Integra modèle de régénération dermique a été gracieusement fournie par Integra Lifesciences Corporation. Sources de fonds soutenant le travail: Ce travail a été partiellement financé par le début Award CIRM-BMBF translationnelle II et le Centre de régulation génomique FONDAP fois à JTE (Nr 15090007.).

1 Préparation des échafaudages <ol><li> Générer des échantillons des échafaudages en utilisant 12 mm de poinçons de biopsie. </li><li> Pour introduire des molécules bioactives ou des cellules dans l&#39;échafaud, égoutter les échafaudages en les pressant doucement avec une gaze stérile. Puis réhydrater les échafaudages en ajoutant 160 ul d&#39;une solution contenant des molécules ou des cellules d&#39;intérêt bioactifs. Double-vérifier le succès de bioactivation avec des cellules par des tests métaboliques tels que le MTT dosages. </li><li> Si nécessaire, fixer les composés ou les cellules à tester à l&#39;intérieur de l&#39;échafaudage, en les délivrant, par exemple dans une solution de fibrine-thrombine ou un hydrogel, de nouveau à travers la réhydratation de la manière décrite dans l&#39;étape 1.2. REMARQUE: Cette étape peut être utile lorsque des composés ou des cellules ne s&#39;attachent pas mécaniquement à la matière. On peut aussi contrôler la dynamique de libération des composés .. </li><li> Préparer les mailles titanisés, qui seront placés sous chaque échafaudage dans chaque groupe d&#39;expérience et de contrôle, par cutting des morceaux en forme de tour, à environ 14 mm de diamètre. REMARQUE: La maille titanisé fonctionne comme une frontière physique requis pour délimiter le tissu régénéré à partir du lit de la plaie. </li></ol> 2. Animaux <ol><li> Avant la mise en œuvre du modèle présenté, consulter les lois de protection des animaux correspondants et obtenir la permission des autorités locales. Dans ce travail, toutes les expériences ont été effectuées avec des souris, selon l&#39;actuelle loi sur la protection des animaux allemand et approuvés par le gouvernement du district de Haute-Bavière (Oberbayern Regierung von). </li><li> Choisissez avec soin l&#39;animal et de la souche. Bien que le modèle de souris a été établi, il peut être converti en d&#39;autres animaux courants, tels que des rats, des lapins ou des cochons. </li><li> Dans le cas où vous travaillez avec des animaux à fourrure, de se raser le dos des animaux avant l&#39;implantation des échafaudages. NOTE: Ce travail utilise des souris de 6 à 8 semaines d&#39;âge avec un poids corporel compris entre 20 g et 25 g, mais différents âges et body poids peuvent également être utilisés. </li></ol> 3. anesthésie <ol><li> Mener l&#39;opération dans des conditions stériles. Afin de maintenir la température normale du corps de l&#39;animal, utilisant une surface de chauffage. </li><li> Avant l&#39;anesthésie par inhalation les animaux reçoivent buprénorphine à une dose de 0,05-0,1 mg / kg de poids corporel, voie sous-cutanée toutes les 8 heures. </li><li> Placez l&#39;animal sous anesthésie par inhalation (isoflurane) ou appliquer des procédures standard équivalent. Confirmez l&#39;anesthésie soit en pressant doucement les orteils de l&#39;animal ou par une pincée de peau. </li><li> Pour éviter exsiccoses, injecter 0,5 ml de solution saline physiologique par voie sous cutanée, au début de