2.6.3. Water Binding Capacity

The water binding capacity (WBC) of SCMs and nc-SCMs was assessed by the evaluation of their weight variation after 1 h soaking in PBS at RT. nc-SCM-F1 and SCM-F1 showed a WBC of 652 ± 35% and of 280 ± 33%, whereas the WBC of nc-SCM-F3 and of SCM-F3 were 701 ± 40% and 420 ± 55%, respectively. Thus, F3-derived membranes showed a slightly higher WBC than F1. Conversely, the commercial not crosslinked collagen membrane Bio-Gide® in the same soaking conditions showed a WBC of 442 ± 41%, which is a value that is significantly lower than the two nc-SCMs, but comparable to the SCMs.

Once hydrated, it was also clearly observable a significant different behavior of the SCMs with respect to the nc-SCMs. The latter, in fact, although much more hydrophilic than their crosslinked counterparts, completely loosed consistency and resulted in being very difficult to manipulate, as shown in Figure 6C.

#### 2.6.4. Biocompatibility

To evaluate the biocompatibility of SCM-derived membranes, a fibroblast cell line and a keratinocyte cell line were tested for their ability to adhere and to grow on FS-coated plates using all of the extracts (F1–F4). Cell adhesion was evaluated 16 h after plating both qualitatively and quantitatively by the crystal violet staining and the MTT assay, respectively (Figure 7A,B). The crystal violet qualitative assessment of both fibroblast and keratinocyte cell adhesion revealed that the cell shape, and thus the interaction with the different matrices, was very similar in all of the conditions for both of the cell lines. A slight, qualitative difference could be observed only for the fibroblasts on F4 coating. In particular, a circular shape seemed to be predominant with respect to the physiological spindle/triangular shape typical of these cells, probably indicating a not preferred interaction with this type of coating (Figure 7A). Similarly, the MTT cell viability assay at 16 h of adhesion indicated a reduction of the attached cells only for fibroblasts (Figure 7B, black bars) and only on the F4 coating (21.75% cell reduction as compared to controls). These results confirm a poorer short-term F4 compatibility with respect to the other FSs and to the control, where cells were seeded onto rat tail collagen coated wells, namely stCol.

**Figure 7.** Cell adhesion evaluation. (**A**) Cell adhesion qualitative evaluation, by optical microscopy, of crystal violet stained L929 fibroblasts (first row) and National Collection of Type Cultures (NCTC) keratinocytes (second row) on the four different FS pre-coated plates (F1–F4) after 16 h incubation. Scale bars, 20 micrometer; (**B**) Cell adhesion quantitative evaluation, by MTT test, of L929 fibroblasts (black bars) and NCTC keratinocytes (grey bars) on the four different FS pre-coated plates after 16 h incubation. Results are expressed as cell percentages with respect to controls that were seeded on standard rat tail collagen-coated (stCol) wells and are the mean ± S.D. of two experiments that were performed in quadruplicate. Asterisks indicate significance in Tukey test (black bars ANOVA, *p <* 0.05; Tukey, F4 vs. stCol *p <* 0.05).

Cell viability and proliferation was also evaluated for longer periods of time on FS-coated plates by using the MTT assay. In particular, both fibroblasts and keratinocytes were evaluated after 3 days, 6 days, and 15 days of cultivation (Figure 8) on the FS-coated plates and compared to control cells that were grown onto rat tail collagen coated wells (stCol). In detail, fibroblasts showed a slight cell number reduction on F4 coating and a slight increase on F1 coating at 3 day with respect to the control (Figure 8A, black bars 19.4% decrease and 12.2% increase, respectively). Conversely, both at 6 day and 15 day of prolonged cell culture (Figure 8A, white bars and grey bars, respectively), no significant differences with respect to the controls grown on rat tail collagen were observed. This indicates a long-term good biocompatibility of the four FS coating for cells of fibroblastic nature. For what concerns keratinocytes, they showed good compatibility and reasonable cell growth, which was comparable to the controls, after 3 day of culture and after prolonged culture for 15 onto all of the FS-coated plates (Figure 8B, black bars and grey bars, respectively). Conversely, a slight, but significant, decrease of cell number at 6 day onto F3 and F4 coatings, as compared to the control, was observed (white bars, 36.0% and 37.2% decrease, respectively), probably indicating that keratinocyte cells undergo a period of adaptation, especially on F3 and F4 coatings, before restarting cell growth.

**Figure 8.** Cell growth evaluation. (**A**) L929 fibroblast cell growth evaluation, by MTT test, on the four

different FS pre-coated plates (F1–F4) after 3 day (black bars), 6 day (white bars) and 15 day (grey bars) incubation. Results are expressed as cell percentages respect to controls seeded on standard rat tail collagen-coated (stCol) wells and are the mean ± S.D. of two experiments performed in quadruplicate. Asterisks indicate significance in Tukey test (black bars ANOVA, *p <* 0.0001; white bars ANOVA, *p <* 0.0001; grey bars ANOVA, *p <* 0.05; Tukey vs. stCol: \* *p <* 0.05, \*\* *p <* 0.001, respectively); (**B**) NCTC keratinocytes cell growth evaluation, by MTT test, on the four different FS pre-coated plates after 3 day (black bars), 6 day (white bars), and 15 day (grey bars) incubation. Results are expressed as cell percentages with respect to controls that are seeded on standard rat tail collagen-coated (stCol) wells and are the mean ± S.D. of two experiments that were performed in quadruplicate. Asterisks indicate significance in Tukey test (black bars ANOVA, *p <* 0.00001; Tukey vs. stCol: \* *p <* 0.05).

