PHBV-based ternary composite by intermixing of magnesium calcium phosphate nanoparticles and zein: In vitro bioactivity, degradability and cytocompatibility

European Polymer Journal 75 (2016) 291–302

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

PHBV-based ternary composite by intermixing of magnesium calcium phosphate nanoparticles and zein: In vitro bioactivity, degradability and cytocompatibility Jun Qian a,⇑, Juan Ma a, Jiacan Su b,⇑, Yonggang Yan c, Hong Li c, Jung-Woog Shin d, Jie Wei a, Liming Zhao a a Key Laboratory for Ultrafine Materials of Ministry of Education, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China b Department of Orthopaedics Trauma, Changhai Hospital, Second Military Medical University, Shanghai 200433, China c College of Physical Science and Technology, Sichuan University, Chengdu 610041, China d Department of Biomedical Engineering, Inje University, Gimhae 621-749, Republic of Korea

a r t i c l e

i n f o

Article history: Received 23 July 2015 Received in revised form 1 December 2015 Accepted 30 December 2015 Available online 31 December 2015 Keywords: Nano magnesium calcium phosphate Zein Ternary composite Degradability Cytocompatibility

a b s t r a c t Bioactive ternary composite containing nano magnesium calcium phosphate (n-MCP), poly (hydroxybutyrate-co-hydroxyvalerate) (PHBV) and zein (ZN) were fabricated by the solvent casting method. The results showed that incorporation of n-MCP and ZN into PHBV was conducive to enhancing the hydrophilicity, in vitro degradability, and bioactivity of the n-MCP/PHBV/ZN (MPZ) ternary composite. The results of cell experiments revealed that the MPZ ternary composite containing n-MCP and ZN could significantly promote the cell proliferation and enhance alkaline phosphatase (ALP) activity of MC3T3-E1 cells. In addition, the cells with normal phenotype spread well on the MPZ surface, and attached to the substrate, indicating good cytocompatibility of MPZ. The results demonstrated that the MPZ ternary composite with improved physical–chemical and biological performances might be considered as a potential candidate for biomedical applications. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL), poly(butylene succinate) (PBS), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) have been extensively studied and applied in the biomedical fields, owing to their excellent degradability and biocompatibility [1,2]. Among these aliphatic polyesters, PHBV is a biodegradable polymer, which can be produced by numerous bacteria as an intracellular reserve of carbon or energy. In spite that PHBV (bacterial origin) contains lipopolysaccharide (LPS), which induce a proinflammatory response to macrophages, the purified polymer with low-cytotoxicity can be suitable for medical applications [3]. The excellent properties of PHBV, such as absorbability, biologic origin, low-cytotoxicity, piezoelectricity and thermoplasticity, make it promising for biomaterials applications [4]. Nevertheless, several disadvantages have limited its applications as biomaterials: ① poor hydrophilicity, ② lack of bioactivity, ③ slow degradation rate, etc. [5].

⇑ Corresponding authors. E-mail addresses: [email protected] (J. Qian), [email protected] (J. Su). http://dx.doi.org/10.1016/j.eurpolymj.2015.12.026 0014-3057/Ó 2016 Elsevier Ltd. All rights reserved.

