Modified Medial Meniscectomy (MMM) Model to Assess Post-Traumatic Knee Osteoarthritis in Mouse

Abstract

Background/Objectives: Mechanical, physiological, and biochemical changes contribute to post-traumatic osteoarthritis (PTOA). Specific mouse models that are highly reproducible, less invasive, and easy to use are lacking. This limitation hinders the progress of PTOA-related studies on therapeutic applications. The goal of the study was to establish a methodologically innovative, efficient, and less technically challenging surgical model for PTOA. Methods: We developed a modified medial meniscectomy (MMM) model demonstrating high reproducibility and applicability. The MMM model features distinct differences in the execution of transection of the medial meniscus on the lateral side and includes a smaller incision, which enhances reproducibility and is beneficial for studying pain, structure, and function. Results: One month after the MMM surgery, the mice showed increased sensitivity to pain and decreased biomechanical abilities, such as shorter running times and distances. This was further supported by higher Osteoarthritis Research Society International (OARSI) histology scores, a standardized system for determining the severity and extent of OA in cartilage. Additionally, transcriptomic analysis showed an elevated enrichment of immune activity and bone tissue formation gene sets in the knee joint. Conclusions: Overall, functional studies and transcriptomic analyses suggested that the MMM model can be utilized for future biomechanistic and therapeutic applications and could serve as a new resource for studying PTOA.

1. Introduction

Osteoarthritis (OA) has progressed significantly in recent years. The structure and functionality of knee joint cartilage deteriorate due to trauma, inflammation, and other risk factors. Owing to irregularities or loss of cartilage, almost six million people visit hospitals. With an estimated lifetime risk of 14% [1], knee OA is expected to cost USD 185 billion in medical expenses annually [2]. There are no FDA-approved medications; its prevalence is increasing [3]. OA causes synovium, meniscus, and other joint tissue changes and induces pain and disability in preclinical animal models [4,5,6]. Studies have also shown that mouse PTOA can affect Aβ accumulation in the brain, thereby increasing the risk of the development of neurodegenerative disorders [7]. This highlights the need to develop effective therapeutic strategies and experimental models that resemble OA pathophysiology.

Post-traumatic OA (PTOA) affects a significant portion of the US population with various etiological factors, yet there are few reliable animal models. Therefore, identifying a specific, easily reproducible mouse model could help test multiple therapeutic agents and biomarkers for OA prevention and treatment [8,9,10]. Commonly used OA models include chemically induced spontaneous, non-invasive, and surgically induced models. Our earlier studies utilized chemical-induced progressive aging, genetics, human cartilage implants, and other models of OA [11,12,13]. Murine PTOA induces inflammation and pain [14,15,16,17]. Murine PTOA models have been used in mechanistic [18] and therapeutic studies [19,20,21].

The current surgical model utilized in OA studies is destabilization of the medial meniscus (DMM) [22] and the anterior cruciate ligament (ACL) transection model, which requires higher surgical expertise and could be highly invasive. The DMM model has evolved to become the gold standard [22]. Variations in the subluxation/dislocation of the patella in some DMM mice were noted due to forced destabilization during surgery. Additionally, early-stage gait disparity and functional changes were not observed in DMM [23,24]. Non-invasive trauma-induced models do not recapitulate the pathological and inflammatory conditions [25,26]. Notably, OA is more common in females than in males. However, DMM surgery does not recapitulate the sex-linked severity of OA observed in female patients. Refinement of the surgical procedure is required [27]. Thus, given the limitations of the commonly used DMM model, there is an unmet need to develop a less invasive mouse model that progressively develops and mimics pathological changes similar to human knee OA.

In this study, we established a modified medial meniscectomy (MMM) model for PTOA that is less invasive and methodologically innovative for higher reproducibility and which can assess structural and functional changes. The scope and anatomical focus of surgical intervention are the main areas in which the MMM model differs from the conventional DMM model. The MMM model uses controlled meniscectomy, which makes an incision on the lateral part of the medial meniscus. In contrast, the DMM model uses transection of the medial meniscotibial ligament to destabilize the medial meniscus. The purpose of this modification was to accurately resemble the forms of meniscal injuries that commonly occur in post-traumatic OA cases in humans. Slower disease development, common in OA, is also made possible by the avoidance of total instability by the MMM model and preserves joint stability. Therefore, the MMM model can be used for various therapeutic applications. Next, we performed a functional assessment of the knee joint using the von Frey test for allodynia pain assessment and a treadmill exhaustion test to evaluate biomechanical properties and endurance. We also assessed the variation in gene expression patterns and biological processes in MMM models compared with those in mice with sham controls. This functional assessment and molecular variation in the MMM could enhance the evaluation of preclinical OA-related studies.

