Buket S. Bozkurt

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.

Comparison of Er,Cr:YSGG laser and hand instrumentation on the attachment of periodontal ligament fibroblasts to periodontally diseased root surfaces: an in vitro study.

BACKGROUND This study investigates the effects of erbium, chromium:yttrium-scandium-gallium-garnet (Er,Cr:YSGG) laser irradiation and hand instrumentation on the attachment of periodontal ligament (PDL) fibroblasts to periodontally involved root surfaces. METHODS Twenty-four single-rooted periodontally involved human teeth (test groups), and six healthy premolar teeth extracted for orthodontic reasons (control group) were included in this study. A total of 45 root slices were obtained from all selected teeth and assigned to the following five groups: 1) untreated healthy group (+control); 2) untreated periodontally diseased group (-control); 3) hand instrumentation group (scaled Gracey); 4) laser I, Er,Cr:YSGG laser irradiation setting-I (short pulse); and 5) laser II, Er,Cr:YSGG laser irradiation setting-II (long pulse). All of the root slices were autoclaved in phosphate buffered saline and slices were placed onto cell culture inserts. PDL fibroblasts were placed at the density of 80,000 cells on the root plate (5 x 6 mm) and incubated for 48 hours and transferred to 24-well plates. The attachment PDL fibroblasts on the root plates were observed using confocal microscopy (at 12 hours and on days 3 and 7) and scanning electron microscopy (at 12 hours and day 3). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was performed on day 5 for PDL fibroblast survival. RESULTS 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay shows that whereas laser-treated specimens showed a significantly higher cell density, the Gracey-treated group showed a lower cell density compared to the positive control group (P <0.05). Based on confocal microscopy, apparent reduction was observed in the attachment of PDL cells to the periodontally diseased root surfaces. In the laser and Gracey groups, cells looked well-oriented to the root surfaces. Laser-treated groups provided suitable environment for cell adhesion and growth. Laser I treatment was more favorable for the attachment of PDL compared to scaled Gracey, laser II, and even healthy root surfaces. CONCLUSION The results of the study indicate that short-pulse laser setup (laser I) looks more promising regarding the attachment, spreading, and orientation of PDL cells.

Bone morphogenetic protein‐2, ‐6, and ‐7 differently regulate osteogenic differentiation of human periodontal ligament stem cells

The utility of adult stem cells for bone regeneration may be an attractive alternative in the treatment of extensive injury, congenital malformations, or diseases causing large bone defects. To create an environment that is supportive of bone formation, signals from molecules such as the bone morphogenetic proteins (BMPs) are required to engineer fully viable and functional bone. We therefore determined whether BMP-2, -6, and -7 differentially regulate the (1) proliferation, (2) mineralization, and (3) mRNA expression of bone/mineralized tissue associated genes of human periodontal ligament stem cells (hPDLSCs), which were obtained from periodontal ligament tissue of human impacted third molars. hPDLSCs from six participants were isolated and characterized using histochemical and immunohistochemical methods. A real-time cell analyzer was used to evaluate the effects of BMP-2, -6, and -7 on the proliferation of hPDLSCs. hPDLSCs were treated with Dulbeccos modified Eagles medium containing different concentrations of BMP-2, -6, and -7 (10, 25, 50, 100 ng/mL) and monitored for 264 hours. After dose-response experiments, 50 and 100 ng/mL concentrations of BMPs were used to measure bone/mineralized tissue-associated gene expression. Type I collagen, bone sialoprotein, osteocalcin, osteopontin, and osteoblastic transcription factor Runx2 mRNA expression of hPDLSCs treated with BMP-2, -6, and -7, were evaluated using quantitative RT-PCR. Biomineralization of hPDLSCs was assessed using von Kossa staining. This study demonstrated that BMPs at various concentrations differently regulate the proliferation, mineralization, and mRNA expression of bone/mineralized tissue associated genes in hPDLSCs. BMPs regulate hPDLSC proliferation in a time and dose-dependent manner when compared to an untreated control group. BMPs induced bone/mineralized tissue-associated gene mRNA expression and biomineralization of hPDLSCs. The most pronounced induction occurred in the BMP-6 group in the biomineralization of the hPDLSCs. Our data suggest that BMP-2, -6, and -7 are potent regulators of hPDLSC gene expression and biomineralization. Employing BMPs with hPDLSCs isolated from periodontal ligament tissues provides a promising strategy for bone tissue engineering.

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 Dulbeccos modified eagles 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 Dulbeccos modified eagles 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 Dulbeccos 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.

Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes

Abstract The purpose of this study was to investigate adhesion, proliferation and type I collagen (COL I) mRNA expression of gingival fibroblasts on different membranes used in periodontal applications. Collagen (C), acellular dermal matrix (ADM) and polylactic acid; polyglycolic acid; lactide/glycolide copolymer (PLGA) biodegradable membranes were combined with gingival fibroblasts in culture and incubated for 48 h. Cell adhesion was examined with scanning electron and confocal microscopy. MTT assay was used to measure proliferation. COL I mRNA expression was assessed using quantitative-polymerase chain reaction (QPCR). The PLGA group exhibited the lowest cell survival on day 5 and 10, and lowest cell proliferation on days 5, 10 and 14. While cell proliferation was similar in C and ADM groups, the C membrane showed a slightly greater increase in viable cells to day 10. Confocal and scanning electron microscopy confirmed the results of proliferation and MTT assays. The highest COL I mRNA expression was noted in the PLGA membrane group when compared to the C (p < 0.01) and ADM (p < 0.05) membrane groups. These data revealed that adherence and proliferation of primary gingival fibroblasts on collagen-based C and ADM membranes is better than that seen with PLGA membranes, and thus may be preferable in the treatment of gingival recession defects.

Periodontal ligament cell behavior on different titanium surfaces

Abstract 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 cytotoxicity of orthodontic cements on gingival fibroblasts

Introduction: To evaluate the cytotoxicity of four different orthodontic cement materials using the real-time xCELLigence system. Materials and Methods: Four orthodontic glass ionomer cements (GICs) were selected for this study, namely: GC Fuji (GC Cooperation), Ultra Band Lok (Reliance), Multi Cure (3M Unitek), and Meron (Voco). Ten test cylinders (measuring 5 Χ 2 mm) of each material were fabricated, making a total of 40 cylinders. The samples were incubated in Dulbecco modified Eagle medium (DMEM) culture medium for 72 hours. Human gingival fibroblasts (HGFs) were maintained with DMEM containing 10% fetal bovine serum. A real-time cell analyzer (RT-CES, xCELLigence) was used to evaluate cell survival. After seeding 200 μL of the cell suspensions into the wells (10,000 cells/well), gingival fibroblasts were treated with bioactive components released from cement materials and were monitored every 15 minutes for a period of 88 hours. For proliferation experiments, the statistical analyses used were one-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons tests. Results: When the data were evaluated at 24 and 48 hrs, all tested materials showed statistically significant decreases in HGF cell index compared to the control group (P < 0.001). Conclusion: According to the results of this study, all tested cements were found to have cytotoxic effects to the HGFs.

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.