animals in research

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.

740 JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

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

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 741

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

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

742 ALLEN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

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

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 743

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

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.”

744 ALLEN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

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 approvals from the rele- vant regulatory agency in the country in which the work was performed. This approach generally works well, but there continue to be cases in which authors fail to document key steps in the review and approval process, or in which the methods that have been approved in one country are inconsistent with best practices in another. Adoption of the Arrive guidelines by JOR will be beneficial in ensuring consistency.

RECENT INITIATIVES TO INCREASE TRANSPARENCY IN ANIMAL USE Two of the most significant developments in recent years have been the publication of consensus docu- ments on the reporting of animal experiments (AR- RIVE guidelines)1 and the introduction of a framework for openness regarding animal testing in the United Kingdom (the Concordat).18 The implications of these documents are far-reaching, and while further changes are likely, especially with regard to the Concordat, there is hope that they will impact scientific research at a global level. The Journal of Orthopaedic Research will soon be implementing the ARRIVE guidelines into the review process for any manuscript that reports animal data, and a similar approach is likely to percolate down to abstract reviews for conferences. The additional steps required to comply with ARRIVE guidelines are not onerous but they provide a trans- parency that has to date been missing and that will significantly enhance the interpretability and impact and relevance of the published work. Importantly, the introduction of ARRIVE represents an important first step toward reducing the problem of irreproducibility that plagues science in general but animal models in particular.12,13

NEW STRATEGIES TO IDENTIFY AND MANAGE PAIN AND DISTRESS IN LABORATORY ANIMALS Behavioral Scoring/Facial Grimace for Identification of Pain Research in musculoskeletal diseases often requires an intervention that has the potential to cause pain or distress in laboratory animals. Prevention, detection

and relief of pain and distress are paramount in performing good scientific studies. This section will briefly outline some of the methodology for ensuring animal well being during orthopaedic studies.

It is generally understood, and required by regula- tory agencies, that pain should be prevented or alleviated unless it is part of the scientific study, or it will jeopardize the research validity. In the latter case, if the investigator is unable to relieve pain or distress then the patient should be euthanized. Given that most orthopaedic procedures are not examining pain per se, there is rarely a scientific justification for not providing routine analgesia, especially if it is adminis- tered consistently to all study animals as a matter of protocol. There are several analgesic substances avail- able for prevention or treatment of pain. Historically the administration of opioids has been the primary treatment given to humans for pain prevention post- operatively. However, the development of new analge- sics has led to the use of multimodal therapy to reduce the side effects of the high dose of opioids required for post-operative pain control. Multimodal analgesia is the use of multiple agents to act synergistically for more effective pain control with fewer side effects than a single agent. There are several studies that have shown the improved efficacy of multimodal therapy. For example the recently approved intravenous formu- lation of acetaminophen (paracetamol) has been shown in combination with ketorolac (an NSAID) to improve post-operative pain control compared to either drug alone.19,20 In clinical trials, two anticonvulsants, gaba- pentin and pregabalin, have also been shown to be efficacious in reducing post-operative morphine con- sumption with either reduced pain or similar pain levels.21–23 Gabapentin type drugs bind to calcium channels in the spinal cord and brain thus reducing afferent excitatory activity. The use of these drugs also showed a reduction in side effects.21–23 There are other multimodal analgesics that have been shown to improve pain relief, including TRPV1 agonists, NMDA receptor antagonists, and alpha-2 agonists.24 A review article further identified evidence supporting the use of multimodal analgesics for spine surgery. In it they suggested that there is good evidence that gabapenti- noids, acetaminophen, neuraxial blockade and ex- tended-release local anesthetics (in ascending order) reduce postoperative pain and narcotic requirements, fair evidence that preemptive analgesia and non- steroidal anti-inflammatory drugs (NSAID) result in reduced postoperative pain, and insufficient and/or conflicting (Grade I) evidence that muscle relaxants and ketamine provide a significant reduction in post- operative pain or narcotic usage.25

Another relatively recent improvement in pain control is the use of pre-emptive analgesia. Transmis- sion of pain signals evoked by tissue damage leads to hyperalgesia, or sensitization, of the peripheral and central pain pathways. Pre-emptive analgesia is a treatment that is initiated before the surgical

