この作業の一部は、W81XWH-08-1-0503およびW81XWH-12-1-0066に基づく米国陸軍医学研究材料司令部、AHA助成金番号0655441Uおよび15SDG25740015、ならびにNIH R21 HL081892、R21 HL130899、R21 HL089175、RC1 DK087365、R01 DK098526、R01 DK118964、R01 CA140338、R01 CA234428、およびGEヘルスケア(ノルウェー、オスロ)によってサポートされています。

トーマスジェファーソン大学とペンシルベニア大学病院の両方の治験審査委員会は、このプロトコルを承認しました。このプロトコルは、医療保険の相互運用性と説明責任に関する法律に準拠しています。米国食品医薬品局(FDA)は、このプロトコルの治験新薬承認(IND#124,465からF.フォースバーグ)を発行しました。GEヘルスケア(ノルウェー、オスロ)は、この研究で使用されたUCA(ソナゾイド; 表1)。ソナゾイドは、米国での臨床応用についてFDAによって承認されていないため、INDが必要でした。FDA承認1 を有する他のUCAは、潜在的に臨床的に有用であると考えられる場合、治療を行う医師の裁量で適応外使用することができる。
注:完全なプロトコルと統計分析計画は、https:// clinicaltrials.gov/ct2/show/NCT02489045 で入手できます。トライアル登録番号:NCT#02489045。
1.科目の準備
<ol><li>被験者の既知の薬物アレルギーまたは不耐性、特に使用されているUCAに対する既知のアレルギーを確認します。</li>
	<li>心肺状態が不安定な被験者、または一般的に医学的に不安定な被験者を除外します。</li>
	<li>被験者を仰臥位のストレッチャーに置きます。</li>
	<li>UCA注入のために、被験者の右腕または左腕の静脈に18〜22ゲージのカニューレを配置します。</li>
	<li>緊急サービス(クラッシュカートなど)が急性の副作用が発生した場合に備えて、病院内で利用できることを確認してください。. 注:UCAは非常に安全であり、深刻なアナフィラキシー様型反応は0.01%未満の割合で報告されています。20名</li>
</ol>2. UCA製剤(ソナゾイドに特異的)
<ol><li>製造元の指示に従って再懸濁することにより、各被験者に48μLのマイクロバブル(6 mL)を含む3つのバイアルを準備します。UCAは、10 mLの密封バイアル内に乾燥粉末として供給されます。バイアルのヘッドスペースにはペルフルオロブタンが含まれています。<ol><li>UCAバイアルのストッパーにケモスパイクで穴を開けます。</li>
		<li>ケモスパイクのシリンジポートから保護キャップを取り外し、2mLの滅菌水を追加します。</li>
		<li>シリンジをケモスパイクに取り付けたまま、すぐに製品を1分間振って、均質な製品を確保します。.</li>
		<li>製品をシリンジに引き抜き、製品を再びバイアルに再注入します。これは、ケモスパイク内のデッドスペースボリュームによる製品の希釈を回避するためです。</li>
		<li>シリンジポートからシリンジを取り外し、保護キャップを再度取り付けます。再構成されたUCAの濃度は8 μLマイクロバブル/mLです。</li>
		<li>他の2つのバイアルについても再構成手順を繰り返します。</li>
	</ol></li>
	<li>生理食塩水(0.9%NaCl溶液)を使用して、3方向活栓に接続する前に接続チューブを満たします。次に、活栓はカニューレにつながる延長チューブに接続されます。</li>
	<li>吊り下げられたUCAの3つのバイアルすべてを10 mLシリンジに引き込み、患者の同じレベルまたは下のシリンジポンプに入れ、活栓に直接接続します。</li>
	<li>最初の超音波イメージング後、活栓を開いた後、NaCl溶液を120 mL /時間の速度で注入し、ソナゾイドを体重1kgあたり毎分0.024 μLの割合で同時注入します(懸濁液注入速度0.18 mL / kg /時間)。 注:この注入率は、SHAPE 13,14,21を受けている門脈圧亢進症の被験者におけるソナゾイド注入に関する私たちのグループの以前の経験に基づいて選択されました。正確な再懸濁手順と注入方法は、使用するUCAによって異なります。</li>
