感谢上海交通大学医学院耳科研究所的实验室平台和协助。这项工作得到了上海浦江项目（22PJ1409200）的支持;国家自然科学基金（No.11932012）;上海交通大学医学院附属第九人民医院博士后科研基金;上海交通大学医学院附属第九人民医院基础研究项目资助（JYZZ154）。

本文描述的所有实验程序均已获得上海交通大学医学院附属上海市第九人民医院动物护理委员会批准（SH9H-2023-A616-SB）。本研究使用4周龄的C57BL / 6雄性小鼠。所有使用的器械在手术前都经过消毒。
1. 缝合扩张模型的准备
<ol><li>准备两个固定架。<ol><li>使用 0.014 英寸澳焊丝或不锈钢丝（见 材料表）用轻线钳制作螺旋环。环的直径为2 mm，两侧保留1 mm的尾巴（图1A）。</li>
		<li>在高压灭菌器、等离子灭菌器或合适的冷灭菌剂（例如戊二醛）中对手术用的电线和支架进行消毒。</li>
	</ol></li>
	<li>准备弹簧。<ol><li>准备定制的不锈钢弹簧。 注意:本研究使用线径为 0.2 mm、外径为 1.5 mm、间隔空间为 1 mm 且长度为 7 mm 的压力弹簧（图 1B）。每个 1 毫米的弹簧压缩获得约 30 g 的推力。</li>
		<li>在设置之前将弹簧切成可用的长度。在实验前确认专用弹簧的力。 注意:只要平行实验中的力大小相同，弹簧的大小就会变化。如果条件有限，则通过台式单轴测试仪器或小型电子测功机（见材料表）测量力。根据力的大小评估长度变化（图1C-E）。值得注意的是，有必要根据小鼠的年龄、骨骼状况和研究对象来改变弹簧力载荷值。</li>
		<li>准备一根7毫米长的澳大利亚直线或直钢丝，并制作两张直径为2毫米的纸作为屏障（图1F）。</li>
	</ol></li>
</ol>2. 矢状缝扩张手术
<ol><li>麻醉:用替利他明（25mg / kg），唑拉西泮（25mg / kg）和甲苯噻嗪（10mg / kg）麻醉小鼠。同时，在眼睛上涂抹无菌眼膏，以防止角膜干燥。通过脚趾捏来判断小鼠的麻醉深度。 注意:以下特征表明小鼠被适当麻醉:呼吸缓慢而稳定，四肢无力，肌肉松弛，皮肤针灸反射和眼睑反射消失，以及角膜反射较弱。</li>
	<li>毛发去除和消毒:用脱毛膏小心地去除头顶的毛发;避免触摸眼睛。不久之后，交替使用碘伏和 75% 酒精对手术部位进行消毒。通过皮下注射给予美洛昔康（1mg / kg）作为小鼠的镇痛剂。</li>
	<li>身体姿势固定:将鼠标放在前面，用手术胶带将四肢固定在手术台上。</li>
	<li>开放头皮瓣:使用手术剪刀沿着小鼠颈部附近的头皮进行弓形皮瓣，完全暴露矢状缝线和周围的颅骨。之后，在手术台上用 6-0 缝合线固定头皮瓣。</li>
	<li>酸蚀后粘合固定支架。<ol><li>干燥头骨并用37%磷酸蚀刻20秒，并使用生理盐水清理酸蚀刻（图2A）。用橡胶移液器球茎干燥颅骨后，白垩状残留物会很明显。</li>
		<li>用光固化粘合剂将顶骨两侧的两个支架（在步骤1.1中制备）粘合在距矢状缝合线3毫米处（图2B）。</li>
	</ol></li>
