我们感谢杰弗里·卡普兰博士为他的共焦成像的培训和咨询。我们也感谢凯克电子显微镜实验室和超英博士倪扫描电镜的协助。这项工作是由卫生（NIDCD，R01DC008965和R01DC011377）国家研究院​​资助。 ABZ承认美国国家科学基金会综合研究生教育和研究培训实习资助（IGERT）计划。

1，生物反应器组件（视频1） <ol><li>使铝模（圆口模头+隔离销）与预先确定的内部和外部尺寸（ 图1）。 </li><li>从步骤1.1使用模具，制造硅膜（直径:42毫米，厚度:1.5毫米， 图1）与一个根深蒂固的套筒（直径:12毫米，厚度〜0.25毫米，形由图1中的隔离销）在使用市售的有机硅弹性体包的中间。 </li><li>使一对丙烯酸的块（ 图2（4,5））与一个圆形开口（直径:24毫米）的中间，雕刻顶部（1.8厘米厚）和底部（0.9厘米厚）相匹配的凸脊和凹槽的块10 </li><li>夹着一对丙烯酸块之间的有机硅膜。使用微型扭力起子设置为一个恒定的力（35 CN米）四角螺丝将部件固定。因此，水密，24米米，宽18毫米深的振动室被创建（ 图2C）。 </li><li>通过底部亚克力块上另一套角落的螺丝安装一个3"扩展的范围微型低音单元（ 图2D，8Ω/20W）振动室的下方。在这一点上，一个单独的振动模块组装。 </li><li>复制七个额外的振动模块。切成的棒放置在均匀间隔的圆孔（:7厘米，厚度为2厘米直径的）扬声器碱基加盖其中四个到的两个固定铝条（40厘米×10厘米×2.5厘米）1。通过在铝条的侧将螺钉插入各圆孔稳定各扬声器。 </li><li>单独由一个扬声器选择器控制扬声器。通过连接线连接到扬声器主体的正极和负极输入端，然后在选择相应的输出各个扬声器连接到选择器。扬声器选择器允许从t信号他函数发生器，通过功率放大器后，可以一次（ 图2E）到达所有8个扬声器。 </li><li>将两个腔室阵列，扬声器选择器和相关的电子设备中的抗湿度外壳。房子整个组件在商业细胞培养孵化器。 </li><li>输送主电缆（通过医用级PVC管），通过过滤器组件在所述培养箱的背面连接在功率放大器与扬声器选择。 </li></ol> 2，支架制备及表征<ol><li>溶解PCL粒料在氯仿中于15重量％的浓度。加载溶液到10ml注射器上铺21政钝端针。 </li><li>锁定在注射器上一个可编程注射泵和设置流速为1毫升/小时。 </li><li>从针穿过放置铝箔覆盖的集电极水平，具有针尖 - 集电极的距离〜18厘米。 </li><li>钳位正阿利鳄鱼夹，以将针的中间，和接地鳄鱼夹在铝集流体，然后将电压上的高电压电源在15千伏。注意:高电压，保持从针的距离。 </li><li>依次打开注射泵和电源;快速清洁/删除用干纸巾稳定纤维喷射之前围绕所述针的尖端处的残余聚合物溶液和泰勒锥19形成。 </li><li>让纤维积聚在铝集流体的厚度为〜250-300微米（〜当前纺丝条件下7小时）。储存在真空干燥器中得到的支架1-2天以除去任何残留溶剂。 </li><li>图像中的支架，溅镀上金，用扫描电子显微镜显示一致的纤维形态。10 </li></ol> 3，生物反应器组装与表征<ol><li>冲一圆盘（直径:8毫米），四臂（长度:2毫米）出来的初纺PCL垫（ 图2A）由第一使用12毫米直径的活检穿孔器切割圆盘的外直径。然后使用第二个，8毫米活检穿孔，使均匀地分布在圆刀片将比分那里的武器将被切断4个2毫米长的切口。后与8​​毫米冲得分，用手术刀刀片切割臂的边缘向外。将支架放入硅胶膜通过扩展武器槽（视频1）。用平头镊子轻轻按压表面变平的插入支架。 </li><li>附上一小片薄铝箔（8毫米×2毫米，垂直形状， 图2B）到PCL支架来辅助激光反射。 </li><li>固定组装硅油膜/ PCL支架（如在步骤1.4中详述）中的振动室。加入1.5毫升的水在室内，以振动之前滋润PCL支架。 </li><li>使用函数发生器，引入振动标志ALS（ 例如，200赫兹的正弦波的峰值-峰值电压0.1 V的，VPP），以夹心丙烯酸室。