这项工作由 ETH Grant 1-005733 资助

制造小鼠肌腱组合体以研究疾病中的细胞相互作用

作者没有什么可透露的。

总体而言，此处介绍的装配体模型系统有几个关键步骤需要强调。首先，模型系统的好坏取决于其组件的质量。在开始组装过程之前，在显微镜下检查核心外植体和待接种的细胞群至关重要。使用流式细胞术验证分离细胞群的表型至少一次同样重要。特别是当新一批胶原蛋白-1首次使用时，在将细胞包埋之前，在试运行中检查交联速度是有利的。组件组件需要大量的手动操作，这增加了感染的风险。为了最大限度地降低感染风险，请在具有层流气流的无菌生物安全罩中工作，经常更换手套，并用 80% 乙醇对手套和工作空间进行消毒。出于类似的原因，不要多次使用 3D 打印的夹具支架。在包埋过程本身之前，重要的是将所有水凝胶组分（交联溶液、胶原蛋白-1溶液）保持在冰上，以防止过早交联。因此，一旦将细胞添加到交联溶液中，就必须快速工作，以限制由于交联溶液的高pH值和低温而导致的细胞死亡。为了防止核心外植体中与干燥相关的细胞死亡，在将交联溶液与胶原-1溶液混合之前，立即吸出覆盖夹紧的核心外植体的培养基。为了保证核心外植体在水凝胶中的中心位置，理想的做法是将水凝胶浇铸在略微张紧的夹紧核心外植体周围。为此，请使用定位销和 M3 x 16 mm 螺栓螺钉将夹具固定到（3D 打印）板上，该板组具有适当长度的孔。在50分钟的聚合时间后，可以根据所需的培养条件再次对包埋的核心外植体进行拉伸。组装体在培养过程中经历的张力量对实验结果有深远的影响，并且在样品和条件下应保持一致21。
然而，与大多数组织工程替代方案相比，机械（非）加载对实验结果的巨大影响是组装体模型的主要优势，特别是因为核心外植体的基质组成保持不变，也应该在细胞水平上重建复杂的体内加载模式90。虽然在实践中，到目前为止只证明了组装体的线弹性模量、最大拉伸应变和最大拉伸应力的测量，但疲劳强度和应力松弛测量的协议已经在其他地方描述了腱芯外植体，并且应该适用于组装体91,92。除了类似体内的加载模式外，组件的多级模块化可能是其最大的优势。由于有单独的培养室，可以为每个样品单独设置一组可控的生态位条件（即温度、氧张力、葡萄糖浓度、补充剂、刺激剂、抑制剂和板静态拉伸）。接下来，外在区室的基质刚度和基质组成可以通过水凝胶组合物进行定制，例如，通过掺入更多的胶原蛋白-3和细胞纤连蛋白93,94,95来研究日益患病的组织微环境的影响。外源区室中评估的细胞群很容易通过选择要接种的细胞来适应，但也可以通过利用已建立的转基因细胞系和小鼠系（即ScxLin细胞耗竭）在肌腱核心外植体中进行修饰96。两个隔室的不同基质和细胞组成进一步提供了独特的区室化 3D 结构，这是另一个中央肌腱标志 1,30,46。
使用此系统时，重要的是要考虑系统模块化对结果参数粒度的影响。虽然可以单独评估每个隔室的细胞增殖和募集，但机械性能、分泌组成分和降解产物目前只能测量整个组装体。在吞吐量方面，一个经过适当培训的人员可以在一个正常工作日内准备多达 50 个组件，主要瓶颈是夹紧程序。虽然某些读出方法是相互排斥的，但可以重复评估同一样品的机械性能和分泌组成分，以及细胞群组成（流式细胞术）、细胞转录组（RT-qPCR、RNA 测序）或基质和细胞分布（免疫细胞化学/荧光显微镜）。在以前的出版物中，这些方法已被用于广泛表征暴露于病变样生态位的核心//成纤维细胞和核心//巨噬细胞集合体中的细胞间、跨室相互作用84,85。在这项工作中，探索了组合体模型系统在不同微环境刺激下探测核心和外在内皮细胞之间跨区室相互作用的能力。
模型系统的模块化允许将来对方法进行改进，这对于克服当前设计迭代的以下限制是必要的。这项工作中提出的流式细胞术分析和最近发表的单细胞RNA测序数据显示，肌腱核心驻留的腱细胞和跟腱衍生的群体比以前假设的24,34,59,84,97更具异质性24,34,59,84,97。此外，最初驻留在核心或水凝胶驻留的细胞群的迁移行为模糊了培养过程中的组装体区室化。这两个因素共同使得将转录组差异归因于特定细胞类型以及将增殖与基于迁移的过程区分开来具有挑战性。这一局限性可以通过以下方法克服：根据最近体内研究中表征的健康或患病肌腱的细胞组成，使用荧光激活细胞分选 （FACS） 细化输入群体，通过实施单细胞 RNA 测序来改善读数，并在显微镜检查期间整合增殖标志物，例如 EdU（5-乙炔基-2'-脱氧尿苷）染色。
这里介绍的组装体也与目前大多数可用的体外系统有一个共同的弱点，这些系统模拟与身体其他部分断开的患病器官98,99。然而，这里使用的基于培养室的平台将模型系统定位为可以很好地集成到多器官平台中，在该平台中，模拟不同器官的集合体被连接起来，并且可以研究器官间的相互作用。
该模型系统的核心是基于位置啮齿动物肌腱，这导致了其独特的缺点。首先，结果的可翻译性受到未发育或患有肌腱疾病的野生型小鼠的阻碍8,100,101。整合来自人类或新开发的表现出肌腱疾病方面的小鼠品系的组织和细胞可以缓解这个问题102。向基于人类的组合体的转变特别有趣，因为它可以研究来自不同患病肌腱（即肌腱炎、肌腱变性或肌腱膜炎）的患者来源组织，甚至是耐药供体，从而可以解锁更个性化的治疗方案。其次，小鼠尾腱外植体不能很好地处理超负荷诱导的微损伤，这限制了模型系统在急性肌腱损伤研究中的适用性。
由于所有这些原因，外植体//水凝胶组合体在研究肌腱核心生物学、基质结构-功能相互作用以及特定细胞群之间的跨区室相互作用以响应生态位诱导的微损伤方面处于主要地位。从这些相当高通量的研究中收集到的见解可以为 体内 研究和治疗开发提供方向。

