Boric acid alleviates periodontal inflammation induced by IL-1ß in human gingival fibroblasts.

BACKGROUND: Boric acid (BA) has been found to have therapeutic effects on periodontal disease through beneficially affecting antibacterial, anti-viral, and anti-inflammatory actions. METHODS: This study was conducted to determine the effect of BA on cell viability and on mRNA expressions of proinflammatory and anti-inflammatory cytokines and on oxidative stress enzymes induced by IL-1ß (1 ng/mL) in Human Gingival Fibroblasts (HGF) cultured for 24 and 72 h in DMEM media. The BA concentrations added to the media were 0.09 %, 0.18 %, 0.37 %, and 0.75 %. RESULTS: All of the BA concentrations increased the viability of cell cultured in DMEM media only, indicating that these concentrations were not toxic and actually beneficial to cell viability. The addition of 1 ng/m: of IL-1ß decreased cell viability that was overcome by all concentrations of BA at both 24 and 72 h. The IL-1ß addition to the media increased the expressions of the proinflammatory cytokines IL-1ß, IL-6, IL-8, and IL-17; the anti-inflammatory cytokine IL-10; and the oxidative stress enzymes superoxide dismutase (SOD0 and glutathione peroxidase (GPX). The IL-1ß induced increase mRNA expression of IL-1ß was decreased at 24 h by the 0.37 % and 0.75 % BA additions to the media and decreased in a dose-dependent manner by all concentrations of BA at 72 h. The IL-1ß induced increase in the expression of IL-6 was decreased in dose-dependent manner at 72 h by BA. All BA concentrations decreased the IL-1ß induced expression of IL-8 at both 24 and 72 h. The induced increase in IL-17 by IL-1ß was not significantly affected by the BA additions. The increase in the anti-inflammatory cytokine IL10 induced by IL-1ß was increased further by all BA additions in dose dependent manner at both 24 and 72 h. The mRNA expressions of SOD and GPX increased by IL-1ß were further increased by the 0.37 % and 0.75 % BA concentrations at 72 h. CONCLUSIONS: These findings indicate that BA can significantly modulate the cytokines that are involved in inflammatory stress and reactive oxygen species action and thus could be an effective therapeutic agent in the treatment of periodontal disease.

Hyaluronic acid enhances cell migration, viability, and mineralized tissue-specific genes in cementoblasts.

BACKGROUND/OBJECTIVES: It has been repeatedly demonstrated that cementum formation is a crucial step in periodontal regeneration. Hyaluronic acid (HA) is an important component of the extracellular matrix which regulates cells functions and cell-cell communication. Hyaluronic acid/derivatives have been used in regenerative periodontal therapy, but the cellular effects of HA are still unknown. To investigate the effects of HA on cementoblast functions, cell viability, migration, mineralization, differentiation, and mineralized tissue-associated genes and cementoblast-specific markers of the cementoblasts were tested. MATERIALS AND METHODS: Cementoblasts (OCCM-30) were treated with various dilutions (0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) of HA and examined for cell viability, migration, mineralization, and gene expressions. The mRNA expressions of osteocalcin (OCN), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), collagen type I (COL-I), alkaline phosphatase (ALP), cementum protein-1 (CEMP-1), cementum attachment protein (CAP), and small mothers against decapentaplegic (Smad) -1, 2, 3, 6, 7, ß-catenin (Ctnnb1) were performed with real-time polymerase chain reaction (RT-PCR). Total RNA was isolated on days 3 and 8, and cell viability was determined using MTT assay on days 1 and 3. The cell mineralization was evaluated by von Kossa staining on day 8. Cell migration was assessed 2, 4, 6, and 24 hours following exposure to HA dilutions using an in vitro wound healing assay (0, 1:2, 1:4, 1:8). RESULTS: At dilution of 1:2 to 1:128, HA importantly increased cell viability (p < .01). HA at a dilution of 1/2 increased wound healing rates after 4 h compared to the other dilutions and the untreated control group. Increased numbers of mineralized nodules were determined at dilutions of 1:2, 1:4, and 1:8 compared with control group. mRNA expressions of mineralized tissue marker including COL-I, BSP, RunX2, ALP, and OCN significantly improved by HA treatments compared with control group both on 3 days and on 8 days (p < .01). Smad 2, Smad 3, Smad 7, and ß-catenin (Ctnnb1) mRNAs were up-regulated, while Smad1 and Smad 6 were not affected by HA administration. Additionally, HA at dilutions of 1:2, 1:4, and 1:8 remarkably enhanced CEMP-1 and CAP expressions in a dilution- and time-dependent manner (p < .01). CONCLUSIONS: The present results have demonstrated that HA affected the expression of both mineralized tissue markers and cementoblast-specific genes. Positive effects of HA on the cementoblast functions demonstrated that HA application may play a key role in cementum regeneration.

