Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

Sinensetin protects against periodontitis through binding to Bach1 enhancing its ubiquitination degradation and improving oxidative stress.

Periodontitis is a chronic inflammatory and immune reactive disease induced by the subgingival biofilm. The therapeutic effect for susceptible patients is often unsatisfactory due to excessive inflammatory response and oxidative stress. Sinensetin (Sin) is a nature polymethoxylated flavonoid with anti-inflammatory and antioxidant activities. Our study aimed to explore the beneficial effect of Sin on periodontitis and the specific molecular mechanisms. We found that Sin attenuated oxidative stress and inflammatory levels of periodontal ligament cells (PDLCs) under inflammatory conditions. Administered Sin to rats with ligation-induced periodontitis models exhibited a protective effect against periodontitis in vivo. By molecular docking, we identified Bach1 as a strong binding target of Sin, and this binding was further verified by cellular thermal displacement assay and immunofluorescence assays. Chromatin immunoprecipitation-quantitative polymerase chain reaction results also revealed that Sin obstructed the binding of Bach1 to the HMOX1 promoter, subsequently upregulating the expression of the key antioxidant factor HO-1. Further functional experiments with Bach1 knocked down and overexpressed verified Bach1 as a key target for Sin to exert its antioxidant effects. Additionally, we demonstrated that Sin prompted the reduction of Bach1 by potentiating the ubiquitination degradation of Bach1, thereby inducing HO-1 expression and inhibiting oxidative stress. Overall, Sin could be a promising drug candidate for the treatment of periodontitis by targeting binding to Bach1.

Revealing Genetic Dynamics: scRNA-seq Unravels Modifications in Human PDL Cells across In Vivo and In Vitro Environments.

The periodontal ligament (PDL) is a highly specialized fibrous tissue comprising heterogeneous cell populations of an intricate nature. These complexities, along with challenges due to cell culture, impede a comprehensive understanding of periodontal pathophysiology. This study aims to address this gap, employing single-cell RNA sequencing (scRNA-seq) technology to analyze the genetic intricacies of PDL both in vivo and in vitro. Primary human PDL samples (n = 7) were split for direct in vivo analysis and cell culture under serum-containing and serum-free conditions. Cell hashing and sorting, scRNA-seq library preparation using the 10x Genomics protocol, and Illumina sequencing were conducted. Primary analysis was performed using Cellranger, with downstream analysis via the R packages Seurat and SCORPIUS. Seven distinct PDL cell clusters were identified comprising different cellular subsets, each characterized by unique genetic profiles, with some showing donor-specific patterns in representation and distribution. Formation of these cellular clusters was influenced by culture conditions, particularly serum presence. Furthermore, certain cell populations were found to be inherent to the PDL tissue, while others exhibited variability across donors. This study elucidates specific genes and cell clusters within the PDL, revealing both inherent and context-driven subpopulations. The impact of culture conditions-notably the presence of serum-on cell cluster formation highlights the critical need for refining culture protocols, as comprehending these influences can drive the creation of superior culture systems vital for advancing research in PDL biology and regenerative therapies. These discoveries not only deepen our comprehension of PDL biology but also open avenues for future investigations into uncovering underlying mechanisms.

Inhibition of histone deacetylases class I improves adipogenic differentiation of human periodontal ligament cells.

Periodontal ligament stem cells (PDLSCs) show plasticity towards the adipogenic lineage; however, little has been done on the participation of epigenetic mechanisms. Histone acetylation is a dynamic process, though balanced by histone acetyltransferases (HATs) and histone deacetylases (HDACs) activities. This process can be halted by HDACs inhibitors, such as trichostatin A (TSA) and valproic acid (VPA). This study aimed to determine the role of HDACs class I in adipogenic differentiation of PDL cells. PDLSCs were treated with TSA at concentrations of 100, 200, and 250 nM, or VPA at 1, 4 and 8 mM. Cell viability was assessed using MTT assays. Gene expression of pluripotency markers (NANOG, OCT4, SOX2), HAT genes (p300, GCN5), and HDACs genes (HDAC1-3) was analyzed by RT-qPCR. Adipogenic differentiation was evaluated via oil red O staining, and acetylation of histone H3 lysine 9 (H3K9ac) was examined by Western blot. VPA treatment resulted in a 60% reduction in cell proliferation, compared to a 50% when using TSA. Cell viability was not affected by either inhibitor. Furthermore, both TSA and VPA induced adipogenic differentiation, through an increase in the deposition of lipid droplets and in GCN5 and p300 expression were observed. Western blot analysis showed that TSA increased H3K9ac levels on adipogenic differentiation of PDLSCs. These findings highlight the potential of HDAC inhibitors as a tool for modulating H3K9 acetylation status and thus influencing adipogenic differentiation of PDLCs.

