The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

1.钠萘基活性溶液和表面活化制备注:在通风良好的通风橱内开展的反应。按照一般规则处理高可燃性溶剂和腐蚀性的金属，如金属钠。萘具有非常难闻的气味（后备），即使在极少量！如果没有另外说明的反应是在室温下进行。叠氮化钠是剧毒！的THF（99.9％，参见材料的列表）被储存在约20％（体积）分子筛。蒸馏THF有明显的水分含量超过钠。如果痕量水存在不发生萘钠的形成。 <ol><li>到的萘1.4克（10.9毫摩尔）在20毫升四氢呋喃中的溶液（THF中，用3埃分子筛干燥），加入0.25g克（10.9毫摩尔）的金属钠在装有PTFE-一个螺旋盖的100ml玻璃瓶涂覆的磁性搅拌棒。 注:Dissolu（ - 40℃35）和灰可通过切割钠切成小片，并温和加热大大提高。最终溶液具有暗，稍浅绿色并且可以严格干燥的条件下储存。 </li><li> 0.5毫米厚箔材冲压出直径为12毫米的PTFE盘。马克一面（ 例如 ，使用一个字母邮票打孔），清洁的异丙醇。 </li><li> 2分钟使用镊子 - 在活化溶液（步骤1.1）中1单独孵育聚四氟乙烯样品。注:从白色的颜色变化至黑褐色表示成功治疗。 </li><li>随后冲洗两次​​用THF，然后用异丙醇。 注:萘基液耗尽了它的颜色变成水汪汪的浅棕色的时候。 （可能含有金属钠）未使用的萘基溶液必须通过处置前缓慢加入异丙醇分解。 </li><li>氧化在含20％（重量/体积）三氯乙酸3小时的过氧化氢（30％）处理的样品。洗与水和干燥。表面现在有一个轻微的褐色外观。注意:激活和氧化导致表面的急剧增加的润湿性。这一发现进行了详细的研究之前14。 </li><li>治疗用50％（V / V）的六亚甲基在无水THF异氰酸酯（HMDI）氧化光盘2小时，用THF冲洗并离开干燥。 </li><li>水解水异氰酸酯轴承样品2 - 3小时，干燥。注意:最终氨基官能化的PTFE表面比异氰酸酯承载样品更稳定，并与许多标准交联剂进一步耦合兼容。 </li></ol> 2.固定肽<ol><li>在50mM制备的20％（体积/体积）二甘醇二缩水甘油基醚（二环氧化物）中的溶液的碳酸盐缓冲液，pH 9。 </li><li>将带有标记的朝上的胺化光盘单独成24孔板，并添加1.5环氧化物溶液。保证样品的全面覆盖。孵育2小时，洗2次用水和一次用碳酸盐缓冲液。 </li><li>加入50μl0.5毫克/毫升的肽（ 例如 ，REDV）在50mM碳酸盐缓冲液，pH 9含有0.01％NaN 3的的，到一个新的24孔板的各个孔的底部，并小心地将环氧官能化的光盘向上-down（ 即 ，标记面朝下）到肽溶液的下降。确保良好的底部和聚四氟乙烯盘之间的空间被完全润湿由于毛细管作用。 </li><li>孵育在湿室（ 例如，任何可密封塑料盒具有紧闭盖）与湿润气氛（通过将湿纸巾的底部）至少3小时或过夜。 注意:尽管在REDV基序可被视为充分表征，加扰或反向氨基酸序列可以被包括作为一个附加的阴性对照。 </li><li>用水洗涤三次，并在50％的异丙醇/水消毒至少30分钟。至细胞接种，RINS之前E在无菌磷酸盐缓冲盐水（PBS）中的样本。 </li></ol> 3.细胞接种<ol><li>使用标准的细胞培养过程17,18血管内皮细胞生长。 </li><li>放置与改性朝上肽缀合的样品在24孔板中。使用未经处理的PTFE盘作为对照。 </li><li>种子5×10 4的HUVEC以每孔2毫升培养基并孵育在37℃的恒温箱中4小时和5％的CO 2。 </li><li>取出光盘，仔细冲洗去除独立的细胞，并转移到一个新的24孔板。加入2毫升的新鲜培养基中并孵育所需的周期（ 例如，24小时，1个W 等 ）。介质改变每两天。 </li><li>制备1mg / ml的钙黄绿素-AM的二甲亚砜（DMSO）的储备溶液。 </li><li>生长后细胞中除去培养基，用PBS洗并加入PBS中含1微升/毫升钙黄绿素-AM染色（ 例如，1微升每毫升PBS中的储备液）。轻轻搅动和incubat向在37℃下在黑暗中45分钟。 </li><li>用PBS洗涤，并立即在配备标准FITC滤光片的荧光显微镜显微照片（前488纳米，515 EM纳米）。用放大100倍。执行从未修改三份以及处理的样品。 </li><li>使用ImageJ软件确定由细胞定植的区域。或者手动计数细胞或使用ImageJ的。这两种方法都将给出关于各表面上的附着和细胞生长的类似信息。 </li></ol>

