a Department of Orthopaedics and Central Bone Bank Vienna, b Department of Infection Control, Danube Hospital, Langobardenstrasse 122, 1220 Vienna; c Department of Clinical Pharmacology and d Clinical Department of Infections and Chemotherapy, University of Vienna, Währinger Gürtel 1820, 1090 Vienna, Austria
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Abstract |
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Introduction |
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Local application of antibiotics can provide high drug concentrations at the site of infection and can avoid systemic effects.10,11 Impregnated implants have been developed; the most widely evaluated of these is polymethyl methacrylate (PMMA) in combination with gentamicin.1218 These implants have several disadvantages, however: (i) non-resorbable implants have to be removed, leaving empty spaces, which have to be filled, usually requiring further surgery; (ii) gentamicin is not the agent of choice for infections with Gram-positive strains. (iii) although concentrations of locally applied antimicrobial agents initially exceed those achievable by systemic application, these high concentrations are short-lived. Once empty, the carrier may even act as a bed for new colonization with surviving, selected bacteria.19,20
To overcome these problems, an antibiotic carrier is needed which should provide: (i) effective bactericidal activity against all causative pathogens, including MRSA; (ii) a sustained, high concentration at the site of infection without local or systemic toxicity; and (iii) repair and healing of bone defects without further surgery.
Vancomycin is effective against most Gram-positive pathogens and is the agent of choice for infections with MRSA.21 Staphylococci, both coagulase-positive and -negative, are susceptible to vancomycin at concentrations of 15 mg/L,2 making this ideal for treating infection with Gram-positive strains.22 Tobramycin is effective against many Gram-negative and Gram-positive pathogens, covering the majority of the relevant spectrum in orthopaedic surgery.4 Combining vancomycin and/or tobramycin with a resorbable or, even better, osteoconductive carrier, can provide effective antibiotic concentration at the site of infection and avoid the need to remove the carrier.
Polylactates or collagen sponges are used clinically as resorbable carriers, but they do not restore bone stock and are not capable of mechanical loading.2328 Allogeneic cancellous bone has been proven to be effective in restoration of bone stock.29,30 Vancomycin- or tobramycin-impregnated bone grafts have been used clinically for filling infected bone defects after osteomyelitis31,32 and in exchange procedures after replacement of infected joints.33 However, a standardized incubation technique has not yet been established and the concentrations of antibiotics reached inside the graft and at the site of infection have not yet been investigated.
We have developed a preparation technique that gives reproducibly high quantities of vancomycin and tobramycin inside bone grafts. The aim of this study was to measure the kinetics of elution of vancomycin and tobramycin from impregnated bone of human and bovine origin.
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Materials and methods |
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Bone of human origin was obtained from organ donors according to national and international regulations. We only used bone that was unsuitable for transplantation because of incomplete serological testing. Bone was harvested from the femur and the proximal tibia, and was separated into cancellous and cortical parts. After removal of adhering soft tissue it was cut into pieces of 15 mm side length. Fatty bone marrow, cells and small amounts of remaining soft tissue were removed by means of a series of shaking baths in ether, 70%, 50% and 30% ethanol and hydrogen peroxide. The bone was then frozen at 50°C and freeze-dried to a residual moisture of <5% water content. The dry bone was sealed in bottles containing argon and was irradiated using a 60Co source with a dosage of 2530 kGy at room temperature. The specimens were stored at room temperature until incubation.
In a second series, commercially available bone of bovine origin (Lubboc; Ost-Developpement, Clermont-Ferrand, France) registered for clinical transplantation was evaluated. The patented manufacturing process eliminates tissue and cellular elements contained in intratrabecular spaces, but leaves type 1 collagen fibres of the cancellous bone intact. Granules with an average diameter of 3.5 mm and two cubes (5 mm sides) were freeze-dried and irradiated as described above.
Vancomycin and tobramycin were obtained from Eli Lilly, Indianapolis, IN, USA. They were dissolved in distilled water, vancomycin at 1 g/10 mL and tobramycin at 800 mg/10 mL. Grafts were incubated in these solutions for 24 h, then rinsed twice in saline. The remaining solution served as a control.