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 745

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

procedure to reduce this sensitization. The goal of pre- emptive analgesia is to stop pain before it starts, thus, preventing the physiological consequences of nocicep- tive transmission evoked hyperalgesia.26,27 Several clinical trials have been conducted to assess the impact of pre-emptive analgesia versus standard anal- gesic therapy. Unfortunately, despite the scientific rationale supporting pre-emptive analgesia, only NSAIDs have shown a positive effect for reducing post-operative pain compared to giving analgesics only post-operatively.27,28 Although no improvement in the short-term was shown with pre-emptive analgesia in all studies, there was no evidence that it was more painful. In addition, there is a paucity of information on the control of long-term pain using pre-emptive analgesia. Use of pre-emptive analgesia is recom- mended especially in the context of using a multimodal analgesia.

One concern specific to musculoskeletal procedures relates to the possibility that NSAIDs may effect bone healing. A recent review article examining the clinical evidence for this showed that there are conflicting data on the validity of these results.29 Another showed there is fair (Grade B) evidence that short-term use of NSAID result in no long-term reduction in bone healing or fusion rates.25 As the use of NSAIDs is still being debated it should be approached cautiously, as it is imperative that the analgesic protocol does not affect the scientific results of the animal study. Even if NSAIDs are counter indicated another analgesic should be used to provide pain relief.

Current advances with sustained release formula- tions as well as topical analgesics have improved the potential for administration of analgesics to animals without causing handling distress. Examples including the topical use of fentanyl either in a patch or a gel (Recuvyra) show promise in long-term pain relief.30 In addition, a single injection formulation of buprenor- phine is available that provides 72 h of pain relief.31 A recent review of the available topical analgesics found that there are a number of new analgesics being developed.32 It is clear from this work and the absence of a universal analgesic that future analgesic types and combinations are still needed to improve the post- operative welfare of patients.33 As these are developed, transferring this information to our animal models is essential.

Although in human patients self-administration of analgesics is possible, for our experimental animal patients we are required to administer analgesics. Determination of pain is not easily performed because they are prey species that have an instinctive ability to disguise pain. Thus, it is often only subtle behaviors that will alert the investigative team of an animal in potential pain. Careful observation by the animal care staff is often the best method for daily assessments of animal well being, as the individuals who take care of the animals daily will have knowledge of what is normal behavior for the patient. To augment this

assessment, though, there are a few behavioral tests that have and are being developed to determine if there is evidence of pain. One of these is the use of nest building behavior to assess pain in mice.34 This entails addition of a small amount of nesting material into the cage and evaluation of whether the mouse incorporates it into the nest. Mice in pain do not perform this task. Another assessment used in rodents and other species is the facial grimace score. Adapted from children, this scoring system allows assessment of pain based on facial features.35,36 A third potentially exciting new approach involves the use of whole-cage monitoring systems to quantify changes in activity patterns within group-housed animals.37

OBJECTIVE MEASURES OF MUSCULOSKELETAL FUNCTIONAL RECOVERY In a review paper of this type, it is impossible to provide details on all aspects of objective evaluation of musculoskeletal function. This section will present an overview of some of the common outcome measures and some thoughts on how they may be usefully applied to preclinical studies. The main application of the 3Rs in this context is the reduction of animal numbers as well as animal suffering. Furthermore, species selection plays an important role since their level of development, size, trainability, and coopera- tion with the human handlers will impact evaluation methods.

Non-Invasive Diagnostic Imaging Methods In musculoskeletal research the benchmark for assess- ing recovery is usually composed of diagnostic imaging modalities, such as plain radiographs, computed to- mography (CT), magnetic resonance imagine (MRI) and magnetic resonance (MR) spectroscopy, scintigra- phy, ultrasound, and fluoroscopy. All of these methods have the advantage that they allow non-invasive evaluation of the target of assessment over time. For quality and reasons of restraints animals normally have to undergo general anesthesia for these proce- dures, except for possibly radiographs, standing MRI in horses38 and modern fluoroscopy, where ambulation with weight bearing can be visualized.39 In these situations sedation may be required depending on individual animal behavior. It is imperative that SOPs are used to standardize data collection so that datasets that can be compared between and within animals over time.

