我们这项技术的发展过程中，在谢比较医学，寺，得克萨斯州的斯科特与白医院处的工作人员和兽医，提供了宝贵的建议和帮助。这项工作是由再生医学研究所的项目经费，斯科特和怀特RGP批＃90172，NIH 2P40RR017447-07和NIH R01AR066033-01（NIAMS）的部分资助。我们感谢苏珊Zeitouni博士校对书稿。

注：此协议旨在从杰克逊实验室获得的女性裸体（怒江/ J）小鼠（18-25克，6周）。由于小鼠品系略有不同的解剖结构和增长速度方面，我们建议销的制造是优化前给收件人的应变，性别和年龄植入活的科目。如果菌株精心匹配，引脚和骨髓腔之间的过盈配合的高度重复性。住房，饮食和一般的畜牧过程不属于本协议的范围，但所有的老鼠被安置按照指南的护理和使用实验动物（ 第 8版）和体制政策的机构动物护理和设置使用委员会（IACUC）与比较医学系（DCM），在斯科特和白色医院。 1.制造针脚<imgALT =“图2”SRC =“/文件/ ftp_upload / 52368 / 52368fig2highres.jpg”/&gt; 图2：销组件。 （A）中的外科钢管在装配的各个阶段和5毫米厚的明胶海绵片4毫米直径缸截止的照片。（B）的抛光装配销（ 以上 ）后，将明胶海绵圆柱体被修整，定位在钢套环（ 中 ），然后高压灭菌，从而在无菌销涂覆有干燥明胶海绵。这提高了细胞附着在损伤部位，并保持愈合（ 下文 ）。（c）一种切除股骨装有骨髓销钉的方向。（D）的销组件在各种不同的厚度的实施例和长度表明这种方法的灵活性。 （E） - μCT重建的销稳定股骨缺损示延髓销与在近端和股骨远端的小梁骨的相互作用。 <ol><li>切9毫米长度的22克的不锈钢皮下注射管，和19克油管使用安装在旋转工具的直径23.8毫米玻璃纤维增​​强的重型切割轮3毫米的长度。用小细尖钳子固定油管（ 图2A， 上图 ）。穿戴合适的保护眼睛，并小心处理旋转工具。 </li><li>将少量氰基丙烯酸酯类粘合剂（约10微升）到9毫米轴的中间。通过3毫米领过9毫米轴，直到在中心和扭曲均匀分布的领子和轴之间的粘合剂。测量用一对数字卡钳的尺寸，并与图1进行比较。允许至少15小时（ 图2A， 中心 ）来设置。 </li><li>用220金刚砂浸渍盘，去除毛刺和过度胶水。 </li><li>使用旋转工具，抛光用毡抛光盘的管脚。 </li><li>用去离子水冲洗和干燥，COMPREssed空气。 </li><li>通过放置一个19克的皮下注射针头钝了新发针和推动针对白领的轴测试的完整性。确保领子可以抵抗约25克重的。 </li><li>冲洗在无菌过滤的磷酸盐缓冲盐水（ 图2B， 上 ）的位。确保销的轴紧贴地装配到所述股骨髓腔与对切骨（ 图2C）的边缘的领平齐。原型各种配置的研究和收件人（ 图2D）的具体用途。确保销的端部穿透到骨干的骨小梁，以最大限度地固定和提高的干涉配合（ 图2E）。 注：建议的拟合被植入在活体小鼠（ 例如 图2C）之前对骨标本进行测试。 </li><li>使用4毫米直径的冲床活检切割器切断的气缸从5毫米厚的片材的外科明胶海绵。使用解剖刀修剪气缸至3毫米的长度，并通过20G的注射针沿气缸以产生孔（ 图2A， 下图 ）的长度。 注意：我们已经发现，明胶或胶原蛋白支架在缺陷部位的定位提高了单元的保持并诱导沿着骨的轴线纵向生长。 </li><li>通过明胶泡沫容器上的销，以及配合所述轴环（ 图2B， 中心 ）和高压釜上的干燥周期，在120℃下在15磅。明胶海绵将干，颜色变暗（ 图2B，下同）。 </li></ol> 2.手术技术注：以下步骤是写观察的指南实验动物的护理和使用（ 第 8版）中设置的所有准则。请遵守所有附加政策当地IACUC设置，确保了IACUC批准的动物使用的协议（或同等学历）到位继续进行下面的章节之前。 <img alt="图3" src="/files/ftp_upload/52368/52368fig3highres.jpg" /> 图3：手术器械。 （一）手术包。微型钻头（1），切割轮（2），可吸收缝合线（3），外缝线（4），手术刀片（5），解剖刀手柄（6），无菌髓销包裹在箔（7），髓深度计制作形式皮下管（8），精细鼻钳（9），鼠牙钳（10），钝针扩孔（11），针驱动器（12），小止血钳（13），精剪（14），骨膜（15），修改克恩钳（16）（B）：须出示克恩式骨交手老鼠修改。 <ol><li>选择一种外科手术室或清洁实验室具有可关闭的门，没有通流量。消毒一个合适的工作面传热Ë与临床级四元消毒清洁剂。 </li><li>而戴一次性无菌手套，长袍，成立了约60×90平方厘米的无菌区与蒸压布窗帘的消毒工作表面。 注意：最好是有一个助手除去外包装和现在的无菌材料研究者设立现场。 </li><li>放置消毒加热垫安装在被单温水重新循环，盖上无菌一次性窗帘。暖珠灭菌到运行温度（250-265℃，这需要30分钟）。 </li><li>在无菌区，安排手术工具包（ 图3A）提供方便地访问所有组件。此外，提供无菌棉纱布（2×2英寸2），无菌棉签，无菌钢杯（500毫升）含有无菌盐水（0.9％重量/体积），和洗必泰/异丙醇外科消毒剂喷头。 </li><li>组装一小旁边的无菌区域与感应腔和鼻锥体组件按照兽医的指导和用户手册的动物麻醉单元。使用临床级的氧作为气体供给和异氟醚，美国药典。 </li></ol><img alt="图4" src="/files/ftp_upload/52368/52368fig4highres.jpg" /> 图4：手术过程。 （AC）方法和股骨的曝光。（DE）高程和削减。（FK）铰孔和髓质运河的大小。 <img alt="图5" src="/files/ftp_upload/52368/52368fig5highres.jpg" /> 图5：手术过程。 （AB）安装针。（CD）封闭。（E）正确定位针24小时的手术后的典型形象。 <ol start="6"><li>放置鼠标在麻醉系统和本身的感应腔室吨的输出以2升分钟-1 的 O 2和异氟醚浓度为3％（体积/体积）。 <ol><li>检查鼠标在1分钟内昏迷;提高浓度到4％（V / V），如果必要的。用加热垫的下方位置上取出无菌区鼠标，地点和适用的鼻锥。转印按照机构政策流向锥形，并减少异氟烷〜2.5％，等待进一步的20秒，并测试足够外科平面。通常情况下，缺乏后肢反射时，轻轻挤压足以保证足够的麻醉。 </li><li>在整个过程中，有一个助手显示器强大的正常呼吸和四肢和嘴部区域的粉红色彩，以确保氧化而无意识的适当水平。按照兽医的指导和体制政策在必要时调整麻醉。应用无菌人工泪液的润滑剂软膏（15％（体积/体积）的矿物油，83％（体积/体积）的whITE凡士林）的眼睛。 </li></ol></li><li>将鼠标到一边用后肢朝上一个新的一次性悬垂性。如果有必要，去掉毛皮用电动剃刀或脱毛霜。擦拭的部位用无菌生理盐水并删除其他的悬垂性过剩的皮毛。放置一个新的有孔的悬垂比鼠标，以便覆盖所有部分，而是整个后肢（ 图4A），但仍维持图面和爪的监测着色和呼吸。 </li><li>定位在股骨的近端和远端并切开皮肤为5-10毫米沿着纵向轴线（ 图4B）。从筋膜皮肤层用＃15解剖刀分开，经由股二头肌和股外侧露出外侧入到股骨。定位到这两种肌肉的隔满足（这是一排白色的组织对肌肉的粉红色的颜色）。用手术刀，仔细沿直到肌间边界剖析股骨是可见的（ 图4B）。 </li><li>开发切口用钝骨膜电梯以便露出在整个骨干（ 图4C）。使用电梯进一步揭露股骨的中央三分之二同时小心保持内侧上（ 图4D）的后神经血管束。轻轻刮去软组织脱用手术刀骨，并干燥，用无菌棉签或等效物。 </li><li>定位股骨与和卡钳如果需要标记用无菌解剖刀或标记中心，然后向近侧和向远侧马克1.5毫米从中心。轻轻抓住利用一对先前在克恩风格塑造细鼻镊子以防止在该骨过度压力股骨。 注意：在图3B中提供精确的尺寸进行修改。最好是测试钳子上之前的一处以安乐死试样使用，以确保骨压力下不会断裂需要d代表在切割过程中固定。 </li><li>用细钻头配有精美的金刚石粒度的涂层切割轮（8毫米直径×0.1毫米宽），使与电梯的首次降息，以保护组织下方（ 图4E）。提高切割股骨至45°，同时紧紧握着骨干的肢体，使第二切割，拆除3毫米段。 注：面部防护，建议在这个阶段。 </li><li>由钳固定骨头，仔细铰两端的髓腔用钝器23政注射器针头。