Markus Laky

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2

Summary.  Background: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram‐negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. Objectives: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. Methods: Phagocytosis, cell–cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet‐neutrophil‐bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. Results: In the presence of plasma neutrophil‐mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet–neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet–neutrophil aggregate formation but is required for efficient phagocytosis. Conclusions: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.

Periodontopathogens induce soluble P-selectin release by endothelial cells and platelets.

INTRODUCTION Soluble P-selectin plays a pivotal role in inflammation and the development of thrombotic and cardiovascular disease. Accordingly, elevated levels of soluble P-selectin are found in periodontitis and (other forms of) inflammatory diseases. However, the cellular source of soluble P-selectin in periodontitis and the effects of periodontopathogens on P-selectin release are unknown. MATERIAL AND METHODS Soluble P-selectin was determined in 26 patients with periodontitis and 19 controls. Furthermore, human endothelial cells and platelets were investigated for their ability to elicit soluble and surface P-selectin in response to periodontopathogens A. actinomycetemcomitans Y4 and P. gingivalis. Moreover surface E-selectin and ICAM-1 expression as well as NFκB translocation in response to these bacteria were determined on endothelial cells as well as the formation of platelet-leukocyte complexes. RESULTS Plasma levels of soluble P-selectin are significantly elevated in periodontitis and correlate with severity of disease and bacterial infection. Stimulation of endothelial cells with periodontopathogens results in rapid surface expression of P-selectin but does not induce NFκB translocation and subsequent de novo synthesis of P-selectin, E-selectin or ICAM-1. In platelets, bacterial stimulation leads to surface expression of P-selectin and fosters the formation of platelet-leukocyte aggregates within minutes. P-selectin is rapidly shed from the surface of platelets and endothelial cells and results in increased levels of soluble P-selectin. CONCLUSIONS Periodontopathogens are able to directly cause activation of endothelial cells and platelets within minutes. Given that transient periodontitis-associated bacteremia commonly occurs after tooth brushing or chewing, our data suggest that reduction of periodontopathogens might result in potential cardiovascular benefits.

Periodontal Probing of Dental Furcations Compared With Diagnosis by Low-Dose Computed Tomography: A Case Series

BACKGROUND Therapeutic decisions in periodontal surgery are based on the accurate diagnosis of the furcation. Clinical probing is the basic diagnostic tool; however, the accuracy of clinical probing to distinguish Class II and Class III furcation defects is unknown. Therefore, this study compares clinical probing diagnoses to those of computed tomography (CT). METHODS Seventy-five patients with severe periodontal disease were enrolled in this case series study. A total of 582 furcation sites in molars were assigned for the diagnosis of Class II and Class III furcation defects by clinical probing. Diagnosis based on CT served as a reference. RESULTS The degree of furcation involvement on clinical findings was confirmed in 57% of the sites, whereas 20% were overestimated and 23% were underestimated compared with the radiologic analysis. Only 32% of Class III furcations in the CT scan were detected clinically. The best correlation of CT scan and clinical probing was found at buccal furcation sites in the mandible, with a κ-coefficient of 0.52, and buccal furcation sites in the maxilla, κ = 0.38. The κ-coefficient was 0.35 for lingual furcations, 0.29 for mesial furcations, and 0.27 for distal furcations, showing weaker correlations. CONCLUSIONS CT scans offer more detailed information on furcation involvement than clinical probing. Especially before surgical treatment, three-dimensional radiographic imaging can be a useful tool to assess the degree of furcation involvement and optimize treatment decisions.

Periodontopathogens induce expression of CD40L on human platelets via TLR2 and TLR4

INTRODUCTION The outstanding importance of (soluble) CD40L to cardiovascular disease (CVD) is becoming increasingly apparent as CD40L is an important mediator of thrombotic and inflammatory processes. Platelets are the main source for CD40 ligand, linking platelet stimulatory events to inflammation and adverse adaptive immune responses. Periodontitis represents a chronic dental infection by distinct gram negative bacteria that is associated with an increased risk for CVD. However, the effects of periodontopathogens on CD40L expression by platelets have not been determined. MATERIAL AND METHODS Effects of periodontopathogens A. actinomycetemcomitans Y and P. gingivalis on the expression of CD40L were determined and the underlying receptors and pathways were investigated. 26 patients with periodontitis and 19 controls were included in the clinical part of this study. RESULTS Periodontopathogens directly induce surface expression of CD40L in human platelets. This activation depends on plasma factors like CD14 and involves TLR2 and TLR4 but not FcγRII. Inhibition of PI3K and PLC completely abolishes bacteria-induced surface expression of CD40L. TLR2 and TLR4 agonists, for example, are also able to induce expression and release of CD40L in human platelets. In patients with periodontitis, plasma levels of soluble CD40L are elevated and positivity for P. gingivalis is associated with a statistical significant increase of soluble CD40L. CONCLUSIONS Our data indicate an involvement of periodontopathogens in increased plasma levels of soluble CD40L in periodontitis and therefore provide a novel link between periodontitis and increased risk for CVD.

