这项工作是由它的力学生物学研究所，新加坡研究卓越中心，一个管理的新加坡国家研究基金会的支持。 MFAC是1122703037. BKKT是支持的力学生物学研究所机构科学，技术和研究（新加坡）和国家研究机构（法国）项目下的多次联合计划的支持。我们感谢丹尼尔HC黄先生的校对书稿和黎明JH新女士协助视频制作。

1.准备聚电解质溶液的<ol><li>纯化脱乙酰壳多糖，如在Liao 等详细说明。简言之，将创建一个1％（重量/体积）壳聚糖在2％的溶液使用（体积/体积）乙酸和真空过滤级93过滤纸。中和使用5M的NaOH将滤液直到pH稳定至7。离心沉淀的脱乙酰壳多糖在1200×g离心10分钟。滗析出上清液，并添加去离子水以洗涤脱乙酰壳多糖。重复离心和洗涤步骤两次以上。冻结的沉淀壳聚糖在-80℃和冷冻干燥O / N以获得纯化形式。存储纯化壳聚糖在除湿柜。 </li><li>称量1g纯壳聚糖进入无菌组织培养皿。放置在组织培养皿尽可能接近到紫外灯在生物安全柜的脱乙酰壳多糖和暴露于UV光15分钟。使用无菌镊子，将灭菌壳聚糖到玻璃容器中。壳聚糖溶解过滤使用0。15M乙酸至0.5％至1％的终浓度（重量/体积）。 </li><li>称取0.1克藻酸的钠盐和溶解在蒸馏水去离子（DDI）的10ml水，得到1％（重量/体积）溶液。混合的藻酸的钠盐，至少2小时，在涡流混合器以确保完全溶解。通过过滤0.2微米的注射器过滤器的海藻酸钠溶液。储存在4℃的藻酸盐溶液。 </li><li>重组人重组生长因子，如血管内皮生长因子（VEGF）或β - 神经生长因子（NGF），如制造商推荐的。 </li></ol> 2.绘图IPC纤维<ol><li>混合蛋白，生长因子或其它生物分子进入电解质溶液具有类似净电荷10-20微升等份。生物分子与净负电荷（例如，牛血清白蛋白〔BSA〕）应与藻酸盐溶液混合。生物分子的净正电荷（如VEGF）应与混合壳聚糖溶液。 </li><li>将壳聚糖和海藻酸钠的小等份（10-20微升）上覆盖有封口膜平坦光滑的表面。脱乙酰壳多糖和藻酸盐的液滴应该放置在接近但不与彼此相接触。 </li><li>轻轻浸每个尖端上的一对镊子进入壳聚糖和藻酸盐的微滴。一起捏钳把聚电解质的液滴。当液滴接触到彼此接触，慢慢地将钳子垂直向上从两个液滴（ 图1A）的接口绘制了IPC纤维。 </li><li>拉伸的IPC纤维上的集电极，所述镊子的末端小心地例如平坦聚合物支架固定在一个旋转的心轴（见第3和4）。旋转心轴以10mm / sec的固定速度以允许形成均匀的和无珠的IPC纤维。增加绘制了IPC的纤维将形成小珠的速度，这将导致结合的爆发释放生化和过早的光纤终端。10 </li><li>为了确定掺入的效率，收集所有的剩余液体留在封口膜通过用500μl1X磷酸盐缓冲盐水（PBS）中稀释。测量通过BCA测定法（对BSA），酶联免疫吸附（对VEGF和NGF）或适当的测定法来检测结合的生物分子中的残基的蛋白质或生长因子含量。 </li></ol> 3.制作普鲁兰多糖葡聚糖复合水凝胶支架（PD）多糖和IPC纤维<ol><li>制造牺牲普鲁兰框架IPC纤维集合<ol><li>称出支链淀粉的多糖并用蒸馏水去离子（DDI）水混合，以创建一个20％（重量/体积）的水溶液。混合普鲁兰多糖溶液O / N，以保证均匀性。 </li><li>投将15克的支链淀粉溶液倒入一个直径10厘米的组织培养聚苯乙烯（TCPS）菜。干普鲁兰多糖溶液O / N在37℃。切普鲁兰多糖薄膜7成毫米×7毫米的方形框架。 </li></ol></li><li>准备普鲁兰葡聚糖多糖溶液<ol><li>创建一个30％（重量/体积）的多糖类的溶液支链淀粉和葡聚糖:在DDI水（3比1的比例）。混合O / N，以确保多糖溶液的均匀性。慢慢在碳酸氢钠添加到多糖溶液以达到20％的终浓度（重量/体积）。混合O / N，以确保溶液的均匀性。储存在4℃的多糖溶液。 </li></ol></li><li>收集普鲁兰框架IPC纤维<ol><li>贴上使用鳄鱼夹牺牲普鲁兰框架（第3.1节）。粘鳄鱼夹和支链淀粉帧上使用塑料涂覆粘合带的转动心轴的末端。旋转与贴帧芯棒以10毫米/秒的恒定速度。普鲁兰多糖架可贴到所希望的方向旋转芯棒。 </li></ol></li><li>用画一双镊子（第1节）和ATTAC的IPC纤维小时IPC纤维的结束画上旋转普鲁兰框架。