Red-complex bacteria exhibit distinctly different interactions with human periodontal ligament stromal cells compared to Fusobacterium nucleatum.

OBJECTIVE: The red-complex bacteria Porphyromonas gingivalis and Tannerella forsythia together with Fusobacterium nucleatum are essential players in periodontitis. This study investigated the bacterial interplay with human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) which act in the acute phase of periodontal infection. DESIGN: The capability of the bacteria to induce an inflammatory response as well as their viability, cellular adhesion and invasion were analyzed upon mono- and co-infections of hPDL-MSCs to delineate potential synergistic or antagonistic effects. The expression level and concentration of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were measured using qRT-PCR and ELISA. Viability, invasion, and adhesion were determined quantitatively using agar plate culture and qualitatively by confocal microscopy. RESULTS: Viability of P. gingivalis and T. forsythia but not F. nucleatum was preserved in the presence of hPDL-MSCs, even in an oxygenated environment. F. nucleatum significantly increased the expression and concentration of IL-6, IL-8 and MCP-1 in hPDL-MSCs, while T. forsythia and P. gingivalis caused only a minimal inflammatory response. Co-infections in different combinations had no effect on the inflammatory response. Moreover, P. gingivalis mitigated the increase in cytokine levels elicited by F. nucleatum. Both red-complex bacteria adhered to and invaded hPDL-MSCs in greater numbers than F. nucleatum, with only a minor effect of co-infections. CONCLUSIONS: Oral bacteria of different pathogenicity status interact differently with hPDL-MSCs. The data support P. gingivalis' capability to manipulate the inflammatory host response. Further research is necessary to obtain a comprehensive picture of the role of hPDL-MSCs in more complex oral biofilms.

A finite element analysis of periodontal ligament fluid mechanics response to occlusal loading based on hydro-mechanical coupling model.

OBJECTIVE: Considering fluid stimulation is one of the essential biomechanical signals for periodontal tissues, this study aims to characterizing fluid mechanics response during occlusal loading by a hydro-mechanical coupling model for periodontal ligament. DESIGN: Models simulating periodontium with normal bone height and with intraosseous defects were built with three mechanical modules: tooth, periodontal ligament and alveolar bone. Tooth was modeled as linear elastic, and periodontal ligament and alveolar bone as a hydro-mechanical coupling model. Transient analyses under dynamic occlusal loading were performed. Fluid dynamics within periodontal ligament space was simulated and visualized by post-processing module. RESULTS: Reciprocating oscillatory flow occurred within the periodontal ligament under occlusal loading. Higher pore pressure and fluid velocity were observed in furcation and apical regions compared to mid-root and cervical regions. Intraosseous defects increased pore pressure and fluid velocity within the periodontal ligament, most significantly near the defect. CONCLUSION: Based on the results of the hydro-mechanical coupling model, significant oscillatory fluid motion is observed within the periodontal ligament under occlusal loading. Particularly, higher fluid velocity is evident in the furcation and apical areas. Additionally, Intraosseous defects significantly enhance fluid motion within the periodontal ligament.

4-META/MMA-TBB resin containing nano hydroxyapatite induces the healing of periodontal tissue repair in perforations at the pulp chamber floor.

