1. Introduction
Medical materials have to be tested in vitro to choose the most suitable of them for further studies including in vivo tests with animal models and finally clinical trials with human patients. In vitro studies provide a rapid answer whether the material selected for testing—in our experiment graphene substrate –will be non-toxic and have positive effects on cell physiology. Cytotoxicity assessment is the first step in evaluating biocompatibility of the given material. ISO (Organization of Standardization) 10993 standards are well established and give guidance on testing the biocompatibility of materials; especially, ISO 10993 Part 5 describes usual techniques to evaluate cytotoxicity of materials [
1]. Therefore, these methods and techniques have been adopted by researchers to evaluate the biocompatibility of nanomaterials [
2]. This investigation will be helpful in the evaluation of tissue engineering technologies using graphene as a raw material. There are many in vitro cytotoxicity tests, which detect the toxic potential of the material studied [
3]. Results of these tests could be expressed as cell viability, rate of proliferation or rate of metabolism. The most frequently used cells are of rodent or human origin. It should be noted that the more various cell lines are used in biocompatibility tests the wider the answer about medical potential of material will be. Park et al. [
4] suggested that in vitro cytotoxicity test of biomaterials should be compared to other cytotoxicity testing methods with the use of multiple cell lines. Fibroblasts are among the most common cell types in the body and are widely used in cytotoxicity studies [
5]. There are three well established methods of contacting studied material with cell: direct contact, indirect contact, and elution of test material. In direct contact method the cells are seeded on top of the material or material is placed on top of the cells. This method was used in our experiment to evaluate the influence of graphene monolayer substrate on BALB/3T3 murine cells. Our previous study [
6] aimed to assess the biocompatibility of the graphene substrate as a scaffold for L929 cells. The two abovementioned cell lines are the most often used in cytotoxicity tests [
7,
8]. Moreover, it is recommended to perform the in vitro tests on the same type of cells which will be in direct contact with the medical materials in vivo. We suppose that graphene scaffold for fibroblasts or mesenchymal stem cells could be used in the future to aid reconstruction of damaged tissue caused by mechanical injuries, burns or as a result of chronic diseases.
Graphene has unique properties that make it a suitable candidate for several biomedical applications. High electrical conductivity, thermal stability, mechanical strength and antibacterial activity of graphene promote research into its use in the biomedical field [
9]. Graphene and its derivatives are used in research in drug delivery, tissue engineering and anti-cancer therapy [
10]. Because of its properties, the intensity of research on “graphene family” biomaterials and their application in regenerative medicine is rapidly increasing.
Fibroblasts play a crucial role in wound healing and are one of the major cells within the tissue that contact to the implanted device. Therefore, we used BALB/3T3 cells, derived from mouse embryos, for assessment of pristine graphene biocompatibility. Cytocompatibility of graphene monolayer was analyzed based on the assessment of BABL/3T3 cell viability, size and morphology. Additionally, because formation of focal adhesions allows cells throughout integrins to adsorb on the substrate surface and mediate signaling between cell and environment, we also assessed the graphene effect on stabilization of focal cell contacts in fibroblasts. Focal adhesions (FA) are mature opposite to nascent focal complexes, and are generally larger than 1 μm
2 [
11]. FA can also be classified as dot and dash adhesions, which means associated with cell motility or stability, respectively. Depending on the length, FA can be defined as nascent focal complexes (˂1 μm long), focal adhesions (1–5 μm long) and supermature or fibrillar adhesions (>5 μm long) [
12]. Young and Higgs [
13] revealed that FA consists of focal adhesion unit (FAU) 300 nm wide and the FA width is defined by the number of FAU but the FA length is variable and highly dynamic. Focal contacts were analyzed to evaluate the influence of graphene substrate. BALB/3T3 cell morphology (spreading, attachment, cell lysis) was examined under a light optical microscope, viability by trypan blue exclusion test, focal contacts by immunofluorescence staining and mitochondrial activity by colorimetric assay (WST-8) using a spectrophotometer. Moreover, mitochondrial network morphology using fluorescent dyes and membrane potential by flow cytometry was examined.
