Dental pulp stem cells (DPSCs), as one type of mesenchymal stem cells (MSCs), have the capability of self-renewal and multipotency to differentiate into several cell lineages, including osteogenesis, odontoblasts, chondrogenesis, neurogenesis, and adipogenesis. It has found that tumor necrosis factor-α (TNF-α) can promote osteogenic differentiation of human DPSCs in our previous studies. Other experimentation revealed that signal transducer and activator of transcription 3 (STAT3) underwent a rapid activation both in osteogenesis and inflammation microenvironment of MSCs in vitro. MicroRNAs (miRNAs or miRs) have been proved in previous studies to regulate MSCs differentiation in vitro. In this study, we identified miR-21 as a key miRNA contributed the functional axis of odontoblast differentiation induced by STAT3. It is observed that the expression of miR-21 and STAT3 increased gradually in low concentration (1-10 ng/mL) of TNF-α, while they were suppressed in high concentration (50-100 ng/mL). The upregulation of miR-21 may facilitate the odontoblast differentiation of DPSCs coordinating with STAT3. SiSTAT3 or treated by the inhibitor of STAT3, cucurbitacin I (Cuc I), significantly increased primary miR-21 expression along with decreased mature miR-21 expression. Meanwhile, the inhibition of miR-21 (anti-miR-21) decreased the activation of STAT3 as well as suppressed the marker proteins of odontoblast differentiation. The results revealed a new function of miR-21, suggesting that miR-21/STAT3 signal may act as a modulator within a complex network of factors to regulate odontoblast differentiation of human DPSCs. It may provide a novel therapeutic strategy to regulate the odontoblast differentiation of DPSCs.

Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell proliferation, apoptosis, and differentiation. The binding of NRP1 to Sema3A stimulates osteoblast differentiation through the classical Wnt/β-catenin pathway. However, the functions of NRP1 in dental pulp stem cells (DPSCs) are not clear. The aim of our study was to investigate how NRP1 controlled odontoblast differentiation in DPSCs and clarified the underlying mechanisms. NRP1 expression was increased in time-dependent manner along with cell odontoblast differentiation. Overexpression of NRP1 upregulated dentin matrix protein-1, dentin sialophosphoprotein, alkaline phosphatase protein level, and mineralization in DPSCs, while knockdown of NRP1 induced the opposite effects. SiNRP1 similar to DKK1 availably blocked classical Wnt/β-catenin signaling and odontoblast differentiation. In summary, NRP1, as a promoter of odontoblast differentiation, regulates DPSCs via the classical Wnt/β-catenin pathway.

The infiltration of adipocytes in osteoporotic patients' bone marrow suggests an important regulatory function of bone marrow fat on the development of aged bone. Therefore, we have examined the effects of adipocytes derived from bone mesenchymal stem cell (MSC) on osteoblast differentiation using two different co-culture modes (direct mode and indirect mode). Alkaline phosphatase (ALP)-positive areas and mineralized areas of MSC-derived osteoblasts decrease similarly in the two co-culture modes as the amount of MSC-derived adipocytes increases, suggesting that the crosstalk between adipocytes and osteoblasts may be mainly through secretory factors in the medium. To further understand the molecular mechanisms, both mRNA and protein expressions in osteoblasts in the lower layer of the indirect mode were analyzed, leading to identification of 12 differential genes/proteins. Among them, S100A6 and calreticulin are possibly related to bone formation. S100A6 was down-regulated and calreticulin was up-regulated as MSC-derived adipocytes increased. Similarly, differential expression of these proteins was also observed in bone tissue slides from young (1-month-old) and old (6-month-old) mice. The expression level of β-catenin in osteoblasts of bone tissues was lower in 6-month-old mice compared to 1-month-old mice. Total TGF-β analyzed with antibody-based protein microarray and active TGF-β analyzed with ELISA in the co-cultured cell medium increased consistently as the amount of adipocytes increased. Taken together, our results suggest that MSC-derived adipocytes may regulate osteoblast differentiation in the aged bone through TGF-β-mediated canonical Wnt signaling.

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Abnormal odontoblast differentiation of dental pulp stem cells (DPSCs) caused by inflammation is closely related to the development of dental caries. Neuropilin-1 (NRP1) is one of the members of neuropilin family. It can combine with disparate ligands involved in regulating cell differentiation. FYN belongs to the protein-tyrosine kinase family, which has been implicated in the control of cell growth, and the effect can be further strengthened by inflammatory factors. In our studies, we verified that NRP1 can form complexes with FYN and have the correlation changes in odontoblast differentiation of DPSCs. Therefore, we surmise that in the progress of dental caries, NRP1 interacts with FYN, by expanding inflammation and inhibition of odontoblast differentiation of DPSCs through nuclear factor kappa B (NF-κB) signaling pathway. In this subject, we first investigated the expression and interaction of NRP1 and FYN in DPSCs. And then, we researched the effect of this complex controlling downstream signal pathway in normal or inflammation stimulated DPSCs. Finally, we analyzed the relationship between this role and odontoblast differentiation of DPSCs. This research will provide the molecular mechanism of inflammation factors of dental caries through activating NF-κB signal regulating odontoblast differentiation in DPSCs for finding new potential drug targets for the clinical treatment of dental caries.