Abstract
Modification of the surfaces of polymeric scaffolds is often required to make the material suitable for specific tissue engineering applications. Physico-chemical properties of scaffolds can be altered using various methods, such as plasma treatment, laser processing, chemical modifications, grafting with nanoparticles, or surface coating. In this paper physico-chemical modification of polycaprolactone (PCL) surface fibers was performed by exposing PCL samples to simultaneous soft X-ray/extreme ultraviolet (SXR/EUV) radiation and low-temperature, SXR/EUV-induced, nitrogen, and oxygen plasmas. The physical and chemical changes on modified PCL surfaces were examined using a scanning electron microscope and X-ray photoelectron spectroscopy, respectively. The effects of physico-chemical scaffold surface changes were verified with biological tests, i.e., MTT assay and immunofluorescence on murine osteoblast cell line (7F2). It was found that exposure of scaffolds to ionizing radiation and low-temperature plasmas induced strong chemical changes on their surface, i.e., appearance of various new chemical groups. Also, smoothing of the surface of PCL fibers, i.e., disappearance or significant reduction of the size of micropores on their fibers was also observed. Increased viability and adhesion of 7F2 osteoblasts on modified PCL samples after 24 h cell culture compared to non-treated PCL was also confirmed.
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Introduction
Common and well-known life-saving treatment methods for damaged tissues or organs, such as tissue transplants (auto-, allografts) or the use of artificial organs or implants, are sometimes difficult to use. This is primarily due to the poor supply of tissues and organs, the possibility that the transplant is discarded by the body, as well as its lack of integration into the recipient’s tissue, or the limited lifespan of the implant. As support to these traditional methods of treating damaged tissues comes an effective alternative method of treatment in which tissue engineering products are used, the so-called tissue scaffolds, i.e., extracellular matrix (ECM) substitutes [1]. Tissue scaffolds are microporous biomaterials with a very well-developed three-dimensional structure. These materials are to provide adequate structural stability for the development of tissues or organs and conditions that are as similar as possible to those that naturally occur in the body, i.e., stimulate cells to proliferate and differentiate as the functional tissue matures. Most of the research conducted currently focuses on the design and fabrication of scaffolds using various technologies [2, 3]. The cell scaffold must have appropriate physico-chemical and mechanical properties adapted to the parameters of the tissue replaced [4, 5]. The degradation time of such a biomaterial is also of great importance. It should be well matched to the duration of the regeneration process to ensure optimal conditions throughout the healing period [6,7,8]. Therefore, the most attractive candidates for the construction of scaffolds seem to be biodegradable and biocompatible polymeric materials. Scaffolds made of natural polymers, such as collagen, elastin, chitosan, hyaluronic acid, have quite poor mechanical strength. As for the synthetic polymers, scaffolds made of polyglycolide (PGA), polycaprolactone (PCL), polylactide (PLA), or poly(lactide-co-glycolide) copolymer (PLGA) are the ones that are examined the most frequently. Among them, PCL deserves special attention. It is a semi-crystalline linear polymer from the group of aliphatic polyesters, approved by the Food and Drug Administration (FDA) for tissue engineering and regenerative medical applications. This material has quite good plasticity and mechanical strength. It biodegrades in a body to nontoxic products. However, this polymer has poor hydrophilicity as it lacks bioactive surface functional groups that promote cellular adhesion, migration, and proliferation. Therefore, it is very important to properly modify PCL to increase its bioactivity, and thus its potential clinical usefulness. Many techniques of modification of pure PCL scaffolds have been used to improve cell compatibility. These include chemical methods such as hydrolysis or aminolysis [9, 10], laser texturing [11, 12], coating with natural polymers, such as collagen, gelatine, chitosan, etc. [13,14,15], composing or grafting with micro- or nanoparticles (hydroxyapatite (HA), bioglasses (BGs), carbon nanotubes (CNs)) [13, 16,17,18], and finally, the low-temperature plasma treatment which seems to be one of the most versatile and effective technique of bioactivation of polymeric scaffolds [19,20,21]. The exposure of polymeric scaffolds to cold plasma induces various changes in their surface. These include cross-linking, texturing, surface ablation, functional polar group or atom incorporation, etc. As a consequence, the hydrophobic character of the scaffolds may change to a hydrophilic one which directly leads to the improvement of their