Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

中sRANKL MC DNA 1。水动力传递<ol><li>通过小鼠尾静脉流体力学交货<ol><li>尾静脉注射前称量鼠标。中的小鼠体重的10％〜的总体积稀释sRANKL或绿色荧光蛋白（GFP）的MC在林格氏溶液（预暖，在37℃）。 </li><li>热身鼠标在一个笼子10分钟前注射，以扩张血管，使侧脉（LVS）可见。监视鼠标小心，以避免脱水和高热。 </li><li>只要LV的被扩张，可见，鼠标转移到阻挡并将插头远远不够倒入桶内，以抑制运动。监视鼠标正常活动如呼吸。如果需要调整限制器的插头。 </li><li>从尾尖用酒精擦拭消毒注射区的3/4。使用27-30号针头的小鼠尾静脉注射。紧紧握住尾巴用一只手插入针到尾静脉。施加压力的注射器，并在5-7秒完成注射。 </li><li>从尾静脉中取出针头并用棉球将压力施加于注射部位止血。监视鼠标15-30分钟，再用鼠标转回动物饲养，一旦呼吸率降低到正常速度。 </li></ol></li><li>的小鼠血清TRACP5b的帖子基因转移系统性鼠标sRANKL的表达和定量确认<ol><li>热身鼠标在一个笼子里5-10分钟，以扩张血管，使的LV可见。监视鼠标小心，以避免脱水和高热。 </li><li>作一小切口用刀片将鼠标的尾部在一个站点1列从尾部的尖端。 </li><li>收集约100微升的血液中的血清分离管中。 </li><li>孵育血清分离管在室温下放置30分钟。 </li><li>以10,000×g离心5分钟，离心样品并收集血清。 STOR在-80°C，E无血清作进一步的分析。 </li><li>使用市售的试剂盒血清样本进行sRANKL ELISA和小鼠血清TRACP5b的检测。 </li></ol></li></ol> 2，体外破骨细胞生成的鼠标桥梁养护管理<ol><li>隔离和桥梁养护管理的培养<ol><li>胫骨和股骨骨隔离:安乐死的供体小鼠与CO 2，消毒腿用70％乙醇。从耻骨解剖到跟骨，以除去股骨和胫骨完好无损。 </li><li>小心地取出皮肤和肌肉不破坏骨。放置处理股骨和胫骨中含有磷酸盐缓冲盐水（PBS）的培养皿中。 </li><li>去除骨髓:切断对股骨和胫骨的骨骼的一侧的前端和冲洗出骨髓入50毫升的管中，使用1ml注射器用25号针头，装有含1％青霉素/链霉素的α-MEM （破骨细胞培养基/ OCM）。 </li><l我&gt;过程骨髓:混合并通过一个70微米尼龙细胞过滤器传递给它的骨髓悬浮液转移至新的50ml管中。离心将细胞以300×g离心并重新悬浮在α-MEM中含有1％青霉素/链霉素和10％胎牛血清（破骨细胞培养基/ OCM（+））。 </li></ol></li><li>巨噬细胞的培养<ol><li>使用血球或自动细胞计数器计数细胞。 </li><li>板1×10 6每孔细胞的6孔板或3×10 5细胞在玻璃盖玻片（5毫米）或牙本质片放置在96孔板中孵育在37℃下</li><li>吸出物含有非贴壁细胞和培养细胞在37℃下用含有25纳克/毫升鼠M-CSF 48小时新鲜OCM（+）的所有介质。 </li><li>含有25毫微克/毫升鼠标M-CSF和30毫微克/毫升小鼠sRANKL启动破骨细胞吸出所有媒体和培养细胞在37℃的新鲜OCM（+）的媒体。根据需要补充媒体每3天。 </li><li>细胞生长约6-8天以形成能够再吸收骨巨多核细胞。只要多核细胞出现，执行TRAP（抗酒石酸酸性磷酸酶），使用市售的试剂盒染色，而当完全成熟，进行功能分析。直观上用鬼笔环肽染色和图像附着用扫描电子显微镜（SEM）如先前19描述牙本质片的细胞的盖玻片的F-肌动蛋白环。 </li></ol></li></ol> 3， 在体外破骨细胞生成从人PBMC <ol><li>人外周血单个核细胞的分离<ol><li>加入10 ml预热的Histopaque-1077的入50毫升的试管中。 </li><li>从用无菌PBS血库获得到50ml试管（1:1比例的血用PBS）空白细胞过滤器。层中的稀释血液在HISTOPAQUE溶液非常缓慢，并确保血液不与HISTOPAQUE混合。 </li><li>在1000×g离心20分钟，离心样品在18°C。跟随ING离心，小心分离含有白细胞的白色血沉棕黄层和层转移到新的50ml管中。 </li><li>在650×g离心5分钟，再稀释细胞，用PBS离心以收集细胞。 </li><li>在OCM去除上清，重悬细胞（ - ）介质，并使用细胞计数器计数细胞。 </li></ol></li><li>电镀与外周血单个核细胞培养<ol><li>重悬的细胞在OCM（+）介质和板8×10 6个细胞/ ml，每孔中×10 6个细胞/ ml，每孔在96孔板的6孔板或1。根据需要为免疫荧光成像，以及对骨片适于骨吸收测定法作为在玻璃盖玻片（5毫米）的板的细胞先前所描述20。 </li><li>改变培养基，补充细胞与人M-CSF，每2-3天或按要求。然后通过添加人sRANKL的30纳克/毫升连同25毫微克/毫升的人M-CSF在OCM（+）媒体发起osteoclastogenes进行到第5天的分化步骤是。 </li><li>生长细胞为约14至21天，以形成巨型多核细胞。只要多核细胞出现，这些细胞可以被标记为TRAP，并且当完全成熟，功能分析可以被执行。 F-肌动蛋白环可以可视化使用鬼笔环肽染色盖玻片。细胞的形态，可以检查连接在用SEM对牙本质切片的细胞​​。 </li></ol></li></ol>

