Preparation of poly(lactic acid)/sintered hydroxyapatite composite biomaterial by supercritical CO 2

Abstract

Based on a kind of sintered hydroxyapatite (HA) with a good cytocompatibility, a series of polylactic acid (PLA) and PLA/HA with the various PLA:HA weight ratio (5:5, 4:6, 3:7, 2:8, 1:9) were fabricated by supercritical CO₂. The physical and chemical properties were evaluated by pH, degradation, water absorption, porosity, density, mechanical property, and cytotoxicity respectively. With the increase of HA content, the pH value and porosity increased gradually, while weight loss rate and the density showed a gradual downward trend. Existence of HA can drastically improve the hydroscopicity of PLA scaffolds. The compression strength values slightly increased ( $p > 0.05$ ) from 39.96 MPa of PLA to 45.00 MPa of PLA/HA with the ratio of 7:3, subsequently, the values decreased ( $p < 0.05$ ) from 43.29 MPa (8:2) to 19.00 MPa (9:1). While the modulus of elasticity decreased ( $p < 0.05$ ) from 5.89 to 1.84 GPa with increasing HA content. The PLA/HA (8:2) promoted cell proliferation more significantly than any of other groups ( $p < 0.05$ ). Based on the results, the overall properties of porous scaffolds are the optimal when the weight ratio of PLA/HA is 8:2. Its pH, porosity, density, compression strength, and elasticity modulus are 7.39, 83.0%, 0.60g/cm⁻³, 34.1 MPa and 2.63 GPa, respectively. SEM observation presented a homogeneous distribution of HA in PLA matrix and a foam-like structure comprising interconnected pores.

Keywords

Poly(lactic acid)hydroxyapatite composite biomaterial bone repair

1. Introduction

Tumors, trauma, disease and birth defects create a demand for skeletal reconstruction. Although autograft and allograft are the standard treatments, they are sometimes associated with morbidity of donor sites, rejection by the immune system, longer surgical time, and higher costs [1]. Therefore, many investigations have been conducted to find materials for bone substitutes. Among them, hydroxyapatite (HA), with chemical composition of Ca₁₀(PO4)₆(OH)₂ similar to the major mineral component in bone and teeth of the body, is a promising candidate. Due to its excellent biocompatibility, easily available and a lower cost, HA have been widely used in bone grafting and dental devices as bone substitutes in the past decade [2,3]. Two types of HA are currently available: processed materials made from coral or bovine bone and completely synthesized HA. Owing to the properties more similar to HA of nature bone, HA processed from bovine is more used widely [4]. However, the application of processed HA is limited by its brittleness, difficulty in shaping, slow biodegradation [5]. Biodegradable polymers, due to their excellent biocompatibility, biodegradability and processability, have been approved use in medical devices by the US Food and Drug Administration (FDA). Poly(lactic acid) (PLA) is one of the most attractive candidates for scaffold materials among them because of its favorable mechanical properties, processability, biodegradability as well as biocompatibility [6]. However, the inherent drawbacks are poor osteoconduction, highly hydrophobic and acidic degradation products which may cause premature implant failure and local inflammation [7,8]. Combining degradable polymers and HA into composite materials maybe an effective solution to solve these problems [9].

To overcome the inherent drawbacks of HA and PLA, the aim of this study was to prepare a porous PLA/HA composite biomaterial by a novel method, namely supercritical CO₂ technique.

2. Materials and methods

2.1. Materials

PLA with the average molecular weight $M_{w} = 1 \times 10^{5} g/mol$ and polydispersity $M_{w} / M_{n} = 1.6$ was obstained from Nature Works, USA. Fresh bovine cancellous bone were procured from bovine limbs in market. The osteoblast-like cells (MC3T3-E1) were obstained from China Center for Type Culture Collection. DMEM medium and fetal bovine serum were purchased from Hyclone, USA. MTT and DMSO were obstained from Sigmai-Aldrich, USA. All other chemicals (NaOH, phosphate-buffered saline) used in the study were of reagent grade and used as purchased.

