这项工作是支持研究与发展，RR＆D服务，美国退伍军人事务部，NIH COBRE 1P20RR024484 AR02128，NIH K24和国防W81XWH-10-1-0643系办公室。

1。装双轴生物反应器的设计<ol><li>的生物反应器采用由Thor实验室（牛顿，MA）制造的精确引导的激光应用工程化组织施加单轴或双轴的机械应变，负荷剂量以极高的精度（振幅和频率）和各种各样的应用程序的两个阶段组织培养条件从单一到24孔板中（ 图1）。 </li><li>双向轴向载荷是通过“两个TravelMax阶段（LNR50SE）。这些阶段被安装正交在XZ配置。水平阶段提供动态剪切运动，沿X轴振荡。垂直阶段提供动态压缩载荷沿Z轴振荡。这些阶段有一个50毫米的行程范围内，并独立驱动步进电机的致动器（DRV014），控制的一个闭环的步进电机驱动器（BSC102），具有微步进能力，使步长小于50nm以下。 </li><li>该装置被安装在一个刚性的2​​5厘米×30厘米×12.5毫米是作为一个平台，机器部件的组装和安装的组织培养板中使用的铝基板。可调的运动停止，用于锁定到位的组织培养板的铝基板。停止这些运动有微调螺丝，让手，否则无法实现精确对准。底板采用模块化设计，可以灵活放置停止这些运动以适应不同大小和形状的板（培养皿中主场迎战多孔板）。 </li><li>一个定制加工的聚砜加载压板连接到双向轴向移动平台，通过精密加工的直角支架。被选择的聚砜材料由于其生物相容性，易于加工，易于消毒。 </li><li>托尔实验室的先进定位技术（APT）软件控制的阶段变动。步进电机驱动器是我们在与软件的组合，允许独立并同时剪切和压缩的频率和振幅的调整负荷参数。 </li><li> ，附加到每个移动平台，并与软件集成的线性光学编码器提供位置反馈。编码器系统具有双向为0.1μm的可重复性以及分辨率为20nm，翻译准确度小于3μm，在整个50毫米的行程的位置。这些编码器提供必要的位置反馈信号来驱动电子设备，以确保真正的纳米定位功能直接读取的绝对位置。 </li><li>为了提供所需的检测压板和样品之间的接触力反馈和评估加载响应，一个高精度的微型测力传感器（霍尼韦尔31型）加载板的移动平台（ 图2）之间的定位。称重传感器具有很高的精度0.15％至0.25％满量程。在显示单元（SC500）称重传感器还可以提供高达到小数点后5位的负载测量。此外，它有一个RS-232端口的计算机上允许进行数据采集。 </li></ol> 2。小区种子琼脂糖构造<ol><li>准备4％琼脂糖：添加0.8克琼脂糖到20毫升的DMEM（无添加剂）在50ml烧瓶中，煮沸，然后保持在70℃的烘箱中，直到使用。 </li><li>调整细胞悬浮液的体积的期望的细胞接种密度为双倍。此悬浮液与等体积的4％w / v琼脂糖建立所需的接种密度在2％琼脂糖凝胶混合。 </li><li>将细胞悬液和一个10毫升吸管在孵化器。 </li><li>设立凝胶浇注系统。一个1.5毫米和0.75毫米隔板应放在一起，创建一个2.25毫米厚的凝胶。也可以使用其他尺寸的隔离板，以创建不同的凝胶厚度。凝胶注模系统是不是大到足以容纳这些板块toget她，所以他们必须牢固地贴在防止泄漏。 </li><li>接下来的步骤是，包括：混合的细胞悬浮液与预先制备的琼脂糖凝胶和吸液管进入凝胶模在琼脂糖固化之前。从烘箱中取出液体的琼脂糖，其放置无菌的温度计。琼脂糖必须冷却到42-43°C与细胞混合前。热身的细胞悬浮液到37℃。一旦琼脂糖命中43℃，迅速吸取所需的金额，然后立即吸取细胞悬液和混合几次。然后立即吸取整个混合物倒入模具凝胶。 </li><li>使凝胶固化10-15分钟，然后小心地倾斜到水平位置。 </li><li>卸下顶部的玻璃板和活检冲床冲磁盘。磁盘可以拿起一个小的无菌压舌板。根据我们的经验，9毫升凝胶是大到足以使一百多5毫米直径的光盘。 </li></ol> 3。文化磁盘</p&gt; <ol><li> 24非组织培养处理板每孔中的一个磁盘。 </li><li>向每孔中加入2毫升的无血清软骨细胞分化培养基。 </li><li>将板在培养箱（37℃，5％CO 2）。 </li><li>对于媒体的变化，更换1毫升每口井每2-3天。 </li></ol> 4。固定机械装载样品<ol><li>准备（未添加细胞悬浮液）4％的琼脂糖凝胶应该是薄的样品本身（在加载过程中，以防止干扰）。推荐厚度为1.5-1.9毫米（2.25毫米厚的样品）。 </li><li>一旦胶凝，打孔直径16毫米磁盘的24孔板。在每个磁盘中，下冲头一个5 mm的样品孔，如果需要，冲头5毫米的孔的边缘上的吸移管被放置在媒体变更的磁盘。 </li><li>一旦琼脂糖井已被制成，在24孔板中的地方，如在图3中示出。 </li><li>一旦琼脂糖瓦特英语学习者在24孔板，按在每口井的拟合样本。样品应突出从琼脂糖井的顶部。 </li></ol> 5。机械装载<ol><li>消毒台板（ 图2）。 </li><li>铝板固定传感器。安全加载压板/称重传感器/铝板组装阶段。 </li><li>步进电机控制器（开关在背面）打开。 </li><li>打开电脑，并打开“APT用户”程序（ 图4）。 <ol><li>左屏幕控制水平的步进电机。屏幕右侧垂直步进电机控制。在每个屏幕上，“图形控制”选项卡允许手动定位和“移动音序器”选项卡允许自动化。所有单位均为毫米。 </li></ol></li><li>转到“图形控制”选项卡，在两个屏幕上，按“首页/零”按钮。这两个步进电机为50毫米的范围内。