1. İlköğretim Artiküler Kondositler İzolasyonu Hayvan eklemlerin eklem yüzeyleri Hasat 10-15 tam kalınlıkta kıkırdak dilimleri (yerel Elazığ elde iskelet yapısı olgun ineklerin metakarpal-falangeal eklem gibi). <ol><li> 37 °, 2 saat boyunca Ham F-12 (w / v)% 0.5 proteaz, 20 ml içinde bir 100 mm Petri kabı ile inkübe ° C. il kıkırdak dilimleri Ham F-12, kültür ortamı içinde üç kez çalkalayın,% 0.15 collagenase, 20 ml ile bir gece boyunca inkübe 37 Ham&#39;ın F-12 ° C&#39;de kültür ortamı içinde bir </li><li> Temiz bir tabak içine 200 mesh elek filtre yoluyla hücre süspansiyonu filtre. Ham F-12, 5 ml ile yıkanır ve filtre çanak ekleyin. 50 ml&#39;lik bir konik tüpe hücre süspansiyonu aktarın. Ham F-12 10 ml Petri kabı yıkayın ve konik boru ekleyin. </li><li> Oda sıcaklığında 6-8 dakika için 600-800 RCF tüp santrifüjleyin. </li><li> Inci rahatsız etmemek sağlanması, süpernatant aspireE hücre pelet ve Ham F-12 kültür ortamı 40 ml yeniden askıya. </li><li> 6-8 dakika için 600-800 RCF tüp santrifüjleyin. </li><li> 3 kez toplam adımları 1.4 ve 1.5 iki kez daha tekrarlayın. Önce üçüncü kez santrifüj için, ajitasyon yoluyla pelet tekrar süspansiyon ve hücre sayımı için 500 ul kısım edinin. </li><li> Bir hemasitometre ve ters ışık mikroskobu ile Tripan mavi dışlama yöntemi kullanılarak 9 canlı hücreleri saymak. </li><li> Süpernatan aspire ve arzu edilen tohumlama yoğunluk ikiye katlamak için Ham&#39;ın F-12 ortamı bir hacim içinde pelet yeniden askıya (örneğin, 20 x 10 x 10 10 6 hücre / kapsülleme sonra, ml bir nihai konsantrasyon için 6 hücre / ml). </li></ol> 2. Kondrosit-agaroz Hidrojel Kapsülleme <ol><li> Isı 0.8 g otoklavlanmış Tip VII agaroz çözülmüş ° C &#39;ye kadar 120 için ayarlanmış bir sıcak plaka üzerinde steril 1x PBS (pH 7.4), 20 ml. Bir kez çözüldü ° C daha yüksek sıcaklıklar 60 ısı düşürmek will hücre canlılığını etkileyebilir. </li><li> 50 ml&#39;lik konik bir tüp içinde, iyice agaroz (bir son% 2 agaroz hidrojel için örneğin% 4 agaroz süspansiyon) bir çift istenen konsantrasyonuna sahip hücre süspansiyonu eşit hacimde ilave edilir. Onlar hücre canlılığı etkilemez ve yorulma ömrü inşa edebilirsiniz gibi büyük baloncuklar oluşturma kaçının. </li><li> Dikkatlice kısım 10, 60 mm çaplı Petri kabı içine kondrosit-agaroz karışımı ml, büyük baloncuklar oluşturma kaçınarak. </li><li> Jel kadar oda sıcaklığında 30 dakika bekletin. </li><li> Birçok kondrosit-agaroz hidrojel örnekleri olarak 4 mm biyopsi yumruk kullanarak (genellikle 20-30) gerektiği gibi edinin. </li><li> Bir mikrometre kullanarak temsili numuneler fiziksel boyutları (çap ve yükseklik) ölçün ve kaydedin. Oluşumlar örnek yükseklik farkı% 2 den daha büyük olan bir bölgede elde edilen herhangi bir numune atmak, boyuna yaklaşık 5 mm olmalıdır. </li><li> Taze bir 60 mm Petri kabı ve takviyesi için örnekler aktarıntam kültür ortamı 10 mL (20 mM HEPES, 100 ug / ml askorbik asit, ve 2 x antibiyotik / antimikotik ile Ham&#39;ın F-12 ortamı içinde kültür örneğin,% 20 fetal inek serumu) ile. </li></ol> 3. Kondrosit-agaroz Hidrojeller mekanik uyarılması ve radyoaktif <ol><li> Sıkıştırma teçhizat metalik bileşenler otoklavlayınız. % 70 etanol (gece boyunca) ve UV ışığı (en az 15 dakika boyunca) ile plastik bileşenler sterilize edin. </li><li> 24 kuyu kültür plaka kuyu içinde yer 12 steril plastik tutma halkaları (yapı çapından daha büyük iç çapa sahip) (n = 6, örnekleri ve kontroller), üzerine düşen forseps kullanarak yapıları tutmak için. </li><li> Forseps kullanarak, dikkatli bir emniyet halkası her biri emniyet, Petri çanak 24-plaka için kondrosit-agaroz hidrojeller aktarın. </li><li> Tam kültür ortamı 400 ul (örneğin% 20, 20 mM Hepes, 100 ug / ml askorbik asit ile Ham&#39;ın F-12 ortamı içinde kültür FBS, ve aktarmaSıra her bir örnek için) antibiyotik / antimikotik 2x. </li><li> Sterilize sıkıştırma kulesi (Şekil 1) takın ve vida seti merdaneler sabitleyin. 