Yıllar boyunca CT-FIRE ve CurveAlign'e katkıda bulunanlara ve kullanıcılara teşekkür ederiz, dr. Rob Nowak, Dr. Carolyn Pehlke, Dr. Jeremy Bredfeldt, Guneet Mehta, Andrew Leicht, Dr. Adib Keikhosravi, Dr. Matt Conklin, Dr. Jayne Squirrell, Dr. Paolo Provenzano, Dr. Brenda Ogle, Dr. Patricia Keely, Dr. Joseph Szulczewski, Dr. Suzanne Ponik ve Swati Anand ve Curtis Rueden'in ek teknik katkıları. Bu çalışma, Semiconductor Research Corporation, Morgridge Araştırma Enstitüsü ve NIH'nin R01CA19996, R01CA181385 ve U54CA210190'ı K.W.E.'ye hibe etmesiyle desteklendi.

Yazarların açıklayacak bir şeyi yok.

Bu protokol, fibriller kollajen nicelemesi için CT-FIRE ve CurveAlign kullanımını açıklar ve kollajen lifleri veya CT-FIRE veya CurveAlign tarafından analize uygun diğer çizgi benzeri veya fiber benzeri uzun yapılarla herhangi bir görüntüye uygulanabilir. Örneğin, elastin veya elastik lifler bu platformda benzer şekilde işlenebilir. Her iki aracı da hesaplamalı olarak üretilen sentetik lifler üzerinde test ettik21. Uygulamaya bağlı olarak, kullanıcılar verileri için en uy...

