Diese Studie wurde von R01DE023348 (RHK) von NIDCR / NIH und der Fakultät Research Grant (RHK) vom Rat für Forschung des Akademischen Senats der Los Angeles Abteilung der Universität von Kalifornien unterstützt.

Die Mäuse wurden von Jackson Laboratory erworben und in einem keimfreien Vivarium in der UCLA Abteilung für Labortiermedizin (DLAM). Die Experimente wurden aus dem Kanzler Animal Research Committee (ARC # 2016-037) nach den anerkannten Richtlinien des Instituts durchgeführt. 1. Maus Betäubung <ol><li> Verwenden Sie acht Wochen alten weiblichen C57 / BL6-Mäusen (n = 3). </li><li> Anästhesieren die Mäuse unter Verwendung von Ketamin (80-120 mg / kg Mausgewicht) / Xylazin (5 mg / kg Mausgewicht) Lösungen und administrieren intraperitoneal (ip) in einer Dosis von 10 ml / kg. </li><li> Bereiten Ketamin (80 - 120 mg / kg) / Xylazin (5 mg / kg) Lösungen und verwalten sie intraperitoneal (ip) in einer Dosis von 10 ml / kg. </li><li> Bestätigen Sie, dass die Mäuse durch Ausführen einer Zehe Prise vollständig betäubt werden. </li></ol> 2. Zellstoff-Capping Verfahren <ol><li> Platzieren Sie den Mund Halter in der Maus in den Mund. </li><li> Sichern Sie den Mund Halter auf dem Tisch, so dass die erAnzeige nach oben zeigt. </li><li> Platzieren Sie das Mikroskop (10fach) oben auf den Mund, so dass der erste Oberkiefermolaren vollständig sichtbar ist. </li><li> Mit der ¼-Rundbohrer in einem Hochgeschwindigkeits-Handstück bei 200.000 Umdrehungen pro Minute, entfernen Sie den Zahnschmelz Teil des Zahnes in der Mitte, bis der Brei durch das transparente Dentin sichtbar ist. Sie nicht den Brei mit dem bur aussetzen. </li><li> Unter Verwendung eines # 15 Endodontie K-Datei (Durchmesser von 150 &amp; mgr; m), perforieren durch das Dentin und setzen die Zellstoff. HINWEIS: Besondere Vorsicht ist geboten werden, so dass das Dentin Schmutz nicht in den Brei geschoben bekommt. Dies kann vierteljährlich durch Drehen der K-Datei vermieden werden, und dann zieht die K-Datei aus. </li><li> Mischungs MTA mit sterilem H 2 O gemäß den Anweisungen des Herstellers. mit der Spitze des Explorers liefern und den MTA auf die Pulpa platzieren. Verwenden Sie die Rückseite der Papierspitze (fein) durch leichtes Klopfen der MTA in die Pulpa zu packen. Die dickere Seite des Papiers Punkt ist flach und daher erlaubtfür die ordnungsgemäße Kondensation des MTA in die Pulpa. </li><li> Etch den Zahn für 15 s durch die 35% ige Phosphorsäure-Ätzmittel platzieren, wo es nur um den Zahn abdeckt. Besondere Vorsicht die Platzierung des Ätzmittel zu begrenzen, da es Gingivagewebe reizen können. HINWEIS: Das Ätzmittel in einer Spritze kommt, und verwendet wird, um die Zahnflächen aufzurauhen, so dass Dentaladhäsive einströmen kann mikromechanischen Bindung auf den Zahn zu vermitteln. Da sie viskos sind, können sie durch die Anwendung kleiner Mengen direkt auf dem Zahn in sich geschlossen sein. </li><li> Verwenden Sie negative Druck stehenden Saug- das Ätzmittel zu entfernen. Verwenden Sie einen Wattepellet , das leicht mit H 2 O getränkt wird , um die Reste des Ätzmittel zu entfernen. Wiederholen Sie diesen Schritt, bis das Ätzmittel vollständig aus dem Zahn entfernt wird. </li><li> Mit Hilfe eines Druckluftstaubtuch trocknen sanft den Zahn. </li><li> Übernehmen Sie die Dentaladhäsive die Rückseite des Papierspitze verwenden. </li><li> Stellen Sie die Klebeschicht dünn mit Druckluft für 3 s. </li><li> Cure die Dentaladhäsive für 20 s die Aushärtung Lichteinheit. </li><li> Legen Sie das fließfähige Composite in kleinen Mengen auf den Zahn, der mit MTA gekappt wurde. Verwenden