MicroRNA-146a-loaded magnesium silicate nanospheres promote bone regeneration in an inflammatory microenvironment

Abstract

Reconstruction of irregular oral-maxillofacial bone defects with an inflammatory microenvironment remains a challenge, as chronic local inflammation can largely impair bone healing. Here, we used magnesium silicate nanospheres (MSNs) to load microRNA-146a-5p (miR-146a) to fabricate a nanobiomaterial, MSN+miR-146a, which showed synergistic promoting effects on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). In addition, miR-146a exhibited an anti-inflammatory effect on mouse bone marrow-derived macrophages (BMMs) under lipopolysaccharide (LPS) stimulation by inhibiting the NF-κB pathway via targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), and MSNs could simultaneously promote M2 polarization of BMMs. MiR-146a was also found to inhibit osteoclast formation. Finally, the dual osteogenic-promoting and immunoregulatory effects of MSN+miR-146a were further validated in a stimulated infected mouse mandibular bone defect model via delivery by a photocuring hydrogel. Collectively, the MSN+miR-146a complex revealed good potential in treating inflammatory irregular oral-maxillofacial bone defects.

Fig. 1

Diagram of the preparation and function of MSN+miR-146a for bone regeneration with an inflammatory microenvironment. a MSNs were synthesized from SiO₂ nanoparticles and modified with PEI, and miR-146a was then loaded via physical absorption and electrostatic attraction. b The MSN+miR-146a complex significantly accelerated the osteogenesis of hDPSCs, reduced M1-type macrophages, increased M2-type macrophages, and promoted bone regeneration in infected mandibular bone defects in mice

Fig. 2

Preparation and characterization of the MSN+miR-146a complex. a–c ECM and TEM images and EDS of MSNs. d TEM images of MSN-PEI. e CCK-8 assay of hDPSCs after 8 and 24 h of coculture with PEI-modified MSNs at various concentrations. f, g Gel retardation and zeta potential tests of the MSN+miR-146a complex at different weight ratios. h–j Cellular uptake assay of MSN+miR-146a-FAM in hDPSCs after 24 h of coculture. Cells were stained to label the cytoskeleton, cell membrane or lysosome. Yellow arrows indicate colocalization of lysosomes and MSN+miR-146a-FAM. ns no significance. One-way ANOVA with Dunnett’s multiple comparisons to the blank group was used

Fig. 3

MSNs+miR-146a improved the osteogenic differentiation of hDPSCs. a, b ALP staining and SR staining of hDPSCs after 7 days of osteogenic culture. c ARS staining of hDPSCs after 14 days of osteogenic culture. d Enzyme activity assay of intracellular ALP expression in hDPSCs after 7 days of osteogenic culture. e, f IF images and quantitative analysis (n = 4) of RUNX2 (green) in hDPSCs after 7 days of osteogenic culture. g, h qRT‒PCR (n = 3) and WB results of osteogenic biomarker expression in hDPSCs after 7 days of osteogenic culture. i, j WB and qRT‒PCR (n = 3) results of the expression of VEGF-A in hDPSCs after 24 h of coculture with LPS-stimulated BMM-derived conditioned medium. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Tukey’s multiple comparisons test among all groups was used

Fig. 4

MSNs+miR-146a regulated BMM polarization and osteoclast formation. a Statistical results of flow cytometry of CD40-, Arg-1- or CD163-marked BMMs in the four groups after 24 h of LPS stimulation (n = 3). b The mRNA expression of IL-1β, IL-6, Arg-1 and IL-10 after 24 h of LPS stimulation (n = 3). c–e IF images and quantitative analysis of CD86 (red)- and Arg-1 (green)-marked BMMs after 24 h of LPS stimulation (n = 4). f, g qRT-PCR (n = 3) and WB results of the expression of TRAF6 and phosphorylation level of p65 in BMMs after 24 h of LPS stimulation. h, i TRAP staining and quantitative analysis (n = 11) of osteoclasts in the four groups after 6 days of RANKL induction. j The mRNA expression of CTSK and DC-stamp in osteoclasts after 6 days of RANKL induction (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Tukey’s multiple comparisons test among all groups was used

Fig. 5

MSNs+miR-146a accelerated bone regeneration in a stimulated infected mouse mandibular defect model. a, b Micro-CT examinations of mouse mandible samples after 2 weeks (a) and 4 weeks (b) of healing following surgery with representative reconstructed volume and slice images and the mean BV/TV, BMD and Tb.Sp (n = 7–10). c Maximal force detected in the three-point bending test of trimmed mouse mandible samples (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Dunnett’s multiple comparisons to the blank group was used

Fig. 6

MSNs+miR-146a promoted bone regeneration but inhibited osteoclast formation. a HE staining of the mouse mandible samples after 2 and 4 weeks of healing. NB new bone, OB original bone. b Masson staining of the samples after 2 and 4 weeks of healing. Yellow arrows indicate blue-stained type I collagen of new bone. c Statistical analysis of the area proportion of bone tissues in Masson-stained slices of the samples after 2 and 4 weeks of healing (n = 9–10). d TRAP staining and quantitative analysis (n = 10) of the samples after 2 weeks of healing. *P < 0.05, **P < 0.01, ***P < 0.001 One-way ANOVA with Dunnett’s multiple comparisons to the blank group was used

Fig. 7

MSNs+miR-146a regulated the in vivo polarization of macrophages and promoted osteogenic marker expression. a IF images and quantitative analysis (n = 5) of the expression of Arg-1 (green) and Runx2 (red) in the defect area of mouse mandible samples after 2 weeks of healing. b IF images and quantitative analysis (n = 8) of the expression of CD86 (red) and Arg-1 (green) in the defect area of the samples after 2 weeks of healing. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Dunnett’s multiple comparisons to the blank group was used