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International Journal of Molecular... Jan 2018Substantial evidence has indicated that Notch and bone morphogenetic protein (BMP) signaling may regulate odontoblastic differentiation. Hairy/enhancer‑of‑split...
Substantial evidence has indicated that Notch and bone morphogenetic protein (BMP) signaling may regulate odontoblastic differentiation. Hairy/enhancer‑of‑split related with YRPW motif 1 (Hey1), a downstream target gene of Notch and BMP signaling, is expressed in dental pulp tissues and has been demonstrated to be responsible for osteoblast mineralization. The aim of this study was to investigate the effects of Hey1 on odontoblast differentiation. The results of the study demonstrated that Hey1 expression in odontoblast‑lineage cells (OLCs) was upregulated by stimulation of osteoblastic/odontoblastic differentiation medium containing ascorbic acid, β‑glycerol phosphate and dexamethasone. Furthermore, stable Hey1‑overexpressing cells expressed higher levels of dentin sialophosphoprotein (DSPP) and exhibited higher mineralization capabilities following stimulation by differentiation medium. Furthermore, RNA interference‑mediated knockdown of Hey1 downregulated the expression levels of DSPP in OLCs stimulated by differentiation medium. Taken together, the findings indicate that Hey1 may be a positive regulator of odontoblastic differentiation. The present study broadens the understanding of odontoblast differentiation and biomineralization.
Topics: Animals; Ascorbic Acid; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Line; Cell Lineage; Dental Pulp; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Humans; Mice; Odontoblasts; Odontogenesis; Phosphoproteins; Repressor Proteins; Sialoglycoproteins; Signal Transduction
PubMed: 29138798
DOI: 10.3892/ijmm.2017.3254 -
Archives of Oral Biology Jul 2014Heat shock during restorative procedures can trigger damage to the pulpodentin complex. While severe heat shock has toxic effects, fever-range heat stress exerts...
OBJECTIVE
Heat shock during restorative procedures can trigger damage to the pulpodentin complex. While severe heat shock has toxic effects, fever-range heat stress exerts beneficial effects on several cells and tissues. In this study, we examined whether continuous fever-range heat stress (CFHS) has beneficial effects on thermotolerance in the rat clonal dental pulp cell line with odontoblastic properties, KN-3.
METHODS
KN-3 cells were cultured at 41°C for various periods, and the expression level of several proteins was assessed by Western blot analysis. After pre-heat-treatment at 41°C for various periods, KN-3 cells were exposed to lethal severe heat shock (LSHS) at 49°C for 10min, and cell viability was examined using the MTS assay. Additionally, the expression level of odontoblast differentiation makers in surviving cells was examined by Western blot analysis.
RESULTS
CFHS increased the expression levels of several heat shock proteins (HSPs) in KN-3 cells, and induced transient cell cycle arrest. KN-3 cells, not pre-heated or exposed to CFHS for 1 or 3h, died after exposure to LSHS. In contrast, KN-3 cells exposed to CFHS for 12h were transiently lower on day 1, but increased on day 3 after LSHS. The surviving cells expressed odontoblast differentiation markers, dentine sialoprotein and dentine matrix protein-1. These results suggest that CFHS for 12h improves tolerance to LSHS by inducing HSPs expression and cell cycle arrest in KN-3 cells.
CONCLUSIONS
The appropriate pretreatment with continuous fever-range heat stress can provide protection against lethal heat shock in KN-3 cells.
Topics: Adaptation, Physiological; Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Survival; Cells, Cultured; Clone Cells; Heat-Shock Proteins; Heat-Shock Response; Hot Temperature; In Situ Nick-End Labeling; Odontoblasts; Rats; Stress, Physiological
PubMed: 24814171
DOI: 10.1016/j.archoralbio.2014.03.014 -
Oral Diseases Jul 2014Matrix metalloproteinase (MMP)-3 expression increases after pulpectomy and accelerates angiogenesis in rat dental pulp by an uncharacterised mechanism. Odontoblasts, a...
OBJECTIVES
Matrix metalloproteinase (MMP)-3 expression increases after pulpectomy and accelerates angiogenesis in rat dental pulp by an uncharacterised mechanism. Odontoblasts, a major component of dental pulp, could represent a therapeutic target. We investigated whether MMP-3 activity is induced by cytokines and/or is associated with cell proliferation and apoptosis in embryonic stem cell-derived odontoblast-like cells.
