这项工作得到了生物合作研究中心的资金支持, 它是用于细胞治疗制造、澳大利亚研究理事会的添加剂的弧形中心和慕尼黑技术大学的高级研究研究所。这项研究是由澳大利亚研究理事会工业改造培训中心在添加剂生物 http://www.additivebiomanufacturing.org (IC160100026) 进行的。请访问该网站的文章, 书籍, 电视或广播节目, 电子媒体, 或任何其他与该项目相关的文学作品。此外, 作者感激地承认玛丽亚 Flandes Iparraguirre 为支持在摄制, 菲利普哈伯德为声音和 Luise Grossmann 为摄制和编辑。

1. 材料制备
<ol>
	<li>在3毫升塑料注射器与漏斗, 填补2克 PCL 和插入活塞到开放的一端。</li>
	<li>将注射器放在预热的烤箱中, 在65° c 为8小时. 点向上, 使气泡聚集接近开口。</li>
	<li>用一个薄的物体推动活塞, 以释放被困的空气内的熔融材料。</li>
	<li>让它降温到室温, 这是达到时, 聚合物是不透明的, 在10分钟后。</li>
	<li>将 PCL 预装的注射器存放在干燥和黑暗的环境中, 直到使用。</li>
</ol>
2. 硬件和软件设置
<ol>
	<li>在注射器和另一端的枪管接头上附上一个23G 平的尖针 (喷), 将注射器连接到气压系统。</li>
	<li>放置在打印头的注射器, 并按下它, 直到喷尖端脱颖而出1毫米从黄铜部分的底部一侧的头部。</li>
	<li>在舞台上安装一个收集器和清洁的表面以及打印头与 70% (卷/卷) 乙醇清除灰尘或残余聚合物。</li>
	<li>设置工作距离, 在喷和收集器之间定位一个 12 mm 高的对象, 并降低打印头, 直到喷笔尖刚好触及它。</li>
	<li>将电箱上的温度调节器调整到82° c 和65° c, 分别用于喷区域和注射器, 并将其通电以熔化 PCL。</li>
	<li>等待至少10分钟, 直到聚合物熔化, 并启动气压通过设置调节器到1.8 巴。</li>
	<li>准备 G 代码定义的大小和形状, inter-filament 距离和层数的脚手架和收集速度的过程。 注: 在讨论一章 (表 2) 中提供了用于制作扁平和管状脚手架的详细模板。</li>
	<li>手动检查所有接地电缆是否已牢固地连接到机箱和墙上的插头。</li>
	<li>启动计算机上的软件 (例如, 马赫 3) 并上传准备好的 g 代码。</li>
</ol>
3. 脚手架制作
<ol>
	<li>关闭机箱的前门, 连接安全联锁, 并触发高压电源到喷。 注: 一旦门打开, 例如当打印完成或在紧急情况下, 高电压下降和脚手架可以安全地删除。</li>
	<li>在 0.2 kV 步骤中逐渐增加高电压, 直到形成一个泰勒锥, 并向收集器弹出光纤 (参见图 1D中的模范泰勒锥)。</li>