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The authors have nothing to disclose.

肌肉骨骼条件，导致发病率和致残的主要原因，并包含超过150个疾病和证候;影响大约90万美国人。关节的炎症和骨质破坏是骨骼肌肉条件的主要功能，包括关节炎和骨质疏松症。骨质疏松症是骨弱化完整性，常常导致骨的骨折的情况。关节炎是一种慢性的，衰弱的疾病，其特征是变得肿胀，招标和僵硬，限制正常运动，并可能导致残疾的关节发炎。虽然肌肉骨骼疾病的发病机制大大不同于彼此，骨破坏的总体共同特征。在肌肉骨骼疾病的骨破坏主要是由骨吸收和骨形成21,22之间的不平衡造成的。一种治疗方法是试图阻止这些细胞的破坏骨的能力<s高达&gt; 23。此处所述的技术和程序，使破骨细胞在体内和体外的研究。 这里所描述的体内模型使用的minicircles，这是从细菌骨干释放小游离DNA载体（〜4KB），这也作为转基因载体24。的minicircles表达转基因10-1000倍高于正常细菌质粒与稳定表达长达数月。的minicircles较小的分子大小有助于更有效的转染率和大量的表达比较正常的细菌质粒24。因此，的minicircles现在被广泛地应用于临床前基因转移研究25,26。水动力传递是基于在毛细血管动水压力传递的质粒DNA导入小鼠全身发达的方法。经尾静脉注射小鼠流体力学基因递送到肝细胞是行之有效吨Ø通过质粒DNA 6表达转基因。在这些研究中，在体内小鼠模型中过度表达的小鼠sRANKL由sRANKL MC和经尾静脉注射的流体动力输送来实现。鼠标sRANKL ELISA可用于检测小鼠RANKL的血清水平。小鼠血清TRACP5b的，骨吸收标志物27，显著抬高sRANKL基因转移的小鼠观察。值得注意的是，该技术并不局限于sRANKL的过表达;它也可以进一步应用到其他过表达分泌蛋白28。在此过程中的主要技术难点是经尾静脉的流体动力传递。除了定期尾静脉注射，流体动力传输需要交付的minicircles在大量生理溶液进入小鼠在5-7秒。因此，稳定的操控和注射技术是此过程成功的关键。产生sRANKL转基因小鼠的体内 osteoc另一种方法lastogenesis。然而，sRANKL高水平的可导致胚胎致死4以及产生转基因小鼠也是昂贵和费时的。 在OVX啮齿动物模型中，炎性细胞因子如TNF，IL-1和IL-6以及非炎症性细胞因子sRANKL有助于体内破骨细胞和骨损失。 sRANKL基因转移模型导致只有非炎症性细胞因子sRANKL的表达。因此，这里所描述的sRANKL基因转移模型研究人员提供一个功能强大的工具来检查RANKL-RANK轴骨重塑在没有炎症的贡献。 体外破骨细胞培养方法，它涉及到共培养的骨髓细胞和成骨细胞，成立于20世纪80年代之前，RANKL的在1990年底的29,30的发现晚了。重组M-CSF和RANKL诱导的破骨细胞分化有效地在体外次该方法已在其它研究中3,31先前已经描述。 M-CSF是众所周知的刺激RANK表达在早期破骨细胞前体32，并作为对破骨细胞前体和成熟破骨细胞33的存活因子。 RANKL是破骨细胞的分化和活化3是必不可少的。这里， 在体外破骨细胞形成的方法进行说明。成功培养和前体细胞分化为破骨细胞需要无菌细胞分离和适当的电镀技术，以及仔细了解计时单元的收获。细胞计算镀是至关重要的，因为破骨细胞分化需要融合的破骨细胞前体细胞的34和破骨细胞前体细胞的数量不足不会促进融合前体细胞和分化为破骨细胞。类似地，破骨细胞前体细胞的数目过多，会导致结块的形成和最终细胞死亡。拆除非电镀后的细胞贴壁细胞2小时可以帮助维持最佳的手机号码。最后，细胞收获的时间是至关重要的，因为破骨细胞终末分化的短命细胞约48小时35的寿命。因此，电池应在48小时先观察多核细胞后收获。这些细胞然后可以其特征在于，TRAP和F-肌动蛋白染色用免疫组化和骨吸收活性可以通过扫描电子显微镜（SEM）来检测骨。这些实验方法使他们的表型和功能方面破骨细胞的明确定义。 在体内和在这里描述的体外技术将促进目前在临床前研究中的破骨细胞抑制剂的开发，导致对全世界数以百万计的骨质疏松症和关节炎的患者的有效治疗策略。

