Influence of Hypoxic Condition on Cytotoxicity, Cellular Migration, and Osteogenic Differentiation Potential of Aged Periodontal Ligament Cells.

OBJECTIVE: This study aimed to investigate and compare the influence of hypoxic conditions on cytotoxicity, cellular migration, and osteogenic differentiation of aged periodontal ligament (PDL) cells. MATERIALS AND METHODS: Isolated human PDL cells from aged and young subjects were cultured under hypoxic conditions, which were treated with hydrogen peroxide (H2O2) (0, 25, 50, 100, 200, and 500 µM). To assess cytotoxicity, lactate dehydrogenase release was determined by the optical density at 490 nm, and the percentage of cell death was calculated. An in vitro wound healing assay was performed over 24 to 48 hours for cellular migration determination. Osteogenic differentiation was determined by alizarin red staining and osteogenic gene expression, including the expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN). RESULTS: There was a significant difference in the percentage of cell death with high hypoxic condition (200 and 500 µM) compared to low hypoxic conditions on both day 1 and 2. The highest cellular migration was depicted at 50 µM in both young and aged groups of the in vitro wound healing assay. Osteogenic gene expression of RUNX2 in the aged group was increased at 25 and 50 µM hypoxic condition at day 7, but the expression was gradually decreased after 14 days. On the contrary, the expression of ALP and OPN in the aged group was increased at day 14. Only OPN had been found to be statistically significantly different when compared with gene expression at day 7 and 14 (p < 0.05). The results showed no statistically significant differences when compared with the young and aged groups in all genes and all concentrations. CONCLUSION: The concentration of low hypoxic condition (25-50 µM) was proposed to promote cell viability, cellular migration, and osteogenic differentiation in aged PDL cells. We suggested that the potential of aged PDL cells for use in cell therapy for periodontal regeneration might possibly be similar to that of young PDL cells.

Induction of Migration and Collagen Synthesis in Human Gingival Fibroblasts Using Periodontal Ligament Stem Cell Conditioned Medium.

OBJECTIVE: This study aimed to examine the effect of periodontal ligament stem cell conditioned medium (PDLSC-CM) on human gingival fibroblast (HGF) migration and collagen synthesis. MATERIALS AND METHODS: To assess cell viability, we extracted PDLSC-CM, and the total derived protein concentration was adjusted to 12.5 to 200 µg/mL, followed by treatment with HGFs. The viability of HGFs was observed for 24 hours using the MTT assay. Cell migration was monitored for 24 to 48 hours by wound healing and Boyden chamber assays. Collagen synthesis from HGFs was examined by picrosirius red dye and real-time polymerase chain reaction (PCR) to measure collagen type I and III gene expression for 7 to 10 days. A comparison among the groups was assessed using a one-way analysis of variance (ANOVA) and Bonferroni post hoc test, with the exception of the cell viability assay, which was subjected to Welch's test and Dunnett's T3 post hoc test. RESULTS: HGF viability was significantly enhanced by 12.5, 25, and 50 µg/mL PDLSC-CM. The HGFs treated with 50 µg/mL PDLSC-CM promoted cell migration as shown by wound healing and Boyden chamber assays. At this concentration, collagen synthesis increased at 10 days. Collagen type I gene expression increased by 1.6-fold (p < 0.001) and 4.96-fold (p < 0.001) at 7 and 10 days, respectively. Collagen type III gene expression showed an increase of 1.76-fold (p < 0.001) and 6.67-fold (p < 0.001) at the same time points. CONCLUSION: Our study suggested that a low concentration of PDLSC-CM at 50 µg/mL has given an amelioration of HGFs providing for periodontal wound healing and periodontal regeneration, particularly migration and collagen synthesis.

Salivary Oxidative Stress Biomarkers in Thai Adolescents and Young Adults with Type 1 Diabetes Mellitus: A Cross-Sectional Study.

