We are grateful to the Biotechnology and Biological Sciences Research Council (BBSRC) for funding DH (BB/J01446X/1), CF &amp; VEM (Institute Strategic Programme Grant Funding BB/J004316/1). We also acknowledge Arthritis Research UK for funding to KAS (20413). We thank Miss Elaine Seawright for her assistance with the experiments detailed, Dr. Neil Mackenzie for his assistance with the lentiviral techniques, and Mr Darren Smith and Mr Alex Robertson (Roslin Institute BRF) for their assistance with the animal studies.

涉及受试动物程序符合与英国动物（科学程序）1986年法令和动物保持按照内政部公布进行实务守则动物的居所和照顾良种，提供或用于科研目的。 注:使用野生型C57BL / 6J小鼠中进行的实验已经确立并描述如下。来自不同的小鼠品系胚胎跖骨可以类似在体外培养的，虽然它们的生长和矿化速率可能不同于那些本文详述。 1.解剖条件及仪器的研制<ol><li>执行所有媒体的准备，组织解剖和文化工作，层流罩，以确保媒体和胚胎萌芽不育。 </li><li>允许培养和解剖媒体在37℃，5％的CO 2在使用前培养箱平衡至少1小时。 </li><li>清扫消毒剪刀，杜蒙＃5，＃杜蒙4镊子旁边一对超细Vannas微型剪刀（直8厘米，3毫米刀片）通过高压灭菌。高压灭菌后，将其浸入夹层设备在70％乙醇在使用之前。 </li></ol> 2.解剖和文化传媒的制备<ol><li> 准备夹层介质 <ol><li>在磷酸盐缓冲盐水（PBS）（1:13）稀释α-最小必需培养基（无核苷，αMEM）和过滤通过0.22μm33毫米直径的注射器灭菌之前以2mg / M L悬浮牛血清白蛋白（BSA）过滤。 注:夹层介质可制成，过滤灭菌，并在-20℃下以等分试样储存无限期。 </li></ol></li><li> 准备培养基 <ol><li>在300μl的培养基中培养胚胎跖骨（每孔的24孔培养板）。通过加入0.2％w / v的BSA的制备培养基; 5微克/毫升的L- ASCO山梨酸磷酸盐; 1毫米β-甘油（βGP;可选 - 见结果部分）; 0.05毫克/毫升庆大霉素和1.25微克两性霉素B至αMEM和过滤通过0.22μm33毫米直径的注射器过滤消毒。 </li></ol></li></ol> 3.小鼠胚胎跖骨解剖和文化<ol><li>按照英国内政部的指导方针卡尔的孕鼠，窝藏15日龄，颈椎脱位</li><li>放置动物仰卧和通过用70％乙醇喷雾消毒皮肤。用解剖剪使在中线大切口，穿透既皮肤和暴露腹腔腹膜。 </li><li>定位在体腔的背侧区域中的动物的两个子宫角。通过首先从系膜及通过仔细的切口与解剖剪刀释放每个子宫，最后切子宫角自由他们的基地取出子宫角。的PlacË子宫角成夹层介质。 </li><li>沿角子宫着床点之间切割分离每个胚胎和使用镊子从他们的个人囊取出胚胎。卡尔胚胎断头按照英国内政部的指导方针</li><li>通过用70％乙醇喷雾消毒皮肤后，使用Vannas微剪刀通过围绕股骨近端切割以除去由胚胎后肢。尽可能除去这确保尽可能多的后肢。将后肢成前跖骨夹层夹层介质淹没培养皿。 </li><li>在解剖显微镜下，开始以除去围绕胚胎脚皮肤，通过捏和使用＃5镊子同时保持后肢稳定使用＃4镊子牵拉它关闭。这是最有效地通过在围绕胫骨的水平把持皮肤并朝向指骨拉动执行。在这个阶段使用剪刀切割不需要非常薄的皮肤。 </li><li>不放心地胚胎脚与4号镊子的跗骨和使用＃5镊子和丢弃捏掉跗骨附近除去第一和第五跖骨。 </li><li>在同一位置上的脚，使用＃5镊子扰乱剩下的三个指骨和跖骨之间的结缔组织。插入＃5镊子的顶端在趾骨和跖骨之间的接合，以从跖骨除去指骨。 </li><li>最后，使用＃5镊子破坏跗跖骨和之间的结缔组织。再次将＃5镊子尖从跗骨跗骨和跖骨，轻轻免费跖骨之间的关节间隙。 </li><li>轻轻一挑了4号镊子和地方的新鲜夹层介质现在​​免费跖骨。 注:小心拨开应提供从每个胚胎6跖骨。 </li><li>当所有需要跖骨已经被解剖，carefuLLY地方跖骨单独到含有300微升培养基的预热24孔板。在37℃下在5％CO 2的培养箱中长达14天的培养跖骨。 注意:不要更改E15文化的媒体至少到培养第5天，因为这会影响他们的矿化能力26。造成这种情况的原因尚不清楚，但线性生长和基质矿化可以通过从跖骨释放生长因子依赖于刺激。 </li></ol> 4.分析和跖骨文化操纵<ol><li>上剥离（第0天）的天使使用用数码相机附及图像分析软件的显微镜初始纵向测量。测量中央矿化带的长度，当它出现在培养几天。 </li><li>周期性地，根据需要，进行进一步的测量（通常每第二日），直到培养物在该点m的最后一天etatarsal骨头都处理取决于所需要的结果（在引言中讨论）。 </li><li>跖骨器官培养与各种外部因素的补充介质，例如生长因子，激素和从培养22,24的第0天的药理学试剂。在这种所有的研究，控制是必需的未经处理的跖骨。 注:虽然这是可取的控制骨头是从对侧腿相当于雏形，再也没有出现过线性生长或养殖E15 2 日的矿化潜力任何差异，第3和第 4跖骨（见下文， 图2） 。 </li></ol>