	<li>重置头皮瓣。<ol><li>手动放回头皮瓣（图2C）。标记支架的位置，在双侧固定支架的相应位置在头皮上切两个小孔，然后重置拍打的头皮。</li>
		<li>同时，将小环穿过孔，露出皮肤表面。用6-0缝合线缝合弯曲的切口（图2D）。皮肤恢复需要一到三天。每24小时皮下注射美洛昔康（1mg / kg）给小鼠施用1至3天。 注意:手术后，每天检查小鼠头皮的活动以及支架是否脱落。在颜色和厚度方面，皮肤有多种类型;健康的皮肤头皮会保持原来的颜色，缝合后皮肤边缘会逐渐融合在一起。如果发现头皮感染，或者手术装置脱落，请将小鼠从研究中取出并对其进行安乐死。</li>
	</ol></li>
	<li>安装弹簧和导丝。<ol><li>将弹簧切割比两个支架之间的距离长 1 毫米。</li>
		<li>压缩选定的压力弹簧并将其放置在两侧的小线圈之间。</li>
		<li>将不锈钢丝穿过小线圈和弹簧，松开弹簧以获得约 30 g 的起始推力。</li>
		<li>检查鼠标弹簧区域下方的头皮是否没有任何压缩。</li>
		<li>确认粘合牢固无松动后，用光固化粘合剂在弹簧和支架之间粘合两张纸片，以便在弹簧的两端设置屏障（图 2E，F）。</li>
	</ol></li>
</ol>3.矿化骨的双重标记
<ol><li>储备溶液的制备。<ol><li>用含有 0.9% NaCl 和 2% NaHCO3 的蒸馏水制备稀释溶液。</li>
		<li>使用稀释液制备 20 mg/mL 茜素复合物二水合物和 10 mg/mL 钙黄绿素（见 材料表）。</li>
		<li>使用 pH 测量设备将 pH 值调节至 7.4。在两次测量之间冲洗pH探头，以确保读数准确。</li>
		<li>将染料放入无菌容器中，并储存在4°C的冰箱中;箔纸用于防止光线进入。</li>
	</ol></li>
	<li>准备工作溶液。注射前，使用溶解溶液将储备溶液稀释至钙的1mg / mL和茜素复合物的2mg / mL二水合物。</li>
	<li>在两个时间点腹膜内注射5mg / kg钙黄绿素绿和20mg / kg茜素红，以标记矿化骨并分析两次之间的变化。通常，在注射后12-14小时收集样品。 注意:注射前加热染料，并确保注射时间不少于3分钟。注射间隔取决于膨胀时间。在这项研究中，茜素红和钙黄绿素绿分别在扩增前和收集前注射过夜（O/N）。</li>
	<li>在第二次注射后一天收集准备用于组织清除程序的整个颅骨。</li>
</ol>4. EdU染色
<ol><li>腹腔注射:在对小鼠实施安乐死之前2小时腹膜内注射EdU（遵循机构批准的方案）。剂量为1mg / 10g体重。</li>
	<li>标记混合物制备:混合Tris缓冲盐水（最终100 mmol/L，pH 7.6），CuSO4 （最终4 mmol/L），磺基花青3叠氮化物（最终2-5μmol/L）和抗坏血酸钠（最终100 mmol/L，每次使用新鲜）（见 材料表）。</li>
	<li>EdU整体挂片染色:在PEGASOS组织透明化方法（步骤7）中进行组织脱色步骤后，将样品在室温（RT）下放入标记混合物中1天。</li>
</ol>5. 显微计算机断层扫描成像
