Periodontal Ligament Fibroblasts Research Articles

Quantification of Caspase 3 Levels in Patients with Periodontitis with or without Diabetes Mellitus

Background: Diabetes mellitus is a debilitating systemic disease with several major complications affecting the quality and length of life. Periodontal disease has been considered another diabetic complication in addition to cardiovascular disease, nephropathy, neuropathy, peripheral vascular disease. Caspase-3 plays an important role in intracellular signaling pathways that regulate apoptosis. High levels of glucose could induce human periodontal ligament fibroblast apoptosis.
 Aim: The aim of the study is to compare caspase 3 levels in periodontitis patients with or without diabetes mellitus.
 Materials and Methods: 30 patients were included in the study and they are divided into 3 groups: Group a- Periodontal health; Group b- Periodontitis with diabetes mellitus and Group c- Periodontitis patients without diabetes mellitus. Whole unstimulated saliva was collected from 30 patients using expectoration into sterile bulbs. Caspase 3 levels in saliva samples were measured in duplicate using a commercially available Human Caspase-3 (CASP3) enzyme linked immunosorbent assay (ELISA) Kit. Results were analyzed statistically by a one-way analysis of variance (ANOVA) and significant differences between the mean values were measured using Newman-Keuls multiple comparison test using Graph Pad Prism version 5. The results with the p < 0.05 level were considered to be statistically significant.
 Results: From this study, it was observed that there was a significant increase of caspase 3 levels in periodontitis patients with diabetes mellitus (86.29±24.25 pmol/L) when compared to periodontitis patients without diabetes (55.06±12.90 pmol/L). The results showed a positive correlation and high level of significance when compared between periodontally healthy patients and periodontitis patients along with diabetes mellitus (86.29±24.25 pmol/L). Also, relatively significant results were observed in comparison between periodontally healthy patients (43.37±15.35 pmol/L) and patients with periodontitis without diabetes mellitus (55.06±12.90 pmol/L). Please add the most important statistical values.
 Conclusion: The present study showed that caspase-3 concentrations in saliva increases in patients with periodontitis complicated along with diabetes mellitus. Moreover, saliva concentrations of caspase-3 increase with periodontal disease and caspase-3 plays a role as a biomarker of periodontal disease and its progression.

GDF15 Supports the Inflammatory Response of PdL Fibroblasts Stimulated by P. gingivalis LPS and Concurrent Compression.

Periodontitis is characterized by bacterially induced inflammatory destruction of periodontal tissue. This also affects fibroblasts of the human periodontal ligaments (HPdLF), which play a coordinating role in force-induced tissue and alveolar bone remodeling. Excessive inflammation in the oral tissues has been observed with simultaneous stimulation by pathogens and mechanical forces. Recently, elevated levels of growth differentiation factor 15 (GDF15), an immuno-modulatory member of the transforming growth factor (TGFB) superfamily, were detected under periodontitis-like conditions and in force-stressed PdL cells. In view of the pleiotropic effects of GDF15 in various tissues, this study aims to investigate the role of GDF15 in P. gingivalis-related inflammation of HPdLF and its effect on the excessive inflammatory response to concurrent compressive stress. To this end, the expression and secretion of cytokines (IL6, IL8, COX2/PGE2, TNFα) and the activation of THP1 monocytic cells were analyzed in GDF15 siRNA-treated HPdLF stimulated with P. gingivalis lipopolysaccharides alone and in combination with compressive force. GDF15 knockdown significantly reduced cytokine levels and THP1 activation in LPS-stimulated HPdLF, which was less pronounced with additional compressive stress. Overall, our data suggest a pro-inflammatory role for GDF15 in periodontal disease and demonstrate that GDF15 partially modulates the force-induced excessive inflammatory response of PdLF under these conditions.

Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring.

A series of 3,5-bis(benzylidene)-4-piperidones 2a–u were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected.

Impact of FGF1 on human periodontal ligament fibroblast growth, osteogenic differentiation and inflammatory reaction in vitro.

