Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems

Abstract

Purpose In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems. Methods Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period. Results Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro. Conclusion This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology.

Combining biomechanical stimulation and chronobiology: a novel approach for augmented chondrogenesis?

“…Mechanobiology and chronobiological signaling pathways are closely interconnected during cartilage formation and maintenance. Appropriate mechanical stimuli can serve as external timing cues and entrain the circadian clock in developing ( Vago et al, 2022 ) and mature isolated chondrocytes ( Heywood et al, 2022 ).…”

“…Therefore, dynamic mechanical stimulation served as a Zeitgeber for chondroprogenitor clock entrainment, and chondrogenesis was stimulated through the synchronizing ability of the loading regime. When primary articular chondrocytes were exposed to cyclic biaxial tensile stretch, BMAL1 exhibited a sinusoidal expression pattern at the protein level, and the oscillation parameters of BMAL1 followed a daily rhythm which was mimicked by mechanical stimulation ( Heywood et al, 2022 ). Similarly, the molecular clockwork in human dental pulp-derived MSCs could be entrained following rhythmic uniaxial mechanical stretch ( Rogers et al, 2017 ).…”

Circadian Clock Regulation via Biomaterials for Nucleus Pulposus

“…The mammalian circadian clock is a continuous cycle of 24 h. [47] Periods of 24 h oscillation need to be observed for the circadian clock evaluation. [48][49][50] In order to further prove the regulatory effect of ClockMPs on the circadian clock of NPCs, the gene expression changes of Clock within 72 h were detected by real-time quantitative polymerase chain reaction (RT-qPCR). In the Control group, the expression of Clock reached the peak at 12 h and dropped to baseline at 24 h, forming a regular circadian clock (Figure 4A).…”

Importance of mechanical cues in regulating musculoskeletal circadian clock rhythmicity: Implications for articular cartilage

“…Functionality of physiological systems including cardiovascular, nervous, and musculoskeletal systems rely extensively on their ability to respond to mechanical loading including fluid flow, compression and tensile strain, and adapt their cellular behaviors to elicit appropriate responses. Mechanical loading is involved in regulating circadian rhythmicity in tissues including skeletal muscle (Bae et al, 2006; Saracino et al, 2019; Sasaki et al, 2016; Vanmunster et al, 2022; Wang et al, 2021; Wolff & Esser, 2012; Yamanaka et al, 2008), intervertebral disc (Ding et al, 2021), bone (Bouchard et al, 2022), and cartilage (Gossan et al, 2013; Heywood et al, 2022; Kanbe et al, 2006) which can impact tissue homeostasis. Specifically, extrinsic mechano‐regulation of circadian rhythms can promote extracellular matrix (ECM) synthesis; however, circadian dysregulation results in tissue catabolism and onset of pathology (Dudek et al, 2016; Guo et al, 2015; Kc et al, 2015; Snelling et al, 2016).…”

“…However, Kanbe et al (2006) demonstrated that Clock is a mechanosensitive gene, as transcript levels were significantly downregulated following exposure of mouse chondrocytes to tensile strain (5%, 1 Hz, 15 min/h, 4 days) in a 3D sponge model (Kanbe et al, 2006). Heywood et al (2022) recently demonstrated that daily episodes of tensile strain (10%, 0.33 Hz, 12 h) synchronized the chondrocyte clock over 3 days with BMAL‐1 periodicity aligning with diurnal mechanostimulation (Heywood et al, 2022); introduction of a 6 h phase shift in loading resulted in an equivalent shift of the cartilage clock. Furthermore, Bmal1 , Per2/3 , Cry1 , and Rev‐erb transcription was reported to be induced in embryonic chick limb bud‐derived chondroprogenitor cell micromass cultures subjected to compression (0.6 kPa, 0.05 Hz, 1 h daily, 6 days) concomitant with rhythmic expression of Sox6 , Sox9 , and Acan mRNAs (Vago et al, 2022), indicating that mechanical stimulation can induce chondrogenesis via a clock‐dependent mechanism.…”