Proteomic Analysis of Salivary Extracellular Vesicles from COVID-19 Patients Reveals a Specific Anti-COVID-19 Response Protein Signature.

Despite the understanding of the coronavirus disease-19 (COVID-19), the role of salivary extracellular vesicles (sEVs) in COVID-19 remains unclear. Exploring the proteomic cargo of sEVs could prove valuable for diagnostic and prognostic purposes in assessing COVID-19. The proteomic cargo of sEVs from COVID-19(+) subjects and their healthy close contacts (HCC) was explored. sEVs were isolated by ultracentrifugation from unstimulated saliva samples, and subsequently characterized through nanoparticle tracking, transmission electron microscopy, and Western blot analyses. The proteomic cargo of sEVs was processed by LC-MS/MS. sEVs were morphologically compatible with EVs, with the presence of Syntenin-1 and CD81 EV markers. The sEV pellet showed 1417 proteins: 1288 in COVID-19(+) cases and 1382 in HCC. In total, 124 proteins were differentially expressed in sEVs from COVID-19(+) subjects. "Coronavirus-disease response", "complement and coagulation cascades", and "PMN extracellular trap formation" were the most enriched KEGG pathways in COVID-19(+) cases. The most represented biological processes were "Hemoglobin and haptoglobin binding" and "oxygen carrier activity", and the best-denoted molecular functions were "regulated exocytosis and secretion" and "leucocyte and PMN mediated immunity". sEV proteomic cargo in COVID-19(+) suggests activity related to immune response processes, oxygen transport, and antioxidant mechanisms. In contrast, in HCC, sEV signature profiles are mainly associated with epithelial homeostasis.

MicroRNAs: The Missing Link between Hypertension and Periodontitis?

Cardiovascular diseases are the leading cause of death worldwide, and arterial hypertension is a recognized cardiovascular risk factor that is responsible for high morbidity and mortality. Arterial hypertension is the result of an inflammatory process that results in the remodeling and thickening of the vascular walls, which is associated with an immunological response. Previous studies have attempted to demonstrate the relationship between oral disease, inflammation, and the development of systemic diseases. Currently, the existence of an association between periodontitis and hypertension is a controversial issue because the underlying pathophysiological processes and inflammatory mechanisms common to both diseases are unknown. This is due to the fact that periodontitis is a chronic inflammatory disease that affects the interface of teeth and surrounding tissues. However, the most likely explanation for understanding this association is related to low-grade chronic inflammation. An initial path in the study of the relationship between the mentioned pathologies is the possibility of an epigenetic influence, mediated by noncoding RNAs as microRNAs. Thus, in the present review we describe the role of microRNAs related to arterial hypertension and/or periodontitis. In addition, we identified 13 common microRNAs between periodontitis and hypertension. According to the predictions of the DIANA-mirPath program, they can regulate genes involved in 52 signaling pathways.

Obesity is related to maternal periodontitis severity in pregnancy: a cross-sectional study.

OBJECTIVES: To evaluate the relationship between obesity and periodontitis staging compared with periodontal healthy or gingivitis in pregnant women. MATERIALS AND METHODS: An analytical cross-sectional study was conducted on pregnant women between 11 and 14 weeks of pregnancy. Sociodemographic, clinical, obstetric, and periodontal variables were studied. The exposure variable was obesity (body mass index [BMI] ≥ 30), and the primary outcome was periodontitis staging versus periodontal healthy/gingivitis. Data were analysed and estimated by multinomial logistic regression models. RESULTS: The present study screened 1086 pregnancies and analysed 972 women with a median age of 29 years; 36.8% were diagnosed as obese. 26.9% of patients were diagnosed as periodontal healthy or gingivitis, 5.5% with stage I periodontitis, 38.6% with stage II periodontitis, 24% with stage III periodontitis, and 5.1% with stage IV periodontitis. After identifying and adjusting for confounding variables (educational level and plaque index), obesity had a relative risk ratio (RRR) of 1.66 (95% CI: 1.05-2.64; p = 0.03) and 1.57 (95% CI: 1.09-2.27; p = 0.015) for stage III periodontitis compared to periodontal healthy/gingivitis and stage II periodontitis, respectively. CONCLUSION: Besides the already known risk indicators for periodontitis (age, smoking, and educational level), our study suggests a relationship between obesity and periodontitis staging in pregnancy. CLINICAL RELEVANCE: Obesity can alter host immune responses, leading to increased susceptibility to infections and overactive host immunity, which could influence the prevalence and severity of maternal periodontitis in pregnancy.