cytocompatibility. Various studies have been reported on the effect of low-temperature plasma treatment on the physico-chemical properties of scaffolds and the cellular responses they induce. For example, Sankar et. al. [20] treated PCL electrospun fibrous scaffolds with argon and nitrogen plasma and observed that such an approach to modification rendered the surface hydrophilic and led to the appearance of surface functional groups containing oxygen and nitrogen atoms. This in turn had a positive impact on human mesenchymal stem cells (hMSC) spreading, elongation, and proliferation. Interestingly, in this study, the differentiation ability of hMSC toward osteoblast lineage cultured on fibers modified with this technique was also observed. In another study, which is worth mentioning, Prabhakaran et al. [22] examined the cytocompatibility of modified PCL fibrous scaffolds. In this study, the authors tested and compared the cytocompatibility of PCL fiber surfaces modified with plasma (in this case, air plasma) and biocomposite, i.e., a scaffold made of a mixture of PCL and a natural polymer—collagen, commonly added in this type of research. Surprisingly, it turned out that seeded Schwann cells (SC) (glial cells of the nervous system) definitely preferred plasma-modified scaffolds, on which authors observed higher SC proliferation, over PCL/collagen fibers. The results showed the strong potential of a relatively simple plasma modification technique as a valuable method for improving the affinity of polymeric scaffold for cells and, thus, the potential of substrates modified with this method for peripheral nerve regeneration. Other applications of low-temperature plasma in the modification of scaffolds include those whose purpose is to functionalize the surface in order to strengthen their adhesive properties, which are particularly important with regard to matching the specific properties of the coatings to the specific clinical use of regenerative medicine [23,24,25].
This paper aims to present the analysis of the results of physico-chemical changes on the surface of PCL scaffolds exposed to soft X-ray/extreme ultraviolet (SXR/EUV) radiation and low-temperature nitrogen and oxygen plasma. Additionally, the biological response of murine osteoblasts cell line (7F2) on modified scaffolds was examined.
The advantage of the technique presented over standard plasma generators is that it enables simultaneous operation of low-temperature plasma and SXR/EUV radiation, which can be (in some range of parameters) individually adjusted. Plasma created using this technique differs significantly from low-temperature plasmas created using standard methods. Standard plasma generators are usually based on electrical discharge and work in a stationary regime. They deliver plasmas of low-temperature (Te ~ 1 eV) and very low electron density (ne ~ 1011–1013 cm−3) [26]. Even for plasmas created under atmospheric pressure, the electron density is comparatively low and does not exceed these values significantly. In this case, plasmas created by photoionization using SXR/EUV pulses can have electron density several orders of magnitude higher and can exceed ~ 1017 cm−3 [27], which significantly increases the effectiveness of physico-chemical processes connected to modification of the surfaces.
The technique that was used to “bio-functionalize” electrospun PCL scaffolds was presented and described in detail for the first time in our previous work [28]. However, it is worth emphasizing once again that the setup used, which was based on a laser-produced plasma SXR/EUV source driven by a 10 Hz Nd:YAG laser, had no optics for focusing the radiation emitted by the laser-produced plasma. This arrangement allowed the modification of the surface with a diameter of about 10 mm.
In our recent works, the system was used in which the radiation emitted from the laser-plasma was focused by an ellipsoidal collector [29,30,31]. However, the weak point of this system was that in this case the modification area was relatively small, and its diameter was about 2 mm. Also, another problem that appeared was the fact that as a result of the interaction of EUV radiation of, in that case, a fluence of ~ 60 mJ/cm2, with the polymer, the collector mirrors were degrading due to the deposition of ablation products on them. This, in turn, decreased its reflection coefficient in the EUV range. Therefore, it was decided to redevelop the system and not use EUV radiation focusing optics, and then optimize it in such a way that the system can be used for physico-chemical modifications of polymer surfaces.
In this paper, the effect of SXR/EUV radiation and SXR/EUV-induced, oxygen- and nitrogen-based plasmas on chemical surface changes was analyzed using X-ray photoelectron spectroscopy (XPS). Changes in surface morphology were also analyzed using scanning electron microscopy (SEM). The positive results of physico-chemical surface modification were confirmed with biological tests, i.e., MTT assay and immunofluorescence on murine osteoblast cell line (7F2).
