这项研究是由来自NIDCR / NIH R01DE023348（RHK）和理事会学院研究基金（RHK）在加州大学洛杉矶分部的学术评议的研究的支持。

小鼠从杰克逊实验室购买并保存在实验动物医学UCLA司（DLAM）无病原体动物饲养。实验是按照从校长的动物研究委员会（ARC＃2016-037）批准的机构准则进行的。 1.鼠标麻醉<ol><li>使用八周龄雌性C57 / BL6小鼠（n = 3）。 </li><li>麻醉用氯胺酮（80-120毫克/千克小鼠体重）/赛拉嗪（5mg / kg的小鼠体重的）解决方案的小鼠和在剂量为10毫升/千克腹膜内（IP）施用。 </li><li>制备氯胺酮（80 - 120毫克/千克）/赛拉嗪（5mg / kg的）解决方案，并在剂量为10毫升/千克腹膜内（IP）施用它们。 </li><li>确认小鼠完全通过执行脚趾捏麻醉。 </li></ol> 2.盖髓过程<ol><li>放置在鼠标的嘴的嘴保持器。 </li><li>固定口保持器上的表，使得他广告朝上。 </li><li>放置在口的顶部的显微镜（10X），使得第一磨牙完全可见。 </li><li>使用¼ - 圆钻在高速手机为20万转，除去中间的牙釉质一部分，直到纸浆是通过透明牙本质可见。不要暴露与钻纸浆。 </li><li>使用＃15牙髓K-文件（150微米直径），通过穿孔牙本质和揭露浆。 注:特别应注意，使牙本质碎片不会被压入纸浆。这可以通过每季度转动K-文件，然后拉K-鱼贯而出避免。 </li><li>根据制造商的说明用无菌H 2ö混合MTA。交付和MTA放置到露髓的探险家的一角。使用纸张点（精）的背面侧到MTA包入露髓通过轻轻敲击。纸点的较厚侧是平坦的，因此可以对MTA的正常凝结成露髓。 </li><li>通过将35％的磷酸刻蚀剂，其中它只是覆盖了牙齿蚀刻15秒的牙齿。要特别小心，以限制蚀刻剂的位置，因为它可能会刺激牙龈组织。 注:蚀刻剂是在一个注射器中，并用来粗糙化的牙齿表面，使牙科用粘合剂可以流动到调解微机械粘合到牙齿。因为它们是粘性的，它可以是自包含的直接涂布少量到牙齿。 </li><li>使用负压力抽吸去除腐蚀剂。使用所轻轻用H 2 O中浸泡以除去蚀刻液的残差一个棉球。重复此步骤，直至蚀刻液完全从牙齿去除。 </li><li>使用压缩空气除尘器，轻轻擦干牙齿。 </li><li>申请使用的纸张点的背面的牙科粘合剂。 </li><li>使用压缩空气3秒的粘合剂层薄。 </li><li> CURE使用固化光用单元20秒的牙科粘合剂。 </li><li>放入少量的流动树脂到这是与MTA皑皑的牙齿。使用浏览器的尖端复合流入齿槽。 </li><li>固化复合用于使用光固化单元聚合它30秒。确认复合材料完全固化并用坚硬的探险家。 </li></ol> 3.后运护理<ol><li>管理该盖髓过程后立即卡洛芬（5毫克/千克）皮下（SC）。 </li><li>放置在一个加热垫的老鼠在低功耗，以保持温暖的动物，他们醒过来了。 </li><li>返回小鼠动物饲养住房。 </li></ol> 4.组织采购<ol><li>经过5 - 6周，安乐死与异氟醚麻醉完备的条件下通过颈椎脱位的小鼠。 </li><li>小心取出颌骨出头骨的基地，放入50毫升管。修复entir包含纸浆封端齿与在PBS中的4％多聚甲醛，pH值7.4对侧未加帽的齿都在4℃下过夜，然后将其存储在70％的乙醇溶液E颌骨。 注:多聚甲醛是有毒和致癌性。作为标准操作程序（SOP）中列出的正确使用多聚甲醛进行监测。 </li><li>扫描使用扫描μCT鼠标上颌骨。在扫描期间固定上颌骨，用纱布用70％乙醇浸泡包裹样本，将它们放置在15 mL细胞培养管。 </li></ol> 5.μCT扫描<ol><li>准备μCT扫描样本。简言之，将包裹的试样用纱布用70％乙醇浸泡和在通用的15毫升细胞培养物的锥形管固定。安装在管上的μCT扫描阶段，在制造商的说明所述。 </li><li>的X射线源设定为145微安的电流，55 kVp的的一个电压，和2的曝光时间00毫秒。 </li><li>用μCT扫描器在20微米分辨率和0.5mm的铝过滤器执行图像采集。 </li><li>重建图像和可视化是11。 </li><li>一旦μCT扫描完成后，2周开始脱钙，用5％EDTA和PBS中4％蔗糖（pH 7.4）中。 </li></ol> 6.组织处理和染色<ol><li>嵌入在石蜡脱钙组织。之前嵌入，通过矢状精确剪裁上颌骨前壁立即向第一磨牙。而嵌入，向下定位该表面，使得第一磨牙的纵向截面是切割表面。 </li><li>使用切片机，准备5微米厚的幻灯片。纸浆封区域通常与distopalatal（DP）的根，它可被用作一个划时代重合。通过检查在光学显微镜下组织学和比较μCT图像确定感兴趣的精确区域。 </li><li>对于H＆E染色，deparaffinize并用二甲苯（2×）和连续稀释的乙醇（100％EtOH中2倍，95％的EtOH 2x，以及70％乙醇1×）再水合的幻灯片。 </li><li>冲洗用流动的自来水的幻灯片。 </li><li>与2.5分钟苏木素液染色，用自来水冲洗。 </li><li>浸在95％乙醇的幻灯片1分钟。 </li><li>用1分钟伊红染色解决方案，并与自来水冲洗。 </li><li>用连续稀释的乙醇（70％EtOH中1个，95％的EtOH 2x，以及100％乙醇3×）和二甲苯（3×）脱水。 </li><li>安装与安装解决方案的幻灯片。 </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.

