Development of a novel micro-CT based scoring system for the assessment of joint structure in a rat model of inflammatory knee osteoarthritis

Introduction

Osteoarthritis (OA) is a chronic degenerative disease characterized by the degeneration and loss of articular cartilage, subchondral bone sclerosis, and osteophyte formation (1). Inflammation and immune responses are known to be involved in the progression of OA (1). Mono-iodoacetate (MIA) is an agent that induces cartilage degeneration by inhibiting the glycolytic pathway of chondrocytes, creating an inflammatory OA model (2-4). Traditionally, histological evaluation has been used as the main outcome measure in OA research. However, histological evaluation requires animal sacrifice and is time-consuming and labor-intensive. Furthermore, in addition to the necessity of sacrificing animals for sectioning, there are drawbacks such as the limited cross-section of tissue sections, the risk of arbitrarily selecting sections that are advantageous for proving the researcher’s hypothesis, and the difficulty in evaluating the entire joint surface. In addition, the site of cartilage collection can also be arbitrary depending on the researcher’s judgment, despite the significant difference in cartilage thickness between humans and small animals (5).

In recent years, attention has been focused on the evaluation of joint structure using micro-computed tomography (micro-CT) (6-12). Micro-CT can non-invasively visualize the three-dimensional structure of joints, suggesting its potential for in vivo evaluation of OA progression (13). Although micro-CT cannot provide a detailed evaluation of cartilage degeneration, it may be possible to evaluate the overall degenerative process of the knee, roughly where and to what extent degeneration progresses. Although MIA-based knee OA models have been established in rodents, the posture is usually different from that of humans, and the medial tibio-femoral joint surface is not always the most affected, as in humans. As a means of detecting such differences, three-dimensional (3D) imaging may be able to identify areas that are more prone to degeneration.

In this study, we developed a novel micro-CT-based scoring system for the evaluation of joint structure in a rat model of MIA-induced OA and investigated its usefulness. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-101/rc).

Methods

Experimental procedure

All animal experiments were performed under a project license (No. 2024-39) granted by the Animal Ethics Committee of Aichi Medical University. The treatment of all animals complied with the institute regulations and the “3Rs” concept of replacement, reduction and refinement by Japanese policy on animal use (14). A protocol was prepared before the study without registration. A previous study often utilized OA models induced by 1 and 3 mg of MIA (15). Therefore, we aimed to establish a method to clearly distinguish between these two groups, assuming a large effect size of 2. Based on an effect size d=2, the sample size for a power (>0.80) with a two-tailed alpha at a significance level (<0.05) to run a t-test required a minimum of 6 rats per group.

We used 24 male Wistar rats (age, 12 weeks; weight, 280–300 g) that were purchased from SLC (Japan SLC, Inc., Shizuoka, Japan). All rats were housed in a controlled environment at 23±2 ℃ with 55%±10% humidity and a 12-h light/dark cycle; the rats had free access to food and water.

Under isoflurane anesthesia, the rats’ knees were shaved using an electric clipper and OA was induced in the right knee by intra-articular injection of various concentrations of MIA (Sigma-Aldrich, Tokyo, Japan) dissolved in 50 µL sterile saline. The solution was injected through the patellar ligament using a 31G needle. We randomly divided 24 rats into four groups: 0.2 mg MIA group (n=6), 0.5 mg MIA group (n=6), 1 mg MIA group (n=6) and 3 mg MIA group (n=6), respectively. On day 28 after injections of MIA solutions, all rats were sacrificed, and the right lower extremity of each rat was disarticulated at the hip joint and immediately fixed with 10% formalin.

Assessment of joint destruction and micro-CT joint scoring

The removed lower extremity specimen was placed in a silicone tube filled with 10% formalin for imaging. Quantitative assessment of knee joint morphology was conducted using micro-CT. Scans were acquired with a R_mCT2 system (Rigaku Corporation, Tokyo, Japan) employing the following parameters: X-ray voltage, 90 kV; tube current, 160 µA; voxel size, 40×40×40 µm³ for a 20×20 mm² scan field of view (FOV); exposure time, 120 seconds; continuous (non-stepping) rotation. To facilitate visualization of joint structures, three-dimensional volume rendering images (3D-VRIs) were generated using a constant threshold, as described previously by Yamanashi et al. (16). For comprehensive analysis, each 3D-VRI knee joint was virtually segmented into 10 anatomical compartments: (I) patella; (II) patellar groove; (III) anterior lateral femoral condyle; (IV) anterior medial femoral condyle; (V) anterior lateral tibial condyle; (VI) anterior medial tibial condyle; (VII) posterior lateral femoral condyle; (VIII) posterior medial femoral condyle; (IX) posterior lateral tibial condyle; (X) posterior medial tibial condyle. Within each compartment, the severity of degenerative changes was graded on a four-point scale (Figures S1-S11). An overall severity score for each knee joint was calculated by summing the scores of all compartments as a joint score, ranging from 0 (no damage) to 40 (most severe). Two blinded examiners independently evaluated the joint score of the affected knee. Then, interobserver reliability was calculated for the interclass correlation coefficient (ICC) for each group.

