Methods

This study adopted a two-stage preclinical study design consisting of in vitro performance validation and in vivo procedural and safety validation, to systematically evaluate the effectiveness, procedural feasibility, and immediate safety profile of the integrated cerebral protection and active steering directional puncture system. All experimental procedures were strictly performed in accordance with the international guidelines for preclinical development of interventional devices and the ethical requirements for laboratory animal welfare.

In vitro experiments

Experimental materials and equipment

Core devices: integrated cerebral protection and active steering directional puncture system for thoracic aortic ISF (Suzhou Dingke Medical Technology Co, Ltd). The core innovation of this system is the simultaneous realization of full-cycle intraoperative embolic protection and directional puncture fenestration via a single vascular access. Its core functional units include a 120 μm-pore embolic protection filter, an active steering directional puncture assembly, and matched delivery and retrieval assemblies. A commercial thoracic aortic stent graft (Medtronic) was used as the supporting device for in vitro experiments.

Experimental materials

Fluorescent polystyrene microspheres (diameters: 100 , 120 , and 150 μm, Beijing Zhongke Keyou Technology Co, Ltd); glycerol, deionized water, Tween-20 [Youtepu Technology (Suzhou) Co, Ltd]; blood analog solution (glycerol:deionized water = 4:6).

Key equipment

Self-built closed-loop pulsatile flow simulation system (pulsation frequency, 60 beats/min; blood flow velocity, 50-150 mL/s; pressure range, 80-120 mmHg); transparent polymer anatomically accurate human aortic arch model (reproducing the anatomical structures and branch angles of the aortic arch, brachiocephalic trunk, left common carotid artery, and left subclavian artery, including types Ⅰ-Ⅲ aortic arch configurations), fabricated based on computed tomography angiographic data from patients with representative aortic arch anatomies; particle size analyzer (Tianjin Tianhe Analytical Instrument Co, Ltd); ImageJ version 1.8.0 image analysis software (National Institutes of Health).

Simulation system and clinical scenario model construction

The anatomically accurate human aortic arch model was hermetically connected to the circuit of the closed-loop pulsatile flow system, followed by perfusion with the blood analog solution and complete removal of air from the circuit. The pulsatile pump was then activated and calibrated to human physiological hemodynamic parameters (pulsation frequency, 60 beats/min; mean flow velocity, 80 mL/s; mean arterial pressure, 100 mmHg), with a stable circulatory environment maintained for 30 minutes. The commercial thoracic aortic stent graft was accurately implanted into the aortic arch model in strict accordance with the manufacturer's instructions for use, to simulate the standard clinical surgical scenario of stent coverage of the supra-aortic branch ostia after thoracic endovascular aortic repair.

Quantitative analysis of embolus capture efficiency

A randomized controlled design was adopted, with an experimental group (filter deployed) and a blank control group (no filter deployed). For each microsphere size in each group, three independent replicate experiments were performed (n = 3), and the experimental sequence was randomized using a computer-generated random sequence to avoid systematic error.

Experimental pretreatment

Fluorescent microsphere suspensions of 100 , 120 , and 150 μm were prepared at a concentration of 100 particles/mL, followed by sonication to ensure uniform dispersion of the microspheres without sedimentation. The closed-loop flow system and all catheter accesses were rinsed 3 times with deionized water, then preperfused with the blood analog solution to maintain stable hemodynamic parameters. All sample collection tubes were rinsed with deionized water containing 0.1% Tween-20 and air-dried to reduce microsphere adsorption to the tube wall.

Experimental procedure

The integrated system was introduced into the aortic arch model via the simulated brachial artery access. The embolic protection filter was then precisely positioned 5 mm proximal to the origin of the vertebral artery within the left subclavian artery; full deployment, good wall apposition, and stable positioning were confirmed. A 5F single-curve catheter was placed via the simulated femoral artery access, with the catheter tip fixed 1 cm proximal to the filter. After uniform injection of 1 mL of microsphere suspension, 2.5 mL of deionized water was injected at the same flow rate to flush residual microspheres from the circuit. At 2 seconds after microsphere injection (time determined by pre-experiments), the total effluent was hermetically collected at the interface of the vertebral artery outflow tract into a 10 mL graduated tube.

