [email protected];

[email protected].

Tel.: +86-394-8178518; Fax: +86-394-8178518.

#

These authors contributed equally.

KEYWORDS: MoS2, composite nanofibers, electrospinning, BMSCs, tissue engineering

ABSTRACT: In recent years, molybdenum disulfide (MoS2) as a typical class of two-dimensional (2D) material has attracted widely attention due to their various fascinating properties. In this study, we fabricated MoS2 composite nanofibers by ACS Paragon Plus Environment

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electrospinning technology combined with doping method. The as-prepared MoS2 composites nanofibers exhibited excellent biocompatibility. In addition, the detailed investigation about the response of MoS2 composites nanofibers on bone marrow mesenchymal stem cells (BMSCs) indicated that the obtained MoS2 composites nanofibers could promote BMSCs growth behavior, improve BMSCs to contact each other and maintain cellular activity; and also provide positive promotion to regulate cellular proliferation. Moreover, the alkaline phosphatase expression significantly increased along with the MoS2 concentration increasing. Compared with the excellent biocompatibility and natural extracellular matrix-like (ECM-like) structure, we believe that the MoS2 composites nanofibers could provide a new sight for preparing well-defined MoS2 nano-structure materials, and will have promising potential in biomedical applications, such as tissue engineering, photothermal therapy.

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1. INTRODUCTIOIN As a novel class of two-dimensional (2D) nanomaterials, molybdenum disulfide (MoS2) has attracted significant attention due to its unique physical, chemical, optical, electronic and mechanical properties.1-10 There are many class of MoS2 based nanomaterials have been developed for various practical applications, for example, electrospun porous carbon nanofibers designed anodes for lithium-ion batteries.11 In recent years, biomedical application of MoS2 developed much more rapidly compared with other research areas, such as drug delivery,12-14 bio-imaging,15,

16

as well as

biosensors.17-21 Thus, biocompatibility evaluation of MoS2 based nanomaterials is very crucial for their biomedical application. Currently, various methods have been used to develop biomedical nanomaterials using MoS2 nanosheets to gain desirable physicochemical or biochemical properties for special practical applications.22-32 However, most of current methods still have some limits such as the complicated processes, the lack of precise and controllable preparation strategies.3, 33, 34 As we all know, the morphological characters and range of size of MoS2 based nanomaterials are crucial for their applications. These factors not only could affect the performance of MoS2 based nanomaterials, but also affect the response of bio-systems. Thus, preparation of MoS2 biomaterials with suitable structure is very necessary to meet wide range of potential applications in the biomedical area. Electrospinning is a simple yet effective strategy to fabricate ECM-like nanofibrous materials with desirable engineered performances for biomedical application. Thus, incorporating different nanoscale building blocks into electrospun

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nanofibers would greatly improve the performance to meet the various requirements biomedical area, such as MoS2, carbon nanotubes, graphene, nanoparticle, and so on.35-39 For example, fabrication of reduced graphene oxide reinforced composite nanofibers by incorporating graphene nanoscale sheets in nanofibers effectively enhance the mechanical properties, and expanded the application of graphene in biomedical field,36 and MoS2 nanofibers with hexagonal structure were developed by sol-gel method and electrospinning.39 In this study, we fabricated MoS2 composite nanofibers by electrospinning technology combined with doping method. The obtained MoS2 composites nanofibers demonstrated excellent biocompatibility. Subsequently, the effect of MoS2 composite nanofibers with different concentration on BMSCs was observed over a period of 14 days culture, and evaluated according to cellular proliferation, morphology and osteogenic differentiation expression. The results indicated that the obtained MoS2 composites nanofibers could promote BMSCs growth behavior, improve BMSCs to contact each other and maintain cellular activity; and also provide positive promotion to regulate cellular proliferation. Furthermore, the osteogenic differentiation expression significantly increased along with the MoS2 concentration increasing. Compared with the excellent biocompatibility and natural extracellular matrix-like ECM-like structure, we believe the MoS2 composites nanofibers could provide a new sight for preparing well-defined MoS2 nano-structure materials will have promising potential in biomedical applications, such as tissue engineering, photothermal therapy.

