Standardized Instability-induced Animal Models for Preclinical Studies of Osteoarthritis

Summary

This protocol aims to establish suitable animal models for researching osteoarthritis.

Abstract

As the most prevalent joint disorder worldwide, knee osteoarthritis (OA) represents a significant global health burden, prompting extensive research into its pathogenesis and treatment strategies. Despite numerous studies, standardized methodologies for model establishment remain lacking. Thus, to address this gap, we present comprehensive, reproducible protocols for developing instability-induced animal models of osteoarthritis, specifically the Destabilization of the Medial Meniscus (DMM) model in mice and the Medial Meniscus Tear (MMT) model in rats. Furthermore, we describe a straightforward and easily implementable procedure for assessing postoperative pain thresholds and spatial gait stability, aimed at promoting methodological consistency in preclinical OA research. Histological and behavioral validation confirmed the success of both models, as Osteoarthritis Research Society International (OARSI) scores were markedly elevated, stride length was significantly altered, and mechanical pain thresholds were substantially reduced in the DMM and MMT groups compared with controls, indicating pronounced joint degeneration and hypersensitivity. These validation outcomes underscore the reproducibility and translational relevance of the two models. Thus, by providing detailed video documentation, this work promotes procedural standardization and facilitates inter-laboratory consistency, thereby enhancing the reliability and comparability of OA research outcomes.

Introduction

Osteoarthritis (OA) is a highly prevalent degenerative joint disorder and one of the leading causes of disability worldwide^1,2. Despite extensive research, the development of effective disease-modifying OA drugs (DMOADs) has been relatively slow³. It is now widely acknowledged that OA is a multifactorial disease involving complex pathological changes across multiple peri-articular tissues, including articular cartilage, subchondral bone, tendons, and synovium^4,5. Effective preclinical research on the diverse pathological processes of OA requires the use of well-defined and appropriate animal models. Although a variety of OA animal models have been established and reported^{6,7,8,9,10,11}, many studies continue to employ unsuitable animal models, limiting the translational relevance of their findings. To address this issue, we present standardized instability-induced OA models, including reproducible Destabilization of the Medial Meniscus (DMM) and Medial Meniscus Tear (MMT) procedures, in a video format to serve as a practical reference for preclinical OA research and model standardization.

As reported in previous studies, meniscal instability is a major factor contributing to knee joint degeneration in clinical cases. Therefore, surgically induced meniscal instability in mice and rats serves as an effective approach for the experimental induction of OA. In this context, we present two representative instability-induced OA models, including the DMM model in mice and the MMT model in rats. The DMM model in mice is particularly valuable for investigating molecular mechanisms of OA, as mice are amenable to transgenic manipulation, and this model exhibits a gradual and prolonged disease progression, closely mimicking human OA. However, it is important to note that the DMM model is less suitable for studies focusing on pathological alterations in the subchondral bone, where alternative models may provide better translational relevance.

Furthermore, we established the MMT model in rats to complement the mouse DMM model. The MMT procedure induces more extensive structural damage involving both the articular cartilage and subchondral bone, thereby better reflecting the advanced pathological features of OA observed in clinical settings. This makes the rat MMT model particularly suitable for bioengineering and translational research, including the evaluation of biomaterials, regenerative therapies, and drug delivery systems targeting joint repair. Although the MMT and DMM surgical techniques have been described in previous studies, there remains a lack of a comprehensive, step-by-step visual protocol that ensures standardization, reproducibility, and technical precision across research laboratories. Hence, to address this gap, we have developed a detailed video-based demonstration illustrating each critical step of model establishment, from animal preparation to postoperative care. In addition, we introduce two commonly used behavioral evaluation methods, one for assessing pain threshold and another for analyzing spatial gait stability to provide a consistent framework for functional assessment following OA induction. Thus, by integrating surgical and behavioral protocols, this manuscript aims to promote methodological standardization in preclinical OA research, thereby improving data comparability and translational relevance across studies.

Protocol

All experimental procedures were conducted in accordance with the guidelines of the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University and were approved under the protocol number IACUC-20251020-11. For the DMM surgery, male C57BL/6 mice aged 10-12 weeks were used, while male Sprague-Dawley rats weighing 200-225 g were selected for the MMT procedure. All animals were housed under specific pathogen-free (SPF) conditions and allowed a one-week acclimatization period prior to experimentation. Standard husbandry conditions were maintained, with food, water, and bedding replaced at least twice weekly to ensure animal welfare and experimental consistency.

1. Animal preparation

1. Administer appropriate analgesia (e.g., buprenorphine) before surgery according to institutional guidelines. Anesthetize the animal using an inhalational anesthetic (e.g., 2% isoflurane at 0.4 L/min with a 4 L/min fresh gas flow in an induction chamber) and maintain anesthesia throughout the procedure via a nose cone. Confirm the depth of anesthesia by ensuring the absence of a pedal withdrawal reflex.
2. Apply a 2-3 mm strip of lubricating ophthalmic ointment into the conjunctival sac of the lower eyelid and gently massage the eyelid to ensure even distribution of the ointment over the ocular surface.
3. Shave the fur surrounding the right knee joint and disinfect the surgical site using a three-step cleansing procedure with povidone-iodine followed by 70% ethanol. Position the animal on a sterile operating table.

