We gratefully acknowledge funding from the Wellcome Trust Affordable Healthcare for India and an EPSRC Landscape Fellowship for Ilida Ortega as well as contributions from The Electrospinning Company Ltd.

道德声明:在本研究中使用的眼睛是根据上公开​​对动物研究的声明中使用: 1，制作PLGA生物降解膜的配备Micropockets <ol><li>环支架采用的microstereolithography和静电纺丝技术31的组合产生。在本质上，该方法可以归纳为2份（ⅰ）创建的PEGDA模板由microstereolit​​hography以及（ii）电纺丝到模板用于再现的基本​​PEGDA结构（在这种情况下，微制造环）。这两个步骤进行详细说明（ 图1和2）。 <ol><li>的PEGDA模板由microstereolit​​hography制造（microSL） <ol><li>使用2层模型制造的环，与所述第一层（L1）是该结构的基极与所述第二层（L2）的呈递6 micropockets马蹄形态学各种尺寸的ES从300-500微米。 PEGDA戒指的制作是最近这组22中描述。 <ol><li>通过任何合适的画图程序绘制1.2厘米黑圈创造的L1。 </li><li>以相同的方式创建的L2但包括8白0.5×0.35毫米马蹄形结构。黑圈结构中散发白色的小马蹄形状。 </li><li>保存L1和L2为JPEG格式。 </li></ol></li><li>在黑暗的玻璃小瓶中，混合聚乙二醇二丙烯酸酯（PEGDA，锰= 250克/摩尔），用1％w / w的樟脑醌，光引发剂，在磁力搅拌器上20分钟。 </li><li>展开microstereolit​​hography使用伸缩式镜头结构建立的激光束，然后投射到它的计算机的可编程数字多镜装置（紫外线功能的DMD入门套件）。注:DMD的反射图像（在这种情况下的环）上的反射镜，通过10cm的焦距管透镜。的图像，然后由银辅酶定向泰德反映到含有光固化聚合物（PEGDA）小瓶。 </li><li>调整并仔细清洗microstereolit​​hography设立的光学系统。 </li><li>把300微升的-PEGDA水混合成一个12孔培养板的孔中。确保将孔预先涂有特氟隆或其他非粘着材料，便于拆卸的结构的固化后。 </li><li>以前安装在PC上，并上传L1到ALP3，基础软件，交换机上的蓝色激光（150毫瓦MBL-Ⅲ473纳米）。照射所述第一层为60秒。注:ALP3，基本是一个USB接口，提供了PC和数字微镜器件之间的联系。 </li><li>添加到孔中加入250μl更多的PEGDA的，上传L2如上文所述照射L2为60秒。 </li><li>除去未固化的聚合物和洗环异丙醇O 2​​ / N。 </li></ol></li><li>采用静电纺丝可生物降解的PLGA的制备隔膜 注:PEGDA戒指作为模板结束了它的PLGA进行电纺丝与PLGA再现底层的地形，因为它滚动到这些模板。纺丝后，PLGA聚合物的片材从集电极剥离。最终的PLGA膜不含有该被留下附着在金属集电体的PEGDA环。 <ol><li>分布在电镀铝板的PEGDA环（12厘米×20厘米），用于创建一个静态的静电收集器。 </li><li>安装使用导电碳胶带环。请注意:这些一旦形成可以被重新用作用于静电纺丝模板的环。 </li><li>准备纺丝聚合物溶液。溶解的PLGA（50/50 DL-丙交酯（52摩尔％）:乙交酯（48摩尔％），44千克/摩尔）的二氯甲烷（DCM）中的20％w / w的浓度。 </li><li>轰动O 2 / N使用前</li><li>地点4胰岛素注射器（平端，0.8厘米内径针头）在注射泵。注:四个注射器保证比单针筒的工作更快速静电。 </li><li>加载加入2.5ml在每个注射器的PLGA溶液。 </li><li>静电纺丝用30微升/分钟的流速和电压范围从12至15千伏。离开针和15厘米的集电极之间的距离。 </li><li>静电纺丝1小时和30分钟，最后小心剥离从支承PEGDA环集电极的PLGA电纺片材。 </li><li>切静电支架成直径22 mm圆采用圆形打孔和离开位于中心的环结构。 </li></ol></li></ol></li></ol> PLGA微加工膜2。长期储存注:PLGA戒指制作并经认可的外部公司消毒;样品上照射在25-40千戈瑞的外部剂量范围。 <ol><li>装在小容器中的膜（塑料培养皿），并把它放在一个医用级包。 </li><li>使用滤纸来创建小的过滤袋的干燥剂。牛逼o创建袋，折叠滤纸（直径125mm），并削减所产生的半圆一半;然后用胶带封住两端连在一起。 <ol><li>填3滤纸袋用1克二氧化硅橙，钴（II），氯化铜（Ⅱ）硫酸盐，分别。 </li><li>把三袋干燥剂里面的医疗级袋子连同静电膜。 </li></ol></li><li>放入袋子市售6点湿度指示卡检测存储期间的任何水分积聚。 </li><li>使用真空热封机真空并密封袋。 </li><li>发送环膜与外部公司进行γ射线照射。 </li><li>商店γ辐照PLGA膜在很宽的温度范围为-20°C至+37℃，在潮湿的环境中含5％ 二氧化碳培养箱内。 </li><li>交的存储，检查湿度指示器，以确认湿度的水平之下30％。 </li><li> ü本身扫描电子显微镜（SEM）来评估纤维的完整性。 </li></ol> 3，分离角膜缘外植体兔角膜缘外植体兔眼（从农场，兔子繁殖的消费中获得）隔离。 <ol><li>消毒用消毒液（3％）的兔眼。 </li><li>通过消除周围的角膜任何多余的组织清洁眼睛。 </li><li>使用解剖显微镜对角膜的其余的角膜缘区域（标识为角膜（透明）和巩膜（白色）之间的薄的圆形区域）分开。 </li><li>切入解剖显微镜下段（约1.5厘米长）。 </li><li>消毒在1.5％消毒液的角膜缘段1分钟。 </li><li>切缘细分成小块（100-500微米）用手术刀刀片。 </li><li>存储在培养介质中的小片组织（DMEM + Glutamax的:Ham&#39;s F12（1:1），10％胎牛血清，1 U / ml的笔icillin，100毫克/毫升链霉素，2.5微克/毫升的两性霉素，EGF的10纳克/毫升，而胰岛素的5微克/毫升），在37℃和5％CO 2，直到使用（不超过60分钟）。 </li></ol> 4，突增生，从角膜缘外植体细胞兔角膜缘外植体放置在两个新鲜纺丝微制造的膜和薄膜构成，它是真空包装和保存6个月，在-20℃下。 <ol><li>外套环支架用15微升的血纤维蛋白胶:使用细胞刮刀（1 1为18.75毫克/毫升和凝血酶从人血浆中以2.5单位/ ml的浓度的浓度的混合物，从人血浆中的纤维蛋白原），用于分配的纤维蛋白均匀。 </li><li>直接将组织块使用将25g针和解剖显微镜的PLGA micropockets。 </li><li>添加细胞培养基非常轻柔，避免拆卸外植体。 <ol><li>更换介质，每3天（3.7节所描述的媒体配方）和KEEP中培养2周时，在湿润的培养箱中在37℃和5％CO 2。 </li></ol></li><li>固定用3.7％甲醛缓冲液进行10分钟，随后洗涤3次，用PBS中的样品。 </li><li>温育在1微克/毫升的4&#39;染液，6二脒-2 - 苯基吲哚（DAPI）或碘化丙啶（PI）在RT下10分钟。 </li><li>在PBS冲洗3次，存储用铝箔纸覆盖的样本。 </li><li>图片用荧光显微镜在543 nm和800 nm的（双光子）的波长的样品。 </li></ol> 5，设置行动兔角膜受伤的3D模型<ol><li>清洁和上述消毒兔眼。 </li><li>通过浸没于0.14％的氢氧化铵为5分钟，卷绕在兔眼。 </li><li>冲洗眼睛在PBS和刮掉了骨节刀上皮。 </li><li>切开并分离角膜巩膜按钮，清除任何剩余的组织。 </li><li>将角膜上皮面朝下到无菌杯，装满0.5％琼脂的DMEM进行。 </li><li>设置完成后，把角膜上皮面朝上，在小培养皿中，加入培养基（上述配方）到角膜缘区。注意:不要覆盖整个角膜;保持器官培养在气 - 液界面。 </li></ol> 6分离角膜缘外植体和环支架在兔角膜3D模型融入社会<ol><li>涂层具有15微升的血纤维蛋白胶的环膜（1:纤维蛋白原的混合物在1875毫克/毫升和凝血酶以2.5单位/ ml的浓度的浓度）。用细胞刮刀，如上所述。 </li><li>直接将组织块使用将25g针和解剖显微镜的PLGA micropockets。 </li><li>把戒指带在裸露的角膜朝上外植体，在气 - 液界面条件下组织块。 （这些条件先前已有描述22）。 </li><li>维持器官培养模型4周的潮湿培养箱中在37℃和5％CO 2的变化的介质每2天。 </li></ol> 7，评估角膜再生和干细胞维持<ol><li> 4周后，用固定3.7％甲醛的眼角膜。 </li><li>过程中，角膜的组织学传统生产6微米的石蜡切片。 </li><li>染色用苏木精和曙红（H＆E）。 </li><li>对于免疫细胞化学，脱蜡的切片在二甲苯（3分钟）和再水合的100％乙醇（1分钟），70％乙醇（1分钟），和蒸馏水（2分钟）。 </li><li>划定使用Dako公司的笔来界定小区域，避免过度使用抗体的部分。 <ol><li>治疗划定的区域用0.05％胰蛋白酶（100微升）处理20分钟（37℃）。 </li></ol></li><li>用PBS彻底清洗和1小时加入100μl的10％山羊血清（阻塞）。 </li><li>孵育样品与100μl的小鼠单克隆抗体cytokera锡3（CK3）和100微升P63的1％山羊血清在o / n 4℃。 </li><li>用PBS洗涤，并用100微升生物素化的第二抗小鼠抗体（1:1,000在1％山羊血清）中1小时，在室温。 </li><li>添加100μl的FITC-链霉抗（1:100在1％山羊血清），用于在室温30分钟。 </li><li>如上述那样对待该样品用DAPI。 </li><li>图像用荧光显微镜在800纳米（双光子）和488纳米波长下的样品。 </li></ol>