按“首页/零”将发送两个步进电机零位（顶部和最右边的位置）。 </li><li>编写e本样品装载删除一些媒体从每口井的24孔板中。不超过100毫升的媒体应留在每口井在加载过程中，以防止溢出。要确保有足够的介质以及保持留在样本覆盖。 <ol><li>需要注意的是孵化器被保持在低湿度条件下，以防止仪器故障。 </li></ol></li><li>广场24孔板中的生物反应器，仔细排队压板。 <ol><li>该板固定到生物反应器中，使用四个可调的运动定位器。为了使它更容易排队压板，左边的两个定位器已预先定位。拧紧右侧的两个定位器，所以该板块是安全的。确保排队板与前面齐平的生物反应器基地。 </li><li>在“图形控制”选项卡中，具体步进电机位置可以手动点击的位置“框中输入。使用此功能来慢慢放低压板和水平移动板排队。 </li> &lt;/ OL&gt; </li><li>一旦压板接近与样品接触，开始把压板非常缓慢递增（0.1毫米），直到达到预定的起始位置（见第6部分）。 </li><li>一旦达到起始位置，进入“移动音序器”选项卡，并加载所需的移动序列，按“负载”。然后点击“运行”开始。 （ 图5），请参见第7部分。写配料协议。 </li><li>装完时，手动提高压板。如果，任何样品被卡住压板，小心翼翼地把它们放回适当的孔用无菌压舌板。 </li><li>从生物反应器中取出24孔板，并更换介质。 </li><li>小心地取下压板来自称重传感器，然后关闭仪器。 </li></ol> 6。校准装入滚筒 ，以确保适当的菌株应用于样本，每个压板必须仔细校准开始实验前。 <ol><l&gt;执行步骤5.1至5.8。 </li><li>手动将水平步进电机25毫米的位置。 </li><li>压板小心地降低，直到它刚好接触生物反应器的基础上。称重传感器会显示增加的负载，在这一点上。请注意垂直步进电机（尽可能精确，因为所有的压缩应变测量这个值将被计算）的确切位置。 </li><li>记录的位置。这个值将被用来写一个协议，根据样品的尺寸和所需菌株剂量。 </li></ol> 7。写作配料协议<ol><li> ，可以同时或单独的生物反应器是能够施加压缩和剪切应变。三个主要参数必须决定：去皮压应变，动态应变幅度，加载频率。 </li><li>甲皮应变施加，以防止从该样本的压板升空。 </li><li>样品动态应变幅度和加载频率频率被选择。 </li></ol>在这项研究中，我们定义如下：压缩和剪切应变 <img alt="公式1" fo:content-width="3.26in" fo:src="/files/ftp_upload/50387/50387eq1highres.jpg" src="/files/ftp_upload/50387/50387eq1.jpg" />双向配料协议示例 样品厚度：2.25毫米 皮株（压缩）：10％的样品厚度（0.225毫米） 动态应变幅度（压缩）：10％（+ / - 5％的样品厚度） 频率（压缩）：1赫兹 动态应变振幅（剪切）：25％的样品的厚度（0.5625毫米）：剪切应力是 施加在样品上水平移动的压纸卷筒。 频率（剪切）：0.5赫兹 典型的剂量方案是3小时，每天装载。 在这个例子中，动态和剪切载荷的同时施加而不是按顺序。我们相信这种模式更好的MIM集成电路的复杂加载环境中的人的膝盖。 <ol start="4"><li>一旦已经选择了一个定量给料协议，压缩移动序列程序必须被写入。 </li><li>移动序列正是这听起来像，步进电机的位置将移动到指定的加速度和最高速度的列表。 </li><li>计算所需的垂直位置的基础上加药协议和的压板校准值（从第6部分）。 </li><li>滚筒1的计算举例如下： </li></ol><table border="1" fo:keep-together.within-page="always"><tbody><tr><td></td><td> 校准值的差异 </td><td> 垂直位置 </td></tr><tr><td>滚筒校准值（触及生物反应器的底部） </td><td> 0毫米</td><td> 29.7700毫米</td></tr><tr><td>滚筒样品接触（2.25毫米样品） </td><td> 4.4140毫米</td><td> 25.3560毫米</td></tr><tr><td>圣雨（5％的厚度） </td><td> 4.3015毫米</td><td> 25.4705毫米</td></tr><tr><td>应变（10％厚度） </td><td> 4.1890毫米</td><td> 25.5810毫米</td></tr><tr><td>应变（15％厚度） </td><td> 4.0765毫米</td><td> 25.6955毫米</td></tr></tbody></table><ol start="8"><li>一旦位置计算，实验与加速度和最高速度值，以获得正确的频率。应选择，相应的周期数（ 例如 10800个周期为3小时，在1 Hz）。 </li><li>例如动态压缩移动序列计划（10％去皮压缩，10％的动态应变振幅，1赫兹）（ 图5） </li><li>动态剪切移动序列程序：应该选择的周期数，根据所需的频率和持续时间（ 例如 5,400周期为3个小时，在0.5赫兹）。 </li><li>例如动态剪切移动序列计划（去皮压缩10％（25％的厚度），0.5625毫米动剪应变振幅0.5赫兹）（ 图5）。 </li></ol>

