作者要感谢瑞纳Bizios她对稿件宝贵意见。

伦理声明:在本研究中所用小鼠按照欧洲委员会"实验动物护理和使用"（指令63分之2010/欧盟和欧洲公约ETS 123）公布的指导方针进行治疗。实验方案经医学拉里布瓦西埃圣路易学院伦理委员会（CEEA LV / 2010-01-04）。 1.动物<ol><li>使用无胸腺小鼠（10周龄）。使用6小鼠缺陷留空作为阴性对照组的最小数量。 </li></ol> 2.准备支架<ol><li> 同基因移植准备 <ol><li>使用骨同系移植到填充缺损提供对照组6只动物的一最小数量。 </li><li>获取骨同基因移植物被切除收获从鼠标属于任何股骨的"缺陷空或"缺陷充斥着珊瑚支架"组（TH为避免使用额外的动物收集骨基因移植物）11。 </li><li>同花顺磷酸盐缓冲液（PBS）切除的骨头，并保持它用无菌纱布湿润的压缩。 </li></ol></li><li> 珊瑚支架制备方法 <ol><li>使用该支架是由天然珊瑚: 鹿角珊瑚 。珊瑚骨骼立方体，3×3×3mm的作为潜在的骨替代用最少6只动物。 </li><li>用手每个珊瑚立方体刻到圆筒状（3.5高度;直径为2毫米）。 </li><li>消毒通过高压每个支架（121℃，20分钟），用无菌PBS清洗，并浸入其在完全培养基（α-MEM），用于在小鼠中植入前24小时。 </li></ol></li></ol> 3.麻醉操作和镇痛<ol><li>提供预防镇痛，麻醉前15分钟，通过丁丙诺啡皮下注射（0.1毫克/千克动物体重）。 </li><li>在动物应用软膏眼睛，防止干燥每隔30分钟，而动物麻醉下。 </li><li>放置在一个变暖垫鼠标，以防止体温过低。 </li><li> 麻醉和镇痛在手术过程中 <ol><li>注入腹膜内含溶液赛拉嗪（8毫克/千克）和氯胺酮（100mg / kg的）。 </li><li> 经由流由输送氧气（50毫升/分钟）。 </li><li>通过良好的肌肉松弛，缺乏动物反应的存在对有害刺激（ 例如 ，公司脚趾捏）确认麻醉深度足够。 </li><li>注入皮下恩诺沙星单剂量（0.05毫克/公斤），为微生物预防。 </li></ol></li><li> 术后镇痛 <ol><li>通过丁丙诺啡皮下注射（0.1毫克/千克），连续3天，每12小时提供术后镇痛。 </li></ol></li><li> 在诊断成像过程麻醉 <ol><li>放置小鼠在anesthetizinG-框，然后诱导并用约4％和2％异氟烷在氧气中，分别维持麻醉。 </li><li>通过确认良好的动物肌肉松弛和缺乏运动的麻醉深度足够。 </li></ol></li><li> 恢复条件 <ol><li>继续变暖垫老鼠，直到完全恢复</li><li>无人看管，直到它已经恢复了神志充足术后胸骨保持斜卧不要让动物。 </li><li>不要返回已动过手术，以公司的其他动物，直到完全康复的动物。 </li></ol></li><li> 手术后的条件 <ol><li>在第3天分别举办小鼠4在笼子里举办小鼠3天后。 </li><li>提供水和食物适应自由采食 。允许小鼠重熊，而不在整个手术后期间的任何活动限制。 </li></ol></li></ol> 4.手术过程:股骨骨缺损模型11,12 <ol><li>麻醉后，放置在腹侧斜卧每只小鼠用在扩展左后肢。 </li><li>擦洗肢为使用10％聚维酮碘5分钟无菌手术，然后将无菌的悬垂性肢体下创建的无菌表面（无菌透明悬垂是为了用于能够在手术过程中，监测呼吸运动）。小心在手术过程中，以保持手术区域的无菌。 </li><li>做一个15 - 17毫米的纵向皮肤切口在股骨前外侧，从髋关节到膝关节扩展。 </li><li>切开阔筋膜 ，拆股外侧肌和股二头肌肌肉以暴露股骨骨干的全长。注意应采取尾端保留坐骨神经和关节囊远侧（ 图1）。 </li><li>为了提高股骨干博览会当然，横切臀中肌和二头肌从第三转子了大腿 。 </li><li>在骨干的中间执行股骨的圆形清扫。 </li><li>套用一个6孔钛微锁板（10毫米长1.5毫米宽，重量:30毫克）的前股骨侧。 