Aims and Objectives: The primary objectives of this study were to compare salivary oxidative stress (OS) biomarker levels in patients with type 1 diabetes mellitus (T1DM) and without T1DM (non-T1DM) and evaluate the relationships between diabetes, periodontal status, and OS biomarker levels. Materials and Methods: Twenty patients with T1DM and 20 age-matched patients without T1DM were enrolled. All participants were 15-23 years of age and had permanent dentition. Unstimulated whole saliva was collected in a sterile test tube before examination of clinical periodontal parameters, including bleeding on probing (BOP). Salivary levels of OS biomarkers-malondialdehyde, protein carbonyl, total oxidant status (TOS), and total antioxidant capacity-were determined using oxidative and antioxidative assays followed by spectrophotometric measurement at 375-532 nm. The relationships between diabetes, periodontal status, and OS biomarkers were analyzed using multiple linear regression. Results: TOS was significantly lower in the T1DM group compared with the non-T1DM group (5.06 ± 0.39 vs. 6.44 ± 0.51 µmol H2O2 Eq/l, P = 0.035). After adjusting for confounding factors (age, gender, BMI, clinical periodontal parameters, BOP, or diabetes status accordingly), the multiple linear regression showed that T1DM was significantly associated with a reduction of TOS level (P = 0.008). The BOP > 30% group showed a significant correlation with increased TOS levels compared with the BOP ≤ 30% group (P = 0.002). No relationship was found between OS biomarkers and HbA1c levels. Conclusion: Salivary TOS levels were related to both diabetes status and the extent of gingival inflammation. Further studies to elucidate the role of OS in relation of periodontal disease and T1DM are required.

Upregulation of microRNA-223 expression in gingival crevicular blood of women with gestational diabetes mellitus and periodontitis.

Background/Purpose: MicroRNA-223 (miR-223) is involved in several inflammatory diseases, including gestational diabetes mellitus (GDM) and periodontitis. We first described a procedure for purifying miR-223 from gingival crevicular blood (GCB) of pregnant women with or without GDM and periodontitis. This study aimed to determine whether GDM and/or periodontitis modifies miR-223 expression in pregnant women and to analyze miR-223-targeted messenger RNA (mRNA) expression levels in GCB compared to peripheral blood (PB). Materials and methods: Pregnant women were allocated to 4 groups: 10 women with GDM and periodontitis (GDM/P), 10 women with GDM without periodontitis (GDM/NP), 9 women with periodontitis and without GDM (NGDM/P) and 10 women without either condition (NGDM/NP). Clinical parameters of GDM and periodontal status were examined. GCB and PB were collected to assess miR-223, ICAM-1, IL-1ß and ß1-integrin gene expression by quantitative real-time polymerase chain reaction. Results: The GDM/P group demonstrated the highest miR-223 expression levels among the 4 groups in GCB. A significant difference was found between GDM/P and GDM/NP group (P = 0.04). In contrast, the GDM/P showed the lowest miR-223 expression level in PB among the 4 groups. Moreover, ICAM-1 and IL-1ß mRNA expression exhibited the opposite trend of miRNA-223, indicating that miRNA-223 might regulate the mRNA function of those genes by epigenetic events. Conclusion: The upregulation of miR-223 expression in GCB but downregulation in PB, ICAM-1 and IL-1ß genes expression in women with GDM and periodontitis suggest a promising role of miR-223 in the association between GDM and periodontitis.

Association between gestational diabetes mellitus and periodontitis via the effect of reactive oxygen species in peripheral blood cells.

BACKGROUND: Periodontitis (P) has emerged as a risk factor for gestational diabetes mellitus (GDM) through immune cell function alterations, elevated proinflammatory mediators, and increased reactive oxygen species (ROS). The main objective of present study was to determine associations between pregnancy with and without GDM and P. The secondary objective was to compare ROS production in peripheral blood cells (PBCs) of pregnant women with and without GDM. METHODS: This cross-sectional case-control study included 128 pregnant women: 64 with and 64 without GDM. All participants were examined for clinical parameters of GDM and periodontal conditions. Associations between GDM-related periodontal data and GDM risk were evaluated by multiple logistic regression. PBCs were isolated and cultured. ROS productions in each PBCs types was investigated by flow cytometry with ROS antibodies. RESULTS: P was significantly more prevalent in pregnant women with GDM than in those without GDM (57.8% versus 37.5%), with an odds ratio (OR) of 2.28, and a 95% confidence interval (CI) of 1.12 to 4.64 (P = 0.022). The OR (95% CI) was 2.59 (1.19 to 5.65) (P = 0.017) after adjusting for potential confounding factors, including diabetes mellitus (DM) family history, age ≥30 years, body mass index, and maternal age. ROS levels in all PBCs types were significantly higher in the GDM than in the non-GDM group (P < 0.05). CONCLUSION: This study supported the association between P and GDM and indicated that P may be a risk factor for GDM. High levels of ROS production in the PBCs of pregnant women with GDM emphasized the association with GDM.

