作者想感谢 Dr. 的好心提供了转基因小鼠和李 Chen 的组织学帮助。
该出版物的研究报告得到了国立卫生研究院的牙科 & 颅面研究协会的支持, 该研究所的奖项编号 K08DE025914 和美国正畸基金会素密亚达夫。

康涅狄格大学卫生中心的机构动物保育委员会批准了所有的动物程序。
1. 压缩静态颞下颌关节负荷: 口强行打开
注: 四周老的转基因小鼠窝藏荧光记者的胶原蛋白 (Col2a1XCol10a1), 亲切提供由 Dr. 大卫罗 (康涅狄格大学), 被用于本手稿描述的实验 (n = 8; 4 男性和4女性)。Col2a1 青色 (蓝色) 转基因是在 MCC 的 prehypertrophic 区的细胞中表达的, 而 Col10a1 樱桃 (红) 细胞存在于肥厚区8 (图 1)。小鼠平均分为两组: 1) 荷载组, 小鼠受压迫静态颞下颌关节负荷 (描述在步骤 2) 和 2) 控制组, 其中小鼠不接受干预。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55998/55998fig1.jpg"> 图 1。double-collagen 荧光报告鼠 (Col2a1XCol10a1) 的髁突的代表性矢状.缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>制造弹簧 usingbeta 钛合金丝 (0.017 x 0.025 英寸) (图 2A)。</li>
	<li>通过腹腔注射氯胺酮 (90 毫克/千克体重) 和嗪 (13 毫克/千克体重) 的混合物麻醉实验动物。</li>
	<li>轻轻地打开小鼠的嘴巴, 插入弹簧, 使上颌和下颌切牙的循环啮合 (图 2B 和 C)。</li>
	<li>在1小时的麻醉小鼠的门牙中保持弹簧, 从而诱发压缩静态颞下颌关节负荷. 重复这个过程5天。</li>
	<li>注射 5-ethnyl-2-脱氧 (教育; 30 毫克/千克体重) 腹腔细胞增殖分析2天和1天前安乐死的小鼠。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55998/55998fig2.jpg"> 图 2。压缩静态颞下颌关节负荷: 口强迫开放模型.(A)弹簧制作 0.017 x 0.025 β钛合金丝。(B)装满弹簧的鼠标。(C)载入和控制小鼠的 x 射线显示下颌骨定位的差异。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 下颌骨解剖和固定
<ol><li>在强制开放口程序的端点, 通过一种认可的方法安乐实验动物。</li>
	<li>解剖下颌骨, 切断肌肉附着, 而不刮软骨的髁突。</li>
	<li>将清洁的下颌骨放在10% 福尔马林中, 用于24小时固定。 注意: 福尔马林是刺激性的;穿戴适当的个人防护设备。</li>
</ol>3. x 射线成像和形态测量
<ol><li>将下颌骨放在一个扁平的容器中 (例如,一个 55 mm x 16 mm 的培养皿), 并在26伏的 5 s 上使用一个机柜 x 射线系统对样品进行射线照相。 注意: 在保存图像之前放置刻度线。</li>
	<li>使用图像分析软件 (请参见材料表) 对下颌骨进行计量测量。 注意: 在执行任何测量之前, 使用 x 射线的刻度栏来确定单位。这一步是重要的正确测量解剖结构。单击 "单位" 按钮 (图 3A)。</li>
	<li>使用成像软件中的 "标记样式 2" (图 3B) 选择解剖点。要遵循本文中描述的方法, 请选择以下点 (图 3B):<ol>
<li>选择 condylion (点 1), 下颌髁突的最后点;</li>
		<li>选择切牙过程 (点 2);</li>
		<li>选择最深的点在乙状结肠切口 (点 3);</li>
		<li>选取下颌升支凹陷的最深点 (点 4);</li>
		<li>选择髁突关节面最前方的点 (点 5);和</li>
		<li>选择髁突关节面的最后点 (点 6)。</li>
	</ol>
</li>
	<li>在选择解剖点后, 使用 "长度" 工具进行形态学测量, 但对于髁突长度, 使用 "垂直线" 工具从点 (3) 和 (4) (图 3C)。<ol>
<li>测量下颌长度, 从 condylion (1) 到切牙过程 (2)。</li>
		<li>测量髁突长度-从 condylion (1) 的垂直距离, 到从乙状结肠缺口 (3) 的最深点到下颌升支 (4) 凹陷最深点的线。</li>
		<li>测量髁突的头部宽度--从最前面到最后点的髁关节面的距离 (5-6)。</li>
		<li>从屏幕右侧的 "测量列表" 中复制度量值 (图 3C)。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55998/55998fig3.jpg"> 图 3.下颌骨形态测量的表示。(A)使用 x 射线的刻度线确定单位 (以红色圆圈、刻度线:10 mm)。(B)使用 "标记样式 2" (红色圆圈) 选择解剖学点。1) Condylion;2) 切牙工艺;3) 在乙状结肠缺口处最深的点;4) 下颌支的凹陷最深的点;5) 髁突关节面的大部分前点;6) 髁突关节面最后点。缩放栏:10 mm(C)使用 "长度" 和 "垂直" 工具 (用红色圆圈) 执行测量。测量从点1到 2: 下颌长度;从5点到 6: 髁突宽度;垂直从点1到 4-3: 髁突头长度。从 "测量列表" 中保存度量值。缩放条 = 10 mm<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
4. 髁突嵌入
注意: 在拍摄影像后, 下颌骨可以嵌入和分段进行组织学分析。
<ol><li>放置脱下颌骨 (已固定在步骤 2.3) 在30% 蔗糖在 PBS 过夜前嵌入。</li>
	<li>解剖任何多余的软组织, 并仔细切割下颌髁突。</li>
	<li>在一次性塑料模具中倒入一些嵌入树脂 (足够覆盖样品), 并将髁突内侧面与模具底座对中, 将样品与模具的底部平行放置。</li>
	<li>用一块干冰把标本固定在正确的地方。</li>
	<li>用嵌入树脂填充模具, 在冷 2-甲基丁烷中放置固定样品的模具, 可在-20 ° c 或-80 ° c 的冷冻机或干冰中 pre-chilled。</li>
