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

1. Préparation BCM <ol><li> Obtenir mandibules de porcs du boucher local aussi frais que possible. Placez les mandibules sur une surface ferme et libérer un lambeau de pleine épaisseur accordant une attention particulière à ne pas laisser tout tissu doux ou périoste fixé à l&#39;os. Travailler dans un environnement propre, sans la nécessité de travailler sous la hotte à flux. </li><li> Une fois un lambeau de pleine épaisseur est libéré, utiliser un grattoir osseuse pour récolter les éclats d&#39;os du côté buccal. S&#39;il vous plaît noter que le grattoir osseuse doit être forte. Poignée fermement le grattoir osseuse et avec de longs mouvements recueillent l&#39;os. Jeter éclats d&#39;os plus petits que de 1mm. <ol><li> Pour maintenir les éclats d&#39;os natifs, placer directement les éclats d&#39;os dans les plats en plastique de 10 cm de diamètre avec un milieu de Eagle modifié par Dulbecco (DMEM) supplémenté avec 1% d&#39;antibiotiques et antimycosiques ne pas les laisser se dessécher. </li><li> Pour évaluer l&#39;impact du traitement thermique, éclats d&#39;os soumis à une pasteurisation pendant 30 min à 80 ° C ouautoclave pendant 20 min à 121 ° C. </li><li> Pour évaluer l&#39;impact de la déminéralisation, secouez éclats d&#39;os dans 1 M de HCL pour 4-6 heures à 4 ° C et les laver à plusieurs reprises avec un milieu de culture jusqu&#39;à ce que le pH est neutre. </li></ol></li><li> Placez un total de 5 g de copeaux d&#39;os par 10 ml de DMEM frais complété avec 1% d&#39;antibiotiques et antimycosiques dans un nouveau récipient de plastique. </li><li> Placez les plats en plastique dans une atmosphère humidifiée à 37 ° C pendant 24 heures. Ensuite, la récolte BCM. Centrifugeuse la BCM à 200 g pendant 10 min pour éliminer les débris, filtrer stérile (0,2 nm), et de garder aliquotes congelées à -80 ° C. </li><li> Décongeler le BCM de stock immédiatement avant utilisation et d&#39;éviter les cycles de gel et dégel. </li><li> Pour les expériences indiquées, faire tremper les membranes de collagène avec le milieu BCM ou sans sérum pendant 1 heure à température ambiante. Laver vigoureusement les membranes avec du PBS et les placer dans des plaques 96 puits. Membranes humides sont ensemencées avec des cellules. </li><li> processus de préparation du BCM est résumé dans la figure 1. </li&gt; </ol> 2. Bioassays à base de cellules mésenchymateuses <ol><li> les cellules mésenchymateuses humaines graines (par exemple des cellules osseuses, gingivale et les fibroblastes du ligament parodontal) dans une plaque à 12 puits avec une concentration de 30 000 cellules / cm 2. Pour ensemencer les cellules utilisent un milieu de croissance constitué de DMEM, 10% de sérum de veau foetal et des antibiotiques. Laissez les cellules attachent à la plaque O / N. </li><li> Jeter le milieu de culture et laver les cellules avec PBS préchauffé à 37 ° C. Stimuler les cellules en ajoutant préchauffé milieu de culture sans sérum avec et sans 20% BCM. Placer les cellules dans une atmosphère humidifiée à 37 ° C pendant 24 heures. </li><li> Jeter le milieu de culture, rincer les cellules avec PBS préchauffé et extraire l&#39;ARN selon votre protocole préféré. </li><li> Ajuster la concentration de l&#39;ARN afin d&#39;avoir la même quantité d&#39;ARN dans chaque échantillon. Préparer des ADNc et effectuer une qRT-PCR pour analyser les gènes sélectionnés en utilisant les amorces indiquées dans le tableau 1. NOTE: Ce sont les dilutions de chaque composant: 2x SYBR Green, 20x amorce avant, 20x amorce inverse, 5x eau DD stérile, 5x ADNc. Le qRT-PCR est réalisée en 40 cycles de 95 ° C 15 s et 60 ° C 1 min. </li><li> Calculer les niveaux d&#39;expression relatifs en normalisant la GAPDH de gène de ménage en utilisant la méthode Δ (ACt) où ACt est la cible de CT - CT GAPDH et Δ (ACt) est stimulée ACt - ACt contrôle. </li><li> Après ce contrôle de la qualité, BCM ajouter au milieu de culture pour stimuler tous les types de cellules comprenant des cellules mésenchymateuses, des cellules hématopoïétiques ou des cellules endothéliales. </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>

Les auteurs ont rien à révéler. Jordi Caballé-Serrano a reçu une bourse de la Fondation de la recherche dentaire et de l&#39;éducation, de Bâle, Suisse.

