この作品は、ネブラスカ大学医療センターと国立衛生研究所（助成金番号1R15 AR063901-01）からスタートアップ資金から部分的にサポートされていました。

ソリューションの調製<ol><li> 100 mg / mlのおおよその濃度で、ポリ（εカプロラクトン）（PCL）（W M =8万グラム/モル）の溶液を調製する。 4の比率でのジクロロメタン（DCM）およびN、N-dimethlyformamide（DMF）の混合物中でPCLを溶解：1（v / v）の10％の濃度（w / v）の。 </li><li>混合を20 mlのガラスチューブ中の溶液を置き。 30分間超音波洗浄器にガラス管を置き、または溶液は半透明になるまで。 </li></ol> 2.装置の準備<ol><li>付属の21ゲージ鈍針で5ミリリットル注射器に準備されたPCL溶液を加える。 </li><li> 図1によれば、垂直電界紡糸位置にシリンジポンプを配置する。 </li><li> 2cmのX整列繊維基材のための5cmのオープンスペースでステンレス鋼ギャップコレクタを使用してください。針の先端からコレクター12センチメートルを置きます。 </li><li>直流（DC）に高電圧電源を接続し針とコレクタを接地してください。コレクタが個別に他のラボ機器に非接触で接地されていることを確認してください。 </li></ol> 3.エレクトロ<ol><li>取り外したときに、針の先端に形成滴まで1.50ミリリットル/時間に設定したシリンジポンプは、直ちに交換されている。その後、0.50ミリリットル/時に流量を設定。 </li><li> 12 kVのに電圧演算子を回します。 </li><li>エレクトロスピニングは、一軸整列繊維が完全にコレクタ上のギャップをカバーするまで。 </li><li>小さなガラス板の縁部に接着剤を適用し、ガラス板にギャップコレクタから繊維を転送する。ガラスプレートと同じ大きさを有しており、接地されたアルミニウム箔片の上にガラス板を置き。 </li><li> 図3に係る第2のシリンジコレクターを配置します。 </li><li>第2のシリンジポンプにプラスチック製のマスクを取り付け、2ミリメートルコレクタの上にそれを配置する。 </li><li> （w / w）のPCLソリューション-IF NaNに1％のクマリン6を追加します。oencapsulationはdesired-あり、溶液が半透明になるまで混ぜる。 </li><li>ブラント21ゲージ針で5ミリリットルの針にPCLまたはPCL /クマリン6溶液をロードします。 9ミリリットル/時間で、または1ミリメートル/分のおおよその速度で引っ張って0.50ミリリットル/時、水平垂直ポンプを設定します。 </li><li>エレクトロスピニングは、マスクまでほぼ完全にコレクタオフ、それでもマスクの下のエッジ領域に移動した。 </li></ol> 4.ファイバー特性評価<ol><li>両面導電性テープと場所サンプル40ミリアンペアでスパッタコーターを使用して40秒間白金と金属スタッドとを被覆する。 <ol><li>私たちのこれまでの研究17,18に係る走査型電子顕微鏡（SEM）を介して繊維を調べます。 </li><li>加速電圧15kVで画像を収集します。 </li></ol></li><li>繊維配向を測定するために別々の繊維試料に高速フーリエ変換（FFT）解析を行う。 FFTにより、繊維アラインメントを測定に関する詳細情報は、Tを参照することができますこれまでの研究19,20 O。 </li></ol> 5.幹細胞を播種する。 <ol><li> 2時間、70％エタノール溶液に浸漬することによって繊維試料を滅菌する。その後、任意の汚染物質を除去するために蒸留水で繊維を洗浄する。 </li><li> 95％空気/ 5％CO 2の雰囲気中、37℃で、25cm 2のフラスコ中に人間のADSC培養細胞を得る。一日おきに10細胞培養培地を変更します。 </li><li>細胞をトリプシン処理し、細胞数をカウントします。具体的には、細胞培養フラスコから全ての培地を除去し、二リン酸緩衝生理食塩水（PBS）で細胞を洗浄する。その後、細胞単層を覆い、2~3分間、細胞培養インキュベーター中でフラスコをインキュベートする（37℃の水浴中で前加温）、0.25％トリプシン-EDTA溶液1のm リットルを加える。 4ミリリットル培地を追加し、全面にメディアをピペッティングすることにより、表面からすべてのセルを洗い流す。細胞懸濁液を遠心し、再分散番目培養培地中の電子の細胞ペレット。血球計を介して細胞を数える。ナノファイバー足場に1×10 4個の細胞の周りの種子は、35ミリメートルの-culture皿に置かれ、3と7日間インキュベートする。 </li><li>フルオレセインジアセテート（アセトン中5mg / ml）の（FDA）、画像、蛍光顕微鏡を用いて試料を用いて染色細胞。具体的には、培養皿にFDA溶液100μlを添加し、30分間インキュベートする。三回蛍光イメージング前にPBSで細胞を洗浄。私たちのこれまでの研究10に基づいてカスタマイズされたマット·ラボプログラムによってセルの向きを分析します。 </li></ol>

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The authors have nothing to disclose.

