miR615-3p inhibited FBLN1 and osteogenic differentiation of umbilical cord mesenchymal stem cells by associated with YTHDF2 in a m6A-miRNA interaction manner.

To investigate the role and mechanism of FBLN1 in the osteogenic differentiation and bone regeneration by using umbilical cord mesenchymal stem cells (WJCMSCs). We found that FBLN1 promoted osteogenic differentiation of WJCMSCs and WJCMSC-mediated bone regeneration. It was showed that there was an m6A methylation site in 3'UTR of FBLN1 mRNA, and the mutation of the m6A site enhanced the stability of FBLN1 mRNA, subsequently fostering the FBLN1 enhanced osteogenic differentiation of WJCMSCs. YTHDF2 was identified as capable of recognizing and binding to the m6A site, consequently inducing FBLN1 instability and repressed the osteogenic differentiation of WJCMSCs. Meanwhile, miR-615-3p negatively regulated FBLN1 by binding FBLN1 3'UTR and inhibited the osteogenic differentiation of WJCMSCs and WJCMSC-mediated bone regeneration. Then, we discovered miR-615-3p was found to regulate the functions of FBLN1 facilitated by YTHDF2 through an m6A-miRNA regulation mechanism. We demonstrated that FBLN1 is critical for regulating the osteogenic differentiation potentials of WJCMSCs and have identified that miR615-3p mediated the decay of FBLN1 mRNA which facilitated by m6A reading protein YTHDF2. This provided a novel m6A-miRNA epigenetic regulatory pattern for MSC regulation and bone regeneration.

Clinical trials using dental stem cells: 2022 update.

For nearly 20 years, dental stem cells (DSCs) have been successfully isolated from mature/immature teeth and surrounding tissue, including dental pulp of permanent teeth and exfoliated deciduous teeth, periodontal ligaments, dental follicles, and gingival and apical papilla. They have several properties (such as self-renewal, multidirectional differentiation, and immunomodulation) and exhibit enormous potential for clinical applications. To date, many clinical articles and clinical trials using DSCs have reported the treatment of pulpitis, periapical lesions, periodontitis, cleft lip and palate, acute ischemic stroke, and so on, and DSC-based therapies obtained satisfactory effects in most clinical trials. In these studies, no adverse events were reported, which suggested the safety of DSC-based therapy. In this review, we outline the characteristics of DSCs and summarize clinical trials and their safety as DSC-based therapies. Meanwhile, we also present the current limitations and perspectives of DSC-based therapy (such as harvesting DSCs from inflamed tissue, applying DSC-conditioned medium/DSC-derived extracellular vesicles, and expanding-free strategies) to provide a theoretical basis for their clinical applications.

Pleiotrophin inhibited chondrogenic differentiation potential of dental pulp stem cells.

OBJECTIVE: Studies have shown that the levels of pleiotrophin (PTN) are greatly elevated in the synovial fluid and cartilage in osteoarthritis. Therefore, the purpose of this study was to investigate the effect and mechanism of PTN on the chondrogenic differentiation of DPSCs in inflammatory and normal microenvironments. MATERIALS AND METHODS: A lentiviral vector was used to deplete or overexpress PTN in DPSCs. The inflammatory microenvironment was simulated in vitro by the addition of IL-1ß to the culture medium. The chondrogenic differentiation potential was assessed using Alcian Blue staining and the main chondrogenic markers. A dual-luciferase reporter assay was used to explore the relationship between miR-137 and PTN. RESULTS: The results showed that 0.1 ng/mL IL-1ß treatment during chondrogenic induction greatly impaired the chondrogenic differentiation of DPSCs. Supplementation with PTN and PTN overexpression inhibited chondrogenic differentiation of DPSCs, while PTN depletion promoted chondrogenic differentiation. MiR-137 negatively regulated the expression of PTN by binding to the 3'UTR of its mRNA. Moreover, miR-137 promoted chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments. CONCLUSION: Our results suggest that PTN may play an inhibitory role in the chondrogenic differentiation of DPSCs in normal and inflammatory microenvironments, which is regulated by miR-137.

Pleiotrophin attenuates the senescence of dental pulp stem cells.

