一种新的三维打印解光矩阵处理方法
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| Sieve machine | Haver & Boecker Tyler | Ro-Tap RX 29-E Pure | |
| Sieve 90 um | Fisherbrand | 170328156 | No. 170 |
| Sieve 53 um | Fisherbrand | 162513588 | No. 270 |
| Sieve 106 um | Fisherbrand | 162018121 | No. 140 |
| Sputter coater | Leica | n/a | |
| Scanning Electron Microscope | Hitachi, USA | n/a | |
| Filabot EX2 | Filabot.com | FB00061 | |
| Filabot Spooler | Filabot.com | FB00073 | |
| CAPA 6506 | Perstorp | 24980-41-4 | |
| Phosphate buffered saline, PBS | Gibco | 10010023 | |
| 6" Fan | Comfort Zone, Amazon | n/a | |
| Ultrasonic Water Bath | Cole Parmer | SK-08895-13 | |
| Dreamer | FlashForge | n/a | |
| Drum Mixer | Custom made | n/a | Similar piece of equipment: https://www.coleparmer.com/i/argos-technologies-flexiroll-digital-tube-roller-shaker-120-vac/0439744?PubID=UX&persist=true&ip= no&gclid=CjwKCAjw- dXaBRAEEiwAbwCi5khGDMz0 dTjsraEsBGfhMEH7ytx LQWGUPNgUJYQ1p3vj_yxkYoI_ ixoC9GwQAvD_BwE |
| Micro Balance | Mettler Toledo, Fisher Scientific | 01-913-851 | |
| Simplify3D | Simplify3D | n/a | |
| SolidWorks | SolidWorks | n/a | |
| Microspheres | Produced in-house, see concurrently submitted JoVE submission | ||
| p-nitrophenyl phosphate, disodium salt, hexahydrate | Millipore | 4876-5GM | |
| Phosphatase, alkaline | Roche Diagnostics GmbH | 10 713 023 001 | |
| Absorbance Reader | Tecan | Sunrise | |
| Tris-HCl Buffer | Sigma-Aldrich | T6455-100ML | |
| Heated shaker | New Brunswick Scientific | Excella E24 |
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