Diese Arbeit wurde zum Teil durch einen Zuschuss aus NIH R01 GM087609 (HPL), ein Geschenk von Ingrid Lai und Bill Shu zu Ehren von Anthony Shu (HPL), NIH Zuschusses U01 HL099776 (MTL), das Labor für Hagey unterstützt Pediatric Regenerative Medizin und der Oak Foundation (MTL und HPL). GGW wurde von der Stanford School of Medicine, der Stanford Medical Scientist Training Program unterstützt und NIGMS Ausbildungsförderung GM07365. MSH wurde von CIRM Clinical Fellow Ausbildungsförderung TG2-01159 unterstützt. WXH wurde durch Mittel aus dem Sarnoff Herz-Kreislauf-Stiftung.

HINWEIS: Alle in diesem Dokument beschriebenen Verfahren werden nach den Richtlinien der Stanford Administrative Panel on Laboratory Animal Care (APLAC) errichtet wird. 1. Timed Schwangerschaften - Superovulation Technik (Abbildung 1) HINWEIS: Präzise Zeitmessung der Schwangerschaftsdauer von Maus-Embryonen für die fetale Chirurgie an E16.5 und E18.5 ist von entscheidender Bedeutung. In diesem Abschnitt wird detailliert unser Protokoll für Zeit Maus Schwangerschaften mit trächtigen Stuten Serum (PMS) und humanes Choriongonadotropin (HCG) Injektionen Superovulation induziert. <ol><li> Injizieren weiblichen Mäusen (&lt;5 pro Käfig) intraperitoneal (IP) mit 3,0-5,0 internationalen Einheiten (IU) des PMS in einem Volumen von 100 ul PBS 1.00 bis 3.00 für 1. Tag. </li><li> Zwischen 00.00 und 02.00 Uhr des Tages 3 (fünfundvierzig bis 47 Stunden nach der Injektion PMS), spritzen weibliche Mäuse IP mit 3,0-5,0 IU von HCG in einem Volumen von 100 ul PBS. HINWEIS: Die HCG-Injektion induziert<html> Eisprung etwa 12 Stunden nach der Injektion. </li><li> Unmittelbar nach HCG-Injektionen, mate Frauen mit Männern im Alter von 8 bis 16 Wochen. HINWEIS: Wir legen in der Regel zwei Weibchen in einen Käfig einzelner Männchen. </li><li> Separate Weibchen von Männchen am Morgen des 4. Tag (6.00 bis 10.00 Uhr) und Rekord als Fetalalter E0.5. HINWEIS: Vaginal Stecker können zu diesem Zeitpunkt überprüft werden; Allerdings ist die Beobachtung eines Vaginalpfropf nicht garantieren, Schwangerschaft und Frauen können schwanger werden, wenn kein Stecker eingehalten wird. Da die Schwangerschaft ist typischerweise beobachtbaren durch visuelle Inspektion und / oder Abtasten in verschiedenen Schwangerschaftsstadien E16.5 und E18.5, Überprüfung auf Vaginalpfropf am Morgen des Tages 4 ist nicht unbedingt erforderlich. Nach unserer Erfahrung und in Abhängigkeit von der Belastung, etwa 30-50% der super Eisprung Frauen schwanger werden, mit der hier beschriebenen Technik. </li></ol>pg &quot;/&gt; Abbildung 1. Schematische für Superovulation Technik. Schema, das Verfahren für die Superovulation zeitlich Schwangerschaften bei Mäusen. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> 2. Murine Fetale Chirurgie (Rücken Verwundung) auf E16.5 und E18.5 Embryonen (Abbildung 2) <ol><li> Vor dem Eingriff, Reinigen Sie alle Oberflächen des Operationssaals und Ausrüstung mit 70% Isopropylalkohol. Zusätzlich zu sterilisieren alle chirurgisches Material und Instrumente, die im Rahmen des Verfahrens durch Autoklavieren sie verwendet werden soll. Einige Institutionen kann die anschließende Verwendung von heißen Perle Sterilisation zu ermöglichen. Pro Betrieb verwenden steril verpackt, die Gaze und chirurgische Instrumente umfassen. </li><li> Induzieren Anästhesie in schwangere Mütter (fetale Alter E16.5 oder E18.5) unter 2,5% Isofluran / Sauerstoff-Gemisch in 2 l pro Minute, gefolgt von Wartung Anästhesie mit 1 l pro min. </li><li> Um conFirma ordnungsgemäße Betäubung, sicherzustellen, dass die tiefen Pedal Reflexe der Maus unterdrückt werden und die Maus in der Bauchlage. </li><li> Tragen Sie einen Tierarzt Augensalbe wie Puralube, das Augenreizungen oder Trockenheit während des Verfahrens zu verhindern. </li><li> Bereiten Bauch durch Verabreichung einer Licht Anwendung Enthaarungscreme für nicht mehr als 30 Sekunden (Abbildung 2A). </li><li> Bereiten Bauch für aseptische Chirurgie mit PVP-Jod und Alkohol (2B und 2C). </li><li> Führen Mittellinie Bauchschnitt unter dem Mikroskop mit mikrochirurgischen Schere (Abbildung 2D und 2E). </li><li> Aussetzen vorsichtig Gebärmutter und Fötus