Untargeted Metabolomics Analysis of Gingival Tissue in Patients with Severe Periodontitis.

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.

Immunologic burden links periodontitis to acute coronary syndrome: levels of CD4 + and CD8 + T cells in gingival granulation tissue.

OBJECTIVE: To investigate the proportional variation of macrophage and T-lymphocytes subpopulations in acute coronary syndrome (ACS) patients, its association with periodontitis (P), and to compare with control individuals. SUBJECTS AND METHODS: Three groups of subjects participated: one group consisted of 17 ACS patients with P (ACS + P), another group consisted of 22 no ACS + P patients, and a control group consisted of 23 participants with gingivitis (no ACS + G). Macrophage, CD4 + , and CD8 + T-lymphocytes and CD4 + /CD8 + ratio values in gingival tissue were determined histometrically. RESULTS: Significant differences were found among three groups regarding the mean number of macrophage (no ACS + P > ACS + P > no ACS + G; p < 0.05) and CD8 + T-lymphocytes (no ACS + P > ACS + P > no ACS + G; p < 0.05). Significant variations were observed between the groups both CD4 + T-lymphocytes densities (ACS + P > no ACS + P and ACS + P > no ACS + G; p < 0.05) and CD4 + / CD8 + ratio (no ACS + P < no ACS + G and ACS + P < no ACS + G; p < 0.05). CONCLUSIONS: The increased number of CD8 + T-lymphocytes in both group ACS + P and group no ACS + P resulted in a reduction of the CD4 + /CD8 + ratio in gingival tissue when compared with no ACS + G group. CLINICAL RELEVANCE: The decrease of CD4 + /CD8 + ratio in gingival tissue reflects periodontitis and may be associated with severe adverse outcomes in people with ACS.

Increased of IL-18 levels are associated with periodontitis: a systematic review and meta-analysis.

BACKGROUND: The presence of a polymicrobial dysbiotic film in direct and constant contact with periodontal tissues initiates the host immune response. Interleukin 18 (IL-18) triggers up-regulates the production of other proinflammatory cytokines (TNF-α, IL-1ß, IL-6), creating a vicious cycle that expands the inflammatory and destructive process in the periodontal tissue. A systematic review and meta-analysis was carried out with the main propose to investigate IL-18 expression in different biological samples from subjects with chronic periodontitis. METHODS: The protocol followed PRISMA guidelines and was registered in Open Science Framework (OSF): https://doi.org/10.17605/OSF.IO/BS9GM . A digital search was conducted in the databases PubMed, ScienceDirect, Google Scholar, Web of Science and Dentistry & Oral Sciences Source databases were consulted from March 15th, 2005 to February 10th, 2023. Study quality was assessed using the JBI tool for cross-sectional studies and clinical trials. A meta-analysis was performed using a random/fixed effects model to evaluate the concentration of IL-18 in serum, plasma, saliva, gingival tissue and GCF of exposure group compared to control group. RESULTS: The search strategy provided a total of 3,156 articles, of which 18 investigations met the inclusion criteria and 15 articles were quantitatively analyzed. The total number of patients studied was 1,275 (682 cases and 593 controls). The meta-analysis revealed significantly elevated IL-18 levels of serum, saliva and GCF of subjects with chronic periodontitis compared to healthy subjects (Serum: SMD = 62.73, 95%CI: 25.43-100.03, Z = 3.29, p = 0.001*; Saliva: SMD = 243.63, 95%CI: 8.68-478.59, Z = 2.03, p = 0.042*; GCF: SMD = 150.26, 95%CI: 56.86-243.66, Z = 3.15, p = 0.02*). CONCLUSION: IL-18 levels in serum, saliva and GCF could have the potential to be used as complementary diagnostic tools to the clinical and radiographic parameters in subjects with periodontitis.

Magnolia kobus Extract Suppresses Porphyromonas gingivalis LPS-Induced Proinflammatory Cytokine and MMP Expression in HGF-1 Cells and Regulates Osteoclastogenesis in RANKL-Stimulated RAW264.7 Cells.

