The authors have no acknowledgements.

1.聚氨酯（PU）海绵准备为模板<ol><li>使用聚氨酯海绵以产生含有相互连接的孔的羟基磷灰石的模板。使用每个海绵，以提供主小梁用于形成模板支柱以及小梁内形成微通道。 </li><li>切割和修剪80 PPI（每英寸的孔隙）海绵成2桥的形状为3.5厘米的高度×5厘米长×1.5厘米的宽度尺寸。 注意：尺寸和形状可以被选择，根据所希望的主孔径：100 ppi的，80 ppi的，至60ppi。 </li><li>使100毫升的4％（重量/体积）使用150毫升烧杯中的NaOH溶液;然后浸泡和挤压，直到准备好的海绵完全浸透。 </li><li>浸泡后，将与在超声波发生器（42 kHz）的海绵烧杯中。 </li><li>超声波预治疗聚氨酯海绵15-20分钟无热修改表面性质。 </li><li>用蒸馏水冲洗水为5-10分钟。而漂洗，挤压海绵，并允许他们扩大5至7倍，以除去残留的NaOH的海绵内部。 </li><li>挤用纸巾将海绵以除去过量的水;然后在60-80℃下干燥后在炉中。 </li></ol> 2.羟基磷灰石（HA）浆料制备的涂料<ol><li>之前使得HA浆料，测量一个烧杯的重量有磁性搅拌棒。该测量将被用于计算粉末/液体比。 </li><li>测量将10g纳米尺寸的HA粉末。 </li><li>加入20毫升蒸馏水到50ml烧杯中。热为120-140℃，并搅拌用热板磁力搅拌器。 </li><li>添加0.3克每粉末聚乙烯醇（PVA）（89,000-98,000兆瓦）的（3％w / w的）入蒸馏水在300-400转，同时搅拌。 </li><li>搅拌，直到将PVA完全溶解。该解决方案应该是PVA完全溶解后，明确的。 </li><li>打开ØFF的热量，并添加0.1克羧甲基纤维素钠（CMC）（超低粘度）为粉末（1％w / w的）在400-500转，同时搅拌。该解决方案应该是PVA完全溶解后，明确的。 </li><li>搅拌，直至CMC完全溶解并冷却到室温。 </li><li>添加0.3克每粉末聚丙烯酸铵分散剂（3％重量/重量）在300-400转，同时搅拌。搅拌至完全溶解。 </li><li>添加0.2克每粉末甘油（2％w / w的）在300-400转，同时搅拌。搅拌至完全溶解。 </li><li>慢慢分散的HA粉末进入溶液在600至900的转速搅拌的同时，保持搅拌5分钟。 </li><li>使用超声发生器，以确保的HA粉末中的任何结块分散声处理5分钟。 </li><li>添加额外的5毫升蒸馏水加入混合物在600至900转和热搅拌的同时在90-100℃。 </li><li>保持搅拌用磁力搅拌器将混合物在600-800转在90-100℃在奥德呃以蒸发水含量。 </li><li>测量整机重量包括时间烧杯和混合物时获得的1.75-1.8粉/液比，直到。 </li><li>格式化粉末/液体比率，通过将混合物（2.14）的总重量除以粉末重量，包括粉末，试剂，和水，减去烧杯和搅拌器（2.1）的重量，并减去的HA粉末（2.2）。 注：例如：如果A（全部混合物包括粉末，试剂和水）49.05克，B（烧杯中搅拌）33.5克，然后C（HA粉）为10g。 C /（ABC）= 10 /（49.05-33.5-10）= 1.80 </li><li>允许浆料使用用于涂覆前冷却至室温。 </li></ol> 3. HA涂层，烘干和烧结<ol><li>涂层的制备的聚氨酯海绵与使用不锈钢刮铲，直到浆料中的HA涂层的浆液均匀地在整个聚氨酯海绵分布在玻璃板上。 注：除去多余的污点后RY，一些毛孔可能仍与浆因为高浆料粘度堵塞。 </li><li>为了保证互联互通，统一，开放的毛孔，稍微吹用空气压缩机的HA涂层的模板。这一过程可确保模板在PU海绵的内表面和外表面上均匀地涂布两者。 注意：如果一个均匀的涂层是没有实现中，HA涂覆的模板将在烧结过程中塌陷并也可能破裂而由于机械强度低的处理。此外，均匀的涂布是在小梁内产生微通道的关键。 </li><li>干燥的HA涂层模板最少的冷却条件下5小时（20-25℃），轻轻空气流通。然而，延长基于模板的大小的干燥时间。 注意：干燥后，将HA涂覆模板将通常收缩约8％至10％，在每个维度。 </li><li>干燥过程后，将HA在氧化铝坩埚涂覆模板。然后，将它们放置在高温炉，并使用以下8步烧结轮廓。 <ol><li>热火2℃/分钟至230℃。 </li><li>热火1℃/分钟至280℃。 </li><li>热0.5℃/分钟至400℃。 </li><li>热火3℃/分钟至600℃。保持在600℃下1小时。 </li><li>热火5℃/分钟至1230℃。保持1230℃下3小时。 </li><li>凉爽5℃/分钟至室温。 注：烧结将进一步大约22％收缩HA涂层海绵模板 - 25％，在每个维度。 </li></ol></li></ol> 4.入口细胞向模板和现代人居<ol><li>培养前成骨细胞MC3T3细胞在非骨培养基组成的α-MEM的，在37℃下在含有5％ 的 CO 2的潮湿气氛中补充有10％胎牛血清（FBS）和1％抗生素（链霉素和青霉素） 。 </li><li>加入10 mL的细胞悬浮液以2×10 6个细胞密度为单个孔的6孔平板内。 </li><li>放置3厘米×4厘米×1厘米桥形模板垂直插入6孔板。放置模板到含有细胞悬浮液的板，以及另一条腿的一条腿进入相邻的空孔。 </li><li>允许模板吸收细胞悬浮液10分钟。 </li><li>添加5毫升媒体此后最初填充有细胞悬浮液的孔。 </li><li>补充介质在这两个孔中，每2或3天，直到7日子已经过去了。 </li><li>通过苏木精和伊红染色11确定细胞移动性的功效。 <ol><li>通过浸渍于100％乙醇20-30分钟固定细胞和支架材料。 </li><li>染色用苏木1-2分钟。 </li><li>用蒸馏水冲洗1-2分钟两次。 </li><li>通过浸没在70％的脱水，然后95％，然后100％EtOH中的每个1-2分钟。 </li><li>染色与曙红20-30秒。 </li><li>用蒸馏水冲洗12分钟两次。 </li><li>通过浸渍在70％的脱水，80％，90％和100％的乙醇对每个1-2分钟。 </li><li>嵌入在丙烯酸树脂的支架切片和成像。 </li></ol></li><li>确定与3- [4,5-二甲基-2-基] -2,5-二苯基四唑溴化物（MTT）细胞活性分析和活/死测定法（活/死细胞染色试剂盒MPTP）的时间点的细胞存活率的3天和第7天11。 注：骨样模板制作协议，该计划的代表在“代表性的成果”会议。 </li></ol>

