Abstract
The circadian clock contains clock genes including Bmal1 and Period2, and it maintains an interval rhythm of approximately 24 hours (the circadian rhythm) in various organs including growth plate and articular cartilage. As endochondral ossification is involved not only in growth plate but also in fracture healing, we investigated the circadian clock functions in fracture sites undergoing healing. Our fracture models using external fixation involved femurs of Period2::Luciferase knock-in mice which enables the monitoring of endogenous circadian clock state via bioluminescence. Organ culture was performed by collecting femurs, and fracture sites were observed using bioluminescence imaging systems. Clear bioluminescence rhythms of 24-hour intervals were revealed in fracture healing sites. When parathyroid hormone (PTH) was administered to fractured femurs in organ culture, peak time of Period2::Luciferase activity in fracture sites and growth plates changed, indicating that PTH-responsive circadian clock functions in the mouse femur fracture healing site. While PTH is widely used in treating osteoporosis, many studies have reported that it contributes to improvement of fracture healing. Future studies of the role of this local clock in wound healing may reveal a novel function of the circadian timing mechanism in skeletal cells.
Highlights
We showed that molecular clocks maintain circadian rhythms in fracture healing sites by using our external fixator
By real-time monitoring of Per2::Luc activity, clear circadian rhythms were observed in both growth plates and fracture sites from Per2::Luc mice
Per2::Luc activity in the fracture sites and growth plates from the Per2::Luc, Bmal1−/− mice were arrhythmic. These results indicate that Bmal1-dependent tissue-autonomous circadian clocks harbor in the growth plate and in the fracture healing site
Summary
Callus formation was observed at the fracture site on x-ray images from post-surgical day 14 (Fig. 1F). Strong bioluminescence signals were observed in fracture sites, in growth plates and around the screws when the femoral bones of the Per2::Luc mice were isolated with the external fixator at post-surgical day[14] (Fig. 2A,C). When bioluminescence imaging was performed every hour, clear circadian rhythms were observed in these parts for at least 6 days (Fig. 2D,E).
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