Periodontitis is a common oral disease caused by bacteria coupled with an excessive host immune response. Stem cell therapy can be a promising treatment strategy for periodontitis, but the relevant mechanism is complicated. This study aimed to explore the therapeutic potential of mitochondria from human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) for the treatment of periodontitis. The gingival tissues of periodontitis patients are characterized by abnormal mitochondrial structure. Human gingival fibroblasts (HGFs) were exposed to 5 μg/mL lipopolysaccharide (LPS) for 24 h to establish a cell injury model. When treated with hESC-MSCs or mitochondria derived from hESC-MSCs, HGFs showed reduced expression of inflammatory genes, increased adenosine triphosphate (ATP) level, decreased reactive oxygen species (ROS) production, and enhanced mitochondrial function compared to the control. The average efficiency of isolated mitochondrial transfer by hESC-MSCs was determined to be 8.93%. Besides, a therapy of local mitochondrial injection in mice with LPS-induced periodontitis showed a reduction in inflammatory gene expression, as well as an increase in both the mitochondrial number and the aspect ratio in gingival tissues. In conclusion, our results indicate that mitochondria derived from hESC-MSCs can reduce the inflammatory response and improve mitochondrial function in HGFs, suggesting that the transfer of mitochondria between hESC-MSCs and HGFs serves as a potential mechanism underlying the therapeutic effect of stem cells.
Humans ; Gingiva/cytology* ; Fibroblasts/metabolism* ; Mitochondria/physiology* ; Mesenchymal Stem Cells/cytology* ; Animals ; Periodontitis/therapy* ; Mice ; Reactive Oxygen Species/metabolism* ; Inflammation ; Lipopolysaccharides ; Human Embryonic Stem Cells/cytology* ; Cells, Cultured ; Adenosine Triphosphate/metabolism* ; Male

Smoking is a well-established risk factor for periodontitis, yet the precise mechanisms by which smoking contributes to periodontal disease remain poorly understood. Recent advances in spatial transcriptomics have enabled a deeper exploration of the periodontal tissue microenvironment at single-cell resolution, offering new opportunities to investigate these mechanisms. In this study, we utilized Visium HD single-cell spatial transcriptomics to profile gingival tissues from 12 individuals, including those with periodontitis, those with smoking-associated periodontitis, and healthy controls. Our analysis revealed that smoking disrupts the epithelial barrier integrity, induces fibroblast alterations, and dysregulates fibroblast-epithelial cell communication, thereby exacerbating periodontitis. The spatial analysis showed that endothelial cells and macrophages are in close proximity and interact, which further promotes the progression of smoking-induced periodontal disease. Importantly, we found that targeting the endothelial CXCL12 signalling pathway in smoking-associated periodontitis reduced the proinflammatory macrophage phenotype, alleviated epithelial inflammation, and reduced alveolar bone resorption. These findings provide novel insights into the pathogenesis of smoking-associated periodontitis and highlight the potential of targeting the endothelial-macrophage interaction as a therapeutic strategy. Furthermore, this study establishes an essential information resource for investigating the effects of smoking on periodontitis, providing a foundation for future research and therapeutic development for this prevalent and debilitating disease.
Humans ; Gingiva/cytology* ; Smoking/adverse effects* ; Male ; Periodontitis/pathology* ; Single-Cell Analysis ; Female ; Adult ; Middle Aged ; Macrophages ; Fibroblasts ; Endothelial Cells ; Case-Control Studies ; Chemokine CXCL12/metabolism*

