ロブ・ノワク博士を含め、長年にわたりCT-FIREとCurveAlignに多くの貢献者とユーザーに感謝します。 キャロリン・ペールケ博士、ジェレミー・ブレドフェルト博士、グニート・メータ博士、アンドリュー・ライヒト博士、マット・コンクリン博士、ジェイン・リスル博士、パオロ・プロヴェンツァーノ博士、ブレンダ・オグル博士、パトリシア・キーリー博士、ジョセフ・スズルシェフスキ博士、スザンヌ・パウニク博士この研究は、半導体研究公社、モルグリッジ研究所、NIHがK.W.EにR01CA199996、R01CA181385、U54CA210190を助成金として提供しました。

著者らは開示するものは何もない。

このプロトコルは、繊維状コラーゲン定量のためのCT-FIREとCurveAlignの使用を記述し、コラーゲン繊維またはCT-FIREまたはCurveAlignによる分析に適した他の線状または繊維状の細長い構造を有する任意の画像に適用することができる。例えば、エラスチンや弾性繊維は、このプラットフォーム上で同様の方法で処理することができます。我々は、計算生成合成繊維21に関する両方?...

フィブリラコラーゲンは顕著で、構造ECM成分である。彼らの組織は、組織機能に影響を与え、骨形成不全1、心臓機能不全2、および創傷治癒3から乳癌4、5、6、卵巣7、8、腎臓9、および膵臓癌10を含む異なるタイプの癌に至るまで、多くの疾患の進行に関連している可能性が高い。確立された多くのイメージングモダリティは、第2高調波発生顕微鏡11のようなフィブリラコラーゲンを可視化するために使用することができ、光場または蛍光顕微鏡または偏光顕微鏡12、液晶系偏光顕微鏡(LC-PolScope)13、および電子顕微鏡14と共に染色または染料。フィブリラコラーゲン組織の重要性が明らかになり、これらの方法の使用が増加し、コラーゲン繊維分析法の改善の必要性も高まっています。
フィブリルコラーゲンの自動測定のための計算方法の開発には多くの取り組みが行われてきました。自由に利用可能なソフトウェアツールは、主に、画像タイル17のピクセル15、16、またはフーリエ変換ベースのスペクトル解析に対して第1微分または構造テンソルのいずれかを採用することにより、繊維の配向または向きの計算に焦点を当てる。これらのツールはすべて、手動ステップの要件、雑多な背景でのファイバー エッジの検出の不正確さ、またはローカライズされた特徴の特徴付けの欠如などの制限の対象となります。
このプロトコルで説明する他の自由なオープンソースフリーソフトウェアツールと比較して、このプロトコルで説明されている方法では、CT(最適なマルチスケール、指向性画像表現法)を使用して、フィブリラのコラーゲン画像からノイズを除去し、ファイバーエッジを強化または追跡します。位置と方向に関する情報は、ピクセル単位またはウィンドウごとの間接的な情報を使用してファイバー編成のメトリックを推測するのではなく、ファイバーから直接提供できます。このCTベースのフレームワーク18、19、20、21は、主に2つの別々のリンクされた「CT-FIRE」18、21と「CurveAlign」19、21という名前のパッケージを介して、グローバル、ROI、またはファイバベースでファイバー組織を定量化することができます。ソフトウェアの実装に関する限り、CT-FIREでは、複数のスケールのCT係数を使用して、エッジを強化し、ノイズを低減する画像を再構築することができます。次に、CT再構成画像に個別の繊維抽出アルゴリズムを適用して、代表的な中心点を見つけ、中心点から繊維枝を伸ばし、繊維枝をリンクしてファイバーネットワークを形成するためのファイバーを追跡します。CurveAlign では、ユーザ指定のスケールの CT 係数を使用してローカルの繊維配向を追跡し、カーブレットの方向と位置を抽出してグループ化し、対応する位置で繊維の配向を推定します。この結果として生じる定量化フレームワークは、10年以上にわたって開発され、機能、ユーザーインターフェイス、モジュール性など、多くの面で大きく進化してきました。たとえば、このツールは、ローカルの繊維配向を視覚化し、長さ、角度、幅、直線などの個々の繊維特性を含む最大 30 のファイバー フィーチャ、密度や位置合わせなどのバルク測定を測定できます。さらに、ユーザは手動または自動でセグメント化された境界に対して繊維角度を測定することができ、これは、例えば、乳癌22および膵臓癌研究10における画像ベースのバイオマーカーの発達において重要な役割を果たす。このプラットフォームは、ROI分析、自動境界作成、後処理用の機能モジュールを含む、いくつかの機能モジュールを提供します。ROIモジュールはROIのさまざまな形状にアノータを付け、対応するROI分析を行うために使用できます。アプリケーションの例として、自動境界作成モジュールを使用して、ヘマトキシリンとエオシン(H&E)の明るいフィールド画像を第2高調波生成(SHG)画像で登録し、登録されたH&E画像から腫瘍境界の画像マスクを生成することができます。後処理モジュールは、個々の画像からの出力データファイルの処理と統合を容易にして、可能な統計分析を行うことができます。
この定量化プラットフォームは、プログラミングや画像処理の経験を必要とせず、数百または数千の画像を含む大規模なデータセットを処理することができ、生物学的または生物医学的用途のためのコラーゲン組織の効率的な定量化を可能にします。私たちも含め、世界中の多くの研究者によって、さまざまな研究分野で広く利用されています。CT-FIREとCurveAlign18、19、20、21に関する4つの主要な出版物があり、そのうち最初の3つは272回引用されています(Google Scholarによると2020-05-04)。このプラットフォーム(CT-FIREまたはCurveAlign)を引用した出版物のレビューは、約35の出版物が私たちのグループと共同で、他の(〜75)が他のグループによって書かれた分析のために直接使用した約110のジャーナルペーパーがあることを示しています。たとえば、 このプラットフォームは、次の研究のために使用されました: 乳癌22,23,24, 膵臓癌10,25, 腎臓癌9,26, 創傷治癒3,27,28,29,30,卵巣癌8,31,7, 子宮蓋靭帯32,低リンゲン歯腺33, 基底 細胞癌34、低酸素肉腫35、軟骨組織36、心臓機能障害37、ニューロン38、神経膠芽腫39、リンパ収縮40、線維性カッコ軟骨41、胃癌42、微小管43、膀胱線維症44とが挙げられる。図1は、SHG画像から乳癌19の腫瘍関連コラーゲンシグネチャを見つけるためにCurveAlignの癌イメージングアプリケーションを示す。図 2は、このプラットフォームの一般的な概略ワークフローを示しています。これらのツールは技術的には18、19、21、および CurveAlign によるアラインメント分析のための通常のプロトコル20も利用できますが、すべての重要な機能を示すビジュアル プロトコルが役立つ可能性があります。ここで示すように、視覚化されたプロトコルは、このプラットフォームを使用する学習プロセスを容易にし、ユーザーが抱える可能性のある懸念事項や疑問に対してより効率的に対処します。

