Fibroblasts are found in healthy tissue, where they function to maintain the extracellular matrix (ECM), however they also play a pivotal role in the process of wound healing. Once a wound has occurred, fibroblasts are summoned to the damaged area by chemoattractants including PDGF, IL-1β and TNF-α; upon reaching the wound site they support healing through their involvement in processes such as breakdown of the fibrin-containing clot, creation of new ECM, and contraction of the wound.
Although researchers have been aware for decades of the role played by fibroblasts in the wound healing process, a new study published in Science Translational Medicine has provided further proof that these specialised cells exhibit circadian timekeeping, allowing them to function to a rhythmic cycle of approximately 24 hours.
The importance of maintaining a regular circadian rhythm has been the subject of increasing attention recently, with mounting evidence to suggest that disruption to this cycle contributes to conditions including diabetes, cancer and cardiovascular disease, yet the importance of circadian timekeeping at a cellular level requires considerably further understanding.
The authors of this study, Hoyle et al, performed a sophisticated series of experiments to reveal that the time of day at which a wound is sustained has a significant effect on the length of time which is taken for healing. Initially they carried out a proteome-wide screen in extracts from primary fibroblast cultures collected across two circadian cycles, and of 1608 proteins identified over the time course, the abundance of 237 of these changed in accordance with the circadian rhythm.
A significant proportion of these 237 proteins were identified as cytoskeletal regulators, specifically actin regulators, and the team next assessed the polymeric state of actin over two circadian cycles in fibroblast cultures. Through the use of two independent methods they showed the actin polymeric state to be dependent on cycling clock gene activity.
The impact of actin cytoskeletal rhythms on cell migration was next evaluated by employing wound healing assays on synchronised layers of immortalised skin fibroblasts. Wounds were generated at different phases of the circadian cycle, according to expression levels of the circadian oscillator PER2, and while wounds inflicted at the lowest point of PER2 expression demonstrated minimal healing after 16 hours recovery time, wounding at peak PER2 expression resulted in almost complete healing of cell monolayers after the same recovery period.
The team then assessed fibroblast mobilisation in an ex vivo mouse model. Skin explants were harvested at different points of the circadian cycle and were subsequently wounded by biopsy punch before being mounted on to culture membranes. Migration of fibroblasts into the wounds was assayed by immunofluorescence, with approximately twice as many fibroblasts shown to migrate into the wound area of explants collected during the active phase than into that of explants collected during the resting period.
Finally Hoyle et al analysed historic data from the international Burn Injury Database, and calculated the time taken for these wounds to heal to 95%; healing times were approximately 60% longer in the case of burns sustained during the night as compared to those incurred during the day.
This study provides clear evidence to suggest a link between wound healing and the circadian clock, which has the potential to improve surgery, for example by performing operations in synchrony with the circadian rhythm of the patient, or administering wound closure therapies at a particular time of day to increase their effectiveness.