The authors have no acknowledgements.

这些协议遵循我院的人类研究伦理委员会的指导方针。 1.常规角膜胶原蛋白CXL（C-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁眼睛和眼睛周围的皮肤用碘防腐剂两次。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol> 2.上皮去除<ol><li>马可中心9.0毫米用圆圈标记角膜角膜。 </li><li>取出角膜上皮用生硬的抹刀中央7.0至9.0毫米乘机械清除。 </li></ol> 3.核黄素应用<ol><li>适用于日与20％葡聚糖0.1％核黄素<html> Ë角膜每分钟为20分钟。 </li></ol> 4. UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以3毫瓦/厘米2（5.4焦耳/厘米2表面剂量）的辐照，并在一个5厘米的工作距离为30分钟。 </li></ol><img alt="图1" src="/files/ftp_upload/53119/53119fig1.jpg" /> 图1:在C-CXL UVA照射的角膜以3毫瓦/厘米2（5.4焦耳/厘米2表面剂量）的辐照，并在一个5厘米的工作距离30分钟照射370nm的波长的UVA光。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53119/53119fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>在辐照过程中，应用核黄素角膜每隔5分钟滴。 </li><li>在辐照过程中，添加表面麻醉（奥布卡0.4％），如果有必要的。 </li></ol>手术"&gt; 5。完<ol><li>把抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0.18％），到运营的眼睛。 </li><li>将软绷带隐形眼镜在手术结束，直到再上皮化完成。再上皮化通常需要3天。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>后角膜再上皮，发起局部治疗与类固醇（地塞米松局部为1mg / ml），并继续进行3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol> 2.加速角膜胶原CXL（A-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁电子你们和眼睛周围的碘消毒剂皮肤的两倍。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol> 2.上皮去除<ol><li>标记中央9.0毫米角膜的带圈角膜标记</li><li>取出角膜上皮用生硬的抹刀中央7.0至9.0毫米乘机械清除。 </li></ol> 3.核黄素应用<ol><li>在角膜上施加0.1％的核黄素，而不葡聚糖每2分钟进行10分钟。 </li></ol> 4. UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以30毫瓦/厘米2（5.4焦耳/厘米2表面剂量），并在一个5厘米的工作距离为3分钟的辐照。 </li><li>在照射，增加局部麻醉（oxybuproca＆＃239，是ne 0.4％），如果有必要的。 </li></ol> 5.结束手术<ol><li>将抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0<html> 0.18％）进入操作眼睛。 </li><li>将软绷带隐形眼镜在手术结束，直到再上皮化完成。再上皮化通常需要3天。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>后角膜再上皮，发起局部治疗与类固醇（地塞米松局部为1mg / ml），并继续进行3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol> 3，离子透入（I-CXL） 1.患者准备<ol><li>在手术前5天，放了一天的治疗眼1％毛果芸香碱滴两次。 </li><li>在手术室，在无菌条件下，趴在他/她的背部病人。 </li><li>辖表面麻醉，如奥布卡0.4％。 </li><li>清洁眼睛和眼睛周围的皮肤用碘防腐剂两次。 </li><li>使用盖窥器，以保持眼睛睁开。 </li></ol><html> _step"&gt; 2，放置离子电渗装置。 <ol><li>应用粘被动电极的手术视野下的额头。 </li><li>应用活性电极，一个吸环，到打开眼。释放所述抽吸之前围绕角膜的周边上的吸环。 </li></ol><img alt="图2" src="/files/ftp_upload/53119/53119fig2.jpg" /> 图2.离子导入装置，该无源电极施加在手术视野下的额头和活性电极，一个吸环，适用于开放的眼睛。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53119/53119fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.核黄素应用<ol><li>填写吸入环与低渗0.1％核黄素无葡聚糖。 </li></ol><html> JPG"/&gt; 在I-CXL图3.核黄素的应用 。吸入环充满了低渗0.1％核黄素没有葡聚糖。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53119/53119fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>启动电流在0.2 mA和逐步增加至1.0毫安的5分钟（图4），总离子电渗疗法的时间。 </li></ol><img alt="图4" src="/files/ftp_upload/53119/53119fig4.jpg" /> 图4.离子导入设备核黄素渗透的电流最初是0.2 mA和逐渐增加至1.0毫安。总离子导入时间为5分钟。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53119/53119fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> EP"&gt; 4。UVA照射<ol><li>照射具有370nm的波长的UVA光的角膜以10毫瓦/厘米2（5.4焦耳/厘米2表面剂量），并在一个5厘米的工作距离为9分钟的辐照。 </li><li>在照射，增加局部麻醉（oxybuproca＆＃239，是ne 0.4％），如果有必要的。 </li></ol> 5.结束手术<ol><li>将抗生素滴眼液（妥布霉素0.3％）和人工泪液（透明质酸下降0.18％），到运营的眼睛。 </li><li>处方止痛药如对乙酰氨基酚（500毫克）加可待因（30毫克），每日6药片。 </li><li>手术后，发起局部治疗与类固醇（地塞米松局部为1mg / ml）和持续3-4周。此外，使用人工泪液，每天4次，1个月。 </li></ol>