OBJECTIVES: This study aims to explore the potential relationships of cell-free DNA (cfDNA) in gingival crevicular fluid (GCF) with periodontal clinical indicators and the expression of DNA receptor pathway cyclic guanosine phosphate-adenosine phosphate synthase (cGAS)-stimulator of interferon genes (STING) in gingival tissues and human gingival fibroblasts (HGFs). METHODS: GCF and gingival tissue samples were collected from periodontally healthy individuals and patients diagnosed with periodontitis. Periodontal clinical indicators were recorded, including plaque index (PLT), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). The concentration of cfDNA in GCF was quantified, and the correlation between GCF and periodontal clinical indicators was analyzed. Immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to assess the distribution of cGAS, STING, and p-STING in gingival tissues. Additionally, the mRNA expression levels of the key components of the cGAS-STING signaling pathway, namely, cGAS, STING, inhibitory of kappa-B kinase (IKK), nuclear factor kappa-B p65 (NF-κB p65), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), were measured. Furthermore, cfDNA extracted from GCF was employed to stimulate HGFs in the healthy control and periodontitis groups, and the mRNA expression levels of the key molecules of cGAS-STING signaling pathway were detected through Western blot and RT-qPCR. RESULTS: The concentration of cfDNA in GCF was found to be significantly elevated in the periodontitis group compared with the control group. Moreover, cfDNA concentration demonstrated a strong positive correlation with the periodontal clinical indicators. Immunofluorescence analysis revealed considerably increased percentage of fluorescence co-localization of cGAS, STING, and p-STING with the gingival fibroblast FSP-1 marker in the gingival tissues of the periodontitis group. The mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6,and TNF-α were significantly higher in the periodontitis group. In vitro stimulation of HGFs with GCF-derived cfDNA resulted in increased protein expression of cGAS and p-STING and considerably upregulated the mRNA expression levels of cGAS, STING, IKK, NF-κB p65, IL-1β, IL-6, and TNF-α in the healthy and periodontitis groups compared with the blank group. Correlation analysis showed that the concentration of cfDNA at the sampling site was positively correlated with the mRNA expression levels of cGAS, STING, NF-κB p65, and IL-6 in gingival tissues. CONCLUSIONS cfDNA concentrations in the GCF of patients with periodontitis are considerably elevated, and are associated with the activation of the cGAS-STING signaling pathway in HGFs. These findings suggest that cfDNA contributes to the progression of periodontitis.
Humans ; Gingival Crevicular Fluid/metabolism* ; Signal Transduction ; Gingiva/cytology* ; Nucleotidyltransferases/genetics* ; Membrane Proteins/genetics* ; Cell-Free Nucleic Acids/analysis* ; Fibroblasts/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Periodontitis/metabolism* ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Adult ; RNA, Messenger/metabolism* ; Male ; Female

OBJECTIVES: This study was performed to clarify the effects of sitagliptin on METHODS: Healthy gingival samples were collected from the donors. HGFs were isolated with enzymic digestion method and identified. The effects of LPS and sitagliptin on cell viability were detected by cell-counting kit-8 (CCK8). The mRNA levels of inflammatory cytokines, namely, interleukin (IL)-6, IL-8, C-C motif ligand 2 (CCL2), and superoxide dismutase 2 (SOD2), were evaluated by quantity real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA) was used to measure the secretion protein levels of IL-6, IL-8, and CCL2. Western blot analysis was used to further investigate the activation of nuclear factor (NF)-κB signaling pathway. The effect of NF-κB pathway inhibitor BAY11-7082 on LPS-induced HGF inflammatory cytokines at the gene level was verified by qRT-PCR. RESULTS: Low concentrations of sitagliptin (0.1, 0.25, and 0.5 µmol·L CONCLUSIONS Sitagliptin could significantly inhibit LPS-induced HGF inflammatory response by blocking the NF-κB signaling pathway activation.
Fibroblasts ; Gingiva/metabolism* ; Humans ; Lipopolysaccharides ; NF-kappa B/metabolism* ; Signal Transduction ; Sitagliptin Phosphate

