The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

Todos los procedimientos de sangre de dibujo debe ser realizado por profesionales autorizados y certificados. El uso de sujetos humanos de investigación implica la aprobación de la Junta de Revisión Institucional u otra autoridad competente. Precauciones especiales en relación con el consentimiento informado y la protección de la identificación de los participantes tienen que seguir. Todos los experimentos que figuran en este protocolo implican la manipulación de la sangre humana y / o productos de la sangre y el equipo de protección personal adecuado necesario para ser usados ​​en todo momento. La basura debe ser considerado como riesgo biológico y desecharse de acuerdo con las regulaciones. 1. venopunción <ol><li> Identificar al paciente / participante y confirmar con los registros existentes. Explicar el estudio en detalle y obtener un consentimiento informado. </li><li> Tener una bandeja establecido para cada paciente con tubos marcados sobre una superficie plana y estable. </li><li> Explique el procedimiento y hacer el asiento paciente cómodamente con el brazo apoyado. Informar al paciente que él o she sentirá un pequeño pellizco y debe permanecer inmóvil durante todo el procedimiento. </li><li> Coloque la aguja al adaptador. </li><li> Lavarse las manos y use guantes. </li><li> Preparar la fosa antecubital para la venopunción limpiando con un 70% de alcohol isopropílico en círculos concéntricos desde el centro hacia afuera. Permitir el sitio se seque al aire durante 30 segundos. </li><li> Identificar la vena apropiada por palpación. </li><li> Aplicar el torniquete por encima de 3-4 en el sitio de punción asegurarse de que no es demasiado apretado (sin dejar de sentir el pulso radial). </li><li> Realizar la venopunción insertando el bisel de la aguja 15-30 grados a la piel con un movimiento suave. Empuje el tubo de recogida de sangre a través de la aguja y recoger 9 ml de sangre entera. </li><li> Retire el torniquete. Retire el tubo de la aguja. </li><li> Retire la aguja y aplique la plaza gasas en el sitio de punción antes de la extracción de la aguja. Deseche la aguja en un contenedor de riesgo biológico correspondiente. </li><li> Pedir al paciente para mantener pressure en el sitio de la punción. </li><li> Etiquetar los tubos </li><li> Consulte el sitio de punción para asegurarse de sangrado se ha detenido. </li><li> Aplicar vendaje adhesivo o cinta sobre el cuadrado de gasa, pregunte si el paciente se siente bien (sin dolor, hinchazón o mareo) </li><li> Gracias al paciente / participante antes de la descarga. </li></ol> 2. L-PRF Preparación <ol><li> Inmediatamente después de la extracción de sangre venosa en el tubo de vidrio seco rojo rematado, colóquelo en la centrífuga. </li><li> Centrifugar a 400 xg durante 12 minutos a temperatura ambiente después de colocar un contrapeso adecuado. </li><li> Quitar el tubo en el extremo del ciclo. Observe el tres capas: plaquetas plasma pobre (PPP), la fibrina rico en plaquetas (L-PRF) y la base de RBC (Figura 1). </li><li> Aspirar el PPP utilizando una pipeta. Usando pinzas tire suavemente de la L-PRF y la colocan en una malla de metal perforado estéril. </li><li> El uso de bisturí quirúrgico, raspar el grueso de la capa de RBC dejando cuidadosamente la piel de antey la capa intacta. </li><li> Comprimir suavemente el coágulo de L-PRF (utilizando la placa de metal estéril, peso aproximado 225 g) durante 30 seg. Plasma pobre en plaquetas será exprimida. </li><li> Retire la placa y levante suavemente la membrana L-PRF. La membrana L-PRF está listo para su uso en experimentos 12. </li></ol> 3. Ensayo de tracción uniaxial <ol><li> Coloque las membranas L-PRF (n = 6) en un papel de filtro para facilitar su manejo y ponche en &quot;huesos de perro&quot; con el metal a medida muere (2,75 mm de ancho en su punto más estrecho, con una longitud de calibre de 7,5 mm). </li><li> Mida el espesor de cada muestra a tres puntos y tomar la media. </li><li> Comprometerse con cuidado la membrana de L-PRF en el centro de las garras de mordaza del sistema de prueba uniaxial. </li><li> Romper con cuidado el soporte de papel archivador para exponer la membrana L-PRF. </li><li> Programar el instrumento para que el cabezal móvil está funcionando a una velocidad constante (10,0 mm / min) e iniciar el experimento cuando el L-PRF está todavía húmeda. </li><li> Registre el módulo elástico, la energía de romper, y la tensión en la rotura del software que acompaña al sistema de ensayo uniaxial. Estos valores se calculan de forma automática y no se requiere de entrada definido por el usuario. Por favor, vea la Figura 3. </li></ol> 4. Fuerza de retención de sutura <ol><li> Colocar las membranas