Yazarlar osteokondral fişler sürecini sarma katkılarından dolayı UMC Utrecht de gelişme mekaniği grubundan Sn Jeroen van den Berg ve Bay Matthijs Wassink minnettarlıklarını ifade etmek istiyorum. Bu çalışma Hollandalı Artrit Vakfı tarafından Grant tarafından desteklenmiştir.

NOT: Burada sunulan protokol son araştırma makaleleri 6, 8, 9, deneysel ve hesaplamalı prosedürlerden benimsenmiştir. Protokol, Şekil 1 &#39;de gösterilmektedir. kadavra malzemeler Utrecht Üniversitesi Veteriner fakültesi izniyle toplanmıştır. 1. Numune ve Banyo Hazırlama <ol><li> Serin fosfat tamponlu tuzlu su (PBS) püskürtme sırasında aşırı ısınma ve daha sonra kıkırdak hasarı önlemek için ölçüye göre matkap ucu (Şekil 1) kullanılarak kadavra at femoral kondil silindirik osteokondral tapaları (8.5 mm çap) delin. </li><li> üst üste bindirilmiş banyosunun yanal yayılmanın en aza indirmek için kılıfı küçülen bir plastik ile osteokondral fişleri Termo büzülme. <ol><li> th kıkırdak ile osteokondral fiş monteE plastik küçülen yen içinde başlangıçta üst ve ona sıcak hava üfleyerek uygulayın. ısıyla ilgili zarar görmesini önlemek için kıkırdağın yüzeyinde ıslak pamuk parçaları ekleyin. </li></ol></li><li> Ayrıca yüklü (örneğin, 650 uL, 420 mM, iyoksaglat, Molekül Ağırlığı (MA) = 1.269 Da, şarj = 1) ve nötr (650 uL, 420 mM, iyodiksanol, MW = 1,550 Da), çözünen sonlu banyoları hazırlayın. </li><li> Bir şırınga kullanılarak kıkırdak yüzeyinde hazırlanmış sonlu banyoları yük ve oda sıcaklığında (Şekil 2A) ile deneyler sırasında buharlaşmasını önlemek için sarılmış örnek üzerinde mantar fiş yerleştirin. nötr solüt taşınmasını araştırmak üzere iodixanol banyosu yerleştirin ve kıkırdak yüzeyine negatif yüklü çözünmüş madde taşıma yeri iyoksaglat banyosu incelemek için. </li></ol> 2. Görüntüleme ve Görüntü İşleme <ol><li> bir mikro-CT motorlu aşamasına bağlı bir ölçüye tutucu üzerinde mantar tapa ile izole sarılmış numuneler. Yeri to kadar kontrast maddesi çözeltisi yukarı bakacak şekilde kaplı olduğu bir kıkırdak yüzey örnek. </li><li> Mikro-BT (40 x 40 x 40 um 3 voksel, 2 dakika tarama süresini, 90 kV tüp voltajı ve 180 uA tüp akımı) kıkırdak, subkondral plakadan oluşan bir görüş alanını kullanarak tarama ve sonlu denge durumuna (48 saat kadar) bir kaç zaman noktasında banyo (Şekil 2A) ulaşılır. konsantrasyon değerleri zamanla değişmez zaman denge durumuna ulaşılır. </li><li> Üreticinin yazılımını kullanarak İlgi (ROI) Bölge konumlandırılmasını kolaylaştırmak için ilk resme göre farklı zaman noktalarında 3D görüntüler Kayıt (örneğin Analiz). </li><li> Dönüştürme 3D üreticinin yazılımını kullanarak bunları işleme önce 2B Tagged Image File Format (TIFF) yığını haline mikro-BT görüntüleri yeniden. </li><li> Küresel segmentin (ImageJ&gt; ayarlama&gt; Eşiği) subkondral kemik ve kıkırdak yerindenyazılımda banyosu yalan. </li><li> Farklı zaman noktalarında kıkırdak ortalama gri değeri elde edilir önceki adımda oluşturulan kıkırdak maskesi kullanarak (ImageJ&gt;&gt; ölçün analiz). </li><li> kıkırdak ilk banyo konsantrasyonu ve ilk kontrast maddesi konsantrasyonu dikkate alındığında, çözünen maddelerin gerçek konsantrasyonu ortalama gri değerleri dönüştürmek için lineer kalibrasyon eğrisi kullanır. Önceki veriler, gri değerler kontrast ajanlarının konsantrasyonu ile doğrusal bir ilişki muhafaza gerçeğini destekler. </li><li> Deneysel zaman noktalarında karşı çözünen madde konsantrasyonunu çizilir. </li></ol> 3. Sayısal Modelleme NOT: Bu problemde difüzyon deneysel sınır