<ol><li>组织固定和储存:将颅骨固定在4°C O/N的4%多聚甲醛（PFA）中，并在扫描前将其储存在0.5%PFA中。</li>
	<li>扫描和分析:使用高分辨率和体素尺寸为 7 μm 的显微计算机断层扫描 （μCT） 成像系统扫描样品。</li>
</ol>6. PEGASOS组织清理工作液的制备
<ol><li>4% 聚甲醛 （PFA）:将 4 g PFA 粉末溶解在 1× PBS 中至 100 mL。</li>
	<li>心脏灌注液:0.02%肝素，即20mg肝素粉溶于1×PBS至100mL;或者，使用0.05mol/L EDTA，即0.05mol乙二胺四乙酸（EDTA）（见 材料表），溶解在去离子水溶液中至总体积为1L。</li>
	<li>脱钙液:0.5mol/L EDTA，即0.5mol乙二胺四乙酸（EDTA），溶于去离子水溶液中至总体积为1L。</li>
	<li>脱色溶液:25%Quadrol，其是250mL四醇（N，N，N'，N'-Tetrakis（2-羟丙基）乙二胺）（见 材料表）溶于去离子水中至总体积为1L。 注意:由于 Quadrol 的粘度，可以在配置前将其加热到 60 °C 以增加其流动性。</li>
	<li>脱脂液:30%、50%、70%叔丁醇（tB）（见 材料表），按体积分数用水制备。 注意:考虑到纯tB在室温下是结晶的，必须将其加热到60°C直至溶解后才能使用。随后，加入3%至5%（v / v）三乙醇胺（TEA）以调节溶液的pH值>9.5。</li>
	<li>脱水溶液:TB-PEG溶液，70%tB+27%PEG-MMA+3%Quadrol（v/v），其中PEG-MMA是平均分子量为500的聚（乙二醇）甲基丙烯酸甲醚（聚（乙二醇）甲基丙烯酸酯500，PEG-MMA 500）（见 材料表）。</li>
	<li>透明溶液:BB-PEG溶液，75%BB+22%PEG-MMA+3%四醇（v/v），其中BB为苯甲酸苄酯（BB）。</li>
</ol>7. PEGASOS方法的颅骨透明度
<ol><li>通过腹腔注射麻醉剂对小鼠进行麻醉（步骤2.1），并通过捏反应判断小鼠的麻醉深度，以确保心脏灌注前无阻力反应。</li>
	<li>进行心脏灌注和固定。<ol><li>向上握住鼠标的腹部，用胶带固定四肢，并沿着胸部的圆周小心地切开，使其完全暴露。</li>
		<li>用眼科剪刀在右心房切开一个小切口后，立即在左心室的顶端插入灌注22G针。 注意: 仅插入针尖，以避免因过度插入而损坏心脏瓣膜。否则，心脏灌注液会进入肺循环，影响体循环的灌注效果。</li>
		<li>将 30-50 mL 心脏灌注液（步骤 6.2）推入注射器。可以看出，肝脏从鲜红色逐渐变为苍白。</li>
		<li>在右心房流出的液体完全透明后，以等体积注入 4% PFA 溶液。PFA灌注的操作在通风罩中进行。</li>
		<li>解剖并分离组织和器官，并在4°C下用4%PFA固定过夜。</li>
	</ol></li>