2.2. Methods

2.2.1. Preparation of HA

Fresh bovine cancellous bone procured from limbs was stored at below $- 40 ° C$ until treatment. The bone was removed of the soft tissue and cut into strips of $1.0 \times 1.0 \times (1.0 \sim 5.0) cm$ . After cleaning the bone marrow by the ultrasonic washing, the bone strips were defatted in the supercritical CO₂ for 24 hours. Following being immersed in 0.5 M NaOH for 4 hours in the ultrasonic bath, the bone were sintered at 700°C for 4 hours and thoroughly washed three times with excess phosphate-buffered saline ( $pH = 7.0$ ).

2.2.2. Preparation of PLA/HA with the various ratio

PLA with the average molecular weight $M_{w} = 1 \times 10^{5} g/mol$ and polydispersity $M_{w} / M_{n} = 1.6$ and HA was ground and particle size in the range from 50 to 150 μm was sieved. The porous PLA/HA composite biomaterials with the various ratios were fabricated according to the following described method (Fig. 1) [10,11]. Briefly, the PLA and HA was mixed with the PLA/HA weight ratio of 5/5, 4/6, 3/7, 8/2 and 9/1, respectively. The mixture of PLA and HA was loaded into the supercritical cylinder reactor vessel ( $diameter = 1 cm$ ) linked to supercritical-CO₂ reaction apparatus (SFE-2; Applied Separations, Inc., Allentown, PA, USA).The pressure of the container was gradually raised to 20 MPa and the temperature was increased to 37°C. The mixture was kept in $(20 \pm 0.1) MPa$ and the temperature in $(37 \pm 0.5) ° C$ for 30 minutes to saturate the polymer with the supercritical CO₂. The pressure was decreased to ambient pressure at the rate of 5 MPa/min. The samples were removed from the vessel. Following lyophilization, the samples were packed in a three-layer aseptic package and irradiated with 20 kGy ⁶⁰Co (China Institute for Radiation Protection, Taiyuan, China) for sterilization.

Fig. 1.

Schematic of fabrication procedures for the porous PLA/HA composite biomaterials.

2.2.3. Cell affinity of HA

To confirm cell affinity of the prepared HA, Cell affinity of HA was performed. Briefly, the HA used for cell culture tests was sterilized using 20 kGy ⁶⁰Co (China Institute for Radiation Protection, Taiyuan, China). The osteoblast-like cells (MC3T3-E1) were seeded onto the prepared HA in 12-well plates with a density of $1 \times 10^{5} cells/well$ , and the HA were immobilized in culture dishes at 37°C in 5% of CO₂. The DMEM containing 10% FBS was used as the culture medium and changed every other day after culture. The cells cultured for 5 days were fixed with 2.5% glutaraldehyde in PBS for 12 hours at 4°C. The specimens were thoroughly washed three times with PBS, followed by dehydration sequentially through a series of increasing concentrations of ethanol (10, 30, 50, 70, 80, 90, 95, and 100%). Finally, the specimens were coated with gold in a sputtering device for 180 s under vacuum and then observed using a scanning electron microscope (JSM-6360LVV, JEOL, Ltd., Japan) in high vacuum and with an acceleration voltage of 20-25kV.

2.2.4. pH measurement of PLA and PLA/HA with the various ratio

1g of HA, PLA, and PLA/HA with the various ratio were immersed in 50 ml distilled water ( $pH = 6.45$ ) at 37°C water bath respectively. The pH value of solutions were performed with a HS-3C meter (Leici, China) after 7 days. 5 samples were tested each group.

2.2.5. In vitro degradation of PLA and PLA/HA with the various ratio

PBS ( $pH = 7.4$ ) at 37°C was used as the immersion fluid. The PLA/HA samples with the dimension of Ø $10 mm \times 10 mm$ were placed in a 50 ml weighing bottle containing 30 ml PBS, respectively. The immersion fluid was refreshed every 7 days. In vivo, the bone healing time for fracture is typically $5 \sim 12$ weeks [12]. In this work, the immersion times were approximately selected as 1, 2, 3 and 4 weeks to represent the early stage of the bone healing. Three specimens were tested at each time point and the averages were used in the assessment. The mass loss ratio (ω) was calculated by the following equation: $\begin{matrix} ω = (M_{0} - M_{t}) / M_{0} \times 100 % \end{matrix}$ where $M_{0}$ is the initial mass and $M_{t}$ is the final mass of the specimen at time point after drying for 48 h in an oven at 37°C.

