静电生长因子释放到微纤维支架
Summary
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.
Abstract
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 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.
Introduction
其中在神经组织工程的持续挑战是创建一个神经导管（NC），模仿的细胞外基质，其中神经自然生长。有研究表明，细胞多种因素在其环境中，包括机械，地形，粘合剂和化学信号1-3响应。一个在该领域的主要挑战是确定的信号的适当组合，并制造一个系统，可以维持线索长时间，以支持细胞生长4。外周神经元已知喜欢的软衬底，由排列的纤维定向，并且对神经生长因子（NGF）5-7响应。奈米可为周提供化学线索已经示出，以提供改进的功能恢复接近该同种异体移植，目前金标准神经修复8,9。
各种材料和生产方法可用于生产机械和地形人的线索10-13。机械线索是内在所选择的材质，使得选择该应用程序的关键1,13合适的材料。的生产方法，以控制沟槽宽度包括相分离，自组装和电纺丝1,13。对于微型应用程序，微流体，光图案化，蚀刻，盐滤出，或气体的泡沫也可以使用14-17。静电已成为最流行 的方式来设计的纤维基材进行组织培养，由于其灵活性和易于生产13,18-23的。电纺纳米纤维是通过施加高电压的聚合物溶液使其排斥本身并横跨一个短间隙放电24制成。对准支架可以通过收集在纤维上接地的旋转心轴被创建和不结盟支架被收集在固定板25上。粘附信号可以通过涂覆纤维支架机智来实现ħ纤连蛋白或静电26日前缀粘附肽，如RGD，房委会。
化学信号，例如生长因子，是最难以保持在延长的时期，因为它们需要用于控制释放的来源。许多系统已经试图控制释放添加到静电纺丝纤维网具有不同程度的成功。这些方法包括共混静电纺丝，静电纺丝乳液，芯壳电纺和蛋白质结合27。另外，电纺丝是在挥发性溶剂中，可以影响蛋白28的生存能力传统上完成的，因此保持了蛋白质的生物活性必须加以考虑。
这种方法专门针对机械相结合，地形，化学和胶粘剂信号来创建一个可调谐的支架周围神经生长。脚手架力学正是通过合成来控制甲基丙烯酸酯化透明质酸（HA）。该methacrylation站点用来连接光反应的交联剂。交联的材料不再是水溶性的，并且是专门分解通过酶29。交联的量的变化的降解速率，机械和材料的其它物理性能。使用HA以30％methacrylation，其具有〜500 Pa的拉伸弹性模量，创建一个软基质中靠近神经组织的天然机制，并且通常优选的由神经元26,29。静电纺丝在旋转心轴被用来创建排列的纤维的地形线索。使用静电与微球提供了支架在延长期限内的化学信号。支持含有NGF用来建立化学信号的神经突生长的微球体。与大多数静电材料医管局易溶于水，使神经生长因子不会在生产过程中遇到的恶劣的溶剂。要添加粘合剂信号时，SCAffold涂覆有纤连蛋白。完成后的系统包含了所有四种类型的上述信号：软（机械）对齐（地形）纤维释放神经生长因子微球（化学）涂有纤维连接蛋白（胶粘剂）。生产与本系统的测试在此协议中描述。
该过程开始于生产用与水包油包水双乳液的微球。该乳液是稳定用表面活性剂，聚乙烯醇（PVA）。的内水相含有蛋白质。因为它被加入到油相中，含有PLGA壳材料溶解在二氯甲烷（DCM），所述表面活性剂产生保护从DCM中的蛋白质相之间的屏障。此乳液是比分散在含有PVA的制造微球体的外表面上的另一水相。稳定的乳化液进行搅拌，以使DCM蒸发。冲洗和冷冻干燥后，只剩下干微球续癌宁的蛋白质。
后微球完成他们准备静电纺丝成支架。首先，你准备静电解决方案。该溶液的粘度为适当的纤维形成的关键。纯HA的解决方案并不能满足这一要求; PEO中加入作为载体的聚合物，以允许静电。将微球加入到溶液中，并静电纺丝所得的纤维支架与整个分布的微球。
一旦生产完成后，将蛋白进行测试，以验证其可行性。要做到这一点，它响应NGF原代细胞可以被使用。该协议采用背根神经节（DRG）从8-10日龄的鸡胚。单元束被接种到含微球填充有NGF或那些是空的支架。如果NGF仍然是可行的，你应该看到在NGF含支架增强轴突的生长。如果NGF不再可行它将不能促进轴突延伸，应该出现类似的控制。
本文所描述的确切过程但是集中在神经支撑，以简单的修改的材料，静电纺丝法，以及蛋白的系统可以为各种细胞和组织类型进行优化。
Protocol
Representative Results
Discussion
许多研究表明，神经细胞可以由沟槽宽度（纤维取向）和化学信号（生长因子）1,2,10,11,35被引导。静电是一种简便的方法来创建排列的纤维。生长因子促进神经生长，但以它们包括成神经管（NC），需要持续释放的方法。要创建一个更强大的系统，这两个线索，这两个信号应该合并。几种方法已经被先前研究的静电纤维支架用于神经再生中提供蛋白的延长释放，但没有迄今一直能持续释放…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).
