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
Summary
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。
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| BM-LED stereo microscope | MEIJI Techno | Microscope | |
| Optima MCX-LED | Bien Air Dental | 1700588-001 | Electic motor engine |
| isoflurane | Henry schein animal health | NDC 11695-0500-2 | |
| 1/4 round bur | Brasseler | 001092T0 | |
| Endodontic K-file | Roydent | 98947 | |
| ProRoot MTA | Dentsply | PROROOT5W | MTA |
| Paper point | Henry schein | 100-3941 | |
| Ultra-Etch | Ultradent product Inc. | Phosphoric acid etchant | |
| OptiBond SoloPlus | Kerr | 29669 | Adhesives |
| Coltolux LED | Coltene/whaledent Inc. | C7970100115 | Curing light unit |
| Characterization tint | Bisco | T-14012 | Flowable composite |
| Skyscan | Breuker | 1275 | uCT scanner |
| Microm | Thermo | HM355S | Microtome |
| Hematoxyline-1 | Thermo Scientific | 7221 | |
| Eosin-Y | Thermo Scientific | 7111 | |
| Cytoseal 60 | Thermo Scientific | 8310-16 | Mounting solution |
References
- Dye, B., Thornton-Evans, G., Li, X., Iafolla, T. Dental caries and tooth loss in adults in the United States, 2011-2012. NCHS Data Brief. (197), 197 (2015).
- Bagramian, R. A., Garcia-Godoy, F., Volpe, A. R. The global increase in dental caries. A pending public health crisis. Am J Dent. 22 (1), 3-8 (2009).
- Koliniotou-Koumpia, E., Tziafas, D. Pulpal responses following direct pulp capping of healthy dog teeth with dentine adhesive systems. J Dent. 33 (8), 639-647 (2005).
- Tarim, B., Hafez, A. A., Cox, C. F. Pulpal response to a resin-modified glass-ionomer material on nonexposed and exposed monkey pulps. Quintessence Int. 29 (8), 535-542 (1998).
- Tziafa, C., Koliniotou-Koumpia, E., Papadimitriou, S., Tziafas, D. Dentinogenic responses after direct pulp capping of miniature swine teeth with Biodentine. J Endod. 40 (12), 1967-1971 (2014).
- Dammaschke, T., Stratmann, U., Fischer, R. J., Sagheri, D., Schafer, E. A histologic investigation of direct pulp capping in rodents with dentin adhesives and calcium hydroxide. Quintessence Int. 41 (4), 62-71 (2010).
- Jegat, N., Septier, D., Veis, A., Poliard, A., Goldberg, M. Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp. Head Face Med. 3, 40 (2007).
- Paterson, R. C., Radford, J. R., Watts, A. The response of the rat molar pulp of two proprietary calcium hydroxide preparations. Br Dent J. 151 (6), 184-186 (1981).
- Sela, J., Ulmansky, M. Reaction of normal and inflamed dental pulp to Calxyl and zinc oxide and eugenol in rats. Oral Surg Oral Med Oral Pathol. 30 (3), 425-430 (1970).
- Maurice, C. G., Schour, I. Experimental cavity preparations in the molar of the rat. J Dent Res. 34 (3), 429-434 (1955).
- Sohn, S., et al. The Role of ORAI1 in the Odontogenic Differentiation of Human Dental Pulp Stem Cells. J Dent Res. 94 (11), 1560-1567 (2015).
- Kim, S., Shin, S. J., Song, Y., Kim, E. In Vivo Experiments with Dental Pulp Stem Cells for Pulp-Dentin Complex Regeneration. Mediators Inflamm. 2015, 409347 (2015).
- Gronthos, S., Mankani, M., Brahim, J., Robey, P. G., Shi, S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A. 97 (25), 13625-13630 (2000).
- Yu, J., et al. Odontogenic capability: bone marrow stromal stem cells versus dental pulp stem cells. Biol Cell. 99 (8), 465-474 (2007).
- Zhu, X., et al. Transplantation of dental pulp stem cells and platelet-rich plasma for pulp regeneration. J Endod. 38 (12), 1604-1609 (2012).
- Iohara, K., et al. Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2. J Dent Res. 83 (8), 590-595 (2004).
- Saito, K., Nakatomi, M., Ida-Yonemochi, H., Ohshima, H. Osteopontin Is Essential for Type I Collagen Secretion in Reparative Dentin. J Dent Res. , (2016).
- Hunter, D. J., et al. Wnt Acts as a Pro-Survival Signal to Enhance Dentin Regeneration. J Bone Miner Res. , (2015).
- Goldberg, M., Kulkarni, A. B., Young, M., Boskey, A. Dentin: structure, composition and mineralization. Front Biosci (Elite Ed). 3, 711-735 (2011).
- Nascimento, A. B., Fontana, U. F., Teixeira, H. M., Costa, C. A. Biocompatibility of a resin-modified glass-ionomer cement applied as pulp capping in human teeth). Am J Dent. 13 (1), 28-34 (2000).
- Bogen, G., Kim, J. S., Bakland, L. K. Direct pulp capping with mineral trioxide aggregate: an observational study. J Am Dent Assoc. 139 (3), 305-315 (2008).
- Miller, R. A., Nadon, N. L. Principles of animal use for gerontological research. J Gerontol A Biol Sci Med Sci. 55 (3), 117-123 (2000).
- Shah, A., Song, M., Cao, Y., Kang, M. K., Kim, R. H. Osteoclasts are absent in pulpal and periapical inflammatory lesions. J Dent Res. 95, 1503 (2016).
- Williams, D. W., et al. 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). Am J Pathol. 184 (11), 3084-3093 (2014).
- McPherson, J. D., et al. A physical map of the human genome. Nature. 409 (6822), 934-941 (2001).
- Gregory, S. G., et al. A physical map of the mouse genome. Nature. 418 (6899), 743-750 (2002).
- Hilton, T. J. Keys to clinical success with pulp capping: a review of the literature. Oper Dent. 34 (5), 615-625 (2009).
- Holmdahl, R., Bockermann, R., Backlund, J., Yamada, H. The molecular pathogenesis of collagen-induced arthritis in mice–a model for rheumatoid arthritis. Ageing Res Rev. 1 (1), 135-147 (2002).
- Kalu, D. N., Chen, C. Ovariectomized murine model of postmenopausal calcium malabsorption. J Bone Miner Res. 14 (4), 593-601 (1999).
- Yokochi, T. A new experimental murine model for lipopolysaccharide-mediated lethal shock with lung injury. Innate Immun. 18 (2), 364-370 (2012).
- Abe, T., Hajishengallis, G. Optimization of the ligature-induced periodontitis model in mice. J Immunol Methods. 394 (1-2), 49-54 (2013).