Lastly, in the present work we evaluated the impact of graphene monolayer on mitochondria morphology and physiology in BALB/3T3 cells. Mitochondria form a complex network in the cell that is subject to continuous fusion and fragmentation processes [
14]. Mitochondria are involved in cellular processes, i.e., ATP synthesis, apoptosis or buffering of calcium ions in the cell [
15,
16]. It has been shown that graphene oxide in the form of nanoplates can pass into the cytosol, where it can interact with mitochondria, and affect their morphology, function and membrane potential [
10,
17]. However, it should be remembered that the interaction between graphene and the cell membrane is closely related to the physicochemical properties of graphene, including size, shape and chemical forms [
6].
2. Materials and Methods
2.1. Graphene Monolayer Scaffold
In this work, we used graphene supplied from Graphenea company, San Sebastian, Spain. Graphene was transferred onto rounded glass cover slides (1 cm of diameter and 0.17 mm of thickness) from copper substrate. The high-speed (1 mm/s) electrochemical delamination method was used in the transfer process. Cover slides coated with graphene were sterilized by UV (30 min on the both sides) before experiment and then placed into the well of the 24-well plate. The characterization of the properties of graphene transferred onto glass substrate was performed at room temperature by Raman spectroscopy using a Renishaw in Via Raman microscope system with a 532 nm Nd:YAG laser as an excitation source. The Raman spectrum presented in
Figure 1a contains two prominent G and 2D peaks and a negligible low, disorder-related D peak near 1340 cm
−1. The observed symmetric Lorentzian line shape of the 2D peak and its low Full Width at Half Maximum (FWHM about 29 cm
−1) are features confirming the presence of predominantly single layer graphene [
18,
19,
20,
21]. Moreover, the micro-Raman map of the intensity ratio of the 2D to the G peaks (I
2D/I
G) in the area of 40 × 40 μm
2 taken with a step of 1 μm is shown in
Figure 1b. The average value (see inset of
Figure 1b) close to 5 indicates existence of predominantly a monolayer graphene structure on glass substrate.
Micro-Raman map of the I
2D/I
G together with optical image (
Figure 2) of the graphene layer depict that transferred graphene is a continuous layer.
2.2. Cytotoxicity Assessment of Graphene Scaffold
Cytotoxicity was assessed by direct contact method as per ISO-10993-5 guideline [
1]. The BALB/3T3 cells were seeded on the glass cover slide with graphene (experimental group) and without graphene (control group). Three independent experiments were done in triplicate. The degree of toxicity of the graphene monolayer was assessed on the basis of changes in cell morphology, viability (Trypan Blue test) and mitochondrial network morphology (Mito Green and Mito Red), mitochondrial activity (Water Soluble Tetrazolium-WST-8 test) and mitochondrial membrane potential (JC-1).
2.3. Cell Culture and Experimental Design
BALB/3T3 clone A31 cells (ATCC CCL-163) were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA) and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% iron-enriched bovine calf serum (BCS, HyClone). Additionally, culture medium was supplemented with antibiotics: 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, Saint Louis, MO, USA), and antimycotic: 0.25 μg/mL amphotericin B (Sigma-Aldrich). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. Confluent monolayers were detached by incubating with 0.25% trypsin-EDTA solution (Sigma-Aldrich). For experiments, BALB/3T3 cells (3 × 104/well) were seeded onto sterile microscopic glass coverslips (control) and graphene-coated glass coverslips placed in 24-well plates (Falcon, Los Angeles, CA, USA). After 24 h, cells were harvested for further experiments.
2.4. Cell Morphology Evaluation
Morphology of BALB/3T3 fibroblasts was observed at 1, 3, 6, 12 and 24 h of cell seeding on control or graphene-coated microscopic slides. Images were captured with an inverted microscope (Olympus IX71) equipped with Color View III cooled CCD camera and Cell^F software (Soft Imaging System) (Olympus, Tokyo, Japan).
