Resolvin D2 Induces Resolution of Periapical Inflammation and Promotes Healing of Periapical Lesions in Rat Periapical Periodontitis

Abstract

Periapical periodontitis results from pulpal infection leading to pulpal necrosis and resorption of periapical bone. The current treatment is root canal therapy, which attempts to eliminate infection and necrotic tissue. But, in some cases periapical inflammation doesn't resolve even after treatment. Resolvins belongs to a large family of specialized pro-resolving lipid mediators that actively resolves inflammation signaling via specific receptors. Resolvin D2 (RvD2), a metabolite of docosahexaenoic acid (DHA), was tested as an intracanal medicament in rats in vivo. Mechanism was evaluated in rat primary dental pulp cells (DPCs) in vitro. The results demonstrate that RvD2 reduces inflammatory cell infiltrate, periapical lesion size, and fosters pulp like tissue regeneration and healing of periapical lesion. RvD2 enhanced expression of its receptor, GPR18, dentin matrix acidic phosphoprotein 1 (DMP1) and mineralization in vivo and in vitro. Moreover, RvD2 induces phosphorylation of Stat3 transcription factor in dental pulp cells. We conclude that intracanal treatment with RvD2 resolves inflammation and promoting calcification around root apex and healing of periapical bone lesions. The data suggest that RvD2 induces active resolution of inflammation with pulp-like tissue regeneration after root canal infection and thus maybe suitable for treating periapical lesions.

Keywords: DMP1; calcification; periapical lesion; periapical periodontitis; resolution of periapical inflammation; resolvin D2.

Figure 1

Experimental protocol of rat periapical periodontitis model. (A) Root canal treatment was performed 3 weeks after pulp exposure and rats were sacrificed and evaluated 4 weeks after treatment. (B) Cavity preparations of first molar, root canal treatment, and final restoration under microscope guidance.

Figure 2

Molecular imaging analysis examining the effects of RvD2 on periapical inflammation. Images of the signal intensity of MPO activity around RvD2 treated and non-treated tooth are shown (A). Results of comparisons of the levels of signal intensity with respect to total flux are shown (B). Data represent the means of three independent rats (SO on left side and RvD2 on right side for each rat), with error bars indicating standard deviations. *P < 0.05 indicates significant differences compared to the control group, Mann–Whitney test.

Figure 3

Micro-CT analysis of periapical lesions after root canal treatment in rats. (A) Representative image of teeth in the treatment group and SO group. The x-axis (white line) passes through the apical third of the mesial and distal root canal, denoted as the coronal limit of periapical lesion. The y-axis (green line) passes through the center of the mesial and distal root canals of the mandibular first molars. (B,C) Comparison of changes in the size of periapical lesions in mesial and distal canals (*P < 0.05 indicates significant differences compared to the SO group, Mann–Whitney test). Data represent the means of four independent rats (SO on left side and RvD2 on right side for each rat), with error bars indicating standard deviations. The volume of the periapical lesions of the mesial and distal roots for the treatment group were significantly lower than that of the control group after 4 weeks following pulp exposure.

Figure 4

Histological analysis of periapical lesions after root canal treatment in rats. (A) Periapical area of treatment group stained with HE. (B,C) Periapical area of control groups stained with HE. (D) Periapical area of baseline group stained with HE. (E,F,G,H). High magnification views of the solid inset in (A–D), respectively. (I–L). High magnification views of the solid insets in (A–D), stained with a modified Brown and Brenn method. Images are representative for 5 experiments from Group #1 (RvD2 and SO); 3 experiments from Group #2 (VO), and 2 experiments Group #3 (baseline). RC, root canal; CA, closed apex; OA, open apex; AF, apical foramen. The asterisk denotes inflammatory cells and the black arrows specifying some of Gram negative bacteria stain red color and blue arrow specifying some of blue/purple stain Gram positive bacterial cells in canals. Outlined circular images with blue and black colors are higher magnification of specified areas coming from colored dotted circles corresponds to the identifications of Gram positive and negative bacteria.

Figure 5

Immunohistochemical analysis. (A) GPR18 protein expression in the root canal of the treatment group. (B,C) GPR18 protein expression in the root canal of the control groups. (D–F) High magnification views of the solid inset in the panels (A–C), respectively. (G) DMP1 protein was abundantly expressed in the root canals of the treatment group. (H,I) Whereas, DMP1 protein expression was lower in the root canal of the control groups as compared to RvD2 group. (J–L) High magnification views of the solid inset in the panels (G–I), respectively. Images are representative for 5 experiments from Group #1 and 3 experiments from Group #2. (M) Negative control without primary antibody GPR18. (N) Negative control without primary antibody DMP1. RC, root canal; C, cementum. The arrow head indicates GPR18 and DMP1 positive expression.

Figure 6

Alizarin red staining of DPCs stimulated with and without RvD2 (1-200 nM) for 21 days. (A) Obvious differences in the amounts of mineralization among the groups. (B) Quantitative analysis showed that 100 and 200 nM had significantly increased mineralized nodules as compared with control 0 nM (P < 0.05; Tukey-Kramer). *P < 0.05 indicates significant differences compared to the control, post hoc Tukey-Kramer test. Representative data of four independent samples.

Figure 7

Quantification of DMP-1 mRNA from DPCs stimulated with RvD2 (1–100 nM) for 7 and 14 days using real-time RT-PCR. (A) RvD2 induced significant DMP1 mRNA expression at all doses after 7 days culture compared to the control (0 nM) (P < 0.05; Tukey-Kramer). (B) RvD2 induced significant DMP1 mRNA expression at 100 nM (P < 0.05; Tukey-Kramer) after 14 days culture compared to the control (0 nM). *P < 0.05 indicates significant differences compared to the control, post hoc Tukey-Kramer test. Representative data of three to four independent cases.

Figure 8

Western blot analysis. (A) RvD2-induced DMP1 protein expression in 7 days DPC culture at all doses of 1 to 100 nM. (B). RvD2-induced DMP1 protein expression in 14 days DPC culture. DMP1 expression was significantly enhanced at 10 and 100 nM doses compared to at 1 and 0 nM as the control groups (P < 0.05; Tukey-Kramer). Representative data of three independent cases. (C) RvD2 induced phosphorylation of STAT3 in DPCs. Cells were stimulated with RvD2 (100 nM) for 0, 1, 5, 15, and 30min. Western blotting revealed that RvD2 significantly induced phosphorylation of STAT3 after 1-min stimulation compared to non-treated DPCs. In addition, phosphorylation of STAT3 was much higher than in non-treated DPCs after stimulation for 5 and 15 min (*P < 0.05 indicates significant differences compared to the control group, Mann–Whitney test). Representative data of three independent experiments. M, molecular weight marker.