Introduction

Overlapping stent placement for diffuse long coronary artery lesions is a commonly used technique in clinical practice and is performed in 10–30% of patients1,2,3,4,5. Stent overlap is used in patients with edge dissections, excessive lesion length, diffuse stenotic areas, insufficient stent coverage, and recurrent lesions in patients with coronary artery diseases4,6. The conventional overlapping technique follows standard interventional protocols, as described by Finn et al. and Moses et al., with approximately 30% of patients undergoing percutaneous coronary intervention (PCI)1,4,7,8. The overlap geometry can increase wall shear stress, resulting in unfavorable flow conditions that may negatively impact clinical outcomes9. Conventional overlapping stents create mechanical discontinuities, leading to excessive neointimal proliferation and increased thrombogenic risk4,6. Advances in coronary stent technology, including novel antiproliferative drugs with optimized elution dynamics, improved stent architecture, and more biocompatible polymers, have been actively investigated to enhance clinical outcomes in patients with diffuse long coronary artery lesions. Many clinicians recommend using a minimal overlap technique to prolong the luminal patency at the overlapped lesions10.

The bioresorbable scaffold (BRS) offers the potential for future surgical revascularization, enhanced vascular remodeling, restoration of vasomotor function, and accurate non-invasive coronary artery imaging using multislice computed tomography11,12. However, due to their non-radiopaque BRS composition, standardized implantation protocols for overlapping BRSs have not yet been established, making accurate procedural guidance challenging. Furthermore, thicker struts induce greater neointimal development in the microenvironment, particularly in “stacked” BRS struts, and is likely associated with the influence of wall shear stress on neointimal response11,13. Therefore, previous studies have suggested that the overlap regions of BRS should be minimized by positioning them in proximity and ensuring that only a small area of overlap occurs between them10. However, the current overlapping techniques cannot be reliably performed using angiography and are more likely to occur stochastically rather than because of a controlled procedural technique. Here, we propose an optimized overlapping method designed as a theoretical end-to-end deployment method; this can enhance the completion of endothelialization by minimizing the number of stacked struts. This method utilizes balloon and strut markers as reference points during BRS placement to compensate for scaffolds with limited visual information. The end-to-end technique can ensure precise alignment of scaffold ends, maintaining structural continuity and optimizing mechanical integrity to promote vascular healing. Therefore, this proof-of-concept (PoC) study aimed to investigate the efficacy and safety of the end-to-end technique compared with the conventional overlapping technique of BRS placement in long porcine coronary artery lesions.

Results

Characteristics of the BRSs and delivery balloon catheters

The distance between the center of the balloon marker and the distal scaffold strut marker marginally increased following balloon expansion (before: 2.4 ± 0.1 mm; after: 2.5 ± 0.16 mm); however, this difference was not statistically significant. The benchtop stress test results revealed that the stress–displacement response to crush resistance was characterized by a higher radial force in the overlapped BRSs (15.342 N) than in the single BRS (8.058 N) (Fig. 1d and e).

Fig. 1
Fig. 1
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Characterization of the BRS and delivery balloon catheter. (a) schematic illustration shows the marker locations of the BRS and balloon catheter. (b) Photographs show the deflation and inflation status of the BRS-crimped balloon catheter. No difference was observed in the distance between the strut and the balloon markers in the two groups. (c) Schematic illustration shows the technical point of BRS placement in the overlapping and end-to-end groups. (d) Photographs obtained during the measurement of mechanical properties of single and overlapped BRSs. (e) Radial force of overlapped BRSs is significantly higher than that of single BRS.

