Graphene oxide assists polyvinylidene fluoride scaffold to reconstruct electrical microenvironment of bone tissue

Materials and Design 190 (2020) 108564

Contents lists available at ScienceDirect

Materials and Design journal homepage: www.elsevier.com/locate/matdes

Graphene oxide assists polyvinylidene fluoride scaffold to reconstruct electrical microenvironment of bone tissue Cijun Shuai a,b,c, Zichao Zeng a, Youwen Yang a, Fangwei Qi a,⁎, Shuping Peng d,e,⁎⁎, Wenjing Yang a, Chongxian He a, Guoyong Wang a, Guowen Qian a a

Institute of Bioadditive Manufacturing, Jiangxi University of Science and Technology, Nanchang 330013, China State Key Laboratory of High Performance Complex Manufacturing, Central South University, Changsha 410083, China Shenzhen Institute of Information Technology, Shenzhen 518172, China d NHC Key Laboratory of Carcinogenesis and The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Xiangya Hospital, Central South University, Changsha 410078, China e Cancer Research Institute, School of Basic Medical Sciences, Central South University, Changsha 410078, China b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• A novel 3D porous piezoelectric PVDF/ GO scaffold was fabricated by SLS. • The scaffold exhibited good piezoelectric and mechanical properties. • The enhanced electrical charges significantly improved the cell behaviors. • The scaffold presented a huge potential in reconstructing the electrical microenvironment of bone tissue.

a r t i c l e

i n f o

Article history: Received 4 January 2020 Received in revised form 8 February 2020 Accepted 10 February 2020 Available online 11 February 2020 Keywords: Bone regeneration Piezoelectric Polyvinylidene fluoride Graphene oxide

a b s t r a c t Polyvinylidene fluoride (PVDF), as a typical piezoelectric polymer, has a great potential in reconstructing the electrical microenvironment of bone tissue. In present study, graphene oxide (GO) was introduced into PVDF scaffold manufactured via selective laser sintering, aiming to enhance piezoelectric effect of PVDF by increasing β phase content. In detail, the oxygen-containing functional groups of GO could form strong hydrogen bonding with fluorine groups of PVDF. The interaction would force the fluorine groups to be arranged in parallel and perpendicular to the polymer chain, thereby inducing the transformation from α phase to β phase. Results demonstrated that the PVDF/0.3GO scaffold with improved β phase exhibited the maximal output voltage (~8.2 V) and current (~101.6 nA), which were improved by 82.2% and 68.2%, respectively, in comparison with pure PVDF. In vitro cell culture confirmed that enhanced electrical charges could significantly improve cell behavior. Moreover, the scaffold presented a 97.9% increase in

⁎ Corresponding author. ⁎⁎ Correspondence to: S. Peng, NHC Key Laboratory of Carcinogenesis and The Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Xiangya Hospital, Central South University, Changsha 410078, China. E-mail addresses: [email protected] (F. Qi), [email protected] (S. Peng). https://doi.org/10.1016/j.matdes.2020.108564 0264-1275/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).

2 Selective laser sintering

C. Shuai et al. / Materials and Design 190 (2020) 108564

compressive strength and 24.5% increase in tensile strength, which was attributed to GO reinforcements forming strong interaction with PVDF chains. These positive results suggested that the scaffold might have possible application in bone tissue engineering. © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

2. Experiment

Artificial bone scaffold, which enables to provide a mechanical and physiological support to cells for in vitro tissue regeneration and/or in vivo implantation, plays a vital role in bone tissue repair [1,2]. After years of development, the artificial bone scaffold has achieved a breakthrough from short-term “replacement filling” to permanent “replacement repair” [3,4]. However, some problems remain, such as poor tissue-scaffold interface integration, long repair cycles and uncontrollable repair capabilities [5]. To overcome these difficulties, the biomedical materials for artificial bone scaffold are required to be able to mimic the microenvironment of cell and tissues growth, and ultimately achieve the purpose of regulating the cell behavior and improving the tissue regeneration [6]. Piezoelectric biomaterial, as an electroactive material, has ability to reconstruct the electrical microenvironment of cell or tissue growth [7]. It can generate electrical stimulation under the action of body movement without the need of external power source. The electrical stimulation enable to regulate a variety of cellular functions, including reorganization of their cytoskeleton, differentiation, activation of intracellular pathways, secretion of proteins and gene expression. Among the various piezoelectric biomaterials, polyvinylidene fluoride (PVDF) possesses good biocompatibility and moderate mechanical property, which has attracted widespread attention in bone repair [8]. The piezoelectric performance of PVDF is considered to be positively related with its β phase content. In this regard, some strategies have been attempted to enhance the proportion of β phase in PVDF, including nanofiller addition, mechanical drawing, and thermal treatment [9]. Among them, adding nanofillers such as barium titanate [10], zinc oxide [11], and boron nitride [12] et al. has attracted most attentions due to its simplicity and effectiveness. Graphene derivatives like graphene oxide (GO) and reduced graphene oxide (rGO), have lately been considered as a promising material for bone tissue regeneration due to their excellent mechanical properties, biocompatibilities, and high surface-to-volume ratio [13]. Compared with rGO, GO consists of numerous oxygen-containing functional groups including hydroxyl, carboxyl and epoxy groups on its edges as well as basal plane. These oxygen-containing functional groups may form robust hydrogen bonding with the fluorine groups of PVDF. This interaction will force the fluorine groups to be arranged in parallel and perpendicular to the polymer chain, thereby inducing the conversion from α to β phase. Furthermore, GO with the oxygen-containing functional groups possess favorable hydrophilicity and bioactivity [14]. In this work, PVDF/GO scaffolds were fabricated via selective laser sintering (SLS). Compared with other manufacturing techniques, such as solvent casting [15], particulate leaching [16], thermal induced phase separation [17], SLS had an ability to precisely control the external shape of as-built scaffolds, so as to meet the personalization requirement for bone implants [18,19]. Moreover, it enabled to fabricate scaffolds with desired porosity and interconnectivity through computeraided design or 3D reconstruction of the actual defect area [20,21], which were beneficial for the nutrient transmission and cell growth [22]. The wettability, piezoelectric performance, mechanical property and thermal behavior of PVDF/GO scaffolds were systematically explored. The in vitro cell behavior was also evaluated.

