静电纳米纤维支架与渐变的纤维组织
Summary
在这里，我们提出了一个协议，以制造静电纳米纤维支架与纤维的顺层组织，并探索在调节细胞形态/定位他们的应用程序。梯度对于纳米纤维支架的物理和化学性质提供了各种各样的在生物医学领域的应用程序。
Abstract
The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.
Introduction
纳米纤维，因为它们以模仿细胞外基质的结构和相对大小1能力用于组织工程一种流行工具。然而，一些天然组织界面，如腱-骨插入位点，含有胶原纤维，其显示出一个可变的组织结构，增加在对准朝向腱并降低在骨部位2-5。因此，对于有效的组织再生，有必要以制造支架，可以有效地模拟这种结构梯度。
在纤维组合物中的逐渐变化进行以前，已经研究，具体而言，矿物质含量6。然而，重建结缔组织的结构成分在很大程度上仍然未开发。早期的研究调查形态梯度通过研究大鼠颅骨成骨细胞的增殖表面的二氧化硅粒子密度的影响，并发现了一个INVER硅石颗粒密度和细胞增殖7之间本身关系。但介导的细胞增殖前期工作的形态变化，多与表面粗糙度缺乏模仿纤维组织变革7,8的能力。最近的一项研究试图制造的支架，通过使用一种新的收集器，用于电纺丝9模仿的独特胶原纤维取向。虽然本研究成功地制造与两个对准的和随机的纤维的支架，它未能模仿呈现在天然组织中的逐渐变化。另外，在产生了独立的部件，具有从对齐无规取向立即改变，这支架的生物力学特性显著降低。以前没有工作，已经能够生产出适用的纳米纤维支架与对齐和随机连续渐变的纤维取向。我们最近的研究表明纳米纤维支架的成功娱乐在纤维组织层次可以潜在模拟天然胶原组织在腱-骨插入10。这项工作的目的是提出用于生产纳米纤维支架的带结构非常类似于该纤维组织的在天然腱 – 骨组织界面的协议。
梯度纳米纤维结构都可能产生深远跨越各种领域的应用程序。我们集中在应用到腱-骨插入部位的组织工程用脂肪来源的干细胞（ADSC中），它们已经用于组织再生在各种基材上11-14结合我们的支架。此外，脂肪干细胞在性质上骨髓干细胞非常相似于多能的术语和其资源丰富，可以用一个简单的吸脂过程15,16收获。播种这些细胞渐变纳米纤维支架进一步增强他们的那朵通过允许所述细胞可以潜在分化成各种组织的受控分布告工程应用。除了接种干细胞，纳米纤维可以被封装与信号分子对细胞响应的调节。耦合nanoencapsulation这些支架的组织梯度允许的细胞行为或可能的植入物的设计和涂料的研究。的官能分子，如骨形态发生蛋白2（BMP2），这已被证明是诱导成骨细胞分化15,16，封装可以进一步提高这些支架10的组织工程应用。
Protocol
Representative Results
Discussion
The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly.
Fu…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是从内布拉斯加医疗中心和美国国立卫生研究院（授权号1R15 AR063901-01）大学的启动资金部分支持。
Materials
| Polycaprolactone | Sigma-Aldrich | 440744 | |
| N,N-Dimethlyformamide | Fisher Chemical | D-119-1 | |
| Dichloromethane | Fisher Chemical | AC61093-1000 | |
| Coumarin 6 | Sigma-Aldrich | 546283 | |
| Adipose Derived Stem Cells | Cellular engineering Technologies | HMSC.AD-100 | |
| Fetal Bovine Serum | Life Technologies | 26140-111 | |
| Fluorescein Diacetate | Sigma-Aldrich | F7378 | |
| Ethanol | Sigma-Aldrich | E7023 | |
| Trypsin-EDTA | Invitrogen | 25300-054 | |
| α-Modified Eagle's Medium | Invitrogen | a10490-01 | |
| Acetone | Fisher Scientific | s25120a | |
| Phosphate Buffered Saline | Invitrogen | 10010023 | |
| Glass Slides | VWR international, LLC | 101412-842 | |
| Syringe Pump | Fisher Scientific | 14-831-200 | Single syringe |
| Ultrasonic Cleaner | Branson | 1510 | |
| High Voltage DC Power Supply | Gamma High Voltage Research | ES30 | |
| Scanning Electron Microscope | FEI | Nova 2300 | |
| Fluorescence Microscope | Zeiss | Axio Imager 2 |
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