Monitoring bone morphogenetic protein-2 and -7, soluble receptor activator of nuclear factor-κB ligand and osteoprotegerin levels in the peri-implant sulcular fluid during the osseointegration of hydrophilic-modified sandblasted acid-etched and sandblasted acid-etched surface dental implants.

BACKGROUND AND OBJECTIVE: The implant surface plays a major role in the biological response to titanium dental implants. The aim of this study was to investigate levels of soluble receptor activator of nuclear factor-κB ligand (sRANKL), osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2) and -7 (BMP-7) in the peri-implant crevicular fluid (PICF) of different implants during the osseointegration period. MATERIAL AND METHODS: Forty-seven patients (22 females and 25 males, mean age 47.34 ± 10.11) were included in this study. Forty-seven implants from two implant systems (group A1 (sandblasted acid-etched [SLA]-16), group A2 (hydrophilic-modified SLA [SLActive]-16), and group B (sandblasted acid-etched [SLA]-15) were placed using standard surgical protocols. PICF samples, plaque index, gingival index and probing depth measurements were obtained at 1 and 3 mo after surgery. PICF levels of sRANKL, OPG, BMP-2/-7 were analyzed by ELISA. RESULTS: No complications were observed during the healing period. No significant differences were observed in the PICF levels of sRANKL, OPG, BMP-2 and BMP-7 for all groups at any time point (p > 0.05). A significant decrease was observed in BMP-2 levels in group A1 (p < 0.05). A significant increase in BMP-7 levels was observed only for group A2 (p < 0.05). There was a strong negative correlation between OPG and gingival index and a negative correlation between BMP-7 and plaque index (p < 0.05). CONCLUSION: Considering the correlations between clinical and biochemical parameters, the levels of these cytokines in PICF during early healing of implants reflects the degree of peri-implant inflammation, rather than differences in the implant surfaces.

Cytotoxicity of hemostatic agents on the human gingival fibroblast.

BACKGROUND: Achieving of moisture control especially gingival bleeding control is great challenge in clinical practice. Various hemostatic agents and techniques have been promoted for bleeding control during dental operation. But few studies have focused on the cytotoxicity of hemostatic solutions. AIM: The aim of this study was to evaluate cytotoxic effect of hemostatic agents on human gingival fibroblast cells by using real-time cell analysis method. MATERIALS AND METHODS: Two hemostatic solutions, Hemoban (Sultan Healthcare, Hackensack, NJ, USA) and Hemasatic Solutions (W.P. Dental, Hamburg, Germany) that includes mainly aluminum chloride were used with different concentration. Gingival fibroblasts were isolated from gingival connective tissue during crown lengthening surgery of systemically healthy subjects. Gingival fibroblasts were maintained with Dulbecco's modified eagle medium containing 10% fetal bovine serum. A real-time cell analyzer (RT-CES, xCELLigence; Roche Applied Science, Mannheim, Germany, and ACEA Biosciences, San Diego, CA, USA) was used to evaluate cell survival. After seeding 200 mL of the cell suspensions into the wells (10,000 cells/well) of the E-plate 96, gingival fibroblasts were treated with hemostatic solutions (1/2, 1/4 and 1/8 dilutions) and monitored every 15 minutes for 72 hours. For the proliferation experiments, the statistical analyses used were 1-way analysis of variance (ANOVA) and Tukey HSD multiple comparisons tests. RESULTS: According to statistically analysis, when evaluated at 48 and 72 hours, there were significant differences between the cell indexes of the control and all hemostatic agents groups (p < 0.001). Agent reduced cell index value significantly when compared to untreated control group. CONCLUSIONS: The results indicate that using of Hemoban or Hemostatic Solutions as astringent solutions have a significant cytotoxic effect on gingival fibroblast cells.

The response of cementoblasts to calcium phosphate resin-based and calcium silicate-based commercial sealers.