2.5. Trypan Blue Cell Viability Analysis
BALB/3T3 cells grown for 24 h on glass coverslips (control) or graphene-coated microscopic slides (experimental) were detached with trypsin-EDTA solution (HyClone, Logan, UT, USA). After centrifugation, collected cells were resuspended in DMEM and mixed 1:1 with a 0.4% Trypan Blue solution (Sigma-Aldrich), and the mixture was transferred to a Neubauer chamber. Blue (dead) and unstained (live) cells were then counted, and these counts were used to calculate the relative proportion of the dead cells. The results were expressed as a percentage of viable cells.
2.6. Water Soluble Tetrazolium (WST) Assay
Cell Counting Kit-8 (Sigma-Aldrich) is a colorimetric, cytotoxicity assay for measuring mitochondrial activity.
Standard curve was prepared by plating 100 µl of 2-fold serially diluted suspension of BALB/3T3 cells into wells of the sterile flat-bottom 96-well plate (Falcon, Bedford, VA, USA). The ranges of the standard curve sensitivity for BALB/3T3 fibroblasts were approximately 1.5 × 104–1.17 × 102 cells/mL. Negative control containing media only (no cells) was used for measurement of the background. Next, 10 µl of Cell Counting Kit-8 reagent was added to each well of the standard curve and incubate for 2 h at 37 °C in a humidified atmosphere of 5% CO2 in the air. The absorbance of triplicate serial dilutions in 96-well plate was determined at 450 nm using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA). All experimental analysis were performed in parallel with the standard curve.
BALB/3T3 cells grown for 24 h on glass coverslips (control) or graphene-coated glass coverslips (experimental) were detached with trypsin-EDTA solution and resuspended in appropriate culture medium. Cells in the total volume of 100 μL were plated in triplicate into the wells of 96-well plate. 10 µL WST-8 reagent was added to each well (three control and three experimental for both cell lines), and the plates were incubated at 37 °C in a humidified CO2 incubator for 2 h. The absorbance was determined at 450 nm using a spectrophotometer.
2.7. Cell Area (Spreading) Calculation
BALB/3T3 cell area after 12 and 24 h of incubation on glass and graphene substrate was calculated using the ImageJ software (NIH, Bethesda, MD, USA).
2.8. Immunofluorescent Staining and Morphometric Analysis of Focal Contacts
To detect F-actin and vinculin, BALB/3T3 cells were fixed with 4% PFA in PBS for 20 min. Next, the cells were permeabilized with 0.5% Triton X (Sigma-Aldrich) in PBS (15 min) and blocked with 3% bovine serum albumin (BSA, Sigma-Aldrich) in 0.1% Triton X-100-PBS solution (30 min) to prevent nonspecific binding. For F-actin detection, the cells were incubated with 0.5 µg/mL phalloidin-FITC for 20 min in the dark. For immunostaining of focal contacts, anti-vinculin antibody (Sigma) was used. The cells were incubated with primary Abs anti-vinculin for 1 h and next, secondary anti-mouse Abs conjugated with rhodamine Red-X were used (60 min in the dark). DNA was stained with 1 µg/mL Hoechst 33342 for 5 min in the dark. Slides were mounted in ProLong Gold Antifade Reagent (ThermoFisher Scientific, Waltham, MA, USA).
For quantitative analysis of focal contacts, fluorescent images were collected using Color View III cooled CCD camera mounted on a fluorescence microscope (Olympus BX60). Focal contact number per cell and the average focal contact size were evaluated using Cell^F (Olympus, Tokyo, Japan) and ImageJ software (NIH Image, version 1.50i, Bethesda, MD, USA). Focal contacts were manually outlined, and area (size) was calculated automatically. For focal contact number per cell at least 50 cells of each experiment were counted, and for focal contacts size, at least 10 areas of focal contacts per cell were analyzed.
2.9. Mitochondrial Network Morphology Evaluation and Determination of Mitochondrial Membrane Potential
BALB 3T3 cells were seeded on glass coverslips coated or uncoated with graphene (control) in a 24-well plate and mitochondria were labeled with 300 nM Mito Red or Mito Tracker Green for 30 min in the dark at 37 °C with 5% CO2 in a humidified incubator. Next, the staining solution was replaced with fresh prewarmed media, and mitochondrial network was observed under Olympus BX60 microscope.
