The animal study was approved by the Ethics Committee of Chonnam National University Medical School and Chonnam National University Hospital (CNU IACUC-H-2013-36), and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Study animals were castrated male pigs weighing 20- 25 kg. To prevent acute thrombosis after stenting, premedication with aspirin 100 mg and clopidogrel 75 mg per day was given for 5 days before the procedure. On the procedure day, pigs were anesthetized with zolazepam and tiletamine (2.5 mg/kg, Zoletil50^®, Virbac, Caros, France), xylazine (3 mg/kg, Rompun^®, Bayer AG, Leverkusen, Germany), and azaperone (6 mg/kg, Stresnil^®, Janssen-Cilag, Neuss, Germany). They received supplemental oxygen continuously through an oxygen mask. Subcutaneous 2% lidocaine at the cut-down site was administered, the left carotid artery was surgically exposed, and a 7 French sheath was inserted.

Continuous hemodynamic and surface electrocardiographic monitoring was maintained throughout the procedure. Next, 5,000 units of heparin were intravenously administered as a bolus prior to the procedure, the target coronary artery was engaged using standard 7 F guide catheters, and control angiograms of both coronary arteries were performed using nonionic contrast agent in two orthogonal views.

The stent was deployed by inflating the balloon, obtaining a resulting stent-to-artery ratio of 1.3:1. Coronary angiograms were obtained immediately after stent implantation. Then, all equipment was removed and the carotid artery was ligated. Four weeks after stenting, the animals underwent follow-up angiography in the same orthogonal views before they were sacrificed with an intracoronary injection of 20 mL potassium chloride.

The hearts were removed and the coronary arteries were pressureperfusion fixed at 110 mmHg in 10% neutral buffered formalin overnight. All specimens were analyzed by micro- computerized tomography (M-CT) before histopathological testing. Each of the 10 stented arteries in the three groups were step-sectioned, processed routinely for light microscopy, and stained for histological analysis.

The pig was randomly divided into 3 groups: group 1 (sirolimuseluting stent with PLA, SEP, 3.0×16 mm, n=10), group 2 (biolimus A9-eluting stents with PLA, BEP, 3.0×18 mm, n=10), and group 3 (PLA coating stent, PCS, 3.0×16 mm, n=10). A total of 15 pigs were used in this study (15 pigs, 30 coronary arteries, 10 coronary arteries in each group). A SEP, BEP, or PCS was implanted in a randomized manner in the left anterior descending artery and the left circumflex artery of the pigs.

In a previous study, our novel Co-Cr alloy BMS (CNUH stent) showed non-inferior histopathological effects compared to a commercial BMS[9]. PLA was purchased from EVONIK (UK) and sirolimus was purchased LC Laboratories (USA). An ultrasonic spray method was used to apply the coatings to the prepared BMS (3×16 mm). The required amount of polymer PLA and sirolimus were dissolved in 5 mL of tetrahydrofuran (THF). The drug solution was then dissolved in the polymer solution. Stents to be coated were cleaned with ethanol and distilled water and then vacuum-dried for 24 hr. The sprayed liquid consisted of the polymer/drug system dissolved in THF and diluted to 2% by weight. The operating requirements in this application called for flow rates of 50 μL/min. The stents were placed on a mandrel attached to a rotating shaft. The coated stents were vacuum- dried for 24 hr. The surface morphologies of the SEP was examined by scanning electron microscopy (SEM).

The release profiles of sirolimus and biolimus A9 from the SEP and BEP, respectively, were acquired using a total buffer extraction method. The sirolimus-coated stent (140 μg/stent) and the biolimus A9- coated stent (113 μg/stent) were placed in 3 mL of PBS at pH 7.4. The sample in the buffer was held at 37°C while being constantly agitated at 100 rpm in an incubator shaker (JEIO tech, Korea) for 32 days. The buffer in each sample was decanted and replaced with fresh buffer every day. The amount of drug released was determined by the UV-VIS Spectrophotometer at 278 nm. All studies were conducted in triplicates.

Histopathologic evaluation of each artery was performed by an experienced cardiovascular pathologist. The specimens were embedded and sections of 50 to 100 μm thickness were obtained at about 1 mm apart and stained with Hematoxylin-Eosin and Carstairs’ for histological analysis. Measurements of the histopathologic sections were performed using a calibrated microscope, digital video imaging system, and microcomputer program (Visus 2000 Visual Image Analysis System, IMT Tech). Borders were manually traced for lumen area, the area circumscribed by the internal elastic lamina, and the innermost border of the external elastic lamina (external elastic lamina area). Morphometric analysis of the neointimal area for a given vessel was calculated as the measured internal elastic lamina area minus the lumen area. The measurements were made on five cross-sections from the proximal and distal ends and the three midpoints of each stented segment. Histopathologic stenosis was calculated as 100×[1-(lesion lumen area/lesion internal elastic lamina area)][10]. The harvested stent specimen was stored in formaldehyde solution. A 1.5 mL Eppendorf tube was filled with clay, and the clay was tuned with a V shape to hold the stent during contrast agent staining. The stents were taken from the solution and vertically placed in the V-shaped opening in the clay. Each stent had to be fixed with the clay such that there was no movement of the stent inside the Eppendorf tube. The contrast agent used was omnihexol. One milliliter of the contrast agent was taken in a 5 mL syringe and injected through the opening at the center of the stent. The stent was incubated with contrast agent overnight and subjected to micro-computed tomography imaging[11]. All results were interpreted by two independent pathologists in a blind fashion.

