同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述
1Experimental Surgery, GIGA-R & Credec,University of Liège, 2Laboratory of Connective Tissues Biology, GIGA-R,University of Liège, 3Department Argenco,University of Liège, 4Department of Clinical Biology, University Hospital of Liège,University of Liège, 5Physical Medicine and Sport Traumatology Department, FIFA Medical Center of Excellence, University Hospital of Liège,University of Liège
Summary
本协议描述了大鼠经同种异体血小板丰富血浆 (PRP) 或盐水溶液取出部分跟腱后愈合肌腱的评价过程。采用不同类型的分析方法对肌腱愈合的进展进行评价。
Abstract
本文介绍了用于观察 PRP 是否能对肌腱愈合有积极影响的实验程序。有4个主要步骤要遵循: 诱发跟腱损伤;准备 PRP 并注入 (或盐水溶液);取下肌腱;并进行生物力学、分子和组织学评价。在每个步骤中, 都详细描述了所有的过程和方法, 因此可以很容易地进行复制。
跟腱已经手术切片 (切除5毫米长的部分)。然后, 注射 prp 或生理盐水来研究 prp 是否对肌腱愈合有积极作用。三组40只动物 (共120只大鼠在本研究中使用) 分为2组: PRP 注射液组和生理盐水注射控制小组。大鼠在增加的时间点牺牲 (A 组: 5 天;B 组:15 天;C 组:30 天) 和肌腱被删除。90肌腱在进行 transcriptomic 分析前进行生物力学测试, 其余30肌腱提交组织学分析。
Introduction
凝血, 炎症过程和血小板的免疫调节作用是众所周知的1。最近, 已经证明它们还具有恢复性属性2,3。事实上, 在脱粒期间血小板释放各种细胞因子和生长因子 (VEGF、血小板因子、TGF、生长因子 I 和 HGF)。这些生长因子促进血管生成, 组织重塑和伤口愈合 (骨骼, 皮肤, 肌肉, 肌腱)2。离心自体血液产生血小板丰富的血浆 (PRP), 其中含有高血小板浓度取决于隔离方法 (3 和10倍的血液基线浓度)。事实上, 各种 PRP 制备技术不能提供相同的最终产品。到目前为止, 在这一问题上尚未达成国际普遍协议。总的来说, PRP 可能是治疗慢性肌肉骨骼疾病的一种有吸引力的治疗方案, 如肌腱、足底筋膜炎、骨关节炎和不愈合的4。它第一次用于口腔外科和种植4 , 以改善和加速骨愈合后放置一个牙科植入。在本研究中, 我们描述了一种可重现的方法, 允许获取 PRP 的动物实验4。
由于肌腱的损伤在运动员和体力工作者中经常被观察到, 加强愈合过程, 从而减少恢复的时间是非常感兴趣的5。新的治疗方法往往涉及使用生长因子, 而 PRP 的管理是一种简单和微创的方式, 以提供混合的内生生长因子4。
一些体外或动物研究表明, 通过释放生物介质来管理含有高水平血小板的血浆, 可以通过释放生物介质来刺激肌腱和韧带的修复6 ,7,8,9。此外, 其他研究表明, PRP 可刺激肌腱细胞的 i 型和 III. 类胶原合成9,10,11。也有人建议, PRP 可以减少基质金属蛋白酶的活化, 从而减少基体的降解。参与炎症过程的细胞可以产生 MMP-9, 它在炎症12引起的组织重塑 (生理学和病理) 中起着作用。
根据这一信息, 我们假设单个 PRP 注射到大鼠的跟腱, 可以改善修复组织的恢复过程和机械强度。这是通过测量愈合肌腱在恢复过程中的生物力学性质, 并通过进行组织学和分子分析, 以评估新形成的组织胶原重塑的测试。本研究的目的是观察单注射同种异体 PRP 是否会影响切片跟腱的愈合。
Protocol
对动物的护理和处理是按照国家科学院编写并由国家卫生研究院 (美国) 出版的《护理和使用实验动物指南》进行的。欧洲和国家立法得到了仔细的遵循。 1. 动物制剂 使用 132 2 月大的雄性大大鼠重320-450 克 (120 只老鼠进行实验, 12 只老鼠进行血液取样,图 1)。基于 Dell13, 每个组的15只老鼠足够的功率为 0.8 (如果指定的效果存在, 则?…
Representative Results
结果以平均标准偏差表示, 并与方差分析进行了比较。使用了双向方差分析和后端测试 de Scheffé (即参数测试)。 引起大鼠非损伤性跟腱断裂的最终抗拉强度为 42.0, 5.7 n (n = 10)。5天后, 两组抗拉强度显著增加 (p < 0.0001)。对照组比较, 在任何时间测量, 特别是在15和30天, PRP 组的信众均较高。通过测量在进行生物力学评估前被…
Discussion
血小板对肌腱愈合过程的早期炎症期至关重要。当这些血小板暴露于结合组织或诱发凝血的因素时, 它们将释放在α颗粒中储存的生长因子。由于这种相互作用, 胞外基质大分子综合升华, 间充质细胞增殖。血小板也有一个趋化活性的祖细胞在血液循环, 增强血管生成和刺激细胞分化6,20。
生物力学测试是使用专门为体外拉伸试验的大?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了里昂 Frédéricq 基金标准 de 列日和勒琼-Lechien 赠款的支持。
Materials
| Xylazine (Xyl-M) | VMD | none | anesthetic |
| Ketamin (Jétamine 1000 CEVA) | CEVA Santé Animale | none | anesthetic |
| Buprenorphin (Vetergésic Multidosis) | ALSTOE | none | Painkiller |
| iso-Betadine | MEDA-Pharma | none | Desinfectant |
| resorbable yarn Vicryl 6/0 | Johnson & Johnson | ||
| Nembutal | CEVA Santé Animale | none | Anesthetic |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Preserves structure of the tissue |
| Isopropanol 100% | VWR | 20,922,364 | |
| Ethanol 95% | VWR | 20,823,362 | |
| Xylene | VWR | 28973.363 | |
| Paraffin | VWR | LEIC3950.1006 | |
| Hematoxylin | Millipore | 1.15938.0025 | Colorant |
| Eosin | Millipore | 1.15935.0100 | Colorant |
| Eukitt | Sigma-Aldrich | 3989 | Mounting Medium |
| CaCl2 |
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Citazione di questo articolo
Greimers, L., Drion, P. V., Colige, A., Libertiaux, V., Denoël, V., Lecut, C., Gothot, A., Kaux, J. Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description. J. Vis. Exp. (133), e55759, doi:10.3791/55759 (2018).