Improved mechanical strength of porous chitosan scaffold by graphene coatings

Improved mechanical strength of porous chitosan scaffold by graphene coatings

Author’s Accepted Manuscript Improved mechanical strength of porous chitosan scaffold by graphene coatings Shaolin Wen, Zan Wang, Xianliang Zheng, Xin...

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Author’s Accepted Manuscript Improved mechanical strength of porous chitosan scaffold by graphene coatings Shaolin Wen, Zan Wang, Xianliang Zheng, Xin Wang www.elsevier.com

PII: DOI: Reference:

S0167-577X(16)31499-9 http://dx.doi.org/10.1016/j.matlet.2016.09.040 MLBLUE21478

To appear in: Materials Letters Received date: 20 July 2016 Revised date: 25 August 2016 Accepted date: 10 September 2016 Cite this article as: Shaolin Wen, Zan Wang, Xianliang Zheng and Xin Wang, Improved mechanical strength of porous chitosan scaffold by graphene coatings, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.09.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Improved mechanical strength of porous chitosan scaffold by graphene coatings Shaolin Wen1, Zan Wang2, Xianliang Zheng1, Xin Wang1* 1

College of Materials Science and Engineering, Key laboratory of Automobile Materials of MOE, Jilin University,

Changchun, 130012, P.R. China 2

The First Hospital of Jilin University, Changchun, 130021, P.R. China

Abstract Chitosan (CS) scaffolds coated by reduced graphene oxide (rGO) film were prepared and characterized using X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy and a dynamic mechanical analyze. The results exhibited that the rGO coating with wrinkled structure attached on pore wall of CS scaffolds and formed a rough and irregular surfaces. The compressive mechanical strength increased with increasing graphene oxide dispersion (0, 0.02, 0.1, and 1.0 mg/ml) and the maximum value of about 0.707 MPa was achieved at the concentration of 1.0 mg/ml, 1.26-fold greater compared with CS scaffold. The porosity and pore diameter of the scaffold didn’t change much and the composite scaffold might be considered as potential platform for tissue engineering applications. Keywords:

Graphene

coating;

Chitosan

scaffold;

Compressive

strength;

Biomaterials; Carbon materials

1.

Introduction Scaffold or three-dimensional construct is one of the key components of tissue

engineering for cell adhesion, proliferation and differentiation. A high-performance 1

scaffold not only possesses biological properties, regulating cell behavior for tissue development, but also provide enough mechanical support for cell and tissue growth [1, 2]. As a natural biopolymer, chitosan (CS) is a high-molecular-weight linear cationic polysaccharide derived from the extensive deacetylation of chitin, having been served as conventional three-dimensional scaffold in clinical applications [3]. However, weak mechanical strength of chitosan scaffolds prepared by traditional freeze-drying method has limited their further commercialization [4]. Therefore, much effort has been made to enhance their mechanical properties either by optimizing fabrication method to increase crystallinity of chitosan scaffold [5] or through adding nanofillers as a reinforcement such as hydroxyapatite [6] and carbon-based nanomaterials including carbon nanotubes and graphene [7]. Graphene and its chemical derivatives (graphene oxide (GO) and reduced graphene oxide (rGO) sheets) were found to be able to improve mechanical strength of matrix and support cellular proliferation, adhesion, and even induce the differentiation of stem cells [7, 8]. Thus, covering graphene film onto pore wall of relatively soft polymer scaffolds may reinforce the mechanical properties of these scaffolds and change their surface micro-topography. Very recently, Kanayama et al. reported a positive effect of reduced graphene oxide coating on the improvement of a composite scaffold with good bioactivity and enhanced compressive strength [9]. In this letter, graphene oxide coatings were formed by dipping CS scaffolds into GO dispersion and through electrostatic and hydrogen bonding interaction between the oxygen containing functional groups of GO and chitosan amino groups. Then, the prepared samples were heated at 150℃ to obtain rGO-coated CS scaffolds. To our best 2

knowledge, rGO/CS composite scaffolds have been rarely reported up till now.

2.