Effect of enamel matrix derivative on proliferation and differentiation of osteoblast cells grown on the titanium implant surface

OBJECTIVES Enamel matrix derivative (EMD) is widely used in promoting periodontal regeneration, but the mechanisms underlying its effects are not entirely clear. In particular, the effect of EMD on osseointegration of dental implants and its application in the treatment of peri-implantitis are still debatable. The purpose of this study was to investigate the effect of EMD on proliferation and differentiation of osteoblasts grown on the Ti implant surface. STUDY DESIGN Osteoblast-like MG-63 cells were seeded on coarse-grit-blasted and acid-etched surface Ti implant disks and stimulated with various EMD concentrations. Cell proliferation/viability, alkaline phosphatase activity, osteocalcin production, and expression levels of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) were determined. RESULTS EMD inhibited the proliferation/viability of MG-63 cells. Furthermore, EMD significantly increased the alkaline phosphatase activity and osteocalcin production in MG-63 cells grown on Ti surfaces. Finally, EMD enhanced mRNA expression level of OPG and did not influence that of RANKL. CONCLUSION(S) Application of EMD in the dental implantolology may have a positive effect on implant osseointegration, and further studies are required to improve clinical outcome.

Effect of Emdogain on proliferation and migration of different periodontal tissue–associated cells

OBJECTIVES Although Emdogain is widely used as a gel in periodontal therapy, the exact mechanisms underlying its regenerative ability still need to be further investigated. Therefore, we tested in vitro the effect of the product Emdogain on proliferation, viability, and migration of various human cell types of periodontium. STUDY DESIGN Proliferation and viability of alveolar osteoblasts (AOBs), epithelial cell line HSC-2, and human umbilical vein endothelial cells (HUVECs) were measured using [(3)H]-thymidine uptake and 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT)-assay, respectively. Cell migration was investigated in microchemotaxis chamber. RESULTS The proliferation and viability of AOB, HSC-2, and HUVECs were significantly stimulated by Emdogain (12.5-250 microg/mL) in direct relationship with the amount of product present in the cell culture medium. Cell migration was stimulated in AOB and HUVECs depending on Emdogain amount. In contrast, in HSC-2 cells the migration was stimulated only by less than 50 microg/mL of Emdogain, whereas at higher amounts this stimulating effect was either diminished or absent. CONCLUSION Emdogain stimulates proliferation, viability, and migration of AOB, HSC-2, and HUVECs in vitro. This biological versatility of Emdogain could correspond to an essential mechanism underlying its ability to promote periodontal regeneration.

Non-surgical periodontal therapy influences salivary melatonin levels

ObjectivesMelatonin is a hormone, which is involved in the control of the circadian rhythm, but also acts as an antioxidant and immune modulator. Previous studies reported decreased salivary and serum melatonin levels in periodontitis. This prospective cohort trial assessed the effect of non-surgical periodontal therapy on melatonin levels.MethodsSalivary and serum samples of 60 participants (30 patients suffering from a severe generalized form of periodontitis, 30 healthy controls) were collected at baseline and 19 samples of periodontitis patients after treatment. Salivary and serum melatonin levels were determined by a commercially available ELISA kit and serum C-reactive protein (CRP) by a routine laboratory test.ResultsAt baseline, periodontitis patients showed significantly increased serum CRP values and significantly decreased salivary melatonin levels compared to the control group. Clinical periodontal parameters significantly correlated with salivary melatonin levels and serum CRP. Periodontal therapy resulted in a recovery of the decreased salivary melatonin levels and a negative correlation was detected for the changes of salivary melatonin and the inflammatory parameter bleeding on probing. Serum melatonin levels showed no significant differences.ConclusionsSalivary melatonin levels recovered after periodontal therapy and correlated with a decrease of local periodontal inflammation. This may imply the local involvement of melatonin in the pathogenesis of periodontitis due to its antioxidant abilities. However, the exact role of melatonin in periodontal disease remains to be investigated in future trials.Clinical relevanceThe present results suggest salivary melatonin as a risk indicator for the severity of periodontal disease.

