Yazarlar Biyomimetik ve Biyolojik Tabanlı Akıllı Mühendisliği GW Merkezi (COBRE) NSF hibe CBET-0909678 ve finansman desteğini kabul. Biz yardımcı olmak için laboratuar ve Sayın Mathieu Barraja yardım için Bay Christopher Popma Bayan Leanne Penna, Bayan Shannon Callahan, Sayın Shadman Hussain, Sayın Mohammed R. NAJJARI ve Bayan Jessica Hinke öğrencilerimize teşekkür CAD çizimleri.

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Çıkar çatışması ilan etti.

Bu çalışmada sunulan protokol, parçacık görüntü velosimetri tekniği (PIV) ve tutarlı bir yapı algılama yöntemleri, yani. Sürekli dalgacık dönüşümleri kullanarak yüksek sadakat deneysel verilerin edinimi anlatılmaktadır <img alt="denklem 1" src="https://neuron2.jove.com/files/ftp_upload/51288/51288eq1.jpg" /> , Girdap ve kesme-egemen akışlarının tanımlanması için uygundur. idealize edilmiş &quot;Tip IV&quot; kırık varlığında fizyolojik girişlerinin deneysel verilerin analizi ortaya bö...

İkincil akış yapıları kavisli boru ve kanallar gibi eğrilikleri ile iç akış geometrileri meydana girdap akım modellerdir. Bu girdap yapılar nedeniyle merkezkaç kuvveti, ters basınç geçişlerini ve akış özellikleri kombine eylem ortaya çıkar. . 3 İkincil akım morfolojileri ölçüde pulsatilitesini etkilenen - Genel olarak, ikincil akış yapıları ve titreşim girişi koşullarında simetrik Dean- ve Lyne tipi girdaplar 1 sabit girişi altında simetrik Dekanı tipi vortekslerini olarak kavisli boruların düzlemsel kesitler görünür pulsatil ve birden fazla harmonik fizyolojik akış koşulları. Bu yapılar 1 akar fizyolojik olmayan (sabit ve oscillatory) ile karşılaştırıldığında belirgin farklı boyut mukavemetli-şekil özelliklerini elde -. 6 arterlerde Aterosklerotik lezyon gelişimi bölgelerinde yüksek frekans kesme salınımlarının varlığı etkilenen düşük ortalama kesme 27, 28 karşılaşan </sus&gt;. Ikincil akış yapıları, hastalıklar, ateroskleroz ve muhtemelen ilerlemesini etkileyen nedeniyle duvar kayma gerilmeleri ve kanla taşınan parçacıkların kalma sürelerinin değiştirilmesi pulsatif kan akımına endotelyal cevaba aracılık edebilir. ateroskleroz için ortak bir tedavi, obstrüktif lezyonlar arterlerin daralmasına neden olan bir komplikasyon, stent implantasyonu olduğunu. Stent kırıkları daha fazla tıbbi böyle stent restenoz gibi komplikasyonlar (ISR), stent trombozu ve anevrizma oluşumu 9 yol implante stentlerin yapısal hataları vardır -. 13 stent kırıkları çeşitli başarısızlık &quot;Tip I-to-IV&quot; olarak kategorize edilmiştir, burada &quot;Tip IV&quot; en yüksek klinik şiddetini karakterize ve stent parçalarının 14 doğrusal yer değiştirme ile birlikte stent dikmeler tam enine kırığı olarak tanımlanır. Bu çalışmada sunulan protokol bir EXPERIMENTA açıklarkavisli arter modelinde idealize edilmiş &quot;Tip IV&quot; stent kırık aşağı ikincil akış yapılarının görselleştirme l yöntemi. Önerilen protokol şu dört temel özelliklere sahiptir: Tasarım ve laboratuvar ölçekli stent modelleri imalatı: stentlerin Geometrik açıklaması Nitinol teller 29 (nikel ve titanyum alaşımı) kullanarak kendi kendine genişleyebilen spiraller (yaylar veya helezonlar) bir dizi ile iç içe ilişkili olabilir. Stent ve dikme çapının uzunluğu klinik implantasyon 5 sırasında karşılaşılan arteriyel lezyonların uzunluğu ölçeğinde bağlıdır. Parametrik dikme çapı değişimi ve sargı (ya da zift) ve yükseliş çeşitli geometrik konfigürasyonlarda stent yol açar. 3D baskı için seçilen stent tasarım parametrelerinin özeti Tablo 1&#39;de sunulmuştur. Kan analog çalışma sıvısının hazırlanması eşleşenkavisli arter testi bölümüne Optik erişim non-invaziv hız ölçümlerini yapmak için gereklidir: kan kinematik viskozite ve test bölümünün kırılma indeksi ile. Bu duruma göre, insan kanı eşleşen ideal refraktif vasküler modeli indeksi, bir dinamik viskozitesi olan işleme sıvısının bir Newton kan taklit eden 16 doğru bir kan akış ölçümleri elde etmek için kullanılır -. 