	<li>将标本存放在-20 ° c 或-80 ° c, 直到切片。</li>
</ol>5. 髁突矢状切面和滑动准备
<ol><li>创建髁 (5-7 µm) 的冰冻矢状切面, 并使用磁带传输方法10,11将样本传输到幻灯片。</li>
	<li>使用紫外光固化方法10将组织学切片粘附到磁带上。</li>
</ol>6. 组织学染色和显微成像
注意: 大多数组织学染色是通过 Dyment et al10在本文的组织学部分进行描述的。
<ol><li>对于基线扫描, 第一步是对 Col2a1 和 Col10a1 转基因进行映像, 如 Dyment et al10所述。<ol>
<li>将片放在30% 甘油和 PBS 的幻灯片上。执行基线成像 ofthesectionswitha 荧光显微镜和适当的过滤器。</li>
	</ol>
</li>
	<li>执行荧光抗酒石酸酸性磷酸酶 (TRAP) 染色。 注: 陷阱由造血细胞表达, 包括骨吸收细胞, 破骨12。该染色的目的是分析 MCC 和下骨重塑。<ol>
<li>通过在 PBS 中浸泡来去除幻灯片的片。在 PBS 三次洗涤幻灯片5分钟每。</li>
		<li>在蒸馏水的 1 l 中, 用无水 (9.2 克) 和 l-酒石酸钠 (11.4 克) 溶解醋酸钠, 制备疏水阀缓冲1。用乙酸调节 pH 值至 4.2, 并在4° c 处储存陷阱缓冲器1。</li>
		<li>在1毫升蒸馏水中, 用新溶解的亚硝酸盐 (40 毫克) 制备陷阱缓冲器2。</li>
		<li>通过混合 7.5 mL 缓冲器1和150µL 的缓冲 2, 准备陷印基板缓冲 (就在染色前)。将此解决方案 (200 µL) 应用于每张幻灯片, 在室温下为10分钟。</li>
		<li>通过混合1.840 毫升的基底缓冲液和23µL 的荧光基板来制备陷阱反应缓冲器 (参见材料表)。 注: 用于陷阱活动的荧光 substrategeneratesayellowfluorescentsignal。</li>
		<li>通过轻轻移解决方案, 去除多余的陷印基底缓冲器。</li>
		<li>在紫外线源灯泡黑光下, 用200µL 的陷阱反应缓冲液孵育5分钟的幻灯片。</li>
		<li>在 PBS 三次洗涤幻灯片5分钟每。</li>
		<li>将片放在 PBS 的30% 甘油的幻灯片上, 并进行显微成像10。</li>
	</ol>
</li>
	<li>进行细胞增殖染色。 注: 在本试验中, 经改良的胸苷模拟 5-乙炔-2-脱氧 (教育) 被纳入新合成的 DNA: 分裂细胞被标记为荧光染料。<ol>
<li>在对陷阱进行成像后, 取出片并在 PBS 中清洗三次, 每次5分钟。按照 5-乙炔-2 脱氧细胞增殖试剂盒中的步骤来染色组织学切片。</li>
		<li>准备反应鸡尾酒加入430µL 1X 反应缓冲, 20 µL 丘索4, 1.2 µL 荧光染料, 和50µL 的1X 缓冲添加剂 (总体积500µL 是足够的一张幻灯片)。 注: 制造商建议按照此处所述的顺序添加配料。</li>
		<li>用准备好的鸡尾酒在室温下孵育30分钟的幻灯片, 保护其免受光照。</li>
		<li>在 PBS 三次洗涤幻灯片5分钟每。</li>
		<li>将片放在 PBS 的30% 甘油的顶部, 1:1, 000 DAPI。 注: DAPI (4 ", 6-diamidino-2-吲) 是一种蓝色的荧光核染色, 结合在丰富的区域在 DNA。</li>
	</ol>
</li>
	<li>执行甲苯胺蓝 (TB) 染色。 注意: 结核染色显示了 MCC 的蛋白多糖分布。<ol>
<li>冲洗幻灯片 indistilledwater 以删除片。</li>
		<li>去除片后, 在蒸馏水中清洗三次, 每次5分钟, 完全除去 PBS 中的盐。</li>
		<li>通过使溶液 A (在蒸馏水的1升中溶解28.4 克磷酸二钠化合物) 和溶液 B (溶解27.6 克磷酸二在1升蒸馏水中) 制备结核工作缓冲器。混合94.7 毫升溶液 A 与5.3 毫升溶液 b 稀释此溶液1:1 在蒸馏水, 使200毫升0.1 米的工作缓冲。通过在 ph 值固定前加入氢氧化钠, 将 ph 值校正到8.0。</li>
		<li>准备1% 股结核: 在100毫升蒸馏水中混合1克结核。</li>
		<li>在3毫升1% 的储存结核中, 将40毫升的0.1 米工作缓冲液混合, 准备结核工作溶液。</li>
		<li>在14-17 秒内孵育结核工作解决方案中的幻灯片。 注意: 这个污点是非常时间敏感;潜伏期可能需要调整, 以防止 overstaining。</li>
		<li>用蒸馏水冲洗三次, 每次5分钟。</li>
		<li>将片放在30% 甘油的蒸馏水中。执行光学显微镜成像10。 注意: 不要片在甘油 + PBS 中染有结核的幻灯片。PBS 清洗这种类型的污渍。</li>
	</ol>
</li>
</ol>7. 荧光组织学定量
<ol><li>使用图像分析软件对荧光图像进行组织学量化 (请参见材料表)。 注: 该方法的组织学定量的基本原理是将感兴趣的荧光像素的数目除以感兴趣区域的总像素数, 并将所获得的数乘以 100, 从而产生一个正像素的百分比在该区域内。</li>
	<li>对髁矢状段的 Col10a1 表达进行量化。<ol>
<li>使用此方法对所有类型的单元格和污点进行量化时, 请选择要量化的区域。打开图像分析软件中的组织学图像, 然后使用 "套索工具 (L)" (图 4A) 选择区域。对于此处所述的分析, 仅选择 MCC 区域;选择区域后, 将在软件屏幕上的 "直方图" 框 (图 4A) 上显示总的像素数。保存感兴趣区域中的像素数。</li>
		<li>使用采样工具选择 Col10a1 红色像素。单击顶部面板上的 "选择", 然后 "颜色范围"。单击 "吸管工具", 选择图像中感兴趣像素的样本颜色, 然后单击 "确定"。选择的像素颜色的所有区域都将被选中 (图 4B)。复制荧光像素的数量 (显示在屏幕的 "直方图" 框中), 并将它们粘贴到电子表格或统计软件中进行分析。</li>
		<li>将荧光像素的数目除以感兴趣区域的像素数, 并将此数乘以 100: 在感兴趣区域中的荧光像素/像素 * 100。 注意: 其他类型的单元格 (如蓝色 Col2a1) 可以用同样的方法进行量化, 但应选择蓝色像素而不是 Col10a1 红色像素。</li>
