Salivary biomarkers : diagnostic potential in oral and systemic diseases in epidemiological surveys

Human saliva is a fluid with many biological functions, and essential for the maintenance of oral health. Several studies report local and systemic biomarkers appearing in saliva, including electrolytes, blood products, enzymes and tissue destruction molecules, inflammatory markers as well as proteins putatively associated with different diseases. However, the clinical utility of salivary diagnostics for the assessment of oral and systemic disease remains elusive. The general aim of this project was to investigate the utility of salivary biomarkers for diagnostic potential of oral- and systemic diseases in large populations. This thesis consists of two different projects: (i). Skåne cohort - A cross-sectional study (Studies I and II): 451 individuals were randomly selected and enrolled for these investigations, including 51% women. All participants were asked to complete a questionnaire, a medical history was taken, a clinical examination was made and stimulated saliva samples were collected. (ii). PAROKRANK sub-cohort (Periodontal disease and the relation to myocardial infarction) (Studies III and IV): A case-control study comprising 400 subjects. In total 200 consecutive patients with a first acute myocardial infarction (AMI) admitted to coronary care units in Sweden from May 2010 to December 2011, and 200 controls without previous AMI, matched for age, gender, residential area were included during the same time period. Dental examinations were performed, blood and stimulated saliva samples were collected eight to ten weeks after the myocardial infarction (MI). Matched controls were examined within one to two weeks after the MI patients. Study I: The aim of this study was to investigate if selected salivary biomarkers could be used for epidemiological studies for detection of periodontitis. Our findings showed that patients with severe periodontitis had elevated salivary concentrations of interleukin (IL) -1β (IL-1β) and matrix metalloproteinase (MMP) -8 (MMP-8), as well as and increased ratio of MMP-8/ tissue inhibitor of metalloproteinase-1 (TIMP-1). Study II: The aim of this study was to investigate if certain salivary biomarkers could be used for detection of common systemic inflammatory diseases. The results of our study showed that salivary IL-8 concentrations were higher in patients with bowel disease and subjects who had experience of tumor diseases. MMP-8 levels were elevated in saliva from patients with diabetes, muscle and joint diseases or previously had undergone cardiac surgery. Study III: The aim of this study was to investigate whether salivary concentrations of selected cardiovascular biomarkers could be used to identify patients with a previous MI and whether such markers, in plasma and saliva, were related to periodontal status. There was no difference between participants with or without MI in regards of N-terminal prohormone of brain natriuretic peptide (NT- pro BNP) and growth differentiation factor-15 (GDF-15) levels in saliva. However, cystatin C, NT- pro BNP and GDC-15 levels were higher in MI patients with other co-morbidities. The levels of cystatin C were lower in saliva from patients with MI. GDF-15 levels correlated with periodontal status in both groups. Further, there was no correlation between plasma and saliva levels. Study IV: The aim of this study was to explore the levels of the inflammatory markers, MMP-8, MMP-9, TIMP-1 and myeloperoxidase (MPO) in saliva with regards to previous MI and periodontal disease. The analyzed biomarkers correlated significantly with each other and most of the periodontal parameters in both study groups. Salivary MMP-8 and MPO levels were significantly higher in non- MI subjects. In conclusion, the findings in this project indicates that certain salivary biomarkers have the potential to be used for screening purposes in epidemiological studies related to both oral and systemic diseases. In addition, the selected salivary biomarkers in our studies could be seen as markers for increased local and systemic inflammation

Salivary Diagnostics : Point-of-Care diagnostics of MMP-8 in dentistry and medicine

Peer reviewe

Association of peptidoglycan recognition protein 1 to post-myocardial infarction and periodontal inflammation : A subgroup report from the PAROKRANK (Periodontal Disease and the Relation to Myocardial Infarction) study

Background: Peptidoglycan recognition protein 1 (PGLYRP1) is an antimicrobial and proinflammatory innate immunity protein activated during infections. We aimed to investigate whether PGYLRP1 and associated molecules of the immune response in saliva is a cumulative outcome result of both MI and periodontal inflammation. Methods and Results: Two hundred patients with MI and another 200 matched non-MI controls were included. A full-mouthexamination was conducted to assess periodontal inflammation and collection of stimulated saliva was performed 6 to 10 weeks after the first MI. PGLYRP1, triggering receptor expressed on myeloid cells 1 (TREM-1), interleukin-1 beta (IL-1 beta) were analyzed by ELISA. Matrix metalloproteinase (MMP)-8 levels were determined by IFMA. Compared to controls, MI patients showed higher salivary PGLYRP1, but not TRIM-1 levels. The difference in PGLYRP1 levels remained after adjustment for covariates. In MI patients, the PGLYRP1 levels positively correlated with BOP and PPD 4 to 5 mm. Among non-MI subjects, the levels of PGLYRP1 correlated positively and significantly with BOP and total PPD. Salivary PGLYRP1 concentrations also showed strong positive correlations with levels of TRIM-1, IL-1 beta and MM P-8. In multivariate linear regression analysis, in MI patients, BOP and former smokingstatus displayed an association with salivary PGLYRP1 concentration. Conclusion: MI patients showed higher salivary PGLYRP1 levels than healthy controls, also after adjusting for smoking, sex, age and periodontal health status. Salivary levels of PGLYRP1 may reflect the overall inflammatory burden to chronic bacterial exposure, possibly underpinning the observed associations between periodontitis and exposure with MI.Peer reviewe

aMMP-8 Point-of-Care/Chairside Oral Fluid Technology as a Rapid, Non-Invasive Tool for Periodontitis and Peri-Implantitis Screening in a Medical Care Setting

