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

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.

Effect of Nicotine and Porphyromonas gingivalis on the Differentiation Properties of Periodontal Ligament Fibroblasts.

OBJECTIVES: This study aimed to evaluate the effect of Porphyromonas gingivalis and nicotine on the in vitro osteogenic differentiation of periodontal ligament (PDL) fibroblasts. MATERIALS AND METHODS: PDLs were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C under 5% CO2 and 100% humidified atmosphere. Cells were incubated with various concentrations of nicotine and P. gingivalis extracts, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. To study cell differentiation, PDLs (5 × 104cells) were treated with the osteogenic differentiation medium containing 10 mM ß-glycerophosphate, 10 nM dexamethasone, 50 mg/mL ascorbic acid, 1 µM nicotine, and 50 µg/mL P. gingivalis lysate. mRNA samples were collected at 0, 7, and 14 days. Odontogenic-related gene expression, namely, Runt-related transcription factor 2 (Runx2), collagen type I (COL1A1), and alkaline phosphatase (ALP) was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Calcified nodule formation was determined on day 28 using Alizarin Red S. Analysis of variance and Tukey's test were used to compare the difference among groups at significant level of p < 0.05. RESULTS: It showed that 50 µg/mL of P. gingivalis lysate and 1 µM of nicotine showed no toxicity to PDLs. Runx2, COL1A1, and ALP expression were found to decrease significantly after 7 days of treatment, while osteocalcin expression was found to decrease after 14 days. The nodule formation in the control group was much greater in both number and size of nodules than in experimental groups, which implied a positive sign of calcium deposition in controls. CONCLUSION: The results indicated that nicotine and P. gingivalis showed adverse effect on osteogenic differentiation properties of PDLs.

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.