*4.4. IR-Spectroscopy*

The FTIR analysis of the initial components was carried out using a Spectrum Two FT-IR Spectrometer (PerkinElmer, Waltham, MA, USA) in the Attenuated Total Reflectance (ATR) mode. The spectrometer features were as follows: a high-performance, roomtemperature LiTaO3 MIR detector, a standard optical system with KBr windows for the data collection over a spectral range of 8300–350 cm<sup>−</sup><sup>1</sup> at a resolution of 0.5 cm<sup>−</sup>1. All the spectra were initially collected in the ATR mode and converted into the IR transmittance mode. The spectra of collagens were normalized using the intensity of the Amid I band as the internal standard.

### *4.5. Differential Scanning Calorimetry (DSC)*

Differential scanning calorimetry (DSC) measurements were performed using an STA 6000 simultaneous thermal analyzer (PerkinElmer, Waltham, MA, USA). Samples for DSC experiments (about 10 mg) were encapsulated in standard PerkinElmer pans and heated in a nitrogen medium at a gas flow rate of 20 mL/min and a linear heating rate of 10 ◦C/min.

### *4.6. Shrinkage Temperature*

A sample with the sizes of 3 × 15 mm was placed in a special calibrated glass tube and immersed in a vessel filled with distilled water. The vessel was placed in a water bath. The water bath was heated from room temperature to the moment of the sample shrinkage (~60 ◦C). The shrinkage temperature was determined as the temperature at which the beginning of the sample shrinkage was detected. The experiment was repeated thrice.

### *4.7. Histological Study*

Intact and collagenase-treated fragments of the GSCM of *D. gigas* were fixed in a 10% solution of neutral formalin, and 4 μm-thick histological sections were prepared using a standard technique.

The prepared sections were stained with hematoxylin and eosin and picrosirius red to reveal the collagen composition. The prepared slides were studied by optical (bright-field, phase contrast and polarized light) microscopy, and the images were captured with a LEICA DM4000 B LED microscope equipped with a LEICA DFC7000 T digital camera, using the LAS V4.8 software (Leica Microsystems, Heerbrugg, Switserland).

### *4.8. Scanning Electron Microscopy (SEM)*

The GSCM structure was visualized using an EVO LS10 scanning electron microscope (Carl Zeiss Microscopy GmbH Jena, Germany). Two techniques for sample preparation and visualization were used.

The first protocol allowed general images of the samples in the most native state. Naturally dried samples were attached to the microscope stage with a special carbon

adhesive tape. The observations were conducted in the low vacuum regime (EP, 70 Pa) at the accelerating voltage of 20 kV and the current of 94 pA per sample. A detector for back-scattered electrons (BSE) was used. The images were obtained with the resolutions of 473.1 nm/px and 508.8 nm/px. To achieve a satisfactory resolution during back-scattered electrons observations, a working distance of 4.5 mm was used.

For detailed evaluation of the structure, samples were fixed in neutral glutaric aldehyde, dehydrated (battery of alcohols from 20% to 97% and acetone), dried bypassing the critical point of CO2,, and coated with an Au-Pd alloy. The so-prepared samples were attached to the microscope stage providing the charge outflow from the coated surface. The observations were conducted in the high vacuum regime at the accelerating voltage of 21 kV and the sample current of 19 pA. The microtopography images were obtained using the detector for secondary electrons (SE). The 3072 × 2304 px images were captured with the resolutions of 89.89 nm/px and 2697 nm/px.

### *4.9. Laser Scanning Microscopy (Second Harmonics Generation, SHG Signal)*

The study was performed using a LSM 880 NLO laser scanning microscope (Carl Zeiss Microscopy GmbH Jena, Germany) equipped with a tunable Ti:Sa MaiTai HP laser (Spectra-Physics, Milpitas, CA, USA) with a pulse duration of less than 100 fs. The wavelength of 800 nm was used for the study, and the registration of the SHG signal was performed in the range of 370–420 nm. The power of the probing radiation was about 9 mW. The images were obtained using an oil immersion objective with the 40× magnification and numerical aperture of 1.3. The field of view was 212 × 212 μm, and the resolution of images was 1024 × 1024 pixels. A series of images (z-stack) was acquired from the sample surface into the depth with the step of 9 μm, the orientation being parallel to the surface. For the convenience of perception, the acquired images were presented in the green palette.

