Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases. It affects millions of people worldwide and significantly impacts their quality of life [1]. Characterized by the progressive degradation of joint cartilage, synovial inflammation, and subchondral bone remodeling, the multifaceted nature of OA presents challenges in understanding its intricate pathophysiology [2]. This complexity is further highlighted by the variability in pain perception and functional disability among patients, which does not always correlate with the severity of the disease [3].

Given these complexities, it is no surprise that while various pharmacological interventions provide symptomatic relief, they seldom modify disease progression [4]. To develop transformative therapeutic strategies that address the root cause of OA and alleviate pain, a detailed understanding of the underlying mechanisms is paramount. Animal models have been indispensable in this endeavor. From rodents to larger mammals, they offer a controlled environment to mimic disease onset and progression, which facilitates the discovery of new therapeutics.

While animal models provide a simulated environment for in-depth studies, the methodologies available to gage pain and behavioral changes are pivotal in translating the results into the clinic [5]. Moreover, as the scientific community relies heavily on these models, the ethical implications surrounding their use require introspection and careful consideration [6]. In this review, we delve into the diverse landscape of animal models used in OA research, focusing on the methods used for pain assessment and its clinical significance. We provide insight into the pivotal role of these models in enhancing our understanding of OA, while also addressing the ethical contours that envelop their use. In addition to direct pain responses, OA affects a range of behaviors in both animals and humans. Monitoring behavioral changes in animal models provides a deeper understanding of the systemic impact of OA and offers indirect clues regarding pain and discomfort. This section highlights pivotal behavioral tests used in OA research and discusses their clinical implications.

Understanding the intricacies of osteoarthritis (OA) requires models that can recapitulate the pathology of the human disease. Animal models provide valuable insights and aid in the dissection of molecular and biomechanical processes involved in the initiation and progression of OA. They serve as platforms for testing therapeutic interventions and identifying biomarkers for early disease detection (Fig. 1) [7].

Figure 1. Osteoarthritis animal models. (A) Destabilization of the Medial Meniscus (DMM) model: a surgical model in which the medial meniscotibial ligament is transected, leading to joint instability and the subsequent development of OA, primarily in the medial compartment of the knee. (B) Anterior Cruciate Ligament Transection (ACLT) model: this model involves the surgical transection of the anterior cruciate ligament, resulting in joint instability and osteoarthritis, which resembles post-traumatic OA. (C) MIA (Monoiodoacetate) injection: chemical induction of OA through intra-articular injection of monoiodoacetate, which inhibits glycolysis in chondrocytes, resulting in cartilage degeneration.

1. Surgical models: surgical induction of joint instability is commonly used to induce OA in animals

1) Destabilization of the Medial Meniscus (DMM)

In this model, the medial meniscotibial ligament is surgically transected, resulting in joint instability and subsequent development of OA [8]. This method closely resembles post-traumatic OA in humans, making it a highly relevant model for studying secondary OA.

2) Anterior Cruciate Ligament Transection (ACLT)

ACLT involves transection of the anterior cruciate ligament, causing joint instability and OA. This model is particularly useful for studying the early inflammatory response and cartilage degradation post-joint trauma [9].

2. Chemically-induced models: chemicals can induce chondrocyte death, which results in rapid joint degeneration

1) Monoiodoacetate (MIA) injection

MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme. Intra-articular MIA injection results in metabolic disruption in chondrocytes, which results in cartilage degeneration. This model rapidly induces OA-like changes and is especially valuable for pain studies [10].

3. Spontaneous models: some animals naturally develop OA because of genetic predisposition or aging

1) Str/ort mouse

This strain of mice spontaneously develops OA with age, making it a suitable model for studying primary OA. It has expanded our understanding of genetic factors and molecular pathways associated with the spontaneous onset of OA [11].

2) Guinea pigs

Because of their natural aging process and predisposition, guinea pigs spontaneously develop OA. They are useful in studying age-related changes in articular cartilage and the underlying molecular mechanisms [12].

Each of these models has its merits and limitations, and the choice depends on the specific research question being addressed. Although they provide invaluable insights into OA pathology, translating these findings to human OA requires careful consideration of the differences in anatomy, physiology, and biomechanics between species [13].