2. EXPERIMENTAL SECTION

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2.1 Materials. Polyacrylonitrile (PAN, MW=110K) was purchased from Daigang CO. (Jinan, Shandong), MoS2 was obtained Nanfeng CO. (Nanjing, Jiangsu), other chemicals were used without further purification. The DMEM and fetal bovine serum (FBS) were purchased from Sigma-Aldrich. 2.2 Fabrication of MoS2 Nanofibers. The MoS2 composite nanofibers were prepared according to our reported method.36 Various MoS2 composite nanofibers were electrospun on the collector and removed from it, thus the obtained MoS2 composite nanofibers with different concentration of MoS2 were used to evaluate the response of cells. 2.3 Characterization of MoS2 Composite Nanofibers. The SEM images of the different MoS2 composite nanofibers were obtained by a Hitachi S-4800N at an acceleration voltage of 20 kV. The chemical compositions were characterized using Fourier transform infrared (FTIR) spectra (with a VERTEX 70, Bruker Co. Germany, FTIR spectrometer) with

a ATR model and X-ray diffraction (XRD) (Model D8

Avance, Bruker, Germany) using Cu Kα radiation (λ = 0.15406 nm). 2.4 BMSCs Isolation. BMSCs were isolated as previously described from 2-3 weeks old male Sprague-Dawley rats.40 Briefly, the rats were killed by cervical dislocation and disinfected with 70% ethyl alcohol. Surgery took place in the biological safety cabinet, where the femurs of the rats were excised and washed with F12/DMEM USA) with 1000 U/mL penicillin and 1000 mg/mL streptomycin. Then, the bone marrow was flushed out with complete medium (F12/DMEM containing 10%FBS, U/ mL penicillin and 100 mg/mL streptomycin), centrifuged (5 min, 1000 rpm),

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suspended in complete medium, and plated in T-25 flasks, then cultured in a 5% CO2 atmosphere at 37°C. The medium was discarded and the cells were washed with phosphate buffered saline (PBS) (10 mM, pH 7.4) after 24h. The medium was every two days. The adherent cells were allowed to reach approximately 80% confluence (5-7 days for the first passage). Cells were passaged during culture, and passage 3-5 cells were used for experiments. 2.5 Cell Seeding. Various MoS2 composite nanofibers were cut into 14-mm disks and placed into 24-well plates, then sterilized by ultraviolet light for 2h, followed by washing 2-3 times using complete medium. BMSCs were cultured on the various MoS2 composite nanofibers with a density of 1.0×104 cells/well according to previous method.41-43 The resulting nanofibers/cell constructs were placed in an incubator for 6 h to allow for cell attachment, with an additional 0.5 mL complete medium added into each well. The complete medium mentioned, osteogenic medium consisting of a-DMEM, 10% FBS, 0.1 mM dexamethasone (Sigma-Aldrich, USA), 10 mM b-glycerophosphate (Calbiochem, USA) and 50 mM ascorbic acid (Sigma-Aldrich) were used for the osteogenic differentiation studies. Then the cell substrates were cultured in a 5% CO2 atmosphere at 37°C. 2.6 Cell Cytotoxicity and Proliferation The cell cytotoxicity and proliferation were tested at various time points by WST-8 assay using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan). Briefly, when the cells were cultured on these four MoS2 composite nanofibers for 12h, 1d, 3d and 7d, the medium was discarded and 200 uL complete medium supplemented with 10% CCK-8

