Stem cell-derived exosomes from human exfoliated deciduous teeth promote angiogenesis in hyperglycemic-induced human umbilical vein endothelial cells

Abstract Objective To investigate the angiogenesis in human umbilical vein endothelial cells (HUVEC) under high glucose concentration, treated with exosomes derived from stem cells from human exfoliated deciduous teeth (SHED). Methodology SHED-derived exosomes were isolated by differential centrifugation and were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometric assays. We conducted in vitro experiments to examine the angiogenesis in HUVEC under high glucose concentration. Cell Counting Kit-8, migration assay, tube formation assay, quantitative real-time PCR, and immunostaining were performed to study the role of SHED-derived exosomes in cell proliferation, migration, and angiogenic activities. Results The characterization confirmed SHED-derived exosomes: size ranged from 60-150 nm with a mode of 134 nm, cup-shaped morphology, and stained positively for CD9, CD63, and CD81. SHED-exosome significantly enhanced the proliferation and migration of high glucose-treated HUVEC. A significant reduction was observed in tube formation and a weak CD31 staining compared to the untreated-hyperglycemic-induced group. Interestingly, exosome treatment improved tube formation qualitatively and demonstrated a significant increase in tube formation in the covered area, total branching points, total tube length, and total loop parameters. Moreover, SHED-exosome upregulates angiogenesis-related factors, including the GATA2 gene and CD31 protein. Conclusions Our data suggest that the use of SHED-derived exosomes potentially increases angiogenesis in HUVEC under hyperglycemic conditions, which includes increased cell proliferation, migration, tubular structures formation, GATA2 gene, and CD31 protein expression. SHED-exosome usage may provide a new treatment strategy for periodontal patients with diabetes mellitus.

Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells

Abstract Objectives Human dental pulp stem cells (DPSCs) have been used to regenerate damaged nervous tissues. However, the methods of committing DPSCs into neural stem/progenitor cells (NSPCs) or neurospheres are highly diverse, resulting in many neuronal differentiation outcomes. This study aims to validate an optimal protocol for inducing DPSCs into neurospheres and neurons. Methodology After isolation and characterization of mesenchymal stem cell identity, DPSCs were cultured in a NSPC induction medium and culture vessels. The durations of the culture, dissociation methods, and passage numbers of DPSCs were varied. Results Neurosphere formation requires a special surface that inhibits cell attachment. Five-days was the most appropriate duration for generating proliferative neurospheres and they strongly expressed Nestin, an NSPC marker. Neurosphere reformation after being dissociated by the Accutase enzyme was significantly higher than other methods. Passage number of DPSCs did not affect neurosphere formation, but did influence neuronal differentiation. We found that the cells expressing a neuronal marker, β-tubulin III, and exhibiting neuronal morphology were significantly higher in the early passage of the DPSCs. Conclusion These results suggest a guideline to obtain a high efficiency of neurospheres and neuronal differentiation from DPSCs for further study and neurodegeneration therapeutics.

Odontogenic gene expression profile of human dental pulp-derived cells under high glucose influence: a microarray analysis

Abstract Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application. Objective Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved. Methodology hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction. Results Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine-cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1). Conclusions The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.

High glucose affects proliferation, reactive oxygen species and mineralization of human dental pulp cells

