Departments of 1 Orthopaedic Surgery, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam; 2 Oral Cell Biology/ACTA; 3 Oral Biochemistry/ACTA, vd Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
Received 10 March 2004; returned 10 April 2004; revised 31 May 2004; accepted 2 June 2004
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Abstract |
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Methods: Calcium phosphate cement with 50 mg/g hLF1-11 or gentamicin was injected into the femoral canal, after inoculation with Staphylococcus aureus. Three weeks later, slices of the proximal femora were sawn for quantitative bacterial culture and histology.
Results: Treatment with hLF1-11 (P<0.038) or gentamicin (P<0.008) caused a reduction of cfu compared with the untreated control rabbits. The number of sterile cultures was higher in hLF1-11- (3/7) and gentamicin- (5/6) treated animals than in controls (1/7). Radiological and histological analysis showed early bone ingrowth into the cement cracks, and only moderate pathological changes in rabbits with positive cultures.
Conclusions: Local prophylaxis with hLF1-11 effectively reduced development of osteomyelitis in a rabbit model, but gentamicin resulted in a larger number of sterile femora.
Keywords: biodegradable carriers , drug delivery , intramedullary infections , peptide antibiotics , animal models
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Introduction |
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Antimicrobial peptides (AMPs), short positively charged peptides, have been found in virtually all forms of life as a first line of defence against invading microorganisms.11,12 They combine pore formation in negatively charged bacterial cell membranes with intracellular killing events and have a minimal propensity to induce resistance.1315 Their fast killing effect on a broad range of microorganisms offers a great opportunity for the development of new antibiotic agents.14 These peptides have been proposed as a novel class of natural antibiotic agents, and might be used clinically to treat infections caused by resistant bacterial strains.11 Potent antimicrobials have been produced by using natural peptides as a template, and by taking active domains of larger proteins.1618
The antimicrobial peptide hLF1-11 consists of N-terminal amino acids 111 of human lactoferrin, and has a broad antimicrobial spectrum.5,19,20 It has shown in vivo bactericidal activity after systemic administration against a variety of microorganisms, including methicillin-resistant S. aureus (MRSA).20 Biodegradable calcium phosphate (CaP) cements have been studied as a carrier for the controlled release of many different antibiotics.6 We previously shown that hLF1-11 was still able to kill MRSA after being released from CaP carriers in vitro.5 The present study describes the efficacy of hLF1-11-loaded and gentamicin-loaded CaP cement for the prevention of osteomyelitis in a well-characterized rabbit model.79
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Materials and methods |
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S. aureus (Wood 46, ATCC 10832) was used to inoculate the rabbit femora (gentamicin MIC 8 mg/L by broth dilution method; hLF1-11 LC50 6.25 mg/L by cfu assay adapted for salt-sensitive AMPs).5,20 After culture in brain heart infusion, a stock of aliquots for single use was frozen. Pre-operatively, samples containing 107 cfu/mL PBS were prepared; a volume of 0.1 mL (106 cfu) was injected into the femoral canal. Previous studies have shown this dosage to result in a high rate of infection in the presence of PMMA cement.9 Post-operatively, the inoculum was serially diluted and plated on blood agar; colony count after 18 h at 37°C confirmed accurate bacterial content.
Antimicrobial agents
The hLF1-11-peptide (GRRRRSVQWCA, 1375 Da) was manufactured by solid-phase peptide synthesis using Fmoc (9-fluorenyl-methoxycarbonyl) chemistry, as described previously.21 Reanalysis of peak fractions by reversed phase HPLC resulted in one major peak, revealing at least 90% purity; the authenticity of the peptide was confirmed by electrospray ionization quadrupole time-of-flight mass spectrometry (Q-TOF MS, Micromass Inc., Manchester, UK). Gentamicin sulphate powder (GS) was a gift from Biomet Merck BioMaterials (Darmstadt, Germany).