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Fu...

ナノファイバーは、その構造および相対的な大きさ1で、細胞外マトリックスを模倣する能力の組織工学のための人気のユーティリティである。しかし、このような腱から骨挿入部位のようないくつかの天然の組織界面は、腱に向かって整列して増加し、骨部位2-5で減少する可変の組織構造を示すコラーゲン線維を含む。そこで、効果的な組織再生のための効果的な構造勾配を模倣し得る足場を作製する必要がある。 以前は、具体的には、ミネラル含有量6ファイバー組成が徐々に変化に行われた研究があった。しかし、結合組織の構造成分を再現することは、主に未踏のまま。以前の研究は、ラット頭蓋冠骨芽細胞の増殖に対する表面のシリカ粒子密度の効果を研究することによって、形態学的勾配を調べ、インバーを発見シリカ粒子密度及び細胞増殖7とそれの関係。しかし、以前の研究では、細胞増殖を媒介する形態学的変化は、繊維組織変更7,8を模倣で能力を欠いている粗さを表面主に関連していた。最近の研究では、9をエレクトロスピニングするための新規なコレクタを使用して、ユニークなコラーゲン繊維の配向を模倣する足場を作製することを試みた。この研究は、両方の整列とランダム繊維と足場を生産することに成功したが、それはネイティブの組織に展示漸進的な変化を模倣することができなかった。また、ランダムな向きに整列からの即時変更で、個別のコンポーネントを製造する際に、この足場の生体力学的特性が大幅に減少した。以前の仕事は、整列とランダムからの繊維配向で連続階調で適用可能なナノファイバー足場を生成することができなかった。我々の最近の研究では、ナノファイバー足場の成功したレクリエーションを示している潜在的に腱·ツー·ボーンの挿入10で天然コラーゲン組織を模倣することができ、繊維組織内の階調の。この研究は、密接に天然の腱から骨組織界面における繊維組織のそれに似ている構造を有するナノファイバー足場の製造に使用されるプロトコルを提供することを目的とする。 グラデーションナノ構造は、様々な分野での潜在的に遠大な用途がある。我々はすでに様々な基材11-14上の組織再生のために利用されている脂肪由来幹細胞（ADSCを）と私たちの足場を組み合わせることにより、腱·ツー·骨挿入部位の組織工学への応用に焦点を当てた。また、ADSCのは多分化の観点から骨髄幹細胞の性質が非常に類似しており、それらのリソースは、単純な脂肪吸引手順15,16を使用して回収することができる豊富である。さらにぼかしナノファイバー足場にこれらの細胞を播種すると、彼らのTISを強化潜在的に様々な組織に分化できる細胞の分布制御を可能にすることによってエンジニアリングアプリケーションを訴える。幹細胞を播種することに加えて、ナノファイバーは、細胞応答の調節シグナル伝達分子をカプセル化することができる。これらの足場の組織的な勾配とのカップリングナノカプセル化は、細胞挙動または可能なインプラントデザインやコーティングの研究のために可能にする。骨芽細胞分化15,16を誘発することが示された骨形成タンパク質2（BMP2）、のような機能性分子のカプセル化は、さらにこれらの足場10の組織工学アプリケーションを増強できる。

<table><tbody><tr><td>Polycaprolactone</td><td>Sigma-Aldrich</td><td>440744</td><td></td></tr><tr><td>N,N-Dimethlyformamide</td><td>Fisher Chemical</td><td>D-119-1</td><td></td></tr><tr><td>Dichloromethane</td><td>Fisher Chemical</td><td>AC61093-1000</td><td></td></tr><tr><td>Coumarin 6</td><td>Sigma-Aldrich</td><td>546283</td><td></td></tr><tr><td>Adipose Derived Stem Cells</td><td>Cellular engineering Technologies</td><td>HMSC.AD-100</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>26140-111</td><td></td></tr><tr><td>Fluorescein Diacetate</td><td>Sigma-Aldrich</td><td>F7378</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td></td></tr><tr><td>&amp;alpha;-Modified Eagle&#039;s Medium</td><td>Invitrogen</td><td>a10490-01</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>s25120a</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>Glass Slides</td><td>VWR international, LLC</td><td>101412-842</td><td></td></tr><tr><td>Syringe Pump</td><td>Fisher Scientific</td><td>14-831-200</td><td>Single syringe</td></tr><tr><td>Ultrasonic Cleaner</td><td>Branson</td><td>1510</td><td></td></tr><tr><td>High Voltage DC Power Supply</td><td>Gamma High Voltage Research</td><td>ES30</td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>FEI</td><td>Nova 2300</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Zeiss</td><td>Axio Imager 2</td><td></td></tr></tbody></table>