Data quantification

A particle size analyzer was used to count the microspheres in all collected effluent samples, and the capture efficiency was calculated using the following formula: Capture efficiency (%) = [1 – {(Mean number of microspheres in the effluent of the experimental group)/(Mean number of microspheres in the effluent of the control group)} × 100%].

Performance evaluation of active steering directional puncture

Model preparation: In the aortic arch model with an implanted thoracic aortic stent graft, the stent graft regions corresponding to the ostia of the left subclavian artery, left common carotid artery, and brachiocephalic trunk were localized respectively to simulate types Ⅰ to Ⅲ aortic arch anatomical configurations.

Experimental procedure

(1) The integrated system was delivered to the stent graft site corresponding to the target branch vessel, with the tip of the active steering directional puncture catheter positioned against the graft membrane; (2) the force direction and angle of the catheter tip were adjusted via the matched control system to complete simulated puncture; (3) the spatial distribution of all successful puncture sites was recorded.

Area calculation

(1) Accessible puncture area: a convex polygon was formed by connecting all successful puncture sites, and the area was calculated using ImageJ software; (2) target projection area: the geometric projection area of the target vessel ostium on the stent graft membrane was measured; (3) effective puncture area ratio = [(Accessible puncture area)/Target projection area) × 100%].

The effective puncture area ratio is an engineering metric that quantifies how concentrated the puncture attempts are within the projected ostium of the target vessel. A higher ratio suggests that the active steering mechanism can help direct the puncture needle toward the intended target zone. This capability could potentially reduce the likelihood of off-target puncture—a challenge frequently encountered during ISF in complex arch anatomies. However, the clinical relevance of this metric remains to be established.

Qualitative observation of air embolus capture

The embolic protection filter was stably deployed and fully expanded in a straight-tube flow circuit system, with the simulated physiological blood flow environment maintained. 0.1 mL of air was rapidly injected into the flow circuit proximal to the filter to generate air bubbles. The dynamic interaction between the air bubbles and the filter mesh was observed under direct visualization, and the final state of the air bubbles (ie, entrapment, fragmentation, and distal escape) was qualitatively recorded.

In vivo experiments

Experimental animals and ethical approval

All experimental protocols were approved by the Laboratory Animal Ethics Committee of Shanghai Key Laboratory of Vascular Surgery (Approval No. 2025-MS-LAT-D-62). All procedures were performed in strict accordance with the 3R Principles for the Welfare and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, 1996).

Rationale for animal model selection and study design

In this study, an abdominal aortoiliac artery bifurcation model was used to simulate the procedural workflow of thoracic aortic ISF, with the core rationale as follows: (1) the angle between the branch vessel and the aorta in this model is highly consistent with that of the human type Ⅲ aortic arch, which can fully replicate the entire procedural workflow of clinical ISF, including “stent graft coverage of the branch vessel ostium-embolic protection filter deployment-directional puncture-balloon dilation-branch stent implantation”; (2) this model can eliminate the interference of the complex curved anatomy of the aortic arch on the procedural learning curve, enabling more objective and accurate evaluation of the procedural feasibility, efficiency, and immediate safety profile of the device itself; (3) in this study, core performance validation of the device in real clinical anatomical scenarios has been completed via the in vitro anatomically accurate human types Ⅰ to Ⅲ aortic arch models. The core objective of the in vivo experiments is to verify the procedural feasibility and biosafety of the device in a living environment, and this model fully aligns with the objectives of the study design.

Surgical instruments and experimental reagents

Surgical instruments

Abdominal aortic bifurcation stent graft system with iliac limbs (Medtronic); hydrophilic super-stiff guidewires (Terumo), Anplus stiff guidewires (Terumo), hydrophilic guidewires × 6 (Terumo); 5F angiographic catheters × 6 (Johnson & Johnson), 5F single-curve catheters × 6 (Johnson & Johnson); 2 mm Sterling balloon catheters × 6 (Boston Scientific); 4 mm peripheral balloon dilatation catheters × 6 (Dingke); 7 mm peripheral balloon dilatation catheters × 6 (Dingke); indeflators × 6 (Dimark); self-expanding bare metal stents × 6 (Bard).