Although diagnostic imaging offers tremendous pos- sibilities, caution is required for interpretation of results, especially if biomaterials, such as calcium phosphate cements or metallic implants are used. The latter cause artifacts in the immediate environment of CT images and complicate the interpretation of the material to bone-contact-interface (BIC).40 The use of high-field MRI can also be problematic with metal implants due to concerns about implant migration and possible local heating effects. Materials consisting of

746 ALLEN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

calcium aggregates also deliver dubious results in CT scans, since the equipment cannot distinguish between hydroxyapatite of natural bone or calcium scaffolds/ matrices. In addition, results are also dependent on the general threshold of calcium detection set for the scans. Therefore, CT scans used for detection of new bone formation or material resorption should be combined with histology of non-decalcified bone samples.

MRI has the advantage of showing soft tissue struc- tures and can be applied for almost all aspects of musculoskeletal research. However, one has to keep in mind that the power of the MRI equipment may determine the successful detection of treatment differ- ences. For example, a 1.5 Tesla MRI was not reliable for interpretation of changes of hyaline cartilage,41 while 3 or 7 Tesla equipment was more suiTable Spectroscopy can also deliver valuable information about type of tissue molecules (proteoglycan, fat, etc.).42 Ultrasound is feasi- ble for screening tendons for signs of degenerations, ruptures, or fluid accumulation within soft tissue and is routinely used in horses.43

Minimally Invasive Assessment and Sampling Arthroscopy is the most prominent method for “second- look” evaluations in joints following imaging procedures above. The procedure gives direct visualization under good illumination and high magnification, while also allowing for biopsies. Research projects examining cartilage resurfacing can benefit greatly from arthros- copy.44 If performed correctly, arthroscopy results in minimal damage and does not severely disturb the overall course of healing/degeneration of hyaline carti- lage or other associated joint structures.

Serial biopsies are a good method to assess func- tional outcomes following surgery and are easily performed in most cases as long as the biopsy material is removed in a manner (and from a location) that does not negatively influence healing and remodeling of the residual tissue. The same is true for sampling synovial fluid over time with repeated punctures. However, it has to be kept in mind, that even with these minimally invasive methods perioperative inflammation is pro- duced within the joint structure and therefore, these procedures should be temporally spread apart, such that results are not artefactually influenced by the previous procedures.

Biomechanical Methods When considering biomechanical testing, it has to be determined whether tests are to be performed in vivo, potentially with multiple time points, or whether tests are conducted post-mortem to provide only a single time point. This will likely depend on what data you are trying to capture and the equipment available to the investigator. By combining multiple methods a global picture of the structural and material properties can be obtained.

Kinetic analysis of ground reaction forces, whether by traditional force plate or the more recently

developed pressure-sensitive walkways,45 provides for objective functional assessment of overall limb use. Combining kinetic outputs with kinematic data, obtained from motion capture systems, allows for real- time monitoring of changes in both limb use (overall loading) and limb function (changes in range of joint motion).46 These combined data can then be imported into commercial or open source simulation software such as OpenSim to allow for the calculation of joint loads and the development of mathematical models of joint function.47 Muscle activation during activity can also be determined by means of electromyography (EMG) using either invasive (needle electrodes) or surface recording.

Advances in microsensor and telemetry technology are now making it possible to obtain real-time output from tissues or implants in vivo. For example, strain gauges implanted in/on tissues or around joints can be used to record functional loads in vivo.48 Although expensive to deploy, these implantable devices allow for real time collection of serial data from individual animals, allowing for reductions in overall animal numbers.

Measuring indentation is a method to assess me- chanical properties of hyaline cartilage and can be performed in vivo or ex vivo.49 Standardization of the method may be tricky especially in vivo, since setting the instruments at the correct 90˚ angle may not always be easy. More recently, reference point inden- tation has been shown to be a robust, nondestructive method for obtaining quantitative data on the mechan- ical properties of whole bones.50

Ex-vivo methods of biomechanical testing incorpo- rate mostly measuring tensional, compressive and/or shear forces or fatigue of (healed) tissue structures in customized settings. The classic example would be a materials testing machine that can test the mechani- cal properties of whole limbs, or individual elements such as bone or tendons. If working with biomaterials, removal torque or push-out tests are often used to evaluate osseointegration.51

Histology Histology is a valuable tool for assessing functional outcomes after surgical or medical treatment. Structural as well as cellular changes can be observed in detail, although one has to be aware that it is a two dimen- sional method that often suffers from limitations in terms of sampling frequency, making its general applica- bility to the tissue as a whole more limited. However, these limitations can be offset to some extent through the use of stereology, as well as by combining serial sections with 3-dimensional computed tomography and/ or MRI imaging to provide the third dimension.