Osteoinductive Electrospun Scaffold Based on PCL-Col as a Regenerative Therapy for Peri-Implantitis.

Peri-implantitis is a serious condition affecting dental implants that can lead to implant failure and loss of osteointegration if is not diagnosed and treated promptly. Therefore, the development of new materials and approaches to treat this condition is of great interest. In this study, we aimed to develop an electrospun scaffold composed of polycaprolactone (PCL) microfibers loaded with cholecalciferol (Col), which has been shown to promote bone tissue regeneration. The physical and chemical properties of the scaffold were characterized, and its ability to support the attachment and proliferation of MG-63 osteoblast-like cells was evaluated. Our results showed that the electrospun PCL-Col scaffold had a highly porous structure and good mechanical properties. The resulting scaffolds had an average fiber diameter of 2-9 µm and high elongation at break (near six-fold under dry conditions) and elasticity (Young modulus between 0.9 and 9 MPa under dry conditions). Furthermore, the Col-loaded scaffold was found to decrease cell proliferation when the Col content in the scaffolds increased. However, cytotoxicity analysis proved that the PCL scaffold on its own releases more lactate dehydrogenase into the medium than the scaffold containing Col at lower concentrations (PCL-Col A, PCL-Col B, and PCL-Col C). Additionally, the Col-loaded scaffold was shown to effectively promote the expression of alkaline phosphatase and additionally increase the calcium fixation in MG-63 cells. Our findings suggest that the electrospun membrane loaded with Col can potentially treat peri-implantitis by promoting bone formation. However, further studies are needed to assess the efficacy and safety of this membrane in vivo.

Relationship between soluble neuropilin-1 in the gingival crevicular fluid of early pregnant women and different severities of periodontitis: A cross-sectional study.

Background: Pregnancy exacerbates the periodontal inflammation; however, the biological mediators involved are not well characterized. Neuropilins (NRPs) are transmembrane glycoproteins involved in physiological and pathogenic processes such as angiogenesis and immunity but its relationship with periodontal disease in pregnant women has not been studied. Objective: To explore the soluble Neuropilin-1 (sNRP-1) levels in gingival crevicular fluid (GCF) samples during early pregnancy and its association with the periodontitis severity and periodontal clinical parameters. Methods: 80 pregnant women were recruited, and GCF samples were collected. Clinical data and periodontal clinical parameters were recorded. sNRP-1 expression was determined by ELISA assay. The relationship between sNRP-1(+) pregnant women with the severity of periodontitis and periodontal clinical parameters was determined by Kruskal-Wallis and Mann-Whitney tests. Spearman's test estimated the correlation between sNRP-1 levels and periodontal clinical parameters. Results: Periodontitis was classified as mild in 27.5% (n = 22) women, moderate in 42.5% (n = 34), and severe in 30% (n = 24). sNRP-1 expression was higher in the GCF of pregnant with severe (41.67%) and moderate (41.17%) periodontitis compared than in those with mild periodontitis (18.8%). The sNRP-1(+) pregnant had a higher BOP (76.5% v/s 57%; p = 0.0071) and PISA (1199.5 mm2 v/s 880.2 mm2; p = 0.0282) compared with sNRP-1(-). A positive correlation between sNRP-1 levels in GCF and BOP (p = 0.0081) and PISA (p = 0.0398) was observed. Conclusions: The results suggest that sNRP-1 could be involved in periodontal inflammation during pregnancy.

Molecular Biomarkers in Peri-Implant Health and Disease: A Cross-Sectional Pilot Study.

BACKGROUND: The aim of this feasibility study was to investigate the concentration level of CCL-20/MIP-3α, BAFF/BLyS, IL-23, RANKL, and Osteoprotegerin in the Peri-Implant Crevicular Fluid (PICF), from patients diagnosed with peri-implant mucositis and peri-implantitis, and to compare them with PICF from patients with healthy implants. METHODS: Participants with at least one dental implant with healthy peri-implant tissues, peri-implant mucositis, or peri-implantitis were included. PICF was collected using paper strips from healthy and diseased peri-implant sites (n = 19). Biomarker levels were analyzed using a custom Multiplex ELISA Assay Kit. RESULTS: In comparison to peri-implant health, the peri-implant mucositis group showed an increased concentration of CCL-20 MIP-3α, BAFF/BLyS, IL-23, RANKL, and Osteoprotegerin. The peri-implantitis group had the lowest median concentration of Osteoprotegerin (1963 ng/mL); this group had a similar concentration of RANKL (640.84 ng/mL) when compared to the peri-implant health group. BAFF/BLyS (17.06 ng/mL) showed the highest concentration in the peri-implantitis group. CONCLUSIONS: This feasibility study suggests that IL-23 and RANKL may help to elucidate the pathogenesis during the conversion from peri-implant health to peri-implantitis. Further research is required in BAFF/BLyS for the early diagnosis of peri-implantitis.