OBJECTIVES: Pleiotrophin (PTN), a secreted extracellular matrix-associated protein, plays an important role in regulating the osteo/dentinogenic differentiation potential of dental pulp stem cells (DPSCs). Our previous study has demonstrated that PTN expression in young DPSCs was is 10-fold higher than that in aged DPSCs. However, the role of PTN on the in maintaining the stemness of senescent DPSCs remains unclear. The present study aimed to investigate the effect of PTN on senescent DPSCs in vitro. MATERIALS AND METHODS: Dental pulp stem cells were isolated from human third molars. PTN was knocked down using short hairpin RNAs to study the role of PTN on the senescence of DPSCs. DPSCs with aging performance were obtained by a replicative senescence cell model was obtained by the long-term culture of DPSCs to the 15th passage in vitro (P15). We then investigated the effect of PTN on senescent DPSCs (P15 DPSCs). Real-time RT-PCR, western blotting, alizarin red staining, quantitative calcium analysis, SA-ß-Gal staining, CFSE, and cell-counting kit-8 (CCK8) assays were used to study cellular senescence and function. RESULTS: The depletion of PTN increased the ratio of SA-ß-gal-positive cells, upregulated the expression of p16, and down-regulated the expression of TERT and p-p38. Furthermore, 50 pg/ml of PTN recombinant protein rescued these changes the altered ratio of SA-ß-gal-positive cells, decreased the expression of p16, enhanced TERT and p-p38 expression, as well as telomere activity, caused by PTN depletion and long-term culture. The15th passage cells displayed typical aging characteristic, including high ratio of SA-ß-gal-positive cells, increased aging-related gene expression, decreased proliferation rate, high level of Cyclin D expression, and impaired osteo/dentinogenic differentiation potential. However, 50 pg/ml of PTN recombinant protein could partially reverse these alteration rescue these changes. CONCLUSIONS: The present study demonstrated that PTN could protect DPSCs from senescence by improving the proliferation and osteo/dentinogenic differentiation ability, probably through the p38 MAPK pathway.

KDM4D enhances osteo/dentinogenic differentiation and migration of SCAPs via binding to RPS5.

OBJECTIVES: Stem cells of the apical papilla (SCAPs) provide promising candidates for dental pulp regeneration. Despite great advances in the transcriptional controls of the SCAPs fate, little is known about the regulation of SCAP differentiation. MATERIALS AND METHODS: Short hairpin RNAs and full-length RNA were used to deplete or overexpress lysine demethylase 4D (KDM4D) gene expression. Western blotting, real-time RT-PCR, alizarin red staining, and scratch migration assays were used to study the role of KDM4D and the ribosomal protein encoded by RPS5 in SCAPs. RNA microarray, chromatin Immunoprecipitation (ChIP), and co-immunoprecipitation (Co-IP) assays were performed to explore the underlying molecular mechanisms. RESULTS: KDM4D enhanced the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. The microarray results revealed that 88 mRNAs were differentially expressed in KDM4D-overexpressed SCAPs. ChIP results showed knock-down of KDM4D increased the level of H3K9me2 and H3K9me3 in CNR1 promoter region. There were 37 possible binding partners of KDM4D. KDM4D was found to combine with RPS5, which also promoted the osteo/dentinogenic differentiation, migration, and chemotaxis of SCAPs. CONCLUSIONS: KDM4D promoted the osteo/dentinogenic differentiation and migration potential of SCAPs in combination with RPS5, which provides a therapeutic clue for improving SCAPs-based dental tissue regeneration.

PRMT6/LMNA/CXCL12 signaling pathway regulated the osteo/odontogenic differentiation ability in dental stem cells isolated from apical papilla.

