## 3 Results
### Quantitative Biochemical Analysis of Microtissues in the Presence of Different Growth Factors  
Both cell types responded positively to growth factor treatment relative to the no growth factor control (red solid line). For NP microtissues, no significant differences in DNA content were observed after 7 days irrespective of the growth factor type or combination. In contrast, a higher DNA content was observed in NC microtissues when cultured in the presence of GDF5 and all other combinations of growth factors relative to GDF6 (Figure 6A). Total sGAG was highest in NP microtissues stimulated with TGF\(\beta\)3 + GDF5, although there were no significant differences between the groups investigated. Total sGAG levels in the NC microtissues were similar for all growth factor groups and combinations and were significantly higher compared to GDF6. Overall, sGAG levels forNC microtissues were significantly higher compared to NP microtissues (Figure 6B). When normalised by DNA content (sGAG/DNA), no differences were found between growth factor groups for a specific cell type, although NC microtissues still exhibited higher content compared to NP cells (Figure 6C). Figure 3: Biochemical and histological staining for sGAG and collagen of nucleus pulposus (NP) and nasoseptal chondrocyte (NC) microtissues stimulated with TGF\(\beta\)3 on day 7. (**A**) Total sGAG (\(\upmu\)g) content (**B**) Total sGAG/DNA (**C**) Total Collagen (\(\upmu\)g) (**D**) Total Collagen/DNA. Data represent mean \(\pm\) SD of at least three independent experiments performed in triplicate.! (\(p<0.05\)) indicates significance compared to 1000 cells/microtissue. # (\(p<0.05\)) indicates significance compared to the control for the same cell number on day 7. $ (\(p<0.05\)) indicates significance compared to day 0 for the same cell number. * (\(p<0.05\)) indicates significance compared to the NP microtissues for the same experimental group and cell number. (**E**) Histological evaluation with alcian blue staining and picrosirius red to identify sGAG collagen content, respectively. In terms of total collagen content, for NP microtissues, a synergistic effect was observed through co-stimulation of growth factors (TGF\(\beta\)3 + GDF5, TGF\(\beta\)3 + GDF6 and TGF\(\beta\)3 + GDF5 + GDF6) when compared to GDF5 and GDF6 alone. A similar synergistic effect was observed for NC microtissues when cultured in the presence of TGF\(\beta\)3 + GDF5. However, the collagen content in NC microtissues treated with TGF\(\beta\)3 + GDF6 and TGF\(\beta\)3 + GDF5 + GDF6 were significantly higher than those treated with GDF5 alone. NC microtissues cultured in the presence of GDF6, TGF\(\beta\)3 + GDF5 and TGF\(\beta\)3 + GDF5 + GDF6 exhibited significantly higher collagen content compared to NP microtissues exposed to similar conditions (Figure 6D). Total collagen/DNA was significantly higher in NP microtissues cultured in the presence of TGF\(\beta\)3 + GDF6, and no significant difference was observed when compared to TGF\(\beta\)3 (red dashed line). However, for NC microtissues, GDF6-treated groups exhibited significantly higher concentrations compared to TGF\(\beta\)3  
Figure 4: In silico modelling of the nutrient microenvironment within microtissues as a function of cell density. Predicted contour plots and radial profile through the midplane containing 1000, 2500 and 5000 cells for (**A**,**B**) oxygen (%O\({}_{2}\)) (**C**,**D**) glucose (mM) and (**E**,**F**) pH. The radial distance was normalised by the radius of the microtissue for each cell density. (red dashed line), GDF5 and TGF\(\beta\)3 + GDF5 + GDF6 treated groups (Figure 6E). In terms of sGAG/collagen level, which is a surrogate measure of NP-like matrix, with a higher ratio being desirable, no significant difference--irrespective of the growth factor treatment--was observed for NP microtissues. NC microtissues treated with GDF5 showed significantly higher concentrations compared to GDF6, TGF\(\beta\)3 + GDF5 and TGF\(\beta\)3 + GDF6.