Name: multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo
Uploaded: 2013-07-01T12:00:00
Description: Watch this Scientific Journal Video about Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo at JoVE.com

作者想感谢安德烈沃尔德博士和尼古拉斯·佩兰制造钛种植体，K. Benmlih为积聚的聚四氟乙烯模具和G.普雷沃博士对他的帮助与动物实验。我们也承认本地区阿尔萨斯和PMNA的（极MATERIAUX等纳米科学阿尔萨斯）的财务贡献。

1。在孔的金属植入物的制备微孔梯度<ol><li>清洁植入物（如医用级钛有孔玻璃珠的大小范围为400-500微米，法国Neyco SAS，制成的植入物）的乙醇中，然后在丙酮中超声处理15分钟。 </li><li>根据植入物的大小和形状（对于标准的实验中，使用2厘米高度1.5厘米直径的圆筒状的模具）的设计和制造聚四氟乙烯模具。模具应该是模块化的，因此在提取过程中的某些部分可以被删除。这种模具的设计确保?...

<ol>
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孔隙梯度接口组织工程中是重要的工具，这里描述的系统可以单独使用，或与金属植入物一起使用，以形成孔梯度，来研究细胞迁移。该系统并不需要任何额外的设置或除了化学通风柜处理有机溶剂的以外的额外的设备，因此它可以被应用在生物实验室中。可以使用类似的聚合物，如聚（乙醇酸）（PGA），聚（乳酸 - 乙醇酸）共聚物（PLGA），聚（己内酯）（PCL），有轻微的修改。也可以用其他的...