Understanding pain, especially in animal models, requires objective tools that capture the multifaceted nature of this subjective experience [14]. Pain resulting from osteoarthritis is not merely a symptom, but a complex interplay of nociceptive, inflammatory, and neuropathic components, each contributing differently in individual cases [15]. These assessments provide insight into the progression of the disease, the efficacy of therapeutic interventions, and the overall impact of OA on quality of life [16,17]. Here, we explore the key pain tests used in animal models of OA and their clinical relevance.

1. Mechanical sensitivity tests

Mechanical sensitivity or mechanical allodynia, refers to the increased sensitivity to stimuli that would normally be innocuous. In the context of osteoarthritis, it is one of the principal phenotypes studied, because articular degeneration often leads to heightened sensitivity in the affected joint areas. To accurately gage this phenomenon in animal models, the following tests have been formulated:

1) Von frey hair test

This utilizes a series of monofilaments with varying force thresholds, which are applied perpendicular to the animal's paw until they bend. The withdrawal or reaction threshold is recorded [18].

2) Electronic Von Frey

An electronic version of the traditional Von Frey test, which provides a digital reading of the applied force, thus increasing accuracy [19].

3) Randall-Selitto test

Used mainly for larger animals, it measures pain response by applying increasing pressure to a limb until a withdrawal response is observed [20].

4) Gaitpain Analyzer

A cutting-edge method designed for assessing arthritic pain through voluntary locomotion in rats. The Gaitpain Analyzer is an innovative device that precisely measures the weight load on each leg of a walking rat. Integrated with strain gage weight sensors at its base, this device captures real-time changes in weight-bearing, which are indicative of pain or discomfort in the animal. Traditional pain assessment methods primarily focus on reactive behaviors, such as limb withdrawal in response to stimuli. The Gaitpain Analyzer offers a proactive approach, in which the voluntary walking behavior of the animal becomes the main metric for pain assessment. This mimics real-life scenarios more closely, and provides insight into the day-to-day challenges faced by osteoarthritis patients. The continuous measurement of weight load alterations offers a dynamic picture of pain progression and treatment efficacy [21].

2. Thermal sensitivity tests

1) Hargreaves method

This method assesses thermal pain sensitivity by directing a heat source toward the paw and measuring the time taken to withdraw. This method involves placing an animal on a heated glass surface and then directing a focused beam of radiant heat to the paw. The latency period to withdrawal provides an index of thermal nociception [22].

2) Hot plate test

Animals are placed on a heated plate and the latency period to display pain behaviors (licking or shaking paws) is recorded [23].

3) Tail flick test

A radiant heat source is directed toward the tail and the time it takes for the tail to flick away is measured [24].

4) Cold plate test

Contrary to the hot plate test, this test evaluates responses to cold stimuli. The animal is placed on a cold plate and the reaction times are observed and recorded [25].

3. Chemical sensitivity tests

1) Formalin test

A dilute formalin solution is injected into the paw and the subsequent pain behavior (e.g., licking, flinching) is quantified. This test can distinguish between early-phase and late-phase pain responses, which provides insight into inflammatory and neuropathic pain mechanisms [26].

2) Capsaicin test

Capsaicin, an active component of chili peppers, is applied or injected, causing an immediate sensation of burning pain. Observing the subsequent behavior provides insight into neurogenic pain [27].

4. Joint compression and loading tests

1) Knee bend test

The affected joint is moved through its range of motion and resistance or vocalizations are considered indicators of pain [28].

2) Joint loading tests

By applying controlled mechanical loads to the affected joint, researchers can gage pain responses and evaluate joint integrity [28].

5. Gait analysis

1) CatWalk system

This automated gait analysis tool captures the footprints of rodents as they walk on an illuminated glass platform [29]. Parameters such as paw pressure, stance duration, and swing speed help to assess joint pain and mobility issues.

2) Treadmill analysis

By observing the animals' ability and willingness to run or walk on a treadmill, pain levels and joint functionality can be assessed [30].

6. Weight-bearing asymmetry

1) Incapacitance tester

This device measures weight distribution between the two hind limbs of rodents [17]. Animals with joint pain often shift their weight to the less affected limb, which indicates pain and discomfort.

7. Behavioral observation

1) Open field test

This test measures spontaneous activity in a novel environment [31]. Reduced exploration or mobility can indicate pain or discomfort.

2) Grimace scale

Pain can also be assessed by observing the facial expressions of animals, particularly rodents. Specific facial features correlate with pain levels, which provide a non-invasive measure of discomfort [32].

8. Supplementary methods

1) Conditioned place preference

Evaluating an animal's preference for places associated with pain relief can help to assess pain levels and the efficacy of analgesic treatments [33].

