Name: 3d printed porous cellulose nanocomposite hydrogel scaffolds
Uploaded: 2019-04-24T13:00:00
Description: Watch this Scientific Journal Video about 3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds at JoVE.com

这项研究得到了 Knut 和 Alice Walenberg 基金会 (Wallenberg Wood 科学中心)、瑞典研究理事会、VR (Bi顾名思义、DNR 2016-05709 和 DNR 2017-04254) 的资助。

1. 前体的制备
<ol><li>纤维素纳米晶悬浮液的制备 注: 纤维素纳米晶的分离是按照 Mathew 等人报告的程序进行的.<ol>
<li>加入蒸馏水, 将纤维素纳米晶的 17 wt% 悬浮液稀释至 2 wt%, 使其总量彻底为 2 L 混合, 使用超超声, 并使用较小的批次 (250-300 mL) 进行高效混合。</li>
		<li>在 500-600 bar 的压力下, 10次通过均质机通过声纳悬浮液。此时, 得到了 2 wt% 纤维素纳米晶的厚透明凝胶。</li>
		<l...

<ol>
	<li>Moon, R. J., Martini, A., Nairn, J., Simonsen, J., Youngblood, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+nanomaterials+review:+structure,+properties+and+nanocomposites.">Cellulose nanomaterials review: structure, properties and nanocomposites.</a> Chemical Society Reviews. 40 (7), 3941-3994 (2011).</li><li>Dufresne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocellulose:+a+new+ageless+bionanomaterial.">Nanocellulose: a new ageless bionanomaterial.</a> Materials Today. 16 (6), 220-227 (2013).</li><li>Chinga-Carrasco, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+limitations+of+nanocelluloses+as+components+in+biocomposite+inks+for+three-dimensional+bioprinting+and+for+biomedical+devices.">Potential and limitations of nanocelluloses as components in biocomposite inks for three-dimensional bioprinting and for biomedical devices.</a> Biomacromolecules. 19 (3), 701-711 (2018).</li><li>Naseri, N., Poirier, J., Girandon, L., Fr&#246;hlich, M., Oksman, K., Mathew, A. 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L., Choong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+scaffolds+for+tissue+engineering+applications:+role+of+porosity+and+pore+size.">Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size.</a> Tissue Engineering, Part B: Reviews. 19 (6), 485-502 (2013).</li><li>Bru&#382;auskait&#279;, I., Bironait&#279;, D., Bagdonas, E., Bernotien&#279;, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+and+cells+for+tissue+regeneration:+different+scaffold+pore+sizes-different+cell+effects.">Scaffolds and cells for tissue regeneration: different scaffold pore sizes-different cell effects.</a> Cytotechnology. 68 (3), 355-369 (2016).</li><li>Zhang, L., Hu, J., Athanasiou, K. 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3D 打印需要适当的水凝胶油墨流变特性。高粘度油墨的挤出需要极高的压力, 而低粘度油墨在挤出后不会保持其形状。水凝胶油墨的粘度可以通过成分的浓度来控制。与我们之前的工作11相比, 水凝胶油墨的固体含量从 5.4% 增加到 9.9 wt%, 从而产生浓缩的水凝胶油墨, 这有助于提高印刷脚手架的分辨率。可以注意的是, 与长柔性氯化萘不同的是, 由于物理纠缠的缺失, 像氯化萘这样的?...

