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Dentin-induced in vivo Inflammatory Response and in vitro Activation of Murine Macrophages

¹ Department of Stomatology, Pathology, Bauru Dental School, University of São Paulo-Bauru, Rua Sérvio Túlio Carrijo Coube, 3-33, Apto 91-C, Jardim Infante Dom Henrique, 17012-632-Bauru, São Paulo, Brazil;
² Catholic University of Brasília and Brasília Medicine School, University of Brasília-Brasília, Distrito Federal, Brazil; and
³ Department of Pharmacology, Ribeirão Preto Medicine School, University of São Paulo-Ribeirão Preto, São Paulo, Brazil;

View larger version (25K): [in a new window]   Figure 1. (A) Effect of dentin extracts on cell viability. Thioglycolate-elicited macrophages were treated with particulate (D-part) and non-particulate (D-n-part) dentin extracts (5 mg/mL) in the presence or absence of IFN- (60 U/mL), LPS (500 ng/mL), IFN-/LPS, and L-NMMA (200 µM), and cell viability was determined by MTT reduction assay after 48 hrs. *Significantly different from respective control values at P < 0.05. (B) Effect of polymyxin B on nitric oxide production induced by dentin extracts. Peritoneal macrophages were stimulated with particulate dentin extract (D-part) (5 mg/mL) and LPS (500 ng/mL) pre-incubated or not with polymyxin B as described in MATERIALS & METHODS. *Significantly different from respective control values at P < 0.05. #Indicates a level of significance (P < 0.01) relative to polymyxin B-untreated extracts. Data represent the mean + SEM of triplicates.

View larger version (286K): [in a new window]   Figure 2. (A,B,C) Effects of doses of dentin extracts on leukocyte recruitment. Peritoneal cavities were injected with 1 mL of indicated concentrations of dentin extracts and were aspirated with PBS 6 hrs post-injection. The number of leukocytes was assessed as described in MATERIALS & METHODS. Data points represent the mean + SEM from at least 5 mice. *Significantly different from control values at P < 0.05. (D) Time-dependency of dentin-extract-induced leukocyte extravasation. Mice were treated i.p. with particulate dentin extract (D-part) (5 mg/mL) at time zero or were left untreated (control group). Peritoneal cavities were washed at the indicated time points for quantification of polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNs). Data are the mean + SEM of 5 mice. (E,F) Inflammatory responses to dentin extracts in subcutaneous tissue. Mice were injected s.c. with particulate dentin extract (D-part) (5 mg/mL), and the inflammatory cell recruitment was assessed after 5 (E) and 12 days (F) (original magnifications: E, 250x; and F, 400x).

View larger version (46K): [in a new window]   Figure 3. (A,B,C,D) Time-course and effect of IFN- and LPS on nitric oxide production induced by dentin extracts. Thioglycolate-elicited (A,C) or naïve macrophages (B,D) were treated with particulate (D-part), non-particulate (D-n-part) (5 mg/mL), and demineralized (d-Ext) (9 µg/mL) dentin extracts, and silica (30 particles/cell) in the presence or absence of AG (200 µM), IFN- (60 U/mL), LPS (500 ng/mL), or L-NMMA (200 µM) for 48 hrs or at indicated times. *Significantly different from respective control values at P < 0.05. In (A) and (B), # indicates the significance at P < 0.05 vs. condition without AG. In (C) and (D), # indicates the significance at P < 0.05 vs. condition without additional stimulation. (E,F) Effect of dentin treatment on hydrogen peroxide and TNF- production by thioglycolate-elicited macrophages. Cells were stimulated with D-n-part and D-part (5 mg/mL), d-Ext (9 µg/mL), or silica (30 particles/cell) in the presence or absence of PMA (400 ng/mL) and AG (200 µM), and H2O2 production was assessed after 48 hrs. *Significantly different from respective control values at P < 0.05. In E, # indicates the significance at P < 0.05 vs. condition without PMA. In F, # indicates the significance at P < 0.05 vs. condition without additional stimulation. Data are the mean + SEM of triplicates.

View larger version (666K): [in a new window]   Figure 4. Immunocytochemical expression of Mac-1 (A), iNOS (B), IL-1ß (C), and TNF- (D) by dentin-extract-treated macrophages. Thioglycolate-elicited macrophages were treated with particulate dentin extracts (D-part) (5 mg/mL) for 48 hrs. Sections in (E) and (F) are from control groups (original magnifications: A and F, 300x; B and C, 500x; D, 600x; E, 400x). (G) Postulated scheme of participation of dentin in the maintenance of external inflammatory root and bone resorption. In addition to bacteria and their products and inflammatory cell-derived cytokines, dentin may directly cause cell migration and activation (adapted from Susuki et al., 1999).