24 oyuklu kültür plakasına sıkıştırma teçhizat takın. </li><li> Merdane-hidrojel kontakt (sıfır gerilme durumu) kurmak için ayar vidalarını gevşetin ve tekrar sabitleyin. </li><li> Bir Mach-1 Mikromekanik Test Sistemi içine monte sıkıştırma kulesi yükleyin. Ayar vidası ile yer bulma pin ve kilidi ile dikey sahneye Güvenli kulesi. Aralık jig çıkarın. </li><li> Frekansı (örneğin 1 Hz) ve süresi veya devir sayısı (60 dk örneğin zaman aralıklarında kadar), istenen genlik (örneklerin orijinal yüksekliği esas örneğin% 10 gerinim genliği) dinamik basınç yükleme uygulayın. </li><li> Mekanik stimülasyon tamamlanmasıyla, aralık jig yerini bulma pin seti vidasını gevşetin ve Mach-1 Mikromekanik Test Sistemi sıkıştırma teçhizat ve kültür plakasını çıkarın. </li&gt; <li> Radiolabel hücre-hidrojeli arzu edilen izotop (kondrosit-özgü kolajen sentezi ve [35S]-kükürt proteoglikan sentezi için örneğin, [3 H]-prolin protein etiket) 5 μCi ile ortam ile takviye yapıları. </li><li> 37. az 24 saat süre ile radyo işaretlenmiş yapıların inkübe ° C,% 5 2 10 CO. </li><li> Radyoaktif işaretli hidrojel yapıları hasat etmek ve herhangi bir adi izotop kaldırmak için 1 x PBS (pH 7.4) içinde 3 kez yıkayın. Papain (20 mM amonyum asetat içinde 40 mg / ml, 65 mM 1 ethyldiaminetetraacetic asit ve 2 mM ditiotreitol ° C&#39;de 72 saat süre ile) kullanılarak Digest örnekleri. </li><li> PicoGreen boya deneyi 11 ve anonim izotop aktivite, 12,13 hemen aşağıdaki papain sindirim sayma β-sıvı sintilasyon ile, DNA içeriği normalize kullanarak DNA içeriği Testi özet. </li></ol> Not: radyoaktif maddelerin alım ve atıkları (atık izotoplar ve ürüntemas radyoaktif maddeler haline gelmiş olabilir) (ve / veya hükümet) politikalarını ve radyoaktif maddelerin güvenli kullanımı ve imhası için prosedürler ilgili kurumsal izlemeniz gerekir. 4. Temsilcisi Sonuçlar Sığır artiküler kondrosit-agaroz hidrojellerin yapıları (10 x 10 6 hücre / ml kapsüllü hücreleri ile% 2 agaroz) mekanik 20 ila 60 dakika (veya 1,200 3,600 devir için 1 Hz frekansında% 10 basınç gerilme bir genlikte uyarıldı ) ve radyoizotop kuruluş tarafından DNA içeriği ve ekstrasellüler matriks sentezini için tahlil. DNA sentezinin ve hücre dışı matriks doza bağımlı bir şekilde etkilenmiştir. Uyarım 60 dakika (Şekil 2) hiçbir etki göstermiştir, oysa DNA içeriği, uyarım 20 ya da 30 dakika (p &lt;0.01) bir sonucu olarak önemli bir% 35 azalma sergiledi. Kıkırdak-özgü kollajen ve proteoglikan sentezi ([3H]-prolin ve tarafından belirlenirSırasıyla, [35S]-kükürt birleşme), gözlenebilir bir etki sergileyen uyarım 60 dk (Şekil 3) ile birlikte uyarım 20 ya da 30 dakika (p &lt;0.01) cevap olarak yaklaşık% 60 arasında önemli bir artış göstermiştir. <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Şekil 1" /> . Kondrosit-agaroz yapıları teşvik etmek için özel olarak oluşturulmuş bir dinamik kompresyon kulesi Şekil 1 şematik Sol:. Montajlı kulesi Sağ:. Bireysel bileşenleri gösteren görünüm patlamış. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Şekil 2," /> Kondrosit-agaroz DNA içeriğinin Şekil 2. Değişiklikler mekanik olarak 20-60 dakika için 1 Hz,% 10 kompresif gerginlik genlik (ortalama ± SEM, n = 6/group ortalama) altında uyarılan hidrojellerin. Kondrosit-agaroz ekstrasellüler matriks sentezi (kolajen ve proteoglikanlar) Şekil 3. Değişiklikler mekanik 20 ila 60 dakika (± SEM, n = 6/group demek) için 1 Hz&#39;de% 10 basınç gerilme genliği altında uyarılmış hidrojellerin.

