Differentiation Potential Of Dental Pulp Stem Cells Research Articles

Non-cytotoxic Root Canal Dressing with Improved Antimicrobial Efficacy

IntroductionRecurrent endodontic infections are primarily caused by Enterococcus faecalis and are more challenging to treat, compared with primary infection of the root canal system. Calcium hydroxide (CH) is used as an interappointment dressing in endodontics despite its inefficacy against E. faecalis and other pathogens. To improve antimicrobial properties and limit cytotoxicity of CH, we added salicylic acid to CH (CASA) to disinfect the canal. CASA overcomes the main pathogen responsible for recurrent endodontic infections. The aim of this study was to evaluate the antimicrobial activity of CASA and its cytotoxicity against dental pulp stem cells (DPSCs) and its effect on the differentiation potential of DPSCs. MethodsMature E. faecalis biofilm cultured on dentin chips was exposed to CASA and studied using confocal laser scanning microscopy. The dose-dependency of CASA was also studied using the liquid suspension test. The cytotoxicity was tested against DPSCs, and its effect on the expression of osteocalcin and alkaline phosphatase was studied. ResultsCASA produced larger zones of inhibition than CH for all species tested and demonstrated superior efficacy than CH against E. faecalis biofilm. Cytotoxicity studies indicated DPSC's high tolerance for CASA; addition of CASA to DPSCs was observed to increase the expression of biological markers related to mineralization. ConclusionsCASA was proved to have superior antibacterial efficacy against E. faecalis when compared with CH. It also increased the expression of some DPSC differentiation markers involved in mineralization.

MicroRNA-20a elevates osteogenic/odontoblastic differentiation potential of dental pulp stem cells by nuclear factor-κB/p65 signaling pathway via targeting interleukin-8

ObjectiveIn this study, we aimed to explore the mechanism underlying microRNA-20a (miR-20a) in viability, migration and osteogenic/odontoblastic differentiation of dental pulp stem cells (DPSCs). DesignThe results of Targetscan unveiled that interleukin (IL)− 8 might bind to miR-20a, which was confirmed by dual-luciferase reporter assay. MiR-20a expression was up-regulated or down-regulated in DPSCs, followed by measurement of cell viability and migration via MTT and wound healing assays. Then, IL-8 was over-expressed in miR-20a mimic-transfected DPSCs with or without nuclear factor (NF)-κB inhibitor. The effect of miR-20a on osteogenic/odontoblastic differentiation potential of DPSCs was determined by alkaline phosphatase and alizarin red S staining. The expression levels of IL-8, osteogenic differentiation indicators and NF-κB/p65 signaling pathway in cells were detected through quantitative real-time polymerase chain reaction (qRT-PCR) or western blot ResultsContrary to the down-regulated miR-20a, up-regulated miR-20a not only reinforced the viability and migration of DPSCs, but also promoted cell osteogenic/odontoblastic differentiation potential and related gene expressions (alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein (DSPP)). Furthermore, IL-8 was verified to be the target of miR-20a. IL-8 over-expression effectively suppressed the osteogenic/odontoblastic differentiation potential as well as inhibited the activation of NF-κB pathway in DPSCs induced by miR-20a mimic, but these effects could be counteracted by NF-κB inhibitor. ConclusionMiR-20a up-regulation accelerated the viability, migration and osteogenic/odontoblastic differentiation potential of DPSCs by regulating NF-κB/p65 signaling pathway via targeting IL-8, which might be a potential treatment target for dental diseases.