koşulu ile uyumludur (z ekseni boyunca) 1D gerçekleşecek varsayılır. Bu nedenle, geometri keyfi oluşturulabilir. <ol><li> Sonlu banyosu göre kıkırdak çoklu bölge modelini oluşturmak: 1) Kıkırdak yüzeysel bölgesi oluşan<html> (Toplam kıkırdak kalınlığının% 20), orta bölge (toplam kıkırdak kalınlığının% 50) ve derin bölge (toplam kıkırdak kalınlığının% 30) 12 ve FEBio 13, 14 (Şekil 2B 2) sonlu banyosu). </li><li> FEBio kıkırdak ve banyo farklı bölgeleri, mekanik ve fiziksel özellikleri atamak. Young modülü (10 MPa) üstte banyosu ile uygulanan ozmotik basınca dayanıklı ve bu nedenle aşırı deformasyonlar kıkırdak korumak için yeterince yüksek olduğu varsayılmıştır. <ol><li> 10 -3 mm bir hidrolik geçirgenliği kullanım 4 / Ns ve simülasyonlar 8 banyo 0 kullanımı gerçek çözünen yayılma katsayısının Poisson oranı, 9. </li></ol></li><li> Örgü (8-düğüm trilinear altı yüzlü elemanları) oluşturmak ve sınırların yakın hassaslaştırmada (Şekil 2B)class = &quot;xref&quot;&gt; 8, 9. </li><li> Bifazik-çözünen modeli <ol><li> buna tekabül eden banyo ve etkili bir basınç ilk bir eriyen madde konsantrasyonuna uygulanır. 15 9&#39;da etkili basıncın açıklamaya bakın. </li><li> Farklı kıkırdak bölgelerinde öngörülen difüzyon katsayılarına göre zaman eğrileri çözünen konsantrasyonunu elde etmek için geçici bir modda modeli çalıştırın. </li></ol></li><li> multifazik modeli Not: banyo ve doku arasındaki elektrik dalgalanma banyo ve doku hem de iki tek değerli karşıt-iyonlar eklenerek atlatılabilir. <ol><li> kararlı durum modeller için: istenen değere FCD artırırken kıkırdak ve üstte uzanan banyosu içinde aynı etkili sıvı basınçları ve konsantrasyonları kullanır. </li><li> Geçici modeller için: yeterince yüksek banyosunda çözünmüë difüzyon katsayısını tutarak iyice karıştırılmış sonlu banyosu oluşturun. enjekte etmekbanyosuna banyo hava arayüzünden çözünen istenen konsantrasyon değerine ulaşmak için. </li><li> Geçici: Önceki adımda reçete solüt konsantrasyonu sınır koşulu kaldırıp gerçek difüzyon katsayısına sonlu banyosu difüzyon katsayısını dönmek. </li><li> Farklı kıkırdak bölgelerde uygulanan FCDs ve difüzyon katsayılarına göre çözünen konsantrasyonu-zaman eğrilerinin elde edilmesi için bir model çalıştırın. </li></ol></li><li> FEBio-MATLAB arayüzü <ol><li> Otomatik FEBio ve arsa konsantrasyonu-zaman eğrilerinin (FEBio-MATLAB arayüzü) 8, 9, simülasyon gerçekleştirmek için bir MATLAB geliştirir. </li><li> FEBio-MATLAB arayüzünü kullanarak kıkırdak bölgelerinde difüzyon katsayıları ve FCDs değiştirin. FEBio modelleri çalıştırın ve çözünen konsantrasyonu-zaman eğrilerinin 9 8 ekstrakte edin. </li><li> Deneysel elde çözünen konsantrasyonu-zaman eğrileri karşılaştırMinimum kök göre farklı kıkırdak bölgelerinde veriler ve elde difüzyon katsayılarının setleri ve FCDs kare hata (RMSE) 8, 9 anlamına gelir. </li></ol></li></ol><img alt="Şekil 1" class="xfigimg" src="/files/ftp_upload/54984/54984fig1.jpg" /> Şekil 1: Deney Düzeneği. A), ısmarlama bir matkap ucu yardımıyla örnek ekstraksiyon prosedürü. B) Mikro-BT görüntüleme prosedürü difüzyon sürecini izlemek üzere. <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için buraya tıklayın.</a> <img alt="şekil 2" class="xfigimg" src="/files/ftp_upload/54984/54984fig2.jpg" /> Şekil 2: Şematik.A) Deney tasarımı. Sonlu banyo, kıkırdak ve bunlarla ilgili örgü yüzeysel, orta ve derin bölgelerde oluşan B) Çok bölgeli hesaplama modeli. <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için buraya tıklayın.</a>

