Oral and maxillofacial bone defects, stemming from trauma, tumor removal, or infections like periodontitis, pose significant challenges due to their irregular nature and susceptibility to chronic inflammation, which hinders healing. Normal bone repair involves inflammation, regeneration, and remodeling; however, persistent inflammation impairs this process. The oral cavity's complex microbiota increases the risk of infection, further complicating repair. Therefore, biomaterials with immunoregulatory capabilities are crucial for effective treatment. MicroRNAs (miRNAs), such as miR-146a, regulate gene expression and play a vital role in immunoregulation and bone homeostasis. miR-146a inhibits the NF-κB pathway, modulating the immune response to infection and potentially influencing bone formation. Magnesium silicate nanospheres (MSNs) are biocompatible, have high drug-loading capacity, and promote osteogenesis. This study hypothesized that combining miR-146a with MSNs would create a synergistic effect, enhancing bone regeneration in inflammatory environments.

Previous research highlights miR-146a's role in both immunoregulation and bone homeostasis. It negatively regulates NF-κB-mediated immune responses by targeting TRAF6 and IRAK1, key adapter molecules in this pathway. Its expression is altered in periodontitis and linked to conditions like ankylosing spondylitis and age-related bone loss. Effective delivery of nucleic acid drugs is crucial, and MSNs, with their rough surface and mesoporous structure, offer a promising delivery vector. Studies have shown their biocompatibility, cellular uptake efficiency, and ability to promote osteogenic differentiation, possibly by releasing magnesium ions that activate the Wnt/β-actin pathway. The potential synergistic effect of combining miR-146a with MSNs as a delivery system for bone regeneration in an inflammatory environment is the focus of this research.

The study involved synthesizing MSNs using a two-step method, modifying them with polyethyleneimine (PEI) to enhance miR-146a loading, and characterizing the resulting MSN+miR-146a complex using SEM, TEM, and EDS. The optimal miR-146a loading ratio was determined via a gel retardation assay and zeta potential analysis. The biocompatibility of MSNs was assessed using a CCK-8 assay on hDPSCs. Cellular uptake of the MSN+miR-146a complex was evaluated using fluorescence microscopy. In vitro experiments assessed the impact of MSN+miR-146a on osteogenic differentiation of hDPSCs, using ALP staining, SR staining, ARS staining, enzymatic assays, qRT-PCR, and western blotting to analyze relevant markers (ALP, Col1a1, RUNX2, OSX, VEGF-A). The effect on macrophage polarization was investigated using flow cytometry, immunofluorescence (IF), and qRT-PCR to assess M1 and M2 markers (CD40, Arg-1, CD163, CD86, IL-1β, IL-6, IL-10). Osteoclast formation was evaluated using TRAP staining and qRT-PCR. An in vivo study utilized a mouse model with infected mandibular bone defects. MSN+miR-146a, delivered via a photocuring hydrogel (GelMA), was assessed for its effects on bone regeneration using micro-CT, three-point bending tests, histology (HE, Masson's trichrome), and IF staining to evaluate bone volume, mineral density, biomechanical properties, and macrophage polarization and osteogenic marker expression.

The MSN+miR-146a complex showed excellent biocompatibility and efficient cellular uptake by hDPSCs. In vitro, it significantly promoted osteogenic differentiation of hDPSCs, as evidenced by increased ALP and Col1a1 expression, mineralization, and upregulation of RUNX2 and OSX. miR-146a suppressed the expression of pro-inflammatory cytokines (IL-1β, IL-6) and promoted M2 polarization of macrophages, while inhibiting osteoclast formation. In vivo, MSN+miR-146a delivered via GelMA significantly enhanced bone regeneration in infected mandibular defects, increasing BV/TV, BMD, and biomechanical strength. Histological analysis showed increased bone formation and decreased osteoclast numbers. IF staining confirmed reduced M1 macrophages and increased M2 macrophages, along with upregulated Runx2 and Osx in the defect area.

The findings demonstrate the potential of MSN+miR-146a as a therapeutic agent for treating inflammatory bone defects. The synergistic effects of MSNs (promoting osteogenesis and M2 polarization) and miR-146a (anti-inflammatory and osteoclast inhibition) are crucial for this success. The ability of MSNs to deliver miR-146a effectively and the observed in vivo bone regeneration highlight the efficacy of this nanobiomaterial. Although MSNs upregulated VEGF-A, miR-146a counteracted this effect, suggesting a possible need for future modifications to balance angiogenic and anti-inflammatory actions. The results support the hypothesis that targeted miRNA delivery using MSNs offers a promising strategy to treat inflammatory bone defects.

This study successfully demonstrated that the MSN+miR-146a complex effectively promotes bone regeneration in an inflammatory microenvironment. The synergistic actions of MSNs and miR-146a resulted in enhanced osteogenesis, immunomodulation, and improved bone regeneration in a mouse model. Future studies should explore the use of larger animal models with more clinically relevant infection scenarios and investigate the potential of combining MSN+miR-146a with other therapeutic agents to further optimize its effectiveness.

While the study used hDPSCs and a mouse model, further investigation using other stem cell sources and larger animal models is needed to confirm generalizability. The LPS-induced infection model may not fully capture the complexity of clinical oral-maxillofacial infections. The observed upregulation of VEGF-A by MSNs, counteracted by miR-146a, requires further exploration to optimize angiogenic potential. The long-term effects of MSN+miR-146a on bone regeneration also warrant further investigation.