Rahime M. Nohutcu

Validation of reported genetic risk factors for periodontitis in a large-scale replication study.

AIM Many studies investigated the role of genetic variants in periodontitis, but few were established as risk factors. We aimed to validate the associations of recent candidate genes in aggressive periodontitis (AgP). MATERIAL AND METHODS We analysed 23 genes in 600 German AgP patients and 1441 controls on the Illumina custom genotyping array Immunochip. We tested a suggestive association in a Dutch and German/Austrian AgP case-control sample, and a German chronic periodontitis (CP) case-control sample using Sequenom iPlex assays. We additionally tested the common known risk variant rs1333048 of the gene ANRIL for its association in a Turkish and Italian population. RESULTS None of the analysed genes gave statistical evidence for association. Upon covariate adjustment for smoking and gender, in the pooled German-Austrian AgP sample, IL10 SNP rs6667202 was associated with p = 0.016, OR = 0.77 (95% CI = 0.6-0.95), and in the Dutch AgP sample, adjacent IL10 SNP rs61815643 was associated with p = 0.0009, OR = 2.31 (95% CI = 1.4-3.8). At rs61815643, binding of the transcription factor PPARG was predicted. ANRIL rs1333048 was associated in the Turkish sample (pallelic = 0.026, OR = 1.67 [95% CI = 1.11-2.60]). CONCLUSIONS Previous candidate genes carry no susceptibility factors for AgP. Association of IL-10 rs61815643 with AgP is suggested. ANRIL is associated with periodontitis across different populations.

Genetic evidence for PLASMINOGEN as a shared genetic risk factor of coronary artery disease and periodontitis

Background—Genetic studies demonstrated the presence of risk alleles in the genes ANRIL and CAMTA1/VAMP3 that are shared between coronary artery disease (CAD) and periodontitis. We aimed to identify further shared genetic risk factors to better understand conjoint disease mechanisms. Methods and Results—In-depth genotyping of 46 published CAD risk loci of genome-wide significance in the worldwide largest case–control sample of the severe early-onset phenotype aggressive periodontitis (AgP) with the Illumina Immunochip (600 German AgP cases, 1448 controls) and the Affymetrix 500K array set (283 German AgP cases and 972 controls) highlighted ANRIL as the major risk gene and revealed further associations with AgP for the gene PLASMINOGEN (PLG; rs4252120: P=5.9×10−5; odds ratio, 1.27; 95% confidence interval, 1.3–1.4 [adjusted for smoking and sex]; 818 cases; 5309 controls). Subsequent combined analyses of several genome-wide data sets of CAD and AgP suggested TGFBRAP1 to be associated with AgP (rs2679895: P=0.0016; odds ratio, 1.27 [95% confidence interval, 1.1–1.5]; 703 cases; 2.143 controls) and CAD (P=0.0003; odds ratio, 0.84 [95% confidence interval, 0.8–0.9]; n=4117 cases; 5824 controls). The study further provides evidence that in addition to PLG, the currently known shared susceptibility loci of CAD and periodontitis, ANRIL and CAMTA1/VAMP3, are subjected to transforming growth factor-&bgr; regulation. Conclusions—PLG is the third replicated shared genetic risk factor of atherosclerosis and periodontitis. All known shared risk genes of CAD and periodontitis are members of transforming growth factor-&bgr; signaling.

Dexamethasone enhances the effects of parathyroid hormone on human periodontal ligament cells in vitro

Periodontal ligament cells (PDL) are thought to play a major role in promoting periodontal regeneration. Recent studies, focused on characterizing PDL cells, have been directed at establishing their osteoblast-like properties and determining biological mediators and/or factors that induce osteoblastic cell populations in the PDL. The glucocorticoid, dexamethasone (Dex), has been shown to selectively stimulate osteoprogenitor cell proliferation and to induce osteoblastic cell differentiation in many cell systems. In the present study the ability of Dex to modulate parathyroid hormone (PTH)-stimulated cAMP synthesis in cultured human PDL cells was examined. PDL cells, obtained from premolar teeth extracted for orthodontic reasons, were cultured with Dex (0–1000 nM) for 7 days prior to PTH (1–34) stimulation. The exposure of PDL cells to Dex resulted in a dose-dependent increase in cAMP production in response to PTH stimulation. This response was seen in cells obtained from three different patients. The first significant Dex effect was seen on day 7 when compared to day 1 for 100 nM Dex. PTH (1–34) stimulation caused a dose-dependent increase in cAMP synthesis after Dex (1000 nM) treatment for 7 days. Conversely, stimulation of the cells with PTH (7–34) (0–1000 nM) did not increase cAMP production in PDL cells after Dex treatment. Forskolin- (1 μM) and isoproterenol- (1 μM) stimulated cAMP synthesis was not augmented by Dex treatment. Dex treatment did not alter calcitonin-(1 μM) stimulated cAMP production in PDL cells. Glucocorticoid enhancement of PTH-stimulated cAMP synthesis in these cells supports the presence of an osteoblast-like population in the PDL, in vitro.

Bone morphogenetic protein-7 enhances cementoblast function in vitro.

