Sema S. 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.

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.

Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis

Oral lichen planus (OLP) is a chronic inflammatory disease that is frequently detected in oral tissues. The aim of our study was to identify the prevalence of the detection of periodontopathogenic microorganisms (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola in OLP patients and to compare with this prevalence of periodontopathogenic microorganisms in healthy non-OLP patients. Our study included 27 (18 chronic periodontitis (OLPP) and 9 gingivitis (OLPG)) patients diagnosed with OLP along with 26 (13 chronic periodontitis (HP) and 13 gingivitis (HG)) healthy non-OLP patients. The multiplex polymerase chain reaction (PCR) with subsequent reverse hybridization method (micro-IDent) was used for identifying periodontopathogenic microorganisms present in subgingival plaque samples. The percentages of detection for A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola in subgingival plaque samples taken from OLP patients (OLPG and OLPP) were 18.5%, 85.1%, 81.4%, 88.8% and 74%, respectively. Meanwhile, in the non-OLP patients (HG and HP), these values were 7.6%, 50%, 46.1%, 73% and 57.7%, respectively. Thus, comparing the non-OLP groups with the OLP groups, the periodontopathogens’ percentages of detection in the OLP groups were higher than those in the non-OLP groups. According to our study results, OLP patients have higher levels of infection with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola than non-OLP patients. We argue that the high percentages in patients with OLP may help identify the importance of periodontopathogenic microorganisms in the progress of periodontal diseases of OLP.

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.

Periodontal ligament fibroblast response to root perforations restored with different materials – a laboratory study

AIM   To compare the effect of several materials on the attachment of periodontal ligament (PDL) fibroblasts to experimentally perforated root surfaces. METHODOLOGY   Root specimens (size 5 × 5 mm) were obtained from extracted human molar teeth and perforations with a 1 mm diameter were created. One group was kept as a control and the rest were repaired with the following materials: Amalgam, Dyract, IRM, Super Bond C&B and Mineral trioxide aggregate (MTA). PDL fibroblasts were placed at a density of 8 × 10(4) cells on the root specimens, incubated on tissue culture inserts (48 h) and then transferred to 48 well-plates. MTT assays were performed at 48 and 96 h for PDL fibroblast survival. Cell attachment was observed using confocal microscopy on days 2 and 5. Total RNAs from the root specimens were isolated on day 5 and type I collagen (COL I) and Runt-related transcription factor 2 (Runx2) mRNA expressions were checked using Quantitative-Polymerase Chain Reaction (QPCR). For the MTT assay and QPCR, one-way analysis of variance (anova) and Tukey HSD multiple comparison tests were used to compare the groups. RESULTS Mineral trioxide aggregate resulted in a significantly higher cell density (P < 0.001). Dyract, IRM and Super Bond C&B groups had a lower cell density when compared with the control and MTA groups at 48 h (P < 0.001). Confocal microscopy revealed that, among the experimental groups, the MTA group had the largest viable cell population over the restoration site when compared with the other materials; however, reduced cell attachment was noted in all groups when compared with the control. Increased Runx2 mRNA expressions were noted in MTA (P < 0.001) and IRM (P < 0.01) groups when compared with control and other tested materials. COL I transcripts were increased in IRM (P < 0.01), D, C&B and MTA (P < 0.001) when compared with the control. CONCLUSION Mineral trioxide aggregate provided a more favorable environment for PDL cell adhesion and growth.

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.

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.