2.1. Materials PVDF powders (FR906) were supplied by Shanghai 3F New Material Technology Co., Ltd. Graphite oxide with layers b3 and purity higher than 99 wt% was received from Chengdu Organic Chemicals Co., Ltd. of Chinese Academy of Sciences. 2.2. Preparation of PVDF/GO composite scaffolds The detailed preparation process of PVDF/GO composite powders with various GO contents (0, 0.1, 0.3 and 0.5 wt%) are displayed in Fig. 1. A certain amount of spherical PVDF powder (average diameter of 200 nm) and GO powder (average diameter of 0.5–3 μm and average thickness of 0.55–1.2 nm) were weighed according to the abovementioned mass ratio, and then slightly poured into a beaker containing ethanol solution. Subsequently, the mixture was ultrasonically vibrated for 2 h and then mechanically stirred at a speed of 300 rpm for another 10 h. Afterwards, the resultant suspension was filtrated using a funnel. Finally, PVDF/GO composite powders were obtained by drying at 55 °C for 12 h. The PVDF/GO composite scaffolds were fabricated using a selfdeveloped SLS device. The powders on the workbench were melted and adhered under the irradiation of laser beam, which controlled by the predesigned program. The scaffolds were processed with laser power of 2.7 W, scanning speed of 400 mm/s, preheating temperature of 151 °C and scan spacing of 0.14 mm. The three-dimensional porous scaffolds could be obtained through layer by layer construction. For the convenience of description, PVDF scaffold with various GO contents of 0.1, 0.3 and 0.5 wt%, which were named as PVDF/0.1GO, PVDF/0.3GO and PVDF/0.5GO, respectively. 2.3. Microstructural characterization The thermal stabilities of PVDF and PVDF/GO scaffolds were evaluated using thermogravimetric analyzer (TGA, TA Instruments, USA) at a heating rate of 10 °C/min in a nitrogen atmosphere. The melting and crystallization behavior of scaffolds were measured by differential scanning calorimeter (DSC, TA Instruments, USA) in nitrogen atmosphere at a heating/cooling rate of 10 °C/min. The morphologies of samples were detected by scanning electron microscopy (SEM, ZEISS, Germany) using an accelerating voltage of 15 kV, after sputter coating of the samples with gold. The phase structures of the scaffolds were analyzed by utilizing X-ray diffractometer (XRD, Karlsruhe, Germany) over the range of diffraction angle 2θ = 5° to 40° at a scanning speed of 2°/min. The functional groups were evaluated by Fourier transform infrared spectroscopy (FTIR, Tianjin Gang Dong Technology Co. Ltd., China) in the range of 400 ⁓ 2000 cm−1 with 2 cm−1 resolution. Raman spectroscopy of samples was recorded by LeiCA DMLM spectrometer (Renishaw, company, UK) with an excitation wavelength of 532 nm. Contact angle tester (Chengde Dingsheng Test machine Equipment Co., Ltd., China) was used to evaluate the wettability of the bulk samples with a diameter of 12 mm and thickness of 1.5 mm. The electrical output performance of samples was measured using SR570 Stanford measurement system and Keithley 6514 electrometer. Prior to examination, the samples were polarized by a corona discharge polarization method due

C. Shuai et al. / Materials and Design 190 (2020) 108564

3

Fig. 1. Processing diagram of PVDF/GO scaffolds.

to its advantages in cleanliness and could apply higher polarization voltages on porous samples [23]. The porous scaffolds fabricated with the same porosity were polarized at a voltage of 10 kV and a point source height of 60 mm [23]. The compressive and tensile properties of the specimens were evaluated utilizing the mechanical testing equipment (CMTS5205, MTS, USA). The scaffold specimens (L × W × H = 5 × 4 × 5 mm3 ) were placed between two circular platens for the compressive tests. The tensile specimens were fabricated in strict accordance with the standard of non-metallic stretching splines (ISO 604: 2002) with the size of 16 × 5 × 5 mm3. During the compressive and tensile measurements, the loading rate was 0.5 mm/s and each specimen were measured for three times. 2.4. In vitro cell tests MG-63 cells (American Type Culture Collection, Manassas, VA, USA) were incubated in the medium. Then the cells were separated with 0.25% trypsin and 0.03% EDTA and counted for subsequent experiments. Prior to cell seeding, the samples were sterilized in high-temperature sterilization box. Subsequently, the samples were placed in 12-well plates with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. After soaking for 2 h, the cells were seeded on scaffolds with a concentration of 4 × 105 cell/mL in 24-well plate at 37 °C. Meanwhile, the culture medium was replaced every day. Petri dishes were

