Rat Model Of Osteomyelitis Research Articles

Controlled release of vancomycin from thin sol‐gel films on implant surfaces successfully controls osteomyelitis

Peri-prosthetic infection remains a serious complication of joint replacement surgery. Herein, we demonstrate that a vancomycin-containing sol-gel film on Ti alloy rods can successfully treat bacterial infections in an animal model. The vancomycin-containing sol-gel films exhibited predictable release kinetics, while significantly inhibiting S. aureus adhesion. When evaluated in a rat osteomyelitis model, microbiological analysis indicated that the vancomycin-containing sol-gel film caused a profound decrease in S. aureus number. Radiologically, while the control side showed extensive bone degradation, including abscesses and an extensive periosteal reaction, rods coated with the vancomycin-containing sol-gel film resulted in minimal signs of infection. MicroCT analysis confirmed the radiological results, while demonstrating that the vancomycin-containing sol-gel film significantly protected dense bone from resorption and minimized remodeling. These results clearly demonstrate that this novel thin sol-gel technology can be used for the targeted delivery of antibiotics for the treatment of periprosthetic as well as other bone infections.

Scintigraphic imaging using technetium-99m-labeled ceftizoxime in an experimental model of acute osteomyelitis in rats

To investigate inflammatory (zymosan) and infectious (Staphylococcus aureus) processes in experimental models in rats using technetium-99m-labeled ceftizoxime (CFT). Male Wistar rats were used for the development of the inflammatory (zymosan) and infectious (S. aureus) processes in the medullary cavity of the left tibia. Sterile saline was used for the control group. At 48 h after induction of the processes, the animals were anesthetized and scintigraphic images were acquired at 1, 2, 4, and 6 h after intravenous injection of 0.1 ml of 99mTc-CFT (55 MBq). Quantitative analysis of the scintigraphic images was performed by counting the radioactivity in the regions of interest. Samples of tibia were taken for histopathological examination. The images showed that 99mTc-CFT presented higher tropism to infectious foci than with the inflammatory site. The average value of the target/nontarget ratio of the 99mTc-CFT was significantly higher in the infected (2.40+/-0.22) than in the inflamed tibia (1.50+/-0.05) and the control group (1.05+/-0.04) for all of the investigated times. The histological data showed a similar inflammatory response for both the S. aureus and zymosan groups. The 99mTc-CFT presented a high tropism and retention for an infected region in this model of osteomyelitis, thereby constituting an interesting strategy to distinguish aseptic from septic sites.

Role of hypermutability on bacterial fitness and emergence of resistance in experimental osteomyelitis due toStaphylococcus aureus

This study was designed to investigate the role of hypermutability of Staphylococcus aureus on bacterial fitness and antibiotic resistance in a model of chronic bone infection. An isogenic pair of strains, S. aureus RN4220 and its mutator counterpart inactivated in the mutL gene were used in a rat model of osteomyelitis of the tibia. The effect of the mutator phenotype in the emergence of antibiotic resistance was assessed in rats infected by each strain separately and treated with rifampicin for 5 days. No difference between the two strains was found in bacterial growth in vitro and in bacterial survival in the animal model, indicating no fitness defect in the mutator strain. In competition studies performed in rats coinfected with the two strains at a same ratio and sacrificed at different times from day 3 to day 42 postinoculation, the mutator strain was clearly disadvantaged because it was found in all rats and at all study times at lower counts (P<0.05 from day 14 to day 42). Two of the 16 rats infected by the mutator strain and none of the 14 rats infected by the wild-type strain had acquired rifampicin-resistant mutants (P=0.4). Data suggest that the S. aureus mutator phenotype was associated with a decreased bacterial fitness in vivo and did not confer significant advantage in the acquisition of antibiotic resistance in a model of chronic bone infection.

Local Antibiotic Delivery Systems

Local Antibiotic Delivery Systems

Establishment of rat model of acute staphylococcal osteomyelitis: relationship between inoculation dose and development of osteomyelitis

Many animal models of acute and chronic osteomyelitis have been developed. In these models, osteomyelitic lesions are induced using sclerosing agents and foreign bodies with bacterial strains. In the present rat model, these sclerosing agents were not used. We assessed the relationship between inoculation dose and histological, radiological, and microbiological changes in the acute phase (1 week after inoculation) using this rat osteomyelitis model. An experimental rat model of acute osteomyelitis was developed by direct inoculation of the virulent strain BB of Staphylococcus aureus into tibial bone without sclerosants. To examine the relationship between the inoculation dose of the bacteria and the progression of the osteomyelitis, the inoculated lesions were assessed for changes in histological, radiological, and bacteriological parameters at 1 week after infection. Serial dilutions of the bacteria [6 x 10(1) to 6 x 10(5) colony-forming units (CFU)/5 microl] suspended in saline or saline alone were inoculated into the proximal metaphysis of the tibia. Development of significant histological and radiological signs of osteomyelitis required an inoculum of at least 6 x 10(3) CFU/5 microl. The number of viable bacteria at the lesion reached a maximum of 6 x 10(3) CFU/5 microl. These results suggest that strain BB induces the development of acute staphylococcal osteomyelitis with clear infective destruction in the tibia, and that our model may be applied to the identification of virulence factors in studies of posttraumatic osteomyelitis.

