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Antimicrobial Agents and Chemotherapy, July 2008, p. 2335-2339, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01360-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Local Treatment of Experimental Pseudomonas aeruginosa Osteomyelitis with a Biodegradable Dilactide Polymer Releasing Ciprofloxacin{triangledown}

Kyriaki Kanellakopoulou,1* George C. Thivaios,2 Maria Kolia,1,2 Ismini Dontas,3 Lydia Nakopoulou,4 Eleftherios Dounis,2 Evangelos J. Giamarellos-Bourboulis,1 Andreas Andreopoulos,5 Panayotis Karagiannakos,3,6 and Helen Giamarellou1

4th Department of Internal Medicine, University of Athens Medical School,1 Department of Orthopaedics, Laikon Athens General Hospital,2 Department of Experimental Surgery and Surgical Research, University of Athens Medical School,3 1st Department of Pathology, University of Athens Medical School,4 Department of Chemical Engineering, National Polytechnic School,5 Academy of Athens, Institute of Biomedical Research, Athens, Greece6

Received 23 October 2007/ Returned for modification 28 December 2007/ Accepted 5 April 2008


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A biodegradable system of poly-D,L-dilactide releasing ciprofloxacin was assessed in a Pseudomonas aeruginosa osteomyelitis model after inoculation of the test pathogen into the left tibia of 76 New Zealand White rabbits; 31 were controls (group A), and 45 were implanted with the polymer at the infection site (group B). The rabbits were killed on a weekly basis, and cancellous bone was harvested for histopathology and for estimation of bacterial growth and the concentrations of ciprofloxacin. Tibial X ray was performed immediately before the animals were killed. The total number of fistulas with purulent discharge that developed after inoculation of the pathogen was counted, and fistulas with purulent discharge were found in 16 animals in group A (51.6%) and 3 animals in group B (6.7%) (P < 0.0001). The animals in group A had a profound loss of body weight compared to the animals in group B. The main radiological finding was the presence of sequestra in 25 animals (80.6%) in group A and 6 animals in group B (13.3%) (P < 0.0001). The bacterial load in group B was significantly reduced compared to that in group A, possibly due to the prolonged local antibiotic release at concentrations exceeding even 80 times the MIC for the test pathogen. The histology of animals killed after week 49 revealed a mild inflammatory reaction accompanied by diffuse fibrosis and new bone formation in group A animals and the presence of small polymer particles in group B animals. It is concluded that the system described achieved eradication of the pathogen, accompanied by clinical and radiologically confirmed benefits, so this treatment may be a candidate for the management of difficult orthopedic infections.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The management of chronic osteomyelitis typically requires adequate surgical debridement followed by prolonged antibiotic therapy (14). The recommended duration of systemic antibiotic administration is at least 6 weeks, and patients must take multiple doses daily, creating considerable problems like toxicity and a lack of patient compliance. Furthermore, surgical debridement often creates the problem of bone instability due to dead space formation. In order to overcome these drawbacks, the concept of the local administration of high concentrations of antibiotics was proposed. Antibiotic-impregnated implantable materials were first described by Bucholz and Engelbrecht (7). They consisted of polymethylmethacrylate beads that release gentamicin locally. The main problem, however, with antibiotic-impregnated bone cement was the need for surgical removal upon the completion of drug release (8). To alleviate this difficulty, biodegradable materials were developed as antibiotic carriers. Polylactic acid, polyglycolic acid, hydroxyapatite, calcium phosphate, and collagen materials have all been applied as biodegradable carriers for controlled antibiotic release (3, 6, 9).

Among these biodegradable carriers, polymers of D,L-dilactide (PLA) release the largest amount of antibiotics, at least in vitro (10). This material releases ciprofloxacin or pefloxacin at levels exceeding the MICs for most of the pathogens implicated in chronic osteomyelitis. The duration and peak of release depend on the polymer's molecular mass. Intermediate-molecular-weight (26,000) polymers demonstrated better properties, releasing ciprofloxacin for almost 100 days and at levels higher than the levels released from low- and high-molecular-mass polymers. The successful management of experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus was achieved by the local application of a PLA that releases pefloxacin (8).

The objective of this study was to evaluate the in vivo effectiveness of this system as a carrier of ciprofloxacin for the treatment of tibial osteomyelitis caused by Pseudomonas aeruginosa in an experimental model in rabbits.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A total of 76 male New Zealand White rabbits (weight, 2.5 to 3.3 kg) were used in this study. Permission for the study was received from the Veterinary Directorate of the Prefecture of Athens, according to Greek legislation and in conformance with Council Directive 160/81 of the European Union. The animals were housed in single metal cages and had access to tap water and standard balanced rabbit chow ad libitum. The temperature of the room ranged from 18 to 22°C, the relative humidity ranged from 55 to 65% and the light-dark cycle was 6 a.m. with the lights on to 6 p.m. with the lights off.