Mitochondrial membrane potential was measured with the LSR Fortessa flow cytometer (Becton Dickinson Biosciences) by using the dual-emission potential-sensitive probe JC-1 as a fluorescent dye. JC-1 exhibits potential-dependent accumulation in mitochondria where it starts forming J aggregates (red fluorescence with emission at ~590 nm); upon depolarization, it remains as monomer (showing green fluorescence with emission at ~529 nm). Mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.
BALB 3T3 cells grown on graphene-coated or uncoated glass slides in 24-well culture plates were incubated with culture medium supplemented with (6 μL do 1 mL) µM JC-1 dye, for 60 min in the dark at 37 °C with 5% CO2 in a humidified atmosphere. Next, the cells were collected, resuspended in cold (4 °C) PBS and analyzed by flow cytometry (λex = 488 nm). Data were presented as the ratio of red to green fluorescence of JC-1 dye. For positive control of decreased mitochondrial membrane potential, the cells were treated with both JC-1 and 3% hydrogen peroxide.
4. Discussion
Graphene has potential to be used in medical fields and composite enhancement, amongst other uses. Biosafety of nanomaterials has caused increased attention from scientists who are investigating their effects on the cells, animals and environment [
23,
24,
25]. Comparative studies on graphene cytotoxicity help to efficiently apply these materials in medical fields. That is why the main goal of this study was to determine the cytotoxicity of graphene by in vitro tests on murine BALB/3T3 fibroblast. The research provides additional data on the suitability of graphene monolayer for being used as a scaffold for cells in regenerative medicine. Previously, we checked biocompatibility of pristine graphene with L929 fibroblast cells [
6]. The reason for choosing another cell type was to see if the effect of cytotoxicity was cell dependent.
The success of tissue engineering scaffolds highly depends on their interaction with cells. First of all, scaffold cannot be toxic to the tissue in which it is going to be implanted. Furthermore, it is desirable for the scaffold to stimulate physiologic changes leading to, especially in fibroblasts, accelerated cell migration into the wound and increase in proliferation and viability. The exposure of BALB/3T3 cells to graphene scaffold induces a high increase of average cell mitochondrial activity, implying that the graphene scaffold is not cytotoxic and stimulates cells proliferation. Proliferating cells reduce their internal environment more than non-proliferating ones do. The increase in mitochondrial activity by 1/5 compared to control indicates that graphene scaffold stimulates proliferation of cells attached to this nano-material. This is in agreement with results obtained by Gentile et al. [
26], who observed that nano-topography of the material on which cells move affects the physiology of said cells, modifying viability and proliferation. Biggs et al. [
27], on the basis of literature review, concluded that nano-topography plays an essential role in the creation of focal adhesion and subsequent changes in cellular functions. Gentile et al. [
26] noticed that moderately rough (average surface roughness between 10 and 40 nm) surfaces of electrochemically etched silicon substrates increased the proliferation rate of NIH/3T3 fibroblasts from 36 to 60 h after seeding. Mitochondrial activity of BALB/3T3 introduced to graphene raised significantly 24 h after seeding. It can be supposed that the mechanism of graphene action is a result of its topography and roughness, which cause increased effective surface energy. Nanoscale topography of substrates and their stiffness became the subject of many studies trying to estimate the possibility of their use as a platform for cell culture [
26,
28,
29,
30]. They established that cell adhesion, proliferation and differentiation were influenced by the micro- and nano-surface characteristics of biomaterials. Kim et al. [
29] also indicate that graphene oxide may enhance the affinity of cells. In vitro cytotoxicity tests performed in our study reveal excellent cytocompatibility of graphene scaffold to BALB/3T3 cells. Nishida et al. [
30] have published results that confirm our data, but they involve other cells—MC3t3-E1 (mouse osteoblastic cells) as well as a different scaffold, using graphene oxide attached to collagen substrate. The authors noted that proliferation of MC3t3-E1 on the scaffold was stimulated in dose-dependent manner by GO application. Moreover, they concluded that the graphene oxide scaffold exhibited good biocompatibility based on implantation of 1 μg/mL graphene oxide scaffold into the subcutaneous tissue of Wistar rats and to extraction sockets of beagle dogs.
