Name: novel process for 3d printing decellularized matrices
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Novel Process for 3D Printing Decellularized Matrices at JoVE.com

该项目的部分资金来自北美儿科矫形外科协会 (pos形) 和国家卫生研究院的一项赠款 nibb r21eb025378-01 (探索性生物工程研究赠款)。

1. 微球的获取和预处理
<ol><li>生产具有所需矩阵封装 (pla-dm)2的微球。 注: 微球必须是均匀的大小。因此, 在使用前筛过微球是必不可少的。虽然在以前的出版物2中详细介绍了矩阵去电磁化和封装, 但下文对这一过程作了简要总结。<ol>
<li>首先, 从猪后肢收获软骨塞。用0.05% 的胰蛋白酶 0.5 mm 乙二胺四乙酸 (edta)、dulbecco 改性老鹰培养基 (dmem)、1.5% 过氧乙酸和 2....

<ol>
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Y., Whitlock, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspheres+containing+decellularized+cartilage+induce+chondrogenesis+and+remain+functional+after+incorporation+within+a+poly(caprolactone)+filament+useful+for+fabricating+a+3D+scaffold.">Microspheres containing decellularized cartilage induce chondrogenesis and remain functional after incorporation within a poly(caprolactone) filament useful for fabricating a 3D scaffold.</a> Biofabrication. , (2018).</li><li>Partington, L., 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=Biochemical+changes+caused+by+decellularization+may+compromise+mechanical+integrity+of+tracheal+scaffolds.">Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds.</a> Acta Biomaterialia. 9 (2), 5251-5261 (2013).</li><li>Hutmacher, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+tissue+engineering+bone+and+cartilage.">Scaffolds in tissue engineering bone and cartilage.</a> Biomaterials. 21 (24), 2529-2543 (2000).</li><li>Kang, H., Hollister, S. J., La Marca, F., Park, P., Lin, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+biodegradable+lumbar+interbody+fusion+cage+design+and+fabrication+using+integrated+global-local+topology+optimization+with+laser+sintering.">Porous biodegradable lumbar interbody fusion cage design and fabrication using integrated global-local topology optimization with laser sintering.</a> Journal of biomechanical engineering. 135 (10), 101013-101018 (2013).</li><li>Kang, H., Lin, C. Y., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+optimization+of+three+dimensional+tissue+engineering+scaffold+architectures+for+prescribed+bulk+modulus+and+diffusivity.">Topology optimization of three dimensional tissue engineering scaffold architectures for prescribed bulk modulus and diffusivity.</a> Structural and Multidisciplinary Optimization. 42 (4), 633-644 (2010).</li><li>Lin, C. -. Y., 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=Functional+bone+engineering+using+ex+vivo.+gene+therapy+and+topology-optimized,+biodegradable+polymer+composite+scaffolds.">Functional bone engineering using ex vivo. gene therapy and topology-optimized, biodegradable polymer composite scaffolds.</a> Tissue Engineering. 11 (9-10), 1589-1598 (2005).</li><li>Lin, C. -. Y., Hsiao, C. -. C., Chen, P. -. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ornamenting+3D+printed+scaffolds+with+cell-laid+extracellular+matrix+for+bone+tissue+regeneration.">Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration.</a> Biomaterials. 37, 230-241 (2015).</li><li>Zhang, W., 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=The+effect+of+interface+microstructure+on+interfacial+shear+strength+for+osteochondral+scaffolds+based+on+biomimetic+design+and+3D+printing.">The effect of interface microstructure on interfacial shear strength for osteochondral scaffolds based on biomimetic design and 3D printing.</a> Materials Science and Engineering C. 46, 10-15 (2015).</li><li>Williams, J. 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W., 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=A+novel+process+for+optimizing+musculoskeletal+allograft+tissue+to+improve+safety,+ultrastructural+properties,+and+cell+infiltration.">A novel process for optimizing musculoskeletal allograft tissue to improve safety, ultrastructural properties, and cell infiltration.</a> Journal of Bone and Joint Surgery - Series A. 94 (16), 1458-1467 (2012).</li><li>Schultz, G. S., Davidson, J. M., Kirsner, R. S., Herman, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Reciprocity+in+the+Wound+Microenvironment.">Dynamic Reciprocity in the Wound Microenvironment.</a> Wound Repair Regeneration. 19 (2), 134-148 (2012).</li><li>Benders, K. E. M., van Weeren, P. R., Badylak, S. F., Saris, D. B. F., Dhert, W. J. 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脱弹基质和3d 打印 pcl 支架都已独立显示, 可以允许细胞粘附和增殖, 从而验证它们在骨软骨修复10、11、12中的用途。在最近的 2,3, 14, 15, 在工程方法中使用去细胞化矩阵进行组织修复一直是一个非常感兴趣和成功的课题。我们以前?...

