Authors would like to thank Catherine Solioz for her skillful assistance.

1. BCM Hazırlık <ol><li> Mümkün olduğunca taze yerel kasap domuz çene kemiklerini edinin. Sağlam bir yüzeye yerleştirin altçeneleri ve tam kalınlıkta flep herhangi bir yumuşak doku bırakın veya kemiğe bağlı periost değil özel dikkat bırakın. Akış kaputu altında çalışmak gerek kalmadan temiz bir ortamda çalışın. </li><li> Tam kalınlıkta flep yayımlandıktan sonra, bukkal taraftan kemik cips hasat kemik kazıyıcı kullanın. Kemik kazıyıcı keskin olması gerektiğini lütfen unutmayınız. Sıkıca kemik kazıyıcı Kulp ve uzun hareketler kemik toplamak ile. Daha küçük o 1mm kemik cips atın. <ol><li> Doğal kemik parçası muhafaza edilmesi için,% 1 antibiyotik ve bunları kurumasına izin vermeden antimikotik ile takviye edilmiş Dulbecco Modified Eagle ortamı (DMEM) olan 10 cm çaplı plastik tabaklar doğrudan kemik parçası yerleştirin. </li><li> Isıl işlem etkisini değerlendirmek için, söz konusu kemik cipsi, 80 ° C&#39;de 30 dakika boyunca pastörizasyon ya da121 ° C&#39;de 20 dakika süreyle otoklav. </li><li> , Mineralden arındırma etkisini değerlendirmek, 4 ° C&#39;de 4-6 saat boyunca 1 M HCI kemik yongaları çalkalayın ve pH nötr olana kadar, kültür ortamı ile tekrar tekrar yıkamak için. </li></ol></li><li> Yeni bir plastik çanak içine% 1 antibiyotik ve antimikotik ile desteklenmiş 10 ml taze DMEM başına kemik yongaları 5 g toplam yerleştirin. </li><li> 24 saat boyunca 37 ° C&#39;de nemli bir atmosferde plastik tabaklar yerleştirin. Sonra, hasat BCM. Santrifüj, artığın ayrılması steril (0.2 mil) filtre ve -80 ° C&#39;de dondurulur alikotları tutmak için 10 dakika boyunca 200 x g&#39;de BCM. </li><li> Kullanımdan hemen önce, BCM stok çözülme ve dondurma ve eritme tekrar döngülerinden kaçınmak. </li><li> Belirtilen deneyler için, oda sıcaklığında, 1 saat süre ile, BCM ya da serum içermeyen ortam ile kolajen zarları ıslatın. PBS ile kuvvetli bir şekilde membranlar yıkayın ve 96 gözlü levhalar içine yerleştirin. Islak membranlar hücreleri ile tohumlanır. </li><li> BCM hazırlık süreci Şekil 1&#39;de özetlenmiştir. </li&gt; </ol> Mezenkimal Hücreler dayanarak 2. biyolojik tayinler <ol><li> / Cm2 30,000 hücrelik bir konsantrasyonda olan bir 12-çukurlu plaka içinde (örneğin, kemik hücreleri, diş eti ve periodontal ligament fibroblastlar için) Tohum insan mezenkimal hücreler. Hücreler DMEM oluşan büyüme ortamı,% 10 fetal buzağı serumu ve antibiyotiklerin kullanımı tohum. Hücreler plaka O / N eklemek edelim. </li><li> Kültür ortamı atılır ve 37 ° C&#39;de önceden ısıtılmış hücrelerin PBS ile yıkayın. Ve% 20 BCM olmayan önceden ısıtılmış serum içermeyen kültür ortamı eklenerek hücrelerini uyarır. 24 saat boyunca 37 ° C&#39;de nemli bir atmosferde hücreleri yerleştirin. </li><li> , Kültür ortamı atın önceden ısıtılmış PBS ile hücreleri durulayın ve tercih protokole göre RNA ayıklayın. </li><li> Her örnek için RNA ile aynı miktarda olması için RNA konsantrasyonu ayarlayın. CDNA&#39;lar hazırlayın ve Tablo 1 &#39;de gösterilen primerler kullanılarak seçilen genleri analiz etmek için QRT-PCR işlemi gerçekleştirmek. NOT: Bu her bileşenin seyreltmeleri şunlardır: 2x SYBR Green, 20x astar ileri, 20x primer ters, 5x steril DD su, 5x cDNA. QRT-PCR ile 95 ° C 15 saniye 40 döngü, 60 ° C 1 dakika içinde gerçekleştirilir. </li><li> CT GAPDH ve Δ (ACt) ACt uyarılmış olduğunu - - ACt kontrol ACt BT hedef Δ (ACt) yöntemini kullanarak temizlik gen GAPDH&#39;ye normalleştirilmesi ile bağıl ifade seviyelerini hesaplayın. </li><li> Bu kalite kontrolü sonra, mezenkimal hücreler, hematopoietik hücreleri ya da endotel hücreleri dahil olmak üzere bütün hücre tipleri uyarmak için, kültür ortamına BCM ekleyin. </li></ol>