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In order to overcome the shortcomings of PHBV, bioactive inorganic materials, such as hydroxyapatite, calcium phosphate, bioglass, and calcium silicate, have been incorporated into PHBV to prepare inorganic materials/polymer composites [6,7]. Furthermore, it is known that incorporating polypeptides (such as, zein, and gelatin) into aliphatic polyesters was an effective approach for the design of polymer based composite as biomaterials with improved performances for biomedical applications [8]. Among the above polypeptides, zein, a major storage protein of corn, is mainly originated from maize endosperm cells [9]. Previous report demonstrated that zein showed better cytocompatibility and degradability than aliphatic polyesters (such as PHBV and PCL), and both zein and its degradable products exhibited good biocompatibility [10]. Magnesium is an essential mineral element in human body, and about half of it exists in the human bone [11]. Recently, magnesium-based biomaterials have been paid more and more attentions, such as magnesium alloy, magnesium-containing bioactive glass/ceramics, magnesium-substituted biphasic calcium phosphate and magnesium phosphate cement [12,13]. Studies have showed that the Ca and P ions were of great significance to promote osteogenesis both in vitro and in vivo [14]. Therefore, in this study, the nano magnesium calcium phosphate (n-MCP, CaMg2(PO4)2) was synthetized, and a novel ternary composite of MPZ containing n-MCP, ZN and PHBV was prepared and characterized. Furthermore, the in vitro degradability, bioactivity and primary cell responses to the MPZ were also evaluated. 2. Materials and methods 2.1. Preparation of nano magnesium calcium phosphate (n-MCP) Nano-sized magnesium calcium phosphate (n-MCP) was synthesized by the co-precipitation method of Ca(NO3)2, Mg (NO3)2, (NH4)2HPO4 in the molar ratio of 1:2:2 in aqueous solution at pH = 8.0–9.0, followed by iterative centrifugation, solvent exchange and resuspension. Subsequently, the suspension was dried in draught drying cabinet at 80 °C for 48 h. The morphology of n-MCP was observed by using Transmission Electron Microscopy (TEM; JEM2010, JEOL, Japan), and the element composition of n-MCP was monitored by using Energy Dispersive Spectrometer (EDS; INCA Energy, Oxford Instruments, UK) at an accelerating voltage of 15 kV. The functional groups and phase composition of n-MCP was analyzed by using Fourier transform infrared spectroscopy (FTIR; Magna-IR 550, Nicolet, American) and X-ray diffraction (XRD; Geigerflex, Rigaku Co., Ltd., Japan). 2.2. Preparation of MPZ ternary composite Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, Mw = 300,000) containing up to 3% mol of hydroxyvalerate unites was purchased from Tianan Biological Material Co., Ltd (Ningbo, China). Zein with biochemical purity was obtained from CpG Biotech Co., Ltd (Shanghai, China). PHBV (1 g) was dissolved in chloroform (20 mL) at 80 °C to form uniform solution with a concentration of 5% (w/v), followed by the incorporation of n-MCP powders and zein solution dissolved by ethanol. Then the suspension was immediately transferred to disperse by ultrasonication with continuous stirring at room temperature. Next, the homogeneous suspension was casted into stainless steel mold (U12
2 mm) and dried in oven to evaporate the solvent at room temperature to fabricate the samples with the size of 12 mm in diameter and 2 mm in thickness. Finally the n-MCP/ PHBV/ZN (50:40:10, w/w/w, MPZ10) and n-MCP/PHBV/ZN (50:30:20, w/w/w, MPZ20) ternary composite were prepared. The n-MCP/PHBV (50:50, w/w, MP) composite and PHBV prepared by the same method were regarded as controls. All the samples were characterized by XRD and FTIR. 2.3. Hydrophilicity determination The hydrophilicity of PHBV, MP, MPZ10 and MPZ20 (U12
2 mm) was determined by measuring the water contact angles (XG-7501B, Xuanyichuangxi Co., Ltd., China) of these samples, which were implemented by the sessile drop method. Water with a volume of 0.5 lL was dropped on the two points of each sample surface respectively to prevent gravitational distortion of the spherical profile, followed by recording the values after 5 s. Each determination was obtained by averaging the results of five measurements. 2.4. In vitro degradability in Tris–HCl solution The in vitro degradability assessment of PHBV, MP, MPZ10 and MPZ20 (U12
2 mm) was carried out by soaking the samples into Tris–HCl solution (0.05 M, pH = 7.4) with a solid-to-liquid ratio of 1/20 (g/mL) for 12 weeks at 37 °C in the incubator shaker, and the solution was refreshed once a day. At each time point, the samples were taken out and dried in an oven at room temperature for 24 h, and then accurately weighed by electronic analytical balance. The degradation level of the four groups at each time point was expressed as weight loss, which was calculated according to the following equation:

Weight lossð%Þ ¼ ðW 0  W t Þ=W 0
100 where W0 = initial dry weight and Wt = dry weight at time t.

