High-temperature Requirement Protein A1 Regulates Odontoblastic Differentiation of Dental Pulp Cells via the Transforming Growth Factor Beta 1/Smad Signaling Pathway.

Dentinogenesis includes odontoblast differentiation and extracellular matrix maturation as well as dentin mineralization. It is regulated by numerous molecules. High-temperature requirement protein A1 (HtrA1) plays crucial roles in bone mineralization and development and is closely associated with the transforming growth factor beta (TGF-β) signal in osteogenesis differentiation. Simultaneously, the TGF-β1/small mother against decapentaplegic (Smad) signaling pathway is an important signaling pathway in various physiological processes and as a downstream regulation factor of HtrA1. However, the role of HtrA1 and its relationship with the TGF-β1/Smad signaling pathway in dentin mineralization is unknown. We detected the role of HtrA1 and its relationship with the TGF-β1/Smad signaling pathway in odontoblastic differentiation of human dental pulp cells (hDPCs) in this study. First, hDPCs were cultured in mineralized medium, and odontoblastic differentiation was confirmed by investigating mineralized nodule formation, alkaline phosphatase (ALP) activity, and the expression of mineral-associated genes, including ALP, collagen I, and dentin sialophosphoprotein. Then, the expression of HtrA1 and TGF-β1/Smad in hDPCs was investigated in hDPCs during mineralized induction. After HtrA1 knockdown by lentivirus, the mineralized nodule formation, ALP activity, and expression of mineral-associated genes and TGF-β1/Smad genes were investigated to confirm the effect of HtrA1 on odontoblastic differentiation and its relationship with the TGF-β1/Smad signaling pathway. The expression of HtrA1 and TGF-β1 was increased during odontoblastic differentiation of hDPCs along with the messenger RNA expression of downstream factors of the TGF-β1/Smad signaling pathway. In addition, lentivirus-mediated HtrA1 knockdown inhibited the process of mineralization and the expression of HtrA1 and TGF-β1/Smad genes. These findings suggest that HtrA1 might positively regulate odontoblastic differentiation of hDPCs through activation of the TGF-β1/Smad signaling pathway.

Li XY ，Ban GF ，Al-Shameri B ，He X ，Liang DZ ，Chen WX ... -  《-》

Combination of Mineral Trioxide Aggregate and Platelet-rich Fibrin Promotes the Odontoblastic Differentiation and Mineralization of Human Dental Pulp Cells via BMP/Smad Signaling Pathway.

Recent reports have shown that the combined use of platelet-rich fibrin (PRF), an autologous fibrin matrix, and mineral trioxide aggregate (MTA) as root filling material is beneficial for the endodontic management of an open apex. However, the potential of the combination of MTA and PRF as an odontogenic inducer in human dental pulp cells (HDPCs) in vitro has not yet been studied. The purpose of this study was to evaluate the effect of the combination of MTA and PRF on odontoblastic maturation in HDPCs. HDPCs extracted from third molars were directly cultured with MTA and PRF extract (PRFe). Odontoblastic differentiation of HDPCs was evaluated by measuring the alkaline phosphatase (ALP) activity, and the expression of odontogenesis-related genes was detected using reverse-transcription polymerase chain reaction or Western blot. Mineralization formation was assessed by alizarin red staining. HDPCs treated with MTA and PRFe significantly up-regulated the expression of dentin sialoprotein and dentin matrix protein-1 and enhanced ALP activity and mineralization compared with those with MTA or PRFe treatment alone. In addition, the combination of MTA and PRFe induced the activation of bone morphogenic proteins (BMP)/Smad, whereas LDN193189, the bone morphogenic protein inhibitor, attenuated dentin sialophosphoprotein and dentin matrix protein-1 expression, ALP activity, and mineralization enhanced by MTA and PRFe treatment. This study shows that the combination of MTA and PRF has a synergistic effect on the stimulation of odontoblastic differentiation of HDPCs via the modulation of the BMP/Smad signaling pathway.

Woo SM ，Kim WJ ，Lim HS ，Choi NK ，Kim SH ，Kim SM ，Jung JY ... -  《-》

The effect of SIRT6 on the odontoblastic potential of human dental pulp cells.

The aim of this study was to investigate whether SIRT6 is expressed in human dental pulp as well as the effect of SIRT6 on proliferation and odontoblastic differentiation of human dental pulp cells (HDPCs). Immunohistochemical and immunocytochemical assays were used to detect the expression of SIRT6 in human dental pulp tissue and HDPCs. To determine the effect of SIRT6 on odontoblast differentiation, HDPCs with loss (HDPCs SIRT6 knockdown) and gain (HDPCs SIRT6 overexpression) of SIRT6 function were developed, and their proliferation ability was examined. Odontogenic differentiation of HDPCs was determined by alkaline phosphatase (ALP) activity, ALP-positive cell staining, alizarin red staining, and von Kossa staining. Mineralization-related genes, including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1, were determined by real-time quantitative polymerase chain reaction. Western blot analysis was performed to detect the expression of DSPP protein. SIRT6 was found in the dental pulp tissue and HDPCs. SIRT6 knockdown decreased ALP activity in HDPCs; calcium nodule formation ability; and the expression of mineralization-related genes such as ALP, DSPP, and DMP1, whereas these were increased with the overexpression of SIRT6. SIRT6 is expressed in human dental pulp and participates in the odontoblast differentiation of HDPCs.

