1 Department of Chemistry and Biochemistry, Laurentian University, 935 Ramsey Lake Road, Sudbury, Ontario, Canada P3E 2C6; 2 Department of Biomedical Research, The University of Texas Health Center, 11937 US Highway 271, Tyler, TX 75708, USA
Received 17 August 2004; returned 14 September 2004; revised 20 September 2004; accepted 22 September 2004
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Abstract |
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Methods: Gentamicin was incorporated in poly(hydroxybutyric-co-hydroxyvalerate) (PHBV) with 8% or 12% hydroxyvalerate (HV) content at 2:1 or 5:1 (weight to weight) ratio. In conjunction with an elution study, a scanning electron microscopy and a porosity study were carried out to explore physical characteristics of the complexes before and after the leaching effect. The antibacterial effectiveness of the complexes was analysed in a bacterial adhesion assay using clinical isolates of Staphylococcus haemolyticus and Staphylococcus aureus. In addition, the polymers were exposed to pooled human blood to test their biocompatibility in both static and dynamic environments.
Results: We have shown that increasing the HV content from 8% to 12% leads to a faster release of the integrated antibiotic. An increase in antibiotic content enhanced the homogeneity while decreasing the permeability of the complexes and reducing the release rate. A significant reduction in the number of the adherent S. aureus and gentamicin-resistant S. haemolyticus within a 48 h exposure to our formulations confirmed the effectiveness of the PHBV/gentamicin complexes. Finally, these formulations did not alter the haemodynamics of the pooled blood samples after an extended period of time.
Conclusion: Taken together, the PHBV/gentamicin formulations may prove to be effective preventive therapeutic modalities in implant-related Staphylococcus infections.
Keywords: femoral implants , bacterial adhesion , gentamicin , biocompatibility , biomaterials , drug release
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Introduction |
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There is increasing evidence that microbial adhesion and subsequent colonization leading to biofilm development are involved in the aetiology of device-related infections.7,10 Effective treatment may therefore be possible by killing bacteria during the early stages of colonization and the continuous delivery of antibiotics appears to be a promising approach.11 Surgical placement of polymethylmethacrylate (PMMA) spheres at the site of infection, which then release antibiotic for approximately 6 weeks,6,10,12,13 has been the method of choice for the past decade. Although this system functions effectively in terms of antibiotic delivery and eradication of infection, because of their non-biodegradable nature, the spheres must be removed once the vascularity of the region has returned to normal, a process that could be avoided if a biodegradable drug delivery system was used.
Biodegradable synthetic materials such as antibiotic-saturated bone grafts,14 hydroxyapatite-collagen stems, hydroxyapatite-calcium phosphate composites,15 calcium phosphate bone cements,16 polylactic acids and their copolymers with glycolic acids, polyanhydrides,12,14,1719 and polyurethane,2022 have been developed. These biomaterials have also been exploited as coating agents on the femoral stems. The advantage of these synthetic implants is that they are all completely metabolized in humans. However, they exhibit drawbacks such as lowering the pH at the site of implant resulting in localized inflammation, tissue injury, and eventual failure of the femoral implants.23,24 Polyhydroxyalkanoates (PHA) have shown promise when they are used to coat implants used in hard tissue because of a number of unique properties, such as piezoelectricity, thermoplasticity, biodegradability and biocompatibility; and when mixed with antibiotics they have prevented bacterial adhesion.7,11,25
Polyhydroxyalkanoates are natural polymers derived from bacteria. Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) is composed of poly(3-hydroxybutyrate) (PHB) and varying numbers of hydroxyvalerate (HV) molecules. PHBV polymers are biodegradable and biocompatible, and actually promote bone growth.10,11,26,27 In this study, we have evaluated the physiochemical properties and the drug release pattern of a PHBV/gentamicin complex and its impact on the growth pattern of the biofilm-forming Staphylococcus species in vitro.
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Materials and methods |
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Discs from the femoral hip (Total Hip Systemcoated implant for use without cement: DePuy Femoral implant, Warsaw, USA) were created by cutting the femoral hip prosthesis into round discs with a diameter of 9.2 mm and a thickness of 2.7 mm.
Polymer/antibiotic complex preparation and disc coating
PHBV with either 8% or 12% hydroxyvaleric acid was purchased from Across Organics (NJ, USA). One gram of PHBV was dissolved in chloroform and an appropriate amount of gentamicin (Across Organics) was added to obtain a 2:1 or 5:1 (w/w) antibiotic content. The mixture was incubated at 55°C in a shaking water bath and vortexed until fully homogeneous. The polymer/gentamicin mixture was then poured into a metal mould (11.5 mm diameter, 5 mm thickness) containing the orthopaedic discs. The discs were coated and dried for 24 h. Once dried, the discs were measured by an electronic calliper (STM Digimatic Calliper; Dayna, Sudbury, ON, Canada), and weighed to determine the quantity of the coating.
