资助这个项目是由德国研究协会（授予他4578/3-1）和部分欧盟第七框架计划项目“岗巴”NMP3-SL-2010-245993。

A.捐助者兔间充质干细胞的分离制备（手术室） <ol><li>细胞分离雄性新西兰白（NZW）兔岁4个月，体重约3公斤。 </li><li>异丙酚（10毫克/公斤体重IV）麻醉用戊巴比妥钠（100毫克/公斤体重IV）和牺牲。 </li><li>剃须毛皮后肢，背部和腹部，用电动推剪和真空皮草。 </li><li>剃了彻底消毒，用70％乙醇。 </li><li>使用钝钳，锋利的剪刀（或解剖刀）组织及韧带和骨刀。 </li><li>做一个切口沿面颅的腿和小腿。 </li><li>反映尖锐或钝性分离皮肤和皮下组织。 </li><li>独立的胫骨和股骨的肌肉和韧带。保持尽可能靠近骨尽可能地使一个干净的夹层剪切。不要单独股骨胫骨在这个时候。 </li><li>切THRough分离股骨头的髋臼的髋关节。 </li><li>提升胫骨股骨复杂。 </li><li>使用手术刀刀片刮去任何剩余的从骨骼或软组织擦骨头用无菌布组织。在这一点上，股骨和胫骨仍连接。 </li><li>删除髌骨切割，再切分开的骨头终于膝关节韧带。 </li><li>喷雾用70％的乙醇，骨头分离，让空气干燥，到50毫升离心管中，将每个骨细胞培养基（DMEM + 1％青霉素/链霉素（PEN /链球菌）），以保持湿润。 </li><li>现在切换无菌的细胞培养层流罩下。 </li></ol> B.法拉盛兔MSC从骨骼和扩展（细胞培养罩） <ol><li>管和收集骨头，将它们放置到150毫米的菜肴，用无菌镊子。 </li><li>卸下两个骨端用无菌锯和移动件新的150 mm培养皿。 </li><li>填充10毫升注射器中位数嗯（DMEM），附加18号针，插入骨髓开幕。 </li><li>然后，冲洗骨髓腔介质冲洗骨髓入菜。之后，从另一端冲洗，如果可能的话。如果有必要，送行的两端。如果骨破裂，只用清水冲洗里面的骨头。 </li><li>细胞培养基中悬浮液进入注射器吸出，反复冲洗骨髓，直到悬浮液是通过骨髓腔中的自由浮动的，并没有进一步的骨骨髓凝块出现。 </li><li>一旦骨髓已收集所有的骨头，破坏骨髓团块通过18号针：填充注射器针连接到培养基中逼出来的。 </li><li>之后，通过细胞过滤器过滤悬浮液到50毫升管。为了防止细胞的损失，洗培养皿用10毫升培养基的2倍，以及过滤。 </li><li>以500×g离心悬浮液在RT下5分钟。 </li><li>删除上清，重悬细胞佩尔等在10毫升培养基（DMEM + 1％PEN /链球菌）。 </li><li>独立的血细胞从外周血单个核细胞（PBMC）和间充质干细胞（MSC）使用一个Biocoll分离解决方案。 </li><li>填写5毫升到15毫升管中的分离解决方案Biocoll，并小心地加入5毫升细胞悬浮液的顶部，以800×g离心20分钟，在RT（不带制动器）。 </li><li>可能的结果，请参阅图1：依据密度比Biocoll，红血细胞的底部沉积物而外周血单个核细胞和骨髓间充质干细胞的界面处保留。 </li><li>到15毫升管小心地取出接口，并用5毫升PBS冲洗。 </li><li>离心机中所描述的步骤22中，重悬在5ml PBS，并重复2到3倍。 </li><li>然后，以350×g再次离心10分钟，在RT（带制动器）。 </li><li>重悬在10毫升培养基和血球细胞计数。 </li><li>板细胞最初的接种密度约为5×10 6 150毫米的菜肴。 </li><li> 2-3天之后，重新移动非贴壁细胞。您可能需要先用PBS冲洗，以去除细胞碎片。加入新鲜的完全培养基（DMEM + 10％小牛血清（FCS）+ 1％笔/链球菌）之后。 </li><li>喂饱细胞每3-4天（ 图2）。 </li><li> 5-10天之后，通过细胞的第一次。 </li></ol> C. 在体外制备的纤维蛋白凝块<ol><li>在植入的日子，3分钟暴露于0.25％胰蛋白酶-EDTA释放贴壁细胞培养瓶/餐盘。停止胰蛋白酶加入完全培养基。 </li><li>分发在50毫升隼管细胞，并用PBS清洗两次。 </li><li>确定由台盼蓝染色的细胞活力和数字。 </li><li>新增50,000个细胞/ 500 XG为5分钟，在室温的离心管和离心收集颗粒。根据需要，准备一个在盒子的至少一个或多个血块。 </li><li> 17微升PBS重悬细胞沉淀和混合纤维蛋白原成分布甲25微升胶原与这17微升MSC悬挂套件。 </li><li>径与预钻孔（3x3.6月）根据在体内的钻孔（ 图3）的无菌板。 </li><li>首先，4微升的凝血酶溶液（500国际单位/毫升）接种到一个洞，随后立即加入42微升的血纤维蛋白原的细胞悬浮液，再加入4μl的凝血酶溶液的顶部。请勿混用暂停，以避免凝血枪头。首先，将50μl体积的移液的纤维蛋白原的细胞悬浮液，将凸出的预钻孔的边缘不熔化，由于表面张力的情况下。然而，凝血完成后（60分钟后）血块收缩和装入预钻孔。 </li><li>小心使用钝钳，并放入离心管中，用PBS取出血块。准备2凝块/动物。 </li><li>以血栓手术房。 </li></ol> D.植入异体骨髓间充质干细胞的纤维蛋白凝块<ol><li>麻醉兔（NZW，男，体重3.5-4.0公斤，5-6月龄），通过静脉注射丙泊酚（10毫克/公斤体重）。 </li><li>剃须用电动推剪和真空皮草膝盖上进行操作。命名前的所有程序都在术前准备室，手术室的无菌环境，以避免污染。 </li><li>插管后，1.5毫克/公斤/分钟异丙酚和0.05毫克/千克/分钟静脉注射芬太尼维持麻醉。使用二氧化碳监测，脉搏血氧饱和度和脉率监测麻醉。 </li><li>剃了膝盖彻底消毒，并用无菌敷料覆盖其余兔。 </li><li>触诊髌骨与髌骨内侧进行皮肤切口。 </li><li>打开膝关节内侧髌骨关节切开，在无菌条件下。尽量避免切割任何小的浅表血管。 </li><li>横向髌骨置换“（ 图4）。 </li><li>在SPE后的任何伴随的软骨损伤或关节异常的膝关节CTION，创建两个滑车沟的骨软骨缺损（3毫米深，图中呈八字形），用无菌空气工作动力钻（直径3.6毫米）与停止装置（ 图5）。 </li><li>清洁的缺陷，他们用无菌生理盐水冲洗。 </li><li>植入前，填写缺陷和20微升纤维蛋白胶均匀涂于底部的缺陷。 </li><li>然后植入凝块压配合到图中呈八字形缺陷。 </li><li>凝固后，搬迁滑车沟和髌骨内弯曲和伸展膝盖几次。 </li><li>置换髌骨横向再次检查，如果纤维蛋白原细胞凝块是还是很到位的。 </li><li>再次更换髌骨做完手术伤口缝合在单纽扣缝线（4-0薇乔）和连续缝合皮肤（4-0 MONOCRYL）层（可吸收缝合的材料）。 </li><li>最后，密封伤口，喷雾敷料可渗透水蒸汽。 </li><li>对于手术后的护理，每天检查伤口7天。兔子获得手术后镇痛卡洛芬4毫克/公斤SC每24个小时（4天），每12小时（2.5天）Buprenorphin 0.03毫克/千克SC。一个稳定的膝盖（ 如敷料）是没有必要的。 </li></ol>