Experimental reagents

Zoletil 50, for induction of anesthesia (Virbac); Xylazine hydrochloride, for induction of anesthesia (Shengda Animal Pharmaceutical Co, Ltd); Isoflurane, for maintenance of anesthesia (RWD Life Science Co, Ltd); Heparin sodium injection, for anticoagulation (Hepalink Pharmaceutical Group Co, Ltd); Iopamidol contrast medium (Bracco Sine Pharmaceutical Co, Ltd); Lidocaine, for vasodilation (Hualu Pharmaceutical Co, Ltd); Atropine (Changjiang Pharmaceutical Co, Ltd); 0.9% Normal Saline, for fluid resuscitation (Kaile Biotechnology Co, Ltd); 10% Neutral Buffered Formalin, for tissue fixation (Solarbio Science & Technology Co, Ltd); Hematoxylin and Eosin (H&E) Staining Kit (Absin Bioscience (Shanghai) Co, Ltd); Elastic Fiber Staining Kit (Solarbio Science & Technology Co, Ltd).

Surgical procedure

All surgeries were performed by two senior attending vascular surgeons, with the entire procedure monitored under a digital subtraction angiography system. The core workflow is as follows (angiography of key procedural nodes is shown in Fig 1).

Fig 1: Digital subtraction angiography (DSA) images of key procedural nodes of the integrated system during in vivo operation. A, Angiography after precise deployment of the embolic protection filter, confirming favorable deployment morphology and accurate positioning of the filter. The red arrow marks the position of the embolic protection filter; B, Angiography after the active steering catheter was delivered to the target position through the central lumen of the embolic protection filter. The red arrow indicates the position of the steerable catheter; C, Before steering adjustment, the catheter tip presented an obtuse angle with the tangent line of the stent graft; D, After directional steering adjustment, the catheter tip was perpendicular to the tangent line of the stent graft, creating conditions for successful puncture and fenestration; E, Successful puncture and membrane fenestration, with the guidewire smoothly passing through the fenestration and entering the target vessel; F, Final angiography after branch stent deployment, confirming unobstructed blood flow in the target iliac artery without endoleak or stenosis.

Anesthesia and preparation

Intramuscular injection of Zoletil 50 (0.75 mg/kg) combined with xylazine hydrochloride (6 mg/kg) was administered for anesthesia induction. After entering the operating room, the animal underwent endotracheal intubation, and anesthesia was maintained with 1% to 3% inhaled isoflurane. The bilateral inguinal regions were prepped and sterilized; the skin was incised to dissect the external iliac arteries, which were suspended with rubber bands for subsequent use.

Access establishment

Surgical puncture of the suspended external iliac artery was performed under direct vision, and a 6F sheath was inserted to establish the surgical access.

Main stent implantation

An angiographic catheter was advanced via the left access to perform abdominal aortography; the vessel diameter was measured, and a size-matched abdominal aortic stent was selected. The catheter was exchanged for an Anplus stiff guidewire, and the stent was advanced into the abdominal aorta with deployment deferred temporarily.

Integrated system deployment

The integrated system was delivered to the target iliac artery via the right femoral artery access. The filter delivery catheter was fixed, the outer sheath was retracted, and the embolic protection filter was precisely deployed approximately 3 cm proximal to the target vessel ostium.

Directional puncture and fenestration

(1) The puncture adjustment catheter was advanced to the corresponding site of the stent graft membrane via the central lumen of the filter. The stent was deployed along the stiff guidewire, with the main body of the stent positioned in the abdominal aorta, the unilateral iliac limb extended into the ipsilateral iliac artery, and the ostium of the contralateral iliac artery retained as the simulated “target branch vessel.” (2) The tip angle of the active steering directional puncture catheter was adjusted to position it as perpendicular to the graft membrane as possible. (3) Timing of fenestration was initiated, the graft membrane was punctured with a puncture needle, timing was stopped, and a 0.018-inch guidewire was advanced through the fenestration. (4) The puncture needle and active steering directional puncture catheter were withdrawn.

Branch vessel reconstruction

Sequential dilation of the puncture site was performed with 4 and 7 mm peripheral balloons in turn. A self-expanding bare metal stent was advanced over the guidewire and deployed at the fenestration site to complete branch vessel reconstruction.

Endpoint assessment and data collection

Feasibility endpoint

Technical success rate was calculated with the formula below: Technical success rate (%) = [(Number of experimental animals that successfully completed the full workflow of “embolic protection filter deployment-directional puncture-branch stent implantation”)/(Total number of experimental animals) × 100%].

