Azurocidin in gingival crevicular fluid as a potential biomarker of chronic periodontitis.

BACKGROUND AND OBJECTIVE: Azurocidin is a neutrophil-derived protein in gingival crevicular fluid (GCF) which, according to relevant studies, might correlate with periodontal disease. The aim of the present study was to evaluate azurocidin as a potential biomarker for chronic periodontitis. MATERIAL AND METHODS: One hundred and one patients participated in the study, divided into two groups. Forty-eight were included in the periodontally healthy group (HP) and fifty-three in the chronic periodontitis group (CP). Clinical indices included probing depth (PD), recession (REC), clinical attachment level (CAL), bleeding on probing (BOP) and plaque (PL). Pooled GCF samples were collected with paper strips, freezed in liquid nitrogen (-196°C), stored at -80°C, and the levels of azurocidin were analyzed with ELISA. Values were transformed and expressed for comparisons in pg/30 s sample. Statistical comparisons were performed using non-parametric tests (Mann-Whitney) at the 0.05 level. Furthermore, the diagnostic accuracy of the procedure was assessed with receiver operator characteristic curves (ROC), areas under the curve (AUC), and the Youden's J Index calculated. RESULTS: Demographic data were comparable between the two groups. Clinical parameters and the levels of azurocidin were statistically significantly higher in the CP group when compared to the HP group (Mann-Whitney test, P < .05). Quantitative data from ELISA demonstrated a high diagnostic accuracy of azurocidin, with AUC calculated higher than 0.9 at the 0.000 level. CONCLUSION: Azurocidin in GCF is a promising biomarker for periodontal disease. The results of the present study agree with previous studies in the literature showing an up-regulated trend in the levels of azurocidin in periodontitis patients.

Prevalence of undiagnosed diabetes and pre-diabetes in chronic periodontitis patients assessed by an HbA1c chairside screening protocol.

OBJECTIVES: The objective of the present study was to implement a chairside diabetes screening strategy for the identification of undiagnosed hyperglycaemia in periodontal patients. MATERIALS AND METHODS: Measurement of HbA1c was performed in patients (n = 139) diagnosed with periodontal disease to determine possible unknown hyperglycaemia. Patients fulfilled the criteria for screening according to the questionnaire by the Centers for Disease Control and Prevention (CDC). The Cobas® b101 in vitro diagnostic system was used for the measurement of glycosylated haemoglobin (HbA1c) in capillary blood. Body mass index (BMI) and waist circumference were also measured to determine splanchnic obesity. Periodontal parameters were assessed with an automated probe and included probing depth, clinical attachment loss, bleeding on probing and presence/absence of plaque. RESULTS: Most patients had moderate periodontitis. Almost 25% of the subjects tested were found to have unknown hyperglycaemia while 80.5% of them had splanchnic obesity. A significant association was found between HbA1c and BMI (Mann-Whitney test; p = 0.0021) as well as between HbA1c and waist circumference (Spearman rho test; p = 0.0007). No differences were observed regarding periodontal parameters between subjects exhibiting HbA1c ≥ 5.7% and those with HbA1c < 5.7% (Mann-Whitney test; p > 0.05) although those with HbA1c ≥ 5.7% displayed higher proportions of sites with clinical attachment loss > 5 mm (z test with Bonferroni corrections; p < 0.05). CONCLUSIONS: Periodontal patients, especially those with a bigger than normal BMI and waist circumference, are a target group worth screening for diabetes. CLINICAL RELEVANCE: The dental practitioner can contribute significantly to the worldwide effort of health care professionals in diabetes screening and referring for early diagnosis of the disease.

Peripheral blood neutrophil extracellular trap production and degradation in chronic periodontitis.