Materials and methods
Modification of PCL scaffolds
In the experiment, commercial PCL scaffolds (3D Biotek, NJ, USA), with a diameter of 5 mm, a fiber diameter of 300 µm and a pore size of 300 µm, were modified. According to the manufacturer’s information, scaffolds were terminally sterilized by gamma radiation. The modifications were performed using a 10 Hz laser-plasma SXR/EUV source which was presented for the first time and described in detail in our previous work [28]. This source is based on a double stream gas puff target irradiated with ~ 8 ns/2.1 J Nd:YAG laser pulses (NL 129, EKSPLA, Lithuania). The target was created as a result of a pulsed injection of a Xe gas into a hollow stream of nitrogen or oxygen gas (Xe/N2 or Xe/O2 target) using an electromagnetic valve system equipped with a double nozzle setup, synchronized with the laser system. The laser beam focused on the stream of Xe created high-temperature plasma emitting intense SXR/EUV radiation which ionized surrounding nitrogen or oxygen gas. As a consequence, high-temperature Xe plasma was surrounded by low-temperature N2- or O2-plasmas, induced by intense SXR/EUV radiation pulses.
In the experiment, the PCL scaffold sample surface was exposed to 150 pulses of low-temperature plasmas: oxygen, or nitrogen, and simultaneously to the part of SXR/EUV radiation that was not absorbed by the gases. The samples were mounted during exposure on a stand at a distance of ~ 11 mm from the nozzle setup. The experimental arrangement is presented in Fig. 1.
a schematic presentation of the experimental setup for PCL scaffold samples modification with SXR/EUV radiation and low-temperature O2 and N2 plasma, b the most intense part of the spectrum of SXR/EUV radiation emitted from Xe plasma: created in a double stream gas-puff target scheme, with three buffer gases: He, N2 and O2.
Surface characterization of PCL scaffolds
Morphology (SEM)
The morphology of the surfaces of non-treated and treated PCL scaffolds was examined using an SEM (Quanta FEG250, FEI, Oregon, USA) equipped with Everhart–Thornley detector (ETD) working in a high vacuum mode. All the samples studied were firstly coated with a 10 nm thick layer of gold using a sputter coater (Emitech K550X, Quorum Technologies Ltd., UK). During SEM scan acquisition the acceleration voltage was 10 kV and the working distance was ~ 10 mm.
Chemical analysis (XPS)
As a result of the modification, the chemical changes that occurred on the fibers of PCL scaffolds were examined using X-ray photoelectron spectroscopy (XPS). The spectrometer (Prevac, Poland), equipped with SCIENTA R3000 analyser (VG Scienta, Uppsala, Sweden) and X-ray lamp with Al Kα anode (Prevac, Rogów, Poland) was used. During the measurements, the pressure in the ultra-high vacuum analysis chamber of the XPS system was about 3·10−9 mbar. The high-resolution spectra in the narrow ranges of biding energy with a 40 meV step and pass energy of 100 meV for each of the bands: C1s (282–293 eV), N1s (395.5–403.5 eV), and O1s (528–538 eV) were recorded. The peaks related to the C1s, N1s, and O1s bands were fitted using CasaXPS software. The background of Shirley type and Gaussian–Lorentzian (G–L) line shape (G–L 50 for C1s, G–L 60 for N1s, and G–L 55 for O1s) were fitted for all these bands. All XPS spectra collected were shifted in such a way that the maximum of the C–C peak was at 285 eV.
Biological studies
Cell culture
Murine osteoblast cell line (7F2) was purchased from ATCC CRL12557 (ATCC, USA). 7F2 cells (passages 25–27) were cultured in αMEM (Gibco®, USA) together with 10% fetal bovine serum (ATCC, USA) and 1% antibiotics solution containing penicillin (10 000 U/ml) and streptomycin (10 000 µg/ml) (Gibco®, USA). Cells were maintained in humidified air containing 5% CO2 at 37 °C, in sterile cell culture flasks (Greiner, Germany) and passaged every 3–4 days after trypsinization with trypsin/EDTA (Gibco®, USA).
MTT cell viability assay
Cell viability using MTT assay was carried out to evaluate the effect of unmodified and modified substratum on osteoblastic cells. This assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity of viable 7F2 cells, into an insoluble colored formazan product, which can be measured spectrophotometrically after dissolving in DMSO [32].
For the test, the PCL scaffolds were sterilized with ethanol, rinsed with sterile PBS, and moved to the complete cell culture medium in a 96-well plate (Nunc, Denmark) for 48 h. 2.5 × 104 cells per well were seeded into three 96-well plates on previously prepared scaffolds and incubated for 48 h at 37 °C in a fully humidified atmosphere of 5% CO2.
