An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

Acute exposure of white-tailed deer cortical bone to Staphylococcus aureus did not result in reduced bone stiffness.

Staphylococcus aureus (S. aureus) is the main source of osteomyelitis in adults. The end-result of untreated osteomyelitis is bone necrosis and distraction of bone structure. While bone tissue can heal and remodel its structure to ameliorate its mechanical properties, so far no study has tested the mechanical properties of cortical bone tissue exposed to S. aureus. With the increase usage of bone banks as a source of bone graft supply, it is important to screen for any possible pathology that may affect the bone graft success to function normally in the receiving patient. This study tested the effect of acute exposure to S. aureus on cortical bone stiffness. We have postulated that the incubation of cortical bone with S. aureus for 48 h will result in a significant decrease in bone stiffness. Sixty-five bone cubes (2 × 2 × 2 mm) were prepared from the cranial and caudal aspects of four white-tailed deer mid-diaphysis humeri. First, all bone samples were tested to determine their stiffness in the three principle orientations (axial, radial and transverse). Next, bone samples were incubated for 48 h with S. aureus (32 cubes, experimental group) or with sterile distilled water (33 cubes, control group). Finally, all cubes were mechanically tested again and each stiffness value was compared to the original value obtained from the same cube. Our results revealed that overall, acute exposure to S. aureus did not significantly decrease bone stiffness and thus our working hypothesis could not be supported. Therefore, our findings support the current tissue collection screening methods employed by bone-graft banks.

Evidence of Staphylococcus Aureus Deformation, Proliferation, and Migration in Canaliculi of Live Cortical Bone in Murine Models of Osteomyelitis.

Although 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 to 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. © 2016 American Society for Bone and Mineral Research.