破碎散装水凝胶并加工成颗粒状水凝胶，用于生物医学应用
Summary
这项工作描述了通过挤出破碎制造微凝胶，将微凝胶加工成可注射的颗粒水凝胶，并将颗粒水凝胶用作生物医学应用的挤出印刷油墨的简单，适应性强且低成本的方法。
Abstract
颗粒水凝胶是水凝胶微粒（即“微凝胶”）的堵塞组件。在生物材料领域，颗粒水凝胶具有许多有利的性质，包括注射性，微尺度孔隙率以及通过混合多个微凝胶群体的可调性。制造微凝胶的方法通常依赖于油包水乳液（例如，微流体，批量乳液，电喷雾）或光刻，这可能在资源和成本方面提出很高的要求，并且可能与许多水凝胶不相容。这项工作详细介绍了使用挤出破碎法制造微凝胶的简单而高效的方法，并将其加工成对生物医学应用有用的颗粒水凝胶（例如，3D打印油墨）。首先，将块状水凝胶（以光交联透明质酸（HA）为例）通过一系列直径顺序较小的针挤出以形成碎片状的微凝胶。这种微凝胶制造技术快速，低成本且高度可扩展。描述了通过离心和真空驱动过滤将微凝胶卡入颗粒状水凝胶的方法，并可选配用于水凝胶稳定的后交联。最后，由碎片微凝胶制成的颗粒水凝胶被证明为挤出印刷油墨。虽然这里描述的示例使用光交叉链接HA进行3D打印，但这些方法很容易适应各种水凝胶类型和生物医学应用。
Introduction
颗粒状水凝胶是通过水凝胶颗粒（即微凝胶）的填充而制造的，并且是一类令人兴奋的生物材料，具有许多生物医学应用的优势特性1，2，3。由于其颗粒结构，颗粒水凝胶具有剪切变稀和自愈合，允许它们用作挤出印刷（生物）油墨，用于嵌入式印刷的颗粒状载体和注射治疗剂4，5，6，7，8，9。此外，微凝胶之间的空隙空间为细胞运动和分子扩散提供了微尺度的孔隙率8，10，11。此外，多个微凝胶群体可以组合成单个配方，以允许增强的可调性和材料功能8，10，12，13。这些重要的特性推动了近年来颗粒状水凝胶的快速发展。
有一系列方法可用于形成微凝胶以用于颗粒水凝胶制造，每种方法都有其自身的优点和缺点。例如，微凝胶通常由油包水乳液使用液滴微流体4，11，13，14，15，16，17，间歇乳液7，18，19，20，21，22或电喷雾6，23，24，25.这些方法产生具有均匀（微流体）或多分散（间歇乳液，电喷雾）直径的球形微凝胶。这些油包水乳液制造方法存在一些局限性，包括潜在的低通量生产，对低粘度水凝胶前体溶液的需求，以及设置的高成本和资源。此外，这些方案可能需要刺激性油和表面活性剂，必须使用添加处理步骤的程序从微凝胶中清洗，并且可能难以转化为许多实验室中生物医学应用的无菌条件。为了消除对油包水乳液的需求，也可以使用（光）光刻，其中使用模具或光掩模来控制来自水凝胶前体溶液1，26，27的微凝胶的固化。与微流体一样，这些方法的生产吞吐量可能有限，这在需要大量时是一个重大挑战。
作为这些方法的替代方法，散装水凝胶的机械破碎已被用于制造不规则尺寸19，28，29，30，31，32的微凝胶。例如，本体水凝胶可以预先形成并随后通过网状物或筛子以形成碎片状微凝胶，该过程甚至在微凝胶链33，34内的细胞存在下完成。散装水凝胶也已使用诸如用研钵和研杵研磨或通过使用商业搅拌机35，36，37等技术加工成具有机械破坏的微凝胶。其他人还在水凝胶形成过程中使用机械搅拌来制造碎片状微凝胶（即流体凝胶）31。
本文的方法扩展了这些机械破碎技术，并提出了一种以光交联透明质酸（HA）水凝胶为例，通过挤出破碎制造微凝胶的简单方法。挤出破碎仅使用注射器和针头以低成本，高通量且易于扩展的方法制造碎片微凝胶，适用于各种水凝胶19，32。此外，使用离心（低填料）或真空驱动的过滤（高填料）来描述将这些碎片状微凝胶组装成颗粒水凝胶的方法。最后，讨论了这些碎片状颗粒水凝胶的应用，以用作挤出印刷油墨。该协议的目标是引入适用于各种水凝胶的简单方法，并且可以在几乎任何对颗粒水凝胶感兴趣的实验室中实施。
Protocol
1.使用光交联在注射器内制造散装水凝胶 注：使用光交联在注射器内制造散装水凝胶的概述如图 1所示。该方案使用降冰片烯修饰的透明质酸（NorHA）通过光介导的硫醇烯反应制造本体水凝胶。NorHA合成的详细程序在别处38中描述。然而，该方案高度适应任何光交联的水凝胶。有关详细信息，请参阅讨论。 预先确定用…
Representative Results
这些协议的代表性结果如图 3 和 图6所示。挤出破碎产生具有锯齿状多边形形状的微凝胶，直径范围为10-300μm（图3）。此外，圆度范围从0.2（非圆形）到几乎1（完美圆），长宽比范围从1-3（图3）。这些参数描述了由碎裂过程形成的不规则和锯齿状微凝胶形状。 当使用离心或真空驱动的…
Discussion
本文描述了使用挤出碎片微凝胶并通过离心或真空驱动过滤进行包装来制造颗粒水凝胶的方法。与其他微凝胶制造方法（即微流体，间歇乳液，电喷雾，光刻）相比，挤出碎片微凝胶制造非常快速，低成本，易于扩展，并且适用于各种水凝胶系统。此外，该协议具有高度可重复性，具有最小的批次间变异性，其在前面的工作39中具有特征。
该方案使用降冰片…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国家科学基金会通过UPenn MRSEC计划（DMR-1720530）和研究生研究奖学金（V.G.M和M.E.P.）以及美国国立卫生研究院（R01AR077362到J.A.B.）的支持。
Materials
| 15 mL Plastic Conical Centrifuge Tube | Corning | 430766 | |
| 30 G NT Premium Series Dispensing Tip | Jensen Global | JG30-0.5HPX | Catalog Number listed here is for 30 G, 0.5" needle. Various sizes are available. |
| BD Disposable Syringes with Luer-Lok Tips (3 mL) | Fisher Scientific | 14-823-435 | Catalog Number listed here is for 3 mL syringe. Various sizes are available (14-823-XXX). |
| Black folders | Various Vendors | ||