注:该板的通孔，其呈圆锥形的圆筒形部分凹入，容纳钛自攻锁紧螺钉（2毫米长，0.47mmol外径，重量:5毫克，与头部螺钉的底面螺纹，以使内锁所述板孔）连接到一个干，当锁定时它拧断。 </li><li>使用0.3mm的钻头，要么专用发动机功率或在2500转在约500毫瓦12操作非专用发动机功率）钻在板的最基端的孔。 </li><li>插入使用专用螺丝刀第一螺杆，然后将其锁定（ 见图2）。 注意:因为T对齐他板通过该第一螺钉的应用确定，它在插入螺钉时到平行板定位到股骨是重要的。 </li><li>钻板的最远端孔以类似的方式，插入和锁定螺丝（ 图3）。 </li><li>插入，但确实没有锁，在两个外螺丝。 </li><li>放置的0.22毫米吉利在一个内-外方向紧紧看见周围骨中的电线，然后将其插入的夹具（ 图4）的槽。 </li><li>插入上的最后两个螺丝杆的专用夹具和应用它的板（ 图5）的上方。 </li><li>执行使用吉利灌溉看到（用无菌生理盐水），以防止热坏死3.5毫米长的中等骨干股骨截骨。有外科医生的助手拿夹具。有外科医生施加恒定的张力稳定。要小心，不要纠结锯电线使用电线的中间三分之二。避免前为了获得直链骨切塞斯运动（ 图6）。 </li><li>在截骨后，取出吉利看到的。为了避免软组织损伤，切锯丝接近骨一侧。 </li><li>除去夹具和锁定最后两个螺钉（ 图7）。 </li><li>要么离开缺损空或手术通过将内部缺陷被测试的材料填充。 </li><li>丰富地冲洗用无菌生理盐水外科领域。 </li><li>放置股外侧肌松弛地在板上。关闭筋膜和皮下飞机使用简单的连续缝合方式和5.0 glycomer 631缝合;使用4.0 glycomer 631缝合简单间断缝合关闭模式皮肤。或者，它也可以用皮肤胶以关闭皮肤。 </li></ol> 5. 在骨再生的体内评估<ol><li>随着麻醉下的小鼠，进行X射线照相在同时使用常规的X射线的纵向的方式评估（26千伏，10秒; 2X放大; 20线/毫米的空间分辨率）和高清晰度的微型计算机断层扫描（μCT）。 </li><li>对于μCT分析，在36微米的分辨率采集图像（50千伏和第478毫安，在40毫秒的曝光时间，使用0.5mm的铝过滤器，0.7º旋转步骤，以及180º断层旋转）。分析使用常驻软件中的图像。 </li></ol>骨再生的6 前体内评估<ol><li>手术10周后，诱导麻醉在氧气使用异氟醚，然后通过巴比妥的过量（1毫升戊巴比妥）的腹膜内注射牺牲小鼠。 </li><li>切除股骨骨骼，除去覆盖肌肉组织，并固定在4％多聚甲醛（pH 7.4）中的骨标本四天。 </li><li>从多聚甲醛的FIXa后各切除骨标本取出钢板螺钉化。 </li><li> 体外 μCT分析 <ol><li>放置在装满用75％酒精聚乙烯管各切下并固定骨，并使用体外 μCT分析它。 </li><li>在80千伏和100微安（1000毫秒的曝光时间，铝0.5过滤和4微米照相机像素尺寸（2 400点¯x4000为7微米的体素尺寸）获取图像，为0.9º每旋转增量平均四个帧。 </li><li>重建使用海明-滤波反投影与常驻软件的三维图像（13微米平均体素尺寸）。 </li><li>用于骨形成的定量分析，使用驻留软件，以获得对应于缺陷的兴趣的确定和一致的区域矿化组织（45灰度指数下灰度阈值和240灰度索引上灰度阈值）的体积。 </li><li>每只小鼠进行相同的方式分析用相同的章感兴趣的离子。 </li><li>使用单向分析测试（置信区间 - 在95％和当p &lt;0.05的显著水平）比较骨愈合速率和矿化组织中的群体之间感兴趣区域的体积。 </li></ol></li><li> 组织学分析 <ol><li>嵌入各切下并固定在甲基丙烯酸甲酯树脂股骨并处理它脱钙组织学。 </li><li>纵向切成每块骨骼标本成用圆形水冷金刚石锯片粗节（200微米）。 </li><li>磨骨各部分标本到厚度为100微米，打磨，并用Stevenel蓝色和Van Gieson染色picrofuchsin污渍弄脏它。 注:染色后，细胞显示为蓝色，骨骼粉红色，珊瑚褐色光镜。 </li></ol></li></ol>

