Name: 3d imaging of pdl collagen fibers during orthodontic tooth movement in mandibular murine model
Uploaded: 2021-04-15T13:30:00
Description: Watch this Scientific Journal Video about 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model at JoVE.com

这项研究得到了国家卫生研究院（NIDCR R00-DE025053，PI：Naveh）的支持。我们要感谢哈佛生物成像中心的基础设施和支持。所有数字均以 biorender.com 生成。

所有动物实验均符合国家卫生研究院《实验室动物护理和使用指南》和哈佛大学机构动物护理与使用委员会的准则（第01840号议定书）。
1. 矫形牙齿运动
<ol><li>要生成鼠标床，请使用带楔形、45° 角头的扁平塑料平台。头靠面可以通过切割塑料盒生成。<ol>
<li>提升平台的头部端，在头架和桌面平面之间产生大约 30° 角。将弯曲的厚回形针（直径为 0.036"）连接到头部侧端，以?...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tissue,+cellular,+and+molecular+regulation+of+orthodontic+tooth+movement:+100+years+after+Carl+Sandstedt.">The tissue, cellular, and molecular regulation of orthodontic tooth movement: 100 years after Carl Sandstedt.</a> European Journal of Orthodontics. 28, 221-240 (2006).</li><li>Krishnan, V., Davidovitch, Z., molecular, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular,+molecular,+and+tissue-level+reactions+to+orthodontic+force.">Cellular, molecular, and tissue-level reactions to orthodontic force.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 129 (4), 1-32 (2006).</li><li>Shoji-Matsunaga, A., 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=Osteocyte+regulation+of+orthodontic+force-mediated+tooth+movement+via+RANKL+expression.">Osteocyte regulation of orthodontic force-mediated tooth movement via RANKL expression.</a> Scientific Reports. 7 (1), 8753 (2017).</li><li>Oppenheim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+changes,+particularly+of+the+bone,+incident+to+tooth+movement.">Tissue changes, particularly of the bone, incident to tooth movement.</a> European Journal of Orthodontics. 29, 2-15 (2007).</li><li>Unnam, D., 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=Accelerated+Orthodontics-An+overview.">Accelerated Orthodontics-An overview.</a> Journal of Archives of Oral Biologyogy and Craniofacial Research. 3 (1), 4 (2018).</li><li>von Bohl, M., Kuijpers-Jagtman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyalinization+during+orthodontic+tooth+movement+:+a+systematic+review+on+tissue+reactions.">Hyalinization during orthodontic tooth movement : a systematic review on tissue reactions.</a> European Journal of Orthodontics. 31 (1), 30-36 (2009).</li><li>Kirschneck, 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=Comparative+assessment+of+mouse+models+for+experimental+orthodontic+tooth+movement.">Comparative assessment of mouse models for experimental orthodontic tooth movement.</a> Scientific Reports. 10 (1), 1-12 (2020).</li><li>Naveh, G. R. S., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+orthodontic+tooth+movement+of+a+multirooted+tooth:+a+3D+study+of+a+rat+molar.">Initial orthodontic tooth movement of a multirooted tooth: a 3D study of a rat molar.</a> Orthodontics &amp; Craniofacial Research. 18 (3), 134-142 (2015).</li><li>Nakamura, 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=Time-lapse+observation+of+rat+periodontal+ligament+during+function+and+tooth+movement,+using+microcomputed+tomography.">Time-lapse observation of rat periodontal ligament during function and tooth movement, using microcomputed tomography.</a> European Journal of Orthodontics. 30 (3), 320-326 (2008).</li><li>Kawarizadeh, A., Bourauel, C., Jager, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+numerical+determination+of+initial+tooth+mobility+and+material+properties+of+the+periodontal+ligament+in+rat+molar+specimens.">Experimental and numerical determination of initial tooth mobility and material properties of the periodontal ligament in rat molar specimens.</a> European Journal of Orthodontics. 25 (6), 569-578 (2003).</li><li>J&#243;nsd&#243;ttir, S. H., Giesen, E. B. W., Maltha, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+behavior+of+the+periodontal+ligament+of+the+beagle+dog+during+the+first+5+hours+of+orthodontic+force+application.">Biomechanical behavior of the periodontal ligament of the beagle dog during the first 5 hours of orthodontic force application.</a> European Journal of Orthodontics. 28, 547 (2006).</li><li>Lindhe, J., 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=Experimental+breakdown+of+peri-implant+and+periodontal+tissues.+A+study+in+the+beagle+dog.">Experimental breakdown of peri-implant and periodontal tissues. A study in the beagle dog.</a> Clinical Oral Implants Research. 3 (1), 9-16 (1992).</li><li>Salamati, A., 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=Functional+tooth+mobility+in+young+pigs.">Functional tooth mobility in young pigs.</a> Journal of Biomechanics. 104, 109716 (2020).</li><li>Maria, R., 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=An+unusual+disordered+alveolar+bone+material+in+the+upper+furcation+region+of+minipig+mandibles:+A+3D+hierarchical+structural+study.">An unusual disordered alveolar bone material in the upper furcation region of minipig mandibles: A 3D hierarchical structural study.</a> Journal of Structural Biology. 206 (1), 128-137 (2019).</li><li>Wang, 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+miniature+pig:+a+useful+large+animal+model+for+dental+and+orofacial+research.">The miniature pig: a useful large animal model for dental and orofacial research.