l&#39;opération. </li></ol> 4. excision de la peau <ol><li> Placez l&#39;animal dans une position couchée et marquer la ligne médiane du dos de la souris avec un stylo à pointe fine permanent (figure 1A). </li><li> Définir la zone d&#39;excision. Placez les défauts dans la zone indiquée dans Figure 1C. Notez que si les défauts sont placés trop caudale, les animaux sont plus enclins à enlever les pansements et les échafaudages. </li><li> Créer ronde défauts bilatéraux en utilisant un 10 mm biopsie poinçon (figure 1B). Le poinçon doit être soigneusement forcé contre la peau et ne doit pas être utilisé pour couper complètement à travers la peau, mais pour délimiter la zone d&#39;excision (Figures 1C et 1E). </li><li> Soulevez délicatement la peau marquée avec une pince et inciser le long du cercle marqué avec des ciseaux chirurgicaux fines (figure 1F). En cas de saignement, comprimer soigneusement avec de la gaze stérile. Une description étape par étape de l&#39;excision est représenté sur la figure 1. NOTE: Le défaut est créé avec un poinçon plus petit que celui de la génération des échafaudages, pour compenser l&#39;élargissement du défaut dû à l&#39;élasticité de la peau. </li></ol> 5. Échafaudages Implantation <ol><li> Pour faire de la place pour le titanisé maille, de prolonger la séparation de la peau et du tissu sous-jacent aux frontières plaies pour 2-4 mm (figure 2A). </li><li> Placez le titanisés engrener dans le défaut directement sur ​​le lit de la plaie et dans les bords de la plaie (Figures 2A et B). </li><li> Lieu échafaudages directement sur le maillage. </li><li> Suturer les échafaudages sur les bords de la plaie adjacentes de 4 à 6 nœuds simples, laissant les bords un peu plus de l&#39;échafaud (figure 2C). REMARQUE: Évitez l&#39;utilisation de sutures biodégradables. </li><li> Suturer une plaie pansement transparent au-dessus des défauts de protéger l&#39;échafaudage, tout en permettant la surveillance de la zone de la plaie (Figure 2D). </li></ol> 6 Soins postopératoires <ol><li> Gardez une souris par cage pour éviter la manipulation mutuelle de la vinaigrette et les échafaudages. </li><li> Évaluer l&#39;état général de l&#39;animal sur une base quotidienne en observant l&#39;activité motrice, le poids corporel, des signes de douleur, de la toléranceà la vinaigrette et l&#39;auto-mutilation. Surveiller également la surface de la plaie pour un saignement, des signes locaux et systémiques de l&#39;infection et de la position du pansement. </li><li> Afin de minimiser la douleur de l&#39;animal, l&#39;injection buprénorphine par jour à une dose de 0,05 à 0,1 mg de / kg de poids corporel ou d&#39;un médicament analgésique équivalente. </li><li> Si la souris enlève ou endommage le pansement, remplacer ou remettre en place sous anesthésie. </li></ol> 7. euthanasie et explantation de l&#39;échafaud <ol><li> A des moments souhaités, sacrifier les animaux par des surdoses de pentobarbital (150 mg / kg). </li><li> Marquez le site de l&#39;excision de la peau avec un marqueur permanent, qui devrait inclure les échafaudages et autant de la souris peau du dos que possible (figure 3A). </li><li> Inciser la peau le long des lignes marquées avec une paire de ciseaux ou un scalpel. Détachez l&#39;ensemble de la peau, y compris les échafaudages et le