	<li>组织脱钙:对于通过EdU染色处理的样品，将固定的硬组织置于0.5mol/L EDTA（pH 7.0）溶液（10mL）中，在37°C的摇床上约2天，并每天更换液体。 注意:在钙螯合剂标记的骨骼的标准化PEGASOS方法中跳过脱钙过程，以完全保留信号传导。需要脱钙来处理骨骼，以可视化内源性荧光或整体免疫染色信号。</li>
	<li>组织脱色:将固定的颅骨置于25%Quadrol溶液（20mL）中，在37°C的摇床上1天，并更换一次液体。 注意:脱色时间与组织中的血红素含量有关。观察处理液的颜色，因为颜色的深度代表需要继续进行液体置换处理。</li>
	<li>EdU染色:将样品在1×PBS中冲洗5分钟（3次），然后将它们浸入标记混合物中1天。之后，在1×PBS中冲洗样品1小时（三次）。</li>
	<li>组织脱脂和脱水<ol><li>将组织依次置于30%tB，50%tB和70%tB溶液中，以在37°C的振床上进行梯度减小。 以每种浓度放置约2小时。</li>
		<li>随后，在37°C的振荡台上将样品在TB-PEG溶液中脱水6小时。 注意:上述处理时间可以根据组织的数量增加或减少。</li>
	</ol></li>
	<li>组织透明度:将完全脱水的组织置于BB-PEG溶液中，在37°C（2-4小时）的振床上，直至透明。目前，样品可以储存长达 1-2 年。 注意:几乎完全清除后，打开管盖，将样品和介质暴露在空气中，摇晃将进一步提高透明度。该方法适用于提高幼鼠个体组织和器官的透明度，以及整个头部和身体的透明度。然而，为了成年小鼠全身的透明度，上述步骤应使用全周期灌注法进行。</li>
</ol>8. 影像学检查
注:在本研究中，共聚焦显微镜用于透明组织的 3D 可视化。光片显微镜也适用于该协议。之前已经验证了几个操作系统可用。这里以激光共聚焦显微镜操作系统为例（见 材料表）。
<ol><li>根据用户手册，按照正确的步骤打开激光共聚焦和 LAS AF 操作界面。在不同的拍摄通道中打开所需的激光，并调整功率。设置光路和相应的波长，茜素红为561nm，钙黄绿素绿为488nm，其中任何一个都用于根据标记混合物的EdU信号。</li>
	<li>将透明样品放入可携带厚标本的凹玻璃培养皿中，包埋盒，或防水胶等自制放置装置，将透明样品浸入透明液体中。</li>
	<li>选择低倍物镜以快速定位样品。使用快速扫描功能对整个颅骨组织进行概览，并找到成像感兴趣的区域。</li>
	<li>设置输出功率，选择扫描模式，首先调整拍摄系数（分辨率、扫描速度、变焦系数、图像质量、平均背景噪点 等）。 注意: 通常，智能增益范围为 500 到 800（信号和噪声变化）;智能偏移尽可能接近 0，同时确保图像质量（以减少背景噪音）。当PMT增益高于800，或HyD增益大于100%，亮度仍然不足时，可以适当提高激光强度，但原则上越低越好。</li>
	<li>设置轴距Z范围和Z步长，从透明组织的最浅侧向下钻取;也就是说，设置最深和最浅的侧面。 注意: 确认每个镜头的分类和工作距离。仔细设置Z范围，不要操作超过所选镜头限制的工作距离。当需要精细调节时，可以选择"z-Galvo"。</li>
	<li>再次设置拍摄参数，然后开始拍摄。完成后，通过多功能模块对图像进行合并处理。最后，按指示的顺序存储和关闭仪器。</li>
</ol>