The effects of preosteoblast-derived exosomes on macrophages and bone in mice.

The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated in vitro, and bone formation studies were performed in vivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1ß, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For in vivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.

Local application of gingiva-derived mesenchymal stem cells on experimental periodontitis in rats.

BACKGROUND: Stem cell-based approaches in regenerative periodontal therapy have been used in different experimental models. In this study, the effect of local application of gingival mesenchymal stem cells (GMSC) in fibroin/chitosan oligosaccharide lactate hydrogel (F/COS) on periodontal regeneration was evaluated using experimental periodontitis model in rats. METHODS: Mesenchymal stem cells were isolated from the gingiva of rats and characterized. Viability tests and confocal imaging of GMSC in hydrogels were performed. Healthy control without periodontitis (Health; H; n=10), control with periodontitis but no application (Periodontitis; P; n=10), only hydrogel application (F/COS; n=10), and GMSC+F/COS (n=10) four groups were formed for in vivo studies. Experimental periodontitis was created with silk sutures around the maxillary second molars. GMSC labeled with green fluorescent protein (GFP) (250,000 cells/50 µL) in F/COS were applied to the defect. Animals were sacrificed at 2nd and 8th weeks and maxillae of the animals were evaluated by micro-computed tomography (micro-CT) and histologically. The presence of GFP-labeled GMSC was confirmed at the end of 8 weeks. RESULTS: Micro-CT analysis showed statistically significant new bone formation in the F/COS+GMSC treated group compared with the P group at the end of 8 weeks (p < 0.05). New bone formation was also observed in the F/COS group, but the statistical analysis revealed that this difference was not significant when compared with the P group (p > 0.05). Long junctional epithelium formation was less in the F/COS+GMSC group compared with the P group. Periodontal ligament and connective tissue were well-organized in F/COS+GMSC group. CONCLUSION: The results showed that local GMSC application in hydrogel contributed to the formation of new periodontal ligament and alveolar bone in rats with experimental periodontitis. Since gingiva is easly accessible tissue, it is promising for autologous cell-based treatments in clinical applications.

Boric Acid Reverses Nicotine-Induced Cytokine Expressions of Human Gingival Fibroblasts.

Nicotine, the major bioactive ingredient in tobacco, is a major risk factor for periodontal disease and destruction. Nicotine has been shown to stimulate the production of cytokines that are priming agents for inflammation that induces tissue destruction, such as IL-1ß, IL-6, and IL-8, by gingival keratinocytes and human gingival fibroblasts (HGF). Boron as boric acid has been found to decrease pro-inflammatory cytokines and increase anti-inflammatory cytokines in cells with inflammatory stress. Thus, a study was performed to determine whether boric acid reverses negative effects of nicotine on human gingival fibroblasts (HGFs). The viability and cytokine expressions of HGFs cultured for 24 and 72 h in control medium with no nicotine or boric acid added and in media containing only nicotine, only boric acid, or a combination of BA and nicotine were determined. Nicotine in concentrations of 10-1, 10-2, 10-3,10-4, 10-5, and 10-6 mM significantly reduced cell viability compared to the control. Boric acid at 10 and 50 ng/mL in the media partially restored and 100 ng/mL in the media fully restored the nicotine-depressed HGF cell viability to the same level as the control group. Nicotine elevated the expression of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8, and IL-17 and decreased the anti-inflammatory IL-10 in HGFs at 24 and 72 h. Boric acid at 100 ng/mL in the medium prevented the changes induced by nicotine alone. The findings indicate that boric acid can inhibit or reverse nicotine-induced pathology in periodontal tissue and thus may help maintain oral and periodontal health in tobacco users.