To investigate in vitro the impact of fibroblast growth factor1 (FGF1) in comparison to ascorbic acid (AscA) on human periodontal ligament fibroblast (HPdLF) growth, their osteogenic differentiation, and modulation of their inflammatory reaction to mechanical stress. The influence of different concentrations of FGF1 (12.5-200 ng/mL) on growth and proliferation of HPdLF cells was analyzed over 20days by counting cell numbers and the percentage of Ki67-positive cells. Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALPL), Runt-related transcription factor2 (RUNX2), osteocalcin (OCN), and osteopontin (OSP), as well as the fibroblast markers vimentin (VIM) and fibroblast-specific protein1 (FSP1), was performed after 2and 20days of cultivation. Metabolic activity was determined by MTT assay. For comparison with AscA, 50 ng/mL FGF1 was used for stimulation for 2and 20days. Cell number, percentage of Ki67-positive cells, and expression of osteoblast- and fibroblast-specific genes were examined. Alkaline phosphatase activity was visualized by NBT/BCIP and calcium deposits were stained with alizarin red. Cytokine (IL‑6, IL‑8, COX2/PGE2) expression and secretion were analyzed by qPCR and ELISA in 6 h mechanically compressed HPdLF cultured for 2days with FGF1 or ascorbic acid. Higher concentrations of FGF1 promoted cell proliferation upon short-term stimulation, whereas prolonged treatment induced the expression of osteogenic markers even with low concentrations. AscA promotes cell growth more markedly than FGF1 in short-term cultures, whereas FGF1 induced osteogenic cell fate more strongly in long-term culture. Both factors induced an increased inflammatory response of HPdLF to mechanical compression. Our data suggest that FGF1 promotes an osteogenic phenotype of HPdLF and limits inflammatory response to mechanical forces compared to AscA.

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

BackgroundBiocompatibility 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).MethodsCell 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.ResultsThe 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).ConclusionsWithin 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.

Combination of parathyroid hormone pretreatment and mechanical stretch promotes osteogenesis of periodontal ligament fibroblasts

Parathyroid hormone (PTH) potentiates the mechanical loading induced bone formation in fracture healing and orthodontics. This study aimed to gain insight into the underlying mechanisms in periodontal ligament fibroblasts (PDLFs). Human PDLFs were cultured and subjected to uniaxial cyclic stretch at 0.5Hz and 2000μ for 0, 6, 12, and 24hours, respectively. 10nM PTH was preadministered for 30minutes before loading. The expression of PTH1R and osteogenic biomarkers Runx2, osteopontin, collagen type 1, alkaline phosphatase was assessed via immunofluorescence staining, quantitative polymerase chain reaction, or Western blot. Transfection of siPTH1R was applied, and alterations of osteogenic biomarkers were examined by Western blot. The expression of essential Wnt signal components Wnt3a, β-catenin, low-density lipoprotein receptor-related protein 5, Wnt5a, receptor tyrosine kinase-like orphan receptor 2 were examined, and the influence of dickkopf-related protein 1 on osteogenic biomarkers was evaluated. The expression of PTH1R was instantaneously upregulated with PTH pretreatment and maintained a gradual increase until 24hours. PTH synergistically enhanced the increase of Runx2, osteopontin, collagen type 1, and alkaline phosphatase under cyclic stretch, which was substantially attenuated by siPTH1R transfection. As for Wnt signal components, synergistic upregulation was detected on Wnt3a, β-catenin, and low-density lipoprotein receptor-related protein 5, whereas Wnt5a and receptor tyrosine kinase-like orphan receptor 2 increased relatively mildly. Blockage of the canonical Wnt/β-catenin pathway by dickkopf-related protein 1 impaired the boost of osteogenic biomarkers under the combined action of PTH and cyclic stretch. The combination of PTH pretreatment and cyclic stretch promotes osteogenesis of PDLFs synergistically, and the canonical Wnt/β-catenin pathway is crucially involved in the underlying mechanism.