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The authors have nothing to disclose.

小鼠胚胎跖骨的文化提供了软骨的生长和矿化的高度生理模型。早期的研究已经证实，E15小鼠跖骨经受骨骼生长和分化的正常模式。软骨（软骨细胞）和骨（成骨细胞和破骨细胞）细胞和它们各自的胶原基质是从那些在体内观察到的没有区别。不过，也有这种模式的一些公认的局限。破骨细胞分化的速度受损原样骨生长（但不软骨分化）。骨生长是比在体内观察到的大约慢50％，并且在其中影响生产的局部因素（ 例如 ，IGF-1，骨形成蛋白等 ）31全身因素可能是由于缺陷。此外，众所周知，骨是其机械环境敏感，这也是培养跖骨，其中至c响应的情况下hanges在机械负荷32,33。因此，在无负载的情况下，如在该协议中，矿化描述和矿物质吸收可能会受到损害。然而，跖骨模型提供直接测量线性骨生长经由2D和3D是不可能的体外培养系统的能力。 我们已经提供了参与E15鼠跖骨确实的解剖和文化，首次协议的全面描述，确认汇集第2， 第 3 和 第 4跖骨为实验的有效性。 从E17 / E18跖骨通常用于研究骨生长由于它们的巨大潜力的机制，以生长体外的时间（ 即超过14天培养）长时间。他们是一个行之有效的模型来研究胚胎骨的纵向生长的生长因子的作用。此外，产后跖骨的解剖和培养一般用来作为可以理解的是出生后骨骼生长和胎儿骨生长被不同地21调节以描绘周围产后骨生长的机制。然而，在出生后的培养跖骨观察到的骨骼生长比在胚胎萌芽25,34观察显著较少，限制了其在长期研究的潜力，并强调在这个阶段出生后对骨骼生长更加系统性的影响。的确，从小鼠3天旧产后跖骨通常可用于获得可测量的增长34的最新阶段。 在这里，我们描述了胚胎跖骨在E15的隔离我们的协议。由于没有在E15跖骨羟基磷灰石矿物的装置，它们提供了无与伦比模型来研究不仅周围纵向骨生长的机制，而且还引发骨骼矿化。终端肥大软骨细胞区的矿化基质提供了侵入成骨细胞放下一个骨特异性基质（类骨质），这是随后矿化的支架，因此这个初始的矿化是成功和功能骨形成35是至关重要的。事实上，一些利用E15跖骨最近的报告证实了其独特的矿化过程28,36,37的调查能力。此外，研究人员已经描述了使用E15跖骨血管生成38的模型，突出胚胎跖骨作为骨生长/矿化设置外模型的潜力。 我们描述了协议只需要标准的实验室设备，包括一个层流罩，用于执行解剖解剖显微镜，并在 CO 2培养箱中切下的初步知识的培养。此外，一个非常基本的尽头TURE介质含有αMEM，BSA，抗生素，抗真菌剂和L-抗坏血酸（羟脯氨酸和羟赖氨酸合成的辅因子，两个基本氨基酸用于生产胶原39）避免了使用未定义的添加剂，如动物血清的。传统上，研究者已加入βGP用来培养胚胎跖骨媒体，但在我们的研究结果发现，不要求在此培养系统成功矿化使用额外的磷酸盐源的。这是我们的追求，理解支撑ECM矿化机制的一个重要发现。 跖骨先前已感染了含有的Smad2的显性负形式探索长骨发育40的调节转化生长因子β的作用腺病毒。在这里我们也揭示野生型E15跖骨与GFP病毒颗粒成功转染，表明Ť帽子慢病毒技术可以很容易地通过操纵在跖骨的文化基因的表达。同样，跖骨器官培养系统是在其中进行比较的骨头从遗传上改变的小鼠的生长，以更好地理解特定基因在骨生长过程中的作用的优秀模型。我们以前的研究中使用的跖骨器官培养系统，以确定细胞因子抑制信号-2（SOCS2）的软骨内成骨生长的作用。使用从SOCS2缺陷的小鼠跖骨，我们已经表明，生长激素能模拟其纵向生长独立胰岛素样生长因子的（IGF-1），与野生型跖骨不生长激素治疗34反应， 41。这突出到跖骨培养物可以被操纵来检查不同的基因和/或在软骨内骨生长外在因素的影响的程度。 总之，我们有德泰导致胚胎跖骨其可以被操纵的成功提取和培养的方法，以及使用各种分析检查。此体外模型保持细胞-细胞和细胞-基质相互作用，以及包含在软骨的不同阶段的软骨细胞，因此提供了比单层或三维培养的细胞的多种生理模型。因此跖骨器官培养是调查负责软骨内骨化的分子机制的独特模式，并必须进一步推动我们双方骨生理和病理生理的认识