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Part A. 22 (5-6), 534-544 (2016).</li><li>Bos, R. 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=Degradation+of+and+tissue+reaction+to+biodegradable+poly(L-lactide)+for+use+as+internal+fixation+of+fractures:+a+study+in+rats.">Degradation of and tissue reaction to biodegradable poly(L-lactide) for use as internal fixation of fractures: a study in rats.</a> Biomaterials. 12 (1), 32-36 (1991).</li><li>Oest, M. E., Dupont, K. M., Kong, H. J., Mooney, D. J., Guldberg, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+scaffold+and+growth+factor-mediated+repair+of+critically+sized+bone+defects.">Quantitative assessment of scaffold and growth factor-mediated repair of critically sized bone defects.</a> J.Orthop. Res. 25 (7), 941-950 (2007).</li><li>Pihlajamaki, H., Bostman, O., Tynninen, O., Laitinen, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+tissue+response+to+bioabsorbable+poly-L-lactide+and+metallic+screws:+an+experimental+study.">Long-term tissue response to bioabsorbable poly-L-lactide and metallic screws: an experimental study.</a> Bone. 39 (4), 932-937 (2006).</li><li>Rai, B., 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=Combination+of+platelet-rich+plasma+with+polycaprolactone-tricalcium+phosphate+scaffolds+for+segmental+bone+defect+repair.">Combination of platelet-rich plasma with polycaprolactone-tricalcium phosphate scaffolds for segmental bone defect repair.</a> J. Biomed. Mater Res. A. 81 (4), 888-899 (2007).</li><li>Komlev, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+in+vivo+bone+deposition+by+bone+marrow+stromal+cells+into+porous+calcium+phosphate+scaffolds:+an+X-ray+computed+microtomography+study.">Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study.</a> Tissue Eng. 12 (12), 3449-3458 (2006).</li></ol>