Histological evaluation

Following micro-CT assessment, the lower extremity specimens were decalcified by immersion in formic acid for approximately 3 months. The whole knee joints were then bisected sagittally along the midline, dehydrated in a graded ethanol series, and embedded in paraffin wax. This processing allowed for histological visualization of the patellar groove (midline) and condyles (periphery) in the medial sagittal plane. Subsequently, 5 µm-thick sagittal sections were obtained and stained with Safranin-O. Pathohistological evaluation of joint degeneration within the patellar groove at the width of the patella was performed using a modified Mankin grading system (17). This system assesses cartilage degeneration based on parameters including surface integrity (0 to 6 points), cellularity (0 to 3 points), Safranin-O staining intensity (0 to 4 points), and tidemark integrity (0 or 1 points), with scores ranging from 0 to 14. A higher score indicates a greater degree of degeneration. Two blinded examiners independently evaluated the modified Mankin score of the affected knee. Then, interobserver reliability was calculated for the ICC for each group.

Statistical analysis

Continuous variables are represented as median and interquartile range. Variables between the four groups were compared using the Kruskal-Wallis test followed by post hoc Dunn’s comparison. Fleiss’ ICC classification was used to interpret ICC values (18): ICCs above 0.75 indicate excellent reliability, values between 0.40 and 0.75 fair to good reliability, and values below 0.40 poor reliability. We also evaluated correlation coefficients between the total joint score and the modified Mankin score or zone 2 (patellar groove) joint score and the modified Mankin score at the patellar groove using Spearman’s correlation (ρ) test. Analyses were performed using JASP software version 0.19.3 (https://jasp-stats.org/) and SPSS software (version 25, SPSS Inc., Chicago, IL, USA). All results were considered statistically significant at P<0.01.

Results

Figure 1 shows representative images of each group of micro-CT-3D images of the knee, divided into 10 compartments. Figure 2 shows representative pathohistological sagittal specimens stained by Safranin-O at the joint between the patellar groove and the patella. Table 1 shows the mean and total scores for each group of micro-CT-3D images for each of the 10 compartments. Table 2 shows the mean of the modified Mankin scores for each group. The ICC values for the joint of the micro-CT joint scores and Mankin scores were mostly above 0.75 for each, and interobserver reliability was considered excellent (Tables 1,2).

Figure 1 Representative CT images for rats with various amounts of MIA. ①, patella; ②, patellar groove; ③, anterior lateral femoral condyle; ④, anterior medial femoral condyle; ⑤, anterior lateral tibial condyle; ⑥, anterior medial tibial condyle; ⑦, posterior lateral femoral condyle; ⑧, posterior medial femoral condyle; ⑨, posterior lateral tibial condyle; ⑩, posterior medial tibial condyle. CT, computed tomography; MIA, mono-iodoacetate.

Figure 2 Representative histopathological alterations for rats with various amounts of MIA (Safranin-O stainig, ×500). MIA, mono-iodoacetate.

Pathohistological evaluation by the modified Mankin score showed that the 3 mg group was more severely affected than the 0.2 mg group (P<0.01), and the 0.5 mg group (P<0.01). On the other hand, the micro-CT-3D image scoring showed that the 3 mg group had statistically significantly more severe joint scores than the 0.2 mg (P<0.01) and 0.5 mg (P<0.01) groups; in addition, the 1 mg group had statistically significantly more severe joint scores than the 0.2 mg group (P<0.01). The statistical differences between the 0.2 mg group and the 1 mg group were only detected in the micro-CT joint scoring.

The correlation coefficients between total joint score and modified Mankin score or zone 2 (patellar groove) joint score and modified Mankin score at patellar groove were ρ=0.8 (P<0.001) and ρ=0.8 (P<0.001), respectively.