Drug release behaviour
We measured the rate at which gentamicin was released from the antibiotic/polymer complexes on the discs. The gentamicin release was studied in 5 mL of phosphate-buffered saline (PBS, pH 7.4) at 37°C under a mild shaking environment (100 r.p.m.). Aliquots of 50 µL were assayed for gentamicin at the time points of 0, 0.25, 0.5, 1, 3, 6, 12 and 24 h, which was repeated at regular intervals for 6 weeks. Fresh PBS (50 µL) was added to replace the withdrawn amount. Gentamicin concentration was measured by a microbiological assay using Staphylococcus aureus (ATCC 25923; American Type Culture Collection, Manassas, VA, USA) as we described previously.28 A similar polymer/disc complex without antibiotic served as a control to ensure that all material that eluted from the disc had no effect on the inhibition zone.
Antibiotic susceptibility testing
The use of all clinical material in this project was approved by the institutional review board (IRB) at the Sudbury Regional Hospital. Staphylococcus haemolyticus and S. aureus strains used in this study are clinical isolates from femoral orthopaedic implant infections in Sudbury Regional Hospital, Sudbury, Ontario, Canada. The bacterial strains were tested for gentamicin susceptibility by a broth dilution method.29 The minimum inhibitory concentration (MIC) was defined as the lowest concentration of gentamicin that inhibited visible growth of the test bacteria. All MICs were confirmed by the Microscan (WalkAway-40SI System with LabPro; Dade Behring Inc., Toronto, ON, Canada) according to the supplier guidelines.29
Bacterial adhesion
The antibiotic-loaded polymer discs and non-coated metal discs were immersed in bacterial suspensions in PBS (108 cfu/mL) and incubated for 5 h at 37°C, under static conditions.30,31 The samples were transferred into 10 mL of MuellerHinton broth and incubated for 3, 24 and 48 h at 37°C. After each incubation period, samples were washed three times to remove non-adherent bacteria. Adherent bacteria were then removed from the polymeric surfaces by ultrasonication (150 W, 90 s intervals, total of 4.5 min; Fisher sonicator, Toronto, Canada) and the colony counts of viable cells were determined.31,32
Haemocompatibility test
Blood samples were obtained from 10 healthy individuals, pooled and used within 2 h. Duplicate samples (8 mL) of the coated discs, or metal disc alone (negative control) were placed in 2.5 mL of pooled human blood. The samples were maintained at 37°C for 2 h. One group of samples was held at a static state and the other at a dynamic state. We removed samples every 30 min for complete blood count (CBC) analysis (ADVIA 120 Hematology machine; Bayer Inc., Toronto, ON, Canada) to analyse the biocompatibility of the polymer with human blood according to the ISO guidelines.33,34
Scanning electron microscopy
Experiments to assess the physical characteristics of our polymer/antibiotic complexes were carried out by Quantachrome Instruments Laboratory Services (Boynton Beach, FL, USA). The surfaces of polymers were analysed with a field-emission scanning electron microscope (SEM; S-4700, Hitachi Co., Tokyo, Japan) on carbon-sputtered samples.20 The porosity analysis of the polymers was carried out on an Automatic Pore Size Analyzer (PoreMaster 60; Quantachrome Instruments, Boynton Beach, FL, USA). The instrument measures the pressure that is applied to force a non-reactive, non-wetting liquid to penetrate the pores. The relationship between the applied pressure and the pore size into which mercury will intrude is given by the Washburn equation:
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Data analysis
We used Student's t-test (for two samples) or ANOVA (for multiple comparisons) with the two-tailed Dunnett's post-test analysis to compare the mean values. A P value of 0.05 was considered significant. Results are presented as means ± S.E.M. of at least three independent experiments.
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Results |
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We compared the influence of HV content (Figure 1) and gentamicin concentration (Figure 2) on the antibiotic release kinetics in our formulations. After 6 weeks incubation in PBS, PHBV 8% and PHBV 12% with a 5:1 polymer/gentamicin ratio released 33% and 37% of their antibiotic content, respectively (Figure 1a). Statistically significant differences in antibiotic release were evident at all time points (P < 0.05), indicating that the higher the HV content, the more antibiotic will be released. As we increased the antibiotic content to 2:1, however, we found the polymers released less antibiotic in a 6 week period, regardless of their HV content although the differences (3337% versus 29%) were not statistically significant.
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Effects of antibiotic content on morphology and porosity of the polymers
We utilized scanning electron microscopy (SEM) and a pore size analyser to investigate the homogeneity and porosity of the polymer/antibiotic complexes. We have shown that the addition of antibiotic renders the polymer texture more homogeneous. As shown in Figure 3(a, PHBV 12% samples with no antibiotic were the least homogeneous (a) whereas the PHBV 12% 2:1 samples with higher antibiotic content appeared to be the most homogeneous (c). A similar pattern was observed when PHBV 8% 2:1 and PHBV 8% 5:1 images were compared (data not shown). In addition, analysis of the SEM images indicated cracks and surface deformities on the surfaces of the formulations that were left behind by the dissolved drug crystals [arrows on Figure 3(d and e)], the number of which was directly proportional to the gentamicin content of the formula. The size of the post-leaching pore was, however, inversely related to gentamicin concentration used; pores of PHBV 8% 5:1 were significantly larger than the pores of PHBV 8% 2:1 (0.038 µm ± 0.0001 versus 0.015 µm ± 0.0001, P < 0.05), regardless of the HV content.