绘制了IPC纤维以恒定的速度。一旦到达了IPC纤维的末端，干燥纤维上帧构建O / N在室温。 </li><li> IPC嵌入纤维进入PD水凝胶支架</li><li>以交联每克支链淀粉葡聚糖溶液中，添加11％的100微升（重量/体积）三偏磷酸钠水溶液和氢氧化钠将100μl10M钠7使用stirplate为1至2分钟，混合溶液在60rpm。添加的三偏磷酸钠和氢氧化钠之后，将多糖溶液将几乎立即交联。倾粘性多糖溶液到纤维上帧构造完全嵌入的IPC纤维。孵育结合支链淀粉葡聚糖的IPC纤维（PD-IPC），在60℃进行30分钟，形成化学交联的复合支架（ 图1B）。 </li><li>注意:在通风柜执行步骤3.3.2，并使用适当的保护相等pment乙酸是腐蚀性和易燃。 </li><li>为了诱导孔形成在PD-IPC脚手架，淹没整个支架在20％（重量/体积）乙酸20分钟。 </li><li>为5分钟，在1×PBS中洗涤的PD-IPC支架除去未反应的试剂以100rpm摇动。重复此步骤2次。 </li><li>除去多余的PBS，并立即冻结PD-IPC支架在-80°CO / N。冻干支架的任何控制释放生物活性或使用试验前至少24小时。 </li></ol> 4.制作PCL和IPC纤维复合支架注意:二氯甲烷是一种有害物质。二氯甲烷处理时，使用通风橱和个人防护装备。 <ol><li>创建原始和图案的PDMS基板<ol><li>创建一个纯净的聚二甲基硅氧烷（PDMS）的弹性基板采用了一块所需尺寸的TCPS使用软光刻工艺。创建patterned的PDMS衬底 ​​通过使用标准软光刻方法对聚（甲基丙烯酸甲酯）与所需的形貌的模板。12 </li></ol></li><li>制造牺牲PCL框架IPC纤维集合<ol><li>称取PCL和溶解在二氯甲烷中以创建一个0.9％（重量/体积）溶液。每1 平方厘米面积的PDMS衬底 ​​的，滴加入500μl的0.9％的PCL溶液。允许所有的二氯甲烷溶剂完全蒸发，在通风橱。重复铸造0.9％PCL到薄膜变厚至期望的厚度的过程。从PDMS基板取下干燥PCL电影。创建在使用适宜尺寸的冲床在PCL帧的孔。2 </li></ol></li><li>在PCL框架上收集IPC纤维<ol><li>上贴上鳄鱼夹与孔（从4.2.1）牺牲PCL框架。通过使用塑料涂层胶带粘鳄鱼夹到旋转心轴。附加IPC纤维的结束画上PCL FRAM开始旋转，速度为10mm /秒（第2节）以恒定的速度前即IPC纤维拉伸结束后，干燥纤维上的帧构建O / N在4℃。 </li></ol></li><li>嵌入光纤的框架构建成图案PCL基板<ol><li>降500微升0.9％PCL解决方案到PDMS基板打造一个纯净的或有图案的PCL基地，根据需要。施放的0.9％的PCL溶液多层以获得具有所需厚度的脚手架。允许所有的二氯甲烷溶剂完全蒸发，在通风橱。 </li><li>放置在纤维上的帧结构（第4.3.1节）在PCL基座的顶部。加在纤维上帧构造多次0.9％的PCL溶液，以获得所需的厚度和完全嵌入的IPC纤维，制造的PCL-IPC复合支架（ 图1C）。 </li></ol></li></ol> 5.测量生物制剂的释放从复合IPC支架<ol><li>将复合PD-IPC或PCL-IPC支架和单机IPC纤维分别在24孔板。 </li><li>沉浸脚手架和单机IPC纤维用500μl1X PBS的。孵育样品在37℃。收集的PBS在不同时间点（释放介质）和替换用500μl1X PBS中。 </li><li>测量在使用BCA测定法（BSA），酶联免疫吸附（VEGF和NGF）或其他适当的测定法来计算掺入的生物分子的累积释放曲线的释放介质蛋白或生长因子的量。 </li></ol>复合IPC支架6.种子细胞来测试释放生物制剂的生物活性<ol><li>消毒使用UV光在生物安全柜为至少20分钟的冷冻干燥的PD-IPC或PCL-IPC复合支架。 </li><li>使用标准的细胞培养技术，以获得在200μl生长培养基2×10 5个细胞的细胞悬浮液。种子的浓缩细胞悬浮液上的复合支架。 AFTER 20分钟，生长培养基补足体积至完全淹没支架。 </li><li>通过测量如Alamar蓝代谢活性测定，PC12轴突生长法或免疫荧光技术标准的细胞活性。 </li></ol>

