Authors: Brooks JL, Courtney H, Lewis G, Haggard WO, Memphis, TN
Title: Biomechanical study for daptomycin in bone cement
Purpose: The purpose of this study was to evaluate the elution behavior and fatigue properties of bone cement loaded with various concentrations of daptomycin, as well as to characterize of the eluate activity.
Methods: Sample Preparation: Bone cement, Orthoset Type I (Wright Medical, Arlington, TN), was mixed with varying amounts of daptomycin (Cubist, Lexington, MA). The concentrations of daptomycin in bone cement used were 0% (control), 2.25%(Set A), 4.5%(Set B), and 11%(Set D). The cement was then extruded from a cement gun into a silicone rubber mold to produce dog-bone shaped specimens. Once dried, the samples were removed from the mold and selected for testing.
Fatigue Testing: The samples were examined visually and radiographically for defects, and those with no flaws greater than 0.5 millimeter were selected for fatigue testing. Each specimen was immersed in 1X phosphate buffered saline (PBS) and subjected to a fully-reversed tension-compression loading, which corresponds to a stress of +/- 15 Megapascals (MPa) at a frequency of 2 Hertz (ASTM F 2118-03). The number of cycles until failure (Nf) was recorded. Elution Testing: From the remaining specimens, groups of th! ree were randomly chosen for elution testing. Each sample was weighed to account for the various defects. The specimen was placed in a 15 milliliter (mL) centrifuge tube and 10 mL of PBS was added. One mL aliquots were taken at days 1, 2, 5, 7, 10, 14, 21, and 28. PBS was completely refreshed at each time point. The daptomycin concentrations were tested using HPLC.
Activity Testing: Sterile samples were taken in the same manner as the elution samples. After elution testing was completed, a range of daptomycin concentrations of the eluates was tested for activity. Two sets of tubes were prepared with Mueller Hinton II broth supplemented with CaCl2. Aliquots of the samples were added to the broth. One set of tubes was inoculated with Staphylococcus Aureus, while the other set was used as a control. All the tubes were incubated at 37 degrees Celsius overnight. The tubes were then read using a spectrophotometer at 540 nanometers (nm) to determine the optical density.
Results: Fatigue testing results demonstrated a decrease in Nf as the concentration of daptomycin increased (fig1). Bone cement with no daptomycin had an Nf value around 150,000 cycles, whereas bone cement with 11% daptomycin (Set D) had an Nf value less than 50,000 cycles. Elution testing results displayed an initial bolus release within the first 24 hours (fig 2). An initial bolus release from set D (11%) of 430 micrograms (ug)/ml was observed in the first 24 hours. Continued release of daptomycin was seen through day 7. After day 7, the release from all samples measured was negligible. The samples chosen for the activity testing were days 1-7 of sets A, B, and D, day 1 of the control sample, and blank PBS. The results showed that the daptomycin is still active through day 7, even at concentrations as low as 0.04 ug/ml. The control sample and blank PBS showed no activity against the S. Aureus (table 1).
Discuassion and Conclusion: With methicillin-resistant S. Aureus (MRSA) being a major problem in hospital environments, the need for effective uses of daptomycin will continue to grow. Successful incorporation of daptomycin into bone cement could lead to potentially useful treatment applications for infection prevention and eradication. The elution profile demonstrates an initial bolus release of daptomycin, and continued release through day 7. The release of the daptomycin could help to maintain an infection free site. Even after being incorporated into the bone cement, the daptomycin retains its activity against S. Aureus. The fatigue testing data indicates a significant decrease in strength with daptomycin loading at 2.25%. These decreases highlight the necessity for optimization of the concentration of daptomycin to have a final cement with acceptable fatigue testing results while still maintaining high activity against S. Aureus.