</a> Oral Diseases. 10, 1-7 (2007).</li><li>Melsen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+reaction+to+orthodontic+tooth+movement--a+new+paradigm.">Tissue reaction to orthodontic tooth movement--a new paradigm.</a> European Journal of Orthodontics. 23 (6), 671-681 (2001).</li><li>Naveh, G. 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=Direct+MicroCT+imaging+of+non-mineralized+connective+tissues+at+high+resolution.">Direct MicroCT imaging of non-mineralized connective tissues at high resolution.</a> Connective Tissue Research. 55 (1), 52-60 (2014).</li><li>Naveh, G. 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=Nonuniformity+in+ligaments+is+a+structural+strategy+for+optimizing+functionality.">Nonuniformity in ligaments is a structural strategy for optimizing functionality.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (36), 9008 (2018).</li><li>Naveh, G. 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=Tooth+periodontal+ligament:+Direct+3D+microCT+visualization+of+the+collagen+network+and+how+the+network+changes+when+the+tooth+is+loaded.">Tooth periodontal ligament: Direct 3D microCT visualization of the collagen network and how the network changes when the tooth is loaded.</a> Journal of Structural Biology. 181 (2), 108-115 (2013).</li><li>Naveh, G. 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=Tooth+movements+are+guided+by+specific+contact+areas+between+the+tooth+root+and+the+jaw+bone+:+A+dynamic+3D+microCT+study+of+the+rat+molar.">Tooth movements are guided by specific contact areas between the tooth root and the jaw bone : A dynamic 3D microCT study of the rat molar.</a> Journal of Structural Biology. 17 (2), 477-483 (2012).</li><li>Naveh, G. 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=Tooth-PDL-bone+complex:+Response+to+compressive+loads+encountered+during+mastication+-A+review.">Tooth-PDL-bone complex: Response to compressive loads encountered during mastication -A review.</a> Archives of Oral Biology. 57 (12), 1575-1584 (2012).</li><li>Ben-Zvi, 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=Response+of+the+tooth-periodontal+ligament-bone+complex+to+load:+A+microCT+study+of+the+minipig+molar.">Response of the tooth-periodontal ligament-bone complex to load: A microCT study of the minipig molar.</a> Journal of Structural Biology. 205 (2), 155-162 (2019).</li><li>Klingberg, A., 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=Fully+Automated+Evaluation+of+Total+Glomerular+Number+and+Capillary+Tuft+Size+in+Nephritic+Kidneys+Using+Lightsheet+Microscopy.">Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy.</a> Journal of the American Society of Nephrology. 28 (2), 452 (2017).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Taddei, S. R. d. A., 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=Experimental+model+of+tooth+movement+in+mice:+A+standardized+protocol+for+studying+bone+remodeling+under+compression+and+tensile+strains.">Experimental model of tooth movement in mice: A standardized protocol for studying bone remodeling under compression and tensile strains.</a> Journal of Biomechanics. 45 (16), 2729-2735 (2012).</li><li>Nakamura, K., Sahara, N., Deguchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+the+distribution+and+number+of+macrophage-lineage+cells+in+the+periodontal+membrane+of+the+rat+molar+in+response+to+experimental+tooth+movement.">Temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement.</a> Archives of Oral Biology. 46 (7), 593-607 (2001).</li><li>Rygh, P., 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+vascular+system:+A+main+mediator+of+periodontal+fiber+remodeling+in+orthodontic+tooth+movement.">Activation of the vascular system: A main mediator of periodontal fiber remodeling in orthodontic tooth movement.</a> American Journal of Orthodontics and Dentofacial Orthopedics. 89 (6), 453-468 (1986).</li><li>Nagao, 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=Vascular+endothelial+growth+factor+in+cartilage+development+and+osteoarthritis.">Vascular endothelial growth factor in cartilage development and osteoarthritis.</a> Scientific Reports. 7 (1), 13027 (2017).</li><li>Licht, A. H., 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=Endothelium-specific+Cre+recombinase+activity+in+flk-1-Cre+transgenic+mice.">Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic mice.</a> Developmental Dynamics. 229 (2), 312-318 (2004).</li><li>Connizzo, B. K., Naveh, G. R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+AFM-based+nanoscale+rheology+reveals+regional+non-uniformity+in+viscoporoelastic+mechanical+behavior+of+the+murine+periodontal+ligament.">In situ AFM-based nanoscale rheology reveals regional non-uniformity in viscoporoelastic mechanical behavior of the murine periodontal ligament.</a> Journal of Biomechanics. 111, 109996 (2020).</li><li>Connizzo, B. 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由于大小、遗传学和处理优势，在小鼠体内产生OTM是非常理想的。使用可手性在组织解剖以及样品制备和成像方面都提供了一个易于处理的操作。在这里，我们介绍了一种在OTM后7天内产生OTM的方法，即牙齿在骨骼内的转化运动。使用此协议，可以延长牙齿运动的整个持续时间，因为激活的线圈为运动提供了高达 1 mm 的恒定力水平。然而，线圈的中微侧固定在切口上，切口不断喷发。因此，力向量...