肌腱将肌肉力传递到骨骼以实现运动的功能中，肌腱面临着人体中发生的一些最极端的机械应力1,2,3。由于社会老龄化、肥胖患病率的增加以及对机械要求高的体育活动的日益普及，预计发达国家肌腱疾病和损伤的患病率将攀升 4,5,6。目前可用的模型系统的局限性阻碍了新型循证和疾病修饰治疗方案的开发，以应对这种增加1,7,8。
理想情况下，疾病和损伤修复模型将允许研究靶器官如何将一组定义的输入参数（模仿疾病触发因素，表1）处理成可测量的输出参数（代表疾病标志，表2），同时控制混杂因素。然后，使用这种模型系统的研究将能够识别疾病和损伤修复背后的（病理）生理过程，并获得可用于预防或减少临床疾病和伤害特征的知识。将这一原理应用于肌腱，一个有用的模型系统应该概括体内肌腱对疾病和损伤反应的中心部分，其中包括以下特征：微损伤、炎症、新生血管形成、细胞增生、加速基质更新和去区室化 9,10,11,12,13,14,15.以这些标志为基础，可以推断出成功的肌腱疾病和损伤修复模型系统的以下要求。
据推测，机械超载是肌腱损伤和疾病发病机制的核心因素，因此是造成微损伤的常用实验方法16。因此，可控的机械载荷是肌腱疾病和损伤修复模型的首要先决条件。理想情况下，模型系统支持三种主要模式：单拉伸损伤载荷、疲劳载荷和卸载8、17、18。在机械变形时，组织驻留细胞会经历拉力、剪切力（由于细胞周围胶原纤维的滑动）和在卸载期间或附着点附近发生的压缩力的复杂组合19,20。模型系统应尽可能接近地再现这些复杂的加载模式。
引入基质微损伤的另一种方法是利用模拟肌腱疾病和损伤全身易感性的生化应激源，例如（促）炎性细胞因子、氧化应激或高葡萄糖浓度21,22,23。因此，可控的生态位微环境有利于肌腱疾病和损伤修复模型系统。
模型系统能够概括炎症、新生血管形成和细胞增生的一个常见先决条件是驱动这些过程的细胞群的选择性存在24。对于炎症过程，这些群体包括中性粒细胞、T 细胞和巨噬细胞，而内皮细胞和周细胞则需要研究新生血管形成 25,26,27,28,29。肌腱成纤维细胞不仅对肌腱修复至关重要，而且作为增殖和迁移细胞，也部分负责在肌腱疾病中观察到的局部细胞增生 30,31,32,33,34,35,36。
除了常驻细胞群的变化外，肌腱基质组成在肌腱疾病和损伤中也发生了变化7,37,38,39,40。为了呈现正确的与疾病相关的微环境线索，模型系统应该能够整合与目标疾病或损伤阶段相匹配的细胞外基质组成，例如，通过启用胶原蛋白-1、胶原-3和细胞纤连蛋白的相关比例组合41。
健康肌腱分为肌腱核心和外在隔室（即内腱、外皮腱和副腱）是其功能的核心，并且经常在患病或受伤的肌腱中受到干扰 1,42,43,44,45,46,47 .因此，将 3D 肌腱区室化纳入肌腱模型系统不仅需要更紧密地模拟去区室和重区室化的过程，还有助于建立细胞因子和营养物质的正确时空梯度48,49。
最后，模块化是模型系统的另一个核心资产，使研究人员能够在研究过程中将先前描述的压力源的正确相对贡献和相互作用结合起来8,17。
除了选择最佳输入模式外，一个重要的步骤是能够测量、观察和跟踪结果输出的变化。模型系统的力学性能（即脚趾区域长度、线弹性模量、最大拉伸应变、最大拉伸应力、疲劳强度和应力松弛）是这里的核心，因为它们表征了肌腱的主要功能50,51,52。为了将这些功能变化与组织水平的变化联系起来，重要的是启用检测（胶原）结构基质损伤的方法，并跟踪疾病和修复相关细胞群的增殖和募集30,53,54,55,56,57,58,59,60。
为了研究新出现的细胞间和细胞基质串扰，应该能够分离或标记足够量的蛋白质进行定量（即 ELISA、蛋白质组学、免疫组化、流式细胞术）14,21,61,62。群体或至少是区室特异性基因表达分析也应该是可能的（即荧光激活细胞分选 [FACS]、单细胞/批量 RNA 测序和实时定量聚合酶链反应 （RT-qPCR））21,24,27,63。模型系统应允许以足够快的速度测量同一试样和多个试样上尽可能多的上述输出参数，以解锁高通量研究。
在目前可用于研究人体肌腱疾病和损伤修复的模型系统中，人体本身当然是最具代表性的。它也是与实验干预最不相容的。虽然急性肌腱损伤患者可用于临床研究，但早期肌腱病（最常见的肌腱疾病）患者基本上没有症状，并且在临床上通常未被发现，直到出现更严重的变化 14,64,65。这使得很难确定肌腱稳态脱轨的关键时刻以及这种脱轨背后的机制16,66,67,68,69。此外，从健康的肌腱中提取活检在伦理上具有挑战性，因为它可能导致持续的损伤。前交叉韧带重建手术的腘绳肌腱残余通常用作健康对照，但与通常受肌腱疾病和损伤影响的肩袖、跟腱和髌腱相比，可以说在功能、机械性能、细胞群和基质组成方面有所不同70,71,72,73。
体内 动物模型更容易获得和处理，但它们的使用给动物带来了巨大的道德负担，并给研究人员带来了经济成本。此外，大多数流行的模式动物要么不会自发发生肌腱病变（即大鼠、小鼠、兔子），要么缺乏追踪其中涉及的多细胞通讯途径所需的引物和转基因菌株（即马、兔子）。
简单的 2D 体外模型系统位于复杂性/可处理性谱的另一端，并且可以更好地对特定的细胞间通讯通路进行受控、省时的研究，以响应一组更可控的触发因素 8,74。然而，这些简化的系统通常无法概括肌腱功能的核心多维机械载荷（即拉伸、压缩和剪切）。此外，组织培养塑料的（太）高刚度往往会覆盖由旨在模拟感兴趣的疾病状态的涂层提供的任何基质线索75,76。
为了克服这一缺点，已经开发了越来越复杂的组织工程3D模型系统，以提供可加载的基质，其组成至少可以部分匹配到所需的疾病状态77,78,79。尽管如此，这些系统不仅难以准确复制复杂的体内细胞外基质组成和细胞负荷模式，而且通常缺乏长期负荷性和研究协调肌腱疾病和损伤修复的跨室通讯途径所需的室室接口48,49,80。
离体肌腱外植体模型系统具有内置体内样基质组成的明显优势，该基质组成包括细胞周生态位、跨室屏障以及时空细胞因子/营养梯度，并在拉伸时概括复杂的负载模式8。由于大小依赖性营养扩散限制，来自大型动物模型（即马）的外植体很难在肌腱疾病和损伤修复的长期研究中保持活力81,82,83。同时，来自小鼠物种（即跟腱、髌腱）的较小外植体难以重复夹紧和机械载荷。它们的大小还限制了可以收集用于细胞、蛋白质和基因水平读数的材料量，而不会合并样本并降低通量。从这个意义上说，小鼠尾腱束提供了解锁肌腱疾病和损伤修复的高通量研究的潜力，因为它们很容易从单个小鼠中大量获得，保留了复合体内细胞周基质组成，并概括了细胞负荷模式。然而，在提取过程中，它们失去了大部分外在隔室，其中含有血管、免疫和成纤维细胞群，这些群现在被认为可以驱动肌腱疾病和修复 8,18。
