Name: a mouse distraction osteogenesis model
Uploaded: 2018-11-14T12:45:00
Description: Watch this Scientific Journal Video about A Mouse Distraction Osteogenesis Model at JoVE.com

提交人感谢 makiko kato 女士为完成这项研究提供的鼓励。我们还感谢名古屋大学医学研究生院实验动物和医学研究工程系为小鼠提供住房。

所有实验都是根据本机构动物护理和使用委员会批准的协议进行的。在手术前对所有仪器进行消毒。
1. 创建定制分散体的模具的准备
<ol><li>制作两个不完整的圆环 (外径, 20 毫米; 内径, 10 毫米), 这是干扰器的一部分, 与一页石蜡 (145 毫米 x 74 毫米) 使用埃文斯蜡雕刻机。<ol>
<li>做4个相同的部分。使用用燃气燃烧器加热的蜡铲。将四个环堆叠在一起, 厚度为5毫米. 为膨胀螺钉和...

<ol>
	<li>Watson, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distraction+osteogenesis.">Distraction osteogenesis.</a> The Journal of the American Academy of Orthopaedic Surgeons. 14, 168-174 (2006).</li><li>Ilizarov, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tension-stress+effect+on+the+genesis+and+growth+of+tissues:+Part+II.+The+influence+of+the+rate+and+frequency+of+distraction.">The tension-stress effect on the genesis and growth of tissues: Part II. The influence of the rate and frequency of distraction.</a> Clinical Orthopaedics and Related Research. 239, 263-285 (1989).</li><li>Wan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+hypoxia-inducible+factor-1+alpha+pathway+accelerates+bone+regeneration.">Activation of the hypoxia-inducible factor-1 alpha pathway accelerates bone regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (2), 686-691 (2008).</li><li>Fujio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cell-derived+factor-1+enhances+distraction+osteogenesis-mediated+skeletal+tissue+regeneration+through+the+recruitment+of+endothelial+precursors.">Stromal cell-derived factor-1 enhances distraction osteogenesis-mediated skeletal tissue regeneration through the recruitment of endothelial precursors.</a> Bone. 49 (4), 693-700 (2011).</li><li>Tong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+adhesion+kinase+expression+during+mandibular+distraction+osteogenesis:+evidence+for+mechanotransduction.">Focal adhesion kinase expression during mandibular distraction osteogenesis: evidence for mechanotransduction.</a> Plastic and reconstructive surgery. 111 (1), 211-222 (2003).</li><li>Rhee, S. T., El-Bassiony, L., Buchman, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+signal-+related+kinase+and+bone+morphogenetic+protein+expression+during+distraction+osteogenesis+of+the+mandible:+in+vivo+evidence+of+mechanotransduction+mechanism+for+differentiation+and+osteogenesis+by+mesenchymal+precursor+cells.">Extracellular signal- related kinase and bone morphogenetic protein expression during distraction osteogenesis of the mandible: in vivo evidence of mechanotransduction mechanism for differentiation and osteogenesis by mesenchymal precursor cells.</a> Plastic and reconstructive surgery. 117 (7), 2243-2249 (2006).</li><li>Isefuku, S., Joyner, C. J., Simpson, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+distraction+osteogenesis.">A murine model of distraction osteogenesis.</a> Bone. 27 (5), 661-665 (2000).</li><li>Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+and+molecular+analyses+of+distraction+osteogenesis+in+a+mouse+model.">Histochemical and molecular analyses of distraction osteogenesis in a mouse model.</a> Journal of Orthopaedic Research. 16 (5), 636-642 (1998).</li><li>Carvalho, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+angiogenesis+in+a+murine+tibial+model+of+distraction+osteogenesis.">The role of angiogenesis in a murine tibial model of distraction osteogenesis.</a> Bone. 34 (5), 849-861 (2004).</li><li>Osawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+FGFR3+promotes+bone+formation+via+accelerating+endochondral+ossification+in+mouse+model+of+distraction+osteogenesis.">Activated FGFR3 promotes bone formation via accelerating endochondral ossification in mouse model of distraction osteogenesis.</a> Bone. 105, 42-49 (2017).</li></ol>

当大型动物被用作实验模型时, 可以使用现成的延伸装置, 很容易获得良好的固定, 并对延伸手术本身和延伸量进行评估。然而, 当鼠标被用作实验模型时, 有必要开发部分或全部设备。Isefuku等人和 tay等人制造了这个装置, 并制作了一个 7,8的鼠标模型。carvahjo等人采用了一种方法, 将现成的扩张装置 (轨道干扰器: kls martin) 固定在一根结...

