3D spatial distribution of Sost mRNA and Sclerostin expression in response to in vivo mechanical loading

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 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 predominantly at 25% and 37% of the bone length, and in the posterior-lateral side of the tibia after loading. Sclerostin-positive osteocytes in the loaded legs were found to be similar to the contralateral legs after 2 weeks of loading. 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 potential for advancing the development of new treatments targeting mechanosensitivity-related bone disorders.