Fig. 2
Three distinct reservoirs of bacteria in chronic osteomyelitis. Chronic implant-associated osteomyelitis was established in mice with S. aureus as previously described,84,93,117,267 and the bacterial burden: (1) in Staphylococcus abscess communities (SACs) assessed by histology (a–e), (2) on the implant assessed by SEM (f–j), and (3) in cortical bone assessed by TEM (k–m) is shown. Micrographs of orange G/alcian blue-stained histology of tibiae 7 days (a) and 14 days (c) post-infection are shown highlighting SACs (arrows) in the bone marrow and adjacent soft tissues. The boxed regions in (a), (c) are shown in Brown and Brenn-stained parallel section (b, d) to highlight the Gram+ bacteria (dark blue) surrounded by dead and dying neutrophils following NETosis (red cells), which are surrounded by a ring of macrophages (white layer). Chronic infection is clearly established by day 14, as evidenced by the complete replacement of hematopoietic bone marrow (BM) with inflammatory tissue, and the presence of M2 macrophages (brown cells) surrounding the SAC, as seen by immunostaining with antibody against arginase-1 (e). Biofilm formation on the implant commences with planktonic bacterial adhesion (f), as illustrated in this case of in vitro S. aureus attachment onto a stainless-steel wire incubated in a flow chamber system (×10 000). Following transtibial implantation, the planktonic bacteria rapidly transition to biofilm (g), seen as uniform glycocalyx coating the stainless-steel pin 14 days post-op (×200). High power images of the biofilm on the implant reveal cocci adhering to fibrin strands (h, ×2 500), and clusters of S. aureus forming bacterial pods (i, ×5 000). By day 14 post-infection, bacterial emigration from the pod is complete, as evidenced by the empty lacunae (j, ×30 000). S. aureus colonization of cortical bone commences with eradication of bone lining cells to expose canaliculi (blue arrows) leading to an embedded osteocyte (OC) (k, ×6 000). Subsequently, S. aureus invasion and propagation through osteocyte lacuno-canalicular network (OLCN) renders the biofilm bacteria (*) inaccessible to activated neutrophils outside the bone (blue arrows) (l, ×1 800). The uninhibited bacteria demineralize and consume the cortical bone to expands a canaliculus, and propagate into neighboring canaliculi (yellow arrow), to reach a distant osteocyte (red arrow). m High power TEM (×12 000) of the osteocyte in (l) killed by S. aureus bacterial occupation of its lacunar space
