Ca_v1.2 regulated odontogenic differentiation of NG2⁺ pericytes during pulp injury

NG2⁺ pericytes, as the possible precursor cells of mesenchymal stem cells (MSCs), have drawn attention due to their ability to differentiate into odontoblasts. Ca_v1.2 is involved in the differentiation process of stem cells, but its role in the differentiation of NG2⁺ pericytes is not clear. The aim of the present study was to examine the role of Ca_v1.2 in the differentiation of NG2⁺ pericytes into odontoblasts. NG2⁺ pericytes were obtained from human dental pulp cells by magnetic-activated cell sorting. During the odontogenic differentiation of NG2⁺ pericytes, the effects of the Ca_v1.2 inhibitors, nimodipine and Ca_v1.2 knockdown shRNA, were analyzed by real-time polymerase chain reaction and alizarin red staining. NG2CreERT2/Rosa26-GFP lineage-tracing mice were established to further investigate the roles of NG2⁺ pericytes and Ca_v1.2 in incisor self-repair after injury in vivo. At 10 min, 1 day, and 3 days after pulp injuries in transgenic mice, NG2-GFP⁺ and Ca_v1.2 immunofluorescence co-staining was performed on the incisors. Nimodipine treatment and Ca_v1.2 knockdown showed similar inhibition of calcium nodule formation and mRNA levels of osteogenic markers (DSPP, DMP1, and Runx2, p < 0.05). NG2⁺ pericytes migrated from their inherent perivascular location to the odontoblast layers after pulp injury. Ca_v1.2 showed a similar response pattern as NG2⁺ pericytes and gradually returned to normal levels. In addition, many co-stained areas of Ca_v1.2 and NG2⁺ pericytes, both near the perivascular and odontoblast layers, were observed. These results indicate that Ca_v1.2 played a vital role in the odontogenic differentiation of NG2⁺ pericytes, and that it might be closely linked to the NG2⁺ pericytes-mediated repair of dental pulp injury in vivo.