Differential effects of resolvin D1 and resolvin E1 on cementoblast function.

BACKGROUND: Resolvins are endogenous mediators of the resolution of inflammation. They are derived from omega-3 polyunsaturated fatty acid precursors. Resolvin D1 (RvD1) and Resolvin E1 (RvE1) are the best-characterized members for actively promoting periodontal regeneration in experimental animal models. Here, we evaluated the efficacy of RvD1 and RvE1 on cementoblasts, the key cells involved in dental cementum regeneration and the attachment of the tooth to the alveolar bone. METHODS: Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1-1000 ng/mL) of RvD1 and RvE1. Cell proliferation was measured using an electrical impedance-based real-time cell analyzer. Mineralization was evaluated with von Kossa staining. The mRNA expression of mineralized tissue-associated markers of bone sialoprotein (BSP), Type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RunX2), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (NF-κB) (RANK), receptor activator of NF-κB ligand (RANKL), and extracellular matrix-degrading enzymes [matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and their tissue inhibitors (TIMP-1, TIMP-2)], RvE1 receptor (ChemR23) and RvD1 receptor (ALX/PFR2), cytokines (tumor necrosis factor-alpha {TNF-α}, interleukin {IL}-1ß, IL-6, IL-8, IL-10, IL-17), oxidative stress enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPX), and cyclooxygenase-2 (Cox-2)] were analyzed using quantitative polymerase chain reaction (qPCR). RESULTS: Both RvD1 and RvE1 (10-100 ng/mL) significantly increased the proliferation of cementoblasts and mineralized nodules at all concentrations (p < 0.05). RvE1 increased BSP, RunX2, and ALP compared with the RvD1 dose and time-dependently, while RvD1 and RvE1 differentially regulated COL-I. RvE1 increased OPG mRNA expression, whereas RANK-RANKL mRNA expression decreased by RvE1. MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 expressions were reduced by RvE1 compared with RvD1. Treatment of cementoblasts with RvD1 and RvE1 differentially affected cytokine and oxidative stress enzymes while significantly increasing their receptor expressions (ChemR23 and ALX/PFR2). CONCLUSIONS: RvD1 and RvE1 regulate proliferation, mineralization, and gene expression in cementoblasts using similar pathways while differentially affecting tissue degradation, suggesting a targeted therapeutic approach for cementum turnover during periodontal regeneration.

Positive effect of curcumin on experimental peridontitis via suppression of IL-1-beta and IL-6 expression level.

This study examined the effect of curcumin on T-helper (Th17) and T-regulatory (Treg) cells regarding the mRNA of cytokines/mediators in the gingiva. Thirty-five male albino Wistar rats were divided into four groups: Group 1: periodontitis (n = 9); Group 2: periodontitis with curcumin treatment (n = 8); Group 3: periodontally healthy with curcumin treatment (n = 10); and Group 4: periodontally healthy (n = 8). Curcumin was administered via oral gavage (30 mg/kg/day) for a total of 15 days. The gingival tissues were investigated regarding mRNA expressions of Th17/Treg cytokines with qRT-PCR. The distributional properties of the data were evaluated using the Anderson-Darling normality test. Kruskal-Wallis and Mann-Whitney U tests were employed for multiple group comparisons. Partial least squares regression discriminant analysis (PLS-DA) was used to evaluate the degree of contribution of each mRNA to the separation of treatment groups. When the periodontitis groups were compared, curcumin treatment resulted in lower IL-1ß (Group 2 median: 0.002, Group 1 median: 0.12) and IL-6 (Group 2 median: 0.031, Group 1 median: 0.078) and higher IL-17 (Group 2 median: 1.07, Group 1 median: 0.583) relative mRNA expression in Group 2 than in Group 1 (p < 0.001). Group 3 also had higher IL-10 relative expression (median: 0.067) than Groups 1 and 4 (median: 0.028, 0.007, respectively. p < 0.001). These results indicate that curcumin might be a promising agent for the prevention and/or treatment of periodontal diseases due to its decreasing effect on IL-1ß and IL-6 mRNA expression.

Porphyromonas gingivalis-Lipopolysaccharide induces cytokines and enzymes of the mouse cementoblasts.