A new model of implant-related osteomyelitis in rats.

Infection related to osteosynthesis often has dramatic consequences for the patient. Prolonged hospitalization with systemic antibiotic therapy, several revision procedures, possible amputation, and even death may occur. To investigate the pathology of infection in orthopedic surgery, a new rat model of implant related osteomyelitis was developed. Three different concentrations (10(6), 10(3), and 10(2) colony-forming units (CFU)/10 microl) of Staphylococcus aureus were inoculated into the tibial medullary cavity with simultaneous insertion of a titanium Kirschner wire. Controls received phosphate-buffered saline (PBS). Each group consisted of 10 animals. Animals were followed for 4 weeks until sacrifice. X-rays of the tibiae were taken weekly, blood counts were analyzed, and body temperature and weight were determined. After sacrifice, infection was evaluated by histological and microbiological investigations. All animals inoculated with Staph. aureus in either concentration developed microbiological, histological, and radiological signs of osteomyelitis in correlation to the amount of inoculated bacteria. X-rays clearly revealed osseous destruction after 14 days with progression of osteomyelitis during the following weeks. CFU/g bone and bone weight after sacrifice showed dependence on the amount of inoculated CFU. The histological results confirmed the radiological findings. No significant changes in blood counts, body weight, and body temperature between the groups could be observed. The results demonstrate that it is possible to develop a model of implant-related osteomyelitis in rats with dependence on the amount of inoculated bacteria. No other promoters of infection besides intramedullary insertion of titanium Kirschner wires were used in this model.

In Vivo Evaluation of Antibiotic Impregnated Beads in a Rat Osteomyelitis Model

The biocompatibility, release and efficacy of gentamicin released from poly(lactic acid) (PLA) beads in an osteomyelitis model were evaluated. Microsphere particles of PLA containing gentamicin were compressed into 2.5 mm beads. Osteomyelitis was induced in the tibia by a gentamicin resistant Staphylococcus aureus in 32 Sprague-Dawley rats. Following debridement at 8 weeks, rats were randomized into four groups: 1) gentamicin-impregnated PLA bead implant, 2) PLA bead without gentamicin, 3) once-daily gentamicin injections (5 mg/kg) subcutaneously for 14 days, and 4) no treatment (control). Rats were sacrificed at 7 weeks, and the tibias were sampled for quantitative culture, histologic scoring and gentamicin concentration. Radiography and culture in 31 of the 32 rats confirmed osteomyelitis. A low level of gentamicin was measured in the serum of group 1 rats up to day 42. Gentamicin was not detectable in bone samples. There was no significant difference in radiographic scores, histologic scores or quantit...

Application of a Rat Osteomyelitis Model to Comparein Vivoandin Vitrothe Antibiotic Efficacy against Bacteria with High Capacity to Form Biofilms

A rat experimental osteomyelitis model was used to study the efficiency of antibiotics on biofilm bacteria adhered to implants in relation to the efficiency obtainedin vitro.In the osteomyelitis model, 104bacteria of the strain variant used for thein vitrostudies (a slime-producing variant ofStaphylococcus aureus) were inoculated into the rat tibia at surgery, after implanting a stainless steel canula precolonized for 12 h with this strain. After 5 weeks, a 21-day antibiotic treatment was applied (using cefuroxime, vancomycin, or tobramycin). Subsequently, implant and tibia were studied for presence of bacteria. In this osteomyelitis model, cefuroxime inhibited bone colonization and reduced the number of bacteria in metal and bone at a higher degree (P< 0.05) than vancomycin and trobramycin (the latter antibiotic did not have this reduction effect). Thein vitroassay was applied using three concentrations of each antibiotic (8, 100, and 500 μg/ml) and 6-, 24-, and 48-h biofilms. Bacterial viability was evaluated by ATP-bioluminescence after 24 h of antibiotic treatment. In thisin vitroassay, cefuroxime significantly (P< 0.05) reduced in all cases the number of viable bacteria in biofilms, tobramycin did not affect viability, and vancomycin affected viability except at the lowest concentration used (8 μg/ml, i.e., 8× the minimal bactericidal concentration of this antibiotic) when facing the oldest (48 h) biofilm. These results demonstrate the usefulness of the osteomyelitis model applied in providing evidence for a close correlation between thein vitroandin vivofindings on the effect of three antibiotics under study.