</ol>3.初期超音波イメージング
<ol><li>超音波スキャナー(Logiq E10、バージョンR2など)の電源を入れ、C1-6-D曲線プローブを選択します。</li>
	<li>超音波スキャナーで腹部プリセットを選択し、曲線線形アレイ(通常は1-6または2-8 MHzの帯域幅)を使用して、同じイメージング面で同様の深さで門脈と肝静脈の両方のグレースケール画像を取得します(図1)。これは一般に、肋骨下アプローチによって最もよく達成されます。</li>
	<li>グッドクリニカルプラクティスに基づいて画像を最適化し、逆行性の流れの影響を避けるために、下大静脈から離れた肝静脈領域を選択するように注意してください。</li>
</ol>4. SHIとシェイプイメージング
<ol><li>サブハーモニックコントラストタッチパネルボタンを使用して、デュアルディスプレイモード(つまり、リアルタイムBモードとSHIを同時に実行)でSHIコントラストイメージングモードをアクティブにし、コントラストモードをアクティブにします。次に、ロータリーコントロールでSUBH-AMを選択します。<ol><li>送信周波数2.5MHzでSHIを実行し、1.25MHzで受信信号を取得します。</li>
		<li>パルス整形を使用して、ソナゾイドを使用したガウスウィンドウ二項フィルタ付き方形波などのサブハーモニックマイクロバブル信号の生成を最大化しますが21、これはスキャナーとUCAに依存します。17名 注意: イメージング周波数とパルス形状の選択は、エンドユーザーが利用できない場合があります。</li>
	</ol></li>
	<li>ポータルと肝静脈の開存性、および注入開始から最大1〜2分かかるマイクロバブルの存在を確認します。</li>
	<li>SHAPE自動最適化コードをアクティブにして、変化する深さと減衰を補正することでSHAPEを最適化します。22,23タッチパネルでTIC分析を選択し、F6を選択してからkボタンを選択します。</li>
	<li>SHAPE最適化アルゴリズムは、すべての音響出力レベルのサブハーモニックデータを取得します。データ取得が完了したら、コントラストサンプルウィンドウ(TIC分析画面の左上)のポータル静脈にROIを配置します。<ol><li>音響出力の関数としてROI内の平均サブハーモニックデータをプロットし、ロジスティック曲線をデータに適合させます。この曲線の変曲点(または下に示す微分曲線のピーク)を、SHAPE感度が最大の点であることが示されているため、最適化されたパワーとして選択します。22,23 そのような曲線のセットの1つを図2に示します。</li>
	</ol></li>
	<li>音響出力電力を手順4.4.1で特定した値に調整すると、サブハーモニック振幅の最大変化が周囲圧力の関数になります(つまり、SHAPEの感度が最大になります)。</li>
	<li>UCA懸濁液の注入中に、マイクロバブル(つまり、SHAPE)から5〜15秒のセグメントでサブハーモニックデータを取得します(図3)。</li>
</ol>5. シェイプ社のデータ処理
<ol><li>最適化されたSHIシネループを取得したら(ステップ5.6)、タッチパネルの「TIC分析」を選択します。<ol><li>タッチパネルで「モーショントラッキング」が有効になっていることを確認し、各フレームのROI位置を調整して、呼吸やその他の動きを補正します。</li>
		<li>解析ウィンドウのトレースの Y 軸の単位として dB が選択されていることを確認します。</li>
	</ol></li>
	<li>造影剤サンプルウィンドウ(画面の左上)で、肝静脈と門脈内の同一のROI(楕円形領域がデフォルト)を選択します。解析ウィンドウ(右側)では、各容器内のサブハーモニック信号(dB単位)が、約1.25MHzの0.5MHz帯域幅のすべてのフレームで平均化されています。</li>
	<li>最終的なSHAPE勾配(dB単位)を、肝静脈ROIと門脈ROIの平均サブハーモニック信号の差として計算します。現在の調査に基づくと、CSPHを識別するための最適な動作点は-0.11dBであり、線形回帰式はHVPG = 0.81 x SHAPE + 9.43です。14 このカットオフと式は、スキャナーとUCAの両方に依存することに注意することが重要です。</li>
</ol>