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Eng. 122, 252-260 (2000).</li><li>Demarteau, O., Jakob, M., Schafer, D., Heberer, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+bioreactor+for+physical+stimulation+of+engineered+cartilage.">Development and validation of a bioreactor for physical stimulation of engineered cartilage.</a> Biorheology. 40, 331-336 (2003).</li><li>Grad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+motion+upregulates+superficial+zone+protein+and+hyaluronan+production+in+chondrocyte-seeded+three-dimensional+scaffolds.">Surface motion upregulates superficial zone protein and hyaluronan production in chondrocyte-seeded three-dimensional scaffolds.</a> Tissue Eng. 11, 249-256 (2005).</li><li>Schatti, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combination+of+shear+and+dynamic+compression+leads+to+mechanically+induced+chondrogenesis+of+human+mesenchymal+stem+cells.">A combination of shear and dynamic compression leads to mechanically induced chondrogenesis of human mesenchymal stem cells.</a> Eur. Cell Mater. 22, 214-225 (2011).</li></ol>

作者宣称，他们有没有竞争经济利益。

我们设计了一个装载装置，能够制作用于移植的组织工程构建施加单轴或双轴的机械应变。 在体外培养的工程生物复合材料作为生物反应器，或作为测试设备，该设备可以被用于描述的原生组织或其他治疗方法前，后的机械特性。该设备的主体设计的组织结构双轴机械负荷的负荷剂量（振幅和频率）以极高的精度和应用到各种各样的组织培养条件下，从单一的24孔板。 <p class="jove_content"...