Tooth loss and maxillofacial bone defect are common diseases, which seriously affect people's health. Effective tooth and maxillofacial bone tissue regeneration is a key problem that need to be solved. In the present study, we investigate the role of PRMT6 in osteo/odontogenic differentiation and migration capacity by using SCAPs. Our results showed that knockdown of PRMT6 promoted the osteo/odontogenic differentiation compared with the control group, as detected by alkaline phosphatase activity, alizarin red staining, and the indicators of osteo/odontogenic differentiation measured by Western blot. In addition, overexpression of PRMT6 inhibited the osteo/odontogenic differentiation potentials of SCAPs. Then, knockdown of PRMT6 promoted the migration ability and overexpression of PRMT6 inhibited the migration ability in SCAPs. Mechanically, we discovered that the depletion of PRMT6 promoted the expression of CXCL12 by decreasing H3R2 methylation in the promoter region of CXCL12. In addition, PRMT6 formed a protein complex with LMNA, a nuclear structural protein. Depletion of LMNA inhibited the osteo/odontogenic differentiation and CXCL12 expression and increased the intranucleus PRMT6 in SCAPs. To sum up, PRMT6 might inhibit the osteo/odontogenic differentiation and migration ability of SCAPs via inhibiting CXCL12. And LMNA might be a negative regulator of PRMT6. It is suggested that PRMT6 may be a key target for SCAP-mediated bone and tooth tissue regeneration.

Dietary nitrate supplementation prevents radiotherapy-induced xerostomia.

Management of salivary gland hypofunction caused by irradiation (IR) therapy for head and neck cancer remains lack of effective treatments. Salivary glands, especially the parotid gland, actively uptake dietary nitrate and secrete it into saliva. Here, we investigated the effect of dietary nitrate on the prevention and treatment of IR-induced parotid gland hypofunction in miniature pigs, and elucidated the underlying mechanism in human parotid gland cells. We found that nitrate administration prevented IR-induced parotid gland damage in a dose-dependent manner, by maintaining the function of irradiated parotid gland tissue. Nitrate could increase sialin expression, a nitrate transporter expressed in the parotid gland, making the nitrate-sialin feedback loop that facilitates nitrate influx into cells for maintaining cell proliferation and inhibiting apoptosis. Furthermore, nitrate enhanced cell proliferation via the epidermal growth factor receptor (EGFR)-protein kinase B (AKT)-mitogen-activated protein kinase (MAPK) signaling pathway in irradiated parotid gland tissue. Collectively, nitrate effectively prevented IR-induced xerostomia via the EGFR-AKT-MAPK signaling pathway. Dietary nitrate supplementation may provide a novel, safe, and effective way to resolve IR-induced xerostomia.

Head and neck cancers are commonly treated using radiotherapy, where a beam of high-energy radiation is targeted at the tumour. This often severely damages the surrounding salivary glands, leading to chronic dry mouth and impairing a patient's sense of taste, nutrient intake, speech and immune system. Despite this significant impact on quality of life, there is no effective treatment yet for this side effect. In the body, salivary glands are one of the primary users of a compound known as nitrate, which is commonly found in the diet. In the glands, it is ushered into cells thanks to a protein known as sialin. The nutrient supports the activity and maintenance of the glands, before it is released in the saliva. Feng, Wu et al. therefore decided to test whether nitrate could offer protection during neck and head radiotherapy. The experiments used miniature pigs, which have similar salivary glands to humans. The animals that received sodium nitrate before and after exposure to radiation preserved up to 85% of their saliva production. By comparison, without any additional nitrate, saliva production fell to 20% of pre-radiation levels. To understand how this protective effect emerged, Feng, Wu et al. added nitrate to cells from a human salivary gland known as the parotid. This led to the cells producing more sialin, creating a feedback loop which increases the amount of nitrate in the salivary glands. Further examination then showed that the compound promotes growth of cells and reduce their death. These findings therefore suggest that clinical studies may be worthwhile to test if nitrate could be used to prevent dry mouth in head and neck cancer patients who undergo radiotherapy.

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.

BACKGROUND: Epiregulin (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. METHODS: DPSCs 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. RESULTS: EREG 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. CONCLUSIONS: Our 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.

A retrospective analysis of the optimal time and psychological impact of decoronation in children and adolescents.

