MicroRNA (miRNA, miR) is essential in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNA in odontoblastic cell differentiation is still unclear. In this study, we examined the molecular mechanism of miR-27-mediated regulation of odontoblast differentiation in MDPC-23 mouse odontoblastic cells derived from mouse dental papilla cells. The results of the present study demonstrated that the miR-27 expression increases significantly during MDPC-23 odontoblastic cell differentiation. Furthermore, miR-27 up-regulation promotes the differentiation of MDPC-23 cells and accelerates mineralization without cell proliferation. The over-expression of miR-27 significantly increased the expression levels of Wnt1 mRNA and protein. In addition, the results of target gene prediction revealed that Wnt1 mRNA has an miR-27 binding site in its 3’UTR, and is increased by miR-27. These results suggested that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting Wnt1 signaling. Therefore, miR-27 is a critical odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in dental medicine.

MicroRNAs (miRNAs, miRs) are about 21-25 nucleotides in length and regulate mRNA translation by base pairing to partially complementary sites, predominantly in the 3’-untranslated region (3’-UTR) of the target mRNA. In this study, the expression profile of miRNAs was compared and analyzed for the establishment of miRNA-related odontoblast differentiation using MDPC-23 cells derived from mouse dental papilla cells. To determine the expression profile of miRNAs during the differentiation of MDPC-23 cells, we employed miRNA microarray analysis, quantitative real-time PCR (qRT-PCR) and Alizaline red-S staining. In the miRNA microarray analysis, 11 miRNAs were found to be up- or down-regulated more than 3-fold between day 0 (control) and day 5 of MDPC-23 cell differentiation among the 1,769 miRNAs examined. In qRT-PCR analysis, the expression levels of two of these molecules, miR-194 and miR-126, were increased and decreased in the control MDPC-23 cells compared with the MDPC-23 cells at day 5 of differentiation, respectively. Importantly, the overexpression of miR-194 significantly accelerated mineralization compared with the control cultures during the differentiation of MDPC-23 cells. These results suggest that the miR-194 augments MDPC-23 cell differentiation, and potently accelerates the mineralization process. Moreover, these in vitro results show that different miRNAs are deregulated during the differentiation of MDPC-23 cells, suggesting the involvement of these genes in the differentiation and mineralization of odontoblasts.

Cementum is a hard connective tissue, produced by cementoblasts during tooth root formation, which provides for the attachment of the periodontal ligament to the roots and surrounding alveolar bone. Establishment of this attachment is an important event in the regeneration of lost periodontal tissues. We examined whether or not odontoblast conditioned media(CM) have a regulatory influence on the differentiation and mineralization of cementoblasts(murine cementoblastic cell line, OCCM-30) in vitro. To identify the effect of odontoblast conditioned media and dentin non collagenous proteins (dNCPs) on cementoblast differentiation and mineralization, we treated CM and dNCPs to cementoblast then differentiated the cells for 14 days. To evaluate the formation of mineralized nodules alizarin-red S staining was performed at 0,4,7 and 14 days. Expression of cementum matrix genes was measured by RT-PCR. Mineralization of cementoblasts was accelerated with CM from odontoblastic MDPC-23 and OD-11. The expression of BSP, ALP, and OC mRNA in cementoblastic OCCM-30 cells was facilitated by the MDPC-23 and OD-11 cells. The extracted dNCPs had little influence on the proliferation, cell cycle modification, and chemotaxis of OCCM-30 cells. Although the dNCPs did not exhibit chemotactic activities for cementoblasts, the dNCPs promoted the differentiation and mineralization of cementoblasts. In conclusion, the dentin matrix protein, or the secreted products of odontoblast, facilitates cementoblast differentiation and mineralization. This represents a new approach and suggests another avenue for cementum regeneration.