To evaluate cell survival, 10 μl of MTT solution (5 mg/ml in PBS) (Invitrogen™, USA) was added to each well and incubated for 3 h in darkness at 37 °C. Then the media was replaced with 150 μl of DMSO, and complete solubilization of formazan crystals was achieved by repeated pipetting of the solution. Absorbance was then determined at 540 nm wavelength using plate reader Molecular Device SpectraMaxI3x. Each condition was assayed in 4 wells and repeated 3 times. The cytotoxic effect of PCL surface modifications was expressed as the relative viability (% control) and calculated as follows:
where absexp is an experimental absorbance, absb is a background absorbance and abscon is an absorbance of untreated controls.
Staining and fluorescence microscope imaging
Cytoskeleton investigations were done with F-actin staining. Cells were seeded at 1 × 104 cells per well in a 96-well plate on each PCL substrate. After culturing for 24 h, cells were washed with PBS three times and fixed with 4% paraformaldehyde in PBS for 20 min. Fixed cells were rinsed with PBS and permeabilized for 5 min with 0.5% Triton X-100 (Sigma-Aldrich, USA) in PBS. After rinsing with PBS, cells were incubated for 1 h at room temperature with 1% bovine serum albumin (BSA) (Sigma-Aldrich, USA) in PBS/0.01%Tritonx100 to minimize non-specific protein–protein interactions. After rinsing three times, cells were incubated for 30 min at room temperature with Phalloidin-Alexa Fluor 488 (Invitrogen, USA) (5 µl of stock solution per 200 µl PBS). After that, nuclear counterstain with Hoeschst 33342 (4 µl of stock solution 20 mM per ml) (Thermo Fisher Scientific Inc., USA) was performed. Experimental cell images (magnification × 20, NA = 0.8) were performed with the Laser Scanning Confocal System LSM 700 microscope Zeiss Axio Observer.Z1 (Carl Zeiss AG, Germany) with appropriate filters.
Statistical analysis
MTT assay experiments were carried out in 4 parallel attempts and repeated three times, and the data was normalized for comparison. The results are expressed as mean values ± SD. Analysis of Variance (ANOVA) was performed in order to estimate the significance of PCL surface modifications on the viability of investigated cells. To estimate the significant differences among samples, the post-hoc test of Tukey was performed, in which the significance level was set at *p < 0.05.
Results and discussion
Morphological characterization of modified PCL scaffolds
Simultaneous exposure of polymers to SXR/EUV irradiation and low-temperature plasma can result in various morphological changes on their surfaces. The nature of these changes, specifically the shape of the structures obtained, and their orientation or height, are closely related to the SXR/EUV fluence in the single pulse, the number of pulses applied, and the duration of the pulse. It also depends on the physico-chemical properties of polymers such as: their chemical structure (i.e. phase transition temperature), ablation threshold, degree of crystallinity, sensitivity or heat capacity, etc. The effect of 150 pulses of SXR/EUV radiation and low-temperature oxygen and nitrogen plasma on the morphology of PCL scaffold fibers was examined using SEM. Figure 2 shows the surfaces of PCL scaffold fibers unmodified and modified with oxygen and nitrogen plasma, at three various magnifications (× 200, × 800, × 1400). As can be seen in the figure, unmodified PCL scaffold fibers are characterized by a large number of micropores of irregular shapes and various sizes ranging from ~ 1 to ~ 30 μm. It is also clearly visible that the tops of all fibers from the first upper fiber layer seem to be truncated, i.e., top is flattened, smooth, and has no pores. It is possible that during the production process, right after the scaffolds were fabricated, when they were not yet sufficiently hardened, placing them on a smooth surface resulted in imprinting it and, as a result, flattening their upper part. However, the real mechanism of this flattening is not clear and unknown to the authors. Figure 2b and c shows SEM images of PCL scaffolds exposed to simultaneous laser plasma and low-temperature oxygen or nitrogen plasma. It can be seen that for both cases of modification, these factors caused most likely surface melting of the fibers together with their strong surface ablation, and, consequently, led to their strong smoothing. The micropores visible on the unmodified PCL fibers disappeared on the treated samples (the effect was visible primarily for the 1st and ~ 2nd layer of fibers in depth) or their size was radically reduced (in particular: from ~ 2nd to ~ 3rd layer of fibers in depth). The imprint (flattening of the top of the fibers), which was characteristic and visible for the unmodified sample, also became indistinguishable and invisible on the first layer of fibers. It is also worth noting that fibers that were located further from the plasma (deeper in the scaffold, starting from ~ 4th layer of fibers from the surface) or which were shadowed by outer fibers (above them) did not ablate and melted on the surface so much and still had a porous structure. However, the pore size, shape and number on the surface decreased slightly compared to the surface of unmodified PCL fibers.