für Chirurgie (2F) ausgewählt. </li><li> Spülen Operationsfeld mit warmem (38 ° C) phosphatgepufferte Kochsalzlösung (PBS) mit einem stumpfen Nadel-Spitze HINWEIS: Man kann durch vorsichtiges Biegen der Spitze einer großen Injektionsnadel aus vorgenommen werden. (2G und 2H). </li><li> Positionieren Sie den Fötus in einer Weise, die ess vollen Zugriff auf Rücken. </li><li> Übergeben Sie eine Tabaksbeutelstich mit 7-0 Nylonnaht durch die Gebärmutter, die über dem Ort der beabsichtigten Rücken Verwundung (2I). Position Schnürfaden über einen Bereich des Rückens nach links oder rechts von Rückenmark und in einem Bereich der Gebärmutterwand frei von großen Blutgefäßen. </li><li> Machen Sie eine 3 mm Schnitt durch Gebärmutterwand und Fruchtblase in der Mitte der Tabaksbeutel (2J). </li><li> Bewässern Inzisionsstelle mit warmem (38 ° C) PBS. </li><li> Mit mikrochirurgischen Schere, schnitt ein einziges Vollhaut Exzisionswunde, ca. 1 mm in der Länge, in dem Rücken des Fötus. </li><li> Sanft Blot Inzisionsstelle trocknen mit Baumwolle-Spitze-Applikator. </li><li> Injizieren 3 ul Volumen Tusche subkutan in die Wundstelle zu Lage der Wunde (Abbildung 2 K) zu markieren. </li><li> Bewässerung mit warmem (38 ° C) PBS sicherzustellen Tinte innerhalb Wundstelle beibehalten. </li><li> Haben chirurgischen Assistenten inject warm (38 ° C) PBS durch stumpfen Spitze 10 G-Spritze in die Fruchtblase wie die Tabaksbeutelnaht geschlossen ist (Abbildung 2 l). Einfahren Spritze Tabaksbeutelverschluss nähert sich der Fertigstellung (Abbildung 2 M). </li><li> Gebärmutter vorsichtig in die Bauchhöhle (Abbildung 2 N) zurück. </li><li> Evert Haut und Bauchfell. </li><li> Haben chirurgischen Assistenten bewässern Bauchhöhle mit warmem (38 ° C) PBS. </li><li> Schließen Bauch schnell von Heften Haut und Bauchfell geschlossen (Abbildung 2 O). Die Standardverschluss in zwei Schichten durchgeführt wird; Peritoneum und Bauchmuskeln in einer Schicht, Unterhaut und Haut in der zweiten Schicht. Zur sofortigen Opfer und Ernte der Frucht, zeigt unsere Demonstration Schließung in einer Schicht. </li><li> Legen Sie das Tier unter Beobachtung in einem warmen Inkubator bei 37 ° C für 30 Minuten oder bis das Tier gewinnt ausreichend Bewusstsein Brustlage zu halten einstellen. </li><li> Ret nichtUrne das Tier an die Firma von anderen Tieren bis er vollständig von dem Verfahren zurückgewonnen. </li><li> Nach dem Erwachen aus der Narkose und bei der anschließenden 48 Stunden, zu verwalten subkutane Injektion von Buprenorphin (0,05 mg / kg) alle 12 h für die Analgesie nach Bedarf auf Basis von Schmerzbeurteilung. Verwalten Carprofen (5 mg / kg) subkutan injiziert wird für zusätzliche postoperative Schmerzlinderung nach Bedarf. </li><li> Zurück Tiere in den Käfig und sie mit Nahrung und Wasser ad libitum. </li><li> Überwachen Sie sorgfältig auf Manifestationen Schmerzen. </li><li> 48 Stunden nach der Operation, zu opfern schwangere Mutter mit einer Überdosis Isofluran und Ernte verwundet Fötus. Um dies zu tun, passen Isofluran-Konzentration auf 5% oder höher und aufrechtzuerhalten Exposition für 1 min nach dem Atemstillstand. Bestätigen Sie die Sterbehilfe mit Genickbruch. Ernten Sie einen unverletzten Embryo für altersangepassten Kontroll. Späte Embryonen eine separate Methode der Euthanasie im Einklang mit IACUC Empfehlungen, wie haben deKopfpauschale, Genickbruch oder chemische Injektion. </li></ol><img alt="Abbildung 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> Abbildung 2. Schematische für Murine Fetale Chirurgie. Allgemeine Schritte für die Rücken Verwundung in E16.5 und E18.5 Maus-Embryonen. (A) Enthaarung der Maus Bauch. (B und C) Herstellung der Mausunterleib. (D) Mikroskop für chirurgische Verfahren. (E) Mittellinie Laparotomie. (F) Bei der Gebärmutter. (G) Erstellung von stumpfen Spitze Nadel. (H) Bewässerung der Gebärmutter mit warmer Kochsalzlösung. (I) Schaffung von Tabaksbeutelnaht. (J) Incision durch uterine Wand und 1 mm Dicke Voll Exzisionswunde Generation. (K) Die subkutane Injektion von Tusche. (L und M) Schließung der Tabaksbeutelnaht. (N und O)Schließung des Bauches. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a>

