Name: platelet-derived extracellular vesicle functionalization of ti implants
Uploaded: 2021-08-05T14:45:00
Description: Watch this Scientific Journal Video about Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants at JoVE.com

这项研究由卡洛斯三世社会研究所资助，由欧洲社会基金和ERDF欧洲区域发展基金共同资助（MS16/00124;CP16/00124;PI17/01605）、Direcció General d'Investigació、Conselleria d'Investigació、Govern Balear （FPI/2046/2017）和PROJECTA JUNIOR DEL PROJECTE TALENT PLUS， construyendo SALUD， generando VALOR （JUNIOR01/18），由巴利阿里群岛的可持续旅游税资助。

血小板裂解物（PL）按照先前所述使用IdISBa生物库提供的新鲜白蛋白涂层作为起始材料获得，符合机构指南3 。它们在当前项目中的使用得到了其道德委员会的批准（IB 1995/12 BIO）。
1. 电动汽车与PL隔离
<ol><li>较大的身体移除<ol>
<li>在室温下解冻PL。</li>
		<li>在4°C下以1，500× g 离心PL15分钟。 丢弃含有细胞碎片的沉淀。</li>
		<li>收集上清液并在4°C下以1...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+a+nanostructured+titanium+oxide+layer+with+self-assembled+monolayers+for+biomedical+applications:+Surface+properties+and+biomechanical+behaviors.">The potential of a nanostructured titanium oxide layer with self-assembled monolayers for biomedical applications: Surface properties and biomechanical behaviors.</a> Applied Sciences. 10 (2), 590 (2020).</li><li>Jemat, A., Ghazali, M. J., Razali, M., Otsuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modifications+and+their+effects+on+titanium+dental+implants.">Surface modifications and their effects on titanium dental implants.</a> BioMed Research International. 2015, 791725 (2015).</li><li>Damiati, L., 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=Impact+of+surface+topography+and+coating+on+osteogenesis+and+bacterial+attachment+on+titanium+implants.">Impact of surface topography and coating on osteogenesis and bacterial attachment on titanium implants.</a> Journal of Tissue Engineering. 9, 2041731418790694 (2017).</li><li>Chen, L., 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=Self-assembled+human+adipose-derived+stem+cell-derived+extracellular+vesicle-functionalized+biotin-doped+polypyrrole+titanium+with+long-term+stability+and+potential+osteoinductive+ability.">Self-assembled human adipose-derived stem cell-derived extracellular vesicle-functionalized biotin-doped polypyrrole titanium with long-term stability and potential osteoinductive ability.</a> ACS Applied Materials &amp; Interfaces. 11 (49), 46183-46196 (2019).</li><li>Wei, F., Li, M., Crawford, R., Zhou, Y., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-integrated+titanium+oxide+nanotubes+for+targeted+bone+regeneration.">Exosome-integrated titanium oxide nanotubes for targeted bone regeneration.</a> Acta Biomaterialia. 86, 480-492 (2019).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+influence+the+behavior+of+human+mesenchymal+stem+cells+on+titanium+surfaces.">Exosomes influence the behavior of human mesenchymal stem cells on titanium surfaces.</a> Biomaterials. 230, 119571 (2020).</li><li>Lozano-Ramos, I., 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=Size-exclusion+chromatography-based+enrichment+of+extracellular+vesicles+from+urine+samples.">Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples.</a> Journal of Extracellular Vesicles. 4, 27369 (2015).</li><li>Th&#233;ry, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Liu, J., 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=Isolation+and+characterization+of+extracellular+vesicles+from+adult+schistosoma+japonicum.">Isolation and characterization of extracellular vesicles from adult schistosoma japonicum.</a> Journal of Visualized Experiments: JoVE. (135), e57541 (2018).</li><li>JoVE. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Cellular+and+Molecular+Biology.+Using+a+Hemacytometer+to+Count+Cells.">Basic Methods in Cellular and Molecular Biology. Using a Hemacytometer to Count Cells.</a> JoVE Science Education Database. , (2021).</li><li>Chouirfa, H., Bouloussa, H., Migonney, V., Falentin-Daudr&#233;, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+titanium+surface+modification+techniques+and+coatings+for+antibacterial+applications.">Review of titanium surface modification techniques and coatings for antibacterial applications.</a> Acta Biomaterialia. 83, 37-54 (2019).</li><li>C&#243;rdoba, A., Monjo, M., Hierro-Oliva, M., Gonz&#225;lez-Mart&#237;n, M. L., Ramis, J. 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该协议旨在为电动汽车在Ti表面上的功能化提供明确的说明。所提出的方法基于液滴铸造策略，这是一种物理吸附类型的功能化。关于Ti表面上的EV功能化，书目很差，尽管很少有研究表明在Ti10上固定EV具有不同的优势。无论如何，探索的一些策略包括生化结合8，聚合物截留9 或滴铸10。虽然通过共价键使用化学涂层可能会实?...

