# Functionalization Strategies and Fabrication of Solvent-Cast PLLA for Bioresorbable Stents
## 3 Results and Discussion
### HIVECs Adhesion  
In order to biologically characterize the effect of plasma, NaOH, and enzyme hydrolysis of PLLA solvent-cast films, HUVECs adhesion was evaluated after 12 h of cell culture. The cell number was slightly higher on SH films (25,787 \(\pm\) 2288 cells/cm\({}^{2}\)) compared to the TT surfaces (21,297 \(\pm\) 1528 cell/cm\({}^{2}\)) (Figure 6a). Whereas, OP and ET films presented a statistically higher number of HUVECs, 36,958 \(\pm\) 2967 and 33,541 \(\pm\) 4434 cells/cm\({}^{2}\), respectively, compared to the TT and SH samples. SEM images visually confirmed the higher cell density on the activated surfaces and showed a higher spreading on these surfaces compared to the TT films (Figure 7). In particular, high magnification images of the TT surfaces showed round HUVECs, non-expanded, showing an initial adhesion stage with many extended _filopodia_. The low cell affinity of the TT surfaces was expected since PLLA is chemically inert with no reactive side-chain groups that could accelerate cell spreading. On the contrary, HUVECs on OP, SH, and ET films were flat and extended. This higher cell number and spreading on the activated surfaces induced a percentage of area occupied by HUVECs at least four times higher compared to the TT samples (Figure 6b). The plasma and enzyme activation had the highest and statistical equal values with 66.0 \(\pm\) 11.5 and 58.7 \(\pm\) 3.9%, respectively. Surface characteristics such as wettability and functional group density evolved proportionally with the HUVECs adhesion on the three functionalized surfaces, confirming the relationship between the higher functional group densities, hydrophilicity and increased cell adhesion. The reason for this correlation could be attributed to higher protein adsorption on the hydrophilic surfaces, as reported previously [64; 65].  
In summary, concerning the effect of the different activation treatments applied to the PLLA surfaces on the cell response we found that, on the one hand, plasma and enzyme activation increased PLLA biocompatibility by improving HUVECs adhesion and also, created a higher density of surface functional groups that can be used to covalently immobilize biomolecules. On the other hand, sodium hydroxide activation showed a lower density of the activated functional groups on the surface compared to other activation treatments and HUVECs adhesion slightly increased compared to the control. Moreover, the NaOH activation increased the PLLA films roughness, which could dramatically induce a higher platelet adhesion and promote thrombus formation when using this surface for cardiovascular applications [66; 67].  
Figure 6: HUVECs adhesion and spreading on TT and activated films after 12 h of cell culture: (**a**) Cell number quantified by the Lactate Dehydrogenase LDH enzymatic assay; (**b**) percent of area occupied by HUVEC obtained after the SEM images analysis. Images were analyzed with the FJII software. Columns with different letters belong to statistically different groups (_p_-value < 0.05). **TT**: Thermally treated solvent casting films; **OP**: Oxygen plasma TT films; **SH**: Sodium hydroxide etched TT films; **ET**: Enzyme treated TT films.  
Figure 7: SEM images of HUVECs adhesion and spreading on TT and activated surfaces after 12 h of incubation. Bottom images show a high magnification a low spread cell (TT) and a highly spread cell (OP). **TT**: Thermally treated films; **OP**: Oxygen plasma TT films; **SH**: Sodium hydroxide etched TT films; **ET**: Enzyme treated TT films.