Name: synthesis of thermogelling poly(n-isopropylacrylamide)-graft-chondroitin sulfate composites with alginate microparticles for tissue engineering
Uploaded: 2016-10-26T12:30:00
Description: Watch this Scientific Journal Video about Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering at JoVE.com

笔者想衷心感谢詹妮弗Kadlowec博士的协助下在胶粘剂拉伸测试的协议的发展。 研究本出版物中报告是由关节肌肉骨骼及皮肤疾病研究所和生物医学成像和美国国立卫生研究院生物工程的下奖号1R15 AR 063920-01研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1.聚（N-异丙基丙烯酰胺）-g-硫酸软骨素合成<ol><li>之前的生物粘附凝胶的合成，纯化N-异丙基（的NIPAAm）单体和甲基丙烯酸酯硫酸软骨素（CS）。 <ol><li>称出至少10g的NIPAAm和在60℃下溶解在400ml正己烷的单体。定期搅动容器，直到完全溶解。再结晶在-20℃冷冻该溶液24小时。 </li><li>从容器和真空过滤除去结晶单体使用布氏漏斗正己烷。置于在RT真空烘箱单体24小时以除去任何剩余的正己烷。储存在-...

有在合成水凝胶微粒复合材料和评价其粘合强度，溶胀能力和细胞生物相容性几个关键步骤。 PNIPAAm的接枝的CS的自由基聚合需要硫酸软骨素成功methacrylation，单体组分完全溶解，无氧的反应条件。被选择的NIPAAm单体甲基丙烯酸酯化的硫酸软骨素在反应混合物中的比率，因为它已被证明，在我们以前的工作，以产生具有机械性能类似于天然椎间盘组织29共聚物。在水凝胶增加硫酸软骨素的量...

可注射生物材料是那些可方便地输送到身体中作为液体和原位固化。这样的材料已经在再生医学，在那里它们被用来封装细胞递送到受影响的部位1-4，并作为对电池单元5的三维临时的细胞外基质广泛应用。对于患者，因为用于植入的外科手术是微创和固相可以填充不规则形状的组织缺损，消除了对自定义尺寸的植入物的需要注射的生物材料是有利的。 注射能力可以通过各种机制来实现。外部因素，如pH值，已经研究作为用于封装细胞和生物活性分子6-8凝胶的形成的触发器。然而，pH值可能不是最方便的触发器中使用所有生理环境。另一个传统爱特纳略去对于实现可注射使用原位化学聚合或交联。 A组开发过硫酸铵和N，N，N&#39;，N“四甲基乙二胺组成的水溶性氧化还原体系，并用它进行反应的聚乙二醇和聚（丙烯）二醇9,10-组成的大分子单体。咱等人 11发达注射壳聚糖聚乙烯醇网络交联用戊二醛。在这样的系统中，反应性组分的细胞毒性，必须考虑，尤其是对涉及细胞封装的应用程序。另外，放热聚合可以产生足够高的温度妥协周围组织，这已被报道用于聚合的骨水泥12,13。 仍然其它可注射的聚合物系统已经开发显示出从液体到固体的状态的变化与温度作为触发。被称为热胶系统，这些都是aqueo我们不要求化学刺激，单体或交联剂原位形成14来实现聚合物溶液。更确切地说，通常接近生理温度中发生的相变诱导物理交联的三维网络的形成。泊洛沙姆例如Pluronic F127是热胶化药物递送15-17和电池封装18,19研究最广泛的聚合物之一。然而，可以很好地接受，这些凝胶缺乏在生理条件下的稳定性。研究使用的扩链剂20或化学交联剂21,22展示更高的稳定性。然而，使用这些试剂的可能会限制电池封装的材料的潜力。 聚（N-异丙基丙烯酰胺）是已经接收显著关注组织工程和药物输送14的合成热胶凝聚合物。聚的水溶液（N- isoprop基丙烯酰胺）（PNIPAAm的）表现出较低的临界溶液温度（LCST），通常发生约32 - 34°C 23,24。下面的LCST，水保湿PNIPAAm的链。高于转变温度时，聚合物变得疏水，从而导致剧烈的相分离25-27和地层的固体凝胶，无需使用有毒的单体或交联剂。然而，PNIPAAm的均聚物表现出较差的弹性性能，并在生理温度下，由于疏水28容纳少量的水。在这项工作中，我们选择共价结合的硫酸软骨素入PNIPAAm的网络，该网络提供了酶促降解29，抗炎活性30,31，并增加水和营养的吸收32的潜力。通过在甲基丙烯酸酯官能化的CS的存在下，单体的NIPAAm聚合形成接枝共聚物（PNIPAAm的-G-CS）在我们实验室中制备的CS PNIPAAm的共聚物。 BEC该共聚物的低交联密度的澳洲英语，PNIPAAm的接枝CS形成在RT在水中的粘性溶液中，在生理温度下的弹性凝胶由于LCST 29。聚合物溶液由于过渡的可逆性成为再次冷却时低于LCST悬浮剂。 我们已经证明，PNIPAAm的接枝的CS具有以用作组织工程支架，由于机械特性可定制，降解，和人类胚胎肾（HEK）细胞相容293细胞29的潜力。然而，在某些承载区域，如椎间盘，组织工程支架应该具有形成与周围椎间盘组织大幅接口消除错位33的风险的能力。这个接口也是必需的力在整个植入物和组织33之间的界面处的适当的传输。在我们的工作中，我们已经暂停了一在的PNIPAAm-G-CS的水溶液lginate微粒，发现凝胶化本地化的微粒，其提供与周围组织34的粘合性。在本文中，我们列出的步骤准备的热胶，粘性聚合物。生物材料表征，细胞成像的生存力的标准技术，并测定法适于考虑到该聚合物的温度灵敏度和相转变的可逆性。在本文中所述的可注射的聚合物具有那些本文中所描述的以外的用于药物递送和组织工程应用宽的电位。此外，这里所描述的表征方法可以适用于其它的热胶凝系统。

