我们想感谢乔治共享他们的支持和合作，我们的研究和博士院长Vistnes的。支持这项工作是由美国国立牙科和颅面研究所拨款1 R21 DE019274-01，R01EB009689和RC2 DE020771-02，橡树基金会和Hagey来说小儿再生医学博士炫MTL实验室的支持，圣约瑟夫慈善医院GME的。

1。细胞分离和扩增<ol><li>所有病人的同意和实验方案进行了审查，并批准由斯坦福大学的机构审查委员会（议定书＃2188，＃9999）。 </li><li>获取人体皮下脂肪组织从本地/全身麻醉下的的选修lipoaspiration程序。 </li><li>将有两个在lipoaspirate层（ 图1A）。将上清液包含处理后的多孔材料的绝大部分。底层主要是注射生理盐水。 脂肪衍生的基质细胞，可以从任一层收获，但产量从上清液中大得多。 </li><li>要隔离的基质血管级分（SVF），广泛的lipoaspirate洗，用等体积的磷酸盐缓冲盐水（PBS）含有2.5％聚乙烯吡咯酮碘（×2），然后由等体积的1X PBS（×1）不优碘1X。允许在洗涤沉淀。 </li><li>小心地保持无菌TEchnique在整个过程中可能发生污染加工的人体组织。 </li><li>吸的底层，每次冲洗后丢弃。 </li><li>分装15毫升到50毫升BD猎鹰锥形管的脂肪组织。 </li><li>消化的脂肪组织中，与等体积（15毫升），过滤的0.075％II型胶原酶在Hank氏平衡盐溶液（HBSS）中，在37℃下在水浴中60分钟，在恒定搅拌下（大约180摇动/分钟） - 各管通风，每15分钟。 </li><li>与15毫升PBS含有10％（胎儿牛血清）的FBS，并以1,000 rpm离心5分钟，在4℃，以获得一种高密度的SVF颗粒中和酶的活性。沉淀将脂肪衍生的基质细胞和红细胞的混合。 </li><li>弃上清，不破坏颗粒。 </li><li>将沉淀重悬于10毫升传统的生长培养基（Dulbecco改良的Eagle培养基[DMEM] / 10％FBS / 1％青霉素 - 链霉素溶液）。 </li><li>过滤悬浮液<html> SION通过100μm尼龙细胞过滤器，以除去细胞碎片。 </li><li>将3到一个干净的50 mL锥形管。在4℃下以1,000 rpm离心5分钟离心</li><li>弃上清，不破坏颗粒。 </li><li>传统的生长培养基的5毫升，将沉淀重悬于</li><li>结合两个50毫升conicals每1个10公分板或五50毫升的conicals一个15公分板。 </li><li>建立原代培养过夜，在37℃C/21％O 2，5％CO 2。 </li><li>培养过夜后，广泛用PBS清洗板以除去残余的非粘附的红血细胞。将得到的细胞群是脂肪组织衍生的基质细胞。 </li><li>保持在亚汇合的水平，以防止自发分化的细胞在37℃C/21％O 2，5％CO 2在生长介质。细胞通常需要分割在定时的生长培养基接种于50％汇合时，每三到四天。 </li></ol>ve_title"&gt; 2。脚手架准备<ol><li>由李民博士在美国加州大学洛杉矶分校 - 牙科学院的支架。 </li><li> PLGA支架，从85/15的聚（乳酸 - 共 - 乙醇酸）（固有粘度= 0.61分升/克，伯明翰聚合物）制成，通过溶剂浇铸和颗粒状浸出过程。 </li><li> 200-300微米直径的蔗糖，PLGA /氯仿溶液混合，得到92％孔隙率（体积分数），并压制成薄片在Teflon模具。 </li><li>冷冻干燥过夜后，浸渍在双蒸馏水（dd）的H 2 O的三种变化支架以溶解蔗糖，并轻轻地从Teflon板与罚款小费抹刀除去。 </li><li>颗粒浸出后，所有棚架通过浸渍在50％，60％和70％乙醇消毒每次30分钟，然后由三个漂洗DDH 2 O。 </li><li>所有支架下干燥，在层流罩。 </li><li>后支架制作，S涂覆有磷灰石caffolds。 <ol class="ualpha"><li>模拟体液（SBF）制备​​的溶液与离子浓度的5倍，人体血液，血浆。