Human Periodontal Ligament Fibroblasts Research Articles

Synthesis of secretory leukocyte protease inhibitor using cell-free protein synthesis system.

Secretory leukocyte protease inhibitor (SLPI) functions as a protease inhibitor that modulates excessive proteolysis in the body, exhibits broad-spectrum antimicrobial activity, regulates inflammatory responses, and plays an important role in the innate immunity. The purpose of the study was to artificially synthesize a SLPI, an antimicrobial peptide, and investigate its effect on antimicrobial activity against Porphyromonas gingivalis and interleukin-6 (IL-6) production. SLPI protein with a molecular weight of approximately 13kDa was artificially synthesized using a cell-free protein synthesis (CFPS) system and investigated by western blotting and enzyme-linked immunosorbent assay (ELISA). Disulfide bond isomerase in the protein synthesis mixture increased the amount of SLPI synthesized. The synthesized SLPI (sSLPI) protein was purified and its antimicrobial activity was investigated based on the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells. The effect of sSLPI on IL-6 production in human periodontal ligament fibroblasts (HPLFs) was examined by ELISA. Our results showed that sSLPI significantly inhibited the growth of Porphyromonas gingivalis and bacterial adhesion to oral epithelial cells and further inhibited IL-6 production by HPLFs. These results suggested that SLPI artificially synthesized using the CFPS system may play a role in the prevention of periodontal diseases through its antimicrobial and anti-inflammatory effects.

Leukemia inhibitory factor protects against experimental periodontitis through immuno-modulations of both macrophages and periodontal ligament fibroblasts.

To explore the role of leukemia inhibitory factor (LIF) in periodontitis via in vivo and in vitro experiments. The second upper molar of LIF knockout mice and their wild-type littermates were ligated for 8 days. Micro-computed tomography (micro-CT), histological analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) were performed. The expression levels of proinflammatory cytokines were examined in mouse bone marrow derived macrophages and human periodontal ligament fibroblasts (HPDLFs) after lipopolysaccharide (LPS) treatment. LIF deficiency promoted alveolar bone loss, inflammatory cells infiltration, osteoclasts formation and collagen fiber degradation in ligature-induced mouse, along with higher expressions of proinflammatory cytokines, including interleukin-6 (IL6), IL-1β (IL1B), tumor necrosis factor-α (TNFA), matrix metalloproteinase 13 (MMP13), and RANKL/OPG ratio. Additionally, LIF deletion led to higher expression levels of these proinflammatory cytokines in mouse bone marrow-derived macrophages from both femur and alveolar bone and HPDLFs when treated with LPS. Administration of recombined LIF attenuated TNFA, IL1B, and RANKL/OPG ratio in HPDLFs. These findings indicate that LIF deficiency promotes the progress of periodontitis via modulating immuno-inflammatory responses of macrophages and periodontal ligament fibroblasts, and the application of LIF may be an adjunctive treatment for periodontitis to resolute inflammation.

Effect of the antirheumatic medication methotrexate (MTX) on biomechanical compressed human periodontal ligament fibroblasts (hPDLFs)

BackgroundThe aim of this study was to investigate the in vitro effect of the antirheumatic drug methotrexate (MTX) on biomechanically compressed human periodontal ligament fibroblasts (hPDLFs), focusing on the expression of interleukin 6 (IL-6), as its upregulation is relevant to orthodontic tooth movement.MethodsHuman PDLFs were subjected to pressure and simultaneously treated with MTX. Cell proliferation, viability and morphology were studied, as was the gene and protein expression of IL-6.ResultsCompared with that in untreated fibroblasts, IL-6 mRNA expression in mechanically compressed ligament fibroblasts was increased (two to sixfold; ****p < 0.0001). Under compression, hPDLFs exhibited a significantly more expanded shape with an increase of cell extensions. MTX with and without pressure did not affect IL-6 mRNA expression or the morphology of hPDLFs.ConclusionMTX has no effect on IL-6 expression in compressed ligament fibroblasts.

