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Medicina

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Published: March 30, 2018
doi:
10.3791/56810
, , , ,
1College of Veterinary Medicine,Western University of Health Sciences, 2Applied Medical, 3College of Veterinary Medicine,Iowa State University

Summary

本文介绍了大鼠双侧髌腱缺损模型球体脐基质干细胞的制备及评价。该模型与可接受的发病率相关, 并发现检测到未治疗和治疗肌腱之间的差异, 以及两种治疗方法之间的区别。

Abstract

再生医学提供了新的替代条件, 挑战传统治疗。肌腱的发病率和发病率, 加上该组织的愈合特性有限, 促使寻找细胞治疗, 推动实验模型的发展, 以研究其功效。脐带基质源性干细胞 (UCM MSC) 是吸引候选者, 因为它们丰富, 易于收集, 规避了对畸胎瘤形成的伦理关注和风险, 但与原始胚胎干细胞相比, 更接近于成人组织衍生的骨髓间充质干细胞. 对壳聚糖作为一种通过球形形成增强 MSCs 性能的策略, 具有重要的研究价值。本文详细介绍了 UCM-MSCs 的分离技术, 制备了壳聚糖膜球体, 并分析了球形形成对表面标记表达的影响。因此, 本文介绍了在壳聚糖膜上形成的 UCM-MSC 球体的体内植入的大鼠双侧髌腱损伤模型的建立。在研究中没有发现并发症的发病率, 压力上升的影响, 或组织感染。7天大鼠的总功能评分低于正常大鼠, 术后28天内恢复正常。组织愈合的组织学评分证实在治疗缺陷中存在血栓, 7 天, 没有异物反应, 并在28天的进展愈合。这种双侧髌骨肌腱缺损模型通过在每只老鼠体内建立内部控制来控制个体间的变异, 与可接受的发病率相关, 并允许检测未治疗肌腱与治疗之间的差异。

Introduction

肌腱损伤是一个最常见的原因, 严重疼痛和肌肉萎缩跨物种1。在兽医中, 肌腱和韧带损伤对马匹有特殊的兴趣, 因为赛马中82% 的受伤都涉及肌肉骨骼系统, 46% 的人会影响肌腱和韧带2,3。瘢痕组织形成影响愈合肌腱的生物力学特性, 解释屈肌腱损伤后恢复运动的保护预后;再伤害发生在2年之内在67% 匹马保守地对待了4。再生医学提供了新的替代条件, 挑战传统治疗。自体干细胞治疗产生了一些令人鼓舞的结果5,6 , 但受发病率与组织收集, 延迟管理, 由于处理/重新编程的细胞, 和影响患者的健康状况 (如年龄) 对干细胞的属性7,8。这些限制为研究同种异体干细胞作为一种现成的替代方法提供了理论依据。胎儿附件细胞是吸引候选者, 因为他们规避的伦理关注和风险的畸胎瘤形成与胚胎干细胞。在胎儿附件, 脐带基质 (UCM), 也命名沃顿的果冻, 是丰富和容易收集。

无论细胞来源如何, 加强 stemness 是建立同种异体再生医学细胞库的必要条件。从功能的角度来看, stemness 可以定义为自我更新和多谱系分化的可能性9。stemness 的证据依赖于增殖和分化化验, 以及基因标记Oct4、 Sox2、和北美9的表达。增强 stemness 的一个策略是依靠使用生物材料作为空隙填料和载体, 从而增强 UCM-MSCs 的增殖和分化。这种方法消除了对转录因子的操纵, 将成熟细胞重新编程为诱导的多潜能细胞的担忧。在被认为是干细胞潜在载体的生物材料中, 壳聚糖对其生物相容性和降解能力有吸引力10。这种天然 aminopolysaccharide 是由碱性脱乙酰甲壳素, 第二种最丰富的天然多糖, 主要获得作为 subproduct 的贝类10。我们以前研究过 MSCs 和壳聚糖支架之间的相互作用, 观察到球体的形成11,12,13,14,15,16. 我们还报告了软骨在壳聚糖矩阵的优越性12,13,14,15,16,17, 18。最近, 两项独立研究描述了在壳聚糖薄膜上培养的脂肪组织和胎盘组织衍生 MSCs 的球体形成19,20。这种球体的形成不仅增强了 stemness, 而且还改善了体内植入20后干细胞的保留。

