Qimei Gong

The expression of stromal cell-derived factor 1 (SDF-1) in inflamed human dental pulp.

Although dental pulp progenitor/stem cells (DPSCs) are indispensable for repair after pulpal injury, the mechanisms regulating their recruitment and activation remain unknown. To address this issue, we evaluated whether DPSCs in inflamed dental pulp had an upregulation of the chemokine system, a system of proteins known to regulate cellular responses to inflammation. Stromal cell-derived factor 1 (SDF-1), a member of the CXC chemokine subfamily and its receptor CXC chemokine receptor 4 (CXCR4), were evaluated in inflamed dental pulps obtained from extracted human teeth that showed spontaneous pain and/or lingering pain in response to cold and/or heat stimulus and compared with control levels found in normal dental pulps obtained from healthy noncarious third molars. Using immunohistochemistry and real-time reverse-transcription polymerase chain reaction, the results indicated that in inflamed pulps the SDF-1/CXCR4 axis was mostly distributed in inflammatory cells and microvascular endothelial cells rather than in normal pulps. SDF-1 messenger RNA expression levels in clinically inflamed dental pulp were higher than those in healthy dental pulp. These findings suggest that SDF-1 plays an important role in the process of pulpal inflammation via the recruitment of CXCR4-expressing inflammatory cells, and the SDF-1/CXCR4 axis may be involved in the recruitment of dental pulp stem cells at the injury site.

Side Population Increase after Simulated Transient Ischemia in Human Dental Pulp Cell

INTRODUCTION Dental pulp is often exposed to ischemia in case of injury or inflammation because of narrow vascular openings at the apex and poor blood circulation in dental pulp tissue. Resident stem cell populations are thought to contribute to the postischemic regeneration process. The aim of this study was to investigate the influence of simulated ischemia (serum deprivation and hypoxia) on side population (SP) stem cells of human dental pulp cells in order to provide a better understanding of the postischemic tissue repair and regeneration process. METHODS The proliferation of dental pulp cells (DPCs) after exposure to ischemic culture conditions (2% O2, 2% serum) for 24 hours and 48 hours was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The SP fraction was detected by Hoechst 33342 fluorescence flow cytometry, and the expression of SP marker ABCG2 was investigated by immunofluorescence. ABCG2 and OCT4 messenger RNA levels before and after transient ischemia were determined by real-time polymerase chain reaction. RESULTS Proliferation rate of DPCs was lower in 24- and 48-hour ischemic groups than control from day 5 to day 7. SP proportion was significantly higher 24 and 48 hours after simulated ischemic treatment, and immunofluorescence staining of ABCG2 also verified the increasing trend of side population. ABCG2 and OCT4 messenger RNA levels increased more than three folds in 48 hours ischemic group compared with control group. CONCLUSIONS Side population in dental pulp cells increase notably after transient simulated ischemic culture, suggesting that SP may participate in post-ischemic repair and regeneration process of dental pulp.

Regulation of the Stromal Cell–derived Factor-1α–CXCR4 Axis in Human Dental Pulp Cells

INTRODUCTION Although the presence of the stromal cell-derived factor (SDF)-1alpha-CXCR4 axis has been reported in dental pulp tissue, little has been known about the underlying regulation of this axis in dental pulp stem cells (DPSCs). The purpose of this study was to investigate whether inflammation or hypoxia can regulate this axis in cultured human dental pulp cells (DPCs). METHODS Primary cultures of DPCs were stimulated by various concentrations of lipopolysaccharide (LPS) for 48 hours, and the production of SDF-1alpha or CXCR4 was assessed through the enzyme-linked immunosorbent assay and Western blotting, respectively. Additionally, DPCs were incubated in a hypoxic condition (1% O(2)) for 24 hours, and the cell proliferation ability was detected by methylthiazol tetrazolum assay. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was used to observe messenger RNA level changes of hypoxia inducible factor-1alpha(HIF-alpha), SDF-1alpha, and CXCR4. The effects of hypoxia on cell migration ability were further confirmed by transmigration assay. RESULTS All concentrations of LPS inhibited SDF-1alpha production except that 1 microg/mL LPS increased the expression of CXCR4. Hypoxia promoted the proliferation of DPCs in a 24-hour culture period. Quantitative RT-PCR showed that messenger RNA levels of HIF-alpha and CXCR4 increased, whereas SDF-1alpha decreased in hypoxic DPCs. Transmigration assay indicated that hypoxia increased the migration ability of DPCs. CONCLUSIONS These results suggested that inflammation and hypoxia might play an important role in regulating the SDF-1alpha-CXCR4 axis, which further recruits DPSCs to participate in reparative dentinogenesis.