Preparation and optimization of an eggshell membrane-based biomaterial for GTR applications

ObjectivesGuided Tissue Regeneration (GTR) is a popular clinical procedure for periodontal tissue regeneration. However, its key component, the barrier membrane, is largely collagen-based and is still quite expensive, posing a financial burden to the patients as well as healthcare systems and negatively impacting the patient’s decision-making. Thus, our aim is to prepare a novel biomimetic GTR membrane utilizing a natural biomaterial, soluble eggshell membrane protein (SEP), which is economical as it comes from an abundant industrial waste from food and poultry industries, unlike collagen. Additive polymer, poly (lactic-co-glycolic acid) (PLGA), and a bioceramic, nano-hydroxyapatite (HAp), were added to improve its mechanical and biological properties. MethodsFor this barrier membrane preparation, we initially screened the significant factors affecting its mechanical properties using Taguchi orthogonal array design and further optimized the significant factors using response surface methodology. Furthermore, this membrane was characterized using SEM, EDAX, and ATR-FTIR, and tested for proliferation activity of human periodontal ligament fibroblasts (HPLFs). ResultsOptimization using response surface methodology predicted that the maximal tensile strength of 3.1 MPa and modulus of 39.9 MPa could be obtained at membrane composition of 8.9 wt% PLGA, 7.2 wt% of SEP, and 2 wt% HAp. Optimized PLGA/SEP/HAp membrane specimens that were electrospun on a static collector showed higher proliferation activity of HPLFs compared to tissue culture polystyrene and a commercial collagen membrane. SignificanceFrom the results observed, we can conclude that SEP-based nanofibrous GTR membrane could be a promising, environment-friendly, and cost-effective alternative for commercial collagen-based GTR membrane products.

GDF15 Contributes to the Regulation of the Mechanosensitive Responses of PdL Fibroblasts through the Modulation of IL-37.

During orthodontic tooth movement (OTM), areas of compressive and tensile forces are generated in the periodontal ligament (PdL), a mechanoreactive connective tissue between the teeth and alveolar bone. Mechanically stimulated PdL fibroblasts (PdLFs), the main cell type of PdL, express significantly increased levels of growth differentiation factor 15 (GDF15). In compressed PdL areas, GDF15 plays a fundamental role in modulating relevant OTM processes, including inflammation and osteoclast activation. However, the specific function of this factor in tensile areas has not yet been investigated. Thus, the aim of this study was to investigate the role of GDF15 in the mechanoresponse of human PdLFs (hPdLFs) that were exposed to biaxial tensile forces in vitro. Using siRNA-mediated knockdown experiments, we demonstrated that GDF15 had no impact on the anti-inflammatory force response of elongated hPdLFs. Although the anti-inflammatory markers IL1RN and IL10, as well as the activation of immune cells remained unaffected, we demonstrated an inhibitory role of GDF15 for the IL-37 expression. By analyzing osteogenic markers, including ALPL and RUNX2, along with an assessment of alkaline phosphatase activation, we further showed that the regulation of IL-37 by GDF15 modulates the osteogenic differentiation potential of hPdLFs. Despite bone resorption in tensile areas being rather limited, GDF15 was also found to positively modulate osteoclast activation in those areas, potentially by adjusting the IL-37 levels. In light of our new findings, we hypothesize that GDF15 modulates force-induced processes in tissue and bone remodeling through its various intra- and extracellular signaling pathways as well as interaction partners. Potentially acting as a master regulator, the modulation of GDF15 levels may hold relevance for clinical implications.

Er:YAG Laser Alleviates Inflammaging in Diabetes-Associated Periodontitis via Activation CTBP1-AS2/miR-155/SIRT1 Axis.

