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

This study compared the biological changes of lipopolysaccharide (LPS)-treated dental pulp (DP) cells directly cultured on mineral trioxide aggregate (MTA) and calcium silicate (CS) cements. DP cells were treated with LPS for 24 h. Then, the LPS-treated DP cells were cultured on MTA or CS cements. Cell viability, cell death mechanism and interleukin (IL)-1β expressions were analysed. A one-way analysis of variance was used to evaluate the significance of the differences between the means. A significantly higher IL-1β expression (2.9-fold) was found for LPS-treated cells (P<0.05) compared with DP cells without LPS treatment at 24 h. Absorbance values of LPS-treated cells cultured on CS cement were higher than a tissue culture plate. A significant difference (P<0.05) in cell viability was observed between cells on CS and MTA cements 24 h after seeding. At 48 h, a high concentration of Si (5 mM) was released from MTA, which induced LPS-treated DP cell apoptosis. The present study demonstrates that CS cement is biocompatible with cultured LPS-treated DP cells. MTA stimulates inflammation in LPS-treated DP cells, which leads to greater IL-1β expression and apoptosis.
Calcium Compounds ; Dental Cements ; Dental Pulp ; drug effects ; metabolism ; Humans ; Inflammation ; chemically induced ; metabolism ; Interleukin-1beta ; metabolism ; Lipopolysaccharides ; pharmacology ; Silicates

OBJECTIVETo observe the histological and immunohistochemical characteristics of the pulp of teeth after pulpotomy.

METHODSTwenty-nine permanent anterior teeth with completely formed roots after pulpotomy due to dental trauma were selected. Thirty permanent premolars with completely formed roots served as control, which were extracted for orthodontic treatment. HE stain and immunohistochemical study of collagen I and collagen III were performed on the root pulp of these two groups.

RESULTSThere were degenerative changes in root pulp of the teeth after pulpotomy, such as vacuolization and homogenization of the odontoblasts, cell reduction, fibrosis, hyaline degeneration and calcification. In healthy root pulp, collagen I had a dispersed distribution, calcification substance was stained positive, but collagen III weakly stained in the extreme at peripheric pulp, and calcification substance stained negative. While in pulp of teeth after pulpotomy, both types of collagens had increased expression, fibers aggregated forming thick fiber bundles. In the wall of blood vessels collagen I had increased expression, but collagen III decreased.

CONCLUSIONSThe root pulp below dentin bridge after pulpotomy was different from the healthy pulp, and there were some histological degenerative changes in the pulp of the immature anterior teeth after pulpotomy. It is suggested that root canal treatment should eventually be performed on these teeth.

Child ; Collagen ; metabolism ; Dental Pulp ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Male ; Pulpotomy ; Staining and Labeling

OBJECTIVETo investigate the effect of Notch signaling pathway on human dental pulp cells.

METHODSThe γ-secretase inhibitor N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester(DAPT) was applied to inhibit the Notch signaling pathway of human dental pulp cells. The solvent dimethly sulfoxide (DMSO) served as the negative control.Senescence conditions were evaluated by cells morphology changes, the alkaline phosphatase (ALP) expression and its activity, senescence-associated β-galactosidase (SA-β-Gal) expression and the senescence related gene p53 expression.

RESULTSAfter inhibition of the Notch signaling pathway, morphology changes, including flatter cells, larger plasma area, were seen in the 10th passage human dental pulp cells. ALP expression and activity showed a significant decrease at the 8th passage after inhibition (35.36 ± 2.55) U/g, compared with the negative control group[(49.76 ± 4.30) U/g] (t = 4.989, P = 0.008).SA-β-Gal-positive cells could be seen as early as the 8th passage and more positive cells were evident at the 10th passage. The relative expression level of p53 gene was elevated in the 10th passage cells (1.7 ± 0.4) compared with the negative control group(1.0) (t = 3.581, P = 0.012).