Dentin is a mineralized tissue formed by odontoblasts that are differentiated from ectomesenchymal cells , The molecul ar mech anism of odontoblast diffe rentiation remains unclear, Amino acid transporters play an important role in s up plying nutri tion to normal a nd ca ncer cells including odntoblasts, and for cell proliferation , Amino acid transport system L is a maj or nutrient t ransport system responsible for the Na+' -independent transport o[ neutral amino acids incJuding several essentiaJ amino acids , The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2) , In this study, the expression pattern and role of amino acid transport system L were, therefore, investigated in the differentiation of MDPC-23 cells derived from mouse dental papilla celJs , To determi ne the expression Jevel o[ amino acid transport system L participating in intracelJ ular transport of amino acids in the differentiat ion 0 1' MDPC-23 cells, it was examined by RT-PCR, observation of cell morphoJogy‘ A1izaline red-S staining ancl uptake analysis after inclucing experimental differentiation in MDPC-23 cells The res ults were as follows , The LAT1 mRNA was expressed in the early stage of MDPC-23 cell differentiation , The expression leveJ was gradually increased by time course and it was decreased after the late stage, The LAT2 mRNA was not observed in the earJy stage of MDPC-23 cell differentiation, The LAT2 mRNA was expressed at the 11 days 0 1' MDPC-23 cell differentiation and the expression level was gradually decreased by time course, There was no changes in the expression level of 4F2hc mRNA, the cofactor of LAT1 and LAT2, during the differentiation of MDPC-23 cells , The expression of ON mRNA was graduaJJy decreased but the expression of ALP mRNA was increased during differentiation of MDPC-23 cells , The L-Ieucine uptake was increased by time cour se from the early stage to the 9 days in MDPC-23 cell differentiation , The amount of L-Ieucine uptake was maintained to the 11 and 14 days of MDPC-23 cell differentiation As the resul ts‘ it is considered that among neutral amino acid transport system L in differentiation of MDPC-23 cells , the LATl has a key role in cell proliferation in the early stage and middle stage of cell differentiation and the LAT2 has an important roJe in ceJJ differenti ation and mineralization in the Jate stage of cell differentiation for providing cells with neutral a mino acids incJuding several essentiaJ amino acids

The phylogeneticall y conserved nuclear factor 1 (NFI) gene fami ly encodes s ite-specific tra nscription factors essential for the development of a number of organ syst ems. There are four NFI genes in mamma ls (Nfi a , Nfib, Nfi c, and Nfix) and single NFI genes in Drosophila melanogaster, Caenorhabdi t is elegans, Anopheles spP. ‘ and other simpl e animals. It was reported that Nfia-defici ent mice exhi bit agenesis of the corpus call osum and other forebrain defects , wher eas Nfib-defi cient mice possess unique defects in lung ma turation and fo rebrain defect. Recently, it was also found that Nfic-defi cient mice exhibit agenesis of mo l ar서 roots and severe incisor defects. In the present study, we investigat ed the possible role of NFI-C in odon toblast diffe rent ia tion and root dentin formation using the innovative and invalua ble Nfic knockout mice model Nfi c-defi cient mice showed a berrant odontoblast differentiation and consequentl y abnormal dentin formation, while other t issues/organs in the body including ameloblasts of the enamel organ a ppeared to be unaffec ted and normal One of the most st r iking changes observed in these aberrant odontoblasts was t he absence of in tercellular junctions beLween them, r esulting in di ssociation of the cells and loss of th eir cellular polarity a nd organi zation. Surprisingly, these cells became trapped in dentin-like minerali zed t issue and thus their overa ll morphology r esembled osteoblasts and os t eocyt es. There was also an increased apoptotic activity in Nfic-deficient mice. These findings strongly s uggest ed that NFI -C plays a key role in odon tob last differentiation and survival in a cell type-specific manner.

Nuclear factor 1 (NFI) was discovered as a protein required for adenovirus DNA replication in vitro, but it is now clear that NFI protein plays an important role in the expression of many cellular genes. NFI-C null mice demonstrated aberrant odontoblast differentiation, abnormal dentin formation, and thus molar lacking roots while other tissues/or gans in the body, including ameloblasts appear to be unaffected and normal. However, little is known about the mechanism of NFI -C function in odontoblast differentiation and dentin formation. In this study, in order to elucidate the molecular mechanisms of odntoblast differentiation, we examined morphological characteristics of the aberrant odontoblast in NFI-C null mice. we also evaluate the expression of dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) mRNAs in the MDPC-23 cells by northern analysis after over-expression and inactiγation of NFI -C into mouse MDPC-23 cells Odontoblasts of the NFI-C null mouse were round in shape, lost their polarity, organized as a sheet of cells, and trapped in osteodentin-like mineralized tissue. Abnormal odontoblasts of NFI-C null mouse revealed the absence of an intercellular junctional complex known as the t erminal webs. MDPC-23 cells started to express DSPP mRNA beginning from the postnatal day of 14 and showed a steady increase as differentiating into odontoblasts. Over-expression of NFI -C increased the expression of DSPP mRNA. Inactivation of NFI - C induced BSP mRNA expression. These results suggest that NFI-C plays an important role in odontoblast differentiation in a cell-type specific manner and thus in dentin formation