Periodontitis is a significant health concern for individuals with diabetes mellitus (DM), characterized by inflammation and periodontium loss. Hyperglycaemia in DM exacerbates susceptibility to periodontitis by inducing inflammaging in the host immune system. The use of erbium-doped yttrium-aluminum-garnet laser (ErL) in periodontitis treatment has gained attention, but its impact on diabetic-associated periodontitis (DP) and underlying mechanisms remain unclear. In this study, we simulated DP by exposing human periodontal ligament fibroblasts (PDLFs) to advanced glycation end products (AGEs) and lipopolysaccharides from P. gingivalis (Pg-LPS). Subsequently, we evaluated the impact of ErL on the cells' wound healing and assessed their inflammaging markers. ErL treatment promoted wound healing and suppressed inflammaging activities, including cell senescence, IL-6 secretion, and p65 phosphorylation. Moreover, the laser-targeted cells were observed to have upregulated expression of CTBP1-AS2, which, when overexpressed, enhanced wound healing ability and repressed inflammaging. Moreover, bioinformatic analysis revealed that CTBP1-AS2 acted as a sponge for miR155 and upregulated SIRT1. In conclusion, ErL demonstrated the ability to improve wound healing and mitigate inflammaging in diabetic periodontal tissue through the CTBP1-AS2/miR-155/SIRT1 axis. Targeting this axis could represent a promising therapeutic approach for preventing periodontitis in individuals with DM.

Influence of Ultrasound Stimulation on the Viability, Proliferation and Protein Expression of Osteoblasts and Periodontal Ligament Fibroblasts.

Among the adjunctive procedures to accelerate orthodontic tooth movement (OTM), ultrasound (US) is a nonsurgical form of mechanical stimulus that has been explored as an alternative to the currently available treatments. This study aimed to clarify the role of US in OTM by exploring different stimulation parameters and their effects on the biological responses of cells involved in OTM. Human fetal osteoblasts and periodontal ligament fibroblasts cell lines were stimulated with US at 1.0 and 1.5 MHz central frequencies and power densities of 30 and 60 mW/cm2 in continuous mode for 5 and 10 min. Cellular proliferation, metabolic activity and protein expression were analyzed. The US parameters that significantly improved the metabolic activity were 1.0 MHz at 30 mW/cm2 for 5 min and 1.0 MHz at 60 mW/cm2 for 5 and 10 min for osteoblasts; and 1.0 MHz at 30 mW/cm2 for 5 min and 1.5 MHz at 60 mW/cm2 for 5 and 10 min for fibroblasts. By stimulating with these parameters, the expression of alkaline phosphatase was maintained, while osteoprotegerin synthesis was induced after three days of US stimulation. The US stimulation improved the biological activity of both osteoblasts and periodontal ligament fibroblasts, inducing their osteogenic differentiation.

Protective Effects of Collagen Peptide on Periodontal Ligament Fibroblasts

To study the protective effect of collagen peptide on human periodontal ligament fibroblasts (hPDLFs) induced by lipopolysaccharide and its possible mechanism. Lipopoly-saccharide was used as a pathogenic factor to stimulate hPDLFs. Then, interleukin-8 and tumor necrosis factor-α in the supernatant were quantified, and Toll-like receptor 4 expression and the activation of nuclear factor-κB pathway were also studied. The results showed that collagen peptide above 100 mg/L could effectively reduce the toxicity of lipopolysaccharide, and significantly reduce interleukin-8 and tumor necrosis factor-α produced by lipopolysaccharide. Suggesting that collagen peptide can provide effecitve and safe solution to oral cavity repair.

Antibacterial properties of marine algae incorporated polylactide acid membranes as an alternative to clinically applied different collagen membranes

The reconstruction of bony defects in the alveolar crest poses challenges in dental practice. Guided tissue regeneration (GTR) and guided bone regeneration (GBR) procedures utilize barriers to promote bone regeneration and prevent epithelial growth. This study focuses on evaluating the antibacterial properties of marine algae-polylactic acid (PLA) composite membranes compared to commercially available collagen membranes. Marine algae (Corallina elongata, Galaxaura oblongata, Cystoseira compressa, Saragassum vulgare, and Stypopodium schimperi) were processed into powders and blended with PLA to fabricate composite membranes. Cytocompatibility assays using human periodontal ligament fibroblasts (n = 3) were performed to evaluate biocompatibility. Antibacterial effects were assessed through colony-forming units (CFU) and scanning electron microscopy (SEM) analysis of bacterial colonization on the membranes. The cytocompatibility assays demonstrated suitable biocompatibility of all marine algae-PLA composite membranes with human periodontal ligament fibroblasts. Antibacterial assessment revealed that Sargassum vulgare-PLA membranes exhibited the highest resistance to bacterial colonization, followed by Galaxaura oblongata-PLA and Cystoseira compressa-PLA membranes. SEM analysis confirmed these findings and revealed smooth surface textures for the marine algae-PLA membranes compared to the fibrous and porous structures of collagen membranes. Marine algae-PLA composite membranes show promising antibacterial properties and cytocompatibility for guided bone and tissue regeneration applications. Sargassum vulgare-PLA membranes demonstrated the highest resistance against bacterial colonization. These findings suggest that marine algae-PLA composite membranes could serve as effective biomaterials for infection control and tissue regeneration. Further in vivo validation and investigation of biodegradation properties are necessary to explore their clinical potential.Graphical