Nail Mineral Composition Changes Do Not Reflect Bone Mineral Changes Caused by Boron Supplementation.

Nails have been found to be a non-invasive and readily available tissue whose mineral content can change because of a change in dietary mineral intake. Thus, this study was undertaken to determine whether boron (B) supplementation would change the concentrations of some mineral elements in nails and whether these changes correlated with changes induced in bone. Female New Zealand White rabbits (aged 8 months, 2-2.5 kg weight) were fed a grain-based, high-energy diet containing 3.88 mg B/kg. The rabbits were divided into four treatment groups: controls receiving no supplemental B (N: 7; C) and three groups supplemented with 30 mg B/L in drinking water as borax decahydrate (Na2B4O7∙10H2O, N: 10; BD), borax anhydrous (Na2B4O7, N: 7; Bah), and boric acid (H3BO3, N: 7; BA). Boron, calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P), potassium (K), sodium (Na), sulfur (S), and zinc (Zn) concentrations in nails were determined by inductively coupled plasma atomic emission spectroscopy. Parametric and non-parametric multiple group comparisons and post hoc tests were performed and whether a correlation between nail and tibia and femur mineral elements concentrations were determined. A p-value of < 0.05 was considered statistically significant. Boron was not detectable in control nails but was found in the nails of the three B supplemented groups. Boron supplementation markedly increased the Ca concentration in nails with the effect greatest in the BA and BD groups. The P and Mg concentrations also were increased by B supplementation with the effect most marked in the BA group. In contrast, B supplementation decreased the Na concentration with the effect most noticeable in the BD and Bah groups. The Zn concentration in nails was not affected by BA and BD supplementation but was decreased by Bah supplementation. Boron supplementation did not significantly affect the concentrations of Cu, Fe, Mo, K, and S in nails. No meaningful significant correlations were found between nail mineral elements and tibia and femur mineral elements found previously. Nails can be an indicator of the response to boron supplementation but are not useful to indicate changes in mineral elements in bone in response to B supplementation.

Borate and boric acid supplementation of drinking water alters teeth and bone mineral density and composition differently in rabbits fed a high protein and energy diet.

The reported beneficial effects of boron on mineralized tissues in animals and humans vary. Thus, a study was performed to assess whether the variability was the result of different forms of boron supplementation, method of supplementation, and increased adiposity of the rabbit experimental model. Thirty-one female New Zealand White rabbits, (aged 8 months, 2-2.5 kg weight) were fed a grain-based high energy diet containing 11.76 MJ/kg (2850 kcal/kg) and 3.88 mg boron/kg. The rabbits were randomly divided into four treatment groups: Control group was not supplemented with boron (n:7; C), and three groups supplemented with 30 mg boron/L in drinking water in the forms of borax decahydrate (Na2O4B7 10H2O, n:10; BD), borax anhydrous (Na2O4B7, n:7; Bah) or boric acid (H2BO3, n:7; BA). Cone beam micro computed tomographic (micro-CT), histological and elemental analysis was used to evaluate the bones/teeth. Results of the experiments demonstrated that boron supplementation had beneficial effects on mineralized tissue but varied with the type of treatment. Mineral density of the femur was increased by the Bah and BA treatments (p < 0.001), but only BA increased mineral density in the tibia (p = 0.015). In incisor teeth, mineral density of dentin was increased by all boron treatments (p < 0.001), and mineral density of enamel was increased by the BD and Bah treatments. Mineral analysis found that all boron treatments increased the boron concentration in tibia and femur. In the tibia, both the BD and Bah treatments decreased the iron concentration, and the BD treatment decreased the magnesium concentration. Sodium and zinc concentrations in the tibia were decreased by the Bah and BA treatments. The boron treatments did not significantly affect the calcium, copper, molybdenum, potassium phosphorus, and sulfur concentrations. The findings show that boron supplementation can have beneficial effects on mineralized tissues in an animal model with increased adiposity, which is a model of increased inflammatory stress. However, this effect varies with the form of boron supplemented, the method of supplementation, and the mineralized tissue examined.

