部分支持这项研究是由NIH RO3的EB007299-02和NSF EPSCoR第一奖。

1。组织构造的制备组织建设准备过程包括三个主要阶段:扩张的细胞群，细胞生物材料支架上播种，通过使用化学信号分子的分化。结构编写的程序是基于丹尼斯等 。，香港等 。，马里昂和毛泽东8,9,10进行的方法。 <ol><li>经过文化和扩张的细胞株，种子人类间质干细胞（H MSCS）到明胶海绵密度为1×10 6细胞/ ml（直径4毫米，厚度为3.5毫米），骨和3X10 6细胞/ ml脂肪的形成。 </li><li> h 的间充质干细胞分化成脂肪，适用于脂肪诱导培养基组成的1μM的地塞米松，0.5微米异丁基甲基黄嘌呤，10μg/ ml的重组人胰岛素，吲哚美辛和200μM的细胞扩大中等一旦细胞出现在断头台上的融合。三天后，取代10微克/毫升在24小时内扩大媒体的重组人类胰岛素，然后返回到感应媒体的媒体。循环重复三次，然后在维护媒体交换只有每隔两天。 </li><li>诱导成骨，准备作出最后的左旋抗坏血酸2phosphate的50微米，0.1微米的地塞米松，和10毫米的细胞扩增培养基β-甘油浓度的成骨诱导媒体。与新鲜骨媒体每两天更换。 </li></ol> 2。执行机构的表征表征的执行机构是至关重要的一步，为麦格理实验。麦格理依赖于机械横波的传播，以评估当地的力学性能值，因此，这些机械振动，需要生成和组织内使用压电致动器的兴趣特点。一个图文并茂的EXAmple的表征过程如图3所示。此过程的目标是优化执行机构的议案，以产生显著振幅（〜250微米）的无害横波。 <ol><li>在实验之前，适用于0.5％琼脂糖凝胶附上10毫米试管中的构造。凝胶的温度应该是大约37℃，以减少损失构造。 </li><li>允许设置在室温下的琼脂糖凝胶五分钟后，插入凝胶表面弯曲的压电电机的尖端。 </li><li>连接管中样品和执行机构刚性支持，东方束激光多普勒测振仪对机械驱动的尖端。调整定位系统，以优化反射信号，如有必要，使用反光胶带。 </li><li>基于机械驱动的预期共振频率，设置函数发生器，以扫的desired频率范围（即在这个实验中的20至2000赫兹），用白噪声信号20 VPP的工作电压。 </li><li>查看上利达Vibrosoft程序特征光谱识别系统的共振频率，并设置为y轴的方案，以FFT和速度。 </li><li>位移测量，交付使用200 VPP的工作电压在指定的共振频率的连续正弦波驱动器设置，并注意被传递到表面的凝胶产生的位移。设置Vibrosoft显示位移为y轴的FFT。 </li></ol> 3。图像采集<ol><li>完成后，执行器的特性，将在核磁共振成像扫描仪中心的样本和执行机构。对于组织结构的实验中，使用的传输和接收射频信号小，更敏感的射频线圈（即在本实验中的10毫米）。 （显示的程序使用9.4ţ配备了三轴梯度，100克/厘米）垂直孔磁铁。 </li><li>获得侦察图像识别的结构位置。 </li><li>为收购的参数。一个典型的体外矢状扫描，将有20-40毫秒，切片厚度0.5-1.0毫米，和一个128x128像素的矩阵大小为12X10毫米2场的回声时间为1000毫秒，重复时间。 </li><li>为弹性参数，设置驱动器的频率由激光多普勒测振仪特性决定的价值。在目前的研究，需要一个双极对梯度幅值的50克/厘米。其他参数的调整，包括应设置延迟到零毫秒的初始收购。 </li><li>改变函数发生器，突发模式和函数发生器的参数调整，以符合那些在弹性的采集参数，包括频率和周期数。此外，设置的功能TION发生器通过外部触发。 </li><li>为矢状图像，设置的议案敏在积极片的方向，并开始扫描。收购之后，检查图像和宣传，以改变负切片方向。 </li><li>执行MATLAB程序将执行复杂的分裂横波图像生成。 </li><li>评估图像横波和相缠绕的可能，如文物的存在。 </li><li>如果没有图像的调整是必要的，调整参数数组的大小8个等距值范围从零点几秒到特征的共振频率的整段期间。 </li><li>收购中的积极和消极两片方向的扫描。 </li><li>一旦图像被收购，使用MATLAB程序设计产生横波数据从一个图像阵列。 </li></ol> 4。麦格理实验图像处理<ol><li>日E的麦格理的最后一步是计算从横波图像的剪切刚度。放入数据的MATLAB程序，将评估的三维数据集（2的空间，时间1）。 </li></ol> 注 :假设一个平面横波，运动解耦方程，使位移的功能和它的Laplacian复值的剪切模量的估计。该算法接近的空间有限差分的二阶导数和计算上的一个像素由像素的基础上的剪切模量。从这个复杂的数字，许多力学参数可以推断，如剪切波速，波的衰减，剪切刚度，弹性剪切，剪切粘度等算法也可以选择感兴趣的区域的平均值和标准偏差每个参数的计算。 <ol start="2"><li>成像参数需要被指定在程序的开始。此外，日E的上限，elastogram可以调整优化样品的对比度。 </li></ol> 注 :该项目提供的中间结果（低通滤波器后的波，波经过方向滤波，FFT的时间，剖面线等），帮助用户估计复苏的信实。 <ol start="3"><li>基于此信息，如低通滤波器，该议案的时间频率，波的传播方向等参数的标准偏差在特定地区的利益，是各级可以调整一些参数，也计算质量的一个指标。 </li></ol> 5。代表结果图4注意到，在整个四个星期的成骨细胞和脂肪细胞的构造发展变化的力学性能。麦格理进行了730-820赫兹。虽然这两个种子海绵开始约3千帕，osteogeniÇ定向组织，在22千帕的刚度;反之，脂肪定向到1千帕的刚度下降的组织。此外，骨结构从开始到结束的研究表明在比较大小的显着下降。从弹性研究的附加属性如表1所示。 <img src="/files/ftp_upload/3618/3618fig1.jpg" alt="图1"/> 图1。磁共振弹性图像采集过程。在图像采集，脉冲序列（一）控制（二）与双极梯度核磁共振成像扫描仪的脉冲信号发生器的同步。在正面和负面的方向切换双极梯度收购之后，（三）横波图像使用复杂的分工。 <img src="/files/ftp_upload/3618/3618fig2.jpg" alt="图2"/> 图2。麦格理过程的流程图，组织发动机ERED等效结构。首先，细胞（一）首次增长，并扩展到设计项目所必需的人口规模。然后，细胞接种于生物材料支架及化学试剂（二）被应用到信号分化。支架的特点是与麦格理（三）是确定的构造耦合的致动器的共振频率，其第一步。下一步，MRI图像（四）被收购产生横波图像（e）。最后，算法用于产生一个elastogram（F），映射结构的刚度。同时，构造是为了验证分化切片组织学评估（G）。 <img src="/files/ftp_upload/3618/3618fig3.jpg" alt="图3"/> 图3。执行机构鉴定程序。括由0.5％琼脂糖凝胶明胶支架。表征白噪声的议案被转移到样品首先被送入系统（1a）和所产生的运动检测使用激光多普勒测振仪（1B）。一旦确定共振频率，共振连续正弦信号（2A）被发送到确定的位移（2B）转移到明胶环境。 <img src="/files/ftp_upload/3618/3618fig4.jpg" alt="图4"/> 图4。建设了4个星期内开发的地图。脂（A）和骨（O）的结构，从左至右显示相应的规模和横波的图像，elastogram，平均剪切刚度。代表与条形图和错误酒吧的配色方案elastogram对应colormap中的每个结构的感兴趣区域内的标准偏差。 <img src="/files/ftp_upload/3618/3618Table1.jpg" alt="表1"/&gt; 表1。超过了经济增长的四个星期内的脂肪和骨结构的力学性能。

