## 4 Discussion
It has also been reported that Smad2/3 cooperatively is one of the important signalling pathways stimulated by TGF\(\beta\)3 that helps in developing and maintaining a chondrocyte phenotype [57]. Given that increased ECM synthesis was observed in low cell density microtissues, we next explored the effect of growth factors--GDF5, GDF6 in isolation or in combination with TGF\(\beta\)3. A synergistic effect on DNA content (indicative of increased cell number), total sGAG and total collagen was observed for both cell types with higher values observed in groups stimulated with the addition of TGF\(\beta\)3 to GDF5 or GDF6, and this was evident for both cell types. NP microtissues stimulated with TGF\(\beta\)3 + GDF5 or TGF\(\beta\)3 + GDF6 or TGF\(\beta\)3 + GDF5 + GDF6 were comparable to TGF\(\beta\)3 alone while the NC microtissues cultured in the presence of GDF5 alone were equally effective to TGF\(\beta\)3 and had a synergistic effect when combined (TGF\(\beta\)3 + GDF5). When these effects were evaluated on a per cell basis, the effects were diminished, with only a marginal increase observed in microtissues that were co-stimulated with growth factors. This was evident in the NC microtissues, but was not explicit in NP microtissues. Interestingly, [PERSON] et al. showed that MSCs co-stimulated with TGF\(\beta\)3 (10 ng/mL) and GDF5 (150 ng/mL) resulted in a significant increase in sGAG/DNA by day 7 compared to TGF\(\beta\)3 alone [58]. In this study, microtissues cultured in the combination of those growth factors did not result in any significant increase in proteoglycan synthesis on a per cell basis compared to TGF\(\beta\)3 alone, irrespective of the cell type. This could be due to the lower concentration of GDF5 used (100 ng/mL) or could be attributed to the difference in the cell type or culturing conditions with lower oxygen and glucose concentrations. Another study by [PERSON] et al. showed that GDF5 stimulation caused an increase in cell numbers but collagen/DNA was significantly high in MSC scaffolds treated with TGF\(\beta\)1 compared to GDF5 [59]. This trend was also observed in this study for NC microtissues, whereby TGF\(\beta\)3 stimulation resulted in a significant increase in collagen/DNA compared to GDF5 only. This study showed that GDF6 alone had little effect on DNA content (an indicator of cell number) for both NP and NC microtissues. This is in agreement with [PERSON] et al., who reported no increase in cell numbers when articular chondrocytes were cultured in GDF6-supplemented media [60]. [PERSON] et al. showed that GDF6 at a concentration of 400 ng/mL resulted in a significant increase in proteoglycan accumulation in NP cells compared to the non-treated controls [27]. Similar observations were made in this study with both NP and NC microtissues exhibiting significantly higher sGAG/DNA in the presence of GDF6 compared to the non-treated controls. sGAG/collagen was quantified to estimate the potential of the microtissues stimulated with various growth factors to produce the appropriate matrix type normally found in the disc. A higher ratio of sGAG composition of the ECM in comparison to the collagenous matrix is a predominant characteristic of NP tissue [45]. While no significant differences were observed for NP microtissues irrespective of the growth factor used, supplementation of NC microtissues with GDF5 or TGF\(\beta\)3 alone or with the combination of growth factors (TGF\(\beta\)3 + GDF5 + GDF6) yielded a significantly higher concentration. This implies that GDF5 or TGF\(\beta\)3 alone could produce similar effects to the combination of growth factors indicating their potential to induce NC microtissues to form an NP-like matrix. Recent research has shown that a combination of TGF\(\beta\)1 and GDF5 significantly enhanced glycosaminoglycan content of human-derived MSCs, suggesting that this combination is optimal for MSC to NP cell induction [61]. [PERSON] et al.