作者要感谢毛里齐奥教授马西的富有成果的讨论和小姐基娅拉阿莱格雷蒂语言编辑。作者的研究是由阪乔瓦尼Ricercatori 2010部（Ministero della Salute的GR-2010- 2312573）的支持。

注:按照来文中使用的化学品。线性RGD购买，但是它可以通过标准Fmoc固相肽合成16,19来制备。溶剂是分析纯。透析需要与男女截止等于3500大使用膜。所合成的化合物的特征在于记录在400MHz分光计使用氯仿1 H NMR谱（CDCL 3）或氧化氘（D 2 O）作为溶剂，和化学位移被报告为每百万份δ值。此外，水凝胶使用KBr压片技术进行FT-IR分析和他们的物理特征包括使用倒置试管在37℃下评估凝胶化的研究。 1. 4- Azidobutanoyl合成氯化物1 <ol><li>在10ml二氯甲烷和0.5ml二甲基甲酰胺溶解500毫克的4- azidobutanoic酸（3.90毫摩尔）。 </li><li>冷却该溶液在0℃下，使用冰浴。 </li><li>添加505微升草酰氯（5.85毫摩尔）至5毫升二氯甲烷中，并缓慢加入滴加至反应体系中，同时搅拌。 </li><li>用冰浴于0℃1小时后，返回到室温。 </li><li>除去用旋转蒸发器在减压下除去溶剂。 </li><li>通过1 H-NMR谱表征所得产物，在CDCl 3中16的样品溶解。 </li></ol> 2. RGD的叠氮化物衍生物2的合成<ol><li>在1ml的1M NaOH中溶解50毫克RGD的（0.145毫摩尔）。 </li><li>在2ml四氢呋喃中溶解的1（0.16毫摩尔）24毫克。 </li><li>用冰浴的所有RGD溶液添加到溶液1中滴加在0℃。 </li><li>返回到室温并搅拌过夜。 </li><li>加入1ml的1M HCl。 </li><li>除去用旋转蒸发器在减压下除去溶剂。 </li><li>表征OBTained通过1 H-NMR光谱法的产品，将样品溶解在D 2 O中16。 </li></ol> 3. PAA炔修改3 <ol><li>溶解200毫克的35％w / w的在15毫升的蒸馏水中的PAA溶液（2.8毫摩尔）。 </li><li>加入15.4毫克盐酸炔丙基胺（0.20毫摩尔）的。 </li><li> 1体积/体积乙腈:14毫升1溶解42.8毫克1-羟基苯并三唑水合物的三唑（HOBt，0.28毫摩尔）通过加热蒸馏水溶液至50℃。 </li><li>添加所有的HOBt解决方案，以解决PAA在室温下。 </li><li> 53.6毫克ethyldimethylaminopropylcarbodiimide的（EDC，0.28毫摩尔）添加到反应混合物中。 </li><li>使用的1M HCl以调节pH至5.5，并过夜搅拌该反应体系在室温下。 </li><li>透析溶液。溶解11.2克氯化钠于2L蒸馏水，然后添加37％0.2毫升w / w的盐酸。利用透析膜与男女 3.5 kDa的的截止解决方案。 </li><li> PERFORM透析三天。每日用2L含有0.2毫升37％w / w的盐酸新制备的蒸馏水的改变透析溶液。 </li><li>储存在-80℃的最终解决方案。根据制造商的协议在冻干机冻干它。 </li><li>通过1 H-NMR谱表征官能化聚合物，在D 2 O中16中的样品溶解。 </li></ol> 4. PAA-RGD的合成聚合物4 <ol><li>溶解78毫克PAA的10毫升蒸馏水改性炔3（1.083毫摩尔）。 </li><li>在5毫升四氢呋喃溶解25毫克RGD叠氮化物2衍生物（0.0722毫摩尔）的。 </li><li>所有RGD溶液添加到聚合物溶液中。 </li><li>添加2.2毫克的碘化铜（0.0116毫摩尔）和2.2毫克抗坏血酸钠（0.0111毫摩尔）的。 </li><li>回流所得混合物过夜，在60℃，同时搅拌。 </li><li>冷却该混合物至25℃。 </li><li> DialyzË解决方案。溶解11.2克氯化钠于2L蒸馏水，然后添加37％0.2毫升w / w的盐酸。利用透析膜与男女 3.5 kDa的的截止解决方案。 </li><li>三天进行透析治疗。每日用2L含有0.2毫升37％w / w的盐酸新制备的蒸馏水的改变透析溶液。 </li><li>储存在-80℃的最终解决方案。根据制造商的协议在冻干机冻干它。 </li><li>通过1 H-NMR谱表征所得产物，在D 2 O中16中的样品溶解。 </li></ol> 5. RGD功能化合成水凝胶<ol><li>准备PBS。溶解645毫克的PBS盐在50毫升的蒸馏水。 </li><li>混合40毫克卡波姆和9毫升PBS（步骤5.1）的10毫克官能PAA 4，在室温下，直至完全溶解（30分钟）。 </li><li> 400毫克的PEG添加到该溶液中，并保持搅拌45分钟。 </li><li>停止搅拌并允许系统沉降30分钟。 </li><li>使用的1N NaOH以调节pH至7.4。 </li><li>到5ml所获得的混合物中，添加25毫克琼脂糖粉末。 </li><li>照射微波辐射系统在500瓦至沸腾，一时间通常为30秒和1分钟之间，并且电磁加热至80℃。 </li><li>离开暴露于室温下将该混合物直到其温度降低至50℃，加入5毫升的PBS（步骤5.1）的，以便获得在一个1的溶液:1体积比。 </li><li>准备12多孔板含钢瓶用直径1.1厘米</li><li>就拿从溶液500微升等分，并将它们放置到每个钢瓶。 </li><li>离开休息45分钟，直至系统完全凝胶化。 </li><li>除去使用不锈钢镊子以获得水凝胶的圆柱体。 </li></ol> 6.装载的治疗手段（药物或细胞） <ol><li>重复ST每股收益5.1-5.7。 </li><li>当混合物（已经在溶胶状态）达到37℃，加入5毫升含有所需药物溶液或细胞培养物的溶液中，以便在一个1以获得最终系统:1的体积比。 </li><li>重复步骤5.9-5.12以获得与凝胶内的物理包埋biocompounds聚合物网络。 </li></ol> 7.水凝胶表征<ol><li> FT-IR分析<ol><li>后形成凝胶，泡的原料合成的水凝胶中的一个在2.5ml蒸馏水中24小时。 </li><li>除去，其中水凝胶浸入水性介质和冷冻干燥液体N 2。 </li><li>根据KBr压片技术层压水凝胶样品。 <ol><li>添加铲满溴化钾到玛瑙研钵。取的水凝胶样品的少量并用溴化钾粉末混合（KBr压量，或者仅仅足以覆盖刮刀的尖端约0.1-2％）。 </li><li>研磨混合物直到粉末细而均匀。&lt;/ LI&gt; <li>使用KBr压片盒，以形成红外沉淀。按使用手动实验室按粉末:在压力容量等于5吨，然后进行3分钟，在10吨压力容量3分钟。 </li><li>释放压力，得到最终的沉淀为均质和透明的外观。将粒料进红外样本保持器，并运行的频谱16。 </li></ol></li></ol></li><li>凝胶研究<ol><li>填充2ml微量管900微升的PBS并平衡至37℃。 </li><li>添加100微升制备的聚合物溶液，以形成水凝胶，并在37℃下孵育。 </li><li>倒置该管，并且如果凝胶在1,2，5，10和20分钟流向观察。记录在其中凝胶不流动的凝胶化的时间。 </li></ol></li></ol>

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The authors state no conflict of interest and they have not received any payment in preparation of this manuscript.