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	<li>Xu, H., Othman, S. F., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tissue+engineering+using+magnetic+resonance+imaging.">Monitoring tissue engineering using magnetic resonance imaging.</a> J. Biosci. Bioeng. 106, 515-527 (2008).</li><li>Xu, H., Othman, S. F., Hong, L., Peptan, I. A., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+microscopy+for+monitoring+osteogenesis+in+tissue-engineered+construct+in+vitro.">Magnetic resonance microscopy for monitoring osteogenesis in tissue-engineered construct in vitro.</a> Phys. Med. Biol. 51, 719-732 (2006).</li><li>Othman, S. F., Xu, H., Royston, T. J., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+magnetic+resonance+elastography+(microMRE.">Microscopic magnetic resonance elastography (microMRE.</a> Magn. Reson. Med. 54, 605-615 (2005).</li><li>Muthupillai, R., Lomas, D. J., Rossman, P. J., Greenleaf, J. F., Manduca, A., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+elastography+by+direct+visualization+of+propagating+acoustic+strain+waves.">Magnetic resonance elastography by direct visualization of propagating acoustic strain waves.</a> Science. 269, 1854-1857 (1995).</li><li>Othman, S. F., Curtis, E. T., Plautz, S. A., Pannier, A. P., Xu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+elastography+monitoring+of+tissue+engineered+constructs.">Magnetic resonance elastography monitoring of tissue engineered constructs.</a> NMR Biomed. , (2011).</li><li>Oliphant, T. E., Manduca, A., Ehman, R. L., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex-valued+stiffness+reconstruction+for+magnetic+resonance+elastography+by+algebraic+inversion+of+the+differential+equation.">Complex-valued stiffness reconstruction for magnetic resonance elastography by algebraic inversion of the differential equation.</a> Magn. Reson. Med. 45, 299-310 (2001).</li><li>Ringleb, S. I., Chen, Q., Lake, D. S., Manduca, A., Ehman, R. L., An, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+shear+wave:+comparison+to+a+dynamic+shear+material+test.">Quantitative shear wave: comparison to a dynamic shear material test.</a> Magn. Reson. Med. 53, 1197-1201 (2005).</li><li>Hong, L., Peptan, I., Clark, P., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+adipose+tissue+engineering+by+human+marrow+stromal+cell+seeded+gelatin+sponge.">Ex vivo adipose tissue engineering by human marrow stromal cell seeded gelatin sponge.</a> Ann. Biomed. Eng. 33, 511-517 (2005).</li><li>Dennis, J. E., Haynesworth, S. E., Young, R. G., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+marrow-derived+mesenchymal+cell+porous+ceramic+composites+transplanted+subcutaneously:+effect+of+fibronectin+and+laminin+on+cell+retention+and+rate+of+osteogenic+expression.">Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: effect of fibronectin and laminin on cell retention and rate of osteogenic expression.</a> Cell Transplant. 1, 23-32 (1992).</li><li>Marion, N. W., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+tissue+engineering.">Mesenchymal stem cells and tissue engineering.</a> Methods Enzymol. 420, 339-361 (2006).</li><li>Rydberg, J., Grimm, R., Kruse, S., Felmlee, J., McCracken, P., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+spin-echo+magnetic+resonance+elastography+of+the+brain.">Fast spin-echo magnetic resonance elastography of the brain.</a> , 1647-1647 (2001).</li><li>Kruse, S. A., Grim, R. C., Lake, D. S., Manduca, A., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+EPI+based+3D+MR+elastography+of+the+brain.">Fast EPI based 3D MR elastography of the brain.</a> , 3385-3385 (2006).</li></ol>