SDF-1 modulates periodontal ligament-Mesenchymal Stem Cells (pdl-MSCs).

BACKGROUND/OBJECTIVES: In this in vitro study, the effects of Stromal cell-derived factor-1 (SDF-1) was evaluated on the periodontal ligament-Mesenchymal Stem Cells (pdl-MSCs) functions. MATERIAL AND METHODS: Real-time cell analyzer-single plate (RTCA-SP) was employed for proliferation, and RTCA-dual purpose (DP) was utilized for pdl-MSCs migration potential treated with different SDF-1 concentrations (0, 0.1, 1, 10, 100, 200, and 400 ng/ml). Based on the dose-response findings, 10 ng/ml SDF-1 was used for further mRNA experiments. RNAs isolated at 6 and 24 h were checked using quantitative RT-PCR for mineralized tissue-associated genes including type I collagen (COL I), osteocalcin (OCN), osteopontin (OPN), and runt-related transcription factor 2 (Runx2). cRNA was synthesized for 6 h, and whole-genome array analysis was performed for over 47.000 probes. Data were subjected to quantile normalization before analysis. RESULTS: Increased proliferation and migration were observed in pdl-MSCs treated with 0.1, 1, and 10 ng/ml SDF-1. Increased COL I was observed at both time points: 6 and 24 h. While there was no significant change for OCN, OPN, and Runx2 at 6 h, SDF-1 up-regulated OCN and OPN, but down-regulated Runx2 mRNA expressions at 24 h. IL-8 and ESM1 genes were differentially expressed over twofold when the pdl-MSCs were exposed to SDF-1 at whole-genome array analysis. IL-8 induction was confirmed with RT-PCR. CONCLUSION: Findings of this study displayed that SDF-1 modulated pdl-MSCs which were important for periodontal regeneration, inducing migration and proliferation, and regulating extracellular matrix synthesis in favor of the formation of new attachment.

Boron as Boric Acid Induces mRNA Expression of the Differentiation Factor Tuftelin in Pre-Osteoblastic MC3T3-E1 Cells.

The effects of boron on the formation and maintenance of mineralized structures at the molecular level are still not clearly defined. Thus, a study was conducted using MC3T3-E1 cells to determine whether boron affected mRNA expressions of genes associated with bone/alveolar bone formation around the teethMC3T3-E1 (clone 4) cells were cultured in media treated with boric acid at concentrations of 0, 0.1, 10, 100, or 1000 ng/ml. Total RNAs of each group were isolated on day 3. Gene expression profiles were determined by using RT2 Profiler PCR micro-array that included 84 genes associated with osteogenic differentiation. Tuftelin1 mRNA expression was upregulated by all boron treatments. The upregulation was confirmed by quantitative RT-PCR using the tuftelin probe. While 100 ng/ml had no effect on the integrin-α2 (Itga2) transcript and 1 ng/ml boric acid induced Itga2 mRNA expression (2.1-fold), 0.1, 10, and 1000 ng/ml boric acid downregulated the integrin-α2 gene transcript 2.2-, 1.5-, and 2.1-fold respectively. While 0.1 ng/ml boric acid induced BMP6, increased BMP1r mRNA expression (1.5 fold) was observed in 1000 ng/ml boric acid treatment. The findings suggest that boron affects the regulation of the tuftelin1 gene in osteoblastic cells. Further studies are needed to establish that the beneficial actions of boron on alveolar bone and tooth formation and maintenance include an effect on the expression of the tuftelin1 gene.

Effect of diode laser application as an adjunct to nonsurgical periodontal therapy on the reduction of red complex microorganisms in type 2 diabetics with chronic periodontitis.