We aimed to evaluate the materials based on 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate tri-n-butylborane (Super-bond [SB]) and nano hydroxyapatite (naHAp) for the repair of perforation at pulp chamber floor (PPF) in vitro and in vivo models. SB and naHAp were mixed in the mass ratio of 10% or 30% to produce naHAp/SB. Human periodontal ligament stem cells (HPDLSCs) were cultured on resin discs of SB or naHAp/SB to analyze the effects of naHAp/SB on cell adhesion, proliferation, and cementoblastic differentiation. A rat PPF model was treated with SB or naHAp/SB to examine the effects of naHAp/SB on the healing of defected cementum and periodontal ligament (PDL) at the site of PPF. HPDLSCs were spindle-shaped and adhered to all resin discs. Changing the resin from SB to naHAp/SB did not significantly alter cell proliferation. Both 10% and 30% naHAp/SB were more effective than SB in promoting cementoblastic differentiation of HPDLSCs. In the rat PPF model, 30% naHAp/SB was more effective than SB in promoting the formation Sharpey's fiber-like structures with expression of the PDL-related marker and cementum-like structures with expression of cementum-related markers. In conclusion, 30% naHAp/SB can be the new restorative material for PPF because it exhibited the abilities of adhering to dentin and healing of defected periodontal tissue.

In vivo measurement of strain in the periodontal space of pig (Sus scrofa) incisors using in-fiber Bragg sensors.

The incisor teeth in pigs, Sus scrofa, function in association with a disc-shaped snout to explore the environment for potential food. Understanding how mechanical loading applied to the tooth deforms the periodontal ligament (PDL) is important to determining the role of periodontal mechanoreceptors during food exploration and feeding. The objective of this study was to use fiber Bragg (FBG) sensors to measure strain in vivo within the PDL space of pig incisors. The central mandibular incisors of pigs underwent spring loaded lingual tipping during FBG strain recording within the labial periodontal space. FBG sensors were placed within the periodontal space of the central mandibular incisors of ~2-3-month-old farm pigs. The magnitude and orientation of spring loads are expected to mimic incisor contact with food. During incisor tipping with load calibrated springs, FBG strains in vitro (N = 6) and in vivo (N = 6) recorded at comparable load levels overlapped in range (-10-20 µÎµ). Linear regressions between peak FBG strains, that is, the highest recorded strain value, and baseline strains, that is, strain without applied spring load, were significant across all in vivo experiments (peak strain at 200 g vs. baseline, p = .04; peak strain at 2000 g vs. baseline p = .03; peak strain at 2000 g vs. 200 g, p = .004). These linear relationships indicate that on a per experiment basis, the maximum measured strain at different spring loads showed predictable differences. A Friedman test of the absolute value of peak strain confirmed the significant increase in strain between baseline, 200 g, and 2000 g spring activation (p = .02). Mainly compressive strains were recorded in the labial PDL space and increases in spring load applied in vivo generated increases in FBG strain measurements. These results demonstrate the capacity for FBG sensors to be used in vivo to assess transmission of occlusal loads through the periodontium. PDL strain is associated with mechanoreceptor stimulation and is expected to affect the functional morphology of the incisors. The overall low levels of strain observed may correspond with the robust functional morphology of pig incisors and the tendency for pigs to encounter diverse foods and substrates during food exploration.

In-vitro effects of novel periodontal scalers with a planar ultrasonic piezoelectric transducer on periodontal biofilm removal, dentine surface roughness, and periodontal ligament fibroblasts adhesion.

OBJECTIVES: To compare ultrasonic scaler prototypes based on a planar piezoelectric transducer with different working frequencies featuring a titanium (Ti-20, Ti-28, and Ti-40) or stainless steel (SS-28) instrument, with a commercially available scaler (com-29) in terms of biofilm removal and reformation, dentine surface roughness and adhesion of periodontal fibroblasts. MATERIALS AND METHODS: A periodontal multi-species biofilm was formed on specimens with dentine slices. Thereafter specimens were instrumented with scalers in a periodontal pocket model or left untreated (control). The remaining biofilms were quantified and allowed to reform on instrumented dentine slices. In addition, fibroblasts were seeded for attachment evaluation after 72 h of incubation. Dentine surface roughness was analyzed before and after instrumentation. RESULTS: All tested instruments reduced the colony-forming unit (cfu) counts by about 3 to 4 log10 and the biofilm quantity (each p < 0.01 vs. control), but with no statistically significant difference between the instrumented groups. After 24-hour biofilm reformation, no differences in cfu counts were observed between any groups, but the biofilm quantity was about 50% in all instrumented groups compared to the control. The attachment of fibroblasts on instrumented dentine was significantly higher than on untreated dentine (p < 0.05), with the exception of Ti-20. The dentine surface roughness was not affected by any instrumentation. CONCLUSIONS: The planar piezoelectric scaler prototypes are able to efficiently remove biofilm without dentine surface alterations, regardless of the operating frequency or instrument material. CLINICAL RELEVANCE: Ultrasonic scalers based on a planar piezoelectric transducer might be an alternative to currently available ultrasonic scalers.

