[Establishment of a model of endoplasmic reticulum stress response in dental pulp cells induced by tunicamycin].

PURPOSE: The aim of this study was to establish a model of endoplasmic reticulum (ER) stress in dental pulp cells(DPCs) induced by tunicamycin to better understand the molecular mechanism of DPCs related diseases mediated by ER stress. METHODS: DPCs were cultured using modified tissue explant technique in vitro and cultured in presence or absence of tunicamycin. DPCs' viability was measured by methylthiazol tetrazolium (MTT) assay. The mRNA level of ER stress markers was examined by RT-PCR. The data were analyzed with SPSS17.0 software package. RESULTS: The proliferative ability of DPCs decreased when exposed to tunicamycin in a dose-dependent manner. Treatment with tunicamycin resulted in up-regulation of ER stress genes, such as splicing x-box binding protein-1(sXBP1), activating transcription factor 4(ATF4), glucose-regulated protein 78(GRP78) and C/EBP homologous protein (CHOP). CONCLUSIONS: The results indicate that ER stress response is induced in DPCs by tunicamycin, and the ER stress model is successfully established.

[Study of endoplasmic reticulum stress response in osteogenic differentiation of human periodontal ligament cells].

PURPOSE: The aim of this study was to establish whether endoplasmic reticulum (ER) stress is involved in osteogenic differentiation of periodontal ligament cells (PDLCs) and to better understand the mechanism of PDLCs osteogenic differentiation. METHODS: PDLCs were isolated from extracted teeth and cultured in presence or absence of osteogenic medium, which can induce osteogenic differentiation of PDLCs. Alkaline phosphatase and alizarin red S staining were performed to characterize the osteogenic differentiation of PDLCs. The mRNA and protein levels of ER stress markers were examined by RT-PCR and Western blot analysis. The data were analyzed with SPSS 17.0 software package. RESULTS: Cell treated with osteogenic medium showed increased expression of alkaline phosphatase, increased matrix, and mineralized nodule formation compared with untreated controls. Treatment with osteogenic induction resulted in up-regulation of genes, such as splicing x-box binding protein-1 (sXBP1), activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78). The expressions of ER stress protein markers, phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), increased in osteogenic induction cells compared with controls. CONCLUSIONS: The results indicate that ER stress response is involved in the process of osteogenic differentiation of PDLCs.

Effects of deferoxamine on the repair ability of dental pulp cells in vitro.

INTRODUCTION: In previous studies, we found that hypoxia promoted the mineralization of dental pulp cells (DPCs). However, the clinical application of hypoxia as a therapy is questionable or unfeasible. Deferoxamine (DFO), a medication for iron overload, has also been shown to induce hypoxia. The purpose of this study was to investigate the effects of DFO on the repair ability of DPCs. METHODS: DPCs were obtained by using a tissue explant technique in vitro and were treated with different concentrations of DFO or hypoxia culture for 2 days. The viability, proliferation, migration, and odontogenic differentiation of DPCs were assayed and analyzed. The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was assessed through Western blotting. RESULTS: Ten micromolars of DFO enhanced the expression of HIF-1α similarly to hypoxia and did not affect the viability of DPCs for 2 days. Furthermore, the proliferation, migration, and odontogenic differentiation of DPCs were promoted by DFO. CONCLUSIONS: These results suggest that DFO might improve the repair ability of DPCs by HIF-1α.

Hydrogen peroxide induces apoptosis in human dental pulp cells via caspase-9 dependent pathway.

INTRODUCTION: Reactive oxygen species are a group of metabolic intermediates produced during oxidative metabolism in eukaryotic cells. They include superoxide anion (O2(-)), hydrogen peroxide (H2O2), hydroxyl radical (·OH), and (1)O2. Of these intermediates, H2O2 is the most stable. Dental pulp cells can be invaded by tooth bleaching, laser radiation, and dental materials. This can influence the intracellular level of reactive oxygen species. Apoptosis, which is the best-known form of programmed cell death, is pivotal to tissue development and regeneration. Little information is available regarding the relationship between H2O2 and apoptosis of human dental pulp cells (hDPCs). The purpose of this study was to investigate whether H2O2 can induce apoptosis in hDPCs and its signaling way. METHODS: HDPCs were obtained by using a modified tissue explant technique in vitro and cultured at 37°C, 20% O2 (5% CO2, 95% air) in Dulbecco modified Eagle medium. Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell apoptosis was detected by using the annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay and flow cytometry. Expression of activated caspase-3, cleaved caspase-9, and ß-actin was analyzed by using Western blot. RESULTS: Cell viability of hDPCs decreased more in treated groups than in the control group from days 1 to 7. The relative number of apoptotic cells and the expression of activated caspase-3 and cleaved caspase-9 were much higher in groups exposed to 20 and 50 µmol/L H2O2. CONCLUSIONS: These results imply that low concentrations of H2O2 are cytotoxic to hDPCs and induce apoptosis in hDPCs in a caspase-9-dependent way.

