Osteoclast counts in subchondral bone correlate with overlying cartilage degradation in equine
carpal joint osteoarthritis
Bertuglia A1, Girard C2, Lacourt M1, Richard H1, Beauchamp G2, Laverty S*1.
1Comparative Orthopaedic Research Laboratory/Département de Sciences Cliniques/Université de Montréal, Saint-Hyacinthe, Canada.
2Département de Pathologie et Microbiologie Vétérinaires/Université de Montréal, Saint-Hyacinthe, Canada
Introduction. Equine osteoarthritis (OA) is a degenerative joint disease caused by repeated trauma to the joint and is characterised by loss of articular cartilage and subchondral bone remodelling. Cartilage and bone structural changes are well described but the biological events in the bone remain poorly understood. In OA, cartilage and subchondral bone cells respond to local biological signals in the matrix and cross talk may occur between these adjacent tissues.
Osteoclasts (OCs) contribute to subchondral bone loss and may also have a role in overlying cartilage degradation. Our recent investigation of third carpal bone OA in racehorses revealed subchondral bone disorganization on micro-CT and a reduction in bone mineral density that was correlated to the severity of overlying cartilage pathology2. We hypothesize that there is a biological inter-play between the subchondral bone and cartilage in spontaneous equine OA mediated by OCs, similar to that recently described in
experimental rheumatoid arthritis1. Our objectives were to quantify OCs in the subchondral bone and relate these counts to overlying cartilage histological changes.
Material and Methods
Osteochondral cores were harvested from third carpal bones, with varying degrees of intercarpal joint OA, of 15 Standardbred racehorses from an abattoir2. Cores were from the dorsum of the bone in areas with most OA lesions. Cores were decalcified, embedded in paraffin and 5 µm sections were stained with HPS, for cellular and morphologic evaluation, and Safranin O-Fast Green for cartilage histology3. Immunohistochemical localization of cathepsin K (cat-K) in the tissue was performed to identify OCs4.Tartrate-resistant acid phosphatase (TRAP) staining was also performed on selected sections. Complete 2D-images of the
osteochondral sections were captured with a digital slide scanner and processed with a commercially available software5.
Bone Histomorphometric analysis
Complete section analysis: Analysis was performed on 200x view digital sections in a minimum of 100 mm2 of representative subchondral bone for each animal, using consecutive thin sections (Figure 1). The bone from the core was digitally extracted to permit independent assessment (n= 30). Total bone area in the histological sections (L x W), Bone area (mineralized area) and Bone Perimeter (BPm) were measured. BPm was measured computing the length of mineralized surface. Equine OCs were defined as multinucleated giant cells (≥ 3 nuclei) on HPS and cat-K+ cells, lying in corresponding resorbing bays. Total OC counts were performed by 2 independent readers. OC count on the HPS and cat-K stained sections were normalized to each of the Total Area (TA) of the bone section, to the Bone Area (BA) and Bone Perimeter (BPm).
Focal analyses within each section: Each osteochondral section was then divided digitally into 1-mm-width and 3-mm-depth (subchondral bone) regions of interest (ROI). An ROI OC count was performed in each ROI (n=120) and the relative ratio between BA/TA (%) for each ROI was calculated. Values for each ROI obtained were pooled together and considered individually for statistical purposes.
Cartilage histological analysis
A modified Mankin scoring system6 was employed to assess cartilage structural changes. The cartilage was digitally removed from the bone section to permit independent assessment (n=15). An independent cartilage histological ROI assessment (n=120) was performed to provide a cartilage degeneration score co-localized to each of the underlying subchondral bone ROIs described above. Inter-observer agreement was assessed for the cartilage score. Microcracks number/mm and tidemark integrity was also assessed.
Statistical analyses
Inter-observer agreement between the OC counts and cartilage histological assessment was evaluated by Bland-Altman test. OC count was associated with the histological cartilage scores and subchondral bone histomorphometric data employing a linear model with the horse Id as a random effect.
Results
Complete section analysis:
The Bland-Altman plots showed strong inter-observer agreeing for OC count and cartilage scores. OC counts on HPS and Cat-K stained sections were also highly correlated, indicating reliability of assessment. Mean ± SD OC density in the sections was 1.19±1.33 mm2 (n=15) with a range of 0-2.98 cells/mm2. Total cartilage scores in the complete sections ranged from 0 to 15.
ROI analyses:
OC counts ranged from 0 to 8.75 cells/mm2 and cartilage histological scores from 0 to 23 in the ROIs (n=120). The OC counts in ROIs were positively correlated to the cartilage degeneration scores (p<0.001). A focal increased of the OC density is co-localized with the highest inter-individual cartilage score ROIs (p=0.002). The OC counts were highly correlated to microcrack number/mm (p<0.001) and tidemark destruction (p<0.001). An increased OCs density was negatively correlated with the BA/TA in the subchondral bone (n=120) (Fig.1).
Taken together these results suggest that OCs are major players in bone destruction in OA and may also contribute to calcified and hyaline cartilage degeneration in OA.
Conclusion. Our results support the hypothesis that OCs are recruited in the subchondral bone during
progression of spontaneous carpal OA and may participate in and, potentially precipitate, degradation of deep regions of articular cartilage.
References
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