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Öğe BMP-6 regulates osteogenic differentiation of the human periodontal ligament stem cells (hPDLSCs)(ELSEVIER SCIENCE BV, 2012) Hakki, Sema S.; Hakki, Erdogan E.; Bozkurt, S. Buket; Turac, Gizem; Karaoz, Erdal[Abstract not Available]Öğe BMP-6 regulates osteogenic differentiation of the human periodontal ligament stem cells (hPDLSCs)d(ELSEVIER SCIENCE BV, 2012) Hakki, Sema S.; Hakki, Erdogan E.; Bozkurt, S. Buket; Turac, Gizem; Karaoz, Erdal[Abstract not Available]Öğe Bone Morphogenetic Protein-7 Enhances Cementoblast Function In Vitro(Wiley, 2010) Hakkı, Sema S.; Foster, Brian L.; Nagatomo, Kanako J.; Bozkurt, S. Buket; Hakkı, Erdoğan E.; Somerman, Martha J.; Nohutcu, Rahime M.Background: Bone morphogenetic protein (BMP)-7 is a potent bone-inducing factor and was shown to promote periodontal regeneration in vivo and in vitro; however, to our knowledge, the specific effect of BMP-7 on cementoblasts has not been defined. We aimed to investigate the effects of BMP-7 on cementoblasts, which are cells responsible for tooth root-cementum formation. We hypothesized that BMP-7 would regulate mineralized tissue-associated genes in cementoblasts and influence the expression profile of genes associated with cementoblast extracellular matrix (ECM) and cell adhesion molecules (CAMs). Methods: A murine immortalized cementoblast cell line (OCCM.30) was cultured with and without 50 ng/ml BMP-7. After 72 hours, total RNA was isolated, and mRNA levels for bone/cementum markers, including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor-2 (Runx2), were investigated by real-time quantitative reverse transcription-polymerase chain reaction (Q-PCR). In vitro mineral nodule formation was assayed on day 8 using von Kossa staining. A pathway-specific gene-expression array was used to determine BMP-7-responsive ECM and CAM genes in cementoblasts. Results: Mineralized tissue markers were strongly regulated by BMP-7, with an almost three-fold increase in BSP and OCN transcripts and significant increases in OPN and Runx2 mRNA expressions. BMP-7 treatment markedly stimulated cementoblast-mediated biomineralization in vitro compared to untreated cells at day 8. BMP-7 treatment altered the OCCM.30 expression profile for ECM and CAM functional gene groups. BMP-7 tended to increase the expression of collagens and matrix metalloproteinases (MMPs), mildly decreased tissue inhibitors of MMPs (TIMPs), and had mixed regulatory effects on integrins. Using Q-PCR, selected array results were confirmed, including a significant BMP-7-induced increase in MMP-3 and a decrease in TIMP-2 mRNA expression. Conclusion: These results support the promising applications of BMP-7 in therapies aimed at regenerating periodontal tissues lost as a consequence of disease.Öğe Comparison of Different Sources of Mesenchymal Stem Cells: Palatal versus Lipoaspirated Adipose Tissue(KARGER, 2017) Hakki, Sema S.; Turac, Gizem; Bozkurt, S. Buket; Kayis, Seyit Ali; Hakki, Erdogan E.; Sahin, Eren; Subasi, CansuObjectives: The purpose of this study was to compare the proliferation and differentiation potential of mesenchymal stem cells (MSCs) derived from palatal adipose tissue (PAT) and lipoaspirated adipose tissue (LAT). Materials and Methods: PATs were obtained from 2 healthy female patients undergoing surgery for gingival recession, and LATs were obtained from 2 healthy female patients undergoing plastic surgery. LAT-and PAT-derived MSCs were confirmed by flow cytometry using MSC-specific surface markers. The multilineage differentiation capacity of the MSCs was analyzed. The expression of immunophenotyping, embryonic, and differentiation markers was compared between both MSC lines. The proliferation of PAT-and LAT-MSCs was evaluated using a real-time cell analyzer, and telomerase activity was determined using an ELISA-based TRAP assay. Stem cells isolated from PAT and LAT were analyzed by real-time PCR and whole genome array analysis. Results: The cells isolated from PAT had MSC characteristics. In addition, PAT-MSCs had significantly higher alkaline phosphatase activity and osteogenic potential than LAT-MSCs. Although the proliferation and telomerase activities of LAT-MSCs were higher than those of PAT-MSCs, the difference was not statistically significant. The level of embryonic stem cell markers (Oct4 and Nanog) was higher in LAT-MSCs than in PAT-MSCs. The whole