作者感谢得克萨斯州A与M大学工程试验站（TEES）对这项研究的财政支持。林赛指甲非常感谢来自得克萨斯州A与M大学路易斯·斯托克斯联盟少数人参与（LSAMP）和美国国家科学基金会（NSF）研究生研究奖学金计划（GRFP）的支持。张大伟感谢得克萨斯州A与M大学论文奖学金。

1.合成PCL-DA大分子单体<ol><li>运行丙烯酸酯化反应。 <ol><li>重20克的PCL二醇（Mn为=〜10,000g / mol的）的成250ml圆底烧瓶装配有聚四氟乙烯覆盖的磁力搅拌棒。 </li><li>化解PCL二醇在DCM。 <ol><li>添加120 mLlof DCM至烧瓶（浓度= 0.17克/毫升）。 </li><li>放置一个橡胶隔膜松散到烧瓶的颈部，以避免压力积聚，同时也防止DCM中蒸发。 </li><li>在〜250rpm下为约30分钟搅动溶液以完全溶解聚合物。 </li></ol></li><li>添加〜6.6​​毫克的4-二甲基氨基吡啶（DMAP）到溶液中，溶解并搅拌。 </li><li>放置一个橡胶隔膜牢固地插入烧瓶颈部。让溶液继续搅拌。 </li><li>通过橡胶隔膜，轻轻用正的 N 2压力针入口和一开放针吹扫烧瓶用N 2〜3分钟作为出口。 </li><li>取出N 2入口和出口。 </li><li>通过配备有穿过橡胶隔片插入一针的玻璃注射器添加0.56毫升（4.0毫摩尔）三乙胺（的Et 3 N）的溶液。 </li><li>通过配备有穿过橡胶隔片插入一针的玻璃注射器添加0.65 ml的丙烯酰氯滴加（8.0毫摩尔）。 </li><li>返回的 N 2入口到烧瓶中，并允许内容物在正N 2压力搅拌〜30分钟。 </li><li>预加热的油浴至55ºC。 </li><li>所分配的〜30分钟后，除去N 2入口和有冷凝器替换隔膜。 </li><li>淹没烧瓶到预加热的油浴中。 </li><li>允许烧瓶的内容物搅拌20小时。 </li><li>所分配的20小时后，除去从油浴中的烧瓶中，并允许内容​​物冷却至室温。 </li><li>使用旋转蒸发器，从烧瓶中除去DCM溶剂。 </li></ol></li><li> Purify粗PCL-DA产品。 <ol><li>向该烧瓶中，添加〜135毫升乙酸乙酯和溶解粗的PCL-DA。 </li><li>重力过滤通过滤纸将溶液倒入干净250ml圆底烧瓶中。 （注：溶液可增稠在滤纸上，不容易穿过如果是这样，仔细地施加温和热用加热枪。） </li><li>使用旋转蒸发器，除去乙酸乙酯从烧瓶溶剂。 </li><li>向该烧瓶中，添加140〜毫升DCM并溶解粗的PCL-DA。 </li><li>传送内容至500ml分液漏斗中。 </li><li>到漏斗中，添加13.5毫升2M碳酸钾（K 2 CO 3）。 </li><li>盖上漏斗。轻轻倒置漏斗和打旋轻轻一次或两次，照顾经由旋塞以释放压力混合两层。重复3次。 </li><li>替换用一层石蜡膜的盖，并允许该混合物分离O / N（〜12小时）。 </li><li>收集底部或加尼奇层放入250毫升锥形瓶中。 </li><li>添加〜5克无水硫酸镁（ 硫酸镁 ）到烧瓶中，轻轻地旋转。 </li><li>重力过滤通过定性滤纸和到一个干净的250ml圆底烧瓶中的混合物。 </li><li>使用旋转蒸发器，从烧瓶中除去DCM溶剂。 </li><li>在高真空下干​​燥，以除去残留的DCM。 （注：PCL-DA应避光保存。） </li><li>确认丙烯酸化用 1 H-NMR 22,23。 </li></ol></li></ol> 2.准备SMP脚手架（图1） <ol><li>制备熔盐模板。 <ol><li>使用425微米的筛子，直径获得的氯化钠（盐）颗粒〜460±70微米。 （注：平均粒径可以由扫描电子显微镜[SEM]图片包含的ImageJ软件确认。）14 </li><li>向3毫升的玻璃小瓶（ID = 12.9Hz MM），添加1.8克本预先过筛的NaCl。 </li><li>慢慢添加，分四份，7.5重量％（基于重量盐）去离子水（0.146克）到小瓶中。混合通过加入水每个部分的后一个金属刮刀。 </li><li>帽小瓶中，包裹在组织和放置垂直插入一个离心管中。离心15分钟，在3220×g下。 </li><li>取下盖子，让空气干燥O / N（〜12小时）。 </li></ol></li><li>在一个新的玻璃小瓶，准备了“单体溶液”相结合，0.15克每毫升DCM的PCL-DA的。 （注意：对于一种脚手架，〜1毫升溶液应制备）盖上盖子并混合溶液在高速在涡旋混合器上为约1分钟。 </li><li>在一个新的3毫升玻璃小瓶中，准备基于10重量％的2,2-二甲氧基-2-苯基苯乙酮（DMP）中的1-乙烯基-2-吡咯烷酮（NVP）一个“光引发剂溶液”。结合0.115克DMP在1ml的NVP。 （注：其一支架，〜180微升是必需的。）帽，包装在铝箔（阻挡光）和混合解决方案，在高速上的旋涡混合器对于〜1分钟。 （注意：如果在先进制备，该溶液应冷藏并避光。） </li><li>包裹含有大分子单体溶液（不包括帽）用铝箔小瓶（方框光），并通过移液管添加15体积％（基于大分子单体溶液的总体积）的光引发剂的溶液。