Technical feasibility of overlapping and end-to-end techniques

The overlapping and end-to-end BRS placement was technically successful in all trials (Fig. 2a and b). The distances of the overlap lesions were 2.4 mm ± 0.15 and 0 mm ± 0.11 in the overlapping and end-to-end groups, respectively (Fig. 2c). SEM image analysis revealed that the markers of the overlapping BRSs were aligned in the same line at the overlap lesion, and the overlap lesion was formed with a length of 2.4 mm in the overlapping group. While both ends of the BRSs in the overlapping group were well-attached, multiple strut fractures were observed (Fig. 2d). A fracture was observed at the overlap lesion of the initially deployed scaffold, which was positioned on the outer layer. In the end-to-end group, the BRSs maintained structural continuity with only the edges in contact, and the overlap was observed to be less than 0.1 mm long in the SEM images. Additionally, the strut morphologies in the end-to-end group showed no evidence of strut damage.

Fig. 2
Fig. 2
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Technical feasibility of the overlapping and end-to-end techniques. Representative radiographic images show the technical steps in the (a) overlapping and (b) end-to-end groups (white arrows, first balloon markers; white arrowheads, first BRS markers; yellow arrows, second balloon markers; yellow arrowheads, second BRS markers). (c) Graph shows the mean distances between both ends of the BRSs at the overlap lesion in the two groups. (d) SEM images show multiple strut fractures (white arrows) in the overlapping group but no strut damage in the end-to-end group.

Procedural outcomes, follow-up coronary angiography, and OCT findings

BRS placement was technically successful in all the study groups. All the pigs survived until the end of the study without procedure-related complications and humane endpoints. The follow-up coronary angiographic and OCT findings are represented in Fig. 3 and summarized in Table 1. Follow-up coronary angiography revealed that the stent patency was well-maintained 4 weeks after BRS placement in the end-to-end group. However, luminal narrowing was observed in the overlapping group (Supplemental Fig. S2). The mean luminal diameters of the overlapped lesion and the first BRS segment in the overlapping group were significantly greater than those in the end-to-end group (all p < 0.05, two-sample t-test). The most pronounced difference between the two groups was observed at the overlap lesion (24.82% ± 1.07 vs. 8.22% ± 0.32, p < 0.01, two-sample t-test). However, no significant difference was observed in the mean luminal diameters of the two groups at the second BRS segment (p = 0.218, two-sample t-test). OCT analysis revealed that the BRS was minimally overlapped or completely fit both ends of the BRSs in the end-to-end group, whereas two layers of overlapped struts were observed in the overlapping group. The mean neointimal thickness at the overlapped lesion was significantly higher in the overlapping group than that in the end-to-end group (0.58 mm ± 0.11 vs. 0.22 mm ± 0.03; p < 0.001, Mann–Whitney U test). Furthermore, the percentage stenosis area in the overlapped lesion was significantly higher in the overlapping group than that in the end-to-end group (65.7% ± 1.02 vs. 20.62% ± 0.53; p < 0.001, two-sample t-test). However, the neointimal thickness and percentage stenosis area in the first and second BRS segments were not significantly different between the two groups.

Fig. 3
Fig. 3
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Follow-up coronary angiography and OCT findings in a porcine coronary artery model. (a) Representative coronary angiography and OCT images of the porcine coronary artery immediately and 4 weeks after the overlapping and end-to-end procedures (white arrowhead, first BRS; yellow arrowhead, second BRS). Graphs show changes in the (b) luminal diameter, (c) neointimal thickness measurements, and (d) percentage stenosis area in the two study groups. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

Table 1 Angiographic, OCT, and histological findings in the overlapping and end-to-end groups at one month.
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Histological findings

The histological findings are represented in Figs. 4 and 5. The mean degrees of inflammatory cell infiltration in all segments were significantly higher in the overlapping group than in the end-to-end group (all p < 0.05, Mann–Whitney U test). The fibrin deposition at the overlap lesion was significantly higher in the overlapping group than that in the end-to-end group (p < 0.001, Mann–Whitney U test). The mean thickness of the neointima in the first BRS and overlapped lesion was significantly higher in the overlapping group than in the end-to-end group (all p < 0.05, Mann–Whitney U test). The EL change at the overlap lesion was significantly greater in the overlapping group than in the end-to-end group (p < 0.001, Mann–Whitney U test). Additionally, the degree of presence of apoptotic cells at the overlap lesion was significantly higher in the overlapping group than that in the end-to-end group (p < 0.001, Mann–Whitney U test).