使用预先制成的深度计从22克油管的长度放置在19克油管制成（ 图3A， 我 TEM 8），评估该铰髓腔的深度，并确保它为3毫米（ 图4F-K）的。如果髓腔抵抗22克深度计，用钝22克注射器针头再次令。 </li><li>小心地将髓销插入近端远端则髓Çavities使股骨背出其原始长度，建立稳定3毫米间隙（ 图5A，B）。如果需要的话，可以使用少量手动压力来实现杆的良好干涉配合股骨的皮质骨。确保销正好符合双方的髓状空腔并切割骨头的边缘与轴环齐平。如果还有一定的差距，铰再次用22克钝针。 </li><li>重新定位的肌肉和外周组织在销和关闭与一个连续吸收5-0缝合（ 图5C）。关闭皮肤切口5-7平结尼龙5-0缝线和密封，粘接手术（ 图5D）。 </li></ol> 3.术后程序<ol><li>关闭切开皮肤后，退出麻醉，但允许O 2保持流动，直到鼠标开始移动;这花费时间不超过1分钟。如果5分钟后，鼠标仍然一动不动O 2的管理，是指对兽医机构的干预政策。 </li><li>当鼠标开始移动，轻轻将其传送到最好含干，蒸压床上用品笼子里。 <ol><li>发布冰屋型窝在每个网箱和鼠标将撤退到它，从而减少运动。检查鼠标恢复后肢的流动性意识恢复后，5-10分钟。 </li><li>执行日常术后监测按照机构政策。提供在笼的地板上糊化水化和食品的第5-7天及施用镇痛和术后监测按照制度上批准策略。 注意：我们管理0.05-0.1毫克公斤-1丁丙诺啡24 0.25ml的生理盐水皮下注射，每日两次，第3天，此后如果鼠标没有恢复正常的效用。 </li></ol></li><li>复苏的第一个24小时后，理解过程的现场表演动物X射线成像ř麻醉可视化管脚布局（ 图4E）。如果脚脱臼，考虑立即翻修手术。 </li><li>在7天的手术后，取出在麻醉状态下，房屋缝线组按照机构牧政策。 </li><li> 2-5周后，人道地安乐死动物按照美国兽医医学协会批准用于安乐死25的方法。 注：腹腔注射市售巴比妥组合安乐死鸡尾酒是有效的，人性化，但安乐死地方机构的政策应遵循。 </li><li>仔细解剖掉后肢暴露股骨近端和内侧的骨盆。轻轻地从肢体的侧面按关节向内而拆卸股骨头从髋臼 （髋插座）用手术刀。切剩下肌肉和皮肤用锋利的解剖刀或微型剪刀，释放整个肢体形成的骨盆。用锋利的一对咬骨钳或剪刀沉重的，切下后肢（胫骨/腓骨）以下的膝关节约5毫米。 注：建议所有标本分析之前储存在一个相同的方式进行。 </li><li>去除皮肤，但留下的肌肉到位。固定的组织，在10％缓冲的福尔马林中补充了10mM的CaCl 2固定液1周后贮存在磷酸盐缓冲盐水补充了10mM的CaCl 2长达1个月前成像。在4℃下进行固定和存储。另外，扫描标本没有立即固定。 </li></ol>标本4.分析注：骨愈合后可通过多种方法，超出本协议的范围进行评估。以下是我们已经成功地利用标本显微（μCT）成像采用的方法。所以建议下列参数ARê测试最初，那么对于该项目的具体需要优化。 <ol><li>塑料密封膜，它垂直位置的仪器室一层薄薄的包装样品。确保股骨垂直于试料台在整个扫描过程中。设置扫描到下列参数;电压= 29千伏，电流= 661μA，功率= 19 W，图像像素大小（微米）= 21.00; 360度旋转= YES;帧平均=（5）;在旋转步骤（度）= 1.00，随机运动=。图像保存为JPEG文件在每次扫描一个目标文件夹（ 如图6A）。 注：扫描应该是完整的57分钟。 </li><li>利用重建的软件以基于以下参数的轴向图像;平滑=（4）上，不对补偿=上环神器减少=（5）上，束硬化校正（40％），CS旋转（度）= 0.00。设置输出2,000-15,000菲尔德单位。图像保存为JPEG文件在每次扫描一个目标文件夹。 </li><li>使用轴向图像和分析软件，通过首先设定的近端和原有缺陷远侧边限定的感兴趣区域（ROI）的区域。通过选择只包括领子的部分实现这一目标。因为轴环比所述骨髓销钉较厚，这些部分容易形成（ 图6B， 左 ）。通过绘制其周围的隔离区（带有100微米余量）和区转移到每个部分中的ROI（ 图6B， 右 ）排除从计算的衣领。 注：惯性，三维重建和计算极性的时刻，如新骨体积（ 图6C，D），可以容易地使用的软件实现。 </li></ol>

