Fig. 1 shows a schematic illustration of the Ta implantation treatment of a PLLA stent. PLLA tubes (Poly[L-lactide], 38; 3 mm outer diameter; 2.75 mm inner diameter; length, 10 mm) were purchased from Biogeneral, Inc. (San Diego, CA, USA). Femtosecond laser processing (Spitfire Ace; Spectra Physics, Andover, MA, USA) was utilized to manufacture a PLLA polymer stent under optimized conditions of the pulse energy, scan speed, and spot size. Before the surface treatment, the stent samples provided were ultra-sonicated in ethanol for 10 minutes. Using a DC magnetron sputtering device (Ultech Co. Ltd., Daegu, Korea), Ta was implanted into the stent surface. The chamber was maintained in a vacuum state up to 5×10^-4 Pa for 90 minutes and argon gas was then flowed until the inner pressure reached 0.6 Pa. Three different parts of the stent were marked at constant circumferential intervals for a uniform surface treatment. The three sections were attached to the substrate sequentially and sputtering onto the section was conducted for 20 seconds in each case. High negative voltage of 2000 V was applied to the substrate, and the Ta target power was 12 W.

For an in vivo animal experiment, a radio-opaque marker was adopted at the end of the stent using gold wire (0.1 mm thickness; Alfa Aesar, Ward hill, MA, USA). The PLLA stent was then mounted onto a 4×20 mm non-compliant coronary balloon (Genoss, Suwon, Korea) by means of a crimping process (Fig. 2). Various sizes of balloons (3–5 mm) and different pressure levels (8–12 atm) were tested in order to deploy the stent successfully while maintaining its stable structure. The balloon size (4 mm) and pressure (10 atm) used in this study were selected as a result. The arterial coverage ratio by the stent was 27.3%. The control samples were a bare PLLA stent without a surface treatment.

The stents were observed via optical microscopy to confirm the maintenance of the structure after the surface treatment. The surface morphology and roughness (Ra) of the bare and Ta-implanted PLLA stents were examined using a scanning electron microscope (SEM) and a confocal laser microscope, respectively. The wettability of the surface was evaluated by dropping 3.0 µL of distilled water onto it and fitting the droplet in a circle using a water contact angle (WCA) analyzer. For a WCA test of the luminal surface of a Ta-implanted sample, a PLLA tube, only vertical half of which had undergone a laser cutting process, was treated via Ta implantation to mimic a luminal side of a Ta-implanted stent. Ta-implanted PLLA stents were fabricated as transmission electron microscope (TEM) specimens using a focused ion beam after carbon deposition to evaluate the content and depth of the implanted Ta on the surface with an energy-dispersive spectroscopy line scan analysis by TEM. For a mechanical test, a flat plate test was conducted by referring to an earlier method [12]. The stent was placed between two parallel plates and compression force was applied until the gap distance reached 50% of the diameter of the stent. Human umbilical vein endothelial cells (HUVECs; CRL-1730), purchased from American Type Culture Collection, were utilized to evaluate the endothelialization capabilities. All samples were sonicated in 70% ethanol for 10 minutes and then exposed to ultraviolet light for 1 hour. The cells were seeded at a density of 5×10^-4 cells/mL in an endothelial cell basal medium-2 (Lonza, Walkersville, MD, USA) with 10% fetal bovine serum and 1% penicillin-streptomycin. They were cultured in a humidified incubator containing 5% CO₂ at 37°C and obtained after 1, 4, and 7 days. To visualize the spreading morphology and area of the cells on each specimen through confocal laser scanning microscopy (CLSM), they were treated with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), 0.1% Triton X-100 (Sigma-Aldrich) and 1% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline and then stained by phalloidin (Invitrogen, Waltham, MA, USA) and 4, 6-diamidino-2-phenylindole (Invitrogen) for the cytoplasm and nuclei, respectively. As a proliferation test, the seeding density was 3×10⁴ cells/mL and the absorbance at 492 nm was measured according to a methoxyphenyl tetrazolium salt assay with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

An in vitro platelet adhesion test was conducted as the sterilized stents were incubated in diluted platelet-rich plasma (PRP) for 1 hour. After washing out the remaining platelets, the numbers of platelets adhering to each stent were quantified using a lactate dehydrogenase assay by Multiskan GO (Thermo, Vantaa, Finland). For a SEM analysis, the samples An in vitro platelet adhesion test was conducted as the sterilized stents were incubated in diluted platelet-rich plasma (PRP) for 1 hour. After washing out the remaining platelets, the numbers of platelets adhering to each stent were quantified using a lactate dehydrogenase assay by Multiskan GO (Thermo, Vantaa, Finland). For a SEM analysis, the samples then dehydrated.

In total, four specific-pathogen-free male swine of about 30 kg were used for the in vivo test. Antiplatelet drugs (aspirin 100 mg and clopidogrel 75 mg daily) were administered 1 week before stent placement and were maintained until the animals were sacrificed.