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was added into each well. After incubation for 1h and shaking for 2 min, the supernatant was taken to another 96-well plate, and the optical density (OD) was measured on a microplate reader at a wavelength of 450 nm. The cell cytotoxicity and proliferation was measured by OD value. 2.7 Cell Fluorescence Detection The cell viability on these MoS2 composite nanofibers was assessed by fluorescence images. DAPI (Beyotime, China) and Actin-Tracker Green (Beyotime, China) were used to visualize cellular nuclei and cytoplasm, respectively. Briefly, all cell substrates were fixed by 3.7% paraformaldehyde for 30 min, followed by permeabilizing the cells in 0.1% Triton-PBS for 15min. Block non-specific binding using 5% albumin from bovine serum (BSA) in PBS for 30 min prior to staining. After stained, the fluorescence images were obtained using a confocal microscope (LSM700, Zeiss). 2.8 Cell Morphology The cell morphology on these MoS2 composite nanofibers was evaluated by scanning electron microscopy (SEM) images. All cell substrates were fixed with 3.7% paraformaldehyde for 2 h and dehydrated by incremental concentrations of ethanol– water including 25%, 50%, 70%, 80%, 90%, 95% and absolute ethanol for 30 min each, subsequently they were dried in the air. And then, the cell substrates were sputter-coated by gold and palladium with a thickness of 10 nm, and SEM images were obtained using a Hitachi Model S-4800 system. 2.9 Alkaline Phosphatase (ALP) Activity

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The ALP activity of the cells was detected by osteogenic differentiation using an ALP assay kit (Jiancheng, China); each cell substrate was measured at day 3, 7 and 14. At various points, all cell substrates were washed with PBS three times, and lysed with 1% Triton X-100 solution under 4°C all night. The ALP activity of the lysates was determined using p-nitrophenyl phosphate as a substrate. The absorbance was measured on a microplate reader at a wavelength of 520 nm. 2.10 Glycosaminoglycans (GAGs) detection Alcian blue (Solarbio, China) assay was used for measuring the content of glycosaminoglycan in cell culture supernatant. Briefly, cell culture medium was collected at day 3,6 and 9. After centrifuged (5 min, 1500 rpm), the supernatant was mixed with 1.5% Alcian blue solution for 10 minutes at room temperature. The optical density (OD) was measured on a microplate reader at a wavelength of 480 nm.

3. RESULTS AND DISCUSSION 3.1 Fabrication, Structural and Chemical Characterization of MoS2 Composite Nanofibers In this study, we prepared 10mg, 20mg and 40mg mL-1 MoS2 and DMF solution through strongly ultrasonication by a cell disrupter 100 W of power in 10mL batchers for 10 min, the TEM image (Figure S1) showed that the prepared MoS2 with suitable size (< 200nm) for fabrication composite nanofibers. And then, PAN was added into the obtained solution with a 100mg/mL concentration. The MoS2 composite nanofibers were fabricated according to previous method (Figure 1A).35 As shown in Figure 1B, the color of optical images of MoS2 composite nanofibers were gradually

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darken as the MoS2 content increased, the content of MoS2 was up to 40%, the color shows obvious metallic luster. The morphology of MoS2 composite nanofibers was tested by SEM images (Figure 2), the results indicated that the composite nanofibers with a low concentration of MoS2 (10%) maintain a smooth surface as the raw nanofibers, however, as the content of MoS2 increasing, the surface topography also became rough, some bumps with needle-like structure could be seen clearly (Figure. 2C, D). Moreover, the high magnification SEM image also showed that MoS2 sheets were obviously observed (Figure S2 and Figure S3). The chemical characterization was tested by X-ray diffraction (XRD) and FTIR spectrum. Figure 3 A showed the patterns of MoS2 and MoS2 composite nanofibers. As shown in the XRD patterns, MoS2 reveals its characteristic peaks. After MoS2 embedded into nanofibers, a broad characteristic reflection was observed in MoS2 composite nanofibers.44 To further investigate the chemical composition, we performed FTIR spectra test on the MoS2 composite nanofibers. As shown in Figure 3B, the spectra of MoS2 was completely consistent with previous reported data,45, 46 meanwhile, after incorporating MoS2 in the nanofibers, the obtained composite nanofibers also reveals characteristic peaks of MoS2. These results combined with the SEM images indicated that the MoS2 composite nanofibers were successfully fabricated in this study. 3.2 Cell Cytotoxicity and Proliferation In order to evaluate the cellular response of MoS2 composite nanofibers, the BMSCs were cultured on the different MoS2 composite nanofibers for a period of 7