	<li>允许聚合物熔体在静止的收集器板上挤压, 以稳定喷嘴, 不移动5分钟。在开始新的打印之前, 请先除去这堆材料。</li>
	<li>使用键盘上的光标将打印头移到 G 代码将启动的点上方。</li>
	<li>在计算机上的软件中启动 G 代码。</li>
</ol>
4. 纤维直径调整
<ol>
	<li>将工作距离 (12 mm) 和温度调节器 (82 ° c 和65° c 分别用于喷区域和注射器) 保持恒定的水平, 如步骤2.4 和2.5 所述。 注: 在表 3中给出了调整不同直径的摘要。</li>
	<li>打印直径小的纤维 (3-10 µm)。将气压级别降低到 0.8 bar, 将应用电压调整到8伏, 并将收集器速度设置为 1700 mm/分钟。</li>
	<li>打印中等大小直径的纤维 (10-20 µm). 调整气压等级为1.5 巴, 设置电压为 11 kV, 并降低收集速度为1200毫米/分。</li>
	<li>打印大直径的纤维 (20-30 µm)。将气压水平提高到 2.6 bar, 将应用电压改变为12伏, 并将采集速度降低到700毫米/分。</li>
</ol>
5. 射流优化
<ol>
	<li>照亮了从外壳外部的强 LED 灯的喷气机, 以提高能见度。</li>
	<li>观察纤维的行为1分钟, 并调整系统参数以在小步骤中优化过程,即0.1 kV 的应用电压, 100 毫米/分钟的收集速度和0.1 酒吧的气压。 注意: 在表 4中给出了一个摘要。</li>
	<li>通过减少气压, 增加速度和最小化电压来稳定周期性偏转行为, 直到纤维的飞行路径类似于稳定的接触网曲线超过3分钟。</li>
	<li>通过增加电压、减少空气压力和降低收集器的速度来修正滞后于射流的飞行路径。应用这些措施, 直到纤维的飞行路径回到一个接触网曲线形状。</li>
	<li>避免纤维垂直向收集器移动, 减少施加的电压, 增加收集器的速度, 增加空气压力, 直到飞机的飞行路径再次保持了接触网曲线的形状。</li>
</ol>
6. 脚手架收藏
<ol>
	<li>当打印完成时打开门, 使用光标将收集器板移到门上以获得更好的可访问性。</li>
	<li>用乙醇 70% (卷/卷) 喷洒支架, 等待10秒, 直到它与收集器明显分离。</li>
	<li>收集完成的脚手架, 抓住一个边缘与镊子和解除它的外壳。</li>
</ol>
7. 故障排除
<ol>
	<li>如果在喷可见或有裂纹的声响之间有火花, 请立即降低应用电压或打开门。</li>
	<li>将所有危险物质和液体, 如乙醇 70% (卷/卷) 从外壳内移走, 以防起火, 可能引发火灾。</li>
	<li>程序的 G 代码相应的喷移动距离的地方, 脚手架是在所有层完成后打印。这避免了物质堆积在喷最后停止的点之上。</li>
	<li>检查放大镜下的喷并验证喷没有损坏, 因为这将显著影响泰勒锥的均匀性。</li>
</ol>