Fibriller kollajenler belirgin, yapısal ECM bileşenleridir. Organizasyonları etki dokusu fonksiyonunu değiştirir ve muhtemelen osteogenez kusurlu 1 , kardiyak disfonksiyon 2 ve yara iyileşmesi3'tenmeme 4 , 5 ,6 , yumurtalık 7,8,böbrek9ve pankreas kanserleri10dahil olmak üzere farklı kanser türlerine kadar birçok hastalığın ilerlemesi ile ilişkilidir. İkinci harmonik nesil mikroskopi11,parlak alan veya floresan mikroskopisi veya polarize ışık mikroskopisi 12, sıvı kristal bazlı polarizasyon mikroskopisi (LC-PolScope)13ve elektron mikroskopisi14ilebirlikte lekeler veya boyalar gibi fibriller kollajenleri görselleştirmek için birçok yerleşik görüntüleme yöntemi kullanılabilir. Fibriller kollajen organizasyonunun önemi netleştikçe ve bu yöntemlerin kullanımı arttıkça, geliştirilmiş kollajen lif analizi yaklaşımları ihtiyacı da artmıştır.
Fibriller kollajenin otomatik ölçümü için hesaplama yöntemleri geliştirmek için birçok çaba olmuştur. Serbestçe kullanılabilen yazılım araçları esas olarak, görüntü karoları için15, 16veya Fourier dönüşüm tabanlı spektrum analizi için ilk türev veya yapı tensörünü benimseyerek fiber hizalama veya oryantasyon hesaplamasına odaklanmıştır17. Tüm bu araçlar, manuel adımların gerekliliği, gürültülü arka planda fiber kenarın algılanmasındaki yanlışlık veya yerelleştirilmiş özellik karakterizasyonu eksikliği gibi sınırlamalara tabidir.
Serbestçe kullanılabilen diğer açık kaynaklı özgür yazılım araçlarıyla karşılaştırıldığında, bu protokolde açıklanan yöntemler, fibriller kollajen görüntülerindeki gürültüyü gidermek ve fiber kenarlarını geliştirmek veya izlemek için en uygun, çok ölçekli, yönlü görüntü gösterimi yöntemi olan CT'yi kullanır. Konum ve yönlendirme hakkındaki bilgiler, fiber organizasyon ölçümlerini çıkarmak için dolaylı piksel veya pencere açısından bilgileri kullanmak yerine doğrudan bir fiberden sağlanabilir. Bu CT tabanlı çerçeve18,19,20,21, fiber organizasyonuni küresel, yatırım getirisi veya fiber bazında ölçebilir, esas olarak iki ayrı, ancak bağlantılı, "CT-FIRE" 18,21ve "CurveAlign"19,21adlı paketler aracılığıyla. Yazılımın uygulanması söz konusu olduğunda, CT-FIRE'da, kenarları geliştiren ve gürültüyü azaltan bir görüntüyü yeniden oluşturmak için birden fazla ölçekte CT katsayıları kullanılabilir. Daha sonra, temsili merkez noktalarını bulmak, fiber dalları merkez noktalarından uzatmak ve fiber dalları fiber ağ oluşturmak için bağlamak için lifleri izlemek için BT tarafından yeniden oluşturulan görüntüye ayrı bir fiber ekstraksiyon algoritması uygulanır. CurveAlign'de, kullanıcı tarafından belirtilen ölçekteki CT katsayıları, eğrilerin yönünün ve konumlarının çıkarıldığı ve ilgili konumlardaki fiber yönünü tahmin etmek için gruplandığı yerel fiber yönünü izlemek için kullanılabilir. Ortaya çıkan bu nicelik çerçevesi on yıldan fazla bir süredir geliştirilmiştir ve işlevsellik, kullanıcı arayüzü ve modülerlik gibi birçok açıdan büyük ölçüde gelişmiştir. Örneğin, bu araç yerel fiber yönünü görselleştirebilir ve kullanıcının uzunluk, açı, genişlik ve düzlük gibi bireysel fiber özelliklerinin yanı sıra yoğunluk ve hizalama gibi toplu ölçümler de dahil olmak üzere otuz fiber özelliğini ölçmesine olanak tanır. Ek olarak, kullanıcı lif açısını manuel veya otomatik olarak bölünmüş sınırlara göre ölçebilir, bu da örneğin meme kanseri22 ve pankreas kanseri çalışmalarında görüntü tabanlı biyobelirteç gelişiminde önemli bir rol oynar10. Bu platform, yatırım getirisi analizi, otomatik sınır oluşturma ve işlem sonrası modüller de dahil olmak üzere çeşitli özellik modülleri sağlar. Yatırım getirisi modülü, farklı yatırım getirisi şekillerine açıklama eklemek ve karşılık gelen yatırım getirisi analizi yapmak için kullanılabilir. Uygulama örneği olarak, otomatik sınır oluşturma modülü, hematoksilin ve eosin (H&E) parlak alan görüntülerini ikinci harmonik nesil (SHG) görüntülerle kaydetmek ve kayıtlı H&E görüntülerinden tümör sınırlarının görüntü maskesini oluşturmak için kullanılabilir. İşlem sonrası modül, olası istatistiksel analizler için tek tek görüntülerden çıktı veri dosyalarının işlenmesini ve entegrasyonunu kolaylaştırmaya yardımcı olabilir.
Bu niceleme platformu, önceden programlama veya görüntü işleme deneyimi gerektirmez ve yüzlerce veya binlerce görüntü de dahil olmak üzere büyük veri kümelerini işleyebilir ve biyolojik veya biyomedikal uygulamalar için kollajen organizasyonunun verimli bir şekilde ölçülmesini sağlar. Biz de dahil olmak üzere tüm dünyada birçok araştırmacı tarafından farklı araştırma alanlarında yaygın olarak kullanılmaktadır. CT-FIRE ve CurveAlign18,19,20,21' de dört ana yayın vardırvebunlardan ilk üçü 272 kez alıntılanmıştır (Google Scholar'a göre 2020-05-04 itibarıyla). Bu platforma (CT-FIRE veya CurveAlign) atıfta bulunan yayınların gözden geçirilmesi, yaklaşık 35 yayının grubumuzla işbirliği içinde olduğu ve diğerlerinin (~ 75) diğer gruplar tarafından yazıldığı analizleri için doğrudan kullanan yaklaşık 110 dergi makalesi olduğunu göstermektedir. Örneğin, bu platform aşağıdaki çalışmalar için kullanılmıştır: meme kanseri22,23,24, pankreas kanseri10,25, böbrek kanseri9,26, yara iyileşmesi3,27,28,29,30, yumurtalık kanseri8,31,7, uterosacral ligament 32 , hipofosfatemik dentin3233, bazal hücreli karsinom34, hipoksik sarkom35, kıkırdak doku36, kardiyak disfonksiyon37, nöronlar38, glioblastoma39, lenfatik kasılmalar40, lifli cacffolds41, mide kanseri42, mikrotübül43ve mesane fibrozis44. Şekil 1, SHG görüntüsünden meme kanseri19'un tümörle ilişkili kollajen imzalarını bulmak için CurveAlign'in kanser görüntüleme uygulamasını göstermektedir. Şekil 2, bu platformun tipik bir şematik iş akışını açıklar. Bu araçlar teknik olarak18,19,21ve CurveAlign ile hizalama analizi için normal bir protokol20 gözden geçirilmiş olsa da, tüm temel özellikleri gösteren görsel bir protokol yararlı olabilir. Burada sunulan görselleştirilmiş bir protokol, bu platformu kullanmanın öğrenme sürecini kolaylaştıracak ve kullanıcıların sahip olabileceği endişeleri ve soruları daha verimli bir şekilde ele alacaktır.