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Yazarlar ifşa hiçbir şey yok.

Biz eklem kıkırdağı boyunca nötr ve yüklü çözünenlerin difüzyon çalışması için bir sonlu elemanlar modelleme prosedürü ile kombine deneysel protokol sundu. Son çalışmalara göre, önerilen modeller doğru eklem kıkırdağında 8, 9 farklı bölgeleri arasında nötr (bifazik-çözünen) her ikisinin de taşıma ve negatif yüklü (çok fazlı) çözünmüş tarif olabilir. Yaygın eklem kıkırdağı işlevsel böyle OA ilerlemesi <sup clas...

<html> Moleküler taşıma dile eklemlerin homeostazı hayati bir rol oynar, artiküler tedavilerin teslim kıkırdak ve kontrastlı kıkırdak görüntüleme 1, 2, 3. Bu tür kıkırdak entegrasyonu ve eksiklik, çözünen yük ve boyut olarak osmolalitesi ve nakil oranı 4, 5, 6 etkileyebilir kıkırdak ile temas içinde banyo konsantrasyonu gibi faktörler. Her bölge farklı konsantrasyonlarda ve büyük ekstrasellüler matris moleküllerin yönelimlerinin oluşur, çünkü nötr ya da yüklü, ya çözünen, nakliyesi, eklem kıkırdağı bölgeleri arasında farklı olabilir, yani proteoglikanlar (PG) ve kollajen tip II, 1, 7, 8, 9,lass = &quot;xref&quot;&gt; 10, 11. Daha da önemlisi, yüklü çözünen taşıma eklem kıkırdağının 8, 9 boyunca artar hücre dışı matris içinde negatif sabit yük ihtiva proteoglikanlar konsantrasyonuna büyük ölçüde bağlı olabilir. Osteoartrit (OA), böylece kıkırdak boyunca difüzyonu çalışmanın önemini gösteren, ilerledikçe Bu parametreler özellikle de sabit yük yoğunluğu (FKH), kolajen fibrilleri ve kıkırdak arasında su içeriği varyasyon yönünü değişiklik olabilir. Bu çalışmada, bir protokol 6, 8, 9 doğru difüzyon sonlu banyosu modelinde nötr ve yüklü çözünen maddeleri kullanılarak çeşitli sınır şartları altında difüzyon araştırmak için önerilmektedir daha önce kurulmuş deney ve bilgisayar çalışmalara dayanmaktadır. To yöntemler kıkırdak ve gelişmiş iki fazlı-çözünen ve çok fazlı sonlu eleman modelleri ile desteklenen bir sonlu banyosunda içeren bir sistemin, mikro-bilgisayarlı tomografi görüntüleme (mikro-CT) oluşmaktadır önerilmiştir. Bu modeller, eklem kıkırdağının çeşitli dilimleri arasında nötr ve yüklü moleküllerin yanı sıra FCDs difüzyon katsayılarını elde etmek sağlar. bu modelleri kullanarak, bir kıkırdak ve bindirilirken sonlu banyosu arasındaki etkileşimleri incelemek için kullanılabilir difüzyon nötr ve yüklü moleküllerin davranışlarının daha iyi anlayabilirsiniz.