<ol>
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J., Deshpande, P., MacNeil, S., Claeyssens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+microfabrication+and+electrospinning+to+produce+3-D+architectures+for+corneal+repair.">Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair.</a> Acta Biomaterialia. 9, 5511-5520 (2013).</li><li>Blackwood, K. A., 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=Development+of+biodegradable+electrospun+scaffolds+for+dermal+replacement.">Development of biodegradable electrospun scaffolds for dermal replacement.</a> Biomaterials. 29, 3091-3104 (2008).</li></ol>

作者什么都没有透露。

这项研究描述了静电膜含在其中微特征的制造及（b）如何准备这类膜用于临床使用的真空包装，γ辐照，然后贮存在使用前（A）的技术。在这个特定的应用程序，我们已经开发出含micropockets的模仿角膜缘干细胞小生境的物理特性PLGA膜。本研究的目的是:（i）描述的方法，为读者提供设计和制作含有微特征的研究棚架成的干细胞小生境组织再生及（ii）提供一个更好地了解读者的贡献所需要的知识如何存储电纺支架的很长一段时间。 在临床应用方面，环膜的存储是极其重要的。在这项工作中，该环的降解进行了研究历时6个月。退化膜是通过水解只须保持无水分的过程中暂停该膜从动左右。黑木等人报告说，通过改变聚乳酸的比例PGA，膜的降解发生变化32。本研究还表明，通过提高PGA的量，电纺膜的降解速率在 ​​体内 22增加。这里已经示出了用真空包装的膜连同一些干燥剂和照射它们，并将它们存储在低温下为6个月，没有在纤维完整性和降解没有发生变化。目前，6个月是因为我们已经研究了用含有micropockets但存储数据这些膜1年已报道在普通静电膜在-20℃下22，现在我们有未公布的数据的储存于-20℃下尽可能2年未降解的迹象。因此，对于长期贮存将被推荐给存储它们干燥，在-206，C，但有可能甚至在印度将它们存储在RT至少6个月（可能更长）。列入湿度指标给出检查包装一直保持膜干在这种情况下，他们将适合为目的的一种简单方法。 从这些环到3D模型角膜细胞的转移micropockets内放置角膜缘外植体时，被证明。该小组最近通过将植在普通PLGA膜（无膜环结构）24日报道转移细胞到体外兔角膜模型。使用本微构支架细胞移植已经采取了一步，因为我们现在特别是微特征中定位组织块。在壁龛内直接将外植体的能力，也可以让医生直接在手术影院避免了洁净室的需要首先扩大角膜缘干细胞使用的膜。虽然这片WOR的k具有集中在用于角膜疾病设备的发展，这种微细加工技术还可以应用于开发了许多其它的应用程序的设备。未来的工作将探索构建体的制造对其它组织如皮肤和骨的再生。 而PEGDA微结构的设计和最初的制造可能是耗时的，一旦制造的结构可以在不降低反复使用多次。因此，PLGA微结构的生物降解膜的通过电纺丝的后续制造可以以相同的速度收集器的装配如下进行，以生产纯（"非结构化"）膜。虽然在本工作中，我们已经使用microstereolit​​hography用于制造模具，其它制造方法，例如三维印刷或注射成型，可以也使用。因此，底层的模具可以由PE制成的出其它的聚合物或金属的，而不是GDA。因此这种技术是非常灵活，研究人员可以很容易地适应方法，以符合自己的需求和设施。 在内部 microstereolithography设置在该研究中使用将不会允许与下30μm的特征的制备构建体;这不是对这里所描述的角膜应用的限制，但它可以是在其它型号的设计是至关重要的。在这种情况下，其他技术如2光子聚合（2PP）可能会感兴趣但是静电纺丝技术可能不允许在亚微米尺度的结构的再现（这是目前正在研究通过我们的组）。 制造过程中的关键步骤是:（i）避免的PEGDA模板，可以通过调整时间和光引发剂的量来控制的overcuring。 （ⅱ）控制电纺丝条件，例如温度和湿度。 （三）妥善保存的electrospu采用真空包装，干燥剂Ñ环膜。 总之，通过在膜的微特征内放置角膜缘组织的外植体，我们已经表明，从上的适当位置区域，小区转移的外植体的细胞生长到兔子受伤角膜和随后的再上皮化的角膜。存储在不同的温度下，膜的降解也进行了研究，并包装协议，它允许长期贮存的膜已被开发出来，后者是在发展中的膜用于临床使用是必不可少的。