<ol>
	<li>Brinker, M. R., O'Connor, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+fractures+and+dislocations+referred+for+orthopaedic+services+in+a+capitated+population.">The incidence of fractures and dislocations referred for orthopaedic services in a capitated population.</a> J Bone Joint Surg Am. 86, 290-297 (2004).</li><li>Cheung, 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+future+of+bone+healing.">The future of bone healing.</a> Clin Podiatr Med Surg. 22, 631-641 (2005).</li><li>Rosemont, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> United States Bone and Joint Decade: The burden of musculoskeletal diseases and musculoskeletal injuries. , (2008).</li><li>Tzioupis, C., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+long-bone+non-unions.">Prevalence of long-bone non-unions.</a> Injury. 38, S3-S9 (2007).</li><li>Marsh, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+of+fracture+union,+delayed+union,+and+nonunion.">Concepts of fracture union, delayed union, and nonunion.</a> Clin Orthop Relat Res. , S22-S30 (1998).</li><li>Spicer, P. 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=Evaluation+of+bone+regeneration+using+the+rat+critical+size+calvarial+defect.">Evaluation of bone regeneration using the rat critical size calvarial defect.</a> Nat Protoc. 7, 1918-1929 (2012).</li><li>Green, E., Lubahn, J. D., Evans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors,+treatment,+and+outcomes+associated+with+nonunion+of+the+midshaft+humerus+fracture.">Risk factors, treatment, and outcomes associated with nonunion of the midshaft humerus fracture.</a> J Surg Orthop Adv. 14, 64-72 (2005).</li><li>Kanakaris, N. K., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+economics+of+the+treatment+of+long-bone+non-unions.">The health economics of the treatment of long-bone non-unions.</a> Injury. 38, S77-S84 (2007).</li><li>Dimitriou, R., Mataliotakis, G. I., Angoules, A. G., Kanakaris, N. K., Giannoudis, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+following+autologous+bone+graft+harvesting+from+the+iliac+crest+and+using+the+RIA:+a+systematic+review.">Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review.</a> Injury. 42, S3-S15 (2011).</li><li>Boer, H. 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+history+of+bone+grafts.">The history of bone grafts.</a> Clin Orthop Relat Res. , 292-298 (1988).</li><li>Aro, H. T., Aho, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+use+of+bone+allografts.">Clinical use of bone allografts.</a> Ann Med. 25, 403-412 (1993).</li><li>Burstein, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+substitutes.">Bone substitutes.</a> Cleft Palate Craniofac. J. 37, 1-4 (2000).</li><li>Kao, S. T., Scott, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+bone+substitutes.">A review of bone substitutes.</a> Oral Maxillofac Surg Clin North Am. 19, 513-521 (2007).</li><li>Boden, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+biology+of+lumbar+spine+fusion+and+principles+for+selecting+a+bone+graft+substitute.">Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute.</a> Spine. (Phila Pa 1976). 27, S26-S31 (1976).</li><li>Hollinger, J. O., Kleinschmidt, 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=The+critical+size+defect+as+an+experimental+model+to+test+bone+repair+materials).">The critical size defect as an experimental model to test bone repair materials).</a> J Craniofac Surg. 1, 60-68 (1990).</li><li>Key, J. <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+local+calcium+depot+on+osteogenesis+and+healing+of+fractures.">The effect of local calcium depot on osteogenesis and healing of fractures.</a> J. Bone Joint Surg. (Am). 16, 176-184 (1934).</li><li>Holstein, J. 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=Advances+in+the+establishment+of+defined+mouse+models+for+the+study+of+fracture+healing+and+bone+regeneration).">Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration).</a> J Orthop Trauma. 23, S31-S38 (2009).