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J., 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=Fabrication+of+poly(ethylene+glycol)+hydrogel+microstructures+using+photolithography.">Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography.</a> Langmuir. 17 (18), 5440-5447 (2001).</li><li>Yim, E. K. F., Liao, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+compatibility+of+interfacial+polyelectrolyte+complexation+fibrous+scaffold:+evaluation+of+blood+compatibility+and+biocompatibility.">Tissue compatibility of interfacial polyelectrolyte complexation fibrous scaffold: evaluation of blood compatibility and biocompatibility.</a> Tissue Eng. Part A. 13 (2), 423-433 (2007).</li><li>Chaouat, M., Le Visage, C., Autissier, A., Chaubet, F., Letourneur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evaluation+of+a+small-diameter+polysaccharide-based+arterial+graft+in+rats.">The evaluation of a small-diameter polysaccharide-based arterial graft in rats.</a> Biomaterials. 27, 5546-5553 (2006).</li><li>Eshraghi, S., Das, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+microstructural+properties+of+polycaprolactone+scaffolds+with+one-dimensional,+two+dimensional,+and+three-dimensional+orthogonally+oriented+porous+architectures+produced+by+selective+laser+sintering.">Mechanical and microstructural properties of polycaprolactone scaffolds with one-dimensional, two dimensional, and three-dimensional orthogonally oriented porous architectures produced by selective laser sintering.</a> Acta Biomater. 6, 2467-2476 (2010).</li></ol>

The authors have nothing to disclose.

IPC纤维通过两个带相反电荷的聚电解质的相互作用形成。该方法使用从聚电解质的界面的复杂的提取，便利了一种用于纤维形成稳定的自组装过程。 IPC的纤维形成的机制可确保任何生物分子加入到类似电荷的聚电解质可以在络合过程中引入。10,11相反，除了生物分子的成带相反电荷的聚电解质将导致瞬间沉淀。用于IPC纤维的简单的制造方法借给多功能性在将各种生物材料如细胞，生长因?...