肌腱的患病率和发病率促使实验模型的发展, 以研究 tendinopathies 的病理生理学和试验新的治疗方法, 如干细胞注射。在马匹中, 胶原酶诱导的炎是一种常用的模型, 用于证明在肌腱修复中使用 MSCs 的有效性21。这种方法的相关性是有限的, 因为注射引起急性炎症变化, 而临床 tendinopathies 通常是由慢性致使22,23造成的。此外, 化学诱导肌腱疾病诱发愈合反应, 并没有复制受损愈合过程中存在的临床病例22,23。切除的表面数字屈肌腱部分已被描述为一个外科模型炎在马24。最近, 一种微创的方法被用来限制外伤损伤的中心核心的表面数字屈肌腱25。手术模型不模拟可能导致自然肌腱疾病的疲劳机制, 而且在造成的损害程度上往往缺乏重现性25。无论模型, 与马模型肌腱疾病的发病率和成本是额外的限制, 这证明了一个兴趣的啮齿动物模型作为在体内评估新的疗法的第一步。

啮齿类动物实验模型的主要优点之一是控制个体间变异的成本和能力。啮齿类动物可以标准化的各种生理因素, 由于其快速生长速度和相对较短的寿命, 限制变异的来源, 因此减少了检测差异所需的动物数量。在啮齿动物中诱导肌腱疾病的策略依赖于化学诱导, 同时也用于部分肌腱缺损的外科创建21。手术模型可以模拟自然 tendinopathies 比化学模型更好, 但可能导致更高的发病率和灾难性破坏肌腱。在这方面, 老鼠似乎比老鼠更适合这些模型, 因为它们的大小允许产生更大的缺陷, 从而有助于评价组织愈合。大大鼠已被用于四主要肌腱组的 tendinopathies 实验研究: 肩袖、屈肌、阿基里斯和髌腱26。其中, 涉及髌骨肌腱的模型特别吸引人, 因为这种肌腱的体积较大, 并且易于访问27。髌腱将股四头肌与胫骨股骨。在这个伸肌机制中, 髌骨是一种籽骨, 它引导四头肌的作用, 并勾勒出髌腱的近端程度。髌腱近端和远端骨锚的存在有利于生物力学试验。涉及髌骨肌腱的模型通常依赖于单边外科缺损, 而对侧完整肌腱作为控制28,29。最常见的髌骨肌腱缺损模型包括切除髌骨远端的髌腱中央部分 (1 毫米) 至胫骨股骨的插入, 而对侧髌腱保持完好。结果的措施包括组织学, 无损生物力学测试或对失败的生物力学测试, 超声成像,活体荧光成像, 总观察和功能测试28,30 ,31。单边模型不允许比较建议的治疗与保守的管理类似的伤害在同一动物。同样, 几种治疗方法之间的比较需要单独的动物。双边模式将消除个体间的变异, 减少研究所需的动物数量32。然而, 双边伤害可能增加发病率, 双侧跛行可能阻碍治疗评估。几项研究简要报告了双侧髌腱缺损在大鼠中的应用, 但侧重于治疗的效果而非围手术期管理和模型3334的发病率。

这项研究的长期目标是制定一个战略, 以改善 stemness 和在体内生存的 UCM-MSCs, 注定到同种异体移植。为了实现这一目标, 我们最近报告了在缺氧环境下, 球体在壳聚糖膜上的形成和孵化的 stemness 改善了 UCM-MSCs 的结构35。这些体外属性与改善的髌骨肌腱缺损的生物力学性能相关. 基于这些结果, 大鼠双侧髌腱缺损模型似乎适合于考生肌腱损伤的治疗方法36。本研究报告的目的是为 UCM-MSCs 的分离和鉴定提供详细的协议, 为干细胞制备生物递质系统, 建立和治疗双侧髌骨肌腱缺损, 以及术后缺损内组织愈合的恢复和评估。

Protocol

这里描述的所有方法都已被西方健康大学机构动物护理和使用委员会 (IACUC) 批准。 1. 马脐基质中 MSCs 的分离和扩增 从成年母马 (怀孕) 中获得胎盘, 观察 foaling, 灌装从胎盘中分离脐带。将脐在磷酸盐缓冲盐水 (PBS) 与1% 青霉素-链霉素 (P/秒) 在4°c 在转移, 直到处理。 用室温 PBS 在50毫升管中清洗脐带两次, 1% P/秒。将脐带分为2英寸长的碎片在150毫米板和洗涤室温 PB…