角膜，眼睛的缺血性中心外大多数组织，是涉及视力1的最重要的组织之一。有几种类型的细胞维持角膜的​​功能。角膜的顶部最外层包括上皮细胞，可以是在厚度2约5-7层。这层防止细菌侵入到角膜3，并允许氧气4的条目。据报道，该角膜上皮在于龛或隐窝（具有120-150微米大小）在被称为角膜缘5,6角膜的周边区域的干细胞。由于干细胞分裂，子细胞也被称为短暂扩充细胞行驶出龛，并为司继续在细胞移动向心向内和向上导致终末分化细胞在角膜中央区7,8。这些细胞经常擦去眼睛E的眨眼xposing新的细胞下方9。 除了 ​​是上皮干细胞的位置，角膜缘也起着从角膜区域10保持血管的结膜走了作用。损害角膜缘可以通过热/化学烧伤，辐射和也遗传疾病10引起的。当此情况发生时，角膜缘屏障被分解使结膜细胞移动到角膜，血管化的区域，引起疼痛和失明的情况。该情况被称为角膜缘干细胞缺陷（LSCD）10。 不同的天然底物已被报告为可能的干细胞的载体用于辅助在角膜再生。例如，胶原基膜使用Dravida 等 11罗摩和他的同事12日报道在112例患者的研究中使用的纤维蛋白。在治疗的本但是最常用的方法是利用人类羊膜从组织库和文化角膜缘上皮细胞在其表面13,14。一旦单层形成，羊膜粘细胞面朝上到受损的角膜有所有的结膜细胞和疤痕组织从它这个细胞移植14前手术切除。星期内的羊膜降低到几个月留下附着在裸露面积以再生上皮15,16的上皮细胞。该技术已成功地恢复视力但仍然有其临床上限制了它的广泛摄取一些实际问题。由于羊膜是人体组织，它需要进行良好的用组织库的程序被用于对患者的细胞移植前筛查。这种筛选不仅降低了疾病传播的风险，但不能完全消除17。除了这已经有变化的，在p报告羊膜由于跨施主变异18,19和加工方法不同19,20 erformance。除了疾病传播的风险小有用于外科中心能够获得良好的运行组织库，而不是提供给所有的要求。 虽然羊膜是比较成功的，有必要对角膜疾病的治疗的新合成的生物可降解的细胞载体的替代品的开发。综合运营商将克服需要银行程序，以及消除疾病传播和跨捐助变性的风险小。在这个意义上说，材料，如聚乙二醇21,22和PLGA 23,24进行了研究。 在发展中的合成替代羊膜也有设计成其期望的功能，希望帮助培养的细胞的存活的可能性。政府间谈判在生物材料设备微特征为细胞行为的具体控制lusion感兴趣的新兴领域。许多作者报道工作，争取人工干细胞的发展龛25-30。这个小组最近报告创立了微加工PEGDA纤维连接蛋白biofunctionalized人工角膜缘的角膜缘上皮细胞22的交付和含微加工口袋角膜缘上皮细胞31的支持静电的生物降解膜的制作的方法。 这个工作的目的是开发一种新的制造技术用于容纳微特征的模拟的程度，其中干细胞存在于身体的微环境中的生物材料的设备的开发。我们已经开发出结合microstereolit​​hography和静电纺丝的技术，它允许显示格雷亚可生物降解的微结构化的膜的制造中吨潜力的组织再生程序。 注意到，虽然在此工作此技术已经被应用到环角膜再生的制造中，该技术可以应用于器件的制造为各种上皮组织的再生， 例如 ，皮肤，口腔是很重要粘膜，肠，呼吸，和膀胱上皮细胞。具体地说，在该研究中，我们开发了一种合成的生物可降解的膜以相同的方式发挥功能的羊膜来提供细胞对角膜。该膜含有大约300微米micropockets（大于沃格特的Pallisades的角膜缘隐窝（约150微米））。最后，我们建立了封装协议，它允许存储这些膜在-20℃，6个月以上没有显示出崩溃的迹象。