Bactericidal and detoxification effects of diode laser (DL) have been reported in periodontal treatment. The objective of this study was investigating the additional effect of DL with nonsurgical periodontal treatment on the red complex bacteria in type 2 diabetes mellitus (DM) patients with chronic periodontitis (CP). Sixty type 2 DM patients with chronic periodontitis (CP) were randomly assigned in two parallel groups to receive scaling root planning (SRP, n = 30) or SRP followed by DL periodontal pocket irradiation (SRP + DL, n = 30). Recording of clinical parameters and subgingival plaque sampling were performed at baseline, and post therapy (1 and 3 months after treatment). Amounts of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia were evaluated with quantitative RT-PCR. Significant reductions for numbers of all three bacterial species were observed at 1 and 3 months compared with baseline for both treatments (p < 0.001), but no significant differences were found between two groups regarding bacterial reductions at these follow-up time points. No additional benefit of DL as an adjunct to nonsurgical periodontal therapy was recognized in the reduction of P. gingivalis, T. denticola, and T. forsythia for type 2 DM patients with CP. Further studies are required to clarify the effects of diode laser on the other periodontopathogens.

Salivary Del-1, IL-17, and LFA-1 levels in periodontal health and disease.

OBJECTIVE AND BACKGROUND: Developmental endothelial locus-1 (Del-1), lymphocyte function-associated antigen-1 (LFA-1), and interleukin 17 (IL-17) play critical roles in transendothelial migration of neutrophils in periodontal diseases. The aim of this study was to evaluate salivary Del-1, IL-17, and LFA-1 protein levels in patients with gingivitis (G), chronic periodontitis (CP), and generalized aggressive periodontitis (GAP). METHODS: A total of 180 systemically healthy, non-smoking patients (45 periodontally healthy (H) and 45 G, 50 CP, and 40 GAP) individuals (between March 2014 and February 2016) were included in this study according to Armitage's (1999) classification. Clinical periodontal parameters, including clinical attachment level, probing depth, plaque index, and gingival index, were recorded. Del-1, IL-17, and LFA-1 protein expression levels were measured in unstimulated saliva samples collected from patients by using enzyme-linked immunosorbent assays. Kruskal-Wallis and Mann-Whitney U tests were used for multiple comparisons and post hoc statistical analyses, respectively. ROC curve analysis was used to evaluate the sensitivity and specificity of Del-1, IL-17, and LFA-1 in distinguishing periodontal disease from health and gingivitis. RESULTS: It was found a high level of IL-17 and a low level of Del-1 in the CP and GAP, as compared to the G and H groups (P < .001). Nevertheless, we found LFA-1 levels were higher in the GAP than in the CP or G groups (P = .00). Consistently, LFA-1 levels were lower in the H and G groups than in the CP and GAP groups (P = .00). The combination of three biomarkers was found as the best predictor yielded exhibited the highest AUC [0.893, 0.845-0.94 (%95 CI) P < .001] in discriminating periodontal disease from health and gingivitis. CONCLUSION: Salivary Del-1, LFA-1, and IL-17 levels might be useful markers for determining the clinical health and disease status of patients with periodontitis. However, further studies that evaluate the level of salivary Del-1, LFA-1, and IL-17 before and after periodontal therapy are required to understand the exact roles of these cytokines during the periodontal healing period.

The Association Between Some Macro and Trace Elements in Saliva and Periodontal Status.

