Bu çalışma NIDCR / NIH R01DE023348 (RHK) ve University of California, Los Angeles Bölümü Akademik Senatosu Araştırma Konseyi Fakültesi Araştırma Bursu (RHK) tarafından desteklenmiştir.

Fareleri Jackson Laboratory&#39;den satın alındı ​​ve Deney Hayvanları UCLA Tıp Bölümü (DLAM) bir patojen içermeyen vivaryum tutuldu. Deneyler Başbakan&#39;ın Hayvancılık Araştırma Komitesi (ARC # 2016-037) onaylanmış kurumsal kurallarına göre yapılmıştır. 1. Fare Anesthetization <ol><li> Sekiz haftalık dişi C57 / BL6 fareler kullanın (n = 3). </li><li> ketamin (fare ağırlığı 80-120 mg / kg) / ksilazin (5 mg / fare ağırlığındaki her bir kg) çözeltileri kullanılarak fareler anestezi ve 10 ml / kg&#39;lık bir dozda intraperitonal (ip) verilmesi. </li><li> ketamin hazırlanması (80-120 mg / kg) / ksilazin (5 mg / kg) çözeltiler ve 10 mL / kg bir dozda intraperitonal (ip) olarak onları yönetmek. </li><li> fareler tamamen ayak tutam yaparak anestezi onaylayın. </li></ol> 2. Selüloz-kapatma İşlemi <ol><li> Farenin ağzına ağız tutucu yerleştirin. </li><li> O böyle masada ağız yuvasını Güvenlireklam yukarı bakacak. </li><li> İlk maksiller molar tamamen görünür böylece ağız üstüne mikroskobu (10X) yerleştirin. </li><li> hamuru şeffaf dentin aracılığıyla görünür oluncaya kadar 200.000 rpm&#39;de yüksek hızda piyasemene ¼ yuvarlak bur kullanarak, ortada diş minesi parçası kaldırın. bur hamuru maruz bırakmayın. </li><li> Bir 15. endodontik K-dosya (150 mikron çapında) kullanılarak, dentin ile delmek ve hamuru maruz kalmaktadır. NOT: dentin enkaz hamuru içine itilmiş almaz böylece Özel bakım alınmalıdır. Bu üç aylık K-dosyayı dönen ve sonra dışarı K-dosya çekerek önlenebilir. </li><li> Üreticinin talimatlarına uygun olarak, steril H2O ile MTA karıştırın. Sunun ve kaşif ucuyla maruz hamuru üzerine MTA yerleştirin. nazik dokunarak maruz hamuru içine MTA paketi kağıt noktasının (ince) arka tarafını kullanın. Kağıt noktasının kalın tarafı düz ve bu nedenle izin verirmaruz hamuru içine MTA düzgün yoğunlaşması için. </li><li> Sadece diş kapsayan% 35&#39;lik fosforik asit pürüzlendirici koyarak 15 saniye boyunca diş etch. o dişeti dokuları tahriş edebilir gibi etchant yerleştirme sınırlamak için özel özen gösterin. NOT: dağlayıcı bir şırınga gelir ve diş yapıştırıcılar diş üzerine Mikromekanik bağ aracılık etmek akabilmesi için diş yüzeylerinin pürüzlendirin için kullanılır. viskoz olduğundan, kendi kendine yeten bir diş üzerine doğrudan küçük miktarlarda uygulanması ile yapılabilir. </li><li> pürüzlendirici kaldırmak için negatif basınçlı emiş kullanın. Hafifçe etchant ve artıklar kaldırmak için H 2 O ile ıslatılır pamuk bir pelet kullanın. dağlayıcı tamamen diş kaldırılana kadar bu adımı tekrarlayın. </li><li> bir sıkıştırılmış hava silgi kullanarak, yavaşça diş kurutun. </li><li> Kağıt noktasının ters kullanarak diş yapıştırıcıları uygulayın. </li><li> 3 saniye basınçlı hava kullanılarak yapışkan tabaka ince olun. </li><li> Cure kür ışık birimini kullanarak 20 saniye boyunca diş yapıştırıcılar. </li><li> MTA ile kapaklandı diş üzerine küçük miktarlarda akışkan kompozit yerleştirin. diş oluklarına içine kompozit akmaya explorer ucu kullanın. </li><li> Bunu