RESUMO
BACKGROUND: This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis. METHODS: Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88+/+] and Myd88-/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed. RESULTS: P. gingivalis-infected Myd88-/- mice showed alleviated bone loss, TRAP+ osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3+CD4+ T cells in infected Myd88-/- CLNs and a higher frequency of RORγt+CD4+ T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88-/- mice. The Myd88-/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88-/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection. CONCLUSIONS: MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis.
RESUMO
In terms of a novel scaffold with well good osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation. The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro. The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy. The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on preosteoblastic MC3T3-E1 cells was characterized in vitro. An in vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period. In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced alkaline phosphatase (ALP) mRNA expression and ALP activity compared with the control group. Meanwhile, the osteoblast-specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), collagen I (Col I), and osteocalcin (Ocn), were also significantly upregulated. Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared with controls. In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of preosteoblast cells in vitro.
