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.

Calcium Hydroxide Increases Human Umbilical Cord Mesenchymal Stem Cells Expressions of Apoptotic Protease-Activating Factor-1, Caspase-3 and Caspase-9.

PURPOSE: Calcium hydroxide is a gold standard dental material generally used for pulpal and periapical therapy including regenerative endodontic procedures because of its positive properties. However, evaluation about this material on stem cells is limited. Human umbilical cord mesenchymal stem cells (HUCMSCs) are potential to be used in regenerative therapy. Regenerative therapy needs a sustainable cell supply to maintain its regenerative capacity. The aim of this study was to ascertain the apoptosis result of calcium hydroxide on HUCMSCs through the expression of apoptotic protease-activating factor-1 (APAF-1), caspase-3, and caspase-9. MATERIALS AND METHODS: This study used a thawed frozen stock of passage 5 HUCMSCs, grown in minimum essential medium (MEM) alpha containing calcium hydroxide at concentration of 0.1 microgram/mL for 1, 3 and 7 days. Polyclonal antibody with fluorescence isothiocyanate (FITC) label was used to evaluate the expressions. APAF-1, caspase-3, and caspase-9 expressions were recorded and compared on every observation day using fluorescence microscope. Analysis of variance was performed to analyze the significance among the results of treatment groups. The results were concluded significant if p<0.05. RESULTS: The addition of calcium hydroxide in MEM alpha medium increases HUCMSCs expression of APAF-1, caspase-3 and caspase-9 significantly, compared to the control group without calcium hydroxide (p<0.05) in all the times. Day 1 showed the lowest increase followed by higher expressions on day 3 and day 7. CONCLUSION: HUCMSCs express increased APAF-1, caspase-3 and caspase-9 after in-vitro calcium hydroxide exposure. This should be considered when using calcium hydroxide on HUCMSCs for regenerative procedures with regard to other positive properties.

Lipopolysaccharides' Cytotoxicity on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To show the cytotoxicity of Porphyromonas gingivalis lipopolysaccharide (LPS) on human umbilical cord mesenchymal stem cells (HUCMSCs) to better understand the characteristics for its application in regenerative procedures under periodontopathogen LPS influence. Material and Methods: Ultrapure Porphyromonas gingivalis LPS was used in this study. This research used a frozen stock HUCMSCs, previously confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSCs were cultured and divided into two groups, the control group and LPS group with various concentrations from 25 to 0.39 µg/mL. MTT assay was done and the cells were observed and counted. The significance level was set at 5%. Results: The percentage of living HUCMSCs on LPS group were not significantly different among concentrations (p>0.05) from 25 to 0.39 µg/mL, even though there were slight mean decrease between groups, but they were not significant. The duration of 24 hours of exposure of LPS does not significantly lower HUCMSCs viability. Conclusion: LPS does not affect the viability of HUCMSCs. The lower the concentration of LPS, the higher the viability of HUCMSCs.

Cytotoxicity of Calcium Hydroxide on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To examine the cytotoxicity of calcium hydroxide on human umbilical cord mesenchymal stem cells (HUCMSC) to understand the characteristics for use in regenerative dentistry procedures especially regenerative endodontics. Material and Methods: HUCMSC was isolated, cultured, and confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSC was cultured and divided into two groups, the control group (cultured in minimum essential medium (MEM) alpha) and calcium hydroxide group (cultured in MEM alpha and calcium hydroxide). Methyl-thiazole-tetrazolium (MTT) assay was done on different concentrations of calcium hydroxide (0.39 to 25 µg/mL) and the cells were observed and counted. One-way ANOVA test was used with a significance level set at 5%. Results: Flow cytometric analysis confirmed positive of CD73, CD90, CD105, negative of CD45 and CD34. A significant difference was found between the concentration of 6.25 and 3.125 µg/mL (p=0.004). There was no significant difference among 6.25, 12.5 and 25 µg/mL concentrations. There was also no significant difference among 0.39, 0.78, 1.56, and 3.125 µg/mL concentrations. Conclusion: Even though calcium hydroxide is a medicament of choice in clinical endodontics, it decreases the viability of HUCMSC. The lower the concentration of calcium hydroxide, the higher the viability of HUCMSC.

The Comparative Toxicity of Xanthones and Tannins in Mangosteen (Garcinia mangostana Linn.) Pericarp Extract against BHK-21 Fibroblast Cell Culture.

OBJECTIVE: The objective of this study is to compare the toxicity level of xanthones and tannins derived from mangosteen pericarp extract at specific concentrations against BHK-21 fibroblast cell cultures. METHODS: Mangosteen was extracted using a maceration method with ethanol 96%. Xanthones were isolated from the chloroform extract, whereas tannins were isolated using acetone alcohol and serial diluted to 100% concentration. Toxicity levels were monitored after 24 h using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay technique by ELISA reader at 620 nm. RESULTS: Viable cells of BHK-21 against xanthone concentration began to decrease (40.24%) at 3.98% xanthones, whereas viable cells of BHK-21 against tannin concentration began to decrease (68.06%) at 2.2% tannins. CONCLUSION: It is suggested that tannins were more toxic than the xanthones derived from mangosteen pericarp.