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1

ObjectivesOculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. MethodsPeriodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. ResultsRNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. ConclusionsOur findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

In vitro cytotoxicity of Withania somnifera (L.) roots and fruits on oral squamous cell carcinoma cell lines: a study supported by flow cytometry, spectral, and computational investigations.

Oral cancer is a severe health problem that accounts for an alarmingly high number of fatalities worldwide. Withania somnifera (L.) Dunal has been extensively studied against various tumor cell lines from different body organs, rarely from the oral cavity. We thus investigated the cytotoxicity of W. somnifera fruits (W-F) and roots (W-R) hydromethanolic extracts and their chromatographic fractions against oral squamous cell carcinoma (OSCC) cell lines [Ca9-22 (derived from gingiva), HSC-2, HSC-3, and HSC-4 (derived from tongue)] and three normal oral mesenchymal cells [human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF), and human pulp cells (HPC)] in comparison to standard drugs. The root polar ethyl acetate (W-R EtOAc) and butanol (W-R BuOH) fractions exhibited the strongest cytotoxicity against the Ca9-22 cell line (CC50 = 51.8 and 40.1μg/mL, respectively), which is relatively the same effect as 5-FU at CC50 = 69.4μM and melphalan at CC50 = 36.3μM on the same cancer cell line. Flow cytometric analysis revealed changes in morphology as well as in the cell cycle profile of the W-R EtOAc and W-R BuOH-treated oral cancer Ca9-22 cells compared to the untreated control. The W-R EtOAc (125μg/mL) exerted morphological changes and induced subG1 accumulation, suggesting apoptotic cell death. A UHPLC MS/MS analysis of the extract enabled the identification of 26 compounds, mainly alkaloids, withanolides, withanosides, and flavonoids. Pharmacophore-based inverse virtual screening proposed that BRD3 and CDK2 are the cancer-relevant targets for the annotated withanolides D (18) and O (12), and the flavonoid kaempferol (11). Molecular modeling studies highlighted the BRD3 and CDK2 as the most probable oncogenic targets of anticancer activity of these molecules. These findings highlight W. somnifera's potential as an affordable source of therapeutic agents for a range of oral malignancies.

Effects of natural antimicrobial compounds propolis and copaiba on periodontal ligament fibroblasts, molecular docking, and in vivo study in Galleria mellonella

Root canal treatment addresses infectious processes that require control. Occasionally, the radicular pulp is vital and inflamed, presenting a superficial infection. To preserve pulpal remnants, conservative procedures have gained favor, employing anti-inflammatory medications. This study investigated the effects of propolis (PRO), and copaiba oil-resin (COR) associated with hydrocortisone (H) and compared their impact to that of Otosporin® concerning cytotoxic and genotoxic activity, cytokine detection, and toxicity in the Galleria mellonella model. Human periodontal ligament fibroblasts (PDLFs) were exposed to drug concentrations and evaluated by the MTT assay. Associations were tested from concentrations that did not compromise cell density. Genotoxicity was evaluated through micronucleus counting, while cytokines IL-6 and TGF-β1 were detected in the cell supernatant using ELISA. Molecular docking simulations were conducted, considering the major compounds identified in PRO, COR, and H. Increasing concentrations of PRO and COR were assessed for acute toxicity in Galleria mellonella model. Cellular assays were analyzed using one-way ANOVA followed by Tukey tests, while larval survivals were evaluated using the Log-rank (Mantel-Cox) test (α = 0.05). PRO and COR promoted PDLFs proliferation, even in conjunction with H. No changes in cell metabolism were observed concerning cytokine levels. The tested materials induce the release of AT1R, proliferating the PDFLs through interactions. PRO and COR had low toxicity in larvae, suggesting safety at tested levels. These findings endorse the potential of PRO and COR in endodontics and present promising applications across medical domains, such as preventive strategies in inflammation, shedding light on their potential development into commercially available drugs.