maillage titanisé, à partir du tissu sous-jacent à travers la préparation émoussé (Figure 3B). </li><li> Placez le tissu explantée étendu sur une boîte de Pétri (figure 3C et D) </li></ol> 8 Visualisation et quantification du réseau vasculaire <ol><li> Transillumination: <ol><li> Mettre en place un dispositif de radiographie, par le montage d&#39;une boîte de Pétri dessus d&#39;une forte source de lumière blanche (100 Watt ampoule standard). </li><li> Correction d&#39;un appareil photo numérique au-dessus du diaphanoscope pour obtenir des images numériques. </li><li> Placer les échantillons à l&#39;envers sur la boîte de Pétri sur le dispositif de radiographie. </li><li> Prenez des photos des échafaudages complets en mode macro, ainsi que des zones de taille égale de la peau normale. Stocker les images dans un format TIFF pour une analyse plus approfondie numérique. </li><li> Enregistrez le tissu pour plus d&#39;analyse biochimique ou histologique. </li></ol></li><li> Segmentation numérique et la quantification: <ol><li> Télécharger et installer le logiciel VesSeg-outil, qui peut être obtenu free frais au: <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> . </li><li> Ouvrez l&#39;image avec le logiciel VesSeg-Tool. </li><li> Sélectionnez la zone de l&#39;image couverte par l&#39;échafaud pour l&#39;analyse numérique (Image → Select). </li><li> Pour augmenter le contraste des vaisseaux utiliser l&#39;option &quot;seuil d&#39;hystérésis&quot; (image → amélioration de navire filtre → Hystérésis seuil → Top-Hat transformation → amélioration de navire Calculer). </li><li> Procéder à la segmentation de la carte de la cuve (de frontières image → amélioration de navire filtre → → hystérésis seuil de seuil). Sélectionnez le premier seuil («couverture de navire»), de sorte que chaque pixel, ce qui est encore à distance type vaisseau, sera étiqueté comme navire. Sélectionnez le second seuil (&quot;couverture de base&quot;), de sorte que seuls les pixels qui sont particulièrement type vaisseau sera étiqueté comme navires. </li><li> Vérifiez la proposition de segmentation automatique manuellement pour ajouter des navires non capturées et d&#39;éliminer les structures de faux positifs communs, qui sont généralement des structures de navires comme de longues et minces tels que des poils ou des sutures. </li><li> Calculer la longueur des navires et la superficie totale couverte par les navires (statistiques image → Image → statistiques d&#39;images binaires). REMARQUE: Pour le calcul des navires longueurs, les navires de la carte de navire résultant sont éclaircis à des lignes d&#39;une largeur d&#39;un pixel 20. </li></ol></li><li> Analyser la région natale de la peau sous les mêmes paramètres que les échafaudages, l&#39;attribution d&#39;une valeur de 100% pour le tissu natif et rapporter l&#39;échafaud à cette valeur. Par exemple, si le pourcentage de pixels blancs est de 60% dans le tissu normal et 30% dans l&#39;échafaudage, cela représenterait une vascularisation de 50% de l&#39;échafaud. Remarque: Le vaisseaux couverture des cheveux blancs est affectée à la surface d&#39;un carré autour de l&#39;échafaud. Réglez le nombre de pixels blancs couverture considering que la surface du cercle est sensiblement plus petit (π x R 2) que le carré (4 x R 2). </li></ol>