<ol>
	<li>Buser, 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+Stability+of+Early+Implant+Placement+with+Contour+Augmentation.">Long-term Stability of Early Implant Placement with Contour Augmentation.</a> Journal of dental research. , (2013).</li><li>Chiapasco, M., Casentini, P., Bone Zaniboni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=augmentation+procedures+in+implant+dentistry.">augmentation procedures in implant dentistry.</a> The International journal of oral &amp; maxillofacial implants. 24 Suppl. , 237-259 (2009).</li><li>Giannoudis, P. V., Dinopoulos, H., Bone Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=substitutes:+an+update.+Injury.">substitutes: an update. Injury.</a> 36, Suppl 3. S20-S27. , (2005).</li><li>Jensen, S. S., Broggini, N., Hjorting-Hansen, E., Schenk, R., Bone Buser, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=healing+and+graft+resorption+of+autograft,+anorganic+bovine+bone+and+beta-tricalcium+phosphate.+A+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 17, 237-243 (2006).</li><li>Jensen, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+novel+biphasic+calcium+phosphate+in+standardized+bone+defects:+a+histologic+and+histomorphometric+study+in+the+mandibles+of+minipigs.">Evaluation of a novel biphasic calcium phosphate in standardized bone defects: a histologic and histomorphometric study in the mandibles of minipigs.</a> Clinical oral implants research. 18, 752-760 (2007).</li><li>Grabowski, G., Bone Cornett, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=graft+and+bone+graft+substitutes+in+spine+surgery:+current+concepts+and+controversies.">graft and bone graft substitutes in spine surgery: current concepts and controversies.</a> The Journal of the American Academy of Orthopaedic Surgeons. 21, 51-60 (2013).</li><li>Khan, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+bone+grafting.">The biology of bone grafting.</a> The Journal of the American Academy of Orthopaedic Surgeons. 13, 77-86 (2005).</li><li>Bohr, H., Ravn, H. O., Werner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteogenic+effect+of+bone+transplants+in+rabbits.+The+Journal+of+bone+and+joint+surgery.">The osteogenic effect of bone transplants in rabbits. The Journal of bone and joint surgery.</a> British. 50, 866-873 (1968).</li><li>Bone Urist, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=formation+by+autoinduction.">formation by autoinduction.</a> Science. 150, 893-899 (1965).</li><li>Caball&#233;-Serrano, J. D., Buser, D., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+porcine+bone+conditioned+medium.">Proteomic analysis of porcine bone conditioned medium.</a> The International journal of oral &amp; maxillofacial implants. , (2014).</li><li>Peng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-Conditioned+Medium+Inhibits+Osteogenic+and+Adipogenic+Differentiation+of+Mesenchymal+Cells+In+Vitro.">Bone-Conditioned Medium Inhibits Osteogenic and Adipogenic Differentiation of Mesenchymal Cells In Vitro.</a> Clinical implant dentistry and related research. , (2014).</li><li>Brolese, E., Buser, D., Kuchler, U., Schaller, B., Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+chips+release+of+sclerostin+and+FGF-23+into+the+culture+medium:+an+in+vitro+pilot+study.">Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study.</a> Clinical oral implants research. , (2014).</li><li>Caball&#233;-Serrano, J., Bosshardt, D. D., Gargallo-Albiol, J., Buser, D., Bone Gruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=conditioned+medium+enhances+osteoclastogenesis+in+murine+bone+marrow+cultures.+Clin+Oral+Impl+Res;+in.">conditioned medium enhances osteoclastogenesis in murine bone marrow cultures. Clin Oral Impl Res; in.</a> Int J Oral Maxillofac Surg. , (2015).</li><li>Zimmermann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-conditioned+medium+changes+gene+expression+in+bone-derived+fibroblasts).">Bone-conditioned medium changes gene expression in bone-derived fibroblasts).</a> IJOMI accepted. , (2014).</li><li>Fulzele, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelopoiesis+is+regulated+by+osteocytes+through+Gsalpha-dependent+signaling.">Myelopoiesis is regulated by osteocytes through Gsalpha-dependent signaling.</a> Blood. 121, 930-939 (2013).</li><li>Becerra, J., Andrades, J. A., Ertl, D. C., Sorgente, N., Nimni, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demineralized+bone+matrix+mediates+differentiation+of+bone+marrow+stromal+cells+in+vitro:+effect+of+age+of+cell+donor.">Demineralized bone matrix mediates differentiation of bone marrow stromal cells in vitro: effect of age of cell donor.</a> Journal of. 11, 1703-1714 (1996).</li><li>Kupcova Skalnikova, ., H, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+techniques+for+characterisation+of+mesenchymal+stem+cell+secretome.">Proteomic techniques for characterisation of mesenchymal stem cell secretome.</a> Biochimie. , (2013).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cell+secretome:+A+novel+role+for+progranulin+during+risedronate+treatment.">Osteoblastic cell secretome: A novel role for progranulin during risedronate treatment.</a> , (2013).</li><li>Schuldt Filho, ., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+of+demineralized+bone+matrix+activates+TGF-&#946;+signaling+pathways+in+mesenchymal+cells+in+vitro.">Conditioned medium of demineralized bone matrix activates TGF-&#946; signaling pathways in mesenchymal cells in vitro.</a> J Cranio Maxill Surg. , (2015).</li><li>Buser, D., Chen, S. T., Weber, H. P., Belser, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+implant+placement+following+single-tooth+extraction+in+the+esthetic+zone:+biologic+rationale+and+surgical+procedures).">Early implant placement following single-tooth extraction in the esthetic zone: biologic rationale and surgical procedures).</a> The International journal of periodontics &amp; restorative dentistry. 28, 441-451 (2008).</li><li>Stoecklin-Wasmer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absorbable+collagen+membranes+for+periodontal+regeneration:+a+systematic+review.">Absorbable collagen membranes for periodontal regeneration: a systematic review.</a> Journal of dental research. 92, 773-781 (2013).</li></ol>