作者宣称，他们没有竞争的经济利益。

骨科相关的材料和小鼠装置的异位植入通常执行以评估骨形成各种支架13,14的能力。然而，重要的差异异位原位车型，其中包括原生骨信号因子，并与宿主骨形成细胞旁分泌的相互作用之间。 本研究建立了一个可重复的鼠大段，临界尺寸股骨缺损（3.5毫米，股骨长度约为20-25％）。考虑这种缺陷的大小和由所得接骨板提供的稳定性，该模型模仿临床上遇到萎缩骨不连。 在本研究中所选择的手术后的一段时间内，在与前述不愈合模型小鼠线，表示8〜12周4,9,15,16后缺乏足够的愈合。 最重要的是，reprodu无显著发病率和死亡率的1,2-与使用两个锁定板和执行截骨夹具的获得埚和稳定接骨，以及植入骨替代物的稳定性。这一结果也对比时，无论是外固定器或髓内钉用于4,5,17-24结果报告如下。对于外固定器的潜在缺点包括:变异刚度，销束的感染，松动销的，电势由于销与材料（4〜20的小鼠体重的百分比）的重量损伤。对于髓内钉潜在缺点包括:用指甲和关节面的医源性损伤髓腔的填充。 通过接骨板稳定其它鼠节段性，临界尺寸股骨缺损已与由毛刺和范围从1.5至2毫米的长度16,25创建骨缺损说明。在日E存在模式，利用夹具和埋弧焊丝的允许精确3.5毫米长的截骨不显著肌肉创伤。 然而，在执行一个应该考虑的问题几个关键点的过程成功:不要用小的小鼠（裸鼠或者重量低于25 g或未满8周），否则的铭牌应过长。当接近股骨，注意保护两个尾部坐骨神经和关节囊远端。适用于股骨的前侧的板，并且由于板的对准通过该第一螺钉的应用确定，照顾插入该第一螺钉时，平行板定位到股骨。 使截骨术前，照顾到骨干中部执行股骨的圆形解剖，以避免肌肉创伤。当执行截骨术，外科医生的助手必须抓紧指导和河畔GEON必须小心（i）不纠结锯丝，（ⅱ）使用导线的中间三分之二而施加恒定稳定张力，和（iii），以避免过量的运动，以获得直骨切口。 骨愈合是可能在本模型提供了一个骨移植物被使用。此外，该模型允许参与骨替换策略的机制时，无论是人起源移植物或细胞在良好标准化，大，节段性骨缺损用于进一步研究。 此外，在一行需要细化和减少使用在骨科相关的研究的动物目前的趋势，这种模式可在与体内成像技术，例如生物发光一起使用。这种非侵入性技术允许同时监测植入细胞的存活和组织愈合，而不需要动物牺牲26。 本模型的主要限制是两个承重条件和创建的，因为它们不完全模仿那些在人类临床上所遇到的骨缺损的体积。模型的其它局限性是:（i）该板的辐射不透明度，这可能需要移除所述板的离体的μCT分析之前，可能复杂的纵向放射检查结果的解释和，（ii）所述无力调节板刚度其中可能是骨形成27-30的关键力学参数。 必须牢记也，即使用骨同系移植或含矿物组分（具体碳酸钙）等的支架时，有些偏差都在微CT分析的分割过程中引入的，因为新形成的骨的密度部分地重叠无论是同基因移植密度或支架密度。由于这个原因，骨体积由微CT分析获得大多体现矿化组织（新形成的骨的体积加骨替代）11,26,31。

大规模的骨干骨缺损是整形外科医生一个巨大的挑战。骨替代自体骨移植，目前认为是黄金标准治疗，是限量供应，并与收获有关的发病率有关。由于这些原因，组织工程骨构建体骨髓间充质干细胞相结合的骨传导支架已探索作为在整形外科手术自体移植的替代方法。 迄今为止，大多数的研究已经在临床相关的动物模型如狗，猪，羊1-3，但在原位，节段性，关键尺寸骨的小动物模型的缺陷，这些结构的初步评价执行（如只小鼠）可以有几个优点:（ⅰ）低费用，（ⅱ）可被操作大量的动物; （三）在对比大型动物模型中，小鼠品系的均匀限制在支架骨吸收一个个体差异第二骨形成和; （四）最重要的是，特异性抗体和基因靶向动物的可用性使参与骨愈合的生物学过程的评价。最后但并非最不重要的，使用小鼠的免疫缺陷的菌株还可以研究使用任一移植物或人来源的细胞，而不在小鼠的不良免疫反应。 尽管有上述优点，在小鼠体内大量骨干骨缺损的模型是稀疏。大多数这种模型的使用的骨固定用髓内钉，其填埋所述骨髓腔（从而限制材料的体积要测试），也通过不提供旋转和轴向稳定性2,4-7阻碍再现性。 当前研究的目的是（i）模仿临床骨不愈合的情况下，描述一个可重复的，关键的大小，分段，小鼠股骨缺损模型，这是精确和可重复的锁板osteosynth稳定ESIS，提供了高度稳定的生物力学环境8-10; （ⅱ）来说明具有两个潜在的骨代用品本模型和描述的骨形成分析可能被使用。