Hyaluronidase inhibits TGF-ß-mediated rat periodontal ligament fibroblast expression of collagen and myofibroblast markers: An in vitro exploration of periodontal tissue remodeling.

OBJECTIVE: To determine the effect of hyaluronic acid (HA) degradation by hyaluronidase (HYAL) in inhibiting collagen fiber production by rat periodontal ligament cells (rPDLCs). DESIGN: Primary rPDLCs were isolated from the euthanized rats and used for in vitro experiments. The appropriate HYAL concentration was determined through CCK-8 testing for cytotoxicity detection and Alizarin red staining for mineralization detection. RT-qPCR and western blot assays were conducted to assess the effect of HYAL, with or without TGF-ß, on generation of collagen fiber constituents and expression of actin alpha 2, smooth muscle (ACTA2) of rPDLCs. RESULTS: Neither cell proliferation nor mineralization were significantly affected by treatment with 4 U/mL HYAL. HYAL (4 U/mL) alone downregulated type I collagen fiber (Col1a1 and Col1a2) and Acta2 mRNA expression; however, ACTA2 and COL1 protein levels were only downregulated by HYAL treatment after TGF-ß induction. CONCLUSIONS: Treatment of rPDLCs with HYAL can inhibit TGF-ß-induced collagen matrix formation and myofibroblast transformation.

Paracrine- and cell-contact-mediated immunomodulatory effects of human periodontal ligament-derived mesenchymal stromal cells on CD4+ T lymphocytes.

BACKGROUND: Mesenchymal stromal cells (MSCs) isolated from the periodontal ligament (hPDL-MSCs) have a high therapeutic potential, presumably due to their immunomodulatory properties. The interaction between hPDL-MSCs and immune cells is reciprocal and executed by diverse cytokine-triggered paracrine and direct cell-to-cell contact mechanisms. For the first time, this study aimed to directly compare the contribution of various mechanisms on this reciprocal interaction using different in vitro co-culture models at different inflammatory milieus. METHODS: Three co-culture models were used: indirect with 0.4 µm-pored insert, and direct with or without insert. After five days of co-culturing mitogen-activated CD4+ T lymphocytes with untreated, interleukin (IL)-1ß, or tumor necrosis factor (TNF)-α- treated hPDL-MSCs, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were investigated. The gene expression of soluble and membrane-bound immunomediators was investigated in the co-cultured hPDL-MSCs. RESULTS: Untreated hPDL-MSCs decreased the CD4+ T lymphocyte proliferation and viability more effectively in the direct co-culture models. The direct co-culture model without inserts showed a strikingly higher CD4+ T lymphocyte cell death rate. Adding IL-1ß to the co-culture models resulted in substantial CD4+ T lymphocyte response alterations, whereas adding TNF resulted in only moderate effects. The most changes in CD4+ T lymphocyte parameters upon the addition of IL-1ß or TNF-α in a direct co-culture model without insert were qualitatively different from those observed in two other models. Additionally, the co-culture models caused variability in the immunomediator gene expression in untreated and cytokine-triggered hPDL-MSCs. CONCLUSION: These results suggest that both paracrine and cell-to-cell contact mechanisms contribute to the reciprocal interaction between hPDL-MSCs and CD4+ T lymphocytes. The inflammatory environment affects each of these mechanisms, which depends on the type of cytokines used for the activation of MSCs' immunomodulatory activities. This fact should be considered by comparing the outcomes of the different models.

Fucoidan-hybrid hydroxyapatite nanoparticles promote the osteogenic differentiation of human periodontal ligament stem cells under inflammatory condition.