目前用于临床应用的组织工程技术, 如骨骼、软骨、肌腱和韧带重建, 使用自体和同种异体移植修复受损组织。在临床实践中, 每种技术都是作为 "黄金标准" 进行常规的, 首先从患者或尸体匹配中采集供体组织, 然后将供体组织放入缺陷部位2。然而, 这些策略受到捐献者部位发病率、供者部位因缺陷严重、感染风险以及难以找到符合所需几何形状的移植物的限制。此外, 研究表明, 与原生组织3相比, 用于重建的同种异体移植降低了机械和生物特性.考虑到这些因素, 组织工程师最近转向三维 (3d) 生物冲洗, 以产生自定义的, 复杂的几何形状, 生物活性和设计, 以适应缺陷的大小和形状, 同时提供足够的机械性能, 直到生物重塑完成。
理想情况下, 3d 打印的支架将由一个聚合物主干组成, 可以保留原生组织所需的机械稳定性, 而整合的生物制剂则为周围的细胞提供生化线索, 从而导致细胞的迁移、增殖、分化和组织生产2,5。不幸的是, 大多数含有生物成分的结构都是用凝胶或聚合物制成的, 这些凝胶或聚合物太弱, 无法承受目标组织在自体同种异体移植重建中所经历的体内力量。其他聚合物, 如聚乳酸 (pla) 是生物可吸收的, 3d 可打印和结构健全, 但在210°c 或以上的温度下印刷, 使生物制剂在制造过程中不可能共印。聚己内酯 (pcl) 是另一种 fda 清除、生物可吸收的聚合物, 可在较低的温度 (65°c) 下进行3d 打印, 在制造具有复杂形态的复杂形态5, 6 的患者专用植入物方面越来越受欢迎 ,7,8,9。然而, 大多数使用气动技术的生物打印机使得在生物活动不会受到损害的较低温度下打印 pcl 是不可能的。到目前为止, 这些聚合物与自体同种异体移植的整合到一种新的可打印生物材料中的工作尚未完成。在没有这种材料的情况下, 不可能采用真正的组织工程方法进行组织重建。因此, 我们试图将 pla、pcl 和脱核同种异体移植基质 (dm) 结合起来, 利用每种材料的优势, 以制造出能够重建复杂组织的可行结构。这一过程将提供抵抗体内力所需的初始机械强度和热稳定性, 以适应在诱导所需组织形成的结构中的添加剂制造。
在最近的一次尝试中, 我们表明, 将脱细胞化的软骨细胞外基质封装在一个热保护的 pla 屏障中是可行的, 可以在 pcl 细丝中挤出, 从而保持dm 影响周围宿主细胞2。这激励我们寻求临床上有效的组织重建方法。在目前的研究中, 我们利用平台技术构建了包括 pla、dm 和 pcl (pla-dmcl) 在内的一体机支架。
我们的目标是提高同种异体移植的有效性和效用, 使用提出的新生物制备技术, 更准确地重述原生组织, 最终将其用于各种应用。

