Effect of Incorporation of Zeolite Containing Silver-Zinc Nanoparticles into Mineral Trioxide Aggregate on Odontogenic Activity of Human Dental Pulp Stem Cells.

STATEMENT OF THE PROBLEM: The stimulation of odontogenic activity is considered an essential property for biomaterials used in vital pulp therapy. PURPOSE: The present study aimed to evaluate the effect of the incorporation of zeolite containing silver-zinc nanoparticles (Ze-Ag-Zn) into Angelous mineral trioxide aggregate (AMTA) on the odontogenic activity of human dental pulp stem cells (HDPSCs). MATERIALS AND METHOD: In this in vitro study, HDPSCs were treated with 2% wt of synthesized Ze-Ag-Zn particles+AMTA, AMTA and Ze-Ag-Zn disks. The negative control cells did not receive any treatment. Then, cell viability was measured using the MTT assay after 7 and 14 days of the treatment course. The alkaline phosphatase (ALP) activity and calcium ion level were also measured in the supernatant culture media using auto-analyzer kits. The obtained data were analyzed using one-way ANOVA and Student t-test where appropriate. The level of the statistical significance was set at p< 0.05. RESULTS: The results indicated that HDPSCs treated with AMTA and AMTA+Ze-Ag-Zn particles did not show any significant cell death compared with the control cells after 14 days of the treatment course while the ALP activity and calcium ion levels were significantly (p< 0.05) elevated. Also, the addition of AMTA particles to the cell culture media resulted in increased ALP activity and calcium ion level compared with HDPSCs treated with AMTA+ Ze-Ag-Zn particles on day 7 of the treatment course (p< 0.05). CONCLUSION: It seems that the incorporation of Ze-Ag-Zn particles into AMTA did not have any significant positive effect on the biomineralization properties of AMTA.

Correction to: Interaction of opioid with insulin/IGFs signaling in Alzheimer's disease.

The original version of this article unfortunately contained mistake in the Author Group section. Reza Rahbarghazi's family name was inadvertently spelled as "Rahbarghzi".

High Glucose Content Abrogated the Normal Activity of Heat Shock Protein Signaling Pathway in Human Neuroblastoma Cells.

BACKGROUND: Detrimental effects of high glucose content (HGC) were proved in different tissues such as the central nervous system. It seems that diabetic conditions could also alter the functional behavior of stem cells residing in the context of the nervous system. METHODS: The possible effects of 40 and 70 mmol glucose were examined on HSP70 signaling pathways with a specific focus on protein translation, folding values of human neuroblastoma cell line SHSY-5Y after 72 h. Human neuroblastoma cells were exposed to 5, 40 and 70 mmol glucose doses. The transcription level of genes related to HSP70 signaling was also evaluated by PCR array. RESULTS: The data from PCR array showed high glucose especially 70 mmol could potentially modulate the normal function of protein folding, endoplasmic reticulum derived protein folding and synthesis in neuroblastoma cells (p <0.05). CONCLUSIONS: Data showed that high glucose condition makes neuroblastoma cells prone to biochemical insufficiency by affecting the function of HSP70 signaling pathway and protein synthesis.

Interaction of opioid with insulin/IGFs signaling in Alzheimer's disease.

Alzheimer's disease is associated with biochemical and histopathological changes characterized by molecular abnormalities. Due to the lack of effective treatments for Alzheimer's disease, many attempts have been made to find potential therapies to reduce or even return neuronal loss after disease initiation. Alzheimer's disease is also touted as type III diabetes, showing an association with insulin signaling. The large distribution of the insulin receptor on the cell surface and its regulatory role in the central nervous system suggests that the pathogenesis of Alzheimer's disease could be ascribed to insulin signaling. The interference of opioids, such as morphine with insulin signaling pathways, is thought to occur via direct crosstalk between the signaling pathways of the insulin receptor and the mu-opioid receptor. In this review article, we discuss the possible crosstalk between the mu-opioid receptor and insulin signaling pathways. The association of these two signaling pathways with Alzheimer's disease is also debated.

In vitro induction of odontogenic activity of human dental pulp stem cells by white Portland cement enriched with zirconium oxide and zinc oxide components.

