MgO whiskers reinforced poly(vinylidene fluoride) scaffolds

Abstract

In this study, poly(vinylidene fluoride) (PVDF) scaffolds with MgO whiskers were prepared through selective laser sintering, and their properties were studied in terms of mechanical and biological properties. The results indicated that the tensile strength and elastic modulus of the scaffolds were increased by 52.53% and 29.31% respectively when the MgO whiskers content was 2 wt%. The enhancement mechanisms were that MgO whiskers improved the crystallinity by providing nucleating sites for the crystallization of PVDF, absorbed and transferred energy during crack extension via the good interfacial adhesion, and transformed the fracture mode from crazing-tearing/brittle mode to fibrillation/ductile mode. In addition, MG-63 cell culture experiments indicated that better attachment and proliferation, and higher alkaline phosphatase (ALP) activity, were obtained in the case of the PVDF scaffolds with MgO whiskers compared to in the PVDF scaffolds without MgO whiskers.

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1. Introduction

Poly(vinylidene fluoride) (PVDF), a semi-crystalline biopolymer, has been used in the biomedical field and bone tissue engineering owing to its piezoelectric properties, biocompatibility and processability.^1–5 It has four crystalline structures (α, β, δ and γ) where the β-phase PVDF has the largest piezoelectric response.^6,7 Some researchers have found that the piezoelectricity has an important role in cell response.^8–11 However its mechanical properties, such as tensile strength and elastic modulus, are lower than that of natural cortical bones. Meanwhile, it lacks osteoinductivity and osteoconductivity, which makes it hard for the new tissue to bond with PVDF. Therefore, its further application in the biomedical field is limited. Some researchers have used granular fillers with excellent bioactivity, such as calcium carbonate, calcium phosphate and bioactive glass, to improve the properties of PVDF. These particle reinforcement phases could enhance the mechanical properties of PVDF to a certain extent.^12–14

Whiskers, with large length-to-diameter ratio, grow from high-purity single-crystal with highly ordered atomic arrangement. They are nearly internal defect-free such as cavity, dislocation and grain boundary, and the strength of them are close to the theoretical value of the complete crystal.^15–18 In recent years, some researchers have used hydroxyapatite whiskers to reinforce the mechanical properties of PVDF materials.^19,20 MgO has excellent antibacterial properties, good biocompatibilities and bioactive abilities.^21–23 Moreover Mg plays a particularly important role in bone metabolism and stimulating new bone formation, and may improve cell adhesion, proliferation and differentiation.^24–26 Therefore, MgO whiskers as reinforcement phase not only improve the mechanical properties of matrix by interface debonding, load transmitting, pull effect and bridging effect, but also enhance the biological properties.²⁷

Bone scaffolds also required interconnected porous structures which could mimic architecture and function of the extracellular matrix and support cell growth and the ingrowth of new bone tissue.^28–30 Some conventional methods, such as phase separation, gas foaming and gel casting, were used to fabricate scaffolds. However, these methods could only obtain imprecise and finite interconnectivity pore structure.⁴ Selective Laser Sintering (SLS) is an additive manufacturing method, which had the capability to construct complex shape and accurate interconnected porous architecture.^31,32

In this study, MgO whiskers are incorporated into PVDF matrix to enhance the mechanical and biological properties of the scaffolds fabricated with SLS. The effects of the whiskers on the properties were researched. The mechanical properties were analyzed by tensile strength and elastic modulus. The biological properties were evaluated with cell culture experiments. The crystallinity was researched through differential scanning calorimetry (DSC).

2. Experimental

2.1. Materials

MgO whiskers were purchased from Hefei ika new material technology Co., LTD (An Hui, China). PVDF powders were obtained from Huangjiang Huayi Plastics Material Co., LTD (Guang Dong, China). The mixed powders of PVDF/MgO whiskers were prepared as the following process. The MgO whiskers and PVDF powders were weighed according to the weight ratios (100 : 0, 99.5 : 0.5, 99 : 1, 98 : 2, 97 : 3 and 95 : 5). Afterwards, the powders were put into beakers with 30 ml anhydrous alcohol. Subsequently, the mixed solutions were processed with ultrasound 30 min for distribution, and then handled with planetary ball mill 1 h for further distribution. Eventually the solutions were filtered slowly to obtain the mixed powders. The powders were put into electrothermal blowing dry box for drying at 50 °C until the weight didn't change. A series of PVDF/MgO whiskers mixed powders were prepared.