AIM: To investigate cell viability and gene expression of cementoblasts (OCCM.30) exposed to extractable components released by resin-based sealers with different chemical composition Hybrid Root Seal (HRS), SimpliSeal (SS), Real Seal (RS) and AH Plus (AH) and by a MTA-based sealers Tech Biosealer Endo (TBE). METHODOLOGY: Discs of all materials were prepared and allowed to set in humid conditions at 37° for 48 h. The discs were then incubated for 72 h at 37 °C to obtain material extracts (1/1) in DMEM. The extracts containing the components released by the sealers were filtered and other dilutions (1/2, 1/4) were prepared from the original solution (1/1). Original and diluted solutions were tested on the cementoblasts. Impedance-based real-time cell analysis (RTCA) was used to evaluate cell viability, quantitative real-time polymerase chain reaction (QRT-PCR) was used to examine the expression of mineralization-related genes (osteocalcin; OCN, Runt-related transcription factor-2; Runx2, collagen type 1; COL I, alkaline phosphatase; ALP). For statistical analysis, one-way analysis of variance (anova) and Tukey's honestly significant difference (HSD) tests were used. RESULTS: TBE (1/2), RS (1/2, 1/4), and HRS (1/2, 1/4) significantly decreased cell viability (P < 0.001). AH (1/2, 1/4) and SS (1/2, 1/4) had similar cell viability to the control at 30 h. All tested materials significantly decreased cell viability when compared to the control group except AH (1/2, 1/4) and SS (1/4) at 90 h. All of the tested sealers reduced COL I mRNA expressions when compared to the control. SS was associated with significant increases in OCN and Runx2 mRNA expressions when compared to the control (P < 0.001). Whereas all of the dilutions of TBE, RS and HRS significantly decreased BSP mRNA expressions (P < 0,001), 1/2 and 1/4 dilutions of SS increased BSP mRNA expression (P < 0,001). Except the 1/4 dilutions of AH and SS, all the sealer dilutions significantly reduced ALP mRNA expression in cementoblasts (P < 0,001). CONCLUSIONS: SimpliSeal and AH Plus resulted in more favourable response to cementoblasts because of their regulation potential on the mineralized tissue-associated protein's mRNA expressions.

Periodontal ligament fibroblast response to root perforations restored with different materials: a laboratory study.

AIM: To compare the effect of several materials on the attachment of periodontal ligament (PDL) fibroblasts to experimentally perforated root surfaces. METHODOLOGY: Root specimens (size 5 × 5 mm) were obtained from extracted human molar teeth and perforations with a 1 mm diameter were created. One group was kept as a control and the rest were repaired with the following materials: Amalgam, Dyract, IRM, Super Bond C&B and Mineral trioxide aggregate (MTA). PDL fibroblasts were placed at a density of 8 × 10(4) cells on the root specimens, incubated on tissue culture inserts (48 h) and then transferred to 48 well-plates. MTT assays were performed at 48 and 96 h for PDL fibroblast survival. Cell attachment was observed using confocal microscopy on days 2 and 5. Total RNAs from the root specimens were isolated on day 5 and type I collagen (COL I) and Runt-related transcription factor 2 (Runx2) mRNA expressions were checked using Quantitative-Polymerase Chain Reaction (QPCR). For the MTT assay and QPCR, one-way analysis of variance (anova) and Tukey HSD multiple comparison tests were used to compare the groups. RESULTS: Mineral trioxide aggregate resulted in a significantly higher cell density (P < 0.001). Dyract, IRM and Super Bond C&B groups had a lower cell density when compared with the control and MTA groups at 48 h (P < 0.001). Confocal microscopy revealed that, among the experimental groups, the MTA group had the largest viable cell population over the restoration site when compared with the other materials; however, reduced cell attachment was noted in all groups when compared with the control. Increased Runx2 mRNA expressions were noted in MTA (P < 0.001) and IRM (P < 0.01) groups when compared with control and other tested materials. COL I transcripts were increased in IRM (P < 0.01), D, C&B and MTA (P < 0.001) when compared with the control. CONCLUSION: Mineral trioxide aggregate provided a more favorable environment for PDL cell adhesion and growth.

Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by basic fibroblast growth factor and dexamethasone in periodontal ligament cells.