正畸牙齿运动 （OTM） 中的基本生物学过程是骨骼重塑。这种改造过程的触发因素归因于牙周韧带（PDL）结构的变化，如细胞外基质（ECM）应力、坏死以及血管破坏和形成1、2、3。其他可能的紫外骨重塑触发因素与骨细胞在骨骼中的力感应以及骨质疏松体本身的机械变形有关：然而，他们在OTM中的角色仍然没有完全阐明4，5。
尽管许多研究旨在揭示在OTM期间PDL的结构-功能关系，一个明确的功能机制尚未定义6，7。其主要原因是在检索位于两个硬组织（水泥和肺骨）之间的软组织 （PDL） 的数据方面面临挑战。收集结构信息的公认方法通常需要固定和分割，从而破坏和修改 PDL 结构。此外，这些方法大多产生二元数据，即使没有失真，也只提供部分和本地化的信息。由于 PDL 的结构和功能不均匀，因此有必要采用一种处理整个齿-PDL-骨骼复合体完整 3D 结构的方法。
本文将描述在小鼠中生成 OTM 的方法和两种在不分割样品的情况下实现 PDL 中胶原纤维的 3D 可视化的方法。
Murine 模型广泛应用于医学、发育生物学、药物输送和结构研究中的活体实验。它们可以进行基因改造，以消除或增强特定的蛋白质和功能：它们提供快速、可重复和可预测的发展控制;它们也很容易图像，因为他们的小尺寸8。尽管它们有许多优点，但牙科研究中的鼠标模型并不经常使用，尤其是在需要临床操作时，这主要是因为牙齿尺寸小。动物模型，如老鼠9，10，11，狗12，13，猪14，15，16和猴子17比老鼠使用更多。随着高分辨率成像技术的不断发展，利用鼠标模型破译OTM中错综复杂的过程的优点是多种多方面的。本文提出了一种方法，以产生摩尔牙在可塑性与恒定力水平，触发骨重塑的中微运动。大多数在啮齿动物中的OTM实验是在最大值进行的，因为可操纵性和舌头的存在增加了另一个复杂程度。但是，当需要 3D 结构完整性时，可强制具有许多优势。它可以很容易地解剖为整个骨头：在某些物种中，它可以通过纤维共生被分成两个下摆：它是紧凑的，平坦的，只包含没有任何鼻窦空间的牙齿。相比之下，最大骨是头骨的一部分，与其他器官和结构密切相关，因此需要进行广泛的分割，以便用相关的牙齿解剖杏骨。
我们利用室内湿度室与高分辨率微CT内部的加载系统相结合，实现相位增强，开发出一种3D中可视化新鲜纤维组织的方法，如先前描述的9、18、19、20、21、22、23。动物牺牲后立即扫描新鲜组织，无需任何污渍或固定，从而减少组织人工制品以及生物力学特性的改变。这些3D数据可用于分布和方向分析的纤维，如其他地方描述19。
这里介绍的第二个 3D 全组织成像方法基于可拆解的光学清除，使 PDL 纤维在无需任何分割的情况下通过骨骼成像。有趣的是，它也使骨骼本身的胶原纤维可视化，但这里不会讨论这个问题。一般来说，组织清除有两种方法。第一种是水基清除，样品浸入水溶液中，通过简单的浸入、高水化或水凝胶嵌入，折射指数大于 1.4。然而，这种方法在透明度和组织结构保存方面是有限的，因此需要固定组织。第二种方法，产生高度透明的样品，不需要固定是溶剂为基础的清除方法24，25。我们为曼迪布拉样品生成了基于乙基-3-苯丙-2-苯酸酯（乙基肉碱，ECi）的改性溶剂清除方法。该方法具有使用无毒食品级清除剂、最小组织收缩和荧光蛋白保存等优点。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-mL BD Luer-Lok syringe</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>1X phosphate buffered saline</td>
			<td>VWR Life Sciences</td>
			<td>0780-10L</td>
			<td></td>
		</tr>
		<tr>
			<td>200 proof ethanol</td>
			<td>VWR Life Sciences</td>
			<td>V1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum alloy 5019 wire</td>
			<td>Sigma-aldrich</td>
			<td>GF15828813</td>
			<td>0.08 mm diameter wire, length 100th, temper hard. Used as wire ligature around molar.</td>
		</tr>
		<tr>
			<td>Avizo 9.7</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>Used to analyze microCT scans</td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Clr Plan-Apochromat 20x/1.0,CorrVIS-IR M27 85mm</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Used for second harmonic generation imaging</td>
		</tr>
		<tr>
			<td>Cone socket handle, single ended, hand-form</td>
			<td>G.Hartzell and son</td>
			<td>126-CSH3</td>
			<td>Handle of the inspection mirror</td>
		</tr>
		<tr>
			<td>EC Plan-Neofluar 5x/0.16</td>
			<td>Zeiss</td>
			<td>440321-9902</td>
			<td>Used for light-sheet imaging</td>
		</tr>
		<tr>
