Diabetes Medication Metformin Inhibits Osteoclast Formation and Activity in In Vitro Models for Periodontitis

Diabetes and periodontitis are comorbidities and may share common pathways. Several reports indicate that diabetes medication metformin may be beneficial for the periodontal status of periodontitis patients. Further research using appropriate cell systems of the periodontium, the tissue that surrounds teeth may reveal the possible mechanism. Periodontal ligament fibroblasts anchor teeth in bone and play a role in the onset of both alveolar bone formation and degradation, the latter by inducing osteoclast formation from adherent precursor cells. Therefore, a cell model including this type of cells is ideal to study the influence of metformin on both processes. We hypothesize that metformin will enhance bone formation, as described for osteoblasts, whereas the effects of metformin on osteoclast formation is yet undetermined. Periodontal ligament fibroblasts were cultured in the presence of osteogenic medium and 0.2 or 1 mM metformin. The influence of metformin on osteoclast formation was first studied in PDLF cultures supplemented with peripheral blood leukocytes, containing osteoclast precursors. Finally, the effect of metformin on osteoclast precursors was studied in cultures of CD14+ monocytes that were stimulated with M-CSF and receptor activator of Nf-κB ligand (RANKL). No effects of metformin were observed on osteogenesis: not on alkaline phosphatase activity, Alizarin red deposition, nor on the expression of osteogenic markers RUNX-2, Collagen I and Osteonectin. Metformin inhibited osteoclast formation and accordingly downregulated the genes involved in osteoclastogenesis: RANKL, macrophage colony stimulating factor (M-CSF) and osteoclast fusion gene DC-STAMP. Osteoclast formation on both plastic and bone as well as bone resorption was inhibited by metformin in M-CSF and RANKL stimulated monocyte cultures, probably by reduction of RANK expression. The present study unraveling the positive effect of metformin in periodontitis patients at the cellular level, indicates that metformin inhibits osteoclast formation and activity, both when orchestrated by periodontal ligament fibroblasts and in cytokine driven osteoclast formation assays. The results indicate that metformin could have a systemic beneficiary effect on bone by inhibiting osteoclast formation and activity.

Analysis of the cells isolated from epithelial cell rests of Malassez through single-cell limiting dilution

The epithelial cell rests of Malassez (ERM) are essential in preventing ankylosis between the alveolar bone and the tooth (dentoalveolar ankylosis). Despite extensive research, the mechanism by which ERM cells suppress ankylosis remains uncertain; perhaps its varied population is to reason. Therefore, in this study, eighteen unique clones of ERM (CRUDE) were isolated using the single-cell limiting dilution and designated as ERM 1–18. qRT-PCR, ELISA, and western blot analyses revealed that ERM-2 and -3 had the highest and lowest amelogenin expression, respectively. Mineralization of human periodontal ligament fibroblasts (HPDLF) was reduced in vitro co-culture with CRUDE ERM, ERM-2, and -3 cells, but recovered when an anti-amelogenin antibody was introduced. Transplanted rat molars grown in ERM-2 cell supernatants produced substantially less bone than those cultured in other cell supernatants; inhibition was rescued when an anti-amelogenin antibody was added to the supernatants. Anti-Osterix antibody staining was used to confirm the development of new bones. In addition, next-generation sequencing (NGS) data were analysed to discover genes related to the distinct roles of CRUDE ERM, ERM-2, and ERM-3. According to this study, amelogenin produced by ERM cells helps to prevent dentoalveolar ankylosis and maintain periodontal ligament (PDL) space, depending on their clonal diversity.

Comparative evaluation of the effect of cold ceramic and MTA-Angelus on cell viability, attachment and differentiation of dental pulp stem cells and periodontal ligament fibroblasts: an in vitro study