目前可用的金属植入物是适合承载的应用程序，但他们的非降解必须设计保证了强劲的接口与周围组织的其中1个。通过提供结构，促进细胞生长和定植在体内的金属植入物的寿命可以延长。公开多孔金属植入物有前途的组织界面工程材料，也为确保良好的殖民化的植入物。他们一直在积极作为骨科植入物及气管植入3-5。但是，仍然存在一些问题需要解决，如细胞的运动精确地控制孔隙区域。不控制这个过程可能会导致不完全的殖民统治，在一端，并在其他的再狭窄。这些植入物的进一步官能化是必要的，为实现更高的功能，如生长因子，送货，直接血管和不同的细胞类型6-8的同步运动。对于气管植入物，植入血管组织的殖民化是可取的，这一点是至关重要的。然而，不受控制的组织内生长气管管腔是不希望的，因为它降低植入物通畅。 控制细胞运动的可能性之一是尺寸排阻。知道的靶细胞和与一个给定的合成聚合物的能力的大小，有可能开发的孔可有效地确定细胞运动的深度的梯度。例如，通过创建一个孔结构是足够大，可以实现结缔组织细胞如成纤维细胞腔外，但足够小（小于10μm），以防止其移动腔内定植的管状植入物有效控制有关条目。 从可用孔隙创建方法，如冷冻dryi纳克，颗粒浸出，气体发泡9,10;最简单的方法以适应快速形成的孔隙梯度少量必要的设备是冷冻萃取11。在该方法中，聚合物溶液的有机溶剂和水的二元混合物中冻结。之后，将溶剂交换通过预冷的可混溶的液体（如乙醇）萃取。冻结和提取条件确定的孔的形状和大小，和如果提取的提取液的运动可控制的方式进行的，孔的大小和形状可以是定向调制。 第二步是多细胞组织形成不同类型的细胞，以控制它们的相互作用多孔之间的壁垒。不同的细胞类型，根据他们的要求12,13不同的微环境的可用性，这也是必要的。气管是一个管状器官连接喉与支气管（一）它有一个内在的假复层纤毛上皮衬里interdispersed产生粘液的杯状细胞。的三维结构和气管稳定保持在C环的形状的软骨。因此，在一个人工气管内，应该有一个定义的结缔组织和睫状体上皮细胞层之间的交界处。的结缔组织部分的三维结构是必要的，上皮细胞的迁移需要基底膜状的表面，以实现定向运动和关闭伤口。聚电解质多层膜（PEM）等是一个可能的选项来获得基底膜模仿。层 - 层的方法（层层）是一种多功能的过程中获得薄的和功能性的表面涂层。它是基于静电相互作用的两个带相反电荷的聚电解质和积聚在以连续的方式，获得纳米级的表面涂层，其属性可以是多种多样的，通过简单地改变的变量，如聚电解质的种类，pH值，层的数目，此外，覆盖层，交联等，其中的LbL方法的主要优点是它能够以符合下面的衬底的地形。因此，在受控的条件下，该方法也可以被用来获得多孔结构的表面覆盖率。如果胶原被用作聚电解质，它有可能取得的nanofibrillar结构，可以模仿基膜的表面。钛的疏水性使这种结构的发展和fibrillarity可以保存在厚厚的涂料14。这种方式附着和细胞的表面上的运动也可以被控制。通过依次使用冷冻提取和层层薄膜包衣，在细胞的运动，可以控制横向，纵向和圆周方向的结构，可以通过以下方式获得15。 这里，我们描述了两个新的修改的方法，利用其疏水性的行为，它可以是钛种植体扩展到修改的各种多孔植入物：ⅰ）形成的微孔梯度内的大孔与疏水性的，合成的聚合物，ⅱ）形成一个厚厚的支持细胞生长和聚电解质多层膜的衣料形成的植入物表面上的聚合物薄膜层的钛植入物。这些方法可以顺序地或单独地使用。他们提供结构确保控制迁移和空间组织的多细胞组织不同类型的细胞16,17。气管的具体情况下，植入物所期望的结果将是由纤维血管组织内没有再狭窄和纤毛上皮细胞上的聚电解质多层膜的内衬形成微孔梯度定植。 控制整合植入的方法之一是做小的外科手术期间，他们的主机集成在原地 。为了能够吨ö决定干预的定时，重要的是有信息的植入物上的全身效应。 C-反应蛋白（CRP）已被用于监测感染和炎症反应在临床设置。嗜铬粒蛋白A（CGA）也可以以类似的方式使用，并可能提供更准确的结果，观察到的水平，炎症18。观察金属植入物在体内的集成作为一种可能的方式中，我们提出了一种植入物的全身效应的表征动物的血液样品，用高压液相色谱法（HPLC）和随后的蛋白质测序连续监测过程。拟订这种方法可以用来逃避定期终点组织学分析。金属植入物的组织学切割是一个长期的，繁琐的和昂贵的过程，可以只在特定时间点进行。因为这个原因，精心设计提供强大的信息植入健康的血液​​测试最近的欧盟关于动物实验的规则所规定的，是可能的路由，以减少动物实验。 这里介绍的方法可用于提高性能的金属植入物，通过官能化或监测现有的植入物的另一种方式。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Dioxane</td>
 <td>Sigma-Aldrich</td>
 <td>360481</td>
 <td>Toxic material, Strictly under chemical hood</td>
 </tr>
 <tr>
 <td>PLLA 
 i. Poly(L-lactide) inherent viscosity ~0.5 dl/g 
 ii. Poly(L-lactide) inherent viscosity ~2.0 dl/g 
 </td>
 <td>Sigma-Aldrich</td>
 <td>94829, 81273</td>
 <td>The choice of molecular weight and inherent viscosity is application dependent.</td>
 </tr>
 <tr>
 <td>PRONOVA UP LVG (Sodium Alginate)</td>
 <td>Novamatrix</td>
 <td>4200006</td>
 <td>Low viscosity(20-200 mPa.s)</td>
 </tr>
 <tr>
 <td>Collagen type I (Bovine)</td>
 <td>Symatese</td>
 <td>CBPE2US100</td>
 <td></td>
 </tr>
 <tr>
 <td>Pen/Strep, Fungizone </td>
 <td>Promocell</td>
 <td>C42020</td>
 <td></td>
 </tr>
 <tr>
 <td>Genipin</td>
 <td>Wako</td>
 <td>0703021</td>
 <td></td>
 </tr>
 <tr>
 <td>Silicon nitride probes with aspring constant of 0.03 N.m-1. </td>
 <td>Bruker </td>
 <td>MSCT </td>
 <td></td>
 </tr>
 <tr>
 <td>Trifluoroacetic acid for HPLC ,&ge;99.0% </td>
 <td>Sigma-Aldrich</td>
 <td>302031</td>
 <td>Hazardous Material, Please follow MSDS carefully</td>
 </tr>
 <tr>
 <td>Acetonitrile, for HPLC ,&ge;99.9% </td>
 <td>Sigma-Aldrich</td>
 <td>34998</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein-AM</td>
 <td>Invitrogen</td>
 <td>C3100MP </td>
 <td></td>
 </tr>
 <tr>
 <td>PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling </td>
 <td>Sigma-Aldrich</td>
 <td>PKH26GL</td>
 <td></td>
 </tr>
 <tr>
 <td>Rabbit C-Reactive Protein (CRP) ELISA kit</td>
 <td>Genway Bio</td>
 <td>GWB-9BF960 </td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO, Bioreagent, &ge;99.7% </td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Multimode Nanoscope IV Atomic Force microscope</td>
 <td>Bruker</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Procise microsequencer </td>
 <td>Applied Biosystems </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ultima 3000 HPLC system </td>
 <td>Dionex</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope Hitachi TM 100</td>
 <td>Hitachi </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal Scanning Laser Microscope Zeiss LSM 510 </td>
 <td>Zeiss </td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
 </table>
Table 1. List of Materials and Reagents.