Sie die Spitze des Forschers des Verbund in die Zahnrillen zu fließen. </li><li> Heilung der Verbund für 30 s eine lichthärtende Einheit unter Verwendung es zu polymerisieren. Bestätigen Sie, dass der Verbund vollständig ausgehärtet ist und hart den Explorer verwenden. </li></ol> 3. Post-op Pflege <ol><li> Verabreichen Carprofen (5 mg / kg) subkutan (sc) unmittelbar nach der Pulpe-Capping Verfahren. </li><li> Legen Sie die Mäuse auf einem Heizkissen bei geringer Leistung, die Tiere warm zu halten, bevor sie aufwachen. </li><li> Bringen Sie die Mäuse auf das Vivarium für den Wohnungsbau. </li></ol> 4. Gewebebeschaffung <ol><li> Nach 5 - 6 Wochen, die Mäuse durch Genickbruch unter einer vollständigen Betäubung mit Isofluran Zustand einschläfern. </li><li> Entfernen Sie vorsichtig den Oberkiefer aus der Basis des Schädels und steckte es in einen 50-ml-Röhrchen. Befestigen Sie den entire Oberkiefers, die über Nacht in PBS, pH 7,4, bei 4 ° C sowohl die Zellstoff- bedeckten Zahn und die kontralaterale uncapped Zahn in 4% Paraformaldehyd enthält, und es dann speichern Sie in einer 70% igen Ethanollösung. HINWEIS: Paraformaldehyd ist giftig und krebserregend. Die ordnungsgemäße Verwendung Paraformaldehyd sollte in den Standardarbeitsanweisungen (SOP) wie skizziert überwacht werden. </li><li> Scannen Sie die Maus Maxillen die μCT Scan verwenden. Zur Sicherung des Oberkiefers während des Scannens, wickeln Sie die Proben mit Gaze, getränkt mit 70% Ethanol und legen Sie sie in den 15-ml Zellkulturröhrchen. </li></ol> 5. μCT Scanning <ol><li> Bereiten Sie die Proben für μCT Scannen. Kurz gesagt, wickeln Sie die Proben mit Gaze mit 70% Ethanol getränkt und sie in einem allgemeinen 15 ml Zellkultur konischen Röhrchen sichern. Montieren Sie das Rohr auf die μCT Scanstufe, wie in den Anweisungen des Herstellers beschrieben. </li><li> Stellen Sie die Röntgenstrahlenquelle einem Strom von 145 uA, einer Spannung von 55 kVp und einer Belichtungszeit von 200 ms. </li><li> Führen der Bildaufnahme mit dem μCT Scanner mit einer 20-um-Auflösung und mit einer 0,5 mm Al-Filter. </li><li> Rekonstruieren Sie das Bild und visualisieren 11. </li><li> Sobald der μCT Scanvorgang abgeschlossen ist, Entkalkung mit 5% EDTA und 4% Saccharose in PBS (pH 7,4) für 2 Wochen. </li></ol> 6. Gewebeverarbeitung und Färbung <ol><li> Einbetten der entkalkt Gewebe in Paraffin. Vor dem Einbetten, schneiden Sie den Oberkiefer durch einen sagittalen Schnitt sofort auf den ersten Molaren anterioren machen. Während die Einbettung positionieren diese Fläche nach unten, so dass der Längsschnitt des ersten Molaren ist die Schnittfläche. </li><li> Mit dem Mikrotom, bereiten 5 um dicke Folien. Die Zellstoff-Capping Bereiche übereinstimmen in der Regel mit dem distopalatal (DP) Wurzel, die als Orientierungspunkt verwendet werden kann. Bestimmen Sie den genauen Bereich von Interesse durch die Histologie unter dem Lichtmikroskop zu untersuchen und die μCT Bilder zu vergleichen. </li><li> Für H &amp; E-Färbung, Deparaffinize und die Folien mit Xylol (2x) rehydrieren und seriell verdünnt Ethanol (100% EtOH 2x, 95% EtOH 2x, und 70% EtOH 1x). </li><li> Spülen Sie die Folien mit fließendem Leitungswasser. </li><li> Fleck mit Hämatoxylin-Lösung für 2,5 min und Spülen mit Leitungswasser. </li><li> Tauchen der Objektträger in 95% Ethanol für 1 min. </li><li> Fleck mit Eosin-Lösung für 1 min und Spülen mit Leitungswasser. </li><li> Entwässern mit seriell verdünnten Ethanol (70% EtOH 1x, 95% EtOH 2x und 100% EtOH 3x) und Xylol (3x). </li><li> Montieren Sie die Folien mit Befestigungslösung. </li></ol>

