BIOMECHANICAL STIMULI AUGMENT CHONDROGENESIS BY RE-SETTING THE BIOLOGICAL CLOCK

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During cartilage formation, chondroprogenitor cells differentiate into chondrocytes, and produce cartilage-specific extracellular matrix in response to internal or external factors. One of the most relevant external factors in this context is the biomechanical environment. Although mechanical cues play a key role in regulating cartilage formation, current understanding of mechanotransduction pathways in chondrogenic cells is incomplete. Our laboratory has previously shown that temporal cues control chondrogenesis via the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock. Therefore, the aim of this work was to study whether mechanical stimuli synchronise the circadian clock in chondrogenic cells. We used micromass cultures established from the distal limb buds of 4-day-old chicken embryos to set up chondrogenic cultures. Cultures were exposed to uniaxial cyclic compressive force (0.6 kPa, 0.05 Hz) on each culturing day between days 1 and 6 for 60 min using a custom- made mechanical stimulator unit. Samples were harvested every 8 hours between 24 and 72- hours post-stimulation after day 6. Total RNA was isolated, reverse transcribed into cDNA, and the transcript levels of clock genes and chondrogenic marker genes were monitored using RT- qPCR. Cartilage ECM production was studied using metachromatic staining. Cyclic uniaxial mechanical load synchronised the molecular clock in chondrogenic cells in limb bud-derived micromass cultures. In addition to the clock genes, the chondrogenic markers SOX6, SOX9, and ACAN also followed a sinusoidal rhythmic expression pattern. In parallel with this, we observed augmented matrix production following mechanical load. Our results suggest that an optimal biomechanical environment enhances chondrogenesis through synchronising the molecular clock. This novel chondrogenesis-promoting approach should be considered in future cartilage tissue regeneration methods.

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cartilage, biomechanical stimuli, circadian clock, synchronisation, chondrogenesis
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