This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1.孢粉外壁胶囊的提取（秒）从L. clavatum孢子注意:美国证券交易委员会的提取过程涉及的易燃粉（L. clavatum），热腐蚀性酸和易燃溶剂，因此，适当的个人防护装备（护目镜，面罩，手套，实验室外套），使用情况获得批准的风险评估和处置由授权的实验室工作人员的化学品是必不可少的。 <ol><li> 孢子脱脂 <ol><li>通过使用玻璃冷凝器，循环水系统，和水浴（ 图1）准备在通风橱中回流设置。 </li><li>称取百克L. clavatum孢子不要产生灰尘和任何火源。 注意:这里使用的是商业上获得孢子（见材料清单）。 </li><li>转印孢子慢慢到1L聚四氟乙烯（PTFE）的圆底烧瓶中有磁力搅拌棒。 </li><li> 500ml丙酮添加到在孢子聚四氟乙烯烧瓶中并轻轻摇动烧瓶中以形成均匀悬浮液。 </li><li>放置在水浴集聚四氟乙烯烧瓶在50℃下，并连接到回流冷凝器。 </li><li>在丙酮进行回流6小时，以200rpm搅拌，然后允许在室温冷却。 </li><li>使用过滤器真空过滤纸暂停并收集孢子脱脂大（直径15厘米）的玻璃培养皿中。 </li><li>通过用铝箔与溶剂蒸发的孔覆盖干燥，在室温（通风柜）的孢子。 </li></ol></li><li> 酸解 <ol><li>制备85％（V / V）磷酸，用去离子（DI）水和脱脂干孢子转移到一个1升的PTFE烧瓶中。 </li><li> 500毫升的磷酸（85％V / V）添加到脱脂孢子。 </li><li>放置在水浴集聚四氟乙烯烧瓶在70℃，并连接到回流冷凝器。 </li><li>在磷酸执行温和回流（70℃）30小时，然后允许凉爽室温。 </li><li>通过用500毫升温去离子水稀释悬挂和使用滤纸真空过滤收集SEC的。 </li><li>转移SEC的一个3升的玻璃烧杯中进行一系列洗涤的。 </li></ol></li><li> SEC清洗 <ol><li>放置装有肝窦在通风橱中的3升玻璃烧杯中。 </li><li> 800毫升热（50℃）去离子水添加到SEC的温和搅拌10分钟。 </li><li>通过使用滤纸和真空过滤设置进行过滤悬浮液中。 </li><li>收集SEC的在干净的3升的玻璃烧杯中。 </li><li>重复步骤1.3.1。 1.3.4。五次。 </li><li>添加600毫升热（45℃）丙酮SEC的温和搅拌10分钟。 </li><li>通过过滤收集SEC的真空下和SEC的转移到一个干净3升玻璃烧杯中。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（50℃）的2M盐酸。 </li><li>重复步骤1.3.6。 1.3.7和。使用600毫升浩T（50°C）2M的氢氧化钠。 </li><li>重复步骤1.3.1和1.3.4八次。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（45℃）丙酮。 </li><li>重复步骤1.3.6。 1.3.7和。用600毫升热（45℃）乙醇。 </li><li>重复步骤1.3.6。 1.3.7和。使用800毫升热（50℃）去离子水。 </li><li>转移清洁SEC的六个大的玻璃培养皿中，并干燥在通风橱中在室温下放置24小时以除去所有的水含量。 </li><li>干燥在真空烘箱SEC的在60℃和1毫巴下真空10小时或直至恒重。 </li><li>收集干燥的SEC的并转移到聚丙烯瓶中。 </li><li>在干燥的柜子存放SEC的。 </li></ol></li></ol> 2. SEC的表征<ol><li>使用在不同阶段产生的孢子和肝窦进行扫描电子显微镜分析41。 </li><li>使用在不同阶段产生的孢子和SEC的执行元素分析41。 ðRY在60℃元素分析之前至少1小时的所有样品，并计算使用具有6.25乘法系数氮百分比的蛋白质含量。11获取使用用于每个样品一式三份测量的结果。 </li><li>使用动态图像颗粒分析仪进行粉体学性质分析。41 </li><li>使用共聚焦激光扫描显微镜扫描未处理，处理，和FITC-BSA的加载肝窦41 </li></ol> 3.生物大分子封装真空载荷技术<ol><li>准备使用去离子水，并转移到50毫升聚丙烯试管的1.2ml 125毫克/毫升牛血清白蛋白（BSA）溶液。 </li><li>重150毫克肝窦的并传送到BSA溶液。 </li><li>涡管10分钟，以形成均匀的溶液。 </li><li>封面用滤纸筒。 </li><li>在1毫巴管转移到冻干机瓶和干燥4小时真空。 </li><li>执行步骤3.1到3.5。三个独立的批次。 </li><li>收集的BSA加载肝窦并使用研钵和杵除去团块。 </li><li>传送BSA的加载肝窦至50ml管中并加2ml DI水以去除残留的BSA。 </li><li>收集离心SEC的在4704×g离心5分钟并弃上清。 </li><li>重复用DI水洗涤一次。 </li><li>覆盖含有用滤纸BSA加载肝窦管。 </li><li>转移SEC的一个冰箱（-20℃），并冷冻1小时。 </li><li>冷冻干燥SEC的在冷冻24小时，直到店进一步鉴定。 </li><li>准备用同样的步骤不含BSA安慰剂SEC的如在步骤3.1。 3.13。 </li><li>使用相同的程序，在3.1的步骤制备FITC-BSA加载肝窦。 3.13。 </li></ol> 4.测定包封率<ol><li>称量5毫克BSA-加载肝窦在2ml聚丙烯管中。 </li><li>加入1.4毫升磷德缓冲盐水pH为7.4（PBS）中，并涡旋5分钟。 </li><li>探针超声处理在40％振幅的悬浮液10秒的3个循环。 </li><li>过滤用0.45微米的聚醚砜（PES）过滤器解决方案。 </li><li>执行相同的步骤，在步骤4.1。 4.4。使用安慰剂SEC的。 </li><li>测量在使用安慰剂作为空白280nm的吸光度。 </li><li>量化使用在PBS中BSA标准曲线（200至1000微克/毫升）包封的BSA的量。42 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+better+understanding+of+pharmacological+activities+and+uses+of+phytochemicals+of+Lycopodium+clavatum:+A+review.">A better understanding of pharmacological activities and uses of phytochemicals of Lycopodium clavatum: A review.</a> J. Pharmacogn. Phytochem. 3 (1), 207-210 (2014).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+sporopollenin+exine+capsules+from+sunflower+pollen+grains.">Extraction of sporopollenin exine capsules from sunflower pollen grains.</a> RSC Adv. 6 (20), 16533-16539 (2016).</li><li>Mathias, P. M., Chen, C. C., Walters, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Polyethylene+Fractionation+Using+the+Statistical+Associating+Fluid+Theory.">Modeling Polyethylene Fractionation Using the Statistical Associating Fluid Theory.</a> , (2000).</li><li>El-Bayaa, A., Badawy, N., Gamal, A., Zidan, I., Mowafy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+wet+process+phosphoric+acid+by+decreasing+iron+and+uranium+using+white+silica+sand.">Purification of wet process phosphoric acid by decreasing iron and uranium using white silica sand.</a> J. Hazar. Mater. 190 (1), 324-329 (2011).</li><li>Carr, J., Zhang, L., Davis, M., Ravishankar, S., Flieg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+Controlling+Chemical+Additives+for+Phosphoric+Acid+Production+Plants.">Scale Controlling Chemical Additives for Phosphoric Acid Production Plants.</a> Procedia Engineering. 83, 233-242 (2014).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eco-friendly+streamlined+process+for+sporopollenin+exine+capsule+extraction.">Eco-friendly streamlined process for sporopollenin exine capsule extraction.</a> Sci. Rep. 6, 1-14 (2016).</li><li>Smith, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of Fourier transform infrared spectroscopy. , 182 (2011).</li></ol>