Changes in the macro and trace element composition of saliva might be indicative for pathological changes in periodontal tissues. However, there is a lack of evidence in the literature showing associations between mineral elements and periodontal status. The aim of this study was to determine whether such associations occur. Totally, 190 systemically healthy non-smoker participants (mean age 32.2 ± 6.02; 50 periodontally healthy, 50 gingivitis, 50 chronic periodontitis, and 40 aggressive periodontitis individuals) were included in this cross-sectional study. Salivary levels of some macro and trace elements were measured by using inductively coupled plasma mass spectrometry (ICP-MS). Kruskal-Wallis's test was used for statistical analysis. Statistically significant differences were found in sodium (Na), magnesium (Mg), potassium (K), calcium (Ca), vanadium (V), chromium Cr), manganese (Mn), iron (Fe), rubidium (Rb), strontium (Sr), and selenium (Se) concentrations among the groups. Significant increases in the essential minerals Na, Mg, K, Ca, Fe, and Se occurred in both periodontitis groups when compared to the gingivitis and periodontally healthy groups. Lower Se, Sr, Fe, Mn, and V concentrations were found in the aggressive periodontitis group than in the chronic periodontitis group. The results of this study demonstrated that assessment of mineral element concentrations in saliva might be useful in assessing periodontal health and disease. However, further studies are required to determine whether the change in a specific mineral element is the result of periodontal disease or is involved in its pathogenesis.

Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro.

Amelogenin (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.

Comparison of Different Sources of Mesenchymal Stem Cells: Palatal versus Lipoaspirated Adipose Tissue.

OBJECTIVES: 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.

Biostimulation with diode laser positively regulates cementoblast functions, in vitro.

The aim of this study was to evaluate the effects of diode laser biostimulation on cementoblasts (OCCM.30). A total of 40 root plates were obtained from healthy third molar teeth and assigned to the following two groups: (1) control group and (2) laser-treated group. Root plates were placed into the cell culture inserts, and OCCM.30 cells were seeded onto root plates. Cells were irradiated with a low level of diode laser (power: 0.3 W in continuous wave, 60 s/cm2). Proliferation and mineralized tissue-associated gene's and BMP's messenger RNA (mRNA) expressions of cementoblasts were evaluated. Total RNAs were isolated on day 3 and integrin-binding sialoprotein (Ibsp), bone gamma-carboxyglutamate protein (Bglap), Type I collagen (Col1a1), osteoblastic transcription factor, runt-related transcription factor (Runx2), and Bone Morphogenetic Protein (BMP)-2, 3, 4, 6, and 7 mRNA expressions were determined using quantitative RT-PCR. von Kossa staining was performed to evaluate biomineralization of OCCM.30 cells. In the proliferation experiment, while there was no significant difference until 96 h, laser irradiation retarded the decrease in cell proliferation trend after 96 h compared to the untreated control group. Statistically significant increase in Ibsp, Bglap, and BMP-2,3,6,7 mRNA expressions were noted in the laser groups when compared to the untreated control group (p < 0.05). Laser irradiation induced mineralized nodule formation of cementoblasts. The results of this study reveal that the biostimulation setting of diode laser modulates the behavior of cementoblasts inducing mineralized tissue-associated gene's mRNA expressions and mineralization. Therefore, biostimulation can be used during regenerative periodontal therapies to trigger cells with periodontal attachment apparatus.

The effect of different cleaning methods on the surface and temperature of failed titanium implants: an in vitro study.

The aims of this in vitro study are to compare the efficacy of different cleaning methods in removing debris of failed implants and to detect thermal changes of the implants treated by various scaling instruments. Twenty-seven failed implants and two unused implants as control were included to this study-group 1: plastic curette (P), group 2: titanium curette (T), group 3: carbon curette (C), group 4: titanium brush (TB), group 5: Er:YAG laser (laser 1 (L1) 100 mJ/pulse at 10 Hz), group 6: Er:YAG laser (laser 2 (L2) 150 mJ/pulse at 10 Hz), group 7: Er:YAG laser (laser 3 (L3) 200 mJ/pulse at 10 Hz), group 8: ultrasonic scaler appropriate for titanium (US), group 9: air abrasive method (AA) + citric acid, and group 10: implantoplasty (I). The changes on the treated/untreated titanium surfaces and remnant debris were observed by scanning electron microscopy (SEM). Temperature of the implants before and after treatment was detected using a thermocouple. The use of air abrasive and citric acid combination and Er:YAG laser groups was found as the best methods for the decontamination of titanium surfaces of failed implant. When the hand instruments were compared, titanium curette was found better than both the plastic and the carbon curettes which leave plastics and carbon remnants on the titanium surface. The temperature was higher after hand instrumentation when compared to other experimental groups (p < 0.05). Within the limitations of the present in vitro model, it can be concluded that the best method for decontamination of the implant surface is the use of air abrasives and Er:YAG laser.