Inflammation-related bone defects often lead to poor osteogenesis. Therefore, it is crucial to reduce the inflammation response and promote the osteogenic differentiation of stem/progenitor cells to revitalize bone physiology. Here, a kind of hybrid nano-hydroxyapatite was prepared using the confined phosphate ion release method with the participation of fucoidan, a marine-sourced polysaccharide with anti-inflammation property. The physicochemical analyses confirmed that the fucoidan hybrid nano-hydroxyapatite (FC/n-HA) showed fine needle-like architectures. With a higher amount of fucoidan, the crystal size and crystallinity of the FC/n-HA reduced while the liquid dispersibility was improved. Cell experiences showed that FC/n-HA had an optimal cytocompatibility at concentration of 50 µg/mL. Moreover, the lipopolysaccharide-induced cellular inflammatory model with PDLSCs was established and used to evaluate the anti-inflammatory and osteogenic properties. For the 1%FC/n-HA group, the expression levels of TNF-α and IL-1ß were significantly reduced at 24 h, while the expression of alkaline phosphatase of PDLSCs was significantly promoted at days 3 and 7, and calcium precipitates was enhanced at 21 days. In this study, the FC/n-HA particles showed effective anti-inflammatory properties and facilitated osteogenic differentiation of PDLSCs, indicating which has potential application in treating bone defects associated with inflammation, such as periodontitis.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. METHODS AND RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

Effects of surface nanomorphology on the senescence of periodontal ligament stem cells.

OBJECTIVES: The effect of TiO2 nanotube morphology on the differentiation potency of senescent periodontal ligament stem cells was investigated. METHODS: Two types of titanium sheets with TiO2 nanotube morphology (20V-NT and 70V-NT) were prepared via anodic oxidation at 20 and 70 V separately, and their surface morphology was observed. Young periodontal ligament stem cells were cultivated in an osteogenic induction medium, and the most effective surface morphology in promoting osteogenic differentiation was selected. RO3306 and Nutlin-3a were used to induce the aging of young periodontal ligament stem cells, and senescent periodontal ligament stem cells were obtained. The osteogenic differentiation of senescent periodontal ligament stem cells was induced, and the effect of surface morphology on osteogenic differentiation was observed. RESULTS: Nanotube morphology was achieved on the surfaces of titanium sheets through anodic oxidation, and the diameters of the nanotubes increased with voltage. A significant difference in the effect of nanotube morphology was found among nanotubes with different diameters in the young periodontal ligament stem cells. The surface nanotube morphology of 20V-NT had a more significant effect that promoted osteogenic differentiation. Compared with a smooth titanium sheet, the surface nanotube morphology of 20V-NT increased the number of alkaline phosphatase-positive senescent periodontal ligament stem cells and promoted calcium deposition and the expression of osteogenic marker genes Runt-related transcription factor 2, osteopontin, and osteocalcin. CONCLUSIONS: A special nanotube morphology enhances the differentiation ability of senescent periodontal ligament stem cells, provides an effective method for periodontal regeneration, and further improves the performance of implants.

Predicting chronological age of 14 or 18 in adolescents: integrating dental assessments with machine learning.

AIM: Age estimation plays a critical role in personal identification, especially when determining compliance with the age of consent for adolescents. The age of consent refers to the minimum age at which an individual is legally considered capable of providing informed consent for sexual activities. The purpose of this study is to determine whether adolescents meet the age of 14 or 18 by using dental development combined with machine learning. METHODS: This study combines dental assessment and machine learning techniques to predict whether adolescents have reached the consent age of 14 or 18. Factors such as the staging of the third molar, the third molar index, and the visibility of the periodontal ligament of the second molar are evaluated. RESULTS: Differences in performance metrics indicate that the posterior probabilities achieved by machine learning exceed 93% for the age of 14 and slightly lower for the age of 18. CONCLUSION: This study provides valuable insights for forensic identification for adolescents in personal identification, emphasizing the potential to improve the accuracy of age determination within this population by combining traditional methods with machine learning. It underscores the importance of protecting and respecting the dignity of all individuals involved.

[The role of ROS/JNK/caspase 3 axis in apoptosis induction by menthol-favored electronic cigarette liquid in human periodontal ligament stem cells].