<table><tbody><tr><td>&amp;alpha;-MEM , Minimum Essential Medium Eagle</td><td>Sigma-Aldrich, France</td><td>M4526</td><td>500 ml&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Acropora&lt;/em&gt; sp. coral exoskeleton cubes, Biocoral&amp;reg;</td><td>Biocoral&amp;reg;, Inoteb, France</td><td></td><td>3 x 3 x 3 mm cubes, autoclaving (121 &amp;deg;C for 20 min) sterilization</td></tr><tr><td>Buprenorphine, Buprecare&amp;reg;</td><td>Axience, Pantin, France</td><td></td><td>0.3 mg/ml</td></tr><tr><td>Xylazine, Rompun&amp;reg; 2%</td><td>Bayer HealthCare, Puteaux, France</td><td>20 mg/ml</td><td></td></tr><tr><td>Ketamine, Ketamine 500&amp;reg;</td><td>Virbac, Carros, France</td><td></td><td>50 mg/ml</td></tr><tr><td>Isoflurane, For&amp;egrave;ne&amp;reg;</td><td>Abbott, Arcueil, France</td><td></td><td></td></tr><tr><td>Enrofloxacine, Baytril&amp;reg; 5%</td><td>Bayer HealthCare, Puteaux, France</td><td>50 mg/ml</td><td></td></tr><tr><td>Pentobarbital, Dolethal&amp;reg;</td><td>V&amp;eacute;toquinol, Lure, France</td><td></td><td>182.2 mg/ml</td></tr><tr><td>Anesthetizing box</td><td>Ugo Basile, Gemonio, Italy</td><td>7900/10</td><td></td></tr><tr><td>Plastic transparent sterile drape, BusterOpCover 30 x 45 cm</td><td>Buster, Coveto, Montagu, France</td><td>613867</td><td></td></tr><tr><td>10% povidone iodine, V&amp;eacute;t&amp;eacute;dine&amp;reg; Solution</td><td>V&amp;eacute;toquinol, Lure, France</td><td></td><td>100 mg/ml</td></tr><tr><td>Titanium micro- locking plate, MouseFix Plate XL</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>RIS.401.120</td><td>6 holes, 10 mm long and 1.5 mm wide, autoclaving (121 &amp;deg;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td></tr><tr><td>0.3 mm drill bit, Drill Bit 0.30 mm</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>RIS.592.200</td><td>autoclaving (121 &amp;deg;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td></tr><tr><td>Engine power</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>AccuPen</td><td>Cold sterilzation (ethylene oxide)</td></tr><tr><td>Screw driver, Handrill</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>RIS.390.130</td><td>autoclaving (121 &amp;deg;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td></tr><tr><td>Self-tapping locking screws, MouseFix Screw 2 mm</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>RIS.401.100</td><td>2 mm long, 0.47 mm outer diameter and 0.34 mm core diameter, autoclaving (121 &amp;deg;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td></tr><tr><td>Jig, MouseFix XL Drill and Saw Guide</td><td>RISystem AG, Davos, Switzerland, http://www.risystem.com/</td><td>RIS.301.103</td><td>3.5 mm between the slots, autoclaving (121 &amp;deg;C for 20 min) sterilization or cold sterilzation (ethylene oxide)</td></tr><tr><td>0.22-mm Gigli saws (0.22 mm Saws)</td><td>RISystem AG, Davos, Switzerland</td><td></td><td></td></tr><tr><td>5.0 glycomer 631, Biosyn</td><td>Covidien, V&amp;eacute;toquinol, Lure, France</td><td></td><td>Tapper-cut needle</td></tr><tr><td>4.0 glycomer 631, Biosyn</td><td>Covidien, V&amp;eacute;toquinol, Lure, France</td><td></td><td>Tapper-cut needle</td></tr><tr><td>X-ray, MX20</td><td>Faxitron X-ray Corp, Edimex, Le Plessis Grammorie</td><td></td><td></td></tr><tr><td>&lt;i&gt;In vivo&lt;/i&gt; high-resolution microcomputed tomography, Skyscan 1176</td><td>Skyscan, Aartselaar, Belgium</td><td></td><td></td></tr><tr><td>&lt;i&gt;Ex vivo&lt;/i&gt; high-resolution microcomputed tomography, Skyscan 1172</td><td>Skyscan, Aartselaar, Belgium</td><td></td><td></td></tr><tr><td>Resident software: Nrecon (v1.6.9) / Ctan (v.1.14.4)</td><td>Skyscan, Aartselaar, Belgium</td><td></td><td></td></tr></tbody></table>

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.

