## 4 Discussion  
The IVD is a large avascular organ relying mainly on diffusion of nutrients through the CEPs for its energy supply. During disc degeneration calcification of the CEPs hinders the availability of nutrients to cells residing in the centre of the disc and the removal of metabolic by-products of glycolysis, thereby causing acidification of the cellular microenvironment and a shift to an accelerated catabolic state [14; 15]. Moreover, the degenerated disc consists of a wide panel of pro-inflammatory cytokines [22; 29; 43],  
Figure 7: (**A**) Total collagen (\(\mu\)g) produced in hydrogels containing NCs and NP cells in 3D hydrogel culture at different pH (7.1 and 6.5) and inflammatory cytokine (IL-1ß+TNF-\(\alpha\)) supplementation conditions. (**B**) sGAG:Colagen ratio. Both quantities were calculated as respective amounts produced over 14 days by subtracting day 0 content from day 14. * (\(p<0.05\)), ** (\(p<0.01\)), *** (\(p<0.001\)) and *** (\(p<0.0001\)) indicate significant differences between groups, ns (not significant) indicates \(p\geq 0.05\). N = 3 donors, n = 3 samples. (**C**) Collagen type I and collagen type II immunohistochemical staining. Scale bar is 25 \(\mu\)m.  
further contributing to the creation of an extremely challenging biochemical microenvironment. For successful translation of cell-based therapies for disc regeneration, it is imperative that cells remain viable post injection, and function effectively in a challenging microenvironment [8; 33; 34]. In the first part of this study, we investigated the impact of the pro-inflammatory cytokines IL-1\(\beta\) and TNF-\(\alpha\), in combination with low pH media in 2D and 3D hydrogel culture with NCs. It was found that acidic pH affected cell viability and number to a much greater extent in comparison to IL-1\(\beta\) and TNF-\(\alpha\) supplementation. The detrimental effect of acidic pH was also observed in relation to a significant reduction in matrix synthesis in 3D culture, whereas IL-1\(\beta\) and TNF-\(\alpha\) supplementation, either alone or in combination, appeared to have no influence on sGAG synthesis and only a marginal influence on collagen production when NCs were cultured at pH 7.1. Surprisingly, we found that cytokine supplementation had only a marginal influence on cell viability and matrix synthesis. Cellular response to culture in inflammatory conditions has been extensively studied in the last two decades. However, there is still no consensus on the specific molecular mechanisms that are triggered or enhanced by the presence of pro-inflammatory cytokines such as IL-1\(\beta\) and TNF-\(\alpha\), possibly due to the variability in culture conditions and consequently in the results obtained. Some studies have found strong evidence of IL-1\(\beta\) being the key regulator of matrix degradation processes in NP cells [28; 44], and identified the role of TNF-\(\alpha\) more likely to be associated with nerve root irritation rather than influencing matrix catabolism [23]. In contrast, a number of other studies have either not found major differences between the effects of IL-1\(\beta\) and TNF-\(\alpha\) supplementation on NP cells [27; 45], or even reconsidered the central role that was attributed to IL-1\(\beta\) in the process of matrix catabolism [19; 43]. It is possible to find analogous discrepancies in similar studies on articular chondrocytes (ACs); whereby some authors have judged the contributions of both IL-1\(\beta\) and TNF-\(\alpha\) as equal [46], others have found evidence of TNF-\(\alpha\) having a predominant influence on cell death [47] and decreased matrix synthesis [48]. Nevertheless, in contrast with our results, the common denominator of all these findings is that the presence of pro-inflammatory cytokines, either alone or in combination, deeply affects cell fate and gene expression.