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">N-isopropylacrylamide, 99%, pure, stabilized</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">2210-25-5</td>
			<td style="width:347px;">Refrigerate and remove stabilier with hexane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Chondroitin sulfate A sodium salt (from bovine trachea)</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">39455-18-0</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hexanes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">H302-4</td>
			<td style="width:347px;">Store in a flammable cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">50% (w/w) sodium hydroxide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">SS254-1</td>
			<td style="width:347px;">Caustic in nature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methacrylic anhydride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">276685</td>
			<td style="width:347px;">Strong fumes; use in a fume hood</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Acetone</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A18-4</td>
			<td style="width:347px;">Chill in a refrigerator prior to use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Nitrogen Gas</td>
			<td style="width:164px;">Praxair</td>
			<td style="width:120px;">7727-37-9</td>
			<td style="width:347px;">Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tetramethylethylenediamine, 99% extra pure</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">110-18-9</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Ammonium persulfate</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">A3678</td>
			<td style="width:347px;">Hygroscopic and degrades in the presence of water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Phosphate buffered saline tablets</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">BP2944</td>
			<td style="width:347px;">Keep dry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Alginic acid, sodium salt</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">177775000</td>
			<td style="width:347px;">Use heat to aid in dissolving</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Calcium chloride dihydrate</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">C79</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Canola oil</td>
			<td style="width:164px;">Local store</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local store</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tween 20</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">93773</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">70% (v/v) Isopropoanol</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A416-4</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcine ears</td>
			<td style="width:164px;">Haine&#39;s Pork Shop</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local butcher</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Sodium Chloride</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">S271-3</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Human embryonic kidney 293 cells</td>
			<td style="width:164px;">ATCC</td>
			<td style="width:120px;">ATCC CRL-1573</td>
			<td style="width:347px;">Store in liquid nitrogen for long-term use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">DMEM: 1X, high glucose, no pyruvate</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">11965126</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Fetal bovine serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">10082-147</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Penn Strep: 10,000 U/ml</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15140-122</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Trypsin-EDTA: 0.5%, 10X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15400-054</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methanol</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">AAA44571-K7</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Live/Dead Cell viability kit</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">L3224</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">XTT cell viability kit</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">TOX2-1KT</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Clear DMEM: 1X, high glucose, no phenol</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">21063-029</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Dulbecco&#39;s PBS: 1X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">14190136</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Sodium citrate</td>
			<td style="width:164px;">EMD</td>
			<td style="width:120px;">SX0445-1</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Positive displacement pipette</td>
			<td style="width:164px;">BrandTech Scientific, INC</td>
			<td style="width:120px;">2702904</td>
			<td style="width:347px;">Dispenses 100 - 500 &#181;L and comes with attachable tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">No 3. Stainless Steel scalpel handle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">S2896</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Miltex sterile surgical blades</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">12-460-440</td>
			<td style="width:347px;">Size 10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Power gem homogenizer</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">08-451-660</td>
			<td style="width:347px;">Model # 125</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcelain mortar and pestle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">Z247464</td>
			<td style="width:347px;">Holds 50 mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">FreeZone 1 L benchtop freeze dry system</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;">7740020</td>
			<td style="width:347px;">Freeze samples prior to use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Oil sealed rotary vane pump</td>
			<td style="width:164px;">Edwards</td>
			<td style="width:120px;">A65301906</td>
			<td style="width:347px;">Model # RV5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Incubating orbital shaker</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">12620-946</td>
			<td style="width:347px;">Model # 980153</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Benchtop refrigerated centrifuge</td>
			<td style="width:164px;">Forma Scientific, INC</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 5682</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated ovens</td>
			<td style="width:164px;">VWR</td>
			<td></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">2 N force gauge</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGV-0.5XY</td>
			<td style="width:347px;">Model # FGV-0.5XY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">E-force test stand</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGS-200PV</td>
			<td style="width:347px;">Model # FGS-200PV</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture swinging bucket centrifuge</td>
			<td style="width:164px;">Beckman Coulter</td>
			<td style="width:120px;">366830</td>
			<td style="width:347px;">Model #6S-6KR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture microcentrifuge</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model #5415C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hemacytometer set</td>
			<td style="width:164px;">Hausser Scientific</td>
			<td style="width:120px;">3720</td>
			<td style="width:347px;">Requires replacement cover glass slips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Slide warmer</td>
			<td style="width:164px;">Lab Scientific</td>
			<td style="width:120px;">XH-2022</td>
			<td style="width:347px;">Model # XH-2002</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Portable heating lamp</td>
			<td style="width:164px;">Underwriters Laboratories</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Helps to maintain polymer temperature at 37&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Inverted fluorescent microscope</td>
			<td style="width:164px;">Zeiss</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model Axiovert 25 CFL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated water bath</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Rocking platform</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Series 100</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Multiskan FC microtiter plate reader</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Type 357</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Cell culture incubator</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 2350T</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Purifier class II biosafety cabinet</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Delta Series</td>
		</tr>
	</tbody>
</table>

