Tissue engineering applies the principles of cell biology, material science, and biomedical engineering to replace the current function of a tissue or organ. Tissue engineering may overcome the problems of a lack of organ donors and the shortage of functional organs by providing a biological substitute to the bone defect using a scaffold. The scaffold must have certain abilities such as being able to assist localization and delivery to the accurate place in the body, uphold a three-dimensional design that allows the growth of new tissue, and lead to the growth of new tissue with suitable function.1 There are many techniques for fabricating scaffolds for tissue engineering such as solvent casting/particulate leaching,2 gas foaming,3 self-assembly,4 electrospinning,5 phase separation/freeze drying,6 and rapid prototyping.7 Nevertheless, scaffold fabrication using an electrospinning technique has gained wide attention for resembling the extracellular matrix and because of scaffold properties such as a high surface area to volume ratio, high porosity, high mechanical strength, and biodegradability.8
Conductive polymers are polymers that exhibit loosely held electrons in their backbones and the backbone atom is connected to a π bond. The conjugated backbone provides pathway for electron mobility and charge transport and doping π-conjugated polymer, producing high electrical conductivity.9 In biomedical application, conductive polymers are being used in tissue engineering, neural probes, biosensor, drug delivery, and bioactuators.10 Many efforts have been made to develop tissue engineering scaffolds using conductive polymers such as polypyrrole, polyaniline, and polythiophene derivatives such as poly(3,4-ethylenedioxythiophene) (PEDOT) and poly(3-hexylthiophene).11 The benefit of using a conductive polymer is to enhance cell attachment and proliferation.12 Recently, a conductive coated scaffold using the conductive polymer poly(3,4-ethylenedioxythiophene) poly(4-styrenesulfonate) (PEDOT:PSS) has been shown to be biocompatible in vitro.13 Previous research has also proved that this material is suitable for bone tissue engineering.14 The focus of this study is to fabricate a conductive electrospun membrane with commercially available PEDOT:PSS by a dip-coating method. The biodegradable polymers poly(lactic) acid (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were chosen as nanofibrous membrane materials. PLA has wide application in the biomedical area and has been used in sutures, bone fixation material, drug delivery, and tissue engineering. It has received approval from the US Food and Drug Administration and European regulatory system for food and surgical applications.15 Additionally, because of its biodegradability and biocompatibility, PHBV is suitable as a natural bone substitute material. Besides that, the degradation time for PHBV can be controlled by adding inorganic materials, and the product of degradation is the hydroxybutyric acid, which is an element of human blood.16
Calcium phosphate–based bioceramics such as hydroxyapatite (HA) have several advantages when incorporated into polymers membrane. It can reinforce biomineralization process of polymer membrane, as well as higher cell attachment, proliferation, and osteogenic differentiation.17 Only a few studies available are based on conductive polymer PEDOT:PSS with bioactive materials for bone tissue regeneration.18 There is no direct relationship study between conductivity and biomineralization. Because hydrophobicity of conductive polymer is one major drawback limiting its application in bone tissue regeneration, blending with bioactive molecules is a must for mineralization process. A previous study demonstrated that the conductive polymer scaffold, when combined with bioactive molecules under electrical stimulation, can accelerate osteoblast mineralization.19 Biomineralized conducting polymer scaffold also showed enhanced bone differentiation.20
Therefore, a bioactive surface on conductive nanofiber membrane scaffold has been used. The current study aims to enhance the surface property and conductivity of fabricated electrospun membrane by coating with PEDOT:PSS material. The coated membrane was soaking in simulated body fluid (SBF), and its biomineralization effect in term of structural and wettability was investigated. It will be beneficial for an electrospun membrane having the dual property of bioactivity and conductivity and is a potential material for bone tissue regeneration.
HA was obtained from the nanoemulsion freeze-drying technique previously described.21 PHBV (12 mol% PHV, Mw = 121 kDa) and chloroform were purchased from Sigma-Aldrich. PLA (Mw = 220 kDa) was obtained from Biomer, Krailling, Germany (product name Biomer L9000). Dimethylformamide (DMF) was obtained from RCI Labscan Limited. PEDOT:PSS was from Sigma-Aldrich in the form of a water suspension (1 wt%), HA, and 2-propanol (Mw = 60) from QReC (Asia) SDN BHD.