使用电压表来精确地测量电压，每个扬声器的输入。注:从函数发生器的Vpp的读数将不同于最终电压传送到扬声器。 </li><li>组装单点LDV并固定光纤激光传感器头的云台的三脚架。角度传感器头，使得它指向垂直于桌面。通过同轴电缆连接LDV传感器头数据采集模块，然后通过USB模块的笔记本电脑。 </li><li>将激光束聚焦在垂直于硅膜（ 图2B和图3），各预定点。 </li><li>使用数据采集软件，记录中间膜的位移。请从"选项"菜单中的"采集设置";然后改变测量模式为"FFT221;接下来，单击主工具栏中的"连续测量"，然后点击形成在所选择的频率（ 图6D）来记录位移的峰值。 </li><li>绘制正常中期膜的位移（瓦特0）作为横跨基底的相对位置的函数。构建使用地8.5数据分析软件的表面振动轮廓的三维色彩映射表。 </li></ol> 4，振动细胞培养<ol><li>子培养人骨髓来源的MSC在T150组织培养瓶在4,000-5,000细胞/ cm 2在MSC维持培养基的初始接种密度。 </li><li>经过7-8天的细胞培养物（以〜85％汇合），胰蛋白酶消化细胞用细胞解离试剂如的Accutase，计数使用血细胞计数，离心（440×g离心5分钟），并在重悬细胞沉淀新鲜的MSC维持培养基以4.5×10 6细胞/ ml的浓度。 </li><li>沉浸在PCL scaffoLD在70％乙醇的O / N。之后将溶剂蒸发，暴露支架的两侧，以杀菌紫外灯（254纳米）为5-8分钟。 </li><li>浸泡在PCL支架在20微克/毫升纤维连接蛋白溶液在37℃1小时。将纤连蛋白包被的支架插入硅氧烷膜。汇编为在步骤1中所详述的生物反应器。 </li><li>分发40微升的细胞悬液均匀地涂在有抵押的PCL支架。使细胞增加一个额外的1.5毫升新鲜传媒振动腔前1-1.5小时重视。 </li><li>文化海安载货PCL支架体外静态3天，静态培养完成后刷新媒体。 </li><li>选定施加振动政权的蜂窝结构。注:作为一个例子，细胞经受在200赫兹1小时的按1小时-解除（OF）振动与〜40微米AW 0每天12小时为7天。结构受到震动刺激被指定为振样品和那些丘尔红色静态在相同的振动室作为静态控件（统计）。 </li></ol> 5，生物评价<ol><li>从每一个腔室收集200微升的细胞培养基每隔一天（第1天，3，5和7），并从同一个样品池的等分试样一起（每800微升）。 </li><li>量化基质金属蛋白酶1（MMP1）和HA使用MMP1 ELISA开发工具包和透明质酸竞争ELISA试剂盒，分别按照制造商的过程的细胞产量。同时，吸附法生产可溶性弹性蛋白前体继之前报道的ELISA方法。8 </li><li>当最后一个振动周期在7天结束后，迅速取出从振动室使用尖锐镊子蜂窝结构，并简要冷磷酸盐缓冲液（PBS，4℃）冲洗。 </li><li>对于活/死染色，孵化用碘化丙啶（在PBS 1:2,000）的结构和SYTO-13（在PBS中1:1000）同时进行5分钟，在RT。图像染色结构与多光子共聚焦显微镜。 </li><li>另外，嵌入式冻结干冰的PBS漂洗细胞结构和提取继先前报道的协议进行基因分析细胞总RNA。9 </li><li>验证使用紫外可见分光光度计提取的RNA的数量和质量。 RNA样品与1.8-2.2 A260/A280和A260/A230比值被用于随后的定量PCR分析。 </li><li>反转录的RNA（500毫微克/样品）插入使用市售的反转录试剂盒的cDNA。 </li><li>执行上使用市售的PCR主混合物继先前详细过程的实时序列检测系统进行PCR反应。8 </li><li>使用商业定量PCR数据分析软件分析定量PCR结果。为了保证数据分析的可靠性，多参考基因（YWHAZ，磷酸三丁酯，PPIA）被用作整型ernal控件和特异性引物效率的差异考虑在内。8 </li></ol>

