这项工作得到了 CNRS Mecabio 挑战的支持和资助。
作者感谢动物护理技术员在整个过程中照顾动物。作者还通过 IVTV 承认里昂中心。感谢马乔里 Sweetko 的语言版本。
我们感激 Marylène Lallemand、Cécile Génovésio 和帕特里克. 劳伦对这项实验研究的贡献。

所描述的所有程序均由 Aix-马赛机构动物保育和使用委员会和法国研究部批准, 并在马赛医学院的常规动物院进行 (法国). 
 1. 根据以下准则定义外部固定器的功能规范 <ol> <li> 优化骨骼锚固. 
	<ol> <li> 植入 half-threaded 针脚, 其直径 (螺纹部分) 为 1 mm. </li>
	</ol> </li> <li> 选择设计以减少对动物的不适. 
	<ol> <li> 选择适合于 7723 mm 3 (32 mm x 19 mm x 12.7 mm) 的体积的小尺寸的外部固定器。
		<li> 选择一个低重量的外固定器, 不带针脚的13克, 避免干扰动物和 #39; 步态。选择铝作为材料的两个区块由于其低密度. 
		注: 倒块的重量大大降低了两部分的比重. </li>
	</ol> </li> <li> 控制固定器的运动, 使拉伸力的方向与骨骼的方向保持平行, 并确保纯拉伸力。
	<ol> <li> 使用穿过两个块的滑动条。槽和开放的方块, 使每个可以承担两个引脚. </li>
		<li> 使用一个平滑侧的延伸螺钉, 在块中自由转动, 可以用手指夹住, 并通过旋转使第二块偏移的螺纹侧。将两根针固定在骨折的两侧, 使拉力方向与骨骼方向平行。保持纵向轴的拉伸力, 使销钉上的应力分布良好。用锁定螺钉固定四针脚. </li>
	</ol> </li> <li> 选择可以经受灭菌温度的铝、钛和钢等材料. </li>
	<li> 确保针放置对应于外部固定架几何 ( 图 1 B )。
	<ol> <li> 使用一个钻孔导轨, 其中包含在骨骼上拧紧的夹具以保持钻孔位置. </li>
		<li> 缩短夹具的杠杆臂以降低夹紧力。将夹钳放在装置中间, 在以后的骨折处, 以清除两侧. </li>
	</ol> </li> <li> 通过添加花螺钉来拧紧和松开移动块, 使分散操作易于手动调整. 
	<ol> <li> 使用的螺钉宽度足以用手指单独操作. </li>
		<li> 在两个块之间的延伸螺钉中间添加一个钻孔方形螺母, 以便轻松分散. </li>
		<li> 使此方形螺母 (每边 8 mm) 大于两个块, 以允许手动使用细针或任何类型的对象。转动螺丝和 #188; 转动以允许0.125 毫米延长. </li>
	</ol> </li> </ol> 2。手术 注意: 所有外科手术都需要一个助手。四12周大的雄性大大鼠被喂食标准的实验室食谱 ad 随意 . 
<ol> <li> 准备手术工具. 
	<ol> <li> 对以下所有手术器械进行消毒: 1 rugine、2森和 #39 器、1微奥尔森 Hegar 针夹、1梅奥 Hegar 针支架、1梅奥剪刀和1手术刀. </li>
		<li> 消毒外固定架, 钻孔导轨, 4 half-threaded 针, 4 螺丝, 尖端和钻柱, 以及 piezotome 的尖端和串。灭菌135和 #176 对这些仪器进行消毒; C 为 18 min. </li>
		<li> 在无菌场、手术台上安装一个发热垫。将所有仪器或工具放在另一个不育的领域. </li>
	</ol> </li> <li> 麻醉并准备该动物. 
	<ol> <li> 称量大鼠准备麻醉剂和镇痛合剂 </li> <li> 确定并计算 Bupremorphine 和卡洛芬的用量, 准备镇痛合剂即兴。使用丁丙诺啡 (0.03 毫克/毫升) 和卡洛芬 (5 毫克/毫升) 分别在0.05 毫克/千克和5毫克/千克. </li>
		<li> 抑制大鼠和皮下注射镇痛合剂. #160; 等待几秒钟, 然后注射腹腔麻醉合剂. </li>
		<li> 用电动剃须刀剃掉右后肢, 用聚维酮碘溶液消毒肢体. </li>
		<li> 在无菌场上横向 (右侧向上) 放置动物, 以便在 mediolateral 轴上正确定位外固定架. </li>
		在手术过程中, <li> 在头部放置一个不育的压缩来保护眼睛. </li>
	</ol> </li> <li> 将外部固定器植入股骨。
	<ol> <li> 标志性皮肤切口。用一个标记, 从远端部分 (膝部) 和第二个点 (髋部) 在矢状面中中线的后面绘制一个点。然后在这2点之间画一条线. </li>
		<li> 使用手术刀沿绘图线切割拉伸的皮肤. </li>
		<li> 在 股二头肌之间切开 和 股外 , 直到股骨完全暴露在手术刀的帮助下。使用2森和 #39 器 (可能需要助手) 来促进肌肉切开. </li>
		<li> 抬起骨膜并在 rugine 的帮助下从骨骼中断开软组织. </li>
		<li> 检查裸露的股骨是否足够长。
		<ol> <li> 在外部固定器的最近端和远端孔中插入针脚. </li>
			<li> 定位外固定器并检查两个针脚是否可以固定在股骨上. </li>