electrospun nanofiber scaffolds with gradations in fiber organization

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

このプロトコルを使用して、組織的な勾配を有する繊維マットを形成した。 図3は、ナノファイバー足場上の様々な位置で撮影したSEM像を示す。定性的には、これは6mmで（ 図3D）でのランダム繊維品揃え0ミリメートル（ 図3A）で一軸整列した繊維からの進行があると判断することができる。 FFTは繊維配列に定量値を与え、定量的なプロセスに関する詳細...

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

ここでは、繊維のグラデーション組織とのエレクトロスピンナノファイバー足場を作製し、細胞形態/方向を調節することでアプリケーションを探求するプロトコルを提示する。ナノファイバー足場の物理的および化学的性質に関して勾配が生物医学分野での幅広い用途を提供する。

繊維組織における階調の電界紡糸ナノファイバー足場

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

electrospun-nanofiber-scaffolds-with-gradations-in-fiber-organization

Research

JoVE Journal

Bioengineering

9.8K ビュー.  University of Nebraska Medical Center. ここでは、繊維のグラデーション組織とのエレクトロスピンナノファイバー足場を作製し、細胞形態/方向を調節することでアプリケーションを探求するプロトコルを提示する。ナノファイバー足場の物理的および化学的性質に関して勾配が生物医学分野での幅広い用途を提供する。 

ビデオ: Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

エレクトロスピニングの基礎：最適化ソリューションと装置のパラメータ

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

生物工学

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

一次運動とエレクトロポリ- L -乳酸ナノファイバー足場上で定義されているメディアの感覚ニューロンの文化

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

神経科学

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

組織工学のためのエレクトロ繊維のポスト処理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

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

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

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

2 次元エレクトロスピニング ナノファイバー マットの 3 次元足場への拡張

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

遺伝学

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

3次元細胞培養のための自己申告足場

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

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

表面開始アセンブリの使用ECMタンパク質のナノファイバーおよびナノ構造エンジニア

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

繊維状足場に微小球を離すエレクトロ成長因子

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます<em&gt;インビトロ</em&gt;気道壁のモデル

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

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

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

組織工学と細胞培養のための繊維状重合体の足場をエレクトロ

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

繊維組織における階調の電界紡糸ナノファイバー足場

要約

さらに動画を探す

この動画の章

エレクトロスピニングの基礎：最適化ソリューションと装置のパラメータ

一次運動とエレクトロポリ- L -乳酸ナノファイバー足場上で定義されているメディアの感覚ニューロンの文化

組織工学のためのエレクトロ繊維のポスト処理

2 次元エレクトロスピニングナノファイバーマットの 3 次元足場への拡張

3次元細胞培養のための自己申告足場

表面開始アセンブリの使用ECMタンパク質のナノファイバーおよびナノ構造エンジニア

繊維状足場に微小球を離すエレクトロ成長因子

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます

組織工学と細胞培養のための繊維状重合体の足場をエレクトロ

繊維組織における階調の電界紡糸ナノファイバー足場

要約

さらに動画を探す

この動画の章

エレクトロスピニングの基礎：最適化ソリューションと装置のパラメータ

一次運動とエレクトロポリ- L -乳酸ナノファイバー足場上で定義されているメディアの感覚ニューロンの文化

組織工学のためのエレクトロ繊維のポスト処理

2 次元エレクトロスピニング ナノファイバー マットの 3 次元足場への拡張

3次元細胞培養のための自己申告足場

表面開始アセンブリの使用ECMタンパク質のナノファイバーおよびナノ構造エンジニア

繊維状足場に微小球を離すエレクトロ成長因子

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

三層のための3つのユニークな環境を提供するために、エレクトロスピニング法を適応させます

組織工学と細胞培養のための繊維状重合体の足場をエレクトロ

2 次元エレクトロスピニングナノファイバーマットの 3 次元足場への拡張