*4.1. Chemicals*

All reagents were acquired from SIGMA-ALDRICH (Milan, Italy), unless otherwise stated.

### *4.2. Sponge Sampling*

Specimens of *Ircinia oros* (Schmidt, 1864) and *Sarcotragus foetidus* (Schmidt, 1862) were harvested from scuba diving in the area of the Portofino Promontory (Liguria, Italy) at depths of 10–20 m, and were transferred to a laboratory in a thermic bag maintained at 14–15 ◦C. The sponge specimens were frozen at −20 ◦C until further processing.

### *4.3. Sponge Collagenous Filaments and Intercellular Collagen Isolation*

Frozen sponges were extensively washed in running tap water to remove sand residues, and finally washed with distilled water. The sponge tissues were minced in small pieces with scissors and enzymatically digested, as previously described [18]. The flowsheet of the extraction procedure is shown in Scheme 1. A total of 20 g of cut sponge tissues were treated with 0.1% trypsin in 100 mL of ammonium bicarbonate, pH 8.5, overnight at 37 ◦C on a horizontal shaker. Subsequently, the dark fluid was removed by filtration with a metallic strainer, and the solid material was suspended in three volumes of cool deionised water and incubated at 5 ◦C for three days in a rotary disc shaker aliquoted in 50 mL tubes.

**Scheme 1.** Schematic representation of the extraction procedure used to isolate the collagen filaments from *I. oros* and *S. foetidus* tissue.

This treatment efficiently disperses the intercellular collagen fibrils in water, leaving only the brown spongin scaffold combined with the collagenous filaments in the final residue. These last structures, combined with residual intercellular collagen, were finally separated from the fibrous spongin matrix through different rounds of sedimentation steps under gravity, by mild stirring in large volumes of distilled water and repeated decantations. To obtain a homogenous suspension for *S. foetidus* collagenous filaments, the sample was subjected to a short round of homogenisation steps in ice for 15 s at the end of the extraction using Ultra Turrax T25 basic (IKA-WERKE, Staufen im Breisgau, Germany). During all the extraction phases, the filaments' suspension was never subjected to centrifugation steps to avoid knotting the filaments. The filaments were extensively washed with distilled water to allow them to settle by gravity. The extracted sponge collagenous filament suspensions were conserved at 4 ◦C. To establish the concentration of the sponge collagen filaments, 1 mL of each suspension was lyophilised, and the dry material was weighed.

### *4.4. Light Microscopy and Environmental Scanning Electron Microscope (ESEM) Observation*

*I. oros* and *S. foetidus* tissue fragments, isolated sponge collagenous filaments and sponge collagenous filament membranes (SCFMs) were observed in light microscopy through a stereomicroscope (Nikon SMZ1000, Nikon, Tokyo, Japan) equipped with a digital camera (digital Sight DS-SM, Nikon). Isolated sponge collagenous filaments were coloured with picro-sirius red, as described in [53], in order to prove their collagen composition. For ESEM observation, isolated sponge collagenous filaments and SCFMs were firstly completely dehydrated by soaking them in a series of alcoholic solutions with an increasing concentration of ethanol up to 100%, then graphite was covered and examined. Images of the samples were observed and acquired with an ESEM Vega3–Tescan, type LMU (Tescan Brno s.r.o., Brno, Czech Republic) provided with a microanalyser system EDS-Apollo\_x and EDS texture and elemental analytical microscopy software (TEAMTM Analysis System, version 1.0, Coherent Scientific, Thebarton SA). Observation and acquisition of the four SCFMs images were performed with a FESEM Zeiss SUPRA 40 VP (Carl Zeiss AG, Oberkochen, Germany) and its associated software. The fibrillar diameter and the pore areas observed in the collagen membranes were analysed by performing physical measurements on the images of the various membranes acquired with the FESEM, using the ImageJ free software (version 1.53 Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2016). Means ± S.D. were calculated on at least 40 random measurements of fibril diameter or pore areas performed on each membrane.

### *4.5. SCFs Biochemical Characterisation*

### 4.5.1. Amino Acid Composition

Amino acid analysis was performed using a Jasco X- LC system equipped with an autosampler, Xtreme high pressure pumps, degasser, column oven compartment, fluorescence detector connected to a HP ProDesk processor, as described in [54]. In total, 0.5 mL of each sponge collagenous filament suspensions at 2 mg/mL were hydrolysed in NaOH 2 N for 20 min a 121 ◦C at 1 Atm and neutralised with equal volume of HCl 2N.