Arterial injury at each stent strut site was determined by the anatomic structures penetrated by each stent strut. A numeric value was assigned, as previously described by Schwartz et al.[10].: 0=no injury; 1=break in the internal elastic membrane; 2=perforation of the media; 3=perforation of the external elastic membrane to the adventitia. The average injury score for each segment was calculated by dividing the sum of the injury scores by the total number of stent struts at the examined section.

With regard to the inflammation score for each individual stent strut, the grading was as follows: 0=no inflammatory cells surrounding the stent strut; 1=light, noncircumferential lymphohistiocytic infiltrate surrounding the strut; 2=localized, moderate-to-dense cellular aggregate noncircumferentially surrounding the stent strut; 3=circumferential dense lymphohistiocytic cell infiltration of the stent strut. The inflammation score for each cross section was calculated by dividing the sum of the individual inflammation scores by the total number of stent strut at the examined section[12]. Ordinal data for fibrin were collected on each stent section using a scale of 0 to 3, as previously reported[13].

Statistical analysis was performed with the aid of the commercially available software SPSS Version 15 (Chicago, IL, USA). The data were presented as the mean value±SD. The unpaired Student’s t test was used for the comparison of the stent groups. Analysis of variance (ANOVA) was used for comparisons of the four stents groups. Ordinal measurements such as injury score, fibrin score, and inflammation score were analyzed using the Mann Whitney and Kruskal-Wallis test. Non-parametric results were presented as medians with interquartile ranges. A value of P<0.05 was considered statistically significant.

SEM image of the SEP showed a smooth and uniform surface. Bridges and webbings could not be observed in the SEP (Fig. 1).

Fig. 2 showed that approximately 40% of the sirolimus was sustainably released from the stent for 32 days. The BEP indicates a sustained release of 50% of the biolimus A9 over 5 wk. The drug release kinetics of SEP and BEP were very similar.

A total of thirty stents (ten SEP, ten BEP, and ten PCS) were placed in the left anterior descending arteries and left circumflex arteries of 15 swine. Mortality for this study was zero. There were no significant differences in the stent-to-artery ratio among the three stent groups. The stent-to-artery ratio was the proportion of the stent diameter divided by the arterial luminal diameter.

All in-stent restenosis (ISR) patterns after stenting were ISR pattern II (intra-stent)[14] in coronary angiographic analysis. ISR pattern II represents restenosis >10 mm in length within the stent, without extending outside the stent.

There was no significant difference in the injury score [1.0 (range 0.0 to 2.0) in SEP vs. 1.0 (range 0.0 to 2.0) in BEP vs. 1.0 (range 0.0 to 2.0) in PCS, P=NS] and the internal elastic lamina (4.8±0.68 mm² in SEP vs. 5.0±0.48 mm² in BEP vs. 4.8±0.60 mm² in PCS, P=NS). There were significant differences in the lumen area (3.2±0.91 mm² in SEP vs. 3.1±1.10 mm² in BEP vs. 0.5±0.56 mm² in PCS, P<0.0001), in the neointimal area (1.7±0.56 mm² in SEP vs. 1.9±0.95 mm² in BEP vs. 4.3±0.63 mm² in PCS, P<0.0001), in the percent area of stenosis (35.2±13.77% in SEP vs. 39.0±20.00% in BEP vs. 89.4±10.92% in PCS, P<0.0001) (Fig. 3), in the fibrin score [2.0 (range 1.0 to 3.0) in SEP vs. 2.0 (range 2.0 to 3.0) in BEP vs. 1.0 (range 1.0 to 2.0) in PCS, P<0.0001)] (Fig. 4), and in the inflammation score [1.0 (range 1.0 to 2.0) in SEP vs. 1.0 (range 1.0 to 3.0) in BEP vs. 3.0 (range 2.0 to 3.0) in PCS, P<0.0001)] among the three groups (Fig. 5).

Percent area of stenosis using micro-computed tomography showed similar results with the histopathological analysis (36.7±13.77% in SEP vs. 41.2±20.00% in BEP vs. 86.5±10.92% in PCS) (Fig. 6).