*2.5. SDS-PAGE*

SDS-PAGE results present the subunit composition and type of collagen instinctively. As shown in Figure 3, ASC and PSC have very similar protein bands, which have been identified as trimers (γ chains), dimers (β chains), and two alpha chains (α<sup>1</sup> and α2). Up to now, it has been reported that there are two different collagen trimers in tilapia skin, (α1)2α<sup>2</sup> and α1α2α3, and that the two chains of α<sup>1</sup> and α<sup>2</sup> have the same molecular weight, which cannot be distinguished by electrophoresis [65]. According to Sun [41], the structure of Nile tilapia collagen is (α1)2α<sup>2</sup> type. The band intensity of α<sup>1</sup> is twice that of α2, indicating that both ASC and PSC are type I collagen. In addition, the electrophoresis bands are clear, and have no low molecular weight bands, showing that the molecular structure of collagen was well preserved during the extraction process. Although some telopeptides were lost after the treatment of PSC, the banding pattern of PSC was similar to ASC, indicating that the PSC extraction process does not affect the integrity of the triple-helix structure.

**Figure 3.** SDS-PAGE patterns of ASC and PSC from Nile tilapia skin.

#### *2.6. Morphology*

Figure 4 shows the ultrastructure of the cross-section of the lyophilized collagen sponge. The graphs showed that ASC and PSC exhibit slightly different microscopic morphology. From the 50× magnified image, ASC and PSC display a loose porous network structure, but the pores of ASC show a more uniform and less fiber structure pattern than PSC. As shown in the 400× magnified image, ASC is a dense sheet-like film with uniform alignment, and PSC exhibits irregularly arranged curls.

**Figure 4.** Morphological features of Nile tilapia skin collagen using SEM.

The microstructures determine the physicochemical properties and biofunctionality of the materials; they have a significant value to the application of collagen in biomedical materials. The SEM results of ASC present typical characteristics of aquatic collagen, such as miiuy croaker [56] and Acipenser schrenckii [66], while the microstructures of PSC exhibit a fibrous structure similar to that of terrestrial collagen, such as bovine [67]. This difference may be caused by the structure of PSC being changed under the influence of pepsin, forming more collagen fibers in a non-crosslinked state, which is consistent with the results of FTIR. In the field of biomedical materials, greater porosity facilitates the migration of cells into the interior of the scaffolds, which has certain advantages for promoting wound healing. The lower degree of cross-linking is beneficial to the dissolution and re-processing of collagen, and is suitable for many processes such as electrospinning. Therefore, the microstructures of ASC and PSC indicate that they are appropriate for use in different biomedical material fields.