2.2.6. Water absorption of PLA and PLA/HA with the various ratio

The PLA and PLA/HA were pre-wetted to ensure that water permeated through all the pores of material. Three dry samples of each group were placed in a glass bottle filled with 10 ml water for 24 hours. On removal, the samples were carefully wiped with filter paper to remove surface water, followed by measuring their wet weight. Then they were dried in vacuum for 12 hours to determine the mass of the dried samples. The water absorption was calculated using the following equation: $\begin{matrix} Water absorption = (M_{wet} - M_{dry}) / M_{dry} \times 100 % . \end{matrix}$ Where $M_{wet}$ and $M_{dry}$ are the wet and dry mass of the sample, respectively. The water absorption measured includes the water absorbed by both the material and the pores.

2.2.7. Porosity and density measurement of PLA and PLA/HA with the various ratio

The porosity and density of the PLA and PLA/HA were determined using a liquid displacement method [13]. Ethanol was used as the displacing liquid because it penetrated easily into the pores of the scaffold, but not into the composite itself. Briefly, a sample of measured weight W was immersed in a graduated cylinder containing a known volume ( $V_{1}$ ) of ethanol and kept for 5 minutes to allow the ethanol to penetrate into the pores of the scaffolds. The total volume of the remaining ethanol and the ethanol-impregnated scaffolds was then recorded as $V_{2}$ by reading the scale values in graduated cylinder. The volume difference ( $V_{2} - V_{1}$ ) represents the volume of the scafflods. The ethanol-impregnated scaffolds were then removed from the graduated cylinder and the residual ethanol volume was recorded as $V_{3}$ . Hence the total volume of the scaffolds was ( $V_{2} - V_{3}$ ) and the bulk density of the scaffold was formulated as $ρ = W / (V_{2} - V_{3})$ . By measuring the initial and final weights $W_{i}$ and $W_{f}$ , respectively, of the scaffolds immersed in ethanol ( $density = 0.789 {g/cm}^{3}$ ) for 24 hours, the pore volume of the PLA and PLA/HA scaffold can be calculated as $(W_{f} - W_{i}) / ρ_{ethanol}$ on assuming that the ethanol does not penetrate the struts of the scaffold. Then the porosity can be determined using the following equations: $\begin{matrix} porosity = (W_{f} - W_{i}) / ρ_{ethanol} / (V_{2} - V_{3}) \end{matrix}$

2.2.8. Mechanical properties of PLA and PLA/HA with the various ratio

An RGT-20A microcomputer-controlled electronic universal testing machine (Shenzhen Reger Instrument Co. Ltd., Shenzhen, China) was employed to evaluate the compression mechanical properties of the porous PLA and PLA/HA with the various ratio at room temperature. Cylindrical specimens with the length of 10 mm and the diameter of 10 mm were placed vertically between two solid platens and compressed at a rate of 1 mm/min using a 5 N load cell. The modulus of elasticity was estimated from the slope of the initial linear portion of the stress-strain curve. The compression strength was obtained from the peak of the stress-strain curve.

2.2.9. Cytotoxicity test of PLA and PLA/HA with the various ratio

The PLA and PLA/HA composite scaffolds with the various ratio were immersed in serum free DMEM medium in the sealed container at 37°C for 72 hours according to the PLA/HA:DMEM ratio of 0.1 g:1 ml, respectively. After the leaching solution of composite materials were centrifuged, the supernatants were harvested and added 10% fetal bovine serum for cell culture. 100 μl of $10^{4}$ cells/ml MC3T3-E1 cells were seeded in each well of 96-well plate. After 24 hours, the DMEM mediums were removed and 100 μl of the supernatants were added respectively, 15 wells were tested each group. Cells cultured with DMEM medium were analyzed as the normal control and non-seeded well as the blank group. The half of culture mediums were replaced every 2 days. The number of cells was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay to assess the proliferation of the cells on the 2nd, 4th and 7th day. Briefly, to remove culture medium, 30 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4 hours at 37°C. subsequently, to remove MTT solution, 150 μl of DMSO was added to dissolve crystal. The optical density (OD) value was determined using microplate reader ( $λ = 570 nm$ ). The cell proliferation rate (RGR) was calculated according to the following equation. $\begin{matrix} RGR = {OD}_{group} / {OD}_{normal} \times 100 % \end{matrix}$ Where ${OD}_{group}$ and ${OD}_{normal}$ are the OD value of PLA/HA group and normal control group, respectively.