为了弥合这一差距，已经开发了一种模型系统，将小鼠尾腱衍生的核心外植体的优点与基于3D水凝胶的模型系统的优点相结合。该模型系统由在尾腱外植体周围铸造的充满细胞的（胶原蛋白-1）水凝胶组成84,85。在本文中，详细介绍了必要的制造步骤以及有用的读数，这些读数可以通过在内皮细胞负载的 1 型胶原水凝胶（外在隔室）内共培养核心外植体（内在隔室）来获得。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm x 25 mm injection needle (G27)</td>
			<td>Sterican</td>
			<td>9186174</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printing filament: Clear polylactic acid prusament</td>
			<td>Prusa</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>4% formaldehyde</td>
			<td>Roti-Histofix</td>
			<td>P087.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase cell detachment solution</td>
			<td>Sigma-Aldrich</td>
			<td>A6964-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>VWR</td>
			<td>L0009-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Attachable digital C-mount camera: Moticam 2</td>
			<td>Motic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt screw M3 x 16 mm, stainless steel</td>
			<td>RS PRO</td>
			<td>1871235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bolt screw M3 x 6 mm, stainless steel</td>
			<td>RS PRO</td>
			<td>1871207</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td></td>
		</tr>
		<tr>
			<td>CD146 antibody: PE anti-mouse</td>
			<td>BioLegend</td>
			<td>134703</td>
			<td></td>
		</tr>
		<tr>
			<td>CD206 antibody: Alexa Fluor 488 anti-mouse</td>
			<td>BioLegend</td>
			<td>141709</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 antibody: Alexa Fluor 488 anti-mouse</td>
			<td>BioLegend</td>
			<td>102413</td>
			<td></td>
		</tr>
		<tr>
			<td>CD86 antibody: PE anti-mouse</td>
			<td>BioLegend</td>
			<td>105007</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Thermo Fisher Scientific</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV&#160;</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialyzed Fetal Bovine Serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F0392-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>7000183</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>D4693-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160;</td>
			<td>Sigma</td>
			<td>7002211</td>
			<td></td>
		</tr>
		<tr>
			<td>Dowel Pin, 3 mm x 16 mm, stainless steel</td>
			<td>Accu</td>
			<td>HDP-3-16-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dragon Skin 10 Slow/1 silicone</td>
			<td>KauPO</td>
			<td>09301-004-000001</td>
			<td></td>
		</tr>
		<tr>
			<td>Endopan 3 Kit</td>
			<td>Pan-Biotech</td>