Lipopolysaccharide is a potent virulence factor of Porphyromonas gingivalis and has been implicated predominant pathogen in the development and progression of periodontal diseases. The aim of this study was to determine the effect of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on cementoblasts. Cementoblast (OCCM-30) were evaluated proliferation using real-time cell analyzer. In addition, total RNA was isolated at 8, 16, 24 and 72 h from 1000 ng/mL Pg-LPS treated OCCM-30 cells and mRNA expressions of pro/anti-inflammatory cytokine mediators, extracellular matrix enzymes and their tissue inhibitors and of oxidative stress enzymes were studied by real-time polymerase chain reaction. Proliferation analysis indicated that Pg-LPS slightly decreased proliferation of OCCM-30. Pg-LPS had a time-dependent impact on the expression of cytokines and enzymes. There was statistically significant up-regulation of IL-1ß and IL-10 in response to Pg-LPS at 8, 16, 24, 72 h but IL-6 expression was reduced compared to control at 8 h. While IL-8 and IL-17 expressions were determined higher than control group at 16 and 24 h, their expressions were decreased compared to control groups at 72 h (p < 0.01). While MMP-1, MMP-2, MMP-3, TIMP-1, TIMP-2 expressions increased, MMP-9 expression reduced at time-points. Also, a time-dependent up-regulation in mRNA levels for oxidative stress enzymes was detected. These results indicated that up-regulation in the transcripts of inflammation-associated cytokines and degradation enzymes were noted in the cementoblasts exposed to Pg-LPS. Cementoblasts infected with the virulence factors of periodontopathogens might also involve to the induction of inflammation and degradation of the periodontal tissues.

Warm Gutta-Percha Techniques Regulate Cell Viability, Heat Shock, and Mineralized Tissue-associated Proteins of Cementoblasts.

INTRODUCTION: The aim of this study was to determine the effect of the continuous wave of condensation technique (CWCT) and the thermoplastic gutta-percha injection (TGI) technique on the messenger RNA (mRNA) expressions of heat shock proteins (HSPs) and mineralized tissue-associated proteins of the immortalized mouse cementoblasts (OCCM.30). METHODS: Crowns of human premolar teeth with single and straight canals were removed. The root canals were prepared up to the ProTaper Next X5 file (Dentsply Maillefer, Ballaigues, Switzerland) in combination with 2 mL 2.5% sodium hypochlorite solution. Roots (12 ± 2 mm height) were sterilized (121°C for 20 minutes) and placed vertically to the cell culture dishes using a tissue culture insert by opening holes according to the root diameter after the removal of 1 mm from the apex for appropriate adaptation to the petri dish surfaces. Six groups were created: control 1 (without teeth), control 2 (with teeth), AH Plus (Dentsply DeTrey, Konstanz, Germany), single-cone obturation (SC), CWCT, and thermoplastic gutta-percha injection technique (TGI). The viability of the OCCM.30 cells was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell viability experiments at 24 and 96 hours. The mRNA expression of HSP27, HSP70, and HSP90 and mineralized tissue markers (bone sialoprotein, osteocalcin, runt-related transcription factor 2, type I collagen, and alkaline phosphatase) was evaluated by real-time polymerase chain reaction. RESULTS: Reduced OCCM.30 cell viability was observed in all groups except the control groups. When the SC technique and CWCT and TGI groups were compared, it was observed that heat had a significant negative effect on cell viability (P < .05). A reduction in the mRNA expressions of HSP27, HSP70, and HSP90 was recognized in all test groups when compared with the control 1 group (P < .01). When the warm gutta-percha techniques were compared with the SC technique, a decrease in mRNA expression of HSP27 and HSP90 was noted (P < .01). The HSP70 transcript was similar in the CWCT group and the SC group. Higher HSP70 mRNA expression was observed in the TGI group compared with the SC group. In all groups except the control 1 group, bone sialoprotein, osteocalcin, runt-related transcription factor 2, type I collagen, and alkaline phosphatase mineralized tissue markers were affected, but this negative effect was higher in the heat-treated groups (P < .05). CONCLUSIONS: Within the limitations of this study, it was concluded that warm gutta-percha techniques reduced the mRNA expressions of the genes for HSPs and mineralized tissue-associated proteins of cementoblasts. Further animal studies are needed to clarify the effect of heat on the behavior of cementoblasts histologically in short- and long-term periods.

Nicotine suppresses proliferation and mineralized tissue-associated gene expressions of cementoblasts.