2.2.10. Morphology observation of the optimal ratio PLA/HA

Morphology of the porous PLA/HA with the optimal PLA:HA ratio was examined using a scanning electron microscope (JSM-6360LVV, JEOL, Ltd., Japan), operating in high vacuum and with an acceleration voltage of 20–25 kV. Before the observation, the specimens were coated with gold in a sputtering device for 180 s under vacuum. The average pore size of porous scaffolds was calculated by image analysis software.

2.2.11. Statistical analysis

All statistical analyses were performed with SPSS (17.0) software (IBM Corporation,USA). Quantitative data were presented as $mean \pm standard deviation (SD)$ . Significant differences between the groups were determined using analysis of variance (ANOVA). Least Significance Difference (LSD) will be used in multiple comparison. Results were considered significant if $p < 0.05$ .

3. Results

3.1. Preparation and cell affinity of HA

The HA appeared fragile, white cubes (Fig. 2(a)). SEM observation showed HA had a highly porous, well-interconnected pore structure (Fig. 2(b)). HA cultured with MC3T3-E1 showed a large quantity of cells grew both on the surface and in the holes of HA (Fig. 2(c)). The cells appeared polygonal shape and attached well with long processes and more microvilli (Fig. 2(d)).

Fig. 2.

Gross and SEM observation of HA and HA cultured MC3T3-E1 cell. (a) general observation of HA; (b) microstructure of HA; (c) SEM of HA cultured MC3T3-E1 cell ( $\times 170$ ); (d) SEM of HA cultured MC3T3-E1 cell ( $\times 2, 500$ ).

3.2. pH measurement of PLA and PLA/HA with the various ratio

The pH value of PLA/HA showed as in Fig. 3. The pH value of PLA was 5.47, which resulted from lactic acid that PLA degraded into. With the increase of HA content, the pH value increased gradually. When the ratio of PLA/HA was 2:8, the pH value of scaffold increased to 7.39 that stayed in a normal pH range (7.35–7.45) in body fluid. While the pH value of the pure HA (9.47) was far from the normal pH range in body fluid. It indicates that alkaline characterization of HA can neutralize the acidity caused by degradation of PLA.

Fig. 3.

pH value of PLA/HA with the various ratio.

3.3. In vitro degradation of PLA and PLA/HA with the various ratio

Figure 4 shows the percentage changes in weight loss of PLA/HA with the various ratio throughout degradation in PBS at the different time points. Weight loss rate in the PLA group was the most. With the content of HA increase, weight loss rate showed a gradual downward trend. It suggests that an increase of HA content would lead to a lower degradation rate of PLA. PLA/HA with the ratio of 9/1 had the lowest degradation ratio. In all of the groups, the weight loss rate kept in a lower level in 2 weeks. Subsequently, it increased to a higher level after 2 weeks. The accelerating degradation maybe caused by autocatalytic reaction that lactic acid induced.

Fig. 4.

Weight loss of PLA and PLA/HA with the various ratio during degradation in PBS.

3.4. Water absorption of PLA and PLA/HA with the various ratio

Immersion of the scaffolds in water resulted in an increase in mass within 24 h as shown in Fig. 5. It was observed that there was a mean mass increase of 158% for pure PLA scaffold. The mass of the PLA/HA with the ratio of 5:5 increased by 264% ( $p < 0.05$ ). However,there were slightly changes among PLA/HA with the ratio of 5/5, 6/4, 7/3, and 8/2 ( $p > 0.05$ ).When the HA content increased to 90%, the mass declined significantly to 329% ( $p < 0.05$ ). Being an aliphatic polyester, PLA is highly hydrophobic. And HA is hydrophilic material. As a result, existence of HA can drastically improve the hydroscopicity of PLA scaffolds.

Fig. 5.

Water absorption of PLA and PLA/HA with the various ratio.

3.5. Porosity and density measurements of PLA and PLA/HA with the various ratio

The effect of HA content on the porosity and density of the PLA/HA is shown in Fig. 6. As expected, the porosity of the material increased with increase of HA content. On the contrary, the density of the scaffold exhibited a downward trend with HA content. It may be explained by the increasing porosity. The porosity values of pure PLA was 62.7%. With the loading of HA, the porosity increased from 76.0% (5:5) to 83.3% (9:1). However, there was no significant difference among the PLA/HA with 7:3, 8:2 and 9:1 ( $p < 0.05$ ). It indicated the HA ratio over 7:3 can not increase the porosity further. The high porosity presented in all the specimens suggested that the supercritical CO₂ technique is a practical method for preparing porous polymer composite biomaterial.