			<td>P04-0010K</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth supplement&#160;</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf safe-lock plastic tubes (1.5 mL)</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium homodimer, EthD-1, 2 mM stock in DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>46043-1MG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>F4/80 antibody: Apc/fire 750 anti-mouse</td>
			<td>BioLegend</td>
			<td>123151</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon plastic tube (15 mL)</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon plastic tube (50 mL)</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: LSR II Fortessa</td>
			<td>BD Bioscience</td>
			<td>23-11617-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Invitrogen</td>
			<td>D12054</td>
			<td></td>
		</tr>
		<tr>
			<td>Hellmanex III alkaline cleaning concentrate</td>
			<td>Sigma</td>
			<td>Z805939-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyproline assay</td>
			<td>Sigma-Aldrich</td>
			<td>MAK008</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software: Motic Images Plus 3.0 ML</td>
			<td>Motic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid Phosphate Magnesium Salt n-Hydrate</td>
			<td>Wako Chemicals</td>
			<td>013-19641</td>
			<td></td>
		</tr>
		<tr>
			<td>LSE Low Speed Orbital Shaker</td>
			<td>Corning</td>
			<td>6780-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Sigma</td>
			<td>7002231</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse macrophage-stimulating factor (m-CSF)</td>
			<td>PeproTech</td>
			<td>315-02-50ug</td>
			<td></td>
		</tr>
		<tr>
			<td>MSD assay</td>
			<td>Mesoscale Discovery</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh strainer cap, 100 &#181;m</td>
			<td>Corning</td>
			<td>734-2761</td>
			<td></td>
		</tr>
		<tr>
			<td>Original Prusa i3 MK3S 3D printer</td>
			<td>Prusa</td>
			<td>i3 MK3S</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), ph 7.4, sterile, 10 L</td>
			<td>Gibco</td>
			<td>10010001</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>VWR</td>
			<td>786-650</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant m-CSF&#160;</td>
			<td>PeproTech</td>
			<td>315-02</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction kit: Rneasy plus Micro</td>
			<td>Qiagen</td>
			<td>74034</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicing software: PrusaSlicer</td>
			<td>Prusa</td>
			<td>NA</td>
			<td>Version 2.6.0 or higher</td>
		</tr>
		<tr>
			<td>Sterile Cell Strainer 100 &#181;m</td>
			<td>Fisherbrand</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scalpel blade No. 21</td>
			<td>Swann-Morton</td>
			<td>307</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5 %)</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineering tendon assembloids to probe cellular crosstalk in disease and repair

