The aim of this work was to manufacture, using the electrospinning technique, polyacrylonitrile- (PAN-) based carbon nanofibers in the form of mats for biomedical applications. Carbon nanofibers obtained by carbonization of the PAN nanofibers to 1000°C (electrospun carbon nanofibers (ECNF)) were additionally oxidized in air at 800°C under reduced pressure (electrospun carbon nanofibers oxidized under reduced pressure (ECNFV
Modern medicine applies more and more therapeutic solutions based on the achievements of nanotechnology and nanomaterials. Materials with reduced dimensions to nanoscale, i.e., nanomaterials, are often characterized by specific physical and chemical properties, which are of particular interest in terms of potential medical applications [
Otolaryngology, like cardiac surgery or neurosurgery, is looking for new solutions in the field of therapy methods using electric conductive nanomaterials for the construction of both implantable electrodes and nanomaterials allowing the construction of substrates for tissue engineering and stem cell therapy. Hearing loss is a common human disease caused by irreversible damage to hair cells and spiral ganglion neurons in the mammalian cochlea. There are many therapeutic solutions to treat this disease, such as hearing aids and cochlear implants, that can provide good retrieval of the hearing function [
Carbon nanoforms, such as nanotubes, graphene, or carbon nanofibers, have proven to be materials with high potential in the development of new implants and medical devices [
However, all the carbon nanoforms can interact with tissues and cells exhibiting a toxic effect. Recent works on the biocompatibility of CNT have proved a significant influence of the way they were prepared for contact with cells and tissues [
Graphene also finds applications in medicine including materials for biosensors for early detection of cancer and cancer cell imaging/mapping, in targeted drug delivery systems, and in gene therapy [
Another group of carbon nanomaterials are carbon nanofibers produced by the controlled heat treatment of nanometric polymer precursors. This nanomaterial significantly differs in the structure and microstructure from CNT, has a larger diameter, and is generally characterized by a lower degree of structural ordering [
Our earlier study has shown that this form of carbon also requires a specific treatment to remove toxic carbonaceous fractions that may appear in its structure and that can be responsible for its biological behavior [
The aim of this work is to present a new approach to manufacture biocompatible carbon nanofibers that can find applications in medicine for the construction of implants and medical devices as biosensors, microelectrodes, and electrical conductive scaffolds.
The study compares the genotoxicity and cytotoxicity of carbon nanofibers obtained from the electrospun PAN fiber precursor, differing in final chemical treatment, while retaining similar conditions of heat treatment during carbonization.
Copolymer Mavilon Zoltek Company (Hungary) consisting of 93–94 wt% of acrylonitrile mers, 5–6 wt% of methyl acrylate mers, and approximately 1 wt% of sodium alilo-sulfonate mers was used to manufacture polymer nanofibers. The polyacrylonitrile (PAN) nanofibers were spun from the polymer solution using the electrospinning method. The details of the PAN-based nanofiber precursor are described elsewhere [
The following types of carbon nanofibers were prepared for further study: the as-received carbon nanofibers denoted as ECNF (electrospun carbon nanofibers) and carbon nanofibers after oxidation under reduced pressure in the air denoted as ECNFV.
To characterize the morphology of the carbon nanofibers and their surface, a scanning electron microscope (SEM; Nova Nanos 200, FEI COMPANY EUROPE) was used. SEM microphotographs and image analysis software (ImageJ 1.50b) were used to determine the nanofiber diameters as an average diameter of 30 measurements and the porosity of samples in the form of mats. The surface chemical properties were estimated using water contact angle (
Electrical resistance measurements of the carbon nanofiber mats were conducted using a two contact probe (Metex multimeter, model M-3660D). The changes in the resistance of the samples in the temperature range from -190°C to +50°C, in the air atmosphere, were registered. Two copper wire electrodes were fixed on the surface samples (
A FEI Tecnai TF20 X-TWIN high-resolution transmission electron microscope was used to examine the microstructural features of carbon samples.
The Raman spectroscopy measurements were made using a Renishaw inVia Raman microspectrometer in the reflection mode with 50x objective magnifications using 442 nm and 514.5 nm laser lines as the excitation sources. The spectra were obtained with 5% of the laser beam power and with exposure time equal to 10 s. The spectra were collected from the sample with ca. 1-3 mW of the laser beam power. Each sample was analyzed in four different areas to obtain averaged results. The spectra obtained in the range of 100–3200 cm-1 with an argon laser wavelength 514.5 nm were analyzed using Fityk 0.8.0 software [
The deconvolution of the spectra was made using the Pseudo-Voigt function. Deconvolution allowed distinguishing the characteristic bands of the carbon structure. The parameters characterizing the carbon samples, i.e.,
An X-ray diffraction (XRD) study was carried out using an X’Pert Pro Philips X-ray diffractometer. The measurements were made using a copper radiation source lamp (CuK
Infrared (FT-IR) spectra were recorded using a Bio-Rad FTS 165 spectrometer, with a resolution of 2 cm-1, within the range of 4000–400 cm-1.