纤维素是由β (1-4) 连接 d-葡萄糖单位的线性链组成的多糖。它是地球上最丰富的天然聚合物, 从各种来源提取, 包括海洋动物 (如金枪鱼)、植物 (如木材、棉花、小麦秸秆) 和细菌来源, 如藻类 (如 valonia)、真菌, 甚至阿米巴 (原生动物))1,2。纤维素纳米纤维 (CNF) 和纤维素纳米晶 (CNC), 通过机械处理和纤维素酸水解得到, 纳米尺度上至少有一个维度。它们不仅具有化学改性、低毒性、生物相容性、生物降解、可再生等特性, 而且具有高比表面积、高机械性能等纳米级特性、流变和光学特性。这些吸引人的特性使 Cnf 和 Cnc 适合生物医学应用, 主要是以三维 (3D) 水凝胶支架3的形式。这些支架需要定制的尺寸, 具有可控的孔隙结构和相互关联的孔隙率。我们的小组和其他人报告了通过铸造、电纺和冷冻干燥制备的3d 多孔纤维素纳米复合材料 4,5,6, 7,8。然而, 通过这些传统技术, 并不能实现对孔隙结构的控制和复杂几何的制作。
3D 打印是一种添加剂制造技术, 在这种技术中, 3D 对象通过计算机控制的墨迹沉积9逐层创建。与传统技术相比, 3D 打印的优势包括设计自由度、控制的宏观和微观尺寸、复杂架构的制作、定制和可重复性。 此外, Cnf 和 Cnc 的3D 打印还提供了剪切诱导的纳米粒子对齐、首选方向性、梯度孔隙率, 并且可以很容易地扩展到3D 生物印迹10,11, 12,13,14,15. 最近, 据报告, 3d 打印过程中的氯化萘对齐动态为16,17。生物冲洗领域的进步使3D 印刷组织和器官得以实现, 尽管存在相关的挑战, 如活细胞的选择和浓度和生长因子, 载体油墨的组成, 印刷压力和喷嘴直径18 ,19,20。
软骨再生支架的孔隙率和抗压强度是决定其效率和性能的重要特性。孔大小对细胞的粘附、分化和增殖以及营养物质和代谢废物的交换起着重要作用。然而, 目前还没有确定的孔径可以被认为是理想的值, 一些研究表明, 毛孔较小的生物活性较高, 而另一些研究则显示出更大的毛孔的软骨再生较好。大孔隙 (& lt;500 微米) 促进组织矿化、养分供应和废物清除, 而微孔 (15-250μm) 促进细胞附着和更好的机械性能22,23。植入的支架必须具有足够的机械完整性, 从处理、植入到完成其预期目的。据报告, 自然关节软骨的总体压缩模量在 0.1-2 兆帕之间, 具体取决于年龄、性别和测试位置4,24,25,26, 27 ,28,29。
在我们之前的工作11中, 3d 打印被用来制造一个双交联互穿聚合物网络 (ipn) 的多孔生物咖啡因从一个含有增强的 cnc 的水凝胶油墨在海藻酸钠和明胶的矩阵。对三维打印路径进行了优化, 以实现具有均匀梯度孔隙结构 (80-2125μm) 的三维支架, 其中纳米晶最好在打印方向定向 (取向程度在61-76 之间)。在这里, 我们提出了这项工作的延续, 并演示了孔隙率对模拟身体条件下三维印刷水凝胶支架力学性能的影响。这里使用的 Cnc, 我们早些时候报告说, 细胞相容性和无毒 (即, 细胞生长后15天的孵化被确认为 30)。此外, 使用相同的 Cnc、海藻酸钠和明胶通过冷冻干燥制备的支架具有较高的孔隙率、较高的磷酸盐缓冲碱吸收率和细胞与间充质干细胞的相容性5。本工作的目的是展示水凝胶油墨的加工、多孔支架的三维打印和压缩测试。处理路线的原理图如图 1所示。