AIMS: To investigate ex vivo peripheral neutrophil extracellular trap (NET) production and their subsequent degradation by plasma in chronic periodontitis patients, and periodontally and systemically healthy-matched controls. MATERIALS AND METHODS: Chronic periodontitis patient and control (n = 40 pairs) peripheral blood neutrophils (PBN) were stimulated for NET quantification. A subset of patients received non-surgical periodontal therapy (n = 19) and NETs were quantified 3 months later alongside controls. Blood plasma was collected from patients and controls to quantify plasma-induced NET degradation (n = 19 pairs). Subsequent experiments quantified plasma concentrations of DNase-1, immunoglobulin G (IgG), free light chains (FLCs), and cystatin C. RESULTS: No differences were observed in NET production between patients and controls. However, NET production decreased significantly in patients post-treatment. Plasma NET degradation was significantly lower in patients than controls, which may be due to significantly reduced DNase-1 levels as demonstrated, or potentially due to elevated IgG/FLC concentrations in patients. NET degradation post-periodontal treatment was comparable between patients and controls. CONCLUSIONS: NET production was comparable between patients and controls; however, non-surgical therapy causes attenuated NETs. NET degradation by plasma is impaired in untreated chronic periodontitis, potentially increasing the chronic NET burden, which may enhance antimicrobial function, or conversely, increase the risk of autoimmune/inflammatory responses.

Proteomics for the discovery of biomarkers and diagnosis of periodontitis: a critical review.

Periodontitis is a common chronic and destructive disease whose pathogenetic mechanisms remain unclear. Due to their sensitivity and global scale, proteomics studies offer the opportunity to uncover critical host and pathogen activity indicators and can elucidate clinically applicable biomarkers for improved diagnosis and treatment of the disease. This review summarizes the literature of proteomics studies on periodontitis and comprehensively discusses commonly found candidate biomarkers. Key considerations in the design of an experimental proteomics platform are also outlined. The applicability of protein biomarkers across the progression of periodontitis and unexplored areas of research are highlighted.

Oral hygiene reinforcement in the simplified periodontal treatment of 1 hour.

AIM: To compare the clinical and microbiological outcome of the 1-h ultrasonic debridement of chronic periodontitis patients (CPP) with and without frequent sessions of oral hygiene reinforcement. METHODS: Clinical measurements and subgingival plaque were collected from 44 CPP at baseline, 3- and 6-months. The control group received a single session of 1-h full-mouth ultrasonic debridement, while oral hygiene instructions (OHI) were reiterated over four visits. In the test group, OHI were limited in the 1-h treatment session. At 3-months, both groups received additional debridement and OHI. The "Checkerboard" DNA-DNA hybridization technique quantified Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola in plaque. RESULTS: At three months, smaller reductions in plaque and bleeding indices, and in P. gingivalis numbers were noted in the test group, while these differences disappeared at six months. After the 3-month re-treatment visit, the test group presented with a greater probing pocket depth (PPD) reduction. Plaque negatively affected PPD in a similar manner after both treatment approaches. CONCLUSIONS: Lack of oral hygiene reinforcement in the 1-h full-mouth debridement resulted in higher plaque and bleeding scores and numbers of P. gingivalis at three months; professional removal of dental biofilm every three months is beneficial in subjects with compromised plaque control.

Effects of doxycycline on clinical, microbiological and immunological parameters in well-controlled diabetes type-2 patients with periodontal disease: a randomized, controlled clinical trial.

AIM: To evaluate the clinical, microbiological and immunological effects of systemic doxycycline as an adjunct to scaling and root planing (SRP) in chronic periodontitis patients with well-controlled type 2 diabetes. MATERIALS AND METHODS: Sixty-six patients compliant to oral hygiene (Hygiene Index <20%) allocated to either a test (systemic doxycycline for 21 days) or a control (placebo) group participated in the present randomized controlled trial (RCT). Clinical assessments were recorded at baseline, 3 and 6 months after therapy and included clinical attachment level (CAL), set as the primary outcome of the study, probing pocket depth (PPD), recession (RE) and bleeding on probing (BOP). At the same time points, counts of 15 subgingival species were evaluated by "checkerboard" DNA-DNA hybridization, gingival crevicular fluid samples were analysed for matrix metalloproteinase-8 (MMP-8) by ELISA and HbA1c levels were determined. Comparisons between and within groups were performed by non-parametric tests (Mann-Whitney, Wilcoxon signed-ranks and z-test for proportions with Bonferroni corrections) at the 0.05 level. RESULTS: No major differences were noticed in clinical and microbiological parameters of periodontal disease or levels of MMP-8 between the two groups. CONCLUSIONS: Adjunctive systemic doxycycline does not seem to significantly enhance the effects of SRP in well-controlled diabetes type 2 patients.