Clinical prevention is of utmost importance for the management of periodontal diseases. Periodontal disease starts with an inflammatory response in the gingival tissue, and results in alveolar bone destruction and subsequent tooth loss. This study aimed to confirm the anti-periodontitis effects of MKE. To confirm this, we studied its mechanism of action using qPCR and WB in LPS-treated HGF-1 cells and RANKL-induced osteoclasts. We found that MKE suppressed proinflammatory cytokine protein expression by inhibiting the TLR4/NF-κB pathway in LPS-PG-induced HGF-1 cells and blocking ECM degradation by regulating the expression of TIMPs and MMPs. We also confirmed that TRAP activity and multinucleated cell formation were reduced in RANKL-stimulated osteoclasts after exposure to MKE. These results were confirmed by inhibiting TRAF6/MAPK expression, which led to the suppression of NFATc1, CTSK, TRAP, and MMP expression at the gene and protein levels. Our results confirmed that MKE is a promising candidate for the management of periodontal disease based on its anti-inflammatory effects and inhibition of ECM degradation and osteoclastogenesis.

Elevated Foxo3a and Fas-ligand expression in human periapical granulomas as a potential treatment target.

OBJECTIVE: Periapical granuloma is a common periodontitis type involving chronic inflammation; however, the efficacy of current therapies is limited. Its molecular pathogenesis also remains obscure. Forkhead box transcription factor class o3a (Foxo3a) and Fas-ligand (FasL) are associated with chronic inflammation. Therefore, in this study, we aimed to clarify the roles of Foxo3a and FasL in periapical granuloma pathophysiology. SUBJECTS AND METHODS: Periapical lesions were obtained from patients during endodontic surgery and tooth extraction; those diagnosed with periapical granulomas using haematoxylin and eosin staining were further analysed. Immunohistochemical analysis was performed for Foxo3a and FasL, and real-time polymerase chain reaction was performed for FOXO3A, FASL and interleukin (IL)-1ß. Healthy gingival tissues were also examined as controls. RESULTS: Neutrophils, lymphocytes and plasma cells in the periapical granulomas, but not healthy tissues, expressed Foxo3a. Dual-colour immunofluorescence imaging revealed Foxo3a and FasL co-expression in leukocytes. FOXO3A, FASL and IL-1ß mRNA levels in healthy gingival tissues were significantly lower than those in the periapical granulomas. Additionally, FOXO3A and IL-1ß expressions were negatively correlated. CONCLUSIONS: Phosphorylated Foxo3a may reduce IL-1ß release by inhibiting apoptosis through FasL in periapical periodontitis and prevent exacerbation. Thus, Foxo3a is a potential therapeutic agent for periapical periodontitis.

Expression of Wnt signaling agonists and antagonists in periodontitis and healthy subjects, before and after non-surgical periodontal treatment: A systematic review.

Periodontitis is a preventable and treatable multifactorial chronic inflammatory disease that can lead to irreversible periodontal destruction and tooth loss. Wnt signaling and its regulators play an important role in periodontal inflammation, destruction, regeneration, and reconstruction. This systematic review aimed at investigating the involvement of Wnt signaling agonists and antagonists in periodontitis and healthy subjects, before and after periodontal treatment. Electronic searches were carried out using MEDLINE/PubMed, EMBASE, and Cochrane Library databases in addition to hand searches. Studies having different designs assessing the levels of Wnt signaling antagonist and agonist levels in gingival crevicular fluid, serum, and tissue in patients diagnosed with periodontitis or gingivitis, compared with healthy individuals were included. In addition, studies compared these levels in periodontitis patients before and after non-surgical periodontal therapy were also eligible. Sixteen studies met the eligibility criteria. Sclerostin (SOST) has been mainly investigated in the literature (8 publications). Sclerostin (5 studies), Wnt-5a (2 studies), secreted frizzled-related protein 1 (SFRP1) (3 studies), and ß-catenin (3 studies) show increased levels in periodontitis compared with periodontal health. Strong correlations between marker levels and periodontal clinical parameters were identified for SOST (5 studies), SFRP1 (2 studies), and ß-catenin (2 studies). SOST (3 studies) and SFRP1 (1 study) levels significantly decrease following non-surgical periodontal treatment. The present systematic review demonstrated an association between Wnt signaling agonist and antagonist levels and periodontitis. Wnt agonists and antagonists may serve as valuable diagnostic and prognostic markers for periodontitis onset and progression. Further case-control and longitudinal studies should be conducted for different Wnt signaling agonists and antagonists.

The Structure and Function of Next-Generation Gingival Graft Substitutes-A Perspective on Multilayer Electrospun Constructs with Consideration of Vascularization.