<table><tbody><tr><td>Poly (lactic-&lt;em&gt;co&lt;/em&gt;-glycolic acid)</td><td>Purac</td><td>PDLG5004</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich Or Fisher</td><td>270997 Or D/1850/17</td><td>&gt;99.8% contains 50-150 ppm amylene stabilizer</td></tr><tr><td>Digital Micromirror Device (DMD) Discovery 1100 Controller Board &amp;amp; Starter Kit</td><td>Texas Instruments</td><td>1076N732 (UV)</td><td></td></tr><tr><td>473 nm Laser</td><td>Laser 2000</td><td>MBL-III</td><td>150 mW</td></tr><tr><td>Poly (ethylene glycol) diacrylate</td><td>Sigma Aldrich</td><td>475629</td><td>Mn = 250 g/mol, 500 ml</td></tr><tr><td>DEMEM + Glutamax</td><td>Fisher</td><td>12077549</td><td></td></tr><tr><td>Ham&amp;rsquo;s F12</td><td>Labtech biosera</td><td>LMH1236/500</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Labtech biosera</td><td>FB-1090/50</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D</td><td>236-EG-200</td><td></td></tr><tr><td>Insulin</td><td>Sigma Aldrich</td><td>91077C-1G</td><td></td></tr><tr><td>Amphotericin</td><td>Sigma Aldrich</td><td>A2942-100ml</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma Aldrich</td><td>P0781-100ml</td><td></td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>32670</td><td></td></tr><tr><td>Propidium iodide</td><td>Sigma Aldrich</td><td>P4864</td><td></td></tr><tr><td>Thrombin</td><td>Sigma Aldrich</td><td>T9326</td><td></td></tr><tr><td>Fibrinogen</td><td>Sigma Aldrich</td><td>F3879</td><td></td></tr><tr><td>p63</td><td>Sigma Aldrich</td><td>P3737</td><td></td></tr><tr><td>CK3</td><td>Merck Millipore</td><td>CLB218</td><td></td></tr><tr><td>Hematoxylin</td><td>SLS</td><td>HHS16-500ML</td><td></td></tr><tr><td>Eosin</td><td>Sigma Aldrich</td><td>HT110232-1L</td><td></td></tr><tr><td>Medical grade bag (PET/Foil/LDPE) Peelable pouch</td><td>Riverside Medical Ltd. Derby, UK</td><td>Foil laminate PET/Foil/LDPE, (12,7,50)</td><td></td></tr><tr><td>Gamma- irradiation (Sterilization)</td><td>Applied Sterilisation Technologies (Synergy Health Laboratory Services (SHLS), Abergavenny UK) - external dose range of 25-40 KGy</td><td></td><td></td></tr><tr><td>Silica gel orange</td><td>Sigma Aldrich</td><td>10087</td><td></td></tr><tr><td>Cobalt (II) chloride</td><td>Sigma Aldrich</td><td>232696</td><td></td></tr><tr><td>Copper (II) sulfate</td><td>Sigma-Aldrich</td><td>C-1297</td><td></td></tr><tr><td>Six spot humidity indicator card</td><td>SCC, USA</td><td>6HIC200</td><td></td></tr><tr><td>Vacuum heat seal machine</td><td>Andrew James UK Ltd, Bowburn, UK</td><td>VS518</td><td></td></tr><tr><td>Andrew James Vacuum Sealer rolls 28 cm x 40 meter rolls</td><td>Andrew James UK Ltd, Bowburn, UK</td><td>BR2805</td><td></td></tr><tr><td>Scanning Electron Microscope (SEM)</td><td>Philips/FEI XL-20 SEM</td><td></td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss LSM 510 META</td><td></td><td></td></tr><tr><td>Videne Antiseptic Solution</td><td>Ecolab, Swindon, UK</td><td></td><td>3%</td></tr></tbody></table>

combination of microstereolithography and electrospinning to produce membranes equipped with niches for corneal regeneration

角膜问题影响全球数百万人减少了他们的生活质量显著。角膜疾病可以由疾病如无虹膜或史蒂芬约翰逊综合症以及由外部因素如化学烧伤或辐射引起的。目前的治疗是（i）使用的角膜移植物以及（ii）利用扩展在实验室和交付的载体（ 例如 ，羊膜）干细胞;这些治疗都是比较成功的，但不幸的是，他们可以经过3-5年的失败。有必要设计和制造能够详细模拟，其中干细胞位于角膜的生理环境新角膜生物材料的设备。角膜缘干细胞存在于被称为沃格特的山花园具体龛（角膜和巩膜之间的圆形区域）的缘。在这项工作中，我们已经开发出一种新的平台技术，该技术结合了两种尖端的制造技术（microstereolit​​hography和electrospinn荷兰国际集团），用于模仿在一定程度上角膜缘角膜膜的制造。我们的膜含有人工micropockets其目的是为细胞提供保护，沃格特的帕利塞德做的眼睛。

