作者想通过工程技术研究中心革命化金属生物材料（ERC-0812348）和纳米技术本科教育（EEC 1242139）资助的支持，感谢国家科学基金会。

所有协议遵循研究合规与道德的北卡罗莱纳州A＆T州立大学办公室的指导方针。 1.化学制备角蛋白提取4 <ol><li>为了制备1,000ml中2％重量/体积的过乙酸溶液（PAS），在通风橱下20毫升过乙酸加至980毫升去离子（DI）水的。 </li><li>为了制备1,000ml中的100mM Tris碱溶液（TBS）中，添加12.2克Tris碱的达1,000ml去离子水，搅拌至完全溶解。 </li><li>通过倾倒4ml浓盐酸于30ml DI水中制备在通风橱稀盐酸溶液（DHAS）。 </li><li>采购大约20g还没有经过化学处理或改变人的头发丝的剪报。头发可以是任意长度。 </li><li>彻底清洗头发，用手，用温水和肥皂。使用去离子（DI）水进行最后冲洗。 </li><li>把头发分成2台60毫升的烧杯中，并发生在80ºCØVEN 1小时。 </li><li>从烧杯底部排出的液体和放回烘箱直到头发完全干燥。 </li><li>鸿沟干头发均匀地600毫升的烧杯之间，将将10克发到每个烧杯中。不要让头发充满超过500毫升。 </li><li>倒入500毫升PAS到每个烧杯中并确保覆盖所有的头发。用封口膜封住并存储12小时的烧杯。 </li></ol> 2.角蛋白提取液的制备<ol><li>使用500微米筛子分离PAS头发。收集废物的PAS在一个单独的容器中。用去离子水彻底冲洗头发，以消除任何剩余的PAS。 </li><li>放置在一个500ml烧瓶中的漂洗毛发。倾400毫升的TBS到烧瓶中，确保毛发被覆盖。在设置为38ºC，65转1小时的惊天洗澡的地方瓶。 </li><li> 1小时后，从振荡浴中移除，并倒入液体，约400毫升角蛋白提取液（KES），我n要的1000ml烧杯中。 </li><li>倾400毫升DI水到含有头发确保毛发被覆盖并放回振荡浴1小时烧瓶中。从晃动浴中取出烧瓶，并倒入剩余的将400ml KES入1000毫升烧杯中与先前收集的KES。头发不再需要并且可以倾倒出瓶中的并丢弃在最近的垃圾容器。 </li></ol> 3.角蛋白提取液浓度<ol><li>填写rotodistiller浴用蒸馏水顶线，并设置到90℃。设置旋转速度至200转。打开冷却剂冷却器泵和设置为-10ºC。 </li><li>使用pH计来监视KES的pH值，用移液管至1mM DHAS慢慢添加到KES直到达到pH为7.0。慢慢搅拌的溶液中。 </li><li>倾中和的KES到500毫升的圆底烧瓶中直到大约四分之一满。根据制造商的运行蒸馏协议1.25小时。 <ol><li>重复步骤3.3直到所有KES已经蒸馏。确保将烧瓶紧紧相连。 </li></ol></li></ol> 4.透析角蛋白提取液的<ol><li>倒入KES解决方案均分为12个独立14毫升锥形管。离心试管在1050×g离心10分钟。 </li><li>倒入离心KES到一个干净的烧杯中，并确保从收集的碎片倒掉。重复，直到所有的KES已经离心。 </li><li>填写2,000量筒用去离子水。 </li><li>切透析管纤维素膜到24英寸，和夹管的一端保持它关闭。 </li><li>浸管的长度DI水在汽缸中，以使之更柔软和更容易打开。 </li><li>打开透析管纤维素膜的非限幅端和慢慢倒入60毫升离心KES溶液进入管。使用另一个剪辑关闭管道的这一端。 </li><li>把迪所述2000毫升气缸DI水在alysis管并允许放置24小时。改变DI水在气缸中，每3至4小时。 </li><li> 24小时周期之后，清空透析KES溶液放入加盖罐子，应确保在-20℃离开顶部和地点空间在冷冻24小时。 </li></ol> 5.角蛋白提取液冻干<ol><li>冻干机设置为-86ºC。 </li><li>拆下冰箱冷冻含有KES罐子，把帽子脱罐子，并把它们放入冻干机安瓿。广场上的安瓿的密封和转动旋钮创造的0.133毫巴的真空压力。冻干样品约48小时，直到所有的水分已经一去不复返了。 </li></ol> 6.准备静电解决方案（10％重量角蛋白溶液） <ol><li>从冻干机中取出角蛋白粉和称重。 </li><li> DI水添加到角蛋白粉末以创建10％重量/重量角蛋白的解决方案。 </li></ol> 7.准备10％的重量PCL解决方案<ol><li>得到的PCL（ε - 己内酯的聚合物，锰70 - 90 kDa的），和三氟乙醇（TFE）。通过搅拌O / N中的TFE重量的PCL制备10％，将获得均匀的混合物。 </li></ol> 8.制备角蛋白/ PCL解决方案<ol><li>获得预先制备的10％（重量）的PCL溶液和10％（重量）角蛋白溶液。