EV是由任何细胞分泌的膜囊泡（30-200nm），通过传递其货物在细胞间通信中起着关键作用。它们含有多种活性生物分子，其中可能包括核酸、生长因子或生物活性脂质1。由于这些原因，电动汽车已被评估其在治疗中的潜在用途。在骨科和骨再生方面，来自不同来源的EV已经过测试。其中，血小板衍生的EV已被证明可以诱导干细胞的分化作用，同时保持低细胞毒性2，3。因此，需要进一步的研究来探索将EV与生物材料相结合的可能性，以便在日常临床实践中使用它们。
钛基生物材料由于其机械性能、高生物相容性和长期耐用性，被广泛用作骨愈合临床干预的支架4。然而，Ti植入物是一种生物惰性材料，因此与周围骨组织的结合能力较差5。因此，正在研究钛改性，以便通过在其表面实现功能更强大的微环境来提高其性能4，6，7。从这个意义上说，电动汽车可以通过化学8 或物理相互作用与钛锚定9，10。来自干细胞或巨噬细胞的固定化EV通过促进细胞粘附和增殖来增强Ti的生物活性，从而诱导成骨作用8，9，10。
本文将重点介绍使用PL衍生的EV涂覆Ti表面的液滴铸造策略。此外，我们将评估EV在一段时间内从包被表面的释放曲线，并在 体外确认其细胞生物相容性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0,8 &#181;m syringe filter</td>
			<td>Sartorius</td>
			<td>16592K</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Centrifuge tube</td>
			<td>SPL life sciences</td>
			<td>PLC60015</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL syringe</td>
			<td>BD</td>
			<td>303174</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>SPL life sciences</td>
			<td>PLC30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolut ethanol</td>
			<td>Scharlau</td>
			<td>ET0006005P</td>
			<td>Used to prepare 20 %&#160; ethanol with Milli-Q&#174; water</td>
		</tr>
		<tr>
			<td>AKTA purifier System</td>
			<td>GE Healthcare</td>
			<td>8149-30-0014</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-15R Centrifuge</td>
			<td>Beckman Coutler</td>
			<td>392934</td>
			<td>SX4750A swinging rotor</td>
		</tr>
		<tr>
			<td>Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428000210</td>
			<td>F-45-48-11 rotor</td>
		</tr>
		<tr>
			<td>Conical Tube, Conical Bottom, 50ml</td>
			<td>SPL life sciences</td>
			<td>PLC50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytotoxicity Detection Kit (LDH)</td>
			<td>Roche</td>
			<td>11644793001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Syringes 10 ml</td>
			<td>Becton Dickinson</td>
			<td>BDH307736</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Low Glucose Glutamax</td>
			<td>GIBCO</td>
			<td>21885025</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s PBS (1x)</td>
			<td>Capricorn Scientific</td>
			<td>PBS-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) Embrionic Certified</td>
			<td>GIBCO</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtropur S 0.2 &#181;m syringe filter</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep&#160;16/60 Sephacryl&#160;S-400 HR</td>
			<td>GE Healthcare</td>
			<td>28-9356-04</td>
			<td>Precast columns</td>
		</tr>
		<tr>
			<td>human umbilical cord-derived mesenchymal stem cells (hUC-MSC)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000 spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoSight NS300 nanoparticle tracking analysis</td>
			<td>Malvern</td>
			<td>NS300</td>
			<td>Device with embedded laser at &#955;= 532 nm and camera sCMOS</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Terumo</td>
			<td>946077135</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid 69,5%</td>
			<td>Scharlau</td>
			<td>AC16071000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100 XP Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>8043-30-1124</td>
			<td>SW-32Ti Rotor</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100X</td>
			<td>Biowest</td>
			<td>L0022</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Test strips 4.5-10.0</td>
			<td>Sigma</td>
			<td>P-4536</td>
			<td></td>
		</tr>
		<tr>
			<td>Platelet Lysate (PL)</td>
			<td>IdISBa Biobank</td>
			<td></td>
			<td>Obtained from&#160; buffy coats discarded after blood donation</td>
		</tr>
		<tr>
			<td>Polypropylene centrifuge tubs</td>
			<td>Beckman Coutler</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Power wave HT</td>
			<td>BioTek</td>
			<td>10340763</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw cap tube, 15 ml, (Lx&#216;): 120 x 17 mm, PP, with print</td>
			<td>Sarstedt</td>
			<td>62554502</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hidroxide</td>
			<td>Sharlau</td>
			<td>SO04251000</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium implants replicas</td>
			<td>Implantmedia, SA</td>
			<td>NA</td>
			<td>Titanium grade IV. Diameter: 6,2 mm. Height: 1,95 mm</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1 X</td>
			<td>Biowest</td>
			<td>L0930</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryton X100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