	</ol>
</li>
</ol><img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55998/55998fig4.jpg"> 图 4.转基因 Col10a1 量化的表示。(A)使用 "套索工具" (L) 选择感兴趣的区域。对于 Col10a1-positive 细胞, 选择整个下颌骨软骨。保存 "直方图" 框中的像素数。(B)选择感兴趣的像素, 在本例中为红色荧光 Col10a1 像素。请注意, 将只选择感兴趣区域中的红色像素。从 "直方图" 框中保存红色像素的数目。缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="3"><li>对 MCC 和下骨中的陷阱活动进行量化。<ol>
<li>按照步骤8.2 中描述的步骤操作, 但不要仅选择 MCC 区域, 选择软骨和下骨区域 (图 5A)。</li>
		<li>对于陷阱活动分析, 请选择由 ELF97 基板生成的黄色像素, 如步骤 8.2.2 (图 5B) 中所述;复制正像素的个数;并将它们粘贴到电子表格或统计软件中进行分析。</li>
		<li>通过将黄色像素的数目除以下骨区的总像素数并乘以 100, 获取陷阱正像素的百分比。</li>
	</ol>
</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55998/55998fig5.jpg"> 图 5.荧光陷阱量化的表示。(A)选择感兴趣的区域 (下颌软骨和下骨), 并保存该区域的像素数。(B)选择黄色荧光像素, 表示陷阱活动。请注意, 只有陷阱-正像素将被选中。保存所选像素的数目。缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4"><li>对教育阳性细胞进行量化。<ol>
<li>教育染色的幻灯片是1-4 与 DAPI, 因此, 而不是计算总的像素数在感兴趣的区域, 选择 DAPI 阳性像素来计算教育阳性像素的百分比。如步骤8.2 所述, 选择感兴趣的区域, 但不保存总的像素数。选择 DAPI 蓝色像素作为取样颜色, 如步骤8.2.2 所述;复制 DAPI 阳性像素的数目 (图 6A);并将它们粘贴到电子表格或统计软件中进行分析。</li>
		<li>接下来, 选择教育阳性像素 (黄色荧光像素) 并保存 "直方图" 框中的像素数 (图 6B)。</li>
		<li>通过将教育阳性像素的数目除以 DAPI 阳性像素的个数, 并将获得的数字乘以 100, 计算出教育像素的百分比。</li>
	</ol>
</li>
</ol><img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55998/55998fig6.jpg"> 图 6.表示的教育量化。(A)选择 MCC 的增殖区 (软骨的外层)。选择 DAPI-正像素并保存像素数。(B)选择 "教育阳性像素" (黄色荧光) 并保存像素数。缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
8. 软骨厚度和蛋白多糖分布的定量
<ol><li>使用 Digimizer 图像软件分析软骨厚度 (距离映射) 和甲苯胺蓝染色区。 注意: 使用组织学图像中的刻度线来确定单元 (图 7A)。</li>
	<li>执行距离映射测量。 注: 距离测绘测量将提供下颌髁突软骨的厚度。在这里描述的方法中, 选择了 mcc 的五区域, 给出了 mcc 总厚度的平均值。研究人员可以选择测量三不同的区域, 甚至在 MCC 中间的一个单一区域。<ol>
<li>使用 "长度" 工具, 测量软骨从外表面到甲苯胺蓝染色区 (图 7B) 的长度, 在五地区, 或在尽可能多的位置的首选。</li>
		<li>从 "测量列表" 中复制长度度量值。 注意: 该软件还提供了测量的平均值 (图 7B)。</li>
	</ol>
</li>
	<li>测量甲苯胺蓝染色区。 注: 在甲苯胺蓝染色区的测量中, 将得到 MCC 的蛋白多糖区。<ol>
<li>选择 "区域" 工具和轮廓的甲苯胺蓝染色区域 (图 7C)。</li>
		<li>从 "测量列表" 中复制区域测量。</li>
	</ol>
</li>
</ol><img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55998/55998fig7.jpg"> 图 7: 蛋白多糖分布量化的表示。(A)使用组织学图像的刻度线通过单击 "单位" 按钮 (红色圆圈, 选定单位: 500 µm) 来确定单位。(B)使用 "长度" 工具 (用红色圆圈) 测量不同位置软骨的厚度。在右上面板的 "测量列表" 中保存度量值。该软件还提供了右下面板的 "统计", 因此可以直接得到测量的平均值和 SD。(C)使用 "区域" 工具 (用红色圆圈) 测量甲苯胺蓝染色区域。圈出感兴趣的区域, 并从 "测量列表" 中保存度量值。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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	<li>LeResche, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+Temporomandibular+Disorders:+Implications+for+the+Investigation+of+Etiologic+Factors.">Epidemiology of Temporomandibular Disorders: Implications for the Investigation of Etiologic Factors.</a> Crit Rev Oral Biol Med. 8 (3), 291-305 (1997).