<ol>
	<li>Grodzinsky, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+tissue+remodeling+in+response+to+mechanical+forces.">Cartilage tissue remodeling in response to mechanical forces.</a> Annual Review of Biomedical Engineering. 2, 691-713 (2000).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+articular+cartilage+in+health+and+disease.">Biochemistry of articular cartilage in health and disease.</a> Clinical Biochemistry. 25, 155-163 (1992).</li><li>Neu, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+of+functional+biotribology+and+regenerative+medicine+in+synovial+joints.">The interface of functional biotribology and regenerative medicine in synovial joints.</a> Tissue Engineering Part B: Reviews. 14, 235-247 (2008).</li><li>Demarteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+cartilage+constructs+engineered+from+expanded+human+articular+chondrocytes.">Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.</a> Biochemical and Biophysical Research Communications. 310, 580-588 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+compressive+stimulation+improves+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.</a> Tissue Engineering. 10, 1323-1331 (2004).</li><li>Hunter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+chondrocyte-seeded+fibrin+gels:+effects+on+matrix+accumulation+and+mechanical+stiffness.">Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness.</a> Osteoarthritis and Cartilage. 12, 117-130 (2004).</li><li>Buschmann, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+compression+modulates+matrix+biosynthesis+in+chondrocyte/agarose+culture.">Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture.</a> Journal of Cell Science. 108 (Pt 4), 1497-1508 (1995).</li><li>Quinn, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+compression+alters+proteoglycan+deposition+and+matrix+deformation+around+individual+cells+in+cartilage+explants.">Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants.</a> Journal of Cell Science. 111 (Pt 5), 573-583 (1998).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cartilage+matrix+by+mammalian+chondrocytes+in+vitro.+I.+Isolation,+culture+characteristics,+and+morphology.">Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.</a> The Journal of Cell Biology. 93, 743-750 (1982).</li><li>Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+loading+of+chondrocytes+embedded+in+3D+constructs:+in+vitro+methods+for+assessment+of+morphological+and+metabolic+response+to+compressive+strain.">Mechanical loading of chondrocytes embedded in 3D constructs: in vitro methods for assessment of morphological and metabolic response to compressive strain.</a> Methods in Molecular Medicine. 100, 307-324 (2004).</li><li>McGowan, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+quantification+of+DNA+in+human+articular+and+septal+cartilage+using+PicoGreen+and+Hoechst+33258.">Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.</a> Osteoarthritis and Cartilage. 10, 580-587 (2002).</li><li>Fan, J. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intermittent+static+biaxial+tensile+strains+on+tissue+engineered+cartilage.">The effect of intermittent static biaxial tensile strains on tissue engineered cartilage.</a> Annals of Biomedical Engineering. 38, 1672-1682 (2010).</li><li>Kaupp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+vibrations+increase+the+proliferation+of+articular+chondrocytes+in+high-density+culture.">Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture.</a> Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine. 222, 695-703 (2008).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+shear+deformation+improves+the+quality+of+cartilaginous+tissue+formed+in+vitro.">Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro.</a> Journal of Orthopaedic Research. 21, 590-596 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+application+of+cyclic+loading+can+accelerate+matrix+deposition+and+enhance+the+properties+of+tissue-engineered+cartilage.">A single application of cyclic loading can accelerate matrix deposition and enhance the properties of tissue-engineered cartilage.</a> Osteoarthritis and Cartilage. 14, 323-330 (2006).</li><li>Kisiday, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dynamic+compressive+loading+on+chondrocyte+biosynthesis+in+self-assembling+peptide+scaffolds.">Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.</a> Journal of Biomechanics. 37, 595-604 (2004).</li><li>Chowdhury, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+chondrocyte+metabolism+in+agarose+constructs+subjected+to+dynamic+compression.">Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression.</a> Archives of Biochemistry and Biophysics. 417, 105-111 (2003).</li></ol>