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The final weight loss was expressed by averaging the results of five parallel samples from each group. The pH values of the solutions containing the samples at each time point were monitored by electrolyte-type pH meter (PHS-2C, JingkeLeici Co., Ltd., Shanghai, China). 2.5. In vitro bioactivity in SBF In vitro bioactivity of PHBV, MP, MPZ10 and MPZ20 (U12
2 mm) was assessed by investigating the apatite formation on their surfaces after immersing the samples into simulated body fluid (SBF, pH = 7.4) at 37 °C in shaking incubator [15]. The SBF solution was not stirred and refreshed throughout. At different soaking time point, five parallel samples from each group were taken out and the samples were slightly washed with deionized water to remove the soluble inorganic ions and dried in vacuum oven at room temperature for 24 h. The surface morphology and composition change of the samples before and after immersion in SBF for 2 weeks were characterized by using Scanning electron microscope (SEM; S-3400N, Hitachi, Japan), EDS and FTIR. In addition, the weight change of the samples before and after soaking into SBF for 2 weeks was monitored by the electronic analytical balance and the weight change ratio was calculated according to the following equation:

Weight changeð%Þ ¼ ðW 2  W 0 Þ=W 0
100 where W0 = samples weight before soaking and W2 = samples weight after soaking for 2 weeks. The change of concentration of Ca, P and Mg ions in the solutions after the samples soaked into SBF at each time point (0.5, 1, 3, 5, 7 and 14 days) was detected with inductively coupled plasma-atomic emission spectrometry (ICP-AES; IRIS 1000, Thermo Elemental, USA), and the results were expressed by average the results of five measurements. 2.6. Cell cytocompatibility 2.6.1. Cell culture MC3T3-E1 cells were cultivated in a cell culture medium comprising a mixture of Dulbecco’s modified eagle medium (DMEM) and 10% fetal bovine serum (FBS) in a 100% humidified atmosphere of 5% CO2 at 37 °C. Before cell culturing, all the samples (PHBV, MP, MPZ10 and MPZ20) were sterilized with 70% alcohol and rinsed with sterile phosphate buffered saline (PBS) thrice. The medium for cell culture was refreshed every 2 days. 2.6.2. Cell proliferation Cell Counting Kit-8 (CCK8) assay was employed to quantitatively assess the level of cell proliferation. Cell suspension was seeded on the samples on the 96-well tissue culture plates at a density of 2
104 cells/well in DMEM with 10% FBS for 1, 3 and 7 days. At each prescribed time point, all the samples were rinsed by PBS for three times and transferred into a new 96-well plate. After CCK8 reagent (Dojindo Molecular Technologies Inc., Japan) was added into every well, the cell-sample systems were maintained for extra 4 h at 100% humidity atmosphere with 5% CO2. Afterwards, the optical density (OD) values of the supernatant were measured using a microplate reader (MULTISKAN MK3, Thermo Electron Co., USA) at the wavelength of 450 nm. 2.6.3. Cell morphology After cultivating the cells on the samples (PHBV, MP, MPZ10 and MPZ20) for 3 days at 100% humidity atmosphere with 5% CO2, the culture medium was taken out and all the samples were washed by PBS for three times to move the unattached cells. Then 4% paraformaldehyde reagent was applied to fix the cells on the sample surface for 15 min. Finally, the samples were rewashed by PBS for three times and dehydrated by ethanol with incremental concentration (10%, 30%, 50%, 70%, 90%, 100%, v/v) for 10 min step by step, respectively. The cell morphology was investigated by scanning electrical microscope (SEM). 2.6.4. Alkaline phosphatase activity Cell differentiation was estimated by measuring alkaline phosphatase (ALP) activity after MC3T3-E1 cells cultured on the samples for 3 and 7 days. At each time point, the culture medium was taken out and all the samples were rinsed by PBS for three times. The cells were trypsinized and scraped into double-distilled water, and then harvested in 10 mL tubes. After the cell suspension was sonicated for 10 min and centrifuged at 2000 rpm, the supernatant was stored at 20 °C until the p-nitrophenyl phosphate (pNPP) assay was performed. The OD value at 405 nm was measured by microplate reader (SPECTR Amax 384, Molecular Devices, USA). Total protein content was determined using the BCA protein assay kits and a series of bovine serum albumin standards. The ALP activity was expressed by OD value per total protein content. 2.7. Statistical analysis All the quantitative experiments were performed in quintuplicate for each group, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by the one-way analysis of variance (ANOVA). A p value less than 0.05 was considered to be statistically significant.