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

Objectives: The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-ß (RANK), receptor activator of nuclear factor kappa-ß ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Materials and Methods: Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. Results: The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Conclusions: Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

Characterization and comparative DNA methylation profiling of four adipogenic genes in adipose-derived stem cells and dedifferentiated fat cells from aging subjects.

Adipose-derived stem cells (ASCs) and dedifferentiated fat (DFAT) cells are alternative cell sources in tissue engineering and regeneration because they are easily obtained and exhibit multilineage differentiation. However, aging may attenuate their regenerative potential and metabolic functions. Reports characterizing DFAT cells derived from aging donors are rare, and comparisons of DNA methylation profiles between aging ASCs and DFAT cells are poorly understood. Therefore, this study aimed to characterize DFAT cells relative to ASCs derived from aging subjects and compare the DNA methylation profiles of four adipogenic genes in these cells. ASCs and DFAT cells from aging donors exhibited characteristics similar to those of stem cells, including colony formation, proliferation, and multilineage differentiation abilities. However, compared with ASCs, DFAT cells exhibited increased proliferation, smooth muscle actin alpha (SMA-α) expression and decreased cellular senescence. DNA methylation profiling of ASCs and DFAT cells by combined bisulfite restriction analysis (COBRA) demonstrated hypermethylation patterns in three potent adipogenic genes-peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid-binding protein 4 (FABP4), and lipoprotein lipase (LPL)-but hypomethylation of CCAAT/enhancer binding protein alpha (C/EBPα) in the aging group. Statistically significant differences were observed between the aging group and the young group. Epigenetic regulation maintains the stability of ASCs and DFAT cells in an age-dependent manner. Our findings suggested that although the DNA methylation patterns of three adipogenic genes correlated with hypermethylation and aging, ASCs and DFAT cells exhibited cellular stability and several stem cell characteristics, offering further opportunities for personalized regeneration and energy maintenance by adipogenesis during aging.

Cellular responses of periodontal ligament stem cells to a novel synthesized form of calcium hydrogen phosphate with a hydroxyapatite-like surface for periodontal tissue engineering.

Calcium hydrogen phosphate with a hydroxyapatite-like surface (CHP-HA) is a novel synthesized compound designed to overcome the limitations of bioactive ceramics. It was originally applied as nano-sized HA strips covering core plates to enhance the degree of interfacial attachment. The objective of the present study was to examine the cellular attachment, proliferation, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) on a CHP-HA substrate in comparison with conventional nanohydroxyapatite (NanoHA). The PDLSCs were cultivated with either CHP-HA or NanoHA for cellular attachment, proliferation, and osteogenic differentiation assay. Osteogenic differentiation was examined using quantitative polymerase chain reaction and immunofluorescence after confirmation by Alizarin red staining. We found that between 14 and 21 days, CHP-HA exhibited a well-organized matrix distribution, a high degree of cell proliferation, and a high level of Alizarin red staining in comparison to NanoHA. Expression of all the osteogenic markers examined was increased significantly relative to NanoHA at 14 days, but no significant differences in some osteogenic genes were found at 21 days. Immunofluorescence revealed stronger staining in the CHP-HA group. In conclusion, PDLSCs cultivated with this novel CHP-HA show enhanced cellular responses. We propose that CHP-HA may be a promising alternative biomaterial for periodontal tissue engineering.

Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro.

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Co-culture with periodontal ligament stem cells enhances osteogenic gene expression in de-differentiated fat cells.

In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.