2. DMM surgery in mice

NOTE: This procedure induces joint destabilization by transecting the medial meniscotibial ligament (MMTL), highlighted in red in Figure 1, which normally anchors the medial meniscus to the tibial plateau.

1. Position the anesthetized mouse in a supine orientation and make a small medial parapatellar skin incision (approximately 3-5 mm) over the right knee.
2. Carefully dissect the overlying soft tissues to expose the joint capsule. Make an incision medial to the patellar tendon and gently displace the patella laterally to visualize the knee joint. Confirm that the MMTL appears as a thin, ligamentous band connecting the anterior horn of the medial meniscus to the tibial plateau. Use a stereomicroscope to enhance visualization and minimize the risk of damaging the articular cartilage.
3. Carefully transect the tibial attachment of the MMTL using micro-scissors, a #11 scalpel blade, or a bent syringe needle. When using a bent syringe needle, the blunt edge helps protect the underlying articular cartilage. Confirm successful transection by observing the resulting destabilization of the medial meniscus.
4. Close the incised joint capsule using 8-0 absorbable sutures, followed by closure of the skin incision with wound clips or 6-0 sutures.
   NOTE: Take care to avoid piercing or damaging the articular cartilage during suturing and ensure that joint tightness is properly maintained while closing the incision.
5. Allow the animal to recover on a heating pad and monitor it closely until full mobility is regained.

3. MMT surgery in the rat

NOTE: This procedure induces OA by creating a complete tear of the medial meniscus following transection of the medial collateral ligament (MCL), thereby generating joint instability and progressive cartilage degeneration.

1. Position the anesthetized rat appropriately and make a medial parapatellar incision, approximately 6-8 mm in length, over the right knee.
2. Perform blunt dissection to separate the muscle layers and clearly expose the MCL (Figure 2).
3. Using surgical scissors, fully transect the MCL at its midpoint.
4. Gently retract the transected MCL to expose the underlying medial meniscus. Using fine forceps, grasp the meniscus and perform a full-thickness transection at its narrowest region to create a complete tear.
5. Reposition the soft tissues and close the incision in layers, using 5-0 absorbable sutures for the muscle and fascia, followed by wound clips or 5-0 non-absorbable sutures for skin closure.
6. Allow the animal to recover by following the same procedure described in step 2.5.

4. Behavioral assessments

1. Conduct baseline assessments prior to surgery and repeat evaluations at designated postoperative time points (e.g., 2, 4, and 8 weeks).
   NOTE: To minimize interoperator variability, behavioral tests were performed by the same trained operator who was blinded to the experimental grouping. Each measurement was repeated three times per animal, and the average value was used for analysis.
   1. Simplified mechanical withdrawal threshold test
      1. Place the mice in individual compartments on a raised wire mesh platform and allow them to acclimate for at least 30 minutes before testing. Mechanical pain sensitivity is then assessed using an electronic von Frey anesthesiometer.
      2. Using a Von Frey filament, apply the probe to the plantar surface of the mouse's right hind paw and gradually increase the force¹².
      3. Record the response, noting that a positive reaction is indicated by sudden paw withdrawal, flinching, or licking. Assess the presence or absence of a consistent response to evaluate changes in sensitivity over time.
   2. Gait analysis
      1. Utilize an automated gait analysis system for assessment.
      2. Allow the animal to voluntarily walk across the glass walkway of the apparatus. Define a valid trial as one in which the animal moves at a steady pace without pausing. Wait for the system software to automatically identify paw prints and compute multiple gait parameters. Analyze key metrics for the right (operated) hindlimb, including stance phase duration, swing speed, and paw pressure, and compare them with those of the contralateral limb and sham-operated controls.
         ​NOTE: All animals were acclimated to the testing apparatus with daily training sessions prior to baseline assessment. For each animal, three runs were recorded per time point, and only trials in which the animal maintained a consistent speed without pausing were considered valid. All measurements were performed by trained operators under a double-blind design to minimize inter-operator variability, and repeated runs were averaged to ensure data reproducibility.

Results

Characterization and validation of the DMM OA mouse model
After a 1 week acclimatization period, the DMM-induced osteoarthritis (OA) model in mice was established following the procedure illustrated in Figure 1. Pathological and behavioral assessments were performed two weeks post-surgery. As shown in Figure 2A, the most pronounced degenerative changes were localized to the medial femorotibial joint. The extent of cartilage degeneration was ...

Discussion

This protocol provides a standardized framework for the induction and evaluation of two widely used surgical OA models. These models are particularly valuable because they closely replicate the pathophysiological features of human post-traumatic OA, thereby offering strong translational relevance. The DMM model involves transection of the medial meniscotibial ligament, whereas the MMT model entails a direct tear of the medial meniscus. In our laboratory, the DMM procedure is routinely performed in mice, as the medial men...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gait analysis system	Shanghai Xin Ruan Information Technology Co., Ltd	XR-FP202
Liposic			ophthalmic ointment
Mice	Nanjing Institute of Model Animals	c57/bl6
Pain testing system	Shanghai Xin Ruan Information Technology Co., Ltd	XR-XZD	electronic von Frey anesthesiometer
Rat	Nanjing Institute of Model Animals	S/D