The use of animals in research is controversial. Investigate this controversy using at least five resources from the Online

Ethical Use of Animal Models in Musculoskeletal Research

Matthew J. Allen,1 Kurt D. Hankenson,2 Laurie Goodrich,3 Gregory P. Boivin,4,5 Brigitte von Rechenberg6

1Department of Veterinary Medicine, Surgical Discovery Centre, University of Cambridge, Madingley Road, Cambridge CB3 0ES, United Kingdom, 2Michigan State University, East Lansing 48824 Michigan, 3Colorado State University, Fort Collins 80523 Colorado, 4Wright State University, Dayton 45435 Ohio, 5Veterans Affairs Medical Center, Cincinnati 45220 Ohio, 6University of Zurich, Zurich, Switzerland

Received 26 September 2016; accepted 16 November 2016

Published online 17 January 2017 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.23485

ABSTRACT: The use of animals in research is under increasing scrutiny from the general public, funding agencies, and regulatory authorities. Our ability to continue to perform in-vivo studies in laboratory animals will be critically determined by how researchers respond to this new reality. This Perspectives article summarizes recent and ongoing initiatives within ORS and allied organizations to ensure that musculoskeletal research is performed to the highest ethical standards. It goes on to present an overview of the practical application of the 3Rs (reduction, refinement, and replacement) into experimental design and execution, and discusses recent guidance with regard to improvements in the way in which animal data are reported in publications. The overarching goal of this review is to challenge the status quo, to highlight the absolute interdependence between animal welfare and rigorous science, and to provide practical recommendations and resources to allow clinicians and scientists to optimize the ways in which they undertake preclinical studies involving animals. � 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:740–751, 2017.

Keywords: preclinical; in vivo; 3Rs; ethics; best practices

The use of laboratory animals remains a critical step in the preclinical evaluation of pharmaceuticals, bio- logics, and biomedical devices. There is increasing public opposition to the use of laboratory animals, and our ability to continue to use animals in research will critically depend on how the field responds to this changing conversation. For many, whether veterinar- ians, physicians, or scientists, the arguments made against the use of animals resonate, and while we may continue to support the use of animals in research, each of us has a particular comfort level regarding what is or is not justifiable in the name of medical research. Independent of this individual view there is no place in science for ill-designed, poorly executed, and inadequately reported studies of any type. When these are cell culture studies, they are financially and scientifically unjustifiable; if they involve animals, they are also ethically unsound, unacceptable, and need to be stopped. It is our contention that the best approach to addressing the public’s concerns over the use of animals in research is to ensure that the scientific community works collectively to regulate itself, and that the steps that are being taken to maintain the highest ethical standards are both trans- parent and consistent.

One of the most significant obstacles to improving the quality of animal research is the lack of uniformity in the training that researchers receive before they start their research careers. This is then compounded by significant variability in the financial and technical resources, including infrastructures, that are available to them on a daily basis. Over the next couple of years, this journal will partner with topic experts to produce a series of “best practice” articles that will drill down into the specifics of some of the core research areas in

which animal models play a central role. In parallel, the Orthopaedic Research Society (ORS) will provide a new forum for researchers using animal models in their research. The goal of the new “Preclinical Models” section (http://www.ors.org/preclinical/) is to help the Society’s members design and perform animal studies to the highest ethical and scientific standards.

This Perspectives article seeks to summarize recent ORS initiatives relating to the use of animals in preclinical research, and to present an overview of the key issues that need to be considered when planning animal studies related to musculoskeletal research. The goal is not to provide a complete how-to guide, rather to stimulate the reader’s curiosity. Much of what we do as researchers is done because “that is how we were told to do it.” When it comes to the use of animals, we are duty bound to challenge the status quo and to critically assess our methodology. By providing new tools to educate researchers about alternatives to using animals, and by training them on new techniques to improve animal study design and technical competence, we hope to fuel a grass-roots process that will lead to substantive improvements in both the quality and the ethical standards of animal studies in musculoskeletal research worldwide.

