Name: preparation and characterization of graphene-based 3d biohybrid hydrogel bioink for peripheral neuroengineering
Uploaded: 2022-05-16T14:00:00
Description: Watch this Scientific Journal Video about Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering at JoVE.com

The graphene used in this study was developed at Kirklareli University, Department of Mechanical Engineering. It was donated by Dr. Karabeyo&#287;lu. The graphene toxicity test was financed by the project titled &#34;Printing and Differentiation of Mesenchymal Stem Cells on 3D Bioprinters with Graphene Doped Bioinks&#34; (Application No: 1139B411802273) completed within the scope of T&#220;B&#304;TAK 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 Dar&#305;c&#305; LAB and YTU The Cell Culture and Tissue Engineering LAB team for productive discussions.

1. Culturing of Wharton&#39;s jelly mesenchymal stem cells
<ol>
	<li>Take the Wharton&#39;s jelly mesenchymal stem cells (WJ-MSCs, from ATCC) out of a &#8722;80 &#176;C freezer. Culture WJ-MSCs in DMEM-F12 medium containing 10% fetal calf serum (FBS), 1% Pen-Strep, and 1% L-glutamine&#160;in a sterile laminar flow at room temperature, as described in Yurie et al.20.</li>
	<li>Cryopreserve some of the cells at 1 x 106 cells/mL with freezing medium containing 35% FBS, 5...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+peripheral+nerve+injury:+A+wartime+review.">Traumatic peripheral nerve injury: A wartime review.</a> Journal of Craniofacial Surgery. 21 (4), 998-1001 (2010).</li><li>Mushtaq, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+peripheral+nerve+injury+in+trauma+in+emergency+settings.">Frequency of peripheral nerve injury in trauma in emergency settings.</a> Cureus. 13 (3), 14195 (2021).</li><li>Allahverdiyev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic Principles of Somatic and Stem Cell Culture Systems, 1st Edition. , (2018).</li><li>Allahverdiyev, A. 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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+clinical+applications+of+Wharton's+jelly-derived+mesenchymal+stromal+cells.">Characteristics and clinical applications of Wharton's jelly-derived mesenchymal stromal cells.</a> Current Research in Translational Medicine. 68 (1), 5-16 (2020).</li><li>Yoo, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+peripheral+nerve+regeneration+through+elastic+nerve+guidance+conduits+prepared+using+a+porous+PLCL+membrane+with+a+3D+printed+collagen+hydrogel.">Augmented peripheral nerve regeneration through elastic nerve guidance conduits prepared using a porous PLCL membrane with a 3D printed collagen hydrogel.</a> Biomaterials Science. 22, 1-12 (2020).</li><li>Jansen, K., Meek, M. F., vander Werff, J. F. A., van Wachem, P. 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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 prolif...