Sun HL ，Wu YR ，Huang C ，Wang JW ，Fu DJ ，Liu YC ... -  《-》

The Effects of M1 and M2 Macrophages on Odontogenic Differentiation of Human Dental Pulp Cells.

M1 (classically activated) and M2 (alternatively activated) macrophages are known to play primary roles in inflammation and tissue regeneration. To investigate the role of macrophages in dentin regeneration, this study examined the effects of M1 and M2 macrophages on the odontogenic/osteogenic differentiation of human dental pulp cells (HDPCs) using the conditioned media (CM) of the activated human monocyte cell line THP-1. M1 and M2 macrophages were induced by lipopolysaccharide/interferon-γ and interleukin-4, respectively, and the phenotypes were confirmed by flow cytometry. Macrophage CM was prepared at 2-day intervals for a period of 6 days, which included the first 2 days of activation. The CM obtained on days 4 (M1CM-4 day and M2CM-4 day) and 6 (M1CM-6 day and M2CM-6 day) were tested for their ability to promote the alkaline phosphatase (ALP) activity of HDPCs. M2CM-4 day was also examined for its effects on the messenger RNA expression of dentin sialophosphoprotein and osteocalcin genes and the matrix mineralization of HDPCs. Tumor necrosis factor alpha and transforming growth factor beta 1 (TGF-β1) in M1CM and M2CM, respectively, were quantified by an enzyme-linked immunosorbent assay. To verify the role of TGF-β1, M2CM-4 day was pretreated by a TGF-β blocking antibody and was examined for its effect on the ALP activity of HDPCs. M2CM-4 day and M2CM-6 day enhanced the ALP activity of HDPCs (P < .05). Furthermore, M2CM-4 day promoted the messenger RNA expression of the dentin sialophosphoprotein gene and matrix mineralization (P < .05), whereas M1CM did not affect ALP activity. The enzyme-linked immunosorbent assay detected large amounts of TGF-β1 in M2CM-4 day and M2CM-6 day. The TGF-β blocking antibody suppressed the ALP-enhancing activity of M2CM-4 day (P < .05). Furthermore, the same amount of TGF-β1 as in M2CM-4 day increased ALP activity to a similar level as M2CM-4day-treated HDPCs. The CM of M2 macrophages enhanced the odontogenic/osteogenic differentiation of HDPCs. M1CM did not affect the ALP activity of HDPCs at least in the absence of M1-type inducers. The effects of M2CM on HDPCs were likely caused by TGF-β1. Therefore, M2 macrophages are expected to support dentin regeneration in dental pulps.

Park HC ，Quan H ，Zhu T ，Kim Y ，Kim B ，Yang HC ... -  《-》

Role of Protein Phosphatase 1 in Angiogenesis and Odontoblastic Differentiation of Human Dental Pulp Cells.

The aims of this study were to examine the immunolocalization of protein phosphatase 1 (PP1) in developing mouse pulp tissue and to explore the role of PP1 in odontoblastic differentiation and in vitro angiogenesis in human dental pulp cells (HDPCs). Immunolocalization of PP1 was assessed in developing mouse pulp tissue. Odontogenic differentiation was examined by alkaline phosphatase activity, alizarin red staining, and reverse transcriptase polymerase chain reaction. Angiogenesis was evaluated by endothelial cell migration and capillary tube formation. Signaling pathways were analyzed by Western blotting and confocal immunofluorescence. PP1 expression was detected in preodontoblasts, odontoblasts, dental pulp cells, and endothelial cells within pulp tissue during the crown formed, root formation, and root completion stages. PP1 messenger RNA (mRNA) and protein levels were up-regulated at the late mineralization stage during odontogenic differentiation of HDPCs. The PP1 activator C2 ceramide increased alkaline phosphatase activity, mineralized nodule formation, and mRNA expression of dentin matrix protein 1 and dentin sialophosphoprotein. In contrast, knockdown by PP1 small interfering RNA inhibited odontoblastic differentiation. Moreover, PP1 activator up-regulated mRNA expression of angiogenic genes in HDPCs and increased the migration and capillary tube formation of endothelial cells, whereas PP1 small interfering RNA showed opposite effects. C2 ceramide increased levels of bone morphogenetic protein 2, phosphorylation of Smad 1/5/8, and mRNA expression of runt-related transcription factor 2 and osterix. This study provides the first evidence that PP1 might be a potent regulator of developing pulp tissue in vivo and odontoblastic differentiation and angiogenesis in HDPCs in vitro and may have clinical implications for pulp/dentin regeneration or reparative dentinogenesis.

Kim JY ，Kim DS ，Auh QS ，Yi JK ，Moon SU ，Kim EC ... -  《-》