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3. Results 3.1. Characterization of n-MCP and n-MCP/PHBV/ZN ternary composite The TEM image of nano magnesium calcium phosphate (n-MCP) is shown in Fig. 1a. It can be seen that the n-MCP with the size of around 20 nm were spherical-shape particles. Fig. 1b shows the EDS of n-MCP, indicating that the n-MCP contained Ca, Mg and P elements with the approximate molar ratio of 1:2:2. The results indicated that the n-MCP (CaMg2(PO4)2) was successfully prepared in this study. Fig. 2a shows the FTIR of n-MCP, it can be seen that the two absorption peaks at 1036.2 and 565.8 cm1 were ascribed to phosphate vibrations, and the absorption peaks at 3442.9 and 1642.5 cm1 were attributed to stretching vibration and blending vibration of absorbed water, respectively [16]. Fig. 2b shows the XRD of n-MCP, the sharp and narrow peaks indicated that n-MCP possessed high crystallinity. Fig. 3a shows the XRD of the PHBV, n-MCP, ZN, MP, MPZ10 and MPZ20. It can be found that the XRD of the MP and MPZ (including MPZ10 and MPZ20) composite contained both the diffraction peaks of PHBV and n-MCP. The diffraction peaks of at 2h = 13.4° was ascribed to PHBV [17]. In addition, the XRD of zein in the MPZ didn’t have obvious diffraction peaks, indicating that zein was amorphous. The FTIR of the PHBV, n-MCP, ZN, MP, MPZ10 and MPZ20 are illustrated in Fig. 3b. The absorption peak at 1036.2 cm1 of 1 n-MCP was attributed to the phosphate ion (PO3 , corresponding to 4 ). The absorption peak of PHBV appeared at 1726.9 cm stretching vibration of C@O bond of ester group [18]. The wide absorption peak at 3430.1 cm1 of zein was ascribed to the stretching vibration of NAH bond in amide group [19]. Obviously, the FTIR of the MPZ included both the absorption peaks of n-MCP and PHBV, and the FTIR of the MPZ (including MPZ10 and MPZ20) also simultaneously presented the absorption peaks of n-MCP, PHBV and zein. All the above descriptions indicated that the MPZ ternary composite containing n-MCP, PHBV and ZN were successfully fabricated by the solvent casting method. 3.2. Hydrophilicity determination The water contact angles represent the hydrophilicity of the samples surfaces. Fig. 4 shows the water contact angles of the PHBV, MP, MPZ10 and MPZ20. It can be seen that the water contact angles of PHBV, MP, MPZ10 and MPZ20 were 75°, 58°, 52° and 48°, respectively. The water contact angles decreased, indicating the improvement of hydrophilicity (hydrophilicity: MPZ20 > MPZ10 > MP > PHBV). It could be suggested that the addition of n-MCP into PHBV resulted in the significant increase of the hydrophilicity of MP as compared with PHBV, and incorporation of zein into the composite further improved the hydrophilicity of MPZ. 3.3. Degradation in Tris–HCl solution Fig. 5a shows the weight loss of the samples with time in Tris–HCl solution. It can be seen that the weight loss ratio of the samples was significantly different (the PHBV was lowest while the MPZ20 was highest). In addition, after soaking for 12 weeks, the final weight loss ratios of PHBV, MP, MPZ10 and MPZ20 were 2.1 wt.%, 36.0 wt.%, 47.1 wt.% and 59.3 wt.%, respectively. Fig. 5b shows the change of pH values of solution after PHBV, MP, MPZ10 and MPZ20 immersed into Tris–HCl solution for different time. The pH value for PHBV decreased with the soaking time while the pH value for the MP, MPZ10 and MPZ20 first rose and then fell. In addition, the solution containing the MP had comparatively higher pH than PHBV, MPZ10 and

(b) O P Mg Ca Ca 0

1

2

3

Energy (KeV) Fig. 1. TEM image (a) and EDS (b) of n-MCP.

