Abstract
Fusobacterium nucleatum is one of the most frequent pathogenic bacteria causing periodontitis. The direct effect of Fusobacterium nucleatum (F. nucleatum) on oral stem cells has rarely been reported. In this study, we aimed to evaluate how gingiva-derived mesenchymal stem cells (GMSCs) respond to a direct challenge with F. nucleatum. GMSCs were isolated by the limiting dilution method and exposed to F. nucleatum at various multiplicities of infection (MOIs; F. nucleatum:cell ratios of 10:1, 50:1, and 100:1) for 24 h to 4 weeks. Our results indicated that F. nucleatum significantly inhibited cell proliferation in a dose-dependent manner and promoted cell migration and the release of chemokines/cytokines, such as CCL2, CXCL1, and IL-6. Additionally, F. nucleatum inhibited GMSC osteogenic differentiation partly by decreasing alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenesis-related gene and protein expression. RNA-sequencing analyses indicated that F. nucleatum time-dependently activated cellular signaling pathways during the process of osteogenic differentiation. A total of 64 cell differentiation-related genes were found to be differentially expressed between non-infected and F. nucleatum-infected GMSCs at 3, 7, 14, and 21 d. Intriguingly, we discovered that the 64 cell differentiation-related differentially expressed genes (DEGs) were significantly enriched in cancer-related pathways, such as bone cancer, osteosarcoma and bone marrow cancer, which provides new insight into tumorigenesis during the process of GMSC osteogenic differentiation. In conclusion, this study demonstrates that persistent exposure to F. nucleatum promotes cell migration and chemokine/cytokine release and inhibits the proliferation and osteogenic differentiation of GMSCs. Our study provides a novel and long-time bacteria-cell co-culture in vitro model and makes a foundation for the future mechanistic studies of GMSCs under F. nucleatum infection.
Highlights
Oral microorganisms are abundant and play key roles in maintaining oral health; periodontitis is a typical chronic infectious disease caused by increased oral pathogenic bacterium (Paster and Dewhirst, 2009)
The results indicated that the mineral deposition of gingival tissuederived mesenchymal stem cells (GMSCs) increased time-dependently from 14 to 28 d, and large quantities of mineralized nodules were detected at day 28 in the GMSCs without infection by F. nucleatum, whereas in GMSCs co-cultured with F. nucleatum, the osteogenic medium-induced mineral deposition formation on days 21 and 28 were dose-dependently inhibited by F. nucleatum (Figures 4B,C)
The stimulatory mechanism of inflammatory bone resorption in periodontitis induced by P. gingivalis has been comprehensive reported (Hanazawa, 1998; Jia et al, 2019), whereas the defined effect of F. nucleatum on oral tissue destruction has rarely been explored
Summary
Oral microorganisms are abundant and play key roles in maintaining oral health; periodontitis is a typical chronic infectious disease caused by increased oral pathogenic bacterium (Paster and Dewhirst, 2009). F. nucleatum is an opportunistic pathogen closely associated with the occurrence and development of periodontitis, and its abundance is positively associated with the periodontal pocket depth (Papapanou et al, 1993; Moore and Moore, 1994; Socransky et al, 1998). F. nucleatum has its unique pathogenic potential and is dominant in the dental biofilm at a later stage, which play critical roles in the destruction of periodontal supporting tissues (Moore and Moore, 1994; Ebersole et al, 1995). If F. nucleatum could directly destruct the periodontal supporting tissues and the potential pathogenic mechanism have not been fully elucidated
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