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			<td height="21" style="height:21px;width:283px;">60 mL syringe</td>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
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			<td height="21" style="height:21px;width:283px;">Alginic acid sodium salt</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">9005-38-3</td>
			<td style="width:551px;"></td>
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			<td height="21" style="height:21px;width:283px;">Anhydrous calcium chloride</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">10043-52-4</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Clamps, three pronged, Talon</td>
			<td style="width:209px;">VWR</td>
			<td style="width:175px;">241-0404</td>
			<td style="width:551px;">102 mm, Dual adjustment clamp, large, clamp extension 127 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cura 2.4.0</td>
			<td style="width:209px;">Ultimaker</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Free slicing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Discov3ry Complete</td>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Gelatin from bovine skin</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">9000-70-8</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glutaraldehyde solution 50 wt. % in H2O</td>
			<td style="width:209px;">Sigma-Aldrich</td>
			<td style="width:175px;">111-30-8</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">homogenizer</td>
			<td style="width:209px;">SPX</td>
			<td style="width:175px;">APV-2000</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Instron 5960</td>
			<td style="width:209px;">Instron</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">Instron 5960, Biopuls Bath, 100 N load cell, 37 &#176;C,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Physica MCR 301 rheometer</td>
			<td style="width:209px;">Anton Paar</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">CP25-2-SN7617, gap height 0.05 mm, 25 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sorvall Lynx 6000 centrifuge</td>
			<td style="width:209px;">AB Ninolab</td>
			<td style="width:175px;">s/n 41881692</td>
			<td style="width:551px;">F12-rotor (6x500 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">stainless steel nozzle</td>
			<td style="width:209px;">Structur3D Printing</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">800, 600 and 400 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">thingsinverse</td>
			<td style="width:209px;">MakerBot&#39;s</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">&#160;sharing and downloading 3D printable things in form of stl files</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">ultra sonication</td>
			<td style="width:209px;">Qsonica, LLC</td>
			<td style="width:175px;">Q500</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Unbarked wood chips</td>
			<td style="width:209px;">Norway spruce(Picea abies)</td>
			<td style="width:175px;"></td>
			<td style="width:551px;">dry matter content of 50&#8211;55%</td>
		</tr>
	</tbody>
</table>

3d printed porous cellulose nanocomposite hydrogel scaffolds

这项工作演示了使用三维 (3D) 打印生产多孔立方支架使用纤维素纳米复合水凝胶油墨, 具有受控制的孔隙结构和机械性能。纤维素纳米晶 (Cnc, 6.962 wt%)开发了以基质 (海藻酸钠和明胶) 为基础的水凝胶油墨, 并将三维印刷成具有均匀梯度孔隙结构 (110-1100μm) 的支架。在体内模拟条件下 (37°c 的蒸馏水) 中进行测试时, 支架的压缩模量在 0.20-0.45 MPa 之间。三维支架的孔径和压缩模量符合软骨再生应用所需的要求。这项工作表明, 油墨的一致性可以通过前体的浓度来控制, 孔隙度可以通过三维打印过程来控制, 而这两个因素反过来都决定了三维打印多孔的机械性能水凝胶脚手架。因此, 这种工艺方法可用于根据患者的具体需要制作结构和组合定制的支架。

基于 Cnc 的纳米复合水凝胶油墨表现出很强的非牛顿剪切变薄行为 (图 2a)。在低剪切速率 (0.001 s-1) 下, 1.55x10 5 pa. s 的表观粘度在50秒-1 (50s-1是3d 打印过程中经历的典型剪切速率) 下降到22.60 帕. s 的5个数量级。.水凝胶油墨表现出粘弹性固体行为, 因为在低剪切应力下, 储存在模量 G ' (4.42x107 pa)大?...

Watch this Scientific Journal Video about 3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds at JoVE.com

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

本协议的三个关键步骤是: (i) 开发纤维素水凝胶油墨的正确成分和一致性, (ii) 将支架三维打印到形状保真度和尺寸良好的各种孔隙结构中, 以及 (三)在模拟身体条件下的机械性能, 用于软骨再生。