</li><li>Chen, 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=Altered+functional+loading+causes+differential+effects+in+the+subchondral+bone+and+condylar+cartilage+in+the+temporomandibular+joint+from+young+mice.">Altered functional loading causes differential effects in the subchondral bone and condylar cartilage in the temporomandibular joint from young mice.</a> Osteoarthr Cartil. 17 (3), 354-361 (2009).</li><li>Pirttiniemi, P., Kantomaa, T., Sorsa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+decreased+loading+on+the+metabolic+activity+of+the+mandibular+condylar+cartilage+in+the+rat.">Effect of decreased loading on the metabolic activity of the mandibular condylar cartilage in the rat.</a> Eur J Orthod. 26 (1), 1-5 (2004).</li><li>Chavan, S. J., Bhad, W. A., Doshi, U. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+temporomandibular+joint+changes+in+Twin+Block+and+Bionator+appliance+therapy:+a+magnetic+resonance+imaging+study.">Comparison of temporomandibular joint changes in Twin Block and Bionator appliance therapy: a magnetic resonance imaging study.</a> Prog Orthod. 15 (57), (2014).</li><li>Dutra, E. 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=Cellular+and+Matrix+Response+of+the+Mandibular+Condylar+Cartilage+to+Botulinum+Toxin.">Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.</a> PLoS ONE. 11 (10), 0164599 (2016).</li><li>Benjamin, M., Ralphs, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+fibrocartilage+cells.">Biology of fibrocartilage cells.</a> Int Rev Cytol. 233, 1-45 (2004).</li><li>Shen, G., Darendeliler, M. 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+Adaptive+Remodeling+of+Condylar+Cartilage-+A+Transition+from+Chondrogenesis+to+Osteogenesis.">The Adaptive Remodeling of Condylar Cartilage- A Transition from Chondrogenesis to Osteogenesis.</a> J Dent Res. 84 (8), 691-699 (2005).</li><li>Utreja, 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=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Kaul, 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=The+Effect+of+Altered+Loading+on+Mandibular+Condylar+Cartilage.">The Effect of Altered Loading on Mandibular Condylar Cartilage.</a> PLoS ONE. 11 (7), 0160121 (2016).</li><li>Dyment, N. 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=High-Throughput,+Multi-Image+Cryohistology+of+Mineralized+Tissues.">High-Throughput, Multi-Image Cryohistology of Mineralized Tissues.</a> J Vis Exp. , e54468 (2016).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch Histol Cytol. 66 (2), 123-143 (2003).</li><li>Hayman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tartrate-resistant+acid+phosphatase+(TRAP)+and+the+osteoclast/immune+cell+dichotomy.">Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy.</a> Autoimmunity. 41 (3), 218-223 (2008).</li></ol>