selected for non-piezoelectric control group. During cultivation, the cells were subjected to ultrasound stimulation for three times a day for 15 s in the ultrasonic frequency of 110 Hz. Cell proliferation on scaffolds was quantitative analyzed using Cell Counting Kit-8 (CCK-8) assays. The cell suspension was slightly poured into a 24-well plate with a scaffold, and 0.5 mL of a 10% CCK-8 solution was added to well and cultured for 2 h. After 4 days of incubation, 100 μL of the culture solution was added dropwise to a 96-well plate, and the absorbance of the solution was measured with a microplate reader at 450 nm. Cell differentiation ability was evaluated by detecting Alkaline phosphatase (ALP) activity. Similarly, the cell suspension was added into 24well plate with a scaffold. After 7 days' culture, the cell-scaffold complex was rinsed with PBS solution for three times and then treated with 0.25% trypsin solution. Then, the cells were fixed with 4% paraformaldehyde for half an hour. Finally, the cells were stained using ALP staining reagents and subsequently detected by a microscope to analysis their cell differentiation.

2.5. Statistical analysis One way analysis of variance (ANOVA) was selected to evaluate the statistical significance. All data were presented as mean ± standard deviations. *p b .05 was recognized to be significant.

4

C. Shuai et al. / Materials and Design 190 (2020) 108564

formation of agglomerates might result in a deterioration of comprehensive performance for scaffolds [27].

3. Results and discussion 3.1. Microstructure

3.2. Thermal properties and crystallization behavior The FTIR spectra of PVDF powder, GO powder and PVDF/GO scaffold were displayed in the Fig. 2a. It could be observed from FTIR spectra of raw PVDF powder that there was a peak at 1178.3 cm−1, which corresponded to the telescopic vibration of fluorine groups (-CF2-) of PVDF [24]. The peak at 1724.06 cm−1 was ascribed to the telescopic vibration of carbonyl groups (C_O) of GO powder [25]. One point should be noted that the peaks of carbonyl groups of GO and fluorine groups of PVDF in composite scaffold were slightly shifted to 1718.28 and 1180.23 cm−1, respectively, which was due to the hydrogen bonding between carbonyl groups of GO and fluorine groups of PVDF [26]. The cryo-fracture surfaces by liquid nitrogen were observed by SEM, as displayed in Fig. 2b-2e. All samples exhibited dense internal structures under the designed processing parameters. Compared with PVDF (Fig. 2b), the scaffold with GO concentration b0.3 wt% (Fig. 2b2d) presented a uniform dispersion of GO throughout PVDF matrix, which was conducive to their comprehensive performance. However, the GO sheets were obviously agglomerated in matrix (Fig. 2e) when its concentration further increased to 0.5 wt%. This result might be attributed to the strong van der Waals force between GO layers. The

The thermal stabilities of PVDF and PVDF/GO composite scaffolds were examined by TG analysis, with TGA and DTG curves showed in Fig. 3a and b. It could be obviously found that all samples present a one-step decomposition process. Particularly, the initial decomposition temperature of PVDF/0.3GO scaffold was significantly increased from 410 to 435 °C. Hence, it was demonstrated that the introduction of GO enhanced the thermal stability of the PVDF composite and further avoid the thermal decomposition of PVDF under the irradiation of high-energy laser beam [28,29]. This enhancement might be ascribed to the inherently high thermal stability of GO sheets [30]. In addition, GO sheets dispersed in polymer matrix could block the penetration of oxygen, thereby preventing the decomposition of PVDF [31]. However, the thermal stability of the scaffolds slightly decreased with GO further increased to 0.5 wt%. This might be attributed to the aggregation of GO, which weakened the barrier effect on the penetration of oxygen [32]. The thermal behavior of PVDF and PVDF/GO scaffolds were determined using DSC, and their corresponding melting and crystallization curves were recorded in Fig. 3c and d, respectively. PVDF scaffold

(a)

(b)

(c) GO

2 μm

2 μm

(d)

(e)

Aggregates

GO

2 μm

2 μm

Fig. 2. FTIR spectra (a), cryo-fractured morphology of PVDF (b), PVDF/0.1GO (c), PVDF/0.3GO (d) and PVDF/0.5GO (e) scaffolds.

C. Shuai et al. / Materials and Design 190 (2020) 108564

(a)

(b)

(c)

(d)

5

Fig. 3. TGA (a), DTG (b), DSC heating (c) and cooling (d) curves of PVDF and PVDF/GO scaffold.

presented a single melting temperature at 166.9 °C, which was corresponded to the melt endothermic behavior of α phase (Fig. 3c) [33]. It was worth noting that a new melting peak presented at 170.8 °C when GO was introduced, which was assigned to the melting of β phase [34]. However, the content of β phase in PVDF was decreased with GO further increased. This was because excessive GO agglomerated in the matrix, which weakened its ability to induce nucleation of β phase. As presented in Fig. 3d, the crystallization temperature peak of PVDF shifted to high temperature with the introduction of GO, which was attributed to the heterogeneous nucleation effect of GO. 3.3. Mechanical properties The mechanical performance of the scaffold plays a critical role in bearing different stresses and offering structural support to the bone tissues [35]. The compressive stress-strain curves and derived bar diagram are presented in Fig. 4a and b, respectively. It could be found that PVDF scaffold exhibited an ultimate compressive strength of 4.9 ± 0.2 MPa, whereas PVDF/0.1GO and PVDF/0.3GO scaffolds increased to 8.1 ± 0.3 MPa and 9.7 ± 0.2 MPa, respectively. Unfortunately, the ultimate compressive strength of PVDF/0.5GO scaffold was decreased to 5.6 ± 0.1 MPa. Even so, the scaffold fabricated in this work were still fulfilled the requirements of natural cancellous bone, which commonly exhibited a compressive strength of 1–10 MPa [36]. It could be seen from Fig. 4b that the compressive strength and modulus of PVDF scaffold were first increased and then decreased with GO content increasing. The PVDF/0.3GO scaffold presented maximum compressive strength of 9.7 ± 0.2 MPa and modulus of 75.6 ± 4 MPa, which were increased by almost 97.9% and 22.3%, respectively, in comparison with PVDF scaffold. This enhancement might be ascribed to the fact that GO could act as crack resistor to prevent the expansion of crack [37]. Additionally,