Çıkar çatışması ilan etti.

<html> Hücre-seeded agaroz hidrojeller için kontrollü mekanik uyaranlara uygulamak için açıklanan yönteme kondrosit metabolizması üzerinde dinamik basınç kuvvetlerinin etkileri içine doğrudan soruşturma için izin verir. Istinat halkalar ile birlikte özel test teçhizat kullanımı örnek devrilme potansiyel sorunları önlemek için yapıları için yanal kısıtlaması sağlanır. Setleri ekipleri tarafından güvenli ölü ağırlıklı yükleme plakaları kullanımı örnek yüksekliği potansiyel fark...

<table border="1"><tbody><tr><td> Reaktif veya ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Ham F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Kollajenaz A </td><td> Sigma Aldrich Ltd </td><td> C0130 </td><td></td></tr><tr><td> Proteaz </td><td> Sigma Aldrich Ltd </td><td> P5147 </td><td></td></tr><tr><td> Fetal Bovine Serum </td><td> Sigma Aldrich Ltd </td><td> F1051 </td><td></td></tr><tr><td> Askorbat </td><td> Sigma Aldrich Ltd </td><td> A4034 </td><td></td></tr><tr><td> Antibiyotikler / antimikotik </td><td> Sigma Aldrich Ltd </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop Canada Ltd </td><td> HEP001 </td><td></td></tr><tr><td> Tripan mavisi </td><td> Sigma Aldrich Ltd </td><td> 93595 </td><td></td></tr><tr><td> Reichert Bright-Line Hemasitometre </td><td> Hausser Bilimsel </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Papaya lateks Papain </td><td> Sigma Aldrich Ltd </td><td> P3125 </td><td></td></tr><tr><td> Amonyum Asetat </td><td> Sigma Aldrich Ltd </td><td> A1542 </td><td></td></tr><tr><td> Ethyldiaminetetraacetic Asit </td><td> Sigma Aldrich Ltd </td><td> E9884 </td><td></td></tr><tr><td> DL-ditiyotretol </td><td> Sigma Aldrich Ltd </td><td> 43819 </td><td></td></tr><tr><td> Düşük Erime Noktası Agaroz, Tip VII </td><td> Sigma Aldrich Ltd </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Mesh Ekran (200) Filtre </td><td> Sigma Aldrich Ltd </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Mikromekanik Tester </td><td> Biomomentum A.Ş. </td><td> V500cs </td><td></td></tr><tr><td> Sıkıştırma Yükleniyor Jig </td><td> Sipariş üzerine yapılmış </td><td></td><td> Benzer ürün Biomomentum A.Ş. tarafından temin edilebilir </td></tr><tr><td> Falcon 24 Şey Kültür Plakalı </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-sıvı sintilasyon sayacı </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] prolin </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Kükürt </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