L-PRF (n = 3) sobre un papel de filtro para facilidad de manejo y cortar en muestras rectangulares de medición (10 mm x 25 mm) utilizando un bisturí quirúrgico. </li><li> Medir el espesor de cada muestra (promedio de 3). </li><li> Hacer un agujero de alfiler en el centro de la muestra usando el alambre de ligadura de ortodoncia de acero inoxidable (220 micras de diámetro). </li><li> Pase el alambre de ligadura a través del agujero para formar un bucle y fijarlo a la máquina de tracción. Coloque el borde de la membrana L-PRF a la empuñadura mandíbula inferior 13. </li><li> Programar el instrumento para que el cabezal móvil está funcionando a una velocidad constante (10 mm / min) y el inicioel experimento. </li><li> Registre el módulo elástico, la energía de romper, y la tensión en la rotura del software que acompaña al sistema de ensayo uniaxial. Estos valores se calculan de forma automática y no se requiere de entrada definido por el usuario. Por favor, vea la Figura 3. </li></ol> 5. El examen morfológico <ol><li> Preparar las muestras L-PRF para el examen SEM utilizando un 10 mm punzón de biopsia cutánea y colocarlos en una placa de 24 pocillos. </li><li> Lavar las muestras con PBS y fijar con glutaraldehído al 2,5% (en PBS) durante 20 min. </li><li> Deshidratar las muestras por inmersión en aumentar secuencialmente concentraciones de etanol (50%, 70%, 80%, 90% y 100%) durante 5 min cada uno. </li><li> Tratar con 0,5 ml de 100% de HMDS (hexametildisilazano) durante 5 min. Airear O / N para eliminar el exceso de HMDS 14. </li><li> Muestras de montaje sobre talones utilizando una cinta de doble cara, pulverización catódica capa de platino por 70 seg y examinan en un microscopio electrónico de barrido que funciona a una aceleracióntensión ración de 20 kV (o lugar adecuados). </li></ol> 6. genipina reticulación de L-PRF, tripsina Susceptibilidad y Ninhidrina Ensayo <ol><li> Para preparar genipina reticulado L-PRF, enjuague membranas con PBS y sumergirse en 4 ml de solución de genipina 1% (en etanol 70%) durante 48 hr. Enjuague con PBS antes de los experimentos para eliminar el exceso genipina 15, 16. </li><li> Evaluar la estabilidad de reticulación genipina de L-PRF por su resistencia a la degradación por tripsina. Membrana Place L-PRF (n = 3) y genipina reticulado L-PRF en 500 l de 0,01% de tripsina y se incubaron a 37 ° C durante 3 días con un cambio diario de tripsina. <ol><li> Pesar muestras en el día 1 antes de la enzima de la exposición y en el día 3. La diferencia en el inicio y al final de peso representa la degradación enzimática 17. </li></ol></li><li> Cuantificar la cantidad de entrecruzamiento en genipina tratado L-PRF (G-PRF) por ninhidrina ensayo. En primer lugar preparar la curv estándare utilizando glicina (mM 1 mM-0.031) curva para establecer la relación entre la concentración de grupos amino ácido (FAA) y la absorbancia. <ol><li> Muestras Heat PRF con 1 ml de 2% (w / v) de ninhidrina durante 15 min a 100 ° C. </li><li> Deje que la solución se enfríe a temperatura ambiente y añadir 1,5 ml de etanol al 50%. </li><li> Analizar la absorbancia a 570 nm usando un espectrofotómetro adecuado. </li><li> Determinar el porcentaje de reticulación utilizando la siguiente fórmula 15 </li></ol></li></ol><img alt="Ecuación 1" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS ensayo de proliferación celular <ol><li> Crecer MC3T3 (ratón preosteoblastos de calota) en la modificación alfa Medio Esencial Mínimo (aMEM) en matraces T-75 hasta que se obtiene una monocapa confluente 80%. </li><li> Preparar membrana fresca, estéril L-PRF (abrir el tubo L-PRF dentro de la campana de cultivo de células) y la transferencia de la membrana a una nueva placa de cultivo celular. </li><li> Medios Aspirar del matraz y enjuagar la monocapa con PBS, añadir 5 ml de tripsina al 0,05% y colocar el matraz en la incubadora a 37 ° durante 5 minutos, pipetear los contenidos del matraz en un tubo de centrífuga y centrifugar a 400 xg durante 5 min. </li><li> Decantar el sobrenadante, golpear suavemente el tubo para romper el sedimento celular y resuspender con 4 ml de MEM α fresca, dispensar una mezcla de suspensión celular (50 l) y azul de tripano (50 l) en el hemocitómetro y contar el número de células </li><li> Semilla de 4x10 5 células en 10 mm anillos de clonación cristal colocadas en la parte superior de las membranas L-PRF para retener las células dentro de las membranas (anillos se pueden quitar después de 24 horas). </li><li> Al día 4, enjuague con PBS tres veces constructos de 10 min. </li><li> Añadir 1 ml de medio libre de suero y 200 l de reactivo MTS a cada pocillo y se incuba durante 2 horas a 37 ° C. </li><li> Medir la absorbancia de 200 alícuotas a 490 nm. </li></ol>