<ol>
	<li>Dye, B., Thornton-Evans, G., Li, X., Iafolla, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+caries+and+tooth+loss+in+adults+in+the+United+States,+2011-2012.">Dental caries and tooth loss in adults in the United States, 2011-2012.</a> NCHS Data Brief. (197), 197 (2015).</li><li>Bagramian, R. A., Garcia-Godoy, F., Volpe, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+increase+in+dental+caries.+A+pending+public+health+crisis.">The global increase in dental caries. A pending public health crisis.</a> Am J Dent. 22 (1), 3-8 (2009).</li><li>Koliniotou-Koumpia, E., Tziafas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulpal+responses+following+direct+pulp+capping+of+healthy+dog+teeth+with+dentine+adhesive+systems.">Pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems.</a> J Dent. 33 (8), 639-647 (2005).</li><li>Tarim, B., Hafez, A. A., Cox, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulpal+response+to+a+resin-modified+glass-ionomer+material+on+nonexposed+and+exposed+monkey+pulps.">Pulpal response to a resin-modified glass-ionomer material on nonexposed and exposed monkey pulps.</a> Quintessence Int. 29 (8), 535-542 (1998).</li><li>Tziafa, C., Koliniotou-Koumpia, E., Papadimitriou, S., Tziafas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentinogenic+responses+after+direct+pulp+capping+of+miniature+swine+teeth+with+Biodentine.">Dentinogenic responses after direct pulp capping of miniature swine teeth with Biodentine.</a> J Endod. 40 (12), 1967-1971 (2014).</li><li>Dammaschke, T., Stratmann, U., Fischer, R. J., Sagheri, D., Schafer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histologic+investigation+of+direct+pulp+capping+in+rodents+with+dentin+adhesives+and+calcium+hydroxide.">A histologic investigation of direct pulp capping in rodents with dentin adhesives and calcium hydroxide.</a> Quintessence Int. 41 (4), 62-71 (2010).</li><li>Jegat, N., Septier, D., Veis, A., Poliard, A., Goldberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effects+of+amelogenin+gene+splice+products+A+4+and+A-4+implanted+in+the+exposed+rat+molar+pulp.">Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp.</a> Head Face Med. 3, 40 (2007).</li><li>Paterson, R. C., Radford, J. R., Watts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+rat+molar+pulp+of+two+proprietary+calcium+hydroxide+preparations.">The response of the rat molar pulp of two proprietary calcium hydroxide preparations.</a> Br Dent J. 151 (6), 184-186 (1981).</li><li>Sela, J., Ulmansky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+normal+and+inflamed+dental+pulp+to+Calxyl+and+zinc+oxide+and+eugenol+in+rats.">Reaction of normal and inflamed dental pulp to Calxyl and zinc oxide and eugenol in rats.</a> Oral Surg Oral Med Oral Pathol. 30 (3), 425-430 (1970).</li><li>Maurice, C. G., Schour, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+cavity+preparations+in+the+molar+of+the+rat.">Experimental cavity preparations in the molar of the rat.</a> J Dent Res. 34 (3), 429-434 (1955).</li><li>Sohn, 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+Role+of+ORAI1+in+the+Odontogenic+Differentiation+of+Human+Dental+Pulp+Stem+Cells.">The Role of ORAI1 in the Odontogenic Differentiation of Human Dental Pulp Stem Cells.</a> J Dent Res. 94 (11), 1560-1567 (2015).</li><li>Kim, S., Shin, S. J., Song, Y., Kim, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Experiments+with+Dental+Pulp+Stem+Cells+for+Pulp-Dentin+Complex+Regeneration.">In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration.</a> Mediators Inflamm. 2015, 409347 (2015).</li><li>Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+human+dental+pulp+stem+cells+(DPSCs)+in+vitro+and+in+vivo.">Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo.</a> Proc Natl Acad Sci U S A. 97 (25), 13625-13630 (2000).</li><li>Yu, 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=Odontogenic+capability:+bone+marrow+stromal+stem+cells+versus+dental+pulp+stem+cells.">Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells.</a> Biol Cell. 99 (8), 465-474 (2007).</li><li>Zhu, 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=Transplantation+of+dental+pulp+stem+cells+and+platelet-rich+plasma+for+pulp+regeneration.">Transplantation of dental pulp stem cells and platelet-rich plasma for pulp regeneration.</a> J Endod. 38 (12), 1604-1609 (2012).