The effect of smoothing the surface of organic polymers in the form of foils treated with low-temperature plasma or pulsed laser radiation was also observed by other authors in their studies [33, 34]. As for polymers with a three-dimensional structure, in particular micro- and nano-fibrous electrospun scaffolds, the ablation effect manifested itself as the disappearance of the thinnest nanofibers from the scaffold structure [35, 36]. We observed such a phenomenon also in our previous research [28], in which an electrospun PCL mat was exposed to the same factors, i.e. laser-plasma and low-temperature plasma. In that case, we noticed that the thinnest fibers with diameters below ~ 0.4 µm were ablated and fiber diameter distributions became more symmetric with slightly higher average diameters.
Chemical analysis of non-modified and modified PCL scaffolds
Due to the fact that ionizing radiation in the SXR/EUV range that is emitted from laser plasma is strongly absorbed in the near-surface layer of the irradiated material, i.e., up to ~ 1 µm deep into the material, all chemical changes that appear as a result of this interaction with polymer samples can be registered only by using X-ray photoelectron spectroscopy (XPS). The photon energies in that range are so high that they easily break the bonds between atoms of the elements that are part of the polymer structure and as an effect lead to a radical chemical decomposition on their surface. Additionally, simultaneous exposure of polymers to low-temperature plasma which is induced by SXR/EUV radiation, may also contribute to the incorporation of additional atoms or functional groups that were not previously present in the polymer.
We have already observed such a phenomenon for many EUV-modified polymers described in our recent works [28,29,30,31].
In this section, registered and modeled high-resolution XPS spectra of PCL scaffolds exposed to simultaneous SXR/EUV radiation and low-temperature nitrogen and oxygen plasma are presented. First, the reference material, i.e., non-modified PCL foil, was analyzed. For the registered C1s and O1s bands of the reference material, the model of peaks corresponding to its chemical structure, shown in Fig. 3, was developed.
Thus, four peaks were modeled in the C1s band: C–C (C1) (carbon atom marked as 1—Fig. 3) at 285.0 eV (FWHM 1.3–1.4 eV), C*–C=O(–O) (C2) (carbon atom marked as 2—Fig. 3; second-order chemical shift) at 285.5 eV (FWHM 1.4–1.5 eV), C–O (C3) (carbon atom marked as 3—Fig. 3) at 286.6 eV (FWHM 1.3–1.4 eV) and C=O(–O) (C4) (carbon atom marked as 4—Fig. 3) at 289.0–289.1 eV (FWHM 1.2–1.4 eV) and 3 peaks were modeled in O1s band: O=C (O1) at 531.7–531.8 eV (FWHM 1.5–1.6 eV), O–C (O2) at 533.0–533.1 eV (FWHM 1.5–17 eV) and O–Hads. (O3) at 534.3–534.4 eV (FWHM 2.3 eV)—Fig. 4.
The model developed appropriately describes the chemical structure of the PCL (Fig. 3), and is consistent with the results presented in the literature concerning pristine PCL [21]. This model was then used to analyze the spectra of the unmodified PCL scaffold. Figure 5 shows the modeled spectra of the C1s and O1s bands of this sample. As can be seen, additional 3 peaks have been added to the C1s band representing the following bonds: C=C (C5) at 284.3–284.5 eV (FWHM 1.3–1.4 eV), C–OH (C6) at 285.8–285.9 eV (FWHM 1.3 eV) and O–C–O (C8) at 288.2 eV (FWHM 1.3 eV).
For the O1s band, two additional peaks were added representing the groups: O=C–O (O4) at 530.7–530.8 eV (FWHM 1.5–1.7 eV) and OH (O5) at 532.2–532.3 eV (FWHM 1.7 eV). The presence of these additional peaks, i.e., C5, C6, C8, O4 and O5, indicating a change in the chemical structure of the unmodified PCL scaffold compared to reference PCL foil, may result from the fact that the scaffolds provided by 3D Biotek were sterilized. As described by the company in their specification, sterilization was carried out by exposing the polymers to gamma radiation. As we know, the energy of a single photon of gamma radiation is sufficiently high to induce permanent changes in the chemical structure of the scaffold. We registered these changes in the C1s and O1s bands exactly in the form of additional peaks. Then, based on the model developed for unmodified PCL, the samples modified with 150 pulses of SXR/EUV radiation and low-temperature oxygen and nitrogen plasmas were analyzed. All the peaks that were modeled, i.e., binding energy, FWHM, and atomic percentage, are given in Table 1. Thus, for the PCL scaffold modified with low-temperature oxygen plasma, two additional peaks were introduced into the C1s band: C–O–C (C7) at 287.4–287.5 eV (FWHM 1.4 eV) and C–O(–O)(–O) (C9) at 289.6–289.9 eV (FWHM 1.3–1.4 eV)—Fig. 6.