There is a shortage of suitable tissue-engineered solutions for gingival recession, a soft tissue defect of the oral cavity. Autologous tissue grafts lead to an increase in morbidity due to complications at the donor site. Although material substitutes are available on the market, their development is early, and work to produce more functional material substitutes is underway. The latter materials along with newly conceived tissue-engineered substitutes must maintain volumetric form over time and have advantageous mechanical and biological characteristics facilitating the regeneration of functional gingival tissue. This review conveys a comprehensive and timely perspective to provide insight towards future work in the field, by linking the structure (specifically multilayered systems) and function of electrospun material-based approaches for gingival tissue engineering and regeneration. Electrospun material composites are reviewed alongside existing commercial material substitutes', looking at current advantages and disadvantages. The importance of implementing physiologically relevant degradation profiles and mechanical properties into the design of material substitutes is presented and discussed. Further, given that the broader tissue engineering field has moved towards the use of pre-seeded scaffolds, a review of promising cell options, for generating tissue-engineered autologous gingival grafts from electrospun scaffolds is presented and their potential utility and limitations are discussed.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Upregulated Vanins and their potential contribution to periodontitis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our study demonstrated upregulation of Vanins in periodontitis and the potential contribution of VNN2 to periodontitis through neutrophils-related pathological processes.

Leptin regulates OPG and RANKL expression in Gingival Fibroblasts and Tissues of Chronic Periodontitis Patients.

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.

Correlations between Width of Keratinized Gingiva and Supracrestal Gingival Tissues Dimensions: A Retrospective Clinical Study.

AIM: The aim of this retrospective study is to assess potential correlation between the width of the keratinized gingiva (KG), and the dimensions of the supracrestal gingival tissue (SGT) components. MATERIALS AND METHODS: On the sample of 259 teeth of 79 patients, the following measurements were collected: width of KG, sulcus depth (SD), SGT, and biological width (BW) dimensions; separate correlations between measured elements were computed for males and females, for anterior and posterior, and for maxillary and mandibular teeth separately. RESULTS: Correlations between buccal KG and BW were present only for the upper anterior teeth and were nonsignificant in the female subsample, whereas the correlation between lingual KG and SGT were present only in females. Additionally, correlations between buccal KG and SD were present in upper anterior teeth only and were absent in the male subgroup. CONCLUSION: The width of the KG cannot routinely be used as an indicator for the dimensions of the SGT components. CLINICAL SIGNIFICANCE: While the width of the KG can hardly be a useful indicator in upper anterior teeth, probing depth and bone sounding prior to prosthetic rehabilitation remains an essential tool to ensure tissue preservation.

Pressure Cycling Technology Assisted Mass Spectrometric Quantification of Gingival Tissue Reveals Proteome Dynamics during the Initiation and Progression of Inflammatory Periodontal Disease.

Understanding the progression of periodontal tissue destruction is at the forefront of periodontal research. The authors aimed to capture the dynamics of gingival tissue proteome during the initiation and progression of experimental (ligature-induced) periodontitis in mice. Pressure cycling technology (PCT), a recently developed platform that uses ultra-high pressure to disrupt tissues, is utilized to achieve efficient and reproducible protein extraction from ultra-small amounts of gingival tissues in combination with liquid chromatography-tandem mass spectrometry (MS). The MS data are processed using Progenesis QI and the regulated proteins are subjected to METACORE, STRING, and WebGestalt for functional enrichment analysis. A total of 1614 proteins with ≥2 peptides are quantified with an estimated protein false discovery rate of 0.06%. Unsupervised clustering analysis shows that the gingival tissue protein abundance is mainly dependent on the periodontitis progression stage. Gene ontology enrichment analysis reveals an overrepresentation in innate immune regulation (e.g., neutrophil-mediated immunity and antimicrobial peptides), signal transduction (e.g., integrin signaling), and homeostasis processes (e.g., platelet activation and aggregation). In conclusion, a PCT-assisted label-free quantitative proteomics workflow that allowed cataloging the deepest gingival tissue proteome on a rapid timescale and provided novel mechanistic insights into host perturbation during periodontitis progression is applied.

JNK Signaling as a Key Modulator of Soft Connective Tissue Physiology, Pathology, and Healing.