Exploring the Expression of Pro-Inflammatory and Hypoxia-Related MicroRNA-20a, MicroRNA-30e, and MicroRNA-93 in Periodontitis and Gingival Mesenchymal Stem Cells under Hypoxia.

Hypoxia associated with inflammation are common hallmarks observed in several diseases, and it plays a major role in the expression of non-coding RNAs, including microRNAs (miRNAs). In addition, the miRNA target genes for hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells-5 (NFAT5) modulate the adaptation to hypoxia. The objective of the present study was to explore hypoxia-related miRNA target genes for HIF-1α and NFAT5, as well as miRNA-20a, miRNA-30e, and miRNA-93 expression in periodontitis versus healthy gingival tissues and gingival mesenchymal stem cells (GMSCs) cultured under hypoxic conditions. Thus, a case-control study was conducted, including healthy and periodontitis subjects. Clinical data and gingival tissue biopsies were collected to analyze the expression of miRNA-20a, miRNA-30e, miRNA-93, HIF-1α, and NFAT5 by qRT-PCR. Subsequently, GMSCs were isolated and cultured under hypoxic conditions (1% O2) to explore the expression of the HIF-1α, NFAT5, and miRNAs. The results showed a significant upregulation of miRNA-20a (p = 0.028), miRNA-30e (p = 0.035), and miRNA-93 (p = 0.026) in periodontitis tissues compared to healthy gingival biopsies. NFAT5 mRNA was downregulated in periodontitis tissues (p = 0.037), but HIF-1α was not affected (p = 0.60). Interestingly, hypoxic GMSCs upregulated the expression of miRNA-20a and HIF-1α, but they downregulated miRNA-93e. In addition, NFAT5 mRNA expression was not affected in hypoxic GMSCs. In conclusion, in periodontitis patients, the expression of miRNA-20a, miRNA-30e, and miRNA-93 increased, but a decreased expression of NFAT5 mRNA was detected. In addition, GMSCs under hypoxic conditions upregulate the HIF-1α and increase miRNA-20a (p = 0.049) expression. This study explores the role of inflammatory and hypoxia-related miRNAs and their target genes in periodontitis and GMSCs. It is crucial to determine the potential therapeutic target of these miRNAs and hypoxia during the periodontal immune-inflammatory response, which should be analyzed in greater depth in future studies.

Histological and Immunohistochemical Analysis of Peri-Implant Soft and Hard Tissues in Patients with Peri-Implantitis.

Currently, researchers are focused on the study of cytokines as predictive biomarkers of peri-implantitis (PI) in order to obtain an early diagnosis and prognosis, and for treatment of the disease. The aim of the study was to characterize the peri-implant soft and hard tissues in patients with a peri-implantitis diagnosis. A descriptive observational study was conducted. Fifteen soft tissue (ST) samples and six peri-implant bone tissue (BT) samples were obtained from 13 patients who were diagnosed with peri-implantitis. All the samples were processed and embedded in paraffin for histological and immunohistochemical analyses. A descriptive and quantitative analysis of mast cells and osteocytes, A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF), osteonectin (ON), and ∝-smooth muscle actin (∝-SMA) was performed. We observed the presence of mast cells in peri-implant soft tissue in all samples (mean 9.21 number of mast cells) and osteocytes in peri-implant hard tissue in all samples (mean 37.17 number of osteocytes). The expression of APRIL-ST was 32.17% ± 6.39%, and that of APRIL-BT was 7.09% ± 5.94%. The BAFF-ST expression was 17.26 ± 12.90%, and the BAFF-BT was 12.16% ± 6.30%. The mean percentage of ON was 7.93% ± 3.79%, and ∝-SMA was 1.78% ± 3.79%. It was concluded that the expression of APRIL and BAFF suggests their involvement in the bone resorption observed in peri-implantitis. The lower expression of osteonectin in the peri-implant bone tissue can also be associated with a deficiency in the regulation of bone remodeling and the consequent peri-implant bone loss.

