用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备
Summary
本方案描述了一种基于非乳液的壳聚糖-杰尼平微凝胶的制造方法。这些微凝胶的大小可以精确控制，它们可以显示pH依赖性肿胀， 在体内降解，并加载治疗分子，这些分子会随着时间的推移而持续释放，使其与组织工程应用高度相关。
Abstract
壳聚糖微凝胶因其应用广泛，成本低廉和免疫原性而在组织工程中引起了人们的极大兴趣。然而，壳聚糖微凝胶通常使用需要有机溶剂冲洗的乳液方法制造，这些方法有毒且对环境有害。本方案提出了一种快速，无细胞毒性，非乳液的方法，用于制造壳聚糖 – 京尼平微凝胶，而无需有机溶剂冲洗。这里描述的微凝胶可以通过精确的尺寸控制来制造。它们表现出生物分子的持续释放，使其与组织工程，生物材料和再生医学高度相关。壳聚糖与京尼平交联形成水凝胶网络，然后通过注射器过滤器产生微凝胶。微凝胶可以被过滤以产生一系列尺寸，并且它们显示出pH依赖性肿胀并随着时间的推移而酶降解。这些微凝胶已被用于大鼠生长板损伤模型，并被证明可以促进软骨组织修复增加， 并在体内28天显示完全降解。由于其低成本，高便利性和易于用细胞相容性材料制造，这些壳聚糖微胶囊在组织工程中呈现出令人兴奋和独特的技术。
Introduction
生长板，也称为骨架，是位于长骨末端的软骨结构，介导儿童的生长。如果生长板受伤，可以形成称为“骨棒”的修复组织，这会中断正常生长并可能导致生长缺陷或角畸形。流行病学数据显示，所有儿童骨骼损伤中有15%-30%与生长板有关。骨条形成发生在高达30%的这些损伤中，使得生长板损伤及其相关治疗成为一个重要的临床表现问题1，2，3，4。当发生骨棒形成时，最常见的治疗途径包括切除骨棒并插入介位材料，例如硅或脂肪组织5。然而，接受骨筋切除手术的患者通常对完全康复的预后较差，因为目前没有治疗方法可以完全修复受损的生长板6，7，8。鉴于这些缺点，迫切需要有效的策略来治疗生长板损伤，无论是在防止骨棒的形成还是再生健康的骨骼软骨组织方面。
水凝胶微粒或微凝胶最近作为可注射支架引起了人们的兴趣，可以提供持续释放的治疗药物9。由于其高可调谐性和生物相容性，微凝胶也非常适合生物活性因子或细胞包封。微凝胶可以由各种材料制成，从合成聚合物（如聚乙二醇（PEG））到天然聚合物，如海藻酸盐或壳聚糖10，11，12。壳聚糖已被证明对组织工程具有几种有益作用，例如其破坏革兰氏阴性菌外膜稳定性的能力，从而提供固有的抗菌活性13，14。此外，壳聚糖具有成本效益，细胞相互作用，并且易于使用其含胺结构进行修饰。基于壳聚糖的微凝胶有望为药物递送和材料信号传导提供一种生物材料策略，可以促进组织再生，同时防止细菌感染。然而，壳聚糖微凝胶通常使用各种技术制造，需要特殊设备，乳液技术或细胞毒性溶剂冲洗。例如，一些研究已经用基于乳液的方法制造了壳聚糖微凝胶，但这些方案需要溶剂冲洗和细胞毒联剂，这可能会否定它们对临床环境的转化15，16。其他研究已经使用微流体或电喷雾方法来制造壳聚糖微凝胶，这需要特殊的设备，制备和培训17，18。壳聚糖微凝胶通常也通过交联剂滴加成壳聚糖溶液的过程制成;然而，该方法高度依赖于溶液粘度、聚合物浓度和流速，使得难以控制微凝胶19，20的大小和分散度。相反，本文中描述的微凝胶制造方法不需要专门的设备或溶剂冲洗，使得这些微凝胶几乎可以在任何实验室或环境中进行制造。因此，这些微凝胶代表了高度相关的生物材料，可为许多应用提供快速，经济高效且易于生产的药物输送载体。