multi-scale modification of metallic implants with pore gradients, polyelectrolytes and their indirect monitoring in vivo

金属植入物，特别是钛植入物，在临床应用中被广泛使​​用。这些植入物在组织中的组织生长和集成是成功的临床结果的重要参数。为了提高组织整合，多孔金属植入物已被开发出来。开口孔隙率的泡沫金属是非常有利的，因为可官能化的孔区域，而不会影响整个结构的机械性能。在这里，我们描述了这样的修改，使用基于钛微球多孔钛种植体。通过使用固有的物理特性，如疏水性的钛，未能取得基于微珠的金属植入物内，并在同一时间有基底膜模仿基于亲水性的，天然的聚合物的疏水性的孔隙梯度。三维孔隙梯度形成的合成聚合物，例如聚-L-乳酸（PLLA）冷冻提取方法。 2D nanofibrillar河畔面形成有通过使用胶原/藻酸盐，然后用天然的交联剂交联步骤（京尼平）。此nanofibrillar电影始建层层沉积法（LBL）两种带相反电荷的分子，胶原蛋白和海藻酸。最后，在不同的区域可以容纳不同类型的细胞，因为这是所必需的许多多细胞组织的植入物，可以通过以下方式获得。通过，这样一来，可以控制由不同的细胞类型的细胞在不同方向的运动。气管再生为特定的情况下，描述了这样的系统，但它可以被修改为其它靶器官。细胞迁移和可能的方法来创建不同孔径梯度分析阐述。这种植入物的分析的下一个步骤是在植入后的表征。然而，金属植入物的组织学分析是一个漫长而繁琐的过程，从而为监控主机的金属植入物的反应在体内是一个基于CGA和不同血液中的蛋白质监测延误保障的方法也作了说明。这些方法可以用于开发量身定制在体外迁移和定植测试的，也可用于官能化的金属植入物在体内无组织学分析。

孔梯度的形成通过改变聚乳酸溶液的浓度，它是可能的植入物上的腔外侧的孔的大小来控制。孔隙的大小和形状显着影响的钛植入物的存在下（ 图1a和1b）。孔径大小范围为40-100微米，利用较低的浓度导致在更小的孔隙。然而，在所述管腔内侧面孔径大小是受限制的提取，约9微米23，成纤维细胞的平均尺寸小于。通过添加，在室温下温育步骤的?...

Watch this Scientific Journal Video about Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo at JoVE.com

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

在这段视频中，我们将演示多孔金属植入物的改性技术，以提高它们的功能和控制细胞迁移。这些技术包括孔梯度发展，控制细胞的移动3D和生产基底膜模仿在2-D控制细胞运动。此外，描述一个基于HPLC的监测方法，通过分析血液中的蛋白质被检体内的植入物的整合。

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 ...

Research

JoVE Journal

Bioengineering

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

Monitoring Protein Adsorption with Solid-state Nanopores

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

科研

生物工程

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

质量流式细胞术：协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

的简便和环保路线制造聚乳酸支架与分级孔径

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...