肌肉骨骼疾病影响到千百万人在美国和现在的严重后果为国家和地方卫生系统1。这些疾病的特点是骨损失和关节功能，需要广泛的治疗和恢复很长时间。通常，在破骨细胞的数量和/或活性的相对增加，细胞专门的重吸收骨骼，在骨质疏松和关节炎，观察2。在生理条件下的破骨细胞的数量和活性是通过核因子κ-B配体（RANKL），它是由成骨细胞产生的受体活化调节。护骨素（OPG），诱饵受体RANKL由成骨细胞3也产生涉及sRANKL全身过度表达，或删除OPG的体内动物模型是非常有价值的骨质疏松症的研究。然而，这些方法需要转基因小鼠4,5的产生。这里，一种新型的替代过表达sRANKL为肌肉骨骼相关的疾病的研究的方法进行说明。具体地，小环（MC）DNA技术和流体动力传递方法被用来实现sRANKL的体内基因转移和过表达小鼠sRANKL全身6。 这种方法也补充其他体内模型的骨质疏松，如由低钙饮食8以下卵巢切除7和饮食干预破骨细胞的激素调节。这些模型是研究的但是他们需要外科手术，可能需要长达数月，在一个显著的成本9肌肉骨骼相关疾病的不同方面非常有用。卵巢切除（OVX）啮齿动物模型的实验动物模型，其中卵巢切除导致雌激素缺乏，从而模仿人类绝经后骨质疏松症10。人绝经期后骨质疏松症，一个条件，其中的雌激素deficiENCY导致骨折的风险增加和骨质疏松症影响约800万妇女在美国就有。虽然绝经后骨质疏松症的去势模型是非常有用的它提供了有限的优势，在一般的研究骨质疏松症。雌激素可以抑制骨质流失，通过诱导破骨细胞，抑制成骨细胞凋亡，因此，在其没有增加破骨细胞活性观察10-12。一个RANKL-OPG比例失衡有利于骨吸收也观察到13。然而， 在体内的雌激素缺乏也伴随着降低的转化生长因子β（TGFβ），增加白细胞介素-7（IL-7）和肿瘤坏死因子，IL-1和IL-6的水平14,15。由于这些细胞因子已知的骨重塑调节功能独立于RANKL通路，这是不可能的属性的任何破骨细胞的活化，仅与RANKL-RANK轴。在本文中描述的模型使研究人员在VIV研究ØRANKL-RANK轴的破骨细胞和骨量丢失无促炎细胞因子相比，去卵巢啮齿类动物模型。 此外， 体外破骨细胞技术是必不可少的工具来研究破骨细胞活化肌肉骨骼疾病的潜在治疗方法。以前的研究还表明，与小鼠巨噬细胞集落刺激因子（M-CSF）和小鼠sRANKL培养小鼠骨髓衍生的巨噬细胞（桥梁养护）能导致破骨细胞分化3,16,17。在此，协议来生成从小鼠骨髓以及从人外周血单核细胞（PBMC） 在体外 18多核破骨细胞样细胞进行了描述。都定义一个成熟的终末分化和功能齐全的破骨细胞所需的基于细胞的测定也简单说明。这些体外技术补充体内的方法小说一起用于为powerful调查工具，研究破骨细胞分化和活化。使用这些系统中，科学家们能够产生在体内和体外的破骨细胞和确定所需的增殖和活化的刺激和信号，以及测试的药理和生物抑制剂的功效。

<table><tbody><tr><td>alpha-MEM</td><td>Life Technologies&amp;nbsp;</td><td>12561-056</td><td></td></tr><tr><td>Human M-CSF</td><td>Miltenyi Biotec</td><td>130-096-492</td><td></td></tr><tr><td>Mouse M-CSF</td><td>Miltenyi Biotec</td><td>130-094-643</td><td></td></tr><tr><td>Human RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-631</td><td></td></tr><tr><td>Mouse RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-076</td><td></td></tr><tr><td>Tailveiner Restrainer for mice</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>MTR00</td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma</td><td>387A</td><td></td></tr><tr><td>MouseTRAP assay&amp;nbsp;</td><td>immunodiagnostic systems</td><td>SB-TR103</td><td></td></tr></tbody></table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

