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

1. Preparación BCM <ol><li> Obtener mandíbulas de cerdos de la carnicería local lo más fresco posible. Coloque las mandíbulas sobre una superficie firme y suelte un colgajo de espesor total con especial atención para no dejar ningún tejido blando o periostio unido al hueso. Trabajar en un ambiente limpio y sin la necesidad de trabajar bajo la campana de flujo. </li><li> Una vez que se dio a conocer un colgajo de espesor total, use un raspador de hueso para recoger los fragmentos de hueso del lado bucal. Tenga en cuenta que el raspador de hueso tiene que ser fuerte. Maneje con firmeza el raspador de hueso y con movimientos largos recogen el hueso. Desechar astillas de hueso más pequeño que 1 mm. <ol><li> Para mantener astillas de hueso nativo, colocar directamente las astillas de hueso en placas de plástico de 10 cm de diámetro con medio de Eagle modificado por Dulbecco (DMEM) suplementado con 1% de antibióticos y antimicóticos no dejar que se sequen. </li><li> Para evaluar el impacto del procesamiento térmico, astillas de hueso sometidos a pasteurización durante 30 minutos a 80 ° C oautoclave durante 20 min a 121 ° C. </li><li> Para evaluar el impacto de la desmineralización, agitar astillas de hueso en 1 M HCL durante 4-6 horas a 4 ° C y lavar varias veces con medio de cultivo hasta que el pH es neutro. </li></ol></li><li> Coloque un total de 5 g de virutas de hueso por 10 ml de DMEM fresco suplementado con 1% de antibióticos y antimicóticos en un nuevo plato de plástico. </li><li> Coloque los platos de plástico en una atmósfera húmeda a 37 ° C durante 24 horas. Entonces, cosecha BCM. Centrifugar el BCM a 200 xg durante 10 minutos para eliminar los residuos, filtrarla estéril (0,2 nm), y tener alícuotas congeladas a -80 ° C. </li><li> Descongelar la población de BCM inmediatamente antes de su uso y evitar ciclos repetidos de congelación y descongelación. </li><li> Para los experimentos indicados, remojar las membranas de colágeno con medio BCM o sin suero durante 1 hora a temperatura ambiente. Lave vigorosamente las membranas con PBS y colocarlos en placas de 96 pocillos. Membranas húmedas se siembran con células. </li><li> BCM proceso de preparación se resume en la Figura 1. </li&gt; </ol> 2. Los bioensayos Basado en células mesenquimales <ol><li> Células mesenquimales humanas de semillas (por ejemplo, células óseas, gingival y fibroblastos del ligamento periodontal) en una placa de 12 pocillos con una concentración de 30.000 células / cm2. Para sembrar las células utilizan el medio de cultivo que consiste en DMEM, 10% de suero fetal bovino y antibióticos. Deja que las células se adhieren a la placa de O / N. </li><li> Deseche el medio de cultivo y se lavan las células con PBS precalentado a 37 ° C. Estimular las células mediante la adición de medio de cultivo libre de suero pre-calentado con y sin 20% BCM. Colocar las células en un ambiente humidificado a 37 ° C durante 24 hr. </li><li> Deseche el medio de cultivo, enjuague las células con PBS precalentado y extraer el ARN de acuerdo con su protocolo preferido. </li><li> Ajustar la concentración de ARN con el fin de tener la misma cantidad de ARN en cada muestra. Preparar ADNc y realizar una QRT-PCR para analizar los genes seleccionados utilizando los cebadores mostrados en la Tabla 1. NOTA: Estas son las diluciones de todos los componentes: 2x SYBR verdes, 20x cebador directo, 20x cebador inverso, 5x agua DD estéril, ADNc 5x. La qRT-PCR se realiza en 40 ciclos de 95 ° C 15 seg y 60 ° C 1 min. </li><li> Calcular los niveles relativos de expresión mediante la normalización de la limpieza gen GAPDH utilizando el método Δ (Ct) donde Ct es objetivo CT - CT GAPDH y Δ (Ct) es estimulada Ct - Ct control. </li><li> Después de este control de calidad, añadir BCM al medio de cultivo para estimular todos los tipos de células, incluidas las células mesenquimales, células hematopoyéticas o células endoteliales. </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>

Los autores no tienen nada que revelar. Jordi Caballé-Serrano recibió una beca de la Fundación de Investigación Dental y Educación, Basilea, Suiza.