The test isolate of P. aeruginosa was derived from a patient with chronic osteomyelitis. The MIC of ciprofloxacin was determined to be 0.5 µg/ml, as assessed by the microdilution technique. After overnight growth, an inoculum of 2 x 108 CFU/ml was prepared and applied for bacterial challenge.

The polymer was prepared as described previously (8) by direct polymerization of lactic acid, with tin(II) octoate used as the catalyst. Ciprofloxacin (Bayer, Leverkusen, Germany) was incorporated at a 1:10 ratio. As a rule, separate amorphous masses of 500 mg containing 50 mg of ciprofloxacin were prepared. They were cut into pieces of 50 mg each. These were given a cylinder shape under sterile conditions and during the operation were implanted in the drilled hole, as described below.

Osteomyelitis was surgically induced in the left upper tibia metaphysis in all rabbits by use of a modification of the model of Norden and Kennedy (17). Briefly, the animals were sedated by the intramuscular injection of 25 mg/kg of body weight of ketamine and 5 mg/kg of xylazine. Anesthesia was maintained by the intramuscular administration of 15 mg/kg of xylazine at 30-min intervals. A 2-cm incision was made in the skin 2.5 cm below the knee joint, which exposed the medial cortex of the tibial metaphysis. A hole was made with a 2-mm drill bit, and 0.1 ml of the prepared bacterial suspension was instilled. A 25-gauge needle was placed through the hole and served as a foreign body. The hub of the needle was broken off. Finally, the hole was plugged with sterile bone wax.

Infection was allowed to progress for 3 weeks, at which time all animals underwent another operation in which the needle and the necrotic tissue were removed. Cancellous bone samples were also obtained from the site of infection, in order to quantitatively culture the test pathogen. Animals were then randomly assigned to two treatment groups. For the animals in group A (n = 31), the hole was again plugged with sterile bone wax. Although it has initially been suggested that bone wax should be avoided due to its promoting effect in experimental osteomyelitis (16), it was applied in the present study to seal the bone defect, as proposed for a more recent model of chronic mandibular osteomyelitis (18). For the animals in group B (n = 45), PLA was implanted into the hole.

The animals were monitored for 70 days. Acetaminophen suppositories were administered to reduce animal suffering. Four animals in group A and five animals in group B were each killed at regular time intervals, i.e., 28, 35, 42, 49, 56, 63, 70, 77, and 91 days postinfection, by the intravenous administration of ketamine. The number of animals to be killed at each time point was designed from the beginning of the experiment. The animals to be killed in each group were selected by use of a randomization chart. The animals killed on days 49, 56, 70, 77, and 91 were weighed immediately before death. All animals were also subject to tibial X ray before they were killed. After the animals were killed, the infected tibias were harvested and specimens of cancellous bone around the site of inoculation of the pathogen were sampled under sterile conditions for quantitative culture; bone specimens were sampled from four different sites from each animal in group B for estimation of the ciprofloxacin concentrations. Latter sites, sites 1 to 4, corresponded to 0 cm, 1 cm, 2 cm, and 3 cm from the site of implantation of the polymer, respectively. Samples were also cut from the muscle and skin adjacent to the infected tibia. At the same times, blood was drawn from the right ear vein under sterile conditions and placed into a sterile tube. The blood was centrifuged, and the serum was kept at –70°C until it was assayed.

The cancellous bone samples were weighed and homogenized with a tissue grinder. Samples were suspended in 1 ml of sterile saline solution, and seven consecutive 1:10 dilutions were quantitatively cultured in sterile water (1 x 10–1 to 1 x 10–8) to avoid the antibiotic carryover effect; a 0.1-ml aliquot of each dilution was plated onto MacConkey agar (Becton Dickinson, Cockeysville, MD). Bacterial growth was expressed as the number of CFU per gram of tissue and given as the log10 value The lowest limit of detection was 10 CFU/ml.

Separate cancellous bone particles harvested from animals killed on days 70, 77, and 91 were fixed in neutral buffered formalin, embedded in paraffin wax, sectioned, and stained with hematoxylin-eosin. Body sections were examined for the presence of an inflammatory reaction in the bone and around the implanted polymer and fibrous tissue.

