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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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 conditio....

Protocol

1. Culturing of Wharton's jelly mesenchymal stem cells

  1. Take the Wharton's jelly mesenchymal stem cells (WJ-MSCs, from ATCC) out of a −80 °C freezer. Culture WJ-MSCs in DMEM-F12 medium containing 10% fetal calf serum (FBS), 1% Pen-Strep, and 1% L-glutamine in a sterile laminar flow at room temperature, as described in Yurie et al.20.
  2. Cryopreserve some of the cells at 1 x 106 cells/mL with freezing medium containing 35% FBS, 55% DMEMF-12, and 10% dimethyl sulfoxide (DMSO). For this, count 1 x 106 cells on a Thoma cell counting slide and add the freezing solution ....

Representative Results

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 g.......

Discussion

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.......

Disclosures

The authors declare that there is no conflict of interest. The project was performed in collaboration with HD Bioink, developer of the 3D bioprinting technology.

Acknowledgements

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 imagi....

Materials

NameCompanyCatalog NumberComments

Centrifugal
HitachiUsed in cell culture and biomaterial step
0.1N CaCl2HD BioinkUsed for crosslinker
0.22 µm membrane filterAιsιmoUsed for sterilization
0.45 µm syringe filterAιsιmoUsed for sterilization
1.5mL conic tubeEppendorfaUsed for bioink drop
15mL Falcon tubeNestUsed in cell culture step
25 cm2 cell culture flasks (Falcon, TPP tissue culture flasksNestUsed for cell culture
3D BioprintingAxolotl Biosystems Bio A2 (Turkey)Bioprinting Step
50 mL Falcon tubeNestUsed in cell culture step
6/24/48/96 well plates (Falcon, TPP microplates)Merck MilliporeUsed in cell culture step
75 cm2 cell culture flasks (Falcon, TPP tissue culture flasksNestUsed for cell culture
Anti mouse IgG-FTIC-rabbitSanta Cruz BiotechnologyJ1514Seconder antibody, used for dye
Anti mouse IgG-SC2781-goatSanta Cruz BiotechnologyC3109Seconder antibody, used for dye
Au coating device EM ACE600Leicafor gold plating of biomaterial section before SEM imaging
AutoclaveNUVE-OT 90LUsed for the sterilization process.
AutoclaveNUVE-OT 90LUsed for the sterilization process.
Cell Cultre CabineHera Safe KSUsed for the cell culture process
Dulbecco's Modified Eagle's Medium/Nutrient Mixture-F12SigmaRNBJ7249Used as cell culture medium
FEI QUANTA 450 FEG ESEM SEMQuantaFEG 450for SEM
Fetal Bovine Serum-FBSCapricornFBS-16AIt was used by adding to the cell culture medium.
Freezer -80°CPanasonicMDF-U5386S-PEWe were used to store cells and the resulting exosomes
Gelatine-Alginate bioink powderHD BioinkUsed for produced bioink step
GFP labelled-WJ-MSCsVirostemUsed 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 CruzUsed for dye
JASCO 6600Tetrafor FTIR
MTT AssaySigmaViability testing
Penicilin/Streptomycin SolutionCapricornPB-SIt was added to the medium to prevent contamination in cell culture.
Thoma slideIsolabUsed for counting the cell
Time-Lapse Imaging SystemZeiss Axio.Observer.Z1Imaging
Tripsin-EDTAMulticellThe flask was used to remove the cells covering the surface.
VorteksBiobaseFor produced bioink step
WJ-MSCsATCCUsed for the cell culture process

References

  1. Kamasak, B., et al. Peripheral Nerve Injuries and Physiotherapy. Clinical Physiotherapy. 19, (2019).
  2. Yegiyants, S., Dayicioglu, D., Kardashian, G., Panthaki, Z. J. Traumatic peripheral nerve injury: A wartime review. Journ....

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Graphene3D BioprintingBiohybrid HydrogelBioinkPeripheral Nerve EngineeringWharton s Jelly Mesenchymal Stem CellsAlginateGelatin3D Cell Culture

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