静电微细加工环用microstereolithography和静电纺丝的组合（ 图1和图2）制造的。不同尺寸的PEGDA环用microstereolithography（ 图3）制造;此技术允许在制造结构中的厘米的顺序和微特征的同时掺入。在这种情况下，直径为环从1.2-1.6厘米含有350-500微米micropockets制备了（ 图4）。 在制造，杀菌和材料，以便将来临床使用的包装而言，发现在医疗级包装袋的真空包装显著改进以实现长期存储的PLGA膜（ 图5）的能力;使用的医疗级袋（PET /铝箔/聚乙烯），0.12毫米的厚度使我们能够实现较长的保质期。这是通过发送membr调查ANES我们在印度和膜的合作者都在-20℃保存数月内，在室温和37℃，故意潮湿的条件下（潮湿孵化器）， 图5显示了使用存储在无理取闹条件37℃，在潮湿条件下，膜是唯一的稳定约1个月的非真空包装的条件下，但在真空包装条件（ 图5和表1）取得储存3个月。 表1显示了在该下也能选择成有利于水的吸收和纤维溶胀如果注重袋的使用的选择条件来实现存储条件的改善。 环支持细胞生长的不同条件（I）环新鲜制作及（ii）戒指保存6个月（ 图6）角膜缘外植体。小区转移是在4周后达到时复表现很优秀的3D模型受伤的PLGA膜。细胞从组织外植体放置在上创建先前裸露的角膜新的上皮细胞（ 图7），所述膜前身。正（角膜未经任何处理的）和阴性对照（受伤的角膜）也维持培养相同的时间周期。阴性对照证实了在不存在任何添加的细胞的缺乏形成的一种新的上皮细胞。免疫细胞化学证实，细胞从外植体生长出是角膜上皮细胞，因为它们呈阳性反应，角膜分化标记CK3（ 图7E）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig1highres.jpg" width="500" /> 图1原理图microstereolithography的建立为建立PEGDA环。 her.within页="总是"&gt; <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig2highres.jpg" width="500" /> 图2原理使用PEGDA微细加工戒指作为模板，静电纺丝的过程。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig3highres.jpg" width="500" /> 图3（A）示出的静态集电极（电镀铝板带PEGDA环），用于微加工的PLGA膜的纺丝的一个例子（B）和（E）示出了不同的静电垫从静态集电极剥离（C）示出了不同尺寸的突出用microstereolithography对于下面的表面的制造的通用性的PEGDA模板（D）示出了PLGA微制造复制品。/files/ftp_upload/51826/51826fig3highres.jpg"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig4highres.jpg" width="500" /> àPEGDA环马蹄microfeature（一）图4 SEM图像;精细加工的口袋里（B）的高倍率SEM图像。以下PLGA环马蹄microfeature（C）的相位对比图像;精细加工的口袋（四）高倍率相位对比图像。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig5highres.jpg" width="500" /><b温度和时间对存储的真空和非真空的 R /&gt; 图5。影响PLGA封装（各占50％）膜（44千克/摩尔）与微机电环比6个月。膜的完整性被评定为完全不变纤维（+ + +），一些纤维肿胀（+ +），纤维合并（+）或不完整纤维（ - ）。扫描电镜图像和三个干燥剂（硅胶橙色，钴（II），氯化铜（Ⅱ））显示纤维的完整性或湿度无明显变化。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig5highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig6highres.jpg" width="500" /> 图6荧光图像显示从新鲜出炉的可生物降解的PLGA环角膜缘外植体的LEC的生长（A，B），并于6个月后储存在-20环℃（C，D），图像（A）和（B）对应于细胞用DAPI染色（蓝色）和碘化丙啶（红色）。图像b是共焦Z堆叠的外植体放置于微加工腔的正交视图。图像（C）和（D）表示阳性染色P63（绿色）。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig6highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig7highres.jpg" width="500" /> 图7（A）中示出了具有位于其上预先涂有纤维蛋白胶支架的环支架和组织外植体兔受伤角膜模型（B）和（C）是阳性和阴性对照。阳性对照是一个新鲜的拉比<html> 。吨角膜和阴性对照角膜其中上皮被故意除去（阴性对照也培养4周）（D）为4周培养后的组织工程角膜的H＆E染色图像;该图中示出了通过将细胞从放置在壁龛外植出来形成的新的多层上皮细胞（E）是免疫细胞图像表示从角膜缘外植体的细胞生长;细胞核用DAPI染色（蓝色）和细胞显示阳性染色细胞角蛋白3，角膜分化标记（绿色）。 <a href="https://www.jove.com/files/ftp_upload/51826/51826fig7highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" width="611"><tbody><tr height="22"><td height="43" rowspan="2" style="height:43px;width:163px;"> PLGA（50/50） </td><html> X;存储在37℃湿膜"&gt;光纤诚信</td></tr><tr height="21"><td height="21" style="height:21px;width:64px;"> 0天</td><td style="width:64px;"> 1个月</td><td style="width:64px;">第2个月</td><td style="width:64px;"> 3个月</td><td style="width:64px;"> 4月</td><td style="width:64px;"> 5月</td><td style="width:64px;"> 6个月</td></tr><tr height="21"><td height="21" style="height:21px;width:163px;">非真空包装</td><td style="width:64px;"> + + + </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr><tr height="67"><td height="67" style="height:67px;width:163px;">真空（袋）（PE，PA复合材料）厚度:0.14毫米</td><td style="width:64px;"> + + + </td><td style="width:64px;"> + </td><td style="width:64px;"> - </td>64px;"&gt; - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr><tr height="61"><td height="61" style="height:61px;width:163px;">真空（袋乙）（PET /铝箔/聚乙烯）厚度:0.75毫米</td><td style="width:64px;"> + + + </td><td style="width:64px;"> + + + </td><td style="width:64px;"> + + + </td><td style="width:64px;"> + </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td><td style="width:64px;"> - </td></tr></tbody></table> 真空和不同的储物袋上的PLGA诚信表1的影响（各占50％）膜（44千克/摩尔）检查6个月以上的存储空间。膜的完整性被评定为完全完整纤维（+ + +），一些纤维肿胀（+ +），纤维合并（+）或不完整纤维（ - ）。