18, 30, bu çalışmada kullanılan çalışma sıvısı bildirilmiştir Deutsch ve ark. (2006), (hacim olarak)% 79 doymuş sulu sodyum iyodür (Nal),% 20 saf, gliserol, ve% 1 su 16 oluşan bu. İki bileşenli, iki boyutlu parçacık görüntüleyerek hız ölçümü (2C-2D PIV) ile tutarlı bir ikincil akış yapılarının saptanması için deney düzeneği: Deneyler alt çeşitli düzlemsel enine kesit bir yerle faz-ortalama ikincil akış hızı verileri almak için tasarlanmış str kombinasyonuaight ve somutlaştıran kavisli stent kesitler idealize &quot;Tip IV&quot; stent kırık 5, 6, 9, 14. parçacık görüntü velosimetri (PIV) tekniği kullanılarak ikincil akış hızı alanlarının edinimi ile ilgili protokol adımlar içeren bir PIV sistemini tutan bir lazer (ışık levha) kaynak, odaklanma ve akışı, özel bir çapraz korelasyon şarj birleştiğinde cihaz (CCD sensörü veya kamera) ve izleyici parçacıkların bölgeleri aydınlatmak için optik ¨t (kısa bir zaman aralığı içinde ışık levha ile aydınlatılacak ; 32, 31) Tablo 4 bakın. Protokolde adımlar varsayalım şunlardır: Birincisi, çift kare, tek maruziyet kayıtları görüntüleri değerlendiren bir iki bileşenli, iki boyutlu (2C-2D) PIV sisteminin kalibre, deneysel kurulum. İkinci olarak, 2C-2D PIV sistemi her kayıt sırasında edinilen iki görüntü kareleri arasındaki çapraz korelasyon yaparak izleyici parçacıkların ortalama değiştirmeler hesaplar. bir brPIV özellikleri ve görüntü elde etme yazılımı ief özeti malzemeleri ve ekipmanları tabloda sunulmaktadır. Üçüncü olarak, lazer çalıştırmak için gerekli tüm güvenlik önlemleri ev sahibi kurum tarafından sağlanan yönergelere göre eğitilmiş laboratuvar personeli tarafından takip edilmektedir. Yazarlar refs öneririz. , Aero hidro ve mikroakışkanlar dinamikleri, korelasyon zirve algılama ve deplasman tahmini, malzeme ve izleyici parçacıkların yoğunluğu ve ölçüm gürültü ve doğruluk içinde uygulanması, işlevsellik ve PIV tekniğin uygulanması bütünsel bir anlayış için 31 ve 32. Ayrıca lazer ve kamera PIV veri toplama bilgisayar (Şekil 3A) ve veri işleme yazılımı tarafından kontrol edilebilir unutmayın. Veri toplama ve post-processing tutarlı bir yapı tespiti için: Bir 2C-2D PIV kullanarak Faz-ortalama ikincil akış hızı ölçümleri aşağıdaki protokol tanımını kullanılarak elde edilmiştir. İşlem sonrası<html> Sürekli dalgacık dönüşümleri: veri ing aşağıdaki üç yöntemi kullanarak uyumlu ikincil akış yapısı algılama dahil <img alt="denklem 1" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5, 6, 19-24, 26. Yazarlar, hız degrade tensör esas olduğunu bir 3 x 3 matris not, <img alt="denklem 2" src="/files/ftp_upload/51288/51288eq2.jpg" /> . protokolü (2C-2D PIV tekniği) iki boyutlu deneysel ölçümler elde bir yöntem sunmaktadır. Bu nedenle, hız gradyanı tensör tam deneysel erişim bu yöntemi kullanarak ulaşılabilir olmayacaktır. her bir piksel için hız gradyanı tensör <img alt="denklem 3" src="/files/ftp_upload/51288/51288eq3.jpg" /> PIV görüntünün <img alt="denklem 4" src="/files/ftp_upload/51288/51288eq4.jpg" /> 2 x 2 matris olmalıdır, <img alt="Denklem 5" src="/files/ftp_upload/51288/51288eq5.jpg" /> . z-bileşenli girdapquation 6 &quot;src =&quot; / files / ftp_upload / 51288 / 51288eq6.jpg &quot;&gt; her bir piksel için / <img alt="Denklem 7" src="/files/ftp_upload/51288/51288eq7.jpg" /> hız degrade tansörünün anti-simetrik bir parçası kullanılarak hesaplanır <img alt="Denklem 8" src="/files/ftp_upload/51288/51288eq8.jpg" /> . Sonuç girdap bir 2D dizi olacak <img alt="Denklem 9" src="/files/ftp_upload/51288/51288eq9.jpg" /> bir kontur arsa görülebilir. Yazarlar kuvvetle Ref öneririz. girdap dağılımı, gerilme oranları ve tutarlı bir yapı tespiti hakkında bilgi artırılması yönünde hız gradyanı tensör bir anlamlı tartışma deneysel erişim için 25. Ayrıca, yazarlar Ref yukarıda belirtilen