Cryogenic non-invasive 3D X-ray phase-contrast imaging of unfixed, intact mouse joints reveals shifting chondrocyte hypertrophy across the endochondral interface

Objectives: i) develop and use a new cryogenically-enhanced phase contrast method to visualise hyaline articular cartilage (HAC); ii) to measure HAC, articular calcified cartilage (ACC) and total articular cartilage thicknesses in male STR/Ort (osteoarthritis, OA) and CBA (healthy) mouse tibial epiphyses, reflecting divergent OA predisposition, at three age timepoints chosen to reflect pre-OA, OA onset and late- progression; iii) to compare HAC, trans-zonal and ACC 3D chondrocyte anatomy in tibial epiphyses. Methods: STR/Ort and CBA mouse knees (n=4 per age and strain group) were synchrotron-CT scanned at high-resolution while fresh frozen, without staining, fixation, dissection or dehydration of the joint capsule. Both cartilage thickness and cellular characteristics (chondrocyte n=420) were manually measured and statistically compared (SPSS). Results: Cryo-enhanced phase contrast allowed cartilage to be seen in full thickness with cellular detail. HAC was thicker in STR/Ort than age-matched CBA mice in 16/24 knee joint compartments and timepoints (all p<0.04). In contrast, HAC was thicker only in the posterior lateral femur of CBA mice at 10weeks (p<0.001, Table 1). ACC and total cartilage were also thicker in STR/Orts. Trans-zonal chondrocytes were smaller than ACC and HAC chondrocytes (p-values<0.001, volumes 878 um3, 1,567 um3 and 1,348 um3 respectively). Conclusions: Cryogenically-enhanced phase-contrast imaging allowed cellular detail to be seen in 3D as never before in HAC in this (or any other) model. Our findings challenge current understanding by associating STR/Ort OA vulnerability with regions of thick, rather than thinning-with-age, cartilage. Our data affirm an association between excessively hypertrophic chondrocytes and OA is present in STR/Ort mice. ### Competing Interest Statement The authors have declared no competing interest.