TWIST1 promotes the odontoblast-like differentiation of dental stem cells

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.     

Pax9调控小鼠帽状期和钟状期牙乳头细胞的增殖和分化的初步研究

目的 观察Pax9对小鼠帽状期和钟状期牙胚牙乳头细胞的作用.方法 培养胚胎14.5d和16.5d小鼠下颌第一磨牙牙胚的牙乳头细胞.转染 Pax9 siRNA至牙乳头细胞中敲低 Pax9的表达,CCK8检测转染后24、48、72和96h细胞的增殖情况,Real-time PCR检测转染后Msx1、Bmp2、Bmp4的表达变化.在转染Pax9 siRNA的同时进行矿化诱导培养7d,然后检测Alp、Dmp1和Dspp的表达情况.结果 敲低Pax9表达后,牙乳头细胞的增殖能力减弱;Msx1的表达水平下降,而Bmp2和Bmp4的表达水平升高;牙本质形成相关基因-Alp、Dmp1和Dspp的表达水平升高,牙乳头细胞的矿化能力增强.结论 Pax9参与调控小鼠帽状期和钟状期牙乳头细胞的增殖和成牙本质分化,同时调控下游基因Msx1、Bmp2和Bmp4的表达.     

小分子整联蛋白结合配体N-连接糖蛋白家族成员在牙髓干细胞分化过程中的作用

体外诱导牙髓干细胞向成牙本质细胞分化并观察其过程,是目前研究牙髓干细胞分化机制和寻找其分化标志的主要方法。在成牙本质细胞分化和矿化过程中,小分子整联蛋白结合配体N-糖蛋白(SBLING)家族成员发挥着重要作用。下面就SBLING家族成员牙本质涎磷蛋白、牙本质基质蛋白-1和细胞外基质磷酸糖蛋白在牙髓干细胞分化过程中的作用作一综述。     

Effects of lipopolysaccharide on newly established rat dental pulp-derived cell line with odontoblastic properties

To clarify mechanisms of pulp wound healing and regeneration, it is important to establish continuous odontoblast-lineage cell lines. In this study, we established the proliferating pulp progenitor cell lines from dental papilla cells of rat incisor. These cell lines showed high levels of alkaline phosphatase (ALP) activity, expression of Runx2 and dentin sialophosphoprotein (DSPP), and extracellular formation of mineralized nodules. By using the cell line with high expression level of DSPP and the prominent mineral deposition, we examined whether bacterial lipopolysaccharide (LPS) had effects on its odontoblastic properties and found that ALP activity, expression of DSPP and Runx2, and the formation of mineralized nodules were suppressed in LPS dose-dependent manner. These results indicate that our established pulp progenitor cell line exhibits odontoblastic properties, which were suppressed by LPS, suggesting that gram-negative bacterial infection might downregulate the odontoblast function.     

MEPE is downregulated as dental pulp stem cells differentiate

Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin. OBJECTIVE: The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation. DESIGN: DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot. RESULTS: Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFbeta-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated. CONCLUSION: DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.     

Cbfa1/Runt2在牙髓组织和牙髓干细胞中的表达研究

目的:检测Cbfa1/Runt2在成人牙髓组织及牙髓细胞的表达,以便进一步探讨其在成牙本质细胞分化过程中所起的作用.方法:选取因正畸拔除的第一前磨牙牙髓,一部分用于免疫酶标检测,一部分用酶消化法进行细胞培养.取第4代细胞进行Cbfa1/Runt2的免疫荧光检测.选取牙龈组织和牙龈成纤维细胞作为对照.结果:Cbfa1/Runt2在牙髓组织的表达集中在成牙本质细胞下层和血管周围.在牙髓细胞的表达集中在细胞核.结论:Cbfa1/Runt2可能参与成牙本质细胞分化的调控,但其在成牙本质细胞分化过程中所起的作用还需进一步研究.     

