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.

Toutes les procédures sang dessin doivent être effectués par des professionnels agréés et certifiés. L&#39;utilisation de sujets humains pour la recherche implique l&#39;approbation de l&#39;Institutional Review Board ou toute autre autorité appropriée. Précautions particulières concernant le consentement éclairé et la protection de l&#39;identification des participants doivent être suivies. Toutes les expériences énoncés dans ce Protocole impliquent la manipulation du sang humain et / ou de produits sanguins et de l&#39;équipement de protection individuelle approprié doivent être portés à tout moment. Les déchets doit être considérée comme Biohazard et éliminés conformément aux règlements. 1. ponction veineuse <ol><li> Identifier le patient / participant et confirmer avec les documents existants. Expliquer l&#39;étude en détail et obtenir un consentement éclairé. </li><li> Avoir un plateau mis en place pour chaque patient avec des tubes marqués sur une surface plane et stable. </li><li> Expliquer la procédure et de faire le siège du patient confortablement avec le bras soutenu. Informer le patient qu&#39;il ou she vous sentirez une petite pincée et devrait le rester encore pendant toute la procédure. </li><li> Fixez l&#39;aiguille à l&#39;adaptateur. </li><li> Se laver les mains et porter des gants. </li><li> Préparer le pli du coude pour ponction veineuse par un nettoyage avec 70% d&#39;alcool isopropylique en cercles concentriques à partir du centre vers l&#39;extérieur. Autoriser le site sécher à l&#39;air pendant 30 sec. </li><li> Identifier la veine appropriée par palpation. </li><li> Appliquer le garrot 3-4 au-dessus du site de ponction en vous assurant qu&#39;il est pas trop serré (tout en sentant le pouls radial). </li><li> Effectuer la ponction veineuse en insérant le biseau de l&#39;aiguille 15-30 degrés à la peau dans un mouvement fluide. Appuyez sur le tube de collecte de sang à travers l&#39;aiguille et recueillir 9 ml de sang entier. </li><li> Retirez le garrot. Enlevez le tube de l&#39;aiguille. </li><li> Retirer l&#39;aiguille et appliquer le carré de gaze sur le site de ponction avant le retrait de l&#39;aiguille. Jeter l&#39;aiguille dans un container prévu appropriée. </li><li> Demandez au patient de maintenir prEssure au site de ponction. </li><li> Etiqueter les tubes </li><li> Consultez le site de ponction pour être sûr saignement a cessé. </li><li> Appliquer un bandage ou un ruban adhésif sur la place de la gaze, demander si le patient se sent bien (pas de douleur, un gonflement ou des étourdissements) </li><li> Merci le patient / participant avant de les rejeter. </li></ol> 2. Préparation de L-PRF <ol><li> Immédiatement après le prélèvement de sang veineux dans le tube rouge surmonté verre sec, placez-le dans la centrifugeuse. </li><li> Centrifuger à 400 g pendant 12 min à température ambiante après avoir placé un contrepoids approprié. </li><li> Retirer le tube à la fin du cycle. Remarquez le trois couches: plasma pauvre en plaquettes (PPP), la fibrine riche en plaquettes (L-PRF) et de la base RBC (Figure 1). </li><li> Aspirer le PPP à l&#39;aide d&#39;une pipette. L&#39;aide de pincettes tirez doucement sur le L-PRF et placez-le dans un maillage métallique perforée stérile. </li><li> Utilisation de scalpel, gratter la majeure partie de la couche de RBC laissant soigneusement le buffcouche intacte y. </li><li> Comprimer doucement le caillot L-PRF (en utilisant la plaque de métal stérile, poids approximatif 225 g) pendant 30 secondes. Plasma pauvre en plaquettes sera évincé. </li><li> Retirer la plaque et soulevez délicatement la membrane L-PRF. La membrane L-PRF est prêt pour une utilisation dans des expériences 12. </li></ol> 3. Uniaxial Essai de traction <ol><li> La place membranes L-PRF (n = 6) sur un papier filtre pour la facilité de manipulation et de punch en «os de chien&quot; en utilisant des matrices de métal sur-mesure (2,75 mm de large à son point le plus étroit avec une longueur de jauge de 7,5 mm). </li><li> Mesurer l&#39;épaisseur de chaque échantillon en trois points et prendre la moyenne. </li><li> Engager avec précaution la membrane L-PRF dans le centre des poignées de la mâchoire du système d&#39;essais uniaxiaux. </li><li> Détachez avec soin le support papier de filer pour exposer la membrane L-PRF. </li><li> Programme de l&#39;instrument de telle sorte que la tête mobile fonctionne à une vitesse constante (10,0 mm / min) et commencer l&#39;expérience lorsque le L-PRF est encore humide. </li><li> Enregistrez le module d&#39;élasticité, de l&#39;énergie à briser, et déformation à la rupture à partir du logiciel accompagnant le système de test uniaxial. Ces valeurs sont calculées automatiquement et aucune entrée définie par l&#39;utilisateur est requise. S&#39;il vous plaît voir la figure 3. </li></ol> 4. Suture force de rétention <ol><li> La place membranes L-PRF (n = 3) sur un papier filtre pour faciliter la manipulation et les couper en