A total of 8 male juvenile Chinese pigs with a body weight between 20 and 30 kg were used in the present study. These were were obtained from the Experimental Pig Production Institute (Fangshan District, Beijing, China) and were maintained at 18–25°C, 40–60% humidity, with >10 h of light every day; food was provided twice a day at 2–3% of the pigs' total weight, and free access to water was provided. Nano polymer-free SES (Lepu Medical Technology Co., Ltd., Beijing, China) were randomly implanted in the right coronary artery, left anterior descending or left circumflex coronary artery with two stents in each porcine model (one stent per vessel). In order to assess different stages of neointimal formation, all animals were divided into two groups: Follow-up after stent implantation of 3 (n=4) or 6 months (n=4). At the follow-up time point, the animals were sacrificed following completion of coronary angiography and OCT evaluation to obtain specimens for histological analysis of stented arterial segments. The study protocol was approved by the Institutional Animal Care and Use Committee at Beijing Tiantan Hospital (Beijing, China).

Three days prior to the coronary procedure, animals were administered 300 mg aspirin (Bayer, Newbury, UK) and 75 mg clopidogrel (Sanofi S.A., Paris, France). Thereafter, antiplatelet therapy of 75 mg clopidogrel and 100 mg aspirin was administered daily throughout the study in all animals.

Animals were anesthetized with 0.1–0.2 mg/kg midazolam (Nhwa Pharmaceutical Co., Ltd., Jiangsu, China) and 0.25–0.3 ml/kg xylazine (Sangon Biological Engineering Co. Ltd., Shanghai, China) and an arterial sheath (6 French; Cordis Corporation, Hialeah, FL, USA) was placed in the right femoral artery by cut-down with a sterile technique. Coronary catheterization was performed following administration of intravenous heparin (5,000 units). Baseline angiography was acquired and all stents were placed at a targeted 1.1:1 to 1.2:1 stent-to-vessel ratio compared with the reference vessel diameter to induce a moderate vessel injury and promote neointimal formation. Stents of 14, 15 or 18 mm length and diameters of 2.5, 2.75 and 3.0 mm were implanted according to the coronary artery size.

Frequency domain (FD)-OCT imaging was performed using the C7-XR OCT intravascular imaging system (LightLab Imaging, Inc., Westford, MA, USA). During FD-OCT image acquisition, a continuous non-occlusive contrast as a flush was administrated to replace coronary blood flow and automatic pullbacks were performed at a rate of 20 mm/sec and 100 frames/sec. Off-line OCT analysis was performed with LightLab Imaging software (LightLab Imaging, Inc.).

For quantitative analysis, cross-sectional OCT images were analyzed at 1-mm longitudinal steps throughout the pullback from distal stent edge to proximal stent edge. OCT images were excluded from the analysis if stent struts were not visible on the screen, bifurcation cross-sections had side branches or if residual blood was mistaken for neointimal tissue. The lumen and stent were manually traced and stent struts were positioned manually in the center of the stent strut which showed a bright ‘blooming’ appearance (14). The following parameters were measured: Minimal luminal diameter, luminal area, stent area, neointimal area (stent area - lumen area) and neointimal burden (mean neointimal area / mean stent area × 100%). To analyze neointimal thickness, the distance between the center of each stent strut and the luminal border was measured in the direction of the center of gravity.

Quantitative analyses of all OCT images were performed by two independent investigators who were blinded to the angiographic data and clinical presentations.

Two independent investigators analyzed 50 cross-sectional images without artifacts. Inter-observer agreement was determined by calculating values for differences in measurements of neointimal area and neointimal thickness.

