Immunomodulatory effect of dimethyloxallyl glycine/nanosilicates-loaded fibrous structure on periodontal bone remodeling

Abstract

Background/purpose: Relieving immuno-inflammatory responses is the prerequisite step for treating periodontitis. The angiogenic small molecule, dimethyloxalylglycine (DMOG), and osteoinductive inorganic nanomaterial, nanosilicate (nSi) have a powerful effect on bone regeneration, whereas the roles in osteoimmunomodulation have not been totally uncovered. Our study aimed to explore the immunomodulatory effect of DMOG/nSi-loaded fibrous membranes on periodontal bone remodeling.

Materials and methods: The fibrous membranes were prepared by incorporating DMOG and nSi into poly (lactic-co-glycolic acid) (PLGA) with electrospinning. The morphology features, surface chemical property and biocompatibility of DMOG/nSi-PLGA fibrous membranes were characterized. Thereafter, the fibrous membranes were implanted into rat periodontal defects, bone remodeling potential and immunomodulatory effect were evaluated by micro-computed tomography (micro-CT), histological evaluation and immunohistochemical analysis.

Results: DMOG/nSi-PLGA membranes possessed favorable physicochemical properties and biocompatibility. After the fibrous membranes implanted into periodontal defects, DMOG/nSi-PLGA membranes could relieve immuno-inflammatory responses of the defects (reduction of inflammatory cell infiltration, CD40L and CD11b-positive cells), increased CD206-positive M2 macrophages, and eventually facilitated periodontal bone regeneration.

Conclusion: DMOG/nSi-PLGA fibrous membranes exert protective effects during periodontal bone defect repairing, and steer immune response towards bone regeneration. Consequently, DMOG/nSi-PLGA fibrous membranes may serve as a promising scaffold in periodontal tissue engineering.

Keywords: DMOG; Electrospinning; Immunomodulation; Nanosilicate; Periodontal bone regeneration.

Figure 1

Characterization and cytocompatibility assessment of the fibrous membranes (A, B) SEM images of different fibrous membranes. Scale bar = 100 μm. (C, D) Diameter distribution analysis of different fibrous membranes. (A, C) PLGA (B, D) DMOG/nSi-PLGA. (E) FTIR spectra of the fibrous membranes. (F) Cell activity detected by CCK-8 assay after cultivation on TCP (tissue culture plate) and different fibrous membranes for 72 h. Data represent mean ± standard deviation (n = 3).

Figure 2

Histological analysis of tissue regeneration and inflammatory cell infiltration in five groups. (A) H&E staining of periodontal defect at 1 and 2 weeks post-surgery. The visual fields framed by the black line were magnified in the images below. (B) Quantitative analysis of newly formed bone areas in the five groups at two time points. (C) Quantitative analysis of number of inflammatory cells in new bone regeneration areas. Scale bar = 50 μm. Data represented mean ± standard deviation (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 3

Immunohistochemical analysis of iNOS (A) and CD206 (B) expression level at 1 and 2 weeks post-surgery in five groups. (A) Immunochemical staining of iNOS. (B) Immunochemical staining of CD206. (C) Quantitative analysis of iNOS-positive cells. (D) Quantitative analysis of CD206-positive cells. Scale bar = 50 μm. Data represented mean ± standard deviation (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 4

Expression of inflammatory factors at 1 and 2 weeks post-surgery in five groups. (A, B) Immunofluorescent staining of CD40L. Red indicated CD40L-positive cells. (C) Immunochemical staining of CD11b. (D) Quantitative analysis of CD40L-positive cells. (E) Quantitative analysis of CD11b-positive cells. Scale bar = 50 μm. Data represented mean ± standard deviation (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Figure 5

3D digital Micro-CT analysis of new bone regeneration at 1 and 2 weeks post-surgery in five groups. (A) Reconstructed 3D digital micro-CT images of mandibular bone defects. Green color presented repair area in mandibles after surgery. (B–D) Quantitative analysis of BV/TV, Tb. Th, and Tb. Sp by reconstruction and analysis software. Data represented mean ± standard deviation (n = 6). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.