### *4.10. Atomic Force Microscopy (AFM)*

The morphological AFM studies of the surface were performed using an atomic force microscope (BioScope Resolve, Bruker, Billerica, MA, USA) combined with an Axio Observer inverted optical microscope (Carl Zeiss Microscopy GmbH Jena, Germany). A ScanAsyst Air cantilever (Bruker, Billerica, MA, USA) was used with a nominal spring constant k = 0.4 N/m and a nominal tip radius r = 2 nm, and scanning was performed on air in the PeakForce QNM regime. The collagen structures' periodicity was estimated with the Section function of the NanoScope Analysis v1.9 software (Bruker, Billerica, MA, USA).

### *4.11. Uniaxial Stretching Test*

The uniaxial stretching tests for dry and hydrated samples were conducted using a Mach-1 v500c mechanical tester (Biomomentum, Laval, QC, Canada). For the hydration, samples were immersed in distilled water for 20 min. The measurements were also performed in distilled water. Dumbbell-shaped fragments of the dry and hydrated GSCM were cut both in the tangential and radial directions in respect to the whole material area (with the circle diameter of 30 cm). The working area of the fragments had the length of 15 mm and width of 5 mm. The dry material thickness was 45 μm, while the thickness of the hydrated material was 60 μm. Before the test, the mechanical tester was calibrated using a standard sample provided by the manufacturer. Both ends of the experimental sample were tightly gripped in the clamps followed by gradual elongation at room temperature (25 ◦C) at a constant rate of 0.1 mm/s until rupture. The mechanical parameters were calculated from the stress-strain curves according to the manufacturer's protocol. The data were averaged over 3 or more tests.

### *4.12. Micromechanics by AFM*

The mechanical properties of the samples' surface were studied in fluid (distilled water) at room temperature (25 ◦C), after 20 min of hydration, using an atomic force microscope (BioScope Resolve, Bruker, Billerica, MA, USA). The sample micromechanics was obtained in the regime of nanoindentation over a preset map of 50 × 50 μm with the 32 × 32 pixels resolution, as described in [84]. A ScanAsyst Fluid cantilever (Bruker, Billerica, MA, USA) with a nominal spring constant of 0.95 N/m and a nominal tip radius of 50 nm was precalibrated using a standard titanium sample. The deflection sensitivity was calibrated in the same conditions using a sapphire standard sample. The data were processed using the NanoScope Analysis v1.9 software(Bruker, Billerica, MA, USA) and averaged over 12 measurements.

### *4.13. In Vitro Cytotoxicity Assays*

The biocompatibility and cytotoxicity tests were performed using the primary culture of mesenchymal stromal cells (MSCs) isolated from human gingival mucosa as described in [85]. The cells were cultivated in the medium that contained Dulbecco's Modified Eagle's Medium (DMEM)/F12 (1:1, Biolot, St. Petersburg, Russia), 10% fetal calf serum (HyClone, Logan, UT, USA), L-glutamine (5 mg/mL, Gibco, Gaithersburg, MD, USA), insulin–transferrin–sodium selenite (1:100, Biolot, St. Petersburg, Russia), bFGF (20 ng/mL, ProSpec, Rehovot, Israel), and gentamycin (50 μg/mL, Paneco, Moscow, Russia). Isolated cells were routinely checked with a SH800S microfluidic flow cytometer (Sony Biotechnology, San Jose, CA, USA) for the presence of mesenchymal surface markers (CD90, CD73, CD105) and absence of hematopoietic and endothelial markers (CD45, CD34, CD11b, CD19 and HLA-DR), according to [86]. The cells were cultivated in the standard conditions of 37 ◦C and 5% CO2.

