4.5.2. Water Binding Capacity

The water binding property of SCMs and nc-SCMs, was evaluated according to an already described method [80]. In brief, phosphate buffered saline (PBS, pH 7.4) was used as hydration medium, and the membranes were soaked for 1 h at RT by complete immersion. Then, the surface excess medium was removed by touching to a filter paper and then the SCMs were weighed (wet weight). As a control, the commercial porcine collagen membrane Bio-Gide®, with the same surface area of the SCMs and nc-SCMs, was submitted to hydration as well. The water binding capacity (WBC) was determined using the following Equation:

$$\text{WBC} \text{ (\%)} = (\text{Ww} - \text{Wd}) / \text{Wd} \times 100.$$

Experiments were performed three times in duplicate.

#### 4.5.3. Dynamic Mechanical Tests

Dynamic mechanical analysis was performed with an Anton Paar Physica MCR 301 Rheometer (Anton Paar, GmbH, Ostfildern, Germany), using a Solid Rectangular Fixtures (SFR) system. The temperature was set at 20 ◦C and each sample was tested at least twice. Tests were performed both in Amplitude Sweep and Frequency Sweep modes.

The values of the stress amplitude were checked by means of an amplitude sweep test, with a deformation range (γ) from 0.001 up to 0.1% at a fixed frequency of 0.1 Hz, in order to ensure that all of the measurements were performed within the linear viscoelastic region (LVER).

In order to obtain information about the storage (or elastic) modulus (G ), the loss (or viscous) modulus (G), the complex viscosity (η\*) as a function of the frequency and the Frequency Sweep tests in the range 0.01–10 Hz, at a fixed deformation of 0.01% within LVER, were performed.

The data were collected and analysed using Rheoplus/32 Service V3.40 software.

#### *4.6. SCM Biocompatibility Evaluation*