缝合扩张小鼠拉伸载荷下骨重塑的 3D 可视化

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+posterofrontal/sagittal+suture+mesenchymal+cells+in+vitro.">Isolation and characterization of posterofrontal/sagittal suture mesenchymal cells in vitro.</a> Plastic and Reconstructive Surgery. 119 (3), 819-829 (2007).</li><li>Kong, 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=Isolation+and+characterization+of+human+suture+mesenchymal+stem+cells+in+vitro.">Isolation and characterization of human suture mesenchymal stem cells in vitro.</a> International Journal of Stem Cells. 13 (3), 377-385 (2020).</li><li>Ikegame, 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=Tensile+stress+induces+bone+morphogenetic+protein+4+in+preosteoblastic+and+fibroblastic+cells,+which+later+differentiate+into+osteoblasts+leading+to+osteogenesis+in+the+mouse+calvariae+in+organ+culture.">Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture.</a> Journal of Bone And Mineral Research. 16 (1), 24-32 (2001).</li><li>Liu, S. S., Opperman, L. A., Buschang, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+recombinant+human+bone+morphogenetic+protein-2+on+midsagittal+sutural+bone+formation+during+expansion.">Effects of recombinant human bone morphogenetic protein-2 on midsagittal sutural bone formation during expansion.</a> American Journal of Orthodontics And Dentofacialorthopedics. 136 (6), 768-769 (2009).</li><li>Liang, W., Ding, P., Li, G., Lu, E., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyapatite+nanoparticles+facilitate+osteoblast+differentiation+and+bone+formation+within+sagittal+suture+during+expansion+in+rats.">Hydroxyapatite nanoparticles facilitate osteoblast differentiation and bone formation within sagittal suture during expansion in rats.</a> Drug Design Development and Therapy. 15, 905-917 (2021).</li><li>Morinobu, 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=Osteopontin+expression+in+osteoblasts+and+osteocytes+during+bone+formation+under+mechanical+stress+in+the+calvarial+suture+in+vivo.">Osteopontin expression in osteoblasts and osteocytes during bone formation under mechanical stress in the calvarial suture in vivo.</a> Journal of Bone And Mineral Research. 18 (9), 1706-1715 (2003).</li><li>Hwang, S., Chung, C. J., Choi, Y. J., Kim, T., Kim, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cetirizine,+a+histamine+1+receptor+antagonist,+on+bone+remodeling+after+calvarial+suture+expansion.">The effect of cetirizine, a histamine 1 receptor antagonist, on bone remodeling after calvarial suture expansion.</a> Korean Journal of Orthodontics. 50 (1), 42-51 (2020).</li><li>Jing, 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=Tissue+clearing+of+both+hard+and+soft+tissue+organs+with+the+PEGASOS+method.">Tissue clearing of both hard and soft tissue organs with the PEGASOS method.</a> Cell Research. 28 (8), 803-818 (2018).</li><li>Jing, 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=Tissue+clearing+and+its+application+to+bone+and+dental+tissues.">Tissue clearing and its application to bone and dental tissues.</a> Journal of Dental Research. 98 (6), 621-631 (2019).</li><li>Luo, W., 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=Investigation+of+postnatal+craniofacial+bone+development+with+tissue+clearing-based+three-dimensional+imaging.">Investigation of postnatal craniofacial bone development with tissue clearing-based three-dimensional imaging.</a> Stem Cells Development. 28 (19), 1310-1321 (2019).</li></ol>

作者没有什么可透露的。

我们应用了标准的缝合扩张小鼠模型来观察在整个为期一个月的重塑周期10 中每周发生的规律形态变化。该模型可用于通过扩大颅骨缝合线来研究颅骨重塑和再生，以及研究体内各种缝合细胞。为了充分展示此类研究的结果，需要对染色组织进行三维可视化。因此，PEGASOS技术以其清除硬组织的效率而闻名19,20，与双重标记和EdU?...