Support d&#39;os conditionné reflète l&#39;activité libéré de greffons osseux au cours des premiers stades de la régénération osseuse. Le protocole décrit ici peut être adapté à étudier la réponse de différents types de cellules impliquées dans la régénération osseuse. En outre, le protocole peut être utilisé pour préparer un milieu conditionné à partir de transformés os ou des charges. Les méthodes sont faciles à réaliser et de compter sur un concept simple: les facteurs libérés à partir ...

Osseuse autologue est largement utilisé pour combler les défauts qui ont eu lieu à la suite d&#39;une malformation, une chirurgie d&#39;exérèse, la chirurgie traumatologique reconstructive, et avant le placement de l&#39;implant 1,2. Comprendre les principes biologiques de la façon dont les greffons osseux soutenir le processus de consolidation de la greffe est non seulement la clé pour comprendre pourquoi autogreffes sont considérés comme l&#39;étalon-or en chirurgie reconstructive, il est également bionique pour améliorer la conception des substituts osseux 3. Pourtant, la consolidation de la greffe est plus rapide avec de l&#39;os autologue par rapport à 4,5 substituts osseux. Ainsi, il est impératif de révéler les mécanismes moléculaires et cellulaires qui font osseuse autologue si efficace pour soutenir la régénération osseuse. Il ya trois caractéristiques de manuels de autogreffes qui sont considérés pour soutenir le 6,7 de processus de consolidation. Tout d&#39;abord, l&#39;os autologue est ostéoconducteur, fournissant des orientations pour l&#39;os nouvellement formé à croîtredans le défaut. Deuxièmement, l&#39;os autologue est ostéogénique, ce qui signifie qu&#39;il contient des cellules mésenchymateuses qui peuvent se différencier en ostéoblastes 8. Troisièmement, l&#39;os autologue est ostéoinductrice comme des facteurs de croissance comme les protéines morphogénétiques osseuses ensevelis dans la matrice peuvent initier le processus de endochondral ou même intramembranaire formation osseuse 9. Il ya un autre aspect: éclats d&#39;os fraîchement préparés détiennent une fonction paracrine sur la base des observations in vitro avec «milieu de l&#39;os-conditionné&quot; 10-15. De plus l&#39;impact de la myélopoïèse 16 convient de mentionner. Un terme similaire &quot;milieu de la matrice osseuse déminéralisée conditionné&quot; a déjà été inventé en 1996 et soutient le concept global d&#39;une fonction paracrine de l&#39;os, même lorsque traité par déminéralisation 17. Pour nos fins, BCM peut être préparé à partir de porc fraîche mandibules 10,11. Analyses protéomiques de BCM a révélé la composition complexe, y compris effet de la croissanceors et constituants de la matrice extracellulaire 10, étendant également les connaissances actuelles sur le protéasome de l&#39;os ensemble 18,19. Ainsi, BCM devrait refléter l&#39;activité libéré de diverses modifications de greffes osseuses in vitro. Qu&#39;est-ce qui se passe lorsque les cellules mésenchymateuses, par exemple celles qui sont isolées à partir de fragments d&#39;os ou de tissus mous de la bouche, sont exposés à BCM? In vitro, BCM réduit ostéogénique et la différenciation adipocytaire, et provoque une forte augmentation de l&#39;expression de 11 IL11. Génome large microréseau a révélé plusieurs gènes d&#39;être exprimés de manière différentielle dans des cellules mésenchymateuses en réponse à BCM. Parmi ces gènes sont adrénomédulline (ADM), IL11, IL33, la NADPH oxydase 4 (NOX4), des protéoglycanes 4 (PRG4 ou lubricin) et pentraxine 3 (PTX3) 15. BCM obtenu à partir de copeaux d&#39;os autoclave n&#39;a pas réussi à modifier l&#39;expression des gènes respectifs 14. BCM à partir de fragments d&#39;os qui ont subi la pasteurisation et de congélation a pumodifier l&#39;expression des gènes 14. Aussi milieu conditionné de la matrice osseuse déminéralisée (DBM-CM) modifie l&#39;expression du TGF-β gènes régulés 20. Fait intéressant, les membranes de barrière de collagène utilisées pour protéger les copeaux d&#39;os du tissu mou environnant 21,22, adsorbés les parties de BCM qui sont responsables de l&#39;évolution de l&#39;expression du gène 23. la recherche de BCM peut être étendu à d&#39;autres types de cellules impliquées dans la régénération osseuse tels que les ostéoclastes de résorption osseuse et les cellules endothéliales, pour ne nommer que quelques-uns. Globalement, les données d&#39;accumulation in vitro fournissent la base scientifique pour la conception d&#39;une étude préclinique. Le présent protocole est double: d&#39;abord, il montre comment préparer BCM. Deuxièmement, il montre comment tester son activité biologique à base de cellules mésenchymateuses in vitro.