上述外科手术过程历时45至60分钟。截骨和内固定很容易用一个外科医生的助手的帮助，但不使用任何放大系统来执行。无术中并发症发生。在18只11进行了初步研究，术后放射照片提供的证据表明，骨缺损长度（3.43±0.12毫米），该板的定位（距离扼杀关节腔和板的远端部分之间= 2.65±0.56 MM）是可再现。 麻醉相关的死亡率为约5％。 手术侧肢体的功能恢复是优异所有动物和完全负重手术（动画图1）后一天之内进行了观察。在p中使用的骨接合术（板和螺钉）的重量重发研究是小鼠体重的约0.1％。没有术后并发症（ 如，伤口感染，移植失败;骨移植物迁移等 ）的发生。造成cagemates没有自伤或伤害的发生。 当外科手术造成的骨缺损空，用一致的骨不愈合未见显著骨形成。相反，当所述缺损填充有任一个同系移植或珊瑚支架，新形成的骨没有观察到近端和远端骨边缘延伸。此外，尽管骨形成允许在与同基因移植物（图8），它仅在填充有该材料的缺陷珊瑚支架内观察到治疗的大多数缺陷重新建立骨连续性。事实上，在一个距离没有观察到骨从骨边缘大于1毫米。在所有的组织软骨缺失分析提供的证据结果所获得的骨接合术的稳定性（图9，图10）。 X光片和显微分析提供了证据，骨性愈合没有在缺陷左空组的任何动物发生10周后植入。通过显微分析评估矿化组织的体积为0.8±0.3 mm 3的并且是代表性的新形成的骨的。在同基因移植和珊瑚支架组，于4和4只动物分别获得骨性愈合。矿化组织通过显微分析所评估的体积为4.4±0.9 立方毫米和 8.9±0.7 立方毫米。在这些基团，但是，因为无论是同系移植和珊瑚脚手架含有矿物质，新骨形成不能从剩余的植入材料（同系移植或珊瑚支架）真正区别。盂兰盆联盟的两个速率和矿化组织从同系移植组中得到的体积和从珊瑚支架组均显著（P &lt;0.001），比从缺陷左空组获得的更高。 <img alt="图1" src="/files/ftp_upload/52940/52940fig1.jpg" /> 图1:股骨骨缺损的创作手术暴露 15 - 17毫米的纵向切开皮肤，从髋关节延伸到膝关节，用股骨前外侧进行。 阔筋膜切开; 股外侧肌和股二头肌被分裂暴露股骨干的全长。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52940/52940fig2.jpg" /> 图2: 板定位和近端螺钉放置。施加于前股骨侧的板。该板的最近侧孔被钻出;第一个螺丝插入，然后，被锁定。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52940/52940fig3.jpg" /> 图3:远端螺钉放置在板的最远端孔钻和螺钉插入并锁定。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52940/52940fig4.jpg" /> 图4: </STRONG&gt; 吉利锯的定位。在两个外螺丝插入但不锁定和0.22毫米吉利锯的线是在一个内-外方向周围的骨紧密联系在一起。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52940/52940fig5.jpg" /> 图5: 夹具定位夹具插入上的最后两个螺丝杆和板上面施加和锯的金属丝，然后在夹具的槽插入。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig5large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图6"SRC ="/文件/ ftp_upload / 52940 / 52940fig6.jpg"/&gt; 图6:截骨截骨已执行，吉利锯被撤回。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig6large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52940/52940fig7.jpg" /> 图7:内螺丝锁定除去夹具和最后的两个螺钉锁定。该段骨缺损当时或者空或填充测试的材料。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig7large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img al<html> T ="8"SRC ="/文件/ ftp_upload / 52940 / 52940fig8.jpg"/&gt; 图8: 代表术后X光片和小鼠的股骨矢状μCT重建股骨骨与各自的缺陷或者空（AE），或充满了大量的同源植骨（FJ），或充满了巨大的鹿角珊瑚支架（KO。 ）;手术后立即（A，F，K），手术（B，G，L），手术后6周（C，H，M）和手术10周后（D，E，I，J，N 4周后，O）（板长= 10毫米）。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig8large.jpg" target="_blank">点击此处查看该图的放大版本。</a> 文件/ ftp_upload / 52940 / 52940fig9.jpg"/&gt; 图9:代表X光片，μCT重建和组织学与珊瑚支架目前的研究测试中充满缺陷的大量新骨形成的是介于两者之间观察到的周围骨边缘和珊瑚支架;相反，小骨存在该支架内部。渍:Stevenel蓝和冯Gieson染色picrofuchsin。在这些条件下，骨细胞和珊瑚分别染成红色，蓝色和棕色。比例尺= 500微米。 ACS = 鹿角珊瑚支架; BN =骨。 （转载与组织工程C部分，2013，19（4），271-280许可） <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/52940fig9large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图10" src="/files/ftp_upload/52940/52940fig10.jpg" /> 图10:Repre<html>一个缺陷留空（A），与大规模的同基因骨髓移植（B）填充，并与珊瑚支架（C）填的表性组织学研究。在缺陷空，骨的舍入与髓腔填充和丰富的纤维组织深边缘到缺陷进行观察。在充斥着大量的同基因移植骨缺损，移植物和周围骨边缘之间观察骨连续性;骨髓是贯穿原腔存在。在充满珊瑚支架缺损，周围的骨边缘和珊瑚支架之间观察新骨形成了，但很少骨存在的支架内。渍:Stevenel蓝和冯Gieson染色picrofuchsin。在这些条件下，骨细胞和珊瑚分别染成红色，蓝色和棕色。比例尺= 500毫米。 ACS，珊瑚支架; BN，骨; BM，骨髓; FT，纤维组织。（从组织工程C部分，2013年，经许可后转载19（4），271-280）OAD / 52940 / 52940fig10large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="动画图11" src="/files/ftp_upload/52940/52940animfig1.jpg" /> 动画/视频图1:老鼠一天术后的步态的代表视频。全重观察方位。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52940/Animated_Figure1.MOV" target="_blank">请点击此处观看该视频。</a>