这里， 在体内和细胞培养协议破骨细胞分化的新基因转移技术，用于差分前体细胞在体外破骨细胞作为研究细胞因子对破骨细胞形成的影响的方法进行了描述。在图1中，绿色荧光蛋白和小鼠sRANKL MC小鼠成功的基因转移的代表性结果示。在图2中，示出的小鼠骨髓或前体细胞向破骨细胞通过明场显微术的人PBMC细胞分化时程文化的代表性图像， 图3中，代表性图像从三个独立的方法用于表征小鼠骨髓来源的破骨细胞的示。鼠标sRANKL的存在下激活的TRAP的表达（ 图3A和B）的F-肌动蛋白环（ 图3C和D）的形成。型扫描电微镜（SEM）表明，小鼠sRANKL诱导能力再吸收骨（ 图3E和F）的巨核细胞的形成。 图4，在人PBMC同一表征方法衍生的破骨细胞被示出。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig1highres.jpg" width="500" /> 图1。sRANKL的体内基因转移导致增加的破骨细胞形成。A的前视图，B）的小鼠肝脏和外植体C，D）12周龄的C57BL / 6雄性小鼠的全身图像注射5微克GFP或sRANKL的三菱商事的DNA。箭头指示在体内 GFP表达在GFP基因转移小鼠。 RANKL基因转移的小鼠作为没有背景荧光。由大师2成像仪，一天后基因转​​移（红色和绿色ð拍摄的图像sRANKL MC-DNA结构和F）sRANKL水平1，5分析了血清中的enote背景和GFP，分别）。E）的质粒图和11天后GFP或sRANKL的基因转移。G）小鼠血清TRACP5b的层次分析11天后GFP或sRANKL的基因转移。刻度条表示5mm的A，B，和10毫米，C，D。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig2highres.jpg" width="500" /> 图2。 体外破骨细胞形成在小鼠和人的细胞培养系统。小鼠细胞分离自骨髓并培养在25纳克/毫升鼠M-CSF和30毫微克/毫升小鼠sRANKL的存在呈现出分化的明场显微术时间当然在天A）2 B）5，和C）8。人细胞分离的明视场显微术从外周血中的25毫微克/毫升的人M-CSF和30毫微克/毫升人类sRANKL表示在天D中分化时程），4 E）14，和F）21存在下培养。刻度条表示20μm左右。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig3highres.jpg" width="500" /> 图3。生成和小鼠破骨细胞特征的小鼠细胞分离自骨髓和6天在A的存在）培养的。TRAP染色（粉红色）25毫微克/毫升鼠标M-CSF或B）25毫微克/毫升鼠标M -CSF和30毫微克/毫升小鼠sRANKL。鬼笔环肽（绿色）和小鼠细胞分离自骨髓和8天，在C的存在），25纳克/毫升鼠M-CSF或D）25毫微克/毫升鼠M-CSF和30ng的培养的DAPI（蓝色）染色/ ml小鼠sRANKL显示法的形成CTIN环多核细胞。小鼠细胞从骨髓和8天，在E的存在下培养）25毫微克/毫升鼠M-CSF或F）25毫微克/毫升鼠M-CSF和30毫微克/毫升小鼠sRANKL示出的形成孤立的SEM显微照片由箭头所指示的对骨吸收的区域。比例尺表示40微米的AD，并在E，F。30微米<img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51810/51810fig4highres.jpg" width="500" /> 分离出的PBMC，培养在A的存在15天人类细胞）的图4。代数和人类破骨细胞的表征。TRAP染色（红色）25毫微克/毫升的人M-CSF或B）25毫微克/毫升的人M- CSF和30毫微克/毫升人类sRANKL。鬼笔环肽（绿色）和DAPI（蓝色）染色的人细胞中分离出外周血单核细胞并培养19天以C的存在下） </强&gt; 25纳克/毫升的人M-CSF或D）25毫微克/毫升的人M-CSF和30毫微克/毫升人类sRANKL表示的F-肌动蛋白环的多核细胞的形成。分离PBMC并培养18天的E的存在人类细胞）的SEM显微照片，25毫微克/毫升的人M-CSF或F）25毫微克/毫升的人M-CSF和30毫微克/毫升人类sRANKL表示骨吸收的形成由箭头所指示的骨区域。比例尺表示10微米的A，B，20微米C，D 15微米E，F。

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

Nanyang Technological University

Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K 次观看.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

视频: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

科研

发育生物学

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

医学

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

生物工程

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

股骨窗口商会模型<em&gt;在体内</em在小鼠骨髓&gt;手机跟踪

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

免疫与感染

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

用于刺激骨质复变和分析骨骼重塑功能结果的实验室A芯片平台

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

<meta charset="utf8"></head><body>骨类器官衍生细胞在辐照小鼠中进行有效 HSCT

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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股骨窗口商会模型

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

用于刺激骨质复变和分析骨骼重塑功能结果的实验室A芯片平台

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法

用于收获治疗性造血干细胞/祖细胞的体内骨类器官方法