*3.5. SEM*

SEM analysis was performed to assess the morphological features of the HA/Ch complex [55], after a freeze-drying process. In the first step of freeze-drying, the waterbased solutions (or suspensions) were first frozen—ice nucleated and concentrated the solute (or suspended matter) in the regions between the growing crystals. In the second step, ice was removed via direct sublimation at low pressure, avoiding the intermediate liquid phase, and leaving a porous morphology. The process is widely used to create porous materials using biopolymer [56–58]. The final morphology depends on the specificmaterial, and is controlled by many parameters of the freezing process [59], including sample size and shape, cooling rate, cooling temperature, process time, etc. μ μ

Figure 7 illustrates the specific characteristics of the HA/Ch complex that were compared to the single components of Ch and HA.

**Figure 7.** SEM analysis of (**a**) Ch (magnification 150×); (**a'**) Ch (magnification 500×); (**b**) HA (magnification 150×); (**b'**) HA (magnification 500×); (**c**) Complex (magnification 150×); and (**c'**) Complex (magnification 500×).

Ch (Figure 7a,a') showed a compact structure with a fibrous appearance, causing a rough texture on the Ch surface. HA presents even more clearly a lamellar structure (Figure 7b,b'), which is characteristic of an ice-templated process—the structure was oriented parallel to the temperature gradient (i.e., from the skin to the sample centre), along which the ice crystal grew. HA walls were characterized by a pore regular pattern, with a characteristic diameter of ~10 µm. Such pores might also be related to ice-templating, with holes forming due to the secondary crystal growth, perpendicular to the primary crystal growth direction.

The HA/Ch composite (Figure 7c,c') showed a compact and smooth surface, where fibers were visible on the top of the surface. The material was made of a lamellar structure with the holes between 10 and 20 µm as diameter. Similar to Zhang et al. [60], who investigated the structure of porous chitosan/HA/sodium glycerophosphate hydrogel systems, the material appeared homogeneous overall, and phase separation at the microscale was not observed. As such, we could confirm good compatibility between Ch and HA in the complex.