盖上盖子并混合溶液在高速在涡旋混合器上为约1分钟。 </li><li>用铝箔包装含有熔融盐模板（不包括帽）小瓶（方框光），并通过移液管添加预先制备的大分子单体/引发剂溶液（〜0.6 ml或直到模板被完全覆盖）。 </li><li>帽小瓶中，包裹在组织和放置垂直插入一个离心管中。离心10分钟，在1260×g离心来分发整个模板的大分子单体溶液。 </li><li>取下铝箔，开盖小瓶并暴露于UV光（365纳米，25瓦）下3分钟。风干O / N。 </li><li>删除“含盐的scaffol从具有得分和压裂玻璃瓶后上方镊子小瓶D“。 </li><li>在400毫升烧杯中，准备约200毫升水/乙醇溶剂（1:1体积：体积）。 </li><li>保持浸没在水/乙醇溶剂4天每日溶剂改变该支架。 </li><li>取下溶剂，空气干燥O / N的支架。 </li></ol> 3.应用聚多巴胺涂层以SMP脚手架（图1） <ol><li>在400毫升烧杯中装配有聚四氟乙烯覆盖搅拌棒，准备约200毫升的盐酸多巴胺溶液（在10mM Tris缓冲液2毫克/毫升，pH值= 8.5，25℃）。在搅拌〜150转。 </li><li>一半的距离通过该支架到所述支架，〜;放置一个一次性针（表= 20长度= 40毫米）。环绕所述针毂的线材。 </li><li>由锚定线到烧杯的边缘淹没该支架（与溶液表面上方的针毂）加入搅拌中的多巴胺溶液。 </li><li>通过将注射器插入针毂，并用它拉空气从支架的脱气脚手架。 （注：除气完成时，没有更多的空气可以被除去，该溶液已完全渗入该支架。） </li><li>保持浸没在16小时的搅拌多巴胺溶液中的脚手架。 </li><li>从溶液中除去所述支架和取出针。用DI水和干燥，在真空烘箱中在室温放置24小时冲洗。 </li><li>放置在一个85ºC烘箱该支架1小时。 </li><li>让脚手架冷却至室温。最终的圆柱形支架会〜6毫米直径×约5毫米的高度。 </li></ol> 4.评估“自我装配”行为<ol><li>制作“不规则CMF缺陷模型”使用硬质塑料，其厚度为约5毫米的片材。使用钻头来创建塑料片内的空隙，平均直径大于〜6毫米略少，这表现在图2A中。 </li><li>在AB埃克，热DI水（相当于本临床使用生理盐水）至〜60℃的高温。 </li><li>放置支架到烧杯〜60ºC水。使用镊子推水面下的脚手架，露出所有区域到水中。继续为〜2分钟或直到支架是明显软化（ 图2B）。 </li><li>取下烧杯中的脚手架，并立即按下（手动）到模型的缺陷。 </li><li>允许冷却至室温（〜5-10分钟）（图2C）。 </li><li>从缺陷除去以观察新的，固定的临时形状和相对更刚性的状态（图2D）的返回。 </li></ol> 5.测试形状记忆行为<ol><li>使用动态机械分析仪（DMA;例如TA仪器Q800如本文所用），运行在支架一个应变控制环状热机械压缩测试超过两个周期（N），来确定形状固定性（R f）和形状恢复（R R）（ 图3）。 <ol><li>平衡至60℃（T 高 ）5分钟。 </li><li>压缩到最大应变（ε 米 = 50％）以50％/分钟。 </li><li>保持在ε 米（5分钟）。 </li><li>冷却至25℃（T 低 ），并保持10分钟以固定暂时形状。 </li><li>除去负载。 </li><li>测量在无压力状态下的极限应变（εU）。 </li><li>再加热至60℃（T 高 ），并且维持10分钟，以恢复永久形状。 </li><li>测量回收的应变（εp）的 。 </li><li>同时仍然在60℃（T 高 ），由支架压缩到第 1次循环后回收的高度（N = 1）的50％开始第 2次循环（2 N =）。 </li><li>重复5.1.3-5.1.8对于N = 2。 </li><li>使用以下公式计算R f和R r表示 ，N = 1和2： 的 R F（N）= [εU（N）/εM] R R（N）= [ε 米- εP（N）] / [ε 米- εP（N-1）] </li></ol></li></ol> 6，可视化孔径和孔互连性<ol><li>使用扫描电子显微镜（SEM;例如一个FEI广达SEM如本文所用），观察孔径和孔互连性。 <ol><li>利用镊子以保持SMP支架，淹没在液体N 2 1分钟。 </li><li>从液体的 N 2和断裂以及一个干净的刀片支架的中间除去。 </li><li>使用碳纤维带，加盖SMP支架半一到样品舞台断裂面朝上。 </li><li>溅射涂层与金铂（〜4纳米）。 </li><li>捕获SEM图像在10-15千伏（图4A）推荐的加速电压。 </li></ol></li></ol> 7.测试其体外生物活性的<ol><li>到50毫升离心管中，加入约30毫升1X SBF 24。 </li><li>获得在其原始的，圆筒状成型永久形状的支架。切用干净的刀片一半的支架（跨圆边）。 </li><li>放置一个单独的支架一半到准备好的离心管和帽。 </li><li>保持所述管在37℃下在无SBF更改静态条件下的水浴。 </li><li> 14天后，取出从SBF和空气干燥的支架24小时。 </li><li>使用碳纤维带，加盖支​​架上的断裂面朝上的样品台。 </li><li>溅射涂层与金铂（〜4纳米）。 </li><li>捕获的SEM图像在重赞扬10-15千伏（ 图4B）加速电压。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(&#949;-caprolactone)-based+shape+memory+polymers+with+variable+polydimethylsiloxane+soft+segment+lengths.">Poly(&#949;-caprolactone)-based shape memory polymers with variable polydimethylsiloxane soft segment lengths.</a> J Polym Sci Pol Chem. 49, 754-761 (2011).</li><li>Kokubo, T., Takadama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+useful+is+SBF+in+predicting+in+vivo+bone+bioactivity.">How useful is SBF in predicting in vivo bone bioactivity.</a> Biomaterials. 27, 2907-2915 (2006).</li><li>Murphy, W. L., Dennis, R. G., Kileny, J. L., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+fusion:+an+approach+to+improve+pore+interconnectivity+within+tissue+engineering+scaffolds.">Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.</a> Tissue Eng. 8, 43-52 (2002).</li><li>Yang, Q., Chen, L., Shen, X., Tan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+polycaprolactone+tissue+engineering+scaffolds+by+improved+solvent+casting/particulate+leaching+method.">Preparation of polycaprolactone tissue engineering scaffolds by improved solvent casting/particulate leaching method.</a> J Macromol Sci Phys. 45, 1171-1181 (2006).</li><li>Madbouly, S. A., Kratz, K., Klein, F., L&#252;zow, K., Lendlein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermomechanical+behaviour+of+biodegradable+shape-memory+polymer+foams.">Thermomechanical behaviour of biodegradable shape-memory polymer foams.</a> 1190, (2009).</li><li>Luo, X., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+shape+memory+elastomeric+composites.">Preparation and characterization of shape memory elastomeric composites.</a> Macromolecules. 42, 7251-7253 (2009).</li><li>Lendlein, A., Schmidt, A. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AB-polymer+networks+based+on+oligo(&#603;-caprolactone)+segments+showing+shape-memory+properties.">AB-polymer networks based on oligo(&#603;-caprolactone) segments showing shape-memory properties.</a> Proc Natl Acad Sci. 98, 842-847 (2001).</li></ol>

