Name: preparation and characterization of graphene-based 3d biohybrid hydrogel bioink for peripheral neuroengineering
Uploaded: 2022-05-16T14:00:00
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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。

1. 沃顿商学院果冻间充质干细胞的培养
<ol><li>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。</li>
	<li>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。?...

<ol>
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P., 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=Graphene-based+nanocomposites+for+neural+tissue+engineering.">Graphene-based nanocomposites for neural tissue engineering.</a> Molecules. 24 (4), 658 (2019).</li><li>Othman, S. A., 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=Alginate-gelatin+bioink+for+bioprinting+of+HeLa+spheroids+in+alginate-gelatin+hexagon-shaped+scaffolds.">Alginate-gelatin bioink for bioprinting of HeLa spheroids in alginate-gelatin hexagon-shaped scaffolds.</a> Polymer Bulletin. 78, 6115-6135 (2021).</li><li>Peng, X. 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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...

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。

<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

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。

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 <0.01）时显着降低。其他组与对照组比较差异无统计学意义（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?...

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

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。

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

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

干细胞系统受到其重要成分的影响。如果没有干细胞的微环境的存在，干细胞的保存和分化是不可想象的。这种微环境称为干细胞生态位，由细胞和支架组成。周围神经病变可以是，偶尔，它们可能是臂丛损伤，各种创伤，肿瘤，自主神经异常，免疫定义和代谢性疾病。已经开发了各种3D培养方法，为干细胞提供更好，更自然的生态位。球状体形成和3D生物打印是相对较新和有前途的3D培养方法。3D生物打印也可用于神经工程研究。因此，在这项研究中，开发了基于石墨烯和藻酸盐/明胶的生物墨水，并研究了它们的再生特征。首先，在室温无菌层流下，在含有 10% 胎牛血清或 FBS、1% 笔/链球菌和 1% L-谷氨酰胺的 DMEM F12 培养基中培养沃顿的果冻间充质干细胞或 WJMSC。当培养的细胞在烧瓶中80%汇合时，倒入培养基并用5毫升PBS洗涤细胞。然后加入5毫升0.25%胰蛋白酶和2.21毫摩尔EDTA钠，并在37摄氏度下孵育。五分钟后，向细胞中加入10毫升含有10%FBS的DMEM F12培养基。悬浮细胞，收集培养基并将其转移到离心管中。接下来，离心细胞并弃去上清液，然后将细胞重新接种到含有10%FBS的新鲜营养培养基的新烧瓶中。为了制备不含石墨烯的对照组的生物墨水，称取4.5毫克藻酸盐和1.5毫克明胶，并将它们转移到离心管中。然后将 50 毫升含有 10% FBS 的 DMEM F12 培养基加入试管中。再次称取4.5毫克海藻酸盐和1.5毫克明胶，并将它们转移到离心管中。然后在管中加入50微升0.1%石墨烯，并用含有10%FBS的DMEM F12培养基使体积为50毫升。通过移液和涡旋混合生物墨水，然后在 121 摄氏度下 1.5 下高压灭菌。大气压20分钟。高压灭菌后，离心溶液以去除形成的气泡，并将生物墨水置于37摄氏度，直到准备好细胞。对于细胞生物墨水相互作用，创建生物墨水组。第一组包括用生物墨水打印的3D-B和3D-G，用于生物打印。第二组包括3D-BS和3D-GS生物墨水，生物打印后在其上形成球体。对于第一组，将细胞计数为1乘10至0.5毫升培养基中的第七个细胞。然后加入4.5毫升生物墨水。使用注射器将混合物转移到无菌柜中的墨盒中。将墨盒安装在生物打印机的相应挤出机部分。对于第二组，从每个生物墨水组中取出五毫升生物墨水，并在注射器的帮助下将它们转移到无菌墨盒中。使用带有两个同轴打印头和气动驱动挤出技术的生物打印机。将每微步的XYZ分辨率设置为1.25微米，将挤出宽度设置为400微米，将挤出高度设置为200微米。使用 20 x 20 x 5 毫米的网格创建 3D 模型。使用基于 Web 的开源 CAD 程序创建 3D 模型。对于3D生物打印过程，将打印机的平均压力设置为7.5 psi。然后将墨盒和床身温度设置为 37 摄氏度，速度设置为 60%在写入阶段将系统置于原位。在开始生物打印过程之前，自动定位轴并选择和设置挤出机。打印后，取出样品并将其放在层流柜下。接下来，用0.1普通氯化钙溶液喷洒生物墨水，或在室温下用移液管加入一毫升溶液，等待10至20秒。然后用含有钙和镁的PBS清洗印刷图案两次。在每个含有生物墨水基团的细胞顶部加入两毫升含有10%FBS的DMEM F12培养基，并将板在37摄氏度下与5%二氧化碳孵育30分钟。接下来，向每组的第六个细胞中加入两毫升含有1×10的悬浮培养基，并孵育板。24小时后，在倒置显微镜下观察并拍摄所有批次的生物墨水以形成球状体，以便WJMCs分化为神经元样细胞，对照组除外。每孔加入两毫升神经分化培养基，每两天更新一次。随访七天以观察神经分化。接下来，使用延时成像，检查石墨烯对干细胞的影响并监测生物墨水中的细胞相互作用。这里展示了石墨烯浓度对细胞增殖的影响。与对照相比，观察到0.001%石墨烯浓度显着降低。其他组与对照组之间无显著差异。细胞石墨烯相互作用表明，石墨烯在2D系统中是耐受的，并通过内吞作用被细胞吸收。延时成像显示，在3D石墨烯培养基中存活的细胞通过GFP亮度保持其活力，直到孵育结束。此处显示了 3D-B 和 3D-G 生物墨水组的 SEM 图像和 FIIR 分析。生物墨水细胞相互作用在表面和内部都得到了证实。细胞在形态上是圆形的，并附着在材料上。在3D神经元分化中，认为两组球状细胞的边界透明而活泼，石墨烯组中的球体相对较大，并将石墨烯困在细胞内。此处显示了 2D 和 3D 细胞的免疫染色。绿色图像代表神经元样结构。2D阳性对照样品表达的神经元样结构标志物少于3D样品。我们发现基于石墨烯的生物墨水在干细胞分化为神经元样细胞方面更成功。我们建议基于石墨烯的生物墨水将成为进一步研究中治疗周围神经疾病的优秀工具。今天，干细胞系统可以通过组织工程与天然和合成生物材料创建人造组织，可以取代可用于这些组织再生的活组织，消除损伤并提供功能由组织工程提供。

Research

JoVE Journal

Bioengineering

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

科研

生物工程

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

纤维素碳纤维溶胶单向冷冻干燥制备 Microhoneycomb 石柱: 方法和外延

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

三维印刷多孔纤维素纳米复合水凝胶脚手架

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

用于打印 3D 细胞-拉丹胰腺组织构造的胰腺组织衍生细胞外基质生物墨水

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

在三维生物打印中使用多层水凝胶生物油墨，实现均匀细胞分布

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Quantitative Characterization of Bioink Properties for Continuous DLP Printing

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing

Quantitative Characterization of Liquid Photosensitive Bioink Properties for Continuous Digital Light Processing Based Printing (Video) | JoVE 

基于连续数字光处理的印刷中液体光敏生物墨水特性的定量表征

Precise printing fabrication of bioinks is a prerequisite for tissue engineering; the Jacobs working curve is the tool to determine the precise printing ...