genome array analysis demonstrated that 255 gene sequences were differentially expressed, with more than a twofold change in expression. Conclusions: This is the first comparative analysis of the isolation and characterization of MSCs from PAT and LAT. PAT is an accessible source of MSCs, which could be used in periodontal and craniofacial tissue engineering. (C) 2017 S. Karger AG, BaselÖğe Comparison of Mesenchymal Stem Cells Isolated From Pulp and Periodontal Ligament(WILEY, 2015) Hakki, Sema S.; Kayis, Seyit Ali; Hakki, Erdogan E.; Bozkurt, S. Buket; Duruksu, Gokhan; Unal, Zehra Seda; Turac, GizemBackground: Cell-based therapy using mesenchymal stem cells (MSCs) seems promising to obtain regeneration of dental tissues. A comparison of tissue sources, including periodontal ligament (PDL) versus pulp (P), could provide critical information to select an appropriate MSC population for designing predictable regenerative therapies. The purpose of this study is to compare the proliferation and stemness and the MSC-specific and mineralized tissue-specific gene expression of P-MSCs and PDL-MSCs. Methods: MSCs were obtained from PDL and P tissue of premolars (n = 3) extracted for orthodontic reasons. MSC proliferation was evaluated using a real-time cell analyzer for 160 hours. Telomerase activity was evaluated by a telomeric repeat amplification protocol assay based on enzyme-linked immunosorbent assay. Total RNA was isolated from the MSCs on day 3. A polymerase chain reaction (PCR) array was used to compare the expression of MSC-specific genes. The expression of mineralized tissue-associated genes, including Type I collagen (COL I), runt-related transcription factor 2 (RunX2), bone sialoprotein (BSP), and osteocalcin (OCN) messenger RNA (mRNA), was evaluated using quantitative real-time PCR. Results: Higher proliferation potential and telomerase activity were observed in the P-MSCs compared to PDL-MSCs of premolar teeth. Fourteen of 84 genes related to MSCs were expressed differently in the PDL-MSCs versus the P-MSCs. The expressions of bone morphogenetic protein 2 (BMP2) and BMP6; sex-determining region Y-box 9 (SOX9); integrin, alpha 6 (ITGA6); melanoma cell adhesion molecule (MCAM); phosphatidylinositol glycan anchor biosynthesis, class S (PIGS); prominin 1 (PROM1); ribosomal protein L13A (RPL13A); and microphthalmia-associated transcription factor (MITF) were higher in the P-MSCs compared to the PDL-MSCs, and higher expression of matrix metalloproteinase 2 (MMP2), interleukin (IL)-6, insulin (INS), alanyl (membrane) aminopeptidase (ANPEP), and IL-10 were observed in the PDL-MSCs. However, there was no statistically significant difference in the expression of mineralized tissue-associated genes, including BSP and RunX2, between the P-MSCs and the PDL-MSCs. Higher expression of COL I and lower expression of OCN mRNA transcripts were noted in the PDL-MSCs compared to the P-MSCs. Conclusions: The results of this study suggest that MSCs isolated from P and PDL tissues show different cellular behavior. To increase the predictability of MSC-based regenerative treatment, differences in dental tissue-derived MSCs and favorable aspects of cell sources should be further clarified.Öğe THE EFFECT OF THYMOQUINONE ON THE miRNA PROFILE OF MCF-7 BREAST CANCER CELLS(INT JOURNAL PHARMACEUTICAL SCIENCES & RESEARCH, 2017) Saracligil, Beyza; Ozturk, Bahadir; Bozkurt, S. Buket; Kahveci, YaseminBackground and aim: Thymoquinone (TQ), which is the most bioactive component of Nigella sativa (Black cumin), exhibits anticancer characteristics based on cell culture and experimental animal studies. However, molecular action mechanisms of these effects are not clear. MicroRNAs (miRNAs), small non-coding RNAs of approximately 22 nucleotides, are an emerging class of gene expression modulators with relevant roles in several biological processes, including cell differentiation, development, apoptosis, and regulation of the cell cycle. The purpose of this study was to investigate the potential impact of thymoquinone (TQ) on MCF-7 human breast cancer cell miRNAs. Materials and methods: The expression levels of miRNAs in MCF-7 cell and TQ treated MCF-7 cells were estimated by miRNA sequencing. The expressions of miRNAs were determined real-time qPCR. Results: We detected 10 down-regulated mirRNAs (hsa-miR-1, let 7c-5p, hsa-miR-15b-5p, hsa-mir-202-3p, hsa-miR-214-3p, hsa-miR-210-3p, hsa-miR-31-5p, hsa-miR-424-5p, hsa-miR-497-5p, hsa-miR-98-5p) and 2 up regulated miRNAs (hsa-miR-22-3p, hsa-miR-132-3p) in TQ treated groups, comparing with control group. These findings highlight the effects of TQ miRNA profile and molecular mechanism on MCF-7 cells. Conclusion: Finally, according to computational analyses using validated databases PI3 kinase/AKT (hsa04151), Wnt (hsa04310), MAPK (hsa04010) and p53 (hsa 04115) signaling pathways seem to be the key targets of these TQ groups of miRNA.