作者要感谢巴奥米特3i的为他们继续提供财政支持，尤其是兰迪·古德曼在定制部件的设计和制造的帮助。斯宾塞贝尔是一个收件人的产业研究生奖学金，由国家科学院和加拿大工程研究理事会（NSERC）提供。我们还要感谢John Brunski博士的手稿准备过程中非常有价值的反馈。

1。植入物的设计，制造及表面处理<ol><li>从工业纯钛（CPTI）;（长x宽x高尺寸4毫米×2.5毫米×1.3毫米）制造长方形的植入物。钻一个孔中央下来的植入物（直径为0.7mm）的长轴手术部位和随后的机械测试（ 图1）内以便尽早植入物的稳定性。 </li><li>处理所述植入物的上表面和下表面。 <ol><li>要创建两个不同的面，使用标准的喷砂（GB）的治疗，以创建一个microtopographically复杂的表面。通过叠加磷酸钙（CAP）进一步修改一半的植入物的纳米粒子来创建一个nanotopographically复杂的表面。 </li></ol></li></ol>注意:不同的化学或机械处理可以应用到产生所需的表面形貌和/或化学物质，这些将取决于实验questi的性质上加以解决。在本文提供的例子中，一组纯钛（CPTI）种植体经喷砂（GB） - 扣除方法 - 创建一个复杂的微观形貌。植入物的一半，然后再通过添加磷酸钙（CAP）纳米晶体的修改，以创建一个超级施加纳米形貌（GB-DCD）。 注意:当观看喷砂微面相比，改性后的纳米表面，在10,000倍的放大倍率，有在表面特性无明显差异。然而，在100,000 X放大率观察时，差异变得非常明显（ 图2）。先前已表明，这样的表面变化不会对骨传导14产生深远的影响。 2。动物模型和手术过程<ol><li>用年轻的雄性Wistar大鼠（200-250 g）关于这种模式。所有的程序必须由当地动物保健COM批准mittees。让动物自由饮水和大鼠饲料。 注:Wistar大鼠被选定为这个过程由于与这株鼠以往的经验，虽然大鼠的其他菌株可以采用。获取食物和水可以被改变，这取决于正在解决实验问题的性质。 </li> <li>采用吸入麻醉通过鼻锥稳重给予大鼠:1路2 /分钟的诱导4％异氟醚，在1升一氧化二氮和0.6 LO 2 /分钟的维修2％异氟醚。开展标准的脚趾捏测试，以确保有效镇静的程序，然后再继续。 </li><li>施用镇痛前和手术后通过皮下注射0.01-0.05毫克/公斤丁丙诺啡。 </li><li>通过部分随机分配植入物和放置在双侧股骨大鼠的远端干骺端。这使得不同的植入物，分别从选择不同表面形貌的被比较D，在对侧股骨，优化统计分析。 </li><li>剃须和清洁每个后腿用10％聚维酮碘的安特罗外侧方面准备的动物。为了防止低温，放置一个温水循环垫麻醉大鼠下方。 </li><li>用＃15的外科手术刀，通过沿大腿露出肌肉的外侧面的皮肤做一个切口。使用钝性分离偏转肌肉组织中一种微创的方式暴露股骨远端。 </li><li>刮掉薄薄的一层骨膜覆盖股骨，用骨膜，充分暴露骨皮质钻孔。必须小心，在钝器解剖和切除骨膜不要损坏膝关节的生长板或关节软骨。 </li><li>一旦清洁和检查，旋转股骨横向暴露股骨远端（ 图3A）的前方面。 </li><li>为了准备手术部位，钻双皮质矩形槽下骨的同时通过皮层中间。为了避免过热组织，生理盐水冲洗，必须保持整个钻探手术助理。进行钻孔在三个阶段: <ol><li>首先，钻透前皮层，通过解剖暴露的，使用连接到牙科手持件，以创建两个孔2.5毫米沿着中间的股骨线相距1.3毫米的牙科毛刺。 </li><li>接下来，使用第二个钻头（扭1.3毫米牙科毛刺），通过对方皮质这些孔扩大，导致双皮质平行孔。 </li><li>最后，加入用在近端-远端方向上的第三定制侧切削毛刺，形成现场为植入物（ 图3B）中的孔。 </li></ol></li><li>通过使用附加的针的骨缺损通过一个可生物降解的缝合线，并返回周围的股骨外皮质。 </li><li>主题植入物在缝合线的自由端并将其引导到缺陷，瓦特这里应该是压入式。在这种方式中，植入物的长轴应取向垂直于股骨（ 图3C）的长轴。 </li><li>配合围绕股骨外侧面的缝合手术后的恢复和愈合的早期阶段过程中提供植入物的稳定性。使用剩余的缝合关闭肌肉组织，并用手术钉（9毫米伤口剪辑）reoppose的皮肤组织。 </li><li>检查手术部位感染的迹象，和日常监测动物受损的动态能力。排除不完全恢复行走的动物，或者是那些股骨骨折的牺牲，从分析中。 </li></ol> 3。样品采集<ol><li>牺牲在动物后第9天，手术后由CO 2的曝光颈椎脱位。 </li><li>牺牲后，分离股骨和干净的软组织。在15％蔗糖缓冲溶液立即保存，以保持活体组织Hydration在准备机械测试（ 图4A）。 注:样品被存储在缓冲蔗糖溶液至设施之间的运输期间保持组织水合。标本将准备机械测试期间投入约2-3小时的解决方案。 </li> <li>以制备试样的力学性能测试，修剪骨到使用连接到一个高速系统的圆柱形金刚石毛刺的植入物的宽度。最终的试验样品包括骨的两个拱连接到植入物（ 图4B）的各个面。对于准备或运输过程中脱落的拱门，分配为0牛顿的力学性能测试值 注:修剪样品时要非常轻柔，准确，以免损坏或预应力接口是非常重要的。骨修复性愈伤组织可以围绕所述植入物的长轴和连到纵向孔生长。这样多余的骨骼必须由三被删除mming试样的矩形植入物的精确的尺寸，因为它可以歪斜力学测试的结果。 </li></ol> 4。机械性能测试自定义脱离模具的设计到锅内每个标本，建立准备样品力学性能试验的重复性和准确的方法。在设计允许种植体周围骨一致的测试区的0.5mm的区域的隔离，同时保持在制陶过程为中心的样品和完全水平的，直接垂直于种植体表面允许的的力​​的应用。参见图5为完整的工程图和图6为最终部件。 注:所有行为使用机械测试装置中，以30毫米/分钟的十字头速度操作的测试。对于剩余骨下列测试定性评估，在解剖显微镜可以使用。 <ol><li>灌封该标本和机械测试的<ol><li>从蔗糖缓冲液，轻轻吸干取出标本。 </li><li>位置标本在定制模具。水平地通过在模具的壁上的孔，并通过在植入物的中间的孔滑动销。放置在模具上的后侧稳定板，以稳定植入物（ 图7）。 </li><li>该模具的底部具有可流动的牙科用复合填充并使用高强度的固化的光固化60秒。 注意:选择不设置放热反应的合成，所产生的热量，例如可能影响组织性质是很重要的。 </li><li>固化后，打开模具，取出硬化标本块。画一条细细的黑线在永久性标记上用于识别目的的横向拱。 </li><li>固定在一个副试样的预制复制品，并居中机械测试仪的基座上的单元。 </li><li>使试样成副，并通过孔的植入（ 图8）通过一个尼龙线。附加的松散端向移动横梁的中央。为了保持一致性，始终标签和第一个测试的侧面。与内侧足弓重复上述过程。 </li></ol></li></ol>