Efficiency endpoints

(1) Puncture and fenestration time: the total time from the completion of adjustment of the active steering directional puncture catheter to the passage of the guidewire through the graft membrane was recorded. (2) Operator usability feedback scale was designed with reference to the subjective operation scoring scale for other developed devices10 (as shown in Table I). To collect preliminary user experience, the lead surgeon scored the operational experience of the device postoperatively using a 0-10 point visual analog scale (0 points = unable to operate, 10 points = extremely easy to operate), with assessments across five dimensions: overall usability, directional accuracy, procedural simplification, operability of the cerebral protection device, and confidence in safety.

Assessment dimension | Core question | Scoring reference | Score
Overall usability | Is the overall operation process of the entire device from delivery to retrieval intuitive and smooth? | 0-3 points: Chaotic process with extreme operational difficulty | 
 |  | 4-6 points: Feasible process but requiring extensive adjustments | 
 |  | 7-8 points: Smooth process, basically meeting expectations | 
 |  | 9-10 points: Highly intuitive and efficient process | 
Operability of the cerebral protection device | Is the deployment, positioning, and retrieval process of the embolic protection filter controllable and reliable? | 0-3 points: Uncontrollable, with failed positioning or difficult retrieval | 
 |  | 4-6 points: Controllable but requiring repeated adjustments, with low confidence in reliability | 
 |  | 7-8 points: Controllable and reliable, with successful completion on the first attempt | 
 |  | 9-10 points: Precise and smooth operation with excellent handling experience | 
Directional accuracy of the active steering directional puncture catheter | Is the function of adjusting the catheter tip angle and puncture force line via inflation of different balloons accurate and effective? | 0-3 points: Unable to achieve effective orientation, or extremely unstable | 
 |  | 4-6 points: Able to achieve orientation but with slow response or moderate accuracy | 
 |  | 7-8 points: Able to effectively and stably achieve a vertical puncture force line | 
 |  | 9-10 points: Rapid response, high precision, and excellent handling performance | 
Degree of procedural simplification | Does this integrated device truly simplify the coordinated operation of “cerebral protection” and “precise puncture”? | 0-3 points: No simplification achieved, even more complicated than conventional procedures | 
 |  | 4-6 points: Mild simplification with no obvious advantage | 
 |  | 7-8 points: Significantly simplified workflow and reduced operational burden | 
 |  | 9-10 points: Revolutionary simplification that renders complex steps straightforward | 
Confidence in safety | How confident are you in the safety of the device throughout the entire operation? | 0-3 points: Concerns about safety with clear potential risks | 
 |  | 4-6 points: Generally safe but with residual concerns | 
 |  | 7-8 points: Confident in safety, with comprehensive safety considerations in the device design | 
 |  | 9-10 points: Extremely high confidence in safety with a strong sense of security | 

Safety endpoints

(1) Device integrity: immediately after the procedure, the retrieved active steering directional puncture catheter and embolic protection filter were macroscopically examined, and the presence or absence of coating exfoliation and structural damage was recorded and photographed. (2) Embolus capture analysis: the embolic protection filter was gently flushed, and the flushing fluid was collected, observed and photographed under a microscope, with the presence or absence of captured debris recorded. (3) Histopathological evaluation: immediately after the procedure, the iliac artery segment containing the puncture site was harvested, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned, followed by H&E staining and elastic fiber staining. The integrity of the vascular intima, elastic lamina rupture, and the degree of acute inflammatory reaction were evaluated under a microscope.

Statistical analysis

Statistical analysis was performed using SPSS version 26.0 software (IBM Corp.). Continuous variables conforming to normal distribution were expressed as means ± standard deviation, data with non-normal distribution were expressed as median (interquartile range), and categorical variables were expressed as number (%). For the comparison of capture efficiency of microspheres with different particle sizes, the normality of the data was first verified by the Shapiro-Wilk test, and the homogeneity of variances was verified by Levene test. One-way analysis of variance was applied for data conforming to normal distribution with homogeneous variances, with the Bonferroni method for pairwise comparisons. For data that did not meet the above criteria, the Kruskal-Wallis H test was used, with the Dunn method for pairwise comparisons. Spearman rank correlation analysis was performed to evaluate the correlation between microsphere particle size and capture efficiency. A two-sided P < .05 was considered statistically significant. However, given the small sample size, all statistical analyses are exploratory and results should be interpreted as descriptive rather than definitive.