BACKGROUND/AIMS: Decoronation offers one of the best and most predictable clinical outcomes for dentoalveolar ankylosis. The aim of this study was to determine the factors associated with the efficacy and psychological impact of decoronation for bone preservation. MATERIALS AND METHODS: The study included 42 paediatric patients with 42 infrapositioned replanted permanent teeth. Twelve of these teeth were decoronated. Variables such as the time of injury, stage of root development and the extent of infraposition were analysed. The vertical changes in the alveolar bone level of the decoronated teeth were assessed on radiographs using a three-point scoring system. Parents of 30 patients with teeth that were not decoronated completed a questionnaire addressing their considerations and concerns regarding the treatment of infraposition. RESULTS: Teeth with root development in stages 2 and 3 showed a significantly higher rate of severe infraposition during the follow-up visits. Decoronation was performed on 12 teeth within 1.5-5 years (mean 3.8 ± 1.3 years) after replantation and 11 of these cases developed a considerable alveolar bone level. The alveolar bone levels of boys and girls showed improvements of 2.2 and 3.2 mm, respectively. The optimal age for decoronation to have a considerable increase in bone level was 12.12 ± 0.83 years for boys and 11.25 ± 1.77 years for girls. Complicated treatments, followed by parents' lack knowledge regarding decoronation, children's fear, follow-up times, and cost were the major concerns regarding decoronation. CONCLUSION: The optimal time for decoronation should be decided after considering the age, gender, skeletal growth pattern, and the degree of infraposition at the time of decoronation.

Pleiotropin enhances the osteo/dentinogenic differentiation potential of dental pulp stem cells.

Purpose: Pleiotrophin (PTN) is a heparin-binding growth-associated molecule and expressed in ameloblasts and odontoblasts throughout tooth maturation. Our previous study has shown that PTN expressed more than 20-fold higher in dental tissue than dental stem cells. However, the role of PTN on proliferation and osteo/dentinogenesis of dental pulp stem cells (DPSCs) is unclear. The purpose of the present study was to investigate the role of PTN on the DPSCs' function.Methods: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) was used to knock down the PTN expression in DPSCs. Real-time RT-PCR, alizarin red staining, quantitative calcium analysis, in vivo transplantation and cell counting kit-8 (CCK8) assay were used to study the function of DPSCs. Possible mechanism was studied by RNA sequencing.Results: After PTN depletion, ALP activity and mineralization ability of DPSCs decreased. Expression of DMP-1 and BSP weakened. Proliferation of DPSCs at 48 h and 72 h was inhibited. Furthermore, 50 pg/mL PTN recombinant protein rescued the impaired osteo/dentinogenic differentiation potential and proliferation ability caused by PTN depletion. In addition, RNA sequencing showed 221 genes were downregulated and 233 genes upregulated in PTN depleted DPSCs. Several genes including BMP2 and IGFBP5 might be associated with PTN function on the DPSCs. P53 and the AMPK signaling pathways were involved. LncRNA analysis displayed 47 significantly upregulated lncRNA and 31 downregulated lncRNA comparing PTN depleted DPSCs with the control.Conclusion: Our research demonstrated that PTN has a positive role in maintaining DPSCs proliferation and osteo/dentinogenic differentiation potential.

Rebalancing glucolipid metabolism and gut microbiome dysbiosis by nitrate-dependent alleviation of high-fat diet-induced obesity.

INTRODUCTION: High-fat diet (HFD)-induced obesity is accompanied by compromised nitric oxide (NO) signaling and gut microbiome dysregulation. Inorganic dietary nitrate, which acts as a NO donor, exerts beneficial effects on metabolic disorders. Here, we evaluated the effects of dietary nitrate on HFD-induced obesity and provided insights into the underlying mechanism. RESEARCH DESIGN AND METHODS: To investigate the preventive effect of dietary nitrate on HFD-induced obesity, C57BL/6 mice were randomly assigned into four groups (n=10/group), including normal control diet group (normal water and chow diet), HFD group (normal water and HFD), HFD+NaNO3 group (water containing 2 mM NaNO3 and HFD), and HFD+NaCl group (water containing 2 mM NaCl and HFD). During the experiment, body weight was monitored and glucolipid metabolism was evaluated. The mechanism underlying the effects of nitrate on HFD-induced obesity was investigated by the following: the NO3--NO2--NO pathway; endothelial NO synthase (eNOS) and cyclic guanosine monophosphate (cGMP) levels; gut microbiota via 16SRNA analysis. RESULTS: Dietary nitrate reduced the body weight gain and lipid accumulation in adipose and liver tissues in HFD-fed mice. Hyperlipidemia and insulin resistance caused by HFD were improved in mice supplemented with nitrate. The level of eNOS was upregulated by nitrate in the serum, liver, and inguinal adipose tissue. Nitrate, nitrite, and cGMP levels were decreased in mice fed on HFD but reversed in the HFD+NaNO3 group. Nitrate also rebalanced the colon microbiota and promoted a normal gut microbiome profile by partially attenuating the impacts of HFD. Bacteroidales S24-7, Alistipes, Lactobacillus, and Ruminococcaceae abundances were altered, and Bacteroidales S24-7 and Alistipes abundances were higher in the HFD+NaNO3 group than that in the HFD group. CONCLUSIONS: Inorganic dietary nitrate alleviated HFD-induced obesity and ameliorated disrupted glucolipid metabolism via NO3--NO2--NO pathway activation and gut microbiome modulation.