所有的解决方案是通过0.22μmPES膜（的Nalgene）无菌过滤。立即涂覆前的过程中，干燥，PLGA支架进行辉光放电，氩等离子体蚀刻（Harrick旅游Scientific）的改善润湿性和涂层的均匀性。 </li><li>蚀刻PLGA支架在SBF培养在37°C水套式培养箱中培养12小时内，其次是镁+和HCO 3 - SBF 2张，另有12个小时，在37°C下轻轻搅拌。 </li><li>被覆了的PLGA支架轻轻用无菌DDH 2 O漂洗，洗去多余的氯化钠溶液洗涤，在层流罩中干燥，并用70％乙醇消毒。 </li><li>磷灰石涂层的完整性，用扫描电子显微镜（SEM）分析。磷灰石涂层的SCAffolds被安装在扫描电镜的存根（泰德培拉）和涂覆有提高导电性的碳。的二次电子模式期间施加SEM（FEI /菲利普斯XL-30）观察。能量色散X-射线谱，得到磷灰石结构确认元素的组合物。 </li></ol></li></ol> 3。细胞接种<ol><li>达到合流子的水平后，洗涤细胞，用PBS（×2）和trypsinize。 </li><li>细胞计数，以量化为播种</li><li>种子到预先切割的8.0毫米直径的支架用1.5×10 5个细胞。 <ol class="ualpha"><li>地点个别支架成孔的96 - 孔板</li><li> 重新悬浮细胞定时的生长介质中的浓度大约为1.5×10 5个细胞每20μl的媒体 </li><li>吸取20μL直接上支架，并放入细胞培养孵化器30分钟。 </li><li> 30分钟后，加入200μl的介质，并允许细胞孵育在脚手架上过夜，手术前</li><li>这是没有必要的，以确保粘附播种足够数量的细胞的细胞上的支架，将确保多数将附加到支架。在我们的实验室已证实在体内生物发光和组织学分析。 </li></ol></li></ol> 4。颅骨缺损， 植入体内的创建<ol><li>麻醉成年（60日龄）CD-1裸鼠麻醉剂鸡尾酒。 （氯胺酮/甲苯​​噻嗪/乙酰丙嗪）在浓度为50％（1:1稀释的0.9％NaCl），或每推荐的麻醉方案每个机构协议。这些老鼠是裸鼠，所以hASCs不会引起免疫反应。 <ol class="lalpha"><li>用量:氯胺酮盐酸盐（80毫克/公斤），二甲苯胺噻嗪（2.5毫克/千克）乙酰丙嗪（2.5毫克/公斤） </li><li>效果持续时间:约30分钟</li></ol></li><li>手术垂坠的动物和消毒的手术聚维酮碘和酒精的网站（X3）和鼠标的眼睛软膏之前矢状切口在头皮上的动物暴露的右顶骨。删除从右侧顶骨的骨膜用钝的刮（ 图1B）。 </li><li> 必须格外小心，注意不要打扰基础硬脑膜（图1B）的小鼠颅骨厚度创建一个单方面4毫米的厚度在正确的相关的非缝合顶骨缺损，用无菌环钻金刚石涂层位。是&lt;0.3毫米。 </li><li>在注入之前，冲洗用无菌PBS支架中衍生的生长因子，以防止传输。 </li><li>将支架入缺陷。 </li><li>最后，缝合皮肤和监控动物按照既定术后协议。 </li><li>建立镇痛，手术后- 例如需要动物保健协议。丁丙诺啡0.1毫克/ K克。 </li></ol> 5。类骨质形成的定量分析<ol><li>后微型电脑鼠标上执行，重建三维图像，如先前描述的1使用MICROVIEW软件。 </li><li>使用Adobe Photoshop的图像大小的标准高度。 </li><li>使用"魔术棒"功能，像素测量颅骨缺损。 </li><li>通过随后的CT扫描测量颅骨缺损区域和确定象素数和将它除以原来的缺陷的像素数确定与愈合的缺陷百分率</li><li>因此，如果微型电脑是不可用的，另一种方法是使用Adobe Photoshop和收获颅盖骨在确定的时间点组织学。 </li><li>使用组织学污渍如苯胺蓝及Pentachrome的表示的类骨质形成，多个部分沿跨度的缺陷应该是染色</li><li>使用Adobe Photoshop，出露的所有领域，是不在颅骨缺损</li><li>使用"魔棒"功能，并确定在该地区的缺陷从头骨形成的像素数和管制或其他变量进行比较。 </li></ol> 6。