<ol>
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作者收到的资金和物资支持，巴奥米特3i的（棕榈滩花园，佛罗里达州，美国）。巴奥米特3i的没有参与这个手稿或描述的实验设计的写作。

此处提供可进行试验力模型提供一种改进性方法评价骨锚地到候选人种植体表面的，容许进行精确垂线或是并联随着applied disruption力轴试样协调一致，骨折区极限到内植入物表面的半毫米。该模型是容易纳入比较的化学或机械方式，修饰的表面的任何范围的有效性的研究，但不适合于光滑表面，因为这些都是从骨标本处理过程中容易脱落。该植入物可以从范围广泛的生物材料来制造。数据很容易?...

评估骨锚固到骨内种植体表面一直相当关注的焦点，为此，许多机械测试方法已经被描述1,2。所有这些方法施加一个力，破坏正在使用的骨/种植体模型，大致可分为剪，一般表现为外推式或拉式模型3,4，反向扭矩3,5，和拉伸类型6， 7。通常在这样的测试中，无论是骨8或植入材料（在脆性玻璃和陶瓷9,10的情况下）被破碎，假设某种形式的锚固的发生，骨/植入物界面保持不变（至少部分地）保持不变。这样的实验的结果的意思是不仅使模型的断裂（或中断）所需的力是不分开的骨/植入物界面11,12所需的力，而且还使产生的裂缝平面内的复杂的表面积可以是难治精确的测量。然而，这种测试可以是临床相关的，因为它们提供的别共面的设计植入到被固定在骨能力的比较标尺。然而，还应当指出，这种比较是唯一有效的范围内的实验模型，而实验模型之间的比较是充满着困难，因为研究人员使用不同种类的动物表现出无论是片状或编织骨;小梁或皮质骨愈合模型和不同的机械测试几何形状和条件。 在努力获得的骨/植入物界面的拉伸强度的测定，许多研究者已经使用的植入物的公称表面积而得出一个"拉伸强度"值，因为抗张强度被测量为每单位面积上的力。这显然​​是给定的，如上面所解释的一个近似值，该骨/植入物界面在许多破坏试验采用保持完好编辑。除了 ​​测量植入物，特别是复杂的地形表面的表面区域中，由测量技术的分辨率由Ronald 等人 13但是所讨论的限制，通过Brunski 等 2，作为评论时的植入物的标称表面积是考虑到，在"抗拉强度"与不同的植入物表面的设计相关联的显着的差异被否定，表明植入物表面具有较高的表面面积提供更大的骨/植入物的接触面积，因此需要更大的力来破碎模型。因此，其含义是，地形更复杂的表面可以增加接触成骨，这会导致更大的骨植入物接触（BIC）和由此产生机械测试更高的中断值。接触成骨是两个不同的现象的产品:骨传导和骨形成。事实上，我们已经表明，增加了骨传导的形貌售货机，ically复杂的表面，可以通过测量所得到的BIC 14进行量化，并且这种表面也导致较高的机械破碎值12。 然而，这是有益的是要注意，种植体周围骨可以通过两种机制形成。在间充质起源的接触成骨细胞迁移到植入物表面（骨传导），分化成骨细胞，并阐述从头骨基质在植入物表面（骨形成）。阐述了第一骨基质是在正常骨重塑看到15矿化水泥线（有很多混乱有关该生物矿化结构，有时被认为是未矿化1或一种融合在骨16的所有接口的文献-有关此主题的完整讨论，请参阅戴维斯和侯赛尼17）。联系成骨是骨的现象的一个重要的先决条件接的，但不必要的是对于骨向内生长18。骨的矿化水泥线机械骨骼比19的矿化胶原舱弱。因此，直观地，如果水泥线矩阵与植入物的纳米特征的交错结合与骨组织生长到植入件的宏观特征相比较，然后破坏前所需的机械力会合理地，可以预料到小于后者，并且我们最近已证明了这一点实验12。 种植体周围骨也可以通过距离成骨形成。在这种情况下，骨沉积在旧骨表面并得到逐步接近导致包含无定形基质和成骨细胞20的遗体的接口的植入物表面。一般情况下，距离成骨与光滑，或加工，骨内种植体表面有关，常见于皮质骨的愈合，而microtopographicaLLY复杂曲面与骨的接触是比较典型的骨小梁愈合有关。使用光滑的植入物表面和皮质骨愈合拉伸试验模型已经能够测试此无定形生物基体与地形复杂表面相关联的接触成骨缺失的粘接性能，并且已经表明，发生所谓的"生物化学"接合提供了一个报告与地形复杂的曲面21的"拉伸强度"值较小的组成部分。与此相反，采用了小梁骨愈合模型中，Wong 等人 22表明植入物表面粗糙度和推出故障负载之间"良好的相关性"，并表示化学键合确实起到了骨锚的作用微乎其微到植入物表面。虽然有可能出现两个接触和距离成骨发生，在不同程度上，在所有的骨内围implaNT愈合车厢，microtopographically复杂的表面已经表明自己是特别有利的小梁骨性愈合舱23。后者在牙科文献24列为III级或IV级骨。 我们的目的是要着眼于接触成骨的机制和产生的骨/种植体支抗可在骨小梁愈合环境随之发生。这锚地，这是依赖于植入物表面的形貌（参见上文），可以发生在不同的尺度范围。一方面，只有亚微米植入物的特点有牵连的骨接合 - 所描述的骨水泥线矩阵与这样的表面的交错结合，并看到了生物活性玻璃，陶瓷和网状金属氧化物。另一方面，骨组织（有时完全与血液的血管）可发育成多微米，或宏观尺度，植入物的表面特征18。这两种情况下水库ULT在骨锚固到植入物表面的一种形式，虽然机制明显不同。然而，大多数的上面引用的机械测试方法的通病是使在一个完全垂直或平行的平面上的干扰力，以使植入物表面的（取决于拉伸或剪切模式是否被采用）。我们在此报告来克服这种限制的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Dulbecco&#8217;s Phosphate Buffer solution (DPBS)</td>
			<td >Gibco Life Technologies, Burlington, ON, Canada</td>
			<td >14190-250</td>
			<td ></td>
		</tr>
		<tr >
			<td >10% neutral buffered formalin solution</td>
			<td >Sigma-Aldrich Co. LLC., Canada</td>
			<td >HT501128-4L</td>
			<td></td>
		</tr>
		<tr >
			<td >Custom-designed rectangular implants (commercially pure titanium; dimensions: 4mm x 2.5mm x 1.3mm with a 0.7mm hole drilled centrally down the long axis)</td>
			<td >Biomet 3i, FL, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Custom-designed breakaway mould</td>
			<td >Biomet 3i, FL, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td >Baxter Internationl Inc.</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Buprenorphine</td>
			<td >Bedford Laboratories</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >10% betadine</td>
			<td >Bruce Medical, MA, US</td>
			<td >FR-2200-90</td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >2586-M36-0100</td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel blade #15 (sterile)</td>
			<td >Magna, Medstore, University of Toronto, Canada</td>
			<td >2586</td>
			<td></td>
		</tr>
		<tr >
			<td >Periosteal elevator #24G</td>
			<td >Spectrum Surgical, OH, USA</td>
			<td >EX7</td>
			<td></td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >7747-A10-108</td>
			<td></td>
		</tr>
		<tr >
			<td >Tissue forceps</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >7722-A10-308</td>