</li><li>Histing, T., 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=Small+animal+bone+healing+models:+standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49, 591-599 (2011).</li><li>Cheung, K. 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=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21, 685-690 (2003).</li><li>Hiltunen, A., Vuorio, E., Aro, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+experimental+fracture+in+the+mouse+tibia.">A standardized experimental fracture in the mouse tibia.</a> J Orthop Res. 11, 305-312 (1993).</li><li>Manigrasso, M. B., O'Connor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+closed+femur+fracture+model+in+mice.">Characterization of a closed femur fracture model in mice.</a> J Orthop Trauma. 18, 687-695 (2004).</li><li>Garcia, 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=Rodent+animal+models+of+delayed+bone+healing+and+non-union+formation:+a+comprehensive+review.">Rodent animal models of delayed bone healing and non-union formation: a comprehensive review.</a> Eur Cell Mater. 26, 1-12 (2013).</li><li>Garcia, 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=Development+of+a+reliable+non-union+model+in+mice.">Development of a reliable non-union model in mice.</a> J Surg Res. 147, 84-91 (2008).</li><li>Flecknell, P. 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+relief+of+pain+in+laboratory+animals.">The relief of pain in laboratory animals.</a> Lab Anim. 18, 147-160 (1984).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines on the Euthanasia of Animals. , (2013).</li><li>Neill, K. 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=Micro-computed+tomography+assessment+of+the+progression+of+fracture+healing+in+mice.">Micro-computed tomography assessment of the progression of fracture healing in mice.</a> Bone. 50, 1357-1367 (2012).</li><li>Bagi, C. 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=The+use+of+micro-CT+to+evaluate+cortical+bone+geometry+and+strength+in+nude+rats:+correlation+with+mechanical+testing,+pQCT+and+DXA.">The use of micro-CT to evaluate cortical bone geometry and strength in nude rats: correlation with mechanical testing, pQCT and DXA.</a> Bone. 38, 136-144 (2006).</li><li>Hadjiargyrou, 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=Transcriptional+profiling+of+bone+regeneration.+Insight+into+the+molecular+complexity+of+wound+repair.">Transcriptional profiling of bone regeneration. Insight into the molecular complexity of wound repair.</a> J Biol Chem. 277, 30177-30182 (2002).</li><li>Clough, B. 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=Bone+regeneration+with+osteogenically+enhanced+mesenchymal+stem+cells+and+their+extracellular+matrix+proteins.">Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins.</a> J Bone Miner Res. , (2014).</li><li>Lu, 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=Cellular+basis+for+age-related+changes+in+fracture+repair.">Cellular basis for age-related changes in fracture repair.</a> J Orthop Res. 23, 1300-1307 (2005).</li><li>Jepsen, K. 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=Genetic+variation+in+the+patterns+of+skeletal+progenitor+cell+differentiation+and+progression+during+endochondral+bone+formation+affects+the+rate+of+fracture+healing.">Genetic variation in the patterns of skeletal progenitor cell differentiation and progression during endochondral bone formation affects the rate of fracture healing.</a> J Bone Miner Res. 23, 1204-1216 (2008).</li><li>Thayer, T. C., Wilson, S. B., Mathews, C. E. <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+nonobese+diabetic+mice+to+understand+human+type+1+diabetes.">Use of nonobese diabetic mice to understand human type 1 diabetes.</a> Endocrinol Metab Clin North Am. 39, 541-561 (2010).</li><li>Jee, W. S., Yao, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+animal+models+of+osteopenia+and+osteoporosis.">Overview: animal models of osteopenia and osteoporosis.</a> J Musculoskelet Neuronal Interact. 1, 193-207 (2001).</li></ol>