The type of histology that is performed will depend on tissue type and whether implants are left in situ (e.g., metallic implants). If metallic implants are to be sectioned in situ, non-decalcified tissue samples are embedded in a hard epoxy or acrylic resin such as

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 747

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

polymethylmethacrylate, then sectioned using a bone saw with a diamond band saw.52 The cut sections are then ground and polished to final thickness (usually 100–150 mm) and surface stained with either toluidine blue or Giemsa. Toluidine blue is a convenient histo- chemical staining to assess new bone and/or cartilage formation.

If thin bone sections are required for studying tissue responses at a cellular level, any metal implant will need to be removed. The tissue is embedded in acrylic resin as above, then sectioned on a rotary microtome or annular saw. Thin sections allow for the use of a broader variety of stains, including toluidine blue, Movat pentrachrome, von Kossa/McNeal, hema- toxylin eosin, etc. Staining protocols depend on the specific question that is being asked. Although possi- ble, immunohistology with plastic sections is problem- atic and is most reliable in bone samples from small rodents and there, preferably bone marrow and not cortical bone. Special resins are available, which are mostly too soft for sheep bone and make it almost impossible to get reliable and repeatable results, especially if cortical bone is involved.

If there are no implants in the tissue, or if removal of the implant is feasible, decalcification, and process- ing into paraffin is the preferred technique for bone sections. Decalcification is relatively straightforward requiring hours to days depending on the bone thick- ness and density. Immunohistochemistry is frequently unreliable because of the fixation and decalcification process, thus having reliable validated antibodies is necessary.

Frozen sectioning is technically challenging for bone but feasible for cartilage and soft tissues such as muscle, ligament, tendon, or fibrous tissue. Tendon tissue from larger animals like the horse may be too dense to get good and reliable frozen sections, thus paraffin sections or even plastic sections may be more suitable. For cartilage alone, cut off from the calcified zone, frozen sections and paraffin sections are com- monly used and suitable for most assessments. Also, identification of fat in tissues is best performed on frozen sections because xylene leaches out the fat droplets during processing. Other techniques for iden- tification of fat have proven successful if frozen tissues cannot be used.

Immunohistochemical analysis is highly dependent on the antibody. Tissue preparation can greatly influ- ence whether an antibody is going to be successful, and simply working in western blot analysis is no guarantee that it will also work in tissue sections. Frozen sections are often the most reliable for antigen detection, followed by tissues preserved in a short (less than 24 h) exposure to paraformaldehyde, formalde- hyde, long exposure (greater than a day) in either paraformaldehyde or formaldehyde, and finally the least successful are decalcified tissues. Antigen re- trieval techniques are also available to increase the likelihood of immunohistochemical staining success.

Electron microscopy uses beams with accelerated electrons to study the ultrastructure of specimens. Transmission (TEM), scanning (SEM), reflection (REM), and scanning transmission electron microscope (STEM) are provide high spatial resolution. Confocal light microscope sometimes overlaps with electron microscopy and for each particular research question, pilot studies may be needed to determine the most appropriate method of analysis.

Molecular and Biochemical Methods Modern research in the musculoskeletal area also involved methods of molecular biology in which DNA, RNA, or specific proteins are quantified. The specifics of these techniques are well beyond the scope of this review and will not be presented in detail, beyond reminding the reader that the isolation of intact, high quality RNA from connective tissues can be challeng- ing and requires efficient and rapid processing of tissues following collection from the animal. In addi- tion, the low density of cells in soft tissues such as tendons and ligaments requires extensive processing for retrieval of the DNA and RNA in the vast collagen milieu.