Alteration of MicroRNA Expression of Human Dental Pulp Cells during Odontogenic Differentiation

INTRODUCTION MicroRNAs (miRNAs) play momentous roles in various biological processes including cell differentiation. However, little is known about the role of miRNAs in human dental pulp cells (hDPCs) during odontogenic differentiation. The aims of this study were to investigate the expression of miRNAs in the primary culture of hDPCs when incubated in odontogenic medium. METHODS The potential characteristics of hDPCs were investigated by miRNA microarray and real-time reverse transcriptase polymerase chain reaction. Bioinformatics (ie, target prediction, Gene Ontology analysis, and Kyoto Encyclopedia of Genes and Genomes mapping tools) were applied for predicting the complementary target genes of miRNAs and their biological functions. RESULTS A total of 22 miRNAs were differentially expressed in which 12 miRNAs up-regulated and 10 miRNAs down-regulated in differentiated hDPCs compared with the control. The target genes of differential miRNAs were predicted to associate with several biological functions and signaling pathways including the mitogen-activated protein kinase (MAPK) and the Wnt signaling pathway. CONCLUSIONS The differential expression miRNAs may be involved in governing hDPC odontogenic differentiation, thus contributing to the future investigations of regulatory mechanisms in reparative dentin formation and dental pulp regeneration.

Role of miR-424 on angiogenic potential in human dental pulp cells.

INTRODUCTION Growing evidence shows microRNAs (miRNAs) regulate numerous cellular processes. The purpose of this study was to investigate whether miRNAs can regulate the commitment of human dental pulp cells (hDPCs) to the angiogenic fate. METHODS The hDPCs were induced to differentiate into the vascular lineage. Gene expression of endothelial markers (vWF and CD31) on day 7 after induction was analyzed by using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).The miRNA expression profiling of endothelial differentiation was performed by microarray and was validated by qRT-PCR analysis. The hDPCs were infected by recombinant lentivirus to overexpress or knock down miR-424 stably, and the biological effects of miR-424 on the endothelial differentiation of hDPCs were further investigated. The tube formation ability and the amount of endothelial markers (vWF and KDR) were evaluated by Matrigel assay and Western blotting. Target genes of miR-424 were further determined by bioinformatic algorithms and Western blotting. RESULTS After endothelial differentiation, the expression of vWF and CD31 increased significantly in hDPCs. Microarray data showed that the miR-424 expression level was down-regulated on day 7. The qRT-PCR revealed a time-dependent decrease, with significant differences detected on day 1 and day 7 (P < .05). Knockdown of miR-424 expression in hDPCs promoted endothelial differentiation, with increased tube formation and up-regulated expression of vWF and KDR. In contrast, overexpression of miR-424 inhibited their differentiation. In addition, miR-424 was predicted to target vascular endothelial growth factor and KDR. Overexpression of miR-424 decreased vascular endothelial growth factor and KDR protein levels, whereas miR-424 inhibition significantly elevated them. CONCLUSIONS This study demonstrated that miR-424 may play a negative role in regulating endothelial differentiation of hDPCs, and inhibition of miR-424 may contribute to dental pulp repair and regeneration.

Identification and Characterization of Side Population Cells from Adult Human Dental Pulp after Ischemic Culture

INTRODUCTION Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the side population (SP). Because the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of dental pulp stem cells, this study sought to determine the existence of SP cells and the expression of ABCG2 in human dental pulp and evaluate whether such SP cells had features associated with stem cells. METHODS First, we defined the localization of the SP in healthy and inflammatory human dental pulp. Then, SP cells were isolated from human dental pulp after ischemic culture with flow cytometry and the Hoechst 33342 dye efflux assay. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype, including the colony-forming capacity, the multilineage differentiation ability in vitro, and the expression of stem cell markers. We also evaluated the effect of long-term culture on the marker ABCG2. RESULTS SP cells in human dental pulp possess mesenchymal stem cell characteristics such as colony-forming efficiency, self-renewal, and multilineage differentiation capabilities and are able to differentiate into odontoblast/osteoblast-like cells, adipocytes, neural-like cells, and endothelial cells. However, under the present conditions, ABCG2 expression decreased along with cell passage. CONCLUSIONS SP cells in human dental pulp were enriched in stem cells compared with main population cells after ischemic culture, suggesting a potential use for these subfractions of human dental pulp stem/progenitor cells in tissue engineering, but the culture condition in vitro should be improved before tissue regeneration.

Expression of high mobility group box 1 in inflamed dental pulp and its chemotactic effect on dental pulp cells

High mobility group box 1 protein (HMGB1) is a chromatin protein which can be released extracellularly, eliciting a pro-inflammatory response and promoting tissue repair process. This study aimed to examine the expression and distribution of HMGB1 and its receptor RAGE in inflamed dental pulp tissues, and to assess its effects on proliferation, migration and cytoskeleton of cultured human dental pulp cells (DPCs). Our data demonstrated that cytoplasmic expression of HMGB1 was observed in inflamed pulp tissues, while HMGB1 expression was confined in the nuclei in healthy dental pulp. The mRNA expression of HMGB1 and RAGE were significantly increased in inflamed pulps. In in vitro cultured DPCs, expression of HMGB1 in both protein and mRNA level was up-regulated after treated with lipopolysaccharide (LPS). Exogenous HMGB1 enhanced DPCs migration in a dose-dependent manner and induced the reorganization of f-actin in DPCs. Our results suggests that HMGB1 are not only involved in the process of dental pulp inflammation, but also play an important role in the recruitment of dental pulp stem cells, promoting pulp repair and regeneration.