<ol>
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什么都没有透露。

近年来，组织工程方法处理复杂的关节软骨缺损 - 如那些导致剥脱性骨软骨炎，股骨头坏死和创伤 - 的可能性变得越来越有吸引力。在前面提到的病理实体，延伸到软骨下骨组织损伤，并涉及到两个组织，其特征在于由不同的内源性愈合能力1。有致病的软骨下骨关节软骨损伤的过程，11,23中的作用越来越大的兴趣。关节软骨的功能状况及其配套骨紧密相连。任一组织的损伤的产生...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAN Biotech GmbH</td>
			<td>0401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2210</td>
			<td>1,000 units/10 &mu;g/&mu;l in 0.9% NaCl</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KGaA</td>
			<td>410230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biocoll Separation Sol.</td>
			<td>Biochrom AG</td>
			<td>L6115</td>
			<td>Isotonic solution Density: 1,077 g/ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Invitrogen GmbH</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>DeltaSelectGmBH</td>
			<td>1819340</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>BBraun</td>
			<td>8333A193</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringes (Injekt)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needles (Sterican)</td>
			<td>BBraun</td>
			<td>4657519</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps (blunt/sharp)</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Feather Safety Razor Co</td>
			<td>02.001.30.022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes research</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bone Cutter</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Forma Scientific Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture laminar flow hood Hera Safe</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile saw</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge Megafuge 2.0 R</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Air operated power drill</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TISSUCOL-Kit 1.0 ml Immuno</td>
			<td>Baxter</td>
			<td>2546648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibers (4-0 Monocryl, 4-0 Vicryl)</td>
			<td>Ethicon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
	</tbody>
</table>

treatment of osteochondral defects in the rabbit's knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots

