三维印刷多孔纤维素纳米复合水凝胶脚手架
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 60 mL syringe | Structur3D Printing | ||
| Alginic acid sodium salt | Sigma-Aldrich | 9005-38-3 | |
| Anhydrous calcium chloride | Sigma-Aldrich | 10043-52-4 | |
| Clamps, three pronged, Talon | VWR | 241-0404 | 102 mm, Dual adjustment clamp, large, clamp extension 127 mm |
| Cura 2.4.0 | Ultimaker | Free slicing software | |
| Discov3ry Complete | Structur3D Printing | Ultimaker 2+ 3D printer integrated with Discov3ry paste extruder | |
| Gelatin from bovine skin | Sigma-Aldrich | 9000-70-8 | |
| Glutaraldehyde solution 50 wt. % in H2O | Sigma-Aldrich | 111-30-8 | |
| homogenizer | SPX | APV-2000 | |
| Instron 5960 | Instron | Instron 5960, Biopuls Bath, 100 N load cell, 37 °C, | |
| Physica MCR 301 rheometer | Anton Paar | CP25-2-SN7617, gap height 0.05 mm, 25 °C | |
| Sorvall Lynx 6000 centrifuge | AB Ninolab | s/n 41881692 | F12-rotor (6×500 ml) |
| stainless steel nozzle | Structur3D Printing | 800, 600 and 400 µm | |
| thingsinverse | MakerBot's | sharing and downloading 3D printable things in form of stl files | |
| ultra sonication | Qsonica, LLC | Q500 | |
| Unbarked wood chips | Norway spruce(Picea abies) | dry matter content of 50–55% |
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