Figure 7 shows the XPS spectra of the PCL scaffold modified with SXR/EUV radiation and low-temperature nitrogen plasma.
In the C1s band, 5 additional peaks were introduced representing the following functional groups: as peak C6. As far as PCL_N2 is concerned, due to the similar chemical shift of the C–OH and C–N C–O–C (C7) at 287.4–287.5 eV (FWHM 1.4 eV), C–O(–O)(–O) (C9) at 289.6–289.9 eV (FWHM 1.3–1.4 eV), C–N/C–OH (C10) at 286.0 eV (FWHM 1.5 eV), N–C=O (C11) at 288.1 eV (FWHM 1.7 eV) and C–O(–O)(– N) (C12) at 289.0 eV (FWHM 1.3 eV). As can be seen, two functional groups have been assigned to the C10 peak. One of them: C–OH, occurs also for PCL unmodified samples and PCL treated with oxygen plasma, and has been marked groups, (in this case these groups cannot be treated as two separate peaks), we assumed that the peak at 286.0 eV may represent these two functional groups. Since in this case nitrogen atoms were incorporated into the scaffold structure, a new additional band—N1s—appeared in the spectrum of this sample. This band was modeled with 3 peaks representing the following groups: N–C/N=C (N1) at 399.3 eV (FWHM 2.1 eV), N–C=O (N2) at 400.1 eV (FWHM 1.8 eV) and N–x(N3) at 401.0 eV (FWHM 1.7 eV). The first two groups correspond to the C10 and C11 peaks in the C1s band, respectively, however, it is difficult to unambiguously interpret the N3 peak. Therefore, referring to our previous analysis of the spectra of polymers that contain nitrogen atoms in their structure (e.g., nylon, PU, Kapton) and based on literature, we assumed that this peak may represent a structure in which the nitrogen atom is bonded with a carbon atom, in the vicinity of which there are at least two oxygen atoms. In the O1s band in PCL_N2, one additional peak representing the group was introduced: N–C=O (O6) at 532.3 eV (FWHM 1.6 eV) corresponding to the C11 peak in the C1s band. A similar binding energy was assigned to the O6 peak as to the O5 peak which represents the OH group. However, the O5 peak was not found in PCL_N2.
Summarizing, exposure of PCL scaffolds to 150 pulses of SXR/EUV radiation and low-temperature oxygen and nitrogen plasma resulted in a strong chemical decomposition on the surface of their fibers. As can be seen in Table 2, PCL modification with oxygen plasma led to a slight decrease in the total oxygen content of ~ 1 at.% compared to the PCL unmodified and amounted to 23.80 at.%. However, despite this slight reduction in the oxygen content, the percentage content of OH groups increased significantly (up to 6.34 at.%), and new, additional chemical groups appeared on the PCL surface, containing carbon atoms bonded with one or three oxygen atoms in their structure. As for PCL scaffolds modified with low-temperature nitrogen plasma, the percentage content of nitrogen was as much as 14.15 at.%. Nitrogen atoms incorporated into the structure of PCL fibers replaced oxygen atoms, and the percentage content of oxygen decreased to 13.37 at.%, while the content of carbon remained similar, slightly lower compared to PCL unmodified and PCL treated with oxygen plasma, and was 72.48 at.%.
Cellular morphology and growth studies of 7F2 osteoblasts on modified PCL scaffolds
Surface modification of polymer scaffolds or other biomaterials used in tissue engineering or implantology may be crucial in terms of their biocompatibility and bioactivity [37, 38]. The introduction of alterations associated with surface chemical composition as well as surface morphology may be particularly important for cell behavior and increases their adhesion and proliferation [39, 40]. In this part, the effect of strong chemical and physical surface changes of PCL scaffolds, introduced using SXR/EUV radiation and low-temperature oxygen and nitrogen plasma, and presented in previous sections, was evaluated in terms of growth and morphological changes in 7F2 osteoblasts.
The viability of the 7F2 osteoblast cells on unmodified and modified PCL surfaces is shown in Fig. 8.