Öğe Effects of different setting of diode laser on the mRNA expression of growth factors and type I collagen of human gingival fibroblasts(SPRINGER LONDON LTD, 2012) Hakki, Sema S.; Bozkurt, S. BuketThe aim of this study was to analyze the influence of non-surgical applications of diode laser (940 nm) on the cell proliferation and mRNA expressions of type I collagen and growth factors in human gingival fibroblasts (GF). Gingival fibroblasts were isolated from human gingival connective tissue of systemically healthy individuals. Cells were treated with different laser parameters as follows; (1) Infected pocket setting (power: 2 W, pulse interval: 1 ms, pulse length: 1 ms, 20 s/cm(2)); (2) Perio-pocket setting (power: 1.5 W, pulse interval: 20 ms, pulse length: 20 ms, 20 s/cm(2)); and (3) Biostimulation setting (power: 0.3 W in continuous wave, 20 s/cm(2)). Proliferation of GF was evaluated after different laser applications using a real-time cell analyzer. Total RNA was isolated on day 2 and cDNA synthesis was performed. Type I collagen, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-beta) mRNA expressions were determined with quantitative RT-PCR. In a proliferation experiment, no significant differences were observed in the different laser applications when compared to the control group. Statistically significant increases in IGF, VEGF, and TGF-beta mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). A significant increase in collagen type I mRNA expression was noted in only biostimulation set-up of diode laser (p < 0.05). The results of this study demonstrate that non-surgical laser applications modulate behavior of gingival fibroblasts inducing growth factors mRNA expressions and these applications can be used to improve periodontal wound healing.Öğe Effects of Mineral Trioxide Aggregate on Cell Survival, Gene Expression Associated with Mineralized Tissues, and Biomineralization of Cementoblasts(ELSEVIER SCIENCE INC, 2009) Hakki, Sema S.; Bozkurt, S. Buket; Hakki, Erdogan E.; Belli, SemaThe purpose of this study was to investigate the effects of mineral trioxide aggregate (MTA) on survival, mineralization, and expression of mineralization-related genes of cementoblasts. Immortalized cementoblasts (OCCM) were maintained with Dulbecco modified Eagle medium containing 10% fetal bovine serum. Methyl-thiazol-diphenyl-tetrazolium experiments were performed at 24 and 72 hours to evaluate bioactive components released by MTA (0.002-20 mg/mL) on the cell survival of OCCM. Von Kossa staining was used to evaluate biomineralization of OCCM Cells. Images of cementoblasts were taken on day 3 by using inverted microscopy. Gene transcripts for bone sialoprotein (BSP), OCN, collagen type I (COL I), and osteopontin (OPN) were evaluated on days 3 and 5 by using semi-quantitative reverse transcriptase polymerase chain reaction. The 20 mg/mL concentration of MTA was toxic for OCCM cells, whereas other concentrations of MTA tested exhibited similar cell numbers when compared with control group, and the 0.02 mg/mL concentration of MTA increased OCCM cell survival at 72 hours. Although an apparent decrease n mineralization was observed in the highest 3 concentrations of MTA used, 0.02 and 0.002 mg/mL concentrations of MTA induced greater biomineralization of OCCM cells than seen in the control. Moreover, increased BSP and COL I mRNA expression was observed at 0.02 and 0.002 mg/mL concentrations of MTA. MTA did not have a negative effect on the viability and morphology of cementoblasts and induced biomineralization of cementoblasts at the concentrations of 0.02 and 0.002 mg/mL. Based on these results MTA can be considered as a favorable material regarding cell-material interaction. (J Endod 2009;35:513-519)Öğe Efficacy of Collagen Membrane Seeded With Autologous Gingival Fibroblasts in Gingival Recession Treatment: A Randomized, Controlled Pilot Study(WILEY, 