ORS INITIATIVES RELATING TO THE USE OF ANIMALS IN MUSCULOSKELETAL RESEARCH The ORS has long realized the importance of animal models in the research that its members undertake. Presentations on animal studies can be seen across almost every research theme that comes under the ORS umbrella, both at the Annual Meeting and in this journal. Over the last 5 years, there has been a growing demand from the membership for improve- ments in the way that this work is conducted and, in particular, presented. With a global membership, ORS attracts researchers from many nations, and the regulatory procedures relating to research involving animals vary widely. While, it is not the purview of

Correspondence to: Matthew J. Allen (T: þ44 1223 337642; F: þ44 1223 337610; E-mail: [email protected]) # 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

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the ORS or any other society to dictate the means through which countries regulate animal studies, it is entirely appropriate for the Society to expect its members, as well as non-members who want to present work at our meetings, to ensure that appropri- ate steps are taken to prevent unnecessary pain, suffering or distress, and to follow the central tenet’s of the 3Rs of animal research—reduction, refinement, and replacement. With this in mind, the Journal of Orthopaedic Research has now adopted the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines1 as part of the manuscript submission process; this will ensure that researchers understand and commit to the guiding principles of ethical animal use, and that their methods are clearly reported. The expectation is that by requiring researchers to for- mally commit to meeting ARRIVE requirements, the Society will change the way that researchers approach their research. At the same time the Society will provide enhanced educational content (through “best practice” papers and through the educational offerings of the Preclinical Models section) to equip researchers with the tools to be the agents of change themselves, without the need for changes in the national regula- tory processes. Ultimately, the use of animals in research is a privilege, and like all privileges it comes with responsibility. We are the stewards of the ani- mals that we use in research, and we are ethically bound to ensure that the procedures that are per- formed on these animals are justifiable, ethical, per- formed by individuals with appropriate technical skills, and backed up with clinically proven anesthesia and analgesic protocols to alleviate unnecessary pain or distress.

Veterinarians and others interested in animal mod- els developed an “animal models” research interest group (RIG), and held sessions at both the 2015 and 2016 Annual Meetings. In 2015, the RIG held an early morning session entitled “Good and Bad Animal Models” was organized and chaired by Dr. Stephan Zeiter of the AO Research Institute in Davos. Speakers included Dr. Christopher Little of University of Syd- ney and Dr. Karl Kirker-Head of Tufts University. A second session, a workshop in the main ORS program, was entitled “Animal Welfare in Orthopaedic Re- search: Focus on Refinement and Reduction” and coordinated by Mr. Tim Cooney, a research associate of the University of Pittsburgh Medical Center, Hamot and Dr. Laurie Goodrich of Colorado State University’s College of Veterinary Medicine.

In 2016, the RIG focused on plans to develop an ORS section dedicated to discussions about animal use in musculoskeletal research. As a direct result of that discussion, and with the support of members at the RIG, plans were enacted to develop a new ORS section. The new “Preclinical Models” section is the third to be approved by the ORS Board of Directors, following the paths taken by the very successful “Spine” and “Tendon” sections. Initially, the section

will meet at the Annual Meeting, but future plans include the development and deployment of educa- tional content online, through symposia and at hands- on laboratories that will provide trainees and more senior researchers with cutting-edge skills for perform- ing animal research. It is our hope and expectation that the Preclinical Models section will provide a resource to the entire scientific community—a place where researchers can seek and offer advice, discuss the pros and cons of animal models for a particular research question, identify mentorship and training opportunities, and participate in seminars and labora- tories to develop and hone new technical skills.

EVOLUTION OF THE 3Rs AND IMPLICATIONS FOR MUSCULOSKELETAL RESEARCH In 1959, Russell and Burch published their seminal book on ethical experimentation.2 Since then the use of the term the “3Rs,” referring to the replacement, reduction, and refinement of animal experimentation, has been the foundation for high quality and humane scientific research. Continued understanding and im- plementation of the 3Rs is essential for acceptance of orthopaedic studies using animals. There are multiple resources available to help in the understanding of alternatives for animal research. Organizations from around the world have an online presence that can help with the 3Rs and alternatives. A good site for locating centers is http://caat.jhsph.edu/resources/. The following is a brief introduction to the 3Rs.

Replacement is the most commonly cited of the 3Rs. The goal of replacement is to use alternatives to animals in research whenever possible. Examples in- clude the inclusion of human volunteers, tissues and cells, mathematical and computer models, using estab- lished animal cell lines, invertebrates, or immature forms of vertebrates. An example of the replacement of animal models is the development of a robotic manipu- lator to simulate clinical tests and gait on cadaveric joints.3 In this study, joint biomechanics were first defined in human subjects and then modeled in a robotic simulator. Similarly, mathematical models gen- erated from in-vivo data can also be used for studying mechanical force patterns. Finite element modeling of bone, for example, has been used to explore the cause of hip injuries.4 The successful integration of mathemati- cal or robotic models depends on the availability of valid data to inform the model, and these necessarily come from animals or humans in the first instance. The real strength of computational models lies in their use for parametric studies, where the goal is to isolate single variables (e.g., to study the influence of pre-tensioning on the behavior of ACL grafts). For more complex studies, especially those involving biological processes such as healing or tissue remodeling, it is impossible to replicate the in vivo environment and animal studies of some type are still needed.