作者有没有利益冲突披露。

在此过程中，为组织工程结构的麦格理的过程中表现出细胞制备一个elastogram的代。机械非破坏性评估方法应用组织工程管线，它现在可以评估多个发展阶段，整个工程结构变化。此外，麦格理补充监测组织工程建设，如扩散，磁化传递和化学位移分析1，其他议员的方法。 执行麦格理实验时，应注意一些限制。 体外标本的评估是一个敏感的时间研究。因此，建...

<table border="1"><tbody><tr><td> 材料名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> MSCGM的子弹包</td><td>试剂</td><td>龙沙</td><td> PT-3001 </td><td>保存于4°C间</td></tr><tr><td> 1X DPBS </td><td>试剂</td><td> Invitrogen公司</td><td> 21600-010 </td><td></td></tr><tr><td> 0.05％胰蛋白酶EDTA </td><td>试剂</td><td> Gibco公司，Invitrogen公司</td><td> 25300-054 </td><td>储存在-20°C </td></tr><tr><td>地塞米松</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> D2915 </td><td></td></tr><tr><td> 3 - 异丁基-1 - 甲基黄嘌呤</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> I5879 </td><td>储存在-20°C </td></tr><tr><td>胰岛素牛胰腺</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> I6634 </td><td>储存在-20°C </td></tr><tr><td>吲哚美辛</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> I7378 </td><td></td></tr><tr><td> β-甘油</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> G9891 </td><td></td></tr><tr><td> L-抗坏血酸2 - 磷酸</td><td>试剂</td><td> Sigma-Aldrich公司</td><td> A8960 </td><td></td></tr><tr><td>明胶</td><td>脚手架</td><td>法玛西亚 - 普强公司</td><td> 09-0315-08 </td><td></td></tr><tr><td>人类间质干细胞</td><td>细胞株</td><td>龙沙</td><td> PT-2501 </td><td></td></tr><tr><td> 9.4T议员扫描器</td><td>设备</td><td>安捷伦</td><td></td><td> 400MHz的白平衡</td></tr><tr><td> 10毫米利兹线圈</td><td>设备</td><td>蒂科学</td><td></td><td></td></tr><tr><td>激光多普勒测振仪</td><td>设备</td><td>利达</td><td> PDV-100 </td><td></td></tr><tr><td> vibrosoft（20） </td><td>软件</td><td>利达</td><td></td><td></td></tr><tr><td>函数发生器</td><td>设备</td><td>安捷伦</td><td>的AFG 3022B </td><td></td></tr><tr><td>放大器</td><td>设备</td><td>压电陶瓷公司</td><td>环保局-104-115 </td><td></td></tr><tr><td>压电弯曲机</td><td>设备</td><td>压电INC。 </td><td> T234-A4Cl-203X </td><td></td></tr><tr><td>电脑Linux的</td><td>设备</td><td>处理器:英特尔酷睿2双核E8400 内存:2G </td><td></td><td></td></tr><tr><td>计算机Windows的</td><td>设备</td><td>处理器:英特尔酷睿2双核E8400 内存:2G </td><td></td><td></td></tr><tr><td> MATLAB的</td><td>软件</td><td> MathWorks公司</td><td></td><td> 2009B </td></tr></tbody></table>

magnetic resonance elastography methodology for the evaluation of tissue engineered construct growth

传统的机械测试结果往往破坏样品，并在长期组织工程建设研究的情况下，使用破坏性的评估是不能接受的。拟议的另一种方法是使用称为磁共振弹性成像过程。弹性是一种无损检测方法确定工程测量当地的机械属性值（即，复杂的剪切模量），这是确定一个组织的结构和功能的重要标志成果。作为一种非侵入性手段进行评价，监测工程结构，如磁共振成像（MRI）成像已经看到越来越多的兴趣在过去十年1。例如，磁共振（MR）扩散和relaxometry技术已经能够在化学和物理性质，化学性质的变化特点，在工程组织发展 。在提出的方法下面的协议，采用显微磁共振弹性（μMRE）的议员作为一种非侵入性测量小三软组织的力学性能的基础技术。麦格理是通过耦合声波的机械执行机构利益的组织，并记录与横波传播的磁共振扫描仪4。最近，μMRE已应用于组织工程，以获得增长的重要信息是传统的测量使用破坏性机械5宏观技术。在下面的过程中，弹性是通过与工程结构的改良加上机械执行机构哈恩自旋回波序列成像。如图1所示，修改后的顺序同步图像采集与传输的外部横波;随后，该议案是通过振荡双极对使用致敏。继图像采集与正面和负面的议案sensitizaTION，复杂的数据分工产生横波图像。然后，图像评估使用逆算法来生成剪切刚度地图6。在每个像素的测量结果已显示强烈相关（R&gt; 0.9914）收集的数据用动态力学分析7。在这项研究中，纳入监测人类间质干细胞（H，MSC）分化成脂肪细胞和骨结构如图2所示的组织发展过程中的弹性。

Watch this Scientific Journal Video about Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth at JoVE.com

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

程序演示磁共振弹性的脂肪和骨组织，通过当地的机械性能，使用显微磁共振弹性（μMRE）的无创性评估的工程结构设计的结果监测方法。

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

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12.4K 次观看.  University of Nebraska-Lincoln. 程序演示磁共振弹性的脂肪和骨组织，通过当地的机械性能，使用显微磁共振弹性（μMRE）的无创性评估的工程结构设计的结果监测方法。

视频: Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

科研

生物工程

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

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 animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

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

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

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 ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

聚电解质复合物肝素结合域成骨生长因子交货

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

磁共振显微镜下切除活体脑组织的代谢支持

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

由激光蚀刻大师定制的组织培养模具

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

超声弹性成像验证腹主动脉水凝胶组织模拟幻影的研制

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

小鼠模型中生物合成组织工程气管移植的育苗与植入

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...