Effect Of Mechanical Injury On The Expression Of Notch Signaling And Its Relationship With Apoptosis In Odontoblasts Of Mouse Incisor

The Notch signaling pathway is an evolutionarily conserved signaling mechanism that enables adjacent cells to adopt different fates and participates in the molecular cascade of events governing embryonic development, organ formation, tissue regeneration and repair. Signals exchanged through interaction of Notch receptors and ligands between neighboring cells influence differentiation, proliferation, and apoptotic events. Many studies data suggest that Notch signaling is involved in embryonic tooth development in rodents and humans but absent from adult dental tissues. However, Notch signaling is reactivated and mainly expressed in mesenchymal cells in the repair process of pulp injury. In addition, Notch signaling is also expressed in odontoblasts. Notch up-regulation in pulp cells represents one of the earlier molecular events in the process of dental tissue repair and may be related to promote the repair of dental pulp injury. Odontoblasts are important executive cells of the third dentinogenesis(i.e. reactionary or reparative dentinogenesis) in defense and reparative response. Odontoblasts apoptosis could be induced by bacteria and endotoxins related to the carious lesion, as well as injury. Once apoptosis occurs in odontoblasts, mesenchymal cells migrate to the injury site and differentiate into new odontoblasts, which form reparative dentin and promote repair of dental pulp injury.Mouse incisors grow continuously throughout the life of the animal, which makes it become a good model to study the mechanism of tooth repair of injury. Maintaining the function of tissue regeneration depends on the presence of stem cells. In mouse incisor, stem cells, including mesenchymal stem cells and cervical loop epithelial stem cells, are located in the top of mouse incisor root and ensure the continuous regeneration of incisor. It is found that Notch signaling is expressed in the cervical loop of mouse incisor. So, whether Notch signaling is involved in the regeneration process of mouse incisor? Are there changes in the expression of Notch signaling and the number of apoptotic odontoblasts in the repair process of incisor mechanical injury? And is there relationship between Notch signaling and odontoblasts apoptosis in this process? All of these have not yet been clearly known. Therefore, through establishing experimental animal model of mechanical injury in mouse incisor, the expression of Notch signaling in enamel organ cells and odontoblasts, and apoptosis in odontoblasts located in pulp were observed respectively. Then a comparative observation was made between the expression of Notch signaling and two apoptosis associated proteins to further understand the role of Notch signaling in the repair process of mouse tooth injury. It was hoped that the study can provide new ideas for the repair of tooth injury. The main results are as follows: 1. The effect of mechanical injury on the expression of Notch signaling in enamelorgan cells of mouse incisorEstablishing experimental animal model of mechanical injury by grinding off 1/2 crown of mice mandibular incisor, then incisor area was cut off on three days, five days and seven days after injury. Subsequently, paraffin tissue sections were prepared. The expression of Notch1 and Notch2 in cells of enamel organ was observed by immunohistochemical staining, and the results suggest that Notch signaling in enamel organ cells was activated and showed dynamic changes after mechanical injury. In control group, Notch1 and Notch2 were absent in ameloblasts and weakly expressed in stratum intermedium and stellate reticulum cells. Three and five days after injury, Notch1 and Notch2 were expressed in ameloblasts, and their expressions in stratum intermedium and stellate reticulum cells both increased. However, the expression of Notch2 was more obvious; Seven days after injury, the expression of Notch1 and Notch2 decreased and became weak in all cell layers. The above results suggested that Notch signaling may be involved in the function regulation of enamel organ cells, and the activation of Notch signaling may be related to the repair of incisor injury. 2. The effect of mechanical injury on the expression of Notch signaling inodontoblasts of mouse incisorEstablishing experimental animal model of mechanical injury by grinding off 1/2 crown and cavity preparation in the neck of distal face of mouse mandibular incisor, then incisor area was cut off on one day, three days, five days and seven days after injury. Subsequently, paraffin tissue sections were prepared. The expression of Notch1 and Notch2 in odontoblasts was observed by immunohistochemical staining, and the results suggest that Notch signaling in odontoblasts can be activated by mechanical injury and showed dynamic changes: one day after injury, the expression of Notch1 and Notch2 was positive; three days after injury, Notch1 and Notch2 were expressed quite intensively; five days after injury, the expression of Notch1 and Notch2 became weak and was positive; seven days after injury, Notch1 and Notch2 were weakly expressed. In the control group, Notch1 and Notch2 were weakly expressed. The above results suggested that Notch signaling may be involved in the function regulation of odontoblasts, and the activation of Notch signaling may have a relationship with the repair of tooth pulp injury. 3. The comparative study of Notch signaling activation and odontoblasts apoptosisUsing paraffin tissue sections prepared in the second experiment for TUNEL staining to observe odontoblasts apoptosis, we found that mechanical injury can induce massive apoptosis in dental pulp nearby the injury, apoptosis in odontoblasts was quite obvious one day after injury, and the apoptotic cells decreased gradually from three to seven days after injury. Few apoptotic events were observed in control group. Taking the results compared with that of experiment two, we found that changes on the number of apoptotic odontoblasts were similar to the dynamic changes on the expression of Notch signaling, and their different was that they reached the peak at different time. So we speculated that there may be some correlation between the activation of Notch signaling and the regulation of odontoblasts apoptosis.To prove the speculation, immunohistochemical staining was used to further observe the expression of Notch1, Notch2, anti-apoptotic protein Bcl-2 and apoptotic protein cleaved-Caspase-3 in odontoblasts. According to the staining results, the expression of Notch1 and Notch2 was weak in control group and gradually became strong from one day to three days after injury. Bcl-2 was not expressed both in control group and on one day after injury, but was weakly expressed on three days after injury. The expression of cleaved-Caspase-3 was weakly positive in all groups. Cells expressing cleaved-Caspase-3 were only a little in control group, but increased obviously on one day after injury and reduced remarkably on three days after injury. The above results suggested that the activation of Notch signaling and Bcl-2 may be involved in the regulation of odontoblasts apoptosis.In conclusion, 1. Notch signaling in enamel organ cells was activated after mechanical injury and showed dynamic changes, suggesting that Notch signaling may be involved in the regulation of cells function in enamel organ, and thus regulate the repair of mouse incisor injury. 2. Notch signaling was weakly expressed in odontoblasts of normal mouse incisor and its expression was up-regulated rapidly after mechanical injury, suggesting that Notch signaling may be involved in the function regulation of odontoblasts, and thus play a role in the repair of pulp injury. 3. Mechanical injury can lead to apoptosis in odontoblasts and a similar change trend existed in the number of apoptotic odontoblasts and the expression of Notch signaling, suggesting that some correlation may exist between Notch signaling and odontoblasts apoptosis regulation. According to the results of further research, we speculated that Notch signaling may be associated with Bcl-2, and they may participate to the regulation of odontoblasts apoptosis. However, further researches are needed to confirm the speculation.