作者什么都没有透露。

在这项工作中，从L. SEC提取的系统分析clavatum孢子提出并此报告表明，它能够生产更高质量的胶囊剂，同时实现11的预先存在的常用协议。一个显著精简与此相反的现有协议需要高的处理温度（180℃）和一个较长的过程持续时间（7天）， 第11的当前SEC提取处理优化主要是集中于降低酸解步骤的温度和持续时间。 脱脂，碱处理提供了从最小的孢子?...

有在微胶囊化应用从植物孢子和花粉获得使用天然植物为基础的胶囊显著的兴趣。1-15在自然界，孢子和花粉反对恶劣的环境条件敏感的遗传物质提供保护。植物孢子和花粉的基本结构通常包括外壳层（外壁）中，内壳层（内壁），和内部细胞质材料。外壁是由称为孢粉化学鲁棒生物聚合物1,9,10,13,16和内壁是主要由纤维素材料16-18空胶囊可通过各种方法7,9中分离除去细胞质材料，蛋白质，和内壁层。2,12,16这些孢粉外壁胶囊（秒），由于其窄的尺寸分布和均匀的形态提供令人信服的替代合成密封剂。7,9,13,19,20的标准化的过程，以获得来自不同植物物种，如伸筋草肝窦的发展，打开了一个广泛的药物递送，食品和化妆品等领域的微胶囊的应用的潜力。6,10-13,21 为了获得肝窦，研究人员首先处理孢子和花粉与有机溶剂和碱性溶液回流以除去细胞质内容22-25然而，剩下的胶囊结构被确定为仍然含有纤维素内壁层。为了除去此，研究人员使用盐酸，热硫酸或热磷酸在若干天，22-25用磷酸成为SEC内壁去除的优选方法探讨了使用延长的酸性水解处理2然而，正在进行研究多年来已经表明，各种孢子和花粉具有不同程度的弹性恶劣处理我thods常用。26,27一些孢子和花粉是完全降解并失去在强碱性溶液中的所有结构完整性，或变得严重损坏在强酸性溶液16在SEC对治疗的反应条件的变异性是由于在化学细微变化结构和物种之间的孢粉外壁材料的外壁形态28由于孢粉外壁胶囊（秒）的鲁棒性的可变性，有必要以优化孢子和花粉的各种处理的条件。 从种属植物孢子clavatum已成为肝窦的最广泛研究的单一来源。因此建议，这主要是由于其广泛的可用性，成本低，单分散性，和化学坚固性9,29孢子可以在1分组的形式来方便地收获并含有sporoplasmic内容- 2微米细胞器和biomolecules 11 L. clavatum孢子已被用来作为一种天然粉末润滑剂，30,31一个化妆品的基础上，30和草药32-36为广泛的治疗应用。从L.获得SEC的clavatum已被证明是更有弹性，以来自其它物种的孢子和花粉。2处理之后的处理比肝窦，所得SEC的已显示出保留其错综复杂microridge结构和同时提供用于包囊的大的内部空腔高形态的均匀性。7研究表明，L。 clavatum SEC的可用于药物，10,13疫苗，11种蛋白质，7,14细胞，8油，5-7,9和食品补充剂的封装。5,15观察SEC负载效率相对较高比常规封装材料。7也有一些好处报道SEC的封装诸如掩盖味道，6,10，并提供一定程度的天然保护的抗氧化作用。12在现有的研究的能力，为L.最常用的SEC提取方法clavatum基于四个主要步骤。第一步是在50℃下在丙酮溶剂回流达12小时，脱脂孢子11第二步是在120℃到12小时以除去细胞质和蛋白质材料在6％的氢氧化钾的碱性回流11步骤三是在180℃下在85％的磷酸酸回流至7天以除去纤维素内壁材料11第四步是一个全面的洗涤过程中使用的水，溶剂，酸和碱以除去所有剩余的非外壁材料和化学残留物。 相对于封装应用SEC提取的主要目标是产生胶囊它们是空的细胞质材料制成的，自由往复中号潜在变应原性蛋白质和形态完好。2.37然而，从工业制造的角度来看，它也是重要的考虑附加的经济和环境因素，比如，能源效率，生产持续时间，安全性，和产生的废物。至于能量效率，兼具高的温度和长的处理时间会影响生产成本以及环境的影响。生产时间和周转时间是影响加工盈利的关键因素。特别值得关注的是，高温磷酸处理提高了安全性问题，并已知会导致腐蚀性缩放这导致在基础设施维护显著增加和延迟在批量周转时间。38-40在可能的情况，尽量减少可能会导致所需的步数到显著减少所产生的废物。然而，常用的L的四个步骤clavatum SEC提取具有简单的EV从几十年的研究和olved已很少实际过程的优化。近日，穆恩达尔吉等人 ，41通过系统地评估和优化的最常见的SEC提取技术之一提出在这一领域正在进行的工作显著的贡献。 在这项研究中的第一部分:孢子脱脂论证利用丙酮处理在50℃下6小时; sporoplasm和内壁去除过程证明利用85％的磷酸处理，在70℃，30小时;与水，溶剂，酸和碱充分洗涤用于演示的去除残留sporoplasmic内容;和SEC干燥证明利用对流干燥和真空烘箱干燥。在第三部分中，SEC真空装载证明利用模型蛋白的真空加载，牛血清白蛋白（BSA），其次是BSA-加载-SEC洗涤和冷冻干燥。在第四部分中，determin的BSA包封率通货膨胀论证利用离心，超声探头和紫外/可见光光谱。