作者没有相互竞争的金融利益。

本手稿描述了小鼠下颌髁和下颌骨的形态测量和细胞分析方法。射线照相测量也可以用来分析其他的骨骼从小实验动物。此外, 细胞分析 (细胞定量和软骨距离映射) 不限于啮齿动物的下颌骨髁突, 但可用于量化组织切片的众多组织上。
表达荧光的转基因小鼠模型是视觉理解基因表达变化的绝佳工具。本报告中使用的 double-collagen 荧光报道鼠模型 (Col2a1XCol10a1) 特别适合于 mcc 的研究, 因为转基因在 mcc 的 prehypertrophic 和肥厚区表达, 使胶原蛋白分布可以进行评估。如果研究者没有接触到表达荧光的转基因小鼠模型, 其他的组织学蛋白表达分析方法, 如免疫荧光法, 都可以使用。
对于形态学测量, 采取的关键步骤前的内阁射线图像是删除所有软组织的下颌骨或骨骼进行分析。过多的肌肉或软组织附着在骨骼可以掩盖的实际测量的结构。重要的是要知道的限制, 内阁射线照相系统。像每一个 x 射线, 它是一个3维结构的2维表示, 和结构和工件的重叠应仔细解释。此外, 在影像机柜中的骨骼定位应该是一致的。
通过计数正像素数进行细胞定量是一种有效的方法, 在细胞丰富的区域 (如 MCC) 进行组织学定量化, 但这种方法的局限性在于它不提供精确的细胞数。此外, 由于它只量化选定的像素, 建议使用相同的 "取样" 像素量化所有感兴趣的图像, 以避免在分析不同图像时对不同强度的像素进行量化。
在量化组织学切片时, 一般的建议是分析每个样本的多个序列 (在这篇手稿中, 每一个髁突都有三个连续切片), 因为每个样本中的变异通常被观察到。
我们在实验中提到的方法简单, 易于使用, 可用于任何矿化组织的研究, 其中使用的是记者小鼠。利用现有的方法, 可以可视化细胞的共存, 将被染色的细胞 (Col2a1 或 Col10a1) 或教育 (Col1a1 或 Col2a1) 形象化。