Animals
Twenty-six healthy (specific pathogen free) adult female New Zealand White rabbits (Harlan, Horst, the Netherlands) of 3.23±0.24 kg were kept in group housing. They were assigned to the following treatments: hLF1-11 (n=7), gentamicin (n=7), control (infection, no antibiotic agent, n=9) and sham (operated on but not infected, n=3).
The guidelines according to the Dutch Act on Animal Experiments (1985) have been observed.
Operative procedure
The operation was performed as described by Nijhof et al.7 After sedation, blood samples were drawn for leucocyte count (WBC) and erythrocyte sedimentation rate (ESR) determination. The trochanter tertius of the right femur was surgically exposed and a hole to the medullary canal drilled. Subsequently, 0.1 mL of bacterial suspension (hLF1-11, gentamicin and control groups) or PBS (sham group) was injected, followed by injection of the cement paste. The cement was prepared immediately before inoculation of 1 g of Bonesource cement powder (Stryker-Leibinger, Freiburg, Germany), 50 mg of hLF1-11 (hLF1-11 group) or GS (gentamicin group) and 0.40 mL of mixing solution (Stryker-Leibinger). The control and sham groups received plain cement without antibiotic agent. The cement syringe was weighed before and after injection, to determine the amount of injected cement.
As a baseline image, radiographs of the right femora were made post-operatively. Additional blood samples were drawn on days 7 and 21.
Autopsy and sample acquisition
After 21 days, the animals were killed with a pentobarbital overdose; both femora were aseptically excised and radiographed.7 Signs of osteomyelitis were scored using Norden's system in which a maximum score of seven corresponds with maximal pathogenic changes.22
Of the proximal femoral diaphysis, two transverse slices of ±0.5 cm were sawn; the proximal sample was used for histological and the distal for bacteriological determination. Proximal bone sections were decalcified, embedded in paraffin, H&E stained and mounted on slides. Signs of infection were scored using Smeltzer's system, in which a maximum score of 16 signifies severe osteomyelitis.23 After careful removal of the cement from the distal sample, it was weighed and ground in 50 mL of PBS. This suspension was serially diluted and plated on blood agar; the colony count after 18 h at 37°C was calculated to represent cfu/g bone. Negative cultures were conservatively calculated as 4000 cfu/g (the average detection limit). A bacterial load greater than 105 cfu/g (arbitrarily set at 10% of the inoculum dose) was considered a sign of full-blown infection.
Statistics
The samples from all animals that completed the 21 day follow-up period were randomized in a blinded manner and evaluated by two observers. The differences between groups were analysed using a two-tailed MannWhitney U-test; P<0.05 was considered significant (values are means±SD).
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Results |
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Quantitative culture results indicated that the addition of gentamicin (P=0.008) or hLF1-11 (P=0.038) resulted in a significant decrease in viable bacteria (Figure 1). Table 1 summarizes the results for all treatment groups: cultures, amount of injected cement, body-weight change, histological score and radiological score. Bacteria were cultured from 11 of the 23 operated femora: four of seven in the hLF1-11 group, one of six in the gentamicin group and six of eight in the control group. The non-inoculated sham group and the un-operated left femora had sterile cultures. The amount of cement injected during the operation was 0.83±0.34 g; there was no correlation between amount of injected cement and culture result.
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Overall, the femora with positive culture results had a higher average radiographic score (3.4±1.2, P=0.006) than sterile femora (2.1±1.7). However, this did not result in significant radiological differences between the treatment groups (Table 1). The ESR and WBC values showed no significant post-operative changes within animals or between groups (data not shown).
