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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2205 Stainless steel</td>
			<td>Carpenter Technology Corporation</td>
			<td>N/A</td>
			<td>Round bar stock material</td>
		</tr>
		<tr>
			<td>Abaqus</td>
			<td>Dassault systems</td>
			<td>N/A</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.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011674</td>
			<td></td>
		</tr>
		<tr>
			<td>NdFeB magnet 2&#34; Dia x 1&#34; 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>N/A</td>
			<td>N/A</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.&#160; Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>SpinTJ-020 micro sensor</td>
			<td>MicroMagneitcs Sensible Solutions</td>
			<td>N/A</td>
			<td>Long probe STJ-020 microsensor</td>
		</tr>
		<tr>
			<td>SPION</td>
			<td>Mayo Clinic</td>
			<td>N/A</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

Research

JoVE Journal

Bioengineering

11.4K 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

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

科研

生物工程

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

人卵巢组织与工程化内皮细胞联合移植: 一种以细胞为基础的快速灌注与直接分泌的融合策略

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

超薄聚二甲基硅氧烷微氟化片在保水作用下固定化活线虫个体

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

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

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

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

生物材料膜上人与羊角膜内皮细胞层的生长

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

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, ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

在机械生物学研究的轨道摇床上的 6 井板中分段内皮细胞的生长

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

一种研究炎症期间白细胞-内皮细胞相互作用的微生理系统

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...