Diagnostic value of aMMP-8 and azurocidin in peri-implant sulcular fluid as biomarkers of peri-implant health or disease.

OBJECTIVE: The objective of this study was to investigate the effectiveness of testing for active matrix metalloproteinase-8 (aMMP-8) by a quantitative point-of-care (PoC), chairside lateral flow immunotest and azurocidin, in the peri-implant sulcular fluid (PISF), as biomarkers for the presence or absence of peri-implant diseases. BACKGROUND: Current research indicates that proinflammatory cytokines and extracellular matrix-degrading enzymes may be of value to diagnose and predict peri-implant disease initiation and progression, but more data are needed. METHODS: Eighty patients with implants were recruited. PISF samples were collected and quantitatively analyzed for aMMP-8 (chairside) and azurocidin with ELISA. Radiographic assessments and clinical indices (probing depth, probing attachment level, bleeding on probing, and plaque) were recorded after sampling. Kruskal-Wallis test and pairwise post hoc Dunn-Bonferroni test were used to relate aMMP-8 levels and azurocidin levels to clinical parameters. The diagnostic ability of aMMP-8 (ng/mL) and azurocidin was analyzed by receiver operator curve analysis. Area under the curve (AUC) was calculated and the Spearman's rho, and the coefficient of determination (R2) were used to calculate the correlations between aMMP-8, azurocidin, and periodontal parameters. RESULTS: Statistically significant differences were observed for aMMP-8 levels but not for azurocidin between healthy implants, implants with mucositis, and those with peri-implantitis (13.65 ± 7.18, 32.33 ± 21.20, and 73.07 ± 43.93 ng/mL, respectively), (Kruskall-Wallis test p < .05). The aMMP-8 test with a threshold of 20 ng/mL has a sensitivity of 71.7% and a specificity of 77.8% to identify peri-implantitis and healthy implants, respectively. AUC was found to be 0.814, and the accuracy of the method reaches 73.8%. Above a cutoff value of 33.7 ng/mL of aMMP-8, the accuracy of the test to detect peri-implantitis reaches 77.5% in relation to 62.5% of BoP from the same site. CONCLUSION: Taken collectively, present data indicate that the aMMP-8 PoC lateral flow immunotest can be a beneficial, adjunctive diagnostic quantitative tool for real-time screening for peri-implant diseases.

Immunological and microbiological findings after the application of two periodontal surgical techniques: a randomized, controlled clinical trial.

AIM: To examine microbiological and immunological alterations following two periodontal surgical techniques, over a 6-month period. MATERIALS AND METHODS: A total of 30 chronic periodontitis patients participated in the present randomized controlled clinical trial and were randomized in two groups. Modified Widman flap (MWF) was applied in the control group and apically positioned flap (APF), without intervention to the bone, in the experimental group. Gingival crevicular fluid samples and subgingival plaque samples from the operated sites were collected at baseline, 6th, 12th and 24th post-operative week. RESULTS: No major differences were noticed in immunological and microbiological profile of patients receiving either modified MWF or APF, for a period of 6 months. CONCLUSIONS: The choice of the periodontal surgical procedure does not seem to affect the immunological and the microbiological profile of patients with chronic periodontitis.

Discovery of biomarker combinations that predict periodontal health or disease with high accuracy from GCF samples based on high-throughput proteomic analysis and mixed-integer linear optimization.

AIM: To identify optimal combination(s) of proteomic based biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals and validate the predictions through known and blind test sets. MATERIALS AND METHODS: GCF samples were collected from 96 CP and periodontally healthy subjects and analysed using high-performance liquid chromatography, tandem mass spectrometry and the PILOT_PROTEIN algorithm. A mixed-integer linear optimization (MILP) model was then developed to identify the optimal combination of biomarkers which could clearly distinguish a blind subject sample as healthy or diseased. RESULTS: A thorough cross-validation of the MILP model capability was performed on a training set of 55 samples and greater than 99% accuracy was consistently achieved when annotating the testing set samples as healthy or diseased. The model was then trained on all 55 samples and tested on two different blind test sets, and using an optimal combination of 7 human proteins and 3 bacterial proteins, the model was able to correctly predict 40 out of 41 healthy and diseased samples. CONCLUSIONS: The proposed large-scale proteomic analysis and MILP model led to the identification of novel combinations of biomarkers for consistent diagnosis of periodontal status with greater than 95% predictive accuracy.