<ol>
	<li>Rahaman, M. N., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+composite+tissue+constructs+for+regenerative+medicine.">Stem cell-based composite tissue constructs for regenerative medicine.</a> Biotechnology and Bioengineering. 91 (3), 261-284 (2005).</li><li>Lutolf, M. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biomaterials+as+instructive+extracellular+microenvironments+for+morphogenesis+in+tissue+engineering.">Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering.</a> Nature Biotechnology. 23, 47-55 (2005).</li><li>Machens, H. G., Berger, A. C., Mailaender, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioartificial+skin.">Bioartificial skin.</a> Cells Tissues Organs. 167, 88-94 (2000).</li><li>Priya, S. G., Jungvid, H., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering+for+tissue+repair+and+regeneration.">Skin tissue engineering for tissue repair and regeneration.</a> Tissue Engineering Part B: Reviews. 14, 105-118 (2008).</li><li>Papavasiliou, G., Cheng, M. H., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+vascularization+of+polymer+scaffolds.">Strategies for vascularization of polymer scaffolds.</a> Journal of Investigative Medicine. 58 (7), 838-844 (2010).</li><li>Laschke, M. 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=Angiogenesis+in+tissue+engineering:+breathing+life+into+constructed+tissue+substitutes.">Angiogenesis in tissue engineering: breathing life into constructed tissue substitutes.</a> Tissue Engineering. 12, 2093-2104 (2006).</li><li>Zhong, S. P., Zhang, Y. Z., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+scaffolds+for+skin+wound+healing+and+dermal+reconstruction.">Tissue scaffolds for skin wound healing and dermal reconstruction.</a> Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology. 2 (5), 510-525 (2010).</li><li>Liu, G., Zhang, Y., Liu, B., Sun, J., Li, W., Cui, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+regeneration+in+a+canine+cranial+model+using+allogeneic+adipose+derived+stem+cells+and+coral+scaffold.">Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold.</a> Biomaterials. 34 (11), 2655-2664 (2013).</li><li>Hansson, A., Di Francesco, T., Falson, F., Rousselle, P., Jordan, O., Borchard, G. <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+evaluation+of+nanoparticles+for+directed+tissue+engineering.">Preparation and evaluation of nanoparticles for directed tissue engineering.</a> International Journal of Pharmaceutics. 439 (1-2), 73-80 (2012).</li><li>Sarkar, S. D., Farrugia, B. L., Dargaville, T. R., Dhara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitosan-collagen+scaffolds+with+nano/microfibrous+architecture+for+skin+tissue+engineering.">Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering.</a> Journal of Biomedical Materials Research Part A. 18, (2013).</li><li>Wang, X., 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+roles+of+knitted+mesh-reinforced+collagen-chitosan+hybrid+scaffold+in+the+one-step+repair+of+full-thickness+skin+defects+in+rats.">The roles of knitted mesh-reinforced collagen-chitosan hybrid scaffold in the one-step repair of full-thickness skin defects in rats.</a> Acta Biomaterials. 9 (8), 7822-7832 (2013).</li><li>Rizzi, S. C., Upton, Z., Bott, K., Dargaville, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+dermal+wound+healing:+biomedical+device+approaches.">Recent advances in dermal wound healing: biomedical device approaches.</a> Expert Review of Medical Devices. 1, 143-154 (2010).</li><li>des Rieux, 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=3D+systems+delivering+VEGF+to+promote+angiogenesis+for+tissue+engineering.">3D systems delivering VEGF to promote angiogenesis for tissue engineering.</a> Journal of Controlled Release. 150, 272-278 (2011).</li><li>Reckhenrich, A. K., 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=Bioactivation+of+dermal+scaffolds+with+a+non-viral+copolymer-protected+gene+vector.">Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector.</a> Biomaterials. 32, 1996-2003 (2011).</li><li>Chen, 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=The+Key+Regulatory+Roles+of+the+PI3K/Akt+Signaling+Pathway+in+the+Functionalities+of+Mesenchymal+Stem+Cells+and+Applications+in+Tissue+Regeneration.">The Key Regulatory Roles of the PI3K/Akt Signaling Pathway in the Functionalities of Mesenchymal Stem Cells and Applications in Tissue Regeneration.</a> Tissue Engineering Part B Rev. 19, 516-528 (2013).</li><li>Fedorovich, N. 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=The+role+of+endothelial+progenitor+cells+in+prevascularized+bone+tissue+engineering:+development+of+heterogenous+constructs.">The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogenous constructs.</a> Tissue Engineering Part A. 16 (7), 2355-2367 (2010).