VETERINARY CLINICAL TRIALS AS A BRIDGE BETWEEN PRECLINICAL LABORATORY ANIMAL STUDIES AND HUMAN CLINICAL TRIALS There are many opportunities in the veterinary clini- cal realm to utilize patients to (1) demonstrate efficacy of an orthopaedic surgical procedure or (2) investigate efficacy of a non-surgical treatment modality. Utilizing veterinary patients can potentially bridge the gap between studies in preclinical laboratory animal mod- els and human clinical trials, leading to proof of efficacy and benefits for the veterinary population, as well as providing a “pathway” for drugs and proce- dures to be further studied in humans.53

In the veterinary clinical population there are many patients that provide excellent examples of a naturally occurring disease where intervention may be very similar to those in human patients, including osteoar- thritis (OA)54 and bone cancer.55 A challenge in this regard is that even within a disease category such as OA, there may be significant differences between species in terms of disease onset (acute or chronic), progression (weeks versus months), onset of clinical symptoms versus when the disease began, etc. If the outcomes of the disease is not understood in depth, this could lead to misinterpretations of a disease treatment and false positive or negative results that will not translate successfully into human clinical trials. It therefore, is important for the clinician scientist evaluating new treatments to be familiar with the current standard of care for this condition in human and/or veterinary patients.

Another opportunity in veterinary patients is that issues such as placebo effects are much more rare due to the nature of the patient however the evaluator

748 ALLEN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

(clinician) can certainly be biased. The evaluators of the treatment should remain blinded to the therapy so as to prevent undue bias. Just like in human clinical trials, the use of randomized blinded (it is double- blinded in people) clinical studies is warranted to make the results more robust. Cook et al. published an article with proposed definitions and criteria for reporting time from outcome and complications in veterinary clinical studies and strict definitions are described to aid veterinary clinical scientists in using similar terminology to human clinical trials.56

When outcome parameters are described in veteri- nary clinical patients, variability can be introduced due to pre-existing issues with outcome assessments. For example a horse is subjectively scored based on a typical 0–5 lameness scale and flexion tests but dogs are graded on criteria such as activity, mood, playful- ness etc., which also is very different from how pain is assessed in humans. While more objective analyses are being developed for both the horse and dog in terms of gyroscopic lameness detectors (Lameness Locator1)57 and force plate analyses, these outcome parameters are very different to the pain scales utilized in humans, making it very important to select outcome measures appropriately. The use of validated survey instruments for assessing pain and functional impairment shows considerable promise in this re- gard.58,59

Further challenges that are similar to both veteri- nary and human clinical trials are recruiting patients and getting a population that is similar in disease state. Further, once a treatment or procedure is pursued in a veterinary population, the patients are no longer in a “controlled” environment. Study PI’s rely on owners to interpret recommendations of how to care for the patients but compliance is not always consistent. A more “hybrid” model has been followed recently in which horses with OA were treated and kept within a center for the entire period of assess- ment. This accomplished several objectives that allowed maximum consistency; a controlled environ- ment in which patients had consistent care, access to equipment such as force plate analysis and a lameness locator, and the ability to closely monitor responses that may otherwise have been missed.60 While this approach may initially seem more expensive, the reduction in variability in data collection, combined with the increased compliance of study animals means that the overall cost for these hybrid studies may be lower than with an outpatient field trial (Bertone A, Personal communication).

An opportunity that exists in veterinary patients is that the majority of patients are not covered by insurance policies therefore, owners are often moti- vated to enter their animals onto a clinical trial especially if there is a monetary incentive (to cover some or all of their medical expenses). Conversely, a challenge of these studies is that owners may be unwilling to participate if there is a possibility that

their animal may receive the placebo control treat- ment. To motivate owners to enter their animals onto a clinical study, a crossover design may be an impor- tant incentive needed to partake in a trial where all animals receive treatment eventually (either at the initiation or following treatment with the control).61

As in all clinical trials whether in people or in veterinary patients the importance of accurate power analyses cannot be stressed enough. If these studies are underpowered, the value of the conclusions are meaningless and more importantly, a potentially effec- tive treatment or surgical procedure is assumed falsely effective (dangerous for the patients) or falsely ineffec- tive resulting in a missed opportunity to bring a valuable therapy to fruition. Biostatisticians that are well versed and familiar with biological studies should always be included as valuable team members of clinical trials to ensure proper analyses are performed and that there is no bias associated with the results.