<ol>
	<li>Doctor, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+various+prosthetic+materials+and+newer+meshes+for+hernia+repairs.">Evaluation of various prosthetic materials and newer meshes for hernia repairs.</a> J. Minim. Access Surg. 2, 110-116 (2006).</li><li>Zaghal, 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=Update+on+totally+implantable+venous+access+devices.">Update on totally implantable venous access devices.</a> Surg. Oncol. 21, 207-215 (2012).</li><li>Niu, G., Sapoznik, E., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+blood+vessels.">Bioengineered blood vessels.</a> Exp. Opin. Biol. Th. 14, 403-410 (2014).</li><li>Wang, M. -. J., Tsai, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials in Blood-Contacting Devices: Complications and Solutions. , (2010).</li><li>de Mel, A., Jell, G., Stevens, M. M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalization+of+biomaterials+for+accelerated+in+situ+endothelialization:+a+review.">Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review.</a> Biomacromolecules. 9, 2969-2979 (2008).</li><li>Zdrahala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+caliber+vascular+grafts.+Part+I:+state+of+the+art.">Small caliber vascular grafts. Part I: state of the art.</a> J. Biomat. Appl. 10, 309-329 (1996).</li><li>Cleary, M. 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=Vascular+tissue+engineering:+the+next+generation.">Vascular tissue engineering: the next generation.</a> Trends Mol. Med. 18, 394-404 (2012).</li><li>Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+and+other+recognition+sequences+for+integrins.">RGD and other recognition sequences for integrins.</a> Annu. Rev. Dev. Bi. 12, 697-715 (1996).</li><li>Lei, Y., Remy, M., Labrugere, C., Durrieu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+immobilization+on+polyethylene+terephthalate+surfaces+to+study+specific+endothelial+cell+adhesion,+spreading+and+migration.">Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration.</a> J. Mat. Sci. Mater. M. 23, 2761-2772 (2012).</li><li>Gabriel, 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=Covalent+RGD+Modification+of+the+Inner+Pore+Surface+of+Polycaprolactone+Scaffolds.">Covalent RGD Modification of the Inner Pore Surface of Polycaprolactone Scaffolds.</a> J. Biomat. Sci.. Polym. E. 23, 941-953 (2012).</li><li>Ceylan, H., Tekinay, A. B., Guler, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adhesion+and+growth+of+vascular+endothelial+cells+on+bioactive+peptide+nanofiber+functionalized+stainless+steel+surface.">Selective adhesion and growth of vascular endothelial cells on bioactive peptide nanofiber functionalized stainless steel surface.</a> Biomaterials. 32, 8797-8805 (2011).</li><li>Chlupac, J., Filova, E., Bacakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+replacement:+50+years+of+development+and+tissue+engineering+paradigms+in+vascular+surgery.">Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery.</a> Physiol. Res. / Academia Scientiarum Bohemoslovaca. 58, 119-139 (2009).</li><li>Wise, S. G., Waterhouse, A., Kondyurin, A., Bilek, M. M., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-based+biofunctionalization+of+vascular+implants.">Plasma-based biofunctionalization of vascular implants.</a> Nanomedicine UK. 7, 1907-1916 (2012).</li><li>Mikulikova, R., 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=Cell+microarrays+on+photochemically+modified+polytetrafluoroethylene.">Cell microarrays on photochemically modified polytetrafluoroethylene.</a> Biomaterials. 26, 5572-5580 (2005).</li><li>Gabriel, M., Dahm, M., Vahl, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wet-chemical+approach+for+the+cell-adhesive+modification+of+polytetrafluoroethylene.">Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene.</a> Biomed. Mater. 6, 035007 (2011).</li><li>Gabriel, M., van Nieuw Amerongen, G. P., Van Hinsbergh, V. W., Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+grafting+of+RGD-motif-containing+peptide+on+the+surface+of+polycaprolactone+films.">Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films.</a> J. Biomat. Sci.. Polym. E. 17, 567-577 (2006).</li><li>Larsen, C. C., Kligman, F., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+RGD+fluorosurfactant+polymer+modification+of+ePTFE+on+endothelial+cell+adhesion,+growth,+and+function.">The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.</a> Biomaterials. 27, 4846-4855 (2006).</li><li>Gabriel, M., Nazmi, K., Veerman, E. C., Nieuw Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+LL-37-grafted+titanium+surfaces+with+bactericidal+activity.">Preparation of LL-37-grafted titanium surfaces with bactericidal activity.</a> Bioconjugate Chem. 17, 548-550 (2006).</li><li>Lotz, A., Heller, M., Brieger, J., Gabriel, M., F&#246;rch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Plasma+Polymerized+Thin+Films+and+Attachment+of+Biomolecules+to+Influence+HUVEC-Cell+Adhesion.">Derivatization of Plasma Polymerized Thin Films and Attachment of Biomolecules to Influence HUVEC-Cell Adhesion.</a> Plasma Process Polym. 9, 10-16 (2012).</li></ol>