The concentrations of ciprofloxacin in bone, muscle, skin, and serum were determined in duplicate by a microbiological agar dilution assay with Mueller-Hinton agar (Becton Dickinson) and Escherichia coli ICB 4004 used as the reference strain. Drug levels were estimated from a standard curve created with known concentrations of ciprofloxacin diluted in 10% heat-inactivated horse serum (Merck, Darmstadt, Germany). The standard curve was plotted on a semilogarithmic scale. The lowest detection limit was 0.03 µg/ml, and the interday variation of the assay was 1.2%. The results are expressed as µg/g of tissue.

Animal weights, bacterial growth, and the concentrations of ciprofloxacin were expressed as the means ± standard deviations on each day of sampling. The area under the curve (AUC) for ciprofloxacin was calculated separately for each site of sampling by use of the linear trapezoidal rule and the mean values for each day. Comparisons between groups were done by analysis of variance with post-hoc Bonferroni corrections. Comparisons of the concentrations of ciprofloxacin from sites distinct from the site of implantation of the polymer in group B were performed by Wilcoxon's test. Comparisons of qualitative characteristics between groups were done by Fisher's exact test. Any P value less than 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two weeks after bacterial challenge, swelling was noted at the inoculation site. Fistulas with purulent discharge developed in 16 animals in group A (51.6%) and 3 animals in group B (6.7%) (P < 0.0001 between groups). The changes in the weights of the animals over the period of monitoring are given in Fig. 1. Animals in group A had decreased weights after day 70. Over the same time interval, the body weights of the animals in group B were either increased or remained stable.


Figure 1
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  		FIG. 1. Changes in body weight in animals in groups A (controls; filled circles) and B (treatment; open squares) on the day that the animals were killed compared to the baseline body weight before bacterial challenge. Statistically significant differences are shown. pNS, P is not significant.

 
The main radiological finding among the animals in group A was the presence of sequestra, which were found in 25 animals (80.6%). These were found on day 49 after inoculation of the pathogen. Sequestra were found in only six animals in group B (13.3%; P < 0.0001 between groups). In these animals the main radiological findings were bone remodeling and thickening of the periosteum.

Changes in the growth of the test bacterial pathogen over the period of treatment follow-up compared to the growth at the baseline (the application of therapy) are shown in Fig. 2. The bacterial growth from cancellous bone obtained upon the death of the animals in group B was significantly lower than the growth from cancellous bone obtained from the animals in group A on day 28 (P = 0.001 between groups), day 49 (P = 0.002 between groups), day 56 (P < 0.0001 between groups), day 63 (P = 0.002 between groups), day 70 (P < 0.0001 between groups), day 77 (P < 0.0001 between groups), and day 91 (P = 0.001 between groups). No growth of the test isolate was detected after day 70.


Figure 2
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  		FIG. 2. Growth of the Pseudomonas aeruginosa test isolate in animals in groups A (controls; filled circles) and B (treatment; open squares) on the day after infection.

 
The main histological finding for the animals in group A at week 70 after infection was a mild inflammatory reaction at the infection site accompanied by diffuse fibrosis and new bone formation. Among the animals in group B, the main findings were the presence of small particles of the polymer surrounded by thick fibrous tissue. A mild inflammatory reaction consisting of infiltration by giant cells and macrophages was noted.

The concentrations of ciprofloxacin released at various distances from the site of implantation of the polymer are shown in Table 1. The AUCs for ciprofloxacin were 2,024.3 µg·day/g at site 0, 761.3 µg·day/g at site 1, 524.5 µg·day/g at site 2, and 222.6 µg·day/g at site 3. Overall, antibiotic levels in bone were higher for bone samples closer to the site of implantation. The concentrations of ciprofloxacin in muscle and superficial skin were below the limit of detection.


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  		TABLE 1. Concentrations of ciprofloxacin in cancellous bone at different distances from the polymer implantation site

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The use of biodegradable carriers of antimicrobial agents seems to represent a promising approach to therapy for chronic bone infections (3, 9). Most of these systems use gentamicin as the eluted drug, although there are some reports on the use of vancomycin, teicoplanin, and the fluoroquinolones. However, only a few of them have been tested in animal models of infection.

The concept for the application of PLA as a local carrier of ciprofloxacin for the treatment of chronic bone infection caused by P. aeruginosa is very attractive because (i) ciprofloxacin possesses very potent activity against P. aeruginosa; (ii) newer quinolones are not altered by the heating process mandatory for the generation of the polymer, contrary to other antibiotics, like β-lactams (9); and (iii) it is crucial to achieve high local antibiotic concentrations at the site of infection for a successful therapeutic outcome. A similar system that releases pefloxacin was successfully tested as treatment for methicillin-resistant S. aureus experimental osteomyelitis (8).