PURPOSE: To explore the cytotoxic effect of a menthol-favored E-liquid on human periodontal ligament stem cells (hPDLSCs), as well as the underlying mechanism of electronic cigarette (E-cig)-induced cell apoptosis. METHODS: PDLSCs were isolated and cultured from periodontal ligament tissues of healthy premolars extracted for orthodontic reasons. Cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry. Then the cells were exposed to different doses of menthol-favored E-liquid (at 59 mg/L nicotine concentration) in the culture median (the final nicotine concentrations were 0.1 µg/mL, 1.0 µg/mL, 10 µg/mL, 50 µg/mL, 0.1 mg/mL, 0.2 mg/mL and 0.5 mg/mL, respectively) for different period of times (24, 48 and 72 h). The cell viability was analyzed by CCK-8 assay. Cell apoptosis was evaluated by flow cytometry (7-AAD and Annexin V staining) and TUNEL assay. Reactive oxygen species (ROS) production was detected with fluorescence probe DCFH-DA by confocal microscopy and flow cytometry. The protein expression levels associated with ROS/JNK/caspase 3 axis(p-JNK, JNK, c-Jun, p-c-Jun, Bcl-2, Bax and cleaved-caspase 3) were analyzed by Western blot. Immunocytofluorescense staining was applied to evaluate the expression level of p-JNK. After addition of NAC, a ROS scavenger, and MAPK/JNK specific blocker SP600125, their effects on E-cig-induced cell apoptosis were evaluated. Statistical analysis was performed with Graph Pad 5.0 software package. RESULTS: Human PDLSCs were successfully isolated and cultured and flow cytometry assay showed the mesenchymal stem cell surface biomarkers (CD73, CD90 and CD105) were positively expressed. CCK8 assay indicated cell viability was significantly(P＜0.001) different among all concentration groups at various time points (24, 48 or 72 h), and the difference in apoptosis rate among all concentration groups was also statistically significant (P＜0.001). After exposure to E-liquid with nicotine concentration ≥50 µg/mL, cell viability was significantly reduced, and the proportion of apoptotic cells and the cellular ROS level was significantly increased in a dose-dependent manner as compared with the control group(0.0 mg/mL). Western blot assay showed E-cig exposure could promote MAPK/JNK phosphorylation in a dose-dependent and time-dependent manner. Either NAC or SP600125 could partially rescue the E-cig-induced cell apoptosis via reversing up-regulation of p-JNK and cleaved caspase 3. CONCLUSIONS: ROS/JNK/caspase 3 axis is involved in menthol-favored E-liquid-induced apoptosis of hPDLSCs.

LepR-expressing cells are a critical population in periodontal healing post periodontitis.

Identification of promising seed cells plays a pivotal role in achieving tissue regeneration. This study demonstrated that LepR-expressing cells (LepR+ cells) are required for maintaining periodontal homeostasis at the adult stage. We further investigated how LepR+ cells behave in periodontal healing using a ligature-induced periodontitis (PD) and a self-healing murine model with LepRCre/+; R26RtdTomato/+ mice. Lineage tracing experiments revealed that the largely suppressed osteogenic ability of LepR+ cells results from periodontal inflammation. Periodontal defects were partially recovered when the ligature was removed, in which the osteogenic differentiation of LepR+ cell lineage was promoted and contributed to the newly formed alveolar bone. A cell ablation model established with LepRCre/+; R26RtdTomato/+; R26RDTA/+ mice further proved that LepR+ cells are an important cell source of newly formed alveolar bone. Expressions of ß-catenin and LEF1 in LepR+ cells were upregulated when the inflammatory stimuli were removed, which are consistent with the functional changes observed during periodontal healing. Furthermore, the conditional upregulation of WNT signaling or the application of sclerostin neutralized antibody promoted the osteogenic function of LepR+ cells. In contrast, the specific knockdown of ß-catenin in LepR+ human periodontal ligament cells with small interfering RNA caused arrested osteogenic function. Our findings identified the LepR+ cell lineage as a critical cell population for endogenous periodontal healing post PD, which is regulated by the WNT signaling pathway, making it a promising seed cell population in periodontal tissue regeneration.

Functionalizable bacterial cellulose composite membrane for guided tissue regeneration.