tutarlı bir yapı tespit yöntemleri arasındaki ilişkileri araştırmak ve önermek çalışmayın. bu konuda kapsamlı bir tartışma için 23, 24. protokolde adımların odak ikincil akış (Kıvırma) kantitatif tanımlanmasıdır st(Aynı zamanda kararlı yapıların olarak da bilinir) tiriyoruz. tutarlı yapı tespiti viz üç yöntem., <img alt="Denklem 10" src="/files/ftp_upload/51288/51288eq10.jpg" /> ve dalgacık girdap dönüştürdü <img alt="denklem 11" src="/files/ftp_upload/51288/51288eq11.jpg" /> idealize &quot;Tip IV&quot; stent kırık aşağı ikincil akış yapılarının çok ölçekli, çok mukavemetli olaylar tespiti doğru hız alanı verilerine uygulanır. <img alt="denklem 12" src="/files/ftp_upload/51288/51288eq12.jpg" /> Vortisiti tensörün Öklid normu suşu 19, 23, oranı 24 hayranlarıyla hız gradyanı matris simetrik (suş oranı) ve anti-simetrik (rotasyon) bölüme çürümüş olduğunu hakim olduğu, bir mekansal bölge olarak bir girdap tanımlar. gerilme hızı matrisin özdeğerleri hesaplanır; <img alt="Denklem 13" src="/files/ftp_upload/51288/51288eq13.jpg" /> . gerilme oranı norm sonra hesaplanır; <img alt = &quot;Denklem 14&quot; src = &quot;/ files / ftp_upload / 51288 / 51288eq14.jpg&quot; /&gt;. Vortisiti anti simetrik bölümünden hesaplanır. Enstrophy ya da z-bileşeni girdap kare, <img alt="denklem 15" src="/files/ftp_upload/51288/51288eq15.jpg" /> ) Daha sonra hesaplanır. <img alt="Denklem 16" src="/files/ftp_upload/51288/51288eq16.jpg" /> Son olarak ta olduğu; <img alt="Denklem 17" src="/files/ftp_upload/51288/51288eq17.jpg" /> . tüm grubu bir dış hat planını <img alt="denklem 18" src="/files/ftp_upload/51288/51288eq18.jpg" /> izo-bölgeleriyle <img alt="Denklem 19" src="/files/ftp_upload/51288/51288eq19.jpg" /> Ikincil akış yapıları 19 gösterecektir. <img alt="denklem 20" src="/files/ftp_upload/51288/51288eq20.jpg" /> Ayrıca &#39;dönen gücü&#39; olarak bilinen yerel hız degrade tansörünün kritik nokta analizi ile yapılan bir girdap tanımlama yöntemidir ve karşılık gelen özdeğerler 20-24 <strong&gt;. her pikselin hız gradyanı tensör Özdeğer <img alt="Denklem 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> hesaplanır. özdeğerler, biçimde olmalıdır <img alt="Denklem 22" src="/files/ftp_upload/51288/51288eq22.jpg" /> . bir dış hat planını <img alt="Denklem 23" src="/files/ftp_upload/51288/51288eq23.jpg" /> izo-bölgeleriyle <img alt="Denklem 24" src="/files/ftp_upload/51288/51288eq24.jpg" /> 22 - ikincil akış yapıları 20 gösterecektir. yöntemi dalgacık dönüşümü fiziksel ve spektral alanlarda düzgünlüğü olan bir analiz fonksiyonu (veya dalgacık) kullanır, kabul edilebilir (ya da sıfır ortalama vardır) ve sonlu vardır <img alt="denklem 25" src="/files/ftp_upload/51288/51288eq25.jpg" /> 5, 6, 26. Dilate veya evrişim toplamının ile 2D girdap alanı ile dalgacık sözleşmeli, dalgacık dönüştürülmüş girdap <img alt="Denklem 26" src="/files/ftp_upload/51288/51288eq26.jpg" /> Alan Cı oluşturulurölçekler ve güçlü 5, 6 geniş bir yelpazede, 2D dalgacık-dönüştürülmüş girdap alanının 26. Shannon entropi ile tutarlı yapıların omprising tüm tutarlı yapılar yeterince çözümlenir hangi optimum dalgacık ölçeği tahmin etmek hesaplanır. Bu entropi tahmin olasılıklar bir dizi içerir <img alt="Denklem 27" src="/files/ftp_upload/51288/51288eq27.jpg" /> her bir piksel için <img alt="Denklem 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> öyle ki <img alt="denklem 28" src="/files/ftp_upload/51288/51288eq28.jpg" /> Normalize kare konum m piksel ile birlikte girdap modülü, n 5, 6. Prosedürel adımlar Şekil 6&#39;da grafiksel sunulmuştur. Dalgacık seçimi yerleştirilen kısıtlamalar Ref detaylı olarak sunulmuştur. 26. Bu protokol adımı 2B Ricker dalgacık tutarlı yapı tespiti için prosedür açıklanmaktadır. Bu kullanımı B gerekçesigirdap desen eşleştirme için avelet Ref sunulmuştur. 5, 6 ve ilgili burada belirtilen referanslar.