The extracellular matrix has inherent biochemical and biophysical cues that direct cell behaviors. Mimicking this physiological three-dimensional (3D) microenvironment is a widely explored strategy for regenerative medicine and tissue engineering applications. For example, both naturally-derived and synthetic substrates have been modified with topographical cues as a means to mimic the biophysical cellular environment.1 For example, polycaprolactone (PCL) scaffolds can be easily patterned by casting on patterned PDMS substrates.2 However, most synthetic scaffolds inadequately recapitulate the controlled biochemical environment in vivo. Bulk or surface modification of synthetic materials only present biochemical cues for cell attachment but still lack temporal regulation of biochemical delivery.3 Thus, there is a need for optimal scaffolds that can mimic the temporally regulated biochemical delivery system of the extracellular matrix. 
Biochemical delivery systems such as microspheres are plagued by problems of loss of bioactivity and low incorporation efficiency due to the severity and complexity of multi-step synthesis process.4-6 Alternative methods that use a one-step fabrication and incorporation method were proven to have excellent potential to create a favorable biochemical microenvironment without the accompanying inefficiency in incorporation and loss of bioactivity. One viable solution is the use of interfacial polyelectrolyte complexation (IPC) fibers to deliver and protect biological agents. When two oppositely charged polyelectrolyte aqueous solutions are brought together, IPC fibers can be drawn out from the interface. Virtually any type of hydrophlic biomolecule in aqueous solution can be added into either the negatively- or positively-charged polyelectrolyte solution, thus facilitating the incorporation of useful biomolecules into the IPC fiber during the complexation process. Furthermore, this process only requires aqueous and ambient conditions, thereby decreasing the risk of loss of bioactivity. Using this method, active growth factors2,7 even cells8,9 have been successfully delivered. In addition, the simple method of forming IPC fibers allows molding into any shape or orientation. The stability of such fibers has been advantageous in its incorporation into both hydrophobic2 and hydrophilic polymers7 to create composite scaffolds. These composite scaffolds with IPC fibers are beneficial for creating a physiologically relevant biochemical environment while providing physical anchorage for cells. 
In this study, we show a method to incorporate IPC fibers into a hydrophilic and a hydrophobic scaffold with topography for controlled release of active biomolecules. As a proof-of-concept, we incorporate IPC fibers made from chitosan and alginate into the biocompatible, non-immunogenic and non-antigenic pullulan-dextran hydrophilic hydrogel or the biocompatible polycaprolactone hydrophobic scaffold.

