Name: mitigation of blood borne cell attachment to metal implants through cd47-derived peptide immobilization
Uploaded: 2020-12-03T14:30:00
Description: Watch this Scientific Journal Video about Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization at JoVE.com

本文件提出的协议开发和研究得到了NIH（NBIB）R01基金（# EB023921）对国际基金和SJS的资助，NIH（NHLBI）R01基金（#HL137762）对国际基金和RJL的资助。

根据费城儿童医院的 IRB，获得了所有人工样本。所有动物实验都是经费城儿童医院的亚科中心批准进行的。
1. 用PEI-PDT涂覆裸金属表面
<ol><li>在摇床中用 2 异丙醇（60°C，200 rpm 的速度）清洗不锈钢箔优惠券（1 厘米 x 1 厘米或 0.65 厘米 x 1 厘米）或不锈钢网盘，使用 5 分钟。执行此步骤 2x。然后用氯仿洗2x（60°C，转速200 rpm），每次洗10分钟。</li>
	<li>将清洗过的?...

<ol>
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我们演示并描述了一种相对新颖的化学策略，将治疗性肽莫伊蒂斯附在不锈钢表面，其首要目标是减少表面与血液中的炎症细胞的活性。本文描述的双磷酸盐化学涉及PABT的金属氧化物和双磷酸盐组之间的协调键的形成。在金属表面形成的多磷酸单层厚度不超过5纳米18，因此，对厚聚合物涂层20的潜在刺激作用无关紧要。PABT的脂质侧链中潜在硫醇组的脱保护使金...

皮下冠状动脉介入是治疗冠状动脉疾病（CAD）的第一线，主要涉及支架患病动脉。然而，支架内性休息（ISR）和支架血栓形成是常见的并发症与支架部署1。血-支架界面的血液相互作用的特点是几乎立即吸附金属表面的血浆蛋白，其次是血小板和炎症细胞附着和活化2。从活性炎症细胞释放炎症细胞因子和化疗素导致血管平滑肌细胞 （VSMC） 在 tunica 介质中的表型修饰，并触发其向心迁移到刺激室。活性VSMC在内膜中的扩散导致刺激层增厚，流明变窄和支架内休息3。药物吹风支架（DES）是为了防止VSMC扩散而开发的;然而，这些药物对内皮细胞4，5有非靶对目标细胞毒性作用。因此，后期支架血栓形成是一种常见的并发症与DES6，,7。由可生物降解聚合物（如聚L-乳酸酯）制成的支架在动物实验和初步临床试验中显示出了许多希望，但最终在"现实生活"的临床应用显示出其低于第三代DES8时被召回。因此，有必要提高裸金属支架的生物相容性，以更好的患者结果。
CD47是一种无处不在的表达跨膜蛋白，当与同源受体信号调节蛋白α（SIRP+）9结合时，抑制先天免疫反应。SIRP® 受体具有免疫细胞酪氨酸抑制因子 （ITIM） 域和 SIRP+ - CD47 相互作用时的信号事件最终导致炎症细胞活化10、 11,、12、13的调节。我们实验室的研究表明，重组CD47或其肽衍生物，对应于CD47（pepCD47）细胞外区域的22个氨基酸Ig域，可以降低宿主对一系列临床相关生物材料14、15、16,15的免疫反应。最近，我们已经证明peCD47可以固定在不锈钢支架表面，并显著减少与神经症相关的病理生理反应。值得注意的是，pepCD47改性表面可满足长期储存和环氧乙烷灭菌等相关使用条件。为此，pepCD47可能是一个有用的治疗目标，以解决血管内支架的临床局限性。
pepCD47与金属表面共价附着的策略涉及金属表面的一系列新颖的化学修饰。金属表面首先涂上具有潜在硫醇组 （PABT） 的聚酰胺双磷酸盐，然后对硫醇进行脱保，并附着已安装的二乙酰二硫磷酸组 （PDT）。在反应中未消耗的PET组与脱保护的PABT硫醇发生反应，然后用peCD47在终端半胱氨酸残留物中加入硫醇，从而通过二硫化物键,14、17、18,17将pecd47结合到金属表面。18我们使用荧光结合肽CD47（TAMRA-pepCD47）来确定肽的输入浓度，导致肽的最大表面固定。最后，我们分别评估了pecd47涂层金属表面的急性和慢性抗炎能力，使用钱德勒循环装置进行外体检测，以及单细胞附着/巨噬细胞扩张测定。
本文为硫化肽附着在金属表面提供了系统化方案;确定肽的最大固定密度;并评估pecd47涂层金属表面暴露在全血和孤立的单核细胞中的抗炎特性。

