*3.4. Biological Evaluation*

### 3.4.1. ESEM Microscopy of Hydrogel Membranes

To characterize the microstructural architecture of Ch, Ch/HA and Ch/HAMA membranes and observe the morphology of hMSCs cultured on them, an ESEM analysis was carried out on day 21. It is known that the surface roughness is an important factor in promoting cell attachment [50]. ESEM images of Ch, Ch/HA and Ch/HAMA membranes (Figure 6A) revealed a rougher surface for the Ch/HA membrane compared to those of Ch/HAMA and Ch, corroborating the SEM observations for dried samples (Figure 2) but surfaces of all membranes were able to support cell growth (Figure 6B). ESEM images evidenced the cells covering the surface of Ch, Ch/HA and Ch/HAMA membranes, with good adhesion, spreading, and a homogenous distribution throughout the entire surface. Moreover, ESEM images showed an interconnected cell community that attached to the scaffold which also confirmed the biocompatibility of the membranes [51,52]. *Polymers* **2020**, *12*, x FOR PEER REVIEW 13 of 18

**Figure 6.** ESEM images of Ch, Ch/HA and Ch/HAMA hydrogel membranes. Representative ESEM images of hydrogel membranes before hMSCs culture (**A**). Representative ESEM images of hMSCs growing on hydrogel membranes after 21 days (**B**). **Figure 6.** ESEM images of Ch, Ch/HA and Ch/HAMA hydrogel membranes. Representative ESEM images of hydrogel membranes before hMSCs culture (**A**). Representative ESEM images of hMSCs growing on hydrogel membranes after 21 days (**B**).