BML-111 inhibit H2O2-induced pyroptosis and osteogenic dysfunction of human periodontal ligament fibroblasts by activating the Nrf2/HO-1 pathway

BackgroundPeriodontitis is a common and harmful chronic inflammatory oral disease, characterized by the destruction of periodontal soft and hard tissues. The NLRP3 inflammasome-related pyroptosis and human periodontal ligament fibroblasts (hPDLFs) osteogenic dysfunction are involved in its pathogenesis. Studies have shown that lipoxin A4 is an endogenous anti-inflammatory mediator and BML-111 is a lipoxin A4 analog, which was found to have potent and durable anti-inflammatory effects in inflammatory diseases, but the mechanism remains unclear. The purpose of this study was to investigate whether BML-111 inhibits H2O2-induced dysfunction of hPDLFs, attenuates inflammatory responses, and identifies the underlying mechanisms.MethodsThe oxidative stress model was established with H2O2, and the cell proliferation activity was measured by CCK-8. ALP staining and alizarin red staining were used to detect the osteogenic differentiation capacity of cells; flow cytometry and ELISA were used to detect cell pyroptosis; we explored the effect of BML-111 on hPDLFs under oxidative stress by analyzing the results of PCR and Western blotting. The Nrf2 inhibitor ML385 was added to further identify the target of BML-111 and clarify its mechanism.ResultsBML-111 can alleviate the impaired cell proliferation viability induced by H2O2. H2O2 treatment can induce NLRP3 inflammasome-related pyroptosis, impairing the osteogenic differentiation capacity of hPDLFs. BML-111 can effectively alleviate H2O2-induced cellular dysfunction by activating the Nrf2/HO-1 signaling pathway.ConclusionThe results of this study confirmed the beneficial effects of BML-111 on H2O2-induced NLRP3 inflammasome-related pyroptosis in hPDLFs, and BML-111 could effectively attenuate the impaired osteogenic differentiation function. This beneficial effect is achieved by activating the Nrf2/HO-1 signaling pathway, therefore, our results suggest that BML-111 is a potential drug for the treatment of periodontitis.

The construction of a microenvironment with the vascular network by co-culturing fibroblasts and endothelial cells

IntroductionExtracellular matrix (ECM) synthesis and deposition in fibroblasts, and vascularization via endothelial cells are essential for successful tissue regeneration. Fibroblasts can produce both ECM, physical support for maintaining homeostasis, and bioactive molecules, such as growth factors and cytokines. Endothelial cells can secrete growth factors and form vascular networks that enable the supply of nutrients and oxygen and remove metabolic products. MethodsIn this study, we focused on combining Human Periodontal Ligament Fibroblasts (HPLF) and Human Umbilical Vein Endothelial Cells (HUVEC) for tissue regeneration in clinical applications. ResultsThe fibroblastic and angiogenic phenotypes were promoted in co-culture with HPLF and HUVEC at a ratio of 1:1 compared to HPLF or HUVEC mono-culture. The gene expression of ECM components and angiogenesis-related factors was also enhanced by HPLF/HUVEC co-culture. Despite an apparent increase in the expression of angiogenic factors, the levels of secreted growth factors decreased under co-culture conditions. These data suggest that ECM constructed by HPLF and HUVEC would act as a storage site for growth factors, which can later be released. Our results showed that cell-to-cell interactions between HPLF and HUVEC enhanced collagen synthesis and endothelial network formation, leading to the creation of highly vascularized constructs for periodontal tissue regeneration. ConclusionSuccessful periodontal tissue regeneration requires microenvironmental reconstruction and vascularization, which can be achieved using a co-culture system. In the present study, we found that fibroblastic and angiogenic phenotypes were enhanced by the co-culture of HPLF and HUVEC. The optimal culture conditions (1:1) could potentially accelerate tissue engineering, including ECM synthesis and EC tube formation, and these approaches can improve therapeutic efficacy after transplantation.