<table border="0" cellpadding="0" cellspacing="0" style="width:834px;" width="833">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">1 M Tris-HCL</td>
			<td style="width:184px;">Invitrogen</td>
			<td style="width:129px;">15567-027</td>
			<td style="width:239px;">pH - 7.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">4% Glutaraldehyde</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">16539-07</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">4% Sodium Citrate</td>
			<td style="width:184px;">Sigma</td>
			<td style="width:129px;">S5770</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">ACK lysing buffer</td>
			<td style="width:184px;">Quality Biologicals</td>
			<td style="width:129px;">118-156-721</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD45RA Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">202301</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD5 Ab (mouse anti-rat; clone OX-19)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">203501</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD6 Ab (mouse anti-rat; clone OX-52)</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:129px;">550979</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD68 Ab (mouse anti-rat; clone ED-1)</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:129px;">MCA341</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">anti-CD8a Ab (mouse anti-rat; clone OX-8)</td>
			<td style="width:184px;">Biolegend</td>
			<td style="width:129px;">201701</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Chloroform Certified ACS</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Dimethyl Formammide (DMF)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">39117</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Embra stainless steel grid</td>
			<td>Electron Microscopy Sciences</td>
			<td style="width:129px;">E200-SS</td>
			<td>stainless steel mesh mesh disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Ficoll Hypaque</td>
			<td style="width:184px;">GE Healthcare</td>
			<td style="width:129px;">17-1440-02</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Glacial acetic acid</td>
			<td style="width:184px;">ACROS organic</td>
			<td style="width:129px;">148930025</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">goat anti-mouse IgG Alexa Fluor</td>
			<td style="width:184px;">ThermoFisher</td>
			<td style="width:129px;">A11030</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Heparin sodium</td>
			<td style="width:184px;">Sagent Pharmaceuticals</td>
			<td style="width:129px;">402-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human pepCD47</td>
			<td>Bachem</td>
			<td>4099101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Isopropanol</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">A426P-4</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Metal adapters</td>
			<td style="width:184px;">Leur Fitting</td>
			<td style="width:129px;">6515IND</td>
			<td style="width:239px;">1 way adapter 316 ss 1/4&#34;-5/16&#34; hoes end</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Methanol</td>
			<td style="width:184px;">RICCA chemical company</td>
			<td style="width:129px;">4829-32</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Microscope</td>
			<td style="width:184px;">Nikon Eclipse</td>
			<td style="width:129px;">TE300</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pottasium Bicarbonate (KHCO3)</td>
			<td>Fisher Chemical</td>
			<td style="width:129px;">P184-500</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">PVC tubes</td>
			<td style="width:184px;">Terumo-CVS</td>
			<td style="width:129px;">60050</td>
			<td style="width:239px;">1/4&#34; X 1/16 8&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">sodium cacodylate buffer with 0.1M sodium chloride</td>
			<td style="width:184px;">Electron Microscopy Sciences</td>
			<td style="width:129px;">11653</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodium Dodecyl Sulfate (SDS)</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">161-0302</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Sodum actetate (C2H3NaO2)</td>
			<td style="width:184px;">Alfa Aesar</td>
			<td style="width:129px;">A13184</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Src peptide</td>
			<td>Bachem</td>
			<td style="width:129px;">4092599</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel (AISI 304) cylinder-shaped samples with a lumen</td>
			<td>Microgroup, Medway, MA</td>
			<td style="width:129px;">20097328</td>
			<td>1 cm X 6 mm OD</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Stainless steel foils (AISI 316L)</td>
			<td style="width:184px;">Goodfellow, Coraopolis, PA</td>
			<td style="width:129px;"></td>
			<td style="width:239px;">100 mm X 100 mm X 0.05 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:282px;">Tetramethylrhodamine-conjugated pepCD47 (TAMRA-pepCD47)</td>
			<td>Bachem</td>
			<td style="width:129px;">4100277</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:282px;">TMB (3,3&#8217; ,5,5&#8217; -tetramethylbenzidine) substrate and tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td>
			<td>Thermo Scientific</td>
			<td>PG82089</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:282px;">Tween-20</td>
			<td style="width:184px;">Bio-Rad laboratories</td>
			<td style="width:129px;">170-6531</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vybrant CFDA SE Cell Tracer Kit</td>
			<td>Invitrogen</td>
			<td style="width:129px;">V12883</td>
			<td style="width:239px;"></td>
		</tr>
	</tbody>
</table>

mitigation of blood borne cell attachment to metal implants through cd47-derived peptide immobilization