肌腱通过将肌肉力传递到骨骼来实现运动。它们依赖于由胶原纤维和基质细胞群组成的坚韧肌腱核心。这个承重核心被由外肌腱隔室组成的滑膜样组织层包围、滋养和修复。尽管有这种复杂的设计，但肌腱损伤很常见，临床治疗仍然依赖于物理治疗和手术。现有实验模型系统的局限性减缓了新型疾病修饰疗法和预防复发临床方案的发展。 
体内 人体研究仅限于将健康肌腱与修复手术期间取样的终末期病变或破裂组织进行比较，不允许对潜在的肌腱疾病进行纵向研究。 体内 动物模型在不透明的生理复杂性、动物的伦理负担以及与使用它们相关的巨大经济成本方面也存在重要的限制。此外， 体内 动物模型不太适合系统探测药物和多细胞、多组织相互作用途径。更简单的 体外 模型系统也存在不足。一个主要原因是未能充分复制有意义地研究肌腱细胞及其功能所需的三维机械载荷。 
这里介绍的新3D模型系统通过利用小鼠尾腱核心外植体来缓解其中一些问题。重要的是，这些外植体很容易从单个小鼠大量获得，在细胞水平上保留了 3D 原位 加载模式，并具有类似 体内的细胞外基质。在该方案中，给出了有关如何用富含肌肉来源的内皮细胞、肌腱来源的成纤维细胞和骨髓来源的巨噬细胞的胶原蛋白水凝胶增强肌腱核心外植体的分步说明，以替代外源性肌腱隔室内的疾病和损伤激活细胞群。它展示了如何通过机械或通过定义的微环境刺激来挑战由此产生的肌腱组合体，以研究疾病和损伤期间出现的多细胞串扰。

