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

1. Preparação BCM <ol><li> Obter mandíbulas de porco do talho local o mais fresco possível. Coloque as mandíbulas em uma superfície firme e liberar um retalho de espessura total com especial atenção para não deixar qualquer tecido mole ou periósteo fixados ao osso. Trabalhar em um ambiente limpo, sem a necessidade de trabalhar sob o capô fluxo. </li><li> Uma vez que um retalho de espessura total é liberado, use um raspador de osso para colher as lascas de osso do lado vestibular. Por favor note que o raspador ósseo tem de ser afiado. Manusear com firmeza o raspador ósseo e com movimentos longos recolher o osso. Descarte fragmentos de ossos menores que um milímetro. <ol><li> Para manter fragmentos de ossos nativas, coloque directamente os fragmentos de ossos em pratos de plástico de 10 cm de diâmetro com meio de Eagle modificado por Dulbecco (DMEM) suplementado com 1% antibióticos e antimicóticos não deixá-los secar. </li><li> Para avaliar o impacto do processamento térmico, lascas de osso sujeito a uma pasteurização durante 30 min a 80 ° C ouautoclave durante 20 min a 121 ° C. </li><li> Para avaliar o impacto de desmineralização, agitar fragmentos de ossos em HCl 1 M para 4-6 horas a 4 ° C e lava-se várias vezes com meio de cultura até o pH ser neutro. </li></ol></li><li> Inserir um total de 5 g de pedaços de osso por 10 ml de DMEM fresco suplementado com 1% de antibióticos e antimicóticos para um novo prato de plástico. </li><li> Colocar as placas de plástico em uma atmosfera humidificada a 37 ° C durante 24 h. Então, colheita BCM. Centrifugar o BCM a 200 xg durante 10 min para remover os detritos, filtrá-la estéril (0,2 nM), e manter aliquotas congeladas a -80 ° C. </li><li> Descongele o estoque BCM imediatamente antes da utilização e evitar ciclos repetidos de congelamento e descongelamento. </li><li> Para as experiências indicadas, embeber as membranas de colagénio com meio BCM ou isento de soro durante 1 hora à TA. Lavar vigorosamente as membranas com PBS e colocá-los em placas de 96 poços. Membranas molhadas são semeadas com células. </li><li> Processo de preparação de BCM está resumida na Figura 1. </li&gt; </ol> 2. Os bioensaios Baseado em Células mesenquimais <ol><li> Células mesenquimais semente humana (por exemplo, células ósseas, gengival e fibroblastos do ligamento periodontal) em uma placa de 12 poços com uma concentração de 30.000 células / cm2. Para semear as células usam meio de crescimento consistindo em DMEM, 10% de soro fetal de vitelo e antibióticos. Deixe as células anexar à placa O / N. </li><li> Descartar o meio de cultura e lava-se as células com PBS pré-aquecido a 37 ° C. Estimular as células pela adição de meio de cultura isento de soro pré-aquecido, com e sem 20% de BCM. Colocar as células numa atmosfera humidificada a 37 ° C durante 24 h. </li><li> Descartar o meio de cultura, lavar as células com PBS pré-aquecido e extrai-se o ARN de acordo com o seu protocolo preferido. </li><li> Ajustar a concentração de ARN, de modo a ter a mesma quantidade de ARN em cada amostra. Preparar ADNc e executar um de qRT-PCR para analisar os genes seleccionados utilizando os iniciadores apresentados na Tabela 1. NOTA: Estas são as diluições de cada componente: 2x SYBR verde, 20x iniciador directo, 20x iniciador inverso, 5x DD estéril água, 5x cDNA. O qRT-PCR é realizada em 40 ciclos de 95 ° C, 15 seg e 60 ° C 1 min. </li><li> Calcular os níveis de expressão relativa através da normalização para o gene GAPDH de limpeza utilizando o método Δ (ΔCt) onde é ΔCT alvo TC - CT GAPDH e Δ (ΔCT) é estimulada ΔCT - ΔCT controlo. </li><li> Após este controlo de qualidade, BCM adicionar ao meio de cultura para estimular a todos os tipos de células, incluindo células mesenquimais, células hematopoiéticas ou células endoteliais. </li></ol>

<ol>
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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. 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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>

Os autores não têm nada a revelar. Jordi Caballé-Serrano recebeu uma bolsa da Fundação de Pesquisa Dental e da Educação, Basileia, Suíça.