4

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12000

(a)

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90 60

1642.5

30

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1036.2

-30 4000 3500 3000 2500 2000 1500 1000

Wavenumber

0 5

500

10

15

20

25

30

35

2-Theta (deg.)

(cm-1) Fig. 2. FTIR (a) and XRD (b) of n-MCP.

(b)

(a)

PHBV

PHBV

ZN MP MPZ10

n-MCP

1726.9

Transmittance (%)

Intensity (a.u.)

n-MCP

ZN

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MP

3430.1

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1036.4

MPZ10

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MPZ20

1726.6

1036.1

MPZ20 3429.9

10

15

20

25

30

4000 3500 3000 2500 2000 1500 1000

35

500

Wavenumber ( cm-1)

2-Theta (deg.)

Fig. 3. XRD (a) and FTIR (b) of PHBV, n-MCP, ZN, MP, MPZ10 and MPZ20.

90

Water contact angle ( °)

80

75°

70

58° 60

52° 48° 50

40

PHBV

MP

MPZ10

MPZ20

Fig. 4. Water contact angles of PHBV, MP, MPZ10 and MPZ20.

MPZ20 at each time point. Moreover, during soaking for 12 weeks, it is found that the change of pH for MPZ10 and MPZ20 ranged from 7.4 to 7.55 and 7.47 to 7.3, respectively. Fig. 6 shows the SEM images of the surfaces morphology of PHBV, MP, MPZ10 and MPZ20 before and after immersed in Tris–HCl solution for 4 weeks. It can be found that the cracks appeared on the MP and MPZ composites while the surface of PHBV was relatively flat (no cracks). To some extent, the size of the cracks could represent the degradability of the samples. Obviously, the degradation degree of these samples was as following: MPZ20 > MPZ10 > MP > PHBV.

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80

60

PHBV MP MPZ10 MPZ20

8.0 7.8

(b)

7.6

40

pH

Weight loss (%)

(a)

PHBV MP MPZ10 MPZ20

7.4

20

7.2 7.0

0 1

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Time (w)

7

8

10 12

1

2

3

4

5

6

7

8

10 12

Time (w)

Fig. 5. Weight loss (a) of the samples, and change of pH values (b) of solution after PHBV, MP, MPZ10 and MPZ20 immersed into Tris–HCl solution for different time.

