Six beads were made by different mixing techniques from different orthopaedic surgeons. Beads1 were made of 40g of Zimmer bone cement Zimmer, Inc, USA and 4g of
fosfomycin-sodium powder Fosmicin, MEIJI, Japan. Bone cement was prepared by mixing 40g of Zimmer bone cement powdered polymethylmethacrylate PMMA with 4g of
fosfomycin-sodium powder in a sterile bowl. After both materials are stirred up homogenously by spatula, then 2 ml of methylmethacrylate MMA liquid was poured into
these mixed materials. When the mixture became doughy, then, they poured into three templates and wait until hardening phase of the PMMA cements within ten minutes. Each
template consists of ten round holes to build spherical shapes. It shows that all beads are spherical shape and in same size. These beads were not connected on a small diameter of
stainless steel wire. Beads2 to 6 made of commercially available Simplex® P bone cement Stryker
Howmedica OSTEONICS, Howmedica International S, Limerick, Ireland. Bone cement was prepared by mixing the powdered PMMA with MMA liquid monomer in a bowl with a
spatula. After both materials are mixed homogenously and doughy, then 2g of fosfomycin- sodium was added manually. When the mixture became doughy and non-sticky within four
minutes, spherical shape beads were hand rolled in a various diameters between 5 to 10 mm’s. The beads were threaded onto a small diameter of stainless steel wire and the ends of wire
were knotted see Figure 3. All procedures were carried out under sterile conditions. By different orthopaedic surgeons handrolled fosfomycin-loaded bone cement bead sets were
built in spherical shape. The size and weight of these beads are varied between 1.57 cm and 2.84 cm, with a weight range of 0.51g to 2.44 g.
Figure 3. Hand-made fosfomycin-loaded PMMA beads; ten handrolled beads attached on a
wire.
2.2. Antibacterial efficacy
Beads1 to beads6 and Septopal® beads were immersed in 10 ml of sterile Phosphate Buffer Saline, PBS NaCl 8.76 gl, K
2
HPO
4
43.5 gl, KH
2
PO
4
34.5 gl, pH 7.4 and incubated at 37
C. After 24h of incubation 15 µl samples were taken and these were placed on seeded Trypton Soya Brooth TSB agar plates. The zones of inhibition were established by
measuring the clear areas around one drop of elution fluid. Absence of an inhibition zone was taken as a sign that the antibiotic concentration was to low to inhibit bacterial growth. For this
study we used ten clinical strains isolated from patients with an implant-related infection of the University Medical Center Groningen, The Netherlands. Distribution of the strains are
according to the occurrence of osteomyelitis.
11,12
Each strain was diluted in 4 ml of 0.9 saline to concentration approximately of 10
8
bacteriaml. After overnight incubation at 37 C of
the seeded agar plates the inhibition zone was scored size in mm. The zone of inhibition was considered as clear area around of a drop of elution medium in which bacteria were not able
to grow. In addition, the minimal inhibitory concentration MIC; µgl of the isolated bacteria
against fosfomycin and gentamicin and possible subpopulations see Table 1 were determined by an E-test AB Biodisk, Dälvagen,
Sweden.
Table 1. MICs values of the ten bacterial strains used in this study.
2.3. Antibiotic release
Six different handmade beads and Septopal® beads were immersed in 10 ml sterile PBS NaCl 8.76 gl, K
2
HPO
4
43.5 gl, KH
2
PO
4
34.5 gl, pH 7.4 and incubated at 37 C. At
indicated time points 24h, 48h, 72h, and 144h, the beads were transferred to fresh 10 ml PBS and again incubated at 37
C. Duration of fosfomycin release was established by taking 15 µl samples at the indicated time points and placing this drop on TSB agar plates seeded
with the ten clinical bacterial strains showing any antibacterial efficacy. The antibiotics release was determined by measuring the clear areas in four quadrants on one agar plate. Each
quadrant contained one drop 15 µl of the elution fluid taken at the indicated time points. The
Bacterial strains MICs value µgml
Fosfomycin Gentamicin
Staphylococcus aureus 5298 96
0.75 Staphylococcus aureus 7323
0.19 + subpopulations 1.5
CNS 7368 48
0.38 CNS 7334
24 6
CNS 7391 0.50 + subpopulations
256 Micrococcus 7397
192 0.5
Pseudomonas aeruginosa 5148 1024
2 Pseudomonas aeruginosa 7348
32 4
Escheria coli BS 6206 1.5 + subpopulations
1.0 Klebsiella 333257
1024 1.0
ten bacterial strains were diluted in 4 ml of 0.9 saline to concentration 10
8
bacteriaml. After 24h incubation at 37
C the inhibition zone was scored size in mm. Again, absence of an inhibition zone was taken as a sign that the antibiotic concentration was to low to inhibit
bacterial growth.
3. RESULTS 3.1. Antibacterial efficacy
