1 Orthopaedic Research Unit, Department of Orthopaedic Surgery and Traumatology, University of Turku, Medisiina B4, Kiinamyllynkatu 10, 20520 Turku, Finland; 2 Institute of Biomaterials, Tampere University of Technology, PO Box 589, 33101 Tampere, Finland; 3 Department of Human Microbial Ecology and Inflammation, National Public Health Institute, PO Box 57, 20521 Turku, Finland
Received 22 May 2005; returned 25 July 2005; revised 14 August 2005; accepted 13 September 2005
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Abstract |
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Methods: Cylindrical pellets (2.5 x 1.5 mm) were manufactured from bioabsorbable poly(L-lactide-co-glycolide) (PLGA) matrix, ciprofloxacin [8.3 ± 0.1% (w/w)] and osteoconductive bioactive glass microspheres (90125 µm) [27 ± 2% (w/w)]. In vitro studies were carried out to delineate the release profile of the antibiotic. The antimicrobial activity of the release antibiotic was verified with MIC testing. In a time-sequence study in the rabbit, pellets were surgically implanted in the proximal tibia and the antibiotic concentrations achieved in bone were measured at 1, 2, 3, 4, 5 and 6 months.
Results: In vitro elution studies showed sustained release of ciprofloxacin at a therapeutic level (>2 µg/mL) over a time period of 4 months. The released ciprofloxacin had maintained its antimicrobial capacity against five standard ATCC strains. In vivo, the delivery system produced high local bone concentrations (247.9 ± 91.0 µg/g of bone) for a time period of 3 months with no significant systemic exposure. Histomorphometry and micro-CT imaging confirmed new bone formation around the pellets within 3 months as a sign of an independent osteoconductive property of the composite.
Conclusions: The tested composite seems to be a promising option for local therapy of surgically treated bone infections. The main advantages are the antibiotic release for a definite time period with therapeutic concentrations, which may minimize slow residual release at suboptimal concentrations.
Keywords: antimicrobial delivery , bioactive glass , osteomyelitis
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Introduction |
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Impregnation of antimicrobial agents within osteoconductive biomaterials (calcium sulphate, calcium phosphate, hydroxyapatite or tricalcium phosphate) has been proposed for local treatment of osteomyelitis and to aid dead space management.69 As a common feature, these implants show a rapid release of the antibiotic in a more or less controlled manner.10 An alternative approach is to impregnate the selected antibiotic into a biodegradable polymer and combine it with an osteoconductive material.1113 This type of multifunctional bone defect filler may provide a more sustained release profile of the antibiotic with an independent osteoconductive action for repair of the defect. Recently, we found this type of local therapy efficient in treatment of experimental osteomyelitis.13
In this study, we have further characterized ciprofloxacin release from bone defect filler composed of a poly(L-lactide-co-glycolide) matrix and bioactive glass microspheres. Specifically, we addressed the following questions: (i) Does the composite provide a stable release of ciprofloxacin in vitro and in vivo over an extended time period? (ii) Does the ciprofloxacin that is released retain its antimicrobial properties? (iii) What is the spatial distribution of the released ciprofloxacin within the bone? (iv) Do the bioactive glass microspheres promote new bone formation around the ciprofloxacin-releasing pellets?
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Materials and methods |
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The bone defect filler consisted of three components. The matrix was bioabsorbable poly(L-lactide-co-glycolide) 80:20 (PLGA) (Purasorb PLG, Purac Biochem BV, Gorinchem, The Netherlands) with inherent viscosity (inh) of 6.62 dL/g (chloroform, 25°C, c = 0.1 g/dL). The selected antibiotic was ciprofloxacin (Jinxing Kangle Pharmaceutical Factory, Zhejiang, China). According to the quality certificates provided by the manufacturer, the quality of ciprofloxacin was tested as directed in U.S. Pharmacopoeia 23 specifications.14 Bioactive glass (glass 13-93, Vivoxid Ltd, Turku, Finland) was added to act as the osteoconductive component. The selected bioactive glass is a mixture of six different hydroxides, MgO, CaO, K2O, Na2O, P2O5 and SiO2. The bioactive glass was in the form of spheres and the particle size distribution was in the range 90125 µm. The fillers contained 27 ± 2% (w/w) of bioactive glass microspheres.
