4.7.2. DMA|DMTA

Dynamic mechanical (DMA) and dynamic mechanical-thermal analysis (DMTA) were carried out on the SCFMs via an MCR 301 rheometer (Anton Paar, GmbH, Graz, Austria) equipped with a universal extensional fixture (UXF) geometry and a CDT-450 chamber. Rectangular specimens (40 mm × 10 mm) were prepared from the samples by using a punch cutter, and each sample thickness was measured via a digital micrometre. A static extensional stress (σs) of 2 MPa was applied for all experiments to ensure the correct sample loading and result reliability.

The linear viscoelastic region (LVER) of the samples was first explored via amplitude sweep tests (AS) at T = 25 ± 1 ◦C using a frequency (ν) and oscillatory extensional stress (σ) of 1 Hz and in the range 0.01–10%, respectively. Then, frequency sweep tests (FS) were carried out at T = 25 ± 1 ◦C with a fixed σ = 0.1 MPa, varying the frequency between 0.01 and 10 Hz. Finally, temperature sweep tests (TS) were carried out in the temperature range 25–100 ◦C with a heating rate of 2 ◦C/min.

### 4.7.3. In Vitro Degradation Study

To measure the in vitro degradation, each membrane type was weighed (designated as "Wi") and transferred in a test tube containing 10 mL of PBS (pH = 7.4) alone or added with 0.1 mg/mL collagenase from *Clostridium histolyticum* and kept at 37 ◦C. After 7, 14 and 21 days, the samples were recovered, washed twice with deionised water, dried and weighed again (designated as "Wf"). The percent degradation of the membranes was computed by the following equation:

$$\text{percent degradation (\%)} = \text{(Víi - Wf)} \text{ / } \text{Wí} \times 100\%$$

The procedure was carried in triplicate.

To evaluate the release of organic compounds from the SCFMs in saline or collagenase solution withing 21 days, the absorbance at 280 nm was read in the membrane incubation media after 7, 14 and 21 days using PBS or collagenase solution as a blank sample, respectively.

To detect the percentage of collagen material released in saline solution or hydrolysed by collagenase activity from SCFMs during the stability test, 0.4 mL of the incubation media was recovered at 7, 14 and 21 days, hydrolysed in NaOH 2 M at 120 ◦C at 1 Atm, and finally hydroxyproline content was evaluated as previously described [11].

### 4.7.4. Swelling Test

To evaluate the water binding property of SCFMs, the initial weight of dried membrane (Wi) was measured and then they were soaked in deionised water at 37 ◦C for 20 h. Finally, the wet weight of the scaffolds (designated as "Ww") was again recorded. A second weighing of the samples after 48 h of incubation without registering a further weight gain ensured that the samples had reached their maximum degree of hydration. Finally, the water content was calculated based on the equation:

> water content (%) = (Ww − Wi) / Ww × 100%

Experiments were performed in triplicate.

### 4.7.5. DPPH Radical Scavenging Activity

The radical scavenging activity was evaluated on each type of SCFMs as described in [11]. Briefly, 25 × 28 mm membranes were soaked into 500 μL of deionised water, and then embedded into 250 mL of 0.1 mM DPPH in methanol solution (2,2-diphenyl-1- picrylhydrazyl, Calbiochem ®, Millipore SpA, Milan, Italy). In the same manner, a negative control sample with deionised water was prepared. The samples were left in incubation for 30 min at room temperature in the dark. Then, the membranes were removed with tweezers, and finally the sample solutions were read at 517 nm using a Beckman spectrophotometer (DU 640). The blank sample was prepared by replacing the DPPH solution with methanol. The antioxidant activity of the samples was evaluated by the inhibition percentage of DPPH radical using the following equation:

> DPPH radical scavenging activity (%) = (A0 − A)/A0 × 100%

where A was sample absorbance rate; A0 was the absorbance of the negative control. The procedure was carried out in triplicate.

### *4.8. SCM Biocompatibility Evaluation*

### 4.8.1. Cell Cultures

The L929 mouse fibroblast cell line was obtained by the National Collection of Type Cultures (NCTC), while the human keratinocyte HaCaT cell line (CLS Cell Lines Service, 300493) was obtained by the Cell Lines Service (GmbH, Eppelheim, Germany).

Cells were maintained at 37 ◦C in a humidified, 5% CO2 atmosphere, in high glucose Dulbecco's modified Eagle's medium (D-MEM) with glutamax (Euroclone, Milan, Italy), which was supplemented with 10% FBS (Euroclone) and with the addition of penicillin/streptomycin as antibiotics.

### 4.8.2. Cell Growth and Cell Adhesion

L929 and HaCaT cell lines were seeded at a density of 50,000 cells/well on 96-well plates that were, or not, pre-coated with each type of SCFs, or rat tail standard collagen as described in Section 4.4 for evaluating cell adhesion. Cells were allowed to adhere for 16 h at 37 ◦C in complete medium; the medium was subsequently removed, the adhered cells were washed once with PBS to remove the floating unattached ones, and finally MTT (0.5 mg/mL final concentration) test was performed as well to estimate the amount of attached cells when compared to control cells on uncoated wells. Data are means ± S.D. of four independent experiments.

To evaluate cell growth on SCF-coated plates, experiments were performed on 96-well plates. L929 and HaCaT cell lines were both plated at a density of 5000 cells/well on, or not, pre-coated wells. Cells were cultured for 3 days and 6 days at 37 ◦C in complete medium. At the end of the experiments, the MTT test was once again performed to evaluate cell viability.