Fig. 4
Fig. 4
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Microscopic images of the histological slices stained with hematoxylin-eosin (H&E). (a) Representative microscopic images at the first BRS, overlapped lesion, and the second BRS segments in the overlapping and end-to-end groups (white arrowhead, first BRS; yellow arrowhead, second BRS). Graphs show the (b) degree of inflammatory cell infiltration, (c) fibrin deposition, and (d) thickness of neointima in all the segments in the overlapping and end-to-end groups. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5
Fig. 5
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Verhoeff-Van Gieson staining and immunohistochemistry analysis of TUNEL images. (a) Verhoeff-Van Gieson staining shows the degree of changes in the EL, and TUNEL microscopic images show the apoptotic cells at the overlapped lesion in the study groups (black star, first BRS; yellow star, second BRS). (b and c) The graphs show the degree of elastic lamina and positive deposition of TUNEL staining in the study groups. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

Metal stents with or without a drug coating are often implanted using overlapping techniques to ensure full lesion coverage in long coronary artery disease1,2,3,4,5. This approach increases metal burden, disrupts vascular compliance, and predisposes the vessel to excessive neointimal hyperplasia and thrombosis1,27,28. To mitigate these risks, minimal overlap strategies have been proposed to optimize stent alignment while reducing complications associated with excessive metal stacking10. BRS has been developed to overcome the limitations of metal stents. However, their lack of radiopacity poses technical challenges, making precise implantation difficult and limiting their widespread adoption for additional or multiple stenting. We addressed these challenges by implementing a novel minimal overlapping technique using radiopaque markers in interventional devices, which involves end-to-end alignment of the scaffold, which ensures that they are minimally overlapping or without overlap. Further, we evaluated the efficacy and safety of this technique compared to the conventional overlapping technique. Our findings demonstrate that the BRS-based end-to-end technique is feasible and superior to the overlapping approach, as it improves scaffold integrity, reduces neointimal proliferation, and minimizes inflammatory responses. Although the follow-up duration of the PoC study was only 4 weeks, these results underscore the potential clinical benefits of the end-to-end technique in optimizing multiple BRS implantation using minimal overlapping strategies.

The current study investigated the BRS placement technique for long diffuse coronary lesions, comparing the overlapping and novel techniques. To improve technical accuracy, the BRS-loaded balloon catheter was designed to maintain a constant distance between the BRS strut marker and balloon marker before and after balloon dilation. Technical success rates were 100% in the overlapping and end-to-end groups. Furthermore, the second BRS placement was technically successful in all cases. However, H&E staining confirmed the presence of a strut structure adjacent to the single-layered BRS strut in the end-to-end group. This finding suggests a significant reduction in strut stacking, falling within the technical success rate range of 0 ± 0.24 mm as defined in the end-to-end group, suggesting that minimal overlap can be successfully achieved. These findings emphasize the importance of precise marker-guided positioning of the scaffold and balloon to guarantee accurate deployment of the end-to-end technique.

BRSs have a limited expansion range, beyond which fracture may occur29. Additionally, increased ballooning pressure during stent-in-stent deployment may lead to overexpansion of the BRS. This additional mechanical stress may increase the risk of strut fractures in the first scaffold in the present study; our SEM findings revealed strut fractures predominantly in the overlapping group. Follow-up angiography demonstrated a significant reduction in luminal diameters in the overlapping group, particularly in the overlap lesion, whereas the end-to-end group showed no significant changes. Luminal narrowing and neointimal hyperplasia in the overlap lesion were significantly more pronounced in the overlapping group9,30. These results may be attributed to the mechanical stress exerted on the first BRS during balloon dilation for the second BRS placement. Such strut fractures alter the hemodynamic microenvironment, leading to in-stent restenosis (> 50%), as previously reported31,32. OCT findings supported these results, revealing increased thickness of neointimal tissue and percentage stenosis area in the overlapping group compared with the end-to-end group. Histological analysis further supported these findings, showing increased inflammatory cell infiltration, fibrin deposition, and EL disruption in the overlapping group than in the end-to-end group. Additionally, the apoptotic cell count was significantly higher in the overlapping group, indicating a more pronounced biological response to scaffold implantation. These findings suggest that the end-to-end or minimally overlapping strategy better preserves scaffold integrity and vascular patency, supporting its potential as a preferred approach for BRS implantation in long coronary lesions.