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days, the nanofibers which didn’t contain MoS2 was set as a control. Cell cytotoxicity and proliferation on these nanofibers were analyzed with the CCK-8 kit. The principle of CCK-8 kit is that WST-8 can be reduced by dehydrogenase in mitochondria in the presence of electron coupling reagent, generating highly water-soluble orange formazan. The depth of color is proportional to the cell proliferation, while inversely proportional to the cell toxicity. Therefore, the OD value was measured by microplate reader at a wavelength of 450 nm, which could indirectly reflects the number of living cells. As shown in Figure 4, after 12h and 1d of cell culture, they appeared the same trend, and the cell number on 0% MoS2 composite nanofibers was higher than 20% and 40% MoS2 composite nanofibers. Besides, 40% MoS2 composite nanofibers exhibited lower cell attachment compared to 10% and 20% MoS2 composite nanofibers. However, there was no significant difference after 3d and 7d of cell culture. These results were consistent with some studies that MoS2 induced very low cytotoxicity, even at high concentrations.22, 23 In addition, the obtained results indicated that the cytotoxicity of the composite nanofibers kept the excellent biocompatibility after doping MoS2. Generally, the biocompatibility of 2D materials is evaluated using free standing film. For example, we tested the biocompatible properties of graphene using graphene film.43 In fact, as typical class of 2D materials, the pure MoS2 nanosheets or film cannot provide suitable microenvironment for cellular growth as MoS2 composite nanofibers because they could not mimic natural extracellular matrix (ECM) like the composite nanofibers, which consisted with our previous work. However, after adding MoS2 by

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doping method, the nanoscale size and ECM-like fibrous structure of composite nanofibers help to counteract toxicity of the MoS2 nanosheets or film, thus achieving MoS2 composite nanofibers to effectively guide the focal adhesion, and differentiation.43 3.3 Fluorescence Detection and Morphology of BMSCs The BMSCs were stained by DAPI and Actin-Tracker Green after 3 days of culture for purpose of examining the cell activity on different MoS2 composite nanofibers. As shown in Figure 5, a large number of BMSCs appeared on the different MoS2 composite nanofibers. All of these nanofibers displayed good cell attachment and cell retention, which was consistent with the above mentioned cell proliferation results. A detailed morphology of BMSCs cultured on the 10%, 20%, 40% MoS2 composite nanofibers and the control nanofibers (0% MoS2 nanofibers) was assessed by SEM after 3 days of culture. As shown in Figure 6, cell could be free to stretch on all the different MoS2 composite nanofibers, and cells grown along the nanofibers and showed full extension. Moreover, BMSCs cultured on the MoS2 composite nanofibers have an excellent contact each other compared to the raw PAN nanofibers (Figure S4). 3.4 ALP Activity Osteogenic differentiation of cell was assessed by determining the ALP activity of BMSCs after 3, 7 and 14 days culture. As shown in Figure 7, with the increase of MoS2 concentration, ALP activity was significantly improved at every time point.

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More importantly, MoS2 composite nanofibers contained MoS2 0%, 10% and 20%, the ALP expression had no obvious difference after 14 days culture; however, as the MoS2 content increased to 40%, the ALP value has markedly improved compared to other composite nanofibers. The results indicated that MoS2 combined with nanofibrous morphology could improve osteogenic differentiation of the BMSCs. 3.5 GAGs Test To measure the effect of MoS2 composite nanofibers on GAGs expression of BMSCs, the GAGs were characterized on the day 3, 6, and 9. The optical density (OD) was measured on a microplate reader at a wavelength of 480 nm. The results indicated that MoS2 content of the composite nanofibers had no great effect on the GAGs expression. While the GAGs content was slightly increased as culture time increasing (Figure 8). The obtained results suggested that the MoS2 in the composite nanofibers had a positive impact on the cellular growth behaviour of BMSCs. Compare to the raw PAN nanofibers, the MoS2 composite nanofibers will provide more chemical or physical cues, many parameters and possibility for effect on regulating cellular growth, for example, the size and morphology of MoS2 maybe give some positive cues for response of cells. Based on these latest research results, our MoS2 composite nanofibers holds the possibility to further promote the differentiation of other type cells by provide geometry or chemical cues, at the same time, also preserve the cell survival. Thus, we anticipate that the MoS2 composite nanofibers have great potential for tissue engineering application.