添加角蛋白进入PCL溶液滴在以创造10毫升PCL / 90:10，80:20，70:30角蛋白溶液，和60:40比率明智的。 </li><li>用涡流混合器静电之前获得的PCL /角蛋白溶液均匀的混合物。 </li></ol> 9.生产静电纺丝的PCL /角质纤维<ol><li>放置约8毫升的PCL /角蛋白溶液在装有0.5毫米直径的塑料管10ml的一次性注射器。放置注射器在将流速设定为2.5毫升/小时的注射器泵。 </li><li&gt;应用电压到连接至所述管的针尖（定位从纤维收集鼓〜23厘米和〜从水平30度）。套用19 - 22千伏电压到管，并在约200转旋转滚筒收藏家。 <ol><li>运行示例之前包裹铝箔收集桶。确保该铝箔是通过将标签带在任一端是稳定的。 注:纤维将形成在铝箔上（ 见图 2）中，纤维覆盖箔储存于RT。 </li></ol></li></ol> 10. PCL /角质纳米纤维的力学分析<ol><li> 8 mm切割样品成60个毫米。确保通过使用数字千分尺来记录厚度。对于准备各组成，使用五个样本。 </li><li>附着在拉伸试验机的60×8毫米的样本。使用自定义设计的试样架附加的样本。夹着它是从一个索引卡片使用双制成样品支架之间的样本双面胶带。以10毫米/分钟的位移速率施加10N的测力传感器。 注:样品保持器被设计为有62个毫米乘40毫米，38毫米50mm的内窗。 </li><li>记录延伸，通过软件根据软件协议的负载值。 </li><li>通过测试的样品的区域分割的力计算应力。接着，除以初始长度的长度变化计算应变。 </li><li>产生由在y轴为x轴对应应变值绘制应力的应力 - 应变曲线。 </li><li>从那里斜率等于弹性模量线性区域计算杨氏模量。通过取0.2％应变偏移和绘制平行线到线性域的曲线确定从应力应变图抗拉强度。 </li><li>以前使用分析软件使用8单因素方差分析方法进行一式三份获得的机械数据的统计分析，P值均&lt;0.05，我们再有统计学显著。 </li></ol> 11.表面形貌和结构表征<ol><li>使用具有加速15千伏的电压和5微安的电流来观察电纺丝纤维的形态的参数扫描电子显微镜（SEM）。 <ol><li>用SEM来成像之前，切纤维2厘米2段和使用铜带将其连接到一个SEM阶段。放置溅射镀膜机和涂层用金内部的阶段在15mA进行1分30秒的创建的金层大约11纳米厚。加载样品到SEM的腔室。观察纤维样品。 </li></ol></li><li>使用傅立叶变换红外光谱（FTIR）来表征PCL和角蛋白之间的化学结合。放置静电纳米纤维膜的2 平方厘米部分中的磁夹持器和测试之前吹扫 ​​用干燥空气的系统。获取光谱200扫描和使用STA分析光谱根据软件协议ndard软件。 </li><li>使用标准的软件进行频谱分析，并使用根据制造商的方案的纳米纤维的各个比率的一式三份。 </li></ol> 12.细胞纤维相互作用研究<ol><li>切纤维为16 平方毫米的样品。使用生物相容的，硅氧烷基胶固定每个样本盖玻片用直径为12毫米。 </li><li>胶纤维样品到盖玻片的背面的一端，环绕盖玻片样品，然后粘上样品到盖玻片的背面的自由端，以及，留下覆盖有仅滑移的顶纤维样品。 </li><li>放置纤维样品在24孔板中。通过在80％乙醇中浸渍1小时灭菌样品。 </li><li>冲洗使用去离子水样品中的乙醇。然后，加入2 ml基础培养基上的样品中，并确保以覆盖整个样品。 5分钟后删除网上平台。 </li><li>使用3成纤维细胞T3细胞种子的纤维样品。暂停补充有抗生素和10％胎牛血清（FBS），以便在Dulbecco改良的Eagle培养基（DMEM）的细胞有大约62000个细胞/ cm 2的每1ml DMEM中。 </li><li>滴将1ml 3T3含细胞的DMEM溶液到每个孔板确保每个纤维样品覆盖有大约62000个细胞/ cm 2以下。孵育孔板在37℃下24小时和5％ 的 CO 2。注意:使用5％的CO 2，因为该水平通常可以保持细胞媒体在生理范围的pH值天。 </li></ol> 13.降解纳米纤维矩阵<ol><li>切干PCL /纳米纤维角蛋白膜成方形约30mm×30毫米。 </li><li>消毒900 平方毫米的样品用80％酒精10分钟的潜伏期和去离子水彻底清洗。孵育样品在15毫升PBS中，pH为7.5的，在37ºC。更换BUFFER每3天。 </li><li>取该膜从溶液中在指定的时间间隔，1周和7周，并用DI水冲洗。 </li><li>将膜样品进入冻干机安瓿。放置在安瓿的密封和转动旋钮以产生真空。冻干24小时，直至完全干燥。 </li><li>在干燥的样品将其连接到用铜带的SEM阶段。放置溅射镀膜机和涂层用金内部的阶段在15mA进行1分30秒的。 </li><li>加载样品到SEM的腔室。在1.5千伏和5微安的电流的加速电压观察纤维样品。 </li></ol>

