Name: ferromagnetic bare metal stent for endothelial cell capture and retention
Uploaded: 2015-09-18T15:00:00.000+00:00
Duration: 11 min 1 s
Description: Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

The authors thank Tyra Witt, Cheri Mueske, Brant Newman and Dr. Peter J. Psaltis, MBBS, PhD for their valuable contributions. This study was financially supported by European Regional Development Fund &#8211; FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), American Heart Association Scientist Development Grant (AHA #06-35185N), National Institutes of Health (T32HL007111) and The Grainger Innovation Fund &#8211; Grainger Foundation.

所有的动物研究批准的机构动物护理和利用委员会（IACUC）在梅奥诊所。 1.设计一个2205不锈钢支架与分析<ol><li>设计采用CAD裸金属支架<ol><li>通过选择与墙壁的厚度等于支架支撑厚度&#39;挤压凸台/基"功能生成拉伸中空的圆柱体。 </li><li>设计在不同的草图平面切向挤压筒支架模式。使的平面图案的宽度在挤压空心圆柱体的圆周匹配。 </li><li>传送平面图案设计到使用换行功能的中空圆柱体。 </li><li>保存零件在其原生格式，并在ACIS格式导出的有限元分析。 </li></ol></li><li>对于支架模型的有限元分析<ol><li>导入保存格式ACIS立体几何到有限元分析软件进一步analys的部分模块是。 </li><li>同轴2建模分析气缸在有限元分析软件的一部分建模的支架。外筒具有比所述支架的直径大的初始直径来模拟夹紧器和内筒具有1毫米的最初直径，以模拟一个气球充气。 </li><li>双击组件建模的"实例"树项组装上面说的部件的相对位置。 </li><li>使用有限元分析软件的网模块，指定元素类型为20节点六面体元素缩减积分，指定的元素大小，和网格支架。 </li><li>指定的支架，并分别在模型树的"互动性"的两个气缸之间的摩擦刚性接触对。 </li><li>指定的2205不锈钢弹塑性应力应变行为的支架模型​​。 </li><li>限定边界条件至外部气缸首先卷曲到1毫米它模拟的Crimping支架。除去外筒，以模拟卷曲支架的松弛。扩大内筒至3毫米，以模拟膨胀，最后，取出内筒以模拟支架的反冲。 </li><li>定义仿真参数，包括处理器和RAM容量的分配在"分析"模型树项目的数量和运行模拟。 </li><li>一旦仿真完成后，打开结果文件（filename.odb）和后处理的结果，研究了主应变和迭代地提高支架设计，达到了20％的主应变小于该材料的失效极限。 </li></ol></li></ol> 2.支架制造工艺和检验压着扩建<ol><li>支架制造<ol><li>获取2205不锈钢管通过深钻孔和精密研磨棒料的材料在精密加工公司，如操作精密产品的先锋，OH。 </li><li>转移精密研磨管和平板型式的支架设计，支架切割公司，如Laserage技术公司在沃基根，IL激光切割和电抛光。 </li><li>通过浸没在他们10分钟，然后用碱（10％的NaHCO 3）再过10分钟强酸（50％HCl）的钝化电抛光支架的表面上。小心:处理与适当的防护设备和在通风橱的化学物质。最后，洗净用乙醇和去离子水的支架。这个过程被称为酸洗。 </li></ol></li><li>制造支架的卷边和扩展测试<ol><li>压支架使用上保持压接工具手一个三折的气球。握住支架和压接工具的三折气球。按下手柄径向变形支架上的气球被卷曲。 </li><li>检查用显微镜均匀卷曲和失败的在适当的结构中的任何迹象的卷曲支架塑性变形。 </li><li>通过加压三折气球水其扩大到3毫米的设计直径。检查扩展支架的微观骨折和均匀膨胀。 </li></ol></li></ol>支架的保留磁场3.表征注:直径为2英寸和1英寸高度的圆柱磁体被用于本研究。磁体的磁极沿轴线对齐。磁体的表面磁通量密度为约1 T. <ol><li>磁化支架径向或轴向使用强钕磁铁。握住支架靠近强磁铁的磁化大约1分钟。 </li><li>持在平坦面中的一个的支架，其直径沿着磁场线被径向磁化或保持旁边圆柱形表面的支架，其沿着磁力线轴线轴向磁化它。保留磁FIELD中的支架被发现是稳定至少24小时，但磁化后尽快使用支架。 </li><li>单独安装支架到玻璃心轴，然后装入玻璃心轴的磁探测夹具的精度夹头。磁性微传感器探头可以精确地定位靠近所述支架而不触及使用XYZ表级的磁性探测夹具的装配表面（图4）。 </li><li>测量磁性微传感器的基线读数远离支架，然后通过定位使用磁探测夹具的XYZ阶段探针测量保留磁场在支架的表面。 </li></ol> 4.磁性细胞捕获研究<ol><li>获得的细胞，具有SPION和染色用荧光染料标记<ol><li>在5,7描述来源于猪外周血内皮生长细胞（EOC）。培养在T-75烧瓶unti升大约80％铺满（5×10 6至8×10 6个细胞 ）。 </li><li>合成SPIONs如10纳米直径磁铁矿铁 （Fe 3 O 4）芯如8,9-描述的50纳米厚的聚（乳酸-共-乙醇酸）（PLGA）壳包围。 </li><li>孵育衍生EOC与SPION以200微克/毫升细胞培养基的16小时的浓度在37℃。 </li><li>轻轻吸出细胞培养基。轻轻加入10ml磷酸盐缓冲盐水（PBS）的至烧瓶，摇摆，并且吸取PBS洗涤细胞。 </li><li>将细胞染色用荧光染料（CM-DII）为在实验过程中可视化。这是根据制造商的说明，通过以5微升/毫升的浓度将染料加入至10ml细胞培养基并与细胞在37℃温育30分钟进行。 </li><li>清洗细胞，用PBS作为步骤4.1.4并用3毫升的0.25％胰蛋白酶-EDTA溶液中5分钟，在37℃至解除从烧瓶的细胞。 </li><li>用PBS细胞悬液转移到15毫升锥形管，顶部关闭，离心机在500×g离心5分钟，以形成细胞片状沉淀。 </li><li>重悬在PBS中的细胞沉淀在1-2x10 6细胞 / ml的浓度和在进出锥形管的几次通过移液彻底混合。 </li></ol></li><li> 在体外细胞研究<ol><li>设计和制造（例如，3D打印）一个简单的夹具保持支架只是玻璃盖玻片的表面上方。 </li><li>使用电磁消磁器消磁支架或磁化支架径向或轴向使用强钕磁铁。 </li><li>移液器SPION标记EOC悬浮在PBS到包含轴向磁化或径向磁化或非磁化控制支架的菜。图像使用倒置荧光显微镜与EOC立即用于荧光悬浮在PBS中的支架。 </li></ol></li></ol><p类="jove_title"&gt; 5。 在体动物研究<ol><li>支架植入术<ol><li>绘制外周血4健康约克夏猪-体重约50 KG -分别在5,7描述的前3周支架植入和文化EOC。 </li><li>辖抗血小板药物治疗（每日服用阿司匹林325毫克和氯吡格雷75毫克）开始前3天手术。 </li><li>在支架植入术当天，麻醉猪肌肉注射Telazol，甲苯噻嗪和阿托品（5 / 2-3 / 0.05毫克/千克分别）在适用的机构动物护理和使用规范中规定的。 </li><li>插管，并将猪的吸入1-2.5％异氟醚麻醉。 </li><li>剃猪的腹的颈部区域，并进行中的过程一般在无菌条件。 </li><li>植入物1磁化和1未磁化的支架成使用标准的心导管术的右冠状动脉（RCA）。 <ol><li> Cathete动物化应该由经过培训的介入性心脏病进行。访问右颈动脉用一个9法国鞘。 </li><li>导管插入目标冠状动脉注入碘造影剂，以获得透视图像。 </li><li>发生在动脉中的0.014英寸标准冠状动脉导丝。推进气囊及支架使用该导丝和部署在3-3.5毫米直径的血管支架。 </li></ol></li><li>闭塞的RCA近端内的血流使用具有线上气囊植入支架和递送约2×10 6的自体的EOC标记SPION通过中央导管悬浮在4毫升PBS中在2分钟内。 </li><li>附加闭塞2分钟后恢复血流的RCA。 </li><li>动物转移到康复室，并密切监测动物，直到它恢复意识。 </li><li>继续给予抗血小板药物（阿司匹林325毫克和氯吡格雷75米g）发布操作，直至牺牲。 </li></ol></li><li>支架外植体和组织学<ol><li>安乐死动物手术后7天通过首先麻醉动物如前所述，然后施用静脉内致死剂量的戊巴比妥钠（100毫克/千克）按照适用的机构动物护理和使用指南。 </li><li>手术收获了支架的动脉段。固定在10％福尔马林缓冲液中取出的动脉最少30分钟。留在福尔马林缓冲的样本进行进一步的组织学分析。 </li><li>外包固定样本能够与金属支架进行组织学的设施。在处理过程中，样本被嵌入甲基丙烯酸甲酯，横截面，并分析了组织学上使用Mallory的染色技术与普鲁士蓝染色为铁粒子。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

我们开发了一种磁性支架可以用作裸金属支架，能够吸引SPION标记的内皮细胞。在涉及磁支架先前的研究中，研究人员已经使用由磁性材料制成的镍涂覆的商业支架和线圈或网格，由于强磁性支架5,10-14的不可用性。其它基团也用于靶向纳米颗粒加载内皮细胞3市售304级不锈钢支架的顺磁性性质。镍涂层可以是过敏于接收到所述支架的患者和所述顺磁性支架需要的外部磁场，以吸引?...

Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium &#8211; both bare metal stents (BMS) and drug eluting stents (DES) &#8211; need anti-platelet therapy to prevent thrombus formation. BMS heal rapidly, but are subject to late stage restenosis due to incomplete healing. DES require long term anti-platelet therapy due to delayed healing. Anti-platelet therapy administered to avoid thrombosis as a result of incomplete or delayed healing leads to increased bleeding risk and may not be suitable for certain patients1,2. An ideal stent will heal completely and quickly thus avoiding long-term anti-platelet therapy and late stage restenosis. This complete healing can only be achieved if the stent is rapidly coated with a monolayer of endothelial cells after implantation. Coating the stents with biocompatible materials such as gold or other biopolymers has been shown to improve thrombo-resistance, but none of these techniques achieved ideal blood compatibility as may be possible by coating with endothelial cells3,4.
A stent can be coated with endothelial cells post implantation by attracting circulating progenitor cells. This self-seeding technique can be achieved by utilizing ligands and antibodies. But this technique is limited by the low number of circulating endothelial progenitor cells. A promising strategy is to deliver cells directly to the stent immediately following implantation during a short period of blood flow occlusion3,5. This strategy requires a technique for rapidly capturing cells and retaining them on the stent even after restoring blood flow. We have developed a technique in which a magnetic stent is used to attract and retain magnetically-labeled endothelial cells delivered post implantation. To achieve this, a functional BMS with sufficient magnetic properties to capture and retain magnetically-labeled endothelial cells is required6.
In this paper, we discuss the methods for designing, manufacturing, and testing a 2205 stainless steel stent. The stents were designed using CAD and FEA. The manufactured stents were magnetized using a neodymium magnet and the retained magnetic field was measured using a magneto-resistance microsensor probe. We then tested the stents for magnetically-labeled cell capture in a culture dish during our in-vitro experiments. Finally, the stents were tested in-vivo by implanting magnetic and non-magnetic stents in 4 pigs and histologically analyzing the stented arteries.

<table><tbody><tr><td>2205 Stainless steel</td><td>Carpenter Technology Corporation</td><td></td><td>Round bar stock material</td></tr><tr><td>Abaqus</td><td>Dassault systems</td><td></td><td>Software</td></tr><tr><td>Atropine</td><td></td><td></td><td>Prescription drug.</td></tr><tr><td>Clopidogrel</td><td></td><td></td><td>Commercial name: Plavix. Prescription drug.</td></tr><tr><td>CM-DiI</td><td>Life Technologies</td><td>V-22888</td><td>Molecular Probes, Eugene, OR</td></tr><tr><td>Endothelial growth medium-2</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Hand Held Crimping tool</td><td>Blockwise engineering</td><td>M1-RMC</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Sigma Aldrich</td><td>MFCD00011324</td><td>CAUTION: wear proptective equipment and handle under fume hood</td></tr><tr><td>Isoflurane anesthesia</td><td>Piramal Critical Care, Inc.&amp;nbsp;</td><td></td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma Aldrich</td><td>MFCD00003568</td><td></td></tr><tr><td>NdFeB magnet 2&quot; Dia x 1&quot; thick</td><td>Amazing magnets</td><td>D1000P</td><td>Axially magnetized disc magnet with poles on flat faces</td></tr><tr><td>Over-The-Wire trifold balloon</td><td></td><td></td><td>Any commercially available OTW trifold balloon can be used</td></tr><tr><td>Phosphate buffered saline</td><td>Life Technologies</td><td>10010-023</td><td>Commonly known as PBS</td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Sigma Aldrich</td><td>MFCD00003528</td><td></td></tr><tr><td>Sodium pentobarbital</td><td>Zoetis</td><td></td><td>Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi.&amp;nbsp; Controlled substance to be ordered only by licensed veternarian</td></tr><tr><td>SolidWorks</td><td>Dassault systems</td><td></td><td>Software</td></tr><tr><td>SpinTJ-020 micro sensor</td><td>MicroMagneitcs Sensible Solutions</td><td></td><td>Long probe STJ-020 microsensor</td></tr><tr><td>SPION</td><td>Mayo Clinic</td><td></td><td>Nanoparticles synthesized internally (Ref: Lee, S. J. et al. Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent. J Magn Magn Mater 272, 2432-2433, doi:DOI 10.1016/j.jmmm.2003.12.416 (2004))</td></tr><tr><td>Telazol</td><td>Zoetis</td><td></td><td>Controlled substance to be ordered only by licensed veternarian</td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200-056</td><td>Gibco, Grand Island, NY</td></tr><tr><td>Xylazine</td><td>Bayer Animal Health</td><td></td><td>Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian</td></tr></tbody></table>