</li><li>Iohara, 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=Dentin+regeneration+by+dental+pulp+stem+cell+therapy+with+recombinant+human+bone+morphogenetic+protein+2.">Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2.</a> J Dent Res. 83 (8), 590-595 (2004).</li><li>Saito, K., Nakatomi, M., Ida-Yonemochi, H., Ohshima, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteopontin+Is+Essential+for+Type+I+Collagen+Secretion+in+Reparative+Dentin.">Osteopontin Is Essential for Type I Collagen Secretion in Reparative Dentin.</a> J Dent Res. , (2016).</li><li>Hunter, D. 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=Wnt+Acts+as+a+Pro-Survival+Signal+to+Enhance+Dentin+Regeneration.">Wnt Acts as a Pro-Survival Signal to Enhance Dentin Regeneration.</a> J Bone Miner Res. , (2015).</li><li>Goldberg, M., Kulkarni, A. B., Young, M., Boskey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentin:+structure,+composition+and+mineralization.">Dentin: structure, composition and mineralization.</a> Front Biosci (Elite Ed). 3, 711-735 (2011).</li><li>Nascimento, A. B., Fontana, U. F., Teixeira, H. M., Costa, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+of+a+resin-modified+glass-ionomer+cement+applied+as+pulp+capping+in+human+teeth).">Biocompatibility of a resin-modified glass-ionomer cement applied as pulp capping in human teeth).</a> Am J Dent. 13 (1), 28-34 (2000).</li><li>Bogen, G., Kim, J. S., Bakland, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+pulp+capping+with+mineral+trioxide+aggregate:+an+observational+study.">Direct pulp capping with mineral trioxide aggregate: an observational study.</a> J Am Dent Assoc. 139 (3), 305-315 (2008).</li><li>Miller, R. A., Nadon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+animal+use+for+gerontological+research.">Principles of animal use for gerontological research.</a> J Gerontol A Biol Sci Med Sci. 55 (3), 117-123 (2000).</li><li>Shah, A., Song, M., Cao, Y., Kang, M. K., Kim, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts+are+absent+in+pulpal+and+periapical+inflammatory+lesions.">Osteoclasts are absent in pulpal and periapical inflammatory lesions.</a> J Dent Res. 95, 1503 (2016).</li><li>Williams, D. 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=Impaired+bone+resorption+and+woven+bone+formation+are+associated+with+development+of+osteonecrosis+of+the+jaw-like+lesions+by+bisphosphonate+and+anti-receptor+activator+of+NF-kappaB+ligand+antibody+in+mice).">Impaired bone resorption and woven bone formation are associated with development of osteonecrosis of the jaw-like lesions by bisphosphonate and anti-receptor activator of NF-kappaB ligand antibody in mice).</a> Am J Pathol. 184 (11), 3084-3093 (2014).</li><li>McPherson, J. D., 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=A+physical+map+of+the+human+genome.">A physical map of the human genome.</a> Nature. 409 (6822), 934-941 (2001).</li><li>Gregory, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physical+map+of+the+mouse+genome.">A physical map of the mouse genome.</a> Nature. 418 (6899), 743-750 (2002).</li><li>Hilton, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keys+to+clinical+success+with+pulp+capping:+a+review+of+the+literature.">Keys to clinical success with pulp capping: a review of the literature.</a> Oper Dent. 34 (5), 615-625 (2009).</li><li>Holmdahl, R., Bockermann, R., Backlund, J., Yamada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+pathogenesis+of+collagen-induced+arthritis+in+mice--a+model+for+rheumatoid+arthritis.">The molecular pathogenesis of collagen-induced arthritis in mice--a model for rheumatoid arthritis.</a> Ageing Res Rev. 1 (1), 135-147 (2002).</li><li>Kalu, D. N., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovariectomized+murine+model+of+postmenopausal+calcium+malabsorption.">Ovariectomized murine model of postmenopausal calcium malabsorption.</a> J Bone Miner Res. 14 (4), 593-601 (1999).</li><li>Yokochi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+experimental+murine+model+for+lipopolysaccharide-mediated+lethal+shock+with+lung+injury.">A new experimental murine model for lipopolysaccharide-mediated lethal shock with lung injury.</a> Innate Immun. 18 (2), 364-370 (2012).</li><li>Abe, T., Hajishengallis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+ligature-induced+periodontitis+model+in+mice.">Optimization of the ligature-induced periodontitis model in mice.</a> J Immunol Methods. 394 (1-2), 49-54 (2013).</li></ol>