polimerize bir ışıkla sertleşen birimini kullanarak 30 saniye boyunca kompozit Cure. Kompozit tam kürünü ve sert explorer kullanarak doğrulayın. </li></ol> 3. Ameliyat sonrası bakım <ol><li> Hemen kağıt hamuru kapatma işleminden sonra karprofen (5 mg / kg) deri altından (sc) uygulayınız. </li><li> Onlar uyanmadan sıcak hayvanları tutmak için düşük güçte bir ısıtma pedi üzerinde fareler yerleştirin. </li><li> konut vivari fareler dönün. </li></ol> 4. Doku Alımı <ol><li> 5 sonra - 6 hafta, izofluran ile tam bir anestezi koşulları altında servikal dislokasyon ile farelerin euthanize. </li><li> Dikkatle kafatasının tabanından maksilla çıkarın ve 50 ml tüp içine koydu. entir Fixgece boyunca 4 ° C &#39;de, kağıt hamuru başlıklı diş ve PBS içinde% 4 paraformaldehid, pH 7.4 içinde karşı sınırlanmamış diş hem de içerir, ve daha sonra% 70&#39;lik bir etanol çözeltisi içinde saklayın e maksilla. NOT: Paraformaldehyde toksik ve kanserojen olduğunu. standart operasyon prosedürleri (SOP) &#39;de belirtildiği gibi doğru kullanımı paraformaldehit izlenmelidir. </li><li> uCT tarama kullanarak fare maxillae tarayın. % 70 etanol ile ıslatılmış gazlı bez ile örnekleri sarın ve 15 ml hücre kültürü tüp koyun, tarama sırasında maxillae güvenceye almak için. </li></ol> 5. uCT Tarama <ol><li> uCT tarama için numune hazırlayın. Kısaca,% 70 etanol ile ıslatılmış gazlı bez ile örnekleri sarın ve bir jenerik 15 ml hücre kültürü konik tüp sabitleyin. üreticinin talimatlarında belirtildiği gibi, uCT tarama sahneye tüp monte edin. </li><li> 145 uA bir akıma, 55 kVp bir gerilim ve 2 bir pozlama süresi X-ışını kaynağı Set00 ms. </li><li> 20 mikron çözünürlükte ve 0.5 mm Al filtre ile uCT tarayıcı ile görüntü elde etme gerçekleştirin. </li><li> Görüntüyü yeniden ve 11 görselleştirmek. </li><li> uCT Tarama işlemi tamamlandıktan sonra, 2 hafta içinde% 5 EDTA ve PBS içinde% 4 sakroz (pH 7.4) dekalsifikasyon başlar. </li></ol> 6. Doku İşleme ve Boyama <ol><li> parafin dekalsifiye dokuları gömün. gömmeden önce, hemen birinci molar için ön bir sagital kesim yaparak maksilla kırpın. gömme birlikte, birinci molar uzunlamasına kesit kesme yüzeyi olacak şekilde, aşağı doğru, bu yüzey yerleştirin. </li><li> mikrotom kullanılarak, 5 mikron kalınlığında slaytlar hazırlamak. kağıt hamuru sınırı alanlar genellikle bir dönüm noktası olarak kullanılabilir distopalatal (DP) kök ile örtüşmektedir. Işık mikroskobu altında histoloji incelenmesi ve uCT görüntüleri karşılaştırarak ilgi belirli bir bölgede tespit. </li><li> H &amp; E boyama, depara içinffinize ve ksilen (2x) ve seri olarak seyreltilmiş, etanol (% 100 EtOH 2x,% 95 EtOH 2x,% 70 EtOH 1x) ile slaytlar rehidrate. </li><li> akan musluk suyu ile slaytlar durulayın. </li><li> 2.5 dakika boyunca Hematoksilen çözümü ile Leke ve musluk suyu ile durulayın. </li><li> 1 dakika boyunca% 95 etanol içinde slaytlar batırın. </li><li> 1 dakika süreyle Eosin çözeltisi ile Leke musluk suyu ile yıkayın. </li><li> seri olarak seyreltilmiş, etanol (% 70 EtOH 1x,% 95 EtOH 2x,% 100 EtOH 3x) ve ksilen (3x) ile kurutmak. </li><li> montaj çözümü ile slaytlar monte edin. </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.