Yazarlar ifşa hiçbir şey yok. Jordi Caballé-Serrano Diş Araştırma ve Eğitim, Basel, İsviçre Vakfı burs aldı.

Kemik klimalı ortam kemik rejenerasyonu erken evrelerinde kemik grefti yayımlanan aktivitesini yansıtır. Burada açıklanan protokol kemik rejenerasyonu dahil çeşitli hücre tipleri tepkisini incelemek için adapte edilebilir. Bundan başka, protokol işleme kemik veya kemik dolgu kıvamlandırılmış ortam hazırlamak için de kullanılabilir. Çeşitli yerli ve işlenmiş kemik salınan faktörler: yöntemler gerçekleştirmek ve basit bir kavram güvenmek kolaydır. Nasıl BCM anlamak mezenkimal hücreler gref...

Otolog kemik yaygın bir malformasyon sonucu, rezektif cerrahi, rekonstrüktif travma cerrahisi, ve önceki implant yerleştirme 1,2 olarak meydana gelen kusurları köprü için kullanılır. Kemik greftleri greft konsolidasyon sürecini destekleyen nasıl biyolojik prensiplerini anlamak otogreftleri rekonstrüktif cerrahi altın standart olarak kabul edilir anlamak için tek anahtar değil, aynı zamanda kemik yerine kullanılan 3 geliştirilmiş tasarımı Biyonik olduğunu. Yine, greft konsolidasyon hızlı otolog kemik kemik 4,5 &#39;yerine göre olduğunu. Böylece, kemik rejenerasyonu desteklemek için çok etkili otolog kemik yapmak moleküler ve hücresel mekanizmaları ortaya çıkarmak için zorunludur. Konsolidasyon süreci 6,7 desteklemek için kabul edilen otogreftlerde üç ders kitabı özellikleri vardır. İlk olarak, otolog kemik büyümesi yeni oluşan kemik için rehberlik sağlayarak, osteokondüktif olduğunudefekt içine. İkincisi, otolog kemik o osteoblast 8 ayırt edebilirsiniz mezenkimal hücreler içerdiğini, yani osteojenik olduğunu. Matris içinde Gömülmüş kemik morfogenetik proteinleri gibi büyüme faktörleri endochondral hatta intramembranöz kemik oluşumu 9 sürecini başlatabilir gibi Üçüncüsü, otolog kemik osteoindüktif olduğunu. Başka bir yönü var: taze hazırlanmış kemik fiş &quot;kemik klimalı ortamda&quot; 10-15 ile in vitro gözlemlere dayalı bir parakrin fonksiyon tutun. Ayrıca miyelopoezle etkisi 16 söz edilmelidir. &quot;Demineralize kemik matriks klimalı ortam&quot; Benzer bir terim zaten demineralizasyon 17 tarafından işlenen bile, 1996 yılında icat ve kemik parakrin fonksiyon genel konsepti destekliyor edildi. Bizim amaçlarımız için, BCM 10,11 altçeneleri taze domuz hazırlanabilir. BCM proteomik analizler büyüme aslında da dahil olmak üzere, karmaşık kompozisyon ortayaors ve hücre dışı matris 10 bileşenleri, aynı zamanda bütün kemik 18,19 proteazomun üzerinde mevcut bilgi uzanan. Böylece, BCM in vitro kemik grefti çeşitli değişiklikler yayımlanan aktivitesini yansıtmalıdır. Mezenkimal hücreler, örneğin kemik