graphene oxide composite hydrogels for nerve tissue Engineering

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ScienceDirect Materials Today: Proceedings 5 (2018) 15620–15628

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NTNM2017

Preparation and characterization of chitosan/graphene oxide composite hydrogels for nerve tissue Engineering Mahboubeh Jafarkhania, Zeinab Salehia,*, Tahereh Nematianb a

School of Chemical Engineering, College of Engineering, University of Tehran, Iran b School of Chemistry, College of Science, University of Tehran, Tehran, Iran

Abstract Different hydrogel materials have been designed to improve nerve tissue regeneration. Biomaterials used for nerve tissue have certain restrictions such as lack of proper mechanical strength. Recently, graphene related hydrogels provide appropriate mechanical properties. However, the influence of these engineered constructs on nerve renewal is still unclear and needs to be investigated. In this study, nano graphene oxide/chitosan (NGO/CHT) hydrogels were synthesized via and its physical properties such as morphology, swelling and mechanical properties were separately characterized. The influence of NGO/CHT hydrogel on the adhesion and proliferation rate of nerve cells was assessed. The results indicated that the NGO addition changed the pore structure and improved mechanical strength of hydrogel. Moreover, the addition of NGO increased nerve cells growth up to 20%. The funding showed that an appropriate concentration of NGO could successfully stimulate cell growth. This study may provide a significant experimental basis to design and develop functional hydrogel for nerve regeneration. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017. Keywords: Nerve regeneration; Graphene oxide; Chitosan; hydrogel

1. Introduction Nerve tissue engineering, which is a worldwide clinical problem which extremely affected the quality of patients’ life and increased their economic problem [1]. Hydrogels have received increasing attention in tissue engineering because of similarity to the native tissue [2]. In the field of tissue engineering, biocompatible and bioactive

*

Corresponding author. E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of INN International Conference/Workshop on “Nanotechnology and Nanomedicine’’ NTNM2017.

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hydrogels allow delivery of physical and chemical signals to different type of cells and direct cell’s behaviour including adhesion, proliferation, migration and differentiation [3-6]. Recently, the hydrogels for nerve tissue engineering applications were successfully applied [7]. Graphene oxide, as a derivative of graphene, has been widely used to reinforce biomaterials [8]. The biomaterials containing NGO have plentiful carbonyl group (CO) and hydroxyl (OH) which significantly improved the surface ability to bind with other functional group from biomaterials [9]. Moreover, NGO not only has suitable biocompatibility but also increase hydrogel’s mechanical strength and electrical conductivity [10]. A couple of studies have reported that using NGO in combination with polyacrylamide [11], poly (N-isopropylacrylamide) [12], hyaluronic acid [13], displayed better mechanical strength. For example yang et al. demonstrated that incorporation of 1 wt.% NGO has enhanced the mechanical properties of chitosan scaffolds such as tensile strength, and Young's modulus nearly 122% and 64%, respectively [14]. NGO also has a significant role in nerve tissue engineering because of its outstanding electrical properties. Since, by using NGO the electro-activity of neural cells has been improved [15]. Although, NGOs’ toxicity is a challenge for researchers to incorporate it in biomaterials for nerve regeneration, Volkov et al. [16] reported a number of studies, which used NGO graphene and indicated noteworthy improvement in cell viability. Chitosan (CHT) has been applied in a widespread range of applications in biomedical due to its excellent properties. CHT, as a widely distributed linear copolymer in nature, which is obtained by deacetylation of chitin has great features including biocompatibility, low toxicity, appreciable biodegradability, antimicrobial activity and admirable low immunogenicity [17]. CHT is also appropriate for nerve tissue engineering due to its beneficial qualities such as biocompatibility and biodegradability [18]. For example, Yuan et al. reported that CHT improved Schwann cells behaviours including adhesion, proliferation and migration [19]. Furthermore, Haipeng et al. demonstrated that nervous cells cultured on the chitosan scaffolds could grow successfully and that chitosan tube can promote repair of the peripheral nervous system [20]. However, the application of CHT for nerve tissue engineering was limited due to the poor electrical conductivity. It is well known that electrical stimulation has a critical role in neural cell proliferation in vitro and tissue renewal in vivo. Therefore, blending CHT with NGO can be considered a reasonable idea to fabricate a functional engineered tissue for nerve tissue engineering. Herein, in this study, the NGO-CHT hydrogel was fabricated for nerve regeneration applications and then the morphology, swelling and degradation ratio and mechanical properties of the samples were characterized. Finally, the effect of NGO addition on nerve cell proliferation was evaluated by MTT assay. 2.