Experimental section 2.1 Materials Chitosan, natural powder graphite,sulfuric acid,sodium hydroxide, hydrochloric acid,

acetic acid, sodium nitrate, potassium permanganate, hydrogen peroxide, ethanol. The details of materials are available in Electric Supplementary Material. 2.2 Synthesis of GO colloidal solution Graphite oxide was prepared according to the modified Hummers method [10]. The particular steps are available in Electronic Supplementary Material. Then, graphite oxide solution was mixed with ethanol and ultrasonically treated for 1 h to obtain graphene oxide dispersion (0, 0.02, 0.1, and 1.0 mg/ml). 2.3 Preparation of CS scaffolds A chitosan concentration of 4 wt% was prepared by thorough dissolution of chitosan powder in 0.2 M acetic acid. After that, the solution was poured into 24 well plates and frozen under -20℃, followed by lyophilization in a freeze-dryer to form the CS scaffolds. Lyophilized scaffolds were immersed in sodium hydroxide dispersion and washed with DI water for several times until pH near 7. Finally, they were frozen and lyophilized again for use. 2.4 Preparation of rGO-coated CS scaffolds CS scaffolds were dipped into the GO dispersions for 48 h and dried in air. Finally, the samples were heated at 150℃ for 12 h to get chitosan scaffolds coated by rGO film, 3

which are entitled as CS, 0.02 mg - rGO/CS, 0.1 mg - rGO/CS, and 1.0 mg - rGO/CS. 2.5 Characterization of materials Transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and dynamic mechanical analyzer were used to character materials. The method of "ethanol substitution" were used to measure the porosity of the scaffolds. The details of this part are available in Electronic Supplementary Material.

3. Results and discussion From the digital photographs shown in Fig. 1(a-d), we can see that the yellow-colored CS scaffolds (~25 mm thickness and ~15 mm diameter) get darker with increasing the coated GO concentrations. To characterize the graphene oxide sheet, transmission electron microscopy (TEM) was used. Fig. 1(e) exhibits a successful exfoliation of individual graphene oxide sheets oxide sheet that displays as a thin extended film with a wrinkled surface and a lateral size of about several micrometers. Fig. 1(f) gives the XRD patterns of the as-prepared GO and 150℃-heated GO film coated on circular glasses, which shows the main diffraction peak of 11.98° shifted to 23.50°, confirming the formation of rGO film.

XRD measurements were performed to analyze the crystal structure of the prepared samples. It can be seen from Fig. 2 that all the XRD patterns look similar and two strong diffraction peaks around 14.90° and 20.41° indicate high degree of crystallinity

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of the scaffolds [11, 12]. The rGO coatings hardly disrupt the crystalline constituent of chitosan.

The XPS was used to further confirm the reduction of GO and the combination of rGO and chitosan. In the C1s spectrum of GO (Fig. 3a), five binding energy peaks at 284.4, 285.4, 286.8, 287.7, and 288.6 eV are corresponding to sp2 C=C, sp3 C-C, C-OH, C-O-C, and C=O, respectively. After heating treatment at 150℃ (Fig. 3b), all of the oxygen containing functional groups were reduced largely and a new peak appears at 289.2 eV, which attributes to O=C-OH. Compared these two C1s spectra, we can confirm that GO film coated on chitosan scaffold has been reduced to rGO. In addition, from Fig. 3c and 3d, the C1s spectra of pure CS scaffold and rGO/CS composite scaffold, respectively, we can deduce the covering of rGO on the surface of the scaffold wall, which agrees well with the result of SEM measurement.

SEM images in Fig. 4(a-c) reveal a highly porous, relatively homogeneous and interconnected structure of the pure scaffolds. The pore diameter was in the range of 150~300 μm for all scaffolds including the composites, implying that the incorporation of GO coatings did not obviously alter the porous structure of the scaffolds (porosity keeps nearly constant at about 92%,). From Fig. 4(d-f), it can be seen that not only did the surface of pore wall of rGO/CS scaffolds appear wrinkles, but also the degree of wrinkles be enhanced with the increase of GO dispersion concentration due to the covering of rGO films. The SEM images suggested that rGO had coated on CS scaffolds 5

and the thickness of rGO coatings could be adjusted by the concentration of GO dispersion and thus regulate surface topography of the composite scaffold and influence cell behavior [13].