<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

Greffes osseuses autologues sont largement utilisés dans la chirurgie orale et maxillo-faciale, orthopédie, traumatologie et. Greffes osseuses autologues ne remplacent pas seulement os manquant, ils soutiennent également le processus complexe de la régénération osseuse. Ce comportement favorable des autogreffes est attribuée aux trois caractéristiques: ostéoconductivité, osteogenicity et ostéoinductivité. Cependant, il est un autre aspect: Greffons osseux libérer une multitude de molécules, y compris les facteurs de croissance, qui peuvent cibler des cellules mésenchymateuses impliquées dans la régénération osseuse. Les propriétés paracrines des greffes osseuses peuvent être étudiés in vitro par l&#39;utilisation du support d&#39;os conditionné (BCM). Ici, nous présentons un protocole sur la façon de préparer le milieu de l&#39;os cortical conditionné à partir natif de porc os et l&#39;os qui a subi un traitement thermique ou déminéralisation. Les cellules peuvent être directement exposés à la BCM ou ensemencées sur les biomatériaux, tels que les membranes de collagène, préalablement trempées avec BCM. Nous donnons des exemples pour in vitro bioassays avec des cellules mésenchymateuses sur l&#39;expression des gènes régulés par le TGF-β. Les protocoles présentés devraient inciter à révéler davantage les effets paracrines de greffes osseuses pendant la régénération osseuse et ouvrir un chemin pour la recherche translationnelle dans le vaste domaine de la chirurgie reconstructive.

Os milieu conditionné est préparé à partir de frites fraîches d&#39;os de porc. Aperçu général du processus pour préparer BCM et à utiliser en combinaison avec des biomatériaux BCM est illustré à la figure 1 et la figure 2, respectivement. Lors de la préparation de la BCM, il est important d&#39;obtenir de grandes éclats d&#39;os avec de longs mouvements que les mouvements de courte durée ou de très petits éclats d&#39;os peut affecter la qualité de la BCM finale. Qua...

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

Bone Conditioned Medium: Preparation and Bioassay

Nous décrivons ici comment préparer milieu de l&#39;os de la climatisation (BCM) et de tester son activité in vitro.

Os milieu conditionné: Préparation et essais biologiques

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

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11.0K vues.  School of Dental Medicine, University of Bern.  Nous décrivons ici comment préparer milieu de l'os de la climatisation (BCM) et de tester son activité in vitro. 

Vidéo: 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.

Le maintien de Wolbachia dans la cellule sans moyen

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

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Biologie

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.

La préparation des cultures primaires de cellules hématopoïétiques de la moelle osseuse murine pour l&#39;électroporation

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

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.

Préparation des os décalcifiés pour l&#39;histologie, histomorphométrie et analyse Fluorochrome

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

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.

Analyse par cytométrie en flux de vésicules extracellulaires de médias conditionné par cellule d’écoulement

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.

Prélèvement de tissus et extraction d’ARN de l’articulation ostéoarthritique humaine du genou

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

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Utilisation d’un analyseur de flux métabolique cellulaire en temps réel pour surveiller la bioénergétique des ostéoblastes

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

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.

Un test de chimiotaxie amélioré pour l’identification rapide des chimioattractifs rhizobactériens dans les exsudats racinaires

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.

Essai biologique d’application topique pour quantifier la toxicité des insecticides pour les moustiques et les mouches des fruits

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.

Le bioessai sur cellules Caco-2 pour la mesure de la biodisponibilité du fer alimentaire

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

Implants de cartilage artificiel pour la régénération osseuse par ossification endochondrale

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Pleins feux sur l’auteur : Améliorer la régénération osseuse grâce à l’intégration du cartilage artificiel vascularisé 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Formation osseuse intégrée par ossification endochondrale in vivo à l’aide de cellules souches mésenchymateuses

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...