		</ol> </li> <li> 在股骨中植入4平行的1毫米 half-threaded 针。
		<ol> <li> 采用钻孔导轨, 将2森和 #39 的肌肉分开; 器。拧紧股骨中部的钻孔导轨夹钳. </li>
			<li> 在股骨中钻 4 pre-holes。使用电钻, 钻孔的速度为 2000 rpm 通过一个金属钻头0.6 毫米直径通过4导孔。
			<ol> <li> 从最近端和远端的孔开始, 并完成指南中的两个中间部分。小心地穿过两个皮质, 但不要破坏股骨下方的软组织. </li>
			</ol> </li> <li> 从钻孔导轨上脱下. </li>
			<li> 放大 4 pre-holes 与 0.8 mm half-threaded 针。
			<ol> <li> 通过 0.6 mm 孔来回工作 pin。小心保持垂直于股骨和下沉通过两个皮质. </li>
			</ol> </li> <li> 植入 1 mm half-threaded 针脚。
			<ol> <li> 用针夹抓住 1 mm half-threaded 针脚的头. </li>
				<li> 下沉引脚以放大 pre-hole。检查针脚是否穿透两个皮层, 并不凸出超过1毫米, 与森和 #39; s 牵引器. </li>
			</ol> </li> <li> 将外部固定器连接到 4 half-threaded 针脚。确保偏移量 (固定架与 #39 之间的距离, 两个块和骨表面) 约6毫米, 以便于缝合和系统的良好刚性 18 . </li>
			<li> 固定4锁定螺钉, 使外部固定器锁定在针脚上. </li>
		</ol> </li> </ol> </li> <li> Osteotomize 股骨. 
	<ol> <li> 在2中心针脚与 piezotome 之间执行截骨. </li>
		<li> 关闭禾和使用一个连续针与可缝合线 (5.0) 和梅奥-Hegar 针持有人。确保只有皮肤缝合, 而不是肌肉. </li>
	</ol> </li> <li> 验证手术和监视动物. 
	<ol> <li> 在手术后执行 X 光检查, 而动物仍在麻醉中。检查所有针脚的深度, osteotomized 的肢沿长轴排列. </li>
		<li> 通过皮下注射丁丙诺啡 (0.03 毫克/毫升) 和 Enrofloxacine (50 毫克/毫升) 分别在0.05 毫克/千克和10毫克/千克 #160 确保镇痛和 antibioprophylaxy; </li>
		<li> 确定并计算用于反转麻醉的 antipamezole 量。使用 antipamezole 在1毫克/千克和做皮下注射管理产品。和 #160; 允许动物重拾意识并将它归还给它的笼子. </li>
		<li> 手术后六小时, 分别在 0.05mg/千克和5毫克/千克皮下注射第二剂量的镇痛合剂 (丁丙诺啡和卡洛芬); #160; 
		至少在接下来的3天里, 每天给这些注射两次镇痛合剂, 每天一次, 为 antibioprophylaxy。必须定期进行临床检查以评估镇痛的有效性, 并且必须根据每种动物的行为体征进行修改。和 #160; </li> <li> 检查动物是否可以在手术后的第二天正常行走. </li>
	</ol> </li> </ol> 3。分心 <ol> <li> 在1周内将外固定器保持在中性点。x 射线的肢体在这一阶段结束。检查针脚的位置和骨骼段的对齐方式. </li>
	<li> 手动将方形螺母旋转一半 (0.25 mm), 每12小时顺时针转动一次以执行分心。分心10天, 这将导致一个5mm 的骨骼延长, 代表了12% 延长相对于手术骨段的初始长度. 
	在此步骤中, <ol> <li> 不麻醉动物。x 射线的肢体中途通过和在这段时间结束检查的定位针和对齐骨段. </li>
	</ol> </li> <li> 将外部固定架保持47天。x 线每周检查肢体的进展, 注意力分散的间隙钙化。64天, 取出外固定架, 让动物自由行走2天 (66 天)。总合并期持续7周. </li>
	<li> 安乐所有老鼠经过7周的整合, 过量吸入七氟醚. </li>
	<li> 在没有周围组织的情况下切除分心和对侧股骨. 
	<ol> <li> 在手术刀的帮助下, 在矢状面后的中线上的疤痕上进行皮肤切口。从臀部顶端到膝前部的切口. </li>
		<li> 在 股二头肌 和 股外 与手术刀之间切开直到股骨完全暴露。尽可能地断开附着在骨骼上的肌肉. </li>
		<li> 切断所有膝关节韧带, 并拆除关节. </li>
		<li> 切开髋关节的关节囊. </li>
		<li> 彻底清洁骨骼而不删除 half-threaded 针脚。用手术刀取出所有的软组织. </li>
	</ol> </li> <li> 对对侧股骨重复从3.5.1 到3.5.5 的步骤. </li>
	<li> x 光分心和对侧股骨。卸下 4 half-threaded 针脚, 并将所有股骨存储在-20 和 #176 上; C 用于微 CT 扫描分析 (10 和 #181; m 分辨率). </li>
</ol>

