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>

<ol>
	<li>Annabi, N., Tamayol, A., 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=25th+Anniversary+Article:+Rational+design+and+applications+of+hydrogels+in+regenerative+medicine.">25th Anniversary Article: Rational design and applications of hydrogels in regenerative medicine.</a> Adv. Mater. 26 (1), 85-124 (2014).</li><li>Teo, B. K. K., Tan, G. D. S., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synergistic+effect+of+nanotopography+and+sustained+dual+release+of+hydrophobic+and+hydrophilic+neurotrophic+factors+on+human+mesenchymal+stem+cell+neuronal+lineage+commitment.">The synergistic effect of nanotopography and sustained dual release of hydrophobic and hydrophilic neurotrophic factors on human mesenchymal stem cell neuronal lineage commitment.</a> Tissue Eng. Part A. 20 (15-16), 2151-2161 (2014).</li><li>Lee, K., Silva, E. A., 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=Growth+factor+delivery-based+tissue+engineering:+general+approaches+and+a+review+of+recent+developments.">Growth factor delivery-based tissue engineering: general approaches and a review of recent developments.</a> J. R. Soc. Interface. 8 (55), 153-170 (2011).</li><li>Sun, Q., Chen, R. R., Shen, Y., Mooney, D. J., Rajagopalan, S., Grossman, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+vascular+endothelial+growth+factor+delivery+enhances+angiogenesis+and+perfusion+in+ischemic+hind+limb.">Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb.</a> Pharm. Res. 22 (7), 1110-1116 (2005).</li><li>Rui, J., Dadsetan, M., 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=Controlled+release+of+vascular+endothelial+growth+factor+using+poly-lactic-co-glycolic+acid+microspheres:+in+vitro+characterization+and+application+in+polycaprolactone+fumarate+nerve+conduits.">Controlled release of vascular endothelial growth factor using poly-lactic-co-glycolic acid microspheres: in vitro characterization and application in polycaprolactone fumarate nerve conduits.</a> Acta Biomater. 8 (2), 511-518 (2012).</li><li>King, T. W., Patrick, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+in+vitro+characterization+of+vascular+endothelial+growth+factor+(VEGF)-loaded+poly(DL-lactic-co-glycolic+acid)/poly(ethylene+glycol)+microspheres+using+a+solid+encapsulation/single+emulsion/solvent+extraction+technique.">Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique.</a> J. Biomed. Mater. Res. 51 (3), 383-390 (2000).</li><li>Cutiongco, M. F. A., Tan, M. H., Ng, M. Y. K., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+pullulan-dextran+polysaccharide+scaffold+with+interfacial+polyelectrolyte+complexation+fibers:+A+platform+with+enhanced+cell+interaction+and+spatial+distribution.">Composite pullulan-dextran polysaccharide scaffold with interfacial polyelectrolyte complexation fibers: A platform with enhanced cell interaction and spatial distribution.</a> Acta Biomater. 10 (10), 4410-4418 (2014).</li><li>Yow, S. Z., Quek, C. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-based+fibrous+scaffold+for+spatial+organization+of+encapsulated+and+seeded+human+mesenchymal+stem+cells.">Collagen-based fibrous scaffold for spatial organization of encapsulated and seeded human mesenchymal stem cells.</a> Biomaterials. 30 (6), 1133-1142 (2009).</li><li>Yim, E. K. F., Wan, A. C. A., Le Visage, C., Liao, I. C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+differentiation+of+human+mesenchymal+stem+cell+encapsulated+in+polyelectrolyte+complexation+fibrous+scaffold.">Proliferation and differentiation of human mesenchymal stem cell encapsulated in polyelectrolyte complexation fibrous scaffold.</a> Biomaterials. 27 (36), 6111-6122 (2006).</li><li>Liao, I. C., Wan, A. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+from+fibers+of+polyelectrolyte+complexes.">Controlled release from fibers of polyelectrolyte complexes.</a> J. Control. Release. 104 (2), 347-358 (2005).</li><li>Wan, A. C. A., Liao, I. C., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+fiber+formation+by+interfacial+polyelectrolyte+complexation.">Mechanism of fiber formation by interfacial polyelectrolyte complexation.</a> Macromolecules. 37 (18), 7019-7025 (2004).</li><li>Yim, E. K. F., Reano, R., Pang, S., Yee, A., Chen, C., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopattern-induced+changes+in+morphology+and+motility+of+smooth+muscle+cells.">Nanopattern-induced changes in morphology and motility of smooth muscle cells.</a> Biomaterials. 26 (26), 5405-5413 (2005).</li><li>Sun, H., Xu, F., Guo, D., Yu, H. <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+evaluation+of+NGF-microsphere+conduits+for+regeneration+of+defective+nerves.">Preparation and evaluation of NGF-microsphere conduits for regeneration of defective nerves.</a> Neurol. Res. 34 (5), 491-497 (2012).</li><li>Sim&#243;n-Yarza, T., Formiga, F. R., Tamayo, E., Pelacho, B., Prosper, F., Blanco-Prieto, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEGylated-PLGA+microparticles+containing+VEGF+for+long+term+drug+delivery.">PEGylated-PLGA microparticles containing VEGF for long term drug delivery.</a> Int. J. Pharm. 440 (1), 13-18 (2013).</li><li>Patel, Z. S., Ueda, H., Yamamoto, M., Tabata, Y., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+release+of+vascular+endothelial+growth+factor+from+gelatin+microparticles+and+biodegradable+composite+scaffolds.">In vitro and in vivo release of vascular endothelial growth factor from gelatin microparticles and biodegradable composite scaffolds.</a> Pharm. Res. 25 (10), 2370-2378 (2008).</li><li>Sun, H., Xu, F., Guo, D., Liu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+effects+of+various+additives+and+polymers+on+nerve+growth+factor+microspheres.">In vitro evaluation of the effects of various additives and polymers on nerve growth factor microspheres.</a> Drug Dev. Int. Pharm. 40 (4), 452-457 (2014).</li><li>Lee, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+drug+release+from+hydrogel+matrices.">Kinetics of drug release from hydrogel matrices.</a> J. Control. Release. 2, 277-288 (1985).