OBJECTIVE: The removal of impacted third molars by surgery may occur with a series of complications, whereas limited information about the postoperative pathogenesis is available. The objective of this study is to identify changes in gene expression after flap surgical removal of impacted third molars and provide potential information to reduce postoperative complications.
METHODS: The gingival tissues of twenty patients with flap surgical removal of impacted third molars and twenty healthy volunteers were collected for gene expression testing. The collected gingival tissues were used RNA sequencing technology and quantitative real-time PCR validation was performed. DEG was mapped to protein databases such as GO and KEGG for functional annotation and, based on annotation information, for mining of differential expression genes in patients with mpacted third molars.
RESULTS: A total of 555 genes were differentially expressed. Among the top up-regulated genes, HLA-DRB4, CCL20, and CXCL8 were strongly associated with immune response and signal transduction. Among the top down-regulated genes, SPRR2B, CLDN17, LCE3D and LCE3E were related to keratinocyte differentiation, IFITM5, and BGLAP were related to bone mineralization, UGT2B17 is associated with susceptibility to osteoporosis. KEGG results showed that the DEGs were related to multiple disease-related pathways.
CONCLUSIONS: This first transcriptome analysis of gingival tissues from patients with surgical removal of impacted third molars provides new insights into postoperative genetic changes. The results may establish a basis for future research on minimizing the incidence of complications after flap-treated third molars.

The purpose of this study was to determine potential metabolic biomarkers and therapeutic drugs in the gingival tissue of individuals with periodontitis. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) were used to analyze the gingival tissue samples from 20 patients with severe periodontitis and 20 healthy controls. Differential metabolites were identified using variable important in projection (VIP) values from the orthogonal partial least squares discrimination analysis (OPLS-DA) model and then verified for significance between groups using a two-tailed Student\'s t test. In total, 65 metabolites were enriched in 33 metabolic pathways, with 40 showing a significant increase and 25 expressing a significant decrease. In addition, it was found that patients with severe periodontitis have abnormalities in metabolic pathways, such as glucose metabolism, purine metabolism, amino acid metabolism, and so on. Furthermore, based on a multidimensional analysis, 12 different metabolites may be the potential biomarkers of severe periodontitis. The experiment\'s raw data have been uploaded to the MetaboLights database, and the project number is MTBLS8357. Moreover, osteogenesis differentiation characteristics were detected in the selected metabolites. The findings may provide a basis for the study of diagnostic biomarkers and therapeutic metabolites in severe periodontitis.

Diabetes and periodontitis are prevalent diseases that considerably impact global economy and diabetes is a major risk factor of periodontitis. Mitochondrial dynamic alterations are involved in many diseases including diabetes and this study aims to evaluate their relevance with diabetes aggravated periodontitis. Sixty mice are randomly divided into 4 groups: control, periodontitis, diabetes and diabetic periodontitis. Periodontitis severity is evaluated by alveolar bone loss, inflammation and oxidative stress status. Mitochondrial structural and functional defects are evaluated by the mitochondrial fission/fusion events, mitochondrial reactive oxygen species (ROS) accumulation, complex activities and adenosine triphosphate (ATP) production. Advanced glycation end product (AGE) and Porphyromonas gingivalis are closely related to periodontitis occurrence and development. Human gingival fibroblast cells (HGF-1) are used to investigate the AGE role and lipopolysaccharide (LPS) from Porphyromonas gingivalis (P-LPS) in aggravating diabetic periodontitis by mitochondrial dynamic and function alterations. In vivo, diabetic mice with periodontitis show severe bone loss, increased inflammation and oxidative stress accumulation. Among mice with periodontitis, diabetic mice show worse mitochondrial dynamic perturbations than lean mice, along with fusion protein levels inducing more mitochondrial fission in gingival tissue. In vitro, AGEs and P-LPS co-treatment causes severe.

Although Vanins are closely related to neutrophil regulation and response to oxidative stress, and play essential roles in inflammatory diseases with clinical significance, their contribution to periodontitis remains to be determined. This research was designed to assess the expression of Vanins in human gingiva, and to define the relationship between Vanins and periodontitis.
Forty-eight patients with periodontitis and forty-two periodontal healthy individuals were enrolled for gingival tissue sample collection. Expression levels of VNN1, VNN2 and VNN3 were evaluated by RT-qPCR and validated in datasets GSE10334 and GSE16134. Western blot and immunohistochemistry identified specific proteins within gingiva. The histopathological changes in gingival sections were investigated using HE staining. Correlations between Vanins and clinical parameters, PD and CAL; between Vanins and inflammation, IL1B; and between Vanins and MPO in periodontitis were investigated by Spearman\'s correlation analysis respectively. Associations between VNN2 and indicators of neutrophil adherence and migration were further validated in two datasets.
Vanins were at higher concentrations in diseased gingival tissues in both RT-qPCR and dataset analysis (p < 0.01). Assessment using western blot and immunohistochemistry presented significant upregulations of VNN1 and VNN2 in periodontitis (p < 0.05). The higher expression levels of Vanins, the larger the observed periodontal parameters PD and CAL (p < 0.05), and IL1B (p < 0.001). Moreover, positive correlations existed between VNN2 and MPO, and between VNN2 and neutrophil-related indicators.
Our study demonstrated upregulation of Vanins in periodontitis and the potential contribution of VNN2 to periodontitis through neutrophils-related pathological processes.