2013) Koseoglu, Serhat; Duran, Ismet; Saglam, Mehmet; Bozkurt, S. Buket; Kirtiloglu, Osman S.; Hakki, Sema S.Background: Gingival recession (GR) is one of the most common esthetic concerns associated with periodontal tissues. Recently, tissue engineering technology has been developed and applied in periodontology for the treatment of GR. The aim of this study is to compare the clinical efficacy of collagen membrane with or without autologous gingival fibroblasts under a coronally advanced flap for root coverage. Methods: In this split-mouth, controlled clinical study, 22 sites are selected from 11 patients with Miller Class I recessions affecting canines or premolars in the maxillary arch. One tooth in each patient was randomized to receive either a collagen membrane (CM) (control group) or a collagen membrane seeded with autologous gingival fibroblasts (CM+GF) (test group) under a coronally advanced flap. Thickness of the gingiva, GR, and percentage of root coverage (PRC) were recorded by a calibrated examiner at baseline and 3, 6, and 12 months postoperatively. Furthermore, GR and PRC were evaluated using photogrammetric analysis at baseline and 3, 6, and 12 months. Results: Both treatments resulted in a significant gain in root coverage compared with baseline. A statistically significant increase was detected in PRC in the test group compared with the control group. No significant difference was noted between the test and control sites regarding the thickness of the gingiva. Conclusions: The results indicated that CM+GF prepared by tissue engineering technology can be considered an alternative method for the treatment of Miller Class I recession defects.Öğe Ionizing Radiation Induces Cytokines, MMP-1, TIMP-1 and Supresses Type I Collagen mRNA Expressions in Human Gingival Fibroblasts(AKAD DOKTORLAR YAYINEVI, 2014) Yavas, Guler; Yavas, Cagdas; Bozkurt, S. Buket; Ata, Ozlem; Hakki, SemaWe aimed to evaluate the effects of ionizing radiation on the proliferation of gingival fibroblasts and the expressions of proinflammatory cytokines and matrix metalloproteinase-1 (MMP-1), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and type I collagen (type I Col) mRNA transcripts. Gingival fibroblasts were treated with radiation doses as follows; 0.5 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, and 8 Gy. Expression of interleukin (IL)-1 beta, IL-6, IL-8 and, MMP-1, TIMP-1 and of type I Col mRNA transcripts in human gingival fibroblasts was determined by quantitative polymerase chain reaction (PCR) analysis. Morphology of gingival fibroblasts was evaluated using inverted microscope. Ionizing radiation decreased cell proliferation (p< 0.05) compared to the control group. Expressions of IL-1 beta, IL-6 and IL-8 were stimulated at the highest dosage of radiation (p< 0.001). In parallel to proinflammtory cytokines, MMP-1 and TIMP-1 mRNA expressions were elevated in response to higher dosage of radiation (p< 0.001). Radiation suppressed type I Col mRNA expression in response to all doses at 24 hrs (p< 0.001). In addition to basal epithelial cells of the oral mucosa, gingival fibroblasts have an important role in the pathogenesis of oral mucositis. Results of this study may help to clarify the role of gingival fibroblasts in radiation induced oral mucositis.Öğe Osteogenic Differentiation of MC3T3-E1 Cells on Different Titanium Surfaces(IOP PUBLISHING LTD, 2012) Hakkı, S. Sema; Bozkurt, S. Buket; Hakkı, Erdoğan E.; Korkusuz, Petek; Puralı, Nuhan; Koç, Nursen; Timuçin, Muharrem; Öztürk, Adnan; Korkusuz, FezamRNA expressions related to osteogenic differentiation of MC3T3-E1 cells on electro-polished smooth (S), sandblasted small-grit (SSG) and sandblasted large-grit (SLG) surfaces of titanium alloys were investigated in vitro. Gene expression profiles of cells were evaluated using the RT2 Profiler PCR microarray on day 7. Mineralizing tissue-associated proteins, differentiation factors and extracellular matrix enzymes mRNA expressions were measured using Q-PCR. SLG surface upregulated 23 genes over twofolds and downregulated 3 genes when compared to the S surface. In comparison to the SSG surface, at least a twofold increase in 25 genes was observed in the SLG surface. BSP, OCN, OPN, COL I and ALP mRNA expressions increased in the SLG group when compared to the S and the SSG groups. BMP-2, BMP-6 and TGF-beta mRNA expressions increased in both the SSG and the SLG surfaces. MMP-2 and MMP-9 mRNA expressions increased as the surface roughness increased. This study demonstrated that surface roughness of titanium implants has a significant effect on cellular behavior and SLG surface apparently increased gene expressions related to osteogenesis when compared to the S and the SSG surfaces.