In healthy individuals, the healing of soft tissues such as skin after pathological insult or post injury follows a relatively predictable and defined series of cell and molecular processes to restore tissue architecture and function(s). Healing progresses through the phases of hemostasis, inflammation, proliferation, remodeling, and concomitant with re-epithelialization restores barrier function. Soft tissue healing is achieved through the spatiotemporal interplay of multiple different cell types including neutrophils, monocytes/macrophages, fibroblasts, endothelial cells/pericytes, and keratinocytes. Expressed in most cell types, c-Jun N-terminal kinases (JNK) are signaling molecules associated with the regulation of several cellular processes involved in soft tissue wound healing and in response to cellular stress. A member of the mitogen-activated protein kinase family (MAPK), JNKs have been implicated in the regulation of inflammatory cell phenotype, as well as fibroblast, stem/progenitor cell, and epithelial cell biology. In this review, we discuss our understanding of JNKs in the regulation of cell behaviors related to tissue injury, pathology, and wound healing of soft tissues. Using models as diverse as Drosophila, mice, rats, as well as human tissues, research is now defining important, but sometimes conflicting roles for JNKs in the regulation of multiple molecular processes in multiple different cell types central to wound healing processes. In this review, we focus specifically on the role of JNKs in the regulation of cell behavior in the healing of skin, cornea, tendon, gingiva, and dental pulp tissues. We conclude that while parallels can be drawn between some JNK activities and the control of cell behavior in healing, the roles of JNK can also be very specific modes of action depending on the tissue and the phase of healing.

Human gingiva-derived mesenchymal stem cells alleviate inflammatory bowel disease via IL-10 signalling-dependent modulation of immune cells.

Current evidence indicates that inflammatory bowel disease (IBD) is caused primarily by impaired mucosal immunity, resulting in an imbalance between epithelial barrier function and tissue inflammation. Human gingiva-derived mesenchymal stem cells (GMSCs) exhibit immunomodulatory and anti-inflammatory effects in a variety of immunity- and inflammation-associated diseases. However, the role of GMSCs in treating IBD has not been elucidated. Our study, therefore, examined the therapeutic effect and mechanism of GMSCs in a murine colitis model of IBD. Our results indicate that the infusion of GMSCs significantly prolonged survival and relieved symptoms. Phenotype analyses showed that the frequencies of NK1.1+ and CD11b+ cells, as well as CD4 T cells in the spleen, were suppressed in GMSC-treated mice compared with the PBS- or fibroblast-treated control groups. Additionally, GMSC treatment markedly increased the numbers of interleukin (IL)-10+ regulatory T cells, reduced the secretion of pro-inflammatory cytokines, and increased production of anti-inflammatory cytokines. A mechanistic study revealed that anti-IL-10R antibody abolished the protective effect of GMSCs compared with mice treated with anti-IgG antibody. Thus, our results indicate that GMSCs play a critical role in alleviating colitis by modulating inflammatory immune cells via IL-10 signalling.

Assessment of microRNA-144-5p and its putative targets in inflamed gingiva from chronic periodontitis patients.

BACKGROUND AND 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. MATERIAL AND 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). CONCLUSION: 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.

Macrophage polarization in human gingival tissue in response to periodontal disease.

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. CONCLUSION: 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.

Increased citrullination and expression of peptidylarginine deiminases independently of P. gingivalis and A. actinomycetemcomitans in gingival tissue of patients with periodontitis.

BACKGROUND: A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA. METHODS: Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test. RESULTS: Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes. CONCLUSION: Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Gingival tissue reaction to direct adhesive restoration: A preliminary study.

INTRODUCTION: It is debated whether composite resin marginal/submarginal direct restoration can be usefully performed without inflammatory consequences. This histological study is the first human analysis aimed to compare, in the same tooth, the gingival tissue close to composite resin restorations with gingival tissue close to hard tissue. METHODS: Eight healthy patients with almost a residual strategic tooth needing endodontic therapy, and post-and-core restoration, then indirect prosthetic restoration, were selected. Direct margin relocation with composite resin was necessary to perform endodontic treatment. The crown lengthening with a secondary flap harvested was necessary to perform prosthetic rehabilitation. Three months after marginal relocation, the secondary flap was harvested, embedded in PMMA, 4-µm sectioned, and stained to analyze the inflammation degree. RESULTS: All patients completed post-and-core reconstruction and the planned prosthetic therapy, maintaining the stringent hygienic protocol plan. The inflammation level comparison, slightly lower in gingiva close to the teeth (3.62 ± 0.38) than in gingiva close to the composite (3.75 ± 0.26), results in a p-value of 0.11 after Wilcoxon test. CONCLUSIONS: Results highlight a minimal, statistically not significant difference in the inflammation degree after margin relocation, conceivably due to patients, teeth and cases selection, together with adopted stringent methodological and supportive measures.

Increased levels of soluble CD163 in periodontitis patients.