骨骼是有一系列的运动跨越功能，离子平衡一个高度复杂的，动态的和复杂的器官。由骨和软骨，骨骼是由哪些运行，以维持它的结构，生化和机械完整性1功能不同的细胞群。一个的骨架的主要功能是其在发育和生长的作用。这些过程需要哪些是通过紧密的内分泌和遗传控制调节多种细胞群的编排。 与扁骨例如肩胛骨，颅骨和胸骨的例外，哺乳动物骨架是通过被称为软骨内骨化的方法形成。这个过程中，调节这两个分子和空间，便从主要来自中胚层2间充质细胞的冷凝。下的转录因子SOX9的影响，将细胞在第的冷凝电子内部分化成软骨细胞，表达和分泌软骨特异性细胞外基质（ECM）组分，例如II型胶原和软骨聚集蛋白聚糖。在冷凝的保证金，细胞表达I型胶原，形成软骨膜，划定未来的骨3。以后，软骨膜的骨原分化成成骨细胞，由转录因子，Runx2的和osterix的4的表达定向。这导致成骨细胞的优势，这层被重命名其形成围绕骨的中间部分矿化骨衣领骨膜。软骨支架的骨膜和侵袭的血管发生渗透与它带来，haematopoetically衍生骨再吸收细胞，破骨细胞，从而再吸收软骨细胞残余，大部分软骨基质5。所形成的空间由骨原引起的初级骨化填充中心，逐渐占据了骨干的核心，并与骨膜融合。其他细胞产生骨髓。生左右，血管和骨原在显影骨干的任一侧（骨骺）穿透软骨富基质以形成二次骨化中心。位于骨骺与骨干在长骨的两端之间，软骨带-骨骺生长板-这是负责协调所有长骨6的线性增长。 生长板软骨细胞内增殖最初并随后通过形态不同的区域，通过转录和生长因子的表达顺序协调推进。这一系列事件的顶点是由软骨细胞的细胞周期和肥大在此软骨前体的中心的出口。肥大软骨细胞高效表达X型胶原和基质金属蛋白酶-13（MMP13），并在纵向生长的方向其相当量放大提供在哺乳动物7,8骨生长最大的贡献。此外，肥大软骨细胞通过基质囊泡的释放矿化其周围的ECM，膜为界的结构，通过将其列入磷酸（组织非特异性碱性磷酸酶（TNAP）和PHOSPHO1）和钙窜蛋白质（膜联蛋白专门用于生产无定形的磷酸钙的）9-11。这钙化软骨是由血管日起，破骨细胞招聘和吸收矩阵的基本骨髓侵犯。其次是成骨细胞的迁移和对准到他们放下类骨质的一个层，其迅速矿化钙化软骨残余的表面上。主松质的编织骨稍后改造到辅助松质12的板层骨。骺融合结果在软骨内骨化13停止。 软骨内骨化是根据由许多内分泌和旁分泌激素和生长因子紧调节，以便防止干扰的发展和/或纵向的骨生长14。更好地了解支持这些过程的分子和细胞机制是因此我们的追求，对人类的好处临床进展势在必行。以往的研究进的相似点和不同年龄，地点和物种生长板之间的差异极大地提高了我们的周围生长板的动态15,16的生理机制的理解。然而，分离的软骨细胞的研究中是困难的，因为去除他们的环境的软骨细胞可导致去分化，伴随键标记如的Col2a1 17的表达缺失。 鼠标跖骨器官移植培养，PI伊丽莎白伯格和他的同事在荷兰教授oneered，是研究软骨内骨化和骨骼生长在文化模仿看到体内 18骨骼的生长速度高度生理体外模型。唯一地，跖骨器官培养允许软骨的不同阶段软骨细胞的检查和维护细胞-细胞和细胞-基质相互作用，因此提供比细胞培养19-21接近于体内情况的条件。此外，该模型允许线性骨生长的直接检查，这是不可能在初级软骨细胞培养物。它也允许对全身和局部因素因此允许对生长板的局部效应的具体分析的分离。 跖骨的文化使得众多参数的调查。总长度和矿化的区域中的每日测量雏形可以用标准相差显微镜和图像分析软件来执行。组织学，原位杂交和免疫组织学染色可以在跖骨部分，其允许细胞和分子实体，比传统的细胞培养方法的一个主要优势的空间分辨率可容易地进行。免疫组织化学方法包括由增殖的软骨细胞22的检测和5-溴-2&#39;-脱氧尿苷（BrdU）标记的摄取的量化。此外，可以执行跖骨组织的进一步处理，以允许mRNA的表达（RT-qPCR的），蛋白质和细胞内信号分析（蛋白质印迹）或脂质组合物（质谱法）的下游分析。迄今为止大部分研究使用的胚胎培养跖骨，而不是从跖骨产后的动物。虽然这两种模式可以是信息，胚胎骨骼的研究有几大优势:他们的成长速度更快，可以EXPLO资讯科技教育，研究矿化过程23-25的启动和发展。 这个协议进行详细描述胚胎跖骨从胚胎第15天（E15）小鼠胚胎的后肢的错综复杂的清扫。此外，独特的解剖学跖骨的生长和矿化所示。

