*2.1. Materials*

HA (Ophthalmic grade, 800–1000 kDa, Bioiberica, Barcelona, Spain) and Ch with a degree of deacetylation of 90% (Medical grade, Mw: 200–500 kDa, Altakitin SA, Lisboa, Portugal) were used as received. Methacrylic anhydride (MA), poly (ethylene glycol) dimethacrylate (PEGDMA, Mn: 8000 Da) and the photoinitiator Irgacure 2959 were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received. G1Phy was prepared as previously described by Ana Mora-Boza et al. [24], using phytic acid and glycerol from Sigma Aldrich. Solvents as isopropanol (Scharlau, Barcelona, Spain) and ethanol (BDH Chemicals, Philadelphia, PA, USA) were used as received. Dialysis membranes (3500 Da cut off) were purchased from Spectrum® (Columbia, MO, USA). Additional reagents such as phosphate buffered saline (PBS), calcium chloride, nitric acid 65% (v/v), acetic acid (AA) and sodium hydroxide were purchased from Thermo Fisher Scientific Corporation (Waltham, MA, USA). Tris hydrochloride, 1 M solution (pH 7.5/Mol. Biol.) was purchase from Fisher BioReagents (Waltham, MA, USA).

### *2.2. Synthesis of Methacrylated Hyaluronic Acid*

HAMA was synthesized through an esterification reaction in alkaline conditions following the protocol described by Khunmaneeet et al. [28]. HA (1 g) was dissolved in 100 mL of Milli-Q water in a two necked glass flask for 24 h. MA was added to the HA solution at a MA:HA ratio of 1:1. The mixture was kept at 0 ◦C using an ice bath and the pH was controlled at 8.5 by adding NaOH (5 M) with the help of an automatic titrator (Metrohm, Switzerland) for 24 h. The final product was purified by precipitation in cold ethanol, subsequently centrifuged (Eppendorf centrifuge 5810 R model, Madrid, Spain), dissolved in double distilled water (ddH2O), and dialyzed for 4 days. After freeze drying, a white powder was finally obtained. HAMA was characterized by proton nuclear magnetic resonance (1H-NMR, Bruker AVANCE IIIHD-400, MA, USA) and attenuated total reflection–Fourier transform infra-red (ATR-FTIR, Perkin-Elmer (Spectrum One), Waltham, MA, USA) spectroscopies. HAMA methacrylation degree was determined by its <sup>1</sup>H-NMR spectrum giving a value of 4.5% (Figure S1).

### *2.3. Preparation of Ch Membranes*

Dried Ch was dissolved at a concentration of 2 wt.% in 1% AA water solution containing 13 wt.% CaCl<sup>2</sup> respect to Ch. Once it was dissolved, it was poured into a glass petri dish (internal diameter: 49 mm) and dried under moister conditions at room temperature until constant weight. Then, membranes were detached from the petri dishes after 5 min of incubation in NaOH and subsequently rinsed with Milli-Q water until neutral pH was reached. Finally, membranes were ionically crosslinked by their immersion into a G1Phy water solution at a concentration of 15 mg/mL (30 wt.% respect to chitosan) for 24 h and room temperature. The uncoupled G1Phy was removed by rinsing twice the membranes with Milli-Q water.

### *2.4. Preparation of Ch*/*HA and Ch*/*HAMA Membranes*

Either type of membranes, Ch/HA or Ch/HAMA, were prepared with a content of 75% of Ch and 25% of HA or HAMA, respectively. Either HA or HAMA solution in 1% AA with CaCl<sup>2</sup> (%) was added to the Ch solution together with additional drops of 2 M HCl to achieve the total dissolution of both polymers. For Ch/HAMA membranes, HAMA solution was supplemented with 5% PEGDMA crosslinker and 2% of photoinitiator Irgacure 2959, both respect to HAMA content, in order to trigger

photopolymerization by UV-light irradiation. Therefore, the corresponding solution was poured and irradiated at 365 nm for 15 min using a UVP chamber photoreactor (CL-1000, Thermo Fisher Scientific Corporation, MA, US), equipped with 5 bulbs of 365 nm working at an intensity of 2.9 mW/cm<sup>2</sup> . Finally, membranes were submitted to ionic crosslinking with G1Phy following the same protocol as described in Section 2.3. All membranes were prepared in the form of a circle of 5 cm diameter using a petri dish and with thicknesses between 0.22 ± 0.03 to 0.42 ± 0.06 mm. Those membranes were punched with diameters of 12 mm for the in vitro experiments and biological assays. Figure 1 shows the polymer and crosslinker compositions used for the fabrication of each system along with the digital images of the as-obtained membranes. poured and irradiated at 365 nm for 15 min using a UVP chamber photoreactor (CL-1000, Thermo Fisher Scientific Corporation, MA, US), equipped with 5 bulbs of 365 nm working at an intensity of 2.9 mW/cm2. Finally, membranes were submitted to ionic crosslinking with G1Phy following the same protocol as described in Section 2.3. All membranes were prepared in the form of a circle of 5 cm diameter using a petri dish and with thicknesses between 0.22 ± 0.03 to 0.42 ± 0.06 mm. Those membranes were punched with diameters of 12 mm for the in vitro experiments and biological assays. Figure 1 shows the polymer and crosslinker compositions used for the fabrication of each system along with the digital images of the as-obtained membranes.

