Enterococcus facaelis attenuates osteogenesis in MC3T3-E1 cells

Abstract

OBJECTIVES: Periapical periodontitis is considered an inflammatory lesion around root apex caused by bacterial infection and commonly leads to periapical bone destruction. Enterococcus facaelis, as a pathogen, is usually isolated in primary and persistent endodontic infections, but its role in bone destruction is still unclear. Our study is to explore the mechanism of E. facaelis on bone destruction. METHODS: Pre-osteoblastic MC3T3-E1 cells were infected by E. facaelis ATCC 29212 and clinical E. facaelis P25RC strain, respectively. Cell proliferation was examined by MTT assay up to 8 days. Bone calcium deposition was measured by alizarin red S staining at 7 and 14 days of culture. Alkaline phosphatase (ALP) activity was assayed with BCIP-NBT solution at 7, 14 and 21 days of culture. Gene expression of ALP, osteocalcin (OC), runt related protein 2 (Runx2), osteoprotegerin (OPG) and collagen type 1 (COL1) were detected at 0, 7, 14 and 21 days of culture. MAPK signaling pathway activation was analyzed by western blotting at 3 and 7 days of culture. Culture supernatants from RAW264.7 cells infected with E. facaelis were used to stimulate MC3T3-E1 cells. RESULTS: E. facaelis markedly suppressed proliferation after treatment for 4, 6 and 8 days. Formation of mineralized nodules was significantly inhibited by E. facaelis shown by alizarin red S staining. ALP activity also significantly reduced after E. facaelis treatment. The gene expression of the five osteoblast markers was significantly down-regulated after 21-day E. facaelis treatment. Western blotting results suggested that E. facaelis activated phosphorylation of p38 and ERK1/2. The products from infected RAW264.7 cells contributed to apoptosis of MC3T3-E1 cells. CONCLUSIONS: E. facaelis has an inhibitory effect on osteogenesis, especially for a long time persistent infection. The mechanism of E. facaelis on osteoblasts may become a therapeutic target for persistent periapical periodontitis.