One vancomycin-impregnated human spongy compound sample was freeze-dried again and stored at room temperature for 3 weeks. Evaluation of this specimen started 24 h after rehydration in water.
Six samples, weighing 1 g, were chosen from the granulate specimens, and two blocks of bovine vancomycinincubated bone were placed in 3 mL of 5% human albumin at 37°C. The albumin was replaced completely every 24 h for 10 days, and thereafter on days 13, 15, 20, 22 and 28. All samples collected during the study were stored in liquid nitrogen until assayed for antibiotic content.
Measurement of antibiotics
Vancomycin concentrations were measured using high pressure liquid chromatography (HPLC), and tobramycin concentrations using bioassay. The chromatographic system consisted of a Shimadzu S/L6B autoinjection port, a Shimadzu LC9A pump and a UVvisible 240 nm SPD-10AV Shimadzu LC workstation (Shimadzu, Tokyo, Japan), with a CO1 UDHSE column. The mobile phase consisted of 525 mL of 50 mM KH2PO4 and 1020% (w/w) acetonitrile buffered with H3PO4 to a pH of 2.53. The flow rate was 1 mL/min. The effluent was monitored at 240 nm. A solution of 10 mg ß-hydroxypropyltheophylline in 10 ml of HPLC-grade water served as an internal standard. One millilitre of this stock solution was diluted with acetonitrile to give a total volume of 10 mL, resulting in an internal standard concentration of 100 mg/L. The extraction procedure and chromatographic conditions were as follows: 1 mL of albumin sample was added to 2 mL of acetonitrile (pH 2.53), vortexed for 60 s and centrifuged at 7500g for 150 s. Tissue samples were weighed and diluted with 0.9% saline, so that 1 mL contained 100 mg of tissue. The samples were then homogenized, vortexed in 1.52 mL 50 mM KH2PO4 for approximately 60 s and centrifuged at 14000g for 10 min. Twenty microlitres of the supernatant were injected into the HPLC column through the autoinjector. The lower detection limit was 0.45 mg/L.
Tobramycin concentrations in the supernatant were measured by means of a microagar diffusion test with antibiotic agar 1 (Merck, Darmstadt, Germany) and S. aureus ATCC 65389 as the test organism, used at a final concentration of approximately 106 cfu/mL on the assay plate. Standards were placed into wells of the assay plates at concentrations of 160 to 0.156 mg/L (log2 dilutions). Assay plates with standard and sample were incubated overnight at 37°C and zones of inhibition were read to the nearest 0.1 mm. The lower detection limit of the assay was <0.156 mg/L.
Statistical analysis
The TESTIMATE software package (Test & Estimation; IDV, Gauting) was used for statistical calculations. P values of <0.05 were considered statistically significant. The WilcoxonMannWhitney U-test was used for comparisons between the different groups. The release of active agent on each day and the area under the curve (AUC) was compared from day 1 to day 28. The significance level (i.e. the probability of rejecting H0 when it is true) was 0.05; the type II error was 0.1.
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Results |
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Human cancellous bone.
Human cancellous (spongy) bone in combination with vancomycin generated initial mean vancomycin concentrations of 20904.66 ± 1844 mg/L (range, 18699.5823443.73 mg/L), decreasing to 4.43 ± 0.95 (3.275.95) mg/L after 11 complete exchanges.
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Bovine cancellous bone.
Vancomycin concentrations were higher with bovine cancellous bone than with human bone. After 13 days, the mean concentrations were twice those in human bone, but the overall difference was not statistically significant (P = 0.5887). There was no statistically significant difference between the behaviour of 5 mm cubes of bovine cancellous bone and that of morselized samples (P = 0.2857).
Effect of lyophilization.
Lyophilization and rehydration of morselized human cancellous bone did not have a significant effect on elution (P = 0.0649).
Cancellous human bone.
Cancellous human bone yielded significantly lower concentrations of tobramycin than of vancomycin, especially in the first phase (until day 9) (P = 0.0022). The decrease in concentration, however, was markedly slower for tobramycin. After 13 days the concentrations were similar to those of vancomycin, and after 28 days (15 exchanges) the concentrations were still well above the MIC.
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Bovine cancellous bone.