			<td></td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >Almedic, Medstore, University of Toronto</td>
			<td >7603-A8-240</td>
			<td></td>
		</tr>
		<tr >
			<td >Absorbant Fabric General Purpose Drape (sterile)</td>
			<td >Vitality Medical</td>
			<td >1089</td>
			<td></td>
		</tr>
		<tr >
			<td >Gauze (non-sterile)</td>
			<td >VWR</td>
			<td >89133-260</td>
			<td></td>
		</tr>
		<tr >
			<td >Needles 25G X 5/8&#34; (disposable)</td>
			<td >BD, Canada</td>
			<td >305122</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringes (sterile)</td>
			<td >VWR, Canada</td>
			<td >CABD309653</td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Driver</td>
			<td >Almedic, Medstore, University of Toronto, Canada</td>
			<td >A17-132</td>
			<td></td>
		</tr>
		<tr >
			<td >Dynarex Surgical gloves (sterile)</td>
			<td >Amazon.com</td>
			<td >2475</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgical masks</td>
			<td >Fisherbrand, Medstore, University of Toronto, Canada</td>
			<td >296360759</td>
			<td></td>
		</tr>
		<tr >
			<td >0.9% sterile saline</td>
			<td >House brand, Medstore, University of Toronto, Canada</td>
			<td >1011-L8001</td>
			<td></td>
		</tr>
		<tr >
			<td >Hair clippers</td>
			<td >Remington, US</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >4-0 Polysorb</td>
			<td >Syneture</td>
			<td >SL5627G</td>
			<td></td>
		</tr>
		<tr >
			<td >9mm Wound Clips</td>
			<td >Becton Dickinson, MD, USA</td>
			<td >427631</td>
			<td></td>
		</tr>
		<tr >
			<td >ImplantMED DU 900 and WS-75 dental hand piece&#160;</td>
			<td >W&#38;H Dentalwerk, Austria</td>
			<td >DU1000US</td>
			<td></td>
		</tr>
		<tr >
			<td >1.3 mm twist drill</td>
			<td >Brasseler, GA, USA</td>
			<td >203.21.013</td>
			<td></td>
		</tr>
		<tr >
			<td >1.3 mm dental burr&#160;</td>
			<td >Biomet 3i, FL, USA</td>
			<td >custom</td>
			<td></td>
		</tr>
		<tr >
			<td >1.2 mm cylindrical side-cutting burr</td>
			<td >Biomet 3i, FL, USA</td>
			<td >custom</td>
			<td></td>
		</tr>
		<tr >
			<td >Cylindrical diamond burr</td>
			<td >Brasseler, GA, USA</td>
			<td >H1.21.014</td>
			<td></td>
		</tr>
		<tr >
			<td rowspan="2" >High speed dental drilling system</td>
			<td >Handpiece: KaVo Dental Corporation, IL, USA</td>
			<td rowspan="2" >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Handpiece Control: DCI International, OR, USA</td>
			<td></td>
		</tr>
		<tr >
			<td >99.5% Ultra Pure sucrose</td>
			<td >BioShop Canada Inc., Burlington, ON, Canada</td>
			<td >57-50-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Flowable dental composite</td>
			<td >Filtek Supreme Ultra Flowable Restorative, 3M ESPE, St Paul, Minnesota, USA</td>
			<td >6033XW</td>
			<td></td>
		</tr>
		<tr >
			<td >Sapphire Plasma Arc high intensity curing light</td>
			<td >Den-Mat Holdings, Santa Maria, CA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Instron 4301 with 1000 N load cell</td>
			<td >Instron, Norwood, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td rowspan="2" >Red Wolf 10lb nylon &#64257;shing line</td>
			<td rowspan="2" >Canadian Tire, Canada</td>
			<td rowspan="2" >78-3610-6</td>
			<td></td>
		</tr>
		<tr >
			<td ></td>
		</tr>
		<tr >
			<td >Leica Wild M3Z Stereozoom dissecting microscope</td>
			<td >Leica, Heerbrugg, Switzerland</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >QImaging Micropublisher 5.0 RTV digital camera coupled with QCapture 2.90.1 acquisition software</td>
			<td >QImaging, Surrey, BC, Canada</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Electronic digital caliper&#160;</td>
			<td >Fred V. Fowler Company, Inc., Newton, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
		<tr >
			<td >Mechanical testing instrument</td>
			<td >Instron, Norwood, MA, USA</td>
			<td >N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