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.&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td> 
			Centrifugal</td>
			<td>Hitachi</td>
			<td></td>
			<td>Used in cell culture and biomaterial step</td>
		</tr>
		<tr>
			<td>0.1N CaCl2</td>
			<td>HD Bioink</td>
			<td></td>
			<td>Used for crosslinker</td>
		</tr>
		<tr>
			<td>0.22 &#181;m membrane filter</td>
			<td>A&#953;s&#953;mo</td>
			<td></td>
			<td>Used for sterilization</td>
		</tr>
		<tr>
			<td>0.45 &#181;m syringe filter</td>
			<td>A&#953;s&#953;mo</td>
			<td></td>
			<td>Used for sterilization</td>
		</tr>
		<tr>
			<td>1.5mL conic tube</td>
			<td>Eppendorfa</td>
			<td></td>
			<td>Used for bioink drop</td>
		</tr>
		<tr>
			<td>15mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>25 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>3D Bioprinting</td>
			<td>Axolotl Biosystems Bio A2 (Turkey)</td>
			<td></td>
			<td>Bioprinting Step</td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Nest</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>6/24/48/96 well plates (Falcon, TPP microplates)</td>
			<td>Merck Millipore</td>
			<td></td>
			<td>Used in cell culture step</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture flasks (Falcon, TPP tissue culture flasks</td>
			<td>Nest</td>
			<td></td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Anti mouse IgG-FTIC-rabbit</td>
			<td>Santa Cruz Biotechnology</td>
			<td>J1514</td>
			<td>Seconder antibody, used for dye</td>
		</tr>
		<tr>
			<td>Anti mouse IgG-SC2781-goat</td>
			<td>Santa Cruz Biotechnology</td>
			<td>C3109</td>
			<td>Seconder antibody, used for dye</td>
		</tr>
		<tr>
			<td>Au coating device EM ACE600</td>
			<td>Leica</td>
			<td></td>
			<td>for gold plating of biomaterial section before SEM imaging</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>NUVE-OT 90L</td>
			<td></td>
			<td>Used for the sterilization process.</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>NUVE-OT 90L</td>
			<td></td>
			<td>Used for the sterilization process.</td>
		</tr>
		<tr>
			<td>Cell Cultre Cabine</td>
			<td>Hera Safe KS</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture-F12</td>
			<td>Sigma</td>
			<td>RNBJ7249</td>
			<td>Used as cell culture medium</td>
		</tr>
		<tr>
			<td>FEI QUANTA 450 FEG ESEM SEM</td>
			<td>Quanta</td>
			<td>FEG 450</td>
			<td>for SEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum-FBS</td>
			<td>Capricorn</td>
			<td>FBS-16A</td>
			<td>It was used by adding to the cell culture medium.</td>
		</tr>
		<tr>
			<td>Freezer -80&#176;C</td>
			<td>Panasonic</td>
			<td>MDF-U5386S-PE</td>
			<td>We were used to store cells and the resulting exosomes</td>
		</tr>
		<tr>
			<td>Gelatine-Alginate bioink powder</td>
			<td>HD Bioink</td>
			<td></td>
			<td>Used for produced bioink step</td>
		</tr>
		<tr>
			<td>GFP labelled-WJ-MSCs</td>
			<td>Virostem</td>
			<td></td>
			<td>Used for imaging to cell-bioink interaction</td>
		</tr>
		<tr>
			<td>Graphene nanoplatelets (Graphene-IGP2)</td>
			<td>Grafen Chemical Industries Co.</td>
			<td></td>
			<td>Used for production 3D-G bioink</td>
		</tr>
		<tr>
			<td>Immunofluorescence antibodies (N-CAD; &#946;-III Tubulin)</td>
			<td>Cell Signalling and Santa Cruz</td>
			<td></td>
			<td>Used for dye</td>
		</tr>
		<tr>
			<td>JASCO 6600</td>
			<td>Tetra</td>
			<td></td>
			<td>for FTIR</td>
		</tr>
		<tr>
			<td>MTT Assay</td>
			<td>Sigma</td>
			<td></td>
			<td>Viability testing</td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin Solution</td>
			<td>Capricorn</td>
			<td>PB-S</td>
			<td>It was added to the medium to prevent contamination in cell culture.</td>
		</tr>
		<tr>
			<td>Thoma slide</td>
			<td>Isolab</td>
			<td></td>
			<td>Used for counting the cell</td>
		</tr>
		<tr>
			<td>Time-Lapse Imaging System</td>
			<td>Zeiss Axio.Observer.Z1</td>
			<td></td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Tripsin-EDTA</td>
			<td>Multicell</td>
			<td></td>
			<td>The flask was used to remove the cells covering the surface.</td>
		</tr>
		<tr>
			<td>Vorteks</td>
			<td>Biobase</td>
			<td></td>
			<td>For produced bioink step</td>
		</tr>
		<tr>
			<td>WJ-MSCs</td>
			<td>ATCC</td>
			<td></td>
			<td>Used for the cell culture process</td>
		</tr>
	</tbody>
</table>

preparation and characterization of graphene-based 3d biohybrid hydrogel bioink for peripheral neuroengineering

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.

Graphene toxicity and 2D imaging 
Statistical analysis of the obtained MTT results was conducted with a one-way ANOVA with Tukey&#39;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 &#60; 0.01).. There were no significant differences between the other groups and the control (p &#62; 0.05). Therefore, the optimum g...

Watch this Scientific Journal Video about Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering at JoVE.com

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

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 ...