To determine potential genotoxicity of carbon nanofibers (ECNF, ECNFV), the normal human skin fibroblasts from cell line CCL-110 (American Type Culture Collection (ATCC)) were used. The cells were cultured in MEM medium at 37°C and 5% CO2. The crumbled carbon mats (7.5 mg/4 ml PBS, phosphate-buffered saline) were mixed by means of an ultrasonic probe (Palmer Instruments) for 2 min. Sample’s suspension (500
In addition, two experiments were carried out, in which cell cultures containing both types of nanofibers were exposed to radiation—1 Gy for 1 h and 24 h. In normal cells irradiated with the 1 Gy dose, the damage caused by radiation should be repaired within 24 hours of incubation. Comparison of T-DNA values in such planned experiments, in addition to data related to genotoxicity and cytotoxicity, may provide information on the destruction of repair processes in cells contacted with carbon nanofibers.
The suspension of
The data were presented as mean values and standard error. The statistical analysis was performed using the Student
In order to assess the morphology and viability of the human osteoblast-like MG63 cells (European Collection of Cell Cultures, Salisbury, UK) cultured on two types of nanofibers, live cell imaging employing a double staining of cells with cell-permeable calcein AM (marker of viable cells) and propidium iodide (marker of dead cells) dyes was performed. The fragmented carbon mats (7.5 mg/4 ml PBS) were mixed by means of an ultrasonic probe (Palmer Instruments) for 2 min. Sample’s suspension (500
Parameters characterizing the surface properties and microstructure of carbon samples in the form of mats are presented in Table
Characterization of carbon nanofiber mats.
Sample | Nanofiber diameter (nm) | Material porosity (%) | Wettability |
Electrical conductivity (kΩ/square) |
---|---|---|---|---|
ECNF | 2.21 | |||
ECNFV | 3.69 |
An average thickness of carbon mats was in the range of 40-60
SEM microphotographs of both types of carbon nanofibers are shown in Figure
SEM microphotographs of carbon nanofibers: (a) ECNF, (b, c) ECNF after fragmentation, (d) ECNFV, and (e, f) ECNFV after fragmentation.
As can be seen from the microphotographs, both types of nanofibers are clearly different. The surface of the ECNF is smooth, and their diameter is uniform along their length. In our experiments, the carbon nanofibers were oxidized under polythermal conditions, i.e., from the RT to 800°C. The nanofibers consist of small crystallites connected by intercrystallite boundaries (see further XRD and Raman spectroscopy studies). Such a structure of the nanofibers makes the boundaries lower resistant to oxidation, and the images of the surface topography of these nanofibers after oxidation are a consequence of their diversified microstructure (Figure
High-resolution TEM images of both types of carbon nanofibers are shown in Figure
High-resolution TEM images of carbon nanofibers: (a) ECNF, (b) longitudinal section of ECNF, (c) selected-area electron diffraction patterns (SAED) of ECNF, (d) ECNFV, (e) longitudinal section of ECNFV, and (f) ECNFV-SAED.
The images show the longitudinal sections of ECNF (b) and ECNFV (e), as well as their selected-area electron diffraction patterns (SAED). The ECNF nanofibers represent homogeneous turbostratic carbon crystallites, randomly distributed along the nanofiber length (Figure
The X-ray diffraction patterns of carbon samples with deconvolution of (002) peaks are shown in Figure
XRD patterns of carbon nanofibers and deconvoluted (002) peak of ECNF into three bands and ECNFV into two bands.
The diffractograms show single broad and weak peaks at 23-26°
The microstructural parameter,
Crystallographic data of carbon phases determined from X-ray diffractograms.