The PAA post-polymerization modification with alkyne moieties and the RGD functionalization with the azide group guarantee the formation of a stable bond between the polymer and the peptide. Indeed, triazole serves as a rigid linking unit among the carbon atoms, attached to the 1,4 positions of the 1,2,3-triazole ring and it cannot be cleaved hydrolytically or otherwise. In addition, triazole is extremely difficult to oxidize and reduce, unlike other cyclic structures such as benzenoids and related aromatic heterocycles<...

水凝胶是由亲水性的交联的聚合物，它们是天然或合成的，并且其特征在于，一个独特的三维结构形成的三维网络。这些设备是在药物递送，组织工程，基因的载体和智能传感器1,2的生物医学领域越来越有吸引力。事实上，它们的高水含量，以及它们的流变学和机械性能使之适合候选模拟软组织的微环境，使它们有效工具水溶性细胞因子或生长因子的递送。其中最有希望采用的是作为可注射生物材料携带细胞和生物活性化合物。水凝胶可以通过保持并在生理相关的方式精确地递送干细胞调节信号提高细胞存活和控制干细胞的命运，如在体外和体内实验3,4-观察。这样做的主要优点是有可能保持接种（原位 ）的区域内注射的细胞，减少细胞的叶面积和extravasates进入循环系统洪流，迁移遍布全身，失去靶目标5的量。三维水凝胶网络的稳定性是由于其交联位点，由聚合物链6之间的共价键或内聚力形成。 在此框架下，正交选择的化学品适用于聚合物链能够提高水凝胶演出7的多功能工具。事实上，用适当的化学基团的聚合物的改性可以帮助提供适当的化学，物理和机械性能，以提高细胞生存力以及它们在组织形成使用。以相同的方式，这些技术中以加载细胞或生长因子的凝胶基质中，使用了含RGD肽允许在细胞粘附和存活的改善。 RGD是由三肽精氨酸，甘氨酸和天冬氨酸，这是迄今为止的最有效和最经常使用的三肽，由于其处理多于一个细胞粘附受体的能力和对细胞锚定，行为和生存8,9-其生物学影响。在这项工作中，RGD官能水凝胶的合成进行了研究与设计网络的一个热情细胞微环境特征在于足够的生化特性的目的。 在水凝胶的合成使用微波辐射提供了一个简单的过程，以减少副反应，并在比常规热过程10更短的时间周期得到较高的反应速率和产率。此方法不需要纯化步骤和产量的无菌水凝胶由于聚合物之间的相互作用，并在反应系统11不存在有机溶剂。因此，它可以确保链接到聚合物网络，因为没有模RGD的高百分比ifications需要参与凝胶形成的聚合物的化学基团。羧基，由PAA与卡波姆和羟基，由PEG和琼脂糖，通过缩聚反应产生的水凝胶三维结构。所提到的聚合物可用于水凝胶在脊髓损伤的修复治疗12的合成。这些设备，如报告以前的作品13,14，显示出高生物相容性，以及类似于那些生活的许多组织和触变性质的机械和物理化学性质。此外，它们在原位保持局限性，在注射区域。 在这项工作中，PAA的羧基与炔部分修改（ 图1），和一个RGD叠氮化物化合物合成用的结构（CH 2）N利用该三肽末端基团-NH 2的反应与制备化学化合物- N 3（&lt;STRONG&gt;图2）。随后，将改性的PAA通过CuAAC点击反应15-17（ 图3）的RGD-叠氮化物衍生物进行反应。使用铜（I）催化剂的导致在这两个反应速率和区域选择性的重大改进。该CuAAC反应被广泛用于有机合成和在聚合物科学。它结合了高效率和高耐受性的官能团，并且它是通过使用有机溶剂的影响。高选择性，快速的反应时间和简单的纯化过程允许星形聚合物，嵌段共聚物或链接枝所需部分18的获得。此点击策略使得能够修改聚合物聚合后根据最后的生化应用定制的物理化学性质。所述CuAAC实验条件容易再现（反应不敏感，水，而可以最低限度发生铜氧化），和的性质形成三唑确保了产品的稳定性。使用铜金属可以被认为是一个临界点时，由于对细胞的潜在毒性作用，并在生物微环境，但透析用作纯化方法，以允许完全除去催化剂残留物的。最后，PAA改性RGD在水凝胶的合成（ 图4），将所得网络的物理化学性质使用进行了研究，以检查这些系统如细胞或药物载体的潜在功能。 <img alt="图1" src="/files/ftp_upload/54445/54445fig1.jpg" /> 图1:PAA改性炔合成炔基官能PAA的方案。 "N"表示与羧基与炔丙基反应的单体。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/54445/54445fig2.jpg" /> 图2:。RGD- 叠氮合成 RGD-叠氮衍生物的合成<a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54445/54445fig3.jpg" /> 图3:点击反应 RGD-叠氮衍生物和炔PAA之间的点击反应方案。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54445/54445fig4.jpg" /> 图4:水凝胶SYNThesis。RGD功能化水凝胶合成过程。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

<table><tbody><tr><td>Poly(acrylic acid) solution average M&lt;sub&gt;w&lt;/sub&gt; ~100,000, 35 wt% in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>523925</td><td>CAS 9003-01-4</td></tr><tr><td>Poly(ethylene glycol) 2,000</td><td>Sigma Aldrich</td><td>84797</td><td>CAS 25322-68-3</td></tr><tr><td>Carbomeer 974P</td><td>Fagron</td><td>1387083</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Invitrogen Corp.</td><td>16500-500</td><td>UltraPure Agarose</td></tr><tr><td>RGD peptide</td><td>abcam</td><td>ab142698</td><td></td></tr><tr><td>4-azidobutanoic acid</td><td>Aurum Pharmatech</td><td>Z-2421</td><td>&amp;nbsp;CAS 54447-68-6</td></tr><tr><td>Oxalyl chloride</td><td>Sigma Aldrich</td><td>O8801</td><td>CAS 79-37-8</td></tr><tr><td>Propargylamine hydrochloride 95%</td><td>Sigma Aldrich</td><td>P50919</td><td>CAS 15430-52-1</td></tr><tr><td>Copper(I) iodide</td><td>Sigma Aldrich</td><td>3140</td><td>CAS 7681-65-4</td></tr><tr><td>Sodium ascorbate</td><td>Sigma Aldrich</td><td>Y0000039</td><td>CAS 134-03-2</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td></td></tr><tr><td>Dialysis Membrane</td><td>Spectrum Laboratories, Inc.</td><td>132725</td><td>Spectra/Por 3 Dialysis Membrane&amp;nbsp; Standard RC Tubing&lt;br /&gt;MWCO: 3.5 kD</td></tr></tbody></table>

the synthesis of rgd-functionalized hydrogels as a tool for therapeutic applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

的PAA炔衍生物有效地从聚丙烯酸和炔丙胺合成，如在图1，其中n标签的羧基与胺反应的单体显示。该产物的身份通过1 H-NMR光谱法证实。 图5示出了具有三键改性的PAA的1 H-NMR谱。 <img alt="图5" src="/files/ftp_upload/54445/54445fig5.jpg" /> 图5:1 的PAA的H-NMR谱修改炔</st...

Watch this Scientific Journal Video about The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications at JoVE.com

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

the-synthesis-rgd-functionalized-hydrogels-as-tool-for-therapeutic

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.1K 次观看.  Politecnico di Milano. We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated. 

视频: The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

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

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 ...

生物工程

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫与感染

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

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 ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

破碎散装水凝胶并加工成颗粒状水凝胶，用于生物医学应用

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

强粘性水凝胶明胶O-亚硝基苯甲醛的合成

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...