Although the clinical safety and efficacy of BRS are considered comparable to those of second-generation drug-eluting stents (DESs)33,34, the risk of BRS-related thrombosis remains a concern. Several studies have indicated that overlapping BRS implantation may increase the risk of scaffold thrombosis, possibly due to greater lumen loss caused by the presence of dual-layered struts35,36,37. According to the report by Biscaglia et al., overlapping BRS presents a particular challenge due to increased strut thickness and limited angiographic visualization. To provide sufficient radial strength, BRS struts should have a thickness ranging from 100 to 150 μm38. However, in the overlapped lesion, the stacked struts result in a combined thickness exceeding 200–300 μm, contributing to luminal steric hindrance. This substantial increase in strut thickness has been associated with a high shear rate and expanded surface area for binding adhesion proteins, thereby creating a more thrombogenic surface in the protruding strut39. Findings from Ishibashi et al. suggest that overlapping everolimus-eluting BRS placement is an independent risk factor for periprocedural myocardial infarction40. In the current study, the BRS used a strut thickness of 100 μm. The stress–displacement response in the overlapped areas changed or increased as the thickness of the strut increased due to the overlap. This alteration could affect the vascular environment, leading to acute thrombosis and induced vascular stenosis. Therefore, a minimal overlapping strategy, such as the end-to-end BRS implantation proposed in the PoC study, is necessary and may contribute to lowering the incidence of BRS-related thrombosis in future PCI.

The BRS was developed to achieve enhanced vascular repair and reduced risk of device-related thrombosis compared with permanent DESs in the field of PCI41. However, several clinical trials have raised concerns regarding stent failure, including an increased risk of thrombosis, reduced radial force, and a higher incidence of major adverse cardiac events42,43. Unlike in PCI, the reduced dynamic movement and pulsation in peripheral arteries, along with decreased motion artifacts, make it easier to identify small stent markers, potentially increasing the likelihood of maintaining scaffold integrity. The findings from this study have significant clinical implications for optimizing BRS implantation strategies. Given the challenges of overlapping stents, particularly those made with BRS, minimizing overlap may be a crucial step in reducing procedure-related adverse events such as restenosis and thrombosis. Future preclinical studies are needed to validate these findings with long-term follow-up duration and to refine the procedural guidelines for optimal scaffold placement in long coronary or peripheral artery lesions.

This study has certain limitations. First, it was conducted in a healthy porcine coronary artery model, which may not fully replicate the physiology of a diseased human coronary artery. Second, although variables of study outcomes reached statistical significance, the sample size was small. Additional studies with larger sample sizes should be required to confirm our findings with increased statistical power. Third, this experiment was conducted using a single type of BRS provided by a specific company; therefore, these findings may not be generalizable to all types of BRSs. However, balloon marker-scaffold strut marker and scaffold-scaffold strut markers may help achieve a minimal overlap effect with other BRS products. Fourth, the follow-up period was limited to four weeks, which is insufficient to assess the long-term effects and durability of the end-to-end technique. Given that BRSs undergo complete bioresorption over approximately 36 months, longer-term data are needed to fully evaluate its outcomes44. Lastly, the current study did not include standard-of-care coronary stents as a control group such as metallic DES or bare metal stent (BMS) to demonstrate the clinical context of the study findings. Despite these limitations, the results from angiography, OCT, and histological analyses in our study suggest that the end-to-end technique is superior to the overlapping technique when using BRS in a porcine coronary artery model.