</li><li>Cutiongco, M. F. A., Choo, R. K. T., Shen, N. J. X., Chua, B. M. X., Sju, E., Choo, A. W. L., Le Visage, C., Yim, E. K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composite+scaffold+of+poly(vinyl+alcohol)+and+interfacial+polyelectrolyte+complexation+fibers+for+controlled+biomolecule+delivery.">Composite scaffold of poly(vinyl alcohol) and interfacial polyelectrolyte complexation fibers for controlled biomolecule delivery.</a> Front. Bioeng. Biotechnol. 3 (3), 1-12 (2015).</li><li>Yow, S. Z., Lim, T. H., Yim, E. K. F., Lim, C. T., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3D+Electroactive+Polypyrrole-Collagen+Fibrous+Scaffold+for+Tissue+Engineering.">A 3D Electroactive Polypyrrole-Collagen Fibrous Scaffold for Tissue Engineering.</a> Polymers. 3 (1), 527-544 (2011).</li><li>Leong, M. F., Toh, J. K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+prevascularised+tissue+constructs+by+assembly+of+polyelectrolyte+hydrogel+fibres.">Patterned prevascularised tissue constructs by assembly of polyelectrolyte hydrogel fibres.</a> Nat. Commun. 4, 2353 (2013).</li><li>Di Benedetto, F., Biasco, A., Pisignano, D., Cingolani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+polyacrylamide+hydrogels+by+soft+lithography.">Patterning polyacrylamide hydrogels by soft lithography.</a> Nanotechnology. 16 (5), S165 (2005).</li><li>Revzin, A., Russell, R. 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，生长因子和小分子。还观察到在两个对PD-IPC和PCL-IPC复合支架，其中的生长因子具有不同的物理和生物分子性质（电荷及分子量）掺入具有高效率的IPC纤维的这一关键特征。在禁忌省，使用微球的方法对VEGF和NGF包封率可以低至16％的6和8％13。 这两个PD-IPC和PCL-IPC支架也表现出生物分子的释放时间控制。从PD-IPC支架血管内皮生长因子呈线性释放至少7天。与此相反，报告从聚合物微球的VEGF的释放曲线显示在24小时内的初始突发释放，释放的总的VEGF含量为至少60％。5,14,15同样，神经生长因子被证明从PCL-IPC持续生长因子递送支架，而其它聚合物为基础的增长因子递送系统显示释放的20天释放的平台期。13,16据推测，所述复合支架的不同部件都有助于生长因子释放动力学。聚合物松弛机制，孔隙度和弯曲度可以极大地影响亲水生物分子的释放。17此外，的C所述聚合骨架的hemical特征可以具有静电吸引和排斥向影响生物分子释放的生长因子。因此，该聚合骨架的特点是在确定释放曲线和生物分子以时间上控制释放是至关重要的。例如，当对PD支架呈接近线性的BSA的释放，类似的复合支架使用聚（乙烯醇）水凝胶缺乏渗透性与BSA 18聚合物支架可结合使用IPC纤维可能需要初步试验以确定其渗透性范围。 除了控制生化释放，以保持生长因子的生物活性的能力为任何生物分子递送系统的一个重要特征。对PD溶液的苛刻的碱性环境和有机DCM溶剂中的PCL存在下具有降解任何种类的生物分子的潜力。例如，微球递送system所使用二氯甲烷表现出与增加的时间点，由于NGF的降解减少生物活性的趋势一致。16然而，我们观察到血管内皮生长因子和生长因子的生物活性被保持，进一步重申的IPC纤维的一个重要优势是内复合同化支架。 使用牺牲的，生物相容的多糖或聚合物框架可以容易地并入到散装支架给出的多功能性以创建复合支架具有多个IPC纤维在不同的配置排列。7。因此，要被施加的聚合物支架，有必要与IPC纤维具有进一步同化能力成批量支架。同化过程是完全嵌入IPC纤维进入聚合骨架重要。我们在该牺牲支链淀粉和PCL帧，这是既易于溶解在散装PD的溶剂或观察到这种现象PCL支架。此外，对于IPC的制造和生物分子结合的单步方法提供了灵活性，以能够由单个复合支架递送生物分子的数量。该方法的简单性也允许微调掺入效率和释放的动力学通过改变聚电解质身份或浓度。天然聚电解质壳聚糖和藻酸盐中，由于使用其高电荷密度和相似于动物组织中发现的各种细胞外基质的碳水化合物。然而甲基化胶原和三元共聚物8,19或壳多糖和藻酸盐20还可以用于IPC的纤维形成不同的效果。多的IPC纤维释放不同的生化线索具有不同动力学也可以实现以创建一个多功能复合支架。例如，细胞外基质蛋白，如纤连蛋白加载到IPC纤维可以用于空间控制细胞粘着提供了一个平台。7持续抗生素和生长因子的释放也是理想的组织再生应用程序。这可能通过使用PCL-IPC，其可以掺入疏水性的，小分子药物，并在复合支架的不同区室的亲水性，基于蛋白质的生长因子。2 我们提出的方法也允许与两个地形和生化线索的支架的制造。我们观察到的NP-PCL-IPC支架具有最高的增强的hMSC分化成神经元谱系，这意味着模仿生物物理和生物化学细胞小生的多个方面是有益的指导细胞行为。所提出的方法的便利性使得其应用到其它形成图案的聚合物体系，如聚丙烯酰胺21或聚乙二醇22，其前提是在交联过程中不会显著影响的IPC纤维完整性。例如，PD支架是CHOSEN在这项研究中以其独特的交联机制，发生在环境温度和含水条件24这可以潜在地提供更多的生理学相关底物的体外和体内研究。此外，在一个复合支架嵌入的IPC纤维可以通过提供拉伸强度克服缺乏IPC纤维23的机械强度。的确，PCL 25被选定为它的高机械强度。 总之，用于创建复合支架用于生物分子递送的简单方法进行了说明。我们展示了纤维如何IPC可以用于持续递送生物分子的，而不损害其生物活性和掺入效率。 PD-IPC和PCL-IPC支架:我们通过两种形式创建复合支架表现出这一点。 IPC纤维的适用性不限于掺入PD-和PCL基于支架，但也可以潜在地扩展到其他聚合物体系并提供其它生物分子。