In their function of transferring muscle forces to the bones to enable movement, tendons face some of the most extreme mechanical stresses occurring in the human body1,2,3. Due to aging societies, increasing obesity prevalence, and the growing popularity of mechanically demanding sports activities, the prevalence of tendon diseases and injuries is projected to climb in developed countries4,5,6. The development of novel evidence-based and disease-modifying treatment regimens to combat this increase has been hindered by limitations of currently available model systems1,7,8.
Ideally, disease and injury repair models would allow studying how the target organ processes a defined set of input parameters (imitating disease triggers, Table 1) into measurable output parameters (representing disease hallmarks, Table 2) while controlling for confounding factors. Studies using such model systems would then be able to identify the (patho-) physiological processes underlying disease and injury repair and gain knowledge that could be exploited to prevent or reduce disease and injury hallmarks in the clinics. Applying this principle to tendons, a useful model system should recapitulate central parts of the in vivo tendon response to disease and injury, which encompass the following hallmarks: microdamage, inflammation, neovascularization, hypercellularity, accelerated matrix turnover, and decompartmentalization9,10,11,12,13,14,15. Using these hallmarks as a base, the following requirements for a successful tendon disease and injury repair model system can be inferred.
Mechanical overloading is hypothesized to be a central factor in tendon injury and disease pathogenesis and is thus a commonly used experimental approach to create microdamage16. Controllable mechanical loadability is, therefore, a prime prerequisite for tendon disease and injury repair models. Ideally, the model system enables three main modes: single stretch-to-damage loading, fatigue loading, and unloading8,17,18. Upon mechanical deformation, tissue-resident cells experience a complex combination of tensional forces, shear forces (due to the sliding of collagen fibers surrounding the cells), and compression forces occurring during unloading or near the enthesis19,20. Model systems should recreate these complex loading patterns as closely as possible.
An alternative way to introduce matrix microdamage is to leverage biochemical stressors that mimic systemic predispositions for tendon disease and injury, such as (pro-)inflammatory cytokines, oxidative stress, or high glucose concentrations21,22,23. Consequently, a controllable niche microenvironment is advantageous for a tendon disease and injury repair model system.
A common prerequisite for model systems to be able to recapitulate inflammation, neovascularization, and hypercellularity is the selective presence of cell populations that drive these processes24. For inflammatory processes, these populations include neutrophils, T-cells, and macrophages, while endothelial cells and pericytes would be needed to study neovascularization25,26,27,28,29. Tendon fibroblasts are not only vital for tendon repair but, as proliferative and migrating cells, also partially responsible for the local hypercellularity observed in tendon disease30,31,32,33,34,35,36.
Besides changes in resident cell populations, the tendon matrix composition is altered in tendon disease and injury as well7,37,38,39,40. To present the right disease-relevant microenvironmental cues, model systems should be able to integrate an extracellular matrix composition matched to the targeted disease or injury stage, for example, by enabling relevant proportional combinations of collagen-1, collagen-3, and cellular fibronectin41.
The compartmentalization of healthy tendons into the tendon core and the extrinsic compartments (i.e., endotenon, epitenon, and paratenon) is central to their function and often disturbed in diseased or injured tendons1,42,43,44,45,46,47. Incorporating 3D tendon compartmentalization into tendon model systems is therefore not only required to simulate the processes underlying de- and re-compartmentalization more closely but also helps to establish the correct spatiotemporal gradients of cytokines and nutrients48,49.
Finally, modularity is another central asset of model systems, allowing researchers to combine the correct relative contribution of and interplay between the previously described stressors during the investigated processes8,17.
Besides selecting the optimal input modalities, an important step is being able to measure, observe, and track changes in the resulting output. The mechanical properties of the model system (i.e., toe region length, linear elastic modulus, maximum tensile strain, maximum tensile stress, fatigue strength, and stress relaxation) are central here, as they characterize the tendon&#39;s main function50,51,52. To link these functional changes to tissue-level changes, it is important to enable methods detecting (collagen) structural matrix damage and tracking proliferation and recruitment of disease- and repair-relevant cell populations30,53,54,55,56,57,58,59,60.