<ol>
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S., 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=Pharmacokinetics+and+safety+of+transcorneal+iontophoresis+of+tobramycin+in+the+rabbit.">Pharmacokinetics and safety of transcorneal iontophoresis of tobramycin in the rabbit.</a> Invest Ophthalmol Vis Sci. 29 (9), 1397-1401 (1998).</li><li>Vinciguerra, P., 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=Transepithelial+iontophoresis+corneal+collagen+cross-linking+for+progressive+keratoconus:+initial+clinical+outcomes.">Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: initial clinical outcomes.</a> J Refract Surg. 30 (11), 746-753 (2014).</li><li>Caporossi, 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=Riboflavin-UVA-induced+corneal+collagen+cross-linking+in+pediatric+patients.">Riboflavin-UVA-induced corneal collagen cross-linking in pediatric patients.</a> Cornea. 31 (3), 227-231 (2012).</li><li>Buzzonetti, L., Petrocelli, G., Valente, P., Larossi, G., Ardia, R., Petroni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iontophoretic+transepithelial+corneal+cross-linking+to+halt+keratoconus+in+pediatric+cases:+15-month+follow-up.">Iontophoretic transepithelial corneal cross-linking to halt keratoconus in pediatric cases: 15-month follow-up.</a> Cornea. 34 (5), 512-515 (2015).</li><li>Baiocchi, S., Mazzotta, C., Cerretani, D., Caporossi, T., Caporossi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+crosslinking:+riboflavin+concentration+in+corneal+stroma+exposed+with+and+without+epithelium.">Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium.</a> J Cataract Refract Surg. 35 (5), 893-899 (2009).</li><li>Wollensak, G., Iomdina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histological+changes+after+corneal+crosslinking+with+and+without+epithelial+debridement.">Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement.</a> J Cataract Refract Surg. 35 (3), 540-546 (2009).</li><li>Soeters, N., Wisse, R. P., Godefrooij, D. A., Imhof, S. M., Tahzib, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transepithelial+versus+epithelium-off+corneal+cross-linking+for+the+treatment+of+progressive+keratoconus:+a+randomized+controlled+trial.">Transepithelial versus epithelium-off corneal cross-linking for the treatment of progressive keratoconus: a randomized controlled trial.</a> Am J Ophthalmol. 159 (5), 821-828 (2015).</li></ol>

The authors have nothing to disclose.

用UVA照射和核黄素CXL的标准治疗为阻止圆锥角膜的进展。核黄素是光敏剂诱导化学共价键（交联）时的UVA 3照射。在角膜上，这种现象造成的胶原纤维能够提高角膜刚度之间交联。虽然这种现象被很好地描述，到现在为止已有角膜内交联的直接证据。然而，一些研究已经报道了的Kmax的稳定化从而显示出的C-CXL 3-8的有效性的步骤之后。这个效果是否是由于交联或在角膜基质等微观结...

圆锥角膜是一个双边和渐进的角膜扩张通常在一般人群1导致角膜形状的变形例中报告1 2000，从而视力下降2。圆锥角膜通常存在于早期青春期的进展，直到第三次生命不惑之年发病时通常趋于稳定，但进展可以在整个病人的生命变量。通过阻止圆锥角膜进展，交联的目的是推迟或避免角膜移植术。 到目前为止，唯一的有效和安全的治疗进步圆锥角膜证明在临床研究中是常规的角膜胶原交联（C-CXL）协议，其目的是提高刚度，从而制止圆锥角膜级数3-8。为了减少操作时间和对C-CXL其它可能的危险因素，如感染性角膜炎或基质混浊9，几种改进协议具有已有描述。首先，在加速CXL（A-CXL），UVA的更高的辐照度被传递到角膜通过减少的时间10。其次，为了避免的必要性上皮清创，跨上皮的方法已被采用。遗憾的是，他们已经时比现有协议11有限的成功。最近的跨上皮方法CXL期间角膜核黄素递送是离子电渗疗法（Ⅰ-CXL），但这种治疗方法的严格评价还未执行的12。离子电渗疗法是一种在其中一个小电流被施加穿过组织来提高离子化的药物的渗透非侵入性技术。在CXL通过离子电渗，核黄素被离子化通过上皮渗透角膜。 体内共焦显微镜（IVCM）是成像，可以突出显示异常的角膜中的疾病，如圆锥角膜13的细胞改变角膜的方法。事实上，IVCM已经证明改建在圆锥角膜的角膜所有层与在子基底神经丛的密度特定还原和基质角膜13-15。加，IVCM已被证明是对后的C-16 CXL角膜微观结构分析非常方便。 角膜分界线被描述为一个高反射线见于眼前段光学相干断层扫描（OCT AS）1个月后的C-CXL在300微米17,18的深度。 IVCM下面的C-CXL提供有关角膜的结构改变，包括缺乏角膜基​​质的至300μm的深度信息。此无细胞区域的深度，以及在角膜基质内的分界线的深度显示在AS OCT中，似乎与CXL治疗19和角膜分界线深度的测量的有效深度的AS OCT中1到相关联的一个月后CXL已被提出作为一种有效的临床方法的CXL效力18的评价。 在本研究中，我们使用角膜基质分界线测量由AS OCT和共聚焦显微镜研究了三种不同的角膜胶原交联的协议（常规的，加速，离子电渗疗法）的效率。此外，我们使用IVCM定量分析的三种治疗后，角膜组织变化。

