用于刺激骨质复变和分析骨骼重塑功能结果的实验室A芯片平台
Summary
在这里，我们提出了用于在芯片实验平台内分析骨骼重塑的协议。3D 打印机械装载装置可与平台配对，通过变形细胞基质诱导骨质异质异体转导。该平台还可用于量化骨细胞和成骨细胞（再吸收/形成）的骨骼重塑功能结果。
Abstract
骨骼重塑是骨骼生长和修复以及适应机械环境变化所需的严格监管过程。在这个过程中，对心肌敏感性骨细胞调节骨细胞和合成代谢性骨细胞之间的对立反应。为了更好地了解调节这一过程的高度复杂的信号通路，我们的实验室开发了一个基础的片上实验室（LOC）平台，用于分析小型系统中骨骼重塑的功能结果（形成和再吸收）。由于骨骼重塑是一个漫长的过程，发生在数周到数月，我们开发了系统内的长期细胞培养协议。成骨细胞和成骨细胞生长在LOC内的功能活性基质上，并维持长达七周。之后，芯片被拆解，以便定量骨骼形成和吸收。此外，我们还设计了一种3D打印机械装载装置，与LOC平台配对，并可用于通过变形细胞基质诱导骨质异质异化。我们在LOC平台内优化了骨细胞、成骨细胞和成骨细胞的细胞培养方案，并解决了不育和细胞毒性问题。在这里，我们介绍了用于制造和消毒LOC、在功能基板上播种细胞、诱导机械负载和拆卸LOC以量化端点结果的协议。我们认为，这些技术为开发真正的芯片上器官进行骨骼重塑奠定了基础。
Introduction
骨骼是一种高度动态的组织，需要三种主要细胞类型之间的复杂协调：骨细胞、成骨细胞和成骨细胞。这些细胞之间的多细胞相互作用是导致瘫痪和长期不动期间发生的骨质流失，以及因生长和运动而发生的骨骼形成的原因。骨细胞是最丰富的骨细胞类型，对应用于骨骼的机械刺激非常敏感。机械刺激改变骨质代谢活性，导致关键信号分子11、22的增加。通过这个过程，称为二次转导，骨细胞可以直接协调成骨细胞（骨形成细胞）和成骨细胞（骨重组细胞）的活动。保持骨平衡需要骨骼形成和骨吸收率之间的严格调节;然而，这个过程的中断可能导致疾病状态，如骨质疏松症或骨质病。
这三种细胞类型之间相互作用的复杂性非常适合利用微流体和片上实验室 （LOC） 技术进行研究。为此，我们的实验室最近建立了LOC平台的概念证明，用于分析骨骼重塑过程中的骨再吸收和形成（功能结果）。该平台可用于研究细胞相互作用、改变的加载环境以及调查药物筛选。近年来，为研究调节骨重塑的分子信号通路开发了各种微流体装置;然而，这些系统中有许多通过间接标记量化改造，这些标记指示功能活动33、4、5、6、7。4,5,6,7我们的系统的一个优点是，它可用于直接量化功能结果。骨骼重塑是一个长期的过程。因此，直接定量骨吸收和形成需要一个培养系统，可以维持至少几个星期到月88，9，10，11。9,10,11因此，在开发LOC平台时，我们建立了形成和吸收所需的长期培养协议，并在系统内保持了长达7周的11周细胞。此外，我们还将两种细胞类型的适当培养基板纳入平台;骨细胞直接在骨骼上培养，骨细胞，已知是塑料粘附物，在聚苯乙烯盘上培养。此外，我们讨论了有关不育、长期细胞毒性和芯片拆卸的问题，以便进行改造分析11，12。11,
LOC平台还可用于通过矩阵变形诱导骨质异质转导。开发了一个3D打印机械装载装置，与LOC配对，并应用静态平面外散回来拉伸细胞13。为了适应这种机械负载，LOC 内的井深度增加。这种小规模、简单的机械装载装置可以很容易地由具有有限工程经验的实验室生产，我们之前曾共享过3D打印组件13的图纸。在目前的工作中，我们展示了成功使用LOC所需的一些新技术。 具体来说，我们演示了芯片制造、功能基板的细胞播种、机械加载和芯片拆卸，用于重塑定量。我们认为，这些技术的解释得益于视觉格式。
Protocol
Representative Results
Discussion
本文介绍了为培养骨细胞、骨细胞和骨细胞构建骨骼改造LOC平台奠定基础。通过改变芯片内井的深度和大小，开发了多种配置，以机械载荷刺激骨质和量化骨骼重塑的功能结果（图1B）。
在芯片组装过程中，优化等离子氧化协议对于消除泄漏问题至关重要。我们发现，将 PDMS 表面暴露在使用中射频功率设置（步骤 4.1）生成的 30 s 氧等离子体…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学基金会（CBET 1060990和EBMS1700299）的支持。此外，本材料还基于国家科学基金会研究生研究奖学金计划（2018250692）支持的工作。本材料中表达的任何意见、结论、结论或建议均为作者的意见、结论或建议，不一定反映国家科学基金会的观点。
Materials
| Acrylic sheet | Optix | — | 3.175 mm thick |
| Angled dispensing tips | Jensen Global | JG18-0.5X-90 | Remove plastic connector prior to use |
| Biopsy punch | Robbins Instruments | RBP-10 | 1 mm diameter |
| Bone wafers | Boneslices.com | 0.4 mm thick | Bovine cortical bone |