SUMMARY AND CONCLUSIONS Our ability to continue to use laboratory animals in biomedical research is under more intense scrutiny than ever before. The balance has shifted so that the burden of evidence now lies squarely with the re- searcher, who must justify the clinical/translational relevance of his/her research and demonstrate that the experimental methods do not cause undue pain, dis- tress, or suffering to the study animals. Recent initia- tives within the ORS and other allied organizations are intended to enhance the training opportunities avail- able to investigators at all career stages, to provide a network of researchers capable of mentoring young investigators, and to offer timely reviews in the form of white papers on best practices in animal model selec- tion, experimental design/conduct, and study reporting. It is our hope that through these initiatives, we will be able to demonstrate to the public that orthopaedic researchers understand the absolute need to consider and then apply the fundamental principles of the 3Rs when undertaking in-vivo research studies in animals.

AUTHORS’ CONTRIBUTIONS All of the authors contributed to the writing of this review article. The corresponding author collated individual contributions and undertook the major editing. The final submitted version has been reviewed and approved by all authors.

REFERENCES 1. Kilkenny C, Browne W, Cuthill IC, et al. 2010. Animal

research: reporting in vivo experiments: the ARRIVE guide- lines. Br J Pharmacol 160:1577–1579.

2. Russell WMS, Burch RL. 1959. The principles of humane experimental technique, 1st ed. Methuen: London (accessible at: http://altweb.jhsph.edu/pubs/books/humane_exp/het-toc).

3. Noble LD, Jr, Colbrunn RW, Lee DG, et al. 2010. Design and validation of a general purpose robotic testing system for musculoskeletal applications. J Biomech Eng 132:025001.

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 749

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

4. Yang L, Palermo L, Black DM, et al. 2014. Prediction of incident hip fracture with the estimated femoral strength by finite element analysis of DXA Scans in the study of osteoporotic fractures. J Bone Miner Res 29:2594–2600.

5. Chen JW, Galloway JL. 2014. The development of zebrafish tendon and ligament progenitors. Development 141:2035–2045.

6. Campos LM, Rios EA, Midlej V, et al. 2015. Structural analysis of alterations in zebrafish muscle differentiation induced by simvastatin and their recovery with cholesterol. J Histochem Cytochem 63:427–437.

7. Mork L, Crump G. 2015. Zebrafish craniofacial development: a window into early patterning. Curr Top Dev Biol 115:235–269.

8. Boivin GP, Elenes EY, Schultze AK, et al. 2014. Biomechani- cal properties and histology of db/db diabetic mouse Achilles tendon. Muscles Ligaments Tendons J 4:280–284.

9. Somineni HK, Boivin GP, Elased KM. 2014. Daily exercise training protects against albuminuria and angiotensin con- verting enzyme 2 shedding in db/db diabetic mice. J Endocrinol 221:235–251.

10. Broadbent E, Petrie KJ, Alley PG, et al. 2003. Psychological stress impairs early wound repair following surgery. Psycho- som Med 65:865–869.

11. Banks RE. 1995. The 4th R of research. Contemp Top Lab Anim Sci 34:43.

12. Collins FS, Tabak LA. 2014. Policy: NIH plans to enhance reproducibility. Nature 505:612–613.

13. Freedman LP, Cockburn IM, Simcoe TS. 2015. The econom- ics of reproducibility in preclinical research. PLoS Biol 13: e1002165.

14. Pearce AI, Richards RG, Milz S, et al. 2007. Animal models for implant biomaterial research in bone: a review. Eur Cell Mater 13:1–10.

15. Huang Y, Zhang Y, Youtie J, et al. 2016. How does national scientific funding support emerging interdisciplinary re- search: a comparison study of big data research in the US and China. PLoS ONE 11:e0154509.

16. Carroll EE. Going GLP: conducting toxicology studies in compliance with good laboratory practices. US Army Med Dep J 3–16:64–69.

17. Annys E, Billington R, Clayton R, et al. 2014. Advancing the 3Rs in regulatory toxicology—carcinogenicity testing: scope for harmonisation and advancing the 3Rs in regulated sectors of the European Union. Regul Toxicol Pharmacol 69:234–242.

18. Jarrett W. 2016. The concordat on openness and its benefits to animal research. Lab Animal 45:201–202.

19. Ong CK, Seymour RA, Lirk P, et al. 2010. Combining paracetamol (acetaminophen) with nonsteroidal antiinflam- matory drugs: a qualitative systematic review of analgesic efficacy for acute postoperative pain. Anesth Analg 110:1170–1179.