CONCLUSIONSHuman dental pulp cells became senescent at earlier passages after inhibition of Notch signaling pathway.Notch signaling pathway may affect life cycle of human dental pulp cells.

Cells, Cultured ; Dental Pulp ; metabolism ; Epithelial Cells ; Humans ; Receptors, Notch ; physiology ; Signal Transduction

OBJECTIVETo investigate the role of pulp in the root resorption of primary teeth without permanent tooth germs.

METHODSThe animal model without permanent tooth germs was established by surgery in Beagle dog. The root resorption was observed by taking periapical radiographs periodically. The samples of mandibular bone and pulp at different resorption stages were collected. The distribution of odontoclasts and the activating factor was analyzed by histological staining and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). The role of pulp in the root resorption of primary teeth was tested by early pulpectomy.

RESULTSIn the root resorption of primary molars without permanent teeth germs, a large number of odontoclasts were present on the pulpal surface of the root canal. Semi-quantification RT-PCR showed that the ratios of the expression of receptor activator of NF-κB ligand (RANKL) mRNA and β-actin in the pulp of permanent teeth and primary teeth without permanent teeth germ during different periods of root resorption are 0.1314, 0.1901, 0.2111 and 0.6058 (P > 0.05). The root resorption of primary teeth without permanent teeth germs in test groups was about 5 weeks later than that of control group.

CONCLUSIONSThe pulp of primary tooth played an important role in the root resorption of primary tooth without permanent tooth germ.

Actins ; metabolism ; Animals ; Dental Pulp ; metabolism ; physiology ; Dental Pulp Cavity ; metabolism ; Dogs ; Male ; Molar ; Osteoclasts ; cytology ; RANK Ligand ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Root Resorption ; metabolism ; Tooth Germ ; Tooth, Deciduous ; physiology

OBJECTIVEThe purpose of this study was to investigate the changes of basic fibroblast growth factor (bFGF) expressed in normal human dental pulp at different root development stages of permanent teeth.

METHODSBased on the teeth root development status, the pulp tissues were classified into three groups: root just starting development, being in development and apical closed. The pulps were immunohistochemically examined by use of bFGF antibody.

RESULTSStaining was strongly positive in immature permanent teeth, especially at the stage of root just starting development. Image analysis indicated that the gray values of positive reaction in three groups were statistically different (P < 0.001). For the first group, the gray value of the outer pulp was higher than that of the pulp core. For the second group, the pulp core has a higher gray value in the coronal pulp, while a lower value in root pulp compared to the outer pulp.

CONCLUSIONWith the development of root formation, the expression of bFGF in dental pulp shows different characteristics. bFGF may play a role in dental pulp development and maturation.

Dental Pulp ; metabolism ; Dentition, Permanent ; Fibroblast Growth Factor 2 ; metabolism ; Humans ; Odontogenesis ; Tooth Root ; growth & development ; metabolism

OBJECTIVETo investigate the expression of CXCR4 in cultured human dental pulp cells (HDPC) in vitro and the corresponding ligand SDF-1alpha level of HDPC supernatants stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha), and to explore the role of SDF-1alpha on the proliferation and the migration of HDPC.

METHODSThe expression of CXCR4 in HDPC was detected by immunocytochemistry technique and indirect immunofluorescence technique. The culture supernatants of HDPC were collected after HDPC had been simulated by LPS and TNF-alpha of different concentrations for 48h and then the SDF-1alpha level was assayed by quantitative sandwich ELISA. Meanwhile, the effects of recombinant human SDF-1alpha (rhSDF-1alpha) on the proliferation and the migration of HDPC at different concentrations were observed by MTT and Boyden Chamber Assay.