ferromagnetic bare metal stent for endothelial cell capture and retention

心血管支架的快速内皮化是必要的，以减少支架血栓，并避免抗血小板疗法可减少出血的危险。利用磁力来捕捉和保留标记与超顺磁性氧化铁纳米颗粒（SPION）内皮细胞生长（EOC）的可行性先前已经证明。但该技术需要一个机械官能支架由磁性和生物相容性材料随后在体外和体内试验证明快速内皮化的发展。我们开发了从2205双相不锈钢弱磁性支架采用计算机辅助设计（CAD）和它的设计采用有限元分析（FEA）进一步完善。支架的最终设计在机械卷边和扩张呈现下面的材料的断裂极限一个主应变。一百支架被制造和它们的子集被用于机械测试，保留ained磁场测量，体外细胞捕获的研究，并在体内植入研究。十支架进行了测试部署，以验证他们是否持续的压接和膨胀循环无故障。另有10支架均采用强大的钕磁磁化，其保留的磁场进行了测量。该支架表明，该保留的磁性足以捕捉SPION标记的EOC 在我国的体外研究 。 SPION标记EOC采集和保留的大动物模型通过植入1磁化支架和各4头1个非磁化控制支架进行了验证。 7天后的支架的动脉植和组织学分析。在这项研究中开发的弱磁性支架有能力吸引和留住SPION标记的内皮细胞可促进快速愈合。

<html>根据有限元分析（图1）的迭代支架设计显示的支架，可以压接并扩大与20％的主应变小于30％的极限应变。卷边和膨胀试验（ 图2）显示骨折的迹象。图片变形支架均与有限元分析计算出变形较好的一致性，也显微图像，显示没有骨折（ 图3）。正如预期的那样，从被保持磁场的测量（ 图4和5），SPION标记的细胞是在轴向磁化的支架优选地吸引...

Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

我们的目标是设计，制造和测试铁支架内皮细胞捕获。十支架进行了测试骨折10多个支架进行了测试保留磁性。最后，10支架体外测试和8个支架植入4头显示细胞捕获和保留。

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. ...

ferromagnetic-bare-metal-stent-for-endothelial-cell-capture

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.5K 次观看.  Mayo Clinic. 我们的目标是设计，制造和测试铁支架内皮细胞捕获。十支架进行了测试骨折10多个支架进行了测试保留磁性。最后，10支架体外测试和8个支架植入4头显示细胞捕获和保留。 

视频: Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Ferumoxytol，一个FDA批准的氧化铁纳米粒子标记干细胞

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

科研

医学

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

颈动脉支架植入再狭窄的研究的小鼠模型

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

生物工程

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

从金属支架表面使用的腺病毒载体栓通过水解的交联剂血管基因转移

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

小口径支架移植物使用静电及球囊扩张裸金属支架的制作

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable experimental animal models to test novel embolization materials such as stents and flow diverters. The aim of the present project was to design a safe, fast, and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.
Saccular aneurysms were created from an arterial graft from the descending aorta.The aneurysms were microsurgically transplanted through end-to-side anastomosis to the infrarenal abdominal aorta of a syngenic male Wistar rat weighing &#62;500 g. Following aneurysm anastomosis, aneurysm embolization was performed using balloon expandable magnesium stents (2.5 mm x 6 mm). The stent system was retrograde introduced from the lower abdominal aorta using a modified Seldinger technique.
Following a pilot series of 6 animals, a total of 67 rats were operated according to established standard operating procedures. Mean surgery time, mean anastomosis time, and mean suturing time of the artery puncture site were 167 &#177; 22 min, 26 &#177; 6 min and 11 &#177; 5 min, respectively. The mortality rate was 6% (n=4). The morbidity rate was 7.5% (n=5), and in-stent thrombosis was found in 4 cases (n=2 early, n=2 late in stent thrombosis).
The results demonstrate the feasibility of standardized stent occlusion of saccular sidewall aneurysms in rats - with low rates of morbidity and mortality. This stent embolization procedure combines the opportunity to study novel concepts of stent or flow diverter based devices as well as the molecular aspects of healing.

Reproducable experimental animal models are needed for the testing of novel embolization materials, which have been designed to treat endovascular occlusion of intracranial aneurysms (IA). The present study aims to develop a safe and standardized surgical technique for stent assisted embolization of saccular aneurysms in a rat animal model.

生物可降解镁支架治疗大鼠囊动脉瘤的实验研究--手术技术介绍

The steady progess in the armamentarium of techniques available for endovascular treatment of intracranial aneurysms requires affordable and reproducable ...

神经科学

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

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

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, ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...