Porphyromonas gingivalis Lipopolysaccharide Induces a Pro-inflammatory Human Gingival Fibroblast Phenotype.

Human 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.

Dietary arginine silicate inositol complex inhibits periodontal tissue loss in rats with ligature-induced periodontitis.

The purpose of this study was to induce experimental periodontitis in rats previously fed diets containing arginine silicate inositol (ASI) complex and examine the biochemical, immunological, and radiological effects. Fifty two 8-week-old female Sprague Dawley rats were equally divided into four groups. The control group included those fed a standard rat diet with no operation performed during the experiment. The periodontitis, ASI I, and ASI II groups were subjected to experimental periodontitis induction for 11 days after being fed a standard rat diet alone, a diet containing 1.81 g/kg ASI complex, or a diet containing 3.62 g/kg ASI complex, respectively, for 8 weeks. Throughout the 11-day duration of periodontitis induction, all rats were fed standard feed. The rats were euthanized on the eleventh day, and their tissue and blood samples were collected. In the periodontitis group, elevated tissue destruction parameters and reduced tissue formation parameters were found, as compared to the ASI groups. Levels of enzymes, cytokines, and mediators associated with periodontal tissue destruction were lower in rats fed a diet containing ASI complex after experimental periodontitis. These results indicate that ASI complex could be an alternative agent for host modulation.

Nonsurgical periodontal therapy with/without diode laser modulates metabolic control of type 2 diabetics with periodontitis: a randomized clinical trial.

In order to evaluate whether nonsurgical periodontal treatment with/without diode laser (DL) decontamination improves clinical parameters, the levels of IL-1ß, IL-6, IL-8, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) in gingival crevicular fluid and metabolic control (HbA1c) in chronic periodontitis (CP) patients with diabetes mellitus type 2 (DM2). Sixty patients with DM2 and CP were randomly assigned into two groups to receive scaling and root planing (SRP, n = 30) or SRP followed by diode laser application (SRP + DL, n = 30). Clinical periodontal and gingival crevicular fluid (GCF) parameters were assessed at baseline, 1, and 3 months after periodontal treatment. HbA1c levels were evaluated at baseline and 3 months post-therapy. Total amounts of cytokines and molecules were analyzed by ELISA. Nonsurgical periodontal treatment with/without DL appeared to improve clinical, biochemical parameters, and glycemic control in DM2 patients (BMI < 25 kg/m(2)) with CP. The SRP + DL group provided better reductions in probing depth (PD) and clinical attachment level (CAL) parameters compared to the SRP group (P < 0.05). Significant reductions were found in the total amounts of GCF levels of IL-1, IL-6, IL-8, ICAM, and VCAM after treatment (P < 0.05). HbA1c levels decreased significantly at 3 months after treatment (P < 0.05). SRP + DL reduced HbA1c levels more significantly compared to SRP alone (0.41 vs. 0.22 %, P < 0.05). SRP, especially in combination with DL, shows improvement of glycemic control for DM2 patients with CP.

Encapsulated boron as an osteoinductive agent for bone scaffolds.

The aim of this study was to develop boron (B)-releasing polymeric scaffold to promote regeneration of bone tissue. Boric acid-doped chitosan nanoparticles with a diameter of approx. 175 nm were produced by tripolyphosphate (TPP)-initiated ionic gelation process. The nanoparticles strongly attached via electrostatic interactions into chitosan scaffolds produced by freeze-drying with approx. 100 µm pore diameter. According to the ICP-OES results, following first 5h initial burst release, fast release of B from scaffolds was observed for 24h incubation period in conditioned medium. Then, slow release of B was performed over 120 h. The results of the cell culture studies proved that the encapsulated boron within the scaffolds can be used as an osteoinductive agent by showing its positive effects on the proliferation and differentiation of MC3T3-E1 preosteoblastic cells.