颅面缝合线是连接颅面骨的纤维组织，在颅面骨的生长和重塑中起着至关重要的作用。缝合线的结构类似于河流，提供细胞资源流来滋养和构建"河岸"，称为成骨前沿，通过膜内成骨促进颅面骨的形成1。
对颅面缝合线的兴趣是由临床需求驱动的，即了解颅缝过早闭合和面部缝合功能障碍，这可能导致颅面畸形，甚至危及儿童生命的疾病。开放缝合切除术是临床治疗的常规应用，但长期随访显示一些患者再骨化复发不完全2.微创开颅手术由扩张弹簧或内窥镜条纹颅骨切除术辅助，可能提供一种更安全的方法来保留潜在的缝合线，而不是丢弃组织3.同样，面罩和扩张器具等骨科疗法已被广泛用于治疗矢状或水平上颌骨发育不全，一些研究将年龄限制扩大到通过微型螺钉辅助腭扩张器治疗成年患者 4,5,6。此外，间充质干细胞 （MSCs） 结合可生物降解材料的颅缝再生是未来的潜在疗法，为相关疾病的治疗提供了新的方向7.然而，缝合线的功能过程或调节机制仍然难以捉摸。
骨重塑主要由成骨细胞进行的骨形成和破骨细胞进行的骨吸收之间的平衡组成，其中机械信号刺激的干细胞成骨分化起着重要作用。经过几十年的研究，人们发现颅面缝合线是高度可塑的间充质干细胞生态位8。缝合干细胞（SuSCs）是一组异质性干细胞，属于间充质干细胞（MSCs）或骨干细胞（SSCs）。SuSCs 在体内 由Gli1、Axin2、Prrx1和Ctsk等4个标记物标记，特别是Gli1+ SuSCs，严格验证了干细胞的生物学特性，不仅表现出典型MSC标志物的高表达，而且表现出优异的成骨和软骨生成潜力9。先前的研究表明，Gli1+ SuSCs 在拉伸力下积极促进新骨形成，将它们确定为支持牵引成骨的缝合干细胞来源10。
过去，通过Flexcell、四点弯曲、微磁体加载系统等在体外研究了干细胞的广泛机械特性。尽管在体外11中已经鉴定出小鼠颅骨缝合来源的间充质细胞，并且最近也分离出人类缝合间充质干细胞12，但缝合细胞的生物力学反应在体外系统中仍不清楚。为了进一步研究骨重塑过程，建立了基于分离颅骨器官培养的缝合扩张模型，为建立有用的体内缝合扩张模型铺平了道路 1,13。兔14 和大鼠15 是缝合扩张基础研究中应用最广泛的动物。然而，小鼠因其与人类高度同源的基因组、众多的基因修饰系和较强的生殖杂交能力，是探索人类疾病的首选动物模型。现有的颅骨缝合扩张小鼠模型通常依靠不锈钢正畸弹簧线对矢状缝线施加拉伸力16,17。在这些模型中，在顶骨的每一侧各打两个孔来固定扩张装置，并将导线嵌入皮肤下，这可能会影响细胞活化模式。
在可视化方法方面，切片在矢状方向上的二维观察已经普遍采用数十年。然而，考虑到骨重塑是一个复杂的三维动态过程，获取完整的三维信息已成为当务之急。PEGASOS组织透明技术的出现是为了满足这一要求18,19。它为硬组织和软组织的透明度提供了独特的优势，使完整的骨重塑过程能够在三维空间中再现。
为了更深入、更全面地了解骨重塑期的生理变化，建立了一个标准的矢状缝合扩张小鼠模型，在手工制作的支架之间设置了弹簧设置10。通过标准化的酸蚀刻和粘合程序，膨胀装置可以牢固地粘合到颅骨上，产生垂直于矢状缝的拉力。此外，在矿化骨扩张后双标记后应用PEGASOS组织清除方法，以充分可视化缝合扩张后的骨骼建模变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>37% Acid etching</td>
			<td>Xihubiom</td>
			<td>E10-02/1807011</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A3882</td>
			<td></td>
		</tr>
		<tr>
			<td>AUSTRALIAN WIRE</td>
			<td>A.J.WILCOCK</td>
			<td>0.014&#39;&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B6630</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcein green</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper(II) sulfate, anhydrous</td>
			<td>Sangon Biotech</td>
			<td>A603008</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamometer</td>
			<td>Sanliang</td>
			<td>SF-10N</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU</td>
			<td>Invitrogen</td>
			<td>E104152</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Confocal Microscope</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sangon Biotech</td>
			<td>E607008</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-MMA 500</td>
			<td>Sigma-Aldrich</td>
			<td>447943</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6148&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Meters</td>
			<td>Mettler Toledo</td>
			<td>S220</td>
			<td></td>
		</tr>
		<tr>
			<td>Quadrol</td>
			<td>Sigma-Aldrich</td>
			<td>122262</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Ascorbate</td>
			<td>Sigma-Aldrich</td>
			<td>A4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sangon Biotech</td>
			<td>A500873</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sangon Biotech</td>
			<td>A610476</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring</td>
			<td>TAOBAO</td>
			<td>0.2*1.5*1*7</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-Cyanine3 azide</td>
			<td>Lumiprobe</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butanol</td>
			<td>Sigma-Aldrich</td>
			<td>360538&#160;</td>
			<td>Protect from light. Do not freeze.</td>
		</tr>
		<tr>
			<td>Transbond MIP 
			Moisture Insensitive Primer</td>
			<td>3M Unitek</td>
			<td>712-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Transbond XT 
			Light Cure Adhesive Paste</td>
			<td>3M Unitek</td>
			<td>712-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine</td>
			<td>Sigma-Aldrich</td>
			<td>V900257</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-buffered saline</td>
			<td>Sangon Biotech</td>
			<td>A500027</td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3-d visualization technique for bone remodeling in a suture expansion mouse model

