Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Summary

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Abstract

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

Introduction

Osteoarthritis (OA), characterized radiographically by joint space narrowing due to the loss of articular cartilage, osteophytes, and subchondral bone (SCB) sclerosis, is the most common form of arthritis^1,2. Although the role of peri-articular bone in the etiology of OA is not fully understood, osteophyte formation and SCB sclerosis are generally thought to be the results of the disease process rather than causative factors, but changes in peri-articular bone architecture/shape and biology may contribute to the development and progression of OA^3,4. Th....

Protocol

All animal procedures included in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kansas Medical Center, in compliance with all federal and state laws and regulations.

1. Creation of knee OA in mice

1. Create a mouse model of knee OA by surgical destabilization of the medial meniscus (DMM) as described by Glasson et al.¹⁸ in 22 wild-type BALB/c mice at 10-11 weeks of age. Perform sham surgery as a control procedure on eight mice with the same background and age.
   NOTE: Both sexes were used for the original project to meet the ....

Results

Reproducibility comparison between visual estimate grading and ImageJ-assisted quantitative measurement:
SCB thickness in 48 regions of interest (ROI) (24 MFC and 24 MTP), defined from a mid-section of each knee from 24 knees/animals was scored by three independent individuals using the existing 0-3 visual scoring scheme as described in the literature^15,23, where 0 = normal (no SCB thickening), 1 = mild, 2 = moderate, and 3 = severe SCB thi.......

Discussion

Measuring SCB thickening is an important component of histologic assessments of OA severity. Most existing OA grading systems focus mainly on cartilage changes^15,16,17. A widely used murine osteoarthritic SCB thickness grading method that categorizes SCB thickening as mild, moderate, and severe is largely subjective, and its reliability has not been fully validated¹⁵. The present study has developed and v.......

Materials

Name	Company	Catalog Number	Comments
Safranin-O	Sigma-Aldrich	S8884
Fast green	Sigma-Aldrich	F7252
Hematoxylin	Sigma-Aldrich	GHS216
Eosin	Sigma-Aldrich	E4382
illustrator	Adobe	Not applicable