the decrease in compressive strength and modulus for PVDF/0.5GO was due to the agglomeration of GO in the matrix. The tensile behavior of samples was measured, with results showed in Fig. 4c and d. The tensile properties of the samples were found to follow the same trend as that of the compressive properties. PVDF/0.3GO scaffold exhibited the maximum tensile strength of 33 ± 1.3 MPa and modulus of 815 ± 18 MPa, which increased by 24.5% and 73.4%, respectively, as compared with that of PVDF. To comprehensively understand the role of GO for the improved mechanical properties, the tensile fractured sections were observed by SEM, as displayed in Figs. 4e-4h. As expected, the PVDF presented relatively smooth fracture morphology. Abundant of ligaments were presented on the tensile fractured surface of PVDF/0.1GO, and the ligaments were increased as GO reached to 0.3 wt%. Meanwhile, GO uniformly distributed in the matrix and exhibited good interface compatibility. However, GO became aggregated with its content further increasing to 0.5 wt%. Therefore, the mechanical enhancement mechanism of GO to PVDF could be clarified as follows. On one hand, GO uniformly dispersed in the PVDF matrix could act as a stress concentration point, which would consume some of energy during the stretching process of samples [38]. On the other hand, there was a strong interaction formed via hydrogen bonding between carbonyl group of the GO and fluorine group of PVDF, as shown in Fig. 4i. The enhanced interface bonding strength would improve the mechanical transfer efficiency of PVDF/GO composites. In this case, more energy would be dissipated during the fracture process by transferring more stress to the GO [39]. 3.4. Wettability and phase structure The hydrophilicity of scaffolds had positive effect on protein adsorption and cell adhesion [40]. The water contact angles on samples with

6

C. Shuai et al. / Materials and Design 190 (2020) 108564

(a)

(b)

(c)

(d)

(e)

(i)

(f) GO 5 μm

GO 5 μm C F H O

(h)

(g)

PVDF

Aggregates GO 5 μm

5 μm

Fig. 4. The compressive (a, b) tensile performance (c, d), tensile fractured images of PVDF/GO (e), PVDF/0.1GO (f), PVDF/0.3GO (g), PVDF/0.5GO (h), and the interaction between GO and PVDF (h).

various GO contents were measured to determine their hydrophilic property, as shown in Fig. 5a. As expected, the water contact angle of PVDF was 105 ± 3°, which was due to its inherent hydrophobic property. The hydrophilic property of the PVDF was significantly improved with the introduction of GO [41]. The water contact angle of PVDF/ 0.1GO, PVDF/0.3GO and PVDF/0.5GO were 94 ± 2°, 87 ± 3° and 75 ± 5°, respectively. These results might be due to the nature hydrophilic property of GO which contains a large amount of hydroxyl functional groups. The phase structures of scaffolds were examined by XRD (Fig. 5b). It could be observed that PVDF exhibited peaks at 17.6°, 18.3°, 19.9° and 26.6°, which were ascribed to the (110), (020), (110) and (021) planes

of α phase, respectively [20]. There was a new peak appeared at 20.4° with the incorporation of GO, which was ascribed to the (110) and (200) planes of β phase in PVDF [42]. Additionally, with increasing of GO content, the β phase characteristic peak intensity at 20.4° was first increased and then decreased. To further confirm the nucleation of β phase nucleation in the GO doped PVDF samples, FT-IR measurement was carried out and the results were displayed in Fig. 5c. There were two characteristic peaks situated at 763, 794 and 975 cm−1, which were assigned to the bending vibration of\\CF2\\and rocking vibration of\\CH2\\, respectively. Obviously, the characteristic peaks located at 840 and 883 cm−1 were enhanced intensively with the introduction of GO. These peaks could be

C. Shuai et al. / Materials and Design 190 (2020) 108564

7

Fig. 5. Contact angles (a), XRD (b), FT-IR (c), and β phase content (d) of samples.

ascribed to the wagging vibration of\\CH2\\and symmetric stretching of\\CF2\\in β phase, respectively. The β phase fraction (Fβ) in the PVDF was calculated according to the Lambert-Beer Law: Fβ ¼

