## 4 Discussion  
In this study, a microwell array system was used to promote the formation of self-assembled homogenous microtissues using two different cell types, nucleus pulposus cells and nasospatial chondrocytes, and were subsequently assessed in response to growth factor stimulation. Growth factors TGF\(\beta\)3, GDF5, GDF6 and a combination of these growth factors were assessed for their ability to induce matrix synthesis. The choice of growth factors selected was based on previous research in the field. For example, TGF\(\beta\)3 is known to support the maintenance of the phenotype of disc cells in a rat lumbar organ culture [19] and MSCs supplemented with TGF\(\beta\)3 support their differentiation towards an NP-like phenotype [37]. In addition, murine IVD explants treated exogenously with TGF\(\beta\)3 have been shown to result in an up-regulation of aggrecan [38]. Studies have also shown that cells primed with TGF\(\beta\)3 promoted higher sGAG and collagen synthesis [31,39] and also mitigated the detrimental effect of the harsh acidic microenvironment [40]. GDF5 (also known as cartilage-derived morphogenetic protein, CDMP1) and GDF6 (CDMP2) are members of the bone morphogenetic protein (BMP) family and are key regulators of cellular condensation and chondrogenesis [41]. GDF5 binds with higher affinity to BMP receptor type-1B (BMPR-IB), which controls the primary stages of cellular condensation and promotes cartilaginous tissue formation [42,43]. GDF6 has been shown to promote chondrogenesis and it positively regulates growth and maintenance of articular cartilage [44]. Both GDF5 and GDF6 have been shown to induce the expression of NP associated genes in MSCs and adipose-derived stem cells (ASCs) [45] with a healthy NP phenotype exhibiting stabilised expression of hypoxia inducible factor HIF-1\(\alpha\), the glucose transporter glut-1, the PG aggrecan (ACAN), type II collagen (COL2A), the signalling factor sonic hedgehog (SHH), the transcription factor Brachyury [T], keratins KRT18, KRT19, carbonic anhydrase CA12, and CD24 [46]. We selected the growth factor concentrations of TGF\(\beta\)3, GDF5, and GDF6, based on previous work stimulating bone marrow-derived MSCs and adipose-derived stem cells  
Figure 7: Immunostaining for collagen types I and II of nucleus pulposus (NP) and nasoseptal chondrocyte (NC) microtissues stimulated with TGF\(\beta\)3, GDF5, GDF6, TGF\(\beta\)3 + GDF5, TGF\(\beta\)3 + GDF6, TGF\(\beta\)3 + GDF5 + GDF6 on day 7. (**A**) Collagen type I and (**B**) Collagen type II. (ADSCs) which reported optimal expression of genes COL2 and ACAN when stimulated with 10 ng/mL TGF\(\beta\)3 and 100 ng/mL GDF5 and GDF6 [28]. This study confirms that the initial cell seeding density of microtissues plays an important role in regulating ECM deposition. Although the amount of sGAG and collagen produced were highest in high cell density microtissues, sGAG and collagen synthesised on a per cell basis (per DNA) were highest in low density microtissues (1000 cells/microtissues). Based on the in silico models, higher cell density microtissues experienced lower nutrient levels and this in turn could alter the metabolism of the cells [32,47], and inhibit or impede matrix synthesis [39,48]. Overall, higher levels of matrix synthesis were observed for NC microtissues compared to NP microtissues, and it was significantly pronounced for NC microtissues stimulated with GDF5 or in combination with TGF\(\beta\)3. In agreement with previous studies, microtissues stimulated with TGF\(\beta\)3 resulted in increased proteoglycan synthesis compared to the controls [49,50]. TGF\(\beta\)3 is widely known to modulate cell proliferation, differentiation and ECM synthesis and in particular supports chondrogenic differentiation of various cell types including mesenchymal stem cells [37,51,52,53,54], through the activation of the Wnt signalling pathway [55,56].