CpG Single-Site Methylation Regulates TLR2 Expression in Proinflammatory PBMCs From Apical Periodontitis Individuals.

Introduction: Apical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown. Aim: To determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP. Methods: Cross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1ß, and IL-12p70 levels were measured by Multiplex assay. Results: PBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1ß compared to controls (p<0.05). Higher TLR2 expression and higher global methylation pattern of the promoter region of the gene were found in AP compared to controls (p<0.05). The CpGs single-sites at positions -166 and -146 were completely methylated, while the site -102 was totally unmethylated, independently of the presence of AP. DNA methylation of CpG single-sites in positions -77 and +24 was positively associated with TLR2 expression. Conclusions: PBMCs from AP subjects show a hyperinflammatory phenotype and TLR2 upregulation in association with single CpG-sites' methylation from the TLR2 gene promoter, thereby contributing to a sustained systemic inflammatory load in individuals with periapical endodontic diseases.

Systemic burden and cardiovascular risk to Porphyromonas species in apical periodontitis.

OBJECTIVE: Porphyromonas (P.) species (spp.) are a major etiological agent of apical periodontitis (AP), which in turn represents a risk factor for cardiovascular diseases. This study explored the associations between endodontic infection with Porphyromonas species, the systemic bacterial burden, and cardiovascular risk, based on high-sensitivity C-reactive protein (hsCRP), in young adults with AP. MATERIALS AND METHODS: Cross-sectional study. Otherwise, healthy individuals with AP and controls (n = 80, ≤ 40 years) were recruited at the University Dental Clinic. Oral parameters and classic cardiovascular risk factors were registered. Endodontic Porphyromonas endodontalis and Porphyromonas gingivalis were identified using conventional PCR. Serum concentrations of anti-P. endodontalis and anti-P. gingivalis antibodies, and endotoxins were determined through ELISA and Limulus-amebocyte assays. Serum hsCRP was determined for cardiovascular risk stratification. RESULTS: Intracanal detection of P. endodontalis and P. gingivalis in AP were 33.3% and 22.9%, respectively. Serum anti-P. endodontalis and anti-P. gingivalis IgG was higher in AP than controls (p < 0.05 and p = 0.057, respectively). Intracanal P. endodontalis associated with higher endotoxemia (p < 0.05). Among endodontic factors, the presence (OR 4.2-5.5, p < 0.05) and the number of apical lesions (OR 2.3, p < 0.05) associated with moderate-severe cardiovascular risk, whereas anti-P. endodontalis IgG were protective (OR 0.3, p > 0.05). CONCLUSIONS: AP and infection with P. endodontalis positively associated with cardiovascular risk based on hsCRP levels and endotoxemia, respectively, whereas anti-P. endodontalis IgG response seems to be protective against low-grade systemic inflammation. CLINICAL RELEVANCE: Apical periodontitis and endodontic P. endodontalis can influence the systemic burden with impact on the surrogate cardiovascular risk marker hsCRP, providing mechanistic links.

Periodontitis and Gestational Diabetes Mellitus: A Potential Inflammatory Vicious Cycle.

Periodontitis is a chronic inflammatory immune disease associated with a dysbiotic state, influenced by keystone bacterial species responsible for disrupting the periodontal tissue homeostasis. Furthermore, the severity of periodontitis is determined by the interaction between the immune cell response in front of periodontitis-associated species, which leads to the destruction of supporting periodontal tissues and tooth loss in a susceptible host. The persistent bacterial challenge induces modifications in the permeability and ulceration of the sulcular epithelium, which facilitates the systemic translocation of periodontitis-associated bacteria into distant tissues and organs. This stimulates the secretion of pro-inflammatory molecules and a chronic activation of immune cells, contributing to a systemic pro-inflammatory status that has been linked with a higher risk of several systemic diseases, such as type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). Although periodontitis and GDM share the common feature of systemic inflammation, the molecular mechanistic link of this association has not been completely clarified. This review aims to examine the potential biological mechanisms involved in the association between periodontitis and GDM, highlighting the contribution of both diseases to systemic inflammation and the role of new molecular participants, such as extracellular vesicles and non-coding RNAs, which could act as novel molecular intercellular linkers between periodontal and placental tissues.

Active MMP-8 Quantitative Test as an Adjunctive Tool for Early Diagnosis of Periodontitis.