BACKGROUND AND OBJECTIVES: In this study, we investigated the effect of basic fibroblast growth factor (bFGF) and dexamethasone (Dex) on mRNA expressions of collagen (COL) type I, III and X, matrix metalloproteinases (MMP)-1, -2, -3 and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2, and also on mineralization and morphology of periodontal ligament (PDL) cells. MATERIAL AND METHODS: Periodontal ligament cells were obtained from premolar teeth extracted for orthodontic reasons. Periodontal ligament cells were cultured with Dulbecco's modified Eagle's medium containing: (1) 5% fetal bovine serum (FBS); (2) 5% FBS + ascorbic acid (AA, 50 microg/mL); (3) 5% FBS + Dex (10(-7) m) + AA; (4) 5% FBS + bFGF (10 ng/mL) + AA; or (5) 5% FBS + Dex (10(-7) m) + bFGF + AA. Cells within each group were evaluated for gene expression profile using semi-quantitative reverse transcriptase-polymerase chain reaction for COL I, III and X, MMP-1, -2, -3 and -9 and TIMP-1 and -2 on days 14 and 21 and for biomineralization by von Kossa stain in vitro on day 21. Images of PDL cells were examined using a phase contrast microscope. RESULTS: Basic fibroblast growth factor stimulated MMP-1, MMP-3 and MMP-9 mRNA expressions and inhibited TIMP-2 mRNA expression. Treatment of cells with Dex + bFGF led to downregulation of MMP-1, MMP-3 and MMP-9 transcripts. Whilst AA alone and Dex alone induced biomineralization of PDL cells, bFGF blocked the mineralization activity of the cells. In the Dex + bFGF group, more mineral nodules were noted when compared to AA alone and Dex alone groups. CONCLUSION: The addition of Dex to culture reversed bFGF-mediated inhibition of mineralization. Use of combined bFGF and Dex to regulate PDL cell function may be a good therapeutic option to obtain periodontal regeneration.

Cytotoxic effects of dental desensitizers on human gingival fibroblasts.

The purpose of this study was to evaluate the effects of three different desensitizers on the cell viability and morphology of human gingival fibroblasts (HGF). Human gingival tissues were obtained from individuals who have clinically, healthy periodontium. HGF were grown at 37 degrees C in humidified atmosphere of 5% CO2 in Dulbecco's modified eagle's medium, supplemented with glutamine, penicillin, streptomycin, and 10% fetal bovine serum. The cells were treated with different concentrations (0.1, 0.3, and 0.5 microL/mL) of desensitizers (Gluma Desensitizer, Seal&Protect, and MicroPrime). After 24- and 48-h exposure to the desensitizer solutions, the viable cells were examined using a hemocytometer. To monitor HGF viability, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was used and cell morphology was also observed at 48 h. Following exposure to concentrations of 0.1 microL/mL of test materials for 24 h, cell survival rates for Gluma Desensitizer (106%) and Micro Prime (62%) were not significantly different from the control, while it was significant for Seal&Protect (50%). Growing cells were significantly inhibited by all tested materials for 48 h (p < 0.05) in survival rates of 51, 47, and 31%, respectively. On the basis of the MTT assay, the cytotoxic effect of MicroPrime was more prominent, especially at high concentrations, than does Gluma Desensitizer and Seal&Protect. After exposure to Seal&Protect and MicroPrime, HGF became retracted, rounded in appearance and had loss of normal organization, leading to enlargement of intercellular space when compared with Gluma Desensitizer. As a conclusion, taking the limitations of an in vitro experiment into consideration, the cytotoxic effects were varied, depending on the chemical composition and exposure periods of the tested desensitizers.

Biocompatibility evaluation of orthodontic composite by real-time cell analysis.

