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

Cellular fibronectin (cFn) is a type of bioactive non-collagen glycoprotein regarded as the main substance used to maintain periodontal attachment. The content of cFn in some specific sites can reflect the progress of periodontitis or peri-implantitis. This study aims to evaluate the expression of cFn messenger RNA (mRNA) in tissues of adult periodontitis and peri-implantitis by real-time fluorescent quantitative polymerase chain reaction (PCR) and to determine its clinical significance. A total of 30 patients were divided into three groups of 10: healthy, adult periodontitis and peri-implantitis. Periodontal tissue biopsies (1 mm×1 mm×1 mm) from each patient were frozen in liquid nitrogen. Total RNA was extracted from these tissues, and the content, purity and integrity were detected. Specific primers were designed according to the sequence, and the mRNA expression levels of cellular fibronectin were detected by real-time PCR. The purity and integrity of the extracted total RNA were both high, and the specificity of amplified genes was very high with no other pollution. The mRNA expression of cFn in the adult periodontitis group (1.526±0.441) was lower than that in the healthy group (3.253±0.736). However, the mRNA expression of cFn in the peri-implantitis group (3.965±0.537) was significantly higher than that in the healthy group. The difference revealed that although both processes were destructive inflammatory reactions in the periodontium, the pathomechanisms were different and the variation started from the transcription level of the cFn gene.
Adult ; Alveolar Bone Loss ; metabolism ; Female ; Fibronectins ; analysis ; genetics ; Gingiva ; metabolism ; Humans ; Male ; Middle Aged ; Peri-Implantitis ; metabolism ; Periodontal Attachment Loss ; metabolism ; Periodontal Index ; Periodontal Pocket ; metabolism ; Periodontitis ; metabolism ; Periodontium ; metabolism ; RNA, Messenger ; analysis ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; genetics ; 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

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