University of Twente Student Theses
Assessing mechanobiology in articular chondrocytes using an Organ-on-Chip platform. Investigating the influence of dynamic compression on YAP/TAZ activation.
Wijngaarden, R. (2023) Assessing mechanobiology in articular chondrocytes using an Organ-on-Chip platform. Investigating the influence of dynamic compression on YAP/TAZ activation.
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| Abstract: | Osteoarthritis (OA) is one of the most prevalent degenerative joint diseases in the Netherlands associated with pain and disability and the incidence is expected to rise rapidly. No treatment is available yet due to a lack of knowledge about the initiation and progression of the disease, as well as a representative research model. Physiological joint loading is necessary for matrix homeostasis and has proven beneficial for attenuating cartilage degradation during OA progression. The mechanobiology behind this could provide invaluable information about homeostatic processes and reveal potential targets for therapeutics, however it is not well understood yet. YAP/TAZ (YAP) transcriptional co-activators have shown potential in attenuating cartilage degradation in OA tissues, but controversy exists about the mechanism. This thesis investigates YAP activity upon dynamic compression in normal and pathological conditions, to eludicate part of the mechanobiology in chondrocytes. A Cartilage-on-Chip platform was used to deliver physiological dynamic compression (300mbar, 1 Hz, 1hr/day) onto human primary chondrocytes embedded in agarose. YAP and SMAD protein nuclear translocation was measured using immunofluorescence to estimate the nuclear-to-cytoplasmic ratio over time and in addition of pro-inflammatory cytokines IL-1β and TNF-α. Additionally, gene expression was measured of matrix components and targets downstream of YAP-SMAD2/3 and YAP-SMAD1/5/9 complexes. The results reveal that YAP, SMAD2/3 and SMAD1/5/9 nuclearisation does not increase upon physiological dynamic compression. CTGF was however upregulated, showing some influence of compression on the transcriptional activity of YAP. Nuclear YAP and SMAD2/3 increased upon addition of cytokines, while compression attenuated the nuclearisation of SMAD2/3. In contrast, nuclear SMAD1/5/9 only increased upon addition of cytokines in combination with compression. Downregulation of CTGF and Pai-1 in both pathological conditions demonstrates the influence of inflammation on the transcriptional activity of YAP-SMAD2/3. Furthermore, compression in pathological condition presents attenuated upregulation of MMP13, which could potentially be related to increased nuclear YAP. This thesis concludes no direct influence of dynamic compression on YAP phosphorylation and nuclear translocation, however an influence on transcriptional activity of YAP is suggested by the data. This research could be continued by investigating the role of YAP in Extracellular Matrix (ECM) development or homeostasis, using YAP inhibitors and stimulators to regulate YAP expression and activity. Future experiments should increase the biological sample size and also include a physiologically relevant ECM to assess the role of mechanotransduction on YAP activation. The findings of this thesis show the possibility to investigate mechanobiology in the Cartilage-on-Chip platform, which could be applied to other signalling pathways involved in matrix homeostasis and potentially study the development of OA. |
| Item Type: | Essay (Master) |
| Faculty: | TNW: Science and Technology |
| Subject: | 42 biology |
| Programme: | Biomedical Engineering MSc (66226) |
| Link to this item: | https://purl.utwente.nl/essays/94300 |
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