我们设计了一个加载的生物反应器，能够制作用于移植的组织工程生物复合材料进行单轴或双轴的机械应变。这个装置的设计主要作为生物反应器的工程关节软骨的替代，它也可以用于其他的承重在人体内的组织。我们在这个生物反应器设计的动机源于Drachman女士和寇罗夫1，谁做出的开创性观察异常形成的关节软骨由于没有运动瘫痪鸡胚。同样，体育锻炼是必不可少的正常的肌肉和骨骼的发展。符合这个概念，许多研究小组调查了不同的模式的物理刺激，在体外培养细胞生物材料复合生物材料和组织植2-7调制生化和机械性能。功能性组织工程的概念涉及在体外使用的机械刺激，以提高组织的功能特性， 即 ，使组织承受预期在体内的应力和应变8,9的机械性能。许多研究报告使用剪切和压缩机械负荷刺激工程化软骨关节关节结构。莫克等10表明，单独的机械负荷，即使在没有被认为是重要的生长因子诱导骨髓间充质干细胞的软骨形成。间歇性的机械负荷，例如在组织培养过程中的压缩或剪切中的应用已被证明能调节软骨和骨形成，然而，加载不同的最佳剂量测定与细胞和组织的性质11。 关节软骨的最重要的功能是内承受压缩力和剪切力的能力联合，因此它必须有高压缩和剪切模量。新软骨在体内关节软骨替换策略的失败的细分功能的机械强度和生理超微结构的工程化软骨已导致缺乏。尽管压缩和剪切已被广泛证明来调节和改善关节软骨的生物复合材料的机械强度，相结合的方式是罕见6,12-15。 16 Wartella和韦恩设计了一个生物反应器应用拉伸和压缩产生半月板软骨替换。瓦尔德曼15 等人设计了一个移动设备以应用在多孔聚磷酸钙基板的软骨细胞的压缩和剪切。边等 17展示了机械性能匹配的本地成年犬软骨细胞在体外培养凝胶和应用双轴机甲软骨anical加载（压缩变形和滑动触点负载）。 双轴机械负荷生物反应器的最初目的是由达尼埃尔楚在我们的实验室的总体目标，诱导组织工程软骨的形态适应构造导致更高的压缩和剪切模量，比现有的18。我们相信，这项研究将大大增加我们更广泛地了解如何可调制机械传导工程师临床相关组织。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS</td>
		 </tr>
		 <tr>
			<td>DMEM, High glucose, pyruvate</td>
			<td>Invitrogen</td>
			<td>11995</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Agarose Type II</td>
			<td>Sigma</td>
			<td>CAS 39346-81-1 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Penicillin Streptomycin Glutamine 100X</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ITS+ Premix</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen Strep Glutamine</td>
			<td>Invitrogen</td>
			<td>10378-016</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Amphotericin B</td>
			<td>Invitrogen</td>
			<td>041-95780</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A-2218</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nonessential Amino Acid Solution 100x</td>
			<td>Sigma</td>
			<td>M-7145</td>
			<td></td>
		 </tr>
		 <tr>
			<td>L-proline</td>
			<td>Sigma</td>
			<td>P-5607</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D-2915</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Recombinant Human Transforming Growth Factor &beta;1</td>
			<td>R&amp;D Systems</td>
			<td>240-B-010</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Model 31 Load Cell (1000 g)</td>
			<td>Honeywell</td>
			<td>AL311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Single Channel Display</td>
			<td>Honeywell</td>
			<td>SC500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 mm Linear Encoded Travelmax Stage with Stepper Actuator</td>
			<td>Thorlabs</td>
			<td>LNR50SE/M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Two Channel Stepper Motor Controller</td>
			<td>Thorlabs</td>
			<td>BSC102</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 mm Trapezoidal Stepper Motor Drive (2)</td>
			<td>Thorlabs</td>
			<td>DRV014</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Adjustable Kinematic Locator (4)</td>
			<td>Thorlabs</td>
			<td>KL02</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Precision Right Angle Plate</td>
			<td>Thorlabs</td>
			<td>AP90/M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Vertical Mounting Bracket</td>
			<td>Thorlabs</td>
			<td>LNR50P2/M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Solid Aluminum Breadboard</td>
			<td>Thorlabs</td>
			<td>MB3030/M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gel Casting System with 1.5 mm and 0.75 mm spacer plates </td>
			<td>BioRad</td>
			<td>#1653312 and #1653310 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Disposable Biopsy Punch, 5 mm</td>
			<td>Miltex, Inc.</td>
			<td>33-35</td>
			<td></td>
		 </tr>
		 <tr>
			<td>16 mm hollow punch</td>
			<td>Neiko Tools</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Non-Tissue Culture Treated Plates, 24 Well, Flat Bottom</td>
			<td>BD Biosciences</td>
			<td>351147</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ultra-Moisture-Resistant Polysulfone sheet for loading platens</td>
			<td>McMaster-Carr</td>
			<td>86735k19</td>
			<td>Custom-machined</td>
		 </tr>
		</tbody>
 </table>