Background . The aim of this in vitro study was to investigate the effect of zinc oxide (ZnO) and zirconium oxide (ZrO2) microparticles (MPs) and nanoparticles (NPs) in combination with white Portland cement (WPC) on odontogenic capacity of human dental pulp stem cells over a period of 21 days. Methods . Synthesized ZnO and ZrO2 particles were characterized using scanning electron microscopy and transmission electron microscopy. The viability of human dental pulp stem cells was measured by a 3-(4,5-dimethylthiazolyl-2-yl)-2,5- diphenyltetrazolium bromide assay at 7-, 14- and 21-day intervals after seeding on WPC disks enriched with ZnO and ZrO2 MPs and NPs. Odontogenic potential of ZnO and ZrO2 particles in combination with WPC was investigated by alkaline phosphatase (ALP) activity and ionized calcium level of supernatant culture media at different time intervals. Data were analyzed using one-way ANOVA and post hoc Tukey tests. Results . All the materials exhibited cell viability over a 21-day period, except for WPC with ZnO NPs on day 7, although it was not statistically significant (P>0.05). The ALP activity and ionized calcium level increased in all the groups compared to the control group (P<0.05). ZnO NPs had superior effect on odontogenic activity and calcium ion release compared to ZnO MPs (P=0.046). There was no significant difference between ZrO2 MPs and NPs in odontogenic activity (P>0.05). Conclusion . WPC enriched with ZnO and ZrO2 increased ALP activity and calcium ion release of human dental pulp stem cells over a period of 21 days in vitro.

Treatment of human neuroblastoma cell line SH-SY5Y with HSP27 siRNA tagged-exosomes decreased differentiation rate into mature neurons.

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2'-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.

Epigenetic mechanisms are behind the regulation of the key genes associated with the osteoblastic differentiation of the mesenchymal stem cells: The role of zoledronic acid on tuning the epigenetic changes.

Mesenchymal stem cells (MSCs) are multipotent stem cells and show distinct features such as capability for self-renewal and differentiation into several lineages of cells including osteoblasts, chondrocytes, and adipocytes. In this study, the methylation status of the promoter region of zinc finger and BTB domain containing 16 (ZBTB16), twist-related protein 1(Twist1), de novo DNA methyltransferases 3A (DNMT3A), SRY-box 9 (Sox9), osteocalcin (OCN), and peroxisome proliferator-activated receptor γ2 (PPARγ2) genes and their messenger RNA (mRNA) expression levels were evaluated during the osteoblastic differentiation of MSCs (ODMSCs). We planned two experimental groups including zoledronic acid (ZA)-treated and nontreated cells (negative control) which both were differentiated into the osteoblasts. Methylation level of DNA in the promoter regions was assayed by methylation-specific-quantitative polymerase chain reaction (MS-qPCR), and mRNA levels of the target inhibitory/stimulatory genes during osteoblastic differentiation of MSCs were measured using real-time PCR. During the experimental induction of ODMSCs, the mRNA expression of the OCN gene was upregulated and methylation level of its promoter region was decreased. Moreover, Sox9 and PPARγ2 mRNA levels were attenuated and their promoter regions methylation levels were significantly augmented. However, the mRNA expression of the DNMT3A was not affected during the ODMSCs though its methylation rate was increased. In addition, ZA could enhance the expression of the ZBTB16 and decrease its promoter regions methylation and on the opposite side, it diminished mRNA expression of Sox9, Twist1, and PPARγ2 genes and increased their methylation rates. Intriguingly, ZA did not show a significant impact on gene expression and methylation levels the OCN and DNMT3A. We found that methylation of the promoter regions of Sox9, OCN, and PPARγ2 genes might be one of the main mechanisms adjusting the genes expression during the ODMSCs. Furthermore, we noticed that ZA can accelerate the MSCs differentiation to the osteoblast cells via two regulatory processes; suppression of osteoblastic differentiation inhibitor genes including Sox9, Twist1, and PPARγ2, and through promotion of the ZBTB16 expression.

Gene expression of TWIST1 and ZBTB16 is regulated by methylation modifications during the osteoblastic differentiation of mesenchymal stem cells.

