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

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate (CHX) on human gingival fibroblasts (HGFs), and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3-7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min (control group), 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8 (CCK-8) assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group (P>0.05). However, there were significant differences between all the other treated groups and the control group (P<0.05). When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.
Adolescent ; Adult ; Anti-Infective Agents, Local ; adverse effects ; toxicity ; Cell Survival ; drug effects ; Cells, Cultured ; Chlorhexidine ; adverse effects ; analogs & derivatives ; toxicity ; Fibroblasts ; drug effects ; Gingiva ; cytology ; Humans ; Plasma ; chemistry ; Root Canal Therapy ; instrumentation ; methods

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 whether signaling through Toll-like receptor-2 (TLR-2) can affect the expression of some cytokines in human gingival fibroblasts.

METHODSThe gingival fibroblasts were isolated and cultured in vivo, divided into blank control group, lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg) group and Escherichia coli (Ec) group. mRNA expression levels were measured by real-time polymerase chain reaction (PCR). The protein expression levels were detected by the enzyme linked immunosorbent assay (ELISA). The data was statistically analyzed by SPSS16.0 software package.

RESULTSLPS from Pg could stimulate the expression of interleukin (IL)-6 and leukemia inhibitory factor (LIF) mRNA and protein, which reached the peak (5.87 ± 0.83) at 10 h, and the expression level increased with the increase of the Pg concentration. IL-11 or oncostatin-M (OSM) mRNA expression was not affected by LPS. After treated with Pg for 48 h, the protein expression of IL-6 and LIF was up-regulated, (962 ± 57) ng/L and (47 ± 18) ng/L respectively.

CONCLUSIONSSignaling through TLR-2 controls the expression of cytokines of IL-6 family in human gingival fibroblasts.

Adolescent ; Adult ; Cells, Cultured ; Child ; Dose-Response Relationship, Drug ; Escherichia coli ; chemistry ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Humans ; Interleukin-11 ; genetics ; metabolism ; Interleukin-6 ; genetics ; metabolism ; Leukemia Inhibitory Factor ; genetics ; metabolism ; Lipopeptides ; pharmacology ; Lipopolysaccharides ; administration & dosage ; isolation & purification ; pharmacology ; Oncostatin M ; genetics ; metabolism ; Porphyromonas gingivalis ; chemistry ; RNA, Messenger ; metabolism ; Signal Transduction ; Toll-Like Receptor 2 ; agonists ; Young Adult

OBJECTIVETo investigate the effect of alloy leaching liquor of four different types of base metal alloy on the expression of prostaglandin E(2) (PGE(2)) and cyclo-oxygenase-2(COX-2) by human gingival fibroblast(HGF) in vitro.

METHODSNi-Cr, Co-Cr, pure Ti and Au ceramic alloys were incubated in Dulbecco's modified Eagle's medium (DMEM) to prepare alloy leaching liquor, and then added in HGF medium. DMEM was prepared as negative control. Aliquots were taken from exposed media after 1, 6, 12, 24 h. Assays for PGE(2) were carried out by enzyme-linked immunosorbent assay (ELISA).

RESULTSIn 6, 12, 24 h, the expression of PGE(2) in Ni-Cr and Co-Cr alloy groups (Ni-Cr: 45.568 ± 0.926, 60.538 ± 0.988, 73.754 ± 0.507; Co-Cr: 40.496 ± 0.693, 53.216 ± 0.327, 65.470 ± 1.086) were significantly higher than those in other experimental groups (Ti: 31.564 ± 0.719, 31.998 ± 0.856, 32.066 ± 0.513; Au alloy: 31.540 ± 0.821, 31.136 ± 0.518, 31.340 ± 0.443) and control group (31.122 ± 0.642, 31.230 ± 0.634, 30.980 ± 0.746) (P < 0.05). No significant difference were found in the expression of PGE(2) among pure Ti, Au alloy groups and the control group (P > 0.05). Immunofluorescence showed dark and uniform COX-2 stain in Ni-Cr and Co-Cr alloy groups, while in pure Ti group, Au alloy group, and negative control group shallow and uneven distribution of COX-2 stain were observed.

CONCLUSIONSOur findings suggested that pure Ti and Au alloy did not cause elevated PGE(2) and COX-2 release from HGF. However, Ni-Cr and Co-Cr alloy caused increase in PGE(2) and COX-2 levels.