AIM: Soluble CD163 (sCD163) has been implicated as a new biomarker in inflammatory conditions. The aim of this cross-sectional study was to assess CD163 levels systemically and locally in patients with chronic periodontitis. METHODS: sCD163 levels were measured by ELISA in serum samples from 70 periodontitis and 70 periodontally healthy subjects, and in saliva samples in a subset of the population. Two gingival biopsies were harvested per subject from 20 periodontitis patients: one from a periodontally affected site, the other from a healthy site, and the relative expression of CD163 mRNA was assessed by real-time PCR. RESULTS: Serum sCD163 was significantly higher in periodontitis patients compared to periodontally healthy subjects (720.0 ± 330.6 ng/ml versus 510.7 ± 219.6 ng/ml, respectively; p < .001). Similarly, sCD163 levels in saliva were significantly increased in periodontitis compared to healthy subjects (3.01 ± 5.07 ng/ml versus 1.98 ± 4.95 ng/ml, respectively; p = .009). Serum and saliva sCD163 levels showed a positive correlation (Kendall's tau .27, p = .018). Importantly, CD163 gene expression was significantly higher in affected sites compared to unaffected sites in periodontitis patients, with a mean fold upregulation of 9.9 (STD: 15.3, p = .010). CONCLUSION: Our findings suggest that CD163 may be involved in the pathogenesis of periodontitis and its link with systemic conditions.

Hard and soft tissue changes following alveolar ridge preservation: a systematic review.

OBJECTIVE: Two focused questions were addressed within this systematic review. Q1) What is the effect of alveolar ridge preservation on linear and volumetric alveolar site dimensions, keratinised measurements, histological characteristics and patient-based outcomes when compared to unassisted socket healing. Q2) What is the size effect of these outcomes in three different types of intervention (guided bone regeneration, socket grafting and socket seal). MATERIALS AND METHODS: An electronic search (MEDLINE, EMBASE, Cochrane Central Register LILACS, Web of Science) and hand-search was conducted up to June 2015. Randomised controlled trials (RCT) and controlled clinical trials (CCT); with unassisted socket healing as controls: were eligible in the analysis for Q1. RCTs, CCTs and large prospective case series with or without an unassisted socket healing as control group were eligible in the analysis for Q2. RESULTS: Nine papers (8 RCTs and 1 CCTs) were included in the analysis for Q1 and 37 papers (29 RCTs, 7 CCTs and 1 case series) for Q2. The risk for bias was unclear or high in most of the studies. Q1: the standardised mean difference (SMD) in vertical mid-buccal bone height between ARP and a non-treated site was 0.739 mm (95% CI: 0.332 to 1.147). The SMD when proximal vertical bone height and horizontal bone width was compared was 0.796mm (95% CI: -1.228 to 0.364) and 1.198 mm (95% CI: -0.0374 to 2.433). Examination of ARP sites revealed significant variation in vital and trabecular bone percentages and keratinised tissue width and thickness. Adverse events were routinely reported, with three papers reporting a high level of complications in the test and control groups and two papers reporting greater risks associated with ARP. No studies reported on variables associated with the patient experience in either the test or the control group. Q2: A pooled effect reduction (PER) in mid-buccal alveolar ridge height of -0.467 mm (95% CI: -0.866 to -0.069) was recorded for GBR procedures and -0.157 mm (95% CI: -0.554 to 0.239) for socket grafting. A proximal vertical bone height reduction of -0.356 mm (95% CI: -0.490 to -0.222) was recorded for GBR, with a horizontal dimensional reduction of -1.45 mm (95% CI: -1.892 to -1.008) measured following GBR and -1.613 mm (95% CI: -1.989 to -1.238) for socket grafting procedures. Five papers reported on histological findings after ARP. Two papers indicated an increase in the width of the keratinised tissue following GBR, with two papers reporting a reduction in the thickness of the keratinised tissue following GBR. Histological examination revealed extensive variations in the treatment protocols and biomaterials materials used to evaluate extraction socket healing. GBR studies reported a variation in total bone formation of 47.9 ± 9.1% to 24.67 ± 15.92%. Post-operative complications were reported by 29 papers, with the most common findings soft tissue inflammation and infection. CONCLUSION: ARP results in a significant reduction in the vertical bone dimensional change following tooth extraction when compared to unassisted socket healing. The reduction in horizontal alveolar bone dimensional change was found to be variable. No evidence was identified to clearly indicate the superior impact of a type of ARP intervention (GBR, socket filler and socket seal) on bone dimensional preservation, bone formation, keratinised tissue dimensions and patient complications.