| Disposable Probe Needle For Use With Syringes and Dispensing Machines (18 G, 0.5") | Grainger | 5FVH5 | Catalog Number listed here is for 18 G, 0.5" needle. Various sizes are available. |
| Disposable Probe Needle For Use With Syringes and Dispensing Machines (23 G, 0.5") | Grainger | 5FVJ3 | |
| Disposable Probe Needle For Use With Syringes and Dispensing Machines (27 G, 1.5") | Grainger | 5FVL0 | |
| Dulbecco's Phosphate Buffered Saline | Fisher Scientific | 14190-250 | Catalog Number listed here is for a case of 10 x 500 mL bottles. |
| Durapore Membrane Filter, 0.22 µm | Millipore | GVWP04700 | |
| Epifluorescent or confocal microscope | Various Vendors | To visualize microgels and granular hydrogels | |
| Eppendorf Snap-Cap Microcentrifuge Safe-Lock Tubes | Fisher Scientific | 05-402-25 | |
| Extrusion printer | Custom-built | Other extrusion printers can be use,d such as commercially available BIOX. | |
| Filter Adapters | Fisher Scientific | 05-888-107 | Catalog Number listed here is for a set of multiple sizes. Various sizes are available (05-888-XXX). |
| Filter Flask | Various Vendors | ||
| Fluorescein isothiocyanate-dextran (2 MDa) | Sigma-Aldrich | 52471 | |
| Glass microscope slide | Various Vendors | ||
| ImageJ | National Institutes of Health | "Analyze Particles" information link: https://imagej.nih.gov/ij/docs/menus/analyze.html | |
| Laptop | Various Vendors | ||
| Luer-Lock Tip Caps | Integrated Dispensin g Solutions | 9991329 | |
| Metal spatula for scooping | Various Vendors | ||
| Microcentrifuge | Various Vendors | Capable of speed up to 18,000 x g | |
| Microscoft Execl | Microsoft | Other programs can be used, such as Google Slides. | |
| OmniCure S2000 Spot UV Curing System | Excelitas Technologies | S2000 | Different light systems may be used to fabricate bulk hydrogels if desired. |
| Porcelain Buchner Funnel with Fixed Perforated Plate | Fisher Scientific | FB966C | Catalog Number listed here is for 56mm diameter plate. Various sizes are available. |
| Radiometer | Various Vendors | ||
| Repetier Host | Hot-World GmbH & Co. KG | 3D printing software | |
| Screw-based extrusion printer | Various Vendors | This study used a custom-modified 3D FDM printer (Velleman K8200). Many alternatives are available. | |
| Solidworks/CAD software | Dassault Systèmes SolidWorks Corporation | Other programs can be used, such as Blender or TinkerCAD. | |
| Tubing to Connect Filter Flask to Vacuum Line | Various Vendors | ||
| UV Eye Protection (i.e., safety glasses) | Various Vendors |
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Citar este artigo
Muir, V. G., Prendergast, M. E., Burdick, J. A. Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications. J. Vis. Exp. (183), e63867, doi:10.3791/63867 (2022).