Establishment of a uniform histological evaluation method for early stage osteophytes in the destabilization of the medial meniscus mouse model

Abstract

Background: Osteophyte formation is attracting attention as an early-stage pathology of knee osteoarthritis (OA). Although osteophyte formation is understood as a defense response to joint instability, its role and impact on OA remain largely unknown. Many studies have been conducted using the surgical destabilization of the medial meniscus (DMM) mouse model, but there are few standard evaluation methods, especially in the histological evaluation of early-stage osteophytes. The purpose of this study was to establish a reproducible and uniform method for histological evaluation of characteristics of early osteophyte formation in the DMM mouse model.

Methods: Male mice were operated with DMM at 12 weeks old and histologically evaluated at 4 days and 1, 2 and 4 weeks after DMM. Osteophyte Width, Osteophyte Area, and Original and Modified Maturity Scores were used to evaluate osteophytes for all sections.

Results: Osteophyte Width, Osteophyte Area and Maturity Scores were all greater anteriorly than posteriorly in the knee joint. The Modified Maturity Score was more strongly correlated with position than the Original Maturity Score, and could be used to evaluate early-stage osteophyte formation.

Conclusion: The Modified Maturity Score as well as Osteophyte Width and Area at the section of the anterior cruciate ligament (ACL) attachment site can provide a reproducible evaluation method to histologically assess the early-stage osteophyte formation in the DMM mouse model.

Keywords: DMM mouse model; Osteoarthritis; Osteophyte; Osteophyte maturity score; Synovium.

Fig. 1

Sectioning of the mouse knee. A, Sectioning range and width between each section. B, The starting point of sectioning stained with Safranin O/Fast green and hematoxylin. The starting point is where the medial articular surface appears. C, The ending point of sectioning stained with Safranin O/Fast green and hematoxylin. The ending point is where the tibial attachment of the posterior cruciate ligament appears. A. Scale bar: 500 ​μm. B, C. Scale bar: 200 ​μm.

Fig. 2

Evaluation methods for osteophytes. A, Osteophyte Width was the maximum transverse diameter parallel to the articular surface of the osteophyte. B, Osteophyte Area was measured by demarcating the outer edge of the osteophyte using Image J. C, The measurement scheme and representative histology for the Modified Maturity Score. Scale bar: 200 ​μm.

Fig. 3

Measurement results for Osteophyte Width and Area in histological coronal sections of the whole knee. A, Correlations between distance from the starting point and Osteophyte Width (n ​= ​5). B, Correlations between distance from the starting point and Osteophyte Area (n ​= ​5). Dots indicate data points for each animal at each distance. d: days, w: weeks.

Fig. 4

Osteophyte maturity scores for histological coronal sections of the whole knee. A, Correlations between distance from the starting point and Original Maturity Score of the four grades reported by Little et al. (n ​= ​5). B, Correlations between distance from the starting point and our Modified Maturity Score of the six grades (n ​= ​5). Dots indicate data points for each animal at each distance. d: days, w: weeks.

Fig. 5

Changes over time in Osteophyte Width, Area, Original Maturity Score, and Modified Maturity Score. The Original Maturity Score is the four grades score reported by Little et al. and the Modified Maturity Score is our six grades score. Dots indicate data points for each animal. The respective values from anterior to posterior were summed and plotted. The data are expressed as means ​± ​SD. d: days, w: weeks.

Fig. 6

Coronal section of mouse knee 4 days and 1, 2, and 4 weeks after DMM. Arrows indicate incisura of the tibial attachment of the anterior cruciate ligament. Scale bars: 200 ​μm. d: days, w: weeks, DMM: destabilization of the medial meniscus.