Bone regeneration capacities of alveolar bone mesenchymal stem cells sheet in rabbit calvarial bone defect.

Mesenchymal stem cells sheets have been verified as a promising non-scaffold strategy for bone regeneration. Alveolar bone marrow mesenchymal stem cells, derived from neural crest, have the character of easily obtained and strong multi-differential potential. However, the bone regenerative features of alveolar bone marrow mesenchymal stem cells sheets in the craniofacial region remain unclear. The purpose of the present study was to compare the osteogenic differentiation and bone defect repairment characteristics of bone marrow mesenchymal stem cells sheets derived from alveolar bone (alveolar bone marrow mesenchymal stem cells) and iliac bone (Lon-bone marrow mesenchymal stem cells) in vitro and in vivo. Histology character, osteogenic differentiation, and osteogenic gene expression of human alveolar bone marrow mesenchymal stem cells and Lon-bone marrow mesenchymal stem cells were compared in vitro. The cell sheets were implanted in rabbit calvarial defects to evaluate tissue regeneration characteristics. Integrated bioinformatics analysis was used to reveal the specific gene and pathways expression profile of alveolar bone marrow mesenchymal stem cells. Our results showed that alveolar bone marrow mesenchymal stem cells had higher osteogenic differentiation than Lon-bone marrow mesenchymal stem cells. Although no obvious differences were found in the histological structure, fibronectin and integrin ß1 expression between them, alveolar-bone marrow mesenchymal stem cells sheet exhibited higher mineral deposition and expression levels of osteogenic marker genes. After being transplanted in the rabbit calvarial defects area, the results showed that greater bone volume and trabecular thickness regeneration were found in bone marrow mesenchymal stem cells sheet group compared to Lon-bone marrow mesenchymal stem cells group at both 4 weeks and 8 weeks. Finally, datasets of bone marrow mesenchymal stem cells versus Lon-bone marrow mesenchymal stem cells, and periodontal ligament mesenchymal stem cells (another neural crest derived mesenchymal stem cells) versus umbilical cord mesenchymal stem cells were analyzed. Total 71 differential genes were identified by overlap between the 2 datasets. Homeobox genes, such as LHX8, MKX, PAX9, MSX, and HOX, were identified as the most significantly changed and would be potential specific genes in neural crest mesenchymal stem cells. In conclusion, the Al-bone marrow mesenchymal stem cells sheet-based tissue regeneration appears to be a promising strategy for craniofacial defect repair in future clinical applications.

Nitrate ameliorates dextran sodium sulfate-induced colitis by regulating the homeostasis of the intestinal microbiota.

Inflammatory bowel disease (IBD) involves chronic inflammation, loss of epithelial integrity, and gastrointestinal microbiota dysbiosis. Effective therapies for IBD have not been established. Accordingly, in this study, we evaluated the effects of inorganic nitrate, a potent nitric oxide (NO) donor and microbiota regulator, in a mouse model of dextran sodium sulfate (DSS)-induced colitis. Mice were pretreated with NaNO3 (2 mM) in their drinking water for 5 days, and NaCl was used as a control. Feces were collected for microbiota analyses. The results showed that oral administration of dietary nitrate could maintained colon consistency, improved colon length, maintained body weight, decreased apoptosis in colon epithelial cells, and ameliorated inflammatory cell infiltration in both the colon and peripheral blood. Microbiota profiling revealed that nitrate regulated dysbiosis. Analysis of the top bacteria at the genus level showed that Bacteroidales_S24-7_group_unidentified, Lactobacillus, Bacteroides, and Prevotellaceae_UCG-001 decreased in the DSS group compared with that in the normal group, whereas Lactobacillus, Ruminococcaceae_UCG-014, and Prevotellaceae_UCG-001 were increased in the DSS + NaNO3 group compared with that in the DSS group. The enriched bacteria in the nitrate group included Gordonibacter, Ureaplasama, and Lachnospiraceae_UCG-006. Moreover, microbiota analysis revealed that nitrate could partially decrease the enriched metabolic pathways (p53 signaling pathway and colorectal cancer pathway) compared with that in the DSS and DSS + NaCl groups. Overall, these findings indicated that nitrate could ameliorate DSS-induced colitis by decreasing inflammation, reducing apoptosis, and regulating the microbiota by activation of the NO3-/NO2-/NO pathway. Nitrate might be a potential treatment for colitis patients in the future clinical application.

