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
1Department of Orthopaedic Sports Medicine,Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology,Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research,Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology,Uniklinik Köln
Summary
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>在体外</em>提供了一个标准化的植入方法。
Abstract
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。
Protocol
A.捐助者兔间充质干细胞的分离制备（手术室） 细胞分离雄性新西兰白（NZW）兔岁4个月，体重约3公斤。 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。 剃须毛皮后肢，背部和腹部，用电动推剪和真空皮草。 剃了彻底消毒，用70％乙醇。 使用钝钳，锋利的剪刀（或解剖刀）组织及韧带和骨刀。 做一个切口沿面颅的腿和小腿…
Representative Results
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?…
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of reagent/equipment | Company | Catalogue Number | Comments |
| DMEM | Biochrom AG | F 0415 | |
| FCS | PAN Biotech GmbH | 0401 | |
| Propofol | Fresenius Kabi | ||
| Penicillin/Streptomycin | Biochrom AG | A 2210 | 1,000 units/10 μg/μl in 0.9% NaCl |
| PBS Dulbecco (1X) | Biochrom AG | L1815 | |
| Ethanol (70%) | Merck KGaA | 410230 | |
| Trypan Blue Solution (0.4%) | Sigma-Aldrich | T8154 | |
| Biocoll Separation Sol. | Biochrom AG | L6115 | Isotonic solution Density: 1,077 g/ml |
| Trypsin-EDTA 0.05% | Invitrogen GmbH | 25300-054 | |
| Fentanyl | DeltaSelectGmBH | 1819340 | |
| NaCl solution (0.9%) | BBraun | 8333A193 | |
| Syringes (Injekt) | BBraun | 4606108V | |
| Needles (Sterican) | BBraun | 4657519 | |
| Forceps (blunt/sharp) | Aesculap | ||
| Scissors | Aesculap | ||
| Scalpels | Feather Safety Razor Co | 02.001.30.022 | |
| Pipettes research | Eppendorf | ||
| Bone Cutter | Aesculap | ||
| Tissue culture dishes 100 mm/150 mm | TPP AG | 93100/93150 | Growth area 60.1 mm2/147.8 mm2 |
| Tissue culture flasks 25/75 mm2 | TPP AG | 90025/90075 | 25 mm2, 75 mm2 |
| Centrifuge Tubes (50 ml) | TPP AG | 91050 | Gamma-sterilized |
| CO2 Incubator | Forma Scientific Inc. | ||
| Cell culture laminar flow hood Hera Safe | Heraeus Instruments | ||
| Sterile saw | Aesculap | ||
| Centrifuge Megafuge 2.0 R | Heraeus Instruments | ||
| Hemocytometer | Brand GmbH+Co KG | 717810 | Neubauer |
| Air operated power drill | Aesculap | ||
| TISSUCOL-Kit 1.0 ml Immuno | Baxter | 2546648 | |
| Fibers (4-0 Monocryl, 4-0 Vicryl) | Ethicon | ||
| Spray dressing (OpSite) | Smith&Nephew | 66004978 | Permeable for water vapor |
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Cite This Article
Berninger, M. T., Wexel, G., Rummeny, E. J., Imhoff, A. B., Anton, M., Henning, T. D., Vogt, S. Treatment of Osteochondral Defects in the Rabbit’s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots. J. Vis. Exp. (75), e4423, doi:10.3791/4423 (2013).