In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.
Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.
The nervous system, which is the mechanism that bridges the internal structure of the organism and the environment, is divided into two parts: the central and peripheral nervous systems. Peripheral nerve damage is a global problem that constitutes 1.5%-5% of the patients who present to the emergency department and develops due to various traumas, leading to significant job loss1,2,3.
Today, cellular approaches to peripheral neuro-engineering are of great interest. Stem cells come first among the cells used in these approaches. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these cells are specialized but can differentiate upon appropriate stimulation in response to injury4,5. According to the stem cell hypothesis, the stem cell system is under the influence of its microenvironment, called the stem cell niche. The preservation and differentiation of stem cells are impossible without the presence of their microenvironment6, which can be reconstituted via tissue engineering using cells and scaffolds7. Tissue engineering is a multidisciplinary field that includes both engineering and biology principles. Tissue engineering provides tools for the creation of artificial tissues that can replace living tissues and can be used in the regeneration of these tissues by removing the damaged tissues and providing functional tissues8. Tissue scaffolds, one of the three cornerstones of tissue engineering, are produced using different methods from natural and synthetic materials9. Three-dimensional (3D) printing is an emerging additive manufacturing technology that is widely used to replace or restore defective tissues via its simple but versatile production of complex shapes using various methods. Bioprinting is an additive manufacturing method that enables the coexistence of cells and biomaterials, called bioinks10. Considering the interaction of nerve cells with each other, studies have shifted to conductive biomaterial candidates such as graphene. Graphene nanoplates, which have properties such as flexible electronics, supercapacitors, batteries, optics, electrochemical sensors, and energy storage, are a preferred biomaterial in the field of tissue engineering11. Graphene has been used in studies where the proliferation and regeneration of damaged tissues and organs were performed12,13.
Tissue engineering consists of three basic building blocks: scaffold, cells, and biosignal molecules. There are deficiencies in the studies on peripheral nerve damage in terms of providing these three structures completely. Various problems have been encountered in the biomaterials produced and used in the studies, such as them containing only stem cells or biosignal molecules, the lack of a bioactive molecule that will enable stem cell differentiation, the lack of biocompatibility of the biomaterial used, and the low effect on the proliferation of cells in the tissue niche, and, thus, nerve conduction not being fully realized2,13,14,15,16. This requires the optimization of nerve regeneration, reducing muscle atrophy17,18, and creating necessary homing19 with growth factors against such problems.At this point, the characterization and analysis of the neuro-activity of a surgical biomaterial prototype, to be transferred to the clinic, are very important.
Accordingly, this methods study investigates the bioink hydrogel patterning with graphene nanoplates formed by a 3D bioprinter and its effectiveness on the neurogenic differentiation of the stem cells it contains. Also, the effects of graphene on neurosphere formation and differentiation are investigated.
1. Culturing of Wharton's jelly mesenchymal stem cells
Created Groups | Reasons to create | Number of reps | ||
2D WJ-MSCs (2D-C) | 2D Control | x 5 | ||
2D WJ-MSCs & Graphene (2D-G) | Graphene toxic dose determination in 2D | x 5 reps to each of different concentrations | ||
WJ-MSCs are included in bioinks (3D-B ) | 3D Control | x 3 | ||
WJ-MSCs & 0.1% Graphene are included in bioinks (3D-G ) | 3D Graphene-Bioink Biohybrid Group | x 3 | ||
WJ-MSCs are in spheroid form on the bioinks (3D-BS ) | 3D Control of Spheroid Form | x 3 | ||
WJ-MSCs & 0.1% Graphene arein spheroid form on the bioinks (3D-GS group) | 3D Graphene-Bioink Biohybrid Group of Spheroid Form | x3 | ||
3D Bioink drop | It is produced for SEM and FTIR characterization analysis. | x5 | ||
3D Graphene drop | It is produced for SEM and FTIR characterization analysis. | x5 | ||
3D Bioink with GFP labeled WJ-MSCs & 0.1% | Observation of the movements of WJ-MsCs in the bioink containing the appropriate dose of graphene. | x3 |
Table 1. Groups in the method. All 2D and 3D groups in the method are included.
2. Graphene toxicity and 2D imaging
3. Graphene – Bioink biohybrid hydrogel production and WJ-MSCs differentiation
4. Graphene-Bioink biohybrid hydrogel characterization
NOTE: Time-lapse imaging, Fourier transform infrared spectroscopy (FT/IR), and scanning electron microscopy (SEM) analyses are performed for the characterization of graphene-bioink biohybrid hydrogel. The samples are created from 3D-B and 3D-G bioink groups by the drip method for FT/IR and SEM analysis.