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Discussion |
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The in vivo killing effect of natural and synthetic AMPs has been demonstrated on a variety of microorganisms, but the number of published animal studies is still relatively small.20,2426 Intravenous injection of low concentrations (ng/mL) of hLF1-11 effectively reduced infection by resistant S. aureus strains in a mouse model of thigh-muscle infection.20 Synthetic and bovine analogues of hLF1-11 have demonstrated antimicrobial activity in mouse models by decreasing Escherichia coli bladder infection, and Toxoplasma gondii-induced brain cysts.24,25 Furthermore, intradermal injection of 16 mg Novispirin G10 effectively reduced infection in rats with Pseudomonas aeruginosa-infected burns.26 Finally, a number of clinical trials underlines the potential of future AMP-based drugs for treatment of superficial infections, oral mucositis and paediatric sepsis.2729
Besides direct antimicrobial killing activity through pore formation and intracellular targeting, AMPs are claimed to have an immunomodulating effect.15,20 This results in higher in vivo than in vitro antimicrobial activity by specific activation of signalling cascades in the host immune system.11,20 The in vivo antimicrobial activity after intravenous administration of hLF1-11 at minimal (ng/mL) concentrations supports the role of an immunomodulating effect of this human-derived peptide.20 Although some AMPs, like the bee venom mellitin, are toxic to mammalian cells, exposure of human erythrocytes to high concentrations of hLF1-11 up to 200 µg/mL did not indicate in vitro cytotoxicity of the peptide (unpublished data). Furthermore, human allergic reactions would not be likely since hLF1-11 consists of a small fragment of the human lactoferrin protein.
Rabbits are among the most frequently used animals for experimental bone infection studies.30 Most studies report complications similar to those described in the current study, either related to infection, to surgery or to cement injection.9,10 Animal models of osteomyelitis prevention require a high rate of infection in the (untreated) control animals in order to detect differences with the treated animals. In the presented study, infection was established in 85% of the (untreated) control animals; similar rates of infection have been reported in other rabbit infection studies.9,31 The infection rate in this study compares well with other studies, and modification of several factors could further improve results and decrease the number of complications.23,30,32 Virulence of the bacterial strain, inoculum dose, presence of a biomaterial to which bacteria preferentially adhere, surgical technique and local tissue trauma all influence the infection rate in untreated animals.8,31,33
The presence of tissue necrosis or a foreign material is essential in the development of osteomyelitis in animal models.8,32 The competition between host cells and bacteria to establish predominance on the implant surface (the race for the surface) determines whether or not the implant becomes infected.34 The result of this conflict depends highly on the material properties of the implant surface. Although speculative, injectable CaP cement may influence bacterial growth by its setting reaction and subsequent osteoconductive properties, which might explain why no infection was detected in one of seven control animals (infected but untreated).
In the present study, the rabbits' clinical results showed mild to moderate signs of infection; the small increase in body weight, the minor variation in ESR and WBC and the absence of signs of systemic illness all indicate that the induced infection was contained within the femur. Furthermore, the relatively small number of pathological changes recorded on histological and radiological observation could be consistent with an infection of intermediate severity. The cement in the proximal femur could have obscured some of the pathological signs on radiographs; nevertheless, the low histological scores appear to confirm the contained and limited nature of the femur infection. However, the short follow-up period (3 weeks) may not have been sufficient for both the disappearance of tissue reaction due to the surgical trauma and the full development of signs of infection in all rabbits.
We used an established technique for histology and culture, but a sample error may still have occurred, which could have resulted in missed cases of infection.7 The difference in infection reduction between hLF1-11 and gentamicin was not significant; adaptation of the drug delivery system may increase the cure rate. Future improvements could include alterations to the AMP dose, the peptide structure and the carrier material to optimize release kinetics in vivo.We previously demonstrated that part of the added hLF1-11 may bind to the cement during the setting reaction, which could limit release. More complete release occurred when higher amounts were added to the cement.4,5 These experiments support using a high dose of hLF1-11; however, there are practical limitations to the scale of experimental peptide production and purification methods.21
In conclusion, both hLF1-11 and gentamicin reduced the number of infected animals and the bacterial count in infected femora. The recent observation of resistant bacterial strains on explanted gentamicin-containing PMMA beads (in 18 of 20 patients) highlights the clinical impact of antibiotic resistance in orthopaedic infections.3 Our results, using a non-resistant strain, support the concept of using hLF1-11 in the prevention of osteomyelitis and thus warrant pre-clinical studies of bone infection caused by resistant strains, comparing the efficacy of AMPs with current antibiotic agents.
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Acknowledgements |
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Footnotes |
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