<table>
	<tbody>
		<tr>
			<td>CT-FIRE</td>
			<td>Univerity of Wisconsin-Madison</td>
			<td>N/A</td>
			<td>open source software available from https://eliceirilab.org/software/ctfire/</td>
		</tr>
		<tr>
			<td>CurveAlign</td>
			<td>University of Wisconsin-Madison</td>
			<td>N/A</td>
			<td>open source software available from https://eliceirilab.org/software/curvealign/</td>
		</tr>
	</tbody>
</table>

quantifying fibrillar collagen organization with curvelet transform-based tools

フィブリラールコラーゲンは、顕著な細胞外マトリックス(ECM)成分であり、そのトポロジー変化は、乳癌、卵巣癌、腎臓癌および膵臓癌を含む幅広い疾患の進行に関連していることが示されている。自由に利用できる繊維定量化ソフトウェアツールは、主に繊維の配置や向きの計算に焦点を当てており、手動ステップの要件、騒々しい背景での繊維エッジの検出の不正確さ、または局在化された特徴の欠如などの制限の対象となります。このプロトコルで説明するコラーゲン繊維定量ツールは、曲線変換(CT)によって可能になる最適なマルチスケール画像表現を使用して特徴付けられる。このアルゴリズムアプローチにより、他のツールから得られた間接的なピクセル単位またはウィンドウごとの情報を使用するのではなく、繊維から直接位置情報と向き情報を提供するために、フィブリラコラーゲン画像からのノイズの除去とファイバエッジの強化が可能になります。この CT ベースフレームワークには、"CT-FIRE" と "CurveAlign" という名前の 2 つの別個のリンクされたパッケージが含まれ、グローバル、関心領域 (ROI)、または個々のファイバ ベースでファイバー組織を定量化できます。この定量フレームワークは、10年以上にわたって開発され、現在では包括的でユーザー主導のコラーゲン定量プラットフォームへと進化してきました。このプラットフォームを使用すると、長さ、角度、幅、直線などの個々のファイバ特性、密度や位置合わせなどのバルク測定など、最大約30のファイバフィーチャを測定できます。また、手動または自動でセグメント化された境界に対して、ファイバ角度を測定することもできます。このプラットフォームには、ROI 分析、自動境界作成、後処理用のモジュールなど、いくつかの追加モジュールも用意されています。このプラットフォームを使用すると、プログラミングや画像処理の経験が必要ではなく、数百または数千の画像を含む大規模なデータセットを処理することができ、生物学的または生物学的医療用途のためのコラーゲン繊維組織の効率的な定量化を可能にします。

これらの方法は、多くの研究で正常に適用されています。.いくつかの典型的な用途は、例えば: 1) Conklinら22 腫瘍関連コラーゲンシグネチャを計算するためにCurveAlignを使用し、コラーゲン線維がより頻繁に、その下で管癌(DCIS)病変において管周囲に垂直に整列することを発見した。2) Drifkaら10 は、CurveAlign の CT-FIRE モードを使用して膵管腺癌および正常/慢?...