UNASSIGNED: Although some studies have taken an interest in the participation of platelets in periodontitis, so far, we know very little about the roles of platelets in periodontitis. The objective of this study is to explore the involvement of platelets in the development of experimental periodontitis in mice.
UNASSIGNED: Twenty C57BL/6 male mice were used for this study. Experimental periodontitis models of mice were constructed by ligating for 1, 3, 7, and 14 days, respectively. Morphological changes in the alveolar bone were assessed by micro-computed tomography (Micro-CT). The gingival crevicular fluid samples of ligation sites were collected and stained by immunocytochemistry. Immunohistochemistry was used to detect platelets infiltration in gingival tissues of mice.
UNASSIGNED: The results of Micro-CT showed that with the extension of ligation time, alveolar bone resorption increased, suggesting that the experimental periodontitis models were established. Immunochemical staining showed that there were almost no platelets in the gingival crevicular fluid of mice ligated for 1 and 3 days. And at 7 and 14 days of ligation, a large number of platelets were present in the gingival crevicular fluid and formed complexes with neutrophils. And with the extension of ligation time, the extent of platelet infiltration increased in mice gingival tissues.
UNASSIGNED: Platelets were infiltrated increasedly in the gingival sulcus and gingival tissues following the experimental time, and may participate in the development of mouse experimental periodontitis.

Objective: Chronic periodontitis is a bone-destructive disease affecting periodontal support structures. Although leptin has a protective effect against periodontitis, the underlying mechanism remains unclear. Therefore, this study aimed to investigate the possible role of leptin by examining its relationship with OPG and RANKL in human gingival tissues obtained from patients with chronic periodontitis. Method: Twenty-two patients with chronic periodontitis were enrolled (10 with moderate periodontitis and 12 with severe periodontitis) in the experimental group, and 12 healthy individuals were enrolled in the control group. Gingival tissue samples were collected, and the protein levels and localization of leptin, OPG, and RANKL were studied using immunohistochemistry (IHC). The staining intensities of leptin, OPG, and RANKL were correlated with the periodontal clinical index. Moreover, real-time quantitative PCR (RT-qPCR) was used to determine OPG and RANKL mRNA levels in gingival fibroblasts stimulated with gradient concentrations of leptin protein in vitro. Result: Leptin, OPG, and RANKL were located in the cytoplasm of gingival epithelial cells and the connective tissue. Leptin was widely and significantly expressed in the control group, whereas it was lightly stained in the severe group. RANKL was lightly stained in the control group, whereas it was widely and significantly expressed in the severe group. The control and the moderate groups had similar OPG levels, which were significantly higher than that in the severe group. Leptin was positively correlated with OPG(r = 0.905, p < 0.01) and negatively correlated with RANKL (r = -0.635, p < 0.01). In vitro low concentrations of leptin led to an increased OPG/RANKL mRNA ratio, whereas the opposite effect was observed at high concentrations. Conclusion: Leptin can regulate OPG and RANKL expression in gingival fibroblasts and may thus play a role in the development of chronic periodontitis by modulating the OPG/RANKL ratio.

This study aimed to investigate the relationship between inflammation-related T-helper cell polarization and the receptor activator for nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio, which is associated with bone resorption or remodeling of chronic periodontitis patients. Gingival crevicular fluid (GCF) and gingival tissues were obtained from periodontally healthy individuals (PH group) and chronic periodontitis patients (CP group). The GCF levels of IFN-γ, IL-4, IL-17, and IL-10 linked to T-helper cell polarization toward the Th1, Th2, Th17, and Treg phenotypes, respectively, were determined by ELISA. The expression levels of these cytokines and the polarized T-helper cells in gingival tissues were assessed through immunohistochemical and immunofluorescence assays. In addition, the RANKL and OPG expression levels in gingival tissues were detected by immunohistochemical assays, and linear regression analysis was used to identify the potential relationship between T-helper cell polarization and the RANKL/OPG ratio. In total, 22 individuals and 35 patients were enrolled in the present study. In both GCF and gingival tissues, increased levels of IL-17 and the decreased levels of IL-4 and IL-10 were observed in the CP group. When polarized T-helper cells were identified in gingival tissues, more Th1 and Th17 cells were found in the CP group, whereas more Th2 and Treg cells were found in the PH group. Although there was no significant difference in OPG expression between the two groups, the RANKL/OPG ratio in the CP group was higher than that in the PH group. The linear regression analysis showed that the presence of more Th1 and Th17 cells correlated with a higher RANKL/OPG ratio, whereas the presence of more Th2 cells correlated with a lower RANKL/OPG ratio. Th1 and Th17 cells are positively correlated and Th2 cells are negatively correlated with the RANKL/OPG ratio. Our data suggest that T-helper cell polarization is closely linked to the RANKL/OPG ratio in gingival tissues from chronic periodontitis patients.