platelet-derived extracellular vesicle functionalization of ti implants

细胞外囊泡（EV）是生物纳米囊泡，在细胞通讯中起关键作用。其含量包括蛋白质和核酸等活性生物分子，在再生医学中具有巨大的潜力。最近，来自血小板裂解物（PL）的EV显示出与PL相当的成骨能力。此外，生物材料经常用于骨科或牙齿修复。在这里，我们提供了一种用PL衍生的EV功能化Ti表面的方法，以改善其成骨性能。
通过尺寸排阻色谱法从PL中分离出EV，然后通过滴铸将Ti表面与PL-EV功能化。功能化通过EV释放和乳酸盐脱氢酶（LDH）释放测定的生物相容性得到证明。

本文中介绍的方法允许获得EV功能化的钛盘。电动汽车与表面物理粘合，这允许随着时间的推移持续释放。NTA可以在第2天，第6天，第10天和第14天测量释放的EV数量。第2天的第一次测量显示，大约有109 辆电动汽车被释放，然后在第6天持续释放（约108 辆电动汽车）;第10天（约107 辆电动汽车）和第14天（约107 辆电动汽车）。这证实了持续释放，尽管随着时间的推移?...

Watch this Scientific Journal Video about Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants at JoVE.com

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

在这里，我们提出了一种分离来自血小板裂解物（PL）的细胞外囊泡（EV）的方法及其用于包衣钛（Ti）植入物表面的方法。我们描述了滴铸涂层方法，电动汽车从表面的释放曲线，以及电动汽车涂层Ti表面的 体外 生物相容性。

血小板衍生的Ti植入物的细胞外囊泡功能化

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as ...

细胞外囊泡与生物材料的组合是再生医学研究的理想方法。因此，这项研究报道了钛功能化与EV作为骨科和骨再生的实用工具。与其他策略（如聚合物截留或生化结合）相比，滴铸功能化是一种简单且低成本的方法。通过在玻璃烧杯中用去离子水洗涤钛盘来开始钛表面功能化。然后在用70%乙醇洗涤圆盘之前丢弃水。倾析溶液，使洗涤的植入物在去离子水中以50°C超声处理五分钟。丢弃水后，将钛植入物在40%氢氧化钠溶液中孵育50°C，搅拌10分钟，然后在去离子水中超声处理植入物，如图所示。接下来，用去离子水对植入物进行至少五次洗涤。直到 pH 指示剂上的 pH 值呈中性。如前所述，通过用50%硝酸代替氢氧化钠来重复超声处理和孵育过程。在将植入物储存在70%乙醇溶液中之前，用去离子水进行洗涤和超声处理。将钛植入物在室温下在30%硝酸溶液中孵育30分钟，轻轻搅拌。孵育后，用去离子水进行至少五次洗涤，直到pH指示剂上的pH值为中性。然后将钛植入物在室温下在去离子水中孵育过夜。第二天，在40°C的真空下干燥植入物10分钟。在细胞培养柜下工作，将钛植入物放置在96孔板中，机器侧面朝上。通过解冻EV溶液来排列细胞外囊泡EV滴铸，并以三秒钟的脉冲涡旋溶液。混合后，根据纳米颗粒跟踪分析确定的浓度，在钛表面上沉积40微升EV溶液，以固定每个植入物最多4 x 10 ^ 11 EV。接下来，将板置于37°C的真空条件下约两个小时，或直到滴剂完全干燥。根据钛植入物的数量和真空室中存在的水来调整干燥时间。通过纳米颗粒跟踪分析测量从钛盘释放的EV的数量。在第2天，大约10 ^ 9辆电动汽车被释放，随后在第6天，第10天和第14天持续释放。在细胞接种到植入物后48小时，用乳酸脱氢酶或LDH释放测定法评估钛EV的体外细胞生物相容性。钛EV表现出的LDH量低于最大可接受的细胞毒性值。然而，钛对照组显示出更高的LDH活性水平。最重要的步骤是涂层植入物的真空干燥。重要的是要确保所有水分都蒸发以获得最佳的物理吸附。

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

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

多尺度孔梯度，聚电解质和间接监测改性金属植入物<em&gt;在体内</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

脂质双层囊泡代使用微流控喷射

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Evaluation of the Storage Stability of Extracellular Vesicles

细胞外囊储存稳定性的评价

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

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

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, ...