Fig. 6.

Porosity and density of PLA and PLA/HA with the various ratio.

3.6. Mechanical properties of PLA and PLA/HA with the various ratio

The mechanical testing results for the PLA and PLA/HA with the various ratio are summarized in Fig. 7. The compression strength values slightly increased ( $p > 0.05$ ) from 39.96 MPa of PLA to 45.00 MPa of PLA/HA with the ratio of 7:3, subsequently, the values decreased ( $p < 0.05$ ) from 43.29 MPa (8:2) to 19.00 MPa(9:1). On the contrary, the modulus of elasticity decreased ( $p < 0.05$ ) from 5.89 to 1.84 GPa with increasing HA content. There were significant differences between PLA/HA with the ratio of 8:2 and 9:1, and between PLA and PLA/HA(5:5) in modulus of elasticity ( $p < 0.05$ ). However, there was no differences among PLA/HA with ratio of 6:4, 7:3 and 8:2 ( $p > 0.05$ ).

Fig. 7.

Compression strength and modulus of elasticity of the PLA and PLA/HA with the various ratio.

3.7. Cytotoxicity test of PLA and PLA/HA with the various ratio

Proliferation rates of MC3T3-E1 cultured with leaching solution of PLA and PLA/HA with the various ratio are shown in Fig. 8. The proliferation rates of PLA/HA (9:1) were the lowest ( $p < 0.05$ ) at all of the time points and decreased gradually over time. It may be resulted from the high pH ( $pH = 8$ ). The proliferation rate second to the PLA/HA (9:1) was the pure PLA with the pH of 5.47. When the content of HA was between 50% and 80%, the cell proliferation rate was positive correlation with HA content. The PLA/HA (8:2) promoted cell proliferation more significantly than any of other groups ( $p < 0.05$ ).

Fig. 8.

Proliferation rate of MC3T3-E1 cultured with leaching solution of PLA and PLA/HA with the various ratio.

3.8. Morphology observation of PLA/HA with the optimal ratio (8:2)

Based on the previous experimental data, PLA/HA with the ratio of 8:2 was considered as the optimal composite ratio. PLA/HA(8:2) showed white porous structure (Fig. 9(a)). SEM observation presented a homogeneous distribution of HA in PLA matrix and a foam-like structure comprising interconnected pores. The well-defined porous structure in three dimensions is throughout in PLA/HA skeleton (Fig. 9(b)). Additionally, the open-pore structure possesses pore with size of a range from $20 \sim 80 μ m$ . Such large pore size is able to provide enough space for cell attachment, growth, and possible vascularization when being implanted. These pores will not only act as the channel to transport nutrient or metabolism product, but also make the pore walls rougher, which is beneficial to improve the interfacial adhesion between cells and scaffold.

Fig. 9.

SEM observation of PLA/HA with the optimal ratio of 8:2. (a) gross observation of PLA/HA; (b) SEM of PLA/HA ( $\times 110$ ); (c) SEM of PLA/HA ( $\times 350$ ); (d) SEM of PLA/HA ( $\times 650$ ).

4. Disscusion

From the biological perspective the natural bone matrix is a combination of organic/inorganic composite materials and consists of a naturally occurring polymer (collagen) and a biological mineral (apatite) [14]. Due to its osteoconductive and biocompatible properties, HA is often a component of orthopedic devices. However, HA is difficult to shape in the specific form required for bone repair and implantation because of its intrinsic hardness, fragility, and lack of flexibility, which limits its use as a load-bearing bone substitute for bone-repair applications [15]. To capitalize on its advantages and simultaneously overcome the drawbacks, polymer/HA composites are a subject of interest to many researchers involved in bone-repair. PLA is the most extensively researched and utilized biodegradable polymer in human history due to its merits. Many studies have proved that combining PLA and HA can take advantage of both and overcome their disadvantages [16]. The resources of HA include natural HA and synthetic HA. Owing to its physical and chemical structure more similar to human bone, natural bone possesses the better biocompatibility and osteoconduction which have be approved by some studies. Some papers have already focus on preparation of PLA and synthetic HA composite by supercritical CO₂ [17,18], however, little experimental data about composite biomaterials of PLA and nature HA have be provided. The traditional methods for fabricating PLA/HA composite biomaterials include solvent casting, the particulate leaching, thermally induced phase separation, and electrospinning techniques [19–21]. These methods need using organic solvents or high temperature in the process which destroy the biocompatibility of materials and some growth factors. Supercritical CO₂ is a novel method for polymer preparation without introduction of high temperature and organic solvent in the process of preparation and particularly suitable for the preparation of biomaterials with good biocompatiblity [10,11].