Genome-Wide DNA Methylation Analysis during Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BMSCs) nowadays are regarded as promising candidates in cell-based therapy for the regeneration of damaged bone tissues that are either incurable or intractable due to the insufficiency of current therapies. Recent studies suggest that BMSCs differentiate into osteoblasts, and that this differentiation is regulated by some specific patterns of epigenetic modifications, such as DNA methylation. However, the potential role of DNA methylation modification in BMSC osteogenic differentiation is unclear. In this study, we performed a genome-wide study of DNA methylation between the noninduced and induced osteogenic differentiation of BMSCs at day 7. We found that the majority of cytosines in a CpG context were methylated in induced BMSCs. Our results also revealed that, along with the induced osteogenic differentiation in BMSCs, the average genomic methylation levels and CpG methylation in transcriptional factor regions (TFs) were increased, the CpG methylation level of various genomic elements was mainly in the medium-high methylation section, and CpG methylation levels in the repeat element had highly methylated levels. The GO analysis of differentially methylated region- (DMR-) associated genes (DMGs) showed that GO terms, including cytoskeletal protein binding (included in Molecular Function GO terms), skeletal development (included in Biological Process GO terms), mesenchymal cell differentiation (included in Biological Process GO terms), and stem cell differentiation (included in Biological Process), were enriched in the hypermethylated DMGs. Then, the KEGG analysis results showed that the WNT pathway, inositol phosphate metabolism pathway, and cocaine addiction pathway were more correlative with the DMRs during the induced osteogenic differentiation in BMSCs. In conclusion, this study revealed the difference of methylated levels during the noninduced and induced osteogenic differentiation of BMSCs and provided useful information for future works to characterize the important function of epigenetic mechanisms on BMSCs' differentiation.

Music stimuli lead to increased levels of nitrite in unstimulated mixed saliva.

Concentration of salivary nitrate is approximately 10-fold to that of serum. Many circumstances such as acute stress could promote salivary nitrate secretion and nitrite formation. However, whether other conditions can also be used as regulators of salivary nitrate/nitrite has not yet been explored. The present study was designed to determine the influence of exposure to different music on the salivary flow rate and nitrate secretion and nitrite formation. Twenty-four undergraduate students (12 females and 12 males) were exposed to silence, rock music, classical music or white noise respectively on four consecutive mornings. The unstimulated salivary flow rate and stimulated salivary flow rate were measured. Salivary ionic (Na+, Ca2+ Cl-, and PO43-) content and nitrate/nitrite levels were detected. The unstimulated salivary flow rate was significantly increased after classical music exposure compared to that after silence. Salivary nitrite levels were significantly higher upon classical music and white noise stimulation than those under silence in females. However, males were more sensitive only to white noise with regard to the nitrite increase. In conclusion, this study demonstrated that classical music stimulation promotes salivary nitrite formation and an increase in saliva volume was observed. These observations may play an important role in regulating oral function.

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway.

BACKGROUND: Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms. METHODS: SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of ß-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs. RESULTS: SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear ß-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential. CONCLUSIONS: The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

Depletion of SHANK2 inhibited the osteo/dentinogenic differentiation potentials of stem cells from apical papilla.

