Circadian rhythm in cartilage mechanobiology

Dintwa, Lekau 2024. Circadian rhythm in cartilage mechanobiology. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Introduction: Cartilage resident cells, chondrocytes are responsible for sustaining tissue homeostasis by maintaining a balance between biosynthesis and degradative activities in response to mechanical loading and other cues. Previous studies revealed that mechanical loading is imperative for normal cartilage function with physiological loads maintaining tissue homeostasis; in contrast, non-physiological loads, i.e., static, insufficient or excessive, induces maladaptive chondrocyte responses, shifting metabolism towards inflammation and extracellular matrix (ECM) catabolism leading to cartilage deterioration and promoting the development of osteoarthritis (OA). OA is a prevalent joint disease characterised by progressive degeneration of articular cartilage, synovial inflammation and bone remodelling. There is currently no disease modifying treatment for OA as available modalities mostly alleviate symptoms. Previous studies identified circadian rhythm as one of the most dysregulated pathways in human OA and other model systems. Circadian rhythm is mediated by circadian clocks which are endogenous cellular oscillators with an ~24-hour rhythmic cycle ensuring that organisms can predict and align their physiology to the daily variations of light and temperature. In mammals, circadian clocks are organised hierarchically with the master clock located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus in the brain. However, articular cartilage and other peripheral tissues possess subordinate clocks. More recently, studies have suggested that non-photic zeitgeber including exercise also entrains circadian rhythmicity. However, at the start of this PhD, very few studies had been performed on mechano-regulation of circadian clocks, particularly in cartilage. Therefore, the aim of the PhD was to investigate whether (i) bovine primary chondrocytes subjected to mechanical load (simulated using centrifugal force) would exhibit an altered circadian rhythm, and (ii) whether mechanical load could reset a disrupted circadian rhythm in an in vitro model of inflammatory OA. Results: This PhD developed and characterised various in vitro 3D pellet models for studying chondrocyte mechanobiology including chondrocyte pellets, with or without pericellular matrix (PCM), exposed to centrifugal force predicted to simulate the compressive, hydrostatic and shear forces observed in vivo. In the absence of PCM, 15- and 60-minutes of centrifugation induced transcription of early mechano-responsive genes c-fos and c-jun, late mechano-responsive catabolic genes adamts-4, adamts-5 and mmp-3, and the chondrocyte phenotype gene marker sox9. However, delayed transcription of the early and late mechano-responsive genes was observed in 3D pellets with PCMsubjected to 15 minutes of load (770xg force). Overall, a 770xg force induced robust mechano-signalling as compared to the 200- and 1000-xg centrifugal forces and 60 minutes mediated stronger responses as evidenced by higher transcript levels of select early and late response genes as compared to the 15 minutes regimen; thus, this regimen (60 minutes of 770xg force) was utilised in subsequent loading experiments. Overall, application of 60 minutes of load induced quicker transcription of the early and late mechano-responsive genes, but dampened the transcription of sox9, delayed adamts-5 transcription and did not alter maximal induction of mmp-3. Assessment of the clock genes in control pellets with no PCM demonstrated rhythmic expression of bmal1, clock, cry1, dbp, npas2, nr1d1, nr1d2, per1 and per2, and the ECM catabolic gene adamts-4 in at least one LD cycle; however, catabolic genes including adamts-5, mmp-3 and mmp-13, and anabolic genes acan and col2a1, and the chondrocyte phenotype gene marker sox9 were not expressed in a rhythmic manner. Circadian expression of bmal1, clock (though a transient disruption was observed only in the first LD cycle) and per2 mRNAs was unaffected in 2 consecutive LD cycles following application of one episode of 770xg force (60 minutes) in the absence and/or presence of PCM whilst acan and col2a1 demonstrated arrhythmicity in both the controls and loaded pellets with or without PCM. However, adamts-4 mRNA rhythmicity in pellets with no PCM was sustained following application of a single episode of load while no circadian oscillations in adamts-4 mRNA were observed in both controls and loaded (a single episode of 770xg force) pellets with PCM. This PhD also utilised an inflammatory OA model, with exposure of 3D pellets with no PCM to 5 ng/ml IL-1α/β in combination with 10 ng/ml OSM; as expected, proinflammatory cytokine stimulus reduced expression of acan and sox9 and increased expression of adamts-4, adamts-5, mmp-3 and mmp-13 at T24 hours; this was concomitant with disruption of circadian rhythmicity of bmal1, clock, cry1, npas2, nr1d1, nr1d2, per1 and per2 genes. Application of dexamethasone, a known anti-inflammatory and clock synchronisation agent, to cytokine-treated cultures synchronised bmal1 and per2 transcription concomitant with the return of acan and mmp-3 mRNA transcripts to control levels at T24 hours. However, application of 770xg force (60 minutes) either once daily for 3-days, twice daily for 1-day or twice daily for 2-days did not reset the cytokine-induced disrupted circadian rhythms of clock gene transcription (bmal1, npas2, per2) and did not return the cytokine-induced reductions in anabolic gene transcription (acan, col2a1) and cytokine-induced increases in catabolic gene transcription to control levels at T24 hours (mmp-3, adamts-4). Conclusion: This thesis demonstrates the capacity of centrifugal force to simulate mechanical load in 3D chondrocyte pellets with or without PCM with 60 minutes of 770xg force inducing stronger mechano-signalling suggestive of its potential suitability for utilisation in mechanobiology investigations though loading regimen may need further optimisation. A single episode of 770xg load did not alter the circadian rhythm of clock gene expression (except for a transient disruption of clock rhythmicity in the first LD cycle in pellets with no PCM) nor a gene marker of a catabolic phenotype in 3D pellets with/without PCM suggesting that the employed load regimens had no impact on circadian clocks in healthy cartilage. This study also corroborates the capacity of proinflammatory cytokines to disrupt circadian clocks in primary chondrocytes and the capability of dexamethasone to reverse this inflammatory phenotype by synchronising these cytokine-disrupted circadian clocks. However, the inability of mechanical load (770xg force either once daily for 3-days, twice daily for 1-day or twice daily for 2-days) to reset circadian oscillations of clock genes in cytokine-treated pellets suggest that these loading regimens have no effect on disrupted circadian clocks, thus they possess no clock synchronisation properties. Moreover, the inability of mechanical load to reverse the inflammatory state of cytokine-treated cultures suggests that these load regimen does not possess anti-inflammatory properties and requires further optimisation. Overall, the findings of this thesis may aid refinement of future studies aimed at exploring mechano-regulation of the cartilage clock to identify potential therapeutic mechanical loading regimens and optimal times for administering exercise or physiotherapy to patients suffering from OA.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	13 May 2025
Last Modified:	13 May 2025 18:23
URI:	https://orca.cardiff.ac.uk/id/eprint/178258

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