Preparation of Solution
PLA/PHBV (1 : 1) solution at a concentration of 20% (wt/vol) was dissolved in chloroform : DMF (9 : 1). The solution was stirred at 50°C. PLA/PHBV/HA solution was prepared by adding 10% HA (wt/wt) into dissolved PLA/PHBV solution, stirring for 15 min, and homogenizing for 2 min.
The prepared solution was transferred into a 15 mL syringe. The electrospinning solution was conducted by setting up the parameters such as tip-to-collector distance of 10 cm, injection flow rate of 1 mL/h, and applied voltage of 25 kV. The temperature of the room (22.5°C) and humidity (45–50%) were recorded during the electrospinning process.
PEDOT:PSS Coating on Electrospun Membranes
The electrospun membrane was cut into a circular disk (diameter = 1 cm). Then, 40% (vol/vol) of PEDOT:PSS solution was prepared from 3 mL propanol added to 2 mL PEDOT:PSS. The electrospun membrane was dipped into 40% PEDOT:PSS solution for 1 h. After that, the sample was rinsed with distilled water and put into a drying oven at 30°C for 3 h.
Morphology of Electrospun Membranes
The nanofibers were examined with a scanning electron microscope (SEM, TM3000) and a field emission scanning electron microscope (FESEM, SU8020, Hitachi) operated at 15 kV. The rectangular specimens were sputter-coated with gold, attached to carbon tape on a stub before imaging. ImageJ was used to measure the diameter of the fibers present. At least 50 diameters of fibers were calculated.
Energy-Dispersive X-Ray Spectroscopy.
Rectangular specimens were cut from the as-fabricated membranes, and the specimens were sputter-coated with a thin layer of gold. The specimens were then examined using an energy dispersive x-ray spectroscopy equipped with a scanning electron microscope (SEM, TM3000) at 15 kV to confirm the presence of elements in the membranes.
The contact angle was used to determine the wettability of the membranes before and after coating with PEDOT:PSS using a contact angle measuring system (VCA Optima, AST Products, Inc). A 1 μL deionized water droplet was dropped onto the membranes, and the measurement was taken for 3 s for each sample. The contact angle also was performed on biomineralized samples after SBF immersion using the video mode.
Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy.
Attenuated total reflectance fourier transform infrared spectroscopy (ATR-FTIR) was used to determine the chemical bonding of the electrospun membranes produced. The analysis was performed using an ATR-FTIR spectroscopy (Perkin-Elmer Series, USA Model, and Thermo Scientific Nicolet iS5) in the range of 4000–350 cm−1. Origin software was used to analyze the graph obtained.
Electrospun PLA/PHBV/HA membranes were cut into a rectangular shape (2 cm × 1 cm) and immersed in 1.5× SBF for 7, 14, and 21 days. The 1.5× SBF was prepared according to a previous study with ion concentrations (in mM): Na+ 243, K+ 7.5, Ca2+ 3.8, Mg2+ 2.6, Cl− 223, HCO3 − 6.3, HPO4 2− 1.5, and SO4 2− 0.8).22
At least three samples were tested for each characterization, and the average and standard deviations were calculated and analyzed.
Results and Discussion
Using electrospinning techniques, electrospun membranes of PLA/PHBV and PLA/PHBV/HA were collected with random nanofiber orientation as in Figure 1. PLA/PHBV membrane had the good formation of the nanofiber structure showing no beads along the fibers (Figure 1A); on the other hand, PLA/PHBV/HA had some beads and agglomeration of HA (Figure 1B). Polymer solution at 20% (wt/vol) was prepared by electrospinning by blending PLA/PHBV using a solvent of chloroform and DMF in a ratio of 9: 1. It was found in a study that a volatile solvent such as chloroform alone leads to needle blockage, resulting in no fiber formation. This is because of the fast evaporation rate of the solution while being ejected from the needle toward the collector.23 By adding a lower volatility solvent such as DMF, the evaporation rate of the solution can be lowered, thus, promoting the formation of electrospun fibers and reducing needle blockage during the electrospinning technique. Long and fine electrospun fibers can be observed from the SEM results. The highest frequency of electrospun fiber diameter measured from the fiber strands was in the range of 700–900 nm, with an average diameter of 846 nm. After adding the 10% (wt/wt) HA, the solution became relatively more viscous. After incorporation of HA, the electrospun PLA/PHBV/HA membrane was obtained as shown in Figure 1B. The highest frequency of diameter distribution for PLA/PHBV/HA was in the range of 400–600 nm with an average diameter of 633 nm. In comparison, the diameter of the electrospun membrane decreased after the addition of HA.