OBJECTIVE: This study aimed to discover the distinctive MicroRNAs (miRNA) functioning in the pathogenesis of periodontal inflammation, which might be potential therapy targets of chronic periodontitis.
METHODS: miRNA profiles of human inflamed gingival tissue from three previous microarrays were re-analysed. Gingival tissues were collected for the validation of overlapping miRNAs, and a network was constructed to show regulatory connection between overlapping miRNAs and periodontitis-associated target genes. Potential miRNAs were screened based on their expression levels and predicted target genes. Correlation analysis and binding site prediction were conducted to reveal the relationship between the potential miRNAs and their target genes.
RESULTS: miR-144-5p, found to be upregulated in all three studies, showed the greatest upregulation (P < 0.0001). Another 16 miRNAs (10 upregulated and six downregulated) overlapped between any two of the three studies. All overlapping miRNAs had expected expression levels except for miR-203 during validation. Ten miRNAs (six upregulated and four downregulated) were found to have periodontal inflammation-associated targets. Cyclooxygenase 2 (COX2) and interleukin-17F (IL17F), predicted target genes of upregulated miR-144-5p, showed significant decreases and were negatively correlated with miR-144-5p in the periodontitis group (r = -0.742 for COX2, r = -0.615 for IL17F).
CONCLUSIONS: This re-analysis of miRNA signatures has implied the potential regulatory mechanism of miR-144-5p and its potential for exploring alternative therapeutic approaches, especially those that use miRNA delivery systems to treat chronic periodontitis. Nevertheless, further study based on larger sample size and homogenous cells is needed to reveal the exact roles of miRNAs in chronic periodontitis.

OBJECTIVE: Although accumulating evidence indicates that macrophages are central players in the destructive and reparative phases of periodontal disease, their polarization states at different stages of periodontal inflammation remain unclear.
METHODS: We collected gingival biopsies from patients with chronic periodontitis (P group), gingivitis (G group), or periodontally healthy individuals (H group). Polarized macrophages were identified through immunofluorescence. M1- and M2-related cytokines were detected by immunohistochemistry.
RESULTS: Compared with the H group, the P group had more M1 cells (higher M1/M2 ratio) and significantly higher TNF-α, IFN-γ, IL-6, and IL-12 levels. Although the G group also exhibited higher TNF-α and IL-12 levels than the H group, they had similar M1/M2 ratios. The M1/M2 ratio and IFN-γ and IL-6 levels were significantly higher in the P than the G group. Among M2-related cytokines, IL-4 levels were significantly higher in the G than the H group. The M1/M2 ratio was positively correlated with clinical probing depth (PD), and both were positively correlated with IFN-γ and IL-6. PD was negatively correlated with IL-4.
CONCLUSIONS: Macrophage polarization in gingival tissue may be responsible for the development and progression of inflammation-induced tissue destruction, and modulating macrophage function may be a potential strategy for periodontal disease management.

OBJECTIVE: This study aimed to evaluate IL-10 producing CD19+ B cells and to examine the correlation between these cells and the expression levels of IL-1β, TNF-α, RANKL, and IL-10 cytokines in the gingival tissues of individuals with and without chronic periodontitis.
METHODS: Data were obtained from 20 patients with chronic periodontitis and 10 healthy controls. The gingival samples were analyzed by immunofluorescence, while real-time PCR and enzyme-linked immunosorbent assays were performed to determine cytokine levels.
RESULTS: The number of IL-10 producing CD19+ B cells and the expression levels of IL-10 were significantly higher in the inflamed gingival tissues than in the healthy tissues. A positive correlation between the expression levels of IL-10 and the number of IL-10 producing CD19+ B cells were observed. IL-1β, TNF-α, and RANKL expression levels were significantly elevated in diseased gingivae compared to healthy tissues, and there was a positive correlation between the expression levels of these pro-inflammatory cytokines and the number of IL-10 producing CD19+ B cells.
CONCLUSIONS: While IL-10 producing CD19+ B cells are present in the gingival tissues of patients with periodontal disease and of those with a healthy periodontium, the diseased gingival tissues had a much greater number of these cells than the healthy. The mRNA and protein levels of IL-10, IL-1β, and RANKL, as well as mRNA levels of TNF-α, were positively correlated with the number of IL-10 producing CD19+ B cells, which highlights the importance of these factors in the development and progression of periodontitis.