synthesis of thermogelling poly(n-isopropylacrylamide)-graft-chondroitin sulfate composites with alginate microparticles for tissue engineering

可注射生物材料被定义为可被引入到体内的液体，并且在原位固化可植入材料。这种材料提供的植入微创侵入性，容易形成不规则形状的缺陷的空间填充物的临床优势。注射生物材料已被广泛研究作为组织工程支架。然而，对于某些承重区在体内的修复，如椎间盘，支架应该具有粘合性能。这将运动期间减少位错的风险，并确保与周围组织紧密接触，提供力的充分传输。在这里，我们描述了一个热敏感聚（N-异丙基）组成的支架的制备和表征接枝 - 硫酸软骨素（PNIPAAM-G-CS）和海藻酸钠微粒。所述的PNIPAAm-G-CS共聚物形成在RT在水中的粘性溶液，在其中alginatË颗粒悬浮以提高附着力。上面的低临界溶液温度（LCST），在30℃左右，该共聚物形成围绕微粒的固体凝胶。我们已经适应标准的生物材料的表征程序考虑到的PNIPAAm-G-CS的可逆相变。结果表明，50％或75毫克/毫升藻酸盐微粒中掺入5％（重量/体积）的PNIPAAm-G-CS溶液四倍的PNIPAAm-GCS的粘合抗拉强度单独（P &lt;0.05）。藻酸盐微粒的掺入也显著增加的PNIPAAm-G-CS的溶胀容量（P &lt;0.05），有助于保持组织缺损内的空间填充凝胶。最后， 体外毒理学测定试剂盒，2,3-双-的结果（2-甲氧基-4-硝基-5-磺苯基）-2H-四唑-5-羧酰（XTT）和活/死活力测定表明，该胶粘剂能够支持的生存和封装的人类胚胎肾细胞增殖（HEK）293ç厄尔超过5天。

一种热响应接枝共聚物已成功合成和表征其生物粘附强度，溶胀性能，并在体外细胞相容性。我们选择调查的褐藻胶因拥有完善粘膜粘附性。藻酸盐微粒，具有59.7±14.9微米的平均直径，用5％的共混（重量/体积）的PNIPAAm-G-CS在25，50，和75毫克/毫升的浓度。这些浓度是基于二分之一，等于，和在水溶液中的PNIPAAm-G-CS的等效干重的两倍。微粒浓度超过75毫克/毫升展出粘...