Watch this Scientific Journal Video about Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration at JoVE.com

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

我们报告的静电膜，用来研究细胞行为中micropockets的制造技术。具体地，我们描述了用于生产的PLGA（聚（丙交酯 - 共 - 乙））配备有微特征角膜生物材料的设备microstereolit​​hography和静电纺丝的组合​​。

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

combination-microstereolithography-electrospinning-to-produce

Research

JoVE Journal

Bioengineering

12.4K 次观看.  University of Sheffield. 我们报告的静电膜，用来研究细胞行为中micropockets的制造技术。具体地，我们描述了用于生产的PLGA（聚（丙交酯 - 共 - 乙））配备有微特征角膜生物材料的设备microstereolit​​hography和静电纺丝的组合​​。 

视频: Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

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

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.

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, ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

角膜组织工程: 人角膜间质神经相互作用的体外模型

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

发育生物学

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

医学

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

用于轴突再生研究的角膜感觉纤维损伤方案

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

作者聚焦：通过创新模型推进角膜神经支配研究 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生

摘要

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静电纤维组织工程的后期处理

静电生长因子释放到微纤维支架

角膜组织工程: 人角膜间质神经相互作用的体外模型

适应静电过程中提供三个独特的环境为一个三层次

生物材料膜上人与羊角膜内皮细胞层的生长

准备可调细胞外基质微环境，以评估施万细胞表型规范

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

创面愈合研究的前活体角膜器官培养模型

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

摘要

探索更多视频

此视频中的章节

静电纤维组织工程的后期处理

静电生长因子释放到微纤维支架

角膜组织工程: 人角膜间质神经相互作用的体外模型

适应静电过程中提供三个独特的环境为一个三层次

生物材料膜上人与羊角膜内皮细胞层的生长

准备可调细胞外基质微环境，以评估施万细胞表型规范

通过三维细胞培养基质的紫外光图案化产生多库细胞微环境

创面愈合研究的前活体角膜器官培养模型

一种由工程成纤维细胞生长因子1刺激的Descemet剥离的人类角膜器官培养模型，仅具有加速愈合

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生