[Detection of reactive oxygen species of human dental pulp cells by flow cytometry].

PURPOSE: To establish a method for detection of reactive oxygen species(ROS) of human dental pulp cells(HDPCs) by flow cytometry. METHODS: HDPCs were obtained using tissue explant technique in vitro. The subcultured cells were exposed to peroxide oxygen(H2O2) of different concentrations from 50 µmol/L to 400 µmol/L for 30 minutes, then incubated with two different concentrations of 2',7'-dichlorofluorescein diacetate (DCFH-DA), which were 10 µmol/L and 20 µmol/L for 20 minutes at 37 degrees centigrade in dark. The fluorescence intensities of intracellular dichlorofluorescein(DCF) were detected by flow cytometry. Statistical analysis was carried out by SPSS 13.0 software software. RESULTS: The positive rate varied with different concentrations of detectors. The fluorescence intensities remained insignificant difference among samples incubated with the same concentration of detector and H(2)O(2),and increased by rising of the incubating concentration of H(2)O(2). CONCLUSIONS: The detector with concentration of 20 µmol/L shows higher detector loading rate(positive rate). The intracellular ROS level changes as the H(2)O(2) treatment concentration rising from 50 to 400 µmol/L. The application of flow cytometry to measure the ROS in HDPCs is simple, reliable and stable.

Isolation and identification of CXCR4-positive cells from human dental pulp cells.

INTRODUCTION: In previous studies, we found expression of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) in human dental pulp and the SDF-1α-CXCR4 axis might play a role in the recruitment of CXCR4-positive dental pulp cells (CXCR4(+) DPCs) toward the damaged sites. However, the specific function of CXCR4(+) DPCs in the injured dental pulp was still unknown. The purpose of this study was to isolate CXCR4(+) DPCs from dental pulp cells in vitro to pave the way for further study of their characteristics. METHODS: CXCR4(+) DPCs were isolated with magnetic-activated cell sorting (MACS). Freshly isolated CXCR4(+) DPCs were identified by immunohistochemistry with light microscopy or confocal microscopy. Then the phenotypes CXCR4, stromal cell surface marker-1 (STRO-1), CD146, and CD34 in 3 groups (ie, CXCR4(+) DPCs, CXCR4(-) DPCs, or non-sorted DPCs) were analyzed by flow cytometry after they were cultured and expanded in vitro. RESULTS: The results indicated the isolated subpopulation of DPCs was enriched with CXCR4(+) DPCs, and the positive rates of STRO-1 and CD146 in CXCR4(+) DPCs group were higher than CXCR4(-) DPCs or non-sorted DPCs groups (P < .05). There was no expression of CD34 in each group. CONCLUSIONS: We can isolate CXCR4(+) DPCs from DPCs with MACS and identify them by immunohistochemistry and flow cytometry.

Proliferation and multilineage potential of CXCR4-positive human dental pulp cells in vitro.

INTRODUCTION: Although the results of our previous studies showed that the stromal cell-derived factor (SDF)-1α-CXC chemokine receptor 4 (CXCR4) axis may play a role in the recruitment of CXCR4-positive dental pulp cells (CXCR4(+) DPCs) toward the damaged sites, the specific function of CXCR4(+) DPCs in the injured dental pulp was still unknown. The purpose of this study was to verify whether CXCR4(+) DPCs possessed stem cells properties so that we can understand their role in area of injury. METHODS: CXCR4(+) DPCs were isolated from normal DPCs with magnetic-activated cell sorting. The characteristics of the cells from the 3 groups of cells (ie, CXCR4(+) DPCs, CXCR4(-) DPCs, or nonsorted DPCs) were analyzed in colony formation, proliferation, and multilineage differentiation including odontogenic and adipogenic lineages. RESULTS: The results showed that CXCR4(+) DPCs were the most dominant population in colony formation, proliferation, alkaline phosphatase activity, calcium content, and adipogenic differentiation among the 3 groups. CONCLUSIONS: CXCR4(+) DPCs may contain more stem cells than nonsorted DPCs.