Watch this Scientific Journal Video about Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering at JoVE.com

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素组成的可注射的组织工程支架，制备（PNIPAAm的接枝CS）含藻酸盐微粒。粘合强度，溶胀性能和体外生物相容性在这项研究中进行分析。这里开发的表征技术可以适用于其他的热胶凝系统。

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

The overall goal of this methodology is to assess both the adhesive properties and cellular biocompatibility of the proposed hydrogel composite for intervertebral disc tissue engineering of the nucleus pulposus. This method can help answer key questions in the field of tissue engineering, like how to design and prepare thermal reversible hydrogels with bioadhesive properties for cell encapsulation. The main advantage of this technique is that we are able to demonstrate in-vitro studies with an injectable, thermally-sensitive composite to evaluate its use as a potential replacement for the nucleus pulposus.In this current study, we evaluate a novel hydrogel scaffold by performing both tensile mechanical tests, and cellular viability assays. Demonstrating the biocompatibility assays will be Emily Schmidt, Mark Dittmer, Edward Goldschmidt, and Frantzeska Giginis, students from the departments of Biological Sciences and Biochemistry. Prior to the synthesis of the bioadhesive hydrogel, purify NIPAAm monomer and mCS, as described in the text protocol.Then, co-dissolve 10 grams of purified NIPAAm and 2.209 grams of mCS in 232 milliliters of deionized water. Purge the solution of oxygen by using inert nitrogen gas. Maintain a vigorous, yet low gas flow rate, to prevent the solution from bubbling over for 15 minutes.Continue purging the solution with nitrogen gas and add 0.976 milliliters of TEMED. Next, initiate the polymerization reaction by mixing 97.6 milligrams of ammonium persulfate. Mix the solution for approximately 20 seconds, and quickly seal the solution to prevent any air from entering the vessel.Allow the solution to polymerize under fluorescent light for 24 hours before processing the sample into a fine powder, as described in the text protocol. Create an oil and water mixture by combining 100 milliliters of canola oil, one milliliters TWEEN 20, and 20 milliliters of 2%of alginate. Emulsify the mixture for 10 minutes with a homogenizer at 15, 000 RPM.Additionally, mix with a magnetic stir bar at 200 RPM to create large microparticles, or 2, 000 RPM to create small microparticles. Aspirate the 2%calcium chloride solution using a syringe with an 18-gauge needle tip. Slowly add the calcium chloride drop-wise into the emulsion.After allowing the microparticles to cross-link for 10 minutes, evenly distribute the batch of microparticles into capped 50 milliliter conical tubes. Centrifuge at 1, 400 times G for two minutes to remove the initial layer of canola oil before washing and lyophilizing the microparticles as described in the text protocol. Use the resulting freeze-dried polymer powder to create a 5%weight volume PNIPPAm graft CS solution by first dissolving 50 milligrams of hydrogel powder in one milliliter of 1X phosphate buffered saline, or PBS.Mix the solution using a vortexer before chilling in a refrigerator at four degrees Celsius for 24 hours. Once the viscous solution forms, create a homogenous composite by adding 25 or 50 milligrams of freeze-dried alginate microparticles to the hydrogel solution and vortexing. Store the composite in a refrigerator at four degrees Celsius for later use.Prior to performing tensile tests, extract cartilage substrate from a porcine ear, as detailed in the text protocol. Then, attach the force gauge to the arm of the test stand. Place a hotplate on top of the force stand, and set the temperature to 50 degrees Celsius.Then, affix a one centimeter by one centimeter piece of cartilage to the stainless steel block with cyanoacrylate glue and a pair of tweezers. Glue an additional piece of cartilage to the Delrin cylinder, and attach the cylinder to the force gauge. It is very important that both cartilage surfaces face each other and contact the hydrogel.Place the plexiglass water bath on top of the hotplate, and insert the stainless steel block with the cartilage substrate inside the bath. Lower the arm of the test stand, and align both cartilage substrates with one another. Then, raise the arm, leaving enough room to apply the hydrogel.With a positive displacement pipette, dispense 200 microliters of the bioadhesive hydrogel to the bottom piece of cartilage. Lower the arm of the force stand at one milliliter per minute until the hydrogel contacts the upper piece of cartilage, and exerts a preload force of 0.001 newtons. Pour the 37-degrees Celsius saline solution into the bath until the volume reaches the designated water level.Check the temperature of the solution with a thermocouple. After allowing the hydrogel to set for a total of five minutes, raise the arm at a speed of two millimeters per minute. The test is completed once the bioadhesive detaches from the cartilage substrate, or when a significant drop in force occurs, signaling failure.Collect the recorded data, and raise the arm of the test stand. Remove the Delrin cylinder from the force gauge. Pour the saline solution into its previous container, and reheat to 37 degrees Celsius.Calculate the stresses, as described in the text protocol. Before performing the Live/Dead assay with PNIPAAm graft CS, prepare human