The aim of this study was to investigate the biological function of SHANK2 on the osteo/dentinogenic differentiation potentials of human stem cells from apical papilla (SCAPs). Real-time RT-PCR was used to detect the expression of SHANK2 in human mesenchymal stem cells (MSCs). Small hairpin RNA (shRNA) was used to knockdown the SHANK2 in SCAPs. The knockdown efficiency was determined by real-time RT-PCR and Western Blot. The in vitro osteo/dentinogenic differentiation potentials of SCAPs were investigated using ALP staining, ALP activity, alizarin red staining, quantitative calcium, the expression levels of DSPP, DMP1, RUNX2 and OSX. In vivo transplantation experiments in immunocompromised mice were used to evaluate the capacity of SCAPs to form bone/dentine-like structure. SHANK2 was highly expressed in dental tissue-derived MSCs compared with cells of other origins. Silencing of SHANK2 inhibited the ALP activity, mineralization, and the expressions of DSPP, DMP1, RUNX2 and OSX in SCAPs. Furthermore, in vivo transplantation experiments indicated that knock-down of SHANK2 in SCAPs generated less bone/dentin-like mineralized tissue compared with the control group. The present study demonstrated that depletion of SHANK2 inhibited the osteo/dentinogenic differentiation potentials in SCAPs, explored the new function of SHANK2, and provided useful information to elucidate the molecular mechanism underlying directed differentiation in dental tissue-derived MSCs.

Analysis of Senescence-Related Differentiation Potentials and Gene Expression Profiles in Human Dental Pulp Stem Cells.

INTRODUCTION: Dental pulp stem cell (DPSC)-mediated dental pulp regeneration is considered a promising method for the treatment of deep caries with pulpitis. However, mesenchymal stem cell (MSC) senescence is an adverse factor from the perspective of cell-based therapies. In this study, we investigated the characteristics and expression profiles of DPSCs from young and old donors. METHODS: DPSCs from young and old donors were cultured in differentiation medium, and their differentiation potentials were assessed. Long noncoding RNA (LncRNA) microarray assays and a bioinformatic analysis were performed to investigate differences in LncRNA and mRNA expression profiles between DPSCs from young and old donors. RESULTS: We found that DPSCs from young donors exhibited more powerful proliferation ability and greater osteogenic and adipogenic differentiation potentials than DPSCs from old donors. In DPSCs from young donors, numerous LncRNAs were significantly up- (n = 389) or down-regulated (n = 172) compared to DPSCs from old donors. Furthermore, 304 mRNAs were differentially expressed, including 247 up-regulated genes and 57 down-regulated genes in DPSCs from young donors. The bioinformatic analysis identified that several pathways may be associated with DPSC characteristics, such as those involved in the cell cycle and RNA transport, and revealed nuclear transcription factor Y subunit ß, general transcription factor IIB, and nuclear receptor subfamily 3 group C member 1 as core regulatory factors and FR249114, FR299091, and ENST00000450004 as core LncRNAs. CONCLUSIONS: Our results indicated that senescence impaired the proliferation and differentiation potentials of DPSCs and that donor age is an important factor that affects their use for tooth regeneration. We also provide insight into the mechanisms responsible for senescence in DPSCs.

miR-21 Modulates the Immunoregulatory Function of Bone Marrow Mesenchymal Stem Cells Through the PTEN/Akt/TGF-ß1 Pathway.

microRNAs (miRNAs) act as regulatory signals for maintaining stemness, self-renewal, and differentiation of mesenchymal stem cells (MSCs), but whether miRNAs modulate the immunoregulatory function of MSCs remains largely unknown. Here, we show that miR-21 negatively regulates the activity of immunoregulatory cytokine transforming growth factor-ß1 (TGF-ß1) in MSCs. Consistently, bone marrow MSCs (BMMSCs) from miR-21(-/-) mice show enhanced immunosuppressive function by more TGF-ß1 secretion and induce more CD4(+) Foxp3(+) regulatory T cells compared with wild-type BMMSCs in vitro, which anti-TGF-ß1 antibody abrogates. Mechanistically, miR-21 inhibits TGF-ß1 expression by targeting phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in BMMSCs. Downstream of PTEN, miR-21 promotes activation of Akt, and consequently increases activation of NF-κB pathway. Importantly, adoptive transfer of miR-21(-/-) BMMSCs into mice with experimental colitis more effectively ameliorates colonic inflammation in a TGF-ß1-dependent manner. Thus, these findings indicate a previously uncovered mechanism of miR-21 control immunoregulatory function of BMMSCs through TGF-ß1 inhibition.

Spatial and temporal expression of c-Kit in the development of the murine submandibular gland.

The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.