			<td>Elipar DeepCure-S LED curing light</td>
			<td>3M ESPE</td>
			<td>76985</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf safe-lock tubes, 1.5mL</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cinnamate, &#62;= 98%</td>
			<td>Sigma-aldrich</td>
			<td>W243000-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle, 27G x 1/2&#39;&#39;</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketathesia 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-0702-1</td>
			<td></td>
		</tr>
		<tr>
			<td>KIMWIPES delicate task wipers</td>
			<td>Kimberly-Clark</td>
			<td>21905-026 (VWR Catalog number)</td>
			<td>Purchased from VWR</td>
		</tr>
		<tr>
			<td>LightSheet Z.1 dual illumination microscope system</td>
			<td>Zeiss</td>
			<td>LightSheet Z.1/LightSheet 7</td>
			<td>Used for lightsheet imaging</td>
		</tr>
		<tr>
			<td>LSM 880 NLO multi-photon microscope</td>
			<td>Zeiss</td>
			<td>LSM 880 NLO</td>
			<td>Used for two-photon imaging</td>
		</tr>
		<tr>
			<td>MEGAmicro, plane, 5mm dia, SS-Thread</td>
			<td>Hahnenkratt</td>
			<td>6220</td>
			<td>Front surface inspectrio mirror</td>
		</tr>
		<tr>
			<td>MicroCT machine, MicroXCT-200</td>
			<td>Xradia</td>
			<td>MICRO XCT-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Colibri</td>
			<td>Fine Science Tools</td>
			<td>17000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PermaFlo Flowable Composite</td>
			<td>Ultradent</td>
			<td>948</td>
			<td></td>
		</tr>
		<tr>
			<td>Procedure platform</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Custom-made from lab materials</td>
		</tr>
		<tr>
			<td>Routine stereo micscope M80</td>
			<td>Leica Micosystems</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Sentalloy NiTi open coil spring</td>
			<td>TOMY Inc.</td>
			<td></td>
			<td>A 0.15mm diameter closed NiTi coil with an inner coil diameter of 0.9mm delivers a force of 10g. Similar products can be purchased from Dentsply Sirona.&#160;</td>
		</tr>
		<tr>
			<td>T-304 stainless steel ligature wire, 0.009&#39;&#39; diameter</td>
			<td>Orthodontics</td>
			<td>SBLW109</td>
			<td>0.009&#39;&#39;(.23mm) diameter, Soft temper</td>
		</tr>
		<tr>
			<td>X-Ject E (Xylazine) 100mg/ml</td>
			<td>Henry Schein Animal Health</td>
			<td>NDC:11695-7085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Z100 Restorative, A2 shade</td>
			<td>3M ESPE</td>
			<td>5904A2</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d imaging of pdl collagen fibers during orthodontic tooth movement in mandibular murine model