<table border="0" cellpadding="0" cellspacing="0" width="894">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Lycopodium clavatum spores (s-type)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">19108-500G-F</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin</td>
			<td style="width:235px;">Sigma</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FITC-conjugated BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">A9771-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Phosphoric acid (85 % w/v)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">438081-2.5L&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hydrochloric acid</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800202&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sodium hydroxide</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">S5881-1KG&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ethanol</td>
			<td style="width:235px;">AcME&#160;</td>
			<td style="width:171px;">C000356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Deionized water</td>
			<td style="width:235px;">Millipore purified water&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:319px;">Qualitative filter paper (grade No. 1, cotton cellulose)</td>
			<td style="width:235px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Polystyrene microspheres (50 &#177; 1 &#181;m)&#160;</td>
			<td style="width:235px;">Thermoscientific (CA, USA)</td>
			<td style="width:171px;">4250A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Vectashield&#160;</td>
			<td style="width:235px;">Vector labs (CA, USA)</td>
			<td style="width:171px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:319px;">Sticky-slides, D 263 M Schott glass, No.1.5H (170 &#956;m, 25 mm x 75 mm) unsterile glass slide</td>
			<td style="width:235px;">Ibidi GmbH (Munich, Germany)</td>
			<td style="width:171px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Commercial Lycopodium SECs (L-type)</td>
			<td style="width:235px;">Polysciences, Inc. (PA, USA)</td>
			<td style="width:171px;">16867-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating plates</td>
			<td style="width:235px;">IKA, Germany</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Scanning electron microscope</td>
			<td style="width:235px;">Jeol, Japan</td>
			<td style="width:171px;">&#160;JFC-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Elemental analyzer&#160;</td>
			<td style="width:235px;">Elementar, Germany</td>
			<td style="width:171px;">VarioEL III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FlowCam: The benchtop system</td>
			<td style="width:235px;">Fluid Imaging Technologies, USA</td>
			<td>FlowCamVS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Confocal laser scanning microscope</td>
			<td style="width:235px;">Carl Zeiss, Germany</td>
			<td style="width:171px;">LSM710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Freeze dryer</td>
			<td>Labconco, USA&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UV Spectrometer</td>
			<td>Boeco, Germany</td>
			<td style="width:171px;">S220</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of plant-based capsules for microencapsulation applications