The cytotoxicity was analyzed via the elution and contact tests. In the first case, the extracts of the GSCM were prepared according to recommendations of ISO 10993-12. Briefly, 5000 cells per well of a 96-well plate were seeded 24 h before adding the extracts. To prepare extracts, GSCM films were incubated in the culture medium for 24 h at 37 ◦C. The thickness of a film was less than 0.5 mm, and, therefore, in accordance with ISO 10993-12, the required sample's area was to be treated in a volume of 1 mL is 6 cm2. Cells were exposed to the maximum concentration of the extract (6 cm2/mL) and its serial twofold dilutions. We used serial two-fold dilutions of 1.5 mg/mL sodium dodecyl sulphate (SDS) in a standard culture medium as a positive control. Cells cultivated in the standard culture medium were applied as a negative control. After 24 h of cultivation with the extract, SDS, or culture medium, the cell viability was assessed either with the AlamarBlue cell viability reagen<sup>t</sup> (Invitrogen, Waltham, MA, USA) or with the Quant-iT PicoGreen kit (Invitrogen, Waltham, MA, USA). For the AlamarBlue metabolic activity assay, the cell culture medium was replaced with a 10% reagen<sup>t</sup> solution and incubated for 2 h. Then, the fluorescence of samples was measured using a Victor Nivo spectrofluorometer (PerkinElmer, Waltham, MA, USA) at a 530 nm excitation wavelength and a 590 nm emission wavelength. The DNA amount was evaluated with the PicoGreen assay after 3 freeze-thaw cycles aimed at releasing DNA, following the manufacturer's instructions. The samples' fluorescence was estimated with the spectrofluorometer at a 480-nm excitation wavelength and a 520-nm emission wavelength.

For the contact cytotoxicity, 20,000 cells were seeded on a surface of the 1 cm<sup>2</sup> GSCM films and cultivated for 3 days. Cells seeded on the culture plastic (monolayer culture) served as a control. Afterwards, the metabolic activity and DNA amount were measured as described above.

The morphology and viability of the cells seeded on the GSCM was visualized with the Live/Dead assay. Briefly, live cells were stained with calcein-AM (Sigma-Aldrich, St. Lois, MO, USA), dead cells were stained with propidium iodide (Thermofisher, Waltham, MA, USA), and nuclei were stained with Hoechst 33,258 (Thermofisher, Waltham, MA, USA). The images were obtained by laser confocal scanning microscopy using a LSM 880 instrument with Airyscan (Carl Zeiss Microscopy GmbH Jena, Germany).

All the samples were triplicated (plate wells for extract cytotoxicity and film samples for the contact cytotoxicity and Live/Dead assay).

### *4.14. Resistance to Collagenase*

The susceptibility to proteolytic degradation was studied in a Collagenase A (from C histolyticum) solution. Approximately 4 mg (dry weight in triplicates) of the sample were weighed. To the weighed samples, 0.5 mL aliquots of a 2.5 mg/mL Collagenase A solution in the Tris buffer (50 mmol/L, pH 7.5) containing 10 mmol/L calcium chloride and 0.02 mg/mL sodium azide (Paneco, Moscow, Russia) were added. The samples were incubated at 37 ◦C for 6 h. Then, the samples were centrifuged at 605 g (3000 RPM) for 90 s (a MiniSpin microcentrifuge by Eppendorf Corporation, Hamburg, Germany). We used a low rotation speed and a short time of centrifugation in order to better preserve the structure integrity for the following histological analysis. Then, the material was washed from the residual collagenase with distilled water. The precipitate was carefully transferred using a micropipette to a coverslip for the following drying in an oven at 50 ◦C for 20 h. Then, the dry residue was weighed using a WXTE ultramicrobalance (Mettler Toledo GmbH Urdorf, Switzerland). Finally, the weight loss was calculated by a paired comparison before and after the treatment.

### *4.15. LAL Test*

The GSCM film was cut into 5\*5-mm pieces under aseptic conditions. The extracts were prepared in 1 mL of endotoxin-free water by continuous shaking for 24 h at 50 ◦C. The endotoxin concentrations were measured using the Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instruction. Briefly, we mixed 50 μL of the extract or the endotoxin standard dilution (0.1, 0.25, 0.5, 0.1 U/mL) and 50 μL of endotoxin-specific Limulus Amebocyte Lysate (LAL) reagen<sup>t</sup> in a well of a 96-well plate. The mixture was incubated for 10 min at 37 ◦C and then 100 μL of the chromogenic substrate was added and incubated for 6 min at 37 ◦C. The reaction was inhibited by adding 100 μL of 25% acetic acid. The absorbance was measured at a wavelength of 405 nm using a microplate Victor Nivo spectrofluorometer (PerkinElmer, Waltham, MA, USA). The minimal detection level of the kit used was 0.1 EU/mL (EU—unit of measurement for endotoxin activity).