Another area of important advancement in replace- ment is the use of less sentient species. Zebrafish and

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insects are rapidly expanding the horizon for research models in multiple fields. This includes orthopaedic research involving tendon, muscle, and bone.5–7 Con- ceptually, the replacement of a mammalian species with fish and insects would lead to less pain and distress and thus, is considered to be more humane, although it should be noted that we understand little of pain perception in fish and insects at the current time, making this an topic of ongoing debate.

Reduction, the second R, seeks to minimize the number of animals used in an experiment. This typically relies on statistical analysis to justify the number of animals used in the experiment. Although difficult to estimate for many studies, there is enough historical data on experimental variables that deter- mining the power of an experiment should be readily achievable. It is important to be mindful of the fact that power calculations have limited validity—they relate to a specific model and to a specific outcome measure. This underpins the importance of research- ers fully disclosing their methods. If complete details are provided, other researchers can duplicate the test methods and use the existing data to support a new power analysis. Reduction of animals can also include sharing of animals between research groups. An exam- ple of this is the joint publications examining the outcome of high fat diet and exercise on a variety of systems. The primary investigator was interested in renal disease associated with diabetes, and was willing to share the musculoskeletal system for use by another investigator.8,9 This sharing of tissues halved the number of animals required if the studies would have been performed separately. Investigators should take full advantage of these and similar opportunities.

Refinement, the third R, refers to strategies designed to minimize the pain, suffering, or distress experienced by animals. In orthopaedic research, sur- gical interventions are common and can most fre- quently benefit from refinements. Refinement can be enacted at multiple steps of the process—by ensuring that the surgical team is technically proficient in the procedure that is to be employed; by having trained personnel assessing animals in the post-operative period; and through the mandatory use of proven anesthetic and analgesic agents to control post-proce- dural pain. One frequently articulated concern of the research teams is that addition of an analgesic during a procedure may alter the biologic process that is being studied. However, pain and distress also can lead to alterations in healing responses,10 compromising the quality of the science. It is our position that analgesics of some form should always be administered when invasive procedures are performed on animals. When considering the use of analgesics, investigators should be aware of the current use in humans so as to best model possible translational opportunities.

The need to improve the design, conduct, and analysis of research using animals is an ongoing process, with increasing emphasis from the research

community on improving animal welfare. It is inter- esting to speculate on the future of the 3R’s as the need for sound scientific research is just as relevant today as it was nearly 60 years ago. Since, the initial description there have been two additional “R’s” that are being proposed as essential components of high quality animal research studies. These are Responsi- bility and Reproducibility. Responsibility takes into account the new performance based outcomes that should reflect integrity, honesty, and scientific cor- rectness in appropriate and reasonable use of labora- tory animals.11 Reproducibility of research results relates to a topic that is touched on earlier in this review—the notion that it is impossible to make valid comparisons between studies when the methods used to derive the data have not been documented appro- priately. Irreproducibility in animal work is inconsis- tent with the tenets of the 3Rs, since we are clearly unable to avoid unnecessary duplication of work, let alone ensure reductions in animal use.12 It is also fiscally unsound in an era of increasing pressure on research funds. It was recently estimated that irre- producibility in preclinical research wastes around $24 billion per year.13

PRACTICALRECOMMENDATIONSFORINTEGRATING THE 3Rs INTO MUSCULOSKELETAL RESEARCH The re-emergence of the 3Rs as a fundamental guiding principle for animal research has led to the develop- ment of emphasis on the practical applications of these principles at every stage of design and execution of an animal study. In this section, we will review the practicalities of implementing the 3Rs in animal-based research and draw upon our collective experience to support this approach and explain why attention to the 3Rs is so important.

Experimental Design The starting point when considering any experimental design is to understand the specific question that is being asked, and the most relevant outcome measures that are to be reported. Is the study intended as a proof of principle study to determine technical feasibil- ity or biological activity, or is it a pivotal preclinical study for the purpose of regulatory submission? Have data been collected from animal models prior to this study? Where do the gaps in knowledge lie? The answers to these questions will impact the choice of animal, the complexity of the experimental matrix, and the selection of an appropriate sample size for the study. The choice of outcome measures will ideally be based on clinical and translational relevance, but will also be influenced by the availability of resources, the technical expertise of the research team, and the budget that is available. Non-invasive imaging, which plays such a critical role in clinical and preclinical orthopaedics, offers tremendous opportunities for both refinement and replacement of animals. However, the trade off is that the instrumentation can be expensive

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to purchase and maintain, while the interpretation of the large and typically complex datasets can be technically challenging and time consuming.