The biomaterial was manufactured by melt-compounding vacuum-dried components in a small laboratory scale mixer and then die-drawn into self-reinforced (SR) rods. Melt-compounding temperatures used (190°C) were much lower than the measured melting point of ciprofloxacin (270°C). Thermal stability of ciprofloxacin was ensured with differential scanning calorimetry (DSC). The machined SR rods were cut into a pellet form. The geometry of the pellets was cylindrical with an average diameter of 2.5 mm and length of 1.5 mm. The pellets were sterilized with
-irradiation using a nominal dose of 25 kGy (Willy Rüsch AG, Kernen-Rommelshausen, Germany).
Antibiotic release in vitro
The initial ciprofloxacin content of the fillers was determined spectrophotometrically. Samples (10 mg) were dissolved in trichloromethane (50 mL) and absorbance values were measured using a UVVis spectrophotometer (UNICAM UV 540, Thermo Spectronic, Cambridge, UK) scanning all samples from 190 to 400 nm. The maximum absorbance value in UV spectra () was 284.5 nm. The ciprofloxacin concentration was calculated according to the BeerLambert law. The initial ciprofloxacin content was expressed as the mean percentage (w/w) of 10 parallel samples.
Detailed in vitro studies were carried out to delineate the release characteristics of the impregnated antibiotic. Samples containing 10 pellets (85 mg) were placed in 20 mL of phosphate buffer solution (NaOH 0.04 mol/L and KH2PO4 0.05 mol/L) at pH 7.4. Five parallel samples in brown drug bottles were kept in an incubator shaker at a temperature of 37°C. After 6, 24, 48, 72 and 96 h and then every 23 days up to a total of 300 days, the buffer solution was replaced with fresh solution. Absorbance values were measured from withdrawn buffer solution using a UVVis spectrophotometer scanning all samples from 190 to 400 nm. The maximum absorbance value in UV spectra (
) was 271 nm. The ciprofloxacin concentration in the buffer solution was calculated according to the BeerLambert law and expressed as the concentration of the drug (µg) per mL. The value was averaged per day by dividing the measured concentration by the number of immersion days (i.e. the number of days between the change of buffer solution).
Antibacterial activity
In parallel with the UV measurements of the ciprofloxacin concentrations, the antibacterial activity of the buffer solutions containing released ciprofloxacin was compared with the activity of non-processed ciprofloxacin (Ciproxin; Bayer AG, Leverkusen, Germany). This was done to explore whether the manufacturing or sterilization processes had had any influence on the antibacterial activity of the impregnated antibiotic (8 h time point) and to investigate whether the released ciprofloxacin had retained its active form during the process of sustained release (40 and 80 day time points). The MIC testing was done using the agar-dilution technique standardized by the NCCLS.15 Briefly, inocula of 107 cfu/mL of studied bacteria were placed on MuellerHinton II (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) agar plates with a Denley Multipoint Inoculator (Denley Instruments Ltd, Billingshurst, UK) delivering final inocula of 1 µL (104 cfu/spot). The plates, which contained doubling dilutions of either processed or non-processed ciprofloxacin, were incubated for 20 h at 35°C in air. The lowest concentrations prohibiting bacterial growth were determined. The tested strains were Staphylococcus aureus (ATCC 25923 and ATCC 29213), Staphylococcus epidermidis (ATCC 35983), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853).
Animals
Twelve adult male New Zealand White rabbits (Harlan, Horst, The Netherlands) weighing a mean of 3750 g (range 33004070 g) were used. Before surgery, the rabbits were acclimatized to their new environment and fed a standard laboratory diet. The animals were caged individually with a constant temperature. The Ethics Committee of the University of Turku and the Provincial State Office of Western Finland approved the study protocol (Registration # 1385/04). All experiments were carried out in accordance with the guidelines of the local Animal Welfare Committee.
Study protocol
Ten cylindrical pellets were surgically implanted into the medullary defect in each animal created in the right proximal tibia. After implantation, sequential monthly analyses were performed to demonstrate the in vivo biodegradation of the composite with peripheral quantitative computed tomography (pQCT) scanning and to monitor the systemic antibiotic exposure by means of determination of ciprofloxacin concentration in the peripheral blood. The animals were euthanized in groups of two at 1, 2, 3, 4, 5 and 6 months. Bone tissue concentrations of ciprofloxacin were determined from serial specimens of the harvested tibia, when histomorphometric, scanning electron microscopic (SEM) and micro computed tomography (micro-CT) studies were also performed to examine the amount and rate of new bone formation around the pellets.