<ol>
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Possible role for inflammatory macrophages and transforming growth factor-beta isoforms.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 2, 104-112 (1994).</li><li>Longaker, M. 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=Studies+in+fetal+wound+healing.+IV.+Hyaluronic+acid-stimulating+activity+distinguishes+fetal+wound+fluid+from+adult+wound+fluid.">Studies in fetal wound healing. IV. Hyaluronic acid-stimulating activity distinguishes fetal wound fluid from adult wound fluid.</a> Annals of surgery. 210, 667-672 (1989).</li><li>Colombo, J. A., Napp, M., Depaoli, J. R., Puissant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+influences+of+human+and+rat+amniotic+fluid+on+neural+tube-derived+rat+fetal+cells.">Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells.</a> International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience. 11, 347-355 (1993).</li><li>Colwell, A. S., Longaker, M. T., Peter Lorenz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+differentially+regulated+genes+in+fetal+wounds+during+regenerative+repair.">Identification of differentially regulated genes in fetal wounds during regenerative repair.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16, 450-459 (2008).</li><li>Hu, M. 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=Gene+expression+in+fetal+murine+keratinocytes+and+fibroblasts.">Gene expression in fetal murine keratinocytes and fibroblasts.</a> The Journal of surgical research. , (2014).</li><li>Colwell, A. S., Krummel, T. M., Longaker, M. T., Lorenz, H. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+induces+scarless+repair+in+mouse+limb+organ+culture.">Hyaluronan induces scarless repair in mouse limb organ culture.</a> Journal of pediatric surgery. 33, 564-567 (1998).</li><li>Chopra, V., Blewett, C. J., Krummel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+from+fetal+to+adult+repair+occurring+in+mouse+forelimbs+maintained+in+organ+culture.">Transition from fetal to adult repair occurring in mouse forelimbs maintained in organ culture.</a> Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society. 5, 47-51 (1997).</li><li>Adzick, N. S., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+study+of+fetal+tissue+repair.">Animal models for the study of fetal tissue repair.</a> The Journal of surgical research. 5, 47-51 (1991).</li><li>Block, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+the+new-born+opossum+(Didelphis+virginianam).">Wound healing in the new-born opossum (Didelphis virginianam).</a> Nature. 187, 340-341 (1960).</li><li>Longaker, M. T., Dodson, T. B., Kaban, L. 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The authors have nothing to disclose.