Depletion of EREG enhances the osteo/dentinogenic differentiation ability of dental pulp stem cells via the p38 MAPK and Erk pathways in an inflammatory microenvironment

BackgroundEpiregulin (EREG) is an important component of EGF and was demonstrated to promote the osteo/dentinogenic differentiation of stem cells from dental apical papilla (SCAPs). Whether EREG can stimulate the osteo/dentinogenic differentiation of dental pulp stem cells (DPSCs) in inflammatory environment is not clear. The purpose of the present study is to investigate the role of EREG on the osteo/dentinogenic differentiation ability of DPSCs in inflammatory environment.MethodsDPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) were used to knock down EREG expression in DPSCs. Recombinant human EREG (rhEREG) protein was used in the rescue experiment. TNF-α was employed to mimic the inflammatory environment in vitro. Alkaline phosphatase (ALP) staining, Alizarin red staining, quantitative calcium analysis, and real-time RT-PCR were performed to detect osteo/dentinogenic differentiation markers and related signalling pathways under normal and inflammatory conditions.ResultsEREG depletion promoted the ALP activity and mineralization ability of DPSCs. The expression of BSP, DMP-1, and DSPP was also enhanced. Moreover, 50 ng/mL rhEREG treatment decreased the osteo/dentinogenic differentiation potential of DPSCs, while treatment with 10 ng/mL TNF-α for 4 h increased the expression of EREG in DPSCs. Conversely, EREG knockdown rescued the impaired osteo/dentinogenic differentiation ability caused by TNF-α treatment. Further mechanistic studies showed that EREG depletion activated the p38 MAPK and Erk signalling pathways in DPSCs under normal and inflammatory conditions.ConclusionsOur results demonstrated that EREG could inhibit the osteo/dentinogenic differentiation potential of DPSCs via the p38 MAPK and Erk signalling pathways. Under inflammatory environment, EREG depletion enhanced osteo/dentinogenic differentiation potential of DPSCs by improving the expression of p-p38 MAPK and p-Erk.

Age of the donor affects the nature of in vitro cultured human dental pulp stem cells

ObjectivesThe dental pulp stem cells (DPSCs) of six donors (three young donors aged < 19 years and three adult donors aged > 25 and < 30 years) were characterized for their stem cell marker expression and differentiation potential to study the effect of donor age on DPSCs in vitro. MethodsDPSCs were cultured in αMEM supplemented with 20% fetal calf serum (conventional conditions) or on fibronectin-coated flasks with neurobasal medium supplemented with B27, bFGF and EGF (alternative conditions). DPSCs were characterized by immunofluorescence staining to detect the neural crest/mesenchymal stem cells markers P75 and CD146, respectively. The differentiation potential was tested by the induction of DPSCs into osteogenic, adipogenic and glial lineages and then by detecting the corresponding markers osteocalcin, lipidtox and S100ß, respectively. ResultsThe DPSCs of the young donors expressed CD146 only under the conventional conditions and expressed P75 regardless of the culture conditions. However, the DPSCs of adult donors expressed CD146 only under the alternative conditions and expressed P75 only under conventional conditions. Only the DPSCs of the young donors differentiated into the glial linage. The DPSCs of the adult donors differentiated more efficiently into the adipogenic linage. Osteogenic differentiation was comparable. ConclusionDonor age affects the expression of stem cell markers and differentiation potential of DPSCs. Moreover, the effect of culture conditions on DPSCs is age dependent.

Retinoic Acid Signal Negatively Regulates Osteo/Odontogenic Differentiation of Dental Pulp Stem Cells.

Retinoic acid (RA) signal is involved in tooth development and osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) are one of the useful MSCs in tissue regeneration. However, the function of RA in osteo/odontogenic differentiation of DPSCs remains unclear. Here, we investigated the expression pattern of RA in miniature pig tooth germ and intervened in the RA signal during osteo/odontogenic differentiation of human DPSCs. Deciduous canine (DC) germs of miniature pigs were observed morphologically, and the expression patterns of RA were studied by in situ hybridization (ISH). Human DPSCs were isolated and cultured in osteogenic induction medium with or without RA or BMS 493, an inverse agonist of the pan-retinoic acid receptors (pan-RARs). Alkaline phosphatase (ALP) activity assays, alizarin red staining, quantitative calcium analysis, CCK8 assay, osteogenesis-related gene expression, and in vivo transplantation were conducted to determine the osteo/odontogenic differentiation potential and proliferation potential of DPSCs. We found that the expression of RARβ and CRABP2 decreased during crown calcification of DCs of miniature pigs. Activation of RA signal in vitro inhibited ALP activities and mineralization of human DPSCs and decreased the mRNA expression of ALP, osteocalcin, osteopontin, and a transcription factor, osterix. With BMS 493 treatment, the results were opposite. Interference in RA signal decreased the proliferation of DPSCs. In vivo transplantation experiments suggested that osteo/odontogenic differentiation potential of DPSCs was enhanced by inversing RA signal. Our results demonstrated that downregulation of RA signal promoted osteo/odontogenic differentiation of DPSCs and indicated a potential target pathway to improve tissue regeneration.