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フォースバーグ博士、グプタ博士、ウォレス博士、アイゼンブリー博士は、SHAPE技術に関する特許を出願中です。ウォレス博士はGEの従業員です。

人体内の圧力を非侵襲的かつ正確に測定することは、長い間重要ではあるがとらえどころのない臨床目標でした。ここで紹介するSHAPE測定のプロトコルは、この目標を達成します。SHAPE手順の最も重要なコンポーネントは最適化アルゴリズムであり、最適な音響出力で取得されないサブハーモニックデータは静水圧との相関が不十分であるためです。17,22,23 Logiq 9スキ...

世界中の心臓病学(特に左心室混濁)および放射線学(特に成人および小児の肝病変の特性評価)における臨床使用のために、多くの異なる超音波造影剤(UCA)が承認されています。1超音波イメージングの感度と特異性は、血管系全体を通過し、信号を最大30dB増強するUCAとして脂質またはタンパク質の殻に封入されたガス充填マイクロバブル(直径10μm)<静脈内(IV)注入によって改善できます。1これらのUCAは、後方散乱超音波信号を強化するだけでなく、十分な音圧(> 200 kPa)で非線形発振器としても機能します。したがって、サブハーモニックおよびハーモニックから超高調波周波数の範囲の受信エコーで重要なエネルギー成分が生成されます。1,2これらの非線形信号成分は、組織および線形バブルエコーから抽出し(パルス反転などを使用して)、送信周波数の半分(つまり、f 0/2)で受信するサブハーモニックイメージング(SHI)などのコントラスト固有のイメージングモダリティを作成するために使用できます。3 私たちのグループは、ヒト臨床試験で、SHIがさまざまな腫瘍や組織に関連する新血管や細動脈の血流を検出できることを実証しています。4、5、6、7、8、9
我々は、UCAを血管トレーサーとしてではなく、低調波コントラスト気泡振幅変動をモニタリングすることにより、循環器系の非侵襲的圧力推定のためのセンサとして使用することを提唱しています。10 サブハーモニック支援圧力推定(SHAPE)と呼ばれるこの革新的な手法は、表1に要約されているように、ほとんどの市販UCAでin vitroで測定されたサブハーモニック信号の振幅と静水圧(最大186 mmHg)との間の逆線形相関に依存しています(r2 > 0.90)。10,11ただし、すべてのUCAがこの動作を示すわけではないことに注意してください。最も注目すべきは、UCA SonoVue(米国ではLumasonとして知られている)からのサブハーモニック信号は、静水圧の増加とともに最初に上昇し、その後、プラトーと減少位相が続くことが示されています。12それにもかかわらず、SHAPEは、心臓および心血管系全体の圧力勾配、ならびに腫瘍の間質液圧を非侵襲的に得ることを可能にする可能性を提供します。 13,14,15,16,17 最近、市販の超音波スキャナーにSHAPEアルゴリズムのリアルタイムバージョンを実装し、SHAPEが患者の左心室と右心室の誤差が3mmHg未満の生体内圧力推定値を提供できるという概念実証を提供しました。16.17
これまでのSHAPEで最も多くの経験は門脈圧亢進症の診断であり、220人以上の被験者が登録され、多施設試験で最初の所見が確認されました。13,14門脈圧亢進症は、門脈と肝静脈の間の圧力勾配の増加、または5 mmHgを超える下大静脈の増加として定義されますが、臨床的に重要な門脈圧亢進症(CSPH)には、勾配またはそれに相当する10 mmHg≥肝静脈圧勾配(HVPG)が必要です。18 CSPHは、胃食道静脈瘤、腹水、肝代償不全、術後代償不全、および肝細胞癌のリスク増加と関連しています。18,19腹水を発症した患者は50%の3年死亡率を有し、腹水の自然感染を発症した患者は70%の1年死亡率を有する。肝硬変の患者は、胃食道静脈瘤形成の年間発生率が5〜10%、出血の発生率が年間4〜15%である。各出血エピソードは、最大20%の死亡リスクを伴います。18.19
この原稿では、市販の機器とUCAを使用して、患者の肝臓の門脈圧亢進症を特定することに重点を置いたSHAPE研究の実施方法について説明します。圧力変化の推定に対して最高の感度を達成するために必要な重要な校正手順について詳しく説明します。

<table>
	<tbody>
		<tr>
			<td>2 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>309637</td>
			<td>Used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>10 mL saline-filled syringe</td>
			<td>Becton Dickinson</td>
			<td>306545</td>
			<td>Used for flushing line to verify IV access</td>
		</tr>
		<tr>
			<td>500 mL saline bag</td>
			<td>Baxter Healthcare Corp</td>
			<td>2131323</td>
			<td>Used for co-infusion with Sonazoid</td>
		</tr>
		<tr>
			<td>C1-6-D curvi-linear proble</td>
			<td>GE Healthcare</td>
			<td>H40472LT</td>
			<td>Used for liver imaging</td>
		</tr>
		<tr>
			<td>Chemoprotect Spike</td>
			<td>Codan USA</td>
			<td>C355</td>
			<td>Chemospike used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>Discofix C Blue</td>
			<td>B. Braun Medical Inc</td>
			<td>16494C</td>
			<td>3-way stopcock</td>
		</tr>
		<tr>
			<td>Intrafix Safeset 180 cm</td>
			<td>B. Braun Medical Inc</td>
			<td>4063000</td>
			<td>Infusion tubing</td>
		</tr>
		<tr>
			<td>Logiq E10 ultrasound scanner</td>
			<td>GE Healthcare</td>
			<td>H4928US</td>
			<td>Used for conventional ultrasound imaging as well as for SHI and SHAPE</td>
		</tr>
		<tr>
			<td>Luer lock 10 mL syringe</td>
			<td>Becton Dickinson</td>
			<td>300912</td>
			<td>For infusion of Sonazoid</td>
		</tr>
		<tr>
			<td>Medfusion 3500 syringe pump</td>
			<td>Smiths Medical</td>
			<td>3500-500</td>
			<td>Used for infusing Sonazoid at 0.18 mL/kg/hour</td>
		</tr>
		<tr>
			<td>Perfusor-leitung tubing 150 mm</td>
			<td>B. Braun Medical Inc</td>
			<td>8722960</td>
			<td>Extension line enabling syringe connection to patient&#39;s IV access</td>
		</tr>
		<tr>
			<td>SHI/SHAPE software</td>
			<td>GE Healthcare</td>
			<td>H4920CI</td>
			<td>Contrast-specific imaging software</td>
		</tr>
		<tr>
			<td>Sigma Spectrum infusion system</td>
			<td>Baxter Healthcare Corp</td>
			<td>35700BAX</td>
			<td>Pump used for co-infusing saline at 120 mL/hour</td>
		</tr>
		<tr>
			<td>Sonazoid</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>Gas-filled microbubble based ultrasound contrast agent</td>
		</tr>
		<tr>
			<td>sterile water, 2 mL</td>
			<td>B. Braun Medical Inc</td>
			<td></td>
			<td>Used for reconstituting Sonazoid</td>
		</tr>
		<tr>
			<td>ultrasound gel</td>
			<td>Cardinal Health</td>
			<td>USG-250BT</td>
			<td>Used for contact between probe and patient</td>
		</tr>
		<tr>
			<td>Venflon IV cannula 22GA</td>
			<td>Becton Dickinson</td>
			<td>393202</td>
			<td>Cannula needle for obtaining IV access</td>
		</tr>
	</tbody>
</table>