Bone medio acondicionado refleja la actividad liberada de injertos óseos durante las primeras etapas de la regeneración ósea. El protocolo se describe aquí puede adaptarse para estudiar la respuesta de diferentes tipos de células implicadas en la regeneración ósea. Además, el protocolo se puede utilizar para preparar el medio de rellenos óseos o hueso procesado condicionado. Los métodos son fáciles de realizar y se basan en un concepto simple: los factores liberados de varios hueso nativo y procesados. La com...

El hueso autólogo es ampliamente utilizado para cerrar defectos que se produjeron como consecuencia de la malformación, la cirugía de resección, la cirugía reconstructiva trauma, y antes de la colocación del implante 1,2. La comprensión de los principios biológicos de cómo injertos óseos apoyan el proceso de consolidación del injerto no sólo es clave para entender por qué los autoinjertos son considerados como el estándar de oro en la cirugía reconstructiva, también es biónico para la mejora del diseño de los sustitutos óseos 3. Sin embargo, la consolidación del injerto es más rápido con hueso autólogo en comparación con sustitutos óseos 4,5. Por lo tanto, es imperativo para revelar los mecanismos moleculares y celulares que hacen hueso autólogo tan eficaz para apoyar la regeneración ósea. Hay tres características de libros de texto de autoinjertos que se consideran para apoyar el proceso de consolidación 6,7. En primer hueso, autólogo es osteoconductiva, proporcionar orientación para el hueso de nueva formación para creceren el defecto. En segundo lugar, el hueso autólogo es osteogénico, lo que significa que contiene células mesenquimales que pueden diferenciarse en osteoblastos 8. Hueso Tercero, autólogo es osteoinductivo como factores de crecimiento, como las proteínas morfogenéticas óseas sepultados en la matriz pueden iniciar el proceso de endocondral o incluso la formación de hueso intramembranosa 9. Hay otro aspecto: astillas de hueso recién preparados tienen una función paracrina sobre la base de las observaciones in vitro con &quot;medio hueso acondicionado&quot; 10-15. También el impacto de mielopoyesis debe mencionarse 16. Un término similar &quot;medio de matriz ósea desmineralizada acondicionado&quot; ya fue acuñado en 1996 y es compatible con el concepto general de una función paracrina del hueso, incluso cuando procesada por desmineralización 17. Para nuestros propósitos, BCM puede prepararse a partir de cerdo fresca mandíbulas 10,11. Análisis proteómico de BCM revelaron la composición compleja, incluyendo hecho de crecimientoors y componentes de la matriz extracelular 10, que también se extiende el conocimiento existente sobre el proteasoma de toda ósea 18,19. Por lo tanto, BCM debe reflejar la actividad de lanzamiento de varias modificaciones de injertos óseos in vitro. ¿Qué ocurre cuando las células mesenquimales, por ejemplo los aislados de virutas de hueso o de tejido blando oral, están expuestos a BCM? In vitro, BCM reduce osteogénico y la diferenciación adipogénica, y provoca un fuerte aumento de la expresión de IL11 11. Genoma amplia microarrays reveló más genes que se expresó diferencialmente en las células mesenquimales en respuesta a BCM. Entre estos genes son adrenomedulina (ADM), IL11, IL33, NADPH oxidasa 4 (NOX4), proteoglicanos 4 (PRG4 o lubricina) y pentraxina 3 (PTX3) 15. BCM obtenido a partir de astillas de hueso en autoclave no pudo cambiar la expresión de los genes respectivos 14. BCM de astillas de hueso que se sometieron a la pasteurización y la congelación fue capaz decambiar la expresión del gen 14. Medio de la matriz ósea desmineralizada (DBM-CM) También acondicionado cambia la expresión de TGF-β genes regulados 20. Curiosamente, las membranas de barrera de colágeno utilizados para proteger los chips de hueso a partir del tejido blando circundante 21,22, adsorbidos aquellas partes de BCM que son responsables de los cambios en la expresión génica 23. Investigación BCM se puede extender a otros tipos de células que participan en la regeneración ósea, tales como los osteoclastos de resorción ósea y células endoteliales, para nombrar unos pocos. En general, los datos se acumulan in vitro proporcionan la base científica para el diseño de un estudio preclínico. El presente protocolo es doble: Primero, muestra cómo preparar BCM. En segundo lugar, muestra cómo probar su actividad biológica basado en células mesenquimales 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