颞下颌关节是一个独特的 load-bearing 关节位于颅面地区, 是由软骨。颞下颌关节的 MCC 对于关节功能是必不可少的, 包括在说话和咀嚼时不受阻碍的颚运动, 但它通常受到退行性疾病的影响, 包括骨关节炎1。颞下颌关节有能力适应外部刺激和负荷改变, 导致结构和细胞的变化的组成部分的 MCC2,3,4,5。MCC 的 load-bearing 性能可以通过其成分之间的相互作用来解释, 包括水、胶原网络和密被包装的蛋白。MCC 有四独特的细胞区, 表达不同类型的胶原蛋白和 non-collagen 蛋白: 1) 表面或关节区;2) 增殖区, 由未分化的间充质细胞组成, 并对负荷需求作出反应;3) prehypertrophic 区, 由成熟的软骨细胞表达2型胶原;和 4) 肥厚区, 该地区的肥大软骨细胞表达型胶原10死亡, 并经历钙化。non-mineralized 地区是丰富的蛋白, 提供抗压缩力6。
在 MCC 的肥厚区有连续的矿化, 在那里从软骨到成骨的转变发生, 保证下颌髁突的下骨的健壮的矿物结构7。unmineralized 和矿化区的细胞变化最终导致下颌骨髁突和下颌骨的形态学和结构改变。维持 MCC 所有细胞区域的稳态和下部分的矿化, 对颞下颌关节的健康、load-bearing 能力和完整性至关重要。
多胶原蛋白转基因小鼠模型 (如 Utreja et al.所述)8是一个很好的工具, 可以用来理解胶原蛋白表达的变化, 因为所有的转基因都是在 MCC 中表达的。对于 in-depth 的组织学评价, 组织学染色用于研究基质沉积、矿化、细胞增殖和凋亡, 以及在 MCC 不同细胞层的蛋白质表达。
在这篇手稿中, 组织学和形态学分析被用来评价细胞和结构的变化, 在 MCC 和下骨的下颌髁突的小鼠。此外, 还描述了用于分析荧光组织学图像和绘制光学显微镜幻灯片的细胞定量方法。压缩静态颞下颌关节加载方法, 导致细胞和形态学的变化, 在 MCC 和下骨9, 也说明了验证我们的方法。
本文所述方法可用于确定鼠下颌骨髁突和下颌骨的形态和组织学变化, 或分析其他软骨骨化区和其他矿化组织的形态。

<table><tbody><tr><td>MX20 Radiography System</td><td>Faxitron X-Ray LLC&amp;nbsp;</td><td></td><td></td></tr><tr><td>Digimizer Image software&amp;nbsp;</td><td>MedCalc Software</td><td></td><td></td></tr><tr><td>Shandon Cryomatrix embedding resin</td><td>Thermo Scientific</td><td>6769006</td><td></td></tr><tr><td>Manual microscope Axio Imager Z1</td><td>Carl Zeiss</td><td>208562</td><td></td></tr><tr><td>yellow fluorescent protein filter&amp;nbsp; - EYFP</td><td>Chroma Technology Corp</td><td>49003</td><td></td></tr><tr><td>cyan fluorescent protein filter - ECFP</td><td>Chroma Technology Corp</td><td>49001</td><td></td></tr><tr><td>red fluoresecent protein filter - Cy5</td><td>Chroma Technology Corp</td><td>49009</td><td></td></tr><tr><td>sodium acetate anhydrous</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>sodium L-tartrate dibasic dihydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>228729</td><td></td></tr><tr><td>sodium nitrite&amp;nbsp;</td><td>Sigma-Aldrich</td><td>237213</td><td></td></tr><tr><td>ELF97 substrate</td><td>Thermo Fisher Scientific</td><td>E6600</td><td></td></tr><tr><td>ClickiT EdU Alexa Fluor 594 HCS kit</td><td>Life Technologies</td><td>C10339&amp;nbsp;</td><td>includes EdU (5-ethynyl-2&#039;-deoxyuridine)&amp;nbsp;</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Scientific</td><td>D1306</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>Sodium phosphate monobasic&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71505</td><td></td></tr><tr><td>Toluidine Blue O&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Adobe Photoshop&amp;nbsp;</td><td>Adobe Systems Incorporated</td><td></td><td></td></tr><tr><td>Phosphate buffered saline tablets (PBS)</td><td>Research Products International</td><td>P32080-100T</td><td></td></tr><tr><td>CNA Beta III Nickel-Free Archwire</td><td>Ortho Organizers, Inc.</td><td></td><td></td></tr><tr><td>GraphPad Prism&amp;nbsp;</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr></tbody></table>