Materials and methods

Chitosan, glutaraldehyde, sulfuric acid (H2SO4), phosphoric acid (H3PO4), acetic acid and potassium permanganate (KMnO4) were purchased from Sigma-Aldrich. Fine grade of natural graphite powder was obtained from Fluka. Furthermore, Dulbecco's Modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Biowest. 2.1. Preparation of graphene oxide sheets Graphene oxide (NGO) was synthesized from natural graphite powder using the modified Hummers’ method. Briefly, 1g of graphite flake was suspended in a 9:1 mixture of concentrated H2SO4/H3PO4 and stirred for 30 min. Then 6g of KMnO4 was slowly added to the mixture. It was heated up to 50 °C and agitated for 24 h. The mixture was cooled to room temperature and subsequently, 200 ml distilled water was added to it. For further oxidation, the suspension treated with 5 mL 30% H2O2. The mixture was centrifuged and washed twice with hydrochloric acid (1 M) and finally, washed with deionized water several times until neutralized pH.

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2.2. Preparation of chitosan-graphene oxide hydrogels An aqueous graphene dispersion was prepared and chitosan powder was added to obtain a 2 %w/v solution. NGO-CHT hydrogel were labelled as NGO-0, NGO-0.1, NGO-0.5, NGO-1.5 and NGO-3, based on the weight percentage of the NGO content per chitosan. Then lactic acid was added under stirring for 1 hour. A homogenous dispersion was produced by sonication for 2 hours and cast onto a petri dish and withered at 55°C. 2.3. Graphene oxide characterization We performed transmission electron microscopy (TEM, CM120FEG TEM philipss) analysis for thin films of NGO suspension at 200 kV to study the number and the stacking state .Moreover, the applied force on NGO was measured versus cantilever tip position by atomic force microscopy using Park Scientific model CP-Research (VEECO) at 25 °C. The layers of NGO was observed via AFM twenty times. 2.4. Scaffold characterizations 2.4.1. Swelling Measurements The dried scaffolds were weighted (Wd), submerged in Phosphate Buffered Saline (PBS) solution, PH = 7.4 and kept at 37 °C for 90 minutes. Then the swollen scaffolds were taken out from the solution and their extra of liquid was omitted using filter paper. Then the weight of the swollen scaffolds was measured (Ws). The swelling rate of the NGO-CHT hydrogels were determined by following equation: %=

× 100

(1)

Where Ws and Wd are the weights of the swollen and dry hydrogels, respectively. The results from each scaffold was obtained using three times repetition. 2.4.2.

Mechanical properties

We studied the mechanical properties of the scaffolds via measuring their compression strength and Young modulus. The samples were cut to cylindrical shape with appropriate size (10 mm in diameter and 20 mm in thickness), and 25 kN load cell at a strain rate of 10 mm/min was applied on the scaffold’s cross-section. All of these data was obtained by three times repetitions. 2.4.3.

FTIR analysis

We utilized fourier transform infrared (FTIR) spectroscopy (Nicolet 170SX, USA) in the wavenumber range of 400 to 4000 cm−1to analyse the composite of scaffolds prepared with of KBr. 2.4.4.

Electrical conductivity

We measured the conductivity of the dried samples via an instrument based four-point probe method at a constant current of 0.5 mA and ambient temperature. 2.5. Cell culture C132 was purchased from Pasteur institute (Iran). C132s were maintained in DMEM with (FBS) and penicillin/streptomycin solution. Cells were retained until around 80% confluent in incubator at 37 °C with a wet atmosphere of 95% air and 5% CO2. C132s were cultured on the all of scaffolds and RPMI media was poured on the samples.

