# Functionalization Strategies and Fabrication of Solvent-Cast PLLA for Bioresorbable Stents
## 3 Results and Discussion
### PLLA Film Properties  
To evaluate potential cytotoxic effects of the solvent employed for the synthesis of the PLLA films and/or the different activation treatments, an indirect cytotoxicity assay was performed (Table 1). The ISO 10993-5 standard specifies that a material is cytotoxic if the percentage of cell survival is inferior to 80%. The results indicated that SC samples were cytotoxic but the thermal treatment increased cell viability reaching non-cytotoxic values, higher than 92%, as observed for the TT samples at different dilutions. Activation treatments with oxygen plasma (OP) or enzymatic treatment (ET) did not affect the cytotoxicity of TT films. However, SH samples presented cytotoxicity in some cases probably due to NaOH residues in the obtained films. The presence of chlorine residues from solvent casting fabrication was verified by EDS (Table 2). SC was the only film presenting chlorine residues. As chloroform toxicity is well known [47; 48], the cytotoxicity of SC observed previously was probably due to this residual chloroform in the film. Moreover, the absence of chlorine in TT films suggested that the thermal treatment helped PLLA solvent-cast films to be biocompatible by evaporating chloroform residues. PLLA PL65 from Purac was chosen for its particularly high molecular weight and crystallinity in order to fabricate PLLA films with mechanical and degradation properties similar to the actual commercial PLLA BRS. The potential reduction in molecular weight and crystallinity due to solvent casting were investigated by GPC and DSC (Table 2). Generally, all the processed films presented M\({}_{\text{w}}\) values in the same range, from 0.89 up to 0.98 \(\times\) 10\({}^{6}\) g/mol. The REMEDY stent presented a M\({}_{\text{w}}\) five times lower than SC films. SC and TT films presented a statistically similar molecular weight of 0.95 \(\pm\) 0.04 and 0.98 \(\pm\) 0.03 10\({}^{6}\) g/mol, respectively. Therefore, the thermal treatment did not significantly \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{5}{c}{**Cell Viability (\%)**} \\ \cline{2-6}
**Sample** & **1/1** & **1/2** & **1/10** & **1/100** & **1/1000** \\ \hline SC & 45 \(\pm\) 26 & 70 \(\pm\) 13 & 67 \(\pm\) 12 & 79 \(\pm\) 16 & 94 \(\pm\) 12 \\ TT & 95 \(\pm\) 3 & 95 \(\pm\) 4 & 92 \(\pm\) 4 & 102 \(\pm\) 15 & 96 \(\pm\) 7 \\ OP & 101 \(\pm\) 4 & 113 \(\pm\) 6 & 122 \(\pm\) 16 & 116 \(\pm\) 12 & 113 \(\pm\) 18 \\ SH & 95 \(\pm\) 15 & 92 \(\pm\) 19 & 76 \(\pm\) 12 & 86 \(\pm\) 7 & 123 \(\pm\) 13 \\ ET & 98 \(\pm\) 11 & 112 \(\pm\) 24 & 111 \(\pm\) 25 & 112 \(\pm\) 12 & 121 \(\pm\) 21 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Cell viability on the different solvent-cast PLLA films. Results correspond to the percentage of living cells after an indirect cytotoxicity assay. Extracts were obtained by submerging the different fabricated PLLA films in a complete cell medium for 72 h. The extracts were serially diluted 1/1, 1/2, 1/10, 1/100, 1/1000 with a fresh complete cell medium. Values \(<\) 80% indicate human umbilical vein endothelial cells (HUVEC) cytotoxic surfaces. **SC**: Solvent casting PLLA films; **TT**: Thermally treated SC films; **OP**: Oxygen plasma TT films; **SH**: Sodium hydroxide etched TT films; **ET**: Enzyme treated TT films. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample** & **Cl Residues** &
\begin{tabular}{c} **Mw** \\ **(10\({}^{6}\) g/mol)** \\ \end{tabular} & **\% Crystallinity** \\ \hline REMEDY stent & No & 0.19 \(\pm\) 0.00 & 50.5 \(\pm\) 2.7 \\ SC & Yes & 0.95 \(\pm\) 0.04 & 12.5 \(\pm\) 3.1 \\ TT & No & 0.98 \(\pm\) 0.03 & 26.6 \(\pm\) 2.7 \\ OP & No & 0.95 \(\pm\) 0.01 & 25.1 \(\pm\) 1.7 \\ SH & No & 0.89 \(\pm\) 0.01 & 24.2 \(\pm\) 2.7 \\ ET & No & 0.97 \(\pm\) 0.02 & 25.8 \(\pm\) 0.4 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Bulk properties of the commercial REMEDY stent and all the different solvent-cast films: Presence of chloride (Cl) residues, Mw: Molecular weight obtained by HPLC and % of crystallinity following DSC.