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F., Luxenhofer, R., Groll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+definition+fibrous+poly(2-ethyl-2-oxazoline)+scaffolds+through+melt+electrospinning+writing.">High definition fibrous poly(2-ethyl-2-oxazoline) scaffolds through melt electrospinning writing.</a> Polymer. 55 (20), 5017-5023 (2014).</li><li>Chen, F., 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=Additive+Manufacturing+of+a+Photo-Cross-Linkable+Polymer+via+Direct+Melt+Electrospinning+Writing+for+Producing+High+Strength+Structures.">Additive Manufacturing of a Photo-Cross-Linkable Polymer via Direct Melt Electrospinning Writing for Producing High Strength Structures.</a> Biomacromolecules. 17 (1), 208-214 (2016).</li><li>Hochleitner, G., 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=Additive+manufacturing+of+scaffolds+with+sub-micron+filaments+via+melt+electrospinning+writing.">Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing.</a> Biofabrication. 7 (3), 035002 (2015).</li></ol>

作者没有什么可透露的。

整合 AM 为医疗领域的挑战找到创新的解决方案为 21st世纪提供了一个新的范例。"生物制造" 的 so-called 领域正在兴起, 制造技术的创新使得为 TE 应用生产高度复杂的体系结构。聚合物在直接写入模式下的静电纺丝被认为是最有希望的制造候选者之一, 以满足 TE 社区的需要, 在微米到纳米级的生物相容性材料的有序结构必需的27。
本教程旨在通过解释物理原理和演示使用此技术制造可再生支架的操作步骤, 来生成对水电部操作的基本知识。
由于水电部的一般原则与常规的添加剂制造技术相媲美, 即以层状的方式将挤压材料的靶向沉积, 因此控制头部与器.根据我们的经验, 我们建议使用可保持固定头的水电部设备, 而收集器的各自运动则由阶段 (X 和 Y) 进行。一个固定的头保持在一个稳定的位置, 并没有产生运动力, 这将采取行动的泰勒锥, 并可能导致干扰期间, 它的创作。此外, 与高压和加热器相关的布线不受持续重复运动的限制。收集器运动由 G 代码定义, 需要在软件中上载。该代码也被称为 RS-274, 广泛应用于计算机辅助制造领域, 以控制工具的通路。对于具有平面收集器的水电部应用程序, G 代码文件决定了 X 和 Y 方向的移动和速度;对于圆柱形收集器或芯轴应用, G 代码文件定义了平移 (X 方向) 和旋转速度。表 2详细说明了 G 代码的编程。
与其他添加剂制造技术相比, 水电部可以通过调整系统参数温度、采集速度和应用电压来制造各种直径的纤维, 如协议所述。
为了实现小纤维 (3-10 µm), 建议使用低压力, 中等电压和高收集速度。一般来说, 减压会导致挤压质量降低。这是伴随着相应的减少的表面面积的喷气机。因此, 需要更小的静电力来加速纤维向收集器的质量, 即需要应用较低的电压。此外, 可比较高的收集速度导致纤维的拉伸增强, 从而导致最终纤维直径的进一步降低。
增加压力会引起更多的熔融聚合物流动, 从而导致较大的纤维直径 (10-20 µm)。在这种情况下, 需要更大的静电力来补偿放大的聚合物表面 (较厚的纤维)。为了获得稳定的聚合物射流, 必须改变电压, 降低收集速度。
大纤维直径 (20-30 µm) 要求增强聚合物挤出, 即更高的气压。这激起相对较厚的纤维, 并建议与较高的电压结合使用, 以提供足够的静电在纤维上的力量。另外, 减少的收集速度导致较少纤维舒展。在表 3中给出了摘要。