a morphometric and cellular analysis method for the murine mandibular condyle

颞下颌关节具有适应外部刺激的能力, 负荷变化会影响髁的位置, 以及下颌髁突软骨的结构和细胞成分。本文介绍了分析这些变化的方法和改变小鼠颞下颌关节负荷的方法 (即,压缩静态颞下颌关节负荷)。这里的结构评估是一个简单的形态学方法, 使用 Digimizer 软件, 并在小骨骼的射线照相中进行。此外, 分析了细胞的变化导致胶原蛋白表达改变, 骨重塑, 细胞分裂, 和蛋白多糖分布的 MCC。在组织学切片的这些变化的量化-通过计数的阳性荧光像素使用图像软件和测量的距离测绘和染色面积与 Digimizer-也被证明。这里所示的方法不仅限于小鼠颞下颌关节, 还可用于小型实验动物和其他软骨骨化地区的骨骼。

进行描述性统计, 检查形态学测量的分布 (下颌长度, 髁突长度, 髁突宽度) 和组织学分析。结果被比较了被装载的小组 (即,小鼠受压缩装载与 beta 钛春天) 和控制小组 (即,匹配控制小鼠没有接受任何过程)。统计学意义上的差异之间的方法是由不配对 t 检验, 和 p 值的 < 0.05 被认为是有统计学意义的。
如步骤4和图 3所述, 在加载和控制组的下颌骨中进行了形态学测量。被装载的小组提出以明显地增加的下颌长度 (装载: 16.62 ±0.23 毫米与控制: 16.21 ±0.2 毫米; p < 0.05) 和髁突头长度 (装载: 4.6 毫米, SD 0.08 毫米与控制: 4.4 毫米;SD 0.06;p < 0.05) 与对照组比较。然而, 在髁突宽度上没有显著差异 (载: 3.06 ±0.12 毫米与控制: 2.9 ± 0.11; p = 0.09)。
使用步骤8中描述的方法对胶原蛋白分布的定量显示在负载组的髁的 MCC 中 Col10a1 表达明显增加 (加载: 21% ±4.46% 与控制: 7.50% ± 2.03%; <0.005;图 8A 和 D)。另一方面, 在负载和控制组之间的 Col2a1 表达上没有统计学上的差异 (加载: 4.85% ±1.95% 与控制: 2.92% ± 1.89%; p = 0.13;图 8B 和 E)。此外, 对诱捕器活动的分析显示, 荷载小鼠髁下区的诱捕阳性区增加 (载: 5.28% ±1.45% 与对照: 2.41% ± 1.39%; p < 0.05;图 8C 和 F)。同样, 我们观察到增加的细胞增殖, 如增加的教育阳性细胞在负载组的 MCC 对照小鼠 (加载: 6.23% ±1.89% 与控制: 1.90% ± 1.03%; p < 0.05;图 9A 和 C)。
通过对甲苯胺蓝染色区和距离图的评价, 对负载和控制小鼠的蛋白多糖分布进行量化, 如步骤9和图 7所述。我们发现, 相比控制 (加载: 210.22 µm ±4.11 µm 与控制: 187.36 µm ±8.64 µm; p < 0.005;图 9B 和 D)。然而, 蛋白多糖染色的区域在装载和控制组之间没有统计学上的不同 (加载: 203897.93 µm2 ±10171.00 µm2 vs 控制: 202875.09 µm2 ±33419.09 µm2; p = 0.94;图 9B 和 E)
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/55998/55998fig8.jpg"> 图 8: 有代表性的结果: (A)从控制和荷载小鼠的髁 Col10a1-positive 的矢状段中的细胞。加载的组(D)中的 Col10a1-positive 单元格数增加。(B)在控制和加载的小鼠中 Col2a1-positive 细胞。在(E)组之间的 Col2a1-positive 单元格中没有差异。(C)陷阱染色髁控制和加载小鼠。与控制(F)相比, 已加载组中的陷阱活动增加。直方图(d-F)代表 n = 4每个组的方法± SD。** 在控制组和载入群之间有显著差异 (p < 0.005)。缩放栏 = 200µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/55998/55998fig9.jpg"> 图 9:代表性结果: (A)髁控制和荷载小鼠的染色。增加的细胞增殖在被装载的小组, 代表由增加的教育阳性细胞(C)。(B)甲苯胺蓝染色在髁的控制和负载小鼠。增加的小鼠软骨厚度, 如在实验组(D)中增加的距离映射所示。在控件和加载的组(E)之间的多糖染色区域之间没有差异。直方图(C-E)表示为 n = 4每个组的方法为± SD。** 在控制组和载入群之间有显著差异 (p < 0.005)。缩放栏 = 200 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55998/55998fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle at JoVE.com