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	<li>Huang, Z. -. M., Zhang, Y. Z., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+polymer+nanofibers+by+electrospinning+and+their+applications+in+nanocomposites.">A review on polymer nanofibers by electrospinning and their applications in nanocomposites.</a> Compos Sci Technol. 63, (2003).</li><li>Li, W. J., Laurencin, C. T., Caterson, E. J., Tuan, R. S., Ko, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+structure:+a+novel+scaffold+for+tissue+engineering.">Electrospun nanofibrous structure: a novel scaffold for tissue engineering.</a> J Biomed Mater Res. 60, 613-621 (2002).</li><li>Liu, W., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibers+for+regenerative+medicine.">Electrospun nanofibers for regenerative medicine.</a> Adv Healthc Mater. 1, 10-25 (2012).</li><li>Edwards, A., Jarvis, D., Hopkins, T., Pixley, S., Bhattarai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(-caprolactone)/keratin-based+composite+nanofibers+for+biomedical+applications.">Poly(-caprolactone)/keratin-based composite nanofibers for biomedical applications.</a> J Biomed Mater Res B. 103, 21-30 (2015).</li><li>Dowling, L. M., Crewther, W. G., Parry, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+structure+of+component+8c-1+of+alpha-keratin.+An+analysis+of+the+amino+acid+sequence.">Secondary structure of component 8c-1 of alpha-keratin. An analysis of the amino acid sequence.</a> Biochem J. 236, 705-712 (1986).</li><li>Yamauchi, K., Maniwa, M., Mori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+fibroblast+cells+on+keratin-coated+substrata.">Cultivation of fibroblast cells on keratin-coated substrata.</a> J Biomat Sci-Polymer. 9, 259-270 (1998).</li><li>Shea, L. D., Wang, D., Franceschi, R. T., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Bone+Development+from+a+Pre-Osteoblast+Cell+Line+on+Three-Dimensional+Scaffolds.">Engineered Bone Development from a Pre-Osteoblast Cell Line on Three-Dimensional Scaffolds.</a> Tissue E. 6, 605-617 (2000).</li><li>Fortin, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Biological+Software.">New Biological Software.</a> Q Rev Biol. 71, 169-170 (1996).</li><li>Bhattarai, N., Edmondson, D., Veiseh, O., Matsen, F. A., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+chitosan-based+nanofibers+and+their+cellular+compatibility.">Electrospun chitosan-based nanofibers and their cellular compatibility.</a> Biomaterials. 26, 6176-6184 (2005).</li><li>Yang, S., Leong, K. F., Du, Z., Chua, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+scaffolds+for+use+in+tissue+engineering.+Part+I.+Traditional+factors.">The design of scaffolds for use in tissue engineering. Part I. Traditional factors.</a> Tissue E. 7, 679-689 (2001).</li><li>Bhattarai, N., 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=Natural-Synthetic+Polyblend+Nanofibers+for+Biomedical+Applications.">Natural-Synthetic Polyblend Nanofibers for Biomedical Applications.</a> Adv Mater. 21, 2792-2797 (2009).</li></ol>

