Localization of interleukin-6 signaling complex in epithelialized apical lesions of endodontic origin.

OBJECTIVES: We aimed to determine the immunolocalization patterns of the interleukin (IL)-6 signaling complex in epithelialized and non-epithelialized apical lesions of endodontic origin (ALEOs). MATERIALS AND METHODS: Epithelialized (n = 8) and non-epithelialized (n = 7) ALEOs were obtained from teeth with indication of extraction in patients with clinical diagnosis of apical periodontitis. All tissues were subjected to routine processing for histopathologic examination and primary antibodies for IL-6, IL-6 receptor (R), and glycoprotein (gp)-130 were used for immunohistochemistry and double immunofluorescence co-localization. RESULTS: IL-6, IL-6R, and gp-130 were immunolocalized in endothelial cells and mononuclear leukocytes in a diffuse pattern within the connective tissue of epithelialized and non-epithelialized ALEOs. In the epithelialized lesions, two different patterns were identified: IL-6 signaling complex was localized within the proliferating epithelium in a diffuse intracellular pattern and in a cell membrane localization pattern within the mature epithelial lining, showing a decreased intensity towards the surface layers. CONCLUSIONS: IL-6, IL-6R, and gp-130 localized to mononuclear inflammatory cells, vascular endothelial cells, and immature proliferating epithelia in a diffuse pattern and in mature lining epithelia in a localized cell membrane pattern, supporting a role for epithelial proliferation during cyst formation. Additional cell membrane co-localization of IL-6 receptor complex suggests classic signaling involvement in addition to trans-signaling.

Molecular signatures of extracellular vesicles in oral fluids of periodontitis patients.

OBJECTIVE: To characterize extracellular vesicles (EVs) in gingival crevicular fluid (GCF) and saliva samples from healthy/gingivitis and periodontitis patients and correlate them with clinical inflammatory periodontal parameters. MATERIAL AND METHOD: An exploratory study, including 86 subjects, was conducted. Clinical and periodontal data were recorded, and oral fluid samples were obtained. EVs were precipitated by ExoQuick-TC™ and characterized by nanoparticle tracking (NanoSight™), Western blot (WB), transmission electron microscopy (TEM), and ELISA analysis. RESULTS: TEM showed nanoparticles morphologically compatible with EVs, and WB analysis revealed bands of specific EV markers (CD9, TSG101, and Alix) in both oral fluids of periodontitis and healthy/gingivitis subjects. The total concentration of EVs in GCF was increased in periodontitis patients compared to healthy/gingivitis subjects (p = .017). However, we did not observe differences in the EV concentration of saliva samples (p = .190). The size of GCF-EVs was 144.2 nm in periodontitis and 160.35 nm in healthy/gingivitis patients (p = .038). The CD63 exosome marker was increased in GCF of periodontitis patients (p = .00001). The total concentration of EVs in GCF was correlated with bleeding on probing (rho = 0.63, p = .002), periodontal probing depth (rho = 0.56, p = .009), and clinical attachment level (rho = 0.48, p = .030). CONCLUSION: Periodontitis patients have an increased concentration of EVs in GCF, and their role in periodontitis should be clarified.

Levels of Myeloperoxidase and Metalloproteinase-9 in Gingival Crevicular Fluid from Diabetic Subjects with and without Stage 2, Grade B Periodontitis.

OBJECTIVE: The present study aimed to compare levels of matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) in gingival crevicular fluid (GCF) from subjects with controlled and noncontrolled Type 2 Diabetes Mellitus (T2D), with and without stage 2 grade B periodontitis (POD2B) versus healthy (H) subjects. METHODS: The levels of both enzymes, from 80 GCF samples collected with PerioPaper strips, were analyzed by a Multiplex/Luminex assay. Five groups were formed, all current patients at the Institutional Dentistry Service, and distributed as follows: two groups of diabetics (one controlled and one poorly controlled); two groups with the previous conditions and diagnosed with POD2B; and one H group. RESULTS: The highest concentration of MMP-9 corresponded to the H group, while the lowest corresponded to the T2D controlled group. Regarding MPO levels, the highest levels were associated with the T2D controlled with POD2B group and the lowest with the T2D controlled group. CONCLUSIONS: No apparent relationship between the elevation of MMP-9 and MPO levels was observed among subjects with T2D, with and without POD2B, compared to H subjects.