Periodontitis is a host-mediated bacterial disease that affects the tooth attachment apparatus. Metalloproteinase-8 (MMP-8), a validated biomarker, could aid in clinical diagnosis. This study aimed to evaluate the diagnostic performance of active (a) MMP-8 immunotest versus total (t) MMP-8 ELISA for quantitative real-time diagnosis and assessment of periodontitis severity at the site level. Gingival crevicular fluid (GCF) was sampled from 30 healthy, 42 mild, and 59 severe periodontitis sites from thirty-one volunteers. MMP-8 concentrations were determined by time-resolved immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using the STATA package. Both active and total MMP-8-based methods discriminated among sites according to periodontal diagnosis and severity, with a positive correlation between the two tests (p < 0.001). (a) MMP-8 models showed the best performance in receiver operating characteristic (ROC) curves to discriminate between healthy and periodontitis sites (area under the curve [AUC] = 0.89), while (t) MMP-8 demonstrated a high diagnostic precision in the detection of mild from severe periodontitis sites (AUC ≥ 0.80). The use of (a) MMP-8 and (t) MMP-8 could represent a useful adjunctive tool for periodontitis diagnosis and severity. These results support the applicability of new point-of-care methods in the monitoring of high-risk periodontal patients.

Collagen quantification in peri-implant soft tissues in human peri-implantitis lesions / Cuantificación de colágeno en tejidos blandos periimplantarios en lesiones de periimplantitis en humanos

SUMMARY: Peri-implantitis is an inflammatory lesion of bacterial etiology characterized by inflammation of the mucosa and bone loss. Chronic inflammation is characterized by neovascularization and collagen neoformation. Mast cells have been shown to participate in the inflammatory process by releasing mediators and proteases such as chymase and tryptase, important in the collagen neoformation process. Although a higher percentage of collagen has been described in periodontal disease, the literature is scarce about the percentage of collagen in peri-implantitis. The aim of this study was to quantify the percentage of collagen fibers present in the peri- implant soft tissue of patients with peri-implantitis lesions. A descriptive observational cross-sectional study was performed. Samples of peri-implant soft tissue were collected from eleven patients with peri-implantitis and then processed by Masson's Trichrome Technique. In microscopic analysis, collagen fibers were observed in all samples, with an average percentage of 39.89 %, standard deviation of 17.02 %, with a minimum value of 8.99 % and a maximum value of 75.65 % density. From these results, it can be concluded that in human peri-implantitis lesions with bone loss greater than 50 %, there is an important percentage of collagen fibers, which is interpreted as connective tissue in a permanent process of reparative response, in the presence of inflammatory infiltrate.

RESUMEN: La periimplantitis es una lesión inflamatoria de etiología bacteriana caracterizada por inflamación de la mucosa y pérdida ósea. La inflamación crónica se caracteriza por neovascularización y neoformación de colágeno. Se ha demostrado que los mastocitos participan en el proceso inflamatorio liberando mediadores y proteasas como quimasa y triptasa, importantes en el proceso de neoformación del colágeno. Aunque se ha descrito un mayor porcentaje de colágeno en la enfermedad periodontal, la literatura sobre el porcentaje de colágeno en la periimplantitis es escasa. El objetivo de este estudio fue cuantificar el porcentaje de fibras de colágeno presentes en el tejido blando periimplantario de pacientes con lesiones de periimplantitis. Se realizó un estudio observacional descriptivo transversal. Se recogieron muestras de tejido blando periimplantario de once pacientes con periimplantitis y luego se procesaron mediante la técnica tricrómica de Masson. En el análisis microscópico, se observaron fibras de colágeno en todas las muestras, con un porcentaje promedio de 39,89 %, desviación estándar de 17,02%, con un valor mínimo de 8,99 % y un valor máximo de 75,65% de densidad. De estos resultados se puede concluir que en las lesiones de periimplantitis humana con pérdida ósea superior al 50 %, existe un porcentaje importante de fibras de colágeno, que se interpreta como tejido conectivo en un proceso permanente de respuesta reparadora, en presencia de infiltrado inflamatorio.

Gingival Crevicular Placental Alkaline Phosphatase Is an Early Pregnancy Biomarker for Pre-Eclampsia.