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The authors have nothing to disclose.

需要成功的骨再生和临界尺寸的大的骨缺损的功能恢复的多分量模板包括细胞，生长因子，营养素等等。在这些因素的影响，解剖学符合要求的生物学特性是必不可少的。为了完成生物功能性，模板必须表现出生物相容性，骨传导性，机械完整性，足够的表面积，足够的表面纹理，且所述用于氧气和养分运输。在细胞水平上，下面的功能是为大块骨缺损功能恢复特别重要：促进渗透到模板（主动招聘），整个模板（保留）均匀分布，加速扩散，高生存力（居住）。最后，随后形成大量细胞外基质的和基因表达的触发是在基本生物过程的关键，如快速血管一次成骨。 许多不同类型的合成替代品已被建议更换自动/异体骨移植物。然而，目前的支架的组织没有表现出含微通道和纳米孔的内部微环境，因此不积极地促进细胞浸润，分布和栖居深入到合成替代品是大于10mm。它们不用于开拓细胞有效地，迅速地，且均匀地深深迁移到骨模板提供物理提示。取而代之的是，有限的被动招募细胞产生所述支架的外部和内部区域之间的分布不均匀的细胞群。这不仅加剧到达模板的内芯的单元的最初的挑战，而且阻碍营养素流和与合成的替代的另一端小区通信。这在小区D型不成比例细胞募集和居住结果该支架后eath和不完全的骨生长已经植入到体内14,15。 因此，我们推出了毛细作用的概念作为主要的物理线索，以解决这些障碍。我们已经彻底改造的微通道，在骨髓移植诱导毛细作用，这将占到主拖力的负责任的积极招募细胞深入到骨髓移植。 该PU海绵涂技术提出了一些独特的性质。首先，它允许容易地制备良好控制的多孔骨小梁结构的，它们本身依赖于预定义的模板结构（即，80孔每英寸模板300-400微米）。这对于优化孔径为成骨细胞的浸润15非常重要的。第二，该技术可使互连微通道，占初始化细胞动迁11的显著作用的结构。第三，在创建自定义形状和模板尺寸方面使用聚氨酯海绵的时候几乎没有任何限制。制造商可以用剪刀进行简单的形状，甚至计算激光切割的复杂几何形状。使用这些精确控制技术，我们创建了骨髓移植。 HA被选择，因为它的生物相容性和骨传导容量17作为原料。 在这项研究中，有一些需要强调的几个关键步骤。在HA浆料制备，如果温度太高，搅拌速度太低，则HA浆料将变得卡住在烧杯底部边缘和干涸。吹出过量浆料的HA当涂布过程之后，过高的空气压的可诱导的骨髓移植的表面​​上的裂纹。重要的是要保持空气的压力相对较低，只适当地空出多余的HA浆是很重要的。最后，在第二和第三步骤的烧结过程最关键的（热火1℃/分钟至280℃，加热0.5℃/分钟至400°C）。在这个温度范围内，PU海绵将彻底烧毁而房委会变得密集。如果该协议不紧紧跟随，在骨髓移植将被折叠或烧结后崩溃。 在这项研究中所描述的骨髓移植提供了几个优点。首先，相互连接的大孔（300-400微米）模仿这些人的骨小梁和可实现平滑的骨髓流。第二，模板是由每个小梁隔垫内的微通道（25-50微米）的加速骨细胞通过毛细作用的初始入口。作为使用计算机模拟13，如果模板只有300微米的孔（原生孔隙），并没有显示出微通道，毛细作用将不足以用于与骨髓模板的完全饱和。这将特别举办如此大型的缺陷，将要求采用信用证阿尔赫尺寸模板。微米尺寸的信道表现出高度有效的流体吸收，因此，我们预期的微通道是主要负责在我们的研究中的毛细作用。第三，我们的BMTs有战略地位的纳米孔。从文献数据表明，细胞对纳米图案18,19特别敏感;因此，我们预期的微通道的壁上的纳米孔隙中发挥增加细胞附着的作用。纳米尺寸的孔（100-400纳米）的小梁隔片的表面上允许的固定化细胞以固定。总体上，这三个内部结构的联合作用导致增强的细胞的动员和粘附在整个模板。但是，也有该协议的一些限制和制作完美BMT的关键步骤。例如，存在往往是由于准备保持而涂层的均匀粘度的难度大的量的HA的淤浆。也有在使限制由于工作时间，而涂层的模板大于5 立方厘米的体积。根据制造商的技术而变化，涂层厚度是至关重要的。 我们的研究结果表明，能够吸收并保持细胞将提供比传统的异质（或合成）支架的潜在优势BMT。前瞻性研究正在考虑到随着骨相关生长因子验证BMT对成骨和/或血管发生的好处。因此，我们主张我们独特功能的BMT支架可以解决骨质不足骨髓浸润的主要障碍到合成结构和不完整的骨组织再生大的缺陷。 这项研究的最终目的是通过消除需要时间/劳动密集型的骨髓基质简化生物工程在骨重建和功能恢复的临界尺寸的骨缺损的当前范例L细胞分离和扩增过程。最后，我们的目标是利用符合解剖学的3D构建体与微通道和纳米孔，从而诱发细胞快速吸收的，均匀的分布，和栖居重建骨。

