OBJECTIVETo analyze the difference in protein profiles between human dental pulp cells (DPC) and odontogenic differentiated DPC by using proteomic approach.

METHODSHuman DPC were induced to odontoblast differentiation and total proteins in the cell lysates before and after induction were prepared. Proteins spots were isolated by two-dimensional gel electrophoresis. DeCyder V6.0 software was applied to gel image analysis. Differential protein spots were identified by peptide mass fingerprinting technique.

RESULTSForty-six protein spots were determined to be differentially expressed with twenty identified protein spots. Expression changes of the identified proteins revealed the involvement of various regulation mechanisms in odontoblast differentiation, such as cell cycle, cellular energy regulation and signal transduction.

CONCLUSIONSThe proteomic approach is a high throughput method to screen the candidate proteins involved in odontoblast differentiation of DPC.

Cell Differentiation ; Cells, Cultured ; Dental Pulp ; cytology ; metabolism ; Humans ; Odontoblasts ; cytology ; metabolism ; Proteome ; metabolism ; Proteomics ; methods

OBJECTIVETo investigate the expression of Na(+)/Ca(2+) exchanger 1 (NCX1) channel protein in human odontoblasts (OD) and nervous tissue of dental pulp.

METHODSTwenty intact and healthy third molars extracted for orthodontic purpose were collected. The OD layer and nervous tissue were determined by dentin sialophosphoproteins (DSPP) antibody staining and modified Bielschowsky silver staining respectivelly. The immunohistochemical method was used to detect the expressions of NCX1 protein in human dental pulp tissue. The difference of expression of NCX1 in human OD at different part of dental pulp was statistically analyzed using Image Pro Plus and SPSS software.

RESULTSNCX1 channel protein was mainly expressed on the cell body of OD, and nervous tissue of dental pulp. The expression level of NCX1 on the OD of crown pulp was higher (A = 0.146 ± 0.021) than that on the upper part of root pulp (A = 0.120 ± 0.034), but the expression difference was not significant (P > 0.05).

CONCLUSIONSNCX1 channel protein was expressed on human OD and nervous tissue in dental pulp.

Dental Pulp ; innervation ; metabolism ; Dentin ; chemistry ; Humans ; Molar ; Odontoblasts ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Tooth Crown

OBJECTIVE: To investigate the role of autophagy in lipopolysaccharide (LPS)-induced apoptosis of murine odontoblasts. METHODS: Murine odontoblasts (mDPC-23 cells) were treated with 5 μg/mL LPS for 6, 12 and 24 h, and the changes in cell viability was examined using CCK8 kit and cell apoptosis was detected by TUNEL staining. The changes in the protein levels of LC3, Beclin1, Atg5, AKT, p-AKT, mTOR and p-mTOR were detected using Western blotting. The effect of 3-MA treatment for 24 h on LPS-induced apoptosis of mDPC-23 cells was evaluated by detecting the expressions of apoptosis-related proteins caspase-3 and Bax using Western blotting. RESULTS: Stimulation with LPS for 6 and 12 h did not cause significant changes in the proliferation or apoptosis of mDPC-23 cells, but LPS treatment for 24 h significantly suppressed cell proliferation ( CONCLUSIONS LPS stimulation induces autophagy to promote apoptosis of mDPC-23 cells, and suppression of autophagy attenuates LPS-induced apoptosis. Autophagy may play an important role in the injury of inflamed pulp tissues.
Animals ; Apoptosis ; Autophagy ; Lipopolysaccharides/pharmacology* ; Mice ; Odontoblasts/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction

Multiple signaling pathways are involved in the regulation of cell proliferation and differentiation in odontogenesis and dental tissue renewal, but the details of these mechanisms remain unknown. Here, we investigated the expression patterns of a transcription factor, Krüppel-like factor 6 (KLF6), during the development of murine tooth germ and its function in odontoblastic differentiation. KLF6 was almost ubiquitously expressed in odontoblasts at various stages, and it was co-expressed with P21 (to varying degrees) in mouse dental germ. To determine the function of Klf6, overexpression and knockdown experiments were performed in a mouse dental papilla cell line (iMDP-3). Klf6 functioned as a promoter of odontoblastic differentiation and inhibited the proliferation and cell cycle progression of iMDP-3 through p21 upregulation. Dual-luciferase reporter assay and chromatin immunoprecipitation showed that Klf6 directly activates p21 transcription. Additionally, the in vivo study showed that KLF6 and P21 were also co-expressed in odontoblasts around the reparative dentin. In conclusion, Klf6 regulates the transcriptional activity of p21, thus promoting the cell proliferation to odontoblastic differentiation transition in vitro. This study provides a theoretical basis for odontoblast differentiation and the formation of reparative dentine regeneration.
Animals ; Cell Differentiation/physiology* ; Cell Proliferation ; Mice ; Odontoblasts/metabolism* ; Odontogenesis ; Tooth Germ