*Polymers* **2020**, *12*, x FOR PEER REVIEW 4 of 18


**Figure 1.** Polymer and crosslinker concentrations (wt.%) used for the fabrication of Ch membranes, semi-, and IPNs developed in this work (**A**); Digital images of the developed systems (**B**). **Figure 1.** Polymer and crosslinker concentrations (wt.%) used for the fabrication of Ch membranes, semi-, and IPNs developed in this work (**A**); Digital images of the developed systems (**B**).

### *2.5. Characterisation Techniques 2.5. Characterisation Techniques*

1H-NMR spectra were recorded with a Varian Mercury 400 MHz (Agilent, Santa Clara, CA, USA). The spectra were carried out at 25 °C in D2O (10% w/v) and referenced to the residual proton <sup>1</sup>H-NMR spectra were recorded with a Varian Mercury 400 MHz (Agilent, Santa Clara, CA, USA). The spectra were carried out at 25 ◦C in D2O (10% w/v) and referenced to the residual proton absorption of the solvent, D2O [4.7 ppm].

absorption of the solvent, D2O [4.7 ppm]. ATR-FTIR of samples were carried out on a Perkin-Elmer Spectrum BX spectrophotometer (MA, US). All spectra were recorded from 600 to 4000 cm−1 with a resolution of 4 cm−1 and 32 scans. ATR-FTIR of samples were carried out on a Perkin-Elmer Spectrum BX spectrophotometer (MA, USA). All spectra were recorded from 600 to 4000 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> and 32 scans.

Elemental analysis (EA) was performed with an elemental LECO model CHNS-932 microanalyzer (MI, US). The determination of C and H was carried out with CO2 and H2O specific infrared detectors, while N (N2) was determined by thermic conductivity. The measurements were Elemental analysis (EA) was performed with an elemental LECO model CHNS-932 microanalyzer (MI, USA). The determination of C and H was carried out with CO<sup>2</sup> and H2O specific infrared detectors, while N (N2) was determined by thermic conductivity. The measurements were conducted at 990 ◦<sup>C</sup> using He as transporter gas.

conducted at 990 °C using He as transporter gas. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were carried out in a 4300 DV Perkin-Elmer plasma emission spectrometer (MA, US) under dynamic argon flow at 16 L/min using a Gemcone (Perkin-Elmer, MA, US) nebulizer under dynamic argon flow at Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements were carried out in a 4300 DV Perkin-Elmer plasma emission spectrometer (MA, USA) under dynamic argon flow at 16 L/min using a Gemcone (Perkin-Elmer, MA, USA) nebulizer under dynamic argon flow at 0.8 L/min, and 1300 W of plasma power.

0.8 L/min, and 1300 W of plasma power. Scanning electron microscopy (SEM) images were taken in a Hitachi S-8000 instrument (Tokyo, Scanning electron microscopy (SEM) images were taken in a Hitachi S-8000 instrument (Tokyo, Japan) operating in transmission mode at 100 kV on dry samples.

Japan) operating in transmission mode at 100 kV on dry samples. Atomic force microscopy (AFM) analysis was performed with an apparatus PicoLE (Molecular Imaging) operating in the acoustically driven, intermittent contact ("tapping") mode, using standard silicon AFM probes (NSC11/Cr-Au, Mikromasch, Tallinn, Estonia) having a cantilever spring constant of 48 N/m and a resonance frequency of 330 kHz. 10 × 10 mm2 AFM images were taken on Atomic force microscopy (AFM) analysis was performed with an apparatus PicoLE (Molecular Imaging) operating in the acoustically driven, intermittent contact ("tapping") mode, using standard silicon AFM probes (NSC11/Cr-Au, Mikromasch, Tallinn, Estonia) having a cantilever spring constant of 48 N/m and a resonance frequency of 330 kHz. 10 × 10 mm<sup>2</sup> AFM images were taken on dry samples.

dry samples. Topography was examined by AFM using the WSxM 5.0 Develop 9.1 software. Three

Topography was examined by AFM using the WSxM 5.0 Develop 9.1 software. Three acquisitions were made with roughness parameters analysis for each sample. Data were expressed as mean ± standard deviation (SD).

Water contact angle (WCA) measurements were performed at 25 ◦C on dried membranes, by the sessile drop technique using a KSV instruments LTD CAM 200 Tensiometer (Hertfordshire, UK) and employing Milli-Q water as a liquid with known surface tension. A minimum of 10 measurements were taken and averaged for each sample. Data were expressed as mean ± SD.

Rheological measurements were determined using an advanced rheometer from TA instruments, model AR-G2 (DE, US), equipped with a Peltier and a solvent trap. The last one allows leading the measurement in a water-saturated atmosphere by avoiding water evaporation from the membrane. Samples were previously stabilized by their immersion for 24 h in 7.4 PBS at 37 ◦C. All tests were carried out using a 25 mm diameter steel sand blasted parallel plate. Oscillatory shear tests with strain sweep step were performed at a frequency of 0.5 Hz and a strain ranging from 0.01 to 100% in order to determine the linear viscoelastic region (LVR) of the different membranes. Finally, frequency sweeping tests of membranes were conducted with a frequency scanning from 0.01 to 10 Hz at 0.1% strain and 37 ◦C to determine the elastic (G') and viscous (G") moduli. Three replicates of each sample were evaluated.

The average mesh size ξ was calculated from *G*' based on the rubber elasticity theory (RET) using the following Equation (1) [29]:

$$
\xi = \left(\frac{G'N\_A}{RT}\right)^{-\frac{1}{3}}\tag{1}
$$

where *G*' is the storage modulus, *N<sup>A</sup>* is the Avogadro constant, *R* is the molar gas constant, and *T* is the temperature. Three replicates of each sample were evaluated. Data were expressed as mean ± SD.