BACKGROUND: Previous studies reported that nicotine, which is the prominent constituent of tobacco, has negative effects on periodontium cells. However, the precise role of nicotine in cementoblast functions remains unclear. In the present study, we investigated the effects of nicotine on the functions of cementoblasts (OCCM-30) in terms of proliferation, migration, and mineralized tissue-associated gene expression. METHODS: Immortalized murine cementoblasts were exposed to various concentrations (0, 10-6 , 10-5 , 10-4 , 10-3 , 10-2 , 10-1 , 1, 2.5, 5, and 10 mM) of nicotine, and cementoblast proliferation was then evaluated using a real-time cell analyzer for 142 hours. Using an in vitro wound healing assay, cell migration was evaluated 2, 4, 6, and 24 hours after exposure to different concentrations of nicotine (1, 2.5, 5, and 10 mM). The mRNA expressions of bone sialoprotein (BSP), collagen type I (COL-I), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and alkaline phosphatase (ALP) were assessed in the nicotine-treated (0, 10-3 , 10-2 , 10-1 , 1, 2.5, 5, and 10 mM) OCCM-30 cells by reverse transcription quantitative polymerase chain reaction at 8 and 24 hours exposure. RESULTS: At concentrations of 1 to 10 mM, nicotine significantly reduced cementoblast proliferation (P <0.01). Exposure to nicotine at other concentrations (1, 2.5, and 5 mM) significantly reduced wound healing rates, whereas nicotine at a concentration of 10 mM immediately decreased the viability of OCCM-30 cells. Similar results were observed in inverted microscopy images at the highest nicotine concentrations. All concentrations of nicotine decreased the transcripts of BSP and COL-I in a dose- and time-dependent manner (P <0.001). Nicotine concentrations higher than 1 mM reduced the expression of OCN, RunX2, and ALP in a time-dependent manner (P <0.001). CONCLUSIONS: This study indicated that nicotine inhibited the proliferation, migration, and mineralized tissue-associated gene expression of OCCM-30 cells. These findings suggest that nicotine negatively affects cementoblast function and the formation of new cementum, which is critical for new attachment.

Cytotoxicity Evaluation of Bioresorbable Fixation Screws on Human Gingival Fibroblasts and Mouse Osteoblasts by Real-Time Cell Analysis.

PURPOSE: To evaluate the effects of bioresorbable fixation screws (BFSs) on human gingival fibroblast (HGF) and mouse osteoblast (MC3T3-E1) cell viability. MATERIALS AND METHODS: The KLS Martin SonicPins Rx, Synthes RapidSorb Cortex Screws, and Inion CPS Bioabsorbable Fixation System each were incubated in Dulbecco's Modified Eagle Medium for 72 hours according to ISO 10993-5 standards. A real-time cell analyzer was used to evaluate cell survival. After seeding 200-µL cell suspensions in the wells of an E-plate View 96, HGF and MC3T3-E1 cells were treated with the bioactive components released by the bioresorbable materials and monitored every 15 minutes for 96 hours. Statistical significance was determined using 1-way analysis of variance and Tukey-Kramer tests. RESULTS: There were significant differences in the HGF responses to the untreated control conditions and the Synthes (P < .01), Inion (P < .05), and KLS Martin (P < .05) treatments over 48 hours. The Synthes (P < .01) and Inion (P < .01) treatments produced lower HGF cell index values than the untreated control at 72 hours, whereas the KLS Martin treatment did not. When left to elute for 96 hours, there were no significant differences in values among the control and study groups for HGFs (P > .05). All tested BFSs decreased cell survival rates of M3T3C1 cells for 48 hours (P < .01), 72 hours (P < .001), and 96 hours (P < .001). CONCLUSION: Differences in the sensitivities of the 2 tested cell lines to the different BFSs might be the result of the different materials used to manufacture the screws. These results provide fundamental knowledge and new insights for the future design and development of new biocompatible BFSs for oral and maxillofacial surgery.

Cytotoxicity of temporary cements on bovine dental pulp-derived cells (bDPCs) using realtime cell analysis.

PURPOSE: To evaluate the cytotoxicity of temporary luting cements on bovine dental pulp-derived cells (bDPCs). MATERIALS AND METHODS: Four different temporary cements were tested: Rely X Temp E (3M ESPE), Ultratemp (Ultradent), GC Fuji Temp (GC), and Rely X Temp NE (3M ESPE). The materials were prepared as discs and incubated in Dulbecco's modified eagle's culture medium (DMEM) for 72 hours according to ISO 10993-5. A real-time cell analyzer was used to determine cell vitality. After seeding 200 µL of the cell suspensions into the wells of a 96-well plate, the bDPCs were cured with bioactive components released by the test materials and observed every 15 minutes for 98 hours. One-way ANOVA and Tukey-Kramer tests were used to analyze the results of the proliferation experiments. RESULTS: All tested temporary cements showed significant decreases in the bDPCs index. Rely X Temp E, GC Fuji Temp, and Rely X Temp NE were severely toxic at both time points (24 and 72 hours) (P<.001). When the cells were exposed to media by Ultratemp, the cell viability was similar to that of the control at 24 hours (P>.05); however, the cell viability was significantly reduced at 72 hours (P<.001). Light and scanning electron microscopy examination confirmed these results. CONCLUSION: The cytotoxic effects of temporary cements on pulpal tissue should be evaluated when choosing cement for luting provisional restorations.

Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis.

