## 4 Discussion
It must be noted, however, that in all these previous studies cells have been exposed to concentrations of IL-1\(\beta\) and TNF-\(\alpha\) of the order of ng/mL, which is supraphysiological even for pathological conditions such as the more acute stages of degeneration of the IVD. In a recent study from [PERSON] et al., biopsy specimens from surgically excised discs from patients diagnosed with acute disc disease were shown to contain amounts of IL-1\(\beta\) and TNF-\(\alpha\) in the range of 113-135 pg/mL and 9-26 pg/mL, respectively [29], which are more than three orders of magnitude lower than the concentrations used in previous experiments. Our results suggest that when cell cultures are supplemented with IL-1\(\beta\) and TNF-\(\alpha\) at physiologically relevant concentrations, cellular responses are not as marked as described in previous experiments and that these cytokines affect cell viability and matrix synthesis less dramatically compared to environmental pH. The effects of low pH on cell viability have been explored in the last two decades on different cell sources such as bone marrow stem cells (BMSCs) [49], adipose-derived mesenchymal stem cells (ADMSCs) [50], ACs [51; 52] and NP cells [53]. Results from these studies are in line with our findings, demonstrating a correlation between increasing environmental acidity and decreased cell viability. One possible explanation for this could be that culture in acidic media induces the activation of acid-sensing ion channels (ASICs), which are extracellular pH sensors whose activation has been found to be responsible for acidosis-mediated apoptosis [54]. The expression of this family of receptors has been observed in several cell types, including disc cells and ACs [51; 54; 55; 56]. Although the expression of ASICs has not yet been examined for chondrocytes extracted specifically from the nasal septum, it is reasonable to assume that their response to acidic environments would be driven by the same biochemical mechanisms that drives the response of chondrocytes derived from articular cartilage. Activation of ASICs has been shown to be enhanced under inflammatory conditions, in particular in the presence of IL-1\(\beta\), resulting in increased cell death [57]. However, such a correlation was not observed in our results, which showed no significant differences in cell viability following IL-1\(\beta\) supplementation in 2D or 3D cultures. Acidic pH also had a significant impact on the ability of NCs to synthesise and deposit de novo matrix. We observed a reduction in the synthesis of sGAGs compared to controls (pH 7.1) in NCs cultured in media at pH 6.8 and 6.5, which is not simply due to compromised cell viability. It has previously been reported that cell metabolism can be severely inhibited by low pH [58; 59]. This is due to phosphofractokinases, the rate-limiting enzymes on glycolysis, being sensitive to pH and in particular inhibited by acidosis [60]. As a consequence, cells have less energy available and processes tightly linked to energy consumption such as sGAG synthesis are inhibited or impeded [61]. Interestingly, although we did observe a higher amount of collagen being produced at pH 7.1 without inflammatory factors, the detrimental impact of acidic conditions was less pronounced in comparison to sGAG synthesis, suggesting that the molecular mechanisms regulating collagen synthesis are less dependent on energy availability. A similar finding was reported in a study from [PERSON] et al., where a decrease in energy production in hypoxic conditions promoted hypertrophy in ACs with a decrease in aggrecan and an increase in collagen expression [62]. A second objective of this study was to directly compare the ability of NCs and NP cells to synthesise NP-like matrix in culture conditions mimicking the degenerated disc niche (low glucose, low oxygen, low pH in the presence of pro-inflammatory cytokines), with the aim of determining an appropriate cell source for disc repair strategies.