ECNF | ECNFV | ||||
---|---|---|---|---|---|
Carbon phase 1 | Carbon phase 2 | Carbon phase 3 | Carbon phase 1 | Carbon phase 2 | |
25.272 | 21,725 | 18.692 | 25.403 | 21.635 | |
0.352 | 0.409 | 0.474 | 0.350 | 0.410 | |
1.48 | 1.84 | 4.33 | 1.44 | 1.50 | |
Carbon phase fraction (%) | 78.31 | 19.34 | 2.35 | 82.2 | 17.8 |
The (002) peaks after deconvolution indicate that the ECNF sample consists of three carbon phases, and the ECNFV sample contains two carbon phases differing in structural ordering (Figure
The XRD analysis confirms a turbostratic structure of carbon nanofibers composed of small crystallites connected by intercrystallite boundaries containing also other carbon elements, i.e., hydrogen, nitrogen, and oxygen. Such intercrystallite phases are characterized by a less ordered structure than crystallites. It can therefore be expected that the carbon phases with a less-ordered structure, such as those forming the boundaries between crystallites in the carbon nanofibers, are more prone to the oxidation than a better organized carbon structure, e.g., the crystallites.
FTIR spectra of both nanofibers in Figure
FTIR spectra of carbon nanofibers.
The as-received carbon nanofibers (ECNF) are characterized by low degree of structural ordering, typical for turbostratic structure of carbon materials [
The spectrum of the as-received carbon nanofibers contains broad overlapping bands between 950 cm-1 and 1600 cm-1, coinciding with the band at 1574 cm-1 attributed to C=C vibration bonds of the graphene rings. The broad band is brought about by the presence of the single bonds between carbon and oxygen, hydrogen, and nitrogen. Carbon nanofibers were obtained from the electrospun PAN nanofiber precursor. Due to the stabilization process of the PAN nanofibers which took place in the air atmosphere, carbon nanofibers heat-treated at 1000°C contain some amount of oxygen, nitrogen, and hydrogen. Nitrogen and hydrogen in the carbon nanofiber structure are a residue of the PAN nanofiber precursor, while oxygen, in a small amount, is a consequence of the stabilization process of the PAN nanofibers. Both spectra contain weak bands at 2851 cm-1 and 2919 cm-1, which correspond to the stretching vibrations of the C-H groups. The aromatic and aliphatic CH groups are usually found in the low-carbonized PAN-based carbon fibers (to 1000°C). The presence of hydrogen is also confirmed by the analysis of the Raman spectra described in the further part of the work. The spectrum of the oxidized nanofibers indicates that such a treatment, involving the removal of amorphous carbon, results in the formation of gaseous products and does not lead to the formation of additional chemical groups with oxygen on the nanofiber surface, as is the case of the oxidation of carbon nanofibers with liquid oxidants [
Changes in the resistance of carbon samples as a function of temperature are shown in Figure
Relative resistance changes of carbon nanofibers in function of temperature.
For both samples, the curves showing the resistance changes are similar; i.e., with the temperature increase, the resistance decreases; this is behavior of materials characterized by semiconductor-like properties. A higher RT surface resistivity (
Further differences in the structure of both types of carbon nanofibers were obtained by analysis of the Raman spectra shown in Figure
Raman spectra of carbon nanofibers.
Parameters determined from deconvoluted Raman spectra of ECNF and ECNFV.
Sample | D1 band position (cm-1) | G band position (cm-1) | D3 band position (cm-1) | D4 band position (cm-1) | 2D band position (cm-1) | ||
---|---|---|---|---|---|---|---|
ECNF | 1353.5 | 1595.2 | 1516.8 | 1129.8 | 2786.4 | 4.4 | 4.28 |
ECNFV | 1352.7 | 1588.3 | 1511.1 | 1125.6 | 2875.9 | 7.1 | 2.69 |
Raman spectra reveal distinct differences in the structure of both nanofibers. The intensity ratio,
Deconvoluted Raman spectra of the first-order bands of two types of nanofibers.
The spectrum of ECNF contains peaks related to differently ordered carbon phases centered at 1129.8 cm-1 (D4 band), 1353.5 cm-1 (D1 band), and 1595.2 cm-1 (G band). The poorly organized carbon materials show also band at 1516.8 cm-1 (D3) assigned to defects outside the aromatic layers [
The
The spectra of the nanofibers also show second-order Raman peaks, known as the 2D bands, in the range from about 2500 cm-1 to 3100 cm-1 which can be used to characterize the structure of carbon materials and their susceptibility to graphitization (Figure
Genotoxicity of the carbon nanofibers was assessed by means of the comet test, in terms of the tail DNA, and the results are collected in Tables
Genotoxicity of samples after 1-hour and 24-hour incubation.
Samples | Control fibroblasts | ECNF | ECNFV |
---|---|---|---|
T-DNA | |||
Student |
— | 0.01 | 0.67 |
T-DNA | |||
Student |
— | 0.02 | 0.56 |
Genotoxicity of samples after 1-hour and 24-hour incubation and after 1 Gy dose of X-ray irradiation.
