Cobalt Chloride as a Hypoxia Mimicking Agent Induced HIF-1α and mTOR Expressions of Human Umbilical Cord Mesenchymal Stem Cells

ABSTRACT Objective: To assess the effects of cobalt chloride (CoCl2) as a hypoxia mimicking agent on human umbilical cord mesenchymal stem cells (hUCMSCs) expression of HIF-1α and mTOR for use in regenerative dentistry. Material and Methods: Human umbilical cord mesenchymal stem cells were isolated and then cultured. The characteristics of stemness were screened and confirmed by flow cytometry. The experiment was conducted on hypoxia (H) and normoxia (N) groups. Each group was divided and incubated into 24-, 48-, and 72-hours observations. Hypoxic treatment was performed using 100 µM CoCl2 on 5th passage cells in a conventional incubator (37°C; 5CO2). Then, immunofluorescence of HIF-1α and mTOR was done. Data was analyzed statistically using One-way ANOVA and Tukey's HSD. Results: Significant differences were found between normoxic and hypoxic groups on HIF-1α (p=0.015) and mTOR (p=0.000) expressions. The highest HIF-1α expression was found at 48 hours in the hypoxia group, while for mTOR at 24 hours in the hypoxia group. Conclusion: Hypoxia using cobalt chloride was able to increase human umbilical cord mesenchymal stem cells expression of HIF-1α and mTOR.

Effect of Lipopolysaccharide-Induced Apical Periodontitis in Diabetes Mellitus Rats on Periapical Inflammation.

OBJECTIVES: To evaluate periapical inflammation through immunohistochemical analysis of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-a) expression resulting from lipopolysaccharide (LPS)-induced apical periodontitis in diabetes mellitus rats, observed at 14, 28, and 42 days. MATERIALS AND METHODS: Diabetes model on rats was induced by streptozotocin (STZ). Fifteen rats were injected with low-dose STZ for 5 days and waited for 5 days until the blood glucose level was stable and measured above 300 mg/dL confirmed by a digital glucometer. LPS was used to induce apical periodontitis. After performing access cavity, pulpal and root canal extirpation was done on the right mandibular first molar's root canal space of rats, under anesthesia. LPS of 1 mg/mL dose was induced in the pulpal and root canal space. Apical periodontitis was expected 14 days afterward and then, the rats were randomly allocated to three groups. The first group was terminated 14 days after induction and used as control. The second group was observed 28 days after induction, and the third group was observed 42 days after induction. IL-6 and TNF-a expression was analyzed by immunohistochemistry on macrophages in the periapical area. STATISTICAL ANALYSIS: Data were analyzed using one-way ANOVA and continued with the post hoc Tukey HSD test. Significance was considered if p < 0.05. RESULTS: LPS induced apical periodontitis in diabetes mellitus rats at control (14 days), 28 and 42 days observation showed a significant increase in the expression of IL-6 and TNF-a. There were significant differences between the control and observed groups (p < 0.05). The expression of IL-6 in the apical area was not significant at 14 and 28 days (p > 0.05) but increased significantly at 42 days (p < 0.05). The expression of TNF-a in the apical area was significantly increased after 14 days (p < 0.05) and remained stable at 28 and 42 days (p > 0.05). CONCLUSIONS: The periapical inflammation of LPS-induced apical periodontitis in diabetes mellitus rats increased macrophages' expression of IL-6 at 42 days and TNF-a at 28 days.