Expression of Erythropoietin and Erythropoietin Receptor in Human Dental Pulp

INTRODUCTION In addition to the involvement in erythropoiesis, erythropoietin (EPO) and its receptor (EPO-R) have been shown to be expressed in various nonhematopoietic organs and tissues with diverse biological effects. The purpose of this study was to evaluate the expression patterns of EPO and EPO-R in healthy and inflamed human dental pulp tissues. To gain insight into the possible mechanisms involved in the regulation of EPO and EPO-R expression, we further investigated the hypothesis that their expression in cultured human dental pulp cells (DPCs) may be regulated upon hypoxia, an important factor involved in dental pulp inflammation. METHODS Samples of healthy and inflamed dental pulp tissues were obtained from patients undergoing surgical or orthodontic treatment. The protein localization and messenger RNA levels of EPO and EPO-R in the pulp tissues were detected by immunohistochemistry and quantitative real-time polymerase chain reaction (PCR), whereas the EPO and EPO-R expressions in DPCs in vitro were evaluated by Western blotting and quantitative real-time PCR. RESULTS EPO and EPO-R proteins were detected in inflamed dental pulp, whereas no obvious EPO expression was detected in healthy dental pulp. The EPO messenger RNA level was significantly up-regulated in inflamed pulps compared with healthy pulps. Moreover, the messenger RNA and protein levels of EPO and EPO-R were up-regulated in DPCs under hypoxia in vitro. CONCLUSIONS The up-regulation of EPO might be involved in dental pulp inflammation, which is probably attributed to hypoxia. Further studies are needed to investigate the potential role of EPO and EPO-R in dentin-pulp repair and regeneration.

Lipopolysaccharide stimulation improves the odontoblastic differentiation of human dental pulp cells

Lipopolysaccharide (LPS) is one of the causative agents of pulpitis and previous studies have demonstrated that the LPS stimulation of human aortic valve interstitial cells induces inflammatory mediators and the gene expression of osteogenic factors. Therefore, in the present study, it was hypothesized that LPS affects the odontoblastic differentiation of human dental pulp cells (hDPCs). In order to investigate this, an in vitro study using hDPCs was performed. Increased alkaline phosphatase (ALP) activity was observed in the hDPCs treated with LPS, which was more marked when the cells were costimulated with odontogenic induction medium (OM). LPS also appeared to increase the gene expression levels of dentin sialophosphoprotein and dentin matrix protein‑1 and the protein expression level of dental sialoprotein in the hDPCs, particularly in combination with OM. In addition, the size and the number of nodules formed in the hDPCs exposed to OM and LPS were increased compared to those stimulated by OM alone. To determine the role of nuclear factor κB (NF‑κB) during the LPS‑induced odontoblastic differentiation of hDPCs, immunofluorescence was performed. The nuclear translocation of NF‑κB, induced by LPS was confirmed, suggesting its involvement in the LPS‑induced increase in odontoblastic differentiation of hDPCs. In conclusion, there may be an association between LPS stimulation, with or without OM, and odontoblastic differentiation.

Exosomes with Highly Angiogenic Potential for Possible Use in Pulp Regeneration

Introduction: Angiogenesis is critical for pulp regeneration. Exosomes, a key component of paracrine secretion, have emerged as important players in the modulation of angiogenesis. The role of dental pulp cell–derived exosomes (DPC‐Exos) in angiogenesis has rarely been reported. The proangiogenic properties of DPC‐Exos in human umbilical vein endothelial cells (HUVECs) are investigated in the current study. Methods: Exosomes were isolated from dental pulp cell (DPC) culture supernatants by ultracentrifugation and were characterized by transmission electron microscopy, Western blotting, and nanoparticle tracking analysis. Their roles in HUVEC proliferation, proangiogenic factor expression, and tube formation were examined in vitro. Results: We isolated and characterized exosomes from DPCs and showed that DPC‐Exos promoted HUVEC proliferation, proangiogenic factor expression, and tube formation. Furthermore, we found that p38 mitogen‐activated protein kinase (MAPK) signaling inhibition enhances DPC‐Exos–induced tube formation. Conclusions: Taken together, these results suggest that DPC‐Exos have vital roles in angiogenesis, and p38 MAPK signaling inhibition enhances tubular morphogenesis. HIGHLIGHTSDental pulp cell–derived exosomes (DPC‐Exos) facilitated human umbilical vein endothelial cell growth.DPC‐Exos resulted in increased tube formation and proangiogenic factor expression.Inhibition of p38 mitogen‐activated protein kinase activation enhanced DPC‐Exos–stimulated tubular morphogenesis.