The authors have nothing to disclose.

这个协议描述了一种聚多巴胺涂覆的，PCL-基于支架，其自入行为的制备，以及骨诱导性和生物活性，使得在不规则CMF骨缺损的治疗兴趣它。该协议的各方面可以被改变，以改变各种支架功能。 该协议开始于PCL二醇，允许UV固化的丙烯酸酯化。在所报告的实例中，PCL二醇Mn为是〜10,000g / mol的。然而，通过适当地调整丙烯酰氯量和的Et 3 N的PCL-DA了PCL二醇可以被用?...

目前考虑的颅颌面（CMF）骨缺损治疗的金标准，收获的自体移植的移植是由复杂的移植手术，供体部位发病率和有限1阻碍。一个特别困难被整形并紧紧固定在刚性移植到缺陷，以获得骨整合和预防移植物吸收。组织工程已被调查作为一种替代战略，以自体移植和人工合成骨替代品（ 如骨水泥）2,3。关键的组织工程方法的成功是一组特定属性的支架。第一，为了实现骨整合，支架必须形成与相邻的骨组织4紧密接触。该支架也应该是骨传导，允许细胞迁移，营养扩散和neotissue沉积4,5。这种行为通常是实现了与可生物降解的SCAffolds表现出高度互联的孔结构。最后，该支架应的生物活性，以促进融合和粘接与周围骨组织5。 在这里，我们提出了一个协议，准备组织工程支架具有这些属性。重要的是，这种支架显示能力的“自我适应”到，由于其形状记忆行为6不规则的CMF缺陷。温敏形状记忆聚合物（SMP系统）是已知的在暴露于经受形状改变以加热7,8。中小事务所是由“netpoints”（即化学或物理交联），这决定了永久的形状和“转换段”，它保持暂时形状，并恢复永久的形状。开关段表现出相应于任一所述的玻璃化转变（T 克 ）热转变温度（T 反式）或熔融聚合物的转变（Tm值）。由于其结果，污水收集可以顺序变形为暂时形状在T&gt; T 反式，在T &lt;T 反式固定在暂时形状，并恢复到在T&gt; T 反式的永久形状。因此，SMP支架可以实现“自我装配”中的CMF缺陷如下6。曝光后，以温盐水（T&gt; T 转 ），一个SMP支架将成为延展性，允许一个一般制造的圆筒状的支架是用手压成不规则的缺陷，具有形状恢复促进扩大脚手架的缺陷边界。在冷却（T &lt;T 反式），脚手架将回到其相对更为刚性的状态，具有形状固定性保持缺陷在其新的临时形状。在这个协议中，将SMP支架选自聚己内酯（PCL）中制备，可生物降解的聚合物广泛研究的组织再生和其它生物医学应用9-11。对于形状记忆，日PCL电子Tm值用作经t 反式和43之后60°C之间变化，取决于在PCL 12的分子量。在这个协议中，支架的Ť 反式 （即Tm值）是56.6±0.3ºC6。 为了达到骨传导性，协议的开发，使PCL基的SMP支架与基于溶剂的铸造颗粒淋（SCPL）方法6,13,14高度互连的孔。聚己内酯丙烯酸酯（PCL-DA）（Mn为=〜10,000g / mol的）得到使用，以允许快速，光化学交联和溶解于二氯甲烷（DCM），以允许溶剂浇铸在盐模板。以下光化学固化和溶剂蒸发，盐模板除去浸出到水中。平均盐大小调节支架孔径。重要的是，该盐模板融合用水之前溶剂浇铸实现孔隙interconnectiviTY。 生物活性传授给了SMP的支架原位形成聚多巴胺涂到孔壁6。生物活性通常通过包含玻璃或玻璃-陶瓷填料15引入到支架。但是，这些可能引起不必要的易碎的机械性能。多巴胺已经显示，以形成对各种基材16-19中粘附，薄聚多巴胺层。在这个协议中，将SMP支架进行多巴胺的弱碱性溶液（pH = 8.5），以形成聚多巴胺的所有孔壁表面6 nanothick涂层。除了 ​​增强表面的亲水性以改进细胞粘附和传播，聚多巴胺已被证明是生物活性在形成的羟基磷灰石（HAP）在暴露于模拟体液（SBF）18,20,21的条款。在最后的步骤，将涂覆的支架被暴露在85℃（T&gt;Ť 反式）的wh热处理ICH导致脚手架致密化。热处理是前面提到的是为支架的形状记忆行为所必需的，或许是由于PCL结晶 ​​区域重组，以更接近14。 我们另外描述的方法中的不规则缺陷模型的自装配行为表征，形状记忆行为而言 ​​应变控制环状热机械压缩试验（即形状恢复和形状固定性），孔的形态，并在体外生物活性 。策略定制支架性能也提出了。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td>
			<td>Sigma-Aldrich</td>
			<td>440752</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td>Dried over 4A molecular sieves</td>
		</tr>
		<tr>
			<td>4-dimethylaminopyridine (DMAP)</td>
			<td>Sigma-Aldrich</td>
