44 In Vivo Models for Bone and Joint Infections
Bone and joint infections do not represent a uniform disease, but a heterogeneous group of diseases that require complex treatment algorithms. The integral part is certain highly standardized surgical procedures.
Even in modern times, bone and joint infections are still associated with a high morbidity and mortality rate resulting in high direct and indirect medical costs, which are increasingly important in the course of the rationalization of health care systems in the Western industrialized nations. To meet these issues, joint and bone infections must be prevented and treated more effectively through improved prophylaxis and therapy algorithms. This results in a constantly increasing importance of in vivo animal studies.
The premise for a good animal model is that it reflects as closely as possible a complex clinical situation. Thus, the first crucial step for in vivo animal studies of bone and joint infection is the selection of the best available model or development of a new animal model that closely reflects the clinical scenario. The more precisely an animal model mimics the clinical situation, the more transferable the results will be from the animal model to clinical practice. To achieve this step, it is necessary firstly to avoid systemic complications that lead to the loss of animals and secondly to induce bone and joint infection reliably. The aim of this chapter is to give the reader a guide with simple basic criteria for orientation in the field of in vivo animal models and studies of bone and joint infections.
44.1 General Requirements
Infection experiments have to be approved by the local authorities, particularly regarding ethical aspects. Approval is subject to numerous conditions. This includes the restriction to the minimum number of animals. Infection experiments can be a heavy burden on laboratory animals. As mentioned previously, the inoculation dose is a compromise between reliable induction of the infection and avoiding systemic complications, with the exception that systemic side effects are investigated. For this reason, no animal should be exposed to avoidable pain and suffering. Adequate anesthesia and analgesia must be provided.
Relevant changes in the study design must be reported immediately to the local authorities. Under certain circumstances, you will be asked to submit a new request. A copy of the approval of the local authorities may need to be submitted with the manuscript. Inconsistencies will lead to the rejection of the manuscript. In addition, the authorities will evaluate future projects more critically and possibly refuse permission.
The animals must be housed in a suitable and approved animal laboratory. Animals should be housed individually after the inoculation to prevent mutual chewing and infecting. The cage floor should be covered with absorbent sheets. Classic litter can get into wounds and promote secondary contamination and thus lead to false results.
The laboratory must meet the minimum safety standard S2 due to the work with biological materials that pose a potential danger for the employees. This standard applies throughout the world. Sometimes more strict state-specific certificates are required, and appropriately qualified and certified personnel must be provided. Before an application is sent to the local animal protection authorities, it is advisable to inquire about the structural and personnel standards of the animal laboratory. Failure to meet these standards results in rejection of the application by the local authorities.
Surgical procedures both for operative interventions on the animals and for euthanasia must be carried out under aseptic conditions in order to prevent contamination with other bacteria (Fig. 44.1).
44.2 Guidelines for Study Planning
Before a systematic action guideline is presented, two terms need to be defined: osteitis and osteomyelitis. In osteomyelitis, the bone marrow is infected first; in contrast, in osteitis there is a centripetal infection with spread of germs from outside the bone to inside. This means that with osteitis, the cortex and/or cancellous bone are infected first and a bone marrow infection occurs secondarily, so that some authors use the term secondary osteomyelitis in the case of an osteitis. In German-speaking countries, the term osteitis is commonly used, whereas in the Anglo-American literature no distinction is made between osteitis and osteomyelitis, and it is generally spoken of as osteomyelitis, which is used in this chapter.
44.2.1 Clinical Situation and Research Target
First, the clinical situation of a joint or bone infection has to be characterized in detail to deduce the clinical problem. AlphaThe exact research question can be formulated only when the clinical problem is exactly identified and described in sufficient detail. The research question should then indicate the research target. Therefore, at least one main parameter and maybe one or several secondary outcome parameters have to be quantified to compare the findings of the performed animal study with other animal studies to draw a conclusion and transfer the results back to the clinical situation. In vivo models can either be used to investigate the prevention or treatment of joint and bone infections. This has to be stated in the research question, because this may be associated with a different burden on the animals and thus is relevant for the approval of the animal study.
These infections are first divided into a primary bone infection or primary joint infection; they are then differentiated with respect to their exact etiology, pathology, clinical manifestation, and timeline. A pure joint infection should be considered as an infectious synovitis of the joint membrane that does not involve the adjacent bone (e.g., hematogenous or early postarthroscopic infection). The same is true for bone infections. A pure bone infection should not be complicated with a joint infection. An example is the group of blood-borne bone infections (e.g., acute staphylococcal hematogenous osteomyelitis). Currently, in the industrialized nations, the study of implant-associated bone and joint infections is in the foreground, such as joint prosthesis infections. Foreign materials as an important pathogenetic factor in bone and joint infection significantly affect the progression of infections and clinical outcome. However, there are pathophysiological overlaps between the clinical scenarios.
44.2.2 Bacteria
Both bone infections and joint infections—with the exception of some special forms—are caused by bacteria, viruses, fungi, and parasites of endogenous or exogenous origin, and can progress acutely or chronically according to their pathogenicity and the immune status of the host organism. Most commonly, bone and joint infections are due to bacteria. Staphylococci in general are the most common pathogens in acute bone, chronic bone, and all joint infections. Strains of Staphylococcus aureus are frequently the focus of in vivo animal research due to their increasing antibiotic resistance (Fig. 44.2).
The selection of a suitable strain plays a key role. Besides the use of “own” isolates, a plurality of commercially available strains exists. The most important criterion is the clinical scenario from which the strain originates. Accordingly, in bone and joint infections the used bacterial strain should derive from an equivalent clinical setting.
Before the main study, a pilot study is highly recommended to evaluate an appropriate bacterial strain in a proper inoculation dose. A proper inoculation dose reliably leads to a high infection rate in the control group. This should be titrated to reduce the number of required animals of the entire study: Statistically significant differences are more likely to be found between the control and the treatment group if there is a high infection rate in the control group, and this helps to ensure that the sample size calculation produces the smallest number. However, excessive inoculation doses have to be avoided due to systemic infection complications, including death of animals in the worst case. Thus, because the inoculation dose can vary widely from species to species, a pilot study should not be skipped. In addition, positive effects in the treatment group can be negated by an overcontamination.
Bioluminescent strains of bacteria are available, and they have almost completely replaced radioactive labeling techniques. Bioluminescent strains have a higher generation time than nonbioluminescent strains. Therefore, the induction of an infection is more difficult, requires higher inoculation doses, and increases the probability of the infection failing to persist due to the elimination of the bacteria by the host′s immune system.
44.2.3 Animals
An optimal experimental animal for in vivo study of bone and joint infections does not exist. Most commonly, rats and rabbits are used. Both are large enough to carry out more complex models and thus allow for the use of implants and other materials relevant for clinical use. This leads to a certain degree of standardization of the model and increases the transferability of the results to the clinical situation in humans. Moreover, they can be kept in groups, which is cost-effective. Mice and hamsters are usually too small for implant-related studies, whereas large animals (e.g., sheep or dog) are in most cases too costly and are less ethically accepted than small animals.
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