2.2. Preparation

A scaffold model with interconnected porous architecture was designed. Subsequently, the model was exported into STL format and put into a house-built SLS system to sinter powders into scaffolds.^33,34 The fabrication process was the following steps. (1) The powders were spread onto the elevator to form a powder layer. (2) The paved layer was sintered selectively with the CO₂ laser beam. (3) The work platform was lowered to a thickness of one powder layer. (4) Steps (1–3) were repeated until the ultimate scaffold was obtained completely. (5) The scaffold was brushed off to remove the residual powders and cleaned by compressed air. The scaffolds were designated as PMgO0, PMgO0.5, PMgO1, PMgO2, PMgO3 and PMgO5 respectively. The numbers represented the weight fraction of MgO whiskers.

2.3. Characterization

The X-ray diffraction (XRD) spectra of powders and the scaffolds were conducted on the D/max 2500 X-ray diffractometer (Rigaku, Japan) with CuKα radiation (40 kV, 250 mA) at a scanning speed of 8° min⁻¹. Fourier transformation infrared spectroscopy (FTIR) was carried out using a Nicolet 6700 FTIR spectrophotometer (Thermo Fisher, America). The powders were mixed with KBr according to the ratio 1 : 100 to compress into pellets for the FTIR measurement. The data was recorded from 4000 to 400 cm⁻¹. Scanning electron microscope (SEM) was used to observe the morphology of initial MgO whiskers and PVDF powders, scaffolds, the tensile-fractured surface and the cell-scaffolds. Energy dispersive spectroscopy (EDS, Oxford Inc., UK) was used to analyze chemical composition. All the samples were coated with platinum using a sputter coater (JEOL, Japan) before the SEM and EDS test. The thermal property and crystallization of the scaffolds was analyzed by differential scanning calorimetry (DSC). The samples were heated in the temperature range from 28 to 270 °C at heating rate of 20 °C min⁻¹ under nitrogen atmosphere (Nanjin Dazhan Institute of Electromechanical Technology, STA-200, China). The tensile strength was tested using a universal testing machine (Shanghai Zhuoji Instruments Co. Ltd., China) at 0.5 mm min⁻¹. The test data was recorded too.

2.4. Biological properties

MG-63 cells were used to estimate the biological properties of the scaffolds. MG-63 cells were obtained from Cellular Biology Institute (Shanghai, China) and maintained in culture medium under a 5% CO₂ atmosphere at 37 °C. In this study, MG-63 cells passaged 4 to 6 times were used. Before the cell seeding, all the scaffolds were sterilized. The operation process was described as follow: the scaffolds were immersed into absolute ethyl alcohol for 2 h firstly. Afterwards, they were irradiated with ultraviolet light 15 min. 100 ml cell suspension containing 3 × 10⁵ cells were seeded onto each scaffold in the 12-well plates. After seeding, appropriate DMEM containing 10% fetal bovine serum (FBS) was added to the each well. The culture plates were placed in a 95% humidified/5% CO₂ air atmosphere at 37 °C for 1, 3, and 5 days. The media was changed every two days until the experiment finished.

SEM was used to evaluate the cellular morphology. After 1, 3, and 5 days of culture, the scaffolds were moved from the culture well-plates and rinsed with phosphate-buffered saline (PBS). And then, they were fixed with 2% glutaraldehyde for 2 h at 4 °C. After fixing, the scaffolds were processed with 0.1% osmium tetroxide and dehydrated in an ethanol series (50%, 70%, 90% and 100%). The dehydrated scaffolds were platinum-coated and observed using SEM.

Cell immunofluorescence was used to evaluate the attachment and proliferation of the cells. Briefly, at each predetermined time point (1, 3, and 5 days), MG-63 cells on the scaffolds were washed with PBS (2×). Then, the cells were fixed with 4% paraformaldehyde (Sigma, St. Louis, MI, USA) at 37 °C for 20 min. The cells were then placed in 0.2% Triton X-100 15 min for permeabilizing. The cells were then incubated with anti-4D2 at 4 °C overnight. After incubation with antibodies, the cells were washed with PBS (3×) and incubated with fluorescein isothiocyanate at the human body temperature for 1 h. Finally, the cells were imaged using luminescence microscope.

The levels of alkaline phosphatase (ALP) were measured using spectroscopy to analyze the differentiation of the MG-63 cells. After cultured for 5 days, the cells were washed with PBS precooled at 4 °C (3×, 5 min each), which was processed on the shaking bed. The cells were then fixed with 4% paraformaldehyde for 30 min. Subsequently, the cells were washed with PBS (3×, 5 min each). ALP staining was carried out with 300 μl buffer solution, 1 μl BCIP (5-bromo-4-chloro-3-indolyl phosphate) and 2 μl nitroblue tetrazolium (NBT) for 25 min. The cells were then sealed using neutral gum. Finally the images were obtained with microscopy.