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			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
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			<td style="width:155px;">&#160;LaVision&#160;</td>
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			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
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			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
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experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section

İnsan damarsal arter ağı karmaşık geometriler (şube, eğrilikler ve kıvrımlarında) ile yayg mevcut kan damarlarının oluşmaktadır. İkincil akış yapıları nedeniyle merkezkaç kuvvetleri, olumsuz basınç geçişlerini ve akış özellikleri kombine eylem kavisli arterlerde meydana gelen girdap akım modellerdir. Böyle bir akış morfolojisi ölçüde pulsatilitesini ve fizyolojik akış koşullarının birden harmonikler etkilenen ve 1 akar fizyolojik olmayan (sabit ve oscillatory) ile karşılaştırıldığında boyut mukavemetli-şekil özellikleri büyük ölçüde değişmektedir - 7. Ikincil akış yapıları sonuçta ateroskleroz, restenoz, trombositlerin duyarlılık ve tromboz 4 ilerlemesi doğru kan yoluyla bulaşan parçacıkların duvar kesme stresi ve maruz kalma süresi etkileyebilir - 6, 8-13 Bu nedenle, laboratuar altında bu yapıların tespit ve karakterize etmek yeteneği. hissesi kontrol koşullar precurs olduğunuveya klinik araştırmalar ilerletmek için. ateroskleroz için ortak bir cerrahi tedavi engelsiz kan akışı için daralmış arterleri açmak için stent implantasyonu vardır. Ama nedeniyle stent yüklemeleri için eş zamanlı akış tedirginlikler çok ölçekli ikincil akış morfolojileri 4 neden -. 6 böyle bir bütünlük içinde asimetri ve kayıp olarak Kademeli yüksek dereceli karmaşıklığı vis-à-vis soğukkanlı akışları 5 altında olanlar takip eden stent başarısızlıkları tarafından uyarılan edilebilir. Bu stent arızalar arıza hususlar ve klinik şiddeti 14 dayanan &quot;Türleri I-to-IV&quot; olarak sınıflandırılmıştır. Bu çalışma, bir eğri arter modelinde enine stent kırığı ve kırık parçaların doğrusal deplasman ( &quot;Tip IV&quot;) tamamlamak nedeniyle karmaşık ikincil akış yapılarının deneysel incelenmesi için bir protokol sunar. deneysel yöntem parçacık görüntü velosimetri uygulanmasını içerir (2C-2Bir arketip karotid arter girişi dalga D PIV) teknikleri, bir kırılma indeksi faz ortalama ölçümlerde 15 için sıvı çalışma kan analog eşleşti -. 18 ikincil akış yapılarının Kantitatif kimlik akışı fizik kavramlarını, kritik nokta teorisi ve roman kullanılarak elde edilmiştir 26 - deneysel PIV verilerine 5, 6, 19 uygulanan algoritma dalgacık dönüşümü.