Decreased phosphorylation of platelet vasodilator-stimulated phosphoprotein in periodontitis – a role of periodontal pathogens

INTRODUCTION Epidemiological studies indicate an association between periodontitis and cardiovascular disease, but the underlying mechanisms are poorly understood. Vasodilator-stimulated phosphoprotein (VASP) in its phosphorylated form represents a regulator of platelet function and an indicator for the sensitivity of platelets towards physiologically relevant antagonists of platelet function. As platelets and their activation state play a central role in the development of cardiovascular disease, this study aimed to investigate the influence of periodontal disease and periodontal pathogens on intraplatelet VASP-phosphorylation and platelet function. MATERIAL AND METHODS Besides several markers of platelet activation, basal and PGE(1) induced intracellular VASP-phosphorylation were determined in platelets of periodontitis patients (n = 26) and healthy donors (n = 19). Furthermore, platelets from healthy donors were incubated with distinct periodontal pathogens and basal and PGE(1) induced VASP-phosphorylation was determined. RESULTS Compared to controls, platelets of periodontitis patients showed a significant decrease in basal and PGE(1) induced VASP-phosphorylation. VASP-phosphorylation in platelets from periodontitis patients positive for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis or Tannerella forsythia was significantly decreased compared to patients that were negative for these bacteria. Furthermore, VASP-phosphorylation in platelets isolated from healthy donors was affected by incubation with these periodontal pathogens. CONCLUSIONS Our results provide evidence that periodontitis interferes with VASP-phosphorylation in human platelets, presumably as a consequence of a direct effect of periodontitis-associated bacteria. Decreased basal and PGE(1) induced VASP-phosphorylation might represent a mechanism responsible for enhanced platelet activation in periodontitis.

The Effects of CO2 Laser with or without Nanohydroxyapatite Paste in the Occlusion of Dentinal Tubules

The aim of this study was to evaluate a new treatment modality for the occlusion of dentinal tubules (DTs) via the combination of 10.6 µm carbon dioxide (CO2) laser and nanoparticle hydroxyapatite paste (n-HAp). Forty-six sound human molars were used in the current experiment. Ten of the molars were used to assess the temperature elevation during lasing. Thirty were evaluated for dentinal permeability test, subdivided into 3 groups: the control group (C), laser only (L−), and laser plus n-HAp (L+). Six samples, two per group, were used for surface and cross section morphology, evaluated through scanning electron microscope (SEM). The temperature measurement results showed that the maximum temperature increase was 3.2°C. Morphologically groups (L−) and (L+) presented narrower DTs, and almost a complete occlusion of the dentinal tubules for group (L+) was found. The Kruskal-Wallis nonparametric test for permeability test data showed statistical differences between the groups (P < 0.05). For intergroup comparison all groups were statistically different from each other, with group (L+) showing significant less dye penetration than the control group. We concluded that CO2 laser in moderate power density combined with n-HAp seems to be a good treatment modality for reducing the permeability of dentin.

Smoking influences salivary histamine levels in periodontal disease

OBJECTIVES Histamine, a potent vasoactive amine, is increased in saliva of periodontitis patients. The present study aimed to further investigate the diagnostic potential of histamine for periodontal disease and assessed smoking, a major risk factor of periodontitis, as a possible influencing factor. METHODS Salivary and serum samples of 106 participants (60 periodontitis patients, 46 controls) were collected. Salivary histamine was determined by a commercially available ELISA kit, and serum C-reactive protein was measured by a routine laboratory test. Cigarettes per day and packyears were assessed as smoking exposure parameters. RESULTS Statistically significantly increased levels of salivary histamine and serum C-reactive protein were detected between the patient and control group (P = 0.022 and P = 0.001). Salivary histamine levels were significantly higher in smoking compared with non-smoking patients (P < 0.001), and salivary histamine as well as serum C-reactive protein correlated significantly positively with smoking exposure parameters (P < 0.05). CONCLUSIONS Smoking, an established and common risk factor of periodontitis, was assessed as a possible influencing factor for salivary histamine. Most interestingly, salivary histamine differed highly significantly between smoking and non-smoking periodontitis patients. Our results suggest a possible involvement of histamine in tobacco-exacerbated periodontal disease, but do not suggest salivary histamine as a reliable diagnostic marker for periodontitis.