## 4 Discussion
In the case of ECM hydrogel, other than collagens, proteoglycans and glycosaminoglycans also affected the length of both the lag and growth phases during gelation process as accelerators and decelerators [30]. The lag phase was hastened with the presence of chondro sulfate and keratin sulfate hasten, and impeded by heparin sulfate, but then it was also chondro sulfate that detained the growth phase [30]. As there was no statistical difference in the collagen and sGAG concentration of UBM, sECM and WB, it could be preliminarily explainable that all the gels shared similar gelation tendency after the lag phase. ECM hydrogel formation is a self-assembly process of collagen fibrillogenesis. The polymerization of collagen with long fibrillar structures is regulated by both electrostatic and hydrophobic interactions from glycosaminoglycans, proteoglycans, and other ECM proteins that stimulate the cross-linking of collagen fibers. The ECM digest forms a gel when the liquid is neutralized to physiologic pH (\(7.4\)), salt concentration and temperature (\(37\,^{\circ}\)C) [27]. In practice, the digested ECM, i.e., pregel solution, is neutralized to physiologic pH and salt concentration and kept at a low temperature until follow-up applications [27]. The results from rheology indicated that pregel solutions obtained from different layers of the bladders in our study possessed the same shear thinning property and viscosity. This may be due to the similarity of protein profiles in those tissue types. In pregel solutions, randomly oriented proteins initially bound with sGAG through hydrogen and Van Der Waals interactions, offering the ability to resist flowing at low shear rate. When higher shear was applied, these bonds were broken, leading to the alignment of proteins and GAGs, causing a shear thinning phenomenon [31]. Hydrogels from UBM and WB possessed similar mechanical strength and were stronger than sECM hydrogels despite the fact that all three hydrogels had the same content of soluble collagen and sGAG. In our studies, only collagen types I, III, IV and VI were detected. However, according to the literature, there are at least 28 different types of collagens inside the bladder. Except for collagen types I and III, which represent the bulk proteins in the bladder, accounting for up to 60% of bladder's dry weight, the function of the other less abundant collagens and their localization in the bladder is little studied [15]. Collagen types IX, X, XII, XIV and XVI are classified as fibril associated collagens with interrupted triple helices (FACITs), with the ability to cross-link with other collagens [15]. The interactions between complex groups of collagens might be the cause for difference mechanical strengths among hydrogels. Further study must be conducted to confirm this hypothesis. All the hydrogels obtained from different parts of the bladder exhibited a fibrous surface morphology. The fiber diameter of sECM and WB hydrogels was thicker than that of UBM. This can be theoretically explained by the presence of collagen type VI in the detrusor of sECM, which, according to the literature, can physically associate with collagen I fibrils to increase the thickness of the fibers [15]. These results are also in accordance with studies with liver ECM fibers, in which it was quantitatively found that the skeleton fiber diameter depends on the constituent components [32]. Compared to another study on UBM hydrogel, the UBM hydrogels in this study possessed a thinner fiber diameter with an average thickness of 52 nm compared to 74 nm [19]. The fiber diameters of sECM and WB were also found to be thinner than those from cardiac, SIS, adipose-derived ECM hydrogels, with average fiber diameter of approximately 100 nm [27]. As demonstrated in many studies, ECM hydrogels may share many common characteristics, but the tissue of origin, decellularization process, and protein concentration also affect the structure of the materials [27]. This may be the reason for the variation in fiber diameter in our research compared to other studies.