牵张成骨术 (do) 是一种既定的治疗方法, 治疗各种骨骼疾病, 如肢体长度差异、骨缺损和肢体畸形1。这种独特的治疗策略是基于 ilizarov 提出的 "紧张应激原理"。这种方法需要几天的延迟, 几个星期的主动分心, 和几个月的巩固, 直到成熟的骨骼形成2。
由于血流量堵塞3、4和机械刺激5, 6 引起的局部缺氧条件在 do 的愈合过程中尤为重要。低氧诱导的血管生成携带氧气、营养物质、可溶性因子和细胞, 这些都是组织通过血液流动局部修复所必需的。通过扩展操作进行机械刺激会引起生物反应, 如骨髓间充质干细胞的分化、骨的形成、钙化和重塑。串行 do 治疗不仅可以形成硬组织, 还可以形成软组织, 包括神经、肌肉、血管和皮肤组织, 而不需要干细胞移植。因此, do 模型被认为是分析各种组织再生的一个很好的模型。
兔子和狗是在基础研究中最广泛使用的动物 do;然而, 很少有分析工具可用于这些动物。鼠标 do 模型的使用有助于进行更详细的分析。它特别适用于使用敲除小鼠的实验。然而, 当使用鼠标作为实验动物时, 应创建一个扩展装置。在这里, 我们提出了一个小鼠胫骨 do 模型开发使用定制的牵引器创建使用牙科实验室工具和技术, 这已在以前的研究中使用。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Paraffin wax</td>
			<td style="width:203px;">YAMAHACHI DENTAL MFG. CO.</td>
			<td style="width:147px;">-</td>
			<td style="width:239px;">For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Labocone putty</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td>For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Utility wax</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td>For preparation a mold for resin rings</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Expansion screw</td>
			<td style="width:203px;">Ortho Dentaurum</td>
			<td style="width:147px;">600-301-30</td>
			<td>Component of custom-made distractor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Unifast III</td>
			<td style="width:203px;">GC Corporation</td>
			<td>-</td>
			<td style="width:239px;">Immediate polymerization resin Component of custom-made distractor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Ortho Crystal</td>
			<td style="width:203px;">NISSIN</td>
			<td>-</td>
			<td style="width:239px;">Transparent resin Component of custom-made distractor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">25-gauge needle</td>
			<td style="width:203px;">TERUMO</td>
			<td style="width:147px;">NN-2516R</td>
			<td>For custom-made distractor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">27-gauge needle</td>
			<td style="width:203px;">TERUMO</td>
			<td style="width:147px;">NN-2719S</td>
			<td>For custom-made distractor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">ICR mouse</td>
			<td style="width:203px;">Chubu Kagaku Shizai Corporation</td>
			<td>-</td>
			<td>Experimental animal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:183px;">Somnopentyl</td>
			<td style="width:203px;">Kyoritsu Seiyaku</td>
			<td>-</td>
			<td>Pentobarbital sodium salt</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:183px;">Isoflurane</td>
			<td style="width:203px;">FUJIFILM Wako Pure Chemical Corporation</td>
			<td style="width:147px;">099-06571</td>
			<td>Isoflurane inhalation solution</td>
		</tr>
	</tbody>
</table>