The authors have nothing to disclose.

PTFE的表面改性协议的详细描述由具有消除从聚合物主链的氟开始， 如图6所示的连续步骤，其结果，在形成的层，其中包含的共轭碳-碳双键的充裕量根据深褐色的颜色，在萘基处理开发。用酸性的过氧化氢标准氧化产生伴随着增亮到淡褐色羟基化表面，这反映了双键的损失。该方法显然是通过IR光谱证明。 为了实现更普遍适用的功能性，悬垂OH-基团通过用所述NC...

在医学中使用的各种聚合物已批准了一段时间没有表现出增强的生物相容性， 即缺乏细胞粘合性，纤维化封装和血栓的诱导，仅举几例。生物材料和生物系统之间的相互作用主要发生在植入物的表面上。因此，研究集中于表面改性，以用于期望的应用创建适当的属性，同时留下的材料的整体性质不受影响。聚四氟乙烯（PTFE）作为生理惰性聚合物在许多医学领域中使用，如疝气手术网1，医疗端口2，以及最重要的是，血管移植物3。 特别是在血液接触的情况下的PTFE的疏水性导致血浆成分的后果血小板粘附和作为非特异性吸附，往往造成thrombotiç事件和移植物4的闭塞。此外，PTFE，最喜欢的聚合物，不支持细胞粘附和覆盖这将是非常理想的，诱导血管移植物5的内（管腔）表面上的内皮细胞（EC）的一个有益的层的形成。仿生内皮有望实现其许多天然等价物的功能，尤其是它的抗凝血性能6。一般的仿生修正策略是基于专门的赋予与细胞粘着材料同时使材料整体性能不受影响的概念。另外，血小板的粘附可以通过将抗粘合剂（防污）属性7被减小。各种肽-大多来自细胞外基质的蛋白的-已被描述，通过结合至细胞受体，属于类整8的强烈增强细胞粘附。该会ST-已知在这方面的例子是，与大多数细胞类型相互作用的肽精氨酸 - 甘氨酸 - 天冬氨酸（RGD）。其它氨基酸序列是通过在特定的细胞中只表达整联的认可。例如，精氨酸-谷氨酸-天冬氨酸-缬氨酸（REDV）和Tyr -异亮氨酸-甘氨酸-丝氨酸-精氨酸（YIGSR）已发现结合于内皮以特定方式9。这种肽的共价固定已在固有的非粘性材料，包括金属和聚合物10,11-过多进行。 PTFE多孔，更精确地膨胀型PTFE（膨体聚四氟乙烯） -与聚对苯二甲酸乙酯（PET）的沿-是用于生产血管的最重要的材料移植物12。对适当的治疗建立物理技术，如等离子体改性13或通过光化学方法14，是由以下事实多孔和/或管状的结构没有分别的孔或腔内容易可治疗阻碍。湿化学聚四氟乙烯是一项困难的任务，因为抵抗大多数化学攻击15含氟聚合物的高惰性性质。 在本文中，我们描述了一个共价修饰策略相对简便的方法。从过程适于渲染聚四氟乙烯粘合，官能团分别对材料表面作为生物活性分子的进一步缀合锚固点创建。