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Antibacterial effects of the polymer/gentamicin complexes
We compared bacterial adherence to the polymers at varying time intervals (3, 24 and 48 h). We found that all formulations except PHBV 8% 5:1 reduced the number of adherent S. aureus significantly in the first 3 h of incubation at 37°C compared with antibiotic-free controls (Figure 4a). At the end of a 24 h incubation, S. aureus was eradicated while there remained a few (<100 cfu) live S. haemolyticus (Figure 4b), which were all killed after 48 h. All polymer/gentamicin complexes, regardless of the HV or gentamicin content, were able to reduce the number of adherent S. aureus and S. haemolyticus significantly (<400 cfu) after a 24 or 48 h exposure, respectively. However, PHBV 8% 5:1 was the least effective formulation while PHBV 12% 2:1 was the most effective formulation in this setting.
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We used pooled normal human blood to determine the safety of the polymer/gentamicin complexes in vitro. None of our formulations altered the number or morphology of WBC, RBC, or platelets, regardless of the experimental conditions (static/non-static), HV content, or gentamicin concentration.
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Discussion |
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Increasing the number of drug crystals in PHBV/gentamicin mixtures resulted in an increase in porosity, which is caused by the creation of voids due to the dissolution of the drug crystals. These voids also leave crater-like surface features on the polymer. The effect on porosity due to antibiotic loading was greater than that of HV content on antibiotic elution rates. This can be seen in Figure 1(b) where the cumulative release data for PHBV complexes with two different HV concentrations have almost identical slopes because of their identical gentamicin content. These results confirm that it is possible to control the release of the drug by controlling the concentration of the antibiotic crystals in the polymeric matrix.
In experiments looking at HV content, the higher the HV content the more crystalline and more porous the polymers were, which results in a faster antibiotic release. This phenomenon is due to the altered crystallinity of PHBV by the addition of HV.10,26,27 Thus, the increase in HV from 8% to 12% leads to a more amorphous structure, which allows an increase in water uptake. This speeds up the dissolution of the drug crystals and leads to a faster release as reported by other investigators.7,10
In all experiments, an initial rapid release of gentamicin was seen. This initial burst results from the presence of the antibiotic crystals on the polymer surface. The drug crystals are more soluble than the polymer, therefore, their early dissolution creates pores or channels that allows water to penetrate into the polymers. This speeds up dissolution of the drugs inside the polymeric matrix and consequently leads to the burst effect. The initial burst effect ends because the channel formation ends when all the drugs at the surface, which are in direct contact with each other, are dissolved. After this point, the elution is no longer logarithmic and a constant release pattern is observed. At this point, the breakdown of the polymer controls the release of the antibiotic, allowing the exposure of new drug crystals to the medium. In fact, the rapid burst release may be beneficial from a therapeutic standpoint because the high release will kill any bacteria introduced before or during the operation.7
As well as an increase in homogeneity as the antibiotic content was increased, we also observed that the number of pores increased significantly after elution began. This effect is caused by the antibiotic crystals dissolving in the polymeric matrix and is directly proportional to the concentration of the drug.
The adhesion of S. haemolyticus to PHBV 8% or PHBV 12% alone and the uncoated steel implants was virtually identical (P > 0.05), indicating that neither the polymers nor steel specimens have antimicrobial properties.35,36 However, after a period of 24 h, the gentamicin-containing polymers that released the most gentamicin, had the greatest reduction in adherent bacteria. Adhesion assays also indicated that S. haemolyticus remained on the discs for a longer period of time (48 h) than S. aureus (24 h), possibly because of its higher MIC. Overall, our polymer/gentamicin complexes showed a similar or superior bactericidal effect compared with other biofilm inhibition methods6,22,30,32,37,38 with a good biocompatibility.
In conclusion, PHBV 8% and PHBV 12% exhibit sustained release of gentamicin at a constant rate, and above the MIC for S. haemolyticus and S. aureus for 6 weeks. The HV content and the drug loading influence the release kinetics. These polymer/gentamicin complexes eradicate S. aureus and gentamicin-resistant S. haemolyticus adhering to the surface and show no adverse effects on blood cell integrity. Therefore, our newly developed polymer embedded gentamicin system could potentially reduce infectious complication in hip surgery. In vivo studies are indicated to determine the safety and efficacy of these complexes in a pre-clinical setting.
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Acknowledgements |
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Footnotes |
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