</li><li>Wang, L., 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=Osteogenesis+and+angiogenesis+of+tissue-engineered+bone+constructed+by+prevascularized+&#946;-tricalcium+phosphate+scaffold+and+mesenchymal+stem+cells.">Osteogenesis and angiogenesis of tissue-engineered bone constructed by prevascularized &#946;-tricalcium phosphate scaffold and mesenchymal stem cells.</a> Biomaterials. 36, 9452-9461 (2010).</li><li>Cuadra, 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=Functional+results+of+burned+hands+treated+with+Integra.">Functional results of burned hands treated with Integra.</a> Journal of Plastic, Reconstructive &amp; Aesthetic Surgery. 65 (2), 228-234 (2012).</li><li>Wilcke, I., 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=VEGF(165)+and+bFGF+protein-based+therapy+in+a+slow+release+system+to+improve+angiogenesis+in+a+bioartificial+dermal+substitute+in+vitro+and+in+vivo.">VEGF(165) and bFGF protein-based therapy in a slow release system to improve angiogenesis in a bioartificial dermal substitute in vitro and in vivo.</a> Langenbecks Arch Surg. 392 (3), 305-314 (2007).</li><li>Condurache, A., Aach, T., Grzybowsky, S., Machens, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+and+analysis+in+laboratory+skin+transplant+micro-angiograms.">Vessel segmentation and analysis in laboratory skin transplant micro-angiograms.</a> Proceedings of the Eighteenth IEEE Symposium on Computer-Based Medical Systems. , 21-26 (2005).</li><li>Danner, S., 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+use+of+human+sweat+gland-derived+stem+cells+for+enhancing+vascularization+during+dermal+regeneration.">The use of human sweat gland-derived stem cells for enhancing vascularization during dermal regeneration.</a> Journal of Investigative Dermatology. 132 (6), 1707-1716 (2012).</li><li>Shaterian, 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=Real+Time+Analysis+of+the+Kinetics+of+Angiogenesis+and+Vascular+Permeability+in+an+Animal+Model+of+Wound+Healing.">Real Time Analysis of the Kinetics of Angiogenesis and Vascular Permeability in an Animal Model of Wound Healing.</a> Burns. 35 (6), 811-817 (2009).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nature Medicine. 9 (6), 713-725 (2003).</li><li>Bergeron, L., Tang, M., Morris, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+vascular+injection+techniques+for+the+study+of+perforator+flaps.">A review of vascular injection techniques for the study of perforator flaps.</a> Plastic and Reconstructive Surgery. 117, 2050-2057 (2006).</li><li>Schlatter, P., K&#246;nig, M. F., Karlsson, L. M., Burri, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+intussusceptive+capillary+growth+in+the+chorioallantoic+membrane+(CAM)+of+the+chicken+embryo.">Quantitative study of intussusceptive capillary growth in the chorioallantoic membrane (CAM) of the chicken embryo.</a> Microvascular Research. 54 (1), 65-73 (1997).</li><li>Lehr, H. A., Leunig, M., Menger, M. D., Nolte, D., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+skinfold+chamber+technique+for+intravital+microscopy+in+nude+mice.">Dorsal skinfold chamber technique for intravital microscopy in nude mice.</a> American Journal of Pathology. 143 (4), 1055-1062 (1993).</li><li>Menger, M. D., J&#228;ger, S., Walter, P., Hammersen, F., Messmer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+studies+on+the+microvasculature+of+transplanted+islets+of+Langerhans+in+vivo.">A novel technique for studies on the microvasculature of transplanted islets of Langerhans in vivo.</a> International journal of microcirculation, clinical and experimental. 9 (1), 103-117 (1990).</li><li>Laschke, M. 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=Three-dimensional+spheroids+of+adipose-derived+mesenchymal+stem+cells+are+potent+initiators+of+blood+vessel+formation+in+porous+polyurethane+scaffolds.">Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds.</a> Acta Biomaterials. 9 (6), 6876-6884 (2013).</li><li>Ega&#241;a, J. 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=Use+of+human+mesenchymal+cells+to+improve+vascularization+in+a+mouse+model+for+scaffold-based+dermal+regeneration.">Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.</a> Tissue Eng Part A. 15 (5), 1191-1200 (2009).</li><li>Condurache, A., Aach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+in+angiograms+using+hysteresis+thresholding.">Vessel segmentation in angiograms using hysteresis thresholding.</a> Proceedings of the Ninth IAPR Conference on Machine Vision Applications. , 269-272 (2005).</li><li>Ega&#241;a, J. 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=Ex+vivo+method+to+visualize+and+quantify+vascular+networks+in+native+and+tissue+engineered+skin.">Ex vivo method to visualize and quantify vascular networks in native and tissue engineered skin.</a> Langenbecks Archives of Surgery. 394, 349-356 (2009).</li></ol>