Meio condicionado por óssea reflecte a actividade libertada de enxertos de osso durante os estágios iniciais de regeneração óssea. O protocolo aqui descrito pode ser adaptado para estudar a resposta de diferentes tipos de células envolvidas na regeneração óssea. Além disso, o protocolo pode ser utilizado para preparar o meio condicionado a partir de agentes de enchimento de osso ou ossos processados. Os métodos são fáceis de realizar e contam com um conceito simples: os fatores libertados por várias osso n...

Ósseo autólogo é amplamente utilizado para preencher defeitos que ocorreram como consequência de malformação, a cirurgia ressectivo, a cirurgia reconstrutiva trauma, e antes da colocação do implante 1,2. Compreender os princípios biológicos de como enxertos ósseos apoiar o processo de consolidação do enxerto não é única chave para entender porque auto-enxertos são considerados o padrão-ouro em cirurgia reconstrutiva, também é biônico para o projeto melhorado de substitutos ósseos 3. Ainda assim, a consolidação do enxerto é mais rápido com ósseo autólogo em comparação com osso substitui 4,5. Assim, é imperativo para revelar os mecanismos moleculares e celulares que formam osso autólogo de modo eficaz para apoiar a regeneração do osso. Existem três características exemplares de auto-enxertos que são considerados para suportar o processo de consolidação 6,7. Primeiro osso, autólogo é osteocondutor, fornecendo orientações para o osso recém-formado para crescerno defeito. Em segundo lugar, osso autólogo é osteogénico, o que significa que ele contém células mesenquimais que podem diferenciar-se em osteoblastos 8. Terceiro osso, autólogo é osteoindutora como fatores de crescimento, como proteínas ósseas morfogenéticas sepultados na matriz pode iniciar o processo de endochondral ou mesmo a formação óssea intramembranosa 9. Há um outro aspecto: fragmentos de ossos recém-preparados realizar uma função parácrina baseado nas observações in vitro com &quot;meio-bone condicionado&quot; 10-15. Além disso, o impacto da mielopoese deve ser mencionado 16. Um termo similar &quot;meio condicionado-matriz óssea desmineralizada&quot; já foi cunhado em 1996 e apoia o conceito global de uma função parácrina do osso, mesmo quando processados ​​por desmineralização 17. Para nossos propósitos, BCM pode ser preparado a partir de suíno fresca mandíbulas 10,11. Análises proteômicas de BCM revelou a composição complexa, incluindo facto crescimentoRUP e constituintes da matriz extracelular 10, estendendo-se também ao conhecimento existente sobre o proteassoma de osso total 18,19. Assim, BCM deve refletir a atividade liberada de várias modificações de enxertos ósseos in vitro. O que acontece quando células mesenquimatosas, por exemplo, os isolados a partir de lascas de osso ou de tecidos moles da boca, são expostos para BCM? In vitro, o BCM reduz osteogénico e osteócito, e provoca um forte aumento da expressão IL11 11. Genoma de largura de microarray revelou mais genes a ser expressos diferencialmente em células mesenquimais em resposta ao BCM. Entre estes genes são adrenomedullin (ADM), IL11, IL33, NADPH oxidase 4 (Nox4), proteoglicanos 4 (PRG4, ou lubricin) e pentraxina 3 (PTX3) 15. BCM obtido a partir de fragmentos de ossos autoclavada não conseguiu alterar a expressão dos respectivos genes 14. BCM a partir de fragmentos de ossos que foram submetidos a pasteurização eo congelamento foi capaz dealterar a expressão do gene 14. Meio também condicionado da matriz óssea desmineralizada (DBM-CM) muda a expressão de TGF-β genes regulados por 20. Curiosamente, as membranas barreira de colagénio utilizadas para proteger os fragmentos de ossos do tecido mole circundante 21,22, adsorvido às partes de BCM que são responsáveis ​​para que as alterações na expressão do gene 23. BCM pesquisa pode ser alargado a outros tipos de células envolvidas na regeneração do osso, tais como os osteoclastos de reabsorção óssea e células endoteliais, para citar alguns. No geral, os dados in vitro acumulando proporcionar a base científica para a concepção de um estudo pré-clínico. O presente protocolo é duplo: Primeiro, ele mostra como preparar BCM. Em segundo lugar, como mostra a testar a sua actividade biológica com base em células mesenquimais 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

