## 3 Results
### Acidic pH and Inflammatory Cytokines Have a Greater Impact on NP Cell Viability and Proliferation  
Motivated by our previous results, the effect of acidic media culture in the presence of physiologically relevant inflammatory cytokine concentrations was subsequently assessed for NCs and NP cells in 3D hydrogel culture. pH media groups were reduced to pH 7.1 and pH 6.5 to simulate healthy and acute degeneration conditions, respectively, with or without the combined supplementation of both IL-1\(\beta\)+TNF-\(\alpha\). Live/dead analysis revealed reduced viability for NP cells compared to NCs at pH 7.1 and pH 6.5 in both control and IL-1\(\beta\)+TNF-\(\alpha\) supplemented conditions. Aidity was again observed to be the primary factor responsible for cell death, as shown by the significant decrease in cell viability among cells cultured in acidic media compared to the controls (Figure 5A,B). Quantification of DNA further confirmed the detrimental effect of acidic pH on NCs and NP cells in control and inflamed conditions (Figure 5C). Figure 3: (**A**) Total collagen content (ug) in hydrogels and media at day 14 cultured in pH modified media (pH 7.1, 6.8 and 6.5) either unssupplemented (control) or supplemented with IL1-\(\beta\) and TNF-\(\alpha\) or a combination of both. (**B**) Collagen detected in culture media (%). The values are expressed as a percentage of the total amount of collagen measured at 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 II and collagen type I immunohistochemical staining. Scale bar is 25 um. NCs Can Secrette Higher Amounts of Key Matrix Components Compared to NP Cells in a Degenerated Disc-Like Environment  
Acidity was also observed to have a negative impact on sGAG synthesis. Intense localised sGAG deposition was observed for NCs cultured in pH 7.1 media (control and inflamed conditions), with less intense staining observed for NP cells. At pH 6.5, both NCs and NP cells displayed diminished sGAG staining. There were no appreciable differences in staining intensity between control and inflamed conditions at either pH levels. Biochemical results supported these histological findings, demonstrating that NCs cultured in pH 7.1 media were able to synthesise significantly higher amounts of sGAGs compared to all other groups by day 14. There was no significant impact of culturing in IL-1\(\beta\)+TNF-\(\alpha\) supplemented media for either cell type (Figure 6A,B). Figure 4: **(A)** Sulphated glycosaminoglycan (sGAG) content (\(\upmu\)g) in samples (hydrogels+culture media) at day 14 cultured in pH modified media (pH 7.1, 6.8 and 6.5) either unsupplemented (control) or supplemented with IL1-\(\beta\) and TNF-\(\alpha\) or a combination of both. Dashed line represents day 0 sGAG content. **(B)** sGAG:Collagen ratio. Both sGAG and collagen were calculated as respective amounts produced over 14 days by subtracting day 0 content from day 14. *** (\(p<0.001\)) and *** (\(p<0.0001\)) indicate significant differences between groups. N = 3 donors, n = 3 samples. **(C)** Alcian blue histological evaluation for sGAG accumulation in hydrogels at day 14. Scale bar is 150 \(\upmu\)m.  
In terms of collagen content, cells cultured in pH 7.1 control media showed enhanced collagen production compared to pH 6.5 control media. However, no differences were detected in collagen synthesis when cells were cultured in IL-1\(\beta\)+TNF-\(\alpha\) supplemented media (Figure 7A). Moreover, a similar pattern was found for sGAG:collagen ratios among groups, with NCs exhibiting a higher ratio than NP cells which was higher for groups cultured in pH 7.1 media than pH 6.5. Interestingly, NCs cultured in pH 7.1 media supplemented with IL-1\(\beta\)+TNF-\(\alpha\) exhibited a sGAG:collagen ratio significantly higher than in pH 7.1 control media (Figure 7B), due to diminished collagen deposition.