Salivary miR-150-5p as an indicator of periodontitis severity and regulator of human periodontal ligament fibroblast behavior by targeting AIFM2.

This study aimed to evaluate the role of miR-150-5p in the onset and progression of periodontitis, and reveal the potential molecular mechanism underlying its function and to explore a novel biomarker for periodontitis. Periodontitis is the leading cause of tooth loss in adults, emphasizing the need for a biomarker to improve its early detection and prevention. The association of miR-150-5p with diseases related to Fuscobacterium nucleatum implies its potential involvement in periodontitis. The expression of miR-150-5p in the saliva of patients with periodontitis (n = 77) and healthy individuals (n = 43) was assessed by PCR. Human gingival fibroblasts (HGFs) were induced with an osteogenic culture medium. The regulatory effect of miR-150-5p on the proliferation and migration of HGFs was assessed by CCK8 and transwell assays. Osteogenic differentiation was estimated based on the expression of corresponding factors through western blotting, and the inflammatory response was evaluated by measuring the levels of pro-inflammatory cytokines using ELISA. Significant upregulation of miR-150-5p was observed in patients with periodontitis, which sensitively distinguished them and was closely associated with the severity and periodontal index of the condition. In HGFs, osteogenic induction (OI) resulted in increased miR-150-5p levels, which negatively regulated the expression of AIFM2. Silencing miR-150-5p significantly attenuated OI-induced suppression of proliferation and migration of HGFs. The silencing also alleviated inflammation and osteogenic differentiation, which was reversed upon AIFM2 knockdown. Upregulated miR-150-5p in periodontitis served as a diagnostic biomarker, indicating the occurrence and aggravation of disease condition. Silencing miR-150-5p inhibited the osteogenic differentiation and inflammation of HGFs by negatively modulating AIFM2.

Antioxidant activity and in vitro cytotoxicity of Chitosan/Origanum vulgare essential oil sponges

Essential oils are complex mixtures of volatile organic substances obtained from medicinal plants, their structural diversity determines their pharmacological bioactivity, including their therapeutic applications. Clinical studies have reported favorable health benefits to patients using medicinal plants for ulcerative skin lesions, reducing the discomfort, pain, and duration of ulcers. Chitosan (CHT) is a polymer with potential as a drug carrier in ulcer disease; also has antibacterial, anti-inflammatory, wound-healing, and hemostatic properties, proposed for its use in gels, biofilms, and sponges to cover the ulcerative lesion in the oral mucosa for both drug delivery and physical protection. The objective of this study was to evaluate the biological properties and enzymatic activity of sponges from CHT and Oregano essential oil (OEs). CHT sponges were incubated in OEs eluates (0.01, 0.02 and 0.03 mg/mL). The cell response and enzymatic activity of Super Oxide Dismutase (SOD) and Catalase (CAT) were evaluated in human periodontal ligament fibroblasts (hPLFs). The results showed that CHT-OEs at 0.02 mg/mL and at 0.03 mg/mL significantly increased hPLFs proliferation (p < 0.05). Micrographs demonstrated that the viability and mitochondrial activity of hPLFs were not affected by the eluates from CHT and OEs. SOD activity increased with eluates from OEs and CHT and CAT was significantly increased with CHT+OEs 0.01 mg/mL (p < 0.05). The synergy of CHT with OEs at a concentration of 0.02 mg/mL and 0.03 mg/mL in culture medium increases the proliferative response and stimulates SOD and CAT activity.

Cytotoxicity, Differentiation, and Biocompatibility of Root-End Filling: A Comprehensive Study.