Discussion

Our study successfully developed a novel micro-CT-based 3D imaging scoring system to quantify gross degenerative changes in rat knee joints affected by MIA-induced OA. This method offers a reliable approach for assessing joint structure, demonstrating excellent interobserver reliability with ICC values greater than 0.75 for most compartments, and above 0.8 for the overall system, with only zone 6 showing an ICC of 0.74. This high reliability indicates the objectivity of our scoring system.

A significant finding was the system’s ability to differentiate between various degrees of joint degeneration, specifically distinguishing between the 0.2 mg and the 1 mg MIA groups. This level of differentiation was not consistently observed with traditional histological scores. This highlights a key advantage of our micro-CT scoring system: while histological evaluation requires animal sacrifice and can be time-consuming and labor-intensive with limitations in assessing the entire joint surface and potential for arbitrary section selection, micro-CT provides a non-invasive, 3D visualization of joint structures, enabling a more comprehensive assessment of OA progression. Furthermore, we found a strong correlation between our total micro-CT joint score and the modified Mankin score (ρ=0.8, P<0.001), which supports the criterion-related validity of our method (19). Although micro-CT does not directly assess cartilage damage, its correlation with histological findings suggests its utility as a complementary tool for evaluating overall joint degeneration. This is particularly relevant given that traditional histological methods for evaluating OA often focus on cartilage and can be limited in scope.

Several limitations are acknowledged. First, to date, various induction models—including surgical (such as anterior cruciate ligament transection and/or meniscectomy) and chemical (e.g., MIA)—have been proven suitable for inducing OA in animals, despite pathological differences (20). However, there is currently no gold standard or consensus model that naturally reflects the human disease. In this study, we exclusively employed a chemical model; therefore, this evaluation is applied solely to the MIA-induced OA model.

Second, there is an inherent difficulty in assessing cartilage degeneration using this technique. While MRI is generally superior for quantitative cartilage evaluation in knee OA models, CT offers greater versatility due to its shorter acquisition time and lower equipment costs (21). Prior studies have employed micro-CT with contrast agents for quantitative evaluation of knee cartilage degeneration; however, this approach lacks robustness due to its simplicity and potential influence from agent-specific cartilage affinity (8,12). In addition, grade 3 lesions (massive erosions) appear to be based on changes in adjacent bone rather than true subchondral or articular erosions. While these reflect overall joint degeneration, their relevance to articular pathology is unclear. Consequently, although our method cannot directly evaluate cartilage, its correlation with tissue degeneration severity suggests its utility as a convenient index for estimating tissue degeneration.

The third limitation to consider is the resolution of micro-CT imaging. While this study investigated the feasibility of micro-CT 3D imaging, a FOV of 20 mm × 20 mm was necessary to visualize the articular surfaces with greater clarity, which consequently required euthanizing the rats and excising their legs. Given that a FOV of 30 mm × 30 mm would allow for the longitudinal observation of joint degeneration in vivo, future research should explore the practical application of this larger FOV. Furthermore, previous micro-CT 3D imaging studies investigated trabecular structure parameters (such as bone volume per tissue volume) of rodent knees with OA (22). Although we attempted to examine the bone mass structure of the subchondral bone in parallel with a three-dimensional overall evaluation; however, this assessment could not be performed because the large defects in the subchondral bone in the high-dose (1 and 3 mg) groups made region of interest (ROI) setting difficult in this study.

In future research, concurrent observation of joint structural changes and pain-related behaviors in rodent models of knee OA could facilitate straightforward investigation of the relationship between the extent of structural damage and pain severity.

Conclusions

The novel micro-CT-based 3D imaging scoring system offers a reliable method for quantifying gross degenerative changes in rat knee joints within the MIA-induced OA model. This system demonstrated excellent interobserver reliability, with most compartments showing ICC values greater than 0.75, and the overall system exceeding 0.8. While micro-CT does not directly assess cartilage, its strong correlation with histological findings (total micro-CT joint score and modified Mankin score had suggests its utility as a complementary tool for evaluating overall joint degeneration. This non-invasive 3D visualization provides a more comprehensive assessment of MIA-induced OA progression compared to the limitations of histological evaluation.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-101/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-101/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-101/prf