For stent deployment, a 6 Fr femoral sheath was inserted, and a 6 Fr guiding catheter was positioned in the common carotid artery using a 0.035-inch guide wire under angiographic guidance (Artis zee, Siemens, Germany). Balloon-mounted PLLA stents were deployed in the external carotid artery (ECA) with diameters of 2.6 to 3.2 mm. Two Ta-implanted PLLA stents were deployed in the right ECA and two bare PLLA stents were placed in the left ECA. Ultimately, sixteen PLLA stents were successfully deployed in the four swine. All procedures were performed under general anesthesia by a certified veterinarian. Anesthesia was induced with an intramuscular injection of Zoletil^® (Virbac, Carros, France) 4 mg/kg and Rompun^® (Bayer, Leverkusen, Germany) 2.2 mg/kg. It was maintained using an inhaled agent (sevoflurane) and oxygen during the procedures.

Animals were sacrificed at 1 (n=2), 2 (n=1), and 3 (n=1) months after stent deployment. A follow-up angiography was performed to confirm arterial patency before sacrifice. One Ta-implanted stent at 1-month follow-up was lost during the harvesting procedure and thus 15 stents were harvested. Three paraffin blocks (the proximal, middle, and distal parts) and one resin block were made from each stent sample. For a histologic analysis, hematoxylin and eosin) staining and immunohistochemical staining for CD 31, CD 34, CD 68, and antismooth muscle cell alpha actin were performed. The histologic analysis included luminal thrombus (% = 100 × [luminal thrombus area/luminal area]), neointimal hyperplasia and stenosis, as suggested by Schwartz et al. [18], and inflammation and fibrosis according to a quantitative method to evaluate local biological effects after the implantation of the prosthesis in this case [9,15]. All histologic analyses were performed by an independent, blinded and certified pathologist. SPSS software (version 20; IBM corp., Armonk, NY, USA) and MedCalc for Windows (version 19; MedCalc Software, Ostend, Belgium) were used for the statistical analysis. A value of p<0.05 was considered statistically significant.

After the Ta ion implantation treatment, the PLLA stent had an opaque and colorless appearance, and its complex shape was well maintained without any deformation or distortion (Fig. 3). Despite the several manufactured tracks on the stent surfaces before and after the surface treatment, their average surface Ra values were only approximately 0.028±0.008 µm and 0.028±0.003 µm for the bare and Ta-implanted PLLA stents, respectively, representing excellent micro-scale flatness on both surfaces (Fig. 4). As shown in Fig. 5, the WCAs (º) of the luminal and abluminal sides of the Ta-implanted samples were 60.0º±1.88º and 51.1º±9.12º, respectively, while it was 104.1º±0.44º on the bare PLLA (p=0.000); that is, the Ta treatment increased the hydrophilicity of the stent by introducing a Ta oxide layer. As depicted in Fig. 6, the Ta-implantation process on the surface did not affect the mechanical strength of the PLLA stent, indicating that good mechanical integrity remained.

Fig. 7 shows that Ta ions were successfully implanted into both the luminal and abluminal surfaces of the PLLA stent, forming a Ta-implanted skin layer regardless of the location. Because the distance from the Ta target and physical hindrance could affect the efficiency of Ta implantation on the luminal surface, the atomic concentration of Ta was higher on the abluminal surface. However, even on the luminal surface, the Ta layer was clearly observed at a depth of 10 nm on the topmost surface with the highest atomic concentration of approximately 6%.

The effects of Ta ion implantation on the interaction between the HUVECs, platelets and the PLLA stent surface were examined by visualizing the corresponding adhesion morphologies on bare and Ta-implanted PLLA substrates. Fig. 8A shows representative CLSM images of the adhered HUVECs after 1, 4, and 7 days of culturing. On the Ta-implanted PLLA surface, a significant number of HUVECs were adhered and progressively spread in every direction, forming a typical cobblestone-like morphology with tight cell-cell interaction, whereas the bare PLLA exhibited fewer cells which were adhered individually with a lack of cell-cell junctions. The fractions of cell surface coverage with culturing times of 1, 4, and 7 days were 48%, 61%, and 77% on the Ta-implanted PLLA, respectively, and were 14%, 34%, and 40% on the bare PLLA (Fig. 8B). In addition, HUVECs tended to proliferate more actively on the Ta-implanted PLLA, showing a higher cell viability value when compared to those cultured on bare PLLA (Fig. 8C).

The blood compatibility of each sample was investigated by platelet adhesion tests and the results were compared quantitatively. As shown in Fig. 9A, significantly fewer platelets were found to have adhered when the PLLA surface was treated with Ta ion implantation; the density of the adhered platelets (number/mm²) on the Ta-implanted PLLA was 92.2±95.5, significantly lower than that on the bare PLLA (306.0±10.2, p=0.046) (Fig. 9B and C).

The follow-up angiographic images showed that all ECAs were patent with no evidence of in-stent stenosis (Fig. 10A). However, an arterial lumen collapse was identified in the histologic analysis (Fig. 10B). Therefore, an accurate analysis of percent area stenosis ([1 - luminal area/internal elastic lamina] × 100) based on a histologic examination could not be done. Luminal thrombus formation was significantly suppressed in the Ta-implanted PLLA stent group at 2 months after stent placement (21.2% vs. 63.9%, p=0.005, Fig. 11). Neointimal hyperplasia, luminal stenosis, inflammation and fibrosis showed no differences between the two groups.

The expression levels of CD 31, CD 34, and anti-smooth muscle cell alpha actin showed no significant differences between the two groups in the immunohistochemical analysis. CD 68(+) cells, which represent an inflammatory response, were more frequently identified around the bare PLLA stent during the 1-month follow-up (Fig. 12A and B), and the difference disappeared over time (Fig. 12C).