Cells, Cultured ; Chromium Alloys ; adverse effects ; Cyclooxygenase 2 ; metabolism ; Dental Alloys ; adverse effects ; Dinoprostone ; metabolism ; Fibroblasts ; cytology ; metabolism ; Gingiva ; cytology ; Gold Alloys ; adverse effects ; Humans ; Titanium ; adverse effects

Animals ; Bone Regeneration ; physiology ; Cell Differentiation ; Dental Enamel Proteins ; pharmacology ; Dental Pulp ; cytology ; Fibroblasts ; cytology ; Gingiva ; cytology ; Guided Tissue Regeneration, Periodontal ; methods ; Humans ; Induced Pluripotent Stem Cells ; cytology ; physiology ; Mouth Mucosa ; cytology ; Periodontal Ligament ; cytology ; Tissue Engineering ; methods

OBJECTIVETo construct the co-culture models of salivarya denoid cystic carcinoma (SACC) cells and dorsal root ganglia (DRG) of chickens and investigate the promotive effects of SACC on neural tissue.

METHODSGlass-base culture dish was adopted to construct co-culture model of SACC-83 cells and DRG. SACC-83 cells were seeded in the medium pore with DRG around them. Outgrowth of neuronal processes was observed. Then DRG was cultured in the conditioned medium of SACC-83, with the groups of conditioned medium of MC3T3-E1 and HGF, the group of cell lysis buffer, the groups of serum-free medium and serum-plus medium as the controls. Outgrowth of neuronal processes was also recorded and compared with control groups.

RESULTSIn the co-culture model of tumor and neuronal tissue, SACC-83 cells produced a suitable microenvironment in which neuronal processes remarkably grow. Neuronal processes of most DRG displayed growth tendency toward SACC. The group of conditioned medium from SACC-83 manifested obvious promotive effects on DRG.

CONCLUSIONSCo-culture model of tumor and neuronal tissue was successfully constructed, with which the promotive effects of tumor on outgrowth of neuronal processes could be observed. So hypothesized that SACC could secrete some neurotrophic factors to guide peripheral nerves gemmating and to trigger the cascade of the neural invasion in succession.

Animals ; Carcinoma, Adenoid Cystic ; pathology ; Cell Line ; Cell Line, Tumor ; Chickens ; Coculture Techniques ; Culture Media ; Ganglia, Spinal ; growth & development ; Gingiva ; cytology ; Humans ; Osteoblasts ; cytology ; Salivary Gland Neoplasms ; pathology

OBJECTIVETo investigate the effect of cyclosporin A (CsA) and tumor necrosis factor-α (TNF-α) on cell proliferation of cultured human gingival fibroblasts (GF), and the relationship between gingival inflammation and drug-induced gingival overgrowth.

METHODSHuman GF were cultured in vitro using tissue culture method. then cells from the 4 - 8 th passage were used in the experiment. The cells were cultured and incubated with various concentrations of CsA and TNF-α (A: blank group, B1: 10 µg/L CsA, B2: 50 µg/L CsA, B3: 250 µg/L CsA, B4: 1250 µg/L CsA, C: 5 µg/L TNF-α, D1: 10 µg/L CsA + 5 µg/L TNF-α, D2: 50 µg/L CsA + 5 µg/L TNF-α, D3: 250 µg/L CsA + 5 µg/L TNF-α, D4: 1250 µg/L CsA + 5 µg/L TNF-α) solution for 3, 5 and 7 days. Methyl thiazolyl tetrazolium assay was used to evaluate the cell proliferation in the culture meidiun.

RESULTSThe proliferation of fibroblasts was inhibited when exposed to different concentration of CsA and A value decreased. There was no significant difference between group B1, B2, B3 and the control group, while the A value of group B4 was significantly higher than that of control group (P < 0.01). Fibroblast proliferation was significantly increased while cultured with 5 µg/L TNF-α. A value increased (P < 0.01). When exposed to CsA + TNF-α, A value of group D1, D2, D3 was much higher than that of group A, but was lower than that of group C (P < 0.05). Cell proliferation in group D4 was significantly increased, and significantly different with that in group C (P < 0.01).

CONCLUSIONSCsA did not stimulate the cell proliferation, and high concentration of CsA inhibited cell proliferation. TNF-α can stimulate the cell proliferation. High-concentration CsA + TNF-α can enhance the fibroblast proliferation, which suggests that CsA in certain concentration have amplification effect on TNF-α to stimulate fibroblast proliferation.

Adult ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclosporine ; adverse effects ; pharmacology ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; Gingiva ; cytology ; Gingival Overgrowth ; chemically induced ; Humans ; Immunosuppressive Agents ; adverse effects ; pharmacology ; Tumor Necrosis Factor-alpha ; adverse effects ; pharmacology