###### Abstract  
Pocrine urinary bladder is one of the most used organs to fabricate extracellular matrix (ECM) hydrogel. Although there are two different ECM types inside a bladder, i.e., urinary bladder matrix (UBM) and a subtype ECM (sECM), most studies have only employed UBM for hydrogel fabrication, and overlooked the potential use of sECM. In another aspect, the delamination of UBM from bladders is a time-consuming process; consequently, the use of the whole bladder (WB) will likely increase production yield. Therefore, the objective of this study was to fabricate hydrogels from sECM and WB and compare them to UBM. The results indicated that different layers of the bladder shared almost the same biochemical composition. In terms of gelation kinetics, rheology and morphology, although hydrogels from UBM and sECM exhibited some discrepancies, those from UBM and WB interestingly possessed almost the same characteristics. In in vitro studies, all the hydrogels possessed nearly the same biochemical effects towards L929 viability and C2C12 differentiation. These results could preliminarily indicate that the use of sECM should no longer be ignored, and WB could be a promising substitution for UBM hydrogels, eliminating the need for time-consuming delamination processes, as well as increasing the possibility of mass production. bladder; delamination; extracellular matrix; decellularization; hydrogel; collagen +
Footnote †: journal: Medical Engineering Research Center, National Defense Medical Center, Taipei 11490, Taiwan; 407010125@mail. is a highly organized thin, dense sheet of the ECM, containing primarily collagen (type IV), laminins and fibronectin, which anchor down the epithelium to its underlying lamina propria. The functions of BM are to provide structural support, give the cells physical and functional compartmentalization, and regulate cell behaviors [4]. In contrast, compared to the dense morphology of BM, the interstitial matrix is a loosely organized porous \"3D amorphous lattice\" located next to the BM. The dominant constituent constructing this ECM include collagens type I, III, V, VI, VII, XII, fibronectins, proteoglycans, which direct the organization of the IM structure, and is responsible for mediating cell attachment and function [5]. Among many types of porcine tissues, one of the most studied ECM is porcine urinary bladder [6]. According to literature, there are two types of ECM inside a porcine urinary bladder, namely urinary bladder matrix (UBM) and another lower type of extracellular matrix, in this context called sub-ECM (sECM) [7]. The UBM contains basement membrane of urothelial cells and the lamina propria, offering cells with nutritive and informative support, while sECM is characterized by musculars mucosa (submucosa) and the detrusor, which contain an elastic fiber network, accounting for the distension of the bladder [7]. Additionally, the organized smooth muscle cell bundles of IM are also surrounded by a layer of BM [8]. In our previous study [9], we established a reliable protocol to decellularize UBM using sodium dodecyl sulfate (SDS), and successfully prepared UBM hydrogels. At that point, we also questioned the usage of the rest of the bladder after delamination (i.e., the sECM part), and tried to address this problem. To the best of our knowledge, most studies have only prepared hydrogels from UBM and ignored the potential use of sECM or the whole bladder (WB) to fabricate ECM hydrogels, and there is no study investigating the differences among hydrogels made from those types of tissues [10; 11; 12; 13; 14]. On one hand, since there is a difference in the complicated biochemical compositions inside the UBM and sECM [15], it can accordingly be hypothesized that the ECM hydrogels made from distinct parts of the porcine bladder will possess dissimilar physical and biochemical properties.