échantillons rectangulaires mesure (10 mm x 25 mm) à l&#39;aide d&#39;un scalpel. </li><li> Mesurer l&#39;épaisseur de chaque échantillon (moyenne de trois). </li><li> Ajouter un trou d&#39;épingle dans le centre de l&#39;échantillon en utilisant la ligature orthodontique fil en acier inoxydable (220 um de diamètre). </li><li> Passez le fil de ligature à travers le trou d&#39;épingle pour former une boucle et la fixer à la machine d&#39;essai de traction. Placer le bord de la membrane L-PRF à la poignée inférieure 13 de la mâchoire. </li><li> Programmer l&#39;instrument de telle sorte que la tête mobile fonctionne à une vitesse constante (10 mm / min) et le démarragel&#39;expérience. </li><li> Enregistrez le module d&#39;élasticité, de l&#39;énergie à briser, et déformation à la rupture à partir du logiciel accompagnant le système de test uniaxial. Ces valeurs sont calculées automatiquement et aucune entrée définie par l&#39;utilisateur est requise. S&#39;il vous plaît voir la figure 3. </li></ol> 5. Examen morphologique <ol><li> Préparer les échantillons L-PRF pour examen au MEB en utilisant un 10 mm cutanée biopsie punch et de les placer dans une plaque de 24 puits. </li><li> Laver les échantillons avec du PBS et fixer avec 2,5% de glutaraldéhyde (dans du PBS) pendant 20 min. </li><li> Déshydrater les échantillons par immersion dans l&#39;augmentation des concentrations d&#39;éthanol de façon séquentielle (50%, 70%, 80%, 90% et 100%) pendant 5 minutes chacun. </li><li> Traiter avec 0,5 ml de 100% de l&#39;hexaméthyldisilazane (HMDS) pendant 5 min. Aérer O / N pour éliminer l&#39;excès de HMDS 14. </li><li> Échantillons de montage sur les talons à l&#39;aide d&#39;un ruban adhésif double face, pulvérisation manteau platine pour 70 secondes et d&#39;examiner dans un microscope électronique à balayage fonctionnant à une accélérationtension de ration de 20 kV (ou le réglage approprié). </li></ol> 6. génipine réticulation de la L-PRF, la trypsine et la sensibilité Ninhydrine Assay <ol><li> Pour préparer génipine réticulé L-PRF, rincer les membranes avec du PBS et laisser tremper dans 4 ml de solution de génipine 1% (à 70% d&#39;éthanol) pendant 48 h. Rincer avec du PBS avant expériences pour enlever l&#39;excès génipine 15, 16. </li><li> Évaluer la stabilité de la réticulation de la génipine L-PRF par sa résistance à la dégradation par la trypsine. Lieu membrane L-PRF (n = 3) et la génipine réticulé L-PRF dans 500 ul de 0,01% de trypsine et on incube à 37 ° C pendant 3 jours avec un changement journalière de trypsine. <ol><li> Peser les échantillons au jour 1 avant enzyme exposition et au jour 3. La différence de début et de fin de poids représente la dégradation enzymatique 17. </li></ol></li><li> Quantifier la quantité de réticulation dans génipine traité L-PRF (G-PRF) par ninhydrine dosage. Préparer d&#39;abord la norme curve utilisant la glycine (mm 1 mm-0,031) courbe d&#39;établir la relation entre la concentration de l&#39;acide aminé libre (FAA) et l&#39;absorbance. <ol><li> PRF échantillons de chaleur avec 1 ml de 2% (p / v) de ninhydrine pour 15 minutes à 100 ° C. </li><li> Laisser la solution refroidir à température ambiante et ajouter 1,5 ml d&#39;éthanol à 50%. </li><li> Analyser l&#39;absorbance à 570 nm en utilisant un spectrophotomètre approprié. </li><li> Déterminer le pourcentage de réticulation en utilisant la formule ci-dessous 15 </li></ol></li></ol><img alt="Equation 1" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS prolifération cellulaire Assay <ol><li> Cultiver MC3T3 (préostéoblastes de la voûte crânienne de souris) dans de modification -alpha Minimum Essential Medium (aMEM) dans des flacons T-75 jusqu&#39;à ce qu&#39;une monocouche confluente de 80% est obtenu. </li><li> Préparer membrane L-PRF frais, stérile (ouvrir le tube L-PRF intérieur de la hotte de culture de cellules) et transférer la membrane sur une nouvelle boîte de culture cellulaire. </li><li> Support d&#39;Aspirer à partir de la fiole et rincer la monocouche avec du PBS, ajouter 5 ml de trypsine à 0,05% et placer le ballon dans la 37 o C incubateur pendant 5 min, une pipette le contenu du ballon dans un tube de centrifugation et centrifuger à 400 g pendant 5 min. </li><li> Décanter le surnageant, tapotez doucement le tube de briser le culot de cellules et remettre en suspension avec 4 ml d&#39;α MEM frais, distribuer un mélange de suspension de cellules (50 pi) et bleu trypan (50 pi) dans le hémocytomètre et compter le nombre de cellules </li><li> Graine 4x10 5 cellules à l&#39;intérieur 10 mm des anneaux de clonage en verre placés au-dessus des membranes L-PRF pour retenir les cellules dans les membranes (bagues peuvent être retirées après 24 h). </li><li> Au jour 4, rincer constructions avec PBS trois fois pendant 10 min. </li><li> Ajouter 1 ml de milieu sans sérum et 200 ul réactif MTS pour chaque puits et incuber pendant 2 heures à 37 ° C. </li><li> Mesurer l&#39;absorbance de 200 aliquotes à 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>

