# Matrix Stiffness Influences Tubular Formation in Renal Tissue Engineering
## 4 Discussion  
In this work, we report the generation of tubular structures in micropatterned gels with different elastic moduli or stiffness. Tubular structures are basic in most organs and tissues, such as blood vessels, lungs, and urogenital and digestive systems. Therefore, the capacity to create tubular structures is critical for the bottom-up approach of tissue engineering, which aims to develop basic structures and combine them to create more complex in vivo-like tissues. We previously demonstrated that tube structures could be constructed successfully using micropatterned molds [11]. To see if we can further improve the ratio of formed tubular structures, we investigated tubular formation depending on the substrate stiffness. Native tissues and organs, as well as ECM molecules in vivo, possess different degrees of stiffness [23], from 200 Pa to 2-4 GPa [24], and 1 MPa to 4 GPa [23], respectively. The stiffness of tissues and basement membranes is an essential mechanical factor that can influence the phenotype of a cell in vitro [25,26], and it has been shown to have a role in morphogenesis in vivo [27,28]. Basement membrane stiffness has recently been suggested to be a critical factor in organ development, homeostasis, aging, and diseases [29], such as metastasis [30]. Our current work investigated the influence of matrix stiffness on tube formation by using micropatterned molds that we previously developed for tube formation [11]. We also tested the difference between two substrates, i.e., PDMS and agarose. Changing the fabrication process allowed us to test a range of substrates with an elastic modulus between 79 kPa to 8.9 MPa (Figure 2). We observed that stiffness influences tubular structure formation on both agarose and PDMS molds. Similar to our previous observation, the aggregate starts forming from one end of the micropatterned molds, independently of the substrate stiffness [11]. When cultured in PDMS molds, the percentage of formed tubes is greater when tubes were cultured in molds with a higher PDMS ratio (1:15, 1:20, and 1:30), i.e., a lower elastic modulus, than in molds with a lower PDMS ratio (1:5, and 1:10), i.e., a higher elastic modulus. When cultured in agarose-based micropatterned molds, the rate of tube formation was found to be positively correlated with agarose concentrations, and therefore stiffness, with optimal agarose concentrations, is best at 3% and 5%. Our results are in accordance with results previously reported in a study showing that stiffness regulated the tubulogenesis of lymphatic tubes and components of the vascular system [19,31]. Comparing the rate of tube formation on agarose and on PDMS, we observed that mLMCD cells show optimal tube formation on substrates, with a stiffness ranging between 277 kPa and 2610 kPa. Cells cultured outside this stiffness range failed to form tubes. Figure 7: (**Left**) The diagram represents the measured mean intensity (\(n=5\)) of the stained tubes for polarization marker ZO-1. Tubes were harvested after 24 h from agarose substrates of different stiffness and stained for ZO-1 and measured using ImageJ J. (**Right**) Mean intensities of the ZO-1 staining of tubes (\(n=5\)) from PDMS substrates of different stiffness. Symbols indicate \(p<0.005\) (*) and \(p<0.0005\) (†). A compact letter display is used to indicate non-significant differences from post hoc analysis (where a similar letter indicates no significant differences). Our results could be explained by previous results showing that matrix stiffness seems to regulate tubular cell polarization and proliferation [32], as well as tissue morphogenesis correction [28]. In our experiments, we found that the percentage of fully formed tubes was highest on agarose with an elastic modulus of 439.9 kPa (Figure 5A). Comparing this value to elastic moduli measured in native tissue, we found that this value is similar to the elastic modulus of the kidney tubular basement membrane (BM) (438 \(\pm\) 54 kPa) [33]. [PERSON] et al.