治疗关节软骨缺损已具有挑战性医师多年。更好地理解，在最近几年的关节软骨和软骨下骨的相互作用导致了越来越多的关注，恢复整个骨软骨单元。相比，软骨损伤的再生骨软骨缺损是更加复杂和大得多的手术和治疗的挑战。受损组织不仅包括表面的软骨层，软骨下骨。对于深，骨软骨破坏，因为它会发生与骨软骨病炎例如，需要被替换的缺陷的整个厚度恢复关节面1。合资格的治疗程序必须考虑这两个不同的组织，不同的内在愈合潜力2。在过去的几十年中，一些手术治疗方案已经出现，临床上已经建立了3 -6。 包括关节软骨和软骨下骨的自体或异体骨软骨移植，并允许整个骨软骨单元更换。充满缺陷与的圆柱形软骨移植，旨在提供一个一致的透明软骨覆盖面3,7,8。的缺点是对有限数量的可移植，供体部位的发病率（自体移植）和不协调的表面，从而应用了该方法的特别的限制，大的缺陷。 在组织工程领域的新方法开辟了前途再生软骨治疗的可能性。自体软骨细胞植入标记的第一个单元为基础的生物全层软骨病变的治疗方法，现在世界范围内建立了良好的临床效果，甚至10至20年后植入9,10。然而，达特，这种技术是不适合用于治疗所有类型的病变，如涉及软骨下骨11的深的缺陷。 夹心技术结合植骨与目前的方法在组织工程中5,6。这样的组合似乎是能够克服的局限性，仅见于骨软骨移植。软骨下缺损区域的自体骨移植后，膜与自体软骨细胞接种上述缝合，并促进与损伤部位的拓扑结构相匹配的接枝。当然，以前的骨重建需要额外的手术时间，甚至常常是额外的手术。此外，日期，长期数据丢失12。 无需额外的骨移植的组织工程旨在恢复天然关节软骨的软骨细胞和成骨潜能的移植细胞的复杂结构和性能。 HoweveR中，再次，它通常仅是多还是少再生的软骨组织。额外的软骨损伤，需要一个具体的进一步的治疗。为了实现再生的多层结构的骨软骨缺损，三维组织工程与自体/异体细胞接种的产品可能会提供一个很好的再生能力11。 除了自体软骨细胞，间充质干细胞（MSC）似乎是一个有吸引力的替代全层软骨组织的发展。间充质干细胞在体外和体内研究，在众多的临床前已经显示了出色的组织再生潜能13,14。特别是用于治疗骨软骨缺损的骨髓间充质干细胞的一个重要的优点是它们具有骨细胞和软骨细胞的分化能力。因此，它们可能允许的d的多层再生EFECT。 因此，近年来，一些支架与软骨的再生潜能开发和评估与前景的初步结果1,15-18。此外，纤维蛋白胶作为细胞载体成为实验软骨修复的首选技术之一，已经成功地被用来在多次动物研究19-21，甚至是人类第一次试验22。 以下协议证明隔离从兔子的骨髓间充质干细胞的实验技术，随后在细胞培养增殖，并准备一个标准化的体外模型中的纤维蛋白细胞凝块。最后，将描述一种技术，用于预先建立的血纤维蛋白 - 细胞血栓兔子的膝关节为人造骨软骨缺损的植入。

手术技术允许一个成功的隔离和异体骨髓间充质干细胞植入一个人工软骨缺损。实验装置的植入导致一个成功的整合到周围的软骨。 类似的生物力学特性和类似的耐久性相比周围的软骨缺损填充修复组织。血纤维蛋白细胞凝块在体外制备的无菌板与预钻孔的骨软骨缺损（ 图3），它有相同的大小。其结果是，有植入纤维蛋白凝块和周围的软骨，这将是过早?...

Watch this Scientific Journal Video about Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots at JoVE.com

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

兔子的膝关节中，用于治疗骨软骨缺损的实验技术进行说明。植入异体骨髓间充质干细胞成骨软骨缺损的组织工程领域的发展提供了一个很有前途的。编制纤维蛋白细胞凝块<em&gt;在体外</em&gt;提供了一个标准化的植入方法。

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

treatment-osteochondral-defects-rabbit-s-knee-joint-implantation

Research

JoVE Journal

Medicine

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

17.3K 次观看.  Klinikum rechts der Isar der Technischen Universität München. 兔子的膝关节中，用于治疗骨软骨缺损的实验技术进行说明。植入异体骨髓间充质干细胞成骨软骨缺损的组织工程领域的发展提供了一个很有前途的。编制纤维蛋白细胞凝块在体外提供了一个标准化的植入方法。

视频: Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

纤维蛋白胶对小鼠肺移植研究肺特异性血管生成

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

富血小板血浆注射液治疗膝关节骨关节炎的制备、程序及评价

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

用膝关节关节仪评价大鼠膝关节屈曲收缩的特定组织贡献

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

大鼠骨髓骨髓的隔离和培养

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

骨科机器人辅助股骨颈系统治疗股骨颈骨折

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

使用改进的 Videman 方法在兔子中建立 KOA 模型

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

作者聚焦：研究针尖切开术治疗膝骨关节炎的作用机制

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

针灸术在兔膝骨关节炎模型中的应用

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...