<table><tbody><tr><td></td><td></td><td>Riboflavin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Product number</td><td></td></tr><tr><td>C-CXL</td><td>Sooft SPA, Montegiorgio, Italy</td><td>Ricrolin&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 468465-6</td><td></td></tr><tr><td>A-CXL</td><td>Avedro Inc, Waltham, Massachusetts</td><td>VibeX&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 520-01863-006</td><td></td></tr><tr><td>I-CXL</td><td>Sooft SPA, Montegiorgio, Italy</td><td>Ricrolin+&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 975481-6</td><td>Passive electrode: PROTENS ELITE 4848LE/ Active electrode: IONTOFOR CXL</td></tr><tr><td></td><td></td><td>UVA Machine</td><td></td></tr><tr><td>X-Vega</td><td></td><td></td><td>UVA: 3 mW/cm&lt;sup&gt;2&lt;/sup&gt; 30 min</td></tr><tr><td>KXL System</td><td></td><td></td><td>UVA: 30 mW/cm&lt;sup&gt;2&lt;/sup&gt; 10 min</td></tr><tr><td>X-Vega</td><td></td><td></td><td>UVA: 10 mW/cm&lt;sup&gt;2&lt;/sup&gt; 9 min</td></tr></tbody></table>

three different protocols of corneal collagen crosslinking in keratoconus: conventional, accelerated and iontophoresis

圆锥角膜是双边和渐进的角膜扩张。为了减缓其进展，角膜胶原交联（CXL）最近已推出了作为有效的治疗选择。在生物和化学科学，交联是指活性分子之间形成新的化学键。因此，角膜胶原CXL的目的是合成地增加在角膜基质中的交联的胶原原纤维之间的形成。尽管常规CXL（C-CXL）协议的效率已经在若干临床研究表明，它可能会受益于改进的过程和除去角膜上皮的持续时间。因此，为了提供两个新的和优化的CXL协议一致的评估，我们研究圆锥角膜患者谁经历了三个CXL治疗之一:离子电渗疗法（Ⅰ-CXL），加速CXL（A-CXL），和常规CXL（ C-CXL）。 A-CXL是6时间快CXL程序ü唱了十次提高的UVA辐射，但仍然包括上皮去除。离子电渗疗法是在其中一个小电流施加到改善整个角膜核黄素渗透跨上皮非侵入性技术。使用眼前段光学相干断层扫描（AS OCT）和体内共焦显微镜（IVCM），我们的结论是关于治疗穿透深度，常规CXL协议仍然用于治疗进行性圆锥形角膜的标准。加速CXL似乎是一个快速，有效，安全的替代治疗角膜薄。利用离子导入还在调查中，应该考虑更加谨慎。

<html>角膜分界线是华侨城可见的情况下，92％在301.6微米的平均深度（SD，73.6） <img alt="图5" src="/files/ftp_upload/53119/53119fig5.jpg" /> C-CXL后图5.分界线。高分辨率的角膜眼前段光学相干断层扫描（华侨城）可视化角膜基质分界线在358微米（白色箭头）1个月传统的角膜胶原蛋白交叉后，平均深度链接（C-CXL）。比例尺250微米。 <a href="https:/...

Watch this Scientific Journal Video about Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis at JoVE.com

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

three-different-protocols-corneal-collagen-crosslinking-keratoconus

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

19.8K 次观看.  Quinze-Vingts National Ophthalmology Hospital. Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

视频: Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

科研

发育生物学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

医学

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

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

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 ...

生物工程

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

"全激光"内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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 ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

免疫与感染

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 ...

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

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此视频中的章节

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

供体角膜内皮角膜移植术的组织准备

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

"全激光"内皮移植

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

创面愈合研究的前活体角膜器官培养模型

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

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

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

摘要

探索更多视频

此视频中的章节

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

供体角膜内皮角膜移植术的组织准备

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

"全激光"内皮移植

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

创面愈合研究的前活体角膜器官培养模型

全场光学相干显微镜，用于角膜移植物基质特征的组织学样分析

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