Influence of fixation on CA4+ contrast enhanced microCT of articular cartilage and subsequent feasibility for histological evaluation.

CA4+ is a novel cationic iodinated contrast agent utilized for contrast-enhanced microCT (CECT). In this study, we compared CA4+ CECT for cartilage quantification of unfixed and neutral buffered formalin (NBF)-fixed rabbit distal femur cartilage after 8-, 24- and 30-hours of contrast agent diffusion. The stability of CA4+ binding to cartilage after PBS soak and decalcification was also investigated by CECT. We further assessed the feasibility of cartilage histology and immunohistochemistry after CA4+ CECT. Contrast-enhanced CA4+ labeled unfixed and NBF-fixed cartilage tissues facilitate articular cartilage quantification and accurate morphological assessment. The NBF fixed tissues demonstrate higher cartilage intensity and imaging characteristics distinct from subchondral bone than unfixed tissues while maintaining stable binding even after decalcification with 10% EDTA. The unfixed tissues labeled with CA4+, after CECT imaging and decalcification, are amenable to H&E, Alcian blue, and Safranin O staining, as well as Col2 immunohistochemistry. In contrast, only H&E and Alcian blue staining can be accomplished with CA4+ labeled NBF fixed cartilage, and CA4+ labeling interferes with downstream immunohistochemistry and Safranin O staining, likely due to its positive charge. In conclusion, CA4+ CECT of NBF fixed tissues provides high quality microCT cartilage images and allows for convenient quantification along with feasible downstream H&E and Alcian blue staining after decalcification. CA4+ CECT of unfixed tissues enables researchers to obtain both quantitative microCT as well as cartilage histology and immunohistochemistry data from one set of animals in a cost-, time-, and labor-efficient manner.