Das chirurgische Protokoll hier vorgestellten beschreibt eine excisional Modell des fötalen murinen narbenheil erste im Jahr 2006 von unserem Labor 10 veröffentlicht. Zusätzlich zu anderen etablierten Modelle der excisional Verwundung 11, Narben Modelle von fetalen Maus narbenheil existieren sowie 12,13. Untersuchungen des fetalen narben Wundheilung bei den Affen, Lamm, Kaninchen, Beutelratte, und Ratten wurden berichtet 14-17. Aber Mäuse sind ein ideales Modell für die E...

Fetal Haut Wunden heilen schneller und scarlessly bis spät in der Schwangerschaft ein. Fetal narben Wundheilung wird durch Regeneration der normalen Gewebearchitektur und Funktion. Der Übergang von einer narben Vernarbung Phänotyp tritt im dritten Trimester der Schwangerschaft beim Menschen und um embryonale Tag 18 (E18) in den Mäusen 2,3. Im Vergleich zu Erwachsenen ist fetalen Wundheilung durch schnelle Epithelisierung, Bindegewebsablagerung und Fibroblasten-Migration aus. Viele Studien haben mögliche Erklärungen für das Phänomen der narbenfreien Wundheilung während der frühen fetalen Entwicklung angeboten. Die Entzündung ist eine grundlegende Komponente der Erwachsenen Wundheilung; jedoch fötalen Wunden durch einen Mangel an akuter Entzündung 4 gekennzeichnet. Ob dies eine Folge der funktionellen Unreife des Immunsystems während der fötalen Stadien bleibt unklar. Eine kürzlich durchgeführte Studie zeigte, dass die Unterschiede in der Fülle, matturity und Funktion von Mastzellen in E15 vs. E18 fötalen Haut kann für den Übergang von einer narben Phänotyp, zumindest in der Maus 3 durchgeführt wird. Andere Studien postulieren, dass Unterschiede in den Eigenschaften und Fülle von fötalen und erwachsenen Wund Makrophagen für die Reformierung von normalen extrazellulären Matrix (ECM), während der fetalen Wundheilung 5 zuständig sind. Unterschiede in der Umweltfaktoren während der fetalen und adulten Entwicklung kann auch Auswirkungen auf die Wundheilung. Longaker und Kollegen zeigten, dass Wundflüssigkeit vom Fötus im Vergleich zu keiner in der Erwachsenenwundflüssigkeit 6 besitzt ein hohes Maß an Hyaluronsäure-stimulierende Aktivität. Folglich können höhere Niveaus von Hyaluronsäure, Glycosaminoglycan, das eine Mikroumgebung zuträglich Zellmotilität und Proliferation fördert, in der fetalen Wundumgebung zur narben Phänotyp während der frühen Entwicklung des Fötus gesehen verantwortlich. Andere Beweislinien weisen darauf hin, dass die Fetal Wundumgebung ist relativ hypoxämischen und in sterile Fruchtwasser reich an Wachstumsfaktoren 7 getaucht. Es wurde jedoch keine definitive Antwort für ein kritisches Ereignis oder Faktor während der Embryogenese, die den Übergang von der narbenfreien Regeneration fibrotische Reparatur löst vorgesehen. Das Verständnis der Mechanismen für narbenheil im Fötus verantwortlich erfordert eine präzise und reproduzierbare Modell. Hier werden wir ausführlich ein reproduzierbares Modell der fetalen narben Wundheilung im Rücken E16.5 (narben) und E18.5 (Vernarbung) Maus-Embryonen. Zusätzlich können kleinere Abweichungen von diesem Modell verwendet werden, um eine Reihe von weiteren Untersuchungen wie Genexpressionsanalyse von fötalen Wunden und Haut 8,9 durchzuführen. Da die zeitgenaue Schwangerschaften sind entscheidend für eine erfolgreiche Wiederholung des fetalen narben Wundheilung Modell haben wir auch ausführlich unser Protokoll für die Superovulation zeitlich Schwangerschaften.

