SDF-1 modulates periodontal ligament-Mesenchymal Stem Cells (pdl-MSCs).

BACKGROUND/OBJECTIVES: In this in vitro study, the effects of Stromal cell-derived factor-1 (SDF-1) was evaluated on the periodontal ligament-Mesenchymal Stem Cells (pdl-MSCs) functions. MATERIAL AND METHODS: Real-time cell analyzer-single plate (RTCA-SP) was employed for proliferation, and RTCA-dual purpose (DP) was utilized for pdl-MSCs migration potential treated with different SDF-1 concentrations (0, 0.1, 1, 10, 100, 200, and 400 ng/ml). Based on the dose-response findings, 10 ng/ml SDF-1 was used for further mRNA experiments. RNAs isolated at 6 and 24 h were checked using quantitative RT-PCR for mineralized tissue-associated genes including type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor 2 (Runx2). cRNA was synthesized for 6 h, and whole-genome array analysis was performed for over 47.000 probes. Data were subjected to quantile normalization before analysis. RESULTS: Increased proliferation and migration were observed in pdl-MSCs treated with 0.1, 1, and 10 ng/ml SDF-1. Increased COL I was observed at both time points: 6 and 24 h. While there was no significant change for OCN, OPN, and Runx2 at 6 h, SDF-1 up-regulated OCN and OPN, but down-regulated Runx2 mRNA expressions at 24 h. IL-8 and ESM1 genes were differentially expressed over twofold when the pdl-MSCs were exposed to SDF-1 at whole-genome array analysis. IL-8 induction was confirmed with RT-PCR. CONCLUSION: Findings of this study displayed that SDF-1 modulated pdl-MSCs which were important for periodontal regeneration, inducing migration and proliferation, and regulating extracellular matrix synthesis in favor of the formation of new attachment.

Real-time cell analysis of the cytotoxicity of orthodontic brackets on gingival fibroblasts.

PURPOSE: The aim of this study was to evaluate the cytotoxic effects of 6 different orthodontic bracket types on human gingival fibroblasts (HGFs) using the xCELLigence system.
 METHODS: The orthodontic brackets used in this study were gold-plated steel (Apollo Gold), titanium (Rematitan), stainless steel (Equilibrium 2), lucid ice (Inspire ICE), metal-reinforced ceramic (Clarity) and composite (OrthoFlex). Gingival fibroblasts were isolated from human gingival connective tissue of systemically healthy individuals. Tested brackets were incubated in DMEM culture medium for 72 hours according to ISO 10993-5 standards. Gingival fibroblasts were maintained with Dulbecco modified Eagle medium containing 10% fetal bovine serum. The xCELLigence system was used to evaluate cell survival. The statistical analysis used was ANOVA and Tukey-Kramer multiple comparison tests.
 RESULTS: When the data were evaluated in the 30th hour, Apollo Gold showed significant decreases in cell index (P<0.001). It also showed statistically significant decreases (P<0.001) in the 65th hour, but Clarity and Inspire ICE showed significant increases in cell indices (P<0.001, P<0.01). In the 114th hour, Clarity and Equilibrium 2 showed statistically significant increases in cell indices (P<0.001). Inspire ICE and Rematitan demonstrated significant increases (P<0.05). There were significant decreases in cell index of Apollo Gold (P<0.001). 
 CONCLUSIONS: The tested brackets are suitable for clinical application, but further studies using different test methods are needed for gold-plated brackets.

Periodontal ligament cell behavior on different titanium surfaces.

AIM: The purpose of this study was to investigate proliferation, morphology, mineralization and mRNA expressions of mineralized tissue associated proteins of PDL cells on smooth (S), sandblasted small-grit (SSG), sandblasted large-grit (SLG) and sodium titanate (NaTi) coated titanium alloys, in vitro. METHODS AND MATERIALS: PDL cells were cultured with DMEM media containing 10% FBS on the S, SSG, SLG and NaTi titanium surfaces. PDL cell proliferation, mineralization and immunohistochemistry experiments for Bone Sialoprotein (BSP) were performed. The morphology of the PDL cells was examined using confocal and scanning electron microscopy (SEM). Gene expression profiles of cells were evaluated using a quantitative-polymerase chain reaction (Q-PCR) for type I collagen (COL I), Osteocalcin (OCN), osteopontin (OPN) and Runt-related transcription factor-2 (Runx2) on days 7 and 14. RESULTS: Proliferation results on days 6 and 10 were similar in groups, while those of day 13 revealed a decrease in the NaTi group when compared to the S group. NaTi surface induced BSP mRNA expression which was correlated with mineralization tests and BSP immunostaining results. Increased Runx2 mRNA expression was also noted in the NaTi surface when compared to other surfaces. CONCLUSIONS: This study considers the NaTi surface as a potential alternative to SSG and SLG surfaces. This surface might provide a promising environment for PDL ligament-anchored implants.

Real-time cell analysis of the cytotoxicity of orthodontic mini-implants on human gingival fibroblasts and mouse osteoblasts.