Runx2/Cbfa1在出生后小鼠牙髓牙本质复合体形成中的作用研究

目的　探讨Runx2/Cbfa1在出生后小鼠成牙本质细胞分化及牙本质形成中时间和空间的分布特点及意 义。方法　采用免疫组化SABC法检测Runx2/Cbfa1在出生后BALB/c小鼠牙本质、成牙本质细胞及牙髓细胞中不 同发育时期的表达及特点。结果　Runx2/Cbfa1在成牙本质细胞的发育成熟过程中具有时空特异性,在牙根尚未发 育之前的牙胚钟状晚期,Runx2/Cbfa1只在前期牙本质中有表达,在牙髓细胞及成牙本质细胞中皆为阴性;约从第11 天开始,随着牙根的发育,Runx2/Cbfa1在根部成牙本质细胞、牙髓细胞中表达为阳性,在冠部成牙本质细胞中表达 为阴性。结论　Runx2/Cbfa1参与牙本质的形成和矿化及成牙本质细胞和牙髓细胞的发育,可能对它们起重要作 用。     

Autophagy in the dentin‐pulp complex against inflammation

The dentin‐pulp complex is a highly specialized tissue for protecting the dental pulp. Odontoblasts are long‐lived, hard‐tissue‐forming cells in the dentin‐pulp complex and critically involved in inflammatory responses against invading pathogens. Autophagy is a highly conserved homeostasis mechanism of living cells under various stress conditions. Growing evidence in the literature addresses the role of autophagy in odontoblast differentiation and aging. This review summarizes the current knowledge about autophagy for the dentin‐pulp complex in resisting inflammation.     

Odontoblast-like cell differentiation and dentin formation induced with TGF-β1

Objective

To investigate the inductive potential of scaffold material combing with transforming growth factor-β1 (TGF-β1), and to induce odontoblast differentiation and dentin formation from dental pulp cells both in vitro and in vivo.

Methods

Primarily cultured dental pulp cells were used for MTT, ALP activity assay and Alizarin red staining in the presence of TGF-β1. Pelleted cells were put on the filters combining with or not with TGF-β1 and cultured in vitro or in vivo. The in vitro and in vivo cell response and tissue formation were analysed with Haematoxylin–Eosin (HE), transmission electron microscopy (TEM) and immunohistochemical staining.

Results

TGF-β1 increased the mineralization and ALP activity of dental pulp cells as revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days, cells polarized in the TGF-β1 group and expressed dentin sialoprotein (DSP), osteopotin (OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar odontoblast formed on the surface of filter in experimental group, and tubular dentin expressing DSP formed after 3 months transplantation.

Conclusion

It was concluded that TGF-β1 combining with transfilter could induce odontoblast differentiation and dentin formation. Our results implied that suitable substrate for the progenitors of odontoblast to anchor on and inductive signals to initiate the differentiation of odontoblast should be taken into consideration when designing scaffold material for inducing dentin tissue engineering.     

The Effect of SIRT6 on the Odontoblastic Potential of Human Dental Pulp Cells

Introduction

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).

Methods

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.

Results

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.

Conclusions

SIRT6 is expressed in human dental pulp and participates in the odontoblast differentiation of HDPCs.     

Expression of mineralization markers in dental pulp cells

There is an increasing interest in the utility of dental pulp stem cells (DPSCs) for dentin regeneration. The mechanisms involved in DPSC differentiation remain poorly understood. The purpose of the study was to investigate the mineralization capacity of human dental pulp cells (DPCs) and identify potential markers for odontoblast differentiation. The isolated DPCs expressed mesenchymal stem-cell markers as shown by flow cytometry and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages. Alkaline phosphatase activity of DPCs elevated over time, with significant upregulation on day 21 in odontogenic induction. Quantitative RT-PCR revealed that osteocalcin, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) expression also increased time dependently in the induction cultures. In conclusion, we isolated DPCs with stem cell characteristics. MEPE and DSPP showed a similar regulatory pattern of DPCs mineralization. MEPE along with DSPP may be potential odontogenetic differentiation markers.     