<table><tbody><tr><td>7-O MONOSOF Suture</td><td>eSuture</td><td>SN-1647G</td><td></td></tr><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Autoclip 9 mm</td><td>Texas Scientific Instruments</td><td>205060</td><td></td></tr><tr><td>Insulin Syringe</td><td>Thermo Fisher Scientific</td><td>22-272-382</td><td></td></tr><tr><td>Black Pigment</td><td>AIMS</td><td>242</td><td></td></tr><tr><td>BD Safety-Lok 3 ml Syringe</td><td>BD Biosciences</td><td>309596</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>OPMI-MD Surgical Microscope</td><td>Carl Zeiss Surgical Inc</td><td></td><td></td></tr><tr><td>Pregnant Mares Serum (PMS)</td><td>Millipore</td><td>367222</td><td></td></tr><tr><td>Human Chorionic Gonadotropin (HCG)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr></tbody></table>

a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Für die histologische Analyse sollte Hautwunden in die Rückenhaut von E16.5 und E18.5 Maus-Embryonen 48 h nach der Verletzung, in 4% PFA fixiert und in Paraffin eingebetteten geerntet werden. In fluoreszierende transgenen Modellen kann Kryokonservierung mit Oktober geeignet. Es gibt mehrere Flecken, die verwendet werden, um zelluläre und Bindegewebe Architektur Visualisierung verwendet werden kann. Hämatoxylin und Eosin ist ein Zwei-Farben-Fleck, Kerne blau und eosinophile Strukturen (dh., Zytoplasma und ex...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Eine Maus fetalen Hautmodell von Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

a-mouse-fetal-skin-model-of-scarless-wound-repair

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.2K Aufrufe.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Serie: A Mouse Fetal Skin Model of Scarless Wound Repair

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.

Die dreidimensionalen humanen Haut Rekonstruieren Modell: ein Tool für normale Haut und Melanom Progression Study

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

Forschung

Biotechnik

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

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

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

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Ein Vollhautdefekt Modell zur Vaskularisierung von Biomaterialien Bewerten<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 ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Mausmodell der Wundheilung

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medizin

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.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

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

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Ischämischen Gewebeschädigung des Rückenhautkammer der Maus: Ein Hautlappen Modell zu Akute Persistent Ischämie Untersuchen

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Bewertung der akuten Wundheilung mit den dorsalen subkutanen Polyvinylalkoholschwammimplantation und Exzisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Immunologie und Infektion

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Ein Mausmodell für mechanotransduktionsgetriebene, menschenähnliche hypertrophe Narbenbildung

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Digitale Planimetrie zur Beurteilung der Wundverschlusskinetik in einem Mausmodell

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Ein murines Modell einer Verbranntenwunde, rekonstruiert mit einem allogenen Hautgraft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Biologie

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

Eine Maus fetalen Hautmodell von Scarless Wound Repair

Zusammenfassung

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