MATERIALS AND METHODS
We used reverse transcriptase polymerase chain reaction, western blotting, an MMP-3 activity assay, a BrdU-cell proliferation enzyme-linked immunosorbent assay and DNA fragmentation analysis to evaluate siRNA-mediated downregulation of MMP-3 expression and activity, and any changes in the proliferative and apoptotic responses associated with this reduced expression.
RESULTS
Pro-inflammatory cytokines (interleukin-1β, tumour necrosis factor-α and interferon-γ, at relatively low concentrations) induced MMP-3 mRNA and protein expression, and increased MMP-3 activity and cell proliferation, but not apoptosis. MMP-3 silencing produced a potent and significant suppression of cytokine-induced MMP-3 expression and activity, decreased cell proliferation and increased apoptosis. These effects were rescued by application of exogenous MMP-3.
CONCLUSIONS
Our results suggest that pro-inflammatory cytokines induce MMP-3-regulated cell proliferation and anti-apoptosis effects in odontoblast-like cells derived from embryonic stem cells, in addition to their well-documented destructive role in inflammation.
Topics: Animals; Apoptosis; Blotting, Western; Cell Division; Cell Line; Cell Proliferation; Cytokines; Embryonic Stem Cells; Matrix Metalloproteinase 3; Mice; Odontoblasts; Proteins
PubMed: 23902456
DOI: 10.1111/odi.12165 -
Journal of Photochemistry and... Nov 2016Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various...
Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various clinical applications, molecular mechanisms involved are still poorly understood. The aim of the study was to investigate the ability of laser stimulation to increase cell proliferation through an early activation of three redox sensitive pathways, namely Nrf-2, NF-κB and ERK in a rat odontoblast-like cell line (MDPC-23 cells). MDPC-23 cells were irradiated with different energy settings (0-50J, corresponding to 0-32.47J/cm) and cell proliferation was evaluated by cell counting. Nrf-2, NF-κB and ERK signaling pathways activation was investigated through Western blot analysis. Our results show that a single 25J laser stimulation is able to increase cell proliferation and that this effect could be increased by repeating the stimulation twice with a time lapse of 24h. Western blot experiments demonstrated that laser stimulation is able to induce an early activation response in intracellular signaling, with an overlapping time pattern between the three considered pathways. Results discussed in this paper reveal a complex mechanism underlying near-infrared induced increase in pre-odontoblasts proliferation, involving three survival pathways that can act both separately or through reciprocal crosstalk. In particular, data presented suggest an important role for ERK pathway that could act directly by stimulating cell proliferation but can also induce both Nrf-2 and NF-κB activation, acting as a critical cellular checkpoint in response to imbalanced redox state generated by a laser induced increase in ROS production.
Topics: Animals; Cell Line; Cell Proliferation; Infrared Rays; Lasers; Odontoblasts; Oxidation-Reduction; Rats
PubMed: 27718420
DOI: 10.1016/j.jphotobiol.2016.08.049 -
Archives of Oral Biology 1991The cement produced microcrystals of calcite by reaction with culture medium supplemented with calf serum. Human dental pulp cells seeded on such a substrate...
The cement produced microcrystals of calcite by reaction with culture medium supplemented with calf serum. Human dental pulp cells seeded on such a substrate preferentially adhered and aggregated around the microcrystals. Immunofluorescence and immunogold labelling revealed a high affinity of serum fibronectin molecules for the calcite crystals. At 4 weeks in culture, the cells had various features of differentiated odontoblasts, notably nuclear polarization, typical appearance of the Golgi apparatus, synthesis of type I collagen and absence of type III, and apical accumulation of actin and vimentin. These cells also elaborated a collagenous extracellular matrix which did not mineralize.
Topics: Adolescent; Calcium Hydroxide; Cell Differentiation; Cells, Cultured; Child; Collagen; Crystallography; Dental Cements; Dental Pulp; Electron Probe Microanalysis; Fibronectins; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Microscopy, Electron; Minerals; Odontoblasts; Tubulin; Vimentin; X-Ray Diffraction
PubMed: 2059161
DOI: 10.1016/0003-9969(91)90074-5 -
Archivum Histologicum Japonicum = Nihon... Apr 1983The distribution and organization of odontoblast processes in young human dentin was examined with a scanning electron microscope. By applying the HCl-collagenase... (Comparative Study)
Comparative Study
The distribution and organization of odontoblast processes in young human dentin was examined with a scanning electron microscope. By applying the HCl-collagenase method, the extracellular matrix of dentin was almost completely removed, thereby exposing the odontoblast processes and their branches to direct observation. The odontoblast processes are located close to the dentinoenamel junction. In the middle and outer zones of dentin the processes bear numerous branches. Some of these appeared to bridge the space between the processes and connect them.