Cell viability of 7F2 cells cultured on 3D PCL samples for 48 h post-seeding: unmodified (control sample), O2 and N2 treated PCL. The cytotoxic effect of PCL surface modifications was expressed as the relative viability. Error bars represent the standard deviation of the mean. Results were considered significant when p* < 0.05.
Tests were carried out using MTT assay on 2 days after cell seeding on PCL samples. It was clearly visible that cell viability increased for samples modified using both types of plasma. Oxygen and nitrogen plasma treatment resulted in an increase in the cell viability to 116.3% ± 9.1 and 118.8% ± 12.4, respectively, compared to the unmodified PCL sample (viability 100% ± 12) after 48 h.
The cytoskeleton organization and proliferation of 7F2 osteoblasts cells on unmodified and modified PCL scaffolds are shown in Fig. 9. Cellular F-actin and DNA were stained for cytoskeleton and nuclei analysis. Fluorescence studies were presented using a laser-scanning confocal microscope. Tests were carried out 24 h post seeding. Actin cytoskeleton, an essential component involved in many cellular behaviors, has been reported to be significantly affected by surface topography. CLSM was used to examine the cell interaction with the surface of PCL. In general, actin filaments arranged in more closely packaged arrays in cells growing in 3D substrates were observed. However, better spread morphology with a well-organized actin cytoskeleton was observed in cells on the nitrogen-treated substrate (difference in stress fibers indicated with yellow arrows) providing better cell adhesion. The presented results are consistent with the data on viability. Strong proliferation in both conditions was observed after 24 h of cell culture. The 24-h cultivation of cells on the surfaces tested did not show significant differences in the number of cells.
Visualization of cytoskeleton organization of 7F2 cells cultured for 24 h on PCL scaffolds using immunofluorescent imaging: unmodified (control sample), O2 treated PCL and N2 treated PCL (from left to right). F-actin was stained with Alexa Fluor 488 conjugated Phalloidin (green). DNA was counterstained with Hoechst 33342 (blue). Yellow arrows indicate stress fibers. Scale bar 20 µm.
The effect of PCL plasma modification on various cellular properties to enlarge medical applications was reported in many papers. Yan et al. [41] indicate that generally plasma treatments (gas mixtures: N2 + H2, NH3 + O2, and Ar + O2) introduce polar groups onto the surfaces increasing their hydrophilicity. Furthermore, plasma-treated PCL nanofiber meshes (NFMs) showed a higher proliferation rate and improved cell adhesion of MC3T3-E1 osteoblasts compared with the untreated samples [41]. The effect of O2, NH3, or SO2 plasma treatments of PCL scaffold on HUVEC cells was shown by Recek et al. [42]. The viability and adhesion of HUVEC were the highest for both O2 and NH3 plasma-treated surfaces. Increased endothelialization of PCL was also observed in this condition. Similarly, the positive impact of Ar and N2 plasma treatment of the surface of the PCL scaffold on cell behavior was marked by Sankar et al. [20]. Enhanced hMSCs spreading, elongation, and proliferation, in comparison to the unmodified sample, were shown. Higher cell adhesion of human fibroblasts (HDFs) on O2 plasma-modified PCL nano-fibrous mat in comparison to unmodified and positive control was observed by Sharifi et al. [19].
The impact of treatment on cell viability and adhesion presented in this paper is in accordance with the literature data.
To explain cellular alteration in cell viability and morphology of 7F2 on SXR/EUV and plasma O2 or N2 modified PCL, the role of surface charge and the functional groups located on the polymer surface on cell behavior should be discussed [37, 38]. Numerous studies show that positively charged surfaces increase cell adhesion [43], however negatively charged surfaces control protein adsorption via binding integrin, producing specificity and regulating cellular adhesion to the surface, too [44]. Treatment with oxygen plasma provides additional OH or COOH functional groups enhancing hydrophilicity and promoting binding sites for integrin receptors on cells [45]. Abarrategi et al. [46] indicated an increase in cell adhesion depending on the charged functional group present on the polymer surface as follows: OH > COOH=NH2 > CH3. In Martins’s work [35], electrospun polycaprolactone nanofiber meshes (NFMs) were treated by radio-frequency plasma using Ar or O2 gases. They showed an increase of the oxygen-containing groups (such as: –OH and –C–O) at the plasma-treated surfaces.