上述所有三例, 但仍需要 fine-tuning 和优化, 以维持一个稳定的接触网曲线形状的纤维随着时间的推移, 解释在协议中。在水电部, 只有在确定聚合物质量的流动的力和吸引射流对收集器的力之间的平衡平衡才会最终导致达到一致的支架形态12,28.因此, 射流路径的分歧反映了纤维直径的强烈偏差或不准确的沉积。从我们的经验, 三不同的行为可以得到的变化。
首先, 一个光纤可以脉冲, 一个由道尔顿组12最初报告的现象。不平衡的分布在交付的大量和各自的拖曳力量之间在纤维起因于一个经常吃泰勒锥体, 周期性地发布积累的聚合物。这会引起通路角度的显著变化, 并导致不同直径的结果。
第二, 当收集器的速度高于射流的挤出速度时, 会发生滞后的带电射流。最后的喷射沉积发生在远离垂直方向的喷, 造成一个滞后的喷气流。飞行路径类似于过分强调的曲率, 这也使印刷脚手架的尺寸最小化。
第三, 当收集器速度比射流流出喷的速度慢时, 产生屈曲带电射流的原因是射流对收集器和显示的垂直影响。使用高电压也会导致屈曲, 通过对收集器产生过度的加速度和纤维的直线飞行路径。在这种情况下观察到循环的不受欢迎的沉积。
re-stabilize 的方法在协议中提供, 并显示在表 4中。
从脚手架实施的角度来看, 在使用 PCL 和水电部时存在多重好处, 例如生物相容性、直接写入的再现能力或由此产生的体系结构预先设计的定制。水电部可以在任何传统的实验室工作台上进行, 因为它使用无溶剂聚合物熔体, 因此它不需要昂贵的油烟罩或彻底回收剩余材料29。进入装有水电部设备的房间时没有异味。
此外, 一个可实现的高表面体积比在一个多孔脚手架是非常有利的, 使水电部支架很适合生物应用30。
与著名的3D 印刷技术, 如熔融沉积模拟31相比, 水电部在可打印的有序结构高度上有局限性。原因是在应用静电力的内在过程中看到的, 它在沉积的纤维中捕获移动电荷载流子。一旦脚手架的高度超过约4毫米, 据报道, 在脚手架中累积的超额电荷的总和将对即将到来的纤维32起排斥作用。随后, 在大多数情况下, 产生的顶层被严重扭曲。
另一个不同于传统的3D 印刷技术的事实是, 在过程中的材料沉积不能中断, 并完全停止所有系统参数最终持有材料挤出。这代表了一个设计限制, 并且在编写 G 代码时必须加以看待。虽然 jet 启动可以机械地执行33, 但 G 代码编程需要考虑一种连续的直写方法。
提高水电部的吞吐量和流程效率仍然是一项挑战, 也是我们认为的主要原因, 也是我们的观点和其他人的理由.首先, 由于低流量和有限的采集速度, 在吞吐量方面, 水电部过程本身就很低。这两个方面, 但是, 是必不可少的, 以确保控制沉积的射流和重现性的印刷。实际上, 在印刷过程中, 最大的采集速度限制在所用材料的物理边界上, 即过高的速度会在阻力超过可行极限时导致射流破损。另一项针对高端的策略是使用 multi-extruding 的新设备, 即与多个打印头之间的距离相近的机器;然而, 这些多头会导致每个头部的电场之间的干扰, 并随后扭曲最后的纤维沉积35。针熔体静电纺丝头产生了大量的带电射流36, 虽然控制直接写入纤维的精确位置可能很难实现。然而, 今后在提高水电部效率方面的发展不仅有益于生物医学界, 而且也有利于过滤、纺织或能源应用的工业2。
虽然本教程提供了在建议的参数设置下制作定制脚手架的指南, 但必须指出, 环境条件 (如室温或湿度) 存在较小的依赖性, 可能导致意外偏差37。本教程的结果是基于 know-how 积累的 Hutmacher 组, 在稳定的环境条件下进行控制实验室空间。
PCL 是水电部最突出的候选人。从工程的角度来看, 它的低熔点 (60 ° c) 是有益的, 因为这并不需要有挑战性的实施高温加热器 (和 #62; 和 #62; 100 ° c) 的密切距离, 以高压源。在材料工程的水平, PCL 是导电, 并提供强大的大分子凝聚力, 作为一个流体和固体。尽管机械拉伸强度很大, 但粘性材料在一定程度上会产生明显的纤维细化, 提高了收集器的速度或施加的电压。传统的熔体静电纺丝没有移动收集器已经报告了不同的聚合物, 如聚丙烯, 聚乙烯或尼龙9。直接写作原则的应用然而, 主要地报告了与 pcl 和一些 pcl 混合物与添加剂进一步降低它的黏度38, 虽然有例外39,40。然而, 在未来, 我们预见到水电部处理的材料范围更广。这反过来又意味着这项技术的硬件组件升级, 例如处理聚丙烯 (熔点在 160) 改变了目前的技术要求的硬件的水电部设备。
具有高度精确和可控结构的生物相容性高分子支架的兴趣日益浓厚;迄今为止, 水电部是唯一的技术, 与其他生物技术相比, 它能够在较低微米范围内制造有序体系结构 (在亚微米范围41中有例外)。在过去几年中, 这导致了数量成倍增长的专利和出版物30。因此, 通过实施优化的硬件和建立对水电部的过程控制来解决技术复杂性是非常重要的。这将有助于在今后的广泛应用中制作具有定制结构的脚手架。