an improved mechanical testing method to assess bone-implant anchorage

在材料科学的最新进展已经导致大幅增加种植体表面地形的复杂性，无论是在微观和纳米级。作为描述种植体表面，例如，传统的方法-表面粗糙度，即数值决定因素-是不够的体内性能预测。生物力学测试提供了一个准确和比较平台来分析生物材料表面的性能。一种改进的机械测试方法测试骨锚固候选种植体表面呈现。该方法适用于愈合的早期和晚期阶段，并且可以被用于化学或机械改性的表面的​​任何范围 - 但不是光滑的表面。自定义的矩形植入物在雄性Wistar大鼠的远侧股骨双侧放置并与周围的骨收集。试样制作和使用一种新的分离模具和破坏盆栽测试是使用机械试验机进行的。这种方法精确地垂直或平行，以在植入物表面的平面中允许的干扰力的对准，并提供​​了用于分离一个确切的种植体周围区域进行检测的精确和可重复的方法。

所有的动物下手术的恢复随时间增加他们的活动能力。这很重要，因为负荷对不同的量程范围地形差异的影响，因为我们最近报道12。对试样以下机械试验的代表性力/位移曲线是在图9A，并为每个种植体表面的平均数据列于图9B。由每个试样达到最大力量值，记录和组值平均值进行比较（每组n = 28）。在GB-DCD的表面上，叠加在底层microtopographically复杂曲面亚微米地?...

Watch this Scientific Journal Video about An Improved Mechanical Testing Method to Assess Bone-implant Anchorage at JoVE.com

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

一种改进的方法以机械测试的骨锚定到候选种植体表面呈现。这种方法精确地垂直或平行，以在植入物表面的平面中允许的干扰力的对准，并提供​​了一​​个准确的方法，可直接进行干扰力为确切的种植体周围区域。

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

an-improved-mechanical-testing-method-to-assess-bone-implant-anchorage

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

15.4K 次观看.  University of Toronto. 一种改进的方法以机械测试的骨锚定到候选种植体表面呈现。这种方法精确地垂直或平行，以在植入物表面的平面中允许的干扰力的对准，并提供​​了一​​个准确的方法，可直接进行干扰力为确切的种植体周围区域。 

视频: An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
	Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. 
	We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

小鼠颈动脉的力学性能测试:从新生儿到成人

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

科研

生物工程

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

芯片上的压力诱导抗生素易感性测试

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

方法和股骨骨折测试在横盘整理的秋天在臀部位置检测的夹具

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

基于数字图像相关的骨折检测中股骨皮质应变的估算方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

FR-4 铜包电极板导电微泵性能测试平台

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

测量海洋海绵针力学性能的毫米尺度弯曲试验系统

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

大鼠运动皮质微电极植入所致功能缺损的啮齿动物行为检测

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...