Materials
| DAPI | Invitrogen | D1306 | |
| Irgacure 2959 | BASF | 24650-42-8 | Protect from light |
| PEO 900 kDa | Sigma-Aldrich | 189456 | |
| Methacryloxethyl thiocarbamoyl rhodamine B | Polysciences, Inc. | 23591-100 | Prepare stock solution in DMSO |
| Syringe Pump | KD Scientific | KDS100 | |
| Power Source | Gamma High Voltage | ES30P-5W | |
| Motor | Triem Electric Motors, Inc | 0132022-15 | Must attach to a custom built mandrel |
| Tachometer | Network Tool Warehouse | ESI-330 | Use to monitor mandrel speed |
| Omnicure UV Spot Cure System with collimating adapter | EXFO | S1000 | |
| Needles | Fisher Scientific | 14-825-16H | |
| Coverslips | Fisher Scientific | 12-545-81 | |
| Polyvinyl Alcohol | Sigma-Aldrich | P8136-250G | |
| Isoporopyl Alcohol | Sigma-Aldrich | I9030-500mL | |
| Bovine Serum Albumin (BSA) | Fisher Scientific | BP9703-100 | |
| BSA-FITC | Sigma-Aldrich | 080M7400 | |
| β-Nerve Growth Factor (NGF) | R&D Systems | 1156-NG | |
| 65:35 Poly-Lactic-Glycolic-Acid (PLGA) | Sigma-Aldrich | 1001554270 | |
| Dichloromethane | Sigma-Aldrich | 34856-2L | |
| Coomassie (Bradford) Protein Assay | Thermo Scientific | 1856209 | |
| 3-(Trimethoxysilyl)propyl methacrylate | Sigma-Aldrich | 1001558456 | |
| Fibronectin | Sigma-Aldrich | F2006 | |
| DMEM | Lonza | 12-604F | |
| FBS | Atlanta Biologicals | S11150 | |
| PBS | Hyclone | SH30256.01 | |
| Glutamine | Fisher Scientific | G7513 | |
| Pen-Strep | Sigma-Aldrich | P4333 | |
| Paraformaldehyde | Alfa Aesar | A11313 |
Referenzen
- Wrobel, M. R., Sundararaghavan, H. G. Directed migration in neural tissue engineering. Tissue Eng Part B Rev. , (2013).
- Schmidt, C. E., Leach, J. B. Neural tissue engineering: strategies for repair and regeneration. Annual Review of Biomedical Engineering. 5, 293-347 (2003).
- Madduri, S., di Summa, P., Papaloizos, M., Kalbermatten, D., Gander, B. Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats. Biomaterials. 31, 8402-8409 (2010).
- Madduri, S., Gander, B. Growth factor delivery systems and repair strategies for damaged peripheral nerves. J Control Release. 161, 274-282 (2012).
- Madigan, N. N., McMahon, S., O’Brien, T., Yaszemski, M. J., Windebank, A. J. Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds. Respir Physiol Neurobiol. 169, 183-199 (2009).
- Sundararaghavan, H. G., Monteiro, G. A., Firestein, B. L., Shreiber, D. I. Neurite growth in 3D collagen gels with gradients of mechanical properties. Biotechnol. Bioeng. 102, 632-643 (2009).
- Hudson, T. W., Evans, G. R., Schmidt, C. E. Engineering strategies for peripheral nerve repair. Clin Plast Surg. 26, 617-628 (1999).
- Kokai, L. E., Bourbeau, D., Weber, D., McAtee, J., Marra, K. G. Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair. Tissue Eng Part A. 17, 1263-1275 (2011).
- Bronzino, J. D., Peterson, D. R. . The Biomedical Engineering Handbook, Third Edition – 3 Volume Set: Tissue Engineering and Artificial Organs. , (2006).
- Bell, J. H. A., Haycock, J. W. Next generation nerve guides: materials, fabrication, growth factors, and cell delivery. Tissue Eng Part B Rev. 18, 116-128 (2012).
- Ruiter, G. C. W., Malessy, M. J. A., Yaszemski, M. J., Windebank, A. J., Spinner, R. J. Designing ideal conduits for peripheral nerve repair. Neurosurgical focus. 26, (2009).