<table><tbody><tr><td>Dissection scissors</td><td>World Precision Instruments</td><td>14393</td><td>Autoclave sterilise before use</td></tr><tr><td>Dumont #5 tweezers</td><td>World Precision Instruments</td><td>500342</td><td>Autoclave sterilise before use</td></tr><tr><td>Dumont #4 tweezers</td><td>World Precision Instruments</td><td>500339</td><td>Autoclave sterilise before use</td></tr><tr><td>SuperFine Vannas scissors</td><td>World Precision Instruments</td><td>501778</td><td></td></tr><tr><td>Absolute ethanol</td><td>Fisher Scientific</td><td>E/0650DF/17</td><td>Dilute to 70% v/v in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>&amp;alpha;-MEM without nucleosides</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>22561021</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A3059</td><td></td></tr><tr><td>Syringe filters (0.22 &amp;mu;m) 33 mm diameter</td><td>Merck Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Phosphocholine chloride calcium salt tetrahydrate</td><td>Sigma Aldrich</td><td>P0378</td><td>Add directly to the media prior to experimentation</td></tr><tr><td>L-ascorbic acid-2-phosphate</td><td>Sigma Aldrich</td><td>A8960</td><td>Make up stock soution at 5mg/ml and aliquot at -20 &amp;deg;C</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C5080</td><td>Make up stock soution at 150mM and aliquot at -20 &amp;deg;C</td></tr><tr><td>&amp;beta;-glycerophosphate disodium salt hydrate</td><td>Sigma Aldrich</td><td>G9422</td><td>Make up stock soution at 1mM and aliquot at -20 &amp;deg;C</td></tr><tr><td>Gentamicin</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>15710049</td><td></td></tr><tr><td>Amphotericin B</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>15290018</td><td></td></tr><tr><td>Nunc cell-culture treated 24-well plates</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>142475</td><td></td></tr><tr><td>Polybrene</td><td>Sigma Aldrich</td><td>H9268</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fischer Scientific&amp;nbsp;</td><td>D3571</td><td></td></tr></tbody></table>

culture of murine embryonic metatarsals: a physiological model of endochondral ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