Aβ þ Aβ ð1:26ÞAα

Here, Aα and Aβ represented the absorbance of samples at 763 and 840 cm−1, respectively. The β phase fraction was increased from 44.6% to 49.1% when the GO content reached to 0.3 wt%. However, the β phase fraction was decreased with GO further increasing, which was consistent with the XRD results. The improved β phase fraction in PVDF/GO scaffold could be attributed to the strong interaction between carbonyl group of GO and fluoride group of PVDF. The interaction could drive the fluoride groups to be arranged in parallel and perpendicular to the polymer chain, thus resulting in the conversion from α phase to β phase [43]. Furthermore, GO could provide active sites for the nucleation of PVDF, and induce amorphous regions to β phase [44]. 3.5. Piezoelectric response To determine the piezoelectric output performance of PVDF/GO scaffolds, conductive copper foil was utilized as the electrode attached to the upper and lower surfaces of the scaffolds. Periodic mechanical hitting equipment was used to provide external mechanical pressure. The working principle of piezoelectric responds of scaffolds is shown in Fig. 6a. The scaffolds could not generate electrical current in the absence of external stress (Fig. 6a1). Once vertical mechanical stress was

applied, the dipole in samples would deflect to form polarized charges, which would further induce the same amount of opposite charge on the surface of electrode. In this case, a potential would present between the two electrodes and the electron would flow from one electrode to other across an external load without any external bias (Fig. 6a2). When the pressure was removed, the potential resulted from piezoelectricity would diminish quickly and electrons would migrate to the opposite electrode through external circuit (Fig. 6a3). The open circuit voltage and short circuit current of samples are shown in Fig. 6b and c, respectively. The results indicated that both open-circuit voltage and short-circuit current of samples firstly improved and then reduced with the introduction of GO. The composite scaffolds containing 0.3 wt% GO exhibited the highest output performance of ~8.2 V and ~101.6 nA (Fig. 6d and e), which were improved by 82.2% and 68.3%, respectively, in comparison with that of pure PVDF. The enhancement in output performance could be mainly attributed to the fact that GO could induce the formation of β phase. Meanwhile, good interface bonding between PVDF and GO also had positive effect on improvement of output performance of the composite scaffold by enhancing their mechanical-electrical conversion efficiencies. This implied that the scaffolds could convert more mechanical energy into electrical energy under the same external stress. Furthermore, the enhancement in output performance might also be related to the electrical conductivities of the composite scaffolds. As shown in Fig. 6f, the conductivities of the scaffold were increased with the incorporation of GO. The enhanced conductivity was conducive to the transmission of electrons, thereby improving the output performance of the scaffold. However, the output performance of scaffold decreased with GO

8

C. Shuai et al. / Materials and Design 190 (2020) 108564

(a2)

(a1)

(a3)

e-

e-

Positive electrode Dipole z

I

I

z

PVDF

z

Negative electrode

Original

Pressing

Releasing

(c)

(b)

(d)

8.2 V

101.6 nA

(e)

(f)

releasing

stressing stressing

releasing

Fig. 6. Schematic of the electrical output of samples (a), open-circuit voltage (b) and short-circuit current (c), single cycled voltage (d) and current (e) of PVDF/0.3GO composite scaffold, (f) effect of GO concentration on conductivities of scaffold.

US+ (b)

100 μm

100 μm

100 μm

100 μm

100 μm

100 μm

PVDF/0.3GO

PVDF

Control

US(a)

Fig. 7. (a) Fluorescence images and (b) optimal densities of cells cultured on different substrates for 4 days, with (US+) or without (US-) ultrasounds.

C. Shuai et al. / Materials and Design 190 (2020) 108564

Control

PVDF

9

PVDF/0.3GO

US-

100 μm

100 μm

100 μm

100 μm

100 μm

100 μm

US+

Fig. 8. Fluorescence staining results of cells after 7 days' culture.

content further increasing to 0.5 wt%, which might be ascribed to the aggregation of GO in PVDF matrix. 3.6. Cellular response The fluorescence images of MG-63 cells were shown in Fig. 7a. Obviously, the PVDF/0.3GO scaffolds presented more cells than that of PVDF and control group after 4 days' culture in the absence of ultrasound stimulation. Once ultrasound was applied, the cells on PVDF and PVDF/0.3GO scaffolds were further increased. However, the cell number on the control group basically maintain stable, which suggested that the ultrasound had no effect on the cell proliferation [45]. The cell proliferations on the various substrates after cultivation for 4 days were quantitatively evaluated by CCK-8 assays (Fig. 7b). Similarly, the optical density of cell on control group stayed stable, which was consistent with the fluorescence images observation. This result confirmed that the cell proliferation was independent with ultrasound stimulation [46]. Markedly, the optical densities of scaffolds were greatly improved with the introduction of ultrasound stimulation. More importantly, there was significant difference in optical densities between PVDF and PVDF/GO scaffold under the ultrasound stimulation, which revealed that more charges could promote cell proliferation more effectively. Alkaline phosphatase (ALP) was an exoenzyme of osteoblasts, and its expression activity was a distinct feature of osteoblast differentiation [47]. The cell differentiation of MG-63 cells was shown in Fig. 8. It could be viewed from control group that single ultrasound had almost no impact on ALP staining area, which was consistent with the results as reported by Ma et al. [48]. It was worth noting that ALP staining area of cells on PVDF/0.3GO scaffold was larger than that of pure PVDF scaffold in the presence of ultrasound stimulation. This result might be attributed to the enhanced mechanical-electrical conversion capability of PVDF/0.3GO scaffold, which could generate more electrical stimulation under the same external stress. The enhanced electrical stimulation could more efficiently induce cell differentiation by improving calcium ions concentration, activating signaling pathways and regulating the expressions of growth factors [49]. 4. Conclusions In summary, piezoelectric PVDF/GO composite scaffolds for bone repair were fabricated via SLS technique. Results demonstrated that the