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作者没有什么可透露的。

这项研究描述了一个可复制的协议, 包括一个适当的外固定器设计的大鼠肢体延长, 与详细的手术技术, 允许动物的生理负重后, 去除外固定架。我们的协议导致了一个功能性的再生骨骼。经过47天的巩固, 去除自制的外固定器和2天的生理负重, 动物并没有诱发任何骨折的再生愈伤组织。由于微 CT 重建, 完全架桥的证据证实了再生愈伤组织的功能。先前的研究清楚地证明了钙化桥接的存在与再生愈伤组织能够维持19的负荷之间的正相关关系。此外, vBMD 发现的再生愈伤组织约67% 的 vBMD 发现骨干部分股骨从 controlateral 肢体20。我们还发现了再生愈伤组织的平均 vBMD, 与先前描述的21的值相近。这突出了稳定的环境创造了自制的外部固定器, 导致有效的骨修复。此外, 该固定器是良好的耐受动物。外固定架的定位和重量并没有干扰动物的步态, 所以他们可以在手术后行走。
在整个协议中, 没有折断或松动的针脚发生, 证据表明, 线程程序确保有效和稳定的锚钉在股骨。至关重要的是, 针是来讲的良好的稳定性的微观环境: 松开一个引脚已显示减少一半的刚度的设备18。此外, 老鼠必须单独安置在笼子里, 并有专门设计的扁平盖, 防止动物钩住外固定架。人工和 easy-to-perform 的再生愈伤组织的分散作用限制了针的松动, 但也避免了每12小时要对动物进行镇静, 并有助于自制外固定器的可靠性和效率。另一个重要的检查点是, 针穿透两个皮层和不凸出超过1毫米。这一步是敏感的, 如果 X 射线照片不确定正确定位的针脚, 这需要纠正手动使用梅奥 Hegar 针持有人, 而动物仍然麻醉。该协议有一些限制。这是一个耗时和约束的协议, 它限制了它可以使用的动物数量。此外, 鉴于所需的灵活性和手术的细致, 程序可能需要几个练习跑到掌握。
在整个过程中, osteotomized 四肢的排列保持不变。当在股骨内植入针时, 使用钻孔导轨是非常重要的, 这样 half-threaded 针是平行的, 从而确保在分散和固结过程中不产生剪应力的情况下, 机械应力的良好分布。阶段.每天清洁伤口也是非常重要的, 以防止任何阻碍的方块螺母, 这将使手动分心困难, 这可能会影响到 osteotomized 四肢的对准和正确的锚钉。如何进行截骨是再生的最后一个关键点。截骨术必须用 piezotome, 以确保一个统一和规则的骨段, 并防止热诱导的坏死和周围软组织损伤。此外, 在钻 pre-holes 时, 有时会产生过量的热量。与电钻结合的灌溉系统可用于防止热诱导的坏死。
最后, 我们描述了一个有效的和可重复的协议, 在大鼠股骨模型中, 因为这样做会导致功能性再生骨。根据大鼠股骨的解剖特点设计自制的外固定支架, 使其能够形成一个稳定的环境, 使钙化的再生愈伤组织得到充分巩固, 以支持生理负重的动物。我们的下一个目标是使用这个可重现的协议来确定与功能性再生骨相容的最短固结期。随后的目标将是改进技术, 特别是寻求缩短巩固期的方法。这一可重复性的协议也有助于确定骨修复所涉及的机制。最后, 自制的外固定器的特点使其能在临床上应用于人的手指伸长。

