*2.4. Qualitative Evaluation of the FSs by Histological Methods*

FS composition was also qualitatively evaluated by standard histological methods. Different molecular components were highlighted on each FS, smeared and dried on histological slides, and observed in light microscopy (Figure 3).

Alcian staining (pH 2.5) allowed to highlight in light blue the presence of MPS by reacting with acidic groups. Alcian staining on the smeared FSs showed the following results: in F1 sample (panel A), small and uneven clots of MPS were observable; in the F2 (panel B), a strong coloration of uniformly distributed MPS clots was evidenced; in F3, only a weak MPS staining was detectable in some portions of the sample (panel C); in F4 (panel D), in addition to a weak and diffused coloration, some fibres with an irregular pattern were observable showing the presence of MPS.

**Figure 3.** FS histological staining. The four FSs (F1–F4) smeared on histological slides and stained according to four different staining procedures, as described in Section **??**, and observed by optical/polarized microscopy. (**A**–**D**) Alcian pH 2.5 staining, highlighting mucopolysaccharides (MPS) and glycoproteins in blue, by reacting with acidic groups; (**E**–**H**) Periodic Acid Schiff (Hotchkiss–Mc Manus) (PAS)\_staining highlighting MPS, glycoproteins, glycolipids, mucins, and polysaccharides such as glycogen in pink/red, by reacting with basic groups; (**I**–**L**) Picro-Sirius Red staining of collagen bundles in different shades of green, red, or yellow, depending on the thickness and the packing of fibres, from thinner to thicker, respectively. Each column of panels represents the various histological stainings of each FS. Scale bars in each panel span 20 micrometer.

PAS staining, on the other hand, highlights in pink/red, the presence of MPS by reacting with basic groups. In the F1 sample (panel E), a poorly concentrated staining of basic little clots was observable. In the F2 (panel F), basic MPS clots were still poorly concentrated. In F3 (panel G), a matrix of transparent/pink fibres was clearly visible by DIC (Differential Interference Contrast), with red clots indicating the presence of basic MPS, likely not being associated with fibres. Finally, in F4 samples (panel H) a weak PAS positive staining of fibrous material was observable, similarly to that observed with Alcian and Picro-Sirius Red (PicroS) in panels D and L, respectively, with the presence of MPS distributed in an irregular pattern.

With the PicroS collagen staining, and through the use of polarized microscopy, it is possible to distinguish small-caliber collagen fibres that result in highlighted in shades of green with respect to thicker fibres that appear to be yellow/red. PicroS staining of the F1 preparation (panel I), showed a discontinuous film of small-caliber fibres. Collagen appeared to be distributed non-uniformly, but in bundles with the presence of small lumps of about 20 μm in diameter showing larger caliber collagen fibres (in red/yellow). Conversely, in the F2 preparation, it was possible to observe an uneven distribution of thicker collagen fibres that are highlighted in red (showed in panel J). Finally, in the F3 and F4 preparations (panels K and L, respectively), the method was able to highlight two continuous fibre films, even if with different chromatic characteristics, pointing out smaller caliber fibres in F3, and thicker in F4.

### *2.5. Sponge Collagen Membrane Production*

Sponge Collagen Membranes (SCMs) were produced using 2 mg/mL of F1, F2, F3, and F4 fibrillar suspensions in the presence of EDC/NHS crosslinking solution for 4 h at RT. As shown in Figure 4, the only FSs that were able to generate suitable SCMs were F1 and F3 extracts. In fact, F1- and

F3-derived SCMs looked like thin, clear, films (Figure 4A). Conversely, using F2 and F4 suspensions, even by increasing the FS concentration (data not shown), it was not possible to obtain any suitable membrane. In fact, when dried the derived films resulted lacking texture, extremely fragile and easy to break. Hence, all of the following characterization analyses were performed only on F1- and F3-derived SCMs named SCM-F1 and SCM-F3, respectively.

**Figure 4.** Sponge Collagen Membranes (SCMs). (**A**) SCMs derived from F1–F4 sponge extracts crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/*N*-hydroxysuccinimide (EDC/NHS) solution; (**B**) not crosslinked SCMs derived from F1 and F3 sponge extracts. Scale bars in each panel span 2.0 cm.

As shown in Figure 4B, the membranes were also prepared using not crosslinked F1 and F3 suspensions casting the collagen suspensions without adding the cross-linker. This procedure generated the nc-SCM-F1 and nc-SCM-F3 membranes, respectively, which were macroscopically similar to their crosslinked counterparts.