Previous studies to elucidate the etiology of cyclosporine(Cs)-induced gingival overgrowth in children have not completely excluded all factors that may cause differences among individuals. This study examined the effect of cyclosporine on the metabolism of type 1 collagen(CoL-I) in experimental models that controlled the effects of biological variations on individuals. Five 5-week-old male Sprague-Dawley rats were administered Cs by gastric feeding for 6 weeks. Gingival specimens were harvested from the mandibular posterior area before beginning Cs administration and at 2, 4, and 6 weeks thereafter. Gingival fibroblasts were cultured from all the 20 biopsies collected from the gingiva. Half of the fibroblasts collected prior to the Cs administration were designated as Control. The other half of the fibroblasts were treated with Cs in vitro and called in vitro test group(Tt). The fibroblasts collected 2, 4, and 6 weeks after the Cs administration were called in vivo test groups : T2, T4, T6, respectively. Immunofluorescence microscopy was used to detect CoL-I in all the fibroblasts. CoL-I was analyzed at both the gene and protein expression levels by real-time polymerase chain reaction and western blotting. Changes in CoL-I before and after Cs treatment were evaluated from the gingiva of each rat. There was no significant difference in gene expression of CoL-I in the control and test groups. CoL-I protein expression levels of fibroblasts increased in in vitro Cs treatment for each individual, and also increased in in vivo Cs treatment. In this study, the experimental method that control biological variations that can occur due to differences among individuals was useful. Subsequent studies on other factors besides CoL-I and in-depth studies in humans are needed.
Animals ; Biopsy ; Blotting, Western ; Child ; Collagen Type I ; Cyclosporine ; Fibroblasts ; Gene Expression ; Gingiva ; Gingival Overgrowth ; Humans ; In Vitro Techniques ; Male ; Metabolism ; Methods ; Microscopy, Fluorescence ; Models, Theoretical ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL⁻¹ of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.
Animals ; Gingiva ; drug effects ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; administration & dosage ; Male ; Porphyromonas gingivalis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS⁺) and STRO-1-negative (MACS⁻) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS⁺ and MACS⁻ cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS⁺ and MACS(-) cell fractions showed plastic adherence. MACS⁺ cells, in contrast to MACS⁻ cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS⁺ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS⁻ cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS⁺ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS⁻ cells demonstrated slight osteogenic potential. Unstimulated MACS⁺ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS⁻ cells demonstrated higher expression of osteonectin (P<0.05; Mann-Whitney). The present study is the first to compare gingival MACS⁺ and MACS⁻ cell populations demonstrating that MACS⁺ cells, in contrast to MACS⁻ cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS⁺ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS⁺ cells are a unique renewable source of multipotent stem/progenitor cells.
Base Sequence ; Cell Differentiation ; Cell Lineage ; Cells, Cultured ; DNA Primers ; Gene Expression Profiling ; Gingiva ; cytology ; metabolism ; Humans ; Immunomagnetic Separation ; methods ; Immunophenotyping ; Real-Time Polymerase Chain Reaction

OBJECTIVETo investigate the expression of chemokine stromal cell-derived factor-1 (SDF-1) receptor CXCR4 in human gingival mesenchymal stem cells (GMSCs) and the migration potential of GMSCs stimulated with SDF-1.

METHODSHuman GMSCs were isolated by single-cell cloning method. Their cell surface markers were characterized by flow cytometry, and the rate of colony formation was evaluated. Differentiation assay was used to detect the differentiation potential of GMSCs. The expression of chemokine SDF-l receptor CXCR4 in GMSCs was detected by immunocytochemical staining. The chemotactic effect of SDF-1 on GMSCs was detected using a 24-multiwell Transwell cell culture chamber. The number of net migrated cells was counted in different microscope fields.

RESULTSHuman GMSCs possessed high self-renewal potential and formed single-cell colonies cultured in vitro. GMSCs expressed mesenchymal stem cells-associated markers CD44, CD73, CD90, CD105, and CD166, and the expression of hemopoietic stem cell surface markers CD14, CD34, and CD45 was negative. GMSCs differentiated into osteoblasts and adipocytes under defined culture conditions. The colony forming unit-fibroblastic for GMSCs was 21.4%/±2.8%. Immunocytochemical staining demonstrated that GMSCs expressed chemokine SDF-1 receptor CXCR4. The number of GMSCs migrating at concentrations of 100 ng.mL-1 and 200 ng.mL-1 of SDF-l in the Transwell cell culture chamber was significantly higher than that of the negative control (189.3±4.4, 164.6±4.9 cells/field vs. 47.8±2.5 cells/field, P<0.01). Treatment with the CXCR4 neutralizing antibody, an antagonist for CXCR4, significantly reduced the migratory effect compared with the negative controls (29.0±2.4 cells/field vs. 47.8±2.5 cells/field, P<0.01).

CONCLUSIONHuman GMSCs express chemokine SDF-l receptor CXCR4. SDF-1 may participate in regulating chemotaxis of human GMSCs. Results suggest that the migration induced by SDF-1 is mediated by CXCR4.