Early and innovative diagnostic strategies are required to predict the risk of developing pre-eclampsia (PE). The purpose of this study was to evaluate the performance of gingival crevicular fluid (GCF) placental alkaline phosphatase (PLAP) concentrations to correctly classify women at risk of PE. A prospectively collected, retrospectively stratified cohort study was conducted, with 412 pregnant women recruited at 11-14 weeks of gestation. Physical, obstetrical, and periodontal data were recorded. GCF and blood samples were collected for PLAP determination by ELISA assay. A multiple logistic regression classification model was developed, and the classification efficiency of the model was established. Within the study cohort, 4.3% of pregnancies developed PE. GCF-PLAP concentration was 3- to 6-fold higher than in plasma samples. GCF-PLAP concentrations and systolic blood pressure were greater in women who developed PE (p = 0.015 and p < 0.001, respectively). The performance of the multiparametric model that combines GCF-PLAP concentration and the levels of systolic blood pressure (at 11-14 weeks gestation) showed an association of systolic blood pressure and GCF-PLAP concentrations with the likelihood of developing PE (OR:1.07; 95% CI 1.01-1.11; p = 0.004 and OR:1.008, 95% CI 1.000-1.015; p = 0.034, respectively). The model had a sensitivity of 83%, a specificity of 72%, and positive and negative predictive values of 12% and 99%, respectively. The area under the receiver operating characteristic (AUC-ROC) curve was 0.77 and correctly classified 72% of PE pregnancies. In conclusion, the multivariate classification model developed may be of utility as an aid in identifying pre-symptomatic women who subsequently develop PE.

Soluble neuropilin-1 in gingival crevicular fluid is associated with rheumatoid arthritis: An exploratory case-control study.

BACKGROUND: To explore the soluble Neuropilin-1 (sNRP-1) concentrations in gingival crevicular fluid (GCF) and the periodontal clinical status of patients with Rheumatoid Arthritis (RA). MATERIALS AND METHODS: We conducted an exploratory study with 40 study participants, 20 with RA, and 20 healthy controls. Clinical and periodontal data were recorded, and GCF samples were obtained. sNRP-1 levels in GCF were determined by ELISA assay. Descriptive statistics, Mann-Whitney U test, Unpaired t-test, logistic regression model, and Area Under Receiver Operating Characteristic Curve (AUC-ROC) were made to explore the diagnostic performance accuracy. RESULTS: RA patients had significantly higher levels of sNRP-1 in GCF (p â€‹= â€‹0.0447). The median levels of GCF-sNRP-1 were 208.85 â€‹pg/µl (IQR 131.03) in the RA group compared to 81.46 â€‹pg/µl (IQR 163.73) in the control group. We observed an association between the GCF-sNRP-1 concentrations and the RA diagnosis (OR:1.009; CI 1.00-1.001; p â€‹= â€‹0.047). The diagnosis of chronic periodontitis was also associated with RA (OR: 6.9; CI 1.52-31.37; p â€‹= â€‹0.012). Moreover, the AUC-ROC of GCF-sNRP-1 concentrations combined with periodontal clinical parameters such as periodontal probing depth and periodontal inflamed surface area was 0.80. CONCLUSION: This exploratory case-control study shows that RA patients had significantly higher levels of sNRP-1 in GCF. New longitudinal studies are necessary to evaluate the role of NRP-1 in periodontal tissues and consider it an oral biomarker with clinical value in RA.

Soluble Neuropilin-1 in gingival crevicular fluid from periodontitis patients: An exploratory cross-sectional study.

BACKGROUND: Soluble Neuropilin-1 (sNRP-1) is a glycoprotein with angiogenic and immune regulatory functions, which correspond to processes deeply involved with periodontal diseases. This study's objective was to determine the concentration of sNRP-1 in gingival crevicular fluid (GCF) samples of severe periodontitis (stages III-IV) compared to mild-moderate (stages I-II) periodontitis patients. MATERIALS AND METHODS: An exploratory cross-sectional study was conducted, including 36 adults subjected to a complete periodontal exam, which recorded the following periodontal parameters: periodontal probing depth (PPD), clinical attachment loss (CAL), bleeding on probing (BOP), gingival index (GI) and periodontal inflamed surface area (PISA). Periodontitis was defined by two periodontists using the case definition proposed by the 2017 World Workshop for periodontal diseases. GCF samples were collected to determine the levels of sNRP-1 via ELISA. The results were analyzed using descriptive statistics, Mann-Whitney, Kruskal Wallis, and Spearman tests. RESULTS: The levels of sNRP-1 in patient's GCF with periodontitis in stages III-IV showed a median of 38.385 â€‹ng/mL (iqr 30.98), in comparison with 20.085 â€‹ng/mL (iqr 12.74) for stages I-II (p â€‹= â€‹0.202). Regardless of the periodontitis stage, we observed a positive correlation between the levels of sNRP-1 in BOP (Rho: 0.45; p â€‹= â€‹0.0048), PISA (Rho: 0.50; p â€‹= â€‹0.0019), PD (Rho: 0.3; p â€‹= â€‹0.015) and GI (Rho: 0.37; p â€‹= â€‹0.02). CONCLUSIONS: The GCF-sNRP-1 concentration was positively related to periodontal clinical inflammatory parameters and probably could be involved in pro-inflammatory and angiogenic mechanisms observed in periodontitis. Additional studies are necessary to confirm these preliminary results.