Öğe Porphyromonas gingivalis Lipopolysaccharide Induces a Pro-inflammatory Human Gingival Fibroblast Phenotype(SPRINGER/PLENUM PUBLISHERS, 2017) Bozkurt, S. Buket; Hakki, Sema S.; Hakki, Erdogan E.; Durak, Yusuf; Kantarci, AlpdoganHuman gingival fibroblasts (HGFs) are the major constituents of the gingival tissues responsible for the synthesis and degradation of the connective tissue while actively participating in immune reactions and inflammation. The aim of this study was to test the impact of lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) on human gingival fibroblasts. Human gingival fibroblasts were treated with different P. gingivalis LPS concentrations. Cell survival rate was evaluated with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) after 24 h. Cell proliferation was determined by counting cells on days 3 and 12. Expression of matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs), and pro-inflammatory cytokine transcripts in HGFs was determined by quantitative PCR (Q-PCR) analysis on days 3 and 8. P. gingivalis LPS decreased cell proliferation on day 3 (p < 0.05) compared to the control group without significantly impacting the cell survival (p > 0.05).The experiments showed that P. gingivalis LPS dose-dependently and differentially modulated the expression of MMP-1, 2, and 3 and TIMP-1 and 2 on days 3 and 8. TIMP-1 expression was significantly induced in P. gingivalis LPS-treated cells while TIMP-2 was increased in response to 10 and 30 ng/ml of LPS on day 3. P. gingivalis LPS induced up-regulation of MMP-1/TIMP-1 ratio on day 3 and increased MMP-2/TIMP-2 ratio on day 8 dose-dependently. Expression of interleukin (IL)-6 and IL-8 was stimulated at higher concentrations (1000 and 3000 ng/ml) of LPS. These findings demonstrate that P. gingivalis LPS suppresses cell proliferation and leads to increased pro-inflammatory changes in HGFs, suggesting that P. gingivalis LPS-induced modification of phenotypic and inflammatory characteristics in HGF could potentially be a pathogenic mechanism underlying the tissue destruction.Öğe Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro(NATURE PUBLISHING GROUP, 2018) Hakki, Sema S.; Bozkurt, S. Buket; Tuerkay, Emre; Dard, Michel; Purali, Nuhan; Goetz, WernerAmelogenin (AMG) is a cell adhesion molecule that has an important role in the mineralization of enamel and regulates events during dental development and root formation. The purpose of the present study was to investigate the effects of recombinant human AMG (rhAMG) on mineralized tissue-associated genes in cementoblasts. Immortalized mouse cementoblasts (OCCM-30) were treated with different concentrations (0.1, 1, 10, 100, 1000, 10,000, 100,000 ng . mL(-1)) of recombinant human AMG (rhAMG) and analyzed for proliferation, mineralization and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), collagen type I (COL I), osteopontin (OPN), runt-related transcription factor 2 (Runx2), cementum attachment protein (CAP), and alkaline phosphatase (ALP) genes using quantitative RT-PCR. The dose response of rhAMG was evaluated using a real-time cell analyzer. Total RNA was isolated on day 3, and cell mineralization was assessed using von Kossa staining on day 8. COL I, OPN and lysosomal-associated membrane protein-1 (LAMP-1), which is a cell surface binding site for amelogenin, were evaluated using immunocytochemistry. F-actin bundles were imaged using confocal microscopy. rhAMG at a concentration of 100,000 ng . mL(-1) increased cell proliferation after 72 h compared to the other concentrations and the untreated control group. rhAMG (100,000 ng . mL(-1)) upregulated BSP and OCN mRNA expression levels eightfold and fivefold, respectively. rhAMG at a concentration of 100,000 ng . mL(-1) remarkably enhanced LAMP-1 staining in cementoblasts. Increased numbers of mineralized nodules were observed at concentrations of 10,000 and 100,000 ng . mL(-1) rhAMG. The present data suggest that rhAMG is a potent regulator of gene expression in cementoblasts and support the potential application of rhAMG in therapies aimed at fast regeneration of damaged periodontal tissue.