| Bovine calf serum | Hyclone | SH30072 | |
| Calipers | Global Industrial | T9F534164 | |
| Cell spatula | TPP | 99010 | |
| Chip mask | ProtoLabs | Custom-designed | Print material: Accura SL 5530 |
| Cork borer | Fisher Scientific | 07-865-10B | |
| Cotton tipped applicator | Puritan | 806-WCL | |
| Culture dish (100 mm) | Corning | 430591 | Sterile, Non-tissue culture treated |
| Culture dish (150 mm) | Corning | 430597 | Sterile, Non-tissue culture treated |
| Double sided tape | 3M Company | Scotch 237 | |
| Fetal bovine serum | Hyclone | SH30910 | |
| Forceps | Fisher Scientific | 22-327379 | |
| Leveling box | Custom-made | — | 3D printed |
| Masking tape | 3M Company | Scoth 2600 | |
| MC3T3-E1 preosteoblasts | ATCC | CRL-2593 | Subclone 4 |
| Mechanical loading device | Custom-made | — | 3D printed |
| Minimum essential alpha medium | Gibco | 12571-063 | |
| MLO-Y4 osteocytes | — | — | Gift from Dr. Lynda Bonewald |
| Packaging tape | Duck Brand | — | Standard packaging tape |
| Paraffin film | Bemis Parafilm | PM999 | |
| Penicillin/streptomycin | Invitrogen | p4333 | |
| Plasma cleaner | Harrick Plasma | PDC-001 | Expanded plasma cleaner |
| Polydimethylsiloxane kit | Dow Corning | Sylgard 184 | |
| Polystyrene coverslips | Nunc Thermanox | 174942 | Sterile, tissue culture treated |
| Oven | Quincy Lab | 12-180 | |
| RAW264.7 preosteoclasts | ATCC | TIB-71 | |
| Scalpel | BD Medical | 372611 | |
| Silicone tubing | Saint-Gobain Tygon | ABW00001 | ID: 1/32" (0.79 mm), OD: 3/32" (2.38 mm) |
| SolidWorks software | Dassault Systèmes | — | Used to generate 3D printed models and perform FEA |
| Spray adhesive | Loctite | 2323879 | Multi-purpose adhesive |
| Syringe (5 ml) | BD Medical | 309646 | Sterile |
| Syringe pump | Harvard Apparatus | 70-2213 | Pump 11 Pico Plus |
| Tapered laboratory spatula | Fisher Scientific | 21-401-10 | |
| Two-part expoxy | Loctite | 1395391 | 5 minute quick set |
| Type I collagen | Corning | 354236 | Rat tail collagen |
| Vacuum desiccator | Bel-Art | F42010-0000 | |
| Waterproof sealant | Gorilla | 8090001 | 100% silicone sealant |
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