牵引成骨术 (做) 是一种外科技术广泛使用临床1,2,3,4在人为更低的1,2和上部3肢体延长,治疗后骨不连, 或再生骨缺损后骨肿瘤切除术以及颌面部重建4。在骨内固定支架置入后, 可导致骨再生。osteotomized 四肢通过逐渐分心2 , 以达到预期的伸长距离。巩固期间跟随, 在期间没有更多伸长。
执行过程分为三不同的阶段: 延迟、分心和整合。通常, 7 天的潜伏期是在截骨4之后开始的。这允许骨骼修复开始愈合过程的初始步骤4。潜伏期后, 牵引力被应用到再生愈伤组织和周围软组织的一个分心期1,2,4。当达到预期的伸长率, 分心停止和巩固期开始。在此期间, 外固定器保持, 直到再生骨功能足以支持其去除。
不同的参数对骨修复有影响, 如长度和伸长率, 外固定器的类型, 分心的频率, 巩固期的长短, 或机械应力类型应用于分散的愈伤组织。举例来说, 延长的速度和频率可能导致过早的合并5或通过创建不可恢复的损伤, 如坏死组织或囊肿在胼胝体内,6,7。
许多协议已经应用于不同的动物模型8,9,10 , 以研究骨骼修复过程并最大限度地巩固骨骼。在大鼠中, 大多数研究11、12、13、14、15 , 重点讨论如何通过加速愈伤组织合并来缩短执行协议。其中一些实验研究使用了外部固定架已经商业化可用于人类临床应用的5,13,15,16。然而, 这些类型的外固定器不适合做的大鼠股骨, 这表现出不同的解剖特征, 从人的股骨。此外, 只有少数研究清楚地证明了它们的协议在获得功能性再生骨716中的效率。因此, 很难比较不同的研究结果, 由于他们的不同协议和缺乏有关外部固定器的信息12,13,14,17。
因此, 这项研究的目的是描述, 在大鼠模型, 一个有效的和可重复的协议做股骨, 导致功能性再生骨。为此, 我们设计了一个自制和易于使用的外固定器, 特别是对大鼠股骨, 这是我们在这个协议中详细描述。在起草该装置的技术规格时, 我们考虑到了机械应力分布良好和避免残余应力产生的所有基本约束。技术规格包括一个适当的几何为设备允许纯净的牵引力量在骨头和周围的组织, 适当的重量为动物的步态, 控制骨头伸长的长度和好对骨头段的对准在销钉和骨骼的交叉处不产生剪切应力。此外, 该装置必须在不受干扰、生物相容性和消毒无损伤的情况下使用。经过7周的整合后, 本议定书对大鼠股骨做了一个功能性再生骨, 证明了动物的生理负重没有骨折的再生愈伤组织后外固定架。动物的生理步态与再生愈伤组织和 x 射线分析的显微 CT 分析得到的建筑参数一致。