Les auteurs ont rien à divulguer et confirment qu&#39;il n&#39;y a pas de conflits d&#39;intérêts connus associés à cette publication.

Concentrés plaquettaires autologues sont prometteurs dans le domaine de la médecine régénérative 18 en raison de l&#39;abondance de facteurs de croissance. Cependant, ces préparations manquaient souvent une structure définie qui rend la manipulation chirurgicale très difficile. Plusieurs fois, les suspensions et les gels ne sont pas conservées efficacement au niveau du site de livraison, ce qui entraîne des résultats imprévisibles. L-PRF représente un énorme progrès dans l&#39;évolution de con...

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.

L&#39;image de microscopie électronique à balayage du caillot L-PRF à différentes sections (haut, milieu et bas) couche est illustrée à la figure 2. Comme on le voit, la partie supérieure est composé majoritairement de réseau de fibrine sans cellules. La couche du milieu est enrichi en plaquettes avec la preuve de leur activation et la dégranulation. La couche inférieure a un mélange de leucocytes et les globules rouges piégées dans une matrice de fibrine. <p class="jo...

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.

Caractérisation de leuco-plaquettaire Rich fibrine, un roman biomatériaux

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 vues.  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. 

Vidéo: 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.

L&#39;encapsulation des cardiomyocytes dans un hydrogel de fibrine pour l&#39;ingénierie du tissu cardiaque

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

Recherche

Bio-ingénierie

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Préparation de la 3D pour les applications de fibrine Échafaudages CULTURE DE CELLULES SOUCHES

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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.

Observation et quantification du compactage du gel de fibrine médié par les fibroblastes

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.

Construction et caractérisation d&#39;un pli bioréacteur roman 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.

Préparation et caractérisation de nouvelles nanoparticules imitant HDL pour l&#39;encapsulation du facteur de croissance nerveuse

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.

Ingénierie des surfaces des îlots pancréatiques avec un nanorevêtement StarPEG hépariné

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.

Fabrication et caractérisation des fantômes de tissu optique contenant la Macrostructure

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.

Microdissection de tissu rénal primaire Segments et intégration avec la technologie de nouvelle construction exempte d’échafaudage

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 modèle d’embryon de poisson zèbre pour In Vivo visualisation et analyse Intravitale du biomatériau associée à 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 ...

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.