The effectiveness of the controlled release of gentamicin from polyelectrolyte multilayers in the treatment of Staphylococcus aureus infection in a rabbit bone model

Abstract

While the infection rate of orthopedic implants is low, the required treatment, which can involve six weeks of antibiotic therapy and two additional surgical operations, is life threatening and expensive, and thus motivates the development of a one-stage re-implantation procedure. Polyelectrolyte multilayers incorporating gentamicin were fabricated using the layer-by-layer deposition process for use as a device coating to address an existing bone infection in a direct implant exchange operation. The films eluted about 70% of their payload in vitro during the first three days and subsequently continued to release drug for more than four additional weeks, reaching a total average release of over 550 microg/cm(2). The coatings were demonstrated to be bactericidal against Staphylococcus aureus, and degradation products were generally nontoxic towards MC3T3-E1 murine preosteoblasts. Film-coated titanium implants were compared to uncoated implants in an in vivo S. aureus bone infection model. After a direct exchange procedure, the antimicrobial-coated devices yielded bone homogenates with a significantly lower degree of infection than uncoated devices at both day four (p < 0.004) and day seven (p < 0.03). This study has demonstrated that a self-assembled ultrathin film coating is capable of effectively treating an experimental bone infection in vivo and lays the foundation for development of a multi-therapeutic film for optimized, synergistic treatment of pain, infection, and osteomyelitis.

Fig. 1

Structure of the repeat unit for the hydrolytically degradable Poly 1 cation species.

Fig. 2

(Left) Structure of gentamicin. Sites protonated in fully charged state are indicated. Information on R₁ groups and their relative content percentages can be obtained from Mediatech, Inc. (Lot #: 61098046). (Right) In vitro efficacy of gentamicin sulfate against S. aureus (ATCC 49230) in CMHB over a 16 h incubation period at 37 °C.

Fig. 3

Cumulative amount of gentamicin released from [Poly 1 / Anion / GS / Anion]₅₀ films.

Fig. 4

(Left) Growth curve for [Poly 1/PAA/GS/PAA]_n. SEM images are provided for the circled data points. Thicknesses were measured by profilometry at four predetermined points on each substrate and averaged over three replications. The error bars represent the average root mean squared roughness from triplicate samples and quadruplicate measurements per sample. (Right) SEM images of the growing film on a silicon surface at (A) n = 25, (B) n = 50, (C) n = 100, and (D) n = 200 layers. White arrows mark the film edge. Nota bene: the scale changes between (B) and (C).

Fig. 5

(A) Normalized film erosion from [Poly 1 / PAA / GS / PAA]₂₀₀ films and normalized GS release from [Poly 1 / PAA / GS / PAA]₂₀₀ + [Poly 1 / PAA / GS ]₁ films. Erosion values are normalized to initial film thickness with the error bars representing the normalized rms roughness. Release values are normalized to the final release quantity (582 μg/cm²). Release data have truncated for convenience of comparison. (B) Cumulative antibiotic release from [Poly 1/PAA/GS/PAA]₂₀₀ + [Poly 1/PAA/GS]₁ films in m-SBF at 37 °C before (replotted from (A)) and after an effective dose of ethylene oxide gas. Inset contains data from the first three days.

Fig. 6

The titanium rods coated with [Poly 1/PAA/GS/PAA]₂₀₀ + [Poly 1/PAA/GS]₁ produced a baseline zone of inhibition of 25.6 mm (measured perpendicular to the long axis of the rod) against S. aureus after overnight incubation at 37 °C. As a control, the sample is referenced to a commercially available BD Sensi-Disc. The lighter color at the rod surface is a result of the ruptured agar and not the presence of bacteria. The scale bar is in centimeters.

Fig. 7

(A) MTT metabolic activity of MC3T3 cells after 16-18 hr treatment in elution buffer normalized to negative control (i.e. cells incubated in standard medium). 100x images of MC3T3 cells subjected to 16-18 hr treatment in (B) 100 and (C) 200 tetralayer elution buffer. Live cells are represented by a blue nucleus surrounded by green cytoplasm. Dead cells are represented by a red nucleus. (D) Percentage of dead cells calculated from triplicate images.

Fig. 8

(A) Blood agar plates of explanted titanium rods that were rolled down the center of the plate and streaked for qualitative analysis of device surface colonization. Each plate corresponds to one rabbit. Each white dot corresponds to a single CFU. Explants from the four day treatment group (top row) are compared to explants from the four day placebo group (bottom row). None of the sterile plates are depicted. (B) Explants from the seven day treatment group (top row) are compared to explants from the seven day placebo group (bottom row). (C) Final counts (Mean ± SD) of log-transformed Staphylococcus aureus CFU data in femoral condyles at day zero and after direct exchange. Raw data are available in Supplementary Table 2.