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In addition, our results also indicated that MoS2 composites nanofibers could provide positive promotion to regulate cellular proliferation. It involves two main factors: (1) the nanoscale size and natural extracellular matrix like (ECM-like) fibrous structure of composite nanofibers help to counteract toxicity of the MoS2 nanosheets or film, thus achieving MoS2 composite nanofibers to effectively guide the focal adhesion, and differentiation; (2) Appropriate amount of provide suitable MoS2 microenvironment for cellular growth, meanwhile, the MoS2 composite nanofibers could mimic ECM structure. Thus, we anticipate that the MoS2 composite nanofibers have great potential for tissue engineering application.

4. CONCLUSION In conclusion, we successfully fabricated MoS2 composites nanofibers by electrospinning process. The as-prepared MoS2 composites nanofibers exhibited excellent biocompatibility. In addition, we had a detailed investigation about the response of MoS2 composites nanofibers on BMSCs, the results indicated that the obtained MoS2 composites nanofibers could promote BMSCs growth behavior. The obtained MoS2 composites nanofibers could improve BMSCs to contact each other and maintain cellular activity; also provided positive promotion to regulate cellular proliferation and osteogenic differentiation. Thus, we believe that the obtianed MoS2 composites nanofibers will have broad application in biomedical field, such as tissue engineering, controlled drug release, etc.

ACKNOWLEDGEMENTS

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This research was supported by the National Natural Science Foundation of China (the number of funding are 21404124 and 51572303), and the program of Innovative Talent (in Science and Technology) in University of Henan Province (the number of funding is 17HASTIT007). L. J. acknowledges The project was funded by the Key Laboratory of Polymeric Composite & Functional Materials of Ministry of Education (PCFM-2017-04). S. W. acknowledges The Natural Science Foundation of Guangdong Province, China(No. 2014A030310396)and The Fundamental Research Funds for the Central Universities of China (No. 14ykpy35). Supporting Information Supporting Information is available: [The TEM images of MoS2 sheets (Figure S1) and MoS2 composite nanofiber (Figure S2), the high magnification SEM image of MoS2 composite nanofiber (Figure S3). A detailed morphology of BMSCs cultured on the different MoS2 composite nanofibers (Figure S4)]. REFERENCES

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Figure 1. (A) The schematic illustration of fabrication of MoS2 composites nanofibers. (B) Photo images of MoS2 composites nanofibers contain different MoS2 content.

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Figure 2. SEM images of MoS2 composites nanofibers with various MoS2 (W : W). (A) 0%, (B) 10%, (C) 20%, (D) 40%.

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Figure 3. (A) XRD spectrum and (B) FTIR of MoS2 composites nanofibers

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1.5

OD Value

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** **

*

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Cell culture time (day)

Figure 4. The cytotoxicity and proliferation of BMSCs cultured on different MoS2 composite nanofibers in various incubation periods. Data are mean ± SD, n=4, *P < 0.05, **P < 0.01.

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Figure 5. Fluorescence images of BMSCs cultured on different MoS2 composite nanofibers on day 3.

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A

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Figure 6. SEM images of BMSCs cultured on MoS2 composite nanofibers on day 3: (A) 0% MoS2, (B)10% MoS2, (C)20% MoS2, (D) 40% MoS2.

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Figure 7. The ALP activity of BMSCs cultured on different MoS2 composite nanofibers in various incubation periods. Data are mean ± SD, n=4, *P < 0.05, **P < 0.01, ***P < 0.001.

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Figure 8. The GAGs activity of BMSCs cultured on different MoS2 composite nanofibers in various incubation periods. Data are mean ± SD, n=4, *P < 0.05.

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