contrast-enhanced subharmonic aided pressure estimation (shape) using ultrasound imaging with a focus on identifying portal hypertension

人体内の圧力を非侵襲的かつ正確に測定することは、長い間重要ではあるがとらえどころのない臨床目標でした。超音波イメージング用の造影剤は、血管系全体を横断し、信号を最大30dB増強するガス充填<カプセル化されたマイクロバブル(直径10μm)です。これらのマイクロバブルは、サブハーモニック(送信周波数の半分)から高調波までの周波数で非線形振動も生成します。サブハーモニック振幅は、周囲の静水圧と逆線形の関係にあります。ここでは、リアルタイムの低調波支援圧力推定(SHAPE)を実行できる超音波システムが提示されます。超音波造影剤の注入中に、音響出力を最適化するためのアルゴリズムが作動する。このキャリブレーションに続いて、サブハーモニックマイクロバブル信号(つまり、SHAPE)は、圧力変化に対して最も高い感度を持ち、圧力を非侵襲的に定量化するために使用できます。肝臓の門脈圧亢進症を特定するためのSHAPE手順の有用性はここで強調されていますが、この手法は多くの臨床シナリオに適用できます。

すべての超音波画像検査と同様に、肝臓SHAPEの最初の考慮事項は、標的領域の可能な限り最良のベースライングレースケール画像を取得し、肝内門脈シャントまたは他の血管異常が存在しないことを(ドップラー画像を使用して)確認することです。門脈圧亢進症を診断するための肝画像の場合、門脈と肝静脈の両方を同じ深さで視覚化して、減弱の影響を最小限に抑えることが重要です(<strong ...

Watch this Scientific Journal Video about Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension at JoVE.com

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

注入された造影剤マイクロバブルのサブハーモニック超音波イメージング(適切なキャリブレーション後)を利用して周囲圧力を非侵襲的に推定するためのプロトコルが、慢性肝疾患のヒト患者の例とともに説明されています。

門脈圧亢進症の同定に着目した超音波画像を用いた造影補助圧力推定(SHAPE)

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

contrast-enhanced-subharmonic-aided-pressure-estimation-shape-using

Research

JoVE Journal

Bioengineering

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

2.2K ビュー.  Thomas Jefferson University. 注入された造影剤マイクロバブルのサブハーモニック超音波イメージング(適切なキャリブレーション後)を利用して周囲圧力を非侵襲的に推定するためのプロトコルが、慢性肝疾患のヒト患者の例とともに説明されています。

ビデオ: Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

生物工学

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

医療画像処理アプリケーションのための超音波の広帯域光検出器

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

生理活性、PCLベースの「セルフフィット」形状記憶ポリマー足場の作製

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

高強度のリアルタイムモニタリングの超音波（HIFU）アブレーションを重視しました<em&gt;インビトロ</em集束超音波のための調和運動イメージングの使用&gt;犬の肝臓（HMIFU）

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

ポリジメチルシロキサン（PDMS）同軸フローフォーカス・デバイスを使用したダブルエマルションの生成

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

1 蛍光顕微鏡を用いた複数の画像モードを実施

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

熱と圧力を用いた水性溶媒和による組み換えクモ絹の材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

超音波による患者特異的ポリビニルアルコールファントム作製法とX線コントラストによる脳腫瘍手術計画

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.