该方法的目的是隔离胚胎跖骨和培养它们纵向骨生长和ECM矿化的检查。使用本文中描述我们的协议，胚胎跖骨成功解剖，通过光学显微镜的观察。跖骨， 如图1所示进行的测量，显示出其在纵向长度增长和矿化的ECM的能力（ 图2和3）。矩阵的矿化首先在骨骼雏形的-骨干中期指出，很容易被光镜观察。虽然钙黄绿素的摄取可能会提供新形成的矿物增强的可视化无染色是必需的。 已利用胚胎跖骨最新22,27,28研究汇集解剖的第2， 第 3 和 第 4（CEN扫雷具三级）跖骨，假设在体外生长和矿化是可比的。 在小鼠体内跖骨的发展，但是，揭示了3 和 第 4位有其他跖骨和趾骨29之前，成矿的证据。单独分离，培养E15 第二的生长和矿化潜力评估，第3和第 4跖骨揭示矿化后7天文化（ 图2）的外观或程度无显著差异。从基线的增加百分比没有差异在培养期间被发现。作为矿化潜力没有差异指出汇集2 次 3 次和第 4跖骨的标准协议出现有效期为今后的研究。 传统上，研究者已经添加βGP媒体用于培养胚胎跖骨促进ECM矿化。然而，在细胞培养研究中，已经证明，如果TNAP酶加入到含有βGP成骨培养基，异位羟基磷灰石矿物沉积还甚至在不存在细胞30的发生。检验各种矿化基质和激动剂对跖骨矿化能力的影响，我们培养E15跖骨在培养基中含有各种添加剂，以及图像和长度测量每日记录。结果表明，E15跖骨仍成长和矿化的ECM在没有βGP的。 E15在抗坏血酸培养跖骨仅媒体表明在长度和矿化可比随着跖骨在βGP（ 图3）的存在下培养。 我们还表明，慢病毒可用于转染外源性DNA导入培养metatarsals。在这里，我们成功转染的培养物（夹层即天）用2×10 6个绿色荧光蛋白（GFP）每跖骨病毒颗粒的第0天跖骨。以使病毒转导，聚凝胺同时加入到培养物以1:500。培养物放置7天，没有媒体变化所需E15跖骨矿化发生，用4孵育前&#39;，6-二脒基-2-苯基吲哚（DAPI）染色5分钟，然后直接可视化使用共聚焦显微镜（ 图4）。我们的概念证明实验清楚地显示有效传导。不过这将是审慎的审查整个跖骨的全部章节，以评估其转染和有效的基因操纵。 <img alt="图1" src="/files/ftp_upload/54978/54978fig1.jpg" /> 图1: 标准方法用于测量Ť他长度和胚胎跖骨的矿化带。（A）对文化的第0天E15跖骨的总长度测量（夹层日）通过跖骨中心作出。一个跖骨总纵向长度（B）的测量后在培养七天。请注意，在跖骨的微小曲率。 （C）在培养七天矿化区长度的测量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig1large.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/54978/54978fig2.jpg" /> 图2: 增长和解剖学鲜明E15跖骨成像矿化容量和第二的测量，第三和第四跖骨从E15小鼠的后肢瓦特作为执行。 （ 一 ）第二，第三和第四跖骨整个文化时期的系列图像。在培养的每天（B）的百分比增加，从第0天的长度。 （C）第2，第3和第4跖骨在培养周期长矿物质表示为总跖骨长度的百分比。在数据被表示为平均值的平均值±标准误差（SEM）（N = 6）。 （D）在培养中的每一天之C57B1 / 6J跖骨长度。在数据表示为平均值±标准偏差（n = 6）。黑条= 1毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig2large.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/54978/54978fig3.jpg" /> 图3: 野生型的生长和矿化能力E15跖骨培养各种成骨培养基（A）中唯一的抗坏血酸，1毫βGP，3mM的磷酸，1.5mM的氯化钙或含媒介3 1mM氯化钙培养跖骨串行图像。 （B）从跖骨的第0天长度增加的百分比与各种补品培养。 （C）在各种培养基中培养跖骨矿物长度表示为总跖骨长度的百分比。数据被表示为平均值±平均值的标准误差（SEM），（N = 6）。黑条= 1毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54978/54978fig4.jpg" /> 图4:GFP病毒P E15跖骨的转文章。（A）E15跖骨分别为概念证明GFP病毒颗粒转。 （B）骨头用DAPI进行DNA染色的可视化。 （C）的双图像显示在整个跖骨的整体的GFP病毒的成功转染。白条= 0.5毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54978/54978fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification at JoVE.com

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

小鼠胚胎跖骨文化:软骨内骨化的生理模型

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