In this study, the PLA/HA with the various weight ratio (5:5, 6:4, 7:3, 8:2, 9:1) were prepared by supercritical CO₂ method and the physical, chemical and cytocompatible properties were tested to screen the optimal ratio. To get the best properties of the composite, it is important to minimize the amount of PLA in the composite, as the PLA is highly hydrophobic and poor osteoinductive. On the other hand, if there is too little PLA, there will not be sufficient matrix material to bond HA and neutralize alkaline from HA. When the ratio of PLA/HA was 2:8, the pH value of scaffold increased to 7.39 that was in a normal pH range (7.35–7.45) in body fluid. In vitro degradation showed addition of HA to PLA can decrease the degradation of composite. This is because that addition of HA can not only decrease the proportion of PLA in the composites, but also its alkaline characterization neutralizes the acidity caused by degradation of PLA which may accelerate PLA degradation. As Crow reported [19], weight loss is along with diffusion and dissolution of acidic degradation products into the solution. Water absorption was measured for the assessment of swelling ability of scaffold materials. Suitable water absorption ability is extremely necessary to obtain an ideal material, for the low absorption will result in the lack of water and then affect cell growth. HA can improve the water absorption of the PLA significantly. When the HA content increased to 90%, the mass of PLA/HA declined dramatically. Which may result from its high porosity. On this context, for further modification of HA, a procedure that the HA was placed into the NaOH solution before calcination was adopted. This processing can improve HA hydrophilic by introduction of the hydrophilic groups (-OH) onto the surface. The result that a lot of cells adhered and grew well on the HA also confirmed HA hydrophility. The HA ratio less than 80% had little effect on compression strength of PLA/HA. When the ratio of HA was over 80%, both compression strength and modulus of elasticity dramatically decreased. Which may be closely related with the uneven distribution of HA and higher porosity. The proliferation rate of MC3T3-E1 cultured with leaching solution of PLA/HA (8:2) was higher than any of the other groups. Which may be related with the pH (7.39) of PLA/HA (8:2). The pH within the normal range (7.35–7.45) is beneficial to cells growth. Both the overly basic environment of PLA/HA (9:1) ( $pH = 8.0$ ) and the overly acidic environment of PLA ( $pH = 5.47$ ) inhibited cell proliferation significantly. Due to acid-resistance of cells, PLA/HA (9:1) inhibited cell proliferation more significantly than PLA.

5. Conclusions

From the above analysis, we can draw a conclusion that when the weight ratio of PLA/HA is 8:2, the overall properties of porous scaffolds are the optimal, with the pH of 7.39, the porosity of 83.0%, the density of 0.60 g/cm⁻³, the compression strength of 34.1 MPa, and the elasticity modulus of 2.63 GPa. The mechanical properties (both modulus and strength) are much less than that of cortical bone, which typically has modulus value of $7 \sim 30 GPa$ and strength of $50 \sim 150 MPa$ . However, the PLA/HA fabricated and tested in this report are not intended primarily for load sharing applications but targeted for bone-fillers, maxillofacial applications and orbital implants. Further study is required to confirm osteoinduction and bone repair before using as a bone substitute for repair of bone defect.

Footnotes

Acknowledgements

This work was supported by Natural Science Foundation of Shanxi Province of China (Award No. 201601D011126), Shanxi Scientific Research Fund of Chinese Medicine (Award No. 2016ZYYC02), and Scientific Research Fund for the Doctoral Young Scholars, Shanxi University of Chinese Medicine (Award No. 2014bk05).

Conflict of interest

The authors have no conflict of interest to report.

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