Erdogan E. Hakki

Effects of Mineral Trioxide Aggregate on Cell Survival, Gene Expression Associated with Mineralized Tissues, and Biomineralization of Cementoblasts

The 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 semiquantitative 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 in 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.

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.

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.

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.

Comparison of Mesenchymal Stem Cells Isolated From Pulp and Periodontal Ligament

BACKGROUND 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.

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.

Investigation of matrix metalloproteinase-1--1607 1G/2G polymorphism in a Turkish population with periodontitis.

AIM Matrix metalloproteinase-1 (MMP-1) is a proteolytic enzyme that degrades extracellular matrix and plays a fundamental role during destruction of periodontal tissues. The aim of this study was to examine the association between MMP-1 -1607 1G/2G polymorphism and chronic periodontitis susceptibility in a Turkish population. MATERIAL AND METHODS A total of 180 subjects were enrolled in this study. All the subjects received a periodontal examination including full-mouth clinical attachment loss measurements, probing depths, plaque index scores, gingival index scores and radiographic bone loss ratios. Three groups formed according to periodontal conditions were healthy, moderate periodontitis and severe periodontitis groups. MMP-1 -1607 1G/2G gene promoter polymorphism was genotyped using a polymerase chain reaction-restriction fragment length polymorphism method. RESULTS Analysis of the polymorphism showed no differences in distribution of the MMP-1 -1607 1G/2G polymorphism among healthy, moderate periodontitis and severe periodontitis groups (p>0.05). When the groups were further stratified by smoking status, we found no significant differences in genotype distributions, allele frequencies and carriage rates among any groups either (p>0.05). CONCLUSIONS On the basis of the results, no significant association is found for the MMP-1 -1607 1G/2G polymorphism with susceptibility to periodontitis. Moreover, smoking status did not seem to affect this result.

RT-PCR amplification of a Rhizopus oryzae lactate dehydrogenase gene fragment

No amino acid or DNA sequence information in sequence databases was found for a fungal lactate dehydrogenase (LDH) isozyme. Highly conserved regions in the lactate dehydrogenase enzymes of all taxonomies are found to be betaalphabeta nucleotide binding and substrate binding sites, also catalysis/active site. The conserved regions were selected as PCR primer target regions. The degenerate primers were designed according to the codon usage, determined by analyzing a number of different genes of Rhizopus species. A fragment of the gene (ldh), coding for approximately 72% of the lactate dehydrogenase enzyme from Rhizopus oryzae, was amplified using degenerate primers by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The size of the amplified fragment containing betaalphabeta nucleotide binding site, substrate binding site and catalysis/active site is found to be about 700 bp. The reported degenerate PCR primers and the amplification conditions may lead to the cloning of the lactate dehydrogenase gene of R. oryzae, which is an important organism due to its utilization in lactic acid and enzyme productions in industrial scales.

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.

Boron enhances strength and alters mineral composition of bone in rabbits fed a high energy diet

An experiment was performed to determine whether boron had a beneficial effect on bone strength and composition in rabbits with apparent adiposity induced by a high energy diet. Sixty female New Zealand rabbits, aged 8 months, were randomly divided into five groups with the following treatments for seven months: control 1, fed alfalfa hay only (5.91 MJ/kg); control 2, high energy diet (11.76 MJ and 3.88 mg boron/kg); B10, high energy diet+10 mg/kg body weight boron gavage/96 h; B30, high energy diet+30 mg/kg body weight boron gavage/96 h; B50, high energy diet+50mg/kg body weight boron gavage/96 h. Bone boron concentrations were lowest in rabbits fed the high energy diet without boron supplementation, which suggested an inferior boron status. Femur maximum breaking force was highest in the B50 rabbits. Tibia compression strength was highest in B30 and B50 rabbits. All boron treatments significantly increased calcium and magnesium concentrations, and the B30 and B50 treatments increased the phosphorus concentration in tibia of rabbits fed the high energy diet. The B30 treatment significantly increased calcium, phosphorus and magnesium concentrations in femur of rabbits fed the high energy diet. Principal component analysis of the tibia minerals showed that the three boron treatments formed a separate cluster from controls. Discriminant analysis suggested that the concentrations of the minerals in femur could predict boron treatment. The findings indicate boron has beneficial effects on bone strength and mineral composition in rabbits fed a high energy diet.