FAM96B inhibits the senescence of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are considered a remarkable source for the regeneration of dental pulp tissues, but their therapeutic effectiveness remains limited, especially in elderly people. Previous studies found that senescence has a negative effect on the proliferation and differentiation potential of DPSCs. Moreover, numerous long non-coding RNA (lncRNA) and messenger RNA were significantly differentially regulated in DPSCs from young and elderly donors. However, the changes in DPSCs protein during senescence have not been addressed. In this study, differences in DPSCprotein expression profiles and coexpression of protein and lncRNA were analyzed using proteomics and bioinformatics. The results showed 75 upregulated proteins and 69 downregulated proteins in DPSCs from elderly donors. Vasopressin-regulated water reabsorption, Parkinson's disease, Alzheimer's disease, and protein export were the top four functional pathways associated with DPSCs. High mobility group N1 (HMGN1), HMGN2, UCHL1, and the family with sequence similarity 96 member B homeobox gene (FAM96B) were associated with DPSCs senescence. Then, we investigated FAM96B function in DPSCs. After FAM96B depletion, telomerase reverse transcriptase (TERT) activity decreased, but the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and the protein levels of p16, p53 were significantly increased. Gain-of-function assays suggested that FAM96B overexpression was positively correlated with TERT activity, but negatively correlated with the number of SA-β-gal positive cells and the protein levels of P16 and P53. Moreover, after FAM96B overexpression, the results showed a significant increase in alkaline phosphatase activity and an enhanced mineralization ability of DPSCs. The reverse-transcription polymerase chain reaction results also showed that dentin sialophosphoprotein and osteocalcin were expressed at greater levels. The carboxyfluorescein succinimidyl ester (CFSE) results displayed that FAM96B increased the proliferation potential of DPSCs. Our study revealed candidate proteins that might be related to DPSCs senescence and provided information to elucidate the mechanism of the biological changes in DPSCs' aging. Moreover, FAM96B was demonstrated to play an important role in suppressing DPSCs senescence and promoting osteogenic differentiation and proliferation.

Comparison of two digestion strategies on characteristics and differentiation potential of human dental pulp stem cells

Objective: This study aimed to compare the behavior of dental pulp stem cells (DPSCs) after isolation using solutions containing either collagenase/dispase or collagenase alone. Design: DPSCs were isolated by two digestion methods (collagenase/dispase or collagenase alone) from human third molars. Immunophenotypic features were confirmed by flow cytometry for cell markers STRO-1, cluster of differentiation (CD) 146, CD45, and collagen type-I. The proliferation potential of cells was evaluated by 5-bromo-2′-deoxyuridine (brdU) incorporation assay, and finally they were assessed for multi-lineage differentiation potential. Data were analyzed using one-way analysis of variance and independent t-tests. Results: DPSCs isolated by either method showed similar levels of STRO-1, CD45, and collagen type-I and similar incorporation of brdU (P > 0.05). However, DPSCs obtained by collagenase I/dispase treatment had significantly higher numbers of CD146+ cells and osteogenic and chondrogenic capacities compared to those obtained by treatment with collagenase I alone (P < 0.05). On the other hand, more STRO-1+/CD164-DPSCs were found in the collagenase alone group with higher adipogenic potential. Conclusions: Different enzyme solutions gave rise to different populations of DPSCs. Dispase enhanced isolation of CD146+ DPSCs probably by disrupting the basement membranes of blood vessels and releasing DPCSs embedded in the perivascular niche. Furthermore, the differentiation potential of DPSCs was influenced by the change in enzyme solution.