After coating with PEDOT:PSS, there was no change in the morphology of the membrane (Figure 1C and 1D). However, the average diameter of nanofibers was slightly increased to 931 nm for PEDOT:PSS-coated PLA/PHBV and 669 nm for PEDOT:PSS-coated PLA/PHBV/HA membranes. To confirm there was a coating layer, EDX was conducted on the PLA/PHBV/HA electrospun membrane before and after coating with PEDOT:PSS (Figure 2). The presence of HA in the membranes is shown by the existence of calcium and phosphorus elements in the EDX spectra. The sulfur element in the EDX spectra proved the conductive layer of the membrane.
Figure 3 shows the ATR-FTIR spectra for HA (Figure 3A), PLA/PHBV, and PLA/PHBV/HA (Figure 3B) and the PEDOT:PSS-coated electrospun membrane (Figure 3C). Based on the HA spectrum in Figure 3A, the transmittance band of 1024 cm−1 is shown as the stretching band of the P–O group of HA.24 Thus, it confirmed the presence of HA chemical bonding.
From Figure 3B, the wavenumbers for the PLA/PHBV electrospun membrane were observed as 2995 cm−1, 2956 cm−1, 2941 cm−1 (–CH3 and –CH2 aliphatic compounds), 1749 cm−1 (C=O stretching), 1455 cm−1 (asymmetric bending absorption of CH3), 1381 cm−1 (symmetrical bending), 1281 cm−1 (C–O stretch), 1178 cm−1, 1089 cm−1, 1057 cm−1 (C–O–C stretching), and 982 cm−1 (CH2 in-plane bending). For PLA/PHBV/HA, the transmittance bands were 2991 cm−1, 2935 cm−1 (–CH3 and –CH2 aliphatic compounds), 1758 cm−1, 1725 cm−1, 1720 cm−1 (C=O stretching), 1454 cm−1 (asymmetric bending absorption of CH3), 1381 cm−1 (symmetrical bending), 1280 cm−1 (C–O stretching), 1182 cm−1, 1134 cm−1, 1090 cm−1, 1048 cm−1 (C–O–C stretching), and 976 cm−1 (CH2 in-plane bending). In comparison between the electrospun PLA/PHBV and PLA/PHBV/HA membranes, the stretching vibration of the P–O group of HA was overlapped between 1134 and 1048 cm−1. Because HA was not added in a large quantity, the presence of HA in the sample was not significant.
Figure 3C shows the ATR-FTIR spectra for PEDOT:PSS-coated electrospun PLA/PHBV and PLA/PHBV/HA membranes. Absorbance peaks were observed at 2998 cm−1, 2977 cm−1, 2935 cm−1 (–CH3 and –CH2 aliphatic compounds), 1749 cm−1, 1725 cm−1, 1757 cm−1, 1723 cm−1 (C=O stretching), 1453 cm−1, 1456 cm−1 (asymmetric bending absorption of CH3), 1381 cm−1, 1382 cm−1 (symmetrical bending), 1281 cm−1, 1275 cm−1 (C–O stretching), 1183 cm−1, 1125 cm−1, 1090 cm−1, 1053 cm−1, 1179 cm−1, 1130 cm−1, 1086 cm−1, 1040 cm−1 (C–O-C stretching), and 982 cm−1 (CH2 in-plane bending). The spectra were similar to the uncoated sample graphs because of the low presence of PEDOT:PSS, and the peaks overlapped with those of PLA/PHBV/HA.