Topics: Child; Dental Enamel; Dentin; Humans; Microscopy, Electron, Scanning; Odontoblasts
PubMed: 6882153
DOI: 10.1679/aohc.46.213 -
Experimental Cell Research May 2015Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously...
Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells.
Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Gene Silencing; Induced Pluripotent Stem Cells; Matrix Metalloproteinase 3; Mice; Odontoblasts; Polyphosphates
PubMed: 25662160
DOI: 10.1016/j.yexcr.2015.01.007 -
Connective Tissue Research Dec 2018Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly...
Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca stores and this store depletion is sensed by the ER Ca sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.
Topics: Animals; Antigens, Differentiation; Calcification, Physiologic; Calcium; Cell Differentiation; Cell Line; Mice; Odontoblasts; Osteoblasts; Stromal Interaction Molecule 1
PubMed: 29745808
DOI: 10.1080/03008207.2017.1408601 -
Journal of Endodontics Feb 2017The present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast-like cell line...
INTRODUCTION
The present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast-like cell line (MDPC-23 cells).
METHODS
MDPC-23 cells were cultured on Npnt-coated polystyrene or in the presence of soluble Npnt. Cell proliferation was analyzed using a Cell Counting Kit-8 kit (Dojindo, Kumamoto, Japan). Alkaline phosphatase (ALP) activity was quantified using an ALP activity assay. A reverse-transcription polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression level of odontogenic markers and integrin(s). Alizarin red staining was conducted to quantify the calcium deposition.
RESULTS
Soluble Npnt had no adverse effect on the proliferation of MDPC-23 cells, but it exhibited concentration-dependent inhibitory activity toward differentiation. In contrast, coated Npnt promoted cell proliferation dramatically and significantly up-regulated the mRNA expression of odontogenesis-related genes; moreover, mRNA expression of integrin α1, α3, α5, β1, and β5 was found to be augmented. MDPC-23 cells cultured on Npnt-coated polystyrene displayed markedly higher ALP activity as early as day 3 after inoculation. In addition, mineralization was accelerated on Npnt-coated polystyrene.
CONCLUSIONS
Npnt in its immobilized form enhanced the proliferation of MDPC-23 cells and induced this odontoblastic precursor cell line to differentiate into a mineralizing phenotype.
Topics: Alkaline Phosphatase; Animals; Cell Differentiation; Cell Line; Cell Proliferation; Cell Survival; Extracellular Matrix Proteins; Odontoblasts; Phenotype; Polystyrenes; Rats
PubMed: 28132711
DOI: 10.1016/j.joen.2016.10.028 -
Archives of Oral Biology 1990Cell migration and replication associated with odontoblast replacement occurring soon after pulp exposure in primate teeth were studied. Class 5 cavity preparations...
Cell migration and replication associated with odontoblast replacement occurring soon after pulp exposure in primate teeth were studied. Class 5 cavity preparations resulting in pulp exposures were restored with a calcium hydroxide-containing capping agent and amalgam. Eighty-four and 96 h after this the animals were injected with 0.5 microCi/g body wt tritiated thymidine (sp. act. 6.7 Ci/mM). Teeth were extracted 6, 8, 10 and 12 days after treatment. The number of labelled cells as well as the number of grains per labelled cell were counted for odontoblast-like, fibroblast-like and perivascular cells in three 60 x 260 microns zones. These zones represented the odontoblast and cell-free (zone 1), cell-rich (zone 2) and deep pulp (zone 3) areas of normal pulp tissue. Ten sections centred around the mid-point of the exposure were counted for each tooth. Matrix formation and labelled odontoblast-like cells were observed at the interface between the capping agent and the pulp as early as day 8. Other significant findings were: (1) an increase in labelled odontoblast-like cells in zone 1 over time, suggesting a continual influx of differentiating cells; (2) an increase in labelled cells in zone 1 over time with a concurrent decrease in zone 3, suggesting that the influx of cells in zone 1 was from the deeper pulp; and (3) differences in grain counts between zones, treatment times and cell types, indicating that at least two DNA replications had occurred between initial treatment and final odontoblast-like cell differentiation.
Topics: Animals; Autoradiography; Calcium Hydroxide; Cell Count; Cell Division; Cell Movement; Dental Pulp; Dental Pulp Capping; Fibroblasts; Macaca mulatta; Odontoblasts; Thymidine; Time Factors; Tritium; Wound Healing
PubMed: 2091590
DOI: 10.1016/0003-9969(90)90093-p