Enxertos ósseos autólogos são amplamente utilizados em cirurgia oral e maxilofacial, ortopedia, traumatologia e. Enxertos ósseos autólogos não só substituir osso em falta, eles também suportam o complexo processo de regeneração óssea. Esse comportamento favorável dos auto-enxertos é atribuído às três características: osteocondutividade, osteogenicidade, e osteoindutividade. No entanto, há um outro aspecto: Enxerto ósseo libertar uma miríade de moléculas, incluindo factores de crescimento, que podem direcionar as células mesenquimatosas envolvidas na regeneração óssea. As propriedades parácrinos de enxertos de osso pode ser estudada in vitro através da utilização de meio condicionado de ossos (BCM). Aqui apresentamos um protocolo sobre como preparar o meio-condicionado de osso nativo osso cortical suíno e osso que foram submetidos a tratamento térmico ou desmineralização. As células podem ser directamente exposta à BCM ou semeados em biomateriais, tais como membranas de colágeno, previamente embebidos com BCM. Nós damos exemplos de in vitro bioassays com células mesenquimais sobre a expressão de TGF-β genes regulados. Os protocolos apresentados devem incentivar a revelar ainda mais os efeitos parácrinos de enxertos ósseos durante a regeneração óssea e abrir um caminho para a investigação translacional no amplo campo da cirurgia reconstrutiva.

Osso meio condicionado é preparado a partir frescos fragmentos de ossos suína. Descrição geral do processo para preparar e utilizar BCM biomateriais em combinação com BCM é mostrado na Figura 1 e Figura 2, respectivamente. Durante a preparação BCM, é importante obter grandes fragmentos de ossos com movimentos longos como movimentos curtos ou muito pequenos fragmentos de ossos pode afetar a qualidade do BCM final. Qualidade de o BCM pode ser controlada através da análise da e...

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

Bone Conditioned Medium: Preparation and Bioassay

Descrevemos aqui como preparar meio-bone condicionado (BCM) e testar a sua actividade in vitro.

Osso meio condicionado: Preparação e Bioensaio

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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Biology

11.0K visualizações.  School of Dental Medicine, University of Bern.  Descrevemos aqui como preparar meio-bone condicionado (BCM) e testar a sua actividade in vitro. 

Vídeo: 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.

Manutenção de Wolbachia em Cell-livre Médio

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

Biologia

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Preparação óssea Undecalcified de Histologia, Histomorfometria e Análise 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.

Fluxo Cytometric análise das vesículas extracelulares de célula-condicionado 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 ...

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.

Coleta de tecidos e extração de RNA da articulação do joelho 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.

Usando o analisador de fluxo metabólico de células em tempo real para monitorar bioenergetics osteoblast

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.

Um ensaio de quimiotaxis melhorado para a identificação rápida de quimioattractants rizobacterianos em Exudates 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.

Bioensaio de aplicação tópico para quantificar toxicidade de inseticida para mosquitos e moscas de frutas

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.

O Bioensaio celular Caco-2 para Medição da Biodisponibilidade de Ferro Alimentar

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 cartilagem artificial para regeneração óssea via ossificação 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 em destaque: Melhorando a regeneração óssea com integração de cartilagem artificial vascularizada 

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.

Formação Óssea Integrada Através da Ossificação Endocondral In Vivo Usando Células-Tronco Mesenquimais

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