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

本文介绍了分析啮齿动物下颌骨髁突内形态和细胞变化的方法。

小鼠下颌骨髁突的形态学及细胞分析方法

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...

a-morphometric-cellular-analysis-method-for-murine-mandibular

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K 次观看.  University of Connecticut Health Center. 本文介绍了分析啮齿动物下颌骨髁突内形态和细胞变化的方法。

视频: A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

科研

发育生物学

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

在中短肛解剖，显微扫描和分析形态

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

生物工程

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

医学

Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

鼠股颈悬臂弯曲

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

手术小鼠模型中骨关节炎的标准化形态学评价

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

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

Orthodontic tooth movement is a complex biological process of altered soft and hard tissue remodeling as a result of external forces. In order to understand these complex remodeling processes, it is critical to study the tooth and periodontal tissues within their 3D context and therefore minimize any sectioning and tissue artefacts. Mouse models are often utilized in developmental and structural biology, as well as in biomechanics due to their small size, high metabolic rate, genetics and ease of handling. In principle this also makes them excellent models for dental related studies. However, a major impediment is their small tooth size, the molars in particular. This paper is aimed at providing a step by step protocol for generating orthodontic tooth movement and two methods for 3D imaging of the periodontal ligament fibrous component of a mouse mandibular molar. The first method presented is based on a micro-CT setup enabling phase enhancement&#160;imaging of fresh collagen tissues. The second method is a bone clearing method using ethyl cinnamate that enables imaging through the bone without sectioning and preserves endogenous fluorescence. Combining this clearing method with reporter mice like Flk1-Cre;TdTomato provided a first of its kind opportunity to image the 3D vasculature in the PDL and alveolar bone.

We present a protocol for generating orthodontic tooth movement in mice and methods for 3D visualization of the collagen fibers and blood vessels of periodontal ligament without sectioning.

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 ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

小鼠上颌骨正畸模型骨重塑分析指南

The Establishment of a Murine Maxillary Orthodontic Model

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to provide researchers with a feasible tool to analyze mechanical loading-associated bone remodeling. This study presents a detailed flowchart in addition to different types of schematic diagrams, operation photos, and videos. We performed this protocol on 11 adult wide-type C57/B6J mice and harvested samples on postoperative days 3, 8, and 14. The micro-CT and histopathological data have proven the success of tooth movements coupled with bone remodeling using this protocol. Furthermore, according to the micro-CT results on days 3, 8, and 14, we have divided bone modeling into three stages: preparation stage, bone resorption stage, and bone formation stage. These stages are expected to help researchers concerned with different stages to set sample collection time reasonably. This protocol can equip researchers with a tool to carry out regenerative analysis of bone remodeling.

作者聚焦:深入了解高效的小鼠上颌正畸模型方案

Here we demonstrate step-by-step a manageable, orthodontic tooth movement protocol operated on a murine maxillary model. With the explicit explanation of each step and visual demonstration, researchers can master this model and apply it to their experimental needs with a few modifications.

小鼠上颌骨正畸模型的建立

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

小鼠下颌骨髁突的形态学及细胞分析方法

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此视频中的章节

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

在中短肛解剖，显微扫描和分析形态

一个简单的临界尺寸股骨缺损模型小鼠

使用三维显微计算机断层扫描分析小鼠颅颌面畸形

鼠股颈悬臂弯曲

手术小鼠模型中骨关节炎的标准化形态学评价

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

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

小鼠上颌骨正畸模型的建立

小鼠上颌骨正畸模型的建立