Funding: This research was supported by JSPS KAKENHI (No. 22K09435).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-101/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. 2024-39) granted by the Animal Ethics Committee of Aichi Medical University, in compliance with the institute regulations and the “3Rs” concept of replacement, reduction and refinement by Japanese policy on animal use.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Hunter DJ, Bierma-Zeinstra S. Osteoarthritis. Lancet 2019;393:1745-59. [Crossref] [PubMed]
2. Kalbhen DA. Chemical model of osteoarthritis--a pharmacological evaluation. J Rheumatol 1987;14:130-1.
3. Guingamp C, Gegout-Pottie P, Philippe L, et al. Mono-iodoacetate-induced experimental osteoarthritis: a dose-response study of loss of mobility, morphology, and biochemistry. Arthritis Rheum 1997;40:1670-9. [Crossref] [PubMed]
4. Udo M, Muneta T, Tsuji K, et al. Monoiodoacetic acid induces arthritis and synovitis in rats in a dose- and time-dependent manner: proposed model-specific scoring systems. Osteoarthritis Cartilage 2016;24:1284-91. [Crossref] [PubMed]
5. McCoy AM. Animal Models of Osteoarthritis: Comparisons and Key Considerations. Vet Pathol 2015;52:803-18. [Crossref] [PubMed]
6. Kim YH, Kang JS. Micro-computed tomography evaluation and pathological analyses of female rats with collagen-induced arthritis. J Vet Sci 2015;16:165-71. [Crossref] [PubMed]
7. Schambach SJ, Bag S, Schilling L, et al. Application of micro-CT in small animal imaging. Methods 2010;50:2-13. [Crossref] [PubMed]
8. Piscaer TM, Waarsing JH, Kops N, et al. In vivo imaging of cartilage degeneration using microCT-arthrography. Osteoarthritis Cartilage 2008;16:1011-7. [Crossref] [PubMed]
9. Roemer FW, Mohr A, Lynch JA, et al. Micro-CT arthrography: a pilot study for the ex vivo visualization of the rat knee joint. AJR Am J Roentgenol 2005;184:1215-9. [Crossref] [PubMed]
10. Thote T, Lin AS, Raji Y, et al. Localized 3D analysis of cartilage composition and morphology in small animal models of joint degeneration. Osteoarthritis Cartilage 2013;21:1132-41. [Crossref] [PubMed]
11. McKinney JM, Pucha KA, Bernard FC, et al. Osteoarthritis early-, mid- and late-stage progression in the rat medial meniscus transection model. J Orthop Res 2025;43:102-16. [Crossref] [PubMed]
12. Siebelt M, Waarsing JH, Kops N, et al. Quantifying osteoarthritic cartilage changes accurately using in vivo microCT arthrography in three etiologically distinct rat models. J Orthop Res 2011;29:1788-94. [Crossref] [PubMed]
13. Bouxsein ML, Boyd SK, Christiansen BA, et al. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 2010;25:1468-86. [Crossref] [PubMed]
14. Kurosawa TM. Japanese regulation of laboratory animal care with 3Rs. Alternatives to Animal Testing and EXperimentation (AATEX) 2007;14:317-21.
15. Bryk M, Chwastek J, Mlost J, et al. Sodium Monoiodoacetate Dose-Dependent Changes in Matrix Metalloproteinases and Inflammatory Components as Prognostic Factors for the Progression of Osteoarthritis. Front Pharmacol 2021;12:643605. [Crossref] [PubMed]
16. Yamanashi Y, Ohmichi M, Ohmichi Y, et al. Efficacy of Methotrexate on Rat Knee Osteoarthritis Induced by Monosodium Iodoacetate. J Inflamm Res 2021;14:3247-59. [Crossref] [PubMed]
17. Mankin HJ, Dorfman H, Lippiello L, et al. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg Am 1971;53:523-37.
18. Fleiss JL. Design and analysis of clinical experiments. New York, NY: Wiley; 1999.
19. Ebener MK. Reliability and validity basics for evaluating classification systems. Nurs Econ 1985;3:324-7.
20. Longo UG, Papalia R, De Salvatore S, et al. Induced Models of Osteoarthritis in Animal Models: A Systematic Review. Biology (Basel) 2023;12:283. [Crossref] [PubMed]
21. Oei EH, van Tiel J, Robinson WH, et al. Quantitative radiologic imaging techniques for articular cartilage composition: toward early diagnosis and development of disease-modifying therapeutics for osteoarthritis. Arthritis Care Res (Hoboken) 2014;66:1129-41. [Crossref] [PubMed]
22. Oláh T, Cucchiarini M, Madry H. Temporal progression of subchondral bone alterations in OA models involving induction of compromised meniscus integrity in mice and rats: A scoping review. Osteoarthritis Cartilage 2024;32:1220-34. [Crossref] [PubMed]