embryonic kidney, or HEK 293 cells, as detailed in the text protocol. Create positive control monolayers by pipetting multiple 300-microliter volumes of the cell mixture into a 24-well plate to generate four to six replicate samples.Maintain the same cell concentration by re-suspending the cells in approximately two milliliters of PNIPAAm graft CS for each replicate sample. Pipette the polymer cell mixture up and down using a serological pipette until homogenous. If seeding with alginate microparticles, transfer two milliliters of the PNIPAAm graft CS cell suspension into a 35-millimeter culture dish, and introduce 200 milligrams of particles into the mixture.Pipette the cell mixture up and down using a serological pipette until homogenous. Then, with a serological pipette, dispense multiple 300-microliters volumes of the polymer cell mixture into each of the wells for killed control, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles to generate four to six replicate samples. Incubate the plate at 37 degrees Celsius for 10 to 15 minutes, and allow the PNIPAAm graft CS to transition from a liquid to a gel.The polymer forms an opaque white disc. Position a slide warmer in the hood, and set the temperature to 37 degrees Celsius. Use the slide warmer to maintain well plate temperature, and pipette 600 microliters of pre-warmed media into each well.To further prevent the transitioning of the polymer to a liquid state, position a lamp with a fluorescent bulb above the dish to warm up the air above it. Incubate the cells for five days at 37 degrees Celsius with 5%carbon dioxide. After five days have elapsed, remove the media from the wells of the monolayer, PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles.Wash all of the wells thoroughly, but gently, with PBS, and then, remove the PBS. Allow the hydrogel discs containing alginate microparticles to liquefy at room temperature, and add two milliliters of 50 millimolar sodium citrate to each of the wells. Remove the media from the killed cell wells.Allow the hydrogel discs to liquefy at room temperature, and add 300 microliters of 70%methanol to each well before incubating the plate for 45 minutes at 37 degrees Celsius. Remove the sodium citrate and methanol solutions from the wells, and pipette each of the hydrogel suspensions into individual microcentrifuge tubes. Centrifuge the tubes at 2, 000 times G for 10 minutes to separate the cells from the hydrogel suspension.Carefully remove the suspension by pipetting the solution from the conical tube, and leaving behind the cell pellet. Re-suspend the pellet in 300 microliters of PBS, and transfer to the original well plate. Next, add 300 microliters of the Live/Dead dye mix to each of the wells.Wrap the well plate in aluminum foil, and incubate for 45 minutes on a rocker at room temperature before imaging all of the samples under an inverted fluorescence microscope with a 10X objective. Representative tensile strength results for both PNIPAAm graft CS, and PNIPAAm graft CS with varying alginate microparticle formulations are shown here. The tensile strength of PNIPAAm graft CS quadrupled with the addition of 50 or 75 milligrams per milliliter of alginate microparticles.As expected, PNIPAAm graft CS contracts and shrinks in volume above its lower critical solution temperature. Introducing microparticles within the hydrogel imparts a swelling effect over a seven-day period in PBS. HEK 293 cells encapsulated in both PNIPAAm graft CS, and PNIPAAm graft CS with alginate microparticles showed active cellular fluorescence using Live/Dead dye after a period of five days.These results can be compared to the positive and negative control wells. In parallel, a quantitative assay was used to confirm the Live/Dead imaging, which showed no significant differences in cellular viability between PNIPAAm graft CS, PNIPAAm graft CS with alginate microparticles, and the control monolayer. A hydrogel composite consisting of thermally-sensitive poly(N-isopropylacrylamide)grafted with chondroitin sulfate and alginate microparticles was created for nucleus pulposus replacement.While attempting this procedure, it's important to remember that the composite is temperature-sensitive, and will liquefy below its lower critical solution temperature. Once mastered, tensile mechanical tests can be performed with ease as long as careful precision is practiced. Swelling studies reveal a hydrogel's ability to retain water.However, these tests must mimic the native in-vivo environment. Cellular viability of the thermally-sensitive gels can be confirmed and assessed by a combination of fluorescence-marking and metabolic-producing reagents. After watching this video, you should have a good understanding of how thermally-sensitive injectables are applied in intervertebral discs, tissue engineering, and how composites may enhance certain material properties.

Research

JoVE Journal

Bioengineering

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

心肌组织工程生物衍生的注射材料的制备

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科研

生物工程

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静电纤维组织工程的后期处理

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一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

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温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

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