正畸牙齿运动是一种复杂的生物过程，由于外力而改变软硬组织改造。为了了解这些复杂的改造过程，研究牙齿和牙周组织在其3D上下文中至关重要，因此尽量减少任何分割和组织人工制品。由于小老鼠体积小、代谢率高、遗传学和易处理性，它们经常用于发育和结构生物学以及生物力学。原则上，这也使他们成为牙科相关研究的优秀模型。然而，一个主要的障碍是它们的牙齿尺寸小，尤其是摩尔。本文旨在为产生正畸牙齿运动的逐步协议和小鼠手底摩尔牙周韧带纤维成分的三维成像提供两种方法。提出的第一种方法是基于微CT设置，使新鲜胶原蛋白组织具有相增强成像功能。第二种方法是使用乙基肉桂的骨清除方法，使成像通过骨骼无需分割，并保留内源性荧光。结合这种结算方法与记者鼠标像Flk1-Cre：TdTomato 提供了第一次这样的机会来映像 PDL 和肺骨中的 3D 血管。

本文提出了一种生产OTM的方法，以及两种在PDL内对胶原纤维进行三维成像的方法，无需任何分割。出于动物研究目的，当牙齿不对齐时，如果牙齿运动在所有根部产生对腹膜骨的重塑，则被视为正畸运动。为了生成可靠的 OTM，需要在牙齿上施加恒定的力水平。在这里，激活的形状记忆 NiTi 线圈用于在 7 天及以后的实验时间内产生 10 克的一致力（如果有必要）。此处描述的线圈激活 （<strong class="x...

