手术取出，分离和<em>在体外</em>扩展人类前十字韧带衍生细胞组织工程的应用
1Department of Medical Microbiology, Immunology & Cell Biology,Southern Illinois University School of Medicine, 2Division of Orthopaedics and Rehabilitation, Department of Surgery,Southern Illinois University School of Medicine, 3Department of Electrical and Computer Engineering, Biomedical Engineering Program,Southern Illinois University Carbondale, 4University of Illinois at Springfield
Summary
对于未来的应用作为一个补丁来修复前交叉韧带（ACL）的部分撕裂，人类的ACL的细胞从内重建手术， 体外培养扩增和生长在组织工程支架材料获得的组织分离。然后进行细胞粘附和形态学进行确认的生物相容性支架上表面。
Abstract
伤到ACL是在活跃的人经常遇到的问题。即使是部分的这个关节膝关节韧带导致的损害的功能和生物力学稳定性不足的眼泪。当前选择的部分ACL撕裂的治疗范围从非手术治疗，保守管理多个手术方案，如：热改性，单束修，彻底重建，重建原生韧带的受损部分。一些研究，如果有的话，已经证明管理是一贯出众任何单一的方法，而且在许多情况下，患者继续表现出持续的不稳定等合并症。
本研究的目的是确定可能在部分十字韧带撕裂的修复来实现一个组织工程补丁的发展潜能细胞源的利用率。一种新的协议是从不稳定型心绞痛患者的细胞膨胀发展NTS进行ACL重建。为了分离细胞，ACL重建过程中获得剁碎HACL组织被消化的胶原酶溶液。用补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的DMEM/F12培养基中进行扩展。然后将细胞保存在-80°C或在液氮中包括DMSO，FBS和膨胀介质的冷冻介质。融化后，将HACL衍生细胞，然后接种到组织工程支架，PLAGA（聚乳酸 – 共 – 乙醇酸）和控制组织培养聚苯乙烯（TCPS）。 7天后，扫描电镜进行到细胞粘附比拟的PLAGA与控制TCPS。采用免疫荧光染色细胞形态进行了评估。 SEM（扫描电子显微镜）显微照片表明，细胞生长和粘附这两种PLAGA和TCPS表面上，并分别由7天汇合，在整个表面上。免疫荧光染色显示正常，非胁迫形态学人图案两面上。这种技术是有前途的ACL中再生和重建应用。
Introduction
前交叉韧带（ACL）是膝关节的常见的受伤关节内韧带。约200,000（ACL）损伤是在美国每年报告。超过75％的患者经历ACL损伤齐齐整形重建手术1,2,3,4。手术治疗往往是由于固有的愈合不良潜力3所示。 ACL重建通常是通过自体移植或异体移植腱的方法。自体移植和异体移植代表的金标准重建，因为他们拥有较高的成功率，并缝合修复，其他的治疗方案，已经显示出高达94％的5,6,7故障率。
该ACL部分撕裂占10％的所有ACL的眼泪8 28％。在一项前瞻性研究，诺伊斯等人估计，患者的影响超过了ACL半部分撕裂50％进展到完成的ACL功能不全后非手术治疗9。其他的研究报告持续不稳定，并与患者能够恢复到受伤前的活动水平9,10,11,12,13少于30％下降功能。治疗选择很有限，其中包括保守的方式，其余的ACL的热收缩，或ACL重建。最近，出现了增加在增强初级修复的兴趣。这些技术使用生物制剂，以加强基层缝合修复14。最近的研究已尝试收获的间充质样HACL来源的干细胞，以规避接枝限制，31,32，但这些细胞的效力和疗效尚属未知。理想的细胞来源，组织工程应用似乎非mesechymal HACL衍生的成纤维细胞。
目前的研究重点是确定一个合适的基质材料和细胞源工程支架。这是标准程序十字韧带撕裂被丢弃苏在重建手术rgical浪费，但是，这受损的韧带可能是一个质量的来源，收购，开发及提升工程的ACL替换一个理想的组织所需要的细胞。我们实验室已经开发出一种协议，用于体外扩增这些收获HACL来源的细胞。使用一个精心设计的二维矩阵注入HACL来源的细胞，我们设计了一个修补程序，可能会增加部分ACL修复和加强韧带撕裂。
Protocol
1，手术取出注意：一个IRB的批准，获得在ACL重建手术来收集的ACL残端。 IRB豁免给出所使用的ACL残端一般都弃之如手术废弃物。 20例患者用于收集的ACL残端。 管理全身麻醉和手术前抗生素作为每机构的协议给患者。 应用止血带充气100毫克以上的收缩压。 不要将病人仰卧位。做一个横向切口前外侧膝在30-45°屈曲位为5毫米的门户网站，并插入关节镜套管和…
Representative Results
的工作模式为手术取出，组织消化和人类前十字韧带（HACL）衍生细胞的分离示于图1，从外植体迁移和粘附到T-25培养瓶中的细胞。这些细胞培养3天，然后用光学显微镜（ 图2）下进行了可视化。通过7天而得到的汇合单层。健康，活细胞的存在表明HACL来源的细胞的成功的检索和文化。 细胞附着到PLAGA和TCPS的表面通过扫描电子显微镜定性测定图</s…
Discussion
这hACL/2D支架研究的主要目的是利用所获得的细胞修补，以增加局部ACL撕裂初级修复。部分ACL撕裂非手术治疗可能包括固定，支撑，循序渐进的康复计划，并定期随访评估13,16,17的一个短周期。然而，许多研究表明，在运动员的保守治疗已经效果差和故障有关。巴克利等人对25例局部ACL的眼泪在中间随访发现，只有44％的患者在受伤前的水平恢复运动，72％的活性相关的症状8。</sup…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者要感谢的启动资金，并从南伊利诺伊大学医学院外科研究资助部门;和纪念医学基金会资助。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Petri-dish | Fisher Scientific | 08-757-103C | |
| Phosphate Buffered Saline (PBS) | Fisher Scientific | BP3994 | |
| Collagenase | Gibco | 17018-029 | Store at 40C |