cips veya oral yumuşak dokusundan izole olanlar için, BCM? In vitro maruz kaldığında ne olur, BCM osteojenik ve adipojenik farklılaşma azaltır ve IL11 ifade 11 güçlü bir artış kışkırtır. Genom mikrodizi diferansiyel BCM tepki olarak mezenşimsel hücrelerde ifade edilmesi daha fazla gen ortaya çıkardı. Bu genler arasında adrenomedüllin (ADM) vardır, IL11, IL33, NADPH oksidaz 4 (NOX4), proteoglikan 4 (PRG4 veya lubricin) ve 3 (PTX3) 15 pentraksin. Otoklava kemik cips elde edilen BCM ilgili genlerin 14 ifadesini değiştirmek için başarısız oldu. Pastörizasyon uygulanan ve donma kemik cips BCM başardıGen ekspresyonu 14 değiştirin. Minerali alınmış kemik matrisi (DBM-cm) da koşullandırılmış ortam, TGF-β-ayarlı genlerin 20 ekspresyonunu değiştirir. İlginçtir, çevredeki yumuşak doku 21,22 kemik yongaları korumak için kullanılan kollajen bariyer membranlar, gen ifadesi 23 değişikliklerden sorumlu olan BCM bu parçaların adsorbe. BCM, araştırma birkaç isim, örneğin kemik erimesi ile ilgili osteoklastı ve endotelyal hücreler gibi kemik rejenerasyonu yer alan diğer hücre tipleri için genişletilebilir. Genel olarak, biriken in vitro veriler bir klinik öncesi çalışmanın tasarımı için bilimsel temelini oluşturmaktadır. Mevcut protokol iki yönlüdür: Birincisi, BCM hazırlamak için nasıl gösterir. İkinci olarak, bu in vitro mezenkimal hücreler göre biyolojik etkinliğini test etmek için nasıl kullanılacağını gösterir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pig Mandibles</td>
			<td>Local bucher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bone Scraper</td>
			<td>Hu-Friedy</td>
			<td>PPBUSE2/36</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics &#38; Antimicotics</td>
			<td>All life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Membranes (Bio-Gide)</td>
			<td>Geistlich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Invitrogen Corporation</td>
			<td align="right">16030074</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Corporation</td>
			<td>21885-025</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			High Pure RNA Isolation Kit</td>
			<td>Roche</td>
			<td align="right">11828665001</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcriptor First Strand cDNA Synthesis Kit</td>
			<td>Roche</td>
			<td align="right">4379012001</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>Microsynth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green (for Q-RT-PCR)</td>
			<td>Roche</td>
			<td align="right">4673484001</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Roche</td>
			<td align="right">11666789001</td>
			<td></td>
		</tr>
	</tbody>
</table>