This research aims to develop guided tissue regeneration (GTR) membranes from bacterial cellulose (BC), a natural polysaccharide-based biopolymer. A double-layered BC composite membrane was prepared by coating the BC membrane with mixed carboxymethyl cellulose/poly(ethylene oxide) (CMC/PEO) fibers via electrospinning. The CMC/PEO-BC membranes were then characterized for their chemical and physical characteristics. The 8 % (wt/v) CMC/PEO (1:1) aqueous solution yielded well-defined electrospun CMC/PEO nanofibers (125 ± 10 nm) without beads. The CMC/PEO-BC membranes exhibited good mechanical and swelling properties as well as good cytocompatibility against human periodontal ligament cells (hPDLs). Its functionalizability via carboxyl entities in CMC was tested using the calcium-binding domain of plant-derived recombinant human osteopontin (p-rhOPN-C122). As evaluated by enzyme-linked immunosorbent assay, a 98-99 % immobilization efficiency was achieved in a concentration-dependent manner over an applied p-rhOPN-C122 concentration range of 7.5-30 ng/mL. The biological function of the membrane was assessed by determining the expression levels of osteogenic-related gene transcripts using quantitative real-time reverse-transcriptase polymerase chain reaction. Mineralization assay indicated that the p-rhOPN-C122 immobilized CMC/PEO-BC membrane promoted hPDLs osteogenic differentiation. These results suggested that the developed membrane could serve as a promising GTR membrane for application in bone tissue regeneration.

The impact of Thiopeptide antibiotics on inflammatory responses in periodontal tissues through the regulation of the MAPK pathway.

Periodontitis is a bacteria-induced inflammatory disease that damages the tissues supporting the teeth, gums, periodontal ligaments, and alveolar bone. Conventional treatments such as surgical procedures, anti-inflammatory drugs, and antibiotics, are somewhat effective; however, these may lead to discomfort and adverse events, thereby affecting patient outcomes. Therefore, this study aimed to find an effective method to prevent the onset of periodontal disease and explore the specific mechanisms of their action.The impact of thiostrepton on Porphyromonas gingivalis and periodontal ligament stem cells was evaluated in an inflammatory microenvironment. In vivo experiments were performed using a mouse periodontitis model to assess the effectiveness of locally applied thiostrepton combined with a silk fibroin hydrogel in impeding periodontitis progression. Thiostrepton exhibited significant antimicrobial effects against Porphyromonas gingivalis and anti-inflammatory properties by regulating the MAPK pathway through DUSP2. Locally applied thiostrepton effectively impeded the progression of periodontitis and reduced tissue damage. Thiostrepton treatment is a promising and tolerable preventive strategy for periodontitis, offering antimicrobial and anti-inflammatory benefits. These findings suggest the potential of thiostrepton as a valuable addition to periodontitis management, warranting further research and clinical exploration to improve patient outcomes.

CircPRKD3/miR-6783-3p responds to mechanical force to facilitate the osteogenesis of stretched periodontal ligament stem cells.

BACKGROUND: The mechanotransduction mechanisms by which cells regulate tissue remodeling are not fully deciphered. Circular RNAs (circRNAs) are crucial to various physiological processes, including cell cycle, differentiation, and polarization. However, the effects of mechanical force on circRNAs and the role of circRNAs in the mechanobiology of differentiation and remodeling in stretched periodontal ligament stem cells (PDLSCs) remain unclear. This article aims to explore the osteogenic function of mechanically sensitive circular RNA protein kinase D3 (circPRKD3) and elucidate its underlying mechanotransduction mechanism. MATERIALS AND METHODS: PDLSCs were elongated with 8% stretch at 0.5 Hz for 24 h using the Flexcell® FX-6000™ Tension System. CircPRKD3 was knockdown or overexpressed with lentiviral constructs or plasmids. The downstream molecules of circPRKD3 were predicted by bioinformatics analysis. The osteogenic effect of related molecules was evaluated by quantitative real-time PCR (qRT-PCR) and western blot. RESULTS: Mechanical force enhanced the osteogenesis of PDLSCs and increased the expression of circPRKD3. Knockdown of circPRKD3 hindered PDLSCs from osteogenesis under mechanical force, while overexpression of circPRKD3 promoted the early osteogenesis process of PDLSCs. With bioinformatics analysis and multiple software predictions, we identified hsa-miR-6783-3p could act as the sponge of circPRKD3 to indirectly regulate osteogenic differentiation of mechanically stimulated PDLSCs. CONCLUSIONS: Our results first suggested that both circPRKD3 and hsa-miR-6783-3p could enhance osteogenesis of stretched PDLSCs. Furthermore, hsa-miR-6783-3p could sponge circPRKD3 to indirectly regulate RUNX2 during the periodontal tissue remodeling process in orthodontic treatment.