To study emerging cell-cell and cell-matrix crosstalk, one should be able to isolate or mark proteins in adequate amounts for quantification (i.e., ELISA, proteomics, immunohistochemistry, flow cytometry)14,21,61,62. Population- or at least compartment-specific gene expression analysis should be possible as well (i.e., fluorescence-activated cell sorting [FACS], single-cell/bulk RNA- sequencing, and real-time quantitative polymerase chain reaction (RT-qPCR))21,24,27,63. The model system should allow measuring as many of the aforementioned output parameters on the same specimen and on multiple specimens in a manner fast enough to unlock high-throughput studies.
Among model systems currently available to study human tendon disease and injury repair, the human body itself is, of course, the most representative one. It is also the least compatible with experimental intervention. While patients with acute tendon injuries are abundantly available for clinical studies, patients with early tendinopathy (the most common tendon disease) are largely symptom-free and often go clinically undetected until more severe changes manifest14,64,65. This makes it hard to pinpoint the critical moment when tendon homeostasis derails and the mechanisms behind this derailment16,66,67,68,69. In addition, extracting biopsies from healthy tendons is ethically challenging, as it may result in persisting damage. Hamstring tendon remnants from anterior cruciate ligament reconstruction surgery are often used as healthy controls but arguably differ in function, mechanical properties, cell populations, and matrix composition compared to the rotator cuff, Achilles, and patellar tendons commonly affected by tendon disease and injury70,71,72,73.
In vivo animal models are more accessible and tractable, but their usage imposes a significant ethical burden on the animals and economic cost on the researchers. In addition, most of the popular model animals either do not develop tendinopathic lesions spontaneously (i.e., rats, mice, rabbits) or lack the primers and genetically modified strains necessary to track the multicellular communication pathways involved in it (i.e., horses, rabbits).
Simple 2D in vitro model systems are on the other side of the complexity/tractability spectrum and better allow controlled, time-efficient study of specific intercellular communication pathways in response to a more controllable set of triggers8,74. However, these simplified systems commonly fail to recapitulate the multi-dimensional mechanical loading (i.e., tension, compression, and shear) that is central to tendon functionality. In addition, the (too) high stiffnesses of tissue culture plastic tend to override any matrix cues provided by coatings intended to mimic the disease state of interest75,76.
To overcome this drawback, increasingly sophisticated tissue-engineered 3D model systems have been developed to provide a loadable matrix whose composition can at least be partially matched to the desired disease state77,78,79. Still, these systems not only struggle to accurately replicate the complex in vivo extracellular matrix compositions and cellular loading patterns but generally lack long-term loadability and the compartmental interfaces required to study the cross-compartmental communication pathways that coordinate tendon disease and injury repair48,49,80.
Ex vivo tendon explant model systems have the distinct advantage of a built-in in vivo-like matrix composition that comprises pericellular niches, cross-compartmental barriers, as well as spatiotemporal cytokine/nutrient gradients and recapitulates complex loading patterns when stretched8. As a result of size-dependent nutrient diffusion limits, explants from larger animal models (i.e., horses) are difficult to keep alive for the long-term study of tendon disease and injury repair81,82,83. Meanwhile, smaller explants from murine species (i.e., Achilles tendon, patellar tendon) are challenging to reproducibly clamp and mechanically load. Their size also constrains the amount of material that can be gathered for cell-, protein-, and gene-level readouts without pooling samples and decreasing throughput. In this sense, murine tail tendon fascicles offer the potential to unlock high-throughput study of tendon disease and injury repair as they are readily available in large quantities from a single mouse, preserve the complex in vivo pericellular matrix composition, and recapitulate cellular loading patterns. During the extraction process, however, they lose most of their extrinsic compartment and the therein contained vascular, immune, and fibroblast populations that are now considered to drive tendon disease and repair8,18.
To bridge this gap, a model system combining the advantages of murine tail tendon-derived core explants with the advantages of 3D hydrogel-based model systems has been developed. This model system consists of a cell-laden (collagen-1) hydrogel cast around tail tendon explants84,85. In this paper, the necessary manufacturing steps are provided in detail alongside useful readouts that can be obtained by co-culturing core explants (intrinsic compartment) within an endothelial cell-laden type-1 collagen hydrogel (extrinsic compartment).