Oral lichen planus (OLP) is a chronic inflammatory disease that is frequently detected in oral tissues. The aim of our study was to identify the prevalence of the detection of periodontopathogenic microorganisms (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola in OLP patients and to compare with this prevalence of periodontopathogenic microorganisms in healthy non-OLP patients. Our study included 27 (18 chronic periodontitis (OLPP) and 9 gingivitis (OLPG)) patients diagnosed with OLP along with 26 (13 chronic periodontitis (HP) and 13 gingivitis (HG)) healthy non-OLP patients. The multiplex polymerase chain reaction (PCR) with subsequent reverse hybridization method (micro-IDent) was used for identifying periodontopathogenic microorganisms present in subgingival plaque samples. The percentages of detection for A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola in subgingival plaque samples taken from OLP patients (OLPG and OLPP) were 18.5%, 85.1%, 81.4%, 88.8% and 74%, respectively. Meanwhile, in the non-OLP patients (HG and HP), these values were 7.6%, 50%, 46.1%, 73% and 57.7%, respectively. Thus, comparing the non-OLP groups with the OLP groups, the periodontopathogens' percentages of detection in the OLP groups were higher than those in the non-OLP groups. According to our study results, OLP patients have higher levels of infection with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola than non-OLP patients. We argue that the high percentages in patients with OLP may help identify the importance of periodontopathogenic microorganisms in the progress of periodontal diseases of OLP.

MMP-1, MMP-9, and TIMP-1 levels in oral lichen planus patients with gingivitis or periodontitis.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease of unknown aetiology. The aim of this study was to investigate matrix metalloproteinase (MMP)-1, MMP-9, and MMP inhibitor-1 (TIMP-1) levels in gingival crevicular fluid (GCF) by enzyme-linked immunosorbent assay and to investigate MMP-1, MMP-9, and TIMP-1 levels in gingival tissue by immunohistochemical staining of samples from patients with and without OLP. DESIGN: Twenty-seven patients with OLP (gingivitis, OLPG; chronic periodontitis, OLPP) and thirty healthy non-OLP patients (gingivitis, HG; chronic periodontitis, HP) were included in this study. The MMP-1, MMP-9, and TIMP-1 levels in GCF were determined by enzyme-linked immunosorbent assay. The MMP-1, MMP-9, and TIMP-1 levels in gingival tissue were determined by immunohistochemical staining. RESULTS: The mean levels of MMP-1 and MMP-9 in the GCF of OLPP patients were significantly higher and TIMP-1 was significantly lower than in HP patients; similarly, the mean levels of MMP-1 and MMP-9 were higher and TIMP-1 was significantly lower in OLPG patients than in HG patients. Our findings illustrate that tissue MMP-9 levels were statistically higher and TIMP-1 level were significantly lower in the OLPP group in comparison to the HP group, and the OLPG group in comparison to the HG group. The tissue MMP-1 level in the non-OLP group was found to be lower when compared with the OLP groups. But not statistically significant. CONCLUSIONS: Increased levels of MMP-1 and MMP-9 with decreased levels of TIMP-1 in GCF and increased MMP-1, MMP-9 levels and decreased TIMP-1 levels in the gingival tissue of OLP patients in combination with poor oral hygiene may cause increased tissue breakdown. The results of our study provide information about the effects of the periodontal status on the enzyme profiles in GCF and gingival tissue of OLP and non-OLP patients.

ABM/P-15 modulates proliferation and mRNA synthesis of growth factors of periodontal ligament cells.

OBJECTIVE: Periodontal regeneration is histologically defined as regeneration of the tooth supporting structures, including alveolar bone, periodontal ligament, and cementum. Cells in the remaining periodontal tissues need optimal conditions if they are to perform their functions in the regeneration process. The present study is an investigation of the molecular effects of ABM/P-15 on human periodontal ligament cells (PDL) in vitro. MATERIAL AND METHODS: PDL cells obtained from healthy subjects were used for in vitro experiments. Cell proliferation, morphology, and mineralization using Von kossa staining were evaluated. mRNA expressions for transforming growth factor-beta (TGF-beta), insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), bone morphogenic protein-2 (BMP-2), platelet-derived growth factor (PDGF), and type 1 collagen (COL1) were assessed on days 3 and 7 using RT-PCR. RESULTS: ABM/P-15 enhanced proliferation of cultured PDL cells. It increased the mRNA expression of TGF-beta and BMP-2 in cultured PDL cells on days 3 and 7. IGF-I and b-FGF mRNA expressions showed a slight decrease, while PDGF expression was observed to have increased on day 3. VEGF and COL1 mRNA expressions were found not to be different on days 3 and 7. No differences were observed in the mineralization properties of cultured PDL cells treated with or without ABM/P-15. CONCLUSIONS: Based on the results of this in vitro study, it may be concluded that ABM/P-15 enhanced the regenerative capacity of PDL by regulating specific gene expressions of cells during early wound healing.