Proteomic profiling reveals engineered chitosan nanoparticles mediated cellular crosstalk and immunomodulation for therapeutic application in apical periodontitis

Macrophages (MQ) are major constituents of chronically inflamed periapical tissues in apical periodontitis. This study aimed to investigate the immunomodulatory effect of engineered bioactive chitosan-based nanoparticles (CSnp) antibiofilm medication on MQ cocultured with periodontal ligament fibroblasts (PdLF). Cells viability, spreading, PdLF migration, and intracellular CSnp uptake were characterized. Tandem Mass Tag-based proteomics was applied to analyze MQ global protein expression profiles after interaction with Enterococcus faecalis biofilm, CSnp-treated biofilm, and CSnp. Secreted inflammatory mediators were analyzed. Following bioinformatics analyses, candidate proteins were validated via targeted proteomics. CSnp maintained cells viability, increased MQ spreading, and PdLF migration (p < 0.05). Transmission electron micrographs demonstrated CSnp internalization via macropinocytosis, clathrin-mediated endocytosis, and phagocytosis. Proteomic analysis revealed that CSnp-treated biofilm upregulated proteins (>1.5-folds, p < 0.05) showed functional enrichment in the pathway of metal sequestration by antimicrobial proteins, while downregulated proteins showed enrichment in ferroptosis. CSnp upregulated proteins exhibiting antioxidant and immunoregulatory properties. Upregulation of SERPINB1 by CSnp (>1.5-folds, p < 0.05) was validated. CSnp-treated biofilm reduced pro-inflammatory IL-1β and nitric oxide but enhanced anti-inflammatory IL-10 and TGF-β1 (p < 0.05). Internalized engineered bioactive CSnp reprogrammed MQ proteomic and cytokine profiles to modulate biofilm-mediated inflammation, and prompted PdLF migration, emphasizing its potential to regulate healing process in the treatment of apical periodontitis.

Madecassic acid protects human periodontal ligament fibroblasts against hydrogen peroxide-induced cell damage by maintaining mitochondrial membrane potential

Madecassic acid protects human periodontal ligament fibroblasts against hydrogen peroxide-induced cell damage by maintaining mitochondrial membrane potential

Periodontal ligament fibroblast-derived exosomes induced by compressive force promote macrophage M1 polarization via Yes-associated protein

ObjectivesThis study aimed to investigate the biological roles and mechanisms of compressive force-stimulated periodontal ligament fibroblasts (PDLFs) on polarization of macrophages DesignPDLFs were stimulated with or without static compressive force, and then conditioned medium, high-molecular weight proteins and low-molecular weight proteins were collected to treat THP-1 macrophages. RT-qPCR and flow cytometric analysis were used to evaluate the polarization of macrophages. Exosomes were isolated by ultracentrifugation method and identified via transmission electron microscopy, western-blot and nano-tracking analysis. The protein level of Yes-Associated Protein (YAP) contained in exosomes was detected by western blot. GW4869 and Verteporfin were used to inhibit exosome secretion and YAP- TEA domain transcription factor (TEAD) interaction respectively. ResultsExosomes were successfully purified from PDLFs and could be efficiently incorporated into THP-1 macrophages. conditioned medium, HMW proteins and exosomes derived from compressive force-treated PDLFs significantly induce M1 polarization of macrophages. While inhibiting exosomes secretion by GW4869 treatment eliminated the inductive effect. YAP target genes, connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (CYR61) were upregulated in macrophages when treated with exosomes derived from compressive force-treated PDLFs (F-Exo). YAP level was elevated in the F-Exo. When macrophages were treated with Verteporfin, expression of YAP target genes and M1 polarization were significantly downregulated. ConclusionThese results suggested that exosomes derived from compressive force-treated PDLFs promoted the M1 polarization of the THP-1 macrophages. The elevated level of YAP in the exosomes may be a critical factor for this response.