Şekil 7A-D sunulan sonuçlar (bakınız Şekil 5, 6), Şekil 3A&#39;da gösterilen 2C-2D PIV sistemi elde edilen son işlem ikincil akış hızı verileri, sonra oluşturulmuştur. Idealize edilmiş &quot;Tip IV&quot; stent kırığı olan kavisli arter testi bölümüne verilen giriş koşulu Şekil 4B gösterilen karotid arter dalga oldu. Daha önceki çalışmalar Womersley numaralarının belirli bi...

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Ã‚Â° Curved Artery Test Section at JoVE.com

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

180 ° Kavisli Arter Testi Bölüm Model Tip IV Stent Başarısızlık Mansap İkincil Akış Yapıların Deneysel İncelenmesi

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

experimental-investigation-secondary-flow-structures-downstream-model

Research

JoVE Journal

Bioengineering

11.5K Görüntüleme.  The George Washington University. Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Video: Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180° Curved Artery Test Section

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Protein ve hücre Etrafının İki Boyutlu Desenli Yüzeyler Oluşturma

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Biyomühendislik

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

Laminer Akış Kesme Stres altında endotel progenitör hücrelerin değerlendirin Paralel plakalı Akış Odası ve Sürekli Akış Devre

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Bir Gemi Stent Dağıtım sırasında Duvar Mekaniği izlenmesi

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

İnsan 3D Doku Mühendisliği<em&gt; In vitro</em&gt; Tümör Test Sistemi

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Cilt Tip I Kolajen hazırlanması için geliştirilmiş bir yöntem

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

Çok katmanlı Balık Ölçekler (Of Karakterizasyonu<em&gt; Atractosteus spatula</em&gt;) Nanoindentation, X-ray CT, FTIR ve SEM kullanma

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

Katı, sabit ve lekeli, genel olarak, milimetre ölçekli örnekler için bölüm ücretsiz 3D Histoloji mikro bilgisayarlı tomografi tarafından katıştırma

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Stres birden çok paralel-plaka akışı Chambers kullanarak yamultmak için maruz endotel hücrelerinin gen ifade Analizi

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Multispectral görüntüleme akışı sitometrisi kullanarak In vitro micronucleus assay gerçekleştirmek için otomatik bir yöntem

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

180 ° Kavisli Arter Testi Bölüm Model Tip IV Stent Başarısızlık Mansap İkincil Akış Yapıların Deneysel İncelenmesi

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