Clinical, radiographic, microbiological, and immunological outcomes of flapped vs. flapless dental implants: a prospective randomized controlled clinical trial.

OBJECTIVES: The objective of this study was to compare the placement of flapped vs. flapless dental implants utilizing clinical, radiographic, microbiological, and immunological parameters. MATERIAL AND METHODS: A total of 20 patients received 30 dental implants following a one-stage protocol. The patients were randomly assigned into two study groups: control group with 15 flapped implants and test group with 15 flapless implants. Follow-up examinations were carried out after 1, 2, 6, and 12 weeks. Clinical recordings, sulcular fluid sampling, microbiological analysis, and digital subtraction radiography were utilized to compare the two surgical approaches. RESULTS: Peri-implant sulcus depth was significantly greater in flapped implants at both 6 and 12 postsurgical weeks (P < 0.001). Flapped implants showed crestal bone loss (0.29 ± 0.06 mm), whereas no bone resorption was detected around flapless implants. Matrix metalloproteinase-8 values were higher to a statistically significant level in the control group at 1 (P = 0.003) and 6 weeks (P = 0.007) after placement. In the test group, the presence of Porphyromonas gingivalis was significantly higher at the 2nd postoperative week (P = 0.005), whereas the counts of Tannerella forsythia were significantly elevated at the 1st (P = 0.005), 2nd (P = 0.001), and 12th (P = 0.002) postoperative weeks, possibly indicating an earlier formation and maturation of the peri-implant sulcus. Patients reported more pain after flapped implant placement. CONCLUSIONS: Flapless implant placement yielded improved clinical, radiographic, and immunological outcomes compared with flapped implantation. In addition, patients seem to better withstand flapless implant placement.

Surgical Treatment of Peri-Implantitis Using a Combined Nd: YAG and Er: YAG Laser Approach: Investigation of Clinical and Bone Loss Biomarkers.

The current study aimed to investigate the effect of the combined Nd-Er: YAG laser on the surgical treatment of peri-implantitis by evaluating clinical markers and biomarkers of bone loss (RANKL/OPG). Twenty (20) patients having at least 1 implant diagnosed with peri-implantitis were randomly assigned to two groups for surgical treatment. In the test group (n = 10), Er: YAG laser was used for granulation tissue removal and implant surface decontamination, while Nd: YAG laser was employed for deep tissue decontamination and biomodulation. In the control group (n = 10), an access flap was applied, and mechanical instrumentation of the implant surface was performed by using titanium curettes. The following clinical parameters were evaluated at baseline and six months after treatment: Full-mouth Plaque Score (FMPS), Probing Pocket Depth (PPD), Probing Attachment Levels (PAL), recession (REC), and Bleeding on probing (BoP). Peri-implant crevicular fluid (PICF) was collected at baseline and six months for the evaluation of soluble RANKL and OPG utilizing enzyme-linked immunosorbent assay (ELISA). Baseline clinical values were similar for both groups, with no statistical differences between them. The study results indicated statistically significant improvements in the clinical parameters during the 6-month observation period in both groups. More specifically, PPD, PAL, and REC were improved in the test and control groups with no differences in the between-groups comparisons. However, a greater reduction in the BoP-positive sites was noted for the laser group (Mean change 22.05 ± 33.92 vs. 55.00 ± 30.48, p = 0.037). The baseline and six-month comparisons of sRANKL and OPG revealed no statistically significant differences between the two groups. The combined Nd: YAG-Er: YAG laser surgical therapy of peri-implantitis seemed to lead to more favorable improvements in regard to bleeding on probing six months after treatment compared to the conventional mechanical decontamination of the implant surface. None of the methods was found superior in the modification of bone loss biomarkers (RANKL, OPG) six months after treatment.

Active Matrix Metalloproteinase-8 (aMMP-8) Versus Total MMP-8 in Periodontal and Peri-Implant Disease Point-of-Care Diagnostics.

