Mechanism of mTORC1-regulation in Odontoblast Proliferation and Mineralization In vivo

Objectives: The objectives were to: (1) investigate the mechanism of mTORC1 regulating odontoblastic proliferation and mineralization by activating and inhibiting mTORC1 of odontoblasts in vitro and in vivo; and (2) control the significant protein or mRNA to provide new methods to promote dentin formation and evaluate the etiology of dentin regeneration.
Methods: In vitro, MDPC23 cells were treated with Rapamycin (10 nmol/L) and transfected with shRNA-TSC1 to inhibit or activate mTORC1, respectively. CCK8 assay, flow cytometry, Alizarin red staining, ALP staining, qRT-PCR and Western-blot were performed. In vivo, DMP1-Cre and TSC1^flox/flox mice were used to knockout TSC1 to activate mTORC1 in odontoblasts. Mandibular molars of 1-month-old and 3-month-old wildtype mice and knockout mice were harvested. HE-staining, immunofluorescence and micro-CT scans were used to examine the changes of dentin in vivo.
Results: HE-staining showed that the thickness of pre-dentin and dentin of TSC1- knockout mice mandibular molar was thicker than that of the wild type. The mineralization-related protein (ALP, Runx2, Col1) expressions were up-regulated (p<0.05). The micro-CT results showed that the BV/TV (0.67±0.03) in TSC1-knockout mice was greater than that of wildtype dentin (0.56±0.01). Dentin thickness was (0.36±0.00) in TSC1-knockout mice than wildtype mice (0.24±0.00) (p<0.05). When mTORC1 activity was activated in shRNA-TSC1 group, cell proliferation (2.14±0.01) was up-regulated than control (1.61±0.01). The proportion of cells in S-phase in treated group (30.56±0.23%) was higher than that in control group (21.38±0.45%) (p<0.05). The expression of mineralization-related genes ALP (6.33 ± 0.91), Runx2 (3.67±0.74), DSPP (4.49±0.30), Col1(2.28±0.25) and proteins (ALP, Runx2, Col1) (p<0.05) in shRNA-TSC1 group were up-regulated, compared to those in shRNA-NC group.
Conclusions: The mTORC1 was demonstrated to up-regulate the proliferation and mineralization of odontoblasts. This mechanism is promising to have clinical applications to enhance the formation of the dentin-pulp complex and promote the regeneration of teeth.