3. Results and discussion

3.1. Composition analysis

MgO whiskers and PVDF particles were characterized using XRD, FTIR and SEM (Fig. 1). The XRD spectra of PVDF (Fig. 1a1) showed that the main peaks were located at 18.282°, 20.062° and 26.638° which are characteristic peaks of α-phase, while 2θ = 36.128° is the peak of β-phase of PVDF.³⁵ Generally, the appearance of β-phase might be influenced by the temperature of evaporation and dope solution. The FTIR spectra of PVDF (Fig. 1a2) illustrated that the bands appearing at 878, 1065, 1186 and 1406 cm⁻¹ were attributed to CH₂ in and out-of-plane twisting or wagging mode in PVDF. Besides, vibration bands at 761 cm⁻¹ due to CF₂ bending, and at 841 cm⁻¹ owing to CH₂ rocking corresponded to α-phase and β-phases respectively. The XRD spectra of MgO whiskers (Fig. 1b1) indicated that some diffraction peaks at 36.832°, 42.878° and 62.199° in 2θ appeared, corresponding to the demonstration of the literature.³⁶ The absorption peak at 1454 cm⁻¹ (Fig. 1b2) could be ascribed to the shaking of Mg–O bond. The broad absorption peak at 3445 cm⁻¹ was ascribed to the varying interactions between hydroxyl groups on and the MgO whiskers. The absorption peaks at 1637 and 1064 cm⁻¹ might be due to the vibration of H-bond owing to the entrance of water in air on the MgO whiskers. The PVDF particles were spheroidal and its average diameter was ∼300 nm (Fig. 1a3). The MgO whiskers were rod-shaped and their average diameter and length were ∼2.5 μm and ∼15 μm respectively (Fig. 1b3).

Fig. 1 Characterization of PVDF particles (a) and MgO whiskers (b) using XRD (a1 and b1), FTIR (a2 and b2) and SEM (a3 and b3).

The XRD spectra of the scaffolds were shown in Fig. 2. It could be seen that peaks at 2θ = 42.878° and 62.199° appeared, indicating that MgO whiskers existed in the scaffolds. In addition, the intensity of these peaks increased with the increase of MgO whiskers. Meanwhile, the intensity of the main peaks of PVDF also increased with increasing the MgO whiskers until their content was 2 wt% and then decreased with further increasing. These results indicated that the crystallinity of PVDF was improved by MgO whiskers, while excess MgO whiskers reduced its crystallinity. It might be attributed to that excess MgO whiskers formed agglomeration which prevented the polymer chains from reorganizing and against physical crosslinking, resulting in decrease of crystallinity.^37–39 Compared with the scaffolds without MgO whiskers, the location of the main peaks of PVDF was not changed, indicating that MgO whiskers did not cause the phase transition of PVDF.

Fig. 2 The XRD spectra of the scaffolds.

3.2. DSC analysis

The crystallization of the scaffolds was analyzed by differential scanning calorimetry (DSC). With the increasing of MgO whiskers, the melting temperature of the scaffolds increased until 2 wt% and then decreased with further increasing to 5 wt%, as shown in Fig. 3.

Fig. 3 DSC thermograms of the scaffolds with different MgO whiskers contents.

The DSC parameters of the scaffolds were shown in Table 1. The melting interval could be calculated by the following formula:

ΔT = T_t − T_i

where T_t and T_i represented the terminal and initial temperature of melting respectively. ΔT and T_p represented the melting interval and the temperature of the melting peak respectively. The crystallinity could be estimated by the formula:⁴⁰

X_c = (Δh_m − Δh_o) × 100%

where X_c was the crystallinity, Δh_o (104.7 J g⁻¹) was the standard enthalpies of the PVDF with 100% crystallinity, Δh_m was absorption enthalpies of the scaffolds. The crystallinity of PVDF increased at the beginning and then decreased with the increase of MgO whiskers. The crystallinity obtained the maximum value of 52.34% when the MgO whiskers content was 2 wt%. The results indicated that MgO whiskers could promote the crystalline phase formation of PVDF. It was attributed to that MgO whiskers provided nucleating cites for the crystallization of PVDF. Moreover, it was supported by the XRD results as shown in Fig. 2. Besides, these experimental results were corresponded with the statements in the literature. The literatures have found that nanoparticles, such as ZnO, Al₂O₃ and TiO₂, influenced the crystallinity of PVDF.^41–43 Among these, Zhanjian Liu et al. found that with increasing ZnO loading to 5 wt%, the crystallinity reached a maximum value and then decreased with further increasing ZnO.