三维 (3D) 细胞生物相容结构的制造是在组织工程 (TE) 中添加生物的关键贡献之一, 其目的是通过应用定制材料、细胞、生化因子来恢复组织,或他们的组合。因此, 用于 TE 应用的支架的主要要求包括: 生物相容性材料的可制造性、靶细胞侵袭的可控形态特性和增强细胞相互作用的优化表面特性1。
水电部是一种无溶剂的制造技术, 它将添加剂制造 (通常称为3D 印刷) 和静电纺丝的原理结合起来, 用于生产具有高度有序超薄纤维形态的聚合物网格2。它是一种直接的写作方法, 根据预先编写的代码3准确地存放纤维, 称为 G 代码。熔体纺结构目前准备使用一个平面4,5或一个芯轴6,7收集器, 分别制造多孔扁平和管状脚手架。
这项技术为 TE 和再生医学 (RM) 社区提供了显著的好处, 因为有可能直接打印医用级聚合物, 如聚 (ε-内) (PCL), 其中提出了良好的生物相容性8。其他的优势是有可能定制的大小和分布的孔隙率, 通过沉积纤维的高度有组织的方式来制造高表面积率的脚手架。在可以执行水电部之前, 聚合物首先需要应用热9。一旦处于流体状态, 施加的气压迫使它通过连接到高压源的金属喷流出。在表面张力和静电带电液滴对接地收集器的吸引力之间的力平衡导致泰勒锥的形成, 接着是喷射器的10。
图像和 in-house 构建用于此协议的设备的示意图图如图 1所示。此外, 还说明了使用绝缘胶带避免发热元件与喷周围带电黄铜部件之间的电放电的原理。绝缘不足会导致所实施硬件的内部损坏。
根据三系统参数 (温度、采集速度和气压) 的调整, 水电部可以在讨论部分中解释不同直径的纤维的制造。然而, 在大多数情况下, fine-tuning 和优化的喷气机将需要之前, 稳定的喷气机将被弹出。带电射流的可视化是验证过程一致性和均匀性的有效方法。在理想情况下, 飞行路径类似于由于由系统参数11控制的力平衡而获得的链路曲线。此外, 支架的微观和宏观结构取决于聚合物射流的飞行路径12。讨论部分给出了不同挠度行为和优化措施的详细表。
在本研究中, 我们提出了一个协议, 描述的制造步骤, 生产高控制纤维脚手架利用新技术。在这项工作中, 医疗级 pcl (分子量95-140 公斤/摩尔) 的使用, 因为这个医疗级 pcl 提高纯度超过技术等级, 其机械和加工性能是优良的水电部。PCL 的广泛熔体加工范围来源于其低熔点 (60 ° c) 和高热稳定性。此外, PCL 是一种慢速生物降解聚合物, 它使它成为许多组织工程应用的优秀材料13。
对于这项研究, 温度和收集器距离将保持恒定 (65 ° c 和82° c 为注射器和喷温度 (分别) 和12毫米为收藏家距离);应用电压, 收集器速度和气压, 但是, 将不同的制造有目标直径的纤维。在结果部分中提供了一份详细的研究报告, 并展示了 TE 和 RM 领域的不同应用 (表 1)。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plastic syringe</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012072</td>
			<td>EFD BARREL O 3mL Clear 50</td>
		</tr>
		<tr>
			<td>Medical grade Poly (&#949;-caprolactone) (mPCL)</td>
			<td>Corbion Purac, The Netherlands</td>
			<td>PURASORB PC12</td>
			<td></td>
		</tr>
		<tr>
			<td>23 GA needle</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7018302</td>
			<td>#23GP .013 X .25 ORANGE 50 PC</td>
		</tr>
		<tr>
			<td>Plunger</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012166</td>
			<td>PISTON O 3mL WH WIPER 50</td>
		</tr>
		<tr>
			<td>Pressure adapter</td>
			<td>Nordson Australia Pty Ltd</td>
			<td>7012059</td>
			<td>ADAPTER ASM O 3mL BL 1.8M</td>
		</tr>
		<tr>
			<td>Aluminium collector</td>
			<td>Action Aluminium, Australia</td>
			<td>SHP2</td>
			<td>Sheet 5005 H34</td>
		</tr>
		<tr>
			<td>Acrylic glass</td>
			<td>Mulford Plastics Pty Ltd</td>
			<td>ACC6-13094</td>
			<td></td>
		</tr>
		<tr>
			<td>Mach 3 software</td>
			<td>Art Soft</td>
			<td></td>
			<td>Purchased online</td>
		</tr>
		<tr>
			<td>Safety switch interlock</td>
			<td>RS components Pty Ltd</td>
			<td>12621330</td>
			<td></td>
		</tr>
		<tr>
			<td>High voltage generator</td>
			<td>EMCO High Voltage Co.</td>
			<td>DX250R</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>WATLOW</td>
			<td>PM9R1FJ</td>
			<td></td>
		</tr>
		<tr>
			<td>X and Y positioning slide</td>
			<td>VELMEX Inc.</td>
			<td>XN-10-0020-M011</td>
			<td></td>
		</tr>
	</tbody>
</table>