- Olakowska, E., Woszczycka-Korczyńska, I., Jędrzejowska-Szypułka, H., Lewin-Kowalik, J. Application of nanotubes and nanofibres in nerve repair. A review. Folia Neuropathol. 48, 231-237 (2010).
- Gunn, J., Zhang, M. Polyblend nanofibers for biomedical applications: perspectives and challenges. Trends Biotechnol. 28, 189-197 (2010).
- Sundararaghavan, H. G., Masand, S. N., Shreiber, D. I. Microfluidic generation of haptotactic gradients through 3D collagen gels for enhanced neurite growth. Journal of Neurotrauma. 28, 2377-2387 (2011).
- Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. Electrospun fibrous scaffolds with multiscale and photopatterned porosity. Macromol Biosci. 10, 265-270 (2010).
- Edalat, F., Sheu, I., Manoucheri, S., Khademhosseini, A. Material strategies for creating artificial cell-instructive niches. Current Opinion in Biotechnology. 23, 820-825 (2012).
- Annabi, N., et al. Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro. Biomaterials. 30, 4550-4557 (2009).
- Castaño, O., Eltohamy, M., Kim, H. -. W. Electrospinning technology in tissue regeneration. Methods Mol. Biol. 811, 127-140 (2012).
- Chew, S. Y., Wen, J., Yim, E. K. F., Leong, K. W. Sustained release of proteins from electrospun biodegradable fibers. Biomacromolecules. 6, 2017-2024 (2005).
- Han, D., Gouma, P. -. I. Electrospun bioscaffolds that mimic the topology of extracellular matrix. Nanomedicine. 2, 37-41 (2006).
- Prabhakaran, M. P., et al. Electrospun biocomposite nanofibrous scaffolds for neural tissue engineering. Tissue Eng Part A. 14, 1787-1797 (2008).
- Xie, J., MacEwan, M. R., Schwartz, A. G., Xia, Y. Electrospun nanofibers for neural tissue engineering. Nanoscale. 2, 35-44 (2010).
- Yao, L., O’Brien, N., Windebank, A., Pandit, A. Orienting neurite growth in electrospun fibrous neural conduits. J. Biomed. Mater. Res. Part B Appl. Biomater. 90, 483-491 (2009).
- Sill, T. J., von Recum, H. A. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials. 29, 1989-2006 (2008).
- Ifkovits, J. L., Sundararaghavan, H. G., Burdick, J. A. Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture. Journal of Visualized Experiments: JoVE. , (2009).
- Sundararaghavan, H. G., Burdick, J. A. Gradients with depth in electrospun fibrous scaffolds for directed cell behavior. Biomacromolecules. 12, 2344-2350 (2011).
- Meinel, A. J., Germershaus, O., Luhmann, T., Merkle, H. P., Meinel, L. Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications. Eur J Pharm Biopharm. 81, 1-13 (2012).
- Ji, W., et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm. Res. 28, 1259-1272 (2011).
- Burdick, J. A., Chung, C., Jia, X., Randolph, M. A., Langer, R. Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules. 6, 386-391 (2005).
- Péan, J. M., et al. Optimization of HSA and NGF encapsulation yields in PLGA microparticles. International Journal of Pharmaceutics. 166, 105-115 (1998).
- Cartiera, M. S., Johnson, K. M., Rajendran, V., Caplan, M. J., Saltzman, W. M. The Uptake and Intracellular Fate of PLGA Nanoparticles in Epithelial Cells. Biomaterials. 30, 2790-2798 (2009).
- Hollenbeck, P. J., Bamburg, J. R. . Neurons: Methods and Applications for the Cell Biologist. , (2003).
- Boer, R., et al. Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres. Microsurgery. 31, 293-302 (2011).
- Pujic, Z., Goodhill, G. J. A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen. Journal of Neuroscience Methods. 215, 53-59 (2013).
- Madduri, S., di Summa, P., Papaloïzos, M., Kalbermatten, D., Gander, B. Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats. Biomaterials. 31, 8402-8409 (2010).
- Xu, X., et al. Polyphosphoester microspheres for sustained release of biologically active nerve growth factor. Biomaterials. 23, 3765-3772 (2002).
- Yan, Q., Yin, Y., Li, B. Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration. Biomed Eng Online. 11, (2012).
- Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications. J Biomed Mater Res A. , (2013).
- Li, X., et al. Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning. Colloids Surf B Biointerfaces. 75, 418-424 (2010).
- Wang, C. -. Y., et al. The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration. J Biomater Sci Polym Ed. 23, 167-184 (2012).
- Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, C. -. Y. Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning. J Biomed Mater Res A. 96, 13-20 (2011).