代表性的成果脂肪组织有可能成为一个重要的角色，代祖细胞的临床应用。脂肪组织具有独特的优势，有一个现成的电源，可以在一个相对简单的过程，涉及到最小的发病率和死亡率有所收获。一旦组织被收获和收集的，我们的协议是在图2中列出。脂肪组织衍生的基质细胞是通过一系列的洗涤步骤，胶原酶消化，和离心分离。一旦细胞被接种在培养的，它们可以被扩大，放入不同分化协议在体外 ， 在体内直接或放置。 在Creation 4毫米的关键颅骨缺损提供了一个方便，重现性好， 在体内的模型来测试我们的脂肪基质细胞成骨分化能力的。通过使用的微型电脑，我们能够，按照体内骨组织的形成和跟踪我们的干预的进展情况（ 图3）。关键的颅骨缺损不愈合的动物的生命周期内，我们看到大约90％的缺陷仍然专利8周左右。支架本身具有成骨细胞的电感特性（见讨论），并诱发一些骨再生的能力。支架放置无细胞的典型结果示出，周围的缺陷的三分之一，将有新生骨形成在第8周。随着脂肪组织衍生的基质细胞的增强作用，充分三分之二或更多的缺陷将显示在约8周的骨再生虽然存在的变量应对WTO每个动物和外科手术之间。骨组织的形成可以被量化通过组织学和典型的结果表明:通过苯胺蓝和Pentachrome的染色（ 图3C）的ASCs样品中的类骨质形成增加。此外，我们还表明，植入人体细胞的基本原发骨形成通过使用GFP-的标记hASCs，表明活体染色在2周附近地区的从头颅骨缺损的骨形成（ 图3D） 。此外，我们使用与人类特异性抗体的免疫组化，以显示中的缺陷的区域（ 图3E）的人类细胞的存活和贡献。 <img alt="图1" src="/files/ftp_upload/4221/4221fig1.jpg" /> 图1:A - lipoaspirate有两层。最上方的层中含有的脂肪细胞和大多数的processes多孔材料，而底部的层包含生理盐水期间使用的lipoaspiration程序。 B - 建立颅骨缺损，通过中线切口，在骨膜分离出4毫米的临界大小的颅骨缺损右顶骨，随后创建无中断的基础硬脑膜。 <img alt="图2" src="/files/ftp_upload/4221/4221fig2.jpg" /> 图2概述的收获和应用的lipoaspirate从隔离的脂肪组织衍生的基质细胞，以它们的扩张， 在体外和体内分化和使用。 <img alt="图3" src="/files/ftp_upload/4221/4221fig3.jpg" /> A- 在体内应用先进的结构陶瓷的临界尺寸颅骨缺损愈合的微型电脑显示，通过液压图3。roxyapatitie支架（底行）。控制包括无支架和缺陷（上排）和缺陷位置的支架无细胞（中排）B - 定量分析的微型电脑骨愈合，愈合的ASC组明显增加。 C - 组织学类骨质形成增加的ASC组（下排）通过苯胺蓝和Pentachrome的染色。苯胺蓝的骨样污渍，深蓝色和Pentachrome的骨样污渍，黄色。 D - 与GFP hASCS标记，接种到支架上，并放置到一个颅骨缺损，牺牲在2周。用GFP标记的抗体，以促进再生的缺陷的面积中，显示的人体细胞进行染色。 E -人类细胞核抗原免疫组化显示人体细胞的患病率在该地区的缺陷在2周。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">点击此处查看大图</a> 。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-integrating+knockdown+and+customized+scaffold+design+enhances+human-adipose-derived+stem+cells+in+skeletal+repair.">Non-integrating knockdown and customized scaffold design enhances human-adipose-derived stem cells in skeletal repair.</a> Stem Cells. 29, 21028-21029 (2011).</li></ol>