Conflit d&#39;intérêt: Tous les auteurs: Aucun Liens financiers: Aucun des auteurs a un intérêt financier dans l&#39;un des produits, dispositifs ou médicaments mentionnés dans ce manuscrit.

Il est nécessaire d&#39;établir des méthodes efficaces dans l&#39;amélioration de la perfusion sanguine dans les constructions de l&#39;ingénierie tissulaire, qui exige la mise au point de nouvelles méthodes fiables pour étudier les processus de vascularisation dans les biomatériaux. Les méthodes courantes pour faire échafaud vascularisation ex vivo visible comprennent l&#39;utilisation de la microscopie, qui fournit un outil de haute résolution. Dans la plupart des cas, cependant, cette méthode est...

Dans les dernières décennies, l&#39;ingénierie tissulaire a ouvert une nouvelle option thérapeutique pour remplacer défauts des tissus avec les propres cellules de l&#39;organisme 1. Afin de soutenir le processus physiologique de la régénération des tissus, des échafaudages sont conçus comme une structure biodégradable, qui fournit un scénario où les cellules du lit de la plaie peuvent se développer et de rétablir le défaut 2,3. Vascularisation insuffisante est considéré comme le principal obstacle qui freine la percée clinique d&#39;échafaudages bioartificiel 4. Avec la croissance de cellules, la demande de nutriments et d&#39;oxygène augmente et la vascularisation de la matière devient indispensable. Vascularisation insuffisante ou tardive peut donc conduire à une nécrose centrale de produits de l&#39;ingénierie tissulaire 5. De plus, les vaisseaux sanguins fournissent des cellules immunocompétentes et enlever les résidus métaboliques dans la zone de régénération. Des taux d&#39;infection élevés et une faible régénération ne sontquelques-unes des conséquences de l&#39;insuffisance perfusion sanguine observés dans l&#39;ingénierie tissulaire, qui visent à être évités par l&#39;augmentation de la vascularisation des échafaudages 6,7. Plusieurs stratégies visant à améliorer la vascularisation accent sur le rôle clé du biomatériau lui-même et la microstructure de l&#39;échafaud. Il ya des efforts intensifs de recherche pour développer de nouvelles approches en déplaçant le processus de guérison de la réparation de la régénération, de ce fait (re) produire un tissu avec des propriétés physiologiques les plus proches de celui qui doit être restaurées 8,9. Biomatériaux qui ont été étudiés et évalués en ce qui concerne leur potentiel de régénération inclus collagène, fibrine, le chitosane et l&#39;alginate 10,11. Ces biomatériaux peuvent être utilisés et combinés comme une épine dorsale pour la construction de nouveaux échafaudages en utilisant différentes stratégies telles que décellularisation de tissus, auto-assemblage, prototypage rapide et électrofilature 12. Pour ENHment propre capacité de régénération de l&#39;organisme, les échafaudages peuvent être bioactivé. L&#39;incorporation de croissance angiogénique recombinant facteurs 13 ou génétiques des vecteurs codant pour des facteurs tels 14 a montré pour améliorer la vascularisation de l&#39;échafaud. L&#39;utilisation de cellules souches a été largement démontré être une stratégie prometteuse pour améliorer la vascularisation, où les cellules stromales et les cellules mésenchymateuses progénitrices endothéliales ont obtenu la plus grande attention 15,16. D&#39;autres approches tentent de construire des constructions qui contiennent des réseaux de vaisseaux préfabriqués avant la transplantation 17. Malgré des efforts intensifs dans la conception de l&#39;échafaudage et de leur bio-activation, pas de stratégie a amélioré la vascularisation au niveau cliniquement significative et, à l&#39;exception des remplacements dermiques dans les brûlures massives, la traduction des documents issus du génie génétique dans la routine clinique n&#39;est déroule hésitant 18 . L&#39;une des raisons pour lesquelles la vascularisationde constructions de tissu artificiel est encore un problème non résolu, est la difficulté d&#39;évaluer le succès des nouvelles technologies dans les approches in vivo. Bien que des expériences in vitro peuvent fournir des indications importantes sur le potentiel de la vascularisation des échafaudages, des modèles animaux appropriés sont nécessaires pour étudier les paramètres clés tels que la biocompatibilité du matériau, la sécurité et l&#39;efficacité du traitement et, d&#39;une importance particulière, la vascularisation du tissu construire. Par conséquent, des outils fiables de visualiser et de quantifier les réseaux de vaisseaux sanguins in vivo sont essentiels. Dans cette étude, nous présentons une méthode simple et fiable qui permet la visualisation et la quantification du réseau vasculaire à l&#39;intérieur des échafaudages explantées. Cette méthode est basée sur la transillumination de tissus et de segmentation numérique. Etant donné que cette méthode est non invasif, il permet d&#39;autres analyses moléculaires et histologiques de la matière cible.