The authors have nothing to disclose.

Derzeit gibt es mehrere verschiedene experimentelle Modelle zur Verfügung , um die in vivo - Effekte von Dentalmaterialien, Gerüste oder Wachstumsfaktoren auf odontogene Differenzierung von Zahnpulpa Stammzellen (DPSCs) 13 zu validieren. Diese Modelle sind mit ektopischen autologe Transplantation von DPSCs in ein Organ, wie die Nierenkapsel oder subkutane Transplantation von DPSCs in immunsupprimierten Mäusen mit Gerüsten 14,15. Jedoch sind diese Verfahren begrenzt, daß ihre Wirkung a...

Dental caries are one of the most prevalent oral diseases and the leading cause of surgical interventions to dentitions in almost all individuals1,2. The prognosis of surgical interventions and restorations of a tooth largely depends upon proper pulpal response and successful wound healing. Indeed, dental caries that penetrate deeply through the enamel and dentin frequently lead to the exposure of the underlying pulp tissue that is often "capped" with dental materials, such as calcium hydroxide (Ca(OH)2) or hydraulic calcium-silicate cements (HCSCs), including mineral trioxide aggregates (MTA). The ultimate goal of such a pulp-capping procedure is to expedite pulpal wound healing by regenerating reparative dentin, a physical barrier that functions as a "biological seal" to protect the underlying pulp tissue and to increase the life expectancy of the tooth and the overall oral health. However, the underlying mechanism of pulpal wound healing and reparative dentin formation is not fully understood.
To better understand the mechanisms of pulpal wound healing and reparative dentin formation in vivo, several animals were previously used, including monkeys, dogs, and pigs3-5. Among them, rats are frequently used because they are relatively smaller in sizes compared to the other animals, but their teeth are large enough to perform direct pulp capping without any technical difficulties6-10. These animal models are ideal alternatives to human studies for examining pulpal responses and reparative dentin formation. However, their utilization is limited to observational studies at the cellular level, and they scarcely provide mechanistic insights during reparative dentin formation at the molecular level.
Recent technical advances in genetic engineering provided invaluable and indispensable research tools-mice that harbor a gene that is either overexpressed or deleted-that are instrumental to studying molecular mechanisms of human diseases in vivo. The numbers of different strains of transgenic or knockout mice that are strategically inducible in a cell-specific manner are continually growing in the scientific community. Therefore, examining pulpal wound healing and reparative dentin regeneration in these mice would greatly help to expedite our understanding of these processes at the molecular level. However, the use of mice is significantly dampened, as performing a pulp-capping procedure on a mouse tooth is technically challenging due to its miniature size. Here, we present our reproducible method of performing direct pulp capping in mice for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