Mekanik hakaret veya osteoartrit gibi hastalıklar, tarafından bozulmuş hasar olduğunda Eklem kıkırdağı sınırlı bir tamir kapasitesi muzdarip. Bu eksikliği gidermek için, çeşitli tıbbi müdahaleler geliştirilmiştir. Böyle bir yöntem doku mühendisliği kıkırdak hasarlı bölgeye yeniden su yüzüne olduğunu, ancak mühendislik dokusu genellikle uzun vadeli beka sorgulayan, yerli kıkırdak biyokimyasal özellikleri ve dayanıklılık yoksun. Bu implant materyali korumak için çevre doku dayanarak, küçük odak hasarlarının onarımı için kıkırdak doku mühendisliği uygulaması sınırlar. Gelişmiş doku özelliklerini geliştirmek için, mekanik uyarı kıkırdak ekstraselüler matriksin sentezini gibi mühendislik dokusunun çıkan mekanik özelliklerini arttırmak için kullanılan bir popüler bir yöntemdir. Mekanik stimülasyon dokuya güçleri geçerlidir in vivo bu deneyimli benzer oluşturur. Bumekanik ortamı, kısmen doğal doku 1,2 geliştirme ve bakım düzenler fikrine dayanmaktadır. Kıkırdak doku mühendisliği mekanik stimülasyon en sık uygulanan biçimi 1 Hz 1,3 bir frekansta yaklaşık% 5-20 fizyolojik suşları dinamik sıkıştırma. Çeşitli çalışmalarda, dinamik sıkıştırma etkilerini araştırmış ve sonuçta geliştirilen doku 4-8 fonksiyonel özelliklerini etkileyen, kondrosit metabolizması ve biyosentezi üzerinde olumlu bir etkisi olduğu gösterilmiştir. Bu yazıda, mekanik dinamik basınç altında kondrosit-agaroz hidrojel yapıları canlandırmak ve biyokimyasal ve radyoizotop testleri aracılığıyla biyosentezi değişiklikleri analiz yöntemini göstermek. Bu yöntem aynı zamanda kolayca mekanik uyarılara bir sonucu olarak hücresel tepki olarak herhangi bir potansiyel olarak bağlı değişikliklerin değerlendirilmesi için modifiye edilebilir.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Kondrositler tarafından kıkırdak ekstraselüler matriks biyosentezi mekanik uyaranlara uygulama etkilenebilir. Bu yöntem, 3D yapıları ve kondrosit metabolizması bağlı değişikliklerin değerlendirilmesi kapsüllü kondrositler dinamik basınç suşların uygulama tekniği anlatılmaktadır.

Kondrosit-agaroz Hidrojeller mekanik stimülasyonu

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

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11.6K Görüntüleme.  Queen's University. Kondrositler tarafından kıkırdak ekstraselüler matriks biyosentezi mekanik uyaranlara uygulama etkilenebilir. Bu yöntem, 3D yapıları ve kondrosit metabolizması bağlı değişikliklerin değerlendirilmesi kapsüllü kondrositler dinamik basınç suşların uygulama tekniği anlatılmaktadır.

Video: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Tendon ex vivo Mekanik Yükleniyor

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Biyoloji

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

Devirli Tek Eksenli Gerilme kullanarak Kök Hücre Mekanik Uyarım

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

İnsan denekler üzerinde iltihaplı cilt ısı ve mekanik ağrı eşiğini Belirlenmesi

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Doku Mühendisliği 3D Hidrojeller Hücresel Kapsülleme

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

Tümleşik Optik ve Atomik Kuvvet Mikroskobu eğitimi Mekanik Uyarım Canlı Hücre Tepki

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Matrix Rijitlik Akrilamid tabanlı Hidrojeller kullanarak Hücresel Fonksiyon Üzerindeki Etkilerinin incelenmesi

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Atomik Kuvvet Mikroskobu kullanarak Akciğer Doku Mikro-Mekanik Karakterizasyonu

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Hücre Tek katmanlar mekanik uyarılması bağlı Kalsiyum Dalga Yayılım: Sığır Kornea Endotel Hücreleri Örneği

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA çapraz bağlı poliakrilamid hidrojellerinin hazırlanışı

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

Çok fonksiyonlu, Damlacık Arayüz Bilayers Bakteriyel Mechanosensitive İyon Kanallarının Kuruluş Ve Uyarım için Yöntemi Mikropipet tabanlı

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...