20. Wickerts L, Warr�en Stomberg M, Brattwall M, et al. 2011. Coxibs: is there a benefit when compared to traditional non- selective NSAIDs in postoperative pain management? Mi- nerva Anestesiol 77:1084–1098.

21. Clarke H, Pereira S, Kennedy D, et al. 2009. Gabapentin decreases morphine consumption and improves functional recovery following total knee arthroplasty. Pain Res Manag 14:217–222.

22. Rusy LM, Hainsworth KR, Nelson TJ, et al. 2010. Gabapen- tin use in pediatric spinal fusion patients: a randomized, double-blind, controlled trial. Anesth Analg 110:1393–1398.

23. Zhang J, Ho KY, Wang Y. 2011. Efficacy of pregabalin in acute postoperative pain: a meta-analysis. Br J Anaesth 106:454–462.

24. Buvanendran A, Kroin JS. 2009. Multimodal analgesia for controlling acute postoperative pain. Curr Opin Anaesthesiol Oct 22:588–593.

25. Devin CJ, McGirt MJ. 2015. Best evidence in multimodal pain management in spine surgery and means of assessing postoperative pain and functional outcomes. J Clin Neurosci 22:930–938.

26. Ryu HK, Baek YH, Park YC, et al. 2014. Current studies of acupuncture in cancer-induced bone pain animal models. Evid Based Complement Alternat Med 2014:191347.

27. Dahl JB, Moiniche S. 2004. Pre-emptive analgesia. Brit Med Bull 71:13–27.

28. Joshi GP. 2005. Multimodal analgesia techniques and post- operative rehabilitation. Anesthesiol Clin North America 23:185–202.

29. Pountos I, Georgouli T, Calori GM, et al. 2012. Do nonsteroi- dal anti-inflammatory drugs affect bone healing? A critical analysis. Scientific World J 2012:606404.

30. Freise KJ, Linton DD, Newbound GC, et al. 2012. Popula- tion pharmacokinetics of transdermal fentanyl solution following a single dose administered prior to soft tissue and orthopaedic surgery in dogs. J Vet Pharmacol Ther 35:65–72.

31. Kendall LV, Hansen RJ, Dorsey K, et al. 2014. Pharmacoki- netics of sustained-release analgesics in mice. Am Assoc Lab Anim Sci 53:478–484.

32. Cantisani C, Macaluso L, Frascani F, et al. 2014. New patents on topical anesthetics. Recent Pat Inflamm Allergy Drug Discov 8:125–131.

33. Elmallah RK, Cherian JJ, Pierce TP, et al. 2016. New and common perioperative pain management techniques in total knee arthoplasty. J Knee Surg 29:169–178.

34. Jirkof P. 2014. Burrowing and nest building behavior as indicators of well-being in mice. J Neurosci Meth 234:139–146.

35. Matsumiya LC, Sorge RE, Sotocinal SG, et al. 2012. Using the mouse grimace scale to reevaluate the efficacy of postoperative analgesics in laboratory mice. J Am Assoc Lab Anim Sci 51:42–49.

36. Sotocinal SG, Sorge RE, Zaloum A, et al. 2011. The rat grimace scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. Mol Pain 7:55.

37. Salem GH, Dennis JU, Krynitsky J, et al. 2015. SCORHE: a novel and practical approach to video monitoring of labora- tory mice housed in vivarium cage racks. Behav Res Methods 47:235–250.

38. Porter EG, Werpy NM. 2014. New concepts in standing advanced diagnostic equine imaging. Vet Clin North Am Equine Pract 30:239–268.

39. Jones SC, Kim SE, Banks SA, et al. 2014. Accuracy of noninvasive, single-plane fluoroscopic analysis for measure- ment of three-dimensional femorotibial joint poses in dogs treated by tibial plateau leveling osteotomy. Am J Vet Res 75:486–493.

40. Hoffmann O, Angelov N, Zafiropoulos GG, et al. 2012. Osseointegration of zirconia implants with different surface characteristics: an evaluation in rabbits. Int J Oral Maxillo- fac Implants 27:352–358.

41. Fischbach F, Bruhn H, Unterhauser F, et al. 2005. Magnetic resonance imaging of hyaline cartilage defects at 1.5T and 3.0T: comparison of medium T2-weighted fast spin echo, T1- weighted two-dimensional and three-dimensional gradient echo pulse sequences. Acta Radiol 46:67–73.