Background Biocompatibility and induction of mineralized tissue formation are the properties expected from a material used in vital pulp therapy and repair of perforations. Cold ceramic (SJM, Iran; CC) is a newly introduced calcium silicate-based cement for above mentioned therapeutic applications. This in-vitro study aimed to compare the effect of CC and White MTA-Angelus (MTA) on cell viability, attachment, odontogenic differentiation, and calcification potential of human dental pulp stem cells (DPSCs) and periodontal ligament fibroblasts (PDLFs). Methods Cell viability of DPSCs and PDLFs was assessed using MTT on days 1, 3, 7, and 14 (n = 9) in contact with freshly mixed and set states of CC and MTA. Field emission scanning electron micrographs (FESEM) were taken to evaluate cell-bioceramic interaction (n = 6). Gene expression levels of osteo/odontogenic markers (Dentin sialophosphoprotein, Dentin matrix protein 1, Collagen type I alpha 1, and Alkaline phosphatase (DSPP, DMP1, COL 1A1, and ALP, respectively) (n = 8) were assessed using qrt-PCR. ALP enzymatic activity was evaluated to assess the mineralization potential. A two-way ANOVA test was applied, and p < 0.05 was considered to be statistically significant. Results The effect of freshly mixed and set MTA and CC on the survival of DPSCs and PDLFs in all study groups was statistically similar and comparable to the positive control group (p > 0.05); the only exception was for the viability of PDLFs in contact with freshly mixed cements on day 1, showing a more significant cytotoxic effect compared to the control and the set state of materials (p < 0.05). PDLFs attached well on CC and MTA. The spread and pseudopodium formation of the cells increased on both samples from day 1 to day 14. Contact of MTA and CC with DPSCs similarly increased expression of all dentinogenesis markers studied on days 7 and 14 compared to the control group (p < 0.001), except for DSPP expression on day 7 (p = 0.46 and p = 0.99 for MTA and CC, respectively). Conclusions Within the limitation of this in-vitro study, cold ceramic and MTA-Angelus showed high biocompatibility and induced increased expression of osteo/dentinogenic markers. Therefore, cold ceramic can be a suitable material for vital pulp therapy and the repair of root perforations.

Autophagy Induces Expression of IL-6 in Human Periodontal Ligament Fibroblasts Under Mechanical Load and Overload and Effects Osteoclastogenesis in vitro

Objective: Autophagy is an important cellular adaptation mechanism to mechanical stress. In animal experiments, inhibition of autophagy during orthodontic tooth movement triggered increased expression of inflammation-related genes and decreased bone density. The aim of this study was to investigate how autophagy affects cytokine levels of interleukin 6 (IL-6) in human periodontal ligament (hPDL) fibroblasts under mechanical pressure and the resulting influence on osteoblast communication.Methods: hPDL fibroblasts were subjected to physiologic mechanical load, constant overload, or rapamycin treatment for 16 to 24 h ± autophagy inhibitor 3-MA. Autophagosomes were quantified by flow cytometry. Gene expression of il-6 as well as IL-6 levels in the supernatant were determined with rtPCR and ELISA. To investigate the influence of mechanically-induced autophagy on cell-cell communication, an osteoblast-culture was subjected to supernatant from stimulated hPDL fibroblasts ± soluble IL-6 receptor (sIL-6R). After 24 h, osteoprotegerin (opg) and receptor activator of nuclear factor κB ligand (rankl) gene expressions were detected with rtPCR. Gene expression of a disintegrin and metalloproteinases (adam) 10 and 17 in stimulated hPDL fibroblasts was examined via rtPCR.Results: Autophagy was induced by biomechanical stress in hPDL fibroblasts in a dose-dependent manner. Mechanical load and overload increased IL-6 expression at gene and protein level. Autophagy inhibition further enhanced the effects of mechanical stimulation on IL-6 expression. Mechanical stimulation of hPDL fibroblasts downregulated adam10 and adam17 expressions. Inhibition of autophagy had stimulus-intensity depending effects: autophagy inhibition alone or additional application of physiological stress enhanced adam10 and adam17 expressions, whereas mechanical overload had adverse effects. Osteoblasts showed significantly reduced opg expression in the presence of supernatant derived of hPDL fibroblasts treated with autophagy inhibitor and sIL-6R.Conclusion: IL-6 levels were increased in response to pressure in hPDL fibroblasts, which was further enhanced by autophagy inhibition. This caused a decrease in opg expression in osteoblasts. This may serve as an explanatory model for accelerated tooth movement observed under autophagy inhibition, but may also represent a risk factor for uncontrolled bone loss.