Watch this Scientific Journal Video about Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools at JoVE.com

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

ここでは、正常組織と疾患組織の両方の細胞外マトリックスでフィブリラーコラーゲン組織を定量化するための曲線変換ベースのオープンソースMATLABソフトウェアツールを使用するプロトコルを提示する。このツールは、コラーゲン繊維や他の種類の線状の構造を持つ画像に適用することができます。

カーブレット変換ベースのツールを使用したフィブリラーコラーゲンの定量化

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...

quantifying-fibrillar-collagen-organization-with-curvelet-transform

Research

JoVE Journal

Bioengineering

5.9K ビュー.  University of Wisconsin-Madison. ここでは、正常組織と疾患組織の両方の細胞外マトリックスでフィブリラーコラーゲン組織を定量化するための曲線変換ベースのオープンソースMATLABソフトウェアツールを使用するプロトコルを提示する。このツールは、コラーゲン繊維や他の種類の線状の構造を持つ画像に適用することができます。

ビデオ: Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Quantifying the Mechanical Properties of the Endothelial Glycocalyx with Atomic Force Microscopy

Our understanding of the interaction of leukocytes and the vessel wall during leukocyte capture is limited by an incomplete understanding of the mechanical properties of the endothelial surface layer. It is known that adhesion molecules on leukocytes are distributed non-uniformly relative to surface topography 3, that topography limits adhesive bond formation with other surfaces 9, and that physiological contact forces (&asymp; 5.0 &minus; 10.0 pN per microvillus) can compress the microvilli to as little as a third of their resting length, increasing the accessibility of molecules to the opposing surface 3, 7. We consider the endothelium as a two-layered structure, the relatively rigid cell body, plus the glycocalyx, a soft protective sugar coating on the luminal surface 6. It has been shown that the glycocalyx can act as a barrier to reduce adhesion of leukocytes to the endothelial surface 4. In this report we begin to address the deformability of endothelial surfaces to understand how the endothelial mechanical stiffness might affect bond formation. Endothelial cells grown in static culture do not express a robust glycocalyx, but cells grown under physiological flow conditions begin to approximate the glycocalyx observed in vivo 2. The modulus of the endothelial cell body has been measured using atomic force microscopy (AFM) to be approximately 5 to 20 kPa 5. The thickness and structure of the glycocalyx have been studied using electron microscopy 8, and the modulus of the glycocalyx has been approximated using indirect methods, but to our knowledge, there have been no published reports of a direct measurement of the glycocalyx modulus in living cells. In this study, we present indentation experiments made with a novel AFM probe on cells that have been cultured in conditions to maximize their glycocalyx expression to make direct measurements of the modulus and thickness of the endothelial glycocalyx.

The mechanical characteristics of endothelial glycocalyx were measured by indentation using micron sized spheres on AFM cantilevers. Endothelial cells were cultured in a custom chamber under physiological flow conditions to induce glycocalyx expression. Data were analyzed using a thin film model to determine the glycocalyx thickness and modulus.

原子間力顕微鏡と内皮グリコカリックスの機械的性質の定量化

Our  understanding of the interaction of leukocytes and the vessel wall during  leukocyte capture is limited by an incomplete understanding of the ...

生物工学

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

変性コラーゲンストランドを画像化する<em&gt;インビボ</em&gt;と<em&gt;生体外</em&gt;リテーナ入りコラーゲン模倣ペプチド写真·トリガブリダイゼーション経由

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

脂質ナノチューブ上血液凝固第VIII因子の螺旋組織

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

三次元コラーゲンマトリックス内の細胞移動の解析

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

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 ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

大きな腱欠損を修復する組織設計されたコラーゲン移植片を用いた新規テノルラフィー縫合技術

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Quantifying Intermembrane Distances with Serial Image Dilations

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for electrical conduction between cardiomyocytes. The current method for quantifying this space by manual segmentation is slow and has low spatial resolution.We developed an algorithm that uses serial image dilations of a binary outline to count the number of pixels between two opposing 2 dimensional edges.This algorithm requires fewer man hours and has a higher spatial resolution than the manual method while preserving the reproducibility of the manual process.In fact, experienced and novice investigators were able to recapitulate the results of a previous study with this new algorithm.The algorithm is limited by the human input needed to manually outline the perinexus and computational power mainly encumbered by a pre-existing pathfinding algorithm.However, the algorithm&#39;s high-throughput capabilities, high spatial resolution and reproducibility make it a versatile and robust measurement tool for use across a variety of applications requiring the measurement of the distance between any 2-dimensional (2D) edges.

The purpose of this algorithm is to continuously measure the distance between two 2-dimensional edges using serial image dilations and pathfinding. This algorithm can be applied to a variety of fields such as cardiac structural biology, vascular biology, and civil engineering.

シリアル イメージの肥大と膜間距離を定量化

A recently-described extracellular nanodomain, termed the perinexus, has been implicated in ephaptic coupling, which is an alternative mechanism for ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

軟リソグラフィの改変を用いた根表面微細構造の生物模倣複製

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Methods Article

カーブレット変換ベースのツールを使用したフィブリラーコラーゲンの定量化