According to the obtained XPS results (Tables 1, 2) PCL scaffold surface, treated with low-temperature oxygen plasma, were enriched with a high number of OH (O5) and C–OH (C6) groups that were not present on N2 plasma-treated surfaces. Additionally, the increase in the level of a few polar functional groups was observed compared to the unmodified sample (e.g., OH (O5)—increase from 1.73 to 6.34 at.%; OHads (O3)—from 1.03 to 2.10 at.%; O–C–O (C8)—from 1.95 to 2.40 at.% and C–OH (C6)—from 0.81 to 6.34 at.%). N2 plasma treatment of PCL introduces functional groups that are not present on O2 treated surface and unmodified sample (mainly C–N/C–OH (C10) and N–C=O (C11), C–OON (C12), N–C/N=C (N1), N–C=O (N2) and N–x(N3)). These groups are oxygen and/or nitrogen rich that possess free electron pairs. Therefore free electron pairs provide electrostatic repulsion of the negatively charged species. As examined samples were immersed in a polar medium (cell culturing media) containing electrolytes, polar groups might dissociate and a local negative charge appears. Based on the XPS results, polar groups appear due to plasma modification of PCL both with N2 and O2 plasma assuming a negatively charged surface of the polymer. Thus plasma modification of PCL both with N2 and O2 provides a negatively charged surface of the polymer. Thus plasma modification of PCL both with N2 and O2 provides a negatively charged surface of the polymer. It was mentioned that negatively charged surfaces enhance 7F2 cell adhesion providing higher cell viability and proliferation on desired PCL scaffold based on obtained results (Figs. 8 and 9).
To summarize, increased viability of 7F2 was achieved on both oxygen and nitrogen-modified surfaces of PCL compared to the unmodified (control sample). Increased cell adhesion on surfaces modified with plasma, especially nitrogen plasma, is associated with higher proliferation of 7F2 osteoblasts.
Conclusions
In this paper, the effect of SXR/EUV radiation and low-temperature, SXR/EUV-induced, oxygen and nitrogen plasma on physico-chemical properties and cytocompatibility of PCL scaffold surfaces was presented. It was found that exposure of the scaffolds to ionizing radiation and low-temperature plasmas induced strong morphological changes on their surface, i.e., most likely near-surface layer melting of the fibers together with their strong surface ablation, manifesting as disappearance or significant reduction of the size of micropores on their fibers compared to unmodified PCL scaffold surface. Simultaneous treatment using SXR/EUV radiation and two types of low-temperature plasmas led to strong changes in the chemical composition of the PCL scaffolds surface, too. It was observed that new various chemical groups appeared on the scaffold fibers, such as C*–O–C, C*–O(–O)(–O), C*–N/C*–OH, N–C*=O, C*–OON, N*–C/N*=C, N*–C–O, N*–x and N–C=O*. PCL scaffold modification with oxygen plasma led to a slight decrease of the total oxygen content to ~ 1 at.% (but a strong increase of percentage content of OH groups was observed as well as the appearance of the new functional groups containing carbon atoms bonded with one or three oxygen atoms in their structure), compared to unmodified PCL sample. As for PCL scaffolds modified with nitrogen plasma, the incorporation of up to 14.2 at.% nitrogen was observed. Moreover, the positive impact of these physico-chemical surface modifications on the viability and adhesion of the murine osteoblast cell line (7F2) was confirmed. An increase in viability of 7F2 was achieved on both oxygen and nitrogen plasma-modified surfaces compared to the unmodified scaffold 48 h post seeding. Increased cell adhesion on surfaces modified with plasma was associated with higher proliferation of 7F2 osteoblasts.
The results presented in this paper indicate the high potential of the modification technique of polymers based on a laser plasma SXR/EUV source and low-temperature, SXR/EUV-induced plasmas. However, further detailed research is required, especially in terms of multi-day observations of cell cultures with various cell lines as well as examination of bacteria interaction on such modified surfaces.
Data availability
Not applicable.
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Acknowledgements
This work was supported by the National Science Centre, Poland, grant agreement no. UMO-2019/03/X/ST5/01643 and no. UMO-2020/39/B/ST2/00509.
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Czwartos, J., Nowak-Stępniowska, A., Budner, B. et al. Polycaprolactone scaffold surface modification with soft X-ray/extreme ultraviolet (SXR/EUV) radiation and low-temperature oxygen and nitrogen plasma for biomedical applications. J Mater Sci 59, 11937–11951 (2024). https://doi.org/10.1007/s10853-024-09876-y
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DOI: https://doi.org/10.1007/s10853-024-09876-y