Surgery
The applied animal model was designed to simulate clinical dead space management of a contained bone defect following radical debridement of localized osteomyelitis. Standard surgical techniques were applied, including premedication, anaesthesia, and surgical skin preparation.16 The animals received a single prophylactic dose of 500 000 IU benzylpenicillin (Geepenil, Orion Oyj, Espoo, Finland). A cortical bone window size of 6 mm x 2.7 mm was created in the proximal metaphysis of the tibia, the bone marrow was removed with saline lavage and 10 composite pellets were impacted into the proximal medullary cavity (Figure 1). After surgery, functional activity was not limited and the animals received standard post-operative pain medication for 3 days.
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Under sedation with fentanyl-fluanisone, venous blood samples (68 mL) were taken from the ear vein for the measurement of systemic ciprofloxacin concentration at 12 h (n = 10), 3 days (n = 12), 1 month (n = 12), 2 months (n = 10), 3 months (n = 8), 4 months (n = 6), 5 months (n = 4) and at 6 months (n = 2). Serum was separated at 4000 rpm for 10 min and stored at 20°C before analysis.
pQCT
In each animal, in vivo biodegradation of the pellets and concomitant intramedullary new bone formation was followed by means of sequential pQCT imaging using a Stratec XCT Research M pQCT device (Norland Stratec Medizintechnik GmbH, Birkenfeld, Germany) at 3 days and every month until the animal was killed. Six consecutive cross-sectional images were obtained and the slice with the highest amount of pellet material was selected. The density (mg/cm3) and area (mm2) of trabecular and cortical bone were measured with the pQCT software.
Digital radiography and harvesting of bone specimens
The animals were euthanized with an intravenous administration of sodium pentobarbital (Mebunat, Orion Oyj, Espoo, Finland). Standard digital anteriorposterior and lateral radiographs of the right hind limbs were taken. The harvested bone was aseptically cross-sectioned under continuous saline irrigation and washing out of blood contamination. Seven bone specimens were removed for measurement of ciprofloxacin concentration (Figure 1) and one specimen for histomorphometry.
Histomorphometry
The bone specimens for histomorphometry were fixed in 70% ethanol, dehydrated in a graded series of ethanol, cleared in xylene, and embedded in isobornylmethacrylate (Technovit 1200 VLC, Kulzer, Germany). Sections of 20 µm thickness were prepared with a cutting and grinding technique (Exakt Apparatebau, Hamburg, Germany) and stained with a modified van Gieson method for light microscopy. A bone affinity index was measured using computer-aided image analysis (Corel Photo Paint 9.0, Corel Corporation, Ottawa, Ontario, Canada). The bone affinity index, expressed as a percentage, was defined as the fraction of the perimeter of the pellets covered by new bone. The value was measured for all pellets in a section and averaged.
Micro-CT and SEM
After sectioning for histology, the embedded bone specimens were analysed by a high-resolution micro-CT imaging system (SkyScan 1072 micro-CT system, SkyScan, Aartselaar, Belgium) and backscattered electron imaging (BEI) of SEM. Before the BEI-SEM analysis, the bone blocks were carbon coated with a JEE-4X vacuum evaporator (Jeol Ltd, Tokyo, Japan). The analysis was performed with a SEM (Philips XL-30, Eindhoven, The Netherlands) equipped with a backscattered electron detector for imaging.
Ciprofloxacin concentration measurements
A high performance liquid chromatographic (HPLC) method with fluorescence detection (FLD) and an internal standard method were applied for the determination of concentrations of ciprofloxacin in bone and serum (CRST Bioanalytics, Turku, Finland). Ofloxacin was used as an internal standard.
The bone specimens were ground with a homogenizer (Mikro-Dismembrator S, B. Brown, Melsungen, Germany). Finely ground bone was weighed and mixed with internal standard solution (1.0 µg/mL in methanol), water, methanol and perchloric acid in water. The suspension was centrifuged and the supernatant was filtered with a 0.45 µm membrane filter. Finally, 20 µL of sample was injected into the HPLC column. Standard samples and quality control samples were handled identically, but instead of the pure methanol, solutions containing ciprofloxacin (in methanol) were added.
The frozen serum samples were thawed in a refrigerator, and 0.5 mL of each serum sample was mixed with perchloric acid. After centrifugation, the supernatant was transferred into an autosampler vial from which 20 µL was injected into the HPLC column.