The authors have no competing financial interests.

在此，我们描述一个简单的方法来产生使用标准实验室和兽医器械鼠股骨的临界尺寸的针稳定缺陷。而引脚和手术本身的装配要求的做法，它是指一个训练有素的生物医学研究的科学家或兽医的能力很好。 销被定位成髓管，无需额外的固定，使该过程在技术上更重要的是使用外固定器或互锁螺钉更复杂的方法是可行的。而在愈合的早期阶段，可能会出现一些扭转运动，这是?...

据估计，一半的美国人口经历了骨折的65 1岁。对于那些患者的治疗骨折手术，500000过程涉及使用骨移植2，这个数字预计将上升与人口日趋老龄化3 。虽然骨是少数器官具有完全愈合无疤痕的能力之一，有其中过程失败3,4是实例。根据具体情况和治疗的质量，长骨骨折的2-30％失败，从而导致非工会3,5。虽然仍存在的定义，假关节，临界尺寸或不愈合骨损伤有些争论，一般是指不愈合覆盖主体6的自然寿命的伤害。为实验目的，此持续时间缩短为所需的平均大小的骨损伤完全愈合的平均时间。非工会骨骼发生病变货号危。原因，但主要的因素包括产生了极其大的差距，感染，血管差，烟草使用，或抑制osteoregenerative能力因疾病或7岁极端的创伤。即使非工会的成功治疗，它可以超过60,000美元的过程成本，这取决于损伤的类型和使用的8途径 ​​。 在温和的情况下，自体骨移植是就业。这一战略包括从损伤部位供体部位和植入恢复骨。虽然这种方法是非常有效的，可用的供体来源的骨量是有限的，该过程涉及一个附加的外科手术，这导致持续性疼痛在许多患者9,10。另外，自体骨移植物的功效取决于患者的健康。从合成材料或加工尸体骨制作的骨代用品大量可用11-13，但他们公顷已经显著限制，包括差的宿主-细胞粘着性，减少的骨传导性，以及用于免疫排斥14的潜力。有因此，迫切需要用于骨骼再生的技术，是安全，有效和广泛使用。 我们以改善骨再生战略的能力主要取决于模仿严重骨创伤中的试验动物的能力，但对大的骨病变的产生和稳定化在技术上是有挑战性的。在大多数情况下，严重的长骨创伤是通过建立一个缺陷，即不会自然愈合实验模仿。虽然它可以与物种15而变化，这是通过完全除去一个骨片段，它是骨的横截面16的大于1.5倍的直径的实现。骨然后用金属植入物稳定，以保持骨折边缘的正确取向，并允许流动。由于它们的小尺寸和的脆弱其长骨，建立这样的病变的小鼠都超出了大多数的研究小组的功能。因此，长骨缺损模型仅限于老鼠和较大的动物。然而，小鼠得到显著研究的优点在于，它们可以被遗传修饰并饲养作为免疫妥协菌株不拒绝人类细胞和组织。 对于人体细胞为基础的应用，免疫功能低下小鼠与吸引力，因为他们在生理上良好的特点，易家的工作，成本效益，易于分析影像学和组织学。最重要的是，免疫​​妥协小鼠中不拒绝来自不同物种，包括人类细胞。它们的小尺寸还允许非常小的数量的细胞或实验支架在整形外科应用的卷的测试。几种鼠矫形模型已报道，得到不同程度的骨稳定17,18。这些SYSTE这导致非常高的稳定性水平，如外固定器和锁定板毫秒主要由膜内骨化愈合虽然软骨内愈合已经报道19。与此相反，那些允许一些微观和/或宏观的运动，如那些使用未固定的或部分固定髓针，一般治愈与软骨内骨化20,21的一个优势。延迟愈合或长骨的不愈合缺陷是特别难以实现在小鼠中，由于稳定​​化所需要的额外的水平。然而，一些方法已被报道，包括髓销与髓内钉，锁定板和外固定器22上 。这些系统一般工作良好，但鉴于其复杂的设计，他们可以在技术上具有挑战性的安装。例如，Garcia 等。23设计优雅联锁销系统，用于在小鼠中使用，但该方法包括切口在两个分开的部位s和股骨的大量修改，以容纳插针。这些程序在解剖显微镜下进行的。 在此，我们描述一个简单的股骨骨髓销钉与中央领设计成防止关闭3毫米的骨缺损，也描绘缺陷的原边。而销没有被固定到骨头本身，销直径髓腔导致足够的干扰和扩孔的精确大小，以尽量减少扭转运动（ 图1）。通过仔细挑选的近交的年龄，性别和应变匹配小鼠，其结果是一个高度可再生肥厚非uniondefect 22可以很容易地进行评估放射学。另外的感兴趣的区域可以显微计算机断层摄影术（μCT） 从头骨形成和组织形态学参数的测量之后被再现地限定。引脚用现成的工具原型在我们的实验室。<img alt="图1" src="/files/ftp_upload/52368/52368fig1highres.jpg" /> 图1：实验原理节段性缺损模型的图解总结。一个9-10毫米的鼠股骨的中央3毫米段被手术切除（ 左 ）。 A 3毫米长，19轨距外科钢管越过9毫米长，22克的不锈钢管和固定用粘合剂在准确的中心（ 右 ）。所得销装配到股骨的其余的近端和远端部分的与19克衣领置换骨的3毫米段（ 下面，中心 ）的髓管。