bone conditioned medium: preparation and bioassay

Otolog kemik greftleri yaygın ağız ve çene cerrahisi, ortopedi, travmatoloji ve kullanılmaktadır. Otolog kemik greftleri tek eksik kemik yerini, aynı zamanda kemik rejenerasyonu karmaşık sürecini desteklemek. Otogreftlerde Bu olumlu davranış üç özelliklerine atfedilen: osteoconductivity, osteogenicity ve Osteoindüktivite. Ancak, başka bir yönü vardır: Kemik kemik rejenerasyonu yer mezenkimal hücreler hedefleyebilir büyüme faktörleri de dahil olmak üzere moleküller, sayısız serbest greft. kemik greftleri parakrin özellikleri kemik koşullu ortam (BCM) kullanılması ile in vitro olarak incelenebilir. Burada ısıl işlem veya demineralizasyonuna uygulanan bir yerel domuz kortikal kemikten kemik klimalı ortam hazırlamak için nasıl protokolü ve kemik sunuyoruz. Hücreler doğrudan BCM maruz ya da kolajen membranların, daha önce BCM ile ıslatılmış olarak biyomalzemeler, üzerine numaralı seribaşı olabilir. Biz in vitro bioassa için örnekler verirTGF-β ayarlı genlerin ifadesi üzerindeki mezenkimal hücreler ys. sunulan protokoller ayrıca kemik rejenerasyonu esnasında kemik grefti parakrin etkileri ortaya koymak ve rekonstrüktif cerrahi geniş alanda translasyonel araştırma için bir yol açmak için teşvik etmelidir.

Kemik koşullandırılmış ortam, taze domuz kemik parçacıkları hazırlanır. Işlem genel bakış BCM hazırlamak ve BCM ile kombinasyon halinde biyomalzemeleri kullanımı, Şekil 1, sırasıyla, Şekil 2 &#39;de gösterilmiştir. BCM hazırlanması sırasında, nihai BCM kalitesini etkileyebilir kısa hareketler ya da çok küçük kemik cips sürece hareketlerle büyük kemik cips elde etmek önemlidir. ADM, PTX3, IL11, IL33, NOX4 ve PRG4 (Şekil 3): BCM Kalitesi BCM, hedef genl...

Watch this Scientific Journal Video about Bone Conditioned Medium: Preparation and Bioassay at JoVE.com

Bone Conditioned Medium: Preparation and Bioassay

Biz kemik klimalı ortam (BCM) hazırlamak ve in vitro faaliyetlerini testi nasıl burada açıklamak.

Kemik koşullu ortam: Hazırlama ve Biyo-

Autologous bone grafts are widely used in oral and maxillofacial surgery, orthopedics, and traumatology. Autologous bone grafts not only replace missing ...

bone-conditioned-medium-preparation-and-bioassay

Research

JoVE Journal

Biology

11.0K Görüntüleme.  School of Dental Medicine, University of Bern.  Biz kemik klimalı ortam (BCM) hazırlamak ve in vitro faaliyetlerini testi nasıl burada açıklamak. 