<table><tbody><tr><td>Hexabrix</td><td>Guerbet</td><td>15HX005D</td><td>Negatively-charged contrast agent</td></tr><tr><td>Visipaque</td><td>GE healthcare</td><td>12570511</td><td>Nuetral contrast agent</td></tr><tr><td>PBS (Phosphate-buffered Saline)</td><td>Life technologies</td><td>10010023</td><td>Medium</td></tr><tr><td>micro-CT</td><td>Perkin Elmer</td><td></td><td>Monitoring diffusion</td></tr><tr><td>Freezing-point osmometer</td><td>Advanced instruments</td><td></td><td>Measuring solution osmolality</td></tr></tbody></table>

an experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage

<html> Osteoartrit (OA) kıkırdak ve kondral kemik dejenerasyonu ile ilgili olan bir zayıflatıcı bir hastalıktır. Bu büyük bir kimyasal bozunma örneğin, proteoglikan kaybı ve kolajen fibril bozulmasını karşılaşır olarak eklem kıkırdağı dejenerasyonu ölçüde yük taşıma işlevini bozar. OA sırasında kimyasal zarar mekanizmalarını incelemek için ümit verici bir yol, bir dış çözünen kıkırdak örnekleri ortaya çıkarmak ve moleküllerin difüzyon izlemektir. Sağlıklı kıkırdağa kıyasla farklı difüzyon özelliklerine oluşturur eklem kıkırdağı üzerinde hareket ederken, kıkırdak hasarı (örneğin, konsantrasyon ve temel makromoleküllerin konfigürasyonunda) derecesi dış solutların çarpışma enerji kaybı ile ilişkilidir. Bu çalışmada, çeşitli adımlardan oluşur ve daha önce geliştirilen deneysel mikro C dayanan bir protokol, tanıtmak14px; &quot;&gt; omputed T omography (mikro-CT) ve sonlu eleman modeli ilk olarak iki fazlı-çözünen ve difüzyon katsayılarını elde etmek için çok fazlı sonlu eleman modelleri üzerine tatbik edilmesi izler mikro-CT, kullanılarak kaydedilir yüklenmiş ve yüklenmemiş iyotlu moleküllerin taşınması. ve kıkırdak dilimleri arasında yük yoğunlukları sabit.

Burada sağlanan temsili sonuçları önceki araştırma makaleleri 6, 8, 9, 16 ile kabul edilmiştir. OA olarak, eklem kıkırdağı önemli değişiklikler en önemlisi GAG kaybı ve kolajen fibril hasar 17, 18, <sup class="xref"...

Watch this Scientific Journal Video about An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage at JoVE.com

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Geçenlerde geliştirilen deneysel ve sayısal yöntemlerin yardımıyla eklem kıkırdağının genelinde Yüklü ve yüksüz moleküllerin taşınmasını araştırmak için bir protokol öneriyoruz.

Deneysel ve Sonlu Eleman Protokolü Eklem Kıkırdak genelinde Nötr ve Yüklü Çözünenlerin Ulaştırma Araştırma

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

an-experimental-finite-element-protocol-to-investigate-transport

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.2K Görüntüleme.  Delft University of Technology (TU Delft).  Geçenlerde geliştirilen deneysel ve sayısal yöntemlerin yardımıyla eklem kıkırdağının genelinde Yüklü ve yüksüz moleküllerin taşınmasını araştırmak için bir protokol öneriyoruz. 

Video: An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Gerçek zamanlı görselleştirme ve analiz nedeniyle tam olarak olduğu gibi fare kıkırdak Explants mekanik yükleme kondrosit yaralanma

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Biyomühendislik

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

Erken Osteoartrit tespit etmek için Atomik Kuvvet Mikroskobu Uygulaması

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Sinovya Sıvısı Kültürleme İnsan Eklem kondrosit 3D Sistemi

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biyoloji

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

Katyonik Peptit Taşıyıcılarının Kıkırdak İçi Taşıma Özelliklerinin Karakterizasyonu

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

Kıkırdak Eksplantlarında Göç ve Alımı İzlemek için Hücre Dışı Veziküllerin Etiketlenmesi

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

Koroner Ateroskleroz İlerlemesinin Optik Koherens Tomografi Tabanlı Biyomekanik Akışkan-Yapı Etkileşim Analizi

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

Sinovyal Eklem Biyomekaniği, Mekanobiyoloji ve Fiziksel Regülasyon Çalışmaları için Bir Sürtünme Testi-Biyoreaktör Cihazı

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

İnsan Eklem Kıkırdak Eksplantlarında Atomik Kuvvet Mikroskobu Tabanlı Mikro Girintide Pratik Konuların Ele Alınması

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

Eklem hastalıklarını modellemek ve ilaçları test etmek için çip üzerinde diz eklemi oluşturulması

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

İmplantlar İçin Metale Karşı Kayan Eklem Kıkırdağının Biyotribolojik Test ve Analizi

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...