| 研究課題/領域番号 |
22K21011
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| 研究種目 |
研究活動スタート支援
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| 配分区分 | 基金 |
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| 審査区分 |
0907:口腔科学およびその関連分野
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| 研究機関 | 新潟大学 |
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研究代表者 |
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| 研究期間 (年度) |
2022-08-31 – 2024-03-31
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| 研究課題ステータス |
交付 (2022年度)
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| 配分額 *注記 |
2,860千円 (直接経費: 2,200千円、間接経費: 660千円)
2023年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
2022年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
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| キーワード | dental pulp / odontoblast / immune cells / injuries / CpG-ODNs / dendritic cells |
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| 研究開始時の研究の概要 |
The complex mechanisms of dental tissue natural self-repair and regeneration remain to be clarified. In this study we aim to investigate the cellular cross-talk among odontoblasts, pulpal stem/progenitor cells, and immune cells induced by the effect of a novel and potent immunomodulator (CpG-ODNs) following external injuries to the tooth. Considering the future results, we will propose a new treatment called "dendritic cell activation therapy", aimed to improve the prognosis of severely injured teeth towards their subsequent regeneration and further translation to the clinical setting.
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| 研究実績の概要 |
Using an in vivo model for tooth replantation in mice, we have assessed the effects of two types of synthetic CpG ODNs solutions: Type A (D35, MW:6327.33) and Type B (K3, MW:6349.37) on the healing process of the afflicted dental pulp. The penetration of these solutions in the pulpal tissue was confirmed by using an FITC-conjugated-CpG ODN solution one hour after replantation and 24 hours after replantation without variations on its intensity. For the assessment of the most suitable condition, we tested three different concentrations: 10 mg/ml (1.57 mM), 0.63 mg/ml (0.1 mM) and 5 mg / ml (0.8 mM) for each type of CpG ODN solution. Quantitative analysis of Nestin immunohistochemistry showed that 0.1 mM Type-A and -B CpG ODNs solutions favored the healing of the pulpal tissue two weeks after operation, specially Type B. Conversely, the 1.25 mM of both CpG ODNs induced the highest rate of Nestin-negative hard tissue areas in the dental pulp, suggesting an increase in bone-like tissue formation. The treatment with low-concentration CpG-ODNs solution drastically reduced the rate of proliferative cells in the dental pulp, which is reverted at Week 2, when compared to control groups. Preliminary in vitro data obtained from pulp samples following tooth replantation showed that odontoblast differentiation (Nestin mRNA) is sustainedly induced by CpG ODN type B from days 1-5. Bone-related marker (Osteocalcin mRNA) also increased its expression at day 3 following replantation.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
At present our study have finished the main in vivo stage, and it is currently moving forward to collect the in vitro data to support our main hypothesis. The most suitable conditions for the use of CpG ODN solutions on the injured dental pulp have already been clarified to assess the dynamics of the dental pulp cells at different time points following intentionally delayed tooth replantation. Complimentary in vivo data using doxycycline-induced TetOP-H2B-GFP reporter and WT mice are scheduled following the original plan, however it needs some time to have the adequate number of experimental animals. Although with some difficulties, we have succeeded in establishing a primary cell culture protocol for the assessment of the effects CpG ODNs solutions directly on the dental pulp cells in order to analyze the variations in the gene expression between experimental and control groups and also considering additional time points. The first batch of samples were already collected and are pending to be analyzed by qPRC in the next weeks. Finally, we have already set up the immunohistochemistry protocol of F4/80 and CD206 antibodies as pan macrophage marker and activated M2 macrophages, respectively, which will be useful tools to evaluate the relationship between odontoblast-lineage cells and immune cells following traumatic injuries to the pulpal tissue. In addition, our current results have been presented at the 22nd Annual Meeting of the Japanese Society of Regenerative Medicine in March 2023.
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| 今後の研究の推進方策 |
The first stage of the project is soon to be completed by submitting a manuscript summarizing the findings of our in vivo data and the dynamics of the dental pulp cells during the healing process following severe injuries. We will further perform the intentionally-delayed tooth replantation model using doxycycline-induced TetOP-H2B-GFP reporter and WT (C57BL/6J) mice to assess the differentiation process of odontoblast-like cells by tracking the interaction between pulpal stem/progenitor cells and dendritic and other immune cells. We are ready to carry out immunohistochemistry for the pan-macrophage marker f4/80 and activated M2 macrophage marker CD206 on the samples obtained from this experiment. The next stage of our project is currently on going. Additional data such as in vitro analysis are necessary to understand the biological mechanisms eliciting the pulpal responses caused by these immunomodulatory solutions. At present, the collected samples from cell culture experiments which were treated with CpG ODN solutions at different time points are pending to be evaluated by qPCR. Also, we will analyze in vitro the variations of the gene expression on samples collected following in vivo experiments using the same injury model to compare those results with those from cell culture. The data collected from the experiments of the second stage will be summarized on a second manuscript expected to be submitted by the first months of 2024.
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