For image acquisition, light microscopy cells were seeded at a density of 200,000 cells/well on 12-well plates pre-coated or not, with SCF or rat tail collagen and incubated for 24 h at 37 ◦C. At the end of the experiment, cells were washed once with PBS, fixed for 30 min at room temperature with 4% paraformaldehyde and then dehydrated in a 70% ethanolic solution. After fixation in buffered 4% paraformaldehyde, cells were rinsed for 5 min three times in PBS (0.1 M, pH 7.4), permeabilised for 10 min in 0.2% Triton-X 100 in PBS and thus rinsed again. Then, they were incubated in a blocking solution with 1% bovine serum albumin and 0.1% Tween 20 in PBS. After rinsing, cells were incubated in a moist dark chamber with Alexa Fluor 488-conjugated Phalloidin (1:40 in PBS, Invitrogen) for 30 min at room temperature. For image acquisition, an inverted optical microscope (IX53 Olympus, Tokyo, Japan) equipped with a CCD camera (U-LH100HG Olympus, Tokyo, Japan) was utilised, and the relative software was used.

### 4.8.3. L929 Fibroblast Gene Expression Analysis

L929 mouse fibroblast cells were seeded at a density of 200,000 cell/well on 12-well plates that were, or not, pre-coated with SCFs, or rat tail standard collagen, and were incubated for 24 h at 37 ◦C. At the end of the experiment, total RNA was extracted using the RNeasy Mini Kit, (Qiagen, Milan, Italy) according to the manufacturer's instructions.

The cDNA was synthesised by Revert Aid Reverse Transcriptase (Thermo Fisher Scientific, Milan, Italy) using 1 μg of purified total RNA from each sample. Each PCR reaction was performed in 15 μL containing: 1× master mix iQ SYBR ®Green (Bio-Rad), 0.2 μM of each primer and 3 μL of a 1:5 diluted reverse transcription reaction buffer. Each sample was analysed in triplicate. The following thermal conditions were used: initial denaturation at 95 ◦C for 3 min, followed by 45 cycles with denaturation at 95 ◦C for 15 s and annealing and elongation at 60 ◦C for 60 s. At the end of each elongation step, fluorescence was measured. Values were normalised to GAPDH (reference gene) mRNA expression. All the PCR primers (Table S1) were designed by means of the Beacon Designer 7.0 software (Premier Biosoft International, Palo Alto, CA, USA) and obtained from TibMolBiol (Genova, Italy). Data analyses were acquired by the DNA Engine Opticon ®3 Real-Time Detection System Software program (3.03 version) and, in order to calculate the relative gene expression compared to an untreated (control) calibrator sample, the comparative threshold Ct method was used within the gene expression analysis for iCycler iQ Real Time Detection System Software ®(2004 Bio-Rad, Milan, Italy). Data are means ± S.D. of two independent experiments performed in triplicate.

### 4.8.4. L929 Fibroblast Collagen Synthesis Evaluation

Collagen synthesis by L929 fibroblasts was quantified in the cell medium by applying the SIRCOLTM Soluble Collagen Assay (Biocolor Ltd., Carrickfergus, Northern Ireland, UK). Fibroblasts were seeded in tissue culture 12-well plates at a density of 200,000 cells/well that were, or not, pre-coated with each type of SCF and incubated for 48 h at 37 ◦C.

At the end of the incubation, cell culture media were collected, and the SIRCOL assay was performed according to the manufacturer's instructions. Data are the means ± S.D. of two independent experiments performed in triplicate. Cell culture media alone or preincubated for 48 h a 37 ◦C with the respective coating tested was used as a blank sample for control or for each sample, respectively.

### *4.9. Statistical Analyses*

Statistical analyses were performed using one-way ANOVA plus Tukey's post test (GraphPad Software, Inc., San Diego, CA, USA). *p* values < 0.05 were considered to be significant.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1660-3 397/19/10/563/s1, Figure S1: EDS spectra of *S. foetidus* filaments. Figure S2: *S. foetidus* collagen filament in native tissue. Table S1: S1 primer sequences used in the qPCR analyses.

**Author Contributions:** Conceptualisation, methodology, writing—original draft preparation, M.P.; methodology and software, E.T.; investigation and data curation, A.D., M.C., S.V., S.F., S.A. and D.C.; collection and specie attribution of sponge specimens, M.B.; review, editing and founding acquisition, H.E.; visualisation, I.P.; review and funding acquisition, M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by University of Genova Funding to M.P. and to DAAD Joint Mobility Program 2017 DAAD-Italy Project "Marine Sponges as Sources for Bioinspired Materials Science". Prog. n. 35474 funding by the Italian Ministry of University and Research (MIUR) to M.G. H.E. was partially supported by MAESTRO 12 project (NCN, Poland) and I.P. by Polish National Agency for Academic Exchange (NAWA) Ulam International Programme PPN/ULM/2020/1/00177.

**Institutional Review Board Statement:** The marine sponge specimens used in this study are not protected species, and were sampled in the protected marine area of Portofino promontory with scientific research authorization n. 4/2012 (prot. No. 409 / 2-1-1).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are indebted to Laura Negretti for their precious technical support in ESEM analyses; thank Patrizia Arcidiaco, Centro Grandi Strumenti, University of Pavia, Italy, for amino acid analysis; and Francisco Ardini, Department of Chemistry and Industrial Chemistry, for ICP–AES analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