作者宣称，他们没有竞争的经济利益。

从人发角蛋白的提取成功实现。过乙酸充当对人类的头发氧化剂，允许由Tris碱中提取的角蛋白。生产角蛋白粉的是小规模，由于这样的事实，它为研究目的才被完成。这个程序已经建立在工业的大规模生产。提取的小规模角蛋白的目的是，以控制污染，批次变异性，和成本效益。 角蛋白提取是这个过程的限速步骤。角蛋白粉末的产率是很低的，0.7 - 2％。人的头发丝20克产?...

电纺丝是公认的实现聚合物纳米纤维的一种普遍方法。该纤维可以在纳米级来制备和纤维的性能定制1。这些发展和纳米纤维的特性一直是他们在生物医学工程应用中特别有趣尤其是在组织工程。所述纳米纤维具有相似的细胞外基质，从而促进细胞粘附，迁移和增殖2。由于这种相似的胞外基质（ECM），电纺纤维可以用作材料，以协助在伤口敷料，药物递送，以及用于工程组织如肝，骨，心脏，肌肉3。 各种合成和天然来源的不同聚合物已被用于创建不同的生物医学工程应用4静电纤维。近来，在不断增长terest在复合纳米纤维的发展通过混合合成的和天然的聚合物4。在这些组合物的最终产品通常继承与合成聚合物结合的同时还采用生物线索和性质与天然聚合物的机械强度。 在该实验中，PCL和角蛋白被表示为要使用的复合纳米纤维的合成的合成和天然聚合物。角蛋白是在毛发，羊毛和指甲发现天然聚合物。它包含了许多氨基酸残基;值得注意的兴趣是半胱氨酸4,5。理想的是天然存在的聚合物是生物可再生的，生物相容和可生物降解的。角蛋白具有所有这三种特性，同时也增强细胞增殖和附着到它已在6被纳入的生物材料。 聚己内酯（PCL）是一个可再吸收的，合成的聚合物，在显著组织工程4。这种聚合物先前已经称赞其结构和机械稳定性，但是，它缺乏细胞亲和力并呈现一个冗长的降解速率。 PCL的疏水性质为缺乏细胞亲和性7的可能负责。然而，PCL由是与其他聚合物高度混溶弥补了其局限性。一个PCL /角蛋白复合应当证明PCL的力学性能，并纳入角蛋白的生物特性，使其成为各种生物医学应用的理想选择。

