The Study Of The Role Of Notch Signaling Pathway During Dental Pulp Repair

Tooth organogenesis and dental pulp repair are both precisely regulated by various signaling molecules. According to previous studies, Notch signaling, as a highly conserved mechanism throughout evolution, controls cell proliferation, differentiation, apoptosis through cell-cell interactions, then determines the cell fate during embryonic development and tissue regeneration. In this paper, the role of Notch signaling during dental pulp repair was investigated in vivo and in vitro. First, we use lipopolysaccharide (LPS) derived from cariogenic bacteria to stimulate Mouse odontoblasts-like cells (MDPC-23), and then the effect of LPS on Notch-related genes expression in MDPC-23 and the possible role of Notch signaling pathway in odontoblasts differentiation during dental pulp repair were discussed. Second, we established an experimental animal model of injury-induced pulpitis, and then the time-sequenced alteration of the expression of Notch receptor was observed. Third, we investigated the effects of Notch signaling during the differentiation of odontoblast-like cells by inhibiting Notch signaling pathway.Part 1 Analysis of the expression of Notch-related genes in the dental pulp cell injury model in vitro(1) Establishment of the dental pulp cell injury model.Mouse odontoblasts-like cells (MDPC-23) were exposed to 0～1μg/ ml Escherichia coli LPS. The cell vitality was analyzed by MTT assay. Results showed that, LPS showed mainly toxic effect when the concentration was higher than 1μg/ ml after 72h. After 24h, 10ng/ml～5μg/ ml LPS could obviously accelerate the proliferation of MDPC-23 (P<0.05) with 100ng/ml group had the best effect (P<0.01). After 48h, 10ng/ml～1μg/ml groups have no difference with the control group (P>0.05), while 5μg/ ml LPS expressed an obvious inhibition effect on the cell vitality. After 72h, all the groups of LPS could inhibit cell proliferation, 1μg/ml group and 5μg/ml group obviously depressed cell proliferation (P<0.05). According to the MTT result, we confirmed the appropriate concentration of LPS for this study.(2) Effects of lipopolysaccharide (LPS) on the expression of Notch-related genes in odontoblast-like cell MDPC-23Real-time PCR results showed that, Notch1 expression remained unchanged or showed slightly up-regulation in control group. After adding 10ng/ml～1μg/ml LPS, Notch1 expression showed up-regulation (0～3d) but down-regulation from 3d to 5d. Jagged1 expression showed almost the same as Notch1, it showed down-regulation in control group. After addition of 100ng/ml～1μg/ml LPS, it was up-regulated (1～3d)) but showed down-regulation (3～5d). Delta1 expression seemed opposite to Notch1, it exhibited down-regulation in control group. After adding 10ng/ml～1μg/ml LPS, Delta1 showed down-regulation (0～ 3d) and striking up-regulation (3～5d). Hes1 firstly exhibited slightly up-regulation then remained unchanged. After adding 10ng/ml～100ng/ml LPS, it showed up-regulation during 0～5d.The results verified that the expression of Notch-related genes in MDPC-23 could be changed by LPS. The findings are consistent with the notion that Notch signaling plays an important role during dental pulp repair.Part2 Study on the time-sequenced alteration of immunolocalization of Notch2 expression in rat pulp injuryIn this study, we established experimental animal models of simple injury-induced/LPS-induced pulpitis and then the expression pattern of Notch2 was discussed. For perforation group (simple injury group), 3d post-surgery, weak positive staining of Notch2 was observed in pulp cells but not in odontoblasts. 5d post-surgery, strong Notch2 reactivity was found in subodontoblasts. 7d post-surgery, stronger positive staining was found in odontoblasts. 14d post-surgery, weak Notch2 staining was seen in pulp cells but was absent from odontoblasts. For LPS group, Notch2 staining was much stronger than that of simple injury group from 3d to 7d post-surgery. Notch2 immunoreactivity was completely absent in intact rat dental pulp.The results revealed that, simple injury can induce the expression of Notch2 in odontoblasts and LPS can enhance the expression. Notch signaling pathway plays an important role during pulp repair.Part3 Effects of DAPT on the differentiation potential of MDPC-23.To investigate the effects of Notch signaling during the differentiation of odontoblast-like cells, mouse odontoblasts-like cells (MDPC-23) were cultured in mineralization induction medium (5% serum) with different concentrations DAPT. Then the effects of DAPT were measured by ALP assay and the induction of mineralized nodules. Results showed that, after Notch signaling was inhibited, the expression of ALP and the formation of mineralized nodules of MDPC-23 were both down-regulated. It may suggest that Notch signaling could promote the differentiation of MDPC-23.In summary, the diversity of pulp cells during odontogenesis is regulated by"lateral inhibition"mediated by Notch signaling pathway. When apoptosis is induced in odontoblasts after pulp injury, pulp mesenchyme cells begin to differentiate to odontoblastic cells and then the reparative dentin matrix is secreted. Notch signaling pathway can be activated during dental pulp repair process, this phenomenon reflects a molecular reflection mechanism in the early stage of pulp repair. The current study indicated that, Notch1 and Jagged1 can negatively regulate the differentiation of MDPC-23 in vitro, while Delta1 has a positive regulation effects. Meanwhile, inhibition of Notch signaling can down-regulate the differentiation of MDPC-23. In vivo study showed that, Notch2 exhibited a time-sequenced alteration expression pattern in odontoblastic cells, which reflected a protective reflection during pulp repair. These observations are consistent with the notion that Notch reactivation plays an important role in balancing cell proliferation, differentiation and apoptosis during dental pulp repair process.