The results of the present study revealed that the system applied was very potent at eradicating infection. The bacterial load was reduced within the first 3 weeks of treatment, whereas absolute bacterial killing was achieved by the end of 10 weeks (Fig. 2). Bacterial eradication was accompanied by a clinical benefit, as assessed by the use of both clinical and radiological criteria. More precisely, the occurrence of fistulas was significantly decreased among the treated animals. This was accompanied by weight stability (Fig. 1). A loss of weight was found among the nontreated animals and is probably explained by the lack of food uptake due to the infection or because the animals had difficulty walking and reaching their food (13). This may even have been the situation for the treated animals, which lost weight until day 70, when complete eradication of the bacterial infection was achieved.

The radiological findings for the nontreated animals revealed the frequent occurrence of sequestra; this was significantly decreased upon application of the polymer. The implanted polymer was biodegradable, as assessed by determination of its sequestration into microparticles. Although the present study did not focus on the kinetics of the microparticles after the local implantation of the PLA polymer, previous experimental studies that used lactate microparticles as the carriers of intravenously administered isoniazid and rifampin failed to disclose any sign of hepatotoxicity (1).

It is postulated that the pharmacokinetic properties of each system of local drug release are the cornerstone in eradicating infection (6). Pharmacokinetic analysis of the release of ciprofloxacin by the implanted polymer revealed that sustained release was achieved, since the concentrations at day 7 did not change significantly on consecutive days (Table 1). The concentrations at the site of implantation were almost 80 times higher than the MIC for the test pathogen. Drug levels decreased significantly as the distance from the site of implantation increased. The concentrations 1, 2, and 3 cm away from the site of implantation were almost 40, 12, and 4 times higher than the MIC for the test pathogen, respectively (Table 1).

On the basis of the estimated AUC of ciprofloxacin for each of the sampled sites, the AUC/MIC ratio is well above 125, which is considered the criterion for the antibacterial effects of fluoroquinolones (5). Since the mutant prevention concentration of ciprofloxacin for P. aeruginosa is considered to be equal to 4 µg/ml (5), the AUC/mutant prevention concentration ratio for bone in the present study was kept above 22, indicating that the risk that the implanted polymer would induce the acquisition of resistant strains was low. It may be postulated that the type of pharmacokinetic evaluation applied failed to distinguish between the intracellular and the extracellular concentrations of ciprofloxacin in the bone tissue. This may, however, be of minimal significance, since many pathogens that cause osteomyelitis tend to invade osteocytes (11).

It should be underscored that the drug remained undetectable in the systemic circulation. A probable clinical projection of that finding is the decreased chance for systemic toxicity compared to that during chronic oral treatment and the subsequent effect of oral treatment on the normal flora, which may lead to the acquisition of ciprofloxacin resistance.

PLA has been applied extensively, particularly in various mixtures with coglycolide, for the formation of biodegradable carriers of cefazolin, gentamicin, and ciprofloxacin (12, 15, 19). One of these carriers was implanted in the form of a cylindrical pellet in noninfected rabbit tibiae for estimation of the release of ciprofloxacin (15). Concentrations away from the site of implantation were even lower than those achieved in the present study.

In conclusion, the biodegradable system described here completely eradicates the offending pathogen in an experimental model of osteomyelitis and could play a significant role in the treatment of chronic bone infections caused by P. aeruginosa in humans. The antibiotic-loaded polymeric material tested in the current study should be considered for use in the future because of the absence of side effects from systemic antibiotic treatment and because a second operation is not needed to remove the implant. The fear of systemic toxicity impeded research on the intravenous administration of ciprofloxacin for the therapy of the model of osteomyelitis described here.


   FOOTNOTES
 
* Corresponding author. Mailing address: 4th Department of Internal Medicine, Attikon University Hospital, 1 Rimini Str., Athens 124 62, Greece. Phone: 30210 5831665. Fax: 30210 5326446. E-mail: kyrkanel{at}yahoo.gr Back

{triangledown} Published ahead of print on 14 April 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dutt, M., and G. K. Khuller. 2001. Liposomes and PLG microparticles as sustained release antitubercular drug carriers: an in vitro-in vivo study. Int. J. Antimicrob. Agents 18:245-252.[CrossRef][Medline]
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Antimicrobial Agents and Chemotherapy, July 2008, p. 2335-2339, Vol. 52, No. 7
0066-4804/08/$08.00+0     doi:10.1128/AAC.01360-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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