BACKGROUND: Osteoblastic differentiation of mesenchymal stem cells (MSCs) is the principal stage during the restoration and regeneration of bone tissue. Epigenetic modifications such as DNA methylation play a key role in the differentiation process of stem cells. In this study, the methylation status of the promoter region of ZBTB16 and Twist1 genes and their role in controlling osteoblastic differentiation in MSCs was investigated during the osteoblastic differentiation of MSCs. METHODS: The MSCs were cultured under standard conditions and differentiated into the osteoblasts. We had three treatment groups including 5-azacytidine (methylation inhibitor), metformin (Twist-inhibitor), and procaine (Wnt/ß-catenin inhibitor) and a non-treated group (control). Methylation level of DNA in the promoter regions was monitored by methylation specific-quantitative polymerase chain reaction (PCR). Also, the mRNA levels of key genes in osteoblastic differentiation were measured using real-time PCR. RESULTS: ZBTB16 gene expression was upregulated, and promoter methylation was decreased. For Twist1 messenger RNA (mRNA) level decreased and promoter methylation increased during osteoblastic differentiation of MSCs. 5-Azacytidine caused a significant reduction in methylation and increased the mRNA expression of ZBTB16 and Twist1. Metformin repressed the Twist1 expression, and therefore osteoblastic differentiation was increased. On the opposite side, procaine could block the WNT/ß-catenin signaling pathway, as a consequence the gene expression of key genes involved in osteoblastic differentiation was declined. CONCLUSION: We found that methylation of DNA in the promoter region of ZBTB16 and Twist1 genes might be one of the main mechanisms that controlling the gene expression during osteoblastic differentiation of MSCs. Also, we could find an association between regulation of Twist1 and ZBTB16 genes and osteoblastic differentiation in MSCs by showing the relation between their expression and some key genes involved in osteoblastic differentiation. In addition, we found a connection between the Twist1 expression level and osteoblastic differentiation by using a Twist-inhibitor (metformin).

Mesenchymal stem cells as the game-changing tools in the treatment of various organs disorders: Mirage or reality?

Recently a growing attention in scientific community has been gathered on potential application of mesenchymal stem cells (MSCs) in various fields of medicine. Owing to the fact that they can be easily isolated from different sources, and simply proliferated in large quantities while keeping their original biological characteristics, they can be successfully used as cell-based therapeutics. Engineering MSCs and other type of stem cells to be carriers of therapeutic agents is a new tactic in the targeted gene and cell therapy of cancers and degenerative diseases. Various useful properties of MSCs including tropism toward tumor/injury site(s), weakly immunogenic, production of anti-inflammatory molecules, and safety against normal tissues have made them prone for regenerative medicine, targeted therapy and treating injured tissues, and immunological abnormalities. In this review, we introduce latest advances, methods, and applications of MSCs in gene therapy of various malignant organ disorders. Additionally, we will cover the problems and challenges which researchers have faced with when trying to translate their basic experimental findings in MSCs research to clinically applicable therapeutics.

Impact of morphine on the expression of insulin receptor and protein levels of insulin/IGFs in rat neural stem cells.

Alzheimer's disease is correlated with neuronal degeneration and loss of neuronal precursors in different parts of the brain. It has been found disturbance in the homeostasis neural stem cells (NSCs) can cause neurodegeneration. Morphine, an analgesic agent, can disrupt the dynamic and normal state of NSCs. However, more investigations are required to clearly address underlying mechanisms. The current experiment aimed to investigate the effects of morphine on the cell distribution of insulin factor and receptor and insulin-like growth factors (IGF1, IGF2) in NSCs. NSCs were isolated from rats and stemness feature confirmed by antibodies against nestin and Sox2. The cells were exposed to 100µM morphine, 50µM naloxone and combination of these two drugs for 72h. The neural cell growth, changes in levels of insulin and insulin-like growth factors secreted by NSCs as well as the insulin-receptor-gene expression were assessed by flow cytometry, ELlSA, and real-time PCR, respectively. Cell cycle assay revealed the exposure of cells to morphine for 72h increased cell apoptosis and decreased neural stem cell growth. The biosynthesis of insulin, insulin-like growth factors, and insulin receptor were reduced (p<0.05) after NSCs exposure to morphine at the concentration of 100µM for 24, 48 and 72h. Naloxone is a competitive antagonist which binds MOR where morphine (and endogenous opioids) bind, and reversed the detrimental effects of morphine. It can be concluded that morphine initiated irregularity in NSCs kinetics and activity by reducing the secretion of insulin and insulin-like growth factors and down-regulation of insulin receptor.