melt electrospinning writing of three-dimensional poly(&#949;-caprolactone) scaffolds with controllable morphologies for tissue engineering applications

本教程反映了静电纺丝的基本原理和指南的聚合物熔体, 这是一种具有极大的生物医学应用潜力的添加剂制造技术。该技术促进了生物相容性聚合物纤维的直接沉积, 在亚微米范围内制备有序的支架。建立一个稳定的, 粘弹性, 聚合物射流之间的喷和收藏家是实现使用的应用电压, 可以直接写入。典型的多孔支架的一个显著优点是高表面积比, 它为细胞附着和生长提供了更有效的粘合部位。通过 fine-tuning 系统参数控制印刷工艺, 可使印刷脚手架的质量具有很高的重现性。它还为用户提供了一个灵活的制造平台, 使脚手架的形态结构符合他们的具体要求。为此, 我们提出了一个协议, 以获得不同的纤维直径使用熔融静电纺丝写入 (水电部) 的参数, 包括流量, 电压和收集速度的指导修正。此外, 我们还演示了如何优化 jet, 讨论经常遇到的技术难题, 解释故障排除技术, 并展示各种可打印的脚手架体系结构。

在水电部中常用两种不同的收集方法, 即扁平收集和芯轴收集。生成的体系结构取决于软件执行的 G 代码 (表 2) 的编程。
平面系列 应用平面捕收器是指最常用的方法, 便于直接沉积的材料引用预编程的 G 代码。0/90 和0/60 不同大小的结构在文献中被广泛报道。此外, 直接沉积在收集器上的熔融纤维的能力也便于在使用带图案的平面收集器而不是平滑的一个14时随机组织结构的生成。
管 对于用于 TE 应用的管状结构的脚手架的制造有很大的需求。利用圆柱形集热器是实现具有定制孔隙度的管状支架的有效方法。这些旋转沿他们自己的轴, 而翻译沿轴的主轴。通过对 G 码的微调, 确定了旋转和平移速度, 并对光纤的方向进行了定制。比平动速度更高的转速导致径向取向的孔隙, 反之亦然。孔隙的总层数、分布和形貌将会配置支架的力学性能。管式脚手架的内径由所实施的芯轴的外径决定。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56289/56289fig1.jpg"> 
图 1: 水电部设置。(a) 包括一台 PC、打印单元和电气控制箱 (B) 头和收集器 (C) 中的光纤在一个完全平衡的飞行阶段和 (D) 的示意图的泰勒锥。(E) 显示打印机示意图并列出五最流行的系统参数, 包括 "应用电压" (高压发生器)、"温度" (温度控制器)、"气压" (压力调节器)、"工作距离" (通过 in-house 设计的可移动 z-axis) 和 "采集速度" (X 和 Y 定位幻灯片) 进行调整。(F) 演示通过耐热聚酰胺胶带在打印头内设计保温系统。这样可以防止 "发热元件 1" 和带电的 "黄铜部件" 之间的电弧。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56289/56289fig2.jpg"> 
图 2: 使用平面收集器制作的不同支架(A), 0/90 格 (B) 和同一个格在更大的分辨率 (C) 中。(D) 演示一个0/60 结构和 (E) 一个随机控制的结构。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig2large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56289/56289fig3.jpg"> 
图 3: 从扫描电子显微镜 (SEM) 中展示不同的管状支架和一个各自代表的图像.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>熔融纺丝法在直接书写模式下制备聚 (ε-内) 支架的真皮成纤维细胞浸润 (Farrugia et al., 2013)4 | | 平</td>
		</tr>
		<tr>
			<td>对真皮成纤维细胞种子 PCL 支架进行了细胞浸润的评价。</td>
		</tr>
		<tr>
			<td>人乳腺癌转移骨组织工程化人性化移植模型 (Thibaudeau et al., 2014)15 | |管</td>
		</tr>
		<tr>
			<td>在小鼠模型中, 用管状的新型支架来建立一个可行的异位 "器官" 骨, 以研究人乳腺癌的骨转移。</td>
		</tr>
		<tr>
			<td>组织工程骨中人前列腺癌转移的物种特异归巢机制 (Holzapfel et al., 2014)16 | |管</td>
		</tr>
		<tr>
			<td>用于前列腺癌研究的组织工程骨。</td>
		</tr>
		<tr>
			<td>利用高度组织化的熔体纺纤维结构提高水凝胶的整体性 (Bas et al., 2015)17 | | 平</td>
		</tr>
		<tr>
			<td>采用不同式模式和 poresizes 的支架, 提高了软质水凝胶的机械功能。</td>
		</tr>
		<tr>
			<td>用三维印刷充填 (维瑟 et al., 2015)18 | | 平</td>
		</tr>
		<tr>
			<td>软基水凝胶的增强与 PCL 支架。</td>
		</tr>
		<tr>
			<td>电纺丝在气瓶上的作用: 旋转速度和捕收器直径对管状结构形态的影响 (Jungst et al., 2015)6 | |管</td>
		</tr>
		<tr>
			<td>系统地研究了平动和旋转速度对管状结构的最终形貌的影响。</td>
		</tr>
		<tr>
			<td>分层结构多孔聚 (2-啉) 水凝胶 (约翰乔治黑格 et al., 2016)19 | |平</td>
		</tr>
		<tr>
			<td>在水凝胶中建立一个分层的3D 孔隙网络是一种牺牲模板。</td>
		</tr>
		<tr>
			<td>原发性骨肿瘤的临床前动物模型研究 (瓦格纳等 al., 2016)20 | |管</td>
		</tr>
		<tr>
			<td>在原发骨肿瘤的临床前研究中, 使用了支架材料来创建人性化的组织工程结构。</td>
		</tr>
		<tr>