Representative Results

在目前的研究中, 结果被显示为平均值, 即标准差。细胞从6匹母马的脐带中分离出来, 在标准或壳聚糖条件下表达每个细胞表面标记物的分离细胞系的百分比与弗里德曼试验比较, 作为非参数分析的方差与重复措施.对于肌腱缺损模型的建立, 8 只大鼠进行7天术后评估, 12 只大鼠用于28天的评估。将功能结局的结果显示为均值电子扫描电镜 (平均标准误差), 并与 t 检验进行比较?…

Discussion

为这个项目选择了马细胞, 因为我们最终打算测试在马的自然 tendinopathies 管理的候选方法。事实上, 由于马表面数字屈肌和跟腱在人类中的生物学相似性, 马匹肌腱损伤是肌腱的自然模型 (41。根据国际细胞治疗学会建议的标准, CD44、CD90、CD105、CD34 和 MHC II 的细胞表面标记物为免疫分型细胞选择了42。在最近的一项研究中, 无论培养条件如何, eqUCM-MSCs 对 CD34 和 MHC …

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者要感谢博士, 博士, 她对数据的统计分析。作者还感谢麦克卢尔博士, DVM, 博士 DACLAM, 她的意见, 在麻醉和疼痛管理协议中使用的研究。该项目得到了西方卫生科学大学研究副会长 (12678v) 和美国农业部1433基金 (2090) 的资助。

Materials

PBS 10X Hyclone SH30258.01 Consumable
Collagenase type IA Worthington LS004197 Consumable
DMEM low glucose Hyclone SH30021.FS Consumable
Fetal Bovine Serum Hyclone SH30910.03 Consumable
Penicillin/Streptomycin 100X Hyclone SV30010 Consumable
Trypsin 0.25% Hyclone SH30042.01 Consumable
Accutase Innovative Cell Technologies AT104 Consumable
Trypan blue Hyclone SV30084.01 Consumable
Dimethyl Sulfoxide Sigma D2650 Consumable
Chitosan Sigma C3646 Consumable
Sodium Hydroxide Sigma S8045 Consumable
Bovine Serum Albumin Hyclone SH30574.01 Consumable
Round bottom polystyrene tube Corning 149591A Consumable
Mouse anti-horse CD44 (FITC) AbD serotec MCA1082F Consumable
Mouse anti-rat CD90 (FITC) AbD serotec MCA47FT Consumable
Mouse anti-horse MHC-II (FITC) AbD serotec MCA1085F Consumable
Mouse IgG1 (FITC) – Isotype Control AbD serotec MCA928F Consumable
Mouse monoclonal [SN6] to CD105 (FITC) abcam ab11415 Consumable
Mouse IgG1 (FITC) – Isotype Control abcam ab91356 Consumable
Mouse anti-human CD34 (FITC) BD BDB560942 Consumable
Mouse IdG1 kappa (FITC) BD BDB555748 Consumable
7-AAD BD BDB559925 Consumable
BD Accuri C6 Flow Cytometer BD Equipment
Vacutainer 5ml Med Vet International RED5.0 Consumable
Acid-citrate-dextrose Sigma C3821 Consumable
Calcium Chloride Sigma C5670 Consumable
Sevoflurane JD Medical 60307-320-25 Consumable
Rats Charles River Strain code: 400 Experimental animal
Rat surgical kit Harvard apparatus 728942 Equipment
Surgical Blade #15 MEDLINE MDS15115 Consumable
Rat MD's Baytril (2 mg/Tablet),
Rimadyl (2 mg/Tablet)		 Bio Serv F06801 Consumable
Polyglactin 910, 5-0 Ethicon J436G Consumable
Eosin alchol shandon Thermo scientific 6766007 Consumable
Harris Hematoxylin Thermo scientific 143907 Consumable
DOWNLOAD MATERIALS LIST

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Tags

Stem Cell TherapyBilateral Patellar Tendon InjuryRat ModelOrthopedic ResearchTendinopathyEquine Umbilical CordWharton’s JellyCollagenaseCell Culture
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Wagner, J. R., Taguchi, T., Cho, J. Y., Charavaryamath, C., Griffon, D. J. Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats. J. Vis. Exp. (133), e56810, doi:10.3791/56810 (2018).
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