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Objectives and questions: Accumulating evidence demonstrates that circadian rhythms are present in nearly all tissues of the human body, regulating key physiological processes in synchrony with the environmental day–night cycle. These rhythms are entrained by external cues such as light exposure, food intake, and physical activity. Disruption of circadian timing, as observed in shift work, has been associated with skeletal disorders including osteoporosis and fractures. Rhythmic gene expression has been demonstrated in murine calvarial bone. However, comparable analyses in long bones, particularly those distinguishing between specific bone compartments, are still lacking. Therefore, this prospective experimental animal study investigated circadian gene expression in the murine femoral shaft and growth plate.

Material and methods: The circadian rhythm in the femoral shaft and growth plate of wild-type mice was characterized by gene expression analysis at six time points over a 24-hour period. For each time point six female 12-week-old C57BL/6J mice that were maintained under a 12 hours light/dark cycle were used. Total RNA extracted from the femoral shaft and growth plate was analyzed by transcriptome profiling in parallel with quantitative PCR performed on synthesized cDNA for specific target genes. Rhythmicity was tested using the well-established JTK_CYCLE algorithm implemented in R.

Results: Gene expression analysis of wild-type femur and growth plate showed a robust rhythmic regulation of circadian core clock genes (Bmal1, Per2, Cry1) with peaks and troughs comparable to previous findings in the calvaria. Analysis of bone-specific genes demonstrated that markers of bone formation (Alpl, Runx2, Bmp2) as well as bone resorption markers (Rankl, Ctsk, Fos, Acp5) were predominantly expressed during the light phase, corresponding to the resting phase in mice. Comparative analysis between the femoral shaft and growth plate revealed subtle differences in rhythmic regulation. Notably, the amplitude of rhythmically expressed genes was particularly pronounced in the growth plate, suggesting a strong influence of circadian regulation on chondrocyte function and cartilage homeostasis.

Discussion and conclusions: Our findings demonstrate that circadian rhythms are robustly maintained in distinct compartments of murine long bone, including the femoral shaft and growth plate. The rhythmic expression of core clock genes and bone remodeling markers indicates that bone formation and resorption are temporally coordinated processes, predominantly occurring during the resting phase in mice. The pronounced rhythmic amplitude observed in the growth plate further suggests a strong circadian influence on chondrocyte activity and cartilage homeostasis. Together, these results highlight the importance of temporal regulation in maintaining skeletal integrity and provide a foundation for future chronobiological approaches aimed at improving prevention and treatment strategies for bone disorders associated with circadian disruption.