与裸金属支架和药物椭圆支架相关的主要并发症分别是支架内性恢复和后期支架血栓形成。因此，提高金属支架的生物相容性仍然是一个重大挑战。该协议的目标是描述一种由生物活性肽进行金属表面修饰的强有力技术，以提高血液接触医学植入物（包括血管内支架）的生物相容性。CD47是免疫学物种特异性自我标记，具有抗炎特性。研究表明，22种氨基酸肽与细胞外区域CD47的Ig域（pepCD47）相对应，具有抗炎特性，如全长蛋白。大鼠体内研究，以及我们实验室对兔子和人类血液实验系统的外体研究已经证明，在金属上进行pecd47固定通过防止炎症细胞附着和激活，提高了其生物相容性。本文介绍了金属表面和肽附着功能化的分步协议。使用具有潜在硫醇组（PABT）的聚酰胺双磷酸盐进行改性，然后通过与安装在苯丙胺组（PEI-PDT）的聚乙烯胺发生反应，对硫醇脱保和硫醇反应位点进行扩增。最后，pepCD47通过双8-3，6-二甲苯基分离基器将最终半胱氨酸残留物与核心肽序列连接起来，通过二硫化物键附着在金属表面。这种肽附着在金属表面的方法是有效的，相对便宜，因此可以应用于提高几种金属生物材料的生物相容性。

金属表面通过一系列化学修饰呈现硫醇反应，如图1所示。PABT孵育后PEI-PDT处理使金属表面适合肽附着。肽CD47（pepCD47）含有半胱氨酸残留物在C-术语连接到核心peCD47序列通过一个灵活的双EEAC桥通过硫化物键共价连接到硫醇反应表面。使用此协议，我们已证明pecd47保持稳定连接到金属表面长达六个月，并能承受正常的生理剪切应力和灭菌程序17。
<p class="jov...

Watch this Scientific Journal Video about Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization at JoVE.com

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

此处介绍的是使用多磷酸盐化学将肽 CD47 （pepCD47） 附在金属支架上的规程。使用 pepCD47 使金属支架功能化可防止炎症细胞的附着和激活，从而提高其生物相容性。