<table border="0" cellpadding="0" cellspacing="0" width="799">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:235px;">Name of Equipment/Material*</td>
			<td style="width:207px;">Company</td>
			<td style="width:180px;">Catalog or model</td>
			<td style="width:179px;">Notes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pin Assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dremel rotary tool</td>
			<td>Dremel</td>
			<td>8220</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heavy duty cut off wheel</td>
			<td>Dremel</td>
			<td>420</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 19G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMZ8LY</td>
			<td>OD 1.07mm, ID 0.889mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 21G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMZ8YQ</td>
			<td>OD 0.82mm, ID 0.635mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 22G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FMYLZS</td>
			<td>OD 0.719mm, ID 0.502mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tubing 23G</td>
			<td>Small Parts (Amazon)</td>
			<td>B000FN0SY0</td>
			<td>OD 0.643mm, ID 0.444mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cyanoacrylate adhesive</td>
			<td>Loctite</td>
			<td>1365882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Emery disc</td>
			<td>Dremel</td>
			<td>413</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rubber polishing point</td>
			<td>Dremel</td>
			<td>462</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Felt polishing disc</td>
			<td>Dremel</td>
			<td>414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin sponge</td>
			<td>Surgifoam/Ethicon</td>
			<td>1974</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Punch biopsy cutter</td>
			<td>Miltex</td>
			<td>33-34</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgery/post-operative</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Warm pad and circulator pump</td>
			<td>Stryker/Thermocare</td>
			<td>TP700, TP700C, TPP722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Coverage quaternary spray</td>
			<td>Steris</td>
			<td>1429-77</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bead sterilizer</td>
			<td>Germinator/CellPoint Scentific</td>
			<td>Germinator 500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anesthesia system</td>
			<td>VetEquip Inc</td>
			<td>901806 or 901807/901809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isofluorane anesthetic</td>
			<td>VETone/MWI</td>
			<td>501017, 502017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical disinfectant</td>
			<td>Chloraprep/CareFusion</td>
			<td>260449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical tools</td>
			<td>Fine Science Tools</td>
			<td>various</td>
			<td>recommend German made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Face protection</td>
			<td>Splash Shield</td>
			<td>4505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rechargable high speed drill</td>
			<td>Fine Science Tools</td>
			<td>18000-17</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diamond cutting wheel</td>
			<td>Strauss Diaiond</td>
			<td>361.514.080HP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absorbable sutures&#160;</td>
			<td>Covidien</td>
			<td>UM-213</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Outer sutures</td>
			<td>Ethicon</td>
			<td>668G</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vetbond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydration gel</td>
			<td>Clear H2O</td>
			<td>70-01-1082</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Diet gel</td>
			<td>Clear H2O</td>
			<td>72-01-1062</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>Reckitt and Benckser</td>
			<td>12496-0757-01</td>
			<td>controlled substance</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse igloos</td>
			<td>Bio Serv</td>
			<td>K3328, 3570,3327</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Euthanasia cocktail</td>
			<td>Euthasol/Virbac</td>
			<td>710101</td>
			<td>controlled substance</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live animal imager&#160;</td>
			<td>Orthoscan</td>
			<td>FD Pulse</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-CT unit and software</td>
			<td>Bruker</td>
			<td>Skyscan1174</td>
			<td>or equivalent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sealing film/Parafilm M</td>
			<td>VWR or Fisher</td>
			<td>100501-338, S37441</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Generic sources are suitable for all other items such as gause, drapes, protective clothing, animal care equipment.</td>
		</tr>
	</tbody>
</table>