<table><tbody><tr><td>PTFE foil 0.5 mm</td><td>Cadillac Plastic&amp;nbsp;</td><td>n/a</td><td></td></tr><tr><td>REDV peptide</td><td>Genecust</td><td>n/a</td><td>custom synthesis &gt;95% purity</td></tr><tr><td>iso-propanol</td><td>Sigma Aldrich</td><td>34965</td><td></td></tr><tr><td>tetrahydrofurane (THF)</td><td>Sigma Aldrich</td><td>401757</td><td></td></tr><tr><td>dimethylsulfoxide</td><td>Sigma Aldrich</td><td>D8418</td><td></td></tr><tr><td>molecular sieve 3 &amp;Aring;</td><td>Sigma Aldrich</td><td>208574</td><td></td></tr><tr><td>sodium metal</td><td>Sigma Aldrich</td><td>483745</td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td></td></tr><tr><td>naphthalene</td><td>Sigma Aldrich</td><td>147141</td><td></td></tr><tr><td>hydrogen peroxide 30%</td><td>Sigma Aldrich</td><td>95321</td><td></td></tr><tr><td>trichloroacetic acid</td><td>Sigma Aldrich</td><td>T6399&amp;nbsp;</td><td></td></tr><tr><td>diethylene glycol diglycidyl ether</td><td>Sigma Aldrich</td><td>17741</td><td></td></tr><tr><td>hexamethylene diisocyanate (HMDI)</td><td>Sigma Aldrich</td><td>52650</td><td></td></tr><tr><td>Calcein-AM</td><td>Sigma Aldrich</td><td>56496</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6014&amp;nbsp;</td><td></td></tr><tr><td>sodium azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>24 well plates</td><td>Greiner-Bio-One</td><td>662 160</td><td></td></tr><tr><td>ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td><td>&amp;nbsp;Nicolet</td><td>n/a</td><td></td></tr><tr><td>fluorescence microscope Olympus X-70</td><td>Olympus</td><td>n/a</td><td></td></tr><tr><td>humbilical vein endothelial cells (HUVECs)</td><td>Lonza</td><td>n/a</td><td></td></tr><tr><td>ePTFE vascular graft</td><td>Gore</td><td>n/a</td><td></td></tr></tbody></table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

赋予材料与细胞粘接性能的表面是生物材料研究和组织工程的共同战略。这是对于具有在医学长期使用，因为这些材料是公引进新合成的聚合物可避免相关特征和法律问题已核准的聚合物特别有趣。聚四氟乙烯（PTFE）是用于制造血管移植物的最经常使用的材料之一，但该聚合物缺乏细胞粘合促进功能。内皮，即移植物的内表面与血管内皮细胞的汇合层完全覆盖被认为是关键的最佳性能，主要是通过降低人工接口的血栓形成。 本研究探讨的内皮细胞上的肽，改性PTFE的生长和这些结果未修饰基片上得到的那些进行比较。与耦合内皮细胞粘附肽精氨酸 - 谷氨酸 - 天冬氨酸 - 缬氨酸（REDV）经由使用萘基试剂钠含氟聚合物的活化进行，接着后续缀合的步骤。细胞培养使用在肽 - 固定化材料的人脐静脉内皮细胞（HUVECs）和优异的细胞生长证实超过两个星期内完成。

的关键化学反应步骤的结果是通过IR光谱（ 图1）进行监测。用萘钠初次激活产生双键 - 对一个未成年人的程度 - OH-功能。该信号指示C = C键消失在氧化，得到表面轴承几乎完全的羟基基团。进一步规范结合的步骤分析，这里没有显示。由于激活和氧化的颜色变化是在与所使用的预期化学协议:共轭双键系统预计是褐色及其损失导致增亮（ 图2）。?...

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Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

湿化学和肽固定在聚四氟乙烯为改善细胞粘附

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

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Bioengineering

7.6K 次观看.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

视频: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

保利的微波辅助功能化（乙二醇）和On-树脂链聚合反应，并形成水凝胶的肽使用

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

科研

化学

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生物化学

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

毛细管电泳监视肽接枝壳聚糖膜实时

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

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

生物工程

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

基于AFM的力光谱单分子共价附价

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...

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 ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

通过CD47衍生肽固定，缓解血系细胞附着到金属植入物

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

使用水凝胶图案化技术控制细胞粘附，用于牵引力显微镜的应用

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

评估生物功能自组装肽的细胞粘附和形态学

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

作者聚焦：结直肠癌类器官的超短肽基质优化

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

在超短自组装肽基质中制备和表征来自 SW1222 细胞系的结直肠癌类器官

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...