One of the core elements of the ethical review process is the avoidance of unnecessary duplication, but some degree of duplication will often be necessary in order to ensure relevance and validity of the data. Pilot studies, for example, are intended to develop preliminary data and methods; the same overall experimental design may then be used for a larger follow-up study that will expand on the early data and provide appropriate statistical power for data analysis. Duplication of an existing technique is both necessary and to be encouraged in most cases since the use of an accepted and well characterized animal model will help to reduce the problem of irreproducibility that currently complicates the interpretation of animal studies from different laboratories and different coun- tries. Over time, the adoption of standardized method- ologies and improved reporting mechanisms will make it easier to compare the results from new therapy against those from therapies that have already been evaluated in the same model. This will improve the accuracy of sample size calculations (which are com- monly based on published data) and, as importantly, it may make it possible to reduce overall animal use since comparisons could be made against historical data from earlier studies.

The best approach to avoiding duplication is to remain current with the latest developments in the field of research, through the reading of the latest research articles and attendance at meetings. A de- tailed and up to date literature review should be performed, not just to meet the requirements of the ethical review process but also to challenge and encourage the researcher to consider refinements in the experimental technique, perhaps, through the use of cutting-edge imaging. In addition, with the explo- sion of online resources and social media networks, it is easier then ever to connect to other investigators in the field and to ask for advice regarding model selection. As an example, the veterinary division of AO (www.aovet.aofoundation.org) has recently launched an initiative to develop an online, searchable database for orthopaedic animal models [Kirker-Head C, per- sonal communication]. In the long term, care taken at this early point in study development will help to ensure that the model selection and technical proce- dures are acceptable to the research community and less likely to encounter challenges as they come to peer review for presentation and publication.

The Pilot Study It is impossible to overstate the benefits of a pilot study and its potential positive impact on the quality of the final research product. Pilot studies provide an opportunity to evaluate every aspect of the study, from anesthesia and surgery, through post-operative care to the collection of both in-life and post-mortem endpoint

data. The benefits are perhaps, most obvious when performing complex procedures or experiments where a multidisciplinary team may need to learn to function efficiently together. However, the impact can be equally significant in experiments where a new drug is being evaluated in an established model, offering an opportunity to refine and validate standard operating procedures for drug preparation, administration, and the identification of anticipated or unanticipated treat- ment-related side effects. Many institutional animal care and use committees (IACUCs) actively encourage the use of pilot studies because of the likely benefits of practice in everything that we do, and because the inclusion of a pilot study signals the willingness of the investigators to evaluate, refine, and confirm their procedures ahead of large-scale animal use.

Time Points and Outcome Measures In most cases, the selection of time points for a specific experiment will be based on personal experience, published regulatory guidelines, or a review of previ- ous published data. However, in instances where a new outcome measure is proposed, or in which the purpose of the study is more mechanistic than end- point based, it is extremely important to pay attention to the time points that are to be used. For example, studies on a new surface coating or drug therapy to enhance implant fixation may use an end-point to confirm overall efficacy, but mechanistic information can only come from the study of time points that reflect key biological stages in the healing process. It may well be that overall fixation of the implant is the same as with a predicate device or treatment, but if the rate of healing can be shown to increase, the new approach may well have clinical merit in that it will allow patients to return to function earlier. In plain terms, the destination may be the same, but if the journey is different then there can be clinical impact (positive or negative). Pilot studies can be extremely helpful in this regard, allowing for sampling of small numbers of animals at regular time points in order to develop descriptive (qualitative) data on the healing process. These data can then inform the selection of the most appropriate time points for a larger study that can provide objective data on the mechanisms underlying the different healing rate. By rationalizing the selection of time points and basing the choices on science rather than habit, it is usually possible to achieve significant reductions in overall animal use while maximizing the amount of information gleaned from individual animals. As one moves from animal to human, it is important to recognize that inter-species differences in tissue remodeling rates can significantly impact the translatability of preclinical findings,14 but the expectation would be that mechanisms are more conserved between species.

The outcome measures used in preclinical animal studies can be broadly classified into those with a direct clinical equivalent (e.g., radiography, computed

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tomography, serum, or urinary biomarkers, biopsy material) and those that are limited to preclinical research (e.g., gross anatomical analysis, mechanical testing). In general, the latter tend to be more invasive and/or destructive, while the former tend to be non- invasive or minimally invasive and, as a consequence, more feasible for use in human clinical. While it is hard to make definitive recommendations in this area, early phase studies in rodents and rabbits are more likely to make use of invasive testing and intermediate time points with terminal evaluations, while pivotal large animal studies are more commonly designed with end-points that reflect potential outcome mea- sures that might be of clinical interest in early stage human clinical trials. The use of a parallel set of outcome measures in preclinical and clinical trials offers huge potential value in terms of enhancing the translational relevance of the preclinical work; for example, if preclinical animal studies can be used to define and validate the relationship between magnetic resonance imaging (MRI) appearance and histology for a new cartilage repair strategy, the MRI findings from future human clinical trials will be easier to interpret, allowing for the use of histopathology as a confirma- tory rather than exploratory outcome measure.

