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

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 loss^1,2,3.

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 ....

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.......

Materials

Name	Company	Catalog Number	Comments
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