Construction of micro-grooved PCL/nanohydroxyapatite membranes by non-solvent induced phase separation method and its evaluation for use as a substrate for human periodontal ligament fibroblasts

Micro-patterned membrane constructs based on neat, and nanohydroxyapatite (HA)-blended poly(ε-caprolactone) were developed using the phase separation micro-molding (PSµM) technique. Physical, chemical, thermal, mechanical and in vitro biological properties of the constructs were evaluated. Human periodontal ligament fibroblasts (hPDLFs) were cultured on the surface of the patterned membranes. The effect of micro-patterning on the morphology and osteogenic differentiation capacity of cells was evaluated by SEM and AR-S staining. SEM findings revealed that hPDLFs were able to adhere to ridges, but generally tended to settle along microgroove pattern channels with multiple filopodia. Micro-groove structure led to a narrower elongation of the cytoplasm, orientation and directionality for migration along the groove. After 30 days of culture, the cells had grown to form multiple layers covering the entire patterned surface, while maintaining the alignment of the cells. Extracellular mineralization and osteogenic differentiation of hPDLFs was demonstrated by high AR-S staining, particularly on HA-blended constructs.

UPR attenuates the proinflammatory effect of HPDLF on macrophage polarization.

Human periodontal ligament fibroblast (HPDLF) is a major component of the resident cells in the periodontal microenvironment, and plays important roles in periodontitis through multiple mechanisms. Although lipopolysaccharide (LPS) has been shown to cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) in HPDLF, the mechanisms governing HPDLF function in periodontitis are unclear. In this study, we tested the ability of unfolded protein response (UPR) to regulate HPDLF in vitro and examined the underlying mechanisms. We found LPS-pretreated HPDLF induced macrophage polarization toward M1 phenotype. UPR activation reduced the inflammatory cytokine production and downregulated the expression of TLR4 in HPDLF. The phosphorylation of NF-κB p65 and I-κB was also inhibited by UPR activation. Our findings demonstrate that the connection of LPS, UPR, and HPDLF may represent a new concrete theory of innate immunity regulation in periodontal diseases, and suggest that targeting of UPR in HPDLF may be developed as a potent therapy for periodontitis.

Activity of chlorhexidine formulations on oral microorganisms and periodontal ligament fibroblasts.

Given the importance of microorganisms in the pathogenesis of the two most prevalent oral diseases (i.e. caries and periodontitis), antiseptics are widely used. Among the antiseptics chlorhexidine (CHX) is still considered as gold standard. The purpose of this in-vitro-study was to determine the antimicrobial activity of new CHX digluconate containing formulations produced in Switzerland. Two test formulations, with 0.1% or 0.2% CHX (TestCHX0.1, TestCHX0.2) were compared with 0.1% and 0.2% CHX digluconate solutions (CHXph0.1, CHXph0.2) without additives and with a commercially available formulation containing 0.2% CHX digluconate (CHXcom0.2). The minimal inhibitory concentrations (MIC) of the CHX formulations were determined against bacteria associated with caries or periodontal disease. Then the anti-biofilm activities of CHX preparations were tested regarding inhibition of biofilm formation or against an existing biofilm. Further, the cytotoxicity of the CHX preparations against periodontal ligament (PDL) fibroblasts was measured. There were no or only minor differences of the MIC values between the CHX preparations. Except for 0.1% CHXph, all formulations blocked the formation of the biofilms. When analyzing the effect on an established “cariogenic” biofilm, the TestCHX0.2 was most active (- 3.5 log10 colony forming units (cfu); p<0.001 vs. control). The cfu counts of an established periodontal biofilm were most decreased by CHXcom0.2 (-3.9 log10), followed by TestCHX0.2 (-3.34 log10) and CHXph0.2 (-3.0 log10 cfu) (each p<0.001 vs. control). All CHX preparations reduced the viability of the PDL fibroblasts. Taken together, the Swiss made test mouth rinses have an equal or better antibacterial and anti-biofilm activity as the respective CHX solutions and can be recommended.

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.

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 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.