Since 1990, Kokubo et al. have introduced SBF solution, which has been used as an effective way to study the calcium phosphate and apatite formation or precipitation on the surface of biomaterials. This apatite layer is used for the prediction of in vivo testing of bone bioactivity. Owing to the bone-binding ability of the bone-like apatite layer, SBF solution has been developed.25 The presence of ions such as Na+, K+, Ca2+, Cl−, and HCO3 − may lead to the formation of apatite on HA particles. The concentration of these ions, especially HCO3 −, induces an increased rate of diffusivity by interacting with the hydroxyl groups of the membrane.26 By increasing the amount of SBF solution, which means the 1.5× SBF has a 1.5 times higher concentration of ions than in normal SBF and blood plasma, the ionic activity produced will raise the apatite formation.27 After 21 days, mineralization on the PLA/PHBV/HA and PEDOT:PSS-coated PLA/PHBV/HA membrane was observed (Figure 4). The nucleation of HA grew into a globe shape on the surface of the membranes. Higher magnification of this apatite exhibited flake-like apatite crystals. From Figure 5, the membranes were found to be showing a high distribution of calcium and phosphorus layer on the surface of nanofibers, whereas the higher content of the elements was observed for PEDOT:PSS-coated PLA/PHBV/HA compared with PLA/PHBV/HA membranes. The nucleation may occur from HA nanoparticles present on the surface in contact with SBF solution. This strategy was conducted in several studies using different polymers, which demonstrated incorporation of HA into nanofibers and immersion in 1.5× SBF to obtain apatite membranes.28 , 29 In one study, nanofibers treated with biomimetic HA showed greater biocompatibility but at a different concentration of SBF (10× SBF) and presented increased cell proliferation and differentiation compared with untreated fibers.30 , 31
The contact angle was obtained to determine the wettability of the electrospun membrane. Wettability is a crucial factor involved in biological cell immobilization while the hydrophilicity or hydrophobicity plays a role in the cell adhesion of biological macromolecules.32 A contact angle less than 90° indicates that the wetting of the surfaces is favorable, whereas an angle greater than 90° indicates unfavorable wetting.33
From Figure 6, for the as-fabricated membrane, it can be seen that PLA/PHBV had a hydrophobic surface with a contact angle of 118.77 ± 1.67°. The addition of HA into the membrane slightly increased the contact angle to 122.25 ± 4.56°. Further coating the PLA/PHBV/HA sample with PEDOT:PSS lowered the contact angle to 49.23 ± 8.80°. Based on these results, PLA/PHBV shows hydrophobic properties because the contact angle of more than 90° makes wetting unfavorable. Thus, the addition of HA and the coating of electrospun membranes with the conductive polymer of PEDOT:PSS improved the wettability of the fibers, which might help in cell adhesion.
After SBF immersion for 7, 14, and 21 days, the contact angle decreased to 0°. During the analysis, the contact angle was measured for 3 s for each sample. However, the water droplet was absorbed by the sample after 3 s before the contact angle could be measured, resulting in an angle of 0° and showing that the membrane had become superhydrophilic. The SBF solution used was 1.5× SBF solution, which contains more significant concentrations of sodium and chloride ions compared with other ions in normal SBF.34 However, the higher concentration of SBF may result in the higher wettability of the samples. The results also proved by elemental mapping of the membrane after immersion in SBF, showing a calcium and phosphorus layer on the membranes, and did not hinder the conductivity of the membrane by the existence of sulfur.
The electrospun nanofibrous membrane scaffold of PEDOT:PSS-coated PLA/PHBV/HA was produced through electrospinning and dip-coating techniques. A bioactivity test was conducted to observe the ability of the conductive scaffold to support bone-like apatite formation by using SBF solution. The results of the bioactivity test supported the formation of apatite. Further research could compare the biocompatibility of these conductive-coated nanofibers treated with SBF with non-treated SBF. In conclusion, these nanofibers have the potential to be further studied in the application of bone tissue engineering.
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Keywords:Copyright © 2018 by the American Society for Artificial Internal Organs
biomineralization; conductive materials; hydroxyapatite; PEDOT:PSS; poly(lactic) acid; poly(3-hydroxybutyrate-co-3-hydroxyvalerate)