Table 1 DSC parameters of the scaffolds with different MgO whiskers contents

MgO whiskers content/wt%	Ti/°C	Tp/°C	Tt/°C	ΔT/°C	Δhm/(J g^−1)	Xc/(%)
0	168.6	175.9	182.9	14.3	32.54	31.08 ± 0.60
0.5	169.0	176.4	186.0	17.0	42.64	40.73 ± 1.00
1	167.6	177.1	187.8	20.2	43.99	42.02 ± 1.08
2	166.7	177.4	185.0	18.3	54.80	52.34 ± 1.38
3	165.7	176.5	187.1	21.4	41.89	40.01 ± 0.93
5	168.5	177.0	188.5	20.0	40.45	38.63 ± 1.04

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3.3. The morphology of the scaffolds

The morphology of the scaffolds was characterized, and the composition of the PMgO0 and PMgO0.5 scaffolds was analyzed by EDS in the area marked with red square (Fig. 4). The morphology of the PMgO0 scaffolds was smooth and plane. The rod-shaped objects appeared on the PMgO0.5 scaffolds, and they were analyzed by EDS (Fig. 4b). The appearance peaks of Mg and O confirmed that the rod-shaped objects were MgO whiskers. They distributed on the scaffolds uniformly and combined with PVDF matrix well when the content of them was not exceeded 2 wt%. However, there were some defects appeared on the scaffolds, such as holes and aggregations of MgO whiskers, with further increasing their contents to 5 wt%. It caused the decline of mechanical properties.

Fig. 4 The morphology of the scaffolds (a) PMgO0, (b) PMgO0.5, (c) PMgO1, (d) PMgO2, (e) PMgO3, and (f) PMgO5.

3.4. Mechanical properties

The tensile strength of the scaffolds was tested using a universal testing machine. The tensile strength and the elastic modulus were shown in Fig. 5. The tensile strength and the elastic modulus of the scaffolds attained peak level of 62.17 MPa and 6018.83 MPa respectively when the MgO whiskers dosage increased to 2 wt%, and then decreased with further increasing MgO whiskers. The tensile strength of the PMgO2 was about 1.5 times higher than that of the PMgO0. The results indicated that the tensile strength could be improved by incorporating MgO whiskers into PVDF matrix.

Fig. 5 The mechanical properties of the scaffolds containing different contents of MgO whiskers.

There were some reasons could be used to explain the enhancement effect. The one reason was that the MgO whiskers improved the crystallinity of the scaffolds when their content was appropriate and the PMgO2 obtained the highest crystallinity. The other reason was that the whiskers/polymer interface adhesion confined the molecule mobility. The whisker could restrain strain, absorb and transfer energy during crack extension. When the MgO whiskers contents were larger than 2 wt%, the tensile strength and modulus began to decrease. It was attributed to the reason that excess MgO whiskers formed aggregation, which leaded to non-continuous phase of polymer (Fig. 4e and f). Excess MgO whiskers made the scaffolds weaken and rigider. Therefore, appropriate control of reinforcement phase loading in the scaffolds was critically important to tailor the mechanical properties.

The break elongation of the scaffolds was obtained during the tensile test (Fig. 6). The change trend was that it increased at the beginning and obtained the peak when the content of MgO whiskers was 2 wt%, and then decreased with further increasing to 5 wt%. The results indicated that appropriate MgO whiskers could enhance the tensile strength and at the same time improve the break elongation.

Fig. 6 The break elongation of the scaffolds with different MgO whiskers contents.

The stretching sections of each scaffold were shown in Fig. 7. The morphology of the stretching sections changed obviously. The section of the PMgO0 scaffolds was plane relatively, and some crazing-tear appeared (Fig. 7a). The morphology was the characteristic of the brittle fracture. Compared with the PMgO0 scaffolds, the section of the scaffolds with different content of MgO whiskers had obvious difference. The morphology became rough. Meanwhile, the filament appeared and became longer with increasing the MgO whiskers to 2 wt%. The results confirmed that the appropriate MgO whiskers could strengthen and toughen the PVDF scaffolds. However, the morphology became stiff and filament decreased and became short with further increasing the MgO whiskers to 5 wt%. The results were corresponded with the change of the tensile strength and modulus at Fig. 5. The scaffolds with the optimal mechanical properties were selected to apply in subsequent cell culture experiments.

Fig. 7 The stretching section of each scaffold after fracture from uniaxial tensile testing (a) PMgO0, (b) PMgO0.5, (c) PMgO1, (d) PMgO2, (e) PMgO3, and (f) PMgO5.

