We are pleased to follow-up with our new subsection in our quarterly newsletter, Preclinical Tales, which highlights exciting approaches and initiatives aligned with the 3R principle. The 3R principle (replace, reduce, refine), introduced by Russell and Burch in 1959, serves as a framework for ethically using animals in research and enhancing scientific quality and integrity in studies involving animals. Get inspired, stay informed, and begin implementation!

Are you PREPAREd to ARRIVE?
Brought to you by Annemarie Lang, DVM, PhD

Most researchers conducting experiments involving laboratory animals are well aware of the ARRIVE guidelines, a checklist of recommendations aimed at improving the reporting of research involving animals. At the time of manuscript submission, most journals now require adherence to the ARRIVE guidelines and often request the upload of a completed ARRIVE checklist. This practice ensures the highest quality and reliability of published studies, enabling the scientific community to properly review, evaluate, and reproduce findings from animal experiments.

The Preclinical Models Section underscores the importance of the ARRIVE guidelines by, for example, recognizing abstracts and presentations at the ORS Annual Meeting that demonstrate exemplary adherence to the ARRIVE guidelines with a dedicated award. Additionally, we have contributed to and led discussions on new guidance regarding expectations for animal care, pain control, and experimental design in submissions to the Journal of Orthopaedic Research (JOR) that involve preclinical animal models.

However, we recognize that adherence to ARRIVE represents merely the final stage of the research process, or better yet, the study itself. Sound experimental design, planning, and selection of the most suitable animal model begin much earlier. Proper preparation profoundly impacts the outcomes, quality, and benefits of research for the broader scientific community. We will explore this topic further during an upcoming workshop and session at the ORS 2025 Annual Meeting, and we hope that you can join us for a lively discussion.

If you are interested in this topic, you may want to explore the PREPARE guide, a 15-step framework developed to support the sustainable planning of research studies and experiments.

This guide highlights critical aspects of project design that might otherwise be overlooked and serves as a valuable resource for new and early-career investigators, as well as a useful refresher for all researchers.

For those interested in learning more, we recommend visiting the Norecopa site or reading the full publication here.

Immune Response and Inflammatory Pathways in Orthopedic Models
Claudia Siverino, PhD

Incorporating immune and inflammatory responses into preclinical models is becoming increasingly important, particularly for conditions like osteoarthritis and orthopedic device-related infections (ODRI). Researchers are developing models that integrate musculoskeletal and immune components, which helps evaluate how immune responses affect tissue healing/damage and implant integration/chronic inflammation.

Investigating complex host–pathogen interactions is essential for enhancing our understanding of disease pathogenesis and for developing potential therapeutics, which requires the use of clinically relevant animal models.

In the context of bacterial infection, murine models have significantly contributed to the understanding of bacterial pathogenesis. However, mouse models present several limitations, including altered innate and adaptive immune responses to bacterial infection. In the last two decades, multiple humanized mouse models have been developed to attempt to recapitulate the human immune system in small animal models. There are two approaches to the concept of humanizing mice: the first is to create transgenic mice with a specific human gene, and the second is the combination of immunodeficient mice and human cells and/or tissues.

Bone infections by Staphylococcus aureus (S. aureus) have been modelled in humanized mice showing increased infection susceptibility in humanized mice compared to wild type and non-engrafted mice.

Gowrishankar Muthukrishnan, PhD, and his research group at the University of Rochester, USA, have demonstrated that S. aureus induces a human immune response in a model of implant-associated osteomyelitis. Humanized mice have increased osteolysis, increased local and systemic bacterial burden, and more Staphylococcal Abscess Communities (SACs) at the site of bone infection compared to control animals [1]. Despite this severe infection phenotype, this study revealed proliferating CD3+Tbet+ cells near the infection site, indicating that the infiltration of human T cells is inadequate for achieving bacterial clearance in the bone [1]. Humanized mice have also been recently used to evaluate the role of immunotoxins and virulence factors (including Panton–Valentine leucocidin (PVL) on disease pathogenesis during acute infection. The fewer HLA-DR+ monocytes/macrophages and fewer dead cells in the bone marrow during infection with the ΔPVL mutant suggested that PVL alters innate immune responses in this model [2]. Collectively, these studies highlight the value of humanized mice in investigating an aggressive form of S. aureus osteomyelitis, which closely resembles the condition observed in humans. Research from G. Muthukrishnan, PhD has demonstrated that humanized mice possess the ability to explore the role of specific human T cells in managing S. aureus infections, as well as the human immune responses to S. aureus [3]. This offers a deeper molecular insight into the interactions between the host and the pathogen.

These advancements in preclinical modeling can enhance our understanding of a patient’s in vivo immune response to a pathogen and may offer valuable insights into potential therapeutic interventions. In conclusion, humanized mouse models have significantly enhanced our understanding of host–pathogen interactions. The ongoing development of these models is essential to increasing the translational success rate of orthopedic therapies, thereby ensuring they are more effective and safer for clinical use.

“We need to be using highly clinically translatable models such as humanized mice to study disease pathogenesis and to develop novel therapies,” says Dr. Muthukrishnan.

References:

[1] Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis – PubMed

[2] Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice – PubMed

[3] Humanized Mouse Models of Bacterial Infections – PMC