通过CD47衍生肽固定，缓解血系细胞附着到金属植入物

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

该协议包括一种将治疗肽附在金属表面的新颖化学策略，具有提高各种金属生物材料的生物相容性的潜力。这是一种通用而强大的技术，可将肽和治疗蛋白附在生物植物的裸金属表面。我们利用这项技术通过附加CD47肽来改善金属支架的生物相容性，以防止支架引起的炎症反应。首先，用2异丙醇在摇床中清洗不锈钢箔优惠券或网状盘五分钟。进行两次洗涤，然后用氯仿清洗样品两次，每次洗涤10分钟。将清洗过的不锈钢样品在220摄氏度的烤箱中放置30分钟。将烤箔或网盘浸入 PABT 的 0.5% 水溶液中，并在摇床中孵育一小时。用去维化蒸馏水清洗 PABT 改性样品五次。将试样转移到新瓶中，然后重复洗涤。在摇床的室温下，用 TCEP 处理样品 15 分钟。用真空生成装置（如冻干剂）将蒸馏水除去，用脱气水洗涤铝箔或网状盘五次。将样品转移到新瓶中，然后重复洗涤。通过稀释 212.5 微升的库存 PEI-PDT 和 125 微升 0.4 摩尔醋酸钠在脱气水中，准备 5 毫升 1%的PEI-PDT溶液。用1%的PEI-PDT孵育洗净的不锈钢试样。将空气更换为气，密封小瓶气密，并在摇床上混合一小时。立即进行肽结合，或在四摄氏度下储存样品长达一周。通过溶解脱气50%醋酸中的人类肽CD47粉末，准备每毫升1毫克的人类肽CD47库存溶液，然后通过溶解4.5毫升脱气PBS中的500微升库存来准备工作溶液。在室温下用肽CD47的工作溶液孵育洗涤的PEI-PDT涂层样品，摇动一小时。完成后，如文本手稿中所述，清洗多肽 CD47 涂层样品。在用肽 CD47 或炒多肽涂上金属箔后，将四分之一英寸 PVC 管切成三块 38 厘米长的碎片，并将多达八个未改性、炒多肽或肽 CD47 改性金属箔插入三个不同的管中。从健康的人类献血者那里收集30毫升血液，不含任何抗血小板药物，用一毫升4%柠檬酸钠预装注射器，以防止采集的血液凝固。使用 10 毫升注射器将 10 毫升血液放入每个管中，并使用金属适配器连接两端。将充满血液的管子放在钱德勒循环装置的车轮上，在37摄氏度的车轮旋转下，将血液沿着金属箔传递4小时。将血液从管子中排出，然后根据 IRB 要求进行处理，然后用手术刀切割管中取回箔。用0.1摩尔氯化钠稀释卡迪酸钠缓冲液中的70%溶液，准备2%谷胱甘肽溶液。在2%谷胱甘肽溶液中孵育铝箔15分钟，并在夜间储存在4摄氏度。在分析金属箔之前，用PBS清洗三次。在37摄氏度的水浴中加热8毫升PBS。通过将 90 微升 DMSO 添加到一个 CFDA 小瓶中，准备 10 毫摩尔 CFDA 染料库存溶液，然后通过向温暖的 PBS 中添加 75 微升的库存 CFDA 来准备工作溶液。反转管子几次，用铝箔混合并盖住。将每个铝箔放入一毫升 CFDA 染料中，放入 24 井板中。用铝箔盖住板，在37摄氏度下孵育板15分钟，然后在PBS中清洗铝箔三次，然后用荧光显微镜成像。对共价结合TAMRA结合肽CD47的浓度进行量化，以确定该肽在金属表面的最大固定密度。最大肽保留量约为每厘米180毫微克，其输入浓度为每毫升100微克。在改性金属表面上对TAMRA结合肽CD47的固定性进一步证实了荧光显微镜，该显微镜显示TAMRA结合肽CD47处理网盘表面发出的荧光均匀。在缺乏修饰的控制网格表面只检测到最小的荧光，从而排除了非具体绑定的TAMRA结合肽CD47作为荧光的主要来源。还证实，与炒改性、未经修改的肽对联控制相比，肽CD47涂层表面显示血液传播的细胞附着性大幅减少。从大鼠布衣细胞分离出的单核细胞是在裸金属和肽CD47改性不锈钢箔上培养的。在功能化表面上观察到急性单细胞附着的衰减、其向巨噬细胞的转化和巨噬细胞增殖。在尝试此协议时，执行涉及脱气水或试剂的步骤非常重要，以尽快执行，并尽量减少接触氧气。我们的技术有选择地调节细胞与金属表面的相互作用，典型的细胞类型识别测定，如RT-PCR、流式细胞学和免疫荧光，与该技术兼容。当开发为一系列生物活性肽时，该技术有助于提高组织材料界面的生物相容性。

Research

JoVE Journal

Bioengineering

Methods Development for Blood Borne Macrophage Carriage of Nanoformulated Antiretroviral Drugs

方法Nanoformulated的抗逆转录病毒药物的血源性巨噬细胞运输的发展

Nanoformulated drugs can improve pharmacodynamics and bioavailability while serving also to reduce drug toxicities for antiretroviral (ART) medicines. To ...

科研

生物工程

Bacterial Immobilization for Imaging by Atomic Force Microscopy

原子力显微镜成像的细菌固定化

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Generation and Recovery of ÃƒÅ½Ã‚Â²-cell Spheroids From Step-growth PEG-peptide Hydrogels

产生和回收的β-细胞的细胞球体从逐步增长的PEG-肽水凝胶

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Graphene Coatings for Biomedical Implants

生物医学植入物的石墨涂料

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

通过微接触印刷的定向功能性心肌组织的产生

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

铁磁金属裸支架对血管内皮细胞捕获和保留

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

点化学法现场定向固定骨形态发生蛋白2到固体表面

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

基于多步法链位移光刻的基因功能表面固定化研究

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...