3.5. Biological properties

In order to evaluate the biological properties of the scaffolds, MG-63 cells were seeded onto the PMgO0 (set as control) and the PMgO2. The morphologies of the cells cultured on the different scaffolds for different times were shown in Fig. 8. As shown in Fig. 8A, after 1 day seeding, the cells exhibited a round shape with lamellipodia around them, indicating that the PMgO0 stimulated cell attachment and extracellular matrix formation. The number of MG-63 cells increased with increasing the culture time, and the cells represented typical fibrous-like. After culture 5 days, the cells spread and occupied the most area of the scaffolds. Compared with the cells on the PMgO0, the adhesion and multiplication of the cells on the PMgO2 changed more obviously. At the same culture time point, the numbers of the cells seeded on the PMgO2 were more than that on the PMgO0. And the cells spread and attached better.

Fig. 8 The morphology of MG-63 cells cultured on the scaffolds ((A) the PMgO0 and (B) the PMgO2).

In order to illustrate the attachment and proliferation of the MG-63 cells better, the cell immunofluorescence experiment was carried out and the results were shown in Fig. 9. All the cells presented light green. The morphologies of them changed obviously with the increase of culture time. After culture 1 day, the most cells maintained spherical shape, and only a few cells presented spindle-shaped morphology. With the culture time increasing, the cells grow and spread. After culture 5 days, all the cells maintained a fibroblastic morphology and almost occupied all the scaffolds. Compared with the cells cultured on the PMgO0, the characterization and change trend of the cells cultured on the PMgO2 was more apparently. These results were consistent with the SEM analysis at Fig. 8.

Fig. 9 Confocal fluorescence images of MG-63 cells cultured on the scaffolds ((A) the PMgO0 and (B) the PMgO2).

The reasons were as follows. First, alterative surface of the scaffolds caused by MgO whiskers might cause the results. The MgO whiskers distributing on the scaffolds provided attachment sites for the growth of the MG-63 cells. Second, the Mg²⁺ ions released from the scaffolds might play an important role in causing this phenomenon. Mg²⁺ ions provided the support of energy for the growth of the cells, and enhance the synthesis of fatty acid, protein and nucleic acid. Moreover, they offered positive protection for the growth of cells and prevented all kinds of factors that were harmful to cells, thereby made the growth and proliferation of the cells smoothly.

ALP activities were used to evaluate the differentiation of the MG-63 cells seeded on the different scaffolds for 5 days (Fig. 10). ALP activity of the cells seeded on PMgO2 was higher than that of PMgO0, which might be attributed to the acceleration of MgO whiskers. The release of the Mg²⁺ ions could enhance the differentiation of the cells. Guntur A. R., et al. and Li L., et al. found that phosphatidylinositol 3-kinase and Akt pathway had important effect on the proliferation and differentiation of the osteoblast.^44,45 Chen S. D., et al. and Su N. Y. discovered that appropriate Mg²⁺ ions could activate the phosphatidylinositol 3-kinase and Akt pathway.^46,47 Some researchers have provided the evidence that Mg²⁺ ions could improve the differentiation of cells in recent years.^48,49

Fig. 10 ALP activity of MG-63 cells cultured on the scaffolds cultured for 5 days, ((a) low and (c) high magnified image) the PMgO0, ((b) low and (d) high magnified image) the PMgO2.

4. Conclusions

In conclusion, the scaffolds consisting of MgO whiskers-PVDF were prepared and investigated as bone repair material. The MgO whiskers enhanced the tensile strength and elastic modulus of PVDF, and enhanced crystallinity of PVDF. Moreover, the scaffolds had good biological properties. The scaffolds with MgO whiskers have the potential application value in biomedical field.

Acknowledgements

This work was supported by the following funds: (1) The Natural Science Foundation of China (51575537, 81572577); (2) Overseas, Hong Kong & Macao Scholars Collaborated Researching Fund of National Natural Science Foundation of China (81428018); (3) Hunan Provincial Natural Science Foundation of China (14JJ1006, 2016JJ1027); (4) The Project of Innovation-driven Plan of Central South University (2015CXS008, 2016CX023); (5) The Open-End Fund for the Valuable and Precision Instruments of Central South University; (6) The fund of the State Key Laboratory of Solidification Processing in NWPU (SKLSP201605); (7) The fund of the State Key Laboratory for Powder Metallurgy; (8) The Fundamental Research Funds for the Central Universities of Central South University.

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Footnote

† These authors contributed equally to this work.

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