The ultimate goal of bone tissue engineering with synthetic constructs is the incorporation of the constructs into the host bone, repopulation of the constructs with host cells, and reconstitution of gas and body fluid exchanges to restore normal bone function. Considerable research has been reported over the last decade in the use of polymeric and ceramic biomaterials for producing scaffolds1,2. However, the ideal material and fabrication technique for optimal bone tissue regeneration has yet to be identified. In addition, there is an overall lack of success in bringing these technologies to the clinic, especially for the reconstruction and restoration of large bone defects. Therefore, restoring critical sized bony defects still remains a clinical challenge1-5. 
Ideally, the scaffolds for bone tissue regeneration should exhibit biocompatibility without causing inflammatory responses or foreign body/toxic reactions, have closely matched mechanical properties when compared to those of native bone, and possess a mechanism to allow diffusion and/or transport of ions and nutrients. Strong bonding with the host bone, dynamic bone growth, vascular ingrowth, and biodegradation of the scaffolds are equally desirable. Although the use of biodegradable polymer scaffolds has exhibited progress in terms of tissue ingrowth, there are controversies over their use for bone regeneration. 
Notwithstanding these extensive efforts, the highly organized structural synthetic constructs still have limited potential in overcoming the obstacle of passive cell penetration. Most of these approaches have resulted in the in vitro tissue ingrowth with cross-sections of less than a few &#181;m to several mm from the external surface, an incomplete integration with host bone, and only partial bone regeneration in vivo6,7. The pioneering cells do not migrate deeply into the constructs because of the lack of an initial force that pulls them inside before cell colonization begins. Consequently, cell colonization strictly occurs at the scaffold periphery, becoming an obstruction from the periphery to the center of the scaffold. Thus, the diffusion of oxygen and nutrients into the inner parts of the templates becomes limited8. Therefore, it is clear that the architecture of the scaffolds (pore size, porosity, interconnectivity, and permeability) that affect the transport and diffusion of substances throughout the scaffolds is critical for achieving well-distributed cell proliferation and differentiation9,10. Although calcium phosphates have been used in the past for scaffold fabrication, different processes and procedures have often resulted in calcium phosphate scaffolds with varying architectures. Thus, the selection of the manufacturing process becomes important in dictating the scaffold architecture needed for successful bone tissue regeneration.
In conclusion, there are still two major shortcomings of bone tissue engineering that need to be addressed: the initial cell recruitment into the template prior to cell attachment and colonization and the quality of substance flow both into and out of the template.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>polyurethan sponge</td>
			<td>Plastifoam</td>
			<td>PU-3215</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td align="right">167176</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyapatite Powder</td>
			<td>Ossgen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl Alcoho</td>
			<td>Sigma-Aldrich</td>
			<td align="right">341584</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethyl cellulose sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td align="right">360384</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium polyacrylate</td>
			<td>Vanderbilt</td>
			<td>DARVAN 821A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin</td>
			<td>Sigma-Aldrich</td>
			<td>G2289</td>
			<td></td>
		</tr>
	</tbody>
</table>

distinctive capillary action by micro-channels in bone-like templates can enhance recruitment of cells for restoration of large bony defect