design of a biaxial mechanical loading bioreactor for tissue engineering

我们设计了一个装载装置，其能够应用到用于移植的组织工程生物复合材料的单轴或双轴的机械应变。而该设备主要作为生物反应器，模仿的固有机械株作用的同时，还配备测力传感器，用于提供力反馈或机械测试的构造。该设备主体工程化软骨的结构，以双轴机械负荷负荷剂量（幅度和频率）非常精确和紧凑，足以容纳一个标准组织培养箱内。它装载样品直接在组织培养板，多个板的大小与系统兼容。该设备已设计使用精确制导激光应用组件制造。双向轴向载荷是由两个正交的阶段来完成。该阶段有50 mm的行程范围内，并独立驱动，由步进电机驱动器，控制一个闭环的步进电机驱动器，具有微步的能力，使步长小于50nm。聚砜加载压板被耦合到双向轴向移动平台的。变动的阶段控制由雷神实验室先进定位技术（APT）软件。步进电机驱动器是用软件来调整负载独立并同时剪切和压缩的频率和振幅参数。具有一个双向为0.1μm的可重复性以及分辨率为20nm，翻译的位置精度小于3μm，在整个50毫米的行程通过线性光学编码器提供位置反馈。这些编码器提供必要的位置反馈信号来驱动电子装置，以确保真正的纳米定位能力。为了提供检测力反馈联系和评估加载响应，精密微型称重传感器之间的位置加载压板舞动克平台。称重传感器具有很高的精度为0.15％，0.25％满量程。

通过使用琼脂糖凝胶电泳后种子用20百万个细胞/毫升的软骨细胞和装置进行了测试在单轴（压缩）或双轴（压缩和剪切）机械性负荷的存在下培养。小学猪软骨细胞中分离出2-4月龄猪关节软骨。 5星毫米直径和1.5毫米厚的样品分别在2毫升定义的软骨细胞培养介质中高葡萄糖DMEM培养基（1％ITS +预混物，100Û单/ ml青霉素，100μg/ ml的链霉素，2 mM的L-谷氨酰胺，2.5微克/毫升的培养两性霉素B，50微克/毫升...

Watch this Scientific Journal Video about Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering at JoVE.com

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

我们设计了一种新型的机械负荷生物反应器可应用于单轴或双轴机械应变移植到关节软骨缺损的软骨生物复合材料之前。

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

design-biaxial-mechanical-loading-bioreactor-for-tissue

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Mechanical Engineering

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Unit 1

Analysis and Design of Beams for Bending

SCIENTISTS IN ACTION

14.2K 次观看.  The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital. 我们设计了一种新型的机械负荷生物反应器可应用于单轴或双轴机械应变移植到关节软骨缺损的软骨生物复合材料之前。

视频: Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

一个台式生物反应器的操作

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Agarose-based Tissue Mimicking Optical Phantoms for Diffuse Reflectance Spectroscopy

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a conventional optical system with an integrating sphere. Measuring systems for the acquisition of the diffuse reflectance and total transmittance spectra are constructed with a broadband white light source, a light guide, an achromatic lens, an integrating sphere, a sample holder, an optical fiber probe, and a multi-channel spectrometer. An acrylic mold consisting of two rectangular acrylic pieces and a U-shaped acrylic piece is constructed to create an epidermal phantom and a dermal phantom with whole blood. The application of a sodium dithionite (Na2S2O4) solution to the dermal phantom enables the researcher to deoxygenate hemoglobin in red blood cells distributed in the dermal phantom. The inverse Monte Carlo simulation with the diffuse reflectance and total transmittance spectra measured by a spectrometer with an integrating sphere is performed to determine the absorption coefficient spectrum &#181;a(&#955;) and the reduced scattering coefficient spectrum &#181;s&#39;(&#955;) of each layer phantom. A two-layered phantom mimicking the diffuse reflectance of human skin tissue is also demonstrated by piling up the epidermal phantom on the dermal phantom.

Here, we demonstrate how agarose-based tissue-mimicking optical phantoms are made and how their optical properties are determined using a conventional optical system with an integrating sphere.

基于琼脂糖组织模拟光学幽灵的漫反射光谱研究

This protocol describes how to make agarose-based tissue-mimicking phantoms and demonstrates how to determine their optical properties using a ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

房室心脏瓣膜的双轴力学特性

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...