<table><tbody><tr><td>Human Hair&amp;nbsp;</td><td></td><td></td><td>Obtained from Local Barber Shop in Greensboro</td></tr><tr><td>Peracetic acid</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>PCL (e-caprolactone polymer)</td><td>Sigma Aldrich</td><td>502-44-3</td><td>Mn 70-90 kDa</td></tr><tr><td>Trifluoroethanol (TFE)</td><td>Sigma Aldrich</td><td>75-89-8</td><td></td></tr><tr><td>Tris Base (TrizmaTM Base Powder)</td><td>Sigma Aldrich</td><td></td><td>&gt;99.9% crystalline</td></tr><tr><td>Hydrochloric Acid</td><td>Fischer Scientific</td><td>A144C-212 Lot 093601</td><td>Waltham, MA</td></tr><tr><td>Kwik-Sil</td><td>World Precision Instruments</td><td></td><td>Sarasota, FL</td></tr><tr><td>Cellulose membrane</td><td>Sigma Aldrich</td><td></td><td>12 - 14 kDa molecular cut off</td></tr><tr><td>optical microscope</td><td>Olympus BX51M</td><td>BX51M</td><td>Japan</td></tr><tr><td>scanning electron microscope</td><td>Hitachi SU8000</td><td>SU8000</td><td>Japan</td></tr><tr><td>Table-Top Shimadzu machine</td><td>North America Analytical and Measuring Instruments AGS-X series</td><td>AGS-X Series&amp;nbsp;</td><td>Columbia, MD</td></tr><tr><td>Fourier transform infrared spectroscopy</td><td>Bruker Tensor 2 Instrument&amp;nbsp;</td><td></td><td>Billerica, MA</td></tr><tr><td>Microcal Origin software</td><td></td><td></td><td>Northampton, MA</td></tr><tr><td>X-ray diffraction (XRD)</td><td>Bruker AXS D8 Advance X-ray Diffractometer</td><td></td><td>Madison, WI</td></tr><tr><td>Fibroblast 3T3&amp;nbsp; cell</td><td>American Tissue Type Culture Collection</td><td></td><td>Manassas, VA</td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM</td><td>Invitrogen</td><td></td><td>Grand Island, NY</td></tr><tr><td>Spectra max Gemini XPS microplate reader</td><td>Molecular Devices</td><td></td><td>Sunnyvale, CA</td></tr><tr><td>Student- Newman-Keuls post hoc test</td><td>SigmaPlot 12 software</td><td></td><td></td></tr></tbody></table>

synthesis of keratin-based nanofiber for biomedical engineering

静电纺丝，由于它的多功能性和潜在的在各个领域的应用中，被频繁用于制造纳米纤维。这些多孔纳米纤维的生产是非常感兴趣的，由于其独特的物理化学性质。在这里，我们阐述含有聚（ε -己内酯）角蛋白（PCL）纳米纤维（即，PCL /角蛋白复合纤维）的制造。水溶性角蛋白首次从人类头发萃取，用不同比例的PCL混合。 PCL /角蛋白的混合溶液转化成用实验室设计的静电设立纳米纤维膜。纤维形态，将得到的纳米纤维的机械性能进行了观察和使用扫描电子显微镜和拉伸试验机进行测定。此外，纳米纤维的降解和化学性质用FTIR分析。 SEM图像显示，对于不同的组合物的PCL /角蛋白纤维均匀的表面形貌。这些PCL / keratiÑ​​纤维也表现出优异的机械性能如杨氏模量与故障点。成纤维细胞能够附着并增殖从而证明良好的细胞生存力。根据上面讨论的特征，我们可以强烈认为天然和合成聚合物的共混纳米纤维可以表示可用于不同的生物医学应用的复合材料的优异的发展。

纤维形态 对于所有的纤维组合物得到的纤维的SEM图像。 参见图3。纤维图像证实了纤维被随机取向。 机械测试 机械强度高的纤维一般需要关于各种组织工程应用。这些纤维应保留一定的压力和环境条件9下足够的强度和柔韧性。通常，支架被期望具有接近目标组?...

Watch this Scientific Journal Video about Synthesis of Keratin-based Nanofiber for Biomedical Engineering at JoVE.com

用于生物医学工程的角蛋白纳米纤维的合成|生物工程|视频

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

纳米纤维具有高的表面积与重量比，优异的机械完整性，并支持细胞生长和增殖。这些纳米纤维具有广泛的生物医学应用。在这里，我们制造角蛋白/ PCL纳米纤维，利用静电纺丝技术，和表征纤维用于组织工程可能的应用。

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

synthesis-of-keratin-based-nanofiber-for-biomedical-engineering

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15.3K 次观看.  North Carolina A&T State University. 纳米纤维具有高的表面积与重量比，优异的机械完整性，并支持细胞生长和增殖。这些纳米纤维具有广泛的生物医学应用。在这里，我们制造角蛋白/ PCL纳米纤维，利用静电纺丝技术，和表征纤维用于组织工程可能的应用。 

视频: Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

科研

生物工程

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

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 ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

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.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Griffithsin 修饰纤维支架预防性传播感染的制备与表征

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

制作生物功能纳米纤维的过程

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

生物学

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

<meta charset="utf8"></head><body>FN-Silk 膜的形成和附着到细胞培养物的插入物上

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

<meta charset="utf8"></head><body>作者聚焦：使用重组功能化蜘蛛丝膜增强体外细胞培养模型

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...