从植物为基础的孢子或衍生的微胶囊提供一个多元化的微胶囊化应用提供了强大的平台。当孢子或花粉进行处理，以除去内部sporoplasmic内容孢粉外壁胶囊（秒）获得。得到的中空微胶囊表现出高度的粉体学均匀性并保留相关的特定植物物种错综复杂显微结构特征。在此，我们展示了从伸筋草孢子生产SEC的和为亲水性化合物进入这些SEC的加载一个简化的流程。当前SEC分离过程最近已经优化以显著减少其常规SEC中单独使用的处理的要求，并确保生产完整微囊。自然L. clavatum孢子脱脂用丙酮，用磷酸处理过的，并充分洗涤以除去sporoplasmiC含量。丙酮脱脂后，用85％的磷酸一单个处理步骤已被证明以除去所有sporoplasmic内容。通过限制酸处理时间为30小时，这是可以分离干净肝窦和避免SEC压裂，这已被证明与延长处理时间发生。用清水充分冲洗，稀酸，稀碱和溶剂确保所有sporoplasmic物质和化学残留物被充分去除。真空加载技术被利用来加载一个模型蛋白（牛血清白蛋白），为代表的亲水性化合物。真空装载提供了一种简单的技术来装载的各种化合物，而不需要这往往需要在其他微囊协议苛刻的溶剂或不希望的化学品。根据这些隔离和装载协议，SEC的提供一种在各种不同的微胶囊的应用，如，治疗剂，食品，化妆品使用一个有前途的材料，和个人护理刺UCTS。

对于孢粉外壁胶囊简化提取工艺 该L. clavatum SEC提取由三个主要步骤实现:（1）用丙酮脱脂; （2）酸解使用磷酸85％（体积/体积）;并使用（3）广泛SEC洗涤溶剂。流线型SEC提取处理的流程在图1中提示的-我简言之，该方法涉及在50℃下用丙酮回流一个脱脂步骤6小时，然后将脱脂孢子在?...

Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

Extraction of Plant-based Capsules for Microencapsulation Applications

这个协议/手稿描述了生产由伸筋草芽孢孢粉外壁胶囊（秒）的和为亲水性化合物为这些肝窦的装载一个简化的流程。

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

extraction-of-plant-based-capsules-for-microencapsulation-applications

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.9K 次观看.  Nanyang Technological University. 这个协议/手稿描述了生产由伸筋草芽孢孢粉外壁胶囊（秒）的和为亲水性化合物为这些肝窦的装载一个简化的流程。 

视频: Extraction of Plant-based Capsules for Microencapsulation Applications

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

科研

生物工程

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

宽带超声医疗成像应用，光学检测仪

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

就地 液体生物组件的 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

仿生纳米羟基磷灰石的快速混合制备生物医学应用

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

基于植物的提取胶囊微胶囊化应用

摘要

探索更多视频

此视频中的章节

制备纤维蛋白三维支架干细胞培养应用

宽带超声医疗成像应用，光学检测仪

设计丝绸蚕丝蛋白合金生物医用材料

就地液体生物组件的 TEM

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

制造超疏水高分子材料生物医学应用

加工机械可调和生物活性金属支架的生物医学应用

用于细胞培养应用光介导的形成和构图的水凝胶

仿生纳米羟基磷灰石的快速混合制备生物医学应用

组织工程应用中植物组织去细胞的两种方法

基于植物的提取胶囊微胶囊化应用

摘要

探索更多视频

此视频中的章节

制备纤维蛋白三维支架干细胞培养应用

宽带超声医疗成像应用，光学检测仪

设计丝绸蚕丝蛋白合金生物医用材料

就地 液体生物组件的 TEM

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

制造超疏水高分子材料生物医学应用

加工机械可调和生物活性金属支架的生物医学应用

用于细胞培养应用光介导的形成和构图的水凝胶

仿生纳米羟基磷灰石的快速混合制备生物医学应用

组织工程应用中植物组织去细胞的两种方法

就地液体生物组件的 TEM