通过调节微凝胶的组成和材料特性，研究人员可以精确控制细胞微环境，从而以材料依赖性的方式指导细胞行为。微凝胶可以单独使用或与本体生物材料系统结合使用，以赋予特定的功能，例如生物活性因子的延长释放或天然或外源性细胞的精确特殊信号传导。生物材料和微凝胶已成为生长板损伤的有吸引力的治疗途径。已经投入了大量精力来开发基于海藻酸盐和壳聚糖的生物材料，以治疗生长板损伤21，22，23，24，25。由于生长板骨化和骨伸长的动态时间性质，骨条形成的机制尚不完全清楚。因此，已经开发了几种动物模型来更好地阐明软骨内骨化和骨条形成的机制，例如在大鼠，兔子和绵羊中26，27，28。一个这样的模型是大鼠生长板损伤模型，其利用大鼠胫骨中的钻孔缺陷以可预测和可重复的方式产生骨条，并在生长板29，30的所有三个区域模拟人类损伤。已经使用该模型测试了几种基于生物材料的治疗生长板损伤的策略。此外，已经开发了两种不同的制备壳聚糖微凝胶的方法，其可用作可注射的生物材料系统，其以持续的方式释放治疗药物10，31。这些微凝胶已被用于大鼠生理损伤模型，并且在释放SDF-1a和TGF-b3时显示软骨再生31 有所改善。该协议中提供的技术描述了为制造这些壳聚糖微凝胶而开发的方法，然后可以将其用于各种组织工程应用。例如，最近的研究已经使用热或洋红色响应的壳聚糖微凝胶用于受控肿瘤药物递送应用32，33。
Protocol
Representative Results
Discussion
近年来，微凝胶因其对各种用途的高度适用性（例如药物递送或细胞包封）而得到了广泛的研究 9.微尺度生物材料构建体的易于制造在组织工程中具有重要意义，因为它允许研究人员在特定尺寸和时间尺度上开发基于水凝胶的策略。然而，大多数制造壳聚糖微凝胶的方法需要昂贵的设备和试剂，乳液或细胞毒性溶剂冲洗，这阻止了它们转化为临床用途15，</s…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本出版物中报告的研究得到了美国国立卫生研究院关节炎和肌肉骨骼和皮肤病研究所的支持，奖励号为R03AR068087和R21AR071585，并由Boettcher基金会（#11219）支持MDK。CBE由NIH / NCATS Colorado CTSA拨款号TL1 TR001081支持。
Materials
| Acetic acid | SigmaAldrich | AX0073 | |
| BD Luer-Lock Syringe | Fisher Scientific | 14-823-16E | |
| Büchner Funnel | Fisher Scientific | FB966F | 100 mm diameter |
| Chitosan (low molecular weight) | SigmaAldrich | 448869 | 75-80% deacetylation |
| Dialysis Membrane Tubing | Fisher Scientific | 08-670-5C | 3500 MWCO |
| Ethanol | SigmaAldrich | 493538 | |
| Genipin | SigmaAldrich | G4796 | |
| Heracell 150i Incubator | ThermoFisher | 50116047 | |
| Parafilm | Fisher Scientific | 13-374-12 | |
| Recombinant human SDF-1a | Peprotech | 300-28A | |
| Recombinant human TGF-b3 | Peprotech | 100-36E | |
| Whatman Filter Paper Grade 540 | SigmaAldrich | Z241547 | 8 mm pore size |
| Whatman Filter Paper Grade 541 | SigmaAldrich | WHA1541055 | 22 mm pore size |
| Whatman Filter paper Grade 542 | SigmaAldrich | WHA1542185 | 2.7 mm pore size |
| Wire Mesh Sieve | McMaster-Carr | 9317T86 | No. 100 Mesh |
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