Actividad de metaloproteinasa de matriz extracelular-9 en fluido crevicular gingival durante la respuesta reparativa en periodontitis apical asintomática / Activity of matrix metalloproteinase-9 in gingival crevicular fluid during the reparative phase in asymptomatic apical periodontitis

OBJETIVO: Los procesos destructivos del periodonto apical están en su mayoría mediados por proteasas específicas. Existe evidencia de que los niveles de metaloproteinasa de matriz extracelular-9 (MMP-9) en el fluido crevicular gingival (FCG) podrían reflejar la presencia de periodontitis apical asintomática (PAA). El objetivo de este estudio fue evaluar la actividad de MMP-9 durante la respuesta reparativa periapical en controles postendodóncicos entre una semana y 6 meses y en controles sanos. MATERIALES Y MÉTODOS: En este estudio prospectivo se incluyeron 28 pacientes con periodontitis apical asintomática, y se tomaron muestras de FCG a partir de dientes con PAA en línea base y controles postendodóncicos de una semana, uno, 3 y 6 meses. Adicionalmente se incluyeron controles contralaterales sanos. Las muestras se eluyeron y analizaron mediante gelatinogramas y densitometría. RESULTADOS: En pacientes con periodontitis apical asintomática se identificaron tanto la proforma como la forma activa de la MMP-9. Estas presentaron aumentos significativos en controles postendodóncicos de 3 y 6 meses con signos de reparación periapical. CONCLUSIONES: Los niveles de MMP-9 aumentaron significativamente en dientes con diagnóstico de PAA durante la fase reparativa. Estos resultados sugieren que MMP-9 también cumpliría un papel fisiológico durante la reparación periapical, que es susceptible de ser evaluado a través del análisis del FCG.

OBJECTIVE: Apical destructive periodontal processes are largely mediated by specific proteases. Evidence supports that the levels of extracellular matrix metalloproteinase-9 (MMP-9) could reflect the presence of asymptomatic apical periodontitis (AAP) in gingival crevicular fluid (GCF). The aim of this study was to evaluate the activity of MMP-9 during reparative response in periapical post endodontic controls between 1 week and 6 months later and in healthy controls. MATERIALS AND METHODS: A prospective study was performed on 28 patients with a diagnosis of AAP. GCF samples were taken from AAP teeth at baseline and post-endodontic controls at 1 week, 1, 3 and 6 months. Additional healthy contralateral controls were obtained, and samples were eluted and analyzed by densitometric scanning and gelatin zymography. RESULTS: In patients with asymptomatic apical periodontitis, both the pro form and the active form of MMP-9 were identified. These showed significant increases in post-endodontic controls at three and six months, with signs of periapical repairing. CONCLUSIONS: MMP-9 levels significantly increased in teeth diagnosed with AAP during the reparative phase. These results suggest that MMP-9 might be involved in the healing of apical tissues that might be reflected in GCF.

Proteolytic roles of matrix metalloproteinase (MMP)-13 during progression of chronic periodontitis: initial evidence for MMP-13/MMP-9 activation cascade.

AIM: Matrix metalloproteinases (MMP)-13 can initiate bone resorption and activate proMMP-9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP-13 activity and TIMP-1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy-terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP-13 could potentiate gelatinase activation in diseased gingival tissue. MATERIALS AND METHODS: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP-13 activity and TIMP-1. Diseased gingival explants were cultured, treated or not with MMP-13 with or without adding CL-82198, a synthetic MMP-13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. RESULTS: Active sites demonstrated increased ICTP levels and MMP-13 activity (p<0.05) in progression subjects. The MMP-9 activation rate was elevated in MMP-13-treated explants (p<0.05) and MMP-13 inhibitor prevented MMP-9 activation. CONCLUSIONS: MMP-13 could be implicated in the degradation of soft and hard supporting tissues and proMMP-9 activation during progression of chronic periodontitis. MMP-13 and -9 can potentially form an activation cascade overcoming the protective TIMP-1 shield, which may become useful for diagnostic aims and a target for drug development.