2. Methods

2.1. Animal Experiments

All mouse (C57BL/6J) experiments were performed under the guidance, regulation, and approval of the Boston University Institutional Animal Care and Use Committee (IACUC; approval number AN-15387). Animal studies adhered to the ARRIVE standards. Animals were housed in ventilated microisolator cages with specific pathogen-free (SPF) barriers to prevent contamination and maintain health standards. The environmental conditions were maintained with a 12 h light/dark cycle (7 AM–7 PM) and a constant temperature of 70 °F (~21 °C). The water was acidified to prevent microbial growth, providing clean and safe hydration. Animals received conventional laboratory food directly in their cages, which met their daily nutritional requirements. Nestlets provided environmental enrichment to animals housed separately, encouraging natural behaviors such as nesting and enhancing welfare. Such a controlled environment supports the consistent physiological and behavioral results necessary for reliable research. SPF status was maintained by strict husbandry procedures, including routine monitoring and cleanliness, which also helped provide animals with a stable and humane living environment for the study.

2.2. Surgical Procedure for Modified Medial Meniscectomy (MMM)

To perform the MMM surgery on the knee, the mice were fully anesthetized using isoflurane until they became entirely unconscious, and its administration continued at the same flow rate throughout the procedure, with 2.5% isoflurane in 100% oxygen administered at a flow rate of 1 L/min in the induction chamber. Anesthesia was maintained with a 2.5% mixture at 0.5 L/min throughout the surgery. The right hind limb was shaved and disinfected by alternating povidone-iodine and 70% ethanol scrubbing. The animal was then placed in lateral recumbency, with the knee joint bent to approximately 45°, and the limb was twisted medially to improve surgical accessibility. Surgery was performed under a stereomicroscope to ensure precision and minimal tissue damage. Sterile tools were used consistently throughout this process. The lateral portion of the knee was incised precisely to reveal the joint capsule. Adequate light and magnification under a stereomicroscope offered a clear view of the joint components, enabling correct transection of the medial meniscus with an 11-curved or straight blade. For sham surgery, a small incision was made on the knee capsule. All surgical procedures were performed aseptically, in compliance with the institutional animal care protocols described above. All surgeries were performed by Dr. Manish Bais, an experienced small-animal surgeon. Further procedural details for MMM are described in the Results section.

2.2.1. Treadmill Exhaustion Test

Preclinical behavioral evaluation of chronic pain and inflammation can benefit from treadmill behavior [28]. The treadmill exhaustion test was performed using a standard protocol [29] before and after MMM surgery. Before performance evaluation, mice (n = 8 mice/group) were provided with a day of rest after three days of acclimatization to treadmill running (TSE Systems) [29]. The acclimatization process included five minutes of rest on the treadmill conveyor belt, five minutes of running at 7.2 m/s, and five minutes at 9.6 m/s. On day 0, mice were subjected to a graded maximum running test that began with a 5 min rest period, followed by a running protocol that started at 4.8 m/min and increased by 2.4 m/min every 2 min. At all times, the belt was retained at a 5-degree inclination. The maximum running speed was measured as the fastest rate at which the mice could run for 5 s without hitting the electric shock grid of the treadmill.

2.2.2. Allodynia Test

Allodynia was assessed using the Von Frey hair test, which involves pricking the hind paw 3–5 times with filaments of varying diameters. Specifically, mice (n = 10 mice/group) were allowed to use the test equipment for at least 15 min after being placed in an acrylic chamber above a metal grid floor. Initially, we waited for the mouse to stop its exploratory behavior, and a von Frey filament was pushed onto the plantar surface of the paw until it buckled and then was held there for a maximum of three seconds. A response was noted if the paw was sharply withdrawn when the filament was applied or it flinched when the filament was removed. The experimental groups were not disclosed to the researchers who conducted the experiments.

2.3. Habituation Protocol

To minimize stress-related variables and ensure consistent behavioral responses, a habituation protocol was implemented in which animals were acclimated to the testing environment for three consecutive days prior to the commencement of baseline assessments. During this period, the animals were placed in the testing apparatus for a standardized duration each day, allowing them to become familiar with their surroundings and reduce novelty-induced anxiety. All assessments were conducted during the light phase of the circadian cycle, specifically between 9 AM and 12 PM, to control for potential diurnal variations in activity levels and physiological responses. To minimize bias and maintain the integrity of data collection, all behavioral assessments and analyses were performed by trained individuals blinded to the experimental conditions.