Watch this Scientific Journal Video about Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis at JoVE.com

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

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 ...

establishment-segmental-femoral-critical-size-defect-model-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.0K 次观看.  Université Paris Diderot. 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.

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

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

可调节刚度，外固定支架的大鼠股骨截骨和节段性骨缺损模型

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

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 ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

生物工程

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

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 ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

带稳定针的小鼠股骨横断

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

生物学

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

<meta charset="utf8"></head><body>使用钢板固定研究蝾螈的骨愈合和肢体再生

Stabilizing a Femur Osteotomy with a Plate Fixation in Ambystoma mexicanum

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged organs, including limbs, brain, heart, tail, and others. Studies on axolotl shed light on cellular and molecular pathways ruling progenitor activation and tissue restoration after injury. This knowledge can be applied to facilitate the healing of regeneration-incompetent injuries, such as bone non-union. In the current protocol, the femur osteotomy stabilization using an internal plate fixation system is described. The procedure was adapted for use in aquatic animals (axolotl, Ambystoma mexicanum). &#8805;20 cm snout-to-tail tip axolotls with fully ossified, mouse-size comparable femurs were used, and special attention was paid to the plate positioning and fixation, as well as to the postoperative care. This surgical technique allows for standardized and stabilized bone fixation and could be useful for direct comparison to axolotl limb regeneration and analogous studies of bone healing across amphibians and mammals.

<meta charset="utf8"></head><body>作者聚焦：通过对细胞和分子机制、骨愈合和对人类治疗的影响的见解来探索蝾螈的再生

A protocol for femoral osteotomy surgery with the use of internal plate fixation in mature axolotls is presented. The procedure can be used to perform comparative studies on limb regeneration and fracture healing in aquatic amphibians.

在墨西哥 Ambystoma 中使用钢板固定稳定股骨截骨术

The axolotl (Ambystoma mexicanum) is a promising model organism for regenerative medicine due to its remarkable ability to regenerate lost or damaged ...

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

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此视频中的章节

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

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