#### 2.6.5. Environmental Scanning Electron Microscope (ESEM) Analysis

ESEM analyses were performed on the SCMs that were derived from the four FSs, or fragments thereof for F2 and F4. Furthermore, the analyses were also performed on F1 and F3-derived membranes in the presence of fibroblasts and keratinocytes that were cultured on their sterilized surfaces for 3 day. The ultramicroscopic analysis of the four SCMs alone showed randomly distributed fibril patterns in F1, F3, and F4-derived SCMs (Figure 9A,C,D). Conversely, in F2-derived membranes, the surface that resulted was characterized by an unidentifiable clumped layer (Figure 9B). From F1, F3, and F4-derived SCMs, it was possible to calculate the average of the fibril diameter of *C. reniformis* collagen, which was of 21.08 ± 4.93 nm. Furthermore, in of all the three samples, it was possible to observe either the presence of free single fibrils or bundles of aggregated fibrils forming fibres of higher dimensions. No ultrastructural differences were observed between F1 and F3 crosslinked and not crosslinked membranes (data not shown). In SCM-F1 and F3, the random presence of pores on the surface of the membranes was also observed. SCM-F1 and SCM-F3 pores were measured and they spanned an average value of 6167 ± 2826 nm<sup>2</sup> and 3211 ± 1494 nm2, respectively. The significant difference in the pore dimensions of the two membranes (*p* < 0.005) indicates a higher level of fibre cohesion in F3-derived membranes with respect to F1.

For what concerns the ultrastructural analysis of cells grown onto the SCM-F1 and SCM-F3 membranes, no significant differences could be observed in the degree of cell adherence and surface contact on both of the SCMs, either for fibroblasts (panel E for SCM-F1 and F for SCM-F3), either for keratinocytes (panel G for SCM-F1 and H for SCM-F3). In fact, all of the images that were obtained by the high resolution cell analysis showed a tight adherence to both SCM surfaces, especially in fibroblasts (E,F), and the presence of several cellular elongated processes taking contact with the SCM surface surrounding the cell body in keratinocytes (G,H). These results suggest that both membranes could have a good biocompatibility.

**Figure 9.** Environmental Scanning Electron Microscope (ESEM) analysis. All crosslinked SCMs (**A**–**D**), dehydrated at critical point and graphite covered, were observed with a FESEM Zeiss SUPRA 40 VP instrument, while only SCM-F1 and SCM-F3, in the presence of fibroblasts and keratinocytes (**E**–**H**) dehydrated at critical point and graphite covered, were observed with an ESEM Vega3–Tescan instrument. (**A**) SCM-F1; (**B**) SCM-F2; (**C**) SCM-F3; (**D**) SCM-F4; (**E**,**F**) Visualization of L929 fibroblasts adhesion to the two SCMs; (**E**) SCM-F1; (**F**) SCM-F3; (**G**,**H**) Visualization of NCTC keratinocytes adhesion to the two SCMs; (**G**) SCM-F1; (**H**) SCM-F3. In (**A**–**D**) scale bars span 200 nm; in (**E**–**H**) scale bars span 2 micrometer.

#### *2.7. DPPH Radical Scavenging Activity*

Since it is known that the amino acid residues that are present in collagens show a certain level of antioxidant activity [57], the radical scavenging activity of each FS was evaluated using the DPPH standard assay.

As indicated in Figure 10A, F1 showed the highest radical scavenging activity of 61.78 ± 2.84%, F2 of 14.61 ± 0.61%, whereas for F3 resulted of 26.97 ± 0.23%, and finally F4 showed a radical scavenging activity of 28.89 ± 2.28%. The radical scavenging activity resulted to persist also in F1- and F3-derived membranes (Figure 10B). In this case, the radical scavenging activity was expressed in the function of the membrane surface. In particular, the surface exerting the 50% of the total radical scavenging activity (SA50) measured 242.81 mm2 for SCM-F1, and 665.84 mm<sup>2</sup> for SCM-F3.

**Figure 10.** FS and SCM radical scavenging activity. (**A**) Antioxidant activity of the four different FSs (F1–F4) measured by the spectrophotometric DPPH oxidation assay using a concentration of 1 mg/mL of each FS. Results are expressed as percentages of radical scavenging activity based on the inhibition of DPPH oxidation calculated, as described in Section 4.7, and are the mean ± S.D. of two experiments performed in triplicate (ANOVA, *p <* 0.000001); (**B**) Antioxidant activity of SCM-F1 and SCM-F3 measured by the DPPH assay. Results are expressed as percentages of radical scavenging activity in function of the SCM surface area of the two membranes, and are the mean ± S.D. of two experiments performed in triplicate (ANOVA, *p <* 0.000001).