Active matrix metalloproteinase-8 (aMMP-8) is a promising biomarker candidate for the modern periodontal and peri-implant disease diagnostics utilizing the chairside/point-of-care oral fluid technologies. These rapid biomarker analysis technologies utilize gingival crevicular fluid (GCF), peri-implant sulcular fluid (PISF), or mouth rinse as the oral fluid matrices that can be collected patient-friendly and non-invasively without causing bacteremia. aMMP-8, but not total or latent proMMP-8, has been shown to be a relevant biomarker to be implemented to the latest 2017 classification system of periodontitis and peri-implantitis. Thus, aMMP-8 point-of-care-testing (POCT)-but not total or latent proMMP-8-can be conveniently used as an adjunctive and preventive diagnostic tool to identify and screen the developing and ongoing periodontal and peri-implant breakdown and disease as well as predict its episodic progression. Similarly, aMMP-8 POCT provides an important tool to monitor the treatment effect of these diseases, but also other diseases such as head and neck cancer, where it can identify and predict the rapid tissue destructive oral side-effects during and after the radiotherapy. Additionally, recent studies support aMMP-8 POCT benefitting the identification of periodontitis and diabetes as the escalating risk diseases for COVID-19 infection. Overall, aMMP-8 POCT has launched a new clinical field in oral medicine and dentistry, i.e., oral clinical chemistry.

Novel protein identification methods for biomarker discovery via a proteomic analysis of periodontally healthy and diseased gingival crevicular fluid samples.

AIM: To identify possible novel biomarkers in gingival crevicular fluid (GCF) samples from chronic periodontitis (CP) and periodontally healthy individuals using high-throughput proteomic analysis. MATERIALS AND METHODS: Gingival crevicular fluid samples were collected from 12 CP and 12 periodontally healthy subjects. Samples were trypically digested with trypsin, eluted using high-performance liquid chromatography, and fragmented using tandem mass spectrometry (MS/MS). MS/MS spectra were analysed using PILOT_PROTEIN to identify all unmodified proteins within the samples. RESULTS: Using the database derived from Homo sapiens taxonomy and all bacterial taxonomies, 432 human (120 new) and 30 bacterial proteins were identified. The human proteins, angiotensinogen, clusterin and thymidine phosphorylase were identified as biomarker candidates based on their high-scoring only in samples from periodontal health. Similarly, neutrophil defensin-1, carbonic anhydrase-1 and elongation factor-1 gamma were associated with CP. Candidate bacterial biomarkers include 33 kDa chaperonin, iron uptake protein A2 and phosphoenolpyruvate carboxylase (health-associated) and ribulose biphosphate carboxylase, a probable succinyl-CoA:3-ketoacid-coenzyme A transferase, or DNA-directed RNA polymerase subunit beta (CP-associated). Most of these human and bacterial proteins have not been previously evaluated as biomarkers of periodontal conditions and require further investigation. CONCLUSIONS: The proposed methods for large-scale comprehensive proteomic analysis may lead to the identification of novel biomarkers of periodontal health or disease.

Crestal bone resorption after the application of two periodontal surgical techniques: a randomized, controlled clinical trial.

AIM: To radiographically assess, by means of digital subtraction radiography, crestal bone loss following two periodontal surgical techniques, over a period of 6 months. MATERIAL AND METHODS: A total of 30 chronic periodontitis patients participated in this randomized controlled clinical trial and were separated into two groups. Modified Widman flap was applied in the control group and apically positioned flap, without intervention to the bone, in the experimental group. Clinical measurements (plaque index, gingival bleeding index, probing pocket depth and clinical attachment level) were recorded at baseline, 6 weeks, 3 and 6 months after surgery. Digital radiographs were taken at baseline, 1, 3, 6 weeks, 3 and 6 months after surgical treatment and subtracted digitally in pairs. RESULTS: Both groups showed statistically significant improvement of clinical parameters. Statistically significant differences between the two groups were observed only in probing pocket depth (PPD) at 6 weeks interval, where the test group showed more reduction (smallest statistically significant differences observed, SSSDO = 0.64). Both groups showed the same rate of crestal bone resorption. CONCLUSIONS: Bone resorption of the alveolar crest is a phenomenon that is observed as a consequence of periodontal surgical treatment without significant differences between the two techniques. Furthermore, both surgical techniques lead to satisfactory clinical results, indicating that bone removal during periodontal surgical treatment is not always necessary.