Watch this Scientific Journal Video about 3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model at JoVE.com

3D Imaging of PDL Collagen Fibers during Orthodontic Tooth Movement in Mandibular Murine Model

我们提出了在小鼠中产生正畸牙齿运动的方案，以及无需分割即可对牙周韧带胶原纤维和血管进行3D可视化的方法。

曼迪布勒穆林模型中矫形牙齿运动期间 PDL 胶原蛋白纤维的 3D 成像

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to ...

正畸牙齿运动是一个复杂的生物过程，涉及外部力量和软组织和硬组织的改造。为了了解这些过程，在 3D 环境中研究牙齿和牙周组织至关重要。Murine 模型由于其大小、高代谢率和我们拥有的大量遗传信息，是此类研究的一个很好的候选者。然而，由于小鼠体型小，在小鼠身上产生正畸牙齿运动并不容易。此外，结构分析方法通常涉及分割，这会破坏组织的 3D 结构和上下文。对于 PDL 等非均匀组织而言，保持 3D 结构和上下文至关重要。为了解决这两个障碍，我们提供了一个协议，以产生小鼠第一个手状摩尔的体质正畸牙齿运动，并描述两种方法，使牙周韧带的3D可视化，而无需分割组织。要将麻醉鼠标定位为矫形器设备插入，请将顶部切口钩入回形器环上。使用电源链固定下切口，并用迷你大肠杆菌小鼠缩放器进一步张开嘴。在每只眼睛上涂上一小滴盐水，以防止角膜脱水。这些是设备插入中使用的工具。将一根铝线切成一厘米长。使用显微外科针架，在第一和第二摩尔之间的接触点下方的可亲区域以语言方式从胸针侧滑动导线。将电线从语言侧拉向摩尔的中性侧。将电线的免费端扭曲两到三次以固定。切一块镍钛或镍线圈弹簧约七到九线长。在下部第一摩尔和切口之间插入线圈，通过线圈的两根线将电线的一个自由端传递，然后紧紧地扭动电线的两端，将线圈固定在摩尔上。使用一对钳子拆下电源链。将线圈的两到三个线圈循环到切口上，以锚定线圈。在第一个摩尔和切口之间留下三个活动线。将威胁滑动到切口自由金银边缘。将一层可流动的复合树脂放在线圈的切口边框上，用牙科治疗灯将其固化。固化树脂后更换电源链。修剪摩尔周围的多余电线以避免受伤。使用相同的固化光，加热 NiTi 线圈 20 秒。这将收紧线圈，完成放置应该看起来像这样。解剖的下皮上的软组织可以用无绒擦拭去除。在两根手指之间用样品，以圆周运动摩擦外部，直到取出大部分软组织。其余的可以用一把剪刀切断。清洁的可清洗可在右侧显示。要从微CT扫描将解剖的六溴化物安装在样品室中，将米粒大小的可包装牙科树脂塑造成日志。然后将其放入舞台的示例槽中。将下摆可制于复合材料上。调整位置，直到第一个摩尔以中线槽为中心，遮挡表面为水平。当对定位感到满意时，可以固化复合材料。这是处于适当位置的可强制的示例。拿一小团可包装的牙科树脂。它的直径应该在摩尔的宽度附近。将树脂放在第一个摩尔的遮挡表面上。用水湿润无绒擦拭。将湿巾放在样品阶段的湿度池中。关闭样品室，用螺丝将其贴在微型 CT 上。现在拍摄 2D 图像，同时垂直降低铁锤，直到铁锤尖端被复合材料包围。安全地关闭 X 射线源，打开微型 CT 腔室，并通过透明玻璃窗固化复合材料。现在样品安装正确，可以根据需要进行成像。要生成光学清除的半人掌样品，在 150 万个离心管中准备以下解决方案。4%甲醛，50%和70%乙醇在除离子水中。两管100%乙醇和乙基肉桂。将解剖的半男性化物放在4%的副甲醛中。用铝箔盖住管子，在室温下在柔和的设置上放置在摇杆上六个小时。六小时后，将样品转移到50%乙醇中。将样品放回摇摇器上，在光线覆盖下放置16小时。重复前一步，用70%乙醇取样16小时，然后每次100%乙醇两次，每次16小时。最后将样品移至乙基肉桂至少12小时。可靠的正畸运动是由尼蒂线圈装置在穆林曼迪布拉尔第一摩尔生成的。在9周大的雄性小鼠中，平均通经空间在7天后为40微米。相位增强微CT允许 PDL 的可视化。经过三天的体质正畸运动，PDL密度降低。由于骨表面陨石坑的发展，骨PDL接口更粗糙，这表明骨质疏松活动和骨质吸收。粗糙的表面可以在根的各个层面看到。这表明运动是转化的，而不是小费运动。在第七天，PDL空间比第三天窄，在解剖根表面的骨PDL边界更平滑，这是骨对抗的迹象，如预期的那样，当正畸牙齿运动发生。由于微CT成像时间长，舞台旋转，样品的安全安装至关重要。不稳定的样品，导致模糊扫描，其中可以看到冠周围的剪影和PDL纤维看不到。使用乙基肉桂的光学清除提供了另一种方法来查看 PDL，而无需分割。底板可以通过右侧显示的充分清除样本的拉莫斯看到。这种方法，保留内源性荧光，无需化学固定即可使用。光表显微镜可用于清除样品。通过转基因小鼠，可以观察到荧光血管衬里。如果骨骼未正确清除，PDL 中的血管将不可见，如面板 F. 本协议描述了如何克服在穆林曼迪布勒摩尔牙齿中产生和分析正畸牙齿运动的两个主要挑战。一旦掌握了正畸牙齿运动技术，可以在15分钟内完成。由于小鼠牙齿的微小尺寸和舌头的存在，这个过程具有挑战性。然而，我们提供一切必要的解决办法来克服这些挑战。微型 CT 成像需要相位增强功能。我们使用台式微型 CT 演示了我们的技术。但是，任何能够生成相位增强的设置都会产生同步加速器。ECi 清除结果取决于脱水过程。如果样本不够透明，我们的建议是增加脱水步骤的时间。我们的清除方法也可用于未修复的样品。透明组织可以在微CT中扫描，因为清除过程不影响组织的矿物质含量。因此，我们的方法为荧光和结构信息提供了一种相关的方法，以生成完整的 3D 分析。