<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

Fibriller kollajenler belirgin hücre dışı matris (ECM) bileşenleridir ve topoloji değişikliklerinin meme, yumurtalık, böbrek ve pankreas kanserleri de dahil olmak üzere çok çeşitli hastalıkların ilerlemesi ile ilişkili olduğu gösterilmiştir. Serbestçe kullanılabilen fiber niceleme yazılım araçları esas olarak fiber hizalama veya oryantasyon hesaplamasına odaklanır ve manuel adımların gerekliliği, gürültülü arka planda fiber kenarın algılanmasında yanlışlık veya yerelleştirilmiş özellik karakterizasyonu eksikliği gibi sınırlamalara tabidir. Bu protokolde açıklanan kollajen lif nicelasyon aracı, eğri dönüşümü (BT) tarafından etkinleştirilen en uygun çok ölçekli görüntü gösterimi kullanılarak karakterize edilir. Bu algoritmik yaklaşım, diğer araçlardan elde edilen dolaylı piksel veya pencere açısından bilgileri kullanmak yerine, konum ve yönlendirme bilgilerini doğrudan bir fiberden sağlamak için fibriller kollajen görüntülerinden gürültünün giderilmesine ve fiber kenarlarının geliştirilmesine izin verir. Bu CT tabanlı çerçeve, fiber organizasyonuni küresel, ilgi alanı (ROI) veya bireysel fiber bazında ölçebilen "CT-FIRE" ve "CurveAlign" adlı iki ayrı, ancak bağlantılı paket içerir. Bu nicelik çerçevesi on yıldan fazla bir süredir geliştirilmiştir ve şimdi kapsamlı ve kullanıcı odaklı bir kollajen nicelik platformuna dönüşmüştür. Bu platformu kullanarak, uzunluk, açı, genişlik ve düzlük gibi bireysel lif özelliklerinin yanı sıra yoğunluk ve hizalama gibi toplu ölçümler de dahil olmak üzere yaklaşık otuz fiber özelliğini ölçebilirsiniz. Ayrıca, kullanıcı fiber açısını manuel veya otomatik olarak parçalanmış sınırlara göre ölçebilir. Bu platform ayrıca yatırım getirisi analizi, otomatik sınır oluşturma ve işlem sonrası modüller de dahil olmak üzere çeşitli ek modüller sağlar. Bu platformu kullanmak, önceden programlama veya görüntü işleme deneyimi gerektirmez ve yüzlerce veya binlerce görüntü dahil olmak üzere büyük veri kümelerini işleyebilir ve biyolojik veya biyomedikal uygulamalar için kollajen lif organizasyonunun verimli bir şekilde ölçülmesini sağlar.

Bu yöntemler çok sayıda çalışmada başarıyla uygulanmıştır. Bazı tipik uygulamalar şunlardır: 1) Conklin ve ark.22, tümörle ilişkili kollajen imzalarını hesaplamak için CurveAlign'i kullandı ve kollajen liflerinin duktal karsinom in situ (DCIS) lezyonlarında kanal çevresine daha sık dik olarak hizalandığını buldu; 2) Drifka ve ark.10, pankreas düktal adenokarsinom ve normal / kronik pankreatit dokuları için stromal kollajen hizalamasını ölç...

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

Burada, hem normal hem de hastalıklı dokuların hücre dışı matrisinde fibriller kollajen organizasyonunu ölçmek için kavisli dönüşüm tabanlı, açık kaynaklı matlab yazılım aracını kullanmak için bir protokol sunuyoruz. Bu araç kollajen lifleri veya diğer çizgi benzeri yapı türlerine sahip görüntülere uygulanabilir.

Curvelet Transform Tabanlı Araçlarla Fibrillar Kollajen Organizasyonunu Ölçme

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 Görüntüleme.  University of Wisconsin-Madison. Burada, hem normal hem de hastalıklı dokuların hücre dışı matrisinde fibriller kollajen organizasyonunu ölçmek için kavisli dönüşüm tabanlı, açık kaynaklı matlab yazılım aracını kullanmak için bir protokol sunuyoruz. Bu araç kollajen lifleri veya diğer çizgi benzeri yapı türlerine sahip görüntülere uygulanabilir.

Video: Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Hücresel Daralma miktarının Şikayet Matrisler hazırlanması

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Biyomühendislik

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.

Atomik Kuvvet Mikroskobu ile Endotelyal Glycocalyx Mekanik Özelliklerinin nicel

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

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Fibroblast aracılı Fibrin Jel Sıkıştırmayı Gözlemleme ve Ölçme

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

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.

Doğallığı Kollajen Tellerinin Görüntüleme<em&gt; In vivo</em&gt; Ve<em&gt; Ex vivo</em&gt; Kafesli Kollajen Mimetik Peptidlerinin Fotoğraf-tetiklenen Hybridization yoluyla

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.

Lipid Nanotüpler Kan Pıhtılaşma Faktör VIII Sarmal Organizasyonu

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

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.

Fiber Teşkilatı geçişleri ile electrospun Nanolif iskeleleri

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.

Vasküler Doku Rejenerasyon için Mühendislik 3D cellularized Kollajen jeller

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

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.

Büyük Tendon Kusurlarını Onarmak İçin Doku Mühendisliği Kollajen Greftli Yeni Bir Tenorhaphy Dikiş Tekniği

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.

Seri görüntü Dilations ile Intermembrane mesafeler miktarının

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.

Yumuşak Litografi Değişimi Ile Kök Yüzey Mikroyapısının Biyomimetik Replikasyonu

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