基质辅助重塑和修复软骨缺损的自体软骨细胞移植在兔模型
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
用于治疗在兔子的膝关节软骨缺损的实验技术进行说明。在矩阵上的自体软骨细胞植入是被广泛接受的方法重塑和修复关节软骨病变提供满意的长期效果。基质辅助自体软骨细胞移植（MACT）提供一个标准化的临床植入方法。
Abstract
关节软骨缺损被认为是一个主要的健康问题，因为关节软骨具有自我再生能力是有限的。未处理的软骨病变导致持续的疼痛，生活质量造成不利影响，并易患骨关节炎。在过去的几十年中，一些外科技术已被开发来治疗这种病变。然而，到现在为止，这是不可能实现了全覆盖肺透明关节软骨缺损或提供令人满意的长期恢复2-4维修。因此，关节软骨损伤仍然再生技术，如组织工程的首要目标。相反，其他外科手术技术，这往往导致纤维或纤维软骨组织的形成，组织工程的目的是充分恢复原来的关节软骨的复杂结构和性能的影响通过使用诱导成软骨细胞的移植细胞的潜力。 ŗ最近几个发展开辟了前景的可能性再生软骨疗法。
第一个单元格为基础的全层软骨或骨软骨病变的治疗方法是在1994年由Lars彼得森和Mats Brittberg的的谁率先临床自体软骨细胞移植（ACI）5。如今，该技术在临床上以及建立大型的膝盖软骨缺损的治疗，植入后6甚至10至20年中保持了良好的临床效果。近年来，自体软骨细胞植入经历了快速进展。使用一个人工的三维胶原基质上的细胞，其后再植变得越来越流行7-9。
MACT包括两个手术：首先，为了收集软骨细胞，软骨活检需要执行从非负重软骨区的t他的膝关节。然后，软骨细胞的提取，纯化，并扩展到足够的细胞数量的影响 。软骨细胞，然后接种到一个三维矩阵，并随后可以被重新注入。当准备植入组织工程，增殖率和分化的能力是至关重要的一个成功的组织再生10。一个三维基质作为细胞载体的使用被认为是支持这些细胞的特征11。
以下协议将总结并展示一种技术，用于隔离的软骨细胞软骨活检，其在体外增殖和他们的播种到一个三维矩阵（ 软骨纪德 ，Geistlich生物材料，Wollhusen，瑞士）。最后，植入到人工创造的兔子的膝关节软骨缺损的细胞和基质的构建进行说明。这种技术可以用作一个实验设定进一步的实验中软骨修复。
Protocol
A.软骨活检（外科室;步骤1-5在非无菌制剂室） 执行最终控制体重的兔子（新西兰白兔，女性，体重3.5-4.0公斤，6个月大），以便能够正确剂量的药物，并监测手术后的重量。 兔静脉注射10毫克/公斤的丙泊酚麻醉。 插管后，1.5毫克/公斤/分钟异丙酚和0.05毫克/千克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。 剃须用电动推剪和真空皮?…
Representative Results
手术技术允许一个成功的隔离和自体软骨细胞植入到人工软骨缺损。实验装置的植入导致一个成功的整合到周围的软骨。 12周后， 在体内的 ，均匀的和完整的表面，从而减少了到植入物（ 图4）的剪切应力和损坏软骨缺损修复组织填充。此外，没有肥厚或钙化的植入物被看见了。修复组织呈僵硬和坚实的质量，这是与周围健康软骨组织。这是一个重要的?…
Discussion
该协议提供了一个建立了9,12,13和容易复制的技术，人为制造的兔子膝盖软骨缺损随后的增殖和再植入自体软骨细胞隔离。使用自体软骨细胞的重构和修复关节软骨病变已经在临床使用中提供满意的长期效果（6）。
例如骨膜肥厚和钙化的主要问题，移植物剥离，或与第一代和第二代的自体软骨细胞植入14发生的供体部位的发病率。因此，研究人员已经?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该项目由德国研究协会（DFG，何4578/3-1）资助。
Materials
| Name of reagent/equipment | Company | Catalogue Number | Comments |
| DMEM | Biochrom AG | F 0415 | |
| Collagenase A | Roche | 10 103 586 001 | 0.21 U/mg |
| Fetal calf serum (FCS) | PAN Biotech GmbH | 3702-P103009 | |
| Propofol | Fresenius Kabi | ||
| Penicillin/Streptomycin | Biochrom AG | A 2213 | 10,000 U/ml/10,000 μg/ml |
| PBS Dulbecco (1X) | Biochrom AG | L1815 | |
| Ethanol (70%) | Merck KgaA | 410230 | |
| Trypsin-EDTA 0.25 %/0.02 % | Biochrom AG | L2163 | in PBS w/o Ca2+, Mg2+ |
| Fentanyl | Delta Select GmBH | 1819340 | |
| NaCl solution (0.9%) | Bbraun | 8333A193 | |
| 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 |
| Hemocytometer | Brand GmbH+Co KG | 717810 | Neubauer |
| Trypan Blue Solution 0.4% | Sigma-Aldrich | L8154 | |
| Spray dressing (OpSite) | Smith&Nephew | 66004978 | Permeable for water vapor |
| Chondro-GideÒ | Geistlich Pharma AG | 30915.5 | |
| Biopsy Punch | pfm medical ag | 48351 | |
| Tissucol Duo S | Baxter | 3419627 | 0.5 ml |
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Tags
Matrix-assisted Autologous Chondrocyte TransplantationChondral DefectsRabbit ModelArticular CartilageSelf-regenerationCartilage LesionsSurgical TechniquesHyaline Articular CartilageLong-term RecoveryRegenerative TechniquesChondrogenic PotentialTransplanted CellsRegenerative Cartilage TherapiesAutologous Chondrocyte Implantation (ACI)Large Hyaline Cartilage Defects
Citazione di questo articolo
Berninger, M. T., Wexel, G., Rummeny, E. J., Imhoff, A. B., Anton, M., Henning, T. D., Vogt, S. Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model. J. Vis. Exp. (75), e4422, doi:10.3791/4422 (2013).