Although the conventional overlapping technique using BRSs is relatively simple, it is associated with significant strut fractures with neointimal hyperplasia resulting from severe inflammatory reactions at the overlapped lesions. The minimally overlapping technique using BRSs effectively and safely maintained the luminal patency for 4 weeks at the overlapped lesions in the porcine coronary artery. The end-to-end technique using BRSs seems to be a more favorable strategy in the treatment of long diffuse coronary artery lesions.

Methods

Preparation of BRS

The BRSs used in this study were manufactured by Dotter (BRS35013; Yeonsu-gu, Incheon, Korea) using Poly-L-Lactic Acid (PLLA). The scaffolds were 3.5 mm in diameter and 13 mm long. The strut thickness of the scaffold was 100 μm. The radiopaque strut markers were attached at both ends of the scaffold. The scaffold strut markers were positioned 1.5 mm away from end of balloon markers. The length of balloon markers was 0.7 mm. The scaffold strut markers were positioned 1.2-mm away from both ends of the scaffold. The balloon markers were positioned 0.3 mm away from both ends of the scaffold strut. Therefore, the distance between the balloon marker and the scaffold strut marker was set at 2.4 mm (Fig. 1a). To ensure consistency, the distance from the center of the balloon marker to the distal scaffold strut marker was measured in ten samples before and after expansion using an electronic caliper (Fig. 1b). Everolimus, an antiproliferative agent, was coated onto the surface of the BRS using an ultrasonic spray coating technique. The drug-eluting BRS contained 118 µg/cm2 of everolimus. The everolimus-eluting BRSs were crimped into a balloon catheter (diameter, 3.5 mm; length, 15 mm) using a scaffold crimping machine (J-Crimp; Blockwise Engineering, Tempe, Ariz). The scaffold is currently in a clinical trial for coronary artery disease and awaiting Ministry of Food and Drug Safety (MFDS) approval.

Mechanical properties of single or overlapped BRSs

The mechanical properties of the single or overlapped BRSs were measured. The tester was set to begin reducing the stent diameter from the unconstrained value of 3.5 mm. The crush resistance test was conducted using a universal testing machine (UTM; MINOS-005, MTDI, Daejeon, Korea). To evaluate the mechanical strength specifically at the overlapped region of the BRS, a 3-mm-long 3D-printed tip (CUBICON; Style-220 C, Seongnam-si, Gyeonggi-do, Korea) was fabricated and utilized in the test. The bench-top test was performed at a speed of 0.1 mm/s. Stress and displacement values were recorded at 0.01-s intervals and plotted on stress–displacement graphs. The test was performed using the load-controlled feedback mode with a 60-s linear loading at a peak force of 30 kgf14,15.

Definitions of conventional overlapping and end-to-end techniques

The BRS crimped on the balloon catheter was designed to maintain a consistent distance of 2.4 mm between the balloon marker and the BRS strut marker before and after expansion. In the overlapping group, the two BRSs overlapped by 2.4 mm at their respective ends. The markers attached to the scaffold served as reference points, ensuring that the markers at both ends of the two BRSs were aligned on the same axis or in parallel. The end-to-end deployment method of the BRSs involved minimal overlap between the two BRSs. Markers attached to the scaffolds and balloon were used as reference points, ensuring that two different markers were minimally overlapped along the same axis or parallel to each other (Fig. 1c).