Figure 1: 3D-B and 3D-G bioink groups produced as drops for use in characterization. (A) Bioink samples (pre-characterization image) on a plate with a crosslinker. (B) 3D-B drop image of bioink. (C) 3D-G bioink drop image. The biomaterial to be characterized and the cells it contains can more easily go through processes such as gold plating, sampling, etc. Please click here to view a larger version of this figure.
5. Determination of neurogenic differentiation by immunostaining method
Graphene toxicity and 2D imaging
Statistical analysis of the obtained MTT results was conducted with a one-way ANOVA with Tukey's test in statistical analysis software, and the graph obtained is shown in Figure 2. The graphene percentage compared to control showed a significant decrease only for the 0.001% graphene concentration (**p < 0.01).. There were no significant differences between the other groups and the control (p > 0.05). Therefore, the optimum graphene concentration was determined to be 0.1% since the highest viability rates were observed after exposure to this concentration according to the MTT test results and the stitching images.
Figure 2: Investigation of the effect of graphene concentration on cell proliferation. The graphene percentage compared to control showed a significant decrease only for the 0.001% graphene concentration (**p < 0.01, n = 6). There were no significant differences between the other groups and the control (p > 0.05; n = 6). Statistical analysis of the obtained MTT results was conducted with a one-way ANOVA with Tukey's test in statistical analysis software. The error bars represent the standard deviation. This figure has been modified from28. Please click here to view a larger version of this figure.
This part of the presented method demonstrates that the stem cell-graphene interaction can be used in future studies. It was observed that, in each tested concentration, graphene was tolerated in the 2D system and was uptaken by the cells through endocytosis (Figure 3). It has been determined that the graphene plates move along the cytoplasm inside the cell.
Figure 3: The cell-graphene interactions are shown in two dimensions in stitched images. (A) Control; (B) 0.0001%; (C) 0.001%; (D) 0.01%; (E) 0.1%; and (F) 1% concentrations of graphene. It is obtained by combining time-lapse imaging with high-definition quality settings to obtain a 4 rows x 5 columns collage of multiple photos. This figure has been modified from 28. Please click here to view a larger version of this figure.
Furthermore, in this study, it was observed that the graphene-bioink effect did not create any toxic microenvironment in the 3D system and that the cells were in interaction.
It has been also observed that the use of graphene in 3D systems did not create any toxic microenvironment. Determining the toxic dose for cells is crucial for the use of graphene in long-term conduit implants or injectable hydrogel forms. In addition, since graphene is an effective material in neuronal communication, its use in nerve tissue engineering applications has widely increased, as documented in the literature25.
Production of composite biomaterials and 3D bioprinting
The formation of gelatin-alginate-based bioink biohybrid patterning was performed with a non-toxic concentration of graphene (0.1%). It has been observed that the selected appropriate dose of graphene interacts with the cells in the bioink.
Time-lapse imaging
GFP samples were set at 37 °C for approximately 16 h in time-lapse, and photos and videos were taken (Video 1). GFP signals were lost when cell death was observed. Here, it was detected that cells that survived in the 3D graphene medium maintained their vitalities since GFP brightness was observed until the end of the incubation.
Video 1. Time-lapse video of GFP-labeled WJ-MSCs. The interaction of labeled stem cells with each other is observed. Please click here to download this Video.
Graphene–Bioink biohybrid hydrogel characterization
SEM and FT/IR were used for the characterization of the 3D-B and 3D-G groups created by the drop bioink method. The SEM images of the 3D-B and 3D-G bioink groups are given in Figure 4. Out of the 40 images taken at step 4.2.5., 4 representative images are shown here.
Figure 4: SEM images of the 3D-B and 3D-G bioink groups. (A) Image of a gold-coated 3D-B bioink section with electron microscopy. Scale bar: 200 µm. (B) Image of MSC attached to the 3D-B bioink inner surface. Scale bar: 5 µm (C) Image of 3D-G bioink inner and outer surface. Scale bar: 200 µm.(D) Inner surface cell 3D-G bioink adhesion image. Scale bar: 10 µm. Please click here to view a larger version of this figure.
Accordingly, bioink-cell interactions were demonstrated both on the surface and internally. There was a cell-biomaterial interaction in both bioinks (3D-B; 3D-G). The cells were morphologically round and attached to the material. The FT/IR analysis of the 3D-B and 3D-G bioink drop was compared with the graph in Figure 5.