<ol>
	<li>Marx, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+Growth+factor+enhancement+for+bone+grafts.">Platelet-rich plasma Growth factor enhancement for bone grafts.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 85 (6), 638-646 (1998).</li><li>Rodriguez, I. A., Growney Kalaf, E. A., Bowlin, G. L., Sell, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+plasma+in+bone+regeneration:+Engineering+the+delivery+for+improved+clinical+efficacy.">Platelet-rich plasma in bone regeneration: Engineering the delivery for improved clinical efficacy.</a> BioMed Res In. 2014, (2014).</li><li>Del Corso, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+Knowledge+and+Perspectives+for+the+Use+of+Platelet-Rich+Plasma+(PRP)+and+Platelet-Rich+Fibrin+(PRF).">Current Knowledge and Perspectives for the Use of Platelet-Rich Plasma (PRP) and Platelet-Rich Fibrin (PRF).</a> in Oral and Maxillofacial Surgery Part 1: Periodontal and Dentoalveolar Surgery. Curr Pharm Biotechno. 13 (7), 1207-1230 (2012).</li><li>Mazzucco, L., Balbo, V., Cattana, E., Guaschino, R., Borzini, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+every+PRP-gel+is+born+equal+Evaluation+of+growth+factor+availability+for+tissues+through+four+PRP-gel+preparations:+Fibrinet,+RegenPRP-Kit,+Plateltex+and+one+manual+procedure.">Not every PRP-gel is born equal Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.</a> Vox San. 97 (2), 110-118 (2009).</li><li>Fern&#225;ndez-Barbero, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+and+morphological+characterization+of+platelet-rich+plasma+gel.">Flow cytometric and morphological characterization of platelet-rich plasma gel.</a> Clin Oral Implants Re. 17 (6), 687-693 (2006).</li><li>Li, Z., Guan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+cardiac+tissue+engineering.">Hydrogels for cardiac tissue engineering.</a> Polymer. 3 (2), 740-761 (2011).</li><li>Zhu, J., Cai, B., Ma, Q., Chen, F., Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bricks-enriched+platelet-rich+plasma+gel+for+injectable+cartilage+engineering+&#8211;+an+in+vivo+experiment+in+nude+mice.">Cell bricks-enriched platelet-rich plasma gel for injectable cartilage engineering &#8211; an in vivo experiment in nude mice.</a> J Tissue Eng Regen Med. 7 (10), 819-830 (2013).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+I:+technological+concepts+and+evolution.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e37-e44 (2006).</li><li>Dohan, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+II:+platelet-related+biologic+features.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e45-e50 (2006).</li><li>Choukroun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+V:+histologic+evaluations+of+PRF+effects+on+bone+allograft+maturation+in+sinus+lift.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), 299-303 (2006).</li><li>Dohan Ehrenfest, D. M., Bielecki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+the+Fibrin+Architecture+and+Leukocyte+Content+Influence+the+Growth+Factor+Release+of+Platelet+Concentrates?+An+Evidence-based+Answer+Comparing+a+Pure+Platelet-Rich+Plasma+(P-PRP)+Gel+and+a+Leukocyte-+and+Platelet-Rich+Fibrin+(L-PRF).">Do the Fibrin Architecture and Leukocyte Content Influence the Growth Factor Release of Platelet Concentrates? An Evidence-based Answer Comparing a Pure Platelet-Rich Plasma (P-PRP) Gel and a Leukocyte- and Platelet-Rich Fibrin (L-PRF).