没有利益冲突的声明。

由于隔离脂肪衍生的基质细胞从lipoaspirate 2，这些细胞已分化成各种各样的细胞谱系。脂肪组织是来自中胚层的起源，因此，多能的脂肪组织衍生的基质细胞将可能是最有效的应用向中胚层谱系。生成骨组织的能力是特别重要的，因为短缺的自体移植的供区和合成材料固有的局限性，包括感染，排斥反应，随着时间的推移和故障3。脂肪间充质干细胞提供了一个潜在的自体多能细胞系外科收获有比较大的收益率4％。 的基质血管级分和脂肪细胞组织在该处理期间，重要的是要保持良好的无菌技术。有一个大的污染风险的原代培养的体积性质的lipoaspirate洗涤，消化，和离心的步骤以外的无菌细胞培养罩的多个步骤的结合。一旦颗粒被的培养皿上的菜，可见的单元格的大部分将eyrthrocytes。这些都可以用无菌PBS洗掉一旦脂肪衍生的基质细胞粘附到板或红血细胞溶解缓冲液可以添加。重要的是，所述细胞是相对汇合上，以确保适当的生长和分化的初始电镀步骤。 如何准确地界定脂肪间充质干细胞有没有达成共识。虽然有研究试图确定这些人口的细胞，通过流式细胞仪检测细胞表面标志物的基础上，已经很难找到一组已定义或标记。这可能是由于患者人群中的变异性和表型漂移的经验，这些细胞在体外不同的细胞培养环境下，在PAS的变化圣人的数字。国际细胞治疗协会指出，间充质干细胞在最低限度，必须有一定的特点，包括表达CD105，CD90，CD73，而缺乏CD45，CD34，CD14，CD11b的，CD79α，CD19，和HLA-DR 5。多有力的性质，这些细胞的原始定义来自于他们的能力，分化成中胚层谱系的细胞在体外培养 ，这些协议都是现成的。 的临界尺寸的颅骨缺损模型已经通过验证作为模型来研究骨骼再生使用两个小鼠脂肪组织衍生的基质细胞6和人来源的脂肪衍生的基质细胞1。创造4毫米的缺陷在右侧的顶颅骨的鼠标不会愈合的动物的寿命。因此，看到的是任何形式的治疗由于放置在颅骨缺损的治疗。在手术过程中，尤其重要的是不仁州重的基础硬脑膜。硬脑膜本身可以刺激成骨和任何损伤可显着妨碍愈合过程中的颅骨缺损7。 的使用f支架的作为传送的机制，干细胞是特别重要的，在世界的骨骼工程。骨骼组织的功能是唯一相关的三维结构体的宏观环境。正确的脚手架不仅可以帮助体内的干细胞输送，但也通过增加成骨增强成骨细胞的支架本身的属性。我们的实验室采用羟基磷灰石，钙衍生物，由于该材料的骨诱导和骨传导到PLGA骨干。但是，也有许多其他的选择脚手架创建，可以有很强的骨诱导和骨传导性能8，9。此外，也可以使用的支架来提供强大的佐剂，例如作为小分子，10日，11日和载体击倒或上调的基因目标，12，13。

<table border="1"><tbody><tr><td> 的试剂名称: </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td></td><td></td><td></td><td> Lipoaspirate收获 </td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 10010-023 </td><td></td></tr><tr><td> Hank氏平衡盐溶液</td><td> Cellgro </td><td> 21-023-CV </td><td></td></tr><tr><td>胶原酶</td><td>西格玛</td><td> C6885-500MG </td><td></td></tr><tr><td>细胞过滤100微米</td><td> BD猎鹰</td><td> 352360 </td><td></td></tr><tr><td>无菌前500毫升0.22微米的过滤器</td><td>密理博</td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> 颅骨缺损 </td></tr><tr><td> Z500无刷MicromotorsUM50C </td><td> NSK </td><td> NSKZ500 </td><td></td></tr><tr><td>圆刀4.0毫米的</td><td> Xemax手术</td><td> CK40 </td><td></td></tr></tbody></table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

颅面骨骼的修复和再生的细胞为基础的方法，利用干细胞通过重新组织形成提供了保证。脂肪基质细胞（ASCs）已被证明是一个丰富的来源，能够进行成骨细胞，软骨细胞，脂肪细胞和成肌细胞分化的多能干细胞。许多研究探讨了这些细胞在体内的成骨潜能细胞输送各种脚手架生物材料的使用。它已被证明，通过利用一个骨传导，以羟基磷灰石涂敷的聚（乳酸 - 共 - 羟基乙酸）（HA-PLGA）支架种子与先进的结构陶瓷，颅骨缺损的关键尺寸，其无法进行自发定义的缺陷，这会愈合的动物的整个生命周期，可有效地显示出强劲的骨再生。这在体内模型演示旨在再生的骨组织的基础上的平移的方法-在蜂窝成分和生物基质。这种方法的模型作为最终的临床应用祖细胞对一个特定的组织缺损的修复。

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

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

该协议描述了隔离的脂肪组织的基质细胞从lipoaspirate 4毫米的临界大小的颅骨缺损，以评估骨骼的再生和创造的。

修复的临界大小的颅骨缺损模型脂肪间充质干细胞的收获Lipoaspirate

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

repair-critical-sized-calvarial-defect-model-using-adipose-derived

Nanyang Technological University

Research

JoVE Journal

Medicine

15.8K 次观看.  Stanford University. 该协议描述了隔离的脂肪组织的基质细胞从lipoaspirate 4毫米的临界大小的颅骨缺损，以评估骨骼的再生和创造的。

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

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

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

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

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

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

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

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

一种新的小鼠模型主动脉瘤腔内修复

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

两种方法从子宫切除标本小学建立人子宫内膜基质细胞

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

对无瘢痕修复鼠标胎儿皮肤模型

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

人类肾脏血管周围间质细胞新型临床分级分离方法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...