In vitro interactions between bacteria, osteoblast-like cells and macrophages in the pathogenesis of biomaterial-associated infections

Abstract

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.

Figure 1. Simultaneous growth of bacteria and U2OS cells under flow.

A) Phase contrast images of U2OS cells after 18 h of growth in the presence of adhering bacteria (a – control, b – S. epidermidis ATCC 35983, c – S. aureus ATCC 12600 and d – P. aeruginosa ATCC 27853) on PMMA surfaces. In Fig. 1A-b, U2OS cells are differentiated by a contour line from S. epidermidis biofilm. White arrows in Figs. 1A-c and 1A-d indicate U2OS cell death. The bar denotes 20 µm. B) Percentage change in the number of adhering U2OS cells after 48 h of growth with respect to their initial number immediately after seeding at 1.5 h on PMMA in the absence (no bacteria) and presence of adhering bacteria. Error bars represent the standard deviations over three replicates, with separately cultured bacteria and osteoblast-like cells. For S. aureus and P. aeruginosa the error bars are zero, since all percentage change in adhering numbers of U2OS cells were 100%. Cell number in the presence of bacteria is significantly different (p<0.01) from the cell number in the absence of bacteria. C) Average area per adhering U2OS cell immediately after seeding at 1.5 h (light blue) and after 48 h (dark blue) of growth on PMMA in the absence (no bacteria) and presence of adhering bacteria. Error bar represents the standard deviation over three replicates, with separately cultured bacteria and osteoblast-like cells. Average area by U2OS cells after 48 h in the presence of adhering bacteria (S. aureus and P. aeruginosa) is significantly different (p<0.05) from the absence of bacteria. D) Surface coverage by adhering U2OS cells immediately after seeding at 1.5 h (light blue) and 48 h (dark blue) of growth on PMMA in the absence (no bacteria) and presence of adhering bacteria. Error bar represents the standard deviation over three replicates, with separately cultured bacteria and osteoblast-like cells. Surface coverage by U2OS cells after 48 h in the presence of adhering bacteria are significantly different (p<0.05) from the absence of bacteria.

Figure 2. Surface coverage of U2OS cells as a function of time.

An example of surface coverage by adhering U2OS cells as a function of time on PMMA in the presence of adhering bacteria ((▴) – S. epidermidis 3399, (▪) – S. aureus A20734, (•) – P. aeruginosa DN 7348).

Figure 3. Macrophage migration towards S. epidermidis and phagocytosis under flow.

Phase-contrast images of macrophage activity toward S. epidermidis ATCC 35983 on a PMMA surface in the presence of U2OS cells: macrophage migration towards S. epidermidis (images 1–5), bacterial clearance by phagocytosis (images 6–7) and further migration (images 8–12). The bar denotes 50 µm.

Figure 4. Restriction of bacterial biofilm growth by the presence of macrophages.

The numbers of adhering bacteria on PMMA as a function of time during the simultaneous growth of bacteria and U2OS cells in the absence and presence of macrophages in a parallel plate flow chamber: S. epidermidis ATCC 35983 in the absence (□) and presence of macrophages (▪), S. aureus ATCC 12600 in the absence (○) and presence of macrophages (•), P. aeruginosa ATCC 27853 in the absence (▵) and presence of macrophages (▴).

Figure 5. Adhesion and spreading of U2OS cells in the presence of macrophages.

Phase-contrast images of adhering U2OS cells to PMMA after 24 h of simultaneous growth of bacteria (S. epidermidis ATCC 35983, S. aureus ATCC 12600 and P. aeruginosa ATCC 27853) and U2OS cells in the absence (upper images) and presence (lower images) of macrophages. Macrophages are orange-stained. The bar denotes 50 µm.