3.4. Bioactivity of MPZ in SBF solution Fig. 7 shows the SEM images and EDS of the samples surfaces after soaking into the SBF for 2 weeks. It can be seen that the spherical-shape apatites were found on the surfaces of the MPZ20, MPZ10 and MP (no apatite deposition on the PHBV surface), indicating that the MPZ and MP had good bioactivity (apatite formation ability). In addition, it is found that the size and number of the apatite on these samples were as following: MPZ20 > MPZ10 > MP, indicating that the bioactivity of the samples was as following: MPZ20 > MPZ10 > MP. The results from the EDS analysis showed that the atomic molar ratios (Ca/P) were 1.61 for MP, 1.63 for MPZ10 and 1.62 for MPZ20, which were all approximate to 1.67 of the theoretical value of hydroxyapatite. The results indicated the depositions on the samples surfaces were calcium deficiency apatite [20]. Fig. 8 shows the FTIR of MPZ20 before and after soaking into SBF for 2 weeks, respectively. Obviously, the absorption peaks of phosphate group (PO43) of n-MCP in MPZ20 at 1036.1 cm1 and 565.9 cm1 greatly intensified after soaking into SBF for 2 weeks, indicating more apatite formation on the MPZ20 surface. In addition, the peak at 1726.6 cm1 for PHBV in MPZ20 became extremely weak after immersion for 2 weeks, indicating more apatite formation on the MPZ20 surface, which covered the PHBV. Moreover, the weight gain for PHBV, MP, MPZ10 and MPZ20 was tested after immersion into SBF for 2 weeks. The results showed that no obvious weight change for PHBV was found, indicating no apatite deposition on its surface. However, significant weight increase was found for the MP (5%), MPZ10 (7.8%), and MPZ20 (9.4%) after soaking for 2 weeks, indicating the apatite formation on the composite surfaces. Moreover, it is found that the weight gain for MPZ20 was highest. Fig. 9 shows the change of the concentrations of Ca, P and Mg ions in SBF during the 2 weeks of immersion. It is found that the concentrations of Mg ions increased with time (n-MCP dissolution) while the concentrations of Ca and P ions first rose and then decreased. Clearly, the dissolution of n-MCP would increase the Ca and P ions concentrations in SBF solution while the formation of apatite on the samples surfaces would consume the Ca and P ions. In addition, the concentrations of Ca ions were 73 mg/L, 55 mg/L and 43 mg/L, while the P ion concentrations were 95 mg/L, 87 mg/L and 75 mg/L respectively after the MP, MPZ10 and MPZ20 were soaked for 2 weeks. The results indicated that the quantity of Ca and P ions consumed by the formation of apatite on the samples surfaces was as following: MPZ20 > MPZ10 > MP. Therefore, the number of the apatite on these samples was as following: MPZ20 > MPZ10 > MP. 3.5. In vitro cytocompatibility 3.5.1. Cell proliferation and cell morphology Fig. 10 shows the OD values of MC3T3-E1 cells cultivated on the samples of PHBV, MP, MPZ10 and MPZ20 for 1, 3 and 7 days determined by CCK8 assay. It is found that the OD value for all the samples increased with time, indicating good cytocompatibility. There was no apparent difference of the OD for the four groups at 1 day. After cultivating for 3 and 7 days, the OD value for the MP became higher than PHBV, revealing that the addition of n-MCP into PHBV could facilitate the cell proliferation. Furthermore, at 3 and 7 days, the OD value for the MPZ was higher than MP, indicating that the further introduction of zein into the MP could more effectively promote the cell proliferation, which was zein content dependent. Fig. 11 shows the SEM images of the morphology of MC3T3-E1 cells cultivated on the PHBV, MP, MPZ10 and MPZ20 at 3 days. It can be seen that the MC3T3-E1 cells with normal morphology attached close to the MPZ20 and MPZ10 surfaces and spread better than those on the MP and PHBV. 3.5.2. Alkaline phosphate activity Fig. 12 shows the ALP activity of MC3T3-E1 cells incubated on the PHBV, MP, MPZ10 and MPZ20 at 3 and 7 days. It can be seen that the ALP activity for PHBV, MP, MPZ10 and MPZ20 increased with time, indicating good cytocompatibility. No

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Fig. 6. SEM images of the surface morphology of PHBV (a), MP (b), MPZ10 (c) and MPZ20 (d) before (a1, b1, c1 and d1) and after (a2, b2, c2 and d2) immersed in Tris–HCl solution for 4 weeks.

obvious difference was found for all the samples at 3 days. However, the ALP activity for both the MPZ20 and MPZ10 was significantly higher than MP at 7 days, and the ALP activity for MP was significantly higher than PHBV. 4. Discussions Bioactive inorganic materials, such as hydroxyapatite, calcium phosphate, bioglass, and calcium silicate, have been incorporated into PHBV to prepare biocomposites, and the results showed that PHBV based biocomposite with improved

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Fig. 7. SEM images of the surface morphology of PHBV (a), MP (b), MPZ10 (c) and MPZ20 (d) after soaked into SBF for 2 weeks.