a mouse distraction osteogenesis model

牵张成骨 (do) 是一种无需细胞移植的骨骼组织再生的外科手术。do 模型由以下三个阶段组成: 截骨术后的潜伏期和外部牵引器的放置;分散的阶段, 其中分离的骨端是逐渐和持续的分心;和整合阶段。这种定制的干扰器用于 do 是由两个不完整的丙烯酸树脂环和一个膨胀螺丝。这个过程是通过制造硅胶印模材料, 然后创建定制的牵引器开始的。将牙科树脂倒入硅胶印模材料的模板中, 并允许聚合, 以创建定制的分散体所需的不完整树脂环。这些环是用透明树脂的膨胀螺钉固定的。通过这种方法创建的定制干扰器附着在小鼠胫骨上。胫骨固定在设备上, 近端使用一对 25 g 针, 远端一双 27 g 针, 并使用丙烯酸树脂固定。经过5天的潜伏期, 以 0.2 mm 12h 的速度启动了干扰。延长了 8天, 总间隙为3.2 毫米。老鼠在分心4周后被牺牲。通过影像学和组织学方法证实了牵张间隙的骨形成。

图 1 a 和1b显示不完整环 (外径, 20 毫米; 内径, 10 毫米; 厚度, 5 毫米) 与石蜡。在硅胶印模材料中嵌入了两种蜡图案, 形成了树脂环的模具 (图 1c)。聚合物树脂立即倒入该模具中, 并获得树脂环 (图 1d)。通过将两个树脂环和一个膨胀螺钉结合在一起, 创建了一个定制的牵引器 (<strong...

Watch this Scientific Journal Video about A Mouse Distraction Osteogenesis Model at JoVE.com

A Mouse Distraction Osteogenesis Model

小鼠牵张成骨模型

我们提出了一个小鼠胫骨牵张成骨模型开发使用定制的牵引器。利用鼠标作为分析目标, 有利于推进研究。

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

此方法有助于回答软硬组织再生领域中的关键问题。这种技术的主要优点是，使用鼠标可以详细分析骨骼再生。从完全麻醉的成年小鼠开始。用10%碘溶液仔细剃须和消毒手术区域。然后，在右下肢施用0.5%的盐碱碱。然后，在右小腿用15号手术刀做15毫米的纵向皮肤切口后，钝直分离底层肌肉，注意不要切除所有周长肌。然后，用剪刀切割纤维。用窄而薄的钳子抓住脚踝，用27个量尺的针头在骨头上打个洞，从脚跟大约5毫米。针头应按这种顺序穿透皮肤、骨骼和皮肤。当针头完全穿透时，用钳子切开尖端和根部，使针头大约为 15 毫米。以同样的方式再打一个洞，大约两到三毫米近。接下来，抓住脚踝，以同样的方式在膝盖下打一根25针。针穿透后，用钳子切开尖端和根部。以同样的方式再打一个洞，大约两到三毫米。放置定制的干扰器，使之与扩展方向平行。用足够的聚合牙科树脂固定针头和设备，以填充设备的凹槽。等待聚合完成，大约需要五分钟。然后，在非常小心不损坏周围组织的同时，在施用盐水溶液的同时，使用非常薄的切割盘切割头骨的直径中间。用4-0尼龙缝合线关闭伤口。手术后立即给皮下注射丁丙诺啡进行镇痛。有关于延迟周期和分散注意力率的各种报告，但此处显示了具有代表性的协议。延迟五天后，开始分心过程。对于扩展，请使用连接到快速膨胀螺钉的销。将销朝连接到延长螺钉的黄色箭头方向移动。用小手指和手掌握住尾巴，用食指和拇指固定延长装置。通过向黄色箭头的方向移动针脚 0.2 毫米以四分之一转弯执行扩展。继续延长速度为0.2毫米，每12小时8天，这将导致3.2毫米的总间隙。此图显示了典型的放射成像发现在四周后分心的模型。新形成的骨头在分心现场被看到。色氧林和 eosin 染色结果见于此处。观察了新形成的骨桥，新形成的骨骼很容易与原生骨骼区分开来。我们提出了一个使用定制干扰器开发的鼠标 tibial DO 模型。使用鼠标 DO 模型有助于进行更详细的分析。它特别适合使用敲除小鼠进行实验。

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

科研

发育生物学

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

小鼠胚胎跖骨文化：软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

用小鼠模型对精子发生细胞学分析的精管壁球技术

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

小鼠骨骼肌肉外泄原发性肌基器的分离和分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

从成年小鼠味干细胞中衍生的语言器官的生成与培养

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...