While Staphylococcus aureus osteomyelitis is considered to be incurable, the major bacterial reservoir in live cortical bone has remained unknown. In addition to biofilm bacteria on necrotic tissue and implants, studies have implicated intracellular infection of osteoblasts and osteocytes as a mechanism of chronic osteomyelitis. Thus, we performed the first systematic transmission electron microscopy (TEM) studies to formally define major reservoirs of S. aureus in chronically infected mouse (Balb/c J) long bone tissue. Although rare, evidence of colonized osteoblasts was found. In contrast, we readily observed S. aureus within canaliculi of live cortical bone, which existed as chains of individual cocci and submicron rod-shaped bacteria leading to biofilm formation in osteocyte lacunae. As these observations do not conform to the expectations of S. aureus as non-motile cocci 1.0–1.5 µm in diameter, we also performed immunoelectron microscopy (IEM) following in vivo BrdU labeling to assess the role of bacterial proliferation in canalicular invasion. The results suggest that the deformed bacteria: 1) enter canaliculi via asymmetric binary fission; and 2) migrate toward osteocyte lacunae via proliferation at the leading edge. Additional in vitro studies confirmed S. aureus migration through a 0.5 µm porous membrane. Collectively, these findings define a novel mechanism of bone infection, and provide possible new insight as to why S. aureus implant related infections of bone tissue are so challenging to treat.

In the present study, we successively extracted the Pu-erh tea with acetone, water, chloroform, ethyl acetate, and n-butanol, and the extracts were then isolated by column chromatography. Our study demonstrates that the Pu-erh tea ethyl acetate extract, n-butanol extract, and their fractions had superoxide anion and hydroxyl radical scavenging activity: fractions 2 and 8 from the ethyl acetate extract and fractions 2, 4, and 5 from the n-butanol extract showed protective effects against hydrogen peroxide-induced damage in human fibroblast HPF-1 cells and increased the cells' viability under normal cell culture conditions. In addition, it is found that these fractions, except fraction 5 from the n-butanol extract, decreased the accumulation of intracellular reactive oxygen species in hydrogen peroxide-induced HPF-1 cells. Interestingly, the antioxidant effect of fraction 8 from the ethyl acetate extract on the above four systems was much stronger than that of the typical green tea catechin (-)-epigallocatechin-3-gallate, but there were almost no monomeric polyphenols, theaflavins, and gallic acid in fraction 8.