hydrogen bonding interaction between fluorine group of PVDF and carbonyl group of GO nanosheets induced the transformation from α to β phase, thereby resulting in enhanced electrical output performance. The in vitro cell culture confirmed that the PVDF/0.3GO composite scaffold could efficiently promote the cell behavior by generating electrical stimulation under the ultrasound conditions. Meanwhile, PVDF/0.3GO composite scaffolds exhibited a significant enhancement in tensile strength and compressive strength, which were increased by 24.5% and 97.9%, respectively, in comparison with PVDF scaffolds. This might be attributed to GO reinforcements forming strong interactions with PVDF chains. Moreover, the hydrophilic property of the PVDF scaffold was also significantly improved with the addition of GO. CRediT authorship contribution statement Cijun Shuai: Conceptualization, Methodology, Project administration, Funding acquisition. Zichao Zeng: Investigation, Writing - original draft, Data curation. Youwen Yang: Software, Validation. Fangwei Qi: Writing - review & editing, Supervision, Methodology, Validation, Visualization. Shuping Peng: Writing - review & editing, Supervision, Validation. Wenjing Yang: Formal analysis. Chongxian He: Resources. Guoyong Wang: Data curation. Guowen Qian: Visualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This study was supported by the following funds: (1) The Natural Science Foundation of China (51935014, 51905553, 81871494, 81871498, 51705540); (2) Hunan Provincial Natural Science Foundation of China (2019JJ50774, 2018JJ3671, 2019JJ50588); (3) JiangXi Provincial Natural Science Foundation of China (20192ACB20005); (4) Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2018); (5) The Open Sharing Fund for the Large-scale Instruments and Equipments of Central South University; (6) The Project of Hunan Provincial Science and Technology Plan (2017RS3008). (7) Science and Technology Project of Jiangxi Provincial Department of Education (GJJ180490).