2.4. Histology

Mouse knee joints (n = 6 mice/group) were paraffin-embedded, decalcified, histologically examined, and stained. Safranin-O/Fast Green (MasterTech/StatLab, McKinney, TX 75069, USA) staining was performed as described previously [12]. Histological assessment and Osteoarthritis Research Society International (OARSI) scoring were performed as previously described. The OARSI scoring system, a semi-quantitative method, was used to assess the severity of osteoarthritic cartilage lesions by examining structural changes and the depth and extent of cartilage damage. The scores ranged from 0 (normal cartilage) to 6 (total erosion of cartilage to the calcified layer), with higher scores indicating severe deterioration. The stained tissue slices were scanned using a high-resolution digital slide scanner (Panoramic MIDI, 3D Histech, Budapest, Hungary).

2.5. Sample Collection and RNA Extraction

Four months after the medial meniscal meniscectomy (MMM) surgery, the mice were sacrificed by standard euthanasia guidelines to ensure humane treatment. The knee joints were carefully harvested by cutting the entire leg from the proximal epiphysis of the femur. The surrounding hair, muscle, and fat tissues were meticulously removed using fine scissors and scalpels, followed by gentle rubbing with sterile gauze to ensure tissue cleanliness. Complete knee integrity was preserved, and the joints were collected by cutting the distal femoral metaphysis and proximal tibia. Four mice per experimental group (n = 4 per group) were included for downstream analysis. The harvested samples were snap-frozen immediately in liquid nitrogen and mechanically crushed. Total RNA was extracted from the tissue using TRIzol reagent (QIAGEN, Beverly, MA 01915, USA), and 400 ng of purified total RNA per sample was sent to Novogene (Sacramento, CA 95817, USA) for RNA sequencing.

2.6. RNA Sequencing and Bioinformatics Analyses

All RNA samples were subjected to qualitative assessment before library construction to ensure data reliability and sequencing quality. Only samples that passed the quality control threshold based on their RNA Integrity Number (RIN) were selected for downstream analysis. High-throughput paired-end RNA sequencing was performed using Illumina sequencing technology. Prior to alignment, raw FASTQ reads were processed with an in-house Novogene Perl script to remove low-quality sequences and filtered by GC content, and adapter sequences were trimmed. The cleaned and filtered reads were aligned to the Mus Musculus reference genome (mm10) using the HISAT2 aligner. Gene quantification was conducted using FeatureCounts (v1.5.0-p3), allowing for accurate measurement of transcript abundance.

3. Differential Gene Expression (DGE) Analysis

Differential gene expression (DGE) between the sham and MMM surgery groups was assessed using DESeq2 in the R/Bioconductor environment. DESeq2 uses the Wald test to calculate p-values and the Benjamini–Hochberg method to calculate the adjusted p-values. To explore the biological significance of changes in gene expression, Gene Set Enrichment Analysis (GSEA, version 4.3.2) was performed to identify enriched pathways and biological processes. The differential expression data was visualized by generating a volcano plot using the ggplot2 package in R, highlighting significantly upregulated and downregulated genes. This integrative pipeline provides a comprehensive overview of transcriptional changes associated with the MMM model.

4. Results

4.1. The MMM Model Is Methodologically Innovative and Less Invasive

To study the post-traumatic OA model, the traditional DMM model involves cutting the transection of the medial meniscotibial ligament to destabilize the medial meniscus (Figure 1A). Thus, this model required a significant incision at the midpoint. However, the MMM protocol involves bending the knee medially to access the medial meniscus laterally, which can be dissected under a dissection microscope or a stereomicroscope (Figure 1A,B). This approach is less invasive and does not require complete dissection of the knee capsule. Specifically, at 45°, the knee joint was bent and turned medially, followed by an incision on the lateral side of the knee joint capsule and lateral cutting of the medial meniscus with an 11-curved/straight blade (Figure 1C–E). When the MMM surgery was successful, the femur and tibia at the knee joint were clearly visible (Figure 1C,D), and the medial meniscus was transected and visibly popped out under higher magnification (Figure 1E). Finally, the joint capsule was sutured using Vicryl suture 4.0, followed by skin suturing. Thus, we established a modified meniscectomy model that is methodologically innovative, offering minimal incision size and ease of validation. We performed histological staining, treadmill exhaustion tests, and allodynia assessments to assess the structural and functional changes in mouse knee biomechanics.