RESULTSCXCR4 was expressed in cytomembrane of HDPC and SDF-1alpha was secreted into their normal cell supernatants with a concentration of (4513.55 +/- 962.92) ng/L. The secretion of SDF-1alpha was both significantly decreased by stimulation with LPS and TNF-alpha (P < 0.05). In addition, rhSDF-1alpha stimulated the HDPC proliferation at the concentrations of 50, 100, 200 microg/L (P < 0.01) and increased the chemotactic migration of HDPC significantly after 9h's incubation with the concentrations of 50, 100 microg/L (P < 0.05).

CONCLUSIONSSDF-1alpha accelerated the proliferation and the migration of HDPC which expressed CXCR4. SDF-1-CXCR4 axis may play a role in repair of pulp injury.

Cell Movement ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12 ; metabolism ; Dental Pulp ; cytology ; metabolism ; Humans ; Receptors, CXCR4 ; metabolism

The programmed cell death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) deliver inhibitory signals to regulate immunological tolerance during immune-mediated diseases. However, the role of PD-1 signaling and its blockade effect on human dental pulp stem cells (hDPSCs) differentiation into the osteo-/odontogenic lineage remain unknown. We show here that PD-L1 expression, but not PD-1, is downregulated during osteo-/odontogenic differentiation of hDPSCs. Importantly, PD-L1/PD-1 signaling has been shown to negatively regulate the osteo-/odontogenic differentiation of hDPSCs. Mechanistically, depletion of either PD-L1 or PD-1 expression increased ERK and AKT phosphorylation levels through the upregulation of Ras enzyme activity, which plays a pivotal role during hDPSCs osteo-/odontogenic differentiation. Treatment with nivolumab (a human anti-PD-1 monoclonal antibody), which targets PD-1 to prevent PD-L1 binding, successfully enhanced osteo-/odontogenic differentiation of hDPSCs through enhanced Ras activity-mediated phosphorylation of ERK and AKT. Our findings underscore that downregulation of PD-L1 expression accompanies during osteo-/odontogenic differentiation, and hDPSCs-intrinsic PD-1 signaling inhibits osteo-/odontogenic differentiation. These findings provide a significant basis that PD-1 blockade could be effective immunotherapeutic strategies in hDPSCs-mediated dental pulp regeneration.
B7-H1 Antigen/metabolism* ; Dental Pulp/metabolism* ; Humans ; Programmed Cell Death 1 Receptor/metabolism* ; Regeneration ; Stem Cells

OBJECTIVETo analyze the effects of emdogain(EMD) on the expression of the bone sialoprotein(BSP) gene in human dental pulp cells and to elucidate the molecular mechanism of BSP gene regulated by EMD.

METHODSHuman dental pulp was harvested from premolars freshly extracted for orthodontic purpose and cultured. Cells were divided into different concentrations (25, 50, 100 and 250 mg/L) of EMD and control groups (Dulbecco's modified Eagle's medium). Total RNA of cells was extracted. Human BSP mRNA levels was detected with the real-time PCR. Regulations of EMD on human BSP protein levels were detected with Western blotting.

RESULTSIn the real-time PCR, at the same time point, there were significant differences on BSP mRNA levels between 25, 50, 100 and 250 mg/L EMD groups (7 d:1.79 ± 0.03, 2.03 ± 0.10, 2.67 ± 0.08, 2.94 ± 0.07) and control group (7 d:1.06 ± 0.11) (P < 0.001); at the different time point (1, 3, 5 and 7 d), the same dose(250 mg/L) of EMD stimulated human dental pulp cells, BSP mRNA (2.30 ± 0.06, 2.65 ± 0.05, 2.76 ± 0.05, 2.94 ± 0.07) was increased (P < 0.05). Treatment of human dental pulp cells with EMD (250 mg/L) increased the protein levels.

CONCLUSIONSEMD increases BSP mRNA and protein levels in human dental pulp cells.

Bicuspid ; Cells, Cultured ; Dental Enamel Proteins ; pharmacology ; Dental Pulp ; cytology ; metabolism ; Gene Expression Regulation ; Humans ; Integrin-Binding Sialoprotein ; genetics ; metabolism ; RNA, Messenger ; metabolism