Inflammation-associated proteinase functions are key determinants of inflammatory stromal tissues deconstruction. As a specialized inflammatory pathological process, dental internal resorption (IR) includes both soft and hard tissues deconstruction within the dentin-pulp complex, which has been one of the main reasons for inflammatory tooth loss. Mechanisms of inflammatory matrix degradation and tissue resorption in IR are largely unclear. In this study, we used a combination of Cre-loxP reporter, flow cytometry, cell transplantation, and enzyme activities assay to mechanistically investigate the role of regenerative cells, odontoblasts (ODs), in inflammatory mineral resorption and matrices degradation. We report that inflamed ODs have strong capabilities of matrix degradation and tissue resorption. Traditionally, ODs are regarded as hard-tissue regenerative cells; however, our data unexpectedly present ODs as a crucial population that participates in IR-associated tissue deconstruction. Specifically, we uncovered that nuclear factor-kappa b (NF-κB) signaling orchestrated Tumor necrosis factor α (TNF-α)-induced matrix metalloproteinases (Mmps) and Cathepsin K (Ctsk) functions in ODs to enhance matrix degradation and tissue resorption. Furthermore, TNF-α increases Rankl/Opg ratio in ODs via NF-κB signaling by impairing Opg expression but increasing Rankl level, which utterly makes ODs cell line 17IIA11 (A11) become Trap⁺ and Ctsk⁺ multinucleated cells to perform resorptive actions. Blocking of NF-κB signaling significantly rescues matrix degradation and resorptive functions of inflamed ODs via repressing vital inflammatory proteinases Mmps and Ctsk. Utterly, via utilizing NF-κB specific small molecule inhibitors we satisfactorily attenuated inflammatory ODs-associated human dental IR in vivo. Our data reveal the underlying mechanisms of inflammatory matrix degradation and resorption via proteinase activities in IR-related pathological conditions.
Humans ; Matrix Metalloproteinases/metabolism* ; Minerals/metabolism* ; NF-kappa B/metabolism* ; Odontoblasts/metabolism* ; Osteoclasts/metabolism* ; RANK Ligand/metabolism* ; Tumor Necrosis Factor-alpha/metabolism*

OBJECTIVETo detect the expression of D-E-A-D-box polypeptide 41 (DDX41) in human dental pulp tissues and cells.

METHODSThe mRNA and protein expressions of DDX41 in human dental pulp cells were detected by RT-PCR and immunocytochemistry, and the expression of DDX41 in human dental pulp tissues was investigated by immunohistochemistry.

RESULTSStrong expressions of DDX41 mRNA and protein were detected in dental pulp cells. In dental pulp tissues, DDX41 was expressed in the cytoplasm and nucleus of odontoblasts.

CONCLUSIONDDX41/STING-dependent TBK1-IRF3-IFN-β signaling pathway may play a role in innate immune responses of the dental pulp to caries and pulpitis.

Cell Nucleus ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; DEAD-box RNA Helicases ; metabolism ; Dental Pulp ; metabolism ; Humans ; Immunohistochemistry ; Odontoblasts ; metabolism ; RNA, Messenger ; Signal Transduction

OBJECTIVETo investigate the expression of transient receptor potential vanilloid 3 (TRPV3) ion channel protein in human odontoblasts (OD).

METHODSTwenty intact and healthy third molars extracted for orthodontic purpose were included. The quality of dental tissue sections was determined through HE staining, and the OD layer was further determined by dentin sialophosphoproteins (DSPP) antibody staining, and finally the expression of TRPV3 ion channel protein in human dental pulp tissue was examined by TRPV3 ion channel protein-specific antibody. The expression of TRPV3 channel proteins in human OD at different part of dental pulp was compared using Image Pro Plus (IPP) and SPSS software.