In addition to be able to add elasticity of the matrix scaffolds, graphene-based coating may also increase their compressive strength [9]. In this letter, the compressive strength of chitosan scaffold was found dependent on the concentration of GO dispersions, and the differences in the strength are statistically significant (Fig. 4g). The maximum value of about 0.707 MPa was achieved for sample of rGO/1.0 mg CS scaffold, 1.26-fold greater compared with pure CS scaffold (0.561 MPa). As the strong interfacial interactions between the GO sheets and the polymer matrix and the increase of rGO thickness due to increasing GO dispersion concentration, the stress applied to the composite scaffolds could be efficiently transferred through the rGO coatings, leading to high strength.

4. Conclusions We coated CS scaffolds with GO sheets followed by thermal reduction treatment at low heating temperature. The as-prepared rGO coatings modified the surface morphology of the CS scaffolds and also increased their compressive strength without changing the porous structures. The rGO/CS composite scaffolds may offer a powerful platform for stem cell research and tissue regenerative medicine

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Acknowledgement We would like to thank the Project sponsored by Jilin Province Development and Reform Commission (Grant No.2015Y032-7), the China Scholarship Council (CSC) for a visiting scholar, and the supporting from Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences (16NBI01).

References [1] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials. 2000;21:2529-43. [2] Annabi N, Nichol JW, Zhong X, Ji CD, Koshy S, Khademhosseini A, et al. Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering. Tissue Eng Part B-Rev. 2010;16:371-83. [3] Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv. 2008;26:1-21. [4] Han ZJ, Rider AE, Ishaq M, Kumar S, Kondyurin A, Bilek MMM, et al. Carbon nanostructures for hard tissue engineering. RSC Adv. 2013;3:11058-72. [5] Jana S, Florczyk SJ, Leung M, Zhang MQ. High-strength pristine porous chitosan scaffolds for tissue engineering. J Mater Chem. 2012;22:6291-9. [6] Manjubala I, Scheler S, Bossert J, Jandt KD. Mineralisation of chitosan scaffolds with nano-apatite formation by double diffusion technique. Acta Biomater. 2006;2:75-84.

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[7] Ku SH, Lee M, Park CB. Carbon-Based Nanomaterials for Tissue Engineering. Adv Healthc Mater. 2013;2:244-60. [8] Kang S, Park JB, Lee TJ, Ryu S, Bhang SH, La WG, et al. Covalent conjugation of mechanically stiff graphene oxide flakes to three-dimensional collagen scaffolds for osteogenic differentiation of human mesenchymal stem cells. Carbon. 2015;83:162-72. [9] Kanayama I, Miyaji H, Takita H, Nishida E, Tsuji M, Fugetsu B, et al. Comparative study of bioactivity of collagen scaffolds coated with graphene oxide and reduced graphene oxide. Int J Nanomed. 2014;9:3363-73. [10] Hummers WC, Offeman RE. Preparation of graphitic oxide. J Am Chem Soc. 1958;80:1339. [11] Wang SF, Shen L, Zhang WD, Tong YJ. Preparation and mechanical properties of chitosan/carbon nanotubes composites. Biomacromolecules. 2005;6:3067-72. [12] Han DL, Yan LF, Chen WF, Li W. Preparation of chitosan/graphene oxide composite film with enhanced mechanical strength in the wet state. Carbohydr Polym. 2011;83:653-8. [13] Stevens MM, George JH. Exploring and engineering the cell surface interface. Science. 2005;310:1135-8.

Fig. 1(a-d) Digital photographs of CS scaffold, 0.02 mg - rGO/CS scaffold, 0.1 mg rGO/CS scaffold, and 1.0 mg - rGO/CS scaffold; (e) TEM image of graphene oxide sheets; (f) XRD patterns of GO and rGO coatings.

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Fig. 2 XRD patterns of CS and rGO-coated CS scaffolds.

Fig. 3 XPS spectra of (a) GO, (b) rGO, (c) CS scaffolds and (d) 1.0 mg - rGO/CS scaffold.

Fig. 4 SEM micrographs of (a-c) CS scaffolds, (d) 0.02 mg - rGO/CS scaffold, (e) 0.1 mg - rGO/CS scaffold, (f) 1.0 mg - rGO/CS scaffold, and (g) compressive strengths of the scaffolds.

Highlights 

Graphene coated chitosan scaffolds were fabricated.



The rGO/CS composite scaffolds were characterized.



The rGO/CS exhibited enhanced mechanical strength.

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