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.

小鼠通常恢复意识和后肢的流动性撤离麻醉后5-10分钟。在第5天，最好是单独地容纳小鼠和引入环境丰富，以防止过量使用的肢体。为此，冰屋型巢减少了对鸟巢的建设，鼓励休息。我们还观察到，提供食物和水凝胶上笼的地板上的降低销位移的概率。在5天期间，镇痛应根据需要按照体制批准的政策进行给药。减重高达原始手术前重量的约15％是可能在5天的手术后期间。手术后二十四小时，X射线...

Watch this Scientific Journal Video about A Simple Critical-sized Femoral Defect Model in Mice at JoVE.com

A Simple Critical-sized Femoral Defect Model in Mice

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 ...

a-simple-critical-sized-femoral-defect-model-in-mice

Research

JoVE Journal

Medicine

15.0K 次观看.  Texas A&M Health Science Center. 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. 

视频: A Simple Critical-sized Femoral Defect Model in Mice

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

一个简单的指南螺杆颅内移植瘤小鼠的研究方法

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

科研

医学

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

一个简单的方法小鼠肺插管

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

动脉再狭窄小鼠模型：股导线损伤的技术方面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

重复震荡颅脑损伤模型小鼠

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

一种改良的小鼠心肌梗死诱导简易方法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...