42. Kostyuk O, Birch HL, Mudera V, et al. 2004. Structural changes in loaded equine tendons can be monitored by a novel spectroscopic technique. J Physiol 554:791–801.

43. Gaschen L, Burba DJ. 2012. Musculoskeletal injury in thoroughbred racehorses: correlation of findings using multi- ple imaging modalities. Vet Clin North Am Equine Pract 28:539–561.

750 ALLEN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017

44. McIlwraith CW, Frisbie DD, Rodkey WG, et al. 2011. Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects. Ar- throscopy 27:1552–1561.

45. Kim J, Breur GJ. 2008. Temporospatial and kinetic charac- teristics of sheep walking on a pressure sensing walkway. Can J Vet Res 72:50–55.

46. Atarod M, Frank CB, Shrive NG. 2015. Increased meniscal loading after anterior cruciate ligament transection in vivo: a longitudinal study in sheep. Knee 22:11–17.

47. Lerner ZF, Gadomski BC, Ipson AK, et al. 2015. Modulating tibiofemoral contact force in the sheep hind limb via treadmill walking: predictions from an opensim musculo- skeletal model. J Orthop Res 33:1128–1133.

48. Ledet EH, Tymeson MP, DiRisio DJ, et al. 2005. Direct real- time measurement of in vivo forces in the lumbar spine. Spine J 5:85–94.

49. Hale JE, Rudert MJ, Brown TD. 1993. Indentation assess- ment of biphasic mechanical property deficits in size-depen- dent osteochondral defect repair. J Biomech 26:1319–1325.

50. Gallant MA, Brown DM, Organ JM, et al. 2013. Reference- point indentation correlates with bone toughness assessed using whole-bone traditional mechanical testing. Bone 53:301–305.

51. Berzins A, Sumner DR. 2000. Implant pushout and pullout test. In: An YH, Draughn RA, editors. Mechanical testing of bone and the bone-implant interface, 1st ed. Boca Raton: CRC Press. p 463–476.

52. An YH, Martin Kl. 2003. Handbook of histology methods for bone and cartilage, 1st ed. New Jersey: Humana Press Inc. p 620.

53. Stroud C, Dmitriev I, Kashentseva E, et al. 2016. A one health overview, facilitating advances in comparative

medicine and translational research. Clin Transl Med 5:26.

54. Knazovicky D, Helgeson ES, Case B, et al. 2016. Widespread somatosensory sensitivity in naturally occurring canine model of osteoarthritis. Pain 157:1325–1332.

55. Fenger JM, London CA, Kisseberth WC. 2014. Canine osteosarcoma: a naturally occurring disease to inform pedi- atric oncology. ILAR J 55:69–85.

56. Cook JL, Evans R, Conzemius MG, et al. 2010. Proposed definitions and criteria for reporting time frame, outcome, and complications for clinical orthopaedic studies in veteri- nary medicine. Vet Surg 39:905–908.

57. Schumacher J, Taintor J, Schumacher J, et al. 2013. Function of the ramus communicans of the medial and lateral palmar nerves of the horse. Equine Vet J 45:31–35.

58. Walton MB, Cowderoy E, Lascelles D, et al. 2013. Evalua- tion of construct and criterion validity for the ‘Liverpool Osteoarthritis in Dogs’ (LOAD) clinical metrology instru- ment and comparison to two other instruments. PLoS ONE 8:e58125.

59. Hudson JT, Slater MR, Taylor L, et al. 2004. Assessing repeatability and validity of a visual analogue scale ques- tionnaire for use in assessing pain and lameness in dogs. Am J Vet Res 65:1634–1643.

60. Bertone AL, Ishihara A, Zekas LJ, et al. 2014. Evaluation of a single intra-articular injection of autologous protein solu- tion for treatment of osteoarthritis in horses. Am J Vet Res 75:141–151.

61. Fahie MA, Ortolano GA, Guercio V, et al. 2013. A randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs. J Am Vet Med Assoc 243:1291–1297.

PRECLINICAL MODELS IN MUSCULOSKELETAL RESEARCH 751

JOURNAL OF ORTHOPAEDIC RESEARCH APRIL 2017