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			<td>Supply by the animal house</td>
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			<td>Dental X-ray</td>
			<td>S.A.R.L Innovation m&#233;dicales et dentaires</td>
			<td>WYZ - BLUEX</td>
			<td></td>
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			<td>Winiwix Software</td>
			<td>Softys Dental</td>
			<td>PFT</td>
			<td></td>
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			<td>Micro-CT system</td>
			<td>nanoScan SPECT/CT</td>
			<td>GEIT-31105EN (05/14)</td>
			<td>Subcontract by IVTV central Lyon</td>
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			<td>Word Precision Instruments</td>
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			<td>Blunt version</td>
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			<td>Mundipharma Medical Company</td>
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			<td>Ethicon</td>
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			<td>Mayo scissor</td>
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			<td>Sprague-Dawley</td>
			<td>Janvier</td>
			<td>none</td>
			<td>12 weaks and male</td>
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</table>

an efficient and reproducible protocol for distraction osteogenesis in a rat model leading to a functional regenerated femur

本协议描述了在大鼠股骨模型中使用一种新研制的外固定器牵引成骨术。牵引成骨术 (做) 是一种外科手术技术, 导致骨再生后, 截切。osteotomized 肢通过逐渐的分散而相互移动, 达到预期的伸长率。该方法广泛应用于人下肢延长, 骨不连后治疗, 或骨肿瘤切除术后骨缺损的再生, 以及颌面部重建。只有少数的研究清楚地证明了他们的协议在获得功能性再生骨的效率,即, 在取出外固定架后, 将支持不带骨折的生理负重的骨骼。此外, 由于缺乏信息, 使得不同的研究和重复性的协议受到限制, 使学习比较困难。本研究的目的是制定一个可复制的协议, 包括一个适当的外固定器设计大鼠肢体延长, 与详细的手术技术, 允许动物的生理负重后, 去除外部固定.