Our results presented actin cytoskeleton staining and showed longitudinal filaments along the long axis of the BALB/3T3 cells with colocalization of vinculin on the cell edges. Vinculin is an important FA protein, characteristic for nascent cell-matrix adhesion and mature FA, which bound integrins to cytoplasmic F-actin [
31]. Disruption of the vinculin-F-actin interaction affects cell motility, cell stiffness and adhesion [
32]. Zhou et al. [
31] revealed that vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate and cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and bone marrow-derived stem cells were also reduced in the soft substrate. Kim and Wirts [
33] characterized parameters of FA of mouse embryonic fibroblasts—MEFs and found that FA display an ellipsoidal shape and FA area was approximately 3 μm
2 for glass substrate and decreased with the decreasing of substrate stiffness, reaching about 1.5 μm
2 on soft gel.
Average focal adhesion size for different cell lines (CHO, C2H12 and MDCK) growing on flat surfaces was larger than 1 μm
2; complexes smaller than 1 μm
2 were identified by authors as nascent focal complexes and were located preferentially in the lamellipodium [
11]. Moreover focal adhesion of cells growing on nanopillars was much smaller than for cells growing on a flat surface and was between 0.2 and 0.4 μm
2. Focal contacts in our experiment were also included in these two morphologies: smaller and larger contacts. Young and Higgs [
13] found that focal adhesions initially appear as assemblies of multiple linear units (FAU) of 0.3 μm width, which can split in coordination with FA elongation and that dynamic interactions between FAU control adhesion morphology. The authors have pointed out that, apart from splitting, mature FA can disassemble or forego splitting altogether. Our results indicated larger focal contacts in fibroblasts growing on graphene which could be explained by interactions with FAU. We can assume that focal contact in graphene group consisted of more FAUs than in control group. Mechanism of the FAU connecting on the graphene substrate should be the subject of further studies.
Changes in mitochondrial morphology could be a good indicator of cell condition [
34]. Although mitochondrial morphology is variable and can be described as reticulated or fragmented with spheroid-shaped mitochondria or branched structure. The right proportion of certain types of mitochondria is important to deduce their condition and function. It also depends on the cell type and cell cycle. Mitochondrial shapes fall into four “categories”: point, rod-like, branched, and large and round [
35]. Mitochondria in BALB/3T3 cells growing on graphene-coated or uncoated glass slides exist as a branched network with no significant morphological dysfunction. Symptoms of lesions or defects in mitochondrial morphology include swelling of mitochondria and giant spherical mitochondria [
36]. Mitochondrial morphology also changes as a result of fission and fusion (from fragmented to highly elongated) [
37]. Moreover, mitochondrial shape can change many times over time and as a result of stress factors [
15,
34]. Highly fragmented mitochondria were noted in BALB/3T3 treated with H
2O
2 (positive control). This oxidative stress-induced reagent, which caused mitochondrial fission resulted in fragmented mitochondria. This fragmentation was also reflected in highly reduced mitochondrial potential (Figure x). Jaworski et al. [
17] found that platelets of graphene have dose-dependent cytotoxicity via depletion of the mitochondrial membrane potential against human cancer cells. Monolayer of graphene—used in this study, in contrast to graphene platelets and H
2O
2, has no toxic effect on network and mitochondrial potential.
Cytotoxicity testing of graphene as a biomaterial is mandatory in the assessment of its safety. It should be noted that the biocompatibility of graphene will vary strongly with its type: monolayer graphene, graphene oxide, reduced graphene or few layer graphene [
29]. The cytocompatibility of the graphene scaffold with BALB/3T3 cells suggest its possible use in the healing of tissue damage.