Receptor Activator of Nuclear Factor kappa-B Ligand is Not Regulated During Chronic Osteomyelitis in Pigs.

Bone loss is a major complication of osteomyelitis and from numerous in-vitro studies, it has been concluded that the bone lysis is caused by elevated expression of the receptor activator of nuclear factor κB ligand (RANKL), leading to increased osteoclast activity. However, we failed to find any relationship between bone loss and osseous RANKL expression in a porcine model of acute and chronic implant-associated osteomyelitis (IAO) due to Staphylococcus aureus or in chronic osteomyelitis lesions in slaughter pigs. Surprisingly, we found that the expression of RANKL was reduced during chronic bone infections. This is in line with the few studies conducted on human samples. A significant bone loss was observed in IAO lesions and in lesions from slaughter pigs, but with no indication of osteoclast involvement using histochemistry, immunohistochemistry for RANKL, receptor activator of nuclear factor kappa-B, osteoprotegerin and cathepsin K, and high-throughput quantitative real-time polymerase chain reaction on bone tissue from osteomyelitic lesions. A strong inflammatory response was seen in the infected animals and, therefore, we propose proteolytic enzymes induced by inflammation to be a major component of the bone loss. Furthermore, we found a significant upregulation of the IL26 gene in infected animals, which can inhibit RANKL-induced osteoclastogenesis, but has no homologue in mice. This finding emphasises that neither murine models nor in-vitro studies can mirror human disease development completely. The present study emphasises that the interactions between microorganisms, the immune system and bone cells in osteomyelitis are too complex to be accurately represented by an in-vitro model.

Guideline for Preclinical Studies of Bone Infections in Large Animals Based on a Systematic Review of 316 Non-Rodent Models.

BACKGROUND: In recent years, animal models of bone infections have been used with increased frequency in order to evaluate novel diagnostic and anti-infective technologies, like antibacterial coating of bone implants or local antibiotic carrier products. Therefore, it is highly relevant to evaluate the scientific quality of existing bone infection models. METHODS: We conducted a systematic review of 316 studies of large non-rodent animal models of bone infection (254 rabbit, 16 pig, 23 dog, 11 goat, and 12 sheep) and extracted data on study design, methodological quality, and postmortem evaluation of infection with respect to reporting and quantification of pathology and microbiology. RESULTS: The review demonstrated a substantial lack of study-design information, which hampers reproducibility and continuation of the established work. Furthermore, the methodological study quality was found to be low, as the definition of infection, randomization, power analysis, and blinding were only seldomly reported. The use of histology increased in recent years, but a semi-quantitative scoring of the lesions was often missing, i.e. no objective quantification of outcome. Most of the studies focused on whether the inoculated bacteria were present within the bone tissue post mortem or not. However, very often the bacterial burden was not quantified. In many of the models, different antimicrobial interventions were examined and, although antimicrobial effects were commonly described, a lack of complete sterile outcome was observed in many models. On the basis of the systematic review, we established a study template providing a guideline for the standard reporting of animal models of bone infections, including details related to the animal, pathogen, infected animal, and postmortem analysis that are of crucial importance for validation of results and reproducibility. CONCLUSIONS: As the aim of many bone infection models is to examine the effect of an intervention, the guideline emphasizes the importance of objective quantification of outcome, e.g., blinded quantitative scoring of histological findings and quantification of bacterial burden within tissue and on inserted implants. Less than 5% of the analyzed studies adhered completely to the ideal form presented in the study template. CLINICAL RELEVANCE: Anti-infective interventions must be tested in preclinical animal models before implementation in human patients, and optimal design and validation is essential for a high translational value.