Technical feasibility in a natural rubber latex tube

Two overlapping techniques were evaluated in a natural rubber latex tube (diameter, 3.5 mm; length, 150 mm; thickness, 1.2 mm) to evaluate the technical feasibility of overlapping BRS placement before the in vivo study. BRSs were placed into the latex tube under fluoroscopic guidance (OEC Elite CFD; GE HealthCare, IL, Chicago, USA). The BRS location was determined using the radiopaque strut markers at both the ends of BRSs. Post-balloon dilation was performed after BRS placement to fully expand the BRS in the same way as in clinical practice8. Technical success was defined as overlapped lesion lengths of 2.4 mm ± 0.24 mm and 0 mm ± 0.24 mm in the overlapping and end-to-end groups, respectively. Ten trials were performed in each study group to determine statistical reproducibility.

Surface morphology at the overlapped lesion

After BRS placement, the latex tube was carefully cut and removed. The extent of damage to the strut during the procedure was analyzed using scanning electron microscopy (SEM; ZEISS Sigma-500, Oberkochen, Germany)16. SEM analysis was conducted on the struts of the overlap lesions in the overlapping and end-to-end groups to evaluate strut deformation and strut-to-strut attachment.

Animal study design

This study was conducted in accordance with the guidelines of the US National Institutes of Health for the humane treatment of laboratory animals. This study was approved by the Institutional Animal Care and Use Committee of Asan Institute for Life Sciences (IACUC approval number: 2024-20-086) and conformed to US National Institutes of Health guidelines for handling laboratory animals. A total of eight coronary arteries in four male Yorkshire pigs weighing 32.2–33.45 kg (age, 3–4 months; mean weight, 32.75 kg) were used. These were randomly allocated to one of two groups; each group comprised four coronary arteries, including two right coronary arteries and two left coronary arteries. The overlapping group received two 13-mm BRSs with a 2.4-mm overlap lesion. The end-to-end group received two 13-mm BRSs with a minimally overlapped lesion. All pigs received aspirin (100 mg/day, PO; Bayer AG, Leverkusen, Germany) and clopidogrel (Plavix Table 75 mg/day, PO; HANDOK Inc., Seoul, Korea) as dual antiplatelet maintenance therapy based on the recommendation in standard treatment guidelines. The drugs were administered daily, starting from one day before scaffold placement to the date of their sacrifice. Antibiotics (gentamicin, 7 mg/kg, IM; Shin Poong Pharm Ltd., Seoul, Korea) and analgesia (keromin, ketorolac 1 mg/kg, IM; Hana Pharm Ltd., Seoul, Korea) were administered for 3 days after the procedure. Animals were housed under the same conditions at 24 ± 2 °C with a 12-h day/night cycle and free access to water and food. All pigs were euthanized using an overdose of potassium chloride (75 mg/kg, IV; Daihan Co., Seoul, Korea) four weeks after BRS placement. The humane endpoint was defined as significant weight loss (> 20%), abnormal behavior (e.g., loss of appetite or activity), or any clinical signs of distress or suffering. All animals were monitored daily, and euthanasia was performed if predefined humane endpoints were met prior to the scheduled sacrifice. This study was reported in accordance with the ARRIVE guidelines.

Overlapping BRSs placement in the porcine coronary artery

Animals were premedicated with a mixture of 50 mg/kg zolazepam and 50 mg/kg tiletamine (Zoletil 50, IM; Virbac, Carros, France), along with 10 mg/kg xylazine (Rompun, IM; Bayer HealthCare, Leverkusen, Germany). Anesthesia was maintained through endotracheal intubation with 0.5–2% isoflurane (Ifran®; Hana Pharm. Co., Seoul, Korea) in a 1:1 oxygen ratio (510 mL/kg/min) for an hour using anesthesia machine (Fabius® GS premium; Dräger inc., Lübeck, Germany). A 6-Fr arterial sheath was inserted into the right common femoral artery under ultrasonographic guidance. Heparin (100 units/kg) was administered intra-arterially via the sheath. A 6-Fr guiding catheter with a 0.035” guide wire was advanced into the ascending aorta. The targeted coronary arteries were cannulated, and coronary angiography was performed to determine the location of BRS placement. All BRSs were deployed at 6 atm for 30-s balloon inflation. Post-balloon dilation was performed using a 3.5-mm non-compliant balloon catheter to ensure full expansion in all placed BRSs. The detailed procedure for the overlapping and end-to-end techniques is shown in Supplementary Fig. S1.