			<td>D5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine (Et3N)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A24109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Carbonate (K2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate (MgSO4)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>196118</td>
			<td></td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer (2mol/L)</td>
			<td>Fisher</td>
			<td>BP1759</td>
			<td>Used at 10 mM concentration, pH = 8.5</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>VWR</td>
			<td>47729-972</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Transilluminator (365 nm, 25 W)</td>
			<td>UVP</td>
			<td>95-0426-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments</td>
			<td>Q800</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Sputter Coater</td>
			<td>Cressington</td>
			<td>208HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>FEI</td>
			<td>Quanta 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

将所得的PCL基SMP支架是能够自我装配到一个模型CMF缺陷（图2）的。短暂暴露后温热盐水（〜60℃），圆柱形支架软化允许支架进行手动压入并展开模型缺损内。冷却至RT后，支架被固定到被从缺陷保留在除去其新的临时形状。 一个SMP支架材料的形状记忆行为由应变控制环状热机械压缩试验在形状固定性（R f）和形状恢复（R R）（图3）...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

生物活性，PCL为基础的“自我装配”形状记忆聚合物支架的制备

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

fabrication-bioactive-pcl-based-self-fitting-shape-memory-polymer

Research

JoVE Journal

Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

12.5K 次观看.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

视频: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

科研

生物工程

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

基于几丁聚糖，激光激发薄膜手术胶粘剂，“SurgiLux的制备及示范

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

制造业的鲁棒天然纤维预制体的利用细菌纤维素为粘结剂

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

倒胶体晶体聚（乙二醇）脚手架制作：肝脏组织工程三维细胞培养平台

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

含有宏观结构的光学组织幻影的制备与表征

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

可伸缩、双通道、微流控芯片制造

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...