# Matrix Stiffness Influences Tubular Formation in Renal Tissue Engineering
## 4 Discussion
demonstrated that the tubular BM possesses an instructive potential for tissue elongation [34]. Interestingly, our results differ from those of [PERSON] et al. who observed the apical-basal polarization of Madin Darby Canine Kidney cells (MDCK) at a sub-physiological substrate stiffness of 1 kPa, while polarization was absent at supraphysiological conditions of >10 kPa [35]. Although not specified in their paper, we suppose that they referred to the stiffness of the macroscale kidney (7 kPa) [36]. How stiffness influences cell behavior remains difficult to determine, because the biomaterial potentially contributes to a certain behavior. To test if a specific material or stiffness promotes earlier ZO-1 expression (a marker for tube maturation) than another, we stained ZO-1 in 1P tubes, which, in our grading system, correspond to the earliest and lowest tube maturation stage. We observed differences in the expression of ZO-1, a marker for tube maturation [22], in 1P tubes, depending on the culture substrate. When cultured on agarose, we observed that ZO-1 expression increases with the agarose concentration up to 3%, and then decreases (5%--Figure 6. When compared to the number of 2P and 3P tubes (Figure 4), we observed a similar evolution in function of the agarose concentration, suggesting that the stiffness influences ZO-1 expression, which, in return, influences the number of mature tubes. However, when cultured on PDMS, the PDMS ratio has less influence on the ZO-1 expression, and few differences were observed in the expression intensity of ZO-1 in the function of the PDMS ratio. In addition, no similarity could be found when comparing the quality of tube maturation, as shown by the ZO-1 expression (Figure 6), and the number of mature tubes (2P and 3P), as shown in Figure 5. This suggests to us that PDMS influences tube maturation through other mechanisms that occur after ZO-1 expression and that have yet to be identified. In addition, stiffness, which is different depending on the observation level (molecular, tissue, organ) [23], seems to affect cell behavior on those different levels (molecular, tissue, organ), which could explain the differences between our findings and the findings of [PERSON] et al. [35]. Our results are in accordance with those of others. However, while previous research showed that cell differentiation, growth [32; 37], and morphogenesis [19; 31] are influenced by the stiffness of ECM, our current study suggests that stiffness alone is not the sole factor influencing tubulogenesis in our system. When comparing tube formation on agarose and PDMS holds with similar stiffness, i.e., agarose 3% vs. PDMS 1:30 (439.9 kPa and 680 kPa, respectively) and agarose 5% vs. PDMS 1:20 (1388.2 kPa and 1440 kPa, respectively), significant differences were observed for the formation of 1N, 1P, and 3P tubes. These differences suggest that the composition of the substrate further influences tubulogenesis. Although we can hypothesize that differences in cell or protein attachment onto the substrate or the presence of culture medium components in the agarose gel may have an influence, further experiments will be needed to determine additional factors that influence tubular formation.