Antimicrobial Silver Multilayer Coating for Prevention of Bacterial Colonization of Orthopedic Implants

Abstract

Due to increasing rates of periprosthetic joint infections (PJI), new approaches are needed to minimize the infection risk. The first goal of this study was to modify a well-established infection model to test surface-active antimicrobial systems. The second goal was to evaluate the antimicrobial activity of a silver multilayer (SML) coating. In vitro tests with SML items showed a >4 Log reduction in a proliferation assay and a 2.2 Log reduction in an agar immersion test (7 d). In the in vivo model blank and SML coated K-wires were seeded with ~2 × 10⁴ CFU of a methicillin-sensitive Staphylococcus epidermidis (MSSE) and inserted into the intramedullary tibial canal of rabbits. After 7 days, the animals were sacrificed and a clinical, microbiological and histological analysis was performed. Microbiology showed a 1.6 Log pathogen reduction on the surface of SML items (p = 0.022) and in loosely attached tissue (p = 0.012). In the SML group 7 of 12 SML items were completely free of pathogens (cure rate = 58%, p = 0.002), while only 1 of 12 blank items were free of pathogens (cure rate = 8%, p = 0.110). No silver was detected in the blood or urine of the SML treated animals and only scarcely in the liver or adjacent lymph nodes. In summary, an in vivo infection model to test implants with bacterial pre-incubation was established and the antimicrobial activity of the SML coating was successfully proven.

Keywords: Staphylococcus epidermidis; in vivo osteomyelitis model; infection prophylaxis; periprosthetic joint infections.

Figure 1

(a) Test item: SML-coated AS^®/titanium K-wire, (a’) shows the two coatings in magnification. SML = bronze, AS^® = golden. (b) Blank item: AS^®-coated titanium K-wire, (b’) shows the AS^® coating in magnification. The silver color at the blunt end of each K-wire shows the uncoated titanium surface.

Figure 2

(a) Image of SML coated K-wire implanted in intramedullary canal, (b) Post-operative X-ray image of the rabbit tibia with the K-wire placement.

Figure 3

Results of the proliferation assay. Brutto Onset-OD time and 95% confidence interval for blank items and SML coated items. The netto Onset-OD of 11.2 h relates to a >4 Log reduction.

Figure 4

Results of the agar immersion test. Mean CFU count and 95% confidence interval for blank and SML coated K-wires at time point t = 0 h (t0), t = 24 h (t24), t = 72 h (t72) and t = 168 h (t168).

Figure 5

(a,b) Exemplary images of the knee joint postmortem of animals treated with (a) SML item and (b) blank item. (c,d) Exemplary images of bone marrow after explantation of (c) SML item and (d) blank item. (c) Physiological bone marrow was found in 11 of 12 animals. This equals a cure rate = 8%. (d) Fragmented and hemorrhagic aspects indicating osteomyelitis were found in 4 of 12 animals. This equals to a cure rate of 67%.

Figure 6

Individual value plot of CFU at d0 (implantation, ~2 × 10⁴ CFU) and d7 (explantation). (a) SML coated K-wires, mean CFU = 353 ± 529 CFU. This equals a cure rate of 58% (b) Blank K-wires, mean CFU = 9.282 ± 10.585 CFU. This equals a cure rate of 8%. The black dots represent the mean value of each group with a 95% confidence interval.

Figure 7

Mean values and 95% confidence interval of the CFU count of the in vivo study. (Implantation) = CFU count at implantation on the K-wire surface. (K-wire) = Bacterial suspension derived from the K-wire surface at explantation. (rinsing sol.) = Bacterial suspension derived from rising the K-wire surface to remove attached tissue. (BMstd) = Bacterial suspension derived from the bone marrow (normalized to 1 g). Light grey = Blank items, dark grey = SML items.

Figure 8

(a–c) Histology images of distal tibia of blank item animal. (a) There was a higher incidence of mild focal ongoing osteomyelitis in the distal tibia of blank K-wire implanted animals (b). Green arrows indicate new fibrous tissue (fibroplasia). Osteomyelitis was focal and showed evidence of several days old pus (black arrows) at periphery of implant, in the bone marrow and more recent exudate of intact and degenerated heterophils (red arrows and (c)) in the bone marrow immediately adjacent to the implant imprint (*), indicating active suppurative inflammation.

Figure 9

(a–c) Histology images of distal tibia of test item animal. No evidence of mild ongoing osteomyelitis along the K-wire in most SML K-wire implanted animal and stabilization was often seen to be more significant in the test item at tip of the K-wire imprint (*). Integration was by means of fibroplasia (green arrows) and new fibrous bone formation at host-implant interface ((b), blue arrows), devoid of any heterophilic infiltration (c).

Figure 10

(a–c): Test item SML K-wire-implanted rabbits with no bacterial contamination, showing lack of suppuration and excellent implant stabilization by means of fibrous connective tissue (green arrows inset (b)) and recent new bone formation (see magenta arrows in insets (b,c)). Very recent new fibrous bone formation at host-implant interface (see short magenta arrows in inset (b)), devoid of any heterophilic infiltration, and more mature and older new fibrous bone (see longer magenta arrows in panel (c) from adjacent step section in metaphysis region adjacent the epiphysis EP). At the interface of the removed K-wire and the new connective tissue, there is limited red blood cell extravasation or hemorrhage (red arrows in inset (b)).

Figure 11

Individual value plot of silver levels measured by inductively coupled plasma mass spectrometry (ICP-MS) on day 7 (explantation) in blood, urine, liver and two lymph nodes.