目前，有可用来验证牙科材料，支架，或生长因子对牙髓干细胞（牙髓干）13的牙源性分化的体内作用几个不同的实验模型。这些模型包括牙髓干细胞的异位自体移植到器官，如肾包膜，或牙髓干细胞的皮下移植入与支架14,15免疫功能低下的小鼠。然而，这些方法是有限的，不是在原位纸浆环境中执行对牙髓干细胞的牙源性作用。另一方面，原位移植到牙齿上的纸浆或纸浆?...

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.

<table border="0" cellpadding="0" cellspacing="0" width="641">
	<colgroup>
		<col span="2" />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">BM-LED stereo microscope</td>
			<td style="width:164px;">MEIJI Techno</td>
			<td style="width:140px;"></td>
			<td style="width:173px;">Microscope&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optima MCX-LED&#160;</td>
			<td>Bien Air Dental</td>
			<td>1700588-001</td>
			<td>Electic motor engine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane</td>
			<td>Henry schein animal health</td>
			<td>NDC 11695-0500-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/4 round bur</td>
			<td>Brasseler</td>
			<td>001092T0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Endodontic K-file</td>
			<td>Roydent</td>
			<td>98947</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProRoot MTA</td>
			<td>Dentsply</td>
			<td>PROROOT5W</td>
			<td>MTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paper point</td>
			<td>Henry schein</td>
			<td>100-3941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Etch</td>
			<td>Ultradent product Inc.</td>
			<td></td>
			<td>Phosphoric acid etchant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OptiBond SoloPlus</td>
			<td>Kerr</td>
			<td>29669</td>
			<td>Adhesives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coltolux LED</td>
			<td>Coltene/whaledent Inc.</td>
			<td>C7970100115</td>
			<td>Curing light unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Characterization tint</td>
			<td>Bisco</td>
			<td>T-14012</td>
			<td>Flowable composite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skyscan</td>
			<td>Breuker</td>
			<td>1275</td>
			<td>uCT scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microm</td>
			<td>Thermo</td>
			<td>HM355S</td>
			<td>Microtome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxyline-1</td>
			<td>Thermo Scientific</td>
			<td>7221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin-Y</td>
			<td>Thermo Scientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytoseal 60</td>
			<td>Thermo Scientific</td>
			<td>8310-16</td>
			<td>Mounting solution</td>
		</tr>
	</tbody>
</table>

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.

在这里，我们展示了一步一步的过程来执行盖髓对老鼠牙齿。其中纸浆小鼠封盖的关键方面是有相应的设备。在这方面，具有与10倍放大的显微镜是必要的（ 图1A）。要创建在齿类-I样的准备，我们为20万转（ 图1B）使用1/4轮毛刺在电动高速手机。备选地，任何其他的发动机，包括那些使用压缩空气，可用于制备牙齿。 <p class="jove_content" fo:keep-to...

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

我们描述了执行直接纸浆对小鼠齿封盖为牙髓创伤愈合和体内修复象牙质形成的所述评估的步骤的分步方法。

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

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 次观看.  The UCLA School of Dentistry. 我们描述了执行直接纸浆对小鼠齿封盖为牙髓创伤愈合和体内修复象牙质形成的所述评估的步骤的分步方法。 

视频: 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.

伤口愈合的小鼠模型

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

科研

医学

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导:"电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

选择性化学腰椎交感神经切除术中灰色拉米关节的靶向性

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...