Transmittance (%)

(a)

565.9 1726.6

(b)

1036.1

1726.7 565.6 1036.4 4000

3500

3000

2500

2000

1500

1000

500

Wavenumber ( cm-1) Fig. 8. FTIR of MPZ20 before (a) and after (b) immersion into SBF for 2 weeks.

hydrophilicity, bioactivity and degradability could be used as biomaterials for biomedical applications [6,21]. In this study, the ternary composite of MPZ containing n-MCP, PHBV and ZN were successfully fabricated by the solvent casting method. Our results showed that the addition of n-MCP into PHBV resulted in the significant decrease of water contact angle of MP as compared with PHBV, suggesting the improvement of hydrophilicity. In addition, the water contact angles further reduced after the admixture of zein into the MP, manifesting the further improvement of hydrophilicity (MPZ20 > MPZ10 > MP > PHBV). It is known that the surface hydrophilicity is of great significance to the biological performances of biomaterials [22]. Previous reports have showed that the improvement of hydrophilicity of the biomaterial surface could be helpful to boost the interactions between the matrixes and cells, and then contributed to cell attachment and cell growth [23]. Ideal biocompatible biomaterials as bone substitutes should be desirable to possess the matched speed of degradation with the formation of new bone tissue when implanted in vivo [24]. In this study, during immersion into Tris–HCl solution for 12 weeks, the results showed that the weight loss ratio of the MP composite with n-MCP significantly increased compared with PHBV, and the weight loss ratio of the MPZ obviously increased with the increase of ZN content (the degradability of these samples was as following: MPZ20 > MPZ10 > MP > PHBV). It could be suggested that the addition of n-MCP was conducive to improve the degradability of MP composite, and incorporation of zein further enhanced the degradability of the

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P Mg Ca

(a)

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(b)

P Mg Ca

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Ion concentration (mg/L)

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160 120 80 40

40 0

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2

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(c)

P Mg Ca

200

8

Time (d)

Time (d)

160 120 80 40

0

2

4

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8

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Time (d) Fig. 9. Change of the concentrations of Ca, Mg and P ions in solution after MP (a), MPZ10 (b) and MPZ20 (c) soaked into SBF for different time.

1.6

1.2

PHBV MP MPZ10 MPZ20

∗ ∗

O.D.

∗

∗

∗

0.8

0.4

0.0

1d

3d

7d

Time Fig. 10. OD values of MC3T3-E1 cells cultivated on PHBV, MP, MPZ10 and MPZ20 at 1, 3 and 7 days,

⁄

represents significant difference (p < 0.05).

MPZ ternary composite. Therefore, the intermixing of n-MCP and zein into PHBV could commendably ameliorate the degradability of the MPZ ternary composite, which could be in control by adjusting the mass percentage of n-MCP and zein. The interaction between the biomaterials matrix and osteoblasts would be strongly influenced by the pH value in the inner environment of human body, and whether the pH value is suitable for cell growth or not depends on the integrative action of all the degradation products of the implanted materials [25]. In this study, the result showed that the pH values of PHBV showed the trend of constant declining, which was attributed to its acid degradation products from PHBV [26]. However, the pH value of the solution for MP composite was higher than PHBV, indicating that alkaline ions (Ca and Mg), which were released from n-MCP into the solution, neutralized the acid degradation products. In addition, the acid products produced by the zein resulted in a slight decrease of the pH value (decrease with zein content). Therefore, the change of pH

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ALP activity 405nm OD value/min/mg protein

Fig. 11. SEM images of cell morphology of MC3T3-E1 cells cultivated on PHBV (a), MP (b), MPZ10 (c) and MPZ20 (d) at 3 days.

0.16

PHBV MP MPZ10 MPZ20

∗ ∗

0.12

0.08

0.04

0.00

3d

7d

Time Fig. 12. ALP activity of MC3T3-E1 cells cultivated on PHBV, MP, MPZ10 and MPZ20 at 3 and 7 days,

⁄

represents significant difference (p < 0.05).