Research

JoVE Journal

Medicine

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

科研

医学

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

三维成像伤害表皮内神经纤维在人体皮肤活检

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Determining and Controlling External Power Output During Regular Handrim Wheelchair Propulsion

在常规手部轮椅推进过程中确定和控制外部功率输出

The use of a manual wheelchair is critical to 1% of the world&#39;s population. Human powered wheeled mobility research has considerably matured, which ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

在穆林模型中缺血再灌注损伤后干细胞的心肌内传递延迟

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

多光子活体成像用于监测小鼠后肢模型中动脉生成过程中白细胞募集

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

非酒精性脂肪性肝炎小鼠模型中肝细胞外基质的 3D 成像

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...

Analyzing Alveolar Bone Remodeling to Explore Skeletal Mechanical Biology

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement

Comparing Alveolar and Long Bone Remodeling to Explore OTM Model Potential (Video) | JoVE

使用诱导型成骨细胞谱系特异性 Stat3 敲除小鼠研究正畸牙齿移动过程中的牙槽骨重塑

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial ...

A Murine Maxillary Orthodontic Model Guide to Bone Remodeling Analysis

The Establishment of a Murine Maxillary Orthodontic Model

Author Spotlight: Insights into an Efficient Murine Maxillary Orthodontic Model Protocol

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to ...