## 4 Discussion
Despite having some similarities with respect to the components of ECM, each tissue or organ possesses a distinctive composition, leading to different environments for cells to develop and mature [33]. Typically, when complex compositions of a mixture of single proteins such as collagens, fibronectin, vitronectin or laminin are used as coating substrates, cell behavior can be influenced as a result of the dissimilar ratios of various proteins and polysaccharides compared to the tissue-specific in vivo microenvironment [33]. C2C12 myoblasts have been used as a model to study the effect of ECM materials towards myogenic and osteogenic differentiation for regenerative application [19; 34; 35]. However, the differentiation of this cell line on sECM, WB and UBM remains unknown. Therefore, C2C12 was selected to examine the cell response when cultured and differentiated on three different ECM substrates. According to our LC-MS results, different layers of the bladder possess dissimilar proportions of various biochemical compositions, and the effect of this compositional discrepancy on the differentiation of the C2C12 muscle progenitor cells was examined. The pregel solutions from UBM, sECM, WB and collagen type I were coated on the coverslip or flasks prior to seeding cells. This physical coating technique has been widely employed in other studies using different ECM sources such as meniscus, liver, brain, pancreata, skeletal muscle and bladder [27]. In order for myogenesis to occur, it is necessary for cell proliferation to be hindered. Commonly, 2% horse serum [23] is used to trigger myogenesis. However, in our study, when the cells reached 70% confluence, FBS content was reduced to 0.2% to kindle myotube formation. This method has also been previously employed in other studies, in which the concentration of growth factors was depleted by tenfold for myoblast differentiation [36]. Myosin heavy chain and myogenin were markers of the end-terminal differentiation of myoblasts [37]; therefore, these proteins were chosen for the immunocytochemistry (ICC) and Western blot experiments. Fibroblasts are a cell line commonly used for examining cell viability on ECM-based materials [19]. Therefore, L929 fibroblast was chosen to study the cell viability on UBM, sECM and WB hydrogels. It was observed from the Alamar blue assay that different substrates did not influence the viability of L929 after 5 days. In terms of differentiation, the results from ICC and Western blot demonstrated that C2C12 myoblast differentiated on UBM, sECM and WB coated surface was as efficient as on commercial collagen type I. According to the literature, collagens are the main structural protein in the muscle ECM; other various components of the ECM participate in muscle regeneration as well, not only performing structural support, but also influencing the myoblast cell function and myotube formation [38]. Various studies have also been conducted to investigate the influence of single ECM composition on the differentiation of C2C12, including collagen type I, collagen type IV, fibronectin, laminin, decorin, or mixture of complex composition such as Matrigel [39], dermal, UBM and skeletal muscle ECM [19]. The results from these studies suggest that not only single ECM components, but also a complex ECM composition, offer positive effects with respect to myoblast differentiation. Therefore, the effect of three different ECM substrates on the differentiation of C2C12 was then studied. From our LC-MS results, fibrillin, laminin, fibronectin, etc., were detected in all layers of the bladder, and these glycoprotein/proteoglycans have previously been proven to influence cell behavior, signaling and differentiation [35]. Additionally, heparan sulfate, which plays a pivotal role in in muscle regeneration, was also spotted [35]. However, there was no significant difference in differentiation behavior of C2C12 when cultured on coating substrates prepared from different layers of the bladder.