The stem cell system is under the influence of its important components. The preservation and differentiation of stem cell is unthinkable without the presence of their microenvironment. This microenvironment, called stem-cell niche, consist of cell and scaffolds.Peripheral neuropathy can be and occasionally, they may such as injury to the brachii plexus, various trauma, tumor, autonomic anomalies, immune definition, and metabolic disease. Various 3D culture methods have been developed to provide a better and more natural niche for the stem cells. Spheroid formation and 3D bioprinting are relatively new and promising methods for 3D cultures.3D bioprinting can also be used in neuroengineering studies. Consequently, within this study, graphene-based and alginate/gelatin-based bioinks were developed and studied for their regenerative features. To begin, culture Wharton's jelly mesenchymal stem cells or WJMSCs in DMEM F12 medium containing 10%fetal calf serum or FBS, 1%pen/strep, and 1%L-glutamine under a sterile laminar flow at room temperature.When the cultured cells are 80%confluent in the flask, pour the medium and wash the cells with five milliliters of PBS. Then add five milliliters of 0.25%trypsin and 2.21 millimolar sodium EDTA and incubate at 37 degrees Celsius. After five minutes, add 10 milliliters of DMEM F12 medium containing 10%FBS to the cells.Suspend the cells, collect the medium and transfer it to a centrifuge tube. Next, centrifuge the cells and discard the supernatant before reseeding the cells in a new flask with a fresh nutrient medium containing 10%FBS. To prepare the bioink of the control group without graphene, weigh 4.5 milligrams of alginate and 1.5 milligrams of gelatin and transfer them to a centrifuge tube.Then add 50 milliliters of DMEM F12 medium containing 10%FBS to the tube. Again, weigh 4.5 milligrams of alginate and 1.5 milligrams of gelatin and transfer them to a centrifuge tube. Then add 50 microliters of 0.1%graphene to the tube and make the volume 50 milliliters with DMEM F12 medium containing 10%FBS.Mix the bioinks by pipetting and vortexing before autoclaving at 121 degrees Celsius under 1.5. atmospheric pressure for 20 minutes. After autoclaving, centrifuge the solution to remove formed bubbles and place the bioinks at 37 degrees Celsius until the cells are prepared.For the cell bioink interaction, create the bioink groups. Group one includes 3D-B and 3D-G printed with bioink for bioprinting. Group two includes 3D-BS and 3D-GS bioinks on which spheroids were formed after bioprinting.For group one, count the cells to one by 10 to the seventh cells in 0.5 milliliters of medium. Then add 4.5 milliliters of bioink. Transfer the mixture to the cartridges in the sterile cabinet using syringes.Install the cartridges in the corresponding extruder section of the bioprinter. For the second group, take five milliliters of bioink from each of the bioink groups and transfer them to sterile cartridges with the help of an injector. Use the bioprinter with two coaxial print heads and the pneumatic-driven extrusion technology.Set the XYZ resolution per micro step to 1.25 micrometers, extrusion width to 400 micrometers, and the extrusion height to 200 micrometers. Use a 20 by 20 by 5 millimeter grid to create 3D models. Create 3D models using open source web-based CAD programs.For the 3D bioprinting process, set the average pressure of the printer to 7.5 psi. Then set the cartridge and bed temperature to 37 degrees Celsius and speed to 60%Place the system in the home position during the writing phase. Position the axes automatically and select and set the extruder before starting the bioprinting process.After the printing, remove the sample and place it under a laminar flow cabinet. Next, spray the bioinks with 0.1 normal calcium chloride solution or add one milliliter solution with a pipette at room temperature and wait for 10 to 20 seconds. Then wash the printed patterns twice with PBS containing calcium and magnesium.Add two milliliters of DMEM F12 medium with 10%FBS on top of each cell containing bioink group and incubate the plates at 37 degrees Celsius with 5%carbon dioxide for 30 minutes. Next, add two milliliters of suspension medium containing one by 10 to the sixth cells to each group and incubate the plates. After 24 hours, observe and photograph all batches of bioink for spheroid formation under an inverted microscope for WJMSCs differentiation to neuron-like cells except for the control group.Add two milliliters of neurogenic differentiation medium per well and refresh every two days. Follow for seven days to observe neural differentiation. Next, using timelapse imaging, examine the effects of graphene on stem cells and monitor cell interactions within the bioink.The effect of graphene concentration on cell proliferation is demonstrated here. Compared to the control, significant decrease was observed for the 0.001%graphene concentration. There were no significant differences between the other groups and the control.The cell graphene interactions showed that graphene was tolerated in the 2D system and was taken by the cells through endocytosis. Timelapse imaging showed that cells that survived in the 3D graphene medium maintained their vitalities through GFP brightness until the end of the incubation. SEM images and FIIR analysis of the 3D-B and 3D-G bioink groups are shown here.Bioink cell interactions were demonstrated both on the surface and internally. The cells were morphologically round and attached to the material. In 3D neuronal differentiation, it was considered that the borders of the spheroid cells in both groups were transparent and lively, and the spheroids in the graphene group were relatively larger and trapped the graphene inside the the cell.Immunostaining of 2D and 3D cells is shown here. The green images represent neuron-like structures. The 2D positive control sample expressed fewer neuron-like structure markers than the 3D samples.We discovered that graphene-based bioinks were more successful in terms of differentiation of stem cells into neuron-like cells. We propose that graphene-based bioinks would be excellent tools for treatment of peripheral nerve disorders in further studies. Today, the stem cell system can be created by tissue engineering with natural and synthetic biomaterials The creation of artificial tissues that can replace living tissues which can be used in regeneration of these tissues, removing the damage, and providing function is provided by tissue engineering.

Research

JoVE Journal

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