could be in control by adjusting the mass percentage of n-MCP and zein in the MPZ ternary composite. In addition, the results suggested that the change of pH for MPZ10 and MPZ20 would be suitable for cell/tissue growth, which was appropriate pH range for the human body [27]. In this study, as expected, the apatite could form on the surfaces of the MPZ20, MPZ10 and MP (no apatite deposition on the PHBV surface) after mineralization in SBF for 2 weeks, indicating that the MPZ and MP had good bioactivity (apatite formation ability). In addition, the number of the apatite which formed on these samples was as following: MPZ20 > MPZ10 > MP, revealing that the bioactivity of the samples was as following: MPZ20 > MPZ10 > MP. The potential mechanism of apatite formation on the samples surfaces could be suggested that the dissolution of n-MCP led to the excessive saturation of Ca and P ions concentrations, which induced the apatite formation on the samples surfaces [28,29]. Moreover, the hydrolysis process of zein significantly caused the increase of the content of carboxylic acid and hydroxyl groups on the MPZ composite surfaces, and these hydroxyl groups might ionize in the SBF solution to form negatively charged units, which attracted Ca ions, and then interacted with phosphate anions to form apatite [30]. Therefore, the apatite formation ability might be dependent on the zein content. In short, the incorporation of n-MCP and zein into PHBV can be a prospective approach to prepare the polymer-based bioactive composite.

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Generally, the level of cell proliferation can be expressed by the optical density (OD) values [31]. In this study, the results revealed that the OD value for the MPZ was higher than MP at 3 and 7 days, indicating that the further introduction of zein into the MP could effectively promote the cell proliferation, which was zein content dependent. Moreover, the MC3T3-E1 cells with normal morphology attached to the MPZ20 and MPZ10 surfaces and spread better than those on MP and PHBV, revealing that the MPZ composite with good cytocompatibility could provide advantageous micro-environment for cell attachment and spread. Dong, Sun and Wang assessed the cytocompatibility by using culturing human liver cells and mica fibroblast cells on zein films, and the result showed that the films could provide suitable niches for cell adhesion and growth [32]. Therefore, the MPZ ternary composite containing zein was beneficial to improve the cell attachment and proliferation. In addition, the cell attachment behaviors are significantly influenced by of the surface properties of the biomaterials, and the hydrophilicity of the biomaterial surface is also an important factor for cell adhesion and growth [33]. In this study, the results revealed that the addition of n-MCP and zein into PHBV obviously improved the hydrophilicity of the MPZ ternary composite (MPZ20 > MPZ10 > MP > PHBV), which might be responsible for promoting cell attachment on the samples surfaces. The level of cell differentiation is usually expressed as ALP activity [34]. Our finding showed that the ALP activity for both the MPZ20 and MPZ10 was significantly higher than MP at 7 days, and MP was significantly higher than PHBV. Some studies have shown that ionic products (Si, Mg and Ca) dissolution from bioactive glass could stimulate osteoblast proliferation and differentiation [35]. In this study, both MP and MPZ containing n-MCP could be degradable, and the continuous dissolution of n-MCP might produce a micro-environment containing Ca and Mg ions that might be responsible for stimulating cell responses such as growth, proliferation and differentiation. Therefore, it can be suggested that the first degradation of zein in the MPZ ternary composite would lead to the fast dissolution of n-MCP (increase the surface area of the MPZ), which released more Ca and Mg ions in the micro-environment to stimulated cells responses. In short, the MPZ ternary composite containing n-MCP and zein was conducive to promoting the cell differentiation. 5. Conclusions The bioactive n-MCP/PHBV/ZN ternary composite was prepared by the solvent-casting method. The results demonstrated that the introduction of n-MCP and zein into PHBV led to the improvement of the hydrophilicity, in vitro degradability, bioactivity and cytocompatibility of the MPZ ternary composite. In cell culture experiments, the results showed that the MPZ ternary composite could stimulate cell adhesion, proliferation and differentiation. In conclusion, the incorporation of n-MCP and ZN into PHBV was a useful approach to obtain n-MCP/PHBV/ZN ternary composite with good biocompatibility, which can be applied in biomedical fields for bone defects repair. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (31271031), the International Cooperation Project of the Ministry of Science and Technology of China (2013DFB50280), the National High Technology Research & Development Program of China (863 Program) (2014AA021202), the Major International Joint Research Project between China and Korea (81461148033, 31311140253), and the National Research Foundation of Korea (NRF) Grant (NRF-2014K2A2A7066637). References [1] R. Zhou, W. Xu, F. Chen, C. Qi, B.Q. Lu, H. Zhang, J. Wu, Q.R. Qian, Y.J. 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