10

C. Shuai et al. / Materials and Design 190 (2020) 108564

References [1] X. Gao, J. Song, Y. Zhang, X. Xu, S. Zhang, P. Ji, S. Wei, Bioinspired design of polycaprolactone composite nanofibers as artificial bone extracellular matrix for bone regeneration application, ACS Appl. Mater. Interfaces 8 (41) (2016) 27594–27610. [2] Y. Cao, T. Shi, C. Jiao, H. Liang, R. Chen, Z. Tian, A. Zou, Y. Yang, Z. Wei, C. Wang, L. Shen, Fabrication and properties of zirconia/hydroxyapatite composite scaffold based on digital light processing, Ceram. Int. 46 (2) (2020) 2300–2308. [3] Y. Yang, C. He, E. Dianyu, W. Yang, F. Qi, D. Xie, L. Shen, S. Peng, C. Shuai, Mg bone implant: features, developments and perspectives, Mater. Des. 185 (2020) 108259. [4] C. Gao, M. Yao, S. Li, P. Feng, S. Peng, C. Shuai, Highly biodegradable and bioactive FePd-bredigite biocomposites prepared by selective laser melting, J. Adv. Res. 20 (2019) 91–104. [5] D. Tang, R.S. Tare, L.-Y. Yang, D.F. Williams, K.-L. Ou, R.O.C. Oreffo, Biofabrication of bone tissue: approaches, challenges and translation for bone regeneration, Biomaterials 83 (2016) 363–382. [6] C. Shuai, W. Yang, C. He, S. Peng, C. Gao, Y. Yang, F. Qi, P. Feng, A magnetic microenvironment in scaffolds for stimulating bone regeneration, Mater. Des. 185 (2020), 108275. [7] M. Kitsara, A. Blanquer, G. Murillo, V. Humblot, S. De Braganca Vieira, C. Nogues, E. Ibanez, J. Esteve, L. Barrios, Permanently hydrophilic, piezoelectric PVDF nanofibrous scaffolds promoting unaided electromechanical stimulation on osteoblasts, Nanoscale 11 (18) (2019) 8906–8917. [8] P.K. Szewczyk, S. Metwally, J.E. Karbowniczek, M.M. Marzec, E. Stodolak-Zych, A. Gruszczyński, A. Bernasik, U. Stachewicz, Surface-potential-controlled cell proliferation and collagen mineralization on electrospun polyvinylidene fluoride (PVDF) Fiber scaffolds for bone regeneration, ACS Biomaterials Science & Engineering 5 (2) (2019) 582–593. [9] J. Yan, M. Liu, Y.G. Jeong, W. Kang, L. Li, Y. Zhao, N. Deng, B. Cheng, G. Yang, Performance enhancements in poly(vinylidene fluoride)-based piezoelectric nanogenerators for efficient energy harvesting, Nano Energy 56 (2019) 662–692. [10] X. Zhang, C. Zhang, Y. Lin, P. Hu, Y. Shen, K. Wang, S. Meng, Y. Chai, X. Dai, X. Liu, Nanocomposite membranes enhance bone regeneration through restoring physiological electric microenvironment, ACS Nano 10 (8) (2016) 7279–7286. [11] R. Augustine, P. Dan, A. Sosnik, N. Kalarikkal, N. Tran, B. Vincent, S. Thomas, P. Menu, D. Rouxel, Electrospun poly (vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation, Nano Res. 10 (10) (2017) 3358–3376. [12] G.G. Genchi, E. Sinibaldi, L. Ceseracciu, M. Labardi, A. Marino, S. Marras, G. De Simoni, V. Mattoli, G. Ciofani, Ultrasound-activated piezoelectric P (VDF-TrFE)/boron nitride nanotube composite films promote differentiation of human SaOS-2 osteoblast-like cells, Nanomedicine 14 (7) (2018) 2421–2432. [13] M. He, X. Chen, Z. Guo, X. Qiu, Y. Yang, C. Su, N. Jiang, Y. Li, D. Sun, L. Zhang, Super tough graphene oxide reinforced polyetheretherketone for potential hard tissue repair applications, Compos. Sci. Technol. 174 (2019) 194–201. [14] S. Liu, C. Zhou, S. Mou, J. Li, M. Zhou, Y. Zeng, C. Luo, J. Sun, Z. Wang, W. Xu, Biocompatible graphene oxide–collagen composite aerogel for enhanced stiffness and in situ bone regeneration, Mater. Sci. Eng. C 105 (2019) 110137. [15] S. Mondal, U. Pal, A. Dey, Natural origin hydroxyapatite scaffold as potential bone tissue engineering substitute, Ceram. Int. 42 (16) (2016) 18338–18346. [16] A. Sola, J. Bertacchini, D. D’Avella, L. Anselmi, T. Maraldi, S. Marmiroli, M. Messori, Development of solvent-casting particulate leaching (SCPL) polymer scaffolds as improved three-dimensional supports to mimic the bone marrow niche, Mater. Sci. Eng. C 96 (2019) 153–165. [17] K. Szustakiewicz, M. Gazińska, B. Kryszak, M. Grzymajło, J. Pigłowski, R.J. Wiglusz, M. Okamoto, The influence of hydroxyapatite content on properties of poly (L-lactide)/ hydroxyapatite porous scaffolds obtained using thermal induced phase separation technique, Eur. Polym. J. 113 (2019) 313–320. [18] C. Gao, M. Yao, C. Shuai, S. Peng, Y. Deng, Nano-SiC reinforced Zn biocomposites prepared via laser melting: microstructure, mechanical properties and biodegradability, Journal of Materials Science & Technology 35 (11) (2019) 2608–2617. [19] C. Shuai, Y. Cheng, Y. Yang, S. Peng, W. Yang, F. Qi, Laser additive manufacturing of Zn-2Al part for bone repair: formability, microstructure and properties, J. Alloys Compd. 798 (2019) 606–615. [20] C. Shuai, G. Liu, Y. Yang, W. Yang, C. He, G. Wang, Z. Liu, F. Qi, S. Peng, Functionalized BaTiO3 enhances piezoelectric effect towards cell response of bone scaffold, Colloids Surf. B: Biointerfaces 185 (2020) 110587. [21] C. Shuai, W. Yang, Y. Yang, H. Pan, C. He, F. Qi, D. Xie, H. Liang, Selective laser melted Fe-Mn bone scaffold: microstructure, corrosion behavior and cell response, Materials Research Express 7 (1) (2020), 015404. [22] Y. Yang, J. Zan, W. Yang, F. Qi, S. Huang, S. Peng, C. Shuai, Metal organic framework as compatible reinforcement in biopolymer bone scaffold, Materials Chemistry Frontiers (2020) https://doi.org/10.1039/C9QM00772E. [23] S. Banerjee, K. Cook-Chennault, W. Du, U. Sundar, H. Halim, A. Tang, Piezoelectric and dielectric characterization of corona and contact poled PZT-epoxy-MWCNT bulk composites, Smart Mater. Struct. 25 (11) (2016), 115018. [24] R.D. Simoes, M.A. Rodriguez-Perez, J.A. De Saja, C.J.L. Constantino, Tailoring the structural properties of PVDF and P(VDF-TrFE) by using natural polymers as additives, Polym. Eng. Sci. 49 (11) (2009) 2150–2157.