<table><tbody><tr><td>Pullulan&amp;nbsp;</td><td>Hayashibara Inc Okayama Japan</td><td></td><td>Molecular weight (MW) 200 kDa. This material is pharmaceutical grade pullulan used to make pullulan frames and PD-IPC scaffolds.</td></tr><tr><td>Dextran</td><td>Sigma Aldrich</td><td>D1037</td><td>MW 500 kDa. This material is no longer being produced by Sigma Aldrich. Alternative suggested is catalog number 31392 (Sigma Aldrich). This material is used to make PD-IPC scaffolds.</td></tr><tr><td>Sodium Bicarbonate&amp;nbsp;</td><td>Sigma Aldrich</td><td>S5761</td><td>Sodium bicarbonate must be slowly added to the pullulan-dextran polysaccharide solution. Rapid addition of sodium bicarbonate will result in precipitation.&amp;nbsp;</td></tr><tr><td>Sodium Trimetaphosphate</td><td>Sigma Aldrich</td><td>T5508</td><td>This chemical is hygroscopic and must be stored in the dehumidifying cabinet. Aqueous solution of sodium trimetaphosphate must always be made fresh.</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>This material is hazardous and must be handled with proper protective equipment such as nitrile gloves.</td></tr><tr><td>Chitosan</td><td>Sigma Aldrich</td><td>448877</td><td>MW 190-310 kDa. Acetylation degree of 75% to 85%. Purification of chitosan is required to create stable IPC fibers.</td></tr><tr><td>Acetic Acid</td><td>Merck</td><td></td><td>This can be replaced by another brand type. This material is corrosive and flammable. Protective equipment such as face shield, nitrile gloves, lab coat and shoe cover must be worn when handling this chemical in the fume hood.&amp;nbsp;</td></tr><tr><td>Alginic acid sodium salt from brown algae, low viscosity</td><td>Sigma Aldrich</td><td>A2158</td><td>Dissolve in water overnight. Filter through sterile 0.2&amp;nbsp;&amp;micro;m syringe filter before use. Store at 4 &amp;deg;C.</td></tr><tr><td>Bovine Serum Albumin</td><td>Sinopharm Chemical Reagent</td><td></td><td>Dissolve in sterile PBS and filter using 0.2 &amp;micro;m syringe filter before use.&amp;nbsp;</td></tr><tr><td>BCA assay kit</td><td>Pierce</td><td>23225</td><td>This kit was used to measure BSA release from PD-IPC scaffolds.&amp;nbsp;</td></tr><tr><td>Human Recombinant Vascular Endothelial Growth Factor</td><td>R&amp;amp;D systems</td><td>293-VE</td><td>Dissolve growth factor in 0.2% heparin solution to a final concentration of 5&amp;nbsp;mg/ml.</td></tr><tr><td>Heparin Sodium Salt From Porcine</td><td>Sigma Aldrich</td><td>H3393</td><td>This can be replaced by another brand type. Dissolve heparin salt in sterile water at a final concentration of 1% and filter through 0.2 &amp;micro;m syringe filter before use.&amp;nbsp;</td></tr><tr><td>Human Umbilical Vein Endothelial Cells (HUVEC)</td><td>Lonza</td><td>C2517A</td><td>This primary cell type was used in the assay to determine VEGF bioactivity after release from PD-IPC scaffolds.&amp;nbsp;</td></tr><tr><td>Alamar blue</td><td>Life Technologies</td><td>DAL1025</td><td>This is used to measure cell metabolic activity. Incubate Alamar blue with cells and maintain in standard cell culture conditions for 2 to 4 hours. Measure absorbance at 570 nm to determine Alamar blue percent reduction, which is correlated to the cell activity.&amp;nbsp;</td></tr><tr><td>ScanVac Coolsafe Lyophilizer</td><td>Labogene</td><td>7.001.200.060</td><td>This is a non-programmable freeze dryer that operates at -105 to -110&amp;nbsp;&amp;deg;C. This can be replaced by other standard lab lyophilizers.</td></tr><tr><td>Polycaprolactone (PCL)</td><td>Sigma Aldrich</td><td>181609</td><td>MW 65 kDa. This is no longer being manufactured by Sigma Aldrich. This can be replaced by Sigma Aldrich catalog number 704105.</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>V800151</td><td>This can be replaced by another brand type. This material is hazardous and must be handled in the fume hood. Protective equipment must be worn at all times when handling this chemical.</td></tr><tr><td>Polydimethylsiloxane (PDMS; 184 Silicone Elastomer Kit)</td><td>Dow Corning</td><td>(240)4019862</td><td>The elastomer kit comes with polymer base and crosslinker. Mixing the polymer base and crosslinker in different ratios will result in different stiffness of the PDMS.</td></tr><tr><td>Human Recombinant Beta-Nerve Growth Factor (NGF)</td><td>R&amp;amp;D systems</td><td>256-GF</td><td>Reconstituted in sterile DI water to a final concentration of 100&amp;nbsp;&amp;micro;g&amp;#8288;/&amp;#8288;ml. Aliquot and store in -20 &amp;deg;C until use.</td></tr><tr><td>Human Mesenchymal Stem Cells (hMSC)</td><td>Cambrex</td><td></td><td>This cell type was used in the assay to determine synergistic effect of NGF and nanotopography.</td></tr><tr><td>Rat PC12 Pheochromocytoma Cells&amp;nbsp;</td><td>ATCC</td><td></td><td>This cell type was used in the neurite outgrowth assay to determine bioactivity of NGF. After exposure to release media with NGF, measure number of cells with neurite extensions and normalize to total number of cells.</td></tr><tr><td>Grade 93 filter paper</td><td>Whatman</td><td>Z699675</td><td>This is used for the purification of chitosan after its precipitation with sodium hydroxide at pH&amp;nbsp;7.</td></tr><tr><td>Swing bucket centrifuge</td><td>Eppendorf</td><td>5810R</td><td>To be used during the purification of chitosan using 1,200 x g speed.</td></tr><tr><td>Motor with mandrel rotating at constant speed</td><td>Rhymebus</td><td>RM5E</td><td>The motor is used for the fabrication of IPC fibers on pullulan or PCL frame.</td></tr><tr><td>Phosphate buffered saline</td><td>FirstBase</td><td></td><td>Sterilize through filtration (0.2 &amp;micro;m filter) and autoclave.&amp;nbsp;</td></tr><tr><td>10-mm diameter Tissue Culture Polystyrene Dish (TCPS)</td><td>Greiner</td><td></td><td>The TCPS dish is used for casting of pullulan frame.&amp;nbsp;</td></tr><tr><td>Human VEGF ELISA kit</td><td>R&amp;amp;D systems</td><td>DVE00</td><td>The ELISA kit is used for detection of VEGF in the release medium.</td></tr><tr><td>Human NGF ELISA kit</td><td>R&amp;amp;D systems</td><td>DY256</td><td>The ELISA kit is used for detection of NGF in the release medium.</td></tr><tr><td>Plastic Coated Adhesive Tape</td><td>Bel-Art</td><td>9040336</td><td>The adhesive tape is used to securely stick the alligator clip to the rotating mandrel</td></tr></tbody></table>

composite scaffolds of interfacial polyelectrolyte fibers for temporally controlled release of biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

在这篇文章中，我们试图建立复合支架与IPC纤维各种生物分子的持续释放。在此研究中使用的生物分子的特征见于表1。的IPC纤维第一嵌入到亲水的PD水凝胶来创建的PD-IPC复合支架（ 图1B）。模型分子BSA的第一测试，以确定使用复合支架用于控制生物分子释放的可行性。 BSA的掺入的PD-IPC支架为45±0.97％的效率。 BSA的从PD-瞳距释放呈接近线性的动力学具有初始减毒突释之后?...

Watch this Scientific Journal Video about Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules at JoVE.com

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

composite-scaffolds-interfacial-polyelectrolyte-fibers-for-temporally

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.3K 次观看.  National University of Singapore. Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

视频: Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

密度梯度多层聚合（dGMP的）：创建多室的，可定制的一种新技术，组织工程支架

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

科研

生物工程

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

将二维电纺纳米纤维垫扩展到三维支架中

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

遗传学

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

多尺度孔梯度，聚电解质和间接监测改性金属植入物<em&gt;在体内</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

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.

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 ...

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 ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

生物学

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

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 clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

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

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

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 ...

A Facile and Eco-friendly Route to Fabricate Poly(Lactic Acid) Scaffolds with Graded Pore Size

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of this new field of biomaterials research is due to the necessity to develop implants capable of mimicking the complex functionality of the various tissues, including a continuous change from one structure or composition to another. In this latter context, one topic of main interest concerns the design of appropriate scaffolds for bone-cartilage interface tissue. In this study, three-layered scaffolds with graded pore size were achieved by melt mixing poly(lactic acid) (PLA), sodium chloride (NaCl) and polyethylene glycol (PEG). Pore size distributions were controlled by NaCl granulometry and PEG solvation. Scaffolds were characterized from a morphological and mechanical point of view. A correlation between the preparation method, the pore architecture and compressive mechanical behavior was found. The interface adhesion strength was quantitatively evaluated by using a custom-designed interfacial strength test. Furthermore, in order to imitate the human physiology, mechanical tests were also performed in phosphate buffered saline (PBS) solution at 37 &#176;C. The method herein presented provides a high control of porosity, pore size distribution and mechanical performance, thus offering the possibility to fabricate three-layered scaffolds with tailored properties by following a simple and eco-friendly route.

A Facile and Eco-friendly Route to Fabricate PolyLactic Acid Scaffolds with Graded Pore Size

In this work, poly(lactic acid)/polyethylene glycol (PLA/PEG) scaffolds were prepared by using a combination of melt mixing and selective leaching. The method herein discussed permitted to develop three-layer scaffolds by highly controlling both porosity and pore size. The mechanical properties were also evaluated in a physiological environment.

的简便和环保路线制造聚乳酸支架与分级孔径

Over the recent years, functionally graded scaffolds (FGS) gaineda crucial role for manufacturing of devices for tissue engineering. The importance of ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

生物活性蛋白或多肽在水凝胶上的应用

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...