The sustained release of antibiotic from freeze-dried fibrin-antibiotic compound and efficacies in a rat model of osteomyelitis.

The kinetics of drug release from fibrin adhesive agent (consisting of fibrinogen, factor 8, thrombin, aportinin and calcium chloride)-antibiotic compound and efficacy on rat experimental osteomyelitis were studied. To enhance the slow release activities of antibiotic, a mixture of fibrin clots was freeze-dried. Effects of freeze-drying were to make a fibrin clot an interlinked pore and to increase crosslinking rate containing an antibiotic. A diffusion test from aminoglycoside (Arbekacin Sulfate: 200 mg) compound was carried out. In vitro study freeze-dried antibiotic compound (FFAC: 1 cm3) was placed in saline (3 ml). The saline was replaced every 48 h and the previous solution was stored at -45 degrees C until assay. The result was that a concentration of 0.4 mg/l, sufficiently high to control Staphylococcus aureus strain IM2-42, was maintained within nine exchanges of saline after 18 days. In vivo animal experiments, FFAC (2 x 2 x 3 mm) were tested in rats with established Staphylococcus aureus osteomyelitis in the proximal tibia. The animals were observed for radiographic signs of infection and tissue was examined histopathologically. Bacterial counts by bone cultures were statistically lower in rats implanted with FFAC than in those only given a drug-free FFC and curettage. Radiographical and histological observations also showed beneficial effects of the FFAC. The results suggest that the FFAC provide a simple drug delivery system, and may be a promising alternative treatment for osteomyelitis.

Local expression of tumor necrosis factor alpha in an experimental model of acute osteomyelitis in rats.

The inflammatory response associated with Staphylococcus aureus osteomyelitis results in extensive bone damage characterized by apparent increases in bone resorption and formation. These results suggest an increased local release of agents capable of modulating bone remodelling. Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine proposed to play an important role both in normal bone remodelling and in bone diseases; however, its potential role in osteomyelitis is unclear. This study evaluated changes in bone TNF levels during infection, using a rat model of acute osteomyelitis due to S. aureus. Following direct tibial infection, bacterial counts in bone were persistently high (approximately 6 log10 CFU/g of bone over 63 days) and bone weights increased. TNF activity was undetectable in uninfected bone (<0.01 ng/g of bone) but dramatically higher in infected bone (up to 5.2 +/- 3.5 ng/g of bone). Although TNF-alpha mRNA was weakly detected in uninfected bone, osteomyelitis was associated with up to 37-fold increases in expression of both the 1.6- and 2.4-kb transcripts. Both TNF activity and mRNA transcript levels remained elevated throughout the course of infection. TNF-alpha mRNA detected by in situ hybridization was present in osteoblasts as well as in populations of marrow cells and/or inflammatory infiltrate cells. Histopathology of infected bone indicated extensive bone resorption and adjacent areas of formation that were associated with cells expressing TNF-alpha mRNA. These data suggest that the elevated TNF levels induced by experimental infection may be directly related to changes in the histology of bone during osteomyelitis.

Model of experimental chronic osteomyelitis in rats.

We describe here a Sprague-Dawley rat model for chronic osteomyelitis. Staphylococcus aureus and sodium morrhuate were implanted by either microdrilling or direct needle injection into the tibiae of rats. Of 107 rats, 87 (81%) developed osteomyelitis when a high-speed drill was used for implantation, and 27 (51%) of 53 rats developed osteomyelitis by direct needle inoculation (chi square = 9.81, P less than 0.01). Demonstrated histopathological changes included the presence of resorption bays filled with osteoclasts. Quantitative microbiological monitoring of tibial count confirmed disease chronicity, yielding stable numbers of CFU (10(6.29 +/- 0.27) ) of S. aureus over 70 days. Infected animals became anemic and lost weight. The erythrocyte sedimentation rates and leukocyte counts were not elevated. Roentgenograms provided the best correlation with the number of organisms in infected tibiae (r2 = 0.80). Rats with infected tibiae were treated with either oxacillin (120 mg/kg per day) or ceftriaxone (50 mg/kg per day). Treatment over 14 or 28 days reduced S. aureus counts in tibiae but did not reliably sterilize infected bones, suggesting that this model was resistant to prolonged antimicrobial therapy.