HPLC-FLD analyses were carried out using a Waters 2695 Separation Module, Waters 2475 Multi Fluorescence detector and Millennium version 4.0 software. The column used for separation of the ciprofloxacin was a Nova-Pak C8 150 x 3.9 mm i.d. 60 Å column (Waters Co., Milford, MA, USA). The mobile phase consisted of 9% acetonitrile and 91% buffer. The buffer contained 10 mM sodium dihydrogen phosphate (aq) and 20 mM tetrabutyl ammonium hydrogen sulphate (aq), pH 2.5. The pH was adjusted with 2 M NaOH (aq). The buffer was filtered before use through a 0.45 µm HV filter (Millipore Corporation, Bedford, MA, USA). The flow rate of the mobile phase was 1.0 mL/min. The excitation wavelength was 290 nm, and the emission wavelength 470 nm. A standard curve was generated using weighted (1/x) linear regression. The measured concentration of ciprofloxacin was expressed as per weight of bone tissue (µg/g). The measured concentration of ciprofloxacin in serum was expressed as ng/mL.
Statistical analysis
The data for histomorphometry and measurement of ciprofloxacin bone concentrations were grouped into early (13 months) and late (46 months) phases and the data were tested for normality. In histomorphometry, the unpaired t-test was used in the analysis of the differences observed between the early and late phases. The MannWhitney rank sum test was used in the analyses of ciprofloxacin bone concentrations as the data failed the normality testing. The paired t-test was applied to analyse the significance of differences observed at the monthly pQCT measurements (up to 4 months) compared with the individual initial values determined at day three. The results were expressed as the mean ± SEM. A P value less than 0.05 was considered significant. All statistical analyses were performed with SigmaStat 3.0.1 software (SPSS Inc., Chicago, IL, USA).
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Results |
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The measured initial ciprofloxacin content of the pellets was 8.3 ± 0.1% (w/w). The antibiotic release from the pellets was measured as a function of in vitro immersion time (Figure 2). After the initial burst (39.2 ± 1.7 µg/mL) on the first day of immersion, the concentration of ciprofloxacin in the buffer solution (averaged per measurement days) remained at a stable level throughout the first 120 days (5.4 ± 1.2 µg/mL). Half of the loaded ciprofloxacin was released within 63 days and all loaded ciprofloxacin was released from the pellets within 150 days.
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Based on MIC testing, the activity of the released antibiotic against the tested standard ATCC bacterial strains (S. aureus, S. epidermidis, E. coli, P. aeruginosa) was similar to the non-processed ciprofloxacin. The loaded antibiotic had retained its active form throughout the process of sustained release (from 8 h to 80 days).
Ciprofloxacin concentrations in serum
As a response to composite biodegradation, ciprofloxacin could be detected only in four serum samples. Three of them were measured 12 h after the implantation surgery. The 12 h concentrations were above the detection level (>1 ng/mL) but below the lowest level of quantification (5 ng/mL). Their estimated mean concentration was 1.96 ± 0.19 ng/mL. Ciprofloxacin could only be quantified in one serum sample (6.56 ng/mL) taken 3 days after surgery.
Ciprofloxacin concentrations in bone
The measured individual concentrations in bone are shown in Table 1. During the early phase (13 months), the composite provided high local tissue concentrations of ciprofloxacin (247.9 ± 91.0 µg/g of bone) in bone specimens harvested adjacent to the pellets (Section 1). During the later phase (46 months), the same location showed significantly (P < 0.001) decreased levels of ciprofloxacin in bone (1.9 ± 1.7 µg/g of bone). In more distant sites of the tibia (Sections 27), the concentrations were uniformly lower, but there was a relationship between the measured concentration and the distance from the implantation site. During the early time period (13 months), the concentrations of ciprofloxacin in specimens closer to the implantation area (Sections 24) (1.0 ± 0.4 µg/g of bone) were significantly (P < 0.001) higher than those in more distant specimens (Sections 57) (0.2 ± 0.1 µg/g of bone). During the later time period (46 months), there were only occasional distal bone specimens with measurable (>0.1 µg/g of bone) concentrations of ciprofloxacin.
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Plain radiographs showed an uncomplicated healing process of the cortical defect. In sequential pQCT imaging, the area of trabecular bone increased significantly during the first 3 months. In individual animals, the area of trabecular bone was 6.519.8% higher at 3 months compared with the measured area at day 3 (average change 13.4%, P < 0.001). As a remodelling response, the cortical bone area significantly decreased as a function of time, while its density showed a concurrent increase.
Histomorphometry
There were progressively increased amounts of new bone around and inside the implanted pellets. The implants themselves showed only minor structural changes when examined under a light microscope and no signs of foreign body reaction were visible. One and 2 months after implantation, bioactive glass microspheres showed characteristic reaction layers on their surfaces, whereas there was only modest new bone formation from the endosteal surface to the pellets. Three months after implantation and thereafter, a rim of new bone formation was observed fairly constantly in the outer perimeter of the pellets and occasionally also within the matrix of the pellets. The bone affinity index was 24 ± 8% during the early phase (13 months) and 29 ± 6% during the late phase (46 months).
