## 4 Discussion
The GDF6/TGF-\(\beta\)1/Wnt3a combination did present an overall lower DNA content, suggesting that the GDF6/TGF-\(\beta\)1/Wnt3a combination might have limited cellular proliferation or otherwise comprised cellular viability. Although our initial screening failed to validate GDF5/TGF-\(\beta\)1 as a potent inducer of a chondrogenic and NP cell phenotype, subsequent assessment within a 3D environment did confirm the observations from [PERSON] et al. [47] that the synergy of GDF5/TGF-\(\beta\)1 was able to induce a desirable cell product. Even more, GDF5/TGF-\(\beta\)1 was the only combination able to result in fully chondrogenic pellets. The disagreement between 2D and 3D culture outcomes is not surprising. Cell behavior is strongly altered by such biomechanical cues [56]. Moreover, TGF-\(\beta\)1 is not a specific chondrogenic factor as it has similarly been shown to induce MSC differentiation to other cell lineages, e.g., cardiomyocytes, osteocytes, or adipocytes [57; 58]. The ability to promote a chondrogenic NP cell-like phenotype through TGF-\(\beta\)1 is therefore likely context-dependent. This suggestion is supported by the original work of [PERSON] et al. [47], who showed the requirement of dexamethasone supplementation for a complete NP-like differentiation for MSC. That said, unlike the results of [PERSON] et al., our cells were unable to attain a final expression profile or matrix composition that matched those of naturally obtained human NP cells [47]. This might in part be due to the shorter culture periods that we employed. [PERSON] et al. maintained their pellet cultures for up to 4 weeks, whereas we cultured our pellets for 2 or 3 weeks dependent on the analysis performed. Additionally, while [PERSON] et al. applied adipogenic-derived MSC [47], our study employed bone marrow-derived MSC for the analysis. Distinct differentiation potential of differently sourced MSC is a well-reported phenomenon [46; 59]. Wnt signaling is critical in general joint development, homeostasis, and has also been determined to have a role in disc degeneration [60; 61; 62]. Regardless, the role of Wnt3a in chondrogenesis is less clear [48]. Wnt3a has been shown able to enhance MSC proliferation rates and BMP2-mediated chondrogenesis, within 2D monolayer cultures [63]. Other studies have shown mixed results when applying Wnt3a on MSC for the enhancement of chondrogenic differentiation in 3D conditions [48]. Similarly, our initial screening confirmed these findings, where the highest-ranking growth factor combinations often included Wnt3a in monolayer culture. However, these enhancements were not observed in 3D culture. This could in part be ascribed to the potential role of Wnt3a and its maintenance of a more undifferentiated state, as suggested by [PERSON] et al. [48]. Where 2D culture could induce maturation or loss of potency with extended periods of culture, Wnt3a could potentially play a role in mitigating this loss of cell potency, thereby supporting the chondrogenic potential of additionally added growth factors. This beneficial role is not observed in 3D cultures. To assess this potential application of Wnt3a, we reapplied a pellet culture system, in which MSC were expanded with Wnt3a containing media to possibly maintain their potency, and subsequent differentiation in 3D pellet cultures with chondrogenic factors but without Wnt3a. The histological results were unable to confirm this theory, as the application of Wnt3a during expansion, did not appear to enhance the deposition rate of proteoglycan in the matrix. Another aspect of our study is the direct comparison of growth factor combinations. For example, from our alcian blue assay (Figure 1), we observed that the addition of GDF5 appears to temper alcian blue rates compared to identical combinations without GDF5 or with GDF6 instead. Combinations that combined either Wnt11, Wnt5a, or Shh with GDF5 or GDF6 also did not present strong stimulation of proteoglycan production.