Materials and Methods

Medical grade UHMWPE was molded together with vancomycin at 2, 4, 6, 8, 10 and 14 wt%. Tensile mechanical testing and impact testing were performed to determine the effect of drug content on mechanical properties. Elution of the drug was performed in phosphate buffered saline (PBS) for up to 8 weeks and the detection of the drug in PBS was done by UV-Vis spectroscopy. A combination of vancomycin and rifampin in UHMWPE was developed to address chronic infection and layered construct containing 1 mm-thick drug-containing UHMWPE in the non-load bearing regions was developed for delivery. In a lapine (rabbit) intra-articular model (n=6 each), two plug of the layered UHMWPE construct were placed in the trochlear grove of the rabbit femoral surface and a porous titanium rod with a pre-grown biofilm of bioluminescent S. Aureus was implanted in the tibia. Bioluminescent imaging was employed to visualize and quantify the presence of the bacteria up to 3 weeks.

Methods

Materials and Methods

Skeletally mature adult male New Zealand White rabbits received either two non-antibiotic eluting UHMWPE (CONTROL, n=5) or vancomycin-eluting UHMWPE (TEST, n=5) (3 mm in diameter and 6 mm length) in the patellofemoral groove (Fig. 1). All rabbits received a beaded titanium rod in the tibial canal (4 mm diameter and 12 mm length). Both groups received two doses of 5 × 10⁷ cfu of bioluminescent S. aureus (Xen 29, PerkinElmer 119240) in 50 µL 0.9 % saline in the following sites: (1) distal tibial canal prior to insertion of the rod; (2) articular space after closure of the joint capsule (Fig. 1). None of the animals received any intravenous antibiotics for this study. Bioluminescence signal (photons/second) was measured when the rabbits expired, or at the study endpoint (day 21). The metal rods were stained with BacLight^® Bacterial Live-Dead Stain and imaged using two-photon microscopy to detect live bacteria. Hardware, polyethylene implants and joint tissues were sonicated to further quantify live bacteria via plate seeding.

Methods

A gamma-sterilized, HXL UHMWPE tibial bearing containing VE (E1, Biomet, “VE-PE”) and 100kGy irradiated and melted UHMWPE (“CISM 100”) were cryomilled to particles by Bioengineering Solutions (Oak Park, IL). In the first in vitro study, RAW 264.7 mouse macrophages were exposed (inverted co-culture) to either VE-PE particles or CISM100 particles and lipopolysaccharide (LPS) for 1–7 days. Macrophage viability was measured using a cell counting kit (CCK-8). Control group with no particles and a LPS group were also included. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to determine macrophage apoptosis rate in response to particle exposure over time. In the second study, macrophages were exposed to VE-PE or CISM100 particles for 48h, then exposed to LPS for 30 min. Subsequently, reactive oxygen species (ROS) generation and extracellular regulated protein kinase (ERK) phosphorylation were measured. In a third study, after exposure to particles for 48h, fatigued macrophages were co-cultured with bioluminescent S. aureus strain Xen29 for 3h and 6h. Bioluminescence signal was determined to measure the total amount of bacteria. Bacterial live/dead staining and optical density at 600 nm (OD 600) were also performed to determine S. aureus viability. Statistical analysis was performed using one-way or two-way ANOVA with a post hoc examination. *indicates p<0.05.