Figure 5: FT/IR analysis. (A) 3D-B bioink. (B) 3D-G bioink. Peaks such as 1633.41 cm−1, 1552.42 cm−1, and 1033 cm−1 have been found in alginate-gelatin-based hydrogel studies in the literature26. Also, the 1335.46 cm−1 peak is similar to the peaks seen in graphene biomaterial studies27. Please click here to view a larger version of this figure.
Since the control bioink (3D-B) was based on alginate/gelatin, the most prominent peaks were around 1633.41 cm−1, 1552.42 cm−1, and 1033 cm−1 compared with similar studies in the literature with peaks seen at 1546 cm−1 26. A peak of 1399 cm−1 was observed in the graphene group (3D-G) and a similar peak was found at 1335.46 cm−1 in a study conducted with graphene27.
3D neuronal differentiation
The images of spheroids on the bioinks after the 7th-day post differentiation are represented in Figure 6.
Figure 6: 2D image of control WJ-MSCs and spheroid group samples at day 7 post differentiation. (A) Size of the sphere from the 3D-BS group bioink with diameters of 160 µm and 200 µm. (B) 2D control cells. Spheroids from (C) the 3D-BS group and (D) the 3D-GS group. The black materials in (D) are graphene molecules integrated with spheroids. Please click here to view a larger version of this figure.
It was seen that cells maintained their vitality in both 2D and 3D cultures. It was considered that the borders of the spheroid cells in both groups (3D-B and 3D-G) were transparent and lively, and the spheroids in the graphene group were relatively larger and trapped the graphene inside the cell. The differentiation was also tested by immunostaining. With the double staining used here, the activities of cells in 2D neuronal transformation were compared to 3D culture (Figure 7).
Figure 7: Immunostaining of 2D and 3D cells. (A,B) Immunostaining of cultured spheroids on 3D-BS bioinks. (C,D) 3D-GS bioink spheroid immunostains. (E) Differentiated 2D positive control WJ-MSCs. (F) Undifferentiated 2D negative control WJ-MSCs. Please click here to view a larger version of this figure.
Immunofluorescence images of cells cultured for 7 days are shown in Figure 7. Samples were stained with antibodies specific to N-cadherin (green) and β-III tubulin (red). Additionally, DAPI was used for visualization of the nucleus (blue or purple). Accordingly, the N-cadherins (green) used in the 2D and 3D samples differed over 7 days as it plays an important role in signaling mechanisms and the development of neurons. The green image in Figure 7A–E represents neuron-like structures (Figure 7). Class III β-tubulin is one of the seven isotypes known as neuron markers in the human genome. N-cadherin expression was found to be higher in the samples that were differentiated for 7 days compared to β-III tubulin. According to the results of the present experiment, it was established that the 3D systems created a more suitable microenvironment for the cells to survive and differentiate. The 2D positive control sample (Figure 7E) expressed fewer neuron-like structure markers compared to the 3D samples (Figure 7A–D). This shows us that the microenvironment created with the 3D structure is more effective in the differentiation of stem cells. In addition, instead of cell therapies alone; biomaterial-cell combined treatments appear to be more influential and effective in nerve damage.
The advantages of treatments applied with engineered 3D scaffolds over conventional 2D methods are becoming more and more noticeable every day. Stem cells used alone in these therapies or along with scaffolds produced from various biomaterials with low biocompatibility and biodegradability are usually inadequate in peripheral nerve regeneration. Wharton’s jelly mesenchymal stem cells (WJ-MSCs) seem to be a suitable candidate cell line, especially considering the optimization of the protocols for acquisition, their proliferation ability, and their differentiation capacity29. In this study, we examined the interaction of stem cells with graphene in both 2D and 3D cultures. We also compared the neurogenic differentiation in the generated biohybrid hydrogel groups with the 2D environment. We demonstrated the neurosphere formation on the bioinks by immunostaining. In further studies, we characterized our candidate prototype, which can be injected or implanted as a neural channel, by SEM and FT/IR methods. This study characterizes the properties of the biomaterial produced, the cell-biomaterial interaction, and the neural differentiation of the stem cells in the presence of the biomaterial. In the next stages, the effect on migration will be examined.