BEI-SEM and micro-CT
BEI-SEM analysis showed a limited amount of new bone formation around the pellets during the first 2 months after implantation. At 3 months and thereafter, new bone formation was more evident in the outer perimeters of the pellets. As an indicator of osteoconductivity, trabeculae of new bone were found to be in direct contact with bioactive glass microspheres. Micro-CT imaging demonstrated encapsulation of the pellets by new bone. The images showed long new bone trabeculae, which originated from the endosteal surface and were attached to the pellets.
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Discussion |
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In an optimal local drug delivery system, the antibiotic should be released for a definite time period with therapeutic concentrations. This should not be followed by a slow residual release with suboptimal concentrations, which may increase the risk of overgrowth of antibiotic-resistant organisms.18 For example, polymethylmethacrylate (PMMA) beads may provide slow residual release of antibiotics for undefined periods.19 In this respect, the drug delivery system of this study seems to be safer. Therapeutic concentrations of the released antibiotic were maintained up to 4 months in vitro and the total amount of impregnated antibiotic was released within 5 months. This type of release pattern will minimize the periods of suboptimal antibiotic concentrations.
Bioactive glasses are a group of synthetic silica-based biomaterials, which promote osteogenesis on their surfaces.20,21 As a major indication for their use as osteoconductive components of antibiotic-containing bone defect fillers, a subgroup of bioactive glasses seems to have antibacterial properties. The antibacterial properties of bioactive glasses were first shown against oral pathogens,22 but the antibacterial action might be more general.
There were only minor structural changes of the pellets during the first 6 months of in vivo implantation. Based on the current literature and experience with different orthopaedic implants, the expected degradation rate of implants made of polymers ranges from 1 to 5 years. For the selected composition of the bioactive glass, the expected degradation rate is less than 1 year. However, for both biomaterials, the interaction with surrounding tissues (especially the ingrowth of new bone into the porous structure) may significantly affect the rate of degradation. The implanted pellets did not seem to limit local new bone formation, because the new bone was capable of attaching to the surface of the pellets and growing into the porous spaces of the pellets. The fraction of the pellet surface occupied by bioactive glass microspheres was initially low (less than 510%). However, it is very likely that the bioactive glass microspheres became more exposed during the progression of internal degradation of the polymeric matrix. This was confirmed by the ingrowth of new bone into pellets even during the first 3 months of implantation.
Fluoroquinolones are considered the drug of choice for deep bone infections.23 However, fluoroquinolones as well as several other antibiotics frequently used to treat bone and joint infections, such as rifampicin, gentamicin and vancomycin, have been shown to affect osteoblastic functions in vitro.2430 Fluoroquinolones, but not gentamicin or vancomycin, have also been shown to impair fracture healing in vivo.26,27,31 However, no clinical data have been published to support these experimental findings. In this study, new bone formation around the pellets was evident 3 months after implantation, although the local bone concentration of ciprofloxacin was high.
The antibiotic concentration in bone samples adjacent to the pellets showed a large variation, especially during the early period. To some extent, this might be due to contamination of the bone samples with the residual filler material. The high concentrations may also be partly affected by the ratio of trabecular and cortical bone in the specimens. In clinical studies, a wide variation of bone concentration of antibiotics has also been observed after intravenous administration of antibiotics.32 The reason for the finding is unknown.
The obvious limitations of this study were the lack of different control groups. Therefore, no definite conclusions could be made on whether ciprofloxacin had any retarding effect on the rate of new bone formation or how the bioactive glass microspheres changed the rate of new bone formation. In in vitro testing, for the first 4 months, the concentration of ciprofloxacin remained above the therapeutic level (>2 µg/mL) of ciprofloxacin frequently cited in the literature.24 However, there are obvious uncertainties related to the direct comparison of the in vitro results with the in vivo data. There are a number of factors, such as perfusion, which determine the effective local antibiotic concentration in vivo. Therefore, it is difficult to correlate the measured in vitro concentration of the released antibiotic with the expected concentrations in vivo.
The high local antibiotic concentration was limited only to the tissue areas near the implantation site. Therefore, under clinical conditions of osteomyelitis treatment, there is an obvious need for systemic antibiotics to cover the surrounding bone and soft tissues for a limited time period. New bone formation around the pellets indicated an independent osteoconductive property of the composite. Clinically, this type of filler could be used to obliterate dead space and even substitute secondary bone grafting in favourable cases with contained bone defects.
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Acknowledgements |
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