时不会有良好的分布，并稳定地锚定在插入模板的活性，蓬勃发展的细胞群，呈骨再生不会发生。与传统的模板，由于没有内部微通道导致缺乏细胞浸润，分布和栖居模板深处。因此，高度多孔且均匀地相互连接的小梁骨状模板与微通道（生物微模板; BMT）已经开发来解决这些障碍。小说BMT通过创新的概念（毛细作用）创建，并制造为具有海绵模板涂覆技术。在骨髓移植由几个结构部件：相互连接的初级孔（300-400微米），模仿毛孔骨小梁，每个小梁内的微通道（25-70微米），和纳米孔（100-400纳米）上的表面使细胞固定。此外，骨髓移植已被记录通过机械试验研究有SIM卡ILAR的机械强度性能，这些人骨小梁（〜3.8兆帕）12。 在骨髓移植显示出高的吸收，保留和细胞的居住整个桥形（Π）的模板（3厘米的高度和4厘米长）。该最初的细胞接种到的立即调动到另一端（10厘米的距离）由骨髓移植的细胞培养基毛细作用模板一端。 4小时后，将细胞均匀占领了整个BMT并表现出正常的细胞的行为。毛细作用占细胞悬浮在媒体和分布（活动迁移）在整个骨髓移植的渗透。在观察的骨髓移植的这些能力，我们预计BMTs将吸收的骨髓细胞，生长因子，营养物和从生理条件下的外围。 在骨髓移植可以通过快速渗透，均匀分布和inhabita解决目前的局限性细胞大，体积模板NCE修复大量的骨骼缺陷。