The biohybrid materials formed by combining two or more biocompatible materials are becoming more advantageous in terms of their structural properties compared to homogeneous materials30. In a previous study, implantation of a polyglycolic acid-based neuro tube channel into the facial peripheral nerve was performed. Labeled olfactory stem cells were inserted into this conduit, resulting in the successful regeneration of peripheral surgery and injected stem cell therapy16. In another study, the effectiveness of the 3D structure obtained from multiple cellular spheroids developed using human normal dermal fibroblasts and the silicon nerve canal, which is frequently used in the literature due to its inert feature, was compared in the regeneration of sciatic nerve damage. It was shown that the spheroid-based 3D structure increased tissue regeneration significantly and more rapidly in animal models with sciatic damage as compared to homogenous materials20. However, it is known that silicon-based biomaterials, which are frequently used in the literature, have some important disadvantages, such as high infection risk and low biocompatibility15,30.
In the present study, the production of biocompatible, biodegradable hydrogel biohybrid composite tissue with graphene nanoplatelets, which are known to be effective in nerve conduction, was carried out by a 3D printing technique. There are various studies in which graphene has been used as a biomaterial for nerve conduction25,30. Furthermore, graphene is one of the thinnest and lightest material available, which increases its preference in health technologies13. One of the most desirable properties of biomaterials is biodegradability. Experimental and molecular simulation technologies have been applied to investigate the biodegradation of graphene and its derivatives13. At this point, surface modification-functionalization or production in the form of composite biomaterials can improve or inhibit biodegradation, largely depending on the properties of the additives.
Various enzymes, such as manganese peroxidase (MnP), horseradish peroxidase (HRP), and myeloperoxidase (MPO), are available in the literature for the biodegradation of graphene derivatives. The MPO enzyme, which is a peroxidase released from neutrophils that come to the region of foreign bodies and perform phagocytosis, is associated with graphene biodegradation, especially in the human lung13. The biodegradability levels of composite biomaterials containing graphene instead of graphene nanotubes alone are different from each other. It is easier to achieve this desired property in composite materials. In addition, determining the toxic dose of graphene contributes to the use of clinically applicable biomaterials13.
An important part of the graphene stage of the protocol is sterilizing the graphene when it is to be used. The risk of contamination that may occur during the biohybrid-cell interaction phase is avoided.
The effect of the obtained biohybrid graphene-bioink hydrogel patterning on nerve differentiation was investigated and it was compatible with the literature that the differentiation was higher in the 3D system. The need for the production of biomaterials with tissue-engineered cellular-based therapeutic approaches that can be developed against various neurodegenerative diseases, such as peripheral nerve damage, is increasing with the inadequacy of current treatments day by day, emphasizing the importance of this study.
In a previous study, the interaction of graphene with cells in a 2D cell culture medium was investigated28. With the experience gained from there, here, graphene was added to the alginate-gelatin bioink with the known ratio, and a new and unique biomaterial prototype was produced by a 3D printing method. This new material was used to study cell interaction in two different ways. The first is the co-printing of the cell-composite biomaterial on a 3D printer. The second is the formation of a cellular spheroid from the biomaterial. In addition, the interaction of WJ-MSCs containing the tagged GFP gene with the material was also observed.
In this study, biohybrid bioinks were characterized by selecting FTIR and SEM methods. These enable the sample balls formed by the drip method to be examined during the characterization phase. Especially since we can examine a hard and dry structure during the SEM gold plating stage, SEM provides the physical suitability for getting better, thin sections with a scalpel from the bioink balls we created with this method.
In this method study, cells were added to the biohybrid material in two different ways during the experiments: inside for 3D bioprinting or during spheroid formation. Covering cells with bioinks and using 3D bioprinters allow researchers to create any desired 3D shape for nerve regeneration. On the other hand, it causes more stress on cells due to the printing pressure and, therefore, loss of cell viability. However, it could be a more desirable method to increase cell homing when they are injected or transplanted to a specific area to induce regeneration.
The creation of spheroids on bioinks creates a more usable form of artificial tissue in terms of cell interaction and provides a better niche for cell differentiation. It is also suitable for mimicking the natural microenvironment and, therefore, investigating cellular mechanisms. The low adhesion of the spheroids to the bioinks also allows easier separation from the bioinks and versatility of the applications.
The N-cadherins used (shown in green in Figure 7) are part of cell signaling mechanisms and play an important role in the development of neurons32. Class III β-tubulin is one of the seven isotypes known as neuron markers in the human genome. It shows that the WJ-MSCs used in this study begin to form neuron-like structures. In this context, 3D systems will create a more adequate microenvironment for cells to maintain their viability.