<table border="0" cellpadding="0" cellspacing="0" style="width:877px;" width="877">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Sieve machine</td>
			<td style="width:396px;">Haver &#38; Boecker Tyler</td>
			<td style="width:132px;">Ro-Tap RX 29-E Pure</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 90 um</td>
			<td>Fisherbrand</td>
			<td>170328156</td>
			<td>No. 170</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 53 um</td>
			<td>Fisherbrand</td>
			<td>162513588</td>
			<td>No. 270</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sieve 106 um</td>
			<td>Fisherbrand</td>
			<td>162018121</td>
			<td>No. 140</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sputter coater</td>
			<td>Leica</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanning Electron Microscope</td>
			<td>Hitachi, USA</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filabot EX2</td>
			<td>Filabot.com</td>
			<td>FB00061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filabot Spooler</td>
			<td>Filabot.com</td>
			<td>FB00073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CAPA 6506</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline, PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6&#34; Fan</td>
			<td>Comfort Zone, Amazon</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic Water Bath</td>
			<td>Cole Parmer</td>
			<td>SK-08895-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dreamer</td>
			<td>FlashForge</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Drum Mixer</td>
			<td>Custom made</td>
			<td>n/a</td>
			<td>Similar piece of equipment: https://www.coleparmer.com/i/argos-technologies-flexiroll-digital-tube-roller-shaker-120-vac/0439744?PubID=UX&#38;persist=true&#38;ip= 
			no&#38;gclid=CjwKCAjw- 
			dXaBRAEEiwAbwCi5khGDMz0 
			dTjsraEsBGfhMEH7ytx 
			LQWGUPNgUJYQ1p3vj_yxkYoI_ 
			ixoC9GwQAvD_BwE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro Balance</td>
			<td>Mettler Toledo, Fisher Scientific</td>
			<td>01-913-851</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Simplify3D</td>
			<td>Simplify3D</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SolidWorks</td>
			<td>SolidWorks</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microspheres</td>
			<td>Produced in-house, see concurrently submitted JoVE submission</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">p-nitrophenyl phosphate, disodium salt, hexahydrate</td>
			<td>Millipore</td>
			<td>4876-5GM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase, alkaline</td>
			<td>Roche Diagnostics GmbH</td>
			<td>10 713 023 001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absorbance Reader</td>
			<td>Tecan</td>
			<td>Sunrise</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T6455-100ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E24</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel process for 3d printing decellularized matrices

3d 生物冲洗的目的是创建自定义脚手架, 生物活动, 并适应所需的大小和几何形状。热塑性主干可以提供类似于原生组织的机械稳定性, 而生物制剂则为祖细胞提供合成线索, 导致其迁移、增殖和分化, 从而重建原始组织/器官1,2。不幸的是, 许多3d 打印兼容的生物可吸收聚合物 (如聚乳酸, pla) 打印在210°c 或更高的温度-不利于生物制剂的温度。另一方面, 聚己内酯 (pcl) 是一种不同类型的聚酯, 是一种可生物吸收的3d 打印材料, 打印温度较温和, 为65°c。因此, 假设热保护 pla 屏障中的脱光细胞外基质 (dm) 可以在 pcl 长丝中打印并保留在其功能构象中。在这项工作中, 骨软骨修复是测试该假设的应用。因此, 猪软骨被去细胞化并封装在聚乳酸 (pla) 微球中, 然后用聚己内酯 (porcine) 挤压成长丝,通过熔融沉积建模产生三维结构。对具有或不具有微球 (pla-dmcl 和 pcl (-)) 的结构进行了表面特征差异的评估。

筛分后, 微球应显得均匀, 不受集料的影响。在 sem 下, 被筛过的微球表面可能有小毛孔, 但在其他方面会呈球形和光滑, 如图 1所示。所有挤压细丝应具有均匀的直径和圆形截面。含有微球 (pla-dmp) 的长丝将有一个稍微更多的哑光完成, 而只有 pcl (-) 长丝看起来更有光泽。pla-dmp-pcl 长丝也会比 pcl (-) 长丝更粗糙。脚手架应打印在所需的几何形状, 这是由?...

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Novel Process for 3D Printing Decellularized Matrices