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是伴随稳态（ 图2）。 2个月后，BSA达到97％的总释放。相反，独立的IPC纤维显示出4小时内的80％的BSA的快速释放。 接着，我们检查VEGF的释放曲线和生物活性使用的PD-IPC scaffoLDS。血管内皮生长因子中掺入的75.5±2.7％的效率，并呈持续释放至少1周（ 图3A）。人脐静脉内皮细胞（HUVEC）接种在PD-IPC支架，以确定VEGF的生物活性。血管内皮细胞上的PD-IPC显示支架与普通支架的PD在第1天相比显著增加Alamar蓝和降低代谢活动，表示从PD-IPC支架（ 图3B）被释放后，保存好血管内皮生长因子的功能。阿尔玛蓝在减少3天，第6和第7下降到达到相当水平与普通支架PD（ 图3B）。 在PCL-IPC复合支架还测试控释并与独立的IPC纤维相比。我们结合NGF为代表的分子进入PCL-IPC复合支架与66.38±2.71％的掺入效率。 PCL-IPC复合支架呈线芳缓释和18天后（ 图4A）约80％的累积释放。另一方面，IPC单机纤维显示在24小时内，随后通过一个停滞释放速率的70％的爆发释放。使用的PC12神经突增生测定中，我们检查了释放的NGF（ 图4B）的生物活性。上生长的PCL-IPC复合支架释放介质PC12细胞的神经突增生显示出相似水平与PC12细胞在30毫微克培养/ ml的神经生长因子补充培养基。这表明NGF释放生物活性保持至少7天。 地形的组合和持续的生长因子交货可能模仿了细胞微环境更好。 PCL的-IPC制造的多功能方法允许biochemically-和地形控制的复合支架的制造。我们制作了PCL-IPC复合支架具有纳米尺寸结构光栅（NP-PCL-IPC支架）。我们观察到的协同EF地形FECT和持续的NGF释放通过评估人类间质干细胞（hMSC）的培养（ 图5）的神经元分化。的hMSCs的NP-PCL-IPC复合支架培养表现出较高的表达微管相关蛋白2（MAP2）的，表示神经元的分化。另一方面，MAP2蛋白表达中的hMSCs只有NGF释放或图案化的PCL（NP-PCL）对PCL-IPC培养低得多。 <img alt="图1" src="/files/ftp_upload/53079/53079fig1.jpg" /> 图1. IPC掺入到纤维的亲水性和疏水性支架。IPC纤维（A）水彩在正（壳聚糖）的接口和负（海藻酸钠）电荷的聚电解质溶液。 （B）中示出在亲水性的PD溶液的IPC纤维掺入到创建的PD-IPC组合大示意图<html> Ë脚手架。显示疏水PCL支架IPC纤维的结合（C）示意图创建PCL-IPC复合支架。这个数字是改编自Cutiongco 等人 ，2014年7 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53079/53079fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53079/53079fig2.jpg" /> BSA的从PD-IPC复合支架。BSA的图2的控制释放被并入的PD-IPC复合支架，并使用BCA测定其释放，测定在不同时间点。释放累积的BSA被提供作为BSA（以μg）的在IPC纤维掺入的总量的百分比，并表示为平均值百分比±标准偏差。这个数字是改编自Cutiongco 等人 ，2014年7/www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53079/53079fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53079/53079fig3.jpg" /> 图3.控释和从PD-IPC复合支架的生物活性的VEGF。（A）中的VEGF从PD-IPC复合支架的累积释放曲线。血管内皮生长因子的释放，测定在使用ELISA特异于VEGF的不同的时间点。上生长的PD-IPC复合支架，如通过Alamar蓝代谢试验测定内皮细胞（B）的细胞存活率。使用单向ANOVA与Tukey的事后检验进行统计分析。 *表示P &lt;0.05。这个数字是改编自Cutiongco 等人 ，2014年7 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53079/53079fig3large.jpg" target="_blank">请点击此处查看大图</a><html>这个数字。 <img alt="图4" src="/files/ftp_upload/53079/53079fig4.jpg" /> 图4.控制释放和从PCL-IPC复合支架的生物活性神经生长因子。（A）的 NGF的从PCL-IPC复合支架的累积释放曲线。神经生长因子释放测定在使用ELISA特异于NGF的不同的时间点。插图显示NGF的从PCL-IPC支架的前8小时的累积释放曲线。 NGF（二）生物活性由PC12神经细胞生长测量。 PC12增生是通过图像分析测量。这个数字是改编自张志贤等人 ，2014年2 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53079/53079fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> .JPG"/&gt; 图上的NP-PCL-IPC支架5.分化的hMSC，hMSC的共焦扫描显微镜图像培养在不同的复合支架。 （A）NP-PCL-IPC，（B）NP-PCL，（C）PCL-IPC，（D）精粹PCL支架。绿色染色表示MAP2，神经元谱系标记。红染色表示F-肌动蛋白，表现出细胞骨架。蓝染色表示细胞核。这个数字是改编自张志贤等人 ，2014年2 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53079/53079fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 生化表1.特性用于控释由复合IPC支架。

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 San Sebastian

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

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

专访：干细胞研究的生物反应器和三维支架材料表面改性

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

生物学

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

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 ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

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 ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...