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2.6. Statistical Analysis To analyse statistical significance, we used a one-way ANOVA where appropriate (SPSS Software). Error bar represents the mean ± standard deviation (SD) of measurements performed on each sample group. To determine whether a significant difference exists between specific treatments, we used Tukey’s multiple comparison tests (p < 0.05).

3. Results and discussion SEM and TEM were carried out for synthetized NGO to explore its morphology (Fig.1 A, a and b). As shown, the NGO with nano-scale and paper-like structure with very thin layers is clearly obvious in SEM and TEM micrographs. AFM height profile of the spin coated sample on silicon oxide substrate was shown in Fig. 1, c. The profile revealed a successful exfoliation of graphite layers and demonstrated few layer NGO structure. Therefore, the results confirm synthesis of a nanoscale few layer NGO.

Fig 1. A and B) SEM and TEM images of single layer of OG, c) AFM image of synthesized NGO which indicates the thickness of individual sheets of NGO.

Fig. 2A and B shows the aqueous GO dispersion and CHT and blend dried (NGO-3) hydrogels. Scanning electron microscopy (SEM) was applied to investigate the effect of the NGO addition in CHT matrix. Fig. 2 indicates SEM images of the hydrogels with different NGO contents. Fig. 1,C shows hybrid hydrogels a highly porous microstructure compared to pure CHT gels. As can be seen that the addition of NGO causes no obvious change in the morphology of porosity and there is no agglomeration indicating NGO dispersion of NGO sheets in the polymeric matrix. Moreover, the pore walls of hydrogels were smooth. It confirms that which was an evidence for the well dispersed NGO sheets inside the hybrid hydrogel. However, the porosity of NGO-CHT hydrogel decreased slightly as the NGO concentration increased from 0 to 3 Wt%.

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Fig. 2. A) Photo of synthesised NGO, B) photo of dried hydrogel, C) SEM images of NGO-CHT hydrogels with various concentrations of NGO: a) 0, b) 1.5, c) 0.1, and d) 3 %Wt.

3.1. Swelling analysis Generally, hydrogels are hydrophilic biomaterials due to their functional groups including carboxyl, amide, amino, and hydroxyl groups which can absorb a lot of water and other biological fluids such as PBS without dissolving in these fluids. The importance of having high potential of water absorption is related to the resemblance of swollen hydrogel to native tissues. It can be seen that NGO-0 swells up to 400 % in the first 10 min and up to 500 % in PBS within 90 minutes. As is obviously apparent in Fig. 3, the swelling of the NGO hydrogels could be controlled by NGO due to interaction between the polymer matrix and the hydrophobic graphene-sheets.

Fig. 3. Swelling characteristics of NGO-CHT hydrogels

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3.2. Mechanical properties The tensile strength and modulus of the hybrid hydrogels were provided in table 1. It can be fund that mechanical properties significantly enhanced with increasing NGO content. By addition of only 0.1 wt% NGO, the tensile strength increased by more than 34.8 %, whereas the addition of 3 wt% NGO enhanced the tensile strength by more than 264 % and the Young’s modulus about 156 % (Table 1). The results of mechanical analysis showed the enhancement of young’s modulus and tensile of the hybrid hydrogels established NGOod dispersion of NGO sheets in CHT matrix. Table1: Mechanical properties of chitosan composites with different graphene contents. Samples

Tensile strength [MPa]

Young’s Modulus [MPa]