Notch 1信号蛋白在早期牙髓损伤修复中表达的免疫组化研究

目的 :通过建立大鼠牙髓损伤动物模型 ,观察Notch1信号蛋白在牙髓损伤修复过程中的表达。方法 :在大鼠第一磨牙牙合面开髓 ,分别对损伤后 2h、12h、2 4h、3d、7d及正常大鼠牙髓标本进行HE染色、免疫组化染色 ,检测各组标本中Notch1信号的表达。结果 :Notch1信号蛋白在正常成年大鼠牙髓组织中无表达 ,在牙髓损伤后表达上调。牙髓损伤 12h ,Notch1在成牙本质细胞层、部分牙髓细胞间质中呈弱阳性表达。损伤后 1～ 3d ,Notch1在成牙本质细胞层、部分牙髓细胞间质及接近损伤部位的牙髓细胞中呈阳性着色。损伤后 7d ,Notch1的表达位于成牙本质细胞层和穿髓孔下方的细胞增殖层。结论 :Notch1信号蛋白在大鼠牙髓损伤后早期阶段表达逐渐上调 ,随后在成牙本质细胞区及富细胞区牙髓细胞中表达增强 ,并沉积于穿髓点附近的细胞增殖层。提示它可能在牙髓损伤的早期自身修复和牙本质矿化中起作用     

Toll样受体4在人成牙本质细胞和牙髓组织中的表达

目的 探讨人牙髓组织和成牙本质细胞中Toll样受体4（TLR4）的表达与分布特点，及其在牙髓组织天然免疫防御中的可能作用。方法从新鲜拔除的人第三磨牙获取牙髓组织和成牙本质细胞，扫描电镜观察牙髓摘除后髓腔壁成牙本质细胞形态和附着情况；RT—PCR检测人牙髓组织以及成牙本质细胞中TLR4mRNA的表达情况：采用免疫印迹技术和免疫组织化学方法检测TLR4蛋白的表达与定位。结果扫描电镜观察牙髓摘除后成牙本质细胞大部存留在髓室壁上，细胞形态良好：RT-PCR结果发现健康牙髓、成牙本质细胞均可表达TLR4mRNA，产物大小为278bp；免疫印迹法检测正常牙髓、成牙本质细胞在相对分子质量89000附近出现蛋白条带；免疫组化显示牙髓组织中的成牙本质细胞、血管内皮细胞TLR4免疫反应阳性。正常牙髓中牙髓成纤维细胞不表达TLR4。结论成牙本质细胞通过表达TLR4参与牙髓牙本质复合体的先天免疫反应，TLR4在牙髓抵御外来病原微生物入侵的宿主免疫防御中起重要作用。     

Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.     

Expression of Notch signalling-related genes in normal and differentiating rat dental pulp cells

Notch signalling is of fundamental importance to various processes during embryonic development and in adults. The possible role of Hey1, an important Notch signalling component, in odontoblast differentiation was evaluated in this study. Primary cultured dental pulp cells, derived from upper incisors of 5-week-old Wistar rats, were placed in α-modification of Eagle's minimal essential medium supplemented with 10% Fetal Bovine Serum (FBS), and ascorbic acid (AA) and β-glycerophosphate (β-GP), with or without dexamethasone, and cultured on dishes coated with collagen type IA for 7 days. Conventional and real-time Polymerase Chain Reaction (PCR) was performed to determine the expression of Notch-related genes and dentin sialophosphoprotein as a marker of odontoblast differentiation. Dentin sialophosphoprotein and Hey1 expression was significantly increased and decreased in the presence of AA + β-GP compared with controls, respectively. These findings suggest that Hey1 may be a negative regulator in odontoblast differentiation.