Is There a Link between COVID-19 and Periodontal Disease? A Narrative Review.

The coronavirus disease 2019 (COVID-19) pandemic greatly affected human well-being, social behavior, global economy, and healthcare systems. Everyday clinical practice in dentistry has been adjusted to the increased hazards of aerosol production by routine dental procedures. The objective of this study was to assess the existing literature to determine possible mechanisms of a relationship between COVID-19 and periodontitis, as well as describe findings from relevant epidemiological studies.Scarce data exist in the literature that directly addresses the relationship between the two diseases. However, several data describe the role of the oral cavity and periodontal tissues as portals of entry of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), and the contribution of cytokines known to be produced in periodontal disease to severe forms of COVID-19. It is also suggested from the current literature that periodontal disease, shown to be associated with systemic diseases such as diabetes mellitus, cardiovascular and respiratory diseases, shares common risk factors with-especially-severe forms of COVID-19.Further clinical studies are required to establish the relationship between these diseases. Oral hygiene performance and intact periodontal tissues can assist in mitigating the pandemic, and it is suggested that dental practitioners can contribute to identifying at-risk patients.

Active matrix metalloproteinase-8: A potential biomarker of oral systemic link.

OBJECTIVES: This mini review aims to address some possible gaps in periodontal diagnosis in clinical studies particularly involving the oral-systemic connection with a view to minimize such gaps, and thus improve patient treatment experiences and outcomes. METHODS: The conventional assessment of periodontitis has traditionally been by clinical and radiographic oral parameters. We reviewed numerous studies published mainly within the past decade, to affirm the oral-systemic link, the contribution of periodontitis to the inflammatory burden in various systemic diseases and conditions, and the potential role of active matrix metalloproteinase-8 (aMMP-8). RESULTS: While it is established that periodontal pathogens in dental plaque biofilm are the primary initiating agents in periodontitis, it has become clear from the appraisal of recent studies that the host inflammation, including biomarkers such as aMMP-8 play a major role, being the driving underlying pathological mechanism in both periodontitis and systemic diseases. CONCLUSIONS: The apparent limitations of conventional diagnostic tools have led researchers to seek alternative methods of evaluation such as the quantification of biomarkers including aMMP-8, which can be a bridge between oral/periodontal and systemic diseases; aMMP-8 can form a mouth-body connection.

aMMP-8 Oral Fluid PoC Test in Relation to Oral and Systemic Diseases.

The manuscript uses the previously published literature and highlights the benefits of active-matrix metalloproteinase (aMMP)-8 chairside/point-of-care (PoC) diagnostic tools as adjunctive measures in oral and systemic diseases. Previous studies suggest that as a biomarker, aMMP-8 is more precise than total MMP-8, MMP-9, MMP-2, MMP-3, MMP-13, MMP-7, MMP-1, calprotectin, myeloperoxidase (MPO), human neutrophil elastase (HNE), tissue inhibitor of matrix metalloproteinase (TIMP)-1, and bleeding of probing (BOP). Therefore, aMMP-8 could be implemented as the needed key biomarker for the new disease classification for both periodontitis and peri-implantitis. With a sensitivity to the tune of 75-85% and specificity in the range of 80-90%, lateral flow aMMP-8 PoC testing is comparable to catalytic protease activity assays for aMMP-8. The test can be further applied to estimate the glycemic status of an individual, to ascertain whether a person is at risk for COVID-19, in managing the oral side effects of radiotherapy carried in head and neck cancers, and in selected cases pertaining to reproductive health. In the future, aMMP-8 could find application as a potential systemic biomarker in diseases affecting the cardiovascular system, cancers, bacteremia, sepsis, diabetes, obesity, meningitis, as well as pancreatitis. The aMMP-8 PoCT is the first practical test in the emerging new dental clinical field, that is, oral clinical chemistry representing oral medicine, clinical chemistry, peri-implantology, and periodontology.