[25] A.B. Chen, C.C. Zhao, Y.F. Yu, J.H. Yang, Graphene quantum dots derived from carbon fibers for oxidation of dopamine, Journal of Wuhan University of TechnologyMaterials Science Edition 31 (6) (2016) 1294–1297. [26] W. Jang, J. Yun, K. Jeon, H. Byun, PVDF/graphene oxide hybrid membranes via electrospinning for water treatment applications, RSC Adv. 5 (58) (2015) 46711–46717. [27] X. Li, Y.M. Liu, W.G. Li, C.Y. Li, J.G. Sanjayan, W.H. Duan, Z. Li, Effects of graphene oxide agglomerates on workability, hydration, microstructure and compressive strength of cement paste, Constr. Build. Mater. 145 (2017) 402–410. [28] P. Feng, Y. Kong, L. Yu, Y. Li, C. Gao, S. Peng, H. Pan, Z. Zhao, C. Shuai, Molybdenum disulfide nanosheets embedded with nanodiamond particles: co-dispersion nanostructures as reinforcements for polymer scaffolds, Appl. Mater. Today 17 (2019) 216–226. [29] H. Liang, Y. Yang, D. Xie, L. Li, N. Mao, C. Wang, Z. Tian, Q. Jiang, L. Shen, Trabecularlike Ti-6Al-4V scaffolds for orthopedic: fabrication by selective laser melting and in vitro biocompatibility, Journal of Materials Science & Technology 35 (7) (2019) 1284–1297. [30] Y.-J. Wan, W.-H. Yang, S.-H. Yu, R. Sun, C.-P. Wong, W.-H. Liao, Covalent polymer functionalization of graphene for improved dielectric properties and thermal stability of epoxy composites, Compos. Sci. Technol. 122 (2016) 27–35. [31] Q. Jing, W. Liu, Y. Pan, V.V. Silberschmidt, L. Li, Z. Dong, Chemical functionalization of graphene oxide for improving mechanical and thermal properties of polyurethane composites, Mater. Des. 85 (2015) 808–814. [32] F. Ren, G. Zhu, P. Ren, Y. Wang, X. Cui, In situ polymerization of graphene oxide and cyanate ester–epoxy with enhanced mechanical and thermal properties, Appl. Surf. Sci. 316 (2014) 549–557. [33] A. Islam, A.N. Khan, M.F. Shakir, K. Islam, Strengthening of β polymorph in PVDF/FLG and PVDF/GO nanocomposites, Materials Research Express 7 (1) (2019), 015017. [34] P. Martins, A.C. Lopes, S. Lanceros-Mendez, Electroactive phases of poly(vinylidene fluoride): determination, processing and applications, Prog. Polym. Sci. 39 (4) (2014) 683–706. [35] L. Wang, J. Kang, C. Sun, D. Li, Y. Cao, Z. Jin, Mapping porous microstructures to yield desired mechanical properties for application in 3D printed bone scaffolds and orthopaedic implants, Mater. Des. 133 (2017) 62–68. [36] D. Mao, Q. Li, D. Li, Y. Tan, Q. Che, 3D porous poly(ε-caprolactone)/58S bioactive glass–sodium alginate/gelatin hybrid scaffolds prepared by a modified melt molding method for bone tissue engineering, Mater. Des. 160 (2018) 1–8. [37] S. Zhang, X. Huang, J. Feng, F. Qi, E. Dianyu, Y. Jiang, L. Li, S. Xiong, J. Feng, Structure, compression and thermally insulating properties of cellulose diacetate-based aerogels, Mater. Des. (2020) 108502. [38] R. Aradhana, S. Mohanty, S.K. Nayak, Comparison of mechanical, electrical and thermal properties in graphene oxide and reduced graphene oxide filled epoxy nanocomposite adhesives, Polymer 141 (2018) 109–123. [39] Y. Wang, G. Colas, T. Filleter, Improvements in the mechanical properties of carbon nanotube fibers through graphene oxide interlocking, Carbon 98 (2016) 291–299. [40] C. Shuai, G. Liu, Y. Yang, F. Qi, S. Peng, W. Yang, Z. Liu, Construction of an electric microenvironment in piezoelectric scaffolds fabricated by selective laser sintering, Ceram. Int. 45 (16) (2019) 20234–20242. [41] C. Shuai, J. Zan, Y. Yang, S. Peng, W. Yang, F. Qi, L. Shen, Z. Tian, Surface modification enhances interfacial bonding in PLLA/MgO bone scaffold, Mater. Sci. Eng. C 108 (2020) 110486. [42] N. Jia, Q. He, J. Sun, G. Xia, R. Song, Crystallization behavior and electroactive properties of PVDF, P(VDF-TrFE) and their blend films, Polym. Test. 57 (2017) 302–306. [43] S. Mishra, R. Sahoo, L. Unnikrishnan, A. Ramadoss, S. Mohanty, S.K. Nayak, Investigation of the electroactive phase content and dielectric behaviour of mechanically stretched PVDF-GO and PVDF-rGO composites, Mater. Res. Bull. 124 (2020), 110732. [44] J.-E. Lee, Y. Eom, Y.-E. Shin, S.-H. Hwang, H.-H. Ko, H.G. Chae, Effect of interfacial interaction on the conformational variation of poly(vinylidene fluoride) (PVDF) chains in PVDF/graphene oxide (GO) nanocomposite fibers and corresponding mechanical properties, ACS Appl. Mater. Interfaces 11 (14) (2019) 13665–13675. [45] L. Feng, X. Liu, H. Cao, L. Qin, W. Hou, L. Wu, A comparison of 1-and 3.2-MHz lowintensity pulsed ultrasound on Osteogenesis on porous titanium alloy scaffolds: an in vitro and in vivo study, J. Ultrasound Med. 38 (1) (2019) 191–202. [46] C. Shuai, L. Yu, W. Yang, S. Peng, Y. Zhong, P. Feng, Phosphonic acid coupling agent modification of HAP nanoparticles: interfacial effects in PLLA/HAP bone scaffold, Polymers 12 (1) (2020) 199. [47] S. He, S. Yang, Y. Zhang, X. Li, D. Gao, Y. Zhong, L. Cao, H. Ma, Y. Liu, G. Li, S. Peng, C. Shuai, LncRNA ODIR1 inhibits osteogenic differentiation of hUC-MSCs through the FBXO25/H2BK120ub/H3K4me3/OSX axis, Cell Death Dis. 10 (12) (2019) 947. [48] B. Ma, F. Liu, Z. Li, J. Duan, Y. Kong, M. Hao, S. Ge, H. Jiang, H. Liu, Piezoelectric nylon-11 nanoparticles with ultrasound assistance for high-efficiency promotion of stem cell osteogenic differentiation, J. Mater. Chem. B 7 (11) (2019) 1847–1854. [49] M. Hoop, X.-Z. Chen, A. Ferrari, F. Mushtaq, G. Ghazaryan, T. Tervoort, D. Poulikakos, B. Nelson, S. Pané, Ultrasound-mediated piezoelectric differentiation of neuron-like PC12 cells on PVDF membranes, Sci. Rep. 7 (1) (2017) 4028.