Early pregnancy levels of gingival crevicular fluid matrix metalloproteinases-8 and -9 are associated with the severity of periodontitis and the development of gestational diabetes mellitus.

BACKGROUND: Gestational diabetes mellitus (GDM) is increasing worldwide and women with a history of GDM are at risk of developing type 2 diabetes which is a risk factor for periodontitis. The aim of this study was to explore the association between the concentrations of matrix metalloproteinase (MMP)-8 and -9 in gingival crevicular fluid (GCF) during early pregnancy with the periodontal diagnosis and the risk of GDM development. METHODS: A prospective cohort study, including 314 women, enrolled at 11 to 14 weeks of pregnancy was conducted. A complete maternal/obstetric and periodontal exam was performed, and GCF samples were obtained for the MMP-8 and -9 determination by Multiplex Elisa Assays. Mann-Whitney test; Spearman's correlation and log-binomial regression model estimated the association between MMPs concentration in GCF and GDM. RESULTS: Fourteen percent of the pregnancies were diagnosed with GDM. An increase in the concentration of MMP-8 and -9 in women with periodontitis stage III and IV compared to periodontitis stage I was observed (99.31 ng/mL [IQR: 85.32] versus 71.95 ng/mL [IQR: 54.04], and 262.4 ng/mL [IQR: 312.55] versus 114.1 ng/mL [IQR: 184.94], respectively). Women who developed GDM showed increased concentrations of MMP-8 and -9 in GCF since the beginning of pregnancy (P = 0.0381; P = 0.0302, respectively). MMP-8 concentration in GCF was associated with GDM (RR: 1.19; P = 0.045; CI 95% 1.00 to 1.40; and RR: 1.20; P = 0.063; CI 95% 0.99 to 1.45 in the adjusted model). CONCLUSION(S): GCF concentrations of MMP-8 and -9 at early of pregnancy are increased in women with severe periodontitis and associated with the GDM development.

Diagnostic potential of peri-implant crevicular fluid microRNA-21-3p and microRNA-150-5p and extracellular vesicles in peri-implant diseases.

BACKGROUND: To explore the diagnostic usefulness of extracellular vesicles (EVs), and their subpopulations (micro-vesicles and exosomes), and microRNAs (miRNA-21-3p, miRNA-150-5p, and miRNA-26a-5p) in peri-implant crevicular fluid (PICF) of subjects with healthy, peri-implant mucositis and peri-implantitis implants. METHODS: A total of 54 patients were enrolled into healthy, peri-implant mucositis, and peri-implantitis groups. PICF samples were collected, EVs subpopulations (MVs and Exo) were isolated and characterized by nanoparticle tracking analysis and transmission electron microscopy. The expression of miRNA-21-3p, miRNA-150-5p and miRNA-26a-5p was quantified by qRT-PCR. Logistic regression models and accuracy performance tests were estimated. RESULTS: PICF samples show the presence of EVs delimited by a bi-layered membrane, in accordance with the morphology and size (< 200 nm). The concentration of PICF-EVs, micro-vesicles and exosomes was significantly increased in peri-implantitis implants compared to healthy implants (P = 0.023, P = 0.002, P = 0.036, respectively). miRNA-21-3p and miRNA-150-5p expression were significantly downregulated in patients with peri-implantitis in comparison with peri-implant mucositis sites (P = 0.011, P = 0.020, respectively). The reduced expression of miRNA-21-3p and miRNA-150-5p was associated with peri-implantitis diagnosis (OR:0.23, CI 0.08-0.66, P = 0.007 and OR:0.07, CI 0.01-0.78, P = 0.031, respectively). The model which included the miRNA-21-3p and miRNA-150-5p expression had a sensitivity of 93.3%, a specificity of 76.5%, a positive predictive value of 77.8%, and a negative predictive value of 92.9%. The positive and negative likelihood ratios were 3.97 and 0.09, respectively. The area under the receiver operating characteristics curve for the model was 0.84. CONCLUSIONS: An increase concentration of EVs with a downregulation expression of miRNA-21-3p and miRNA-150-5p could be related with the peri-implantitis development.