Finally, due to the expense involved and the clinical applicability of cell differentiation systems, it is very important in neuro-engineering to develop systems with controlled release of scaffolds, cells, and differentiation biosignals33 in the future and adapt these to the clinics as combined therapies. Therefore, spheroid and bioprinting methods can also be used in further studies where graphene and other bio-signals are embedded into hydrogels and released in a controlled manner. In vitro studies are an important step on the road to in vivo. When the material, provided here as a prototype, is produced in accordance with Good Laboratory Practice standards, the sterilization standards required for transfer to the clinic can be achieved.
The authors have nothing to disclose.
The graphene used in this study was developed at Kirklareli University, Department of Mechanical Engineering. It was donated by Dr. Karabeyoğlu. The graphene toxicity test was financed by the project titled "Printing and Differentiation of Mesenchymal Stem Cells on 3D Bioprinters with Graphene Doped Bioinks" (Application No: 1139B411802273) completed within the scope of TÜBİTAK 2209-B-Industry-Oriented Undergraduate Thesis Support Program. The other part of the study was supported by the research fund provided by Yildiz Technical University Scientific Research Projects (TSA-2021-4713). Mesenchymal stem cells with GFP used in the time-lapse imaging stage were donated by Virostem Biotechnology. The authors thank Darıcı LAB and YTU The Cell Culture and Tissue Engineering LAB team for productive discussions.
Centrifugal |
Hitachi | Used in cell culture and biomaterial step | |
0.1N CaCl2 | HD Bioink | Used for crosslinker | |
0.22 µm membrane filter | Aιsιmo | Used for sterilization | |
0.45 µm syringe filter | Aιsιmo | Used for sterilization | |
1.5mL conic tube | Eppendorfa | Used for bioink drop | |
15mL Falcon tube | Nest | Used in cell culture step | |
25 cm2 cell culture flasks (Falcon, TPP tissue culture flasks | Nest | Used for cell culture | |
3D Bioprinting | Axolotl Biosystems Bio A2 (Turkey) | Bioprinting Step | |
50 mL Falcon tube | Nest | Used in cell culture step | |
6/24/48/96 well plates (Falcon, TPP microplates) | Merck Millipore | Used in cell culture step | |
75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks | Nest | Used for cell culture | |
Anti mouse IgG-FTIC-rabbit | Santa Cruz Biotechnology | J1514 | Seconder antibody, used for dye |
Anti mouse IgG-SC2781-goat | Santa Cruz Biotechnology | C3109 | Seconder antibody, used for dye |
Au coating device EM ACE600 | Leica | for gold plating of biomaterial section before SEM imaging | |
Autoclave | NUVE-OT 90L | Used for the sterilization process. | |
Autoclave | NUVE-OT 90L | Used for the sterilization process. | |
Cell Cultre Cabine | Hera Safe KS | Used for the cell culture process | |
Dulbecco's Modified Eagle's Medium/Nutrient Mixture-F12 | Sigma | RNBJ7249 | Used as cell culture medium |
FEI QUANTA 450 FEG ESEM SEM | Quanta | FEG 450 | for SEM |
Fetal Bovine Serum-FBS | Capricorn | FBS-16A | It was used by adding to the cell culture medium. |
Freezer -80°C | Panasonic | MDF-U5386S-PE | We were used to store cells and the resulting exosomes |
Gelatine-Alginate bioink powder | HD Bioink | Used for produced bioink step | |
GFP labelled-WJ-MSCs | Virostem | Used for imaging to cell-bioink interaction | |
Graphene nanoplatelets (Graphene-IGP2) | Grafen Chemical Industries Co. | Used for production 3D-G bioink | |
Immunofluorescence antibodies (N-CAD; β-III Tubulin) | Cell Signalling and Santa Cruz | Used for dye | |
JASCO 6600 | Tetra | for FTIR | |
MTT Assay | Sigma | Viability testing | |
Penicilin/Streptomycin Solution | Capricorn | PB-S | It was added to the medium to prevent contamination in cell culture. |
Thoma slide | Isolab | Used for counting the cell | |
Time-Lapse Imaging System | Zeiss Axio.Observer.Z1 | Imaging | |
Tripsin-EDTA | Multicell | The flask was used to remove the cells covering the surface. | |
Vorteks | Biobase | For produced bioink step | |
WJ-MSCs | ATCC | Used for the cell culture process |