| DMEM/F-12 | Cellgro | 10-092-CV | Store at 40C. Warm in 370C water bath before use. |
| Fetal Bovine Serum (FBS) | Gibco | 10082 | Store at -800C |
| Penicillin/Streptomycin | Lonza | 17-602E | Store at 40C |
| Centrifuge Tubes- 15 ml | Corning | 430790 | |
| T-25 flasks | BD Falcon | 3013 | |
| Trypsin-Versene mixture | Lonza | 17-161E | Store at 40C. Warm in 370C water bath before use. |
| DMSO | Fisher Scientific | BP231-100 | Combustible liquid. Can cause skin, eye and respiratory tract irritation. |
| Cryogenic vials | Corning | 430489 | |
| PLAGA | Purac Biomaterials | Purasorb PLG8523 | Store at -800C |
| TCPS disks | Fisher Scientific | 12-545-82 | |
| Dichloromethane | Fisher Scientific | AC36423-0010 | Possible cancer hazard. Store in a dry, cool place. |
| Bytac paper | Saint gobin performance plastics | 1420652 | |
| Scintillation vial | Fisher Scientific | 03-339-21G | |
| Bovine Serum Albumin (BSA) | Sigma Aldrich | A7906 | Store at 40C |
| Tween | Fisher Scientific | BP337-500 | |
| mouse Anti-β-actin antibody (10 Ab) | Sigma Aldrich | A5441 | Store at -200C |
| goat-anti-mouse antibody (20 Ab) | Cell Signaling | 4408 | Store at -200C |
| Hoechst dye | Sigma Aldrich | 14530 | Store at -200C |
| Glycerol | Fisher Scientific | BP229-1 | |
| Glutaraldehyde | Fisher Scientific | BP2547-1 | Toxic by inhalation and if swallowed. Causes burns by all exposure routes. |
| Hexamethyldisilazane | Fisher Scientific | AC43085-1000 | Flammable liquid and vapor. Causes burns by all exposure routes. |
| Sterile Scissors | McKesson | 25-716 | |
| Centrifuge | Eppendorf | 5804R | |
| Light Microscope | Olympus | CK40 | |
| Water Bath | Thermo scientific | 2845 | |
| Vortex | Labnet | VX-200 | |
| Glass Petri plates | fisher Scientific | S31473 | |
| Acu-Punch | Acuderm Inc. | P1225 | Acu-Punch was used to cut 12mm disks |
| Cacodylate buffer | Sigma Aldrich | 97068 | Flammable liquid, carcinogen and irritant. |
| Osmium tetraoxide | Sigma Aldrich | 201030 | Highly toxic |
| Triton X-100 | Sigma Aldrich | T8787 | Harmful if swallowed |
| Ethanol | Decon Labs | 2705 | Keep away from heat, sparks, flame and other form of ignition. |
| General/regional Anesthesia | Amphastar pharmaceuticals | 1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance | |
| Antibiotics | Hospira | 0409-0805-01 | Ancef 1g i.v. |
| Arthroscopy trocar | Smith and Nephew | ||
| Arthroscopy Camera | Smith and Nephew | ||
| Arthroscopic grasper and bitter | Arthrex | ||
| 4.5mm shaver | Arthrex | ||
| Interference Screws | Arthrex | stainless steel screws | |
| Sputter Coater | Polaron | E5400 |
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Citer Cet Article
Gupta, A., Sharif, K., Walters, M., Woods, M. D., Potty, A., Main, B. J., El-Amin III, S. F. Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications. J. Vis. Exp. (86), e51597, doi:10.3791/51597 (2014).