4.2. The MMM Model Could Be Used to Study Joint Biomechanics, Pain, and PTOA

To determine whether the new model caused functional effects, sham and MMM surgeries were performed on 6-month-old C57BL/6J mice (n = 6–10; equal numbers of males and females). Functional outcomes were evaluated using the treadmill exhaustion test and the Von Frey hair test, conducted two days before surgery and again 31 days (four weeks) post-surgery. This time point was selected based on prior studies indicating that acute inflammatory and pain responses typically subside within the first three weeks post-injury. By five weeks, animals generally transition into a chronic pain phase characterized by joint degeneration and persistent behavioral alterations, making this an appropriate time point to assess early chronic PTOA-related changes [30]. Results from the treadmill exhaustion test showed no significant differences in running time or distance between the groups before surgery (Figure 2A,B). However, 31 days after MMM, the mice in the MMM group exhibited significantly reduced running time and distance compared with the sham-operated controls. Control mice maintained their performance, whereas MMM mice showed clear impairments in endurance and mobility. In the Von Frey test, the hind paw withdrawal threshold was similar across all groups prior to surgery, with all mice responding to a 1.5 g filament (Figure 2C). After MMM surgery, the MMM group demonstrated increased mechanical sensitivity, with responses to 0.4 g filaments, indicating the development of mechanical allodynia. In addition to these functional changes, histological analysis using Safranin-O staining revealed marked structural changes consistent with OA. Four months post-MMM, mice in the MMM group showed significantly increased cartilage damage, as reflected by elevated total OARSI scores, compared with sham controls (Figure 2D). Together, these findings demonstrate that MMM surgery induces functional impairment (reduced physical performance and increased pain sensitivity) and structural joint degeneration. These results support using the MMM model to mimic OA-like pathology and functional decline, validating it as a relevant model for studying osteoarthritis mechanisms and potential treatments.

4.3. The MMM Model Shows OA- and Inflammation-Related Gene Expression

To better understand the molecular impact of MMM surgery, RNA sequencing was conducted on the knee joints of both sham-operated and MMM mice (n = 4 mice/group). Differential gene expression analysis revealed that 503 protein-coding genes were significantly altered in expression following MMM surgery compared to the sham controls, using a p-value threshold of 0.05 (Figure 3A; Supplementary Table S1). These differentially expressed genes provided insights into the biological pathways influenced by this innovative surgical procedure. Gene ontology analysis, performed using the GSEA software, identified several enriched biological processes. Notably, processes related to cartilage development and extracellular structure organization were significantly upregulated, suggesting that the joint initiated a compensatory anabolic response to repair or maintained tissue integrity following mechanical disruption (Figure 3B). In parallel, gene sets associated with immune system activation were also markedly enriched (Figure 3B), indicating that MMM surgery induced a strong inflammatory response. This immune activation supports the utility of the MMM model as a reliable experimental system for studying inflammatory mechanisms in OA. Furthermore, ossification and bone development genes were among the most significantly enriched pathways (Figure 3B). This enrichment aligns with the known OA pathophysiology, where abnormal bone remodeling contributes to disease progression. Taken together, the transcriptional profile observed after MMM surgery mirrors the key features of human OA, including cartilage matrix disruption, inflammation, and bone remodeling. These findings validate the MMM model as a valuable tool for studying the molecular underpinnings of OA and testing potential therapeutic strategies to target inflammatory and degenerative changes in joint tissues.

5. Discussion

The modified medial meniscectomy (MMM) mouse model represents a significant advancement in post-traumatic osteoarthritis research. DMM has been used extensively in various studies [31,32,33,34,35].

The MMM model differs from the DMM model in the anatomical focus of the surgical operation. While the DMM model requires transection of the medial meniscotibial ligament to destabilize the medial meniscus, the MMM technique comprises controlled meniscectomy focusing on the lateral part of the medial meniscus. This modification was made to more precisely resemble the types of meniscal injuries observed in human OA, particularly in post-traumatic situations. Furthermore, the MMM model avoids complete destabilization and maintains joint stability, allowing for more gradual disease progression, as seen in human OA. Mechanistically, this modified technique allows for a more physiologically appropriate process of joint deterioration, such as delayed cartilage erosion, subchondral bone alterations, and synovitis. These factors improve the ability of the model to represent human diseases accurately. Furthermore, the MMM approach produces more uniform joint pathology because of the consistent extent of meniscal resection.

