This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1，水/油/水双重乳液微球生产<ol><li>首先准备2％和0.5％重量/在去离子水中的聚乙烯醇（PVA）诉的解决方案。搅拌溶液，在50℃，直到清楚，这可能需要几个小时。制备的2％体积/体积异丙醇在去离子水中的溶液。 </li><li>制备所需的亲水性蛋白的水溶液。下表提供了示例制剂。 </li></ol><img alt="表1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> 表1：实施例的蛋白质解下列蛋白的解决方案已经成功地封装和使用该协议静电。根据需要，其它亲水蛋白溶液可被使用。 <ol start="3"><li>放置40毫升的0.5％PVA溶液到50ml离心管中，放在一边。 </li><li>在15毫升离心管中溶解300毫克65:35聚在3ml二氯甲烷中（乳酸 - 共 - 乙醇酸）（PLGA）。一种涡流混合器可用于加速的PLGA溶解。 </li><li>结合200微升的蛋白质溶液和4微升2％的PVA溶液。倒入蛋白混合物进入PLGA溶液（步骤1.4）。该解决方案将保持大部分独立。 </li><li>将管放入冰水中的烧杯中。用魔杖超声仪在约10瓦（RMS），搅拌几秒钟（5-10）溶液，直至形成均匀的乳白色乳液。 </li><li>倾乳液进50毫升的试管含0.5％的PVA（步骤1.3）。混合溶液在高速涡流混合器〜20秒。该解决方案将制定一个浑浊的外观。 </li><li>传送乳液到200ml烧杯中并置于搅拌板上，在350 rpm离心2分钟。加入50毫升2％的异丙醇的搅拌板的烧杯中。使混合物继续搅拌至少1小时，以允许DCM蒸发，PLGA硬化。 </li><li>转让吨他微球溶液到离心管中。 </li><li>离心机在425×g离心3分钟。微球将收集在试管的底部并出现白色。小心地从管除去上清液，将微球上，并储存在500ml的瓶子。 </li><li>通过填充管四分之三满并摇动它以重新分布的微球体在液体中用去离子水冲洗该微球。 </li><li>重复步骤1.10和1.11的四倍。 </li><li>以下最后的漂洗，与其它样品再次和地方除去在500ml瓶中的上清液。冻结收集在离心管中的微球，然后冷冻干燥至少24小时。 </li><li>可视化在光学显微镜下，或用扫描电子显微镜，微球。微不超过60微米的大都是有效的静电。如果微球太大，再超声处理或涡旋倍，可能需要在步骤1.6或1.7。 </li> &lt;LI&gt;存放干燥的微球在-20℃的冰箱中。 </li><li>可选：使用蛋白质测定中，按照制造商的说明，从步骤1.10 30测试在500毫升瓶的蛋白质的量。这被用来计算百分数蛋白包封在微球中，减去的量在从什么在生产过程中所用的溶液。 </li></ol>注意：要在可视化微球蛋白质位置添加罗丹明2微克/毫升的PLGA溶液31和封装一FITC标记的蛋白质图1显示了一个例子。 2，静电与微<ol><li>之前制备电纺丝溶液，通过溶解在37℃下创建0.5％w / v的溶液的光引发剂，在去离子水中。这个过程可能需要几个小时。 </li><li>创建一个2％w / v的甲基丙烯酸酯化的透明质酸（的MeHA）（见的Burdick 等人的合成）29，3％w / v的900 kD的聚（环氧乙烷）（PEO）和0.05％的重量/在去离子水中v光引发剂溶液。 <ol><li>计算的MeHA和PEO的正确量为所需的体积。例如，将10毫升静电纺丝溶液需要200毫克的MeHA和300毫克的PEO。 </li><li>溶解于去离子水中的PEO的90％所需的最终体积（9毫升这个例子）。这可能需要几个小时，在37℃下加热搅拌盘或水浴可以加速这一进程。 </li><li>接下来添加的MeHA并用旋涡混合器搅拌溶液，直到清楚。这将只需要几分钟的时间。 </li><li>最后加入0.5％的光引发剂溶液，以填补剩下的10％的体积（1毫升本示例）。 </li></ol></li><li>添加微球体在所需要的浓度高达400毫克/毫升。混在涡旋混合器上的溶液，直到微球均匀地分布在该溶液中。 </li><li>将溶液转移到一个注射器，并附加一个6英寸的18号BLUNT尖的针。 </li><li>发生在注射泵的注射器，并把它设置为免除1.2毫升/小时。 </li><li>胶带铝箔上收集板或型芯的层。这允许容易清理成品支架和存储。一种旋转心轴被用来创建排列的纤维。的平板或固定芯棒将导致随机排列的纤维。 </li><li>从高电压电源的收集装置连接的接地线。正极导线连接到针。 </li><li>调整注射器泵和收集表面，以便有针尖和收集表面的15公分。 </li><li>启动该聚合物泵送，当溶液是在注射器的端部可见，接通电压源和设置电压24千伏注意 ：一旦电压接通时不要触摸系统中的任何金属部分。负责人也可以跳很短的距离，从带电部位的皮肤。 </li><li>运行解决方案，直到ðesired脚手架厚度。当完全关闭电压源和注射器泵。 </li><li>删除箔支架连接。含蛋白质已完成支架储存在-20℃的冰箱中。 </li></ol> 3，蛋白质的生物活性测试<ol><li>制备的细胞培养介质。加入10％体积/体积的胎牛血清，1％体积/体积L-谷氨酰胺和1％体积/体积的青霉素 - 链霉素，以Dulbecco改良的Eagle培养基。 </li><li>选择盖玻片适合完全陷入了孔板。 </li><li>用3（三甲氧基硅基）丙基甲基丙烯酸酯如制造商所述治疗的盖玻片。 Methacrylation增强脚手架坚持盖玻片。 </li><li>静电纺丝之前，请安装甲基丙烯酸盖玻片与可拆卸的双面胶带electrospinner的收集区。纺纱到盖玻片简化了操作和观看。 </li><li>静电纺丝到所需的厚度，如上所述。 </li><li> á压脚提升静电纺丝小心地从心轴上取下盖玻片。放置在支架涂覆的盖玻片成清晰氮气室，并确保所有的氧气被吹洗。 </li><li>放置在10毫瓦/厘米2 365纳米的光的腔室和支架15分钟。后交联处成适当大小的孔板。确保棚架的一面朝上。 </li><li>在杀菌灯30分钟的地方支架进行消毒。如果需要，纤连蛋白或其它蛋白质被用作涂层以增强细胞附着。按照制造商的说明大衣支架。 </li><li>嘉实背根神经节（DRG）由霍伦贝克32如前所述。一DRG将需要为每个支架覆盖盖玻片进行测试。 </li><li>将100-200微升媒体在孔板每个支架。小心将一个背根神经节的媒体液滴每个支架。对于厚的支架可能需要更多的媒体; DRG需要被完全浸没，而不是FLOA婷。 </li><li>孵育所述支架和背根神经节，在37℃下搅拌4小时，以使细胞附着在支架上。 </li><li>培养基灌装到适当的水平井，并将进入孵化器。继续温育4-6天。 </li><li>潜伏期后，小心地从每一个媒体好，用PBS轻轻洗一次。固定的细胞用4％w / v的多聚甲醛固定30分钟。 </li><li>用抗体染色为神经丝染色的细胞。这将允许可视化神经突向外生长的定量。 DAPI也可以用来查看细胞核。一个例子染色协议是由Sundararaghavan和他的同事14描述。 </li><li>使用可视化荧光显微镜细胞。 <ol><li>放置孔平板上的显微镜的阶段。 </li><li>找到了用DAPI过滤器和励磁设置的细胞团。 </li><li>一旦该细胞是切换滤波器来FITC以可视化的扩展的突起。 ü唱在显微镜的线圈功能收集和组合一样多的图像，必要时看到整个结构。重复DAPI，FITC和亮场。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurons: Methods and Applications for the Cell Biologist. , (2003).</li><li>Boer, R., 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=Rat+sciatic+nerve+repair+with+a+poly-lactic-co-glycolic+acid+scaffold+and+nerve+growth+factor+releasing+microspheres.">Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres.</a> Microsurgery. 31, 293-302 (2011).</li><li>Pujic, Z., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+compartment+diffusion+chamber+for+studying+axonal+chemotaxis+in+3D+collagen.">A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen.</a> Journal of Neuroscience Methods. 215, 53-59 (2013).</li><li>Madduri, S., di Summa, P., Papalo&#239;zos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Xu, X., 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=Polyphosphoester+microspheres+for+sustained+release+of+biologically+active+nerve+growth+factor.">Polyphosphoester microspheres for sustained release of biologically active nerve growth factor.</a> Biomaterials. 23, 3765-3772 (2002).</li><li>Yan, Q., Yin, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+new+PLGL-RGD-NGF+nerve+conduits+for+promoting+peripheral+nerve+regeneration.">Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.</a> Biomed Eng Online. 11, (2012).</li><li>Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+growth+factor+loaded+microspheres+into+polymeric+electrospun+nanofibers+for+tissue+engineering+applications.">Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.</a> J Biomed Mater Res A. , (2013).</li><li>Li, X., 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=Encapsulation+of+proteins+in+poly(L-lactide-co-caprolactone)+fibers+by+emulsion+electrospinning.">Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning.</a> Colloids Surf B Biointerfaces. 75, 418-424 (2010).</li><li>Wang, 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=The+effect+of+aligned+core-shell+nanofibres+delivering+NGF+on+the+promotion+of+sciatic+nerve+regeneration.">The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration.</a> J Biomater Sci Polym Ed. 23, 167-184 (2012).</li><li>Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, 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=Peripheral+nerve+regeneration+using+composite+poly(lactic+acid-caprolactone)/nerve+growth+factor+conduits+prepared+by+coaxial+electrospinning.">Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning.</a> J Biomed Mater Res A. 96, 13-20 (2011).</li></ol>

The authors have nothing to disclose.

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...

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。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

微球50±14微米直径的85％以上的蛋白质封装一贯生产，静电成支架。大小是由成像微球的样本来自三个不同的生产批次确定。 图1，其中捕获在哪里使用商业实验室软件测定在光学显微镜和长度的图像。表示粒度分布的直方图。包封率也由三个单独的微球的批次进行测试，通过量化在生产过程中逃脱的蛋白质。 图2示出了代表性的微球与罗丹明（2微克...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

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, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

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Bioengineering

12.2K 次观看.  Wayne State University. 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.

视频: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

静电基础：优化解决方案和设备参数

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

科研

生物工程

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 ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长：神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

保利的静电纤维支架（甘油 - 十二双）工程神经组织从小鼠胚胎干细胞

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

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

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

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 ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...