NGO-0

28.54±1.7

654±23

NGO-0.1

34.78±2.1

787±26

NGO-0.5

57.68±2.5

986±31

NGO-1.5

75.45±1.3

1254±69

102.77±1.12

1678±76

NGO-3

3.3. FTIR analysis Chemical structure of graphene oxide (NGO) was explored using FTIR spectroscopy. Fig. 4 shows FTIR spectrum of synthetized NGO. The broad peaks appeared in FTIR spectroscopy of CHT at 3421 cm-1 , 2935 cm-1 and 1650 cm-1 belong to O-H, C-H and C=O bonds, respectively and the bands around 2800cm−1 match to C–H bonds. The absorption peaks from 1037, 1153 cm-1 and 1573 cm-1 are related to primary and secondary alcohol groups and N−H bonds, respectively. Moreover, Fig. 4 shows FTIR spectrum of synthetized GO and its peak at 3400 cm-1 and 1720 cm-1 which shows O-H and C=O bonds, respectively. Peaks at 1225 cm-1 and 1060 cm-1 show the presence of C-O bonds. All these peaks in GO FTIR spectrum approved oxygenation of the structure. Furthermore, the presence of C=C bonds in aromatic rings on GO plates was demonstrated with the peak appeared at 1621 cm-1. From the FTIR spectrum of NGO-3, it also can be seen that the peaks at 1410 cm−1, 1065 cm−1 are related to COO−, and C-O-C bonds, respectively.

Fig. 4. FTIR spectroscopy of CHT, NGO and NGO-3 hydrogel

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3.4. conductivity It is well known that one of the most important components for neural communication in vivo is the action potential produced at the synapse. It means that a functional engineered construct for neural tissue engineering application should have appropriate electrical conductivity to improve neurite extension and improve nerve regeneration. CHT as a weak cationic polyelectrolyte with conductivity of about 1×10-8 S m-1 is considered as nonconductive material [21]. However, the hydrogels conductivity was increased when NGO was added to CHT composites (Fig. 5). It can be found that the addition of only 0.1 % NGO enhances the conductivity more than 1×104 S m-1 and its enhancement increases to about 0.1 when NGO content enhances to 3% wt.%.

Fig. 5. Electrical conductivity measurement for different samples

3.5. Cell growth The proliferation rate of the cells next was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay by using a scanning multiwall spectrophotometer (ELISA reader). Briefly, the cells were cultured into a 96-well plate (Nunc, Denmark) at a density of 2 × 104 cells/well and then culture media was replaced by 90 μl powder extracts plus 10 μl FBS after 24 h. The extract media was removed after 1, 3, 7 and 14 days and 100 μl of MTT solution (0.5 mg ml−1) was poured into every well. The plate was incubated for 4 h at 37 ◦C and the purple formazan crystals were observed. To dissolve these crystals 100 μl of isopropanol (Sigma, USA) was added per well. After incubation for 15 min the optical wavelength density (OD) of formazan in the solution was obtained at two 570 and 630 nm using a multiwall microplate reader (STAT FAX 2100, USA). The results were presented in Fig. 6. As it can be seen for 1 there is no significant difference among all of the samples and control groups. After day 3, only two of the samples (NGO-0.5 and NGO-1.5) showed higher proliferation rate than other sample. The highest degree of cell growth was obtained for the NGO-1.5 sample for all extract intervals. According to MTT assay the optimum concentration of NGO is 1.5 Wt%.

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Fig. 6. The nerve cells viability using MTT assay for different samples

Cell viability assay of the samples also was performed using Acridine orange (AO) staining. 2× 106 C123s were seeded on the surface of hydrogels and placed into a 24-well plate. Then the samples were incubated at 37°C for 30 minutes. After nearly 3 days, the hydrogels including cells were incubated with the 0.01 mM AO diluted in 10 ml DMEM mixture for 10 minutes. Finally the samples were observed under a fluorescent microscope. As Fig. 7 shows live cells were stained green (AO) [23]. Results of cell staining showed that C123s has more proliferation rate for the samples containing 1.5 Wt% NGO. It can be seen that NGO supported cell proliferation and growth.

Fig 7. Cell viability of the samples, A) 0 Wt% NGO, B) 0.1 Wt% NGO, C) 0.5 Wt% NGO and D) 1.5 Wt% NGO (alive cell: green, magnification 10x)

4. Conclusions In this study, graphene-chitosan composite hydrogels were prepared by using a simple and quick approach and lactic acid as a cross-linker. Analysis showed excellent dispersion of NGO sheets in the matrix of chitosan/lactic acid matrix. These graphene composites indicated great improvements in mechanical properties, and controllable swelling behavior of the polymer matrix. Moreover, MTT assay showed these large advances supported nerve cells growth.

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