Comparison of two contrast-enhancing staining agents for use in X-ray imaging and digital volume correlation measurements across the cartilage-bone interface

Objective: The pathogenesis of osteoarthritis (OA) is associated with subchondral bone changes, which is linked to abnormal strain distribution in the overlying articular cartilage. This highlights the importance of understanding mechanical interaction at the cartilage-bone interface. The aim of this study is to compare solutions of two contrast-enhancing staining agents (CESA) for combining high-resolution Contrast-Enhanced X-ray microfocus Computed Tomography (CECT) with Digital Volume Correlation (DVC) for full-field strain measurements at the cartilage-bone interface.

Design: Bovine osteochondral plugs were stained with phosphotungstic acid (PTA) in 70% ethanol or 1:2 hafnium-substituted Wells-Dawson polyoxometalate (Hf-WD POM) in PBS. Mechanical properties were assessed using micromechanical probing and nanoindentation. Strain uncertainties (from CECT data) were evaluated following two consecutive unloaded scans. Residual strains were computed following unconfined compression (ex situ) testing.

Results: PTA and Hf-WD POM enabled the visualisation of structural features in cartilage, allowing DVC computation on the CECT data. Residual strains up to ∼10,000 μɛ were detected up to the tidemark. Nanoindentation showed that PTA-staining caused an average ∼6-fold increase in articular cartilage stiffness, a ∼19-fold increase in reduced modulus and ∼7-fold increase in hardness, whereas Hf-WD POM-stained specimens had mechanical properties similar to pre-stain tissue. Micromechanical probing showed a 77% increase in cartilage surface stiffness after PTA-staining, in comparison to a 16% increase in stiffness after staining with Hf-WD POM.

Conclusion: Hf-WD POM is a more suitable CESA solution compared to PTA for CECT imaging combined with DVC as it allowed visualisation of structural features in the cartilage tissue whilst more closely maintaining tissue mechanical properties.