			<td>原位骨膜组织工程在活体平台中的应用 (鲍德温 et al., 2017) 21 | |管</td>
		</tr>
		<tr>
			<td>为骨膜组织再生应用开发了一种由多网和水凝胶组成的支架。</td>
		</tr>
		<tr>
			<td>3D 微尺度纤维基质中细胞-基质相互作用的尺寸测量 (Tourlomousis 和 2017)22 | |平</td>
		</tr>
		<tr>
			<td>对不同结构的大棚支架进行了细胞-基质相互作用研究。</td>
		</tr>
		<tr>
			<td>内状细胞外基质在熔融纺书写支架上的表达 (Muerza-Cascante et al., 2017)23 | |平</td>
		</tr>
		<tr>
			<td>PCL 支架用于发展一种内的骨样组织, 促进初级人类造血干细胞的生长。</td>
		</tr>
		<tr>
			<td>3D 印刷晶格作为 T 细胞治疗的活化和扩展平台 (Delalat et al., 2017)24 | |平</td>
		</tr>
		<tr>
			<td>不同纤维间距的支架 (200 µm、500µm 和1000µm) 是表面 functionalised, 用 T 细胞播种以进行扩张。</td>
		</tr>
		<tr>
			<td>软骨组织工程 Biofabricated 软网络复合材料 (下 al., 2017)25 | |平</td>
		</tr>
		<tr>
			<td>报道了由水凝胶基质和用于关节软骨修复的增强滤网组成的仿生软网络复合材料。</td>
		</tr>
		<tr>
			<td>通过精密的界面工程对生物复合材料的改进3D 印刷加工和机械性能 (Hansske et al., 2017)26 | |平</td>
		</tr>
		<tr>
			<td>设计并开发了用于骨组织工程的氟化镁纳米颗粒增强 PCL 支架。</td>
		</tr>
	</tbody>
</table>
表 1: 参考了一份研究报告清单, 其中为生物应用制作了一种水电部支架.该清单提供了实施扁平和管状支架的结果。
<img alt="Table 2" src="/files/ftp_upload/56289/56289tbl2.jpg"> 
表 2: 对用于平面和管状支架的 G 代码的编程解释, 使用文本文件 (.txt) 上载到软件中.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl2large.jpg" target="_blank">请单击此处查看此表的较大版本.</a>
<img alt="Table 3" src="/files/ftp_upload/56289/56289tbl3.jpg"> 
表 3: 参数的代表值气压、电压和采集速度 (温度和采集距离常数) 达到三不同直径范围 (小, 中和大).红色箭头在各自的类别之内提出确切的价值到达纤维直径。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl3large.jpg" target="_blank">请单击此处查看此表的较大版本。</a>
<img alt="Table 4" src="/files/ftp_upload/56289/56289tbl4.jpg"> 
表 4: 在水电部可能出现的纤维沉积的不同情况和真实图像的示意图说明以及优化方法.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56289/56289tbl4large.jpg" target="_blank">请单击此处查看此表的较大版本.</a>

Watch this Scientific Journal Video about Melt Electrospinning Writing of Three-dimensional PolyÃŽÂµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications at JoVE.c

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

本议定书是一个全面的指导方针, 以直接的写作方式通过静电纺丝和聚合物熔体制造脚手架。我们系统地概述了该过程, 并定义了适当的参数设置, 以实现有针对性的脚手架结构。

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

melt-electrospinning-writing-three-dimensional-poly-caprolactone

Nanyang Technological University

Research

JoVE Journal

Bioengineering

15.0K 次观看.  Queensland University of Technology (QUT). 本议定书是一个全面的指导方针, 以直接的写作方式通过静电纺丝和聚合物熔体制造脚手架。我们系统地概述了该过程, 并定义了适当的参数设置, 以实现有针对性的脚手架结构。

视频: Melt Electrospinning Writing of Three-dimensional Poly(ε-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

科研

生物工程

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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 inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

静电生长因子释放到微纤维支架

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作:肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三维肽支架中哺乳动物细胞的培养

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...