Samples | Control fibroblasts | ECNF | ECNFV |
---|---|---|---|
T-DNA | |||
Student |
— | 0.03 | 0.34 |
T-DNA | |||
Student |
— | 0.01 | 0.23 |
Results obtained for fibroblasts treated for 1 h with ECNF, without and with X-ray exposure, showed a significantly higher level of DNA damage for both nonirradiated (
Microphotographs of MG63 cell morphologies in contact with two types of carbon nanofibers and control sample are shown in Figure
Fluorescence staining of MG 63 cells using calcein AM (green cells, viable cells) and propidium iodide (red cells, dead cells) after 24 h incubation in contact with the control sample (PS culture dish) (a), ECNF (b), and ECNFV (c).
Analyzing the morphology of MG63 cells in contact with the nanofibers’ significant differences in their behavior depending on the type of nanofibers can be observed. In case of the ECNF (Figure
Number of dead fibroblast cells in contact with control, ECNF, and ECNFV samples.
The results of a nanofiber study in contact with both osteoblast-like cells and fibroblasts indicate the negative effect of ECNF nanofibers on the cellular response, suggesting a certain cytotoxic effect of this material in the studied period of time.
The study showed that carbon nanofibers after the carbonization process have unfavorable characteristics as a biomaterial for biological use. Although carbon samples in the form of nanofibrous mat perform many advantageous properties for medical use including conductible structures for tissue engineering, the as-received samples assessed in genotoxicity and cytotoxicity tests behaved like toxic materials. Oxidation in the air is a simple way of modifying the biological properties of carbon nanofibers without deteriorating other physical and chemical properties important for use in medicine. Under the controlled surface treatment in air of nanofibers, significant changes in their surface morphology were observed. Raman spectroscopy has shown that the carbon structure is composed of several phases that vary in the degree of crystallinity. Raman spectra revealed that due to surface modification, the intensity of the (D) band associated with the less ordered carbon phase decreased, which resulted from the greater susceptibility of this phase to oxidation. For this reason, in the ECNFV sample the intensity of the G band increased. It is also worth mentioning that the surfaces of these nanofibers (Figure
The surface properties of carbon mats were studied by measuring the water contact angle. As is apparent from Table
The applied oxidation treatment caused a slight increase in the resistivity of nanofibers, but they were still conductive materials. In addition, their microstructural parameters (porosity, nanofiber diameter) in the form of mats exhibited similar parameters as carbon mats prior to the oxidative modification. Thus, nontoxic oxidized mats can be the subject of further study for applications as electrode elements for electrical stimulation, as well as substrate elements of electrically activated cell cultures.
Carbon nanofibers in the form of mats were obtained from the electrospun PAN nanofiber precursor. The stabilized PAN nanofibers were carbonized up to 1000°C (ECNF) and additionally oxidized in air at 800°C under vacuum pressure (ECNFV). The final oxidized treatment enabled to decrease contact angles to provide a more hydrophilic character of the nanofibers’ surface. The treatment in air under reduced pressure significantly changed the surface morphology of ECNFV and decreased the carbon phase fraction containing disordered carbon crystallites. A genotoxicity study of both types of carbon nanofibers showed differences in comet assay tests. The T-DNA test revealed that the surface-oxidized carbon nanofibers were not genotoxic, whereas the as-received carbon nanofibers indicated the increase in DNA strand damage and the number of dead cells as compared to control. ECNFV introduced into the cell culture did not affect the repair processes in the cells contacting them. The results demonstrated the enhancement in biocompatibility of the surface-oxidized carbon nanofibers determined by the T-DNA tests. Due to the removal of a part of carbon phase from the near-surface region, the carbon nanofibers with the specific surface nanotopography were manufactured. Such nanofibers, thanks to their conductive properties, specific surface nanostructure, and biocompatibility, may be considered as promising substrates for electrical field stimulation regulating the behaviors of cells and as fibrous scaffolds for cell cultures. However, an assessment of the biological behavior of such a nanofiber requires further research on their biocompatibility including
The methods used to characterize the physical, chemical, structural, and biological properties of the samples and resulting data in the form of tables, figures, and images used to support the findings of this study are included within the article.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This work has been supported by the Polish National Science Center, project no: 2017/25/B/ST8/02602, and by the Laryngology Department, School of Medicine, Medical University of Silesia in Katowice (Statute Found no.: KNW-1-102/K/8/0 and no.: KNW-1-043/K/7/0).