Hydrolysed amino acids were then derivatised by ortho-phthalaldehyde (OPA) and fluorenylmethylchloroformate (FMOC), leading to the formation of derivatives from primary amino acids and secondary amino acids, respectively. Derivatisation was performed accordingly wiyh Jasco autosampler program. The derivatives were detected with a fluorometric detector (emission λ = 446 nm – excitation λ = 340 nm for OPA derivatives and emission λ = 268 nm – excitation λ = 308 nm for FMOC derivatives). Amino acid identification was performed via elution times of the obtained derivatives and compared to a mixture of standard amino acids submitted to derivation in identical test conditions. Data processing software (ChromNav, version 2.0, JASCO, Inc., Easton, MD, USA) allowed integration of peak areas for the assessment of the amount of amino acids occurring in the sample.

### 4.5.2. Glycosaminoglycans (GAGs) Quantification

The quantitative evaluation of the GAGs content in sponge collagen filaments was obtained using the Alcian blue assay as described in [11] and expressed as μg of dry weight of collagen filaments.

### 4.5.3. Quantitative Analysis of Iron Content

The μg of iron present in sponge collagen filaments was obtained by inductively coupled plasma atomic emission (ICP–AES), as described in [55].

### 4.5.4. Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE)

Protein patterns of sponge collagenous filament samples were analysed by SDS–PAGE using Mini-Protean 3 (Bio-Rad Laboratories, Hercules, CA, USA), according to the method previously described [56]. A total of 0.5 mL of each sponge collagen filament suspension normalised at 4 mg/mL were added to an equal to volume of acid-washed glass beads (0.5 mm diameter) and vortexed 3 times for 30 s. Samples were mixed at 1:1 (*v/v*) ratio with 2× gel loading buffer (1 M Tris–HCl buffer (pH 6.8), 10% 2-mercaptoethanol, 40% glycerol, 0.2% bromophenol blue and 20% Sodium Dodecyl Sulfate solution) and was heated at 90 ◦C for 5 min, and 40 μL were loaded in a 7% of polyacrylamide gel and run at 60 mA with constant amperage. After electrophoresis, the gel was fixed for 1 h a room temperature in a solution containing 10% (*v/v*) acetic acid and 40% (*v/v*) ethanol), washed twice for 10 min at room temperature with distilled water and stained over night at room temperature with a staining solution obtained combing 80 mL of colloidal Coomassie solution (0.1% (*w/v*) Coomassie Brilant blue G250, 2% (*w/v*) ortho-phosphoric acid, 10 (*w/v*) ammonium sulphate) with 20 mL of methanol. Finally, the gel was destained with 5% acetic acid solution and acquired with ChemiDoc Imaging System (Bio-Rad, Milan, Italy). Rat tail type I collagen (0.5 mg/mL) was run alongside as control.

### *4.6. SCFMs Production*

SCFMs were obtained by casting 2 mg/mL of sponge collagenous filament suspension in silicone moulds as rectangular (25 × 28 mm) sheets that were filled with 3.3 mL of 2 mg/mL of each suspension, and as rectangular (10 × 45 mm) sheets for mechanical tests, filled with 2.25 mL of 2 mg/mL of each SCF suspension and let completely dry at 37 ◦C overnight. For DSC analysis, 3 mg of sponge collagenous filaments were left to dry directly on metallic melting pot. For biocompatibility tests, 2 mg/mL of sponge collagenous filament suspension derived from each sponge species and a standard rat tail collagen were used to directly coat 24-well and 96-well plates. In total, 300 μL (for 24-well plates) or 50 μL (for 96-well plates) were placed on the plates and were left to dry at 37 ◦C overnight. The coated plates were then washed thrice with 100% ethanol, UV sterilised for 20 min and conserved at 4 ◦C until use.

### *4.7. SCFMs Characterisation*

### 4.7.1. Differential Scanning Calorimetry

DSC was performed using a DSC1 STARe System (Mettler-Toledo, Switzerland). About 3 mg dry SCFs were placed in aluminium crucibles and analysed with increasing heat from 0 to 200 ◦C at a heating rate of 5 ◦C/min. During the DSC runs, a nitrogen flow at a rate of 20 mL/min was constantly applied.

As a control, 3 mg of a commercial porcine collagen membrane Bio-Gide ®(Geistlich Pharma AG, Wolhusen, Switzerland) was dried on a metallic melting pot and analysed on the DSC following the same procedure outlined above.