Abstract Diabetes is a group of metabolic disorders that can lead to damage and dysfunction of many organs including the dental pulp. Increased inflammatory response, reduction of dentin formation and impaired healing were reported in diabetic dental pulp. Hyperglycemia, which is a main characteristic of diabetes, was suggested to play a role in many diabetic complications. Therefore our aim was to investigate the effects of high glucose levels on proliferation, reactive oxygen species (ROS) production and odontogenic differentiation of human dental pulp cells (HDPCs). HDPCs were cultured under low glucose (5.5mM Glucose), high glucose (25 mM Glucose) and mannitol (iso-osmolar control) conditions. Cell proliferation was analyzed by MTT assay for 11 days. Glutathione and DCFH-DA assay were used to assess ROS and antioxidant levels after 24 h of glucose exposure. Odontogenic differentiation was evaluated and quantified by alizarin red staining on day 21. Expression of mineralization-associated genes, which were alkaline phosphatase, dentin sialophosphoprotein and osteonectin, was determined by RT-qPCR on day 14. The results showed that high glucose concentration decreased proliferation of HDPCs. Odontogenic differentiation, both by gene expression and mineral matrix deposit, was inhibited by high glucose condition. In addition, high DCF levels and low reduced glutathione levels were observed in high glucose condition. However, no differences were observed between mannitol and low glucose conditions. In conclusion, the results clearly showed the negative effect of high glucose condition on HDPCs proliferation and differentiation. Moreover, it also induced ROS production of HDPCs.

Resumo O diabetes abrange um grupo de distúrbios metabólicos que podem levar a danos e disfunções de muitos órgãos, incluindo a polpa dentária. Aumento da resposta inflamatória, redução da formação de dentina e comprometimento da cicatrização foram relatados na polpa dentária diabética. A hiperglicemia, que é uma característica determinante do diabetes, desempenha um papel importante em muitas complicações diabéticas. Portanto, nosso objetivo foi investigar os efeitos dos altos níveis de glicose na proliferação, produção de espécies reativas de oxigênio (ROS, em inglês) e diferenciação odontogênica das células da polpa dental humana (HDPCs, em inglês). As HDPCs foram cultivadas em condições de baixa glicose (glicose 5,5 mM), alta glicose (glicose 25 mM) e manitol (controle iso-osmolar). A proliferação celular foi analisada pelo ensaio MTT por 11 dias. Glutationa e DCFH-DA foram utilizados para avaliar os níveis de ROS e antioxidantes após 24 h de exposição à glicose. A diferenciação odontogênica foi avaliada e quantificada pela coloração com vermelho de alizarina no dia 21. A expressão de genes associados à mineralização, que eram fosfatase alcalina, sialofosfoproteína de dentina e osteonectina, foi determinada por RT-qPCR no dia 14. Os resultados mostraram que a alta concentração de glicose diminuiu a proliferação de HDPCs. A diferenciação odontogênica, tanto pela expressão gênica quanto pelo depósito da matriz mineral, foi inibida pela condição de alta glicose. Além disso, altos níveis de DCF e níveis reduzidos de glutationa foram observados na condição de alta glicose. No entanto, não foram observadas diferenças entre o manitol e as condições de baixa glicose. Em conclusão, os resultados mostraram claramente o efeito negativo da condição de alta glicose na proliferação e diferenciação de HDPCs. Além disso, essa condição também induziu a produção de ROS em HDPCs.

Quinone reductase inducers in Azadirachta indica A. Juss flowers, and their mechanisms of action.

We have previously shown that the flowers of neem tree (Azadirachta indica A. Juss, family Meliaceae), Thai variety, strongly induced the activity of glutathione S-transferase (GST) while resulting in a significant reduction in the activities of some cytochrome P(450)-dependent monooxygenases in rat liver, and possess cancer chemopreventive potential against chemically-induced mammary gland and liver carcinogenesis in rats. In the present study, 2 chemicals possessing strong QR inducing activity were fractionated from neem flowers using a bioassay based on the induction of QR activity in mouse hepatoma Hepa 1c1c7 cultured cells. Spectroscopic characteristics revealed that these compounds were nimbolide and chlorophylls, having CD (concentration required to double QR specific activity) values of 0.16 and 3.8 mug/ml, respectively. Nimbolide is a known constituent of neem leaves, but was found for the first time here in the flowers. Both nimbolide and chlorophylls strongly enhanced the level of QR mRNA in Hepa 1c1c7 cells, as monitored by northern blot hybridization, indicating that the mechanism by which these constituents of neem flowers induced QR activity is the induction of QR gene expression. These findings may have implication on cancer chemopreventive potential of neem flowers in experimental rats previously reported.