<ol>
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K., Webb, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering+Therapies+for+the+Vocal+Fold+Lamina+Propria.">Tissue Engineering Therapies for the Vocal Fold Lamina Propria.</a> Tissue Eng. Part B: Rev. 15, 249-262 (2009).</li></ol>

没有竞争的经济利益存在。

功能性声带组织在体外成功的工程需要声带般的微环境介导多能细胞的行为的娱乐。人们普遍认为组织或器官的结构反映他们需要执行的功能。对于22声带组织，提出发声过程中发生的高频振动来对组织成熟的重要。在我们的研究中，PCL支架是用来提供一个带样结构支撑而声带生物反应器被设计成引入生理学相关的机械信号到培养的干细胞。这里描述的（J2）的生物反应器，?...

人类的声带，上皮层构成，固有层（LP）和声带肌肉，是一种将来自肺部的气流进入的声波产生声音的专门的软组织。1声带定期正常发声期间振荡，表现出高达30％的菌株在基本频率范围从100至300赫兹。2成人声带唱片是肤浅（SLP）组成的梯度结构，中间（ILP）和深（DLP）层。进一步分类组的上皮和SLP为粘膜层，并结合ILP和DLP到声韧带。3 SLP层主要包含一种无定形矩阵稀疏分散的胶原纤维，而韧带富含成熟的胶原蛋白和弹性纤维提供足够的强度。4的结构和新生儿声带力学从他们成熟的同行有所不同显著。虽然机制•调控声带发育和成熟都还没有完全理解，实验证据指出，以发声来源的机械应力的决定性作用。 几个医疗条件，包括滥用嗓音，感染，化学刺激和手术过程中，可能会损坏声带。声带疾病影响美国人口估计有3-9％。目前的治疗方法声带疾病是有限的5和基于干细胞的组织工程方法已经成为一种很有前途的战略恢复声带功能。间充质干细胞（MSCs）是一个合适的替代品主要声带成纤维细胞的声带组织工程。6-9干细胞的命运规范和随后的组织发展是由它们驻留在特定的利基，其中的机械条件是介个至关重要的因素。10机械力的组织形态发生一个重要的监管机构第二平衡，特别是对于那些定期进行装载11从组织工程的角度来看的组织，它已被证明，暴露于生理相关的机械刺激促进干细胞分化和组织特异性基质重塑。12-15 组织培养物的生物反应器被设计来模拟所需的生理环境，细胞或组织生长的体外 。对声带组织工程，这是特别重要的，以重新创建phonating声带的机械环境。一个理想的声带生物反应器应有效地提供振动提示来培养细胞，让轻便的控制频率，振幅和振动持续时间。 Titze和同事设计了一种声带生物反应器（T1生物反应器 ）16，结合了静态拉伸与高频（20-200赫兹）振动来刺激基质蛋白的细胞产量。使用设置克该生物反应器，Webb和同事研究了17的10天，100赫兹的振动对皮肤成纤维细胞中透明质酸（HA）为基础的水凝胶培养的影响。构建体进行振动表现出升高的表达HA合酶-2（HAS2），核心蛋白聚糖，纤维调节和基质金属蛋白酶1（MMP1），相对于静态控件。被发现的刺激作用是时间依赖性的。最近，使用了功率放大器，一个函数发生器，一个封闭的扬声器和一个沿圆周锚硅油膜，该振荡空气传送到贴壁细胞我们组18组装声带生物反应器（J1生物反应器 ）。培养在生物反应器中的J1新生儿包皮成纤维细胞，进行1小时的振动，在60，110或300赫兹，带有一个在平面应变高达0.05％。的定量PCR结果显示，一些细胞外基质基因的表达被适当地改变以响应变化的振动频率和幅度。 这些生物反应器的设计，而耐人寻味，有一些局限性。例如，在T1的系统需要大量的连接器和棒的机械耦合的，从而限制了最大频率达到的。此外，细胞可以经受不期望的机械搅拌和流体扰动变得复杂的数据解释。 J1的生物反应器，在另一方面，具有相对较低的能量转换效率，而不是用户友好。此外，频繁的振动分离从底层硅油膜细胞载货结构。这里报道的J2声带生物反应器，基于相同的原理为J1版本设计的，被优化的一致性和可重复性。在空气动力学中单独装配的振动室，其中的MSC-纤维状填充的聚（ε-己内酯）（PCL）支架是effectiv产生的发声-模拟振动伊利抵押。激光多普勒Vibrometry（LDV）允许用户以验证膜/支架组件的振动模式。在我们的演示中，干细胞暴露在200 Hz的正弦振动与1小时的按1小时 - 解除（OF）模式，共12小时，每天的7天。细胞反应所施加的振动线索进行了系统研究。总体而言，J2声带提供了最人性化的功能，允许动态细胞培养的研究，高通量和可重复的方式进行。

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="16">
			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

construction and characterization of a novel vocal fold bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

通过静电纺丝制备的PCL支架包含微米尺寸的空隙孔和具有4.7微米（ 图4A）的平均直径的随机缠结纤维。在更高的放大倍率，纳米级凹槽和孔是在单根纤维（ 图4B）可见。与纤连蛋白支架的涂层提高了亲水性，有利于初始细胞粘附/在PCL支架（未发表的观察）传播。 具有期望的频率（f，100-300赫兹）和电压正弦的波形（VPP:0-0.125 V）被引入到每个振...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

一种新型声带生物反应器的构建与鉴定

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Research

JoVE Journal

Bioengineering

9.1K 次观看.  University of Delaware. A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(ε-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

视频: Construction and Characterization of a Novel Vocal Fold Bioreactor

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

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

合成，多层，自激振荡的声带模型制作

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

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  ...

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 ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

非人灵长类龙脱细胞和Recellularization使用专门的大型器官生物反应器

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

研究模型声乐折叠聚合物引起的三维流量分离

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

白细胞 - 富血小板纤维蛋白，一种新型生物材料的表征

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

用于动态生长测试的温度，光照和pH监测的台式藻类光合生物反应器的构建和设置

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

用于神经生长因子包封的新型HDL模拟纳米颗粒的制备和表征

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...