At the outset of this fellowship, healthy and OA chondrocytes were characterised in culture. This analysis established that baseline expression of healthy anabolic markers was decreased and expression of OA-associated degenerative enzymes (catabolic enzymes) and inflammatory markers was increased in OA chondrocytes compared to healthy chondrocytes. Furthermore, when challenged with a proinflammatory cytokine (IL-1β), the response of OA cells was exaggerated and prolonged compared to healthy cells. Cx43 expression was increased in OA-cells and in response to IL-1β. Next, we determined that through small molecule inhibition of Cx43 channel activity, negative cell responses to IL-1β could be attenuated. This indicated a role for Cx43 in cell responses to cytokine stimulation, to investigate this further we then examined the effects of siRNA knockdown of Cx43. In culture, Cx43 knockdown was observed to increase the baseline expression of healthy chondrocyte markers and decrease the expression of catabolic enzymes. Furthermore, knockdown was able to attenuate responses to IL-1β. Interestingly, cell metabolic responses to IL-1β stimulation were observed to be divergent in cells treated with Cx43 channel inhibitor and Cx43 siRNA, indicating that alternative modes of action may be present.
In other systems Cx43 interacts with yes-associated protein (YAP), an important mechanosensitive molecule, sequestering it to the cytoplasm. Meanwhile, YAP has been demonstrated to be an important regulator of nuclear factor-κB (NF-κB) activity in chondrocytes, an important transcription factor in cell inflammatory responses. We determined that Cx43 interacts with YAP in chondrocytes at the protein level and that increased Cx43 expression coincided with decrease nuclear YAP and increased nuclear P65 (a subunit of NF-κB; nuclear translocation indicates activation).
With a view to developing these findings towards translation, we then optimised a non-viral cell penetrating peptide, the glycosaminoglycan enhanced-binding transfection (GET) peptide, for siRNA delivery. As mesenchymal stem cells (MSCs) are commonly used as a therapeutic cell population for cartilage regeneration, we confirmed that GET-siRNA nanoparticles were able to knockdown Cx43 expression in 3D chondrogenic pellet cultures of MSCs and chondrocytes. In either cell type, this knockdown did not negatively affect the expression of healthy chondrocyte markers and as seen in 2 D cultures, was able to attenuate responses to IL-1β, including increasing the presence of cartilage-like extracellular matrix production in these cultures.
Next, we aimed to combine this GET-siRNA technology with a regenerative collagen and hyaluronic scaffold to create an siRNA-activated scaffold system for cartilage regeneration. GET-siRNA nanoparticles were incorporated into regenerative scaffolds and were demonstrated to effectively knockdown Cx43 expression in both chondrocytes and MSCs. Like in previous pellet culture experiments, this knockdown was again effective at improving cartilage-like extracellular matrix deposition in the presence of IL-1β. The applicability of this system to then deliver this GET-siRNA cargo in vivo and to penetrate the dense extracellular matrix of cartilage tissue was tested through explant cartilage cultures. In these models, it was demonstrated that fluorescently-tagged GET-siRNA nanoparticles were able to penetrate into the cartilage tissue and co-localised with tissue resident chondrocytes.
