## 4 Discussion  
In the field of tissue engineering, to support cell proliferation, morphogenesis and differentiation, it is prevalent to employ reconstituted matrices with purified biomacromolecules originating from animal tissues or artificial extracellular matrices (ECM). These types of materials can be used for coating in 2D culture systems or fabricated into 3D scaffolds to mimic in vivo environments. One of the most used 3D culture systems consists of hydrogels constructed from soluble collagen type I due to their simplicity as macromolecular components [26]. However, compared to that pure collagenous network, hydrogels with more complex compositions derived from mammalian tissues can aid in supporting cell adhesion, migration, and differentiation by the presence of glycosaminoglycans (sGAG) [26]. To study the properties of ECM hydrogels, the basic criteria required for the characterization are decellularization efficacy, biochemical composition, gelation kinetics, surface morphology and rheology [27]. Therefore, those assays were conducted accordingly. Our decellularization method was efficient at eliminating the cellular contents of three different tissue layers of porcine bladder and all met the established criteria for sufficient decellularization. Figure 8: C2C12 differentiation after 28 and 35 days in ODM medium. (**A**) Representative images of cells stained with Alizarin red. (**B**) Calcium content quantified by spectrophotometer (\(n=3\)). (**C**) ALP activity measurement by spectrophotometer (\(n=3\)). *, **, *** denote statistical difference within the same group with \(p<0.05\), \(0.01\), \(0.001\), respectively (\(n=3\)). According to independent studies, the variations in compositions are probably the primary reason for the different characteristics of hydrogels [19]. Interestingly, there was no difference in the collagen and sGAG content of UBM, sECM and WB after decellularization. The collagen retention was four times as high as that of available commercial ECM materials such as TissueMend (fetal bovine dermis), GraftJacket (dermis), Restore (small intestine submuc), and AlloPatch (dermis), and the level of sGAG was also higher than those products [28]. The predominant reason for using UBM is that it has a fully intact basement membrane with good integrity, which may offer advantages for cell adhesion in tissue engineering [29]. Despite not possessing a complete sheet of basement membrane, sECM also contained separate basement membrane component around myofib [8]. UBM is made of basement membrane and loose lamina propria, in which the former contains mainly collagen type IV and VII and the latter is mostly composed of collagen types I, III, and III, IV, VII [15]. On the other hand, sECM is represented by muscularis mucosa matrix and detrusor, in which the first layer comprises collagen types I, IV and the second layer consists collagen types I, III, VI and XII [15]. From the results of LS-MS/MS, it was apparent that UBM and sECM layers shared almost the same types of not only collagens (type I, III, IV, VI), but other compositions as well. In the gelation kinetics study, there were some different patterns observed in the gelation speed and lag phase of hydrogels from different layers of the bladders. Nevertheless, it took almost the same time for all types of hydrogels to gel completely. Due to the complexity of the compositions of ECM materials, the exact gelation mechanism of hydrogels derived from ECM still remains unknown. It is commonly assumed that the overall interaction of collagen, laminin, elastin and proteoglycan are accountable for the fibrillogenesis self-assembling in multiple complex ways [27]. For pure collagen, gelation occurs through a two-step process with formation of thin filaments taking place in the lag-phase nucleation, and fibril growth and interconnections in the growth phase.