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.

bFGF-loaded HA-chitosan: A promising scaffold for periodontal tissue engineering

A scaffold containing growth factors promoting regeneration may be a useful device to maintain periodontal regeneration when applied with appropriate cells. The aim of this study is to evaluate the convenience of chitosan and hydroxyapatite (HA)-chitosan scaffolds loaded with basic fibroblast growth factor (bFGF) for periodontal tissue engineering applications. Scaffolds were fabricated by freeze-drying technique using 2 and 3% chitosan gel in the absence or presence of HA particles. Addition of HA beads to chitosan gels produced a novel scaffold in which the pore sizes and interconnectivity were preserved. The scaffolds were loaded with 100 ng bFGF by embedding technique. HA-chitosan scaffolds provide better controlled release kinetics for bFGF compared with chitosan scaffolds and total release continued up to 168 h. Cell culture studies were carried out with periodontal ligament (PDL) cells and cementoblasts. Both 3-[4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay and confocal laser scanning microscope analysis revealed cells proliferating inside the scaffolds. The results demonstrated that bFGF-loaded HA-chitosan scaffolds provide a suitable three-dimensional environment supporting the cellular structure, proliferation, and mineralization.

Bone Morphogenetic Protein-6-loaded Chitosan Scaffolds Enhance the Osteoblastic Characteristics of MC3T3-E1 Cells

The purpose of this study is to investigate the convenience of bone morphogenetic protein-6 (BMP-6)-loaded chitosan scaffolds with preosteoblastic cells for bone tissue engineering. MC3T3-E1 cells were seeded into three different groups: chitosan scaffolds, BMP-6-loaded chitosan scaffolds, and chitosan scaffolds with free BMP-6 in culture medium. Tissue-engineered constructs were characterized by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay, scanning electron microscopy (SEM), mineralization assay (von Kossa), alkaline phosphatase (ALP) activity, and osteocalcin (OCN) assays. BMP-6-loaded chitosan scaffolds supported proliferation of the MC3T3-E1 mouse osteogenic cells in a similar pattern as the unloaded chitosan scaffolds group and as the chitosan scaffolds with free BMP-6 group. SEM images of the cell-seeded scaffolds revealed significant acceleration of extracellular matrix synthesis in BMP-6-loaded chitosan scaffolds. Both levels of ALP and OCN were higher in BMP-6-loaded chitosan scaffold group compared with the other two groups. In addition, BMP-6-loaded scaffolds showed strong staining in mineralization assays. These findings suggest that BMP-6-loaded chitosan scaffold supports cellular functions of the osteoblastic cells; therefore, this scaffold is considered as a new promising vehicle for bone tissue engineering applications.

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 Dulbeccos modified Eagles 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.

In Vitro Release of Dexamethasone or bFGF from Chitosan/Hydroxyapatite Scaffolds

Chitosan scaffolds containing dexamethasone (Dex) or basic fibroblast growth factor (bFGF) were developed to create alternative drug-delivery systems for possible tissue-engineering applications such as periodontal bone regeneration. Chitosan solutions (2% and 3% (w/v) in acetic acid) were prepared from chitosan flakes with high deacetylation degree (>85%), then these solutions were freeze-dried at –80°C to obtain scaffolds with interconnected pore structures. Dex and bFGF were incorporated into scaffolds by embedding method (solvent sorption method). The initial loading amounts were varied as 300, 600 and 900 ng Dex per dry scaffold (average dry weight is 3 mg) and 50 or 100 ng bFGF per dry scaffold to a range of deliverable doses. Release studies which were conducted in Dulbeccos phosphate-buffered saline (DPBS) showed that 900 ng Dex loaded chitosan scaffolds in both compositions released total Dex during a 5-day period at a nearly constant rate after the initial burst. However, bFGF release from all scaffolds with both loading amounts (50 ng or 100 ng) was completed in 10 or 20 h. In order to prolong the release period of bFGF, composite scaffolds were fabricated in the presence of hydroxyapatite (HA) beads with average particle size of 40 μm. Sustained release of bFGF up to 7 days was achieved due to the electrostatic interactions between HA and bFGF molecules. These results suggested that chitosan scaffolds can be suitable for Dex release; however, the presence of HA in the chitosan scaffold is necessary to achieve the desired release period for bFGF.

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-β (TGF-β), 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-β 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.

Biomaterials in Periodontal Regenerative Surgery: Effects of Cryopreserved Bone, Commercially Available Coral, Demineralized Freeze-dried Dentin, and Cementum on Periodontal Ligament Fibroblasts and Osteoblasts

The ultimate goal of periodontal therapy is to achieve successful periodontal regeneration. The effects of different biomaterials, allogenic and alloplastic, used in periodontal surgeries to achieve regeneration have been studied in vitro on periodontal ligament (PDL) cells and MC3T3-E1 cells. The materials tested included cryopreserved bone allograft (CBA), coralline hydroxyapatite (CH), demineralized freeze-dried dentin (DFDD), and cementum. CBA and CH revealed an increase in initial PDL cell attachment, whereas CH resulted in an increase in long-term PDL cell attachment. Mineral-like nodule formation was observed significantly higher in DFDD compared to other materials tested for osteoblasts. Based on the results of this in vitro study, we conclude that the materials used are all biocompatible with human PDL cells and osteoblasts, which have pivotal importance in periodontal regeneration.