Initial concentrations were markedly higher than with human bone and decreased much more slowly. The difference between bovine and human bone was statistically significant on each day (P = 0.0022). After 28 days, concentrations were still therapeutic (18.09 mg/L). There was no statistically significant difference between structural and morselized bovine grafts in the elution properties of tobramycin (P = 0.0714).
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Discussion |
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It is believed that cleaning the bone to remove fat, bone marrow and soft tissue makes it easier for the solution to penetrate through the canaliculi of the cancellous structures, increasing the surface available for adsorption. Gunal et al.37 investigated the release of gentamicin, ciprofloxacin and penicillin G, from deproteinated xenograft of bovine origin into saline. Eight hours of incubation were found to be sufficient for maximum saturation of the carrier. Concentrations were 5 mg/L after 24 h, decreasing to 0.5 mg/L after 10 days. The authors thought that these concentrations would be sufficient to eradicate bacteria, although their results indicated lower concentrations than those reported by McLaren32 and Hernigou et al.35
In the present study a compound of a carrier and antibiotics was created with the aim of increasing concentrations inside the carrier, to provide high initial concentrations in the surrounding liquid and prolonged release in the following weeks and months. At the same time, the carrier should ideally provide mechanical support and should repair defects. We developed a method consisting of several steps, each of which increases the loading capability. Drying the carrier was considered important for success, as tissue in a completely dry state is likely to absorb much higher quantities of aqueous solution during rehydration than a non-dehydrated tissue. Consequently, a dry tissue will bind water together with agents solubilized therein much more strongly. Of course, a certain amount of hydratable substance is required to provide the desired effect. In the samples described here, the hydratable substance was collagen, embedded in a crystalline scaffolding. It is crucial, therefore, to maintain the amount of natural collagen inside the graft during preparation; this was achieved by lyophilization. All other processing steps were designed to increase further the accessibility of the solution to the carrier as well as binding to it. The presented method increases storage capability by about 100-fold and results in closer binding of substances to the carrier, prolonging the release of vancomycin and tobramycin into the surrounding tissue and increasing the concentrations achieved. In general, vancomycin was released more quickly than tobramycin.
For clinical use in cases of chronic infection of bone, efficacy of antibiotic release is of major importance. Lack of local toxicity is also important, and mechanical properties must be suitable. Edin et al.38 showed that vancomycin 1000 mg/L did not adversely affect osteoblast replication. The same group found that tobramycin 400 mg/L significantly decreased cell replication,39 but there were no detectable differences in the healing characteristics of cancellous bone graft with or without tobramycin as seen on X-rays, microradiographs, bone density analyses, histological examination and biomechanical testing.34 Both antibiotics are more compatible with osteoblast replication than others, such as cefazolin or ciprofloxazin.38,39 Whether the mechanical properties of lyophilized bone are impaired by antibiotic impregnation requires further evaluation, although this might be of minor importance as long as the grafts are not load-bearing.
This study has shown that large amounts of vancomycin and tobramycin can be bound inside bone, provided that the processing method is adequate. The resulting bone antibiotic complexes release antibiotics into the surrounding medium, reaching high peak concentrations initially and then decreasing and becoming steadier for longer periods. This seems to fulfil the conditions needed to treat bone infection, where initial concentrations need to be high to eradicate bacteria in highly contaminated tissue, then after several days the concentrations can be reduced; the continued presence of the antibiotic should prevent re-contamination of the graft and allow antibiotic to reach more distant parts of the operative site. Thus, neo-osteogenesis, which starts only several days after grafting, is not likely to be compromised.38 Both human and bovine bone performed very well as carriers, though minor differences in binding capacity were seen. Cortical bone was less accessible to antibiotics than cancellous bone, resulting in lower initial concentrations of antibiotic. Long-term elution from the two types of bone, however, was comparable. Because of its smaller surface area, cortical bone may not be able to bind so much antibiotic; in contrast, the amount of molecules directly bound to collagen seems to remain more or less equal.
In conclusion, our results suggest that vancomycin or tobramycin in combination with adequately prepared bone graft carriers are well suited for the effective local treatment of bone infection.
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Notes |
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References |
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Received 9 November 1999; returned 22 February 2000; revised 10 March 2000; accepted 19 April 2000