A microCT imaging protocol for reproducible and efficient quantitative morphometric analysis (QMA) of joint structures of the in situ mouse tibio-femoral joint.

Micro-computed tomography (microCT) offers a three-dimensional (3D), high-resolution technique for the visualisation and analysis of bone microstructure. Using contrast-enhanced microCT, this capability has been expanded in recent studies to include cartilage morphometry and whole joint measures, known together as quantitative morphometric analysis (QMA). However, one of the main challenges in quantitative analysis of joint images is sensitivity to joint pose and alignment, which may influence measures related to both joint space and joint biomechanics. Thus, this study proposes a novel microCT imaging protocol for reproducible and efficient QMA of in situ mouse tibio-femoral joint. This work consists of two parts: an in situ diffusion kinetics study for a known cationic iodinated contrast agent (CA4+) for QMA of the cartilage, and a joint positioning and image processing workflow for whole joint QMA. In the diffusion kinetics study, 8 mice were injected at both of their tibio-femoral joints with distinct CA4+ concentrations and diffusion times. The mice were scanned at different time points after injection, and evaluated using attenuation and cartilage QMA measures. Results show that cartilage segmentation and QMA could be performed for CA4+ solution at a concentration of 48 mg/ml, and that reliable measurement and quantification of cartilage were achieved after 5 min of diffusion following contrast agent injection. We established the joint positioning and image processing workflow by developing a novel positioning device to control joint pose during scanning, and a spherical harmonics-based image processing workflow to ensure consistent alignment during image processing. Both legs of seven mice were scanned 10 times, 5 prior to receiving CA4+ and 5 after, and evaluated using whole joint QMA parameters. Joint QMA evaluation of the workflow showed excellent reproducibility; intraclass correlation coefficients ranged from 0.794 to 0.930, confirming that the imaging protocol enables reproducible and efficient QMA of joint structures in preclinical models, and that contrast agent injection did not cause significant alteration to the measured parameters.