All animals were sacrificed following follow-up coronary angiography and OCT imaging; this was performed using intravenously administered 30 ml 10% potassium chloride. Immediately following sacrifice, the hearts were excised and the stented coronary artery segments were harvested from the heart by careful dissection, fixed by immersion in 10% formalin, dehydrated in a graded series of ethanol and embedded in methyl methacrylate resin (Huayi Acrylic Acid Co., Ltd., Shanghai, China). Following polymerization, sections measuring ~1.3 mm were sawed from each stent, beginning at the distal stent edge. The artery-stent specimens were cut on a rotary microtome at 100 µm from the proximal through the distal margin of the stent and stained with hematoxylin and eosin, and elastic Van Gieson stains. There were no histological sections lost due to processing, and all sections were of excellent quality. Morphometric analysis was performed by Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Morphologic analysis of strut-associated inflammation, stent endothelialization and arterial injury were completed using previously described methods (3,15,16). Briefly, the inflammatory score for each individual strut was graded as follows: 0, No inflammatory cells surrounding the strut; 1, light, non-circumferential lymphohistiocytic infiltrate surrounding the strut; 2, localized, moderate-to-dense cellular aggregate surrounding the strut noncircumferentially; and 3, circumferential dense lymphohistiocytic cell infiltration of the strut. The stent endothelialization score was defined as the extent of the circumference of the arterial lumen covered by endothelial cells and was scored between 1 and 3 (1, 25%; 2, 25 to 75%; 3, >75%). The intimal fibrin content was graded as follows: 1, Focal residual fibrin involving any portion of the artery and for moderate fibrin deposition adjacent to the strut involving 25% of the circumference of the artery; 2, moderate fibrin deposition involving 25% of the circumference of the artery or heavy deposition of fibrin adjacent to and between stent struts involving 25% of the circumference of the artery; or 3, heavy deposition of fibrin involving 25% of the circumference of the artery. The intimal smooth muscle cell (SMC) content was scored as follows: 1, Sparse SMC density involving any portion of the artery and for moderate SMC infiltration less than the full thickness of the neointima involving <25% of the circumference of the artery; 2, moderate SMC infiltration less than the full thickness of the neointima involving >25% of the circumference of the artery, or dense SMC content the full thickness of the neointima involving <25% of the circumference of the artery; or 3, dense SMC content the full thickness of the neointima involving >25% of the circumference of the artery.

All histomorphological analyses were performed by a single independent investigator blinded to the study.

Variables were analyzed for normal distribution using the Kolmogorov-Smirnov test. Continuous values following a normal distribution were expressed as the mean ± standard deviation, and those not normally distributed were indicated as the median with interquartile range. Categorical variables were expressed as frequencies and percentages. Differences between the two groups were determined using Student's t-test or Mann-Whitney U test, as appropriate for continuous variables. The χ² test or Fisher exact test was used as appropriate to compare categorical variables. Agreement between the two observers (interobserver variability) was investigated using kappa analysis. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS version 20.0 software (IBM SPSS, Armonk, NY, USA).

A total of 16 nano polymer-free SES were successfully implanted in the coronary arteries of 8 pigs. All pigs survived without problems until sacrifice. The survival time of pigs and stent characteristics are presented in Table I.

The results of OCT quantitative analysis of the stents are presented in Table II. Lumen area (2.73±0.66 vs. 3.00±0.65 mm², P<0.001) and stent area (3.86±0.39 vs. 4.05±0.59 mm², P<0.001) at 6 months were significantly smaller compared with those at 3 months. In addition, at 6 months, neointimal thickness (193.3±109.5 vs. 167.2±119.7 µm, P=0.023) and neointimal burden (29.3±14.3 vs. 24.8±17.4%, P=0.006) significantly increased compared with at 3 months; neointimal area increased from between 3 and 6 months, but this difference was not significant.

Interobserver variability for the quantitative OCT assessment showed good concordance: k=0.91 for neointimal area and k=0.81 for neointimal thickness.

The histomorphometry and semi-quantitative scoring for arterial injury, inflammation and stent endothelialization at 3 and 6 months after nano polymer-free SES implantation are summarized in Table III. Marked stent endothelialization was achieved at 3 and 6 months. However, the endothelialization score was greater at 6 months compared with at 3 months (2.85±0.36 vs. 2.52±0.60, P<0.001). In addition, at 3 months, nano polymer-free SES showed a significantly higher inflammatory score [0 (0, 1) vs. 0 (0, 0), P<0.001] and fibrin score [0 (0, 1) vs. 0 (0, 0), P<0.001] compared with at 6 months. There were no significant differences in injury score and intimal smooth muscle cell content between 3 and 6 months. Representative images of OCT and histomorphologic sections at 3 months after nano polymer-free SES implantation are presented in Fig. 1.