用于外周神经工程的石墨烯基3D生物杂化水凝胶生物墨水的制备与表征
1Vocational School of Health Services,Istinye University, 2Chemical Metallurgy Faculty,Yildiz Technical University, 3Faculty of Medicine, Department of Histology & Embryology,Istinye University, 43D Bioprinting Design & Prototyping R&D Center,Istinye University, 5Stem Cell, and Tissue Engineering R&D Center,Istinye University, 6Vocational School of Health Services,Altinbas University, 7The V. Akhundov National Scientific Research Medical Prophylactic Institute, 8Center for Stem Cells and Regenerative Medicine (LivMedCell),Liv Hospital
Summary
在本手稿中，我们演示了用于外周组织工程的含有石墨烯的生物杂化水凝胶生物墨水的制备方法。使用这种3D生物杂交材料，进行干细胞的神经分化方案。这可能是将类似生物材料带入临床的重要一步。
Abstract
周围神经病可由轴索损伤引起，偶尔也可由脱髓鞘疾病引起。周围神经损伤是一个全球性问题，发生在1.5%-5%的急诊患者中，并可能导致大量失业。如今，随着三维（3D）生物打印技术的发展，基于组织工程的方法（包括支架，适当的细胞系和生物信号）变得更加适用。经常研究各种水凝胶生物材料与干细胞、外泌体或生物信号分子的组合，以克服周围神经再生中存在的问题。因此，可注射系统的生产，如水凝胶，或由各种生物打印方法形成的植入式导管结构，在周围神经工程中变得越来越重要。在正常情况下，干细胞是人体的再生细胞，它们的数量和功能不会随着时间的推移而减少，以保护其种群;这些不是特化的细胞，但可以在损伤反应的适当刺激下分化。干细胞系统受到其微环境的影响，称为干细胞生态位。在周围神经损伤中，特别是在神经病中，即使在手术将切断的神经末梢结合在一起后，也无法完全挽救这种微环境。复合生物材料和联合细胞疗法方法在生物降解性、生物相容性和可加工性等各种特性方面提高了材料的功能和适用性。因此，本研究旨在证明石墨烯基生物杂交水凝胶图案的制备和使用，并检验干细胞向神经细胞的分化效率，这可以成为神经再生的有效解决方案。
Introduction
神经系统是连接生物体内部结构和环境的机制，分为两部分：中枢神经系统和周围神经系统。周围神经损伤是一个全球性问题，占急诊科就诊患者的1.5%-5%，并因各种创伤而发展，导致大量失业1，2，3。
今天，用于外周神经工程的细胞方法引起了极大的兴趣。干细胞在这些方法中使用的细胞中排在第一位。在正常情况下，干细胞是人体的再生细胞，它们的数量和功能不会随着时间的推移而减少，以保护其种群;这些细胞是特化的，但可以根据损伤的适当刺激分化4，5。根据干细胞假说，干细胞系统受到其微环境的影响，称为干细胞生态位。如果没有干细胞的微环境6的存在，干细胞的保存和分化是不可能的，微环境可以使用细胞和支架7通过组织工程重建。组织工程是一个多学科领域，包括工程和生物学原理。组织工程提供了用于创建人造组织的工具，这些人造组织可以替代活组织，并且可以通过去除受损组织和提供功能性组织来用于这些组织的再生8。组织支架是组织工程的三大基石之一，采用天然和合成材料的不同方法生产9。三维（3D）打印是一种新兴的增材制造技术，通过使用各种方法简单但多功能地生产复杂形状，广泛用于替换或恢复有缺陷的组织。生物打印是一种增材制造方法，可实现细胞和生物材料的共存，称为生物墨水10。考虑到神经细胞之间的相互作用，研究已经转向导电生物材料候选物，如石墨烯。石墨烯纳米片具有柔性电子、超级电容器、电池、光学、电化学传感器和储能等特性，是组织工程领域的首选生物材料11。石墨烯已被用于对受损组织和器官进行增殖和再生的研究12，13。
组织工程由三个基本组成部分组成：支架、细胞和生物信号分子。关于周围神经损伤的研究在完全提供这三种结构方面存在不足。在研究中生产和使用的生物材料中遇到了各种问题，例如它们仅包含干细胞或生物信号分子，缺乏能够分化的生物活性分子，所用生物材料缺乏生物相容性，以及对组织生态位中细胞增殖的影响低， 因此，神经传导未完全实现2，13，14，15，16。这需要优化神经再生，减少肌肉萎缩17，18，并创造必要的归巢19与生长因子对抗这些问题。在这一点上，要转移到临床的手术生物材料原型的神经活性的表征和分析非常重要。
因此，该方法研究由3D生物打印机形成的石墨烯纳米板的生物墨水水凝胶图案及其对其所含干细胞神经分化的有效性。此外，还研究了石墨烯对神经球形成和分化的影响。
Protocol
1. 沃顿商学院果冻间充质干细胞的培养 将沃顿商学院的果冻间充质干细胞（WJ-MSCs，来自ATCC）从-80°C冰箱中取出。如Yurie等人20中所述，在含有10%胎牛血清（FBS），1%Pen-Strep和1%L-谷氨酰胺的DMEM-F12培养基中培养WJ-MSC，在室温下无菌层流中。 用含有 35% FBS、55% DMEMF-12 和 10% 二甲基亚砜 （DMSO） 的冷冻培养基以 1 x 106 个细胞 /mL 冷冻保存一些细胞。?…
Representative Results
石墨烯毒性和2D成像在统计分析软件中，使用Tukey检验的单因素方差分析对获得的MTT结果进行统计分析，得到的图形如图 2所示。与对照相比，石墨烯百分比仅在0.001%石墨烯浓度（**p 0.05）。因此，最佳石墨烯浓度被确定为0.1%，因为根据MTT测试结果和拼接图像，暴露于该浓度后观察到最高的活性率?…
Discussion
与传统的2D方法相比，使用工程3D支架应用的处理优势每天都变得越来越明显。在这些疗法中单独使用的干细胞或与由具有低生物相容性和生物降解性的各种生物材料产生的支架一起在周围神经再生中通常不足。沃顿商学院的果冻间充质干细胞（WJ-MSCs）似乎是一种合适的候选细胞系，特别是考虑到获取方案的优化，它们的增殖能力和分化能力29。在这项研究中，我们检查了干细胞…
Divulgations
The authors have nothing to disclose.
Acknowledgements
本研究中使用的石墨烯是在Kirklareli大学机械工程系开发的。它是由卡拉贝约奥卢博士捐赠的。石墨烯毒性测试由名为“使用石墨烯掺杂生物墨水在3D生物打印机上打印和分化间充质干细胞”的项目资助（申请号：1139B411802273），该项目在TÜBİTAK 2209-B-面向行业的本科论文支持计划范围内完成。该研究的另一部分得到了Yildiz技术大学科学研究项目（TSA-2021-4713）提供的研究基金的支持。用于延时成像阶段的具有GFP的间充质干细胞由Virostem Biotechnology捐赠。作者感谢Darıcı LAB和YTU细胞培养和组织工程实验室团队富有成效的讨论。
Materials
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 |
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Citer Cet Article
Zorba Yildiz, A. P., Darici, H., Yavuz, B., Abamor, E. S., Ozdemir, C., Yasin, M. E., Bagirova, M., Allahverdiyev, A., Karaoz, E. Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering. J. Vis. Exp. (183), e63622, doi:10.3791/63622 (2022).