## 4 Discussion  
In this study, we aimed to identify growth factors or factor combinations that showed the highest potency in enhancing MSC to NP cell-like cell differentiation. Starting with monolayer cultures, a large array of factor combinations (86 different growth factors and factor combinations) were screened on their ability to stimulate proteoglycan production, as indicated by alcian blue staining. Unsurprisingly, almost all factor combinations showed the ability to at least enhance proteoglycan production compared to unstimulated MSC, as all factors were selected based on their general chondrogenic potential. Interestingly, of all the combinations identified as high hitters, a large fraction involved a combination with Wnt3a. A selection of the 10 most potent growth factor combinations, plus the combination of GDF5/TGF-\(\beta\)1, were further analyzed on their ability to induce NP cell marker expression. Here we showed the ability of particularly TGF-\(\beta\)2/Wnt3a, TGF-\(\beta\)1/Wnt3a, GDF6/TGF-\(\beta\)1/Wnt3a, and GDF6/Wnt3a to strongly enhance the expression of NP markers [38] CD24 and SOX9. A trend of enhancement in COL2A1 and T was observed for most conditions. Contrary to the impactful findings of [PERSON] et al. [47], our initial screening outcomes (in monolayer culture) did not indicate GDF5/TGF-\(\beta\)1 as a potent chondrogenic factor-combination. Nevertheless, at this stage of the study, differentiation culture was still within the 2D monolayer set-up. MSC tend to change their characteristics relatively quickly in monolayer culture and lose their multipotency capacity [51; 52; 53]. Moreover, for induction of differentiation, the extracellular micro and macro-environment are critical as they can promote or support specific cellular behavior by providing mechanotransduction cues [54], anchor points, and chemical cues through the matrix proteins or by growth factor sequestering [55]. As such, we further validated the findings observed in monolayer cultures in a 3D environment, using a common pellet culture method [47]. Pellet cultures revealed an opposing trend, in which none of the Wnt3a combinations were able to significantly enhance glycosaminoglycan production rates. Although an increase was seen in the expression levels of some NP markers, i.e., CD24, SOX9, and COL2A1, increases in the ACAN expression level were not observed. This in contrast with the pellet cultures treated with GDF5/TGF-\(\beta\)1, which resulted in a significant increase in glycosaminoglycan production rates, and large proteoglycan positive regions within the pellet as indicated by safranin-O staining. Additionally, GDF5/TGF-\(\beta\)1 showed continuously most potent in enhancing NP cell marker expressions following the 3D pellet culture conditions. Although the relative glycosaminoglycan content of the other 4 growth factor combinations did not result in a significant increase, a trend of enhanced glycosaminoglycan production was observed both  
Figure 5: Assessment of Wnt3a pre-culturing for enhancement of chondrogenesis. Human MSC were expanded in standard expansion media without Wnt3a (**A,B**) or with 250 ng mL\({}^{-1}\) Wnt3a (**C,D**). Subsequently obtained MSC were cultured in chondrogenic pellets and media supplemented with GDF5/TGF-\(\beta\)1 (**A,C**) or commercial predefined chondrogenic medium (**B,D**). Subsequent pellets were sectioned and stained by safranin-O/fast-green (left) or hematoxylin/eosin (right) staining. Scale bars represent 250 \(\upmu\)m. Chon.M. : Mesenchymal stem Cell Chondrogenic Differentiation Medium. by the DMMB assay, as well as on histological observation. The negative control revealed no pink/red staining in the safranin-O stained section, the growth factor-stimulated pellets did present lightly pink areas, suggesting the presence of proteoglycans. Interestingly, the combination GDF6/TGF-\(\beta\)1/Wnt3a showed the second-highest rate of glycosaminoglycan production/DNA but did not strongly show safranin-O positive regions.