Assessing the biocompatibility of endodontic root-end filling materials through cell line responses is both essential and of utmost importance. This study aimed to the cytotoxicity of the type of cell death through apoptosis and autophagy, and odontoblast cell-like differentiation effects of MTA, zinc oxide-eugenol, and two experimental Portland cements modified with bismuth (Portland Bi) and barium (Portland Ba) on primary cell cultures. Material and methods: The cells corresponded to human periodontal ligament and gingival fibroblasts (HPLF, HGF), human pulp cells (HPC), and human squamous carcinoma cells from three different patients (HSC-2, -3, -4). The cements were inoculcated in different concentrations for cytotoxicity evaluation, DNA fragmentation in electrophoresis, apoptosis caspase activation, and autophagy antigen reaction, odontoblast-like cells were differentiated and tested for mineral deposition. The data were subject to a non-parametric test. Results: All cements caused a dose-dependent reduction in cell viability. Contact with zinc oxide-eugenol induced neither DNA fragmentation nor apoptotic caspase-3 activation and autophagy inhibitors (3-methyladenine, bafilomycin). Portland Bi accelerated significantly (p < 0.05) the differentiation of odontoblast-like cells. Within the limitation of this study, it was concluded that Portland cement with bismuth exhibits cytocompatibility and promotes odontoblast-like cell differentiation. This research contributes valuable insights into biocompatibility, suggesting its potential use in endodontic repair and biomimetic remineralization.

Mechanical force application and inflammation induce osteoclastogenesis by independent pathways.

To investigate the functional changes of PDL fibroblasts in the presence of mechanical force, inflammation, or a combination of force and inflammation. Inflammatory supernatants were prepared by inoculating human neutrophils with Porphyromonas gingivalis. Primary human PDL fibroblasts (PDLF), gingival fibroblasts (GFs), and osteoblasts (Saos2) were then exposed to the inflammatory supernatants. Orthodontic force on the PDLFs was simulated by centrifugation. Analyses included cell proliferation, cell viability, cell cycle, and collagen expression, as well as osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) expression. Mechanical force did not affect PDLF viability, but it increased the metabolic rate compared to resting cells. Force application shifted the PDLF cell cycle to the G0/G1 phase, arresting cell proliferation and leading to elevated collagen production, mild OPG level elevation, and robust RANKL level elevation. Including an inflammatory supernatant in the presence of force did not affect PDLF viability, proliferation, or cytokine expression. By contrast, the inflammatory supernatant increased RANKL expression in GFs, but not in Saos2 cells. Applying mechanical force significantly affects PDLF function. Although inflammation had no effect on PDLF or Saos2 cells, it promoted RANKL expression in GF cells. Within the limitations of the in vitro model, the results suggest that periodontal inflammation and mechanical forces could affect bone catabolism through effects on different cell types, which may culminate in synergistic bone resorption.

Postbiotics of the Lactiplantibacillus plantarum EIR/IF-1 Strain Show Antimicrobial Activity against Oral Microorganisms with pH Adaptation Capability

Postbiotics offer better properties than probiotics. This study investigated the antimicrobial activity of Lactiplantibacillus plantarum EIR/IF-1 postbiotics against pH-adaptive bacteria, namely Prevotella denticola, Fusobacterium nucleatum, and Streptococcus sanguinis. Cell-free culture media of L. plantarum EIR/IF-1 were used as postbiotics in either crude (acidic) or neutralized form to also understand non-pH-dependent antimicrobial potential. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and viable cell counts were determined for crude and neutralized postbiotics. Culture media adjusted to different pH values were also compared to adjusted media with postbiotics to understand the strength of organic acids in postbiotics. Antibiofilm activity of postbiotics was determined against polymicrobial biofilm formation. Finally, the toxicity of crude postbiotics was tested on human periodontal ligament fibroblast cells (hPDLFCs). MIC values of crude postbiotics were 12.5 mg/mL for all strains. F. nucleatum and P. denticola strains were sensitive to neutralized postbiotics after 48 h of incubation. Moreover, 12.5 and 25 mg/mL postbiotics inhibited biofilm formation and 2.5 mg/mL and lower concentrations of crude postbiotics showed no cytotoxicity in hPDLFCs. This study showed that postbiotics have antimicrobial activity against pH-adaptive oral bacteria and no cytotoxic effect on hPDLFCs depending on the dose. The non-acidic antimicrobial components of postbiotics could also enable their safe use in the oral cavity.

PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.