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

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.