This communication article addresses currently available rapid non-invasive methods to screen and detect periodontitis and dental peri-implantitis. In this regard, oral fluid biomarkers have been researched extensively but self-reported oral health (SROH)-questionnaires have also been developed. Both alternatives may offer a quick and easy way to screen and detect diseased patients. Active matrix metalloproteinase (aMMP-8) is one of the most validated biomarkers for screening and detecting periodontal breakdown related to periodontitis and peri-implantitis and monitoring their treatment effects revealing successful, less- and non-successful treatment results. Currently available aMMP-8 lateral-flow technologies allow this kind of analysis, as demonstrated here, to be conducted quantitatively online and real-time as point-of-care/chairside testing in dental and even medical care settings. In this study, an aMMP-8 peri-implant sulcular fluid point-of-care-test diagnosed peri-implantitis and healthy implants far more accurately than bleeding-on-probing or the other biomarkers, such as polymorphonuclear (PMN)/neutrophil elastase, myeloperoxidase and MMP-9. Although, SROH-questionnaires allow screening in similar settings but they lack the information about the current disease activity of periodontitis and peri-implantitis, which is of essential value in periodontal diagnostics and treatment monitoring. Thus, both methods can be considered as adjunct methods for periodontitis and peri-implant diagnostics, but the value of oral fluid biomarkers analysis does not seem to be substitutable.Peer reviewe

Mucin 4 and matrix metalloproteinase 7 as novel salivary biomarkers for periodontitis

Aim: Periodontitis is a chronic inflammatory disease, characterized by irreversible destruction of tooth-supporting tissue including alveolar bone. We recently reported mucin 4 ( MUC4) and matrix metalloproteinase 7 (MMP7) as highly associated with periodontitis in gingival tissue biopsies. The aim of this study was to further investigate the levels of MUC4 and MMP7 in saliva and gingival crevicular fluid (GCF) samples of patients with periodontitis. Materials and Methods: Saliva and GCF samples were collected from periodontitis patients and healthy controls. The levels of MUC4, MMP7, and total protein concentrations were analysed using ELISA or Bradford assay. Results: MUC4 levels were significantly lower in saliva and GCF from periodontitis patients relative to healthy controls. MMP7 levels were significantly higher in saliva and GCF from periodontitis patients. Multivariate analysis revealed that MUC4 was significantly associated with periodontitis after adjusting for age and smoking habits and, moreover, that the combination of MUC4 and MMP7 accurately discriminated periodontitis from healthy controls. Conclusions: MUC4 and MMP7 may be utilized as possible novel biomarkers for periodontitis.Peer reviewe

The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases

The analysis of the disease-specific oral and systemic biomarkers in saliva and oral fluids (i.e., mouth rinse, gingival crevicular fluid (GCF), and peri-implantitis fluid (PISF)) is demanding. Several hosts and microbial factors may influence their expression, release, and levels. The type of saliva/oral fluids utilized for the diagnostics affects the analysis. High sensitivity and specificities together with sophisticated methods and techniques are essential for valuable outcome. We describe here recently developed practical, convenient, inexpensive, noninvasive, and quantitative mouth rinse and PISF/GCF/chair-side/point-of-care (PoC) lateral-flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyser) to detect, predict, and monitor successfully the course, treatment, and prevention of periodontitis and peri-implantitis, respectively. The tests have been independently and successfully validated to differentiate periodontal and peri-implant health and disease in Finland, Germany, Netherland, Sweden, Turkey, Nigeria, Malawi, and USA. The clinical use of salivary/oral fluid biomarkers to identify oral and systemic conditions requires additional studies utilizing these noninvasive screening, diagnostic, and preventive aMMP-8 PoC/chair-side technologies.Peer reviewe

Salivary Matrix Metalloproteinase-8 and-9 and Myeloperoxidase in Relation to Coronary Heart and Periodontal Diseases : A Subgroup Report from the PAROKRANK Study (Periodontitis and Its Relation to Coronary Artery Disease)

