# Functionalization Strategies and Fabrication of Solvent-Cast PLLA for Bioresorbable Stents
## 2 Materials and Methods
### Characterization of PLLA Films  
Gel permeation chromatography (GPC) analyses were performed in an Agilent HPLC chromatograph 1260 Infinity series (Agilent Technologies, Santa Clara, CA, USA). The separation was carried in a Waters Styragel HR 5E column (USA) using CHCl\({}_{3}\) (HPLC grade) as an eluent. The samples with a concentration between 0.2 to 0.4 mg/mL were analyzed. The used flow was 0.4 mL/min and the amount of injected samples was 20 \(\upmu\)L. Molecular weights were calculated after calibration with PMMA standards from Sigma-Aldrich (Darmstadt, Germany), the molecular weight calibration range was between 2.7 million to 2000 Dalton. The calculations were performed with the Chemstation Software (Agilent Technologies). The crystalline properties of the different PLLA samples were analyzed by differential scanning calorimetry (DSC) with the instrument DSC 2920 Modulated (TA Instruments, USA) and following the ASTM D3418-03 standard [37]. An empty aluminum pan was used as a reference. Ten milligrams of the samples were run in a nitrogen atmosphere at 10 \({}^{\circ}\)C\(\cdot\)min\({}^{-1}\) heating ramp from 20 to 220 \({}^{\circ}\)C. The enthalpy of fusion (\(\Delta\)Hm) and the enthalpy of crystallization (\(\Delta\)Hcc) were obtained from the scan and used to calculate the percentage of crystallinity through the equation \(\mathrm{Xc}\) = (\(\Delta\)Hm \(-\)\(\Delta\)Hcc)/\(\Delta\)H\({}^{0}\)m, where \(\Delta\)H\({}^{0}\)m = 93 J/g is the enthalpy of fusion of a 100% crystalline PLLA [38; 39]. The mechanical properties of the PLLA films were determined by the tensile test (DY 34, [PERSON], France) according to the standard ASTM D882-02 [40] using a 100 N load cell. First, PLLA films were cut following the standard ASTM D6287-98 [41] to obtain five samples of 150 mm length and 10 mm width for each studied condition. The rate of grip separation was set to 15 mm/min and the curve load versus the extension was recorded at RT. From the curve, Young's modulus (E) and the ultimate tensile strength (UTS) were obtained. To detect residues of chloroform in the PLLA films, the energy-dispersive X-ray spectroscopy (EDX) was performed with a Zeiss Neon40 FE-SEM microscope (Carl Zeiss NTS GmbH, Germany). Five measurements at 7 kV were done for each condition. The indirect in vitro cell cytotoxicity of the PLLA films was evaluated from extracts using the lactate dehydrogenase (LDH) enzyme measurement on HUVECs. The extracts were prepared according to ISO 10993-5 [42] by submerging the different fabricated PLLA films in a complete cell medium for 3 days at 37 \({}^{\circ}\)C. Then, the extracts were serially diluted into five concentrations as 1/1; 1/2; 1/10; 1/100, and 1/1000 with a fresh complete cell medium. Previously, PLLA films were sterilized in ethanol 70% during 30 min. The cells were seeded in a 96-well tissue polystyrene (TCPS) cell culture plate at a seeding density of 15,000 HUVECs/well and incubated normally for 24 h to allow an attachment. Afterwards, the cell medium was replaced by 200 \(\upmu\)L of the different extract dissolutions. A complete fresh medium was used as a negative control and a complete fresh medium without cells was used as a positive control. After 24 h, alive HUVECs were quantified by measuring the LDH enzyme. Briefly, the cells were rinsed with PBS and lysed with 350 \(\upmu\)L of the mammalian protein extraction reagent (M-PER) (Thermo Scientific, Darmstadt, Germany). The activity of LDH was determined by measuring the spectrophotometrical absorbance with a commercial LDH kit (Cytotoxicity Detection Kit, Roche Diagnostics, Manheim, Germany) by a microplate reader (Infinite M200 PRO, Tecan Group Ltd., Switzerland) at 490 nm.