INTRODUCTION: The aim of this study was to evaluate the cytotoxic effects of orthodontic mini-implants on gingival fibroblasts and osteoblasts. METHODS: The orthodontic mini-implants used in this study were Orthodontic Mini Implant (Leone, Florence, Italy), MTN (MTN, Istanbul, Turkey), AbsoAnchor (Dentos, Daegu, South Korea), IMTEC Ortho (3M Unitek, IMTEC, Ardmore, Okla), VectorTAS (Ormco, Glendora, Calif). The materials were incubated in Dulbecco's modified eagle's culture medium for 72 hours according to ISO 10993-5 standards (surface area-to-volume ratio of the specimen to cell-culture medium, 3 cm(2)/mL). A real-time cell analyzer (xCELLigence, Roche Applied Science, Mannheim, Germany; ACEA Biosciences, San Diego, Calif) was used to evaluate cell survival. After seeding 200 µL of the cell suspensions into the wells of the E-plate 96, gingival fibroblasts were treated with bioactive components released by the metallic materials and monitored every 15 minutes for 190 hours. For the proliferation experiments, the statistical analyses used were 1-way analysis of variance and Tukey-Kramer multiple comparisons tests. RESULTS: There was no significant differences between the human gingival fibroblast cell indexes of the control and study groups (P >0.05). When evaluated at 27 and 96 hours, only the VectorTAS mini-implants showed statistically significant decreases in the M3T3 cell index (P <0.001) compared with the control group. No significant differences were found among the control and all study groups (P >0.05). Furthermore, the Leone and MTN mini-implants showed statistically significant decreases (P <0.001) at 190 hours. Also, the VectorTAS mini-implants demonstrated a significant decline (P <0.05) at the same time in the M3T3 cell index. CONCLUSIONS: These findings provide fundamental knowledge and new insights for future design and development of new biocompatible titanium alloys for orthodontic mini-implants and temporary anchorage devices.

Real-time cell analysis of the cytotoxicity of the components of orthodontic acrylic materials on gingival fibroblasts.

INTRODUCTION: The aim of this study was to evaluate the cytotoxicity of 3 orthodontic acrylic materials and 2 manipulation methods. METHODS: The orthodontic acrylic materials Orthocryl EQ (Dentaurum, Ispringen, Germany), Orthoplast (Vertex Dental, Zeist, The Netherlands), and O-80 (Imicryl, Konya, Turkey) were prepared with 2 polymerization methods (doughing and spray on). Totally, 60 cylinders (5 × 2 mm), fabricated by using a different acrylic and method, were divided into 6 groups. Gingival fibroblasts were isolated from gingival connective tissue of systemically healthy subjects. Materials were incubated in Dulbecco's modified eagle's medium culture medium (Biological Industries, Beit Haemek, Israel) for 72 hours according to ISO 10993-5 standards (surface area to volume ratio of the specimen to cell-culture medium: 3 cm(2)/mL). 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, Calif) was used to evaluate cell survival. After seeding 200 µL 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 acrylic materials (1/1 and 1/2 dilutions) and monitored every 15 minutes for 121 hours. For the proliferation experiments, the statistical analyses used were 1-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons tests. RESULTS: There was no significant difference between the cell indexes of the control and study groups for the 1/1 and 1/2 dilutions at 21 and 32 hours. When evaluated at 68 hours, all 1/2 dilutions of acrylic materials showed statistically insignificant differences (P >0.05) except for Orthoplast (P <0.05). But all acrylic materials were different from the control group in the 1/1 dilutions (P <0.001). At 121 hours, all test groups were significantly different from the untreated control group (P <0.001). CONCLUSIONS: The results indicate that the long cycle increased the cytotoxicity of the tested materials, and there was no significant difference between the spray-on and doughing methods on cytotoxicity.

Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1).

The aim of this study was to determine the effects of boron (B) on the cell-survival, proliferation, mineralization and mRNA expression of mineralized tissue-associated proteins. Additionally, determination of the effects of B on the BMP-4, -6 and -7 protein levels of pre-osteoblastic cells (MC3T3-E1) was also intended. The effects of B (pH 7.0) concentrations (0, 0.1, 1, 10, 100, 1000, 2000, 4000, 8000 and 10,000 ng/ml) on the survival of the cells were evaluated at 24 and 96 hrs with MTT assay. To evaluate the proliferation in long term, MC3T3-E1 cells were treated with different concentrations of B (0, 0.1, 1, 10, 100 and 1000 ng/ml) and were counted on days 2, 5, and 14. While in short term, decreased cell survival rate was observed at 1000 ng/ml and above, at long term no statistically significant difference was detected in different B concentrations applied. Slight decreases at the proliferation of the B-treated groups were determined on days 5 and 14 but one-way analysis of variance revealed that the difference was statistically insignificant. In mineralization assay, increased mineralized nodules were apparently observed in B treatment (1 and 10 ng/ml concentrations) groups. Based on quantitative RT-PCR results, remarkable regulation in favor of osteoblastic function for Collagen type I (COL I), Osteopontin (OPN), Bone Sialoprotein (BSP), Osteocalcin (OCN) and RunX2 mRNA expressions were observed in B treatment groups in comparison with untreated control groups. Increased BMP-4, -6 and -7 protein levels were detected at 0.1, 1, 10 and 100 ng/ml B concentrations. Results of the study suggest that at the molecular level B displays important roles on bone metabolism and may find novel usages at the regenerative medicine.