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development of a direct pulp-capping model for the evaluation of pulpal wound healing and reparative dentin formation in mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

Hier haben wir gezeigt, die Schritt-für-Schritt-Verfahren Zellstoff zur Durchführung an Mäusen Zähne Capping. Einer der wichtigsten Aspekte der Zellstoff bei Mäusen Capping ist die entsprechende Vorrichtung haben. In dieser Hinsicht mit einem 10fach-facher Vergrößerung des Mikroskops ist von wesentlicher Bedeutung (Abbildung 1A). Um eine Klasse-I-like Vorbereitung in den Zahn zu erstellen, verwendeten wir eine ¼-Runde Grat in einem elektrischen Hochgeschwindigkei...

Watch this Scientific Journal Video about Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice at JoVE.com

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Wir beschreiben eine Schritt- für -Schritt - Methode der direkten Pulpe an Mäusen Zähne für die Bewertung der Pulpa Wundheilung und reparative Dentin Bildung in vivo Capping durchgeführt wird .

Entwicklung eines Direkt Pulp-Kappungsmodell für die Beurteilung von Pulpal Wundheilung und Reparative Dentinbildung bei Mäusen

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

development-direct-pulp-capping-model-for-evaluation-pulpal-wound

Research

JoVE Journal

Medicine

11.2K Aufrufe.  The UCLA School of Dentistry.  Wir beschreiben eine Schritt- für -Schritt - Methode der direkten Pulpe an Mäusen Zähne für die Bewertung der Pulpa Wundheilung und reparative Dentin Bildung in vivo Capping durchgeführt wird . 

Serie: Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

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. ...

Forschung

Medizin

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

Eine Co-Kultur-Methode zur Untersuchung des Neuriten-Auswuchss als Reaktion auf Dental Pulp Paracrine Signale

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Entwicklungsbiologie

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

Murine Excisional Wound Healing Model und histologische morphometrische Wundanalyse

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

Live-Tracking von Zahnzellen mittels Zwei-Photonen-Mikroskopie

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Autor im Rampenlicht: Verständnis des dynamischen zellulären Verhaltens bei der Erneuerung und Reparatur von Zahngewebe bei erwachsenen Mäusen 

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

Verwendung von Ex-vivo-Live-Bildgebung zur Untersuchung von Zellteilungen und -bewegungen während der Zahnerneuerung von Mäusen

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

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

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, ...

Biotechnik

Erstellung eines Mausmodells für eine Pulpaentzündung unter Verwendung eines neuartigen Mundknebels 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

Autor im Rampenlicht: Verbesserung der Zahnpulpaforschung mit verbesserten Mausmodellen

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

Entwicklung eines Direkt Pulp-Kappungsmodell für die Beurteilung von Pulpal Wundheilung und Reparative Dentinbildung bei Mäusen

Zusammenfassung

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Mausmodell der Wundheilung

Eine Co-Kultur-Methode zur Untersuchung des Neuriten-Auswuchss als Reaktion auf Dental Pulp Paracrine Signale

Murine Excisional Wound Healing Model und histologische morphometrische Wundanalyse

Verwendung von Ex-vivo-Live-Bildgebung zur Untersuchung von Zellteilungen und -bewegungen während der Zahnerneuerung von Mäusen

Verwendung von Ex-vivo-Live-Bildgebung zur Untersuchung von Zellteilungen und -bewegungen während der Zahnerneuerung von Mäusen

Verwendung von Ex-vivo-Live-Bildgebung zur Untersuchung von Zellteilungen und -bewegungen während der Zahnerneuerung von Mäusen

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

Digitale Planimetrie zur Beurteilung der Wundverschlusskinetik in einem Mausmodell

Etablierung eines murinen Pulpa-Expositionsmodells mit einem neuartigen Mundknebel für die Pulpitisforschung

Etablierung eines murinen Pulpa-Expositionsmodells mit einem neuartigen Mundknebel für die Pulpitisforschung