Background and Objective Matrix metalloproteinase (MMP) -8, -9 and myeloperoxidase (MPO) are inflammatory mediators. The potential associations between MMP-8, -9, MPO and their abilities to reflect cardiovascular risk remains to be evaluated in saliva. The objective of this study was to investigate the levels and associations of salivary MMP-8, -9, MPO and tissue inhibitors of metalloproteinase (TIMP)-1 in myocardial infarction (MI) patients and controls with or without periodontitis. Materials and Methods 200 patients with a first MI admitted to coronary care units in Sweden from May 2010 to December 2011 and 200 controls matched for age, gender, residential area and without previous MI were included. Dental examination and saliva sample collection was performed 6-10 weeks after the MI in patients and at baseline in controls. The biomarkers MMP -8, -9, MPO and TIMP-1 were analyzed by time-resolved immunofluorescence assay (IFMA), Western blot and Enzyme-Linked ImmunoSorbent Assay (ELISA). Results After compensation for gingivitis, gingival pockets and smoking, the mean salivary levels of MMP-8 (543 vs 440 ng/mL, p = 0.003) and MPO (1899 vs 1637 ng/mL, p = 0.02) were higher in non-MI subjects compared to MI patients. MMP-8, -9 and MPO correlated positively with clinical signs of gingival/periodontal inflammation while TIMP-1 correlated mainly negatively with these signs. The levels of latent and active forms of MMP-8 did not differ between the MI and non-MI groups. Additionally, MMP-8, MPO levels and MMP-8/TIMP-1 ratio were significantly higher in men compared to women with MI. Conclusions This study shows that salivary levels of the analyzed biomarkers are associated with periodontal status. However, these biomarkers could not differentiate between patients with or without a MI. These findings illustrate the importance to consider the influence of oral conditions when analyzing levels of inflammatory salivary biomarkers.Peer reviewe

Active MMP-8 point-of-care (PoC)/chairside enzyme-test as an adjunctive tool for early and real-time diagnosis of peri-implantitis

Objective: The aim of this study was to investigate the utility of the active matrix metalloproteinase (aMMP-8)-point-of-care (PoC) test as a quantitative real-time chair-side diagnostic tool for peri-implant diagnosis, as well as assess the potentially developing and ongoing risk relative to the traditional clinical methods. Background: Current peri-implant and periodontal disease diagnoses rely on clinical arid radiological examinations. This case-control study investigated the applicability of aMMP-8-PoC immunotest for quantitative real-time diagnosis and monitoring of dental implants in health and disease. Methods: Sixty-eight patients visiting a specialist clinic for maintenance following dental implant placement underwent assessment of their peri-implant health. aMMP-8-PoC peri-implant sulcular fluid (PISF) lateral-flow immunotests were performed using ImplantSafe (R) technology quantitated by ORALyzer (R). In addition, the PISF samples were analyzed for total MMP-8, calprotectin, and interleukin (IL)-6 by enzyme-linked immunosorbent assays (ELISA), aMMP-8 by western immunoblot, and MMP-2 and MMP-9 by gelatin zymography. Results: The aMMP-8-PoC test promptly recorded and reflected peri-implant disease, differentiating it clearly from health. X-ray findings (bone loss > 2 mm), peri-implant pocket depth >= 3 mm, and bleeding on probing were significantly more prevalent among implants positive for the aMMP-8-PoC test. aMMP-8/ORALyzer analysis was more precise in recording disease than total MMP-8, calprotectin, IL-6, MMP-2, and MMP-9. Conclusions: The aMMP-8-PoC test can be conveniently implemented to alert for and detect active collagenolysis affecting peri-implant tissues, both in the early and advanced stages of the disease. Active and fragmented MMP-8 exhibits a strong and significant association with peri-implantitis as compared to total MMP-8 arid other biomarkers and can be utilized as the POC/chairside biomarker of choice in the new classification of peri-implantitis.Peer reviewe

Validation of a noninvasive aMMP-8 point-of-care diagnostic methodology in COVID-19 patients with periodontal disease

Objectives: The aim of this study was to validate an active matrix metalloproteinase (MMP-8) point-of-care diagnostic tool in COVID-19 patients with periodontal disease. Subjects, Materials, and Methods: Seventy-two COVID-19-positive and 30 COVID-19-negative subjects were enrolled in the study. Demographic data were recorded, periodontal examination carried out, and chairside tests run for evaluating the expression of active MMP-8 (aMMP-8) in the site with maximum periodontal breakdown via gingival crevicular fluid sampling as well as via a mouth rinse-based kit for general disease activity. In COVID-19-positive patients, the kits were run again once the patients turned COVID-19 negative. Results: The overall (n = 102) sensitivity/specificity of the mouthrinse-based kits to detect periodontal disease was 79.41%136.76% and that of site-specific kits was 64.71%/55.88% while adjusting for age, gender, and smoking status increased the sensitivity and specificity (82.35%/76.47% and 73.53%/88.24, respectively). Receiver operating characteristic (ROC) analysis for the adjusted model revealed very good area under the ROC curve 0.746-0.869 (p < .001) and 0.740-0.872 (p < .001) (the aMMP-8 mouth rinse and site-specific kits, respectively). No statistically significant difference was observed in the distribution of results of aMMP-8 mouth rinse test (p = .302) and aMMP-8 site-specific test (p = .189) once the subjects recovered from COVID-19. Conclusions: The findings of the present study support the aMMP-8 point-of-care testing (PoCT) kits as screening tools for periodontitis in COVID-19 patients. The overall screening accuracy can be further increased by utilizing adjunctively risk factors of periodontitis. The reported noninvasive, user-friendly, and objective PoCT diagnostic methodology may provide a way of stratifying risk groups, deciding upon referrals, and in the institution of diligent oral hygiene regimens.Peer reviewe