作者聚焦：通过开发小鼠组合来了解细胞机制来推进肌腱研究

工程肌腱组件以探测疾病和修复中的细胞串扰

Tendons facilitate movement by transmitting forces from muscle to bone. Although tendon injury is common, these are quite difficult to treat and the outcome for patients is often poor. Currently, all treatments of tendon injury involve some kind of physiotherapy, and this reflects the fact that mechanical forces play such a central role in tendon biology.Good experimental models for studying tendon damage and repair don't really exist, so my lab is actively developing new models that can better capture important features of tendon physiology and pathophysiology. In previous studies, we could show that the tendon core, which represents the load-bearing part of the tendon, has by itself a very limited repair capacity. Combined with other research in the field, we hypothesized that an injured core would recruit cells from the extrinsic tendon compartment to help it heal.Tissue-engineered tendon model system may provide a loadable 3D environment but don't match the intricacies of an in vivo exocellular matrix. Explant model systems do, but they are often difficult to keep alive and mechanically load over longer periods of time or lack the extrinsic compartment that is central for the repair processes. Our unique model system combines the advantages of marine tail tendon-derived core explants with those of 3D hydrogel base systems.It provides a loadable, in vivo-like core matrix, alongside an artificial extrinsic compartment. Its composition can be tuned to the research hypothesis and the biomimetic cross-compartmental barrier in between the two. Our hybrid hydrogel explant assembloids are in a prime position to study tendon core biology, matrix structure function interactions, and cross-compartmental interactions between specific cell populations in a fine-tunable micro environment.Discoveries from the studies conducted with this system will guide in vivo research and treatment development.

Watch this Scientific Journal Video about Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms at JoVE.com

在这里，我们提出了一个组合体模型系统来模拟承重肌腱核心组织与包含由疾病和损伤激活的细胞群的外在隔室之间的肌腱细胞串扰。作为一个重要的用例，我们展示了如何部署该系统来探测外源性内皮细胞的疾病相关激活。

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

肌腱通过将力从肌肉传递到骨骼来促进运动。虽然肌腱损伤很常见，但这些损伤很难治疗，而且患者的预后往往很差。目前，所有肌腱损伤的治疗都涉及某种物理治疗，这反映了机械力在肌腱生物学中起着如此核心的作用。研究肌腱损伤和修复的良好实验模型并不存在，因此我的实验室正在积极开发新的模型，以更好地捕捉肌腱生理学和病理生理学的重要特征。在以前的研究中，我们可以证明代表肌腱承重部分的肌腱核心本身具有非常有限的修复能力。结合该领域的其他研究，我们假设受伤的核心会从外在肌腱隔室招募细胞以帮助其愈合。组织工程肌腱模型系统可以提供可加载的 3D 环境，但与体内外细胞基质的复杂性不匹配。外植体模型系统可以，但它们通常难以在较长时间内保持活力和机械负载，或者缺乏对修复过程至关重要的外在隔室。我们独特的模型系统结合了海洋尾腱衍生的核心外植体与3D水凝胶基础系统的优点。它提供了一个可加载的、类似体内的核心基质，以及一个人工的外在隔室。它的组成可以根据研究假设和两者之间的仿生跨室屏障进行调整。我们的杂交水凝胶外植体组装体在微调微环境中研究肌腱核心生物学、基质结构功能相互作用以及特定细胞群之间的跨室相互作用方面处于有利地位。使用该系统进行的研究的发现将指导体内研究和治疗开发。

engineering-tendon-assembloids-to-probe-cellular-crosstalk-disease

Research

JoVE Journal

Bioengineering

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

422 次观看.  Balgrist, University Hospital Zurich, University of Zurich. 肌腱通过将力从肌肉传递到骨骼来促进运动。虽然肌腱损伤很常见，但这些损伤很难治疗，而且患者的预后往往很差。目前，所有肌腱损伤的治疗都涉及某种物理治疗，这反映了机械力在肌腱生物学中起着如此核心的作用。研究肌腱损伤和修复的良好实验模型并不存在，因此我的实验室正在积极开发新的模型，以更好地捕捉肌腱生理学和病理生理学的重要特征。在以前?...