Injertos óseos autólogos son ampliamente utilizados en la cirugía oral y maxilofacial, ortopedia y traumatología. Injertos óseos autólogos no sólo reemplazan el hueso faltante, también apoyan el complejo proceso de regeneración ósea. Este comportamiento favorable de los autoinjertos se atribuye a las tres características: osteoconductividad, osteogenicidad y osteoinductividad. Sin embargo, hay otro aspecto: Bone injertos de liberar una gran variedad de moléculas, incluyendo factores de crecimiento, que pueden dirigirse a las células mesenquimales implicadas en la regeneración ósea. Las propiedades paracrinos de injertos óseos se pueden estudiar in vitro mediante el uso de medio hueso acondicionado (BCM). Aquí presentamos un protocolo sobre cómo prepararse medio hueso acondicionado de hueso cortical cerdo nativo, y el hueso que sufrió el procesamiento térmico o desmineralización. Las células pueden ser expuestos directamente a BCM o sembradas en biomateriales, tales como membranas de colágeno, previamente remojados con BCM. Damos ejemplos para in vitro bioassays con células mesenquimales en la expresión de genes regulados TGF-β. Los protocolos presentados deben animar a revelar aún más los efectos paracrinos de injertos óseos durante la regeneración ósea y abrir un camino para la investigación traslacional en el amplio campo de la cirugía reconstructiva.

Bone Medium acondicionado se preparó a partir de astillas de hueso porcino frescas. Visión general del proceso para preparar BCM y usar biomateriales en combinación con BCM se muestra en la Figura 1 y la Figura 2 respectivamente. Durante la preparación del BCM, es importante para obtener grandes astillas de hueso con movimientos largos como movimientos cortos o muy pequeñas astillas de hueso puede afectar a la calidad del BCM final. Calidad de BCM se puede controlar mediante el an?...

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

Bone Conditioned Medium: Preparation and Bioassay

Se describe aquí cómo preparar medio hueso acondicionado (BCM) y probar su actividad in vitro.

Bone Medium acondicionado: Preparación y Bioensayo

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 vistas.  School of Dental Medicine, University of Bern.  Se describe aquí cómo preparar medio hueso acondicionado (BCM) y probar su actividad in vitro. 

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.

El mantenimiento de Wolbachia en medio libre de células

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

Investigación

Biología

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 preparación de cultivos primarios de células hematopoyéticas de la médula ósea murina por electroporación

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.

Preparación ósea descalcificados para histología, histomorfometría y Análisis Fluorocromo

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.

Análisis Cytometric del vesículas extracelulares de medios acondicionado de célula de flujo

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.

Recolección de tejidos y extracción de ARN de la articulación de la rodilla osteoartrítica humana

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.

Uso del analizador de flujo metabólico celular en tiempo real para monitorear la bioenergética de osteoblastos

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 ensayo de quimiotaxis mejorado para la identificación rápida de quimioatrayentes rizobacterianos en exudados radiculares

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.

Bioensayo de aplicación tópica para cuantificar la toxicidad de insecticidas para mosquitos y moscas de la fruta

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.

El bioensayo de células Caco-2 para la medición de la biodisponibilidad de hierro en los alimentos

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

Implantes de cartílago artificial para la regeneración ósea mediante osificación endocondral

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.

Autor destacado: Mejora de la regeneración ósea con la integración de cartílago artificial vascularizado 

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.

Formación ósea integrada a través de la osificación endocondral in vivo utilizando células madre mesenquimales

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