INTRODUCTION: The aim of this study was to evaluate the cytotoxic effects of three different light-cured orthodontic composites. MATERIAL AND METHODS: Light Bond (Reliance orthodontic products), Grengloo (Ormco corporation), and Kurasper F (Kuraray Europe GmbH) were selected for the experiment. Specimens were prepared according to the manufacturers' instructions, measuring 5 mm in diameter and 2 mm in thickness. Fibroblast cells were obtained from healthy gingival connective tissues. The composite cylinders were incubated in Dulbecco's modified Eagle's culture medium for 72 h according to ISO 10993-5 standards. The xCELLigence method was used to evaluate fibroblast cell vitality. After seeding 200 mL of the cell suspensions into the wells (20,000 cells/well) of the E-plate 96, gingival fibroblasts were treated with bioactive components released by the orthodontic composite materials and monitored every 15 min for 121 h. RESULTS: There were no significant differences between the human gingival fibroblast (HGF) cell indexes of the control and all testing groups (p > 0.05) at 24 and 48 h. Light Bond demonstrated statistically significant decrease in HGF index (p < 0.05) at 72 h, but there was no significant difference among the Kurasper F, Grengloo, and untreated control groups (p > 0.05). Light Bond (p < 0.001) and Grengloo (p < 0.05) groups had lower HGF cell index values when compared to untreated control group, but Kurasper F demonstrated no significant differences between the control groups at 96 h (p > 0.05). CONCLUSION: Orthodontic composite materials include biologically active components and may change oral tissue. So, biocompatible orthodontic bonding composites should be used.

The effect of enamel matrix protein derivative on follicle cells in vitro.

BACKGROUND: It is thought that during development of the periodontium, dental follicle cells, when appropriately triggered, have the ability to differentiate into periodontal ligament fibroblasts, cementoblasts, and osteoblasts. However, the exact mechanisms/factors responsible for initiating cell differentiation are not defined. The purpose of this in vitro study was to further characterize follicle cells and to determine the effects of an enamel matrix-derived protein (EMD) on these cells. METHODS: Murine follicle cells, transformed with simian virus 40 (SV 40) T antigen-containing virus (SVF cells), were used. SVF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus 2% fetal bovine serum (FBS) or 2% FBS plus EMD (100 microg/ml), with and without ascorbic acid (50 microg/ml). For proliferation assays, cells were plated at 500 cells/cm2 in 24-well plates and counted on days 3, 4, and 5. For Northern analysis, total RNA was isolated on days 8, 12, and 18. Induction of mineral nodules by SVF cells was determined by von Kossa staining. RESULTS: EMD had a significant proliferative effect on SVF cells, when compared with 2% FBS control. Based on investigations in situ, follicle cells at the time point used here do not express key mineral-associated markers, e.g., osteocalcin (OCN) or bone sialoprotein (BSP). Significantly, by day 12 in culture, Northern analysis indicated that the follicle cells expressed transcripts for BSP, OCN, and osteopontin (OPN). EMD increased OPN mRNA and decreased OCN mRNA expression. SVF cells were capable of inducing mineralization on day 18, but EMD blocked this activity. CONCLUSIONS: These results suggest the follicle cells have the capacity to act as cementoblasts or osteoblasts. Furthermore, EMD can regulate follicle cell activity, thus suggesting that epithelial-mesenchymal interactions may be important during development of periodontal tissues.

Epidermolysis bullosa acquisita: clinical manifestations, microscopic findings, and surgical periodontal therapy. A case report.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is an uncommon, acquired, chronic subepidermal bullous disease. This report describes a case of EBA with gingival involvement. A 43-year-old woman with EBA was referred to our clinic for periodontal therapy because of gingival tenderness and bleeding. She has been on cyclosporin A therapy for the last 2 years. METHODS: Clinical findings were analyzed. Anterior gingivectomy operations were performed in 2 stages. The samples obtained during the surgery were examined using histopathologic, immunohistologic, and electronmicroscopic methods. Long-term effects of the surgical periodontal treatment on gingiva were evaluated both clinically and microscopically. RESULTS: The dentition displayed minimal enamel hypoplasia. Decayed, missing, and filled surfaces score was found to be elevated. Periodontal examination showed generalized diffuse gingival inflammation and gingival enlargement localized mainly to the anterior region. Nikolsky's sign was positive. However, wound healing was uneventful after the operations. Microscopic findings were similar to those obtained from the skin. Twenty-one months after the operations, Nikolsky's sign was negative and no remarkable gingival inflammation was noted. Microscopic examination revealed that the blisters were fewer in number and smaller in size. CONCLUSIONS: These results indicate that gingival tissues may also be involved in EBA. Uneventful wound healing after periodontal surgery in this case suggests that periodontal surgery can be performed in patients with EBA. Moreover, both our clinical and histopathologic findings imply that gingivectomy proves useful in maintaining gingival integrity in these patients. Our data may also suggest that the patients with EBA are highly likely to develop dental caries.