Effect of Static Compressive Force on Aldehyde Dehydrogenase Activity in Periodontal Ligament Fibroblasts

Background: The application of static compressive forces to periodontal ligament fibroblasts (PDLFs) in vivo or in vitro has been linked to the expression of biochemical agents and local tissue modifications that could be involved in maintaining homeostasis during orthodontic movement. An approach used for identifying mesenchymal cells, or a subpopulation of progenitor cells in both tumoral and normal tissues, involves determining the activity of aldehyde dehydrogenase (ALDH). However, the role of subpopulations of PDLF-derived undifferentiated cells in maintaining homeostasis during tooth movement remains unclear. Objective: This study aimed at analyzing the effect of applying a static compressive force to PDLFs on the activity of ALDH in these cells. Methods: PDLFs were distributed into two groups: control group (CG), where fibroblasts were not submitted to compression, and experimental group (EG), where fibroblasts were submitted to a static compressive force of 4 g/mm2 for 6 hours. The compressive force was applied directly to the cells using a custom-built device. ALDH activity in the PDLFs was evaluated by a flow cytometry assay. Results: ALDH activity was observed in both groups, but was significantly lower in EG than in CG after the application of a static compressive force in the former. Conclusion: Application of a static compressive force to PDLFs decreased ALDH activity.

Effect of Artemisinin-Loaded Mesoporous Cerium-Doped Calcium Silicate Nanopowder on Cell Proliferation of Human Periodontal Ligament Fibroblasts

Ion doping has rendered mesoporous structures important materials in the field of tissue engineering, as apart from drug carriers, they can additionally serve as regenerative materials. The purpose of the present study was the synthesis, characterization and evaluation of the effect of artemisinin (ART)-loaded cerium-doped mesoporous calcium silicate nanopowders (NPs) on the hemocompatibility and cell proliferation of human periodontal ligament fibroblasts (hPDLFs). Mesoporous NPs were synthesized in a basic environment via a surfactant assisted cooperative self-assembly process and were characterized using Scanning Electron Microscopy (SEM), X-ray Fluorescence Spectroscopy (XRF), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction Analysis (XRD) and N2 Porosimetry. The loading capacity of NPs was evaluated using Ultrahigh Performance Liquid Chromatography/High resolution Mass Spectrometry (UHPLC/HRMS). Their biocompatibility was evaluated with the MTT assay, and the analysis of reactive oxygen species was performed using the cell-permeable ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). The synthesized NPs presented a mesoporous structure with a surface area ranging from 1312 m2/g for undoped silica to 495 m2/g for the Ce-doped NPs, excellent bioactivity after a 1-day immersion in c-SBF, hemocompatibility and a high loading capacity (around 80%). They presented ROS scavenging properties, and both the unloaded and ART-loaded NPs significantly promoted cell proliferation even at high concentrations of NPs (125 μg/mL). The ART-loaded Ce-doped NPs with the highest amount of cerium slightly restricted cell proliferation after 7 days of culture, but the difference was not significant compared with the control untreated cells.

Effects of mechanical strain on periodontal ligament fibroblasts in presence of Aggregatibacter actinomycetemcomitans lysate

Purpose Many adult orthodontic patients suffer from periodontitis, which is caused by oral pathogens such as the gram-negative Aggregatibacter actinomycetemcomitans (Agac). Like orthodontic tooth movement, periodontitis is associated with inflammation and alveolar bone remodelling thereby affecting orthodontic treatment. Interactions of both processes, however, are not sufficiently explored, particularly with regard to oxidative stress. Methods After preincubation with Agac lysate for 24 h periodontal ligament fibroblasts (PDLF) were either stretched or compressed for further 48 h simulating orthodontic forces in vitro. We analysed the expression of genes and proteins involved in the formation of reactive oxygen species (NOX-4, ROS) and nitric oxide (NOS-2), inflammation (TNF, IL-6, PTGS-2) and bone remodelling (OPG, RANKL). Results Agac lysate elevated the expression of NOX-4, NOS-2, inflammatory IL-6 and PTGS-2 and the bone-remodelling RANKL/OPG ratio during compressive, but not tensile mechanical strain. Agac lysate stimulated pressure-induced inflammatory signalling, whereas surprisingly ROS formation was reduced. Pressure-induced downregulation of OPG expression was inhibited by Agac lysate. Conclusions Agac lysate impact on the expression of genes and proteins involved in inflammation and bone remodelling as well as ROS formation, when PDLF were subjected to mechanical forces occurring during orthodontic tooth movement.