该协议描述了用于结构组织工程结构三维打印的嵌入聚乳酸 (pla) 微球的聚己内酯 (pcl) 长丝的生产。

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

此方法允许共同打印结构合成物和生物成分，用于组织工程脚手架。这些支架可以更准确地复制原生组织环境，这对培养细胞是有益的。该技术的主要优点是，它可以打印机械健全的结构，而不会损坏封装在脚手架内的生物材料，在一个一对一的技术组织工程。用所需的基质封装生成微球。这些微球是由猪后肢去细胞化软骨。它们的大小各不相同。使用筛机获得均匀大小的微球。确保三个筛盘在使用前已彻底清洁和干燥。将筛子摇床与顶部较大的网状纸盒组装，在中间组装较小的网状托盘。将筛盘放在底部。将干燥的微球放在最顶的托盘中。然后将盖子放在托盘上。粗筛八到十分钟。然后细筛八到十分钟。在等待时，为筛分球体安排称重纸。筛切完成后，小心取下筛子托盘。将纸盒倒置在称重纸上。轻轻敲击两侧，以确保大部分球体脱落。将所有筛板清空到称重纸上后，丢弃超大尺寸和尺寸不足的球体。其余球体具有正确的大小，适合在后续步骤中使用。将这些球体放入贴有标签的离心管中。将管子存放在20摄氏度的冰柜中，直到需要。称重制作灯丝材料所需的材料。使微球与至少 25 克微球的多丙酮粉末的 1 比 4 重量比。将粉末混合物转移到微型滚动混合器中。每分钟20次旋转混合粉末5分钟。五分钟后，停止旋转并翻转容器。然后在 20 rpm 下再混合五分钟。若要继续，请将混合物放入挤出机和滑阀设置。设置挤出机，使其出口距离滑阀的入口约 60 厘米。然后在挤出机加热元件时，确保没有绝缘护套。将台式风扇放在挤出机和滑阀之间的一半位置，以冷却挤出机。将另一个风扇放在加热护套附近，用环境空气冷却。接下来，确保将正确的喷嘴连接到挤出机并设置加热元件温度。启动冷却风扇，使仪器达到稳定状态。20 到 30 分钟后，获取微球 PCL 混合物并填充挤出料斗。打开滑阀和挤出机螺旋钻，以启动灯丝挤出。使用钳子并手动拉出初始拉伸灯丝。将灯丝馈送至灯丝滑阀。在拉伸时，观察灯丝以确定其组成。一段时间后，灯丝组合物将显得均匀，这是需要的。将胶带缠绕在灯丝上，以标记统一区域的开始。在滑阀滚轮之外，监控灯丝的直径。使用卡钳测试直径是否接近所需的 1.75 mm。根据需要调整滑阀和挤出机的速度和温度。要调整灯丝直径，请首先更改滑阀和挤出机速度。您还可以更改挤出机温度，尽管这通常没有必要。继续重新加注料斗并挤出，直到使用所有粉末。当料斗几乎为空时，将 PCL 粉末添加到料斗中，以冲出微球混合物。添加 PCL 粉末时监控拉伸。首先，微球在拉伸中仍然可见。最终，当看不到更多的微球时，停止添加PCL。在所需浓度内用微球分离丝并标记灯丝。当料斗中粉末最少时，停止挤压并关闭设备。灯丝可用于标准熔融沉积建模打印机。将灯丝装入装有所需直径喷嘴的打印机。加载模型后，设置打印的温度和线性速度，并开始沉积灯丝。自定义灯丝层层沉积。特别注意第一层并根据需要调整设置。这两个 3D 打印脚手架很难在此比例下区分。一个有含有多面体酮的灯丝，仅PCL。另一种是PCL灯丝，内含聚乳酸和去细胞化基质的微球。使用扫描电子显微镜查看时，仅PCL脚手架看起来基本光滑。相比之下，对于其他灯丝，嵌入在PCL中的微球在整个样品中可见。在尝试此过程时，必须记住，超小或尺寸过小的微球会影响挤出机中材料的流动动态。因此，请务必在使用前正确准备微球。为了尽量减少浪费，建议创建所有实验所需的大批量灯丝，而不是更频繁地创建较小的批次。丝丝生产方面的专业知识将随着时间的推移而产生。可对按照本程序制作的脚手架进行体外和体内进一步评估，以回答有关与其他生物印刷脚手架相比，增强感应和机械强度的其他问题。该技术开发后，为组织工程领域的研究人员探索三维脚手架内脱细胞化猪基质的逆向诱导铺平了道路。不要忘记，使用小颗粒物可能会造成呼吸道疾病。鼓励在此过程期间佩戴小颗粒面罩。

Research

JoVE Journal

Bioengineering

Preparation of Complaint Matrices for Quantifying Cellular Contraction

投诉矩阵量化细胞收缩的准备工作

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

科研

生物工程

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

新生儿心脏支架：再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

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 ...

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 ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

通过交替粘性 - 惯性力喷射进行 体外 水凝胶微载体的3D打印

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts

低剂量伽马辐射灭菌治疗脱细胞气管移植物

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation ...