著者は、言語の編集のために実りある議論のための教授マウリツィオ瑪斯とミスキアラAllegrettiに感謝したいと思います。本研究は、坂東GIOVANI Ricercatori 2010（Ministeroデッラサルー​​テGR-2010- 2312573）によってサポートされています。

注：受信のような化学物質が使用されています。線形RGDを購入し、それは標準的なFmoc固相ペプチド合成16,19によって調製することができます。溶媒は分析グレードのものです。透析はカットオフに等しい3500にダワット Mを有する膜を使用する必要があります。合成された化合物は、溶媒、化学シフトとしてクロロホルム（CDCl 3中）または重水（D 2 O）を使用して400 MHzの分光計で記録された1 H NMRスペクトルにより特徴づけられる百万分率でδ値として報告されます。さらに、ヒドロゲルは、KBrペレット技術を使用してFT-IR分析に供し、その物理的特徴付けは、37℃で反転試験管を用いて評価し、ゲル化の研究を含むています。 4-Azidobutanoyl塩化物1の1の合成<ol><li>ジクロロメタン10ml及びジメチルホルムアミド0.5ml中の4- azidobutanoic酸（3.90ミリモル）の500ミリグラムを溶解します。 </li><li> 0°Cで溶液を冷却氷浴を使用して。 </li><li>攪拌しながら、反応系に滴下して加えるゆっくりジクロロメタン5mlに塩化オキサリル（5.85ミリモル）の505μLを加え、。 </li><li>氷浴を用いて0℃で1時間後、室温に戻します。 </li><li>ロータリーエバポレーターを用いて減圧下で溶媒を除去します。 </li><li> CDCl 3中16試料を溶解し、1 H-NMR分光法により得られた生成物を特徴付けます。 </li></ol> RGD-アジド誘導体2の2の合成<ol><li> 1MのNaOH 1mlにRGDを50mg（0.145ミリモル）を溶解させます。 </li><li>テトラヒドロフラン2ml中の1（0.16ミリモル）の24ミリグラムを溶解します。 </li><li>氷浴を用いて0℃で溶液1を滴下してRGDソリューションのすべてを追加します。 </li><li>室温に戻し、一晩攪拌。 </li><li> 1MのHClの1ミリリットルを追加します。 </li><li>ロータリーエバポレーターを用いて減圧下で溶媒を除去します。 </li><li> OBTを特徴づけますained 1 H-NMR分光法による製品、D 2 O 16でサンプルを溶解させます。 </li></ol> 3. PAAアルキン変形例3 <ol><li>蒸留水15ml中のPAA溶液（2.8ミリモル）のw / wの35％の200 mgの溶解。 </li><li>プロパルギルアミン塩酸塩（0.20ミリモル）の15.4 mgの追加。 </li><li> 1（v / v）のアセトニトリル：50°Cに加熱することにより、蒸留水溶液1 14mlの中の1-ヒドロキシベンゾトリアゾール水和物（HOBT、0.28ミリモル）の42.8 mgの溶解。 </li><li>室温でのPAA溶液にHOBtをソリューションのすべてを追加します。 </li><li>反応混合物にethyldimethylaminopropylcarbodiimideの53.6 mgの（EDC、0.28ミリモル）を追加します。 </li><li> pHを5.5に調整し、室温で一晩反応系を撹拌し、1MのHClを使用してください。 </li><li>ソリューションを透析。蒸留水2リットルに塩化ナトリウムの11.2グラムを溶解し、その後、塩酸ワット/ wの37％の0.2ミリリットルを追加します。 3.5キロダルトンのM wのカットオフを有する膜を用いて溶液を透析。 </li><li> Perfo3日間のrm透析。塩酸w / wの37％の0.2ミリリットルを含む新たに調製した蒸留水2Lで毎日透析液を変更します。 </li><li> -80℃での最終溶液を保管してください。製造業者のプロトコルに従って凍結乾燥機でそれを凍結乾燥。 </li><li> D 2 O 16のサンプルを溶解し、1 H-NMR分光法によって官能化ポリマーを特徴付けます。 </li></ol> PAA-RGDポリマー4の4の合成<ol><li> PAAの78 mgを蒸留水10mlにアルキン3（1.083ミリモル）を改変溶かします。 </li><li>テトラヒドロフラン5mlにRGDアジド誘導体2（0.0722ミリモル）25mgを溶解させます。 </li><li>高分子溶液にRGDソリューションのすべてを追加します。 </li><li>ヨウ化銅（0.0116ミリモル）およびアスコルビン酸ナトリウムの2.2ミリグラム（0.0111ミリモル）の2.2 mgの追加。 </li><li>攪拌しながら、60℃で一晩、得られた混合物を還流。 </li><li> 25℃に混合物を冷却。 </li><li> Dialyzソリューションを電子。蒸留水2リットルに塩化ナトリウムの11.2グラムを溶解し、その後、塩酸ワット/ wの37％の0.2ミリリットルを追加します。 3.5キロダルトンのM wのカットオフを有する膜を用いて溶液を透析。 </li><li> 3日間透析を行います。塩酸w / wの37％の0.2ミリリットルを含む新たに調製した蒸留水2Lで毎日透析液を変更します。 </li><li> -80℃での最終溶液を保管してください。製造業者のプロトコルに従って凍結乾燥機でそれを凍結乾燥。 </li><li> D 2 O 16のサンプルを溶解し、1 H-NMR分光法により得られた生成物を特徴付けます。 </li></ol> 5. RGD官能ヒドロゲルの合成<ol><li> PBSを準備します。蒸留水50mlにPBS塩645 mgの溶解。 </li><li>カルボマーの40 mgであり、官能基化PAA 4の10 mgを完全に溶解（30分）まで、室温で、PBSの9ミリリットル（ステップ5.1）でブレンド。 </li><li>溶液に、PEGの400ミリグラムを加え、45分間攪拌し続けます。 </li><li>攪拌を停止し、システムが30分間沈降することを可能にします。 </li><li> pHを7.4に調整するために1NのNaOHを使用してください。 </li><li>得られた混合物の5ミリリットルに、アガロース粉末25mgを追加します。 </li><li>通常は30秒と1分の間の時間のために、沸騰するまで、500 Wのマイクロ波放射を使用してシステムを照射し、電磁的に80℃まで加熱します。 </li><li>その温度が50℃になるまで室温にさらされ、混合物のままにして、1の溶液を得るためには、PBS（ステップ5.1）の5ミリリットルを追加：1の体積比を。 </li><li> 1.1センチメートルの直径を有する鋼製シリンダーを含む12マルチウェルプレートを準備します。 </li><li>溶液から500μlのアリコートを取り出し、各鋼シリンダに配置します。 </li><li>システムを完全にゲル化するまで45分間安静にしておきます。 </li><li>ヒドロゲルを得るために、ステンレス製のピンセットを用いてシリンダーを削除します。 </li></ol>治療ツール（薬物または細胞）の6.のロード<ol><li>目を繰り返しEPS 5.1から5.7まで。 </li><li> 1の体積比（既にゾル状態で）、混合物を37°Cに達すると、1で最終的なシステムを得るために、所望の薬物溶液または細胞培養物を含む溶液5mlを加えます。 </li><li>繰り返しは、物理的にゲル内に捕捉biocompoundsを有するポリマーネットワークを得るために5.9から5.12を繰り返します。 </li></ol> 7.ヒドロゲルキャラクタリゼーション<ol><li> FT-IR分析<ol><li>ゲル形成後、24時間蒸留水2.5 ml中に合成さヒドロゲルのいずれかを浸します。 </li><li>ヒドロゲルが沈んでいる水性媒体を取り出して、凍結乾燥した液体N 2で。 </li><li> KBrペレット法に従ってヒドロゲルサンプルをラミネート。 <ol><li>瑪瑙乳鉢にKBrを完全へらを追加します。ヒドロゲル少量のサンプルを取り、のKBr粉末と混合（臭化カリウム量、またはスパチュラの先端をカバーするのに十分なだけの約0.1から2パーセント）。 </li><li>粉末が微細かつ均一になるまで混合物を粉砕します。&lt;/李&gt; <li> IRペレットを形成するために、KBrペレットキットを使用してください。手動の実験用プレスを使用した粉末を押す：5トンに等しい圧力容量で3分間、その後3分間10トンの圧力容量で。 </li><li>外観のように均質かつ透明な最終ペレットを得るために圧力を解放します。 IRサンプルホルダーにペレットを挿入し、スペクトル16を実行します。 </li></ol></li></ol></li><li>ゲル化研究<ol><li> PBSの900μlの2ミリリットルのマイクロチューブを記入し、37℃に平衡化。 </li><li>ヒドロゲルを形成し、37℃でインキュベートし製造された高分子溶液100μlを加えます。 </li><li>管を反転し、ゲルを1、2、5、10及び20分で流れる場合に観察。ゲルは、ゲル化時間として流れていない時刻を記録します。 </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<...

ヒドロゲルは天然または合成された親水性架橋ポリマーで形成された三次元ネットワークであり、独特の三次元構造によって特徴付けられます。これらのデバイスは、薬物送達、組織工学、遺伝子キャリアとスマート・センサ1,2の生物医学分野でますます魅力的です。実際、その高い水含有量、ならびにそれらのレオロジーおよび機械的性質は、それらの適切な候補が軟組織微小環境を模倣し、水溶性のサイトカインまたは増殖因子の送達のためにそれらの効果的なツールにするために行います。最も有望な使用の一つは、細胞および生物活性化合物を運ぶ注射用生体材料としてです。 インビトロおよびインビボ実験3,4で観察されたようなヒドロゲルは、保持して正確な生理学的関連性の様式で幹細胞の調節シグナルを送達することによって、細胞の生存および制御幹細胞の運命を改善することができます。このの主要な利点は、可能性であります体の上にすべてを移行し、目標の目標5を失って 、循環急流にエリアとextravasatesを離れる細胞の量を最小限に抑え、（ その場で ）接種のゾーン内注入した細胞を維持します。三次元ハイドロゲルネットワークの安定性は、ポリマー鎖の6間の共有結合または凝集力によって形成され、その架橋部位です。 このフレームワークでは、直交選択的化学は、ポリマー鎖に適用されるが、ヒドロゲル公演7を向上させることができる汎用性の高いツールです。実際、適切な化学基を有するポリマーの修飾は、細胞生存率および組織形成におけるそれらの使用を強化するために適切な化学的、物理的および機械的特性を提供するのに役立つ可能性があります。同様に、ゲルマトリックス内の細胞または成長因子をロードするための技術のうち、RGDペプチドの使用は、細胞接着及び生存の改善を可能にします。 RGDは構成されるトリペプチドでありますアルギニン、グリシン、およびはるかに複数の細胞接着受容体と細胞アンカー、行動および生存8,9にその生物学的影響に対処する能力のために最も効果的でしばしば用いられるトリペプチドであるアスパラギン酸の。この作業では、RGD官能ヒドロゲルの合成は、もてなしの細胞微小環境のために十分な生化学的特性によって特徴付けネットワークを設計することを目的として検討されています。 ヒドロゲルの合成におけるマイクロ波照射の使用は、副反応を最小限に抑え、従来の熱プロセス10に比べて短時間でより高い反応速度と収率を得るための簡単な手順を提供します。この方法は、ポリマーとの相互作用及び反応システム11中の有機溶媒の非存在に起因する精製工程及び収率滅菌ヒドロゲルを必要としません。したがって、それは、MODためのポリマーネットワークに結合されたRGDの高い割合を確保しますificationsは、ゲル形成に関与するポリマーの化学基に必要とされます。カルボキシル基は、PAAおよびカルボマー、ならびにヒドロキシル基、PEGから、アガロースから、重縮合反応によりヒドロゲルの三次元構造を生じます。言及されたポリマーは、脊髄損傷の修復処理12におけるヒドロゲルの合成のために使用されます。以前に報告されているようにこれらのデバイスは、高い生体適合性だけでなく、多くの生体組織のとチキソトロピー性のものと似ている機械的および物理化学的特性を示し、13,14を動作します。さらに、それらは、注射ゾーンで、 その場に局在したままです。 この研究では、PAAのカルボキシル基は、アルキン部分（ 図1）を用いて修飾され、及びRGD -アジド化合物は、構造を用いて調製化合物とトリペプチドの末端基-NH 2（CH 2）の反応性を利用するn個合成されます- N 3（&lt;強い&gt;図2）。その後、変更されたPAAはCuAACのクリック反応15-17（ 図3）を介してRGD-アジド誘導体と反応します。銅（I）触媒の使用は、反応速度及び位置選択性の両方の主要な改善につながります。 CuAAC反応は広く、有機合成およびポリマー科学において使用されます。これは、官能基を高効率、高耐性を兼ね備え、かつ、有機溶剤を使用することによって影響を受けません。高選択性、速い反応時間および簡単な精製手順は、所望の部分18を移植する星型ポリマー、ブロックコポリマーまたは鎖の取得を可能にします。このクリック戦略は、最終的な生化学的用途に応じて物理化学的性質をカスタマイズするために重合後のポリマーを修正することが可能となります。 CuAAC実験条件は簡単に（銅の酸化を最小限に発生する可能性があるのに対し、反応は、水の影響を受けない）、再現性、および自然のです形成されたトリアゾールは、製品の安定性を確保します。銅金属の使用は、細胞に対する潜在的な毒性効果および生物学的微小環境において、臨界点と考えることができるが、透析は、触媒残留物の完全な除去を可能にするための精製方法として使用されます。最後に、PAAは、RGD細胞または薬物担体としてのこれらのシステムの潜在的な機能を確認するために、検討されているヒドロゲルの合成（ 図4）、得られたネットワークの物理化学的特性で使用される修飾しました。 <img alt="図1" src="/files/ftp_upload/54445/54445fig1.jpg" /> 図1：PAAは、アルキンの合成を修正アルキン基とPAAの官能化のスキームを;。 「n」はプロパルギルアミンと反応するカルボキシル基を有するモノマーを示しています。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig1large.jpg" target="_blank">ご覧になるにはこちらをクリックしてください。</a>この図の拡大版。 <img alt="図2" src="/files/ftp_upload/54445/54445fig2.jpg" /> 図2：RGD- アジド合成 RGD-アジド誘導体の合成<a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54445/54445fig3.jpg" /> 図3：RGD-アジド誘導体とアルキン-PAA間のクリック反応の反応スキームをクリックしてください 。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54445/54445fig4.jpg" /> 図4：ハイドロゲルSYNThesis。RGD官能ヒドロゲルの合成手順。 <a href="http://ecsource.jove.com/files/ftp_upload/54445/54445fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</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.

nは、カルボキシル基のアミンと反応するモノマーラベル図1に示したようにPAAのアルキン誘導体を効率的に、ポリアクリル酸、およびプロパルギルアミンから合成されます。生成物の同定は、1 H-NMR分光法によって確認される。 図5は、三重結合で変性されたPAAの1 H-NMRスペクトルを示す図 。 <p class="jove_content" fo:keep-together.within-pa...

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.

治療用途のためのツールとしてのRGD官能ヒドロゲルの合成

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

Research

JoVE Journal

Bioengineering

11.1K ビュー.  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. 

ビデオ: 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.

マイクロ波支援ポリの機能化（エチレングリコール）と連鎖重合で使用するためには樹脂ペプチドおよびヒドロゲル形成

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 ...

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.

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

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

生化学

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.

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

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・感染学

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.

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

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

生物工学

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.

自己組織化反応押出、マイクロフルイディクス, 経由で複数の長さスケールでの分解性感温ハイドロゲルを加工・ エレクトロスピニング

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.

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

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.

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

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

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

細胞および薬物送達用途のための注射可能な高分子ポリマーナノ粒子ヒドロゲル

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

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.

バルクヒドロゲルの断片化と生物医学的応用のための粒状ヒドロゲルへの加工

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.

強接着性ハイドロゲルゼラチンO-ニトロソベンズアルデヒドの合成

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

治療用途のためのツールとしてのRGD官能ヒドロゲルの合成

要約

さらに動画を探す

この動画の章

マイクロ波支援ポリの機能化（エチレングリコール）と連鎖重合で使用するためには樹脂ペプチドおよびヒドロゲル形成

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

模様の作成3D細胞培養のための2つの成分のために温度応答性ハイドロゲルリバース金型の印刷

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

自己組織化反応押出、マイクロフルイディクス, 経由で複数の長さスケールでの分解性感温ハイドロゲルを加工・エレクトロスピニング

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

細胞および薬物送達用途のための注射可能な高分子ポリマーナノ粒子ヒドロゲル

バルクヒドロゲルの断片化と生物医学的応用のための粒状ヒドロゲルへの加工

強接着性ハイドロゲルゼラチンO-ニトロソベンズアルデヒドの合成

治療用途のためのツールとしてのRGD官能ヒドロゲルの合成

要約

さらに動画を探す

この動画の章

マイクロ波支援ポリの機能化（エチレングリコール）と連鎖重合で使用するためには樹脂ペプチドおよびヒドロゲル形成

細胞培養用ヒドロゲルの蛋白質ベースのアーキテクチャの簡単な操作

模様の作成3D細胞培養のための2つの成分​​のために温度応答性ハイドロゲルリバース金型の印刷

細胞培養アプリケーションのためのヒドロゲルの光媒介形成とパターニング

自己組織化反応押出、マイクロフルイディクス, 経由で複数の長さスケールでの分解性感温ハイドロゲルを加工・ エレクトロスピニング

生理活性タンパク質や生物学的応用の光化学ハイドロゲルのペプチドをパターニング

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

細胞および薬物送達用途のための注射可能な高分子ポリマーナノ粒子ヒドロゲル

バルクヒドロゲルの断片化と生物医学的応用のための粒状ヒドロゲルへの加工

強接着性ハイドロゲルゼラチンO-ニトロソベンズアルデヒドの合成

模様の作成3D細胞培養のための2つの成分のために温度応答性ハイドロゲルリバース金型の印刷

自己組織化反応押出、マイクロフルイディクス, 経由で複数の長さスケールでの分解性感温ハイドロゲルを加工・エレクトロスピニング