在骨髓移植的整体结构表现出独特的三维模板小梁骨状的内部结构。在BMT含有大孔，微通道，和纳米孔。的完全互连的大孔清晰配置（320微米平均大小），微通道（50微米平均粒径），和纳米孔（100纳米平均粒径）用扫描电子显微镜进行了验证（EVO-40; ZEISS）以及通过微断层摄影术。 图1示出了在创建BMT逐步详细协议。通过从聚氨酯海绵到烧结过程（P1 - P7;图1）的制备方法的协议的精确控制，以下功能可实现：一个高密度和光滑表面HA涂层和干燥后;精确的形状和尺寸的3-D模板;完全互连的多孔小梁网络类似于骨小梁;和微通道机智欣每个模拟内部骨渠道，如哈弗运河和福克曼的运河（ 图2＆3）小梁。此外，相对高的机械强度（〜3.8兆帕）类似于人骨小梁遭到了抗压强度试验测量。与这些人的腰椎骨小梁高度相似组织形态学参数是通过显微CT分析12证实。的毛细作用不同量值通过不同直径的毛细管使用计算机模拟进行了论证于图4。通过这些模拟，我们预计，BMT将初级孔（300-400微米）并基于所述直径的微通道（25-70微米）内表现出不同的吸收率。较小的毛细管表现出较强的吸收能力。这个假设在这个实验证实，如图5。 在骨髓移植展出高效通过微通道结构的毛细作用流体吸收和保留; stevenel的蓝染色用作流体介质可以轻松地跟踪流（图5）。基于计算模拟中，BMT与这些配置被视为吸收和保留细胞悬浮液高达8.5厘米的总距离在10秒内。由于诱发的内部结构一强毛细作用，被染色的介质到达一个3厘米（高度）的相反端×4厘米（长度）×1分钟内，40秒1厘米（宽度）桥形模板。此外，活性细胞动员和掺入BMT观察（图6）。随后，同源细胞动员和附着导致增强的增殖和基质形成一个均匀分布的形成。而且，通过骨髓移植的细胞的长距离（〜10厘米）的迁移被立即验证后的骨髓移植是饱和与细胞悬浮液。硒<html> EDED细胞在模板段被暴露在空气中，而不是浸没在培养基中存活。在该实验中，将培养基中的细胞的孔中接触支架只有腿提供的独占。由微通道呈现的毛细管作用，然后使其为新鲜培养基，以达到该支架的顶部，桥部。培养3天后，模板变得忙于快速增殖细胞。经过7天 ​​的培养，每个小梁包裹由细胞外基质和嵌入单元13。 <img alt="图1" src="/files/ftp_upload/52947/52947fig1.jpg" /> 图1.从预处理的聚氨酯海绵（P1）到最终热处理（P7）的总的骨样模板制造协议。保持精确烧结轮廓后P7是在实现良好的机械强度是至关重要的。F =“htt​​ps://www.jove.com/files/ftp_upload/52947/52947fig1large.jpg”目标=“_空白”&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/52947/52947fig2.jpg" /> 图2代表立体显微镜;一个80 PPI的（AmScope SM-2TZ-M）的图像（4个）的尺寸聚氨酯海绵（左），HA涂覆并干燥BMT（中），和烧结BMT（右）（尺寸：3。身高×4厘米长×1厘米宽）厘米。 <a href="https://www.jove.com/files/ftp_upload/52947/52947fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52947/52947fig3.jpg" /> 一个生物模板图3.扫描电子显微镜和显微CT图像：（A）一个生物模板的整体形象， <st<html>荣&gt;（B，C，D）图像的微通道。为了突出明确的微通道，在小梁，模板进行造粒。 <a href="https://www.jove.com/files/ftp_upload/52947/52947fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52947/52947fig4.jpg" /> 图的毛细管作用与不同的信道直径4.计算的计算在同一时间段（0.4毫秒），而最大的毛细管（D = 300微米：指伯孔隙）吸收的媒体（蓝）至0.16毫米高度，最小的毛细血管（D = 30微米：指的是微信）吸收高达0.415毫米的高度网上平台。 <a href="https://www.jove.com/files/ftp_upload/52947/52947fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> NT“FO：保持together.within页=”总是“&gt; <img alt="图5" src="/files/ftp_upload/52947/52947fig5.jpg" /> 图5.显示图像的毛细作用基于不同尺寸的初级气孔和微通道（初级孔径指的是平均直径的吸收能力的差异：60 ppi的≈470微米，80 ppi的≈320微米，100 ppi的≈ 200微米）。黄线表示诱导初级孔隙和微通道的组合的毛细作用。红色线表示诱发主要是微通道呈现在每个小梁毛细作用。如图（F）中，100 ppi的模板诱发最强的毛细管作用，从而导致在该模板的内39秒的完全饱和。 80生产者价格指数和生产者价格指数60模板，然后进行了测试。 （B）的0秒，（C）的0.5秒，（D）的1.5秒​​，（E）的17.0秒，（F）的39.0秒，（G），50.0秒，和（H）1分钟浸渍后18秒。 （模板尺寸：1厘米×1厘米×4厘米的高度立方）。 <a href="https://www.jove.com/files/ftp_upload/52947/52947fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52947/52947fig6.jpg" /> 图6.初始接种细胞达到非种子选手腿末端的入口和细胞的移民从种子孔（第一部分），以非种子选手的井（第五部分）通过诱导通过毛细作用的生物模板。（第五部分）后，立即完全饱和。后3天，将细胞的汇合是显而易见的整个模板。 7天后，时空的胶原基质的形成发生细胞群（H＆E染色）内。 （模板维数：3厘米的高度×4厘米的长度为x 1厘米宽）。 <a href="https://www.jove.com/files/ftp_upload/52947/52947fig6large.jpg" target="_blank">请CL</a>ICK此处查看该图的放大版本。

Watch this Scientific Journal Video about Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect at JoVE.com

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

独特的毛细管作用通过微通道骨般的模板可以增强细胞的招募大骨缺损修复

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

distinctive-capillary-action-micro-channels-bone-like-templates-can

Research

JoVE Journal

Bioengineering

9.6K 次观看.  Columbia University. A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels. 

视频: Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

在球体哺乳动物细胞大规模扩张营养调控连续通

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

同时进行机电刺激增强细胞心肌成功能的潜能

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...