Şu anda, dental pulpa kök hücrelerin (DPSCs) 13 odontojenik farklılaşma dental materyaller, iskeleler, ya da büyüme faktörlerinin in vivo etkilerini doğrulamak için birkaç farklı deneysel model vardır. Bu modeller gibi böbrek kapsül gibi bir organ DPSCs ektopik otolog transplantasyon, ya da iskeleler 14,15 ile immün sistemi baskılanmış farelere DPSCs deri altına nakli sayılabilir. Bununla birlikte, bu yöntemler DPSCs üzerindeki odontojenik etkisi ortotopik hamur orta...

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.

Burada, fareler dişlerde pulpa kaplama gerçekleştirmek için adım adım uygulandığını göstermiştir. Farelerde kapatma hamuru önemli yönlerinden biri uygun cihazı sahip olmaktır. Bu bağlamda, 10X güç büyütme ile mikroskop olan esas (Şekil 1A) &#39;dir. Diş Sınıf-I-benzeri hazırlık oluşturmak için, biz 200.000 rpm (Şekil 1B) bir elektrik yüksek hızda piyasemene bir ¼ tur çapak kullanılır. Seçenek olarak ise, basınçlı h...

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

Bu pulpa yara iyileşmesi ve in vivo onarım dentin oluşumunun değerlendirilmesi için farenin dişler üzerinde kapatma doğrudan kağıt hamuru yapmayı bir adım adım bir yöntem açıklanmaktadır.

Pulpa Değerlendirilmesi için Doğrudan Selüloz-kapatma Modelinin Geliştirilmesi Yara İyileşmesi ve Farelerde Onarım dentin oluşumu

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 Görüntüleme.  The UCLA School of Dentistry.  Bu pulpa yara iyileşmesi ve in vivo onarım dentin oluşumunun değerlendirilmesi için farenin dişler üzerinde kapatma doğrudan kağıt hamuru yapmayı bir adım adım bir yöntem açıklanmaktadır. 

Video: 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.

Yara Şifa murin Modeli

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

Tıp

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.

Diş Hamuru Parakrin Sinyalleri yanıt olarak Neurite Outgrowth Çalışma Co-Kültür Yöntemi

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

Gelişim Biyolojisi

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 Eksizyonel Yara İyileştirme Modeli ve Histolojik Morfometrik Yara Analizi

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

İki foton mikroskobu ile diş hücrelerinin canlı takibi

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.

Yazar Spotlight: Yetişkin Fare Diş Dokusu Yenileme ve Onarımında Dinamik Hücresel Davranışları Anlama 

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.

Fare Diş Yenileme Sırasında Hücre Bölünmelerini ve Hareketlerini Araştırmak için Ex Vivo Canlı Görüntülemeyi Kullanma

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.

Dorsal Subkutan Polivinil Alkol Sünger İmplantasyonu ve Eksizyonal Kuyruk Deri Yara Modelleri kullanılarak Akut Yara İyileşmesinin Değerlendirilmesi.

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

İmmünoloji ve Enfeksiyon

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.

Bir Fare Modelinde Yara Kapanma Kinetiğini Değerlendirmek için Dijital Planimetri

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

Biyomühendislik

Yeni Bir Ağız Tıkaç Kullanarak Pulpa İltihabı Fare Modeli Oluşturma 

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.

Yazar Spotlight: Geliştirilmiş Fare Modelleri ile Dental Pulpa Araştırmalarını Geliştirme

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

Pulpa Değerlendirilmesi için Doğrudan Selüloz-kapatma Modelinin Geliştirilmesi Yara İyileşmesi ve Farelerde Onarım dentin oluşumu

Özet

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Yara Şifa murin Modeli

Diş Hamuru Parakrin Sinyalleri yanıt olarak Neurite Outgrowth Çalışma Co-Kültür Yöntemi

Murine Eksizyonel Yara İyileştirme Modeli ve Histolojik Morfometrik Yara Analizi

Fare Diş Yenileme Sırasında Hücre Bölünmelerini ve Hareketlerini Araştırmak için Ex Vivo Canlı Görüntülemeyi Kullanma

Fare Diş Yenileme Sırasında Hücre Bölünmelerini ve Hareketlerini Araştırmak için Ex Vivo Canlı Görüntülemeyi Kullanma

Fare Diş Yenileme Sırasında Hücre Bölünmelerini ve Hareketlerini Araştırmak için Ex Vivo Canlı Görüntülemeyi Kullanma

Dorsal Subkutan Polivinil Alkol Sünger İmplantasyonu ve Eksizyonal Kuyruk Deri Yara Modelleri kullanılarak Akut Yara İyileşmesinin Değerlendirilmesi.

Bir Fare Modelinde Yara Kapanma Kinetiğini Değerlendirmek için Dijital Planimetri

Pulpitis Araştırmaları için Yeni Bir Ağız Tıkaç ile Murin Pulpa Maruziyet Modelinin Kurulması

Pulpitis Araştırmaları için Yeni Bir Ağız Tıkaç ile Murin Pulpa Maruziyet Modelinin Kurulması