从外科手术结束到合并结束的 x 线图像显示, 股骨 half-threaded 针没有松动, 表明稳定的锚固。别针是平行和保存完好。在执行过程中, osteotomized 肢沿骨骼的纵轴定向 (图 2)。在潜伏期结束时, 没有钙化区域是可见的 (图 2B)。在分心期结束时, 一些钙化区可见于先前存在的皮层 (图 2C)。经过28天的固结 (45 天), osteotomized 肢间隙的钙化区较小, 我们观察到的骨膜愈伤组织不仅靠近间隙, 而且在针脚的水平 (图 2D)。经过47天的巩固 (天 64), 再生愈伤组织完全桥接 (图 2E)。在取出外固定器和2天的生理负重后, 动物有生理步态, 没有骨折的迹象 (图 2F)。
3D 对再生愈伤组织纵断面的显微 CT 分析表明, 钙化桥接始终存在 (图 3A-D)。在再生愈伤组织 (图 3C-D) 的外围观察到一个连续的外皮质。根据纵廓, 再生愈伤组织完全由骨小网填充。经过49天的固结 (天 66), 一个较少的矿化区域保持在再生骨的中心 (图 3e-F)。微 CT 的结构参数表明, 再生愈伤组织和对侧股骨的平均体积分数 (BV/TV) 分别为55% ±13和97.85% ± 1.7 (表1和表 2)。再生愈伤组织的体积骨密度 (vBMD) 的平均值为750毫克/cm3 ±25。再生愈伤组织的横截面积平均值高于对侧股骨 (17.23 mm2 ± 9.3 vs 9.5 mm2 ± 1.2) (表 1和表 2)。平均愈伤组织皮质厚度小于对侧股骨皮质厚度 (0.317 mm ± 0.04 vs 0.6 mm ± 0.05) (表 1和2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56433/56433fig1.jpg"> 
图 1: 外部固定架和钻孔导轨的计算机辅助设计 (CAD)(A) 带有针脚的外部固定架的 CAD。(B) 钻孔导轨的 CAD。缩放条 = 5 mm<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56433/56433fig2.jpg"> 
图 2: 不同时点的大鼠股骨的 X 射线代表.0、7、17、45、64和66天的分散的愈伤组织的 X 射线照相。(A) 在手术后 (0 天) 拍摄的 X 光片。(B) 在7天的延迟期之后拍摄的 x 射线照相。无钙化愈伤组织在 osteotomized 肢附近明显 (OE)。(C) 在分心的末端, 在 osteotomized 的肢 (长方形区域) 附近有一个小的, 不明显的钙化区。(D) 经过28天的合并 (天 45), osteotomized 四肢 (*) 和第二个皮质之间的间隙是明显的 (黄色箭头)。(E) 射线照相经过47天的固结 (天 64) (F) X 射线照相后49天的固结 (天 66), 具有两天的生理负重。缩放条 = 1 mm<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56433/56433fig3.jpg"> 
图 3: 对大鼠股骨的分散愈伤组织进行3D 微 CT 重建.从黄到蓝的颜色可以说明骨的矿化。(A, B)前后和纵轴后, 分散的愈伤组织的桥皮层的表现。(C, D)近端和远端横断面显露最初的皮质骨 (黑箭头) 和被分心的愈伤组织 (黄色箭头) 的桥接皮层 (E, F) 3D 7 周后, 骨痂愈合后的愈伤组织的纵廓: 愈伤组织由小梁骨网填充。刻度栏 = 1000 µm. 校准条 = 0 到2.54 克/厘米3 (黄色 = 1.73 克/厘米3, 粉红色 = 0.84 克/厘米3, 蓝色 = 0.17 克/cm3)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56433/56433fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Table 1" src="/files/ftp_upload/56433/56433table1.jpg"> 
表 1:合并49天后, 股骨转移后的骨痂参数.选择的感兴趣区域 (ROI) 计算所有参数是 osteotomized 肢之间的区域。每只老鼠的所有值都是在图像栈的每个切片上计算出来的。结果以平均±标准差表示。BV/电视: 体积分数;CSA: 横截面积;vBMD: 骨矿物质密度;ct.: 皮质厚度。
<img alt="Table 2" src="/files/ftp_upload/56433/56433table2.jpg"> 
表 2:在49天的合并后, 对对侧参数进行完整的股骨的.每只老鼠的所有值都是在图像栈的每个切片上计算出来的。结果以平均±标准差表示。BV/电视: 体积分数;CSA: 横截面积;ct.: 皮质厚度。

Watch this Scientific Journal Video about An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur at JoVE.com

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

大鼠牵张成骨的高效可再生性协议

这项研究描述了一个可复制和详细的协议使用新开发的外固定器的牵引成骨 (做) 在股骨大鼠模型, 允许生理负重的动物后, 外固定架。

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

an-efficient-reproducible-protocol-for-distraction-osteogenesis-rat

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.4K 次观看.  Aix Marseille Univ. 这项研究描述了一个可复制和详细的协议使用新开发的外固定器的牵引成骨 (做) 在股骨大鼠模型, 允许生理负重的动物后, 外固定架。

视频: An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

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

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

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

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

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

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

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

无胸​​腺大鼠模型工程前交叉韧带移植评估

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

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal hernia typically requires implantation of a material to serve as a mechanical bridge across the defect in the abdominal wall. Biomaterials, such as porcine small intestinal submucosa (SIS), also serve as a lattice for cell growth into the implant and can naturally incorporate into the host tissue. Development of such repair materials benefits from use of animal models in which experimental abdominal wall defects are easily created and are amenable to repair in a reproducible fashion. The method offered here describes surgical creation and repair of ventral abdominal hernia in a rat model. When SIS is used to repair an experimental ventral abdominal hernia in this model, it is rapidly incorporated into host tissue within 28 days of implantation. Histologically, incorporation of their implanted material into host tissue is characterized by a robust fibrovascular response. Future refinements and applications of the rat abdominal hernia model may likely involve diabetic and/or obese animals as a means to more closely mimic common co-morbidities of man.

This manuscript describes methods associated with creation and repair of a ventral abdominal wall defect (hernia). This model can be used to study repair strategies such as those that use implanted materials. In this manuscript, repair of the experimental hernia with porcine small intestinal submucosa is presented as an example.

大鼠模型在腹腹疝修补术中的应用研究

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...