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			<td style="width:223px;">Ethicon, Norderstedt, Germany</td>
			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
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			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
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			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
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			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
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			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
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			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
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			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
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			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
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			<td></td>
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			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
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			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
			<td></td>
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			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
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			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
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a full skin defect model to evaluate vascularization of biomaterials in vivo

Vascularisation insuffisante est considéré comme l&#39;un des facteurs principaux limitant le succès clinique de constructions du génie tissulaire. Afin d&#39;évaluer de nouvelles stratégies visant à améliorer la vascularisation, méthodes fiables sont nécessaires pour rendre le dans la croissance de nouveaux vaisseaux sanguins dans les échafaudages de bio-artificiel visibles et quantifier les résultats. Au cours des deux dernières années, notre groupe a mis en place un modèle de défaut de peau complet qui permet la visualisation directe des vaisseaux sanguins par transillumination et offre la possibilité de quantification par la segmentation numérique. Dans ce modèle, on crée chirurgicalement défauts complets de la peau dans le dos de souris et les remplace par du matériau testé. Molécules ou cellules d&#39;intérêt peuvent aussi être incorporés dans ces matières à étudier leur effet potentiel. Après un temps d&#39;observation de son choix un, les matériaux sont explantées pour l&#39;évaluation. Blessures bilatéraux prévoient la possibilité de faire des comparaisons internes eà minimiser les artefacts entre les individus ainsi que de diminuer le nombre d&#39;animaux nécessaires à l&#39;étude. En comparaison à d&#39;autres approches, notre méthode offre une analyse simple, fiable et rentable. Nous avons mis en place ce modèle comme un outil de routine pour effectuer le criblage à haute résolution lors du test de la vascularisation de différents biomatériaux et les approches bio-activation.

A défaut fiable pleine peau bilatérale peut être créé chez la souris (Figure 1) où la peau peut être remplacé par un biomatériau à l&#39;étude (Figure 2). Ici, pas de complications majeures sont observées pendant ou après l&#39;intervention chirurgicale, ni signes macroscopiques d&#39;infection ou de réaction à corps étranger. Dans de rares cas, un échafaudage se perd lorsque la souris le supprime. La contraction des plaies n&#39;a jamais été observée (figure...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

La vascularisation est la clé pour les approches en ingénierie tissulaire succès. Par conséquent, des technologies fiables sont nécessaires pour évaluer le développement des réseaux vasculaires dans les tissus-constructions. Nous présentons ici une méthode simple et efficace de visualiser et de quantifier la vascularisation in vivo.

Une peau pleine de défauts modèle pour évaluer la vascularisation des biomatériaux<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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Research

JoVE Journal

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K vues.  Technische Universität München.  La vascularisation est la clé pour les approches en ingénierie tissulaire succès. Par conséquent, des technologies fiables sont nécessaires pour évaluer le développement des réseaux vasculaires dans les tissus-constructions. Nous présentons ici une méthode simple et efficace de visualiser et de quantifier la vascularisation in vivo. 

Vidéo: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

La peau Three-Dimensional Human Reconstruire Modèle: un outil pour étudier la peau normale et la progression du mélanome

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Recherche

Bio-ingénierie

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Conception d&#39;un bioréacteur pour la pression cyclique Ex Vivo Étude des valves cardiaques aortiques

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Génération d&#39;une épaisseur en trois dimensions équivalent de peau et automatisé Blessant

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microenvironnements sandwich à exploiter cellulaires / Interactions matérielles

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Génie tissulaire par Intrinsic vascularisation dans un<em&gt; In Vivo</em&gt; Tissue Chambre Ingénierie

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Une méthode d&#39;évaluation de rapidité et la précision des réponses Volitional moteur à vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

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 ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Un modèle fiable et reproductible taille critique segmentaire fémorale défaut chez les Rats stabilisé avec un fixateur externe personnalisée

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

In Vivo Deux-Couleur 2-Photon Imagerie des cellules de reporter génétiquement marqués dans la peau

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

Modèle calvarial de l'augmentation osseuse dans le lapin pour l'évaluation de la croissance osseuse et la néovascularisation dans les matériaux de substitution osseuse

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...