The key benefits of MMM include reduced invasiveness and suitability for investigating pain-related behaviors and structural changes. It consistently replicates medial meniscus injury-related changes and facilitates the examination of PTOA progression from the initiation to late stages. These features have enhanced its utility in therapeutic and mechanistic studies.

Specific models, such as monosodium iodoacetate (MIA)-induced OA, which produces pain-depressed wheel running [36], have been used to assess knee joint function. Treadmill behavior is applicable for preclinical behavioral assessments of biomechanical properties, chronic pain, and inflammation [28]. Traditional OA models, including chemically induced methods such as MIA-induced osteoarthritis, are helpful in assessing pain but fail to capture the complete clinical spectrum of osteoarthritis, particularly structural degeneration. MMM overcomes these limitations by integrating functional validations, such as treadmill exhaustion tests, allodynia assessments, and histological analysis, within a single experimental setup. This comprehensive approach allows researchers to study both functional impairments and underlying structural damage simultaneously.

Innovative aspects of the MMM model include reproducibility, simplified surgical procedures, and versatility for functional studies. It minimizes non-physiological inflammatory changes observed in other models and promotes a more natural progression of PTOA-like pathology. Its minimally invasive nature may enhance consistency in experimental outcomes. Functional assessments, such as the treadmill exhaustion test and von Frey hair test, demonstrated a substantial reduction in running time and distance and enhanced sensitivity in the MMM group compared with sham controls. These findings indicated that the MMM model can effectively elicit functional alterations associated with PTOA. Histological analysis with safranin-O staining revealed higher OARSI scores in the MMM group, validating the model-induced structural alterations.

Gene expression analysis using RNA sequencing and downstream gene ontology analysis demonstrated that the MMM model reflects inflammatory OA-related molecular alterations. Moreover, there was compensatory enrichment of cartilage growth gene sets, which a larger enrichment of activated immune responses and bone tissue formation in the mouse knee joint might offset. Our RNA-seq analysis corroborated this mitigation, which was performed four months after MMM surgery and revealed that biological processes linked to activated immune response and ossification were strongly enriched in gene ontology analysis. Further studies are required to evaluate this model under various conditions, including aging, obesity, and inflammation, and to understand its translational relevance under specific conditions better.

Beyond PTOA, the MMM model has the potential for broader applications, such as exploring osteochondral progenitors, stem cells, and cartilage–bone metabolism. It offers a promising platform for investigating biomarkers and therapeutic agents, including novel chondroprotective candidates such as lysyl oxidase-like 2 [11,12,13]. Additionally, its suitability for studying collagen degradation and repair mechanisms makes it highly applicable in osteoarthritis research.

Although the MMM has several benefits, it is essential to acknowledge its limitations. The present study lacks direct comparative data on our proposed surgical and established OA models, such as DMM, ACL transection, and partial meniscectomy. Moreover, the study used a limited number of mice and requires further validation with a large cohort of small animals. Although the present study emphasizes the technical and conceptual rationale for developing this innovative model, we admit that direct comparisons could strengthen the data by demonstrating its benefits. To determine the relative advantages of our strategy more conclusively, future research is necessary to rigorously assess disease progression and translational relevance across models. Another critical factor is the frequently observed separation between structural damage and pain-related behaviors in mouse models. Although we have documented considerable damage to joint pathology, how these links are associated with functional outcomes or nociceptive behavior is unknown. This is a prevalent issue in rodent OA research and highlights the significance of including multimodal assessments such as micro-CT, cytokine profiling, analysis of cartilage degradation products, and immunostaining with OA-specific markers such as MMP13 or IL1B in future investigations.

While the MMM model design, including features such as tibial rotation and visualization of the medial meniscus, facilitates reproducibility, the procedure’s reliability is also contingent on the surgeon’s technical proficiency. Research indicates that surgeons’ technical skills are associated with lower rates of complications and improved patient outcomes [37]. Therefore, both the model design and the surgeon’s expertise contribute to the overall success of the procedure.

In conclusion, the MMM model bridges the gaps in preclinical studies by combining structural and functional analyses. This study provides valuable insights into the mechanisms of PTOA and opens new avenues for developing therapeutic interventions. Optimizing this model for aging mice and enhancing its relevance to human osteoarthritis will further strengthen its role in translational research.

Meniscus injuries are common in clinical orthopedic treatments. This MMM model could provide a more precise and efficient method for testing new therapies and interventions for early-to-late-stage OA resulting from knee injuries caused by accidents, sports, or military training.