</a> Curr Pharma Biotechno. 13 (7), 1145-1152 (2012).</li><li>Dohan Ehrenfest, D. M., Rasmusson, L., Albrektsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+platelet+concentrates:+from+pure+platelet-rich+plasma+(P-PRP)+to+leucocyte-+and+platelet-rich+fibrin+(L-PRF).">Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF).</a> Trends Biotechno. 27 (3), 158-167 (2009).</li><li>Mine, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suture+Retention+Strength+of+Expanded+Polytetrafluoroethylene+(ePTFE)+Graft.">Suture Retention Strength of Expanded Polytetrafluoroethylene (ePTFE) Graft.</a> Acta Med Okayam. 64 (2), 121-128 (2010).</li><li>Braet, F., De Zanger, R., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drying+cells+for+SEM+,+AFM+and+TEM+by+hexamethyldisilazane:+a+study+on+hepatic+endothelial+cells.">Drying cells for SEM , AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.</a> J Micros. 186 (1), 84-87 (1997).</li><li>Yuan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cross-linking+of+chitosan+microspheres+with+genipin+on+protein+release.">The effect of cross-linking of chitosan microspheres with genipin on protein release.</a> Carbohydr Poly. 68 (3), 561-567 (2007).</li><li>Sell, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. The Role of Platelet Gel in Regenerative Medicine.</a> Advances In Regenerative Medicine. , 319-349 (2011).</li><li>Dohan Ehrenfest, ., Lemo, D. M., Jimbo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selecting+a+relevant+animal+model+for+testing+the+in+vivo+effects+of+Choukroun&#8217;s+platelet-rich+fibrin+(PRF):+Rabbit+tricks+and+traps.">Selecting a relevant animal model for testing the in vivo effects of Choukroun&#8217;s platelet-rich fibrin (PRF): Rabbit tricks and traps.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 110 (4), 413-416 (2010).</li><li>Guilak, F., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+tissue+engineering:+Ten+more+years+of+progress.">Functional tissue engineering: Ten more years of progress.</a> J Biomec. 47 (9), 1931-1932 (2014).</li><li>Guilak, F., Butler, D. L., Goldstein, S. A., Baaijens, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+mechanobiology+in+functional+tissue+engineering.">Biomechanics and mechanobiology in functional tissue engineering.</a> J Biomec. 47 (9), 1933-1940 (2014).</li><li>Collet, J. P., Shuman, H., Ledger, R. E., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elasticity+of+an+individual+fibrin+fiber+in+a+clot.">The elasticity of an individual fibrin fiber in a clot.</a> Proc Natl Acad Sci. 102 (26), 9133-9137 (2005).</li><li>Liu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+fibers+have+extraordinary+extensibility+and+elasticity.">Fibrin fibers have extraordinary extensibility and elasticity.</a> Science. 313 (5787), 634 (2006).</li><li>American National Standard. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8:+Test+methods+for+vascular+prostheses.">Chapter 8: Test methods for vascular prostheses.</a> Association for the Advancement of Medica lnstrumentation Guidance document: Cardiovascular Implants - Tubular vascular prostheses. , 33-34 (2001).</li></ol>