颅面缝合线除了是连接颅面骨的纤维关节外，还起着至关重要的作用;它们也是颅骨和面部骨骼生长的主要生态位，容纳间充质干细胞和骨祖细胞。由于大多数颅面骨是通过膜内骨化发育的，因此缝合线的边缘区域充当起始点。由于这一重要性，这些缝合线已成为骨科治疗中有趣的靶标，如弹簧辅助颅穹窿扩张、上颌骨快速扩张和上颌骨牵伸。在骨科示踪力的作用下，缝合干细胞被迅速激活，成为扩增过程中骨重塑的动态来源。尽管它们很重要，但骨重塑期间的生理变化仍然知之甚少。传统的切片方法，主要是矢状方向，无法捕捉到整个缝合线中发生的全面变化。本研究建立了矢状缝扩张的标准小鼠模型。为了充分可视化缝合扩张后的骨重塑变化，PEGASOS组织清除方法与全安装EdU染色和钙螯合双标记相结合。这使得在扩张后在整个颅骨上看到高度增殖的细胞和新的骨形成。该协议提供了标准化的缝合扩张小鼠模型和3D可视化方法，揭示了拉伸力载荷下缝合和骨重塑的机械生物学变化。

使用该协议，已经建立了矢状缝合线扩张的小鼠模型（图1-2）。对于缝合线扩张后骨骼建模变化的 3D 可视化，将 PEGASOS 组织清除方法应用于扩张后的整个颅骨。灌注后，分离颅骨（图3A），并继续适当的PEGASOS过程（表1和表2）。值得注意的是，无论是否进行脱钙，在完整的PEGASOS过程后，颅骨几乎变得?...

Watch this Scientific Journal Video about PEGASOS Tissue Clearing Technique to Visualize Bone Remodeling at JoVE.com

作者聚焦:PEGASOS 组织透明化技术可视化骨骼重塑 

该协议提出了一个标准化的缝合扩张小鼠模型和3-D可视化方法，以研究拉伸力载荷下缝合线和骨重塑的机械生物学变化。

缝合扩张小鼠模型中骨重塑的 3D 可视化技术

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...

对颅面缝合的兴趣是由了解正畸和骨科治疗机制的临床需求所驱动的。我们的研究范围是建立标准化的缝合扩张小鼠模型，以及3D可视化方法，以研究缝合和扩张时骨重塑的机械生物学变化。PEGASOS组织透明化技术与多种标记方法相结合，从空间角度获得缝合和粘结重塑的多功能信息。该模型适用于不同年龄的转基因小鼠的各种强度。方便随时调整拉应力的大小和拉力。通过PEGASOS技术，我们可以观察骨重塑过程中颅缝干细胞的三维时空分布。通过PEGASOS技术，我们将进一步探索缝合干细胞与机械应力之间的关系，以及其具体机制。

a-3-d-visualization-technique-for-bone-remodeling-suture-expansion

Research

JoVE Journal

Biology

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

1.3K 次观看.  Shanghai Jiao Tong University School of Medicine. 对颅面缝合的兴趣是由了解正畸和骨科治疗机制的临床需求所驱动的。我们的研究范围是建立标准化的缝合扩张小鼠模型，以及3D可视化方法，以研究缝合和扩张时骨重塑的机械生物学变化。PEGASOS组织透明化技术与多种标记方法相结合，从空间角度获得缝合和粘结重塑的多功能信息。该模型适用于不同年龄的转基因小鼠的各种强度。方便随时调整拉应力的大小和拉力。?...

视频: 作者聚焦:PEGASOS 组织透明化技术可视化骨骼重塑 

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

一个现场的固定化酵母细胞可视化的快速技术

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

在哺乳动物细胞中的内质网定位的mRNA的可视化

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

小鼠模型的验证扰乱LINC配合物在细胞特异性的方式

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

牛膝外生软骨重塑的外骨组织培养模型

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

离体 聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...