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, β-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 β-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

Osteogenic differentiation of MC3T3-E1 cells on different titanium surfaces

mRNA expressions related to osteogenic differentiation of MC3T3-E1 cells on electro-polished smooth (S), sandblasted small-grit (SSG) and sandblasted large-grit (SLG) surfaces of titanium alloys were investigated in vitro. Gene expression profiles of cells were evaluated using the RT2 Profiler PCR microarray on day 7. Mineralizing tissue-associated proteins, differentiation factors and extracellular matrix enzymes mRNA expressions were measured using Q-PCR. SLG surface upregulated 23 genes over twofolds and downregulated 3 genes when compared to the S surface. In comparison to the SSG surface, at least a twofold increase in 25 genes was observed in the SLG surface. BSP, OCN, OPN, COL I and ALP mRNA expressions increased in the SLG group when compared to the S and the SSG groups. BMP-2, BMP-6 and TGF-beta mRNA expressions increased in both the SSG and the SLG surfaces. MMP-2 and MMP-9 mRNA expressions increased as the surface roughness increased. This study demonstrated that surface roughness of titanium implants has a significant effect on cellular behavior and SLG surface apparently increased gene expressions related to osteogenesis when compared to the S and the SSG surfaces

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). 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

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., A protein with its roots in dental development stimulates the proliferation and gene expression of cells linked to regeneration. Amelogenin is a mediator of enamel and tooth root formation, and the main component of a recently-developed medicine for periodontal regeneration. An international research group led by Sema Hakki, of Selcuk University, Turkey, has now elucidated the effects of amelogenin on cementoblasts, a type of cell responsible for producing the vital, mineralized layer on surface of the tooth root. Hakki’s team found that the bacteria-derived amelogenin increased the rate of mouse cementoblast proliferation and mineralization in vitro, and increased the expression of genes related to bone and tissue generation. The team also demonstrated the presence of a likely amelogenin receptor on the cells used in their study. These findings support further investigation into amelogenin’s therapeutic potential.PubMedWoSScopu

Depths of magma chambers at three volcanic provinces in the Karlıova region of Eastern Turkey

The size of a volcanic eruption, and thus the associated potential hazards, depends partly on the depth, geometry, and size of the source magma chamber. To estimate magma chamber depths and sizes, we apply a newly developed analytical method, based on the aspect ratio (length/thickness) of dikes, to three volcanoes in the Karlıova region of Eastern Turkey, namely Turnadağ, Varto, and Özenç. The results indicate that the depths of the source chambers are between 2 and 4 km at Turnadağ, 2 to 5 km at Varto, both of which are located in transtensional tectonic regimes, but from 22 to 27 km at Özenç, which is located in a convergent tectonic regime. A similar reservoir depth at Özenç is indicated by seismic tomography, and this data also suggests that the reservoir is laterally continuous for more than 40 km. The large volume of ignimbrites (> 40 km3) associated with Varto, a collapse caldera, indicates that caldera subsidence may have maintained the excess magmatic pressure (through tectonic forcing) in the chamber over a longer time than during normal pyroclastic eruptions. The dike aspect ratios further indicate magmatic overpressures of 13–21 MPa for Varto, 13–17 MPa for Turnadağ, and 26–31 MPa for Özenç. The combined results from seismic tomography, analytical models and magma compositions indicate that both Turnadağ and Varto volcanoes, which are typical stratovolcanoes composed of mostly intermediate, and more rarely, acidic magmas, were fed by two very shallow and comparatively small magma chambers (2–5 km depth). Whereas less evolved magmas were erupted from Özenç, which hosts predominantly basaltic and intermediate lavas and dikes that were fed by a deep reservoir at 22–27 km depth. Our tomographic models show that none of the volcanoes are located directly over the center of a deep magma reservoir. Our data also indicates that the magma in the reservoir has migrated between 34 and 40 km in a right lateral motion (to the east) below Varto and Turnadağ, respectively, and 23 km in a left lateral motion (to the west) at Özenç over the past 3 Ma. This lateral propagation of magma can be explained by tectonic escape of the Anatolian block to the west through the Northern Anatolian Fault and the Varto Fault Zone over the last 6 Ma