Video: Bone Conditioned Medium: Preparation and Bioassay

Maintaining Wolbachia in Cell-free Medium

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ascertain the viability of the purified Wolbachia and (3) maintain the now extracellular Wolbachia in cell-free medium.  Purified Wolbachia remain alive in the extracellular phase but do not replicate until re-inoculated into eukaryotic cells.  Extracellular Wolbachia purified in this manner will remain viable for at least a week at room temperature, and possibly longer.  Purified Wolbachia are suitable for micro-injection, DNA extraction and other applications. 


This video describes a method for purifying Wolbachia pipientis from an Anopheles gambiae cell line and then culturing the endosymbiont in cell-free medium. An assay for viability of the bacterium is demonstrated.

Hücre Orta Wolbachia bakımı

In this video protocol, procedures are demonstrated to (1) purify Wolbachia symbionts out of cultured mosquito cells, (2) use a fluorescent assay to ...

Biyoloji

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Hazırlık Elektroporasyon murin Kemik İliği itibaren İlköğretim Hematopoetik Hücre Kültürleri

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

Nosiseptif ve İnflamatuar Arabulucular Doku Özel Ölçüm İnsan In-Vivo Biyoassay

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. 
Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Histoloji, histomorfometri ve Florokrom Analiz Undecalcified Kemik Hazırlık

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Konak Bitki uçucu için bir tarama aracı olarak Electroantennographic Biyoassay

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Hücre şartına ortamdan hücre dışı veziküller sitometrik analizinde akışı

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

İnsan Osteoartrit Diz Ekleminden Doku Toplama ve RNA Ekstraksiyonu

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Kök Eksüdalarında Rhizobacterial Kemoattractants'ın Hızlı Tanımlanması için Geliştirilmiş Bir Kemotaksi Tahlili

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

Topical Application Bioassay to Quantify Insecticide Toxicity for Mosquitoes and Fruit Flies

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. Insecticide resistance surveillance of mosquito populations is typically done through Centers for Disease Control and Prevention (CDC) bottle bioassays or World Health Organization (WHO) tube tests. However, these methods can result in a high degree of variability in mortality data due to variable insecticide contact with&#160;the insect, the relatively small numbers of organisms tested, extensive variation in mass between populations, and constantly changing environmental conditions, leading to variable outcomes. This paper presents the topical application bioassay, adapted as a high-throughput phenotypic bioassay for both mosquitoes and fruit flies, to test large numbers of insects along&#160;a range of insecticide concentrations.
This assay 1) ensures consistent treatment and insecticide contact with every organism, 2) produces highly specific dose-response curves that account for differences in average mass between strains and sexes (which is particularly important for field-collected organisms), and 3) allows for the calculation of statistically rigorous median lethal doses (LD50), which are necessary for resistance ratio comparisons-an alternative surveillance approach from diagnostic dose mortality, which is also used for larvicide resistance surveillance. This assay will be a complementary tool for accurately phenotyping mosquito populations and, as illustrated using fruit flies, is easily adaptable for use with other insects. We argue that this assay will help fill the gap between genotypic and phenotypic insecticide resistance in multiple insect species.

We describe the methodology and importance of the topical application bioassay to measure insecticide susceptibility in mosquitoes and fruit flies. The presented assay is high-throughput, utilizes insect mass-thus allowing for calculating a mass-relativized lethal dose instead of concentration-and likely has lower variability than other similar methods.

Sivrisinekler ve Meyve Sinekleri için İnsektisit Toksisitesini Ölçmek için Topikal Uygulama Biyotahlili

The continued use of insecticides for public health and agriculture has led to widespread insecticide resistance and hampering of control methods. ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Gıda Demir Biyoyararlanımının Ölçümü için Caco-2 Hücre Biyotahlili

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Methods Article

Kemik koşullu ortam: Hazırlama ve Biyo-