Galectin-7 promotes proliferation and wound healing capacities in periodontal ligament fibroblasts by activating ERK signaling

Periodontitis is a progressive inflammation condition and a primary cause of tooth loss in adults. As one of the abundant cell types in the periodontium, periodontal ligament fibroblasts (PDLFs) play an integral role in the maintenance and regeneration of periodontal tissue. Our previous work has shown that the application of Er:YAG laser increased the cell proliferation and migratory capacity of PDLFs via induction of galectin-7. In the present study, we aimed to evaluate if the forced expression of galectin-7 directly affected the cellular phenotypes of PDLFs. Our results showed that the cell proliferation, transwell migration, invasion, and wound healing capacities were all upregulated in PDLFs with the ectopic expression of galectin-7. These results suggest that therapeutic approaches to enhance the expression of galectin-7 in periodontium may accelerate tissue regeneration by recruiting more PDLFs to the injured site.

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

PurposeMany 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.MethodsAfter 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).ResultsAgac 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.ConclusionsAgac 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.

Bio-Morphological Reaction of Human Periodontal Ligament Fibroblasts to Different Types of Dentinal Derivates: In Vitro Study.

Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape; the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions; and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material; vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin’s degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.

PPARγ-Induced Global H3K27 Acetylation Maintains Osteo/Cementogenic Abilities of Periodontal Ligament Fibroblasts.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

The role of 25-hydroxyvitamin-D3 and vitamin D receptor gene in human periodontal ligament fibroblasts as response to orthodontic compressive strain: an in vitro study

BackgroundThis study aimed to investigate, if different physiological concentrations of vitamin D (25(OH)D3) and single nucleotide polymorphisms in vitamin D receptor (VDR) gene have an impact on gene expression in human periodontal ligament (hPDL) fibroblasts induced by simulated orthodontic compressive strain.MethodsA pool of hPDL fibroblasts was treated in absence or presence of 25(OH)D3 in 3 different concentrations (10, 40 and 60 ng/ml). In order to evaluate the role of single nucleotide polymorphisms in the VDR gene, hPDL fibroblasts from 9 patients were used and treated in absence or presence of 40 ng/ml 25(OH)D3. Each experiment was performed with and without simulated orthodontic compressive strain. Real-time PCR was used for gene expression and allelic discrimination analysis. Relative expression of dehydrocholesterol reductase (DHCR7), Sec23 homolog A, amidohydrolase domain containing 1 (AMDHD1), vitamin D 25-hydroxylase (CYP2R1), Hydroxyvitamin D-1-α hydroxylase, receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), cyclooxygenase-2 (COX-2) and interleukin-6 (IL6) was assessed. Three single nucleotide polymorphisms in VDR were genotyped. Parametric or non-parametric tests were used with an alpha of 5%.ResultsRANKL, RANKL:OPG ratio, COX-2, IL-6, DHCR7, CYP2R1 and AMDHD1 were differentially expressed during simulated orthodontic compressive strain (p < 0.05). The RANKL:OPG ratio was downregulated by all concentrations (10 ng/ml, 40 ng/ml and 60 ng/ml) of 25(OH)D3 (mean = 0.96 ± 0.68, mean = 1.61 ± 0.66 and mean = 1.86 ± 0.78, respectively) in comparison to the control (mean 2.58 ± 1.16) (p < 0.05). CYP2R1 gene expression was statistically modulated by the different 25(OH)D3 concentrations applied (p = 0.008). Samples from individuals carrying the GG genotype in rs739837 presented lower VDR mRNA expression and samples from individuals carrying the CC genotype in rs7975232 presented higher VDR mRNA expression (p < 0.05).ConclusionsSimulated orthodontic compressive strain and physiological concentrations of 25(OH)D3 seem to regulate the expression of orthodontic tooth movement and vitamin-D-related genes in periodontal ligament fibroblasts in the context of orthodontic compressive strain. Our study also suggests that single nucleotide polymorphisms in the VDR gene regulate VDR expression in periodontal ligament fibroblasts in the context of orthodontic compressive strain.