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

这是关于3D打印纳米纤维素水凝胶脚手架的第一份报告，我们有一个梯度孔隙结构和机械性能，以模仿自然结构作为软骨。使用 3D 打印技术的两个主要优点是定制和设计自由。这为制造新的和未开发的几何设计提供了无限的机会。该协议是用户友好的，新手可以很容易地重现结果。切片软件的选择和喷嘴的移动对最终产品有显著影响。首先，在容器中混合11%重量CNC，6%按重量含盐酸钠，12%按重量明胶混合40毫升水凝胶油墨。将混合物加热至40摄氏度，并用铲子混合，直到获得光滑的糊状物。将混合物转移到60毫升注射器中。接下来，在机械夹的帮助下，将混合物通过一系列不同直径的喷嘴进入另一个 60 毫升的注射器中。重复该工艺，直到获得水凝胶油墨的平滑挤出丝。以 4，000 倍 g 轻轻将装满水凝胶墨水的注射器离心，以清除被困的空气。从 SD 卡中，选择保存的文件以用于均匀和梯度孔隙基架，然后开始打印。如果需要，相应地调整速度和流速。要在 3D 打印完成后将脚手架交叉链接，请轻轻将重量氯化钙溶液滴下 3%添加到脚手架中，直到其完全湿润。等待五分钟。非常仔细地将脚手架从打印机床转移到一个 50 毫升的容器中，容器中填充了 3% 的重量氯化钙溶液。留一夜。用蒸馏水彻底清洗，将脚手架转移到一个50毫升的容器中，用重量填充3%的谷胱甘肽溶液。留一夜。彻底清洗，将 3D 打印脚手架存放在蒸馏水中。对于压缩测试，将装有潜水压缩底板的容器加满两升水，并启动加热系统达到37摄氏度。初始化蓝山通用软件，并设置测试方法。选择矩形试样几何体，并选择在测试每个样本之前输入尺寸的选项。将应变速率设置为每分钟 2 毫米，结果为 80% 压缩应变以及 90 牛顿力。在"测量"部分中，选择"力"、位移、压缩应力和压缩应变。选择将数据导出为文本文件以用于将来绘图的选项。使用慢跑控制装置将横头板尽可能靠近底板，设置零延长点。测量并记录要测试的样品的尺寸。当水温达到37摄氏度时，将样品放在底板上。通过移动横头板固定样品，以便它开始接触样品。将水浴向上移动，使其中包含样品的盘子浸入水中。输入示例名称和维度，然后开始测试。测试完成后，先将水浴向下移动，然后升起横头板。取出样品及其碎片（如果有）清洁两个板，并加载新样品。测试所有样本后，导出原始数据。绘制压缩应力与压缩应变曲线，并确定基于 1 到 5% 和 25 至 30% 的基于 CNC 的纳米合成水凝胶油墨的压缩切线模量，显示出一种强烈的非牛顿剪切变薄行为，其明显粘度下降为 5 个数量级。水凝胶油墨表现出粘弹性固体行为，因为存储模量比低剪切应力下损耗模数大数量级。在1至5%的低应变率下，与无孔隙的参考基架相比，所有类型的多孔基架的压缩模量相似，表明即使存在微孔，水凝胶油墨的弹性性质也保留下来。然而，在25至30%的高应变率下，为无孔隙的参考脚手架获得了最高的模量。一旦孔径增大，模量会因密度降低而减小，表明脚手架的孔隙度与相应的机械性能之间的预期关系。此外，3D 水凝胶支架的压缩模组随着压缩率的增加而增加，表现出并模仿天然软骨组织的粘弹性。在 3D 打印过程中，油墨的均匀和连续流动是最重要的。研究人员将采用纳米纤维素水凝胶作为3D可打印平台，将这种方法扩展到其他应用领域。例如，我们已经开发了基于纳米纤维素的杂交种，用于按照相同的程序进行控制药物释放。

Research

JoVE Journal

Bioengineering

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

科研

生物工程

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 ...

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 ...

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 ...

Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

使用患者专用的 3D 打印肺模型评估区域肺沉积

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

3D Cell-Printed Hypoxic Cancer-on-a-Chip for Recapitulating Pathologic Progression of Solid Cancer

3D 细胞打印的芯片上低氧癌症，用于回顾固体癌症的病理进展

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Gelatin Methacryloyl Granular Hydrogel Scaffolds: High-throughput Microgel Fabrication, Lyophilization, Chemical Assembly, and 3D Bioprinting

明胶甲基丙烯酰基颗粒水凝胶支架：高通量微凝胶制造、冻干、化学组装和 3D 生物打印

The emergence of granular hydrogel scaffolds (GHS), fabricated via assembling hydrogel microparticles (HMPs), has enabled microporous scaffold formation ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels (Video) | JoVE

3D生物打印光可调水凝胶研究成纤维细胞活化

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...