Follow-up coronary angiography and optical coherence tomography

The coronary angiography and intracoronary optical coherence tomography (OCT, Aurios™; Dotter Inc.) were performed immediately after BRS placement and before sacrifice to verify the BRS position and patency. The OCT catheter was advanced beyond the stented distal coronary artery over a 0.014” guidewire. OCT pullback was performed with contrast injection through the guiding catheter (with typical flush rates of 4.0–5.5 mL/s and 3.0–4.0 mL/s for the left and right coronary arteries, respectively, at 300–400 psi)17. Analyses of continuous cross-sections were performed at 180-µm longitudinal intervals with adjustment for pullback speed, in line with previously validated methodologies11,12,18,19. A total of ten cross-sections per segment were obtained to analyze luminal diameters. The stented coronary artery was portioned into three segments: the center of the first BRS, overlap lesion, and center of the second BRS. The luminal diameters of the stented coronary arteries were measured using RadiAnt DICOM viewer (version 1.1.20, Medixant Company, Poland). The luminal diameter changes were calculated using the calculation formula given below.

$$\:Luminal\:diameter\:change\%\:=\left(\frac{1\:month\:after\:lumimal\:diameter-immediately\:luminal\:diameter}{immediately\:luminal\:diameter}\right)\times\:100$$

The axial- and longitudinal-sectioned OCT images were analyzed with the Aurios™ ORW software system (Dotter Inc.). The thickness of the neointimal tissue and the percentage stenosis area were measured20. The thickness of neointimal tissue was defined as the distance between the outer strut of the BRS and the luminal boundary21. The percentage of stenosis area was calculated using the calculation formula given below22.

$$\:SA\%=(1-\:[lumen\:area/\:outer\:stented\:area\left]\right)\:\times\:100$$

Histological examination

The stented coronary arteries were surgically extracted after sacrifice. The arteries were flushed with heparinized saline, and the samples were fixed in 10% formaldehyde. The stented segments were embedded in paraffin and sectioned into 5-µm-thick slices. Fixed tissue samples were sectioned transversely at the center portion of the first BRS, the overlapped lesion, and the center portion of the second BRS. The sectioned specimens were subsequently stained with hematoxylin and eosin (H&E) to evaluate degrees of inflammation and fibrin deposition4. The degrees of inflammatory cell infiltration and fibrin deposition were evaluated according to the density and distribution around the scaffold struts as follows: 1, mild; 2, mild to moderate; 3, moderate; 4, moderate to severe; 5, severe15,23. Eight points were randomly selected for each sample and analyzed using ImageJ software to determine statistical reproducibility. Verhoeff Van Gieson staining was performed to evaluate changes in the elastic lamina (EL). EL changes were assessed based on the specific vascular layers traversed by each individual strut. A numeric value was assigned as follows: 0 = no injury; 1 = break in the internal elastic membrane; 2 = perforation of the media; 3 = perforation of the external elastic membrane to the adventitia24. For each analyzed segment, the injury score was calculated by dividing the sum of the individual scores of all struts by the total number of struts present in the corresponding Sect25.

Immunohistochemical analysis

Immunohistochemical analysis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The deposition of TUNEL-positive cells in the stented coronary artery was subjectively determined according to the density and distribution of apoptotic cells (1, mild; 2, mild to moderate; 3, moderate; 4, moderate to severe; and 5, severe)15,26. The analyses of the histologic findings were accessed on the basis of the consensus of three observers blinded to group assignment.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). Differences between groups were analyzed using a two-sample t-test and Mann–Whitney U test, as appropriate. Results with *p < 0.05 were considered statistically significant. Statistical analyses were performed using SPSS software (version 27; IBM, Chicago, IL, USA).