2) Acoustic analysis

Vocal changes, especially ultrasonic vocalizations in rodents, can indicate distress or discomfort and serve as a supplementary measure of pain [34].

Osteoarthritis (OA) remains a leading cause of disability worldwide. It affects millions of individuals. Its impact on quality of life, coupled with the economic burden it causes to healthcare systems underscores the need for a comprehensive understanding of the disease and the development of effective interventions [35,36].

Animal models of OA offer unique insights into disease onset, progression, and the underlying molecular mechanisms responsible. Surgical, chemically-induced, and spontaneous models mirror various OA subtypes observed in humans, which range from post-traumatic to age-related forms [37]. These models enable the dissection of OA's intricate biomechanical and molecular interplay, which is challenging to study in humans because of ethical and methodological constraints.

Pain and behavioral test methods provide a dynamic view of the symptomatic evolution of OA. Techniques, such as gait analysis, weight-bearing asymmetry, and mechanical sensitivity tests, not only chart the progression of pain, but also serve as essential tools to evaluate the efficacy of therapeutic agents [38]. In the clinical setting, understanding the nuances of pain is important, because pain management remains one of the primary challenges in OA treatment.

Translating these findings from bench to bedside holds significant promise. The knowledge derived from animal models can lead to the development of targeted therapies that address the root causes of OA, rather than merely managing its symptoms. Furthermore, the behavioral assays used in these models may lead to novel diagnostic tools and methodologies in the clinic, which will aid in early OA detection and timely intervention [39].

In conclusion, although animal models and behavioral tests are foundational to OA research, their true value lies in their potential to revolutionize clinical practice. By bridging the gap between laboratory research and patient care, we move closer to a future in which the impact of OA is mitigated, both symptomatically and economically.

Osteoarthritis (OA) represents a complex interplay of biological, biomechanical, and environmental factors, thus its study and management remain a challenge for clinicians and researchers. Exploring animal models, pain, behavioral test methods, and their clinical significance reveals the strengths and limitations of current methodologies, while highlighting potential future directions in OA research.

Animal models, including surgical, chemical, or spontaneous variants, capture the heterogeneity of human OA and recreate the biomechanical and molecular intricacies of this disease. The wide variety of models is invaluable as they enable the examination of distinct OA etiologies and their respective progression pathways [40]. With respect to pain assessment, innovative behavioral assays, such as the CatWalk system and the Von Frey Hair test, have revolutionized our understanding of OA symptoms [41]. These tools have not only refined our understanding of the effects of OA on patients, but also serve as barometer for therapeutic efficacy.

Translating insights from animal models into the clinic has the potential to reshape therapeutic paradigms for OA [42]. The knowledge derived from these models will lead to targeted interventions and shift the focus from just symptomatic relief. This transition will markedly improve OA management, in which early detection and intervention will significantly decrease the debilitating impact. However, as with all complex research endeavors, limitations persist. Although animal models provide invaluable insights, replicating the complexity of human OA remains challenging. Differences in biomechanics, lifespan, and molecular pathways between species may result in discrepancies when translating findings to human OA [43]. Similarly, behavioral assays, although robust, may not capture the entire spectrum of pain experienced by OA patients, which requires continuous refinement and validation.

Future studies should focus on enhancing the fidelity of animal models to human OA through genomic editing technologies or sophisticated biomechanical simulations. In addition, integrating cutting-edge technologies, such as artificial intelligence and machine learning, may refine behavioral assays and offer more nuanced insights into the issues of pain and mobility [44].

In conclusion, the landscape of OA research, enriched by animal models and behavioral test methodologies, holds immense promise. By continuously refining these tools, guided by clinical insights, we will eventually alleviate the global burden of OA on patients and society.

Osteoarthritis (OA), with its intricate biological and biomechanical roots, remains a significant challenge worldwide. The use of animal models that encompass various OA etiologies, has enhanced our understanding of the underlying mechanisms and progression. Advancements in pain and behavioral test methods have provided insight into the symptomatic profile and the impact on quality of life. Although these techniques offer valuable insight, translation from animal studies to the clinic requires careful consideration because of inherent species-specific variations. In the future, refining these models and methods, integrating advanced technologies, and aligning preclinical studies with clinical findings are paramount. These endeavors will reduce the burden of OA by improving patient outcomes and quality of life.

No potential conflict of interest relevant to this article was reported.