RESULTSTRPV3 channel protein expressed on the cell body of OD in the coronal and root pulp, and the expression in the coronal pulp was significantly higher than that in the root pulp. The TRPV3 protein also expressed at the odontoblastic process, with the higher expression in the crown (IA = 2516 ± 162) than in the root (IA = 2224 ± 150) and external root (IA = 2121 ± 92) (P < 0.05), but the expression between the lateral root area and external root area was not significantly different (P > 0.05).

CONCLUSIONSHuman odonoblasts expressed TRPV3 ion channel protein and the expression level was different at different part of dental pulp OD.

Adolescent ; Adult ; Dental Pulp ; cytology ; Humans ; Immunohistochemistry ; Odontoblasts ; cytology ; metabolism ; TRPV Cation Channels ; metabolism ; Tooth Crown ; metabolism ; Tooth Root ; metabolism ; Young Adult

OBJECTIVETo observe the transcriptional expression of enamelin in developing postnatal rat first mandibular molar germs, for further studies of functions of enamelin in enamel development and mineralization.

METHODSTissue slices of first mandibular molar germ of rat 1, 3, 7, 10, 14 days after birth were prepared. The enamelin mRNA expression was identified by in situ hybridization.

RESULTSEnamelin mRNA was observed in both ameloblast and odontoblast in 1-10 day old rat postnatal first mandibular molar germs. Enamelin mRNA appeared very weakly at 1st day, and increased through 3rd day, reached the maximum at 7th day, and reduced at 10th day and became negative at 14th day postnatally; while the expression of enamelin mRNA in odontoblast maintained lower from 1st to 10th day and negative at 14th day postnatally.

CONCLUSIONEnamelin gene transcriptional expression lasts from preameloblast to maturation ameloblast, which suggests that enamelin may participate in the development of enamel and mantle dentin.

Ameloblasts ; metabolism ; Animals ; Dental Enamel Proteins ; biosynthesis ; genetics ; Gene Expression Regulation ; In Situ Hybridization ; Molar ; embryology ; Odontoblasts ; metabolism ; RNA, Messenger ; analysis ; Rats ; Tooth Germ ; growth & development ; metabolism ; Transcription, Genetic

OBJECTIVETo investigated the effect of Escherichia coli (Ec) LPS on alkaline phosphatase (ALP) activity and expression of dentin sialophosphoprotein (DSPP) and osteocalcin (OCN) genes in vitro differentiation human dental pulp cell.

METHODSOdontoblast-like cells were cultured, cells exposed to Ec LPS for 12 h, total RNA was isolated and DSPP, OCN transcripts were examined by real-time RT-PCR. ALP kit were used to assessed the changes of ALP activity.

RESULTSReal-time RT-PCR analysis indicated that Ec LPS induced about a 3.6-fold decrease for DSPP gene and a 1.6-fold decrease for OCN gene in odontoblast-like cells as compared with controls. At the same time, cells treated with LPS could depress ALP activity from (1156.10 +/- 100.60) pmol x h(-1) x ng(-1) down to (884.80 +/- 26.72) pmol x h(-1) x ng(-1).

CONCLUSIONSThese results indicate that exposure of odontoblast-like cells to LPS can alter cells function by downregulating cell markers of odontoblastic activity.

Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dental Pulp ; cytology ; Escherichia coli ; Humans ; Lipopolysaccharides ; pharmacology ; Minerals ; metabolism ; Odontoblasts ; drug effects ; Osteocalcin ; metabolism

OBJECTIVETo investigate the regulation effects of upstream stimulatory factor 1 (USF1) on osteopontin expression in odontoblasts.

METHODSOdontoblast MDPC-23 was cultured and stably transfected with PCMV-USF1 or A-USF plasmids. Total RNA was extracted and osteopontin expression examined by semi-quantitative RT-PCR. Gray value of osteopontin was measured and statistic analysis performed.

RESULTSClones of stable PCMV-USF1 and A-USF plasmids transfection were obtained. Compared with the control, osteopontin was upregulated in PCMV-USF1 transfection group, and downregulated in A-USF transfection group.

CONCLUSIONSUpstream stimulatory factor 1 could regulate the osteopontin expression in odontoblasts, which could be blocked partly by A-USF.

Cell Line, Tumor ; Humans ; Odontoblasts ; metabolism ; Osteopontin ; genetics ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Upstream Stimulatory Factors ; genetics