culture-murine-embryonic-metatarsals-physiological-model-endochondral

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.8K 次观看.  The University of Edinburgh. We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development. 

视频: Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

科研

发育生物学

Culturing and Measuring Fetal and Newborn Murine Long Bones

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy human bones makes particularly valuable the use of mammalian models, such as mouse and rat, to look into different aspects of bone growth and homeostasis. Additionally, the development of sophisticated genetic tools in mice allows more complex studies of long bone growth and asks for an expansion of techniques used to study bone growth. Here, we present a detailed protocol for ex vivo murine bone culture, which allows the study of bone and cartilage in a tightly controlled manner while recapitulating most of the in vivo process. The method described allows the culture of a range of bones, including tibia, femur, and metatarsal bones, but we have focused mainly on tibial culture here. Moreover, it can be used in combination with other techniques, such as time-lapse live imaging or drug treatment.

Here, we present a method for ex vivo culture of long murine bones at both fetal and newborn stages, suitable for analyzing bone and cartilage development and homeostasis in controlled conditions while recapitulating the in vivo process.

胎儿和新生儿小鼠长骨的培养与测量

Long bones are complex and dynamic structures, which arise from endochondral ossification via a cartilage intermediate. The limited access to healthy ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

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

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 ...

使用三系荧光小鼠的精确生长板损伤研究

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

作者聚焦:在小鼠中使用三系荧光报告基因推进生长板损伤模型

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

用于研究生长板损伤的 Tricolor 转基因小鼠模型

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

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 ...

小鼠胚胎跖骨文化:软骨内骨化的生理模型

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