Adipocytes ; Cell Culture Techniques ; Cell Differentiation ; Chemokine CXCL12 ; metabolism ; Chemotaxis ; Flow Cytometry ; Gingiva ; physiology ; Humans ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; Receptors, CXCR4 ; Signal Transduction

OBJECTIVEThis paper aimed to determine the mRNA expression of osteoclast-related factors interleukin-6 (IL-6), interleukin-12 (IL-12) p35, IL-12p40, matrix metalloproteinase-9 (MMP-9), nuclear factor of activated T-cells cytoplasmic 1 (NFATcl), receptor activator of nuclear factor-KB (RANK), and tumor necrosis factor-α (TNF-α) mRNA in murine macrophages infected by a periodontitis patient's own tissue nucleic acid. Another aim was to investigate the effects of a periodontitis patient's own tissue nucleic acid on the differentiation of macrophages into osteoclasts.

METHODSInflammatory periodontal tissue samples of chronic periodontitis patients were taken during periodontal flap surgery, and healthy gingival tissue samples were taken from orthodontic patients during tooth extractions. Total RNA from periodontal tissue was extracted and reversely transcribed into cDNA and then cryo-preserved until further use. First, specific sequence oligodeoxynucleotide MT0I at a concentration of 1 µg · mL⁻¹ was added in murine macrophage RAW264.7, and the cells were incubated for 3 hours. Cells with PBS (1 µg · mL⁻¹) were used as negative controls. The inflammatory periodontal tissue cDNA and healthy periodontal tissue cDNA (1 µg · mL⁻¹) was added subsequently. There were four experimental groups: healthy periodontal tissue cDNA+ RAW264.7, inflammatory periodontal tissue cDNA+RAW264.7, MT01+healthy periodontal tissue cDNA+RAW264.7, and MT01+inflammatory periodontal tissue cDNA+RAW264.7. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of osteoclast-related factors IL-6, IL-12p35, IL-12p4O, MMP-9, NFATcl, RANK, and TNF-α mRNA after 3, 6, 12, and 24-hours.

RESULTSThe mRNA levels of osteoclast-related factors NFATc1, MMP-9, TNF-a, IL-6, IL-12p40, IL-12p35, and RANK in RAW264.7 were markedly upregulated with the treatment of periodontitis patient's own tissue nucleic acid. However, the mRNA expression of osteoclast-related factors was inhibited by use of an immunosuppressant MT01.

CONCLUSIONThe periodontitis patient's own tissue nucleic acid could promote the differentiation of murine macrophage into osteoclasts.

Animals ; Cell Differentiation ; Cytokines ; metabolism ; Gene Expression ; Gingiva ; Humans ; Interleukin-12 Subunit p40 ; Interleukin-6 ; Macrophages ; Matrix Metalloproteinase 9 ; Mice ; Osteoclasts ; metabolism ; Periodontitis ; RNA, Messenger ; Tumor Necrosis Factor-alpha

OBJECTIVEThis study investigates the immune status of T helper (Th) 17 cells in mice with periodontitis.

METHODSSeven-week-old C57BL/6 female mice were randomly divided into the control and periodontitis groups. Each group comprisedfour mice. Experimental periodontitis was induced in mice by oral infection with Porphyromonas gingivalis in the periodontitis group. Phosphate-buffered saline solution was used in the control group. All mice were sacrificed 4 weeks after the last P. gingivalis infection. CD4⁺retinoid-related orphan receptor (ROR) γτ⁺(Th17) cells were analyzed by flow cytometry. The protein expression of Th17 cell-related cytokine interleukin (IL)-17A was detected by enzyme-linked immunosorbentassay (ELISA).

RESULTSFlow cytometry showed that the percentage of CD4⁺RORγτ⁺(Thl7) cells in all CD4⁺ cells and the cell number in gingival tissues, cervical lymph nodes (CLNs), and the peripheral blood obviously increased (P < 0.01) in the periodontitis group. ELISA showed that compared with the control group, the periodontitis group exhibited an obvious increase in the protein expression of IL-17A (P < 0.05).

CONCLUSIONTh17 cell-mediated cell response is enhanced, and the gingival tissues, CLNs, and the peripheral blood are probably the main locations of Thl7 cell-mediated cell response during the development of periodontitis.

Alveolar Bone Loss ; Animals ; Cytokines ; Female ; Flow Cytometry ; Gingiva ; Interleukin-17 ; metabolism ; Mice ; Mice, Inbred C57BL ; Periodontitis ; metabolism ; Porphyromonas gingivalis ; Random Allocation ; T-Lymphocytes, Helper-Inducer