3D spatial distribution of Sost mRNA and sclerostin protein expression in response to in vivo tibia loading in female mice

Bones adapt to external mechanical loads through a process known as mechanoadaptation. Osteocytes are the bone cells that sense the mechanical environment and initiate a biological response. Investigating the changes in osteocyte molecular expression following mechanical loading has been instrumental in characterizing the regulatory pathways involved in bone adaptation. However, current methods for examining osteocyte molecular expression do not preserve the three-dimensional structure of the bone, which plays a critical role in the mechanical stimuli sensed by the osteocytes and their spatially controlled biological responses. In this study, we used WISH-BONE (Whole-mount In Situ Histology of Bone) to investigate the spatial distribution of Sost-mRNA transcripts and its encoded protein, sclerostin, in 3D mouse tibia midshaft following in vivo tibia loading. Our findings showed a decrease in the percentage of Sost-positive osteocytes, after loading, along the posterior-lateral side of the tibia. The number of sclerostin-positive osteocytes were found to significantly decrease at a very specific 2D location of the tibia after loading. However, in 3D, the total number of sclerostin-positive osteocytes was similar between loaded and control legs. This work is the first to provide a 3D analysis of Sost and sclerostin distribution in loaded versus contralateral mouse tibia midshafts. It also highlights the importance of the bone region analyzed and the method utilized when interpreting mechanoadaptation results. WISH-BONE represents a powerful tool for further characterization of mechanosensitive genes regulation in bone and holds the potential for advancing the development of new treatments targeting mechanosensitivity-related bone disorders.