Conducting the Study With the move toward the era of large interdisciplinary research teams and “big science,” there is a much greater emphasis on inter-disciplinary teamwork in research.15

As a result, it is commonplace to see investigators from engineering, cell biology, and medicine working together on an experimental study. There are clear benefits to the development of this team-based approach, but it also creates challenges, especially with regard to experience in, and attitudes toward, animal research. It is vital that the team discusses the logistics of working together on an animal study to ensure that everyone is on the same page with regard to experimental design and study conduct. Whenever possible, it can be extremely beneficial—we would argue that it should in fact be standard practice—to involve an experienced veterinarian as either a co-investigator or a consultant to provide input on best practices in drug administration, anesthesia and analge- sia, post-operative care, and euthanasia. It is usually very helpful to engage the institution’s animal care staff by presenting an overview of the work, so that they can better understand the goals of the work, the potential for complications, and the steps that need to be taken to manage those complications.

Whether undertaken under Good laboratory Practice (GLP) guidelines16 or not, it is important that every animal procedure is conducted under the umbrella of one or more standard operating procedures (SOP). Ideally, the SOPs should be developed following consultation with individuals with prior training and experience with a given procedure; the draft SOP can then be evaluated and refined in a pilot study at your institution, and the definitive SOP is then used for all future studies.

Deviations from the SOP should be recorded and reported when the work is presented and published (see below). Use of SOPs will reduce variation in procedural methodol- ogies, reduce the number of animals needed to achieve statistical power for a given study design and decrease the risk of irreproducibility by ensuring that other groups can make use of the same experimental design.

Reporting the Study As mentioned previously, the impact of any scientific study can be critically limited by deficiencies in experimental design and study execution, but it is often in the reporting of the work that the greatest deficiencies are seen. Whether by accident or intent, failure to accurately document experimental proce- dures, post-surgical complications, and clinical out- come has a significant negative impact on the quality of the resulting manuscript. More importantly, it becomes impossible to repeat that experiment, or to relate the findings from that study to any other. Taken as a whole, failure to fully disclose the research methodologies significantly decreases the translational impact of the research because it is impossible for the reader to determine the relevance or the robustness of the science. For preclinical science to be relevant, it must be designed and conducted appropriately, but it must also be reported and disseminated in an efficient, timely and trans- parent manner. The publication of a set of recom- mendations regarding appropriate reporting of animal research, the ARRIVE guidelines1 represent an important step in the right direction.

THE ETHICAL REVIEW PROCESS The ethical review of scientific research will always be a potentially contentious topic. While most if not all agree on the need for oversight, each of us brings personal experience and bias (conscious or unconscious) to discussions on this topic. Ethical review does neces- sarily delay researchers who want to be getting on with their experiments, but we would argue that appropriate and efficient ethical review is actually central to doing great science. If we are to make use of animals in our research, it is our duty (not that of a committee) to ensure that we do so in an ethical and humane manner. The purpose of ethical review should be to provide external guidance to facilitate this, and of course to identify and block research that is inconsistent with ethical and humane principles. The review process therefore, needs to be formative, timely, unbiased and based on current best practices. The committee charged with undertaking ethical review should be approach- able, knowledgeable and responsive both to investigator needs and to changes in best practices in animal care, veterinary medicine, and research methodologies. If ethical review functions in this way, it will be seen as being a valuable and important part of the process, not an obstacle that one must clear before being able to get on with the “real work.”

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Although the specific procedures for ethical review vary by country, the primary goal of the ethical review process should be to undertake a cost-benefit assess- ment to determine whether it is justifiable to make use of animals for a particular line of research. Additionally, steps need to be taken to ensure that investigators are appropriately trained and make use of procedures that minimize pain, distress, and suffer- ing as much as is practical while undertaking their research. The review process may be managed cen- trally by national agencies (such as the Home Office in the UK) or locally through institutional structures (such as the Institutional Animal Care and Use Committee in the US). Attempts at international harmonization are ongoing,17 but until agreement has been reached it has been the policy of most publishers to accept data from animal studies as long as there is documentation of appropriate appro

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