In situ endoscopic analysis of bone microstructure and vascularization in post-extraction sites immediately after a minimally invasive vertical tooth extraction in teeth with different periodontal status / Análisis endoscópico in situ de la microestructura ósea y vascularización en alvéolos post-extracción inmediatamente después de una extracción dental mínimamente invasiva en dientes con diferente estado periodontal

SUMMARY: The aim of this study was to perform an in situ endoscopic analysis of the vascularization of post-extraction sites immediately after a non-traumatic extraction in terms of the number of blood vessels per field (NBV), relative area of blood vessels (RABV) and relative area of unmineralized bone (RAUB) in teeth with different periodontal status (PS). This assessment was performed using short distance support immersion endoscopy (SD-SIE). Ten patients (4 men/ 6 women, aged between 25 and 44) were selected. From them, 10 teeth were extracted due to periodontal reasons or other motives. These teeth were then categorized into 2 groups according to their PS, either as periodontally compromised (PC) (clinical attachment loss (CAL) > 7 mm and probing depth (PD) > 5 mm) or periodontally healthy (PH) (CAL < 7 mm and PD < 5 mm, without bleeding or suppuration during periodontal probing), and mobile (M) (> 1 mm horizontally) or immobile (I) (< 1 mm horizontally). The minimally invasive vertical tooth extractions were performed using the Benex ® extractor. Immediately after extraction, a rigid immersion endoscope with a diameter of 2.7 mm was introduced, and a video-alveoloscopy was carried out. This video was analyzed by ImageJ software for the quantification of NBV, RABV and RAUB per field of the post-extraction sites with different PS (PC, PH, M, I) were quantified. In the PC group, significantly greater values for RAUB were observed (33.45 %) compared to those from the PH group (19.65 %). Compared with the M group, the I group did not show significant differences in terms of RAUB or RABV. There were also no differences in NBV in both groups (Means: 33.8 vs. 30.5, respectively).

RESUMEN: El objetivo de este estudio fue realizar un análisis endoscópico in situ de la vascularización de los alvéolos post-extracción inmediatamente después de una extracción atraumática en términos de número de vasos sanguíneos por campo de observación (NBV), área relativa de vasos sanguíneos (RABV) y el área relativa de espacios no mineralizados (RAUB) en dientes con diferente estado periodontal (PS). Esta evaluación se realizó mediante endoscopía de inmersión de corta distancia (SD-SIE). Se seleccionaron diez pacientes (4 hombres / 6 mujeres, con edades comprendidas entre 25 y 44). De ellos, se extrajeron 10 dientes debido a razones periodontales u otros motivos. Estos dientes se clasificaron en 2 grupos según su PS, ya sea como periodontalmente comprometidos (PC), los que presentaban un nivel de inserción clínica (CAL) ≥ 7 mm y una profundidad de sondaje (PD) ≥ 5 mm; o periodontalmente sanos (PH) (CAL <7 mm y PD <5 mm, sin sangramiento o supuración durante el sondaje periodontal). También se categorizaron según su movilidad como móvil (M) (≥ 1 mm horizontalmente) o inmóvil (I) (<1 mm horizontalmente). Las extracciones verticales mínimamente invasivas se realizaron con el extractor Benex ®. Inmediatamente después de la extracción, se introdujo un endoscopio rígido de inmersión con un diámetro de 2.7 mm, con el cual se realizó una video-alveoloscopía. Este video fue analizado por el software ImageJ para la cuantificación de NBV, RABV y RAUB por campo, de los alvéolos post-extracción con diferente estado periodontal. En el grupo de dientes PC, se observaron valores significativamente mayores para RAUB (33.45%) en comparación con los del grupo PH (19.65 %). En comparación con el grupo M, el grupo I no mostró diferencias significativas en términos de RAUB o RABV. Tampoco hubo diferencias en el NBV en ambos grupos (Media: 33.8 frente a 30.5, respectivamente).