The authors have nothing to disclose and confirm that there are no known conflicts of interest associated with this publication.

Autologous platelet concentrates are promising in the field of regenerative medicine18 because of the abundance of growth factors. However, these preparations often lacked a defined structure that makes surgical manipulation very difficult. Many times, the suspensions and gels are not retained effectively at the site of delivery, resulting in unpredictable outcomes. L-PRF represents a huge advance in the evolution of platelet concentrates in that it is essentially a firm fibrin membrane with entrapped platelet...

The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4. 
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used. 
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Needle 19G</td>
			<td>BD</td>
			<td>305186</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Disposal Container</td>
			<td>Fisherbrand</td>
			<td>14-827-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Red-Topped Glass Collection Tube</td>
			<td>BD</td>
			<td>8020129</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze Pads</td>
			<td>Tyco</td>
			<td>5750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandage</td>
			<td>Johnson &#38; Johnson</td>
			<td>5005989</td>
			<td></td>
		</tr>
		<tr>
			<td>Surshield</td>
			<td>Terumo</td>
			<td>SV*S19BL</td>
			<td>Safety winged infusion set</td>
		</tr>
		<tr>
			<td>Blood Collection Assembly</td>
			<td>BD</td>
			<td>303380</td>
			<td></td>
		</tr>
		<tr>
			<td>Tourniquets</td>
			<td>BD</td>
			<td>367203</td>
			<td></td>
		</tr>
		<tr>
			<td>Brand Luer Adapter</td>
			<td>Vacutainer</td>
			<td>L42179</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-Spin System&#160;</td>
			<td>Intra-Lock International</td>
			<td>ISS110</td>
			<td>Centrifuge and Xpression L-PRF FabricationKit&#160;</td>
		</tr>
		<tr>
			<td>Pipettes (Serological &#38; Micro)</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Exelint</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Bionix 200</td>
			<td>MTS Systems Corporation</td>
			<td></td>
			<td>Material testing systems</td>
		</tr>
		<tr>
			<td>MTS Test Works 4</td>
			<td>MTS Systems Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Filter Paper</td>
			<td>Whatman</td>
			<td>1004 070</td>
			<td></td>
		</tr>
		<tr>
			<td>SS Orthodontic ligature wire</td>
			<td>Patterson Dental</td>
			<td>628-4228</td>
			<td></td>
		</tr>
		<tr>
			<td>200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Aldrich</td>
			<td>440191</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminium Mounting Stubs</td>
			<td>Ted Pella</td>
			<td>16324</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Sided Carbon Tape</td>
			<td>PELCO Tabs</td>
			<td>16084-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>LV 5610</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>51026282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimicotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Genipin</td>
			<td>Wako</td>
			<td>078-03021</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Media</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>Minimum Essential Medium-Alpha</td>
		</tr>
		<tr>
			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G1118</td>
			<td></td>
		</tr>
		<tr>
			<td>PMS Reagent</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td></td>
			<td>Epoch Spectrophotometer</td>
		</tr>
		<tr>
			<td>10mm Glass Cloning Rings</td>
			<td>Corning</td>
			<td>3166-10</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Corning</td>
			<td>55-031-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Ninhydrin 98%</td>
			<td>Aldrich</td>
			<td>454044</td>
			<td></td>
		</tr>
		<tr>
			<td>24 Well Plate</td>
			<td>Corning</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>Acu Punch</td>
			<td>P1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Micrometer</td>
			<td>Pittsburgh</td>
			<td>68305</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well Plate</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterization of leukocyte-platelet rich fibrin, a novel biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

The scanning electron microscope image of the L-PRF clot at different sections (top, middle and bottom) layer is illustrated in Figure 2. As can be seen, the top portion is composed predominantly of fibrin network with no cells. The middle layer is enriched with platelets with evidence of their activation and degranulation. The lower layer has a mixture of leukocytes and red blood cells entrapped within a fibrin matrix.
The mechanical properties were evaluated in two modes: un...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Caracterización de los leucocitos-plaquetas Rich fibrina, una novela de Biomateriales

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.8K vistas.  School of Dentistry, Virginia Commonwealth University. Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. 

Video: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Investigación

Bioingeniería

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Cine Cultura de la Seda del sistema para In vitro Análisis y Diseño de Biomateriales

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Observación Y Cuantificación De Fibroblastos Mediada Por Fibrina Gel Compactamiento

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Construcción y caracterización de un Fold biorreactor Novela Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Preparación y caracterización de nuevas nanopartículas que imitan las HDL para la encapsulación del factor de crecimiento nervioso

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Ingeniería de superficies de islotes pancreáticos con un nanocobertura StarPEG heparinizada

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricación y caracterización de tejido óptico fantasmas que contiene macroestructura

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Microdisección de segmentos de tejido Renal primaria y la incorporación con la tecnología de construcción de andamio-libre nuevo

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Un modelo de embrión de pez cebra para In Vivo visualización y análisis Intravital de Biomaterial asociados Staphylococcus aureus infección

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.