Morphological analysis of cadaveric porcine ET
This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of the Asan Institute for Life Sciences and conformed to the ARRIVE guidelines for humane handling of laboratory animals (IACUC-2020-12-189). All the experiments were performed in accordance with relevant guidelines and regulations. Six ETs of three fresh cadaveric porcine heads (Yorkshire, 33.4–36.7 kg at 3 months; Orient Bio, Seongnam, Korea) were used in this study. The porcine heads were mid-sagittally sectioned to identify the nasopharyngeal ostium. A porcine ET phantom was fabricated using the fabrication process of a cadaveric ET phantom in a previous study19. A 100 μl pipette tip was connected to a 10 ml syringe, and the end tip of the pipette was inserted into the nasopharyngeal ostium. Silicone (Otoform AK®, Dreve Otoplastik GmbH, Unna, Germany) was injected through the pipette into the porcine ET until it completely covered the nasopharyngeal ostium, and constant pressure was then applied in the ET (Fig. 1). The silicone-filled porcine heads were kept at 4 °C for 12 h, and the cured silicone was later separated from the porcine ET.
Fabrication of the cadaveric porcine ET phantom. (a) The nasopharyngeal ostium (arrows) of the cadaveric porcine head was identified following mid-sagittal sectioning. (b) Silicone (arrow heads) was injected until completely filling the ET. (c,d) The two lateral and (e) anterior projections of the silicone phantom extracted from the porcine ET.
Image analysis of porcine ET phantom
A micro-CT imaging system (NanoPET/CT, Mediso Ltd–Bioscan Inc., Arlington, Texas, USA) was used to quantitatively analyze the size of the porcine ET phantom. CT images were reconstructed to analyze the morphology of the porcine ET phantoms. Three-dimensional-reconstructed images were obtained from the portions axially sectioned at 4 mm intervals from the nasopharyngeal ostium to the isthmus. A total of six sections were obtained every 4 mm, and points were defined from P0 to P5. The height of the axially sectioned ET was measured as the length from the top to bottom of the ET lumen. The width of the axially sectioned ET was measured as the maximum distance between the anterior and posterior sides. The overall length of the ET was measured as the distance from the nasopharyngeal ostium to isthmus.
Stent preparation
All SEMSs (S&G Biotech Co., Ltd, Yongin, Korea) used in this study were designed and manufactured based on the morphological findings of the porcine ET phantom. A total of 32 nitinol wires with a thickness of 0.09 mm were interwoven using a braiding machine. The SEMS optimized for the porcine ET had a tapered structure. When fully expanded, the T-SEMS was 2 mm in diameter at the distal end and 5 mm in diameter at the proximal end of the T-SEMS and 16 mm in length (Fig. 2a). The C-SEMS had a tubular structure and was 3 mm in diameter and 16 mm in length (Fig. 2b).
Self-expandable metallic stents (SEMSs) with the delivery system and the technical steps for stent placement into the porcine ET. Photographs showing (a) the tapered SEMS (T-SEMS) and (b) conventional SEMS (C-SEMS). (c) The steering control threads (arrows) were used to modulate the angle of the proximal end of the stent delivery system for easy access to ET orifice (maximum curved angle was 45°). (d) Endoscopic image showing the nasopharyngeal ostium (arrows) and inserted steerable delivery system. (e) The angled delivery system was inserted into the nasopharyngeal ostium. (f) The T-SEMS (arrow heads) was successfully placed into the ET.
Steerable stent delivery system
The SEMS delivery system consisted of a 9 Fr steerable sheath (OSYMED. Co., Ltd, Yongin, Korea), steering control threads, and a pusher catheter (Fig. 2c). The maximum curved angle of the SEMS delivery system was 45°. The steerable SEMS delivery system was developed for easy access to the nasopharyngeal ostium of the porcine ET.
Animal study
This part of the experiment was also approved by IACUC and conformed to the ARRIVE guidelines. A total of six ETs of three pigs (Yorkshire; Orient Bio) weighing 34.2–36.5 kg was divided into two groups that received the T-SEMS or C-SEMS. All pigs were supplied with water and food ad libitum and were maintained at a temperature of 24 ± 2 °C with a 12-h day–night cycle. Subsequently, all pigs were euthanized 4 weeks after the stent placement by intravenously injecting potassium chloride (DAI HAN PHARM CO., Seoul, Korea).
Stent placement into the porcine ET and endoscopic examination
All pigs were anesthetized immediately before stent placement using a mixture of 50 mg/kg zolazepam, 50 mg/kg tiletamine (Zoletil 50; Virbac, Carros, France), and 10 mg/kg xylazine (Rompun; Bayer HealthCare, Leverkusen, Germany). An endotracheal tube was then placed, and anesthesia was administered by inhalation of 0.5–2% isoflurane (Ifran®; Hana Pharm. Co., Seoul, Korea) with 1:1 oxygen (510 ml/kg per min). Endoscopic examination (VISERA 4K UHD Rhinolaryngoscope; Olympus, Tokyo, Japan) was performed to check the nasopharyngeal ostium of the ET. The steerable delivery system loaded with the T-SEMS or C-SEMS was advanced through the nostril to the nasopharyngeal ostium of the ET under endoscopic guidance (Fig. 2d). The steerable delivery system was bent toward the nasopharyngeal orifice and carefully inserted into the ET until it met with resistance in the isthmus portion of the ET (Fig. 2e). The stent was placed into the ET by withdrawing the delivery system, while the pusher catheter in place (Fig. 2f). Post-procedure endoscopic examination was performed to evaluate any procedure-related complications and to determine the location of the proximal end of the stent. An endoscopic examination was conducted on the pigs at 4 weeks after the stent placement to evaluate the stent position, patency, and secretion presence around the stent.
Histological examination
A histological examination was conducted on the basis of previous ET stent studies14,15. The stented ET tissues were extracted. The ET tissue samples were fixed in 10% neutral-buffered formalin for 3 days. After fixation, the samples were embedded in a resin block. The resin blocks were sliced from the proximal and distal portions of the segment using the grinding system (Apparatebau GmbH, Hamburg, Germany). The slides were stained with hematoxylin–eosin for the histological evaluation. The histological evaluations were performed to assess the percentage of stent-induced tissue hyperplasia and the degree of inflammatory cell infiltration. The percentage of tissue hyperplasia of ET was calculated using the following equation:
$$100 \times \left(1- \frac{Stenotic \, area \, of \, stent \, \left({\mathrm{mm}}^{2}\right)}{Original \, area \, of \, stent \, \left({\mathrm{mm}}^{2}\right)}\right).$$
The degree of inflammatory cell infiltration is determined based on the distribution and density of inflammatory cells. Measurement indicators 1, 2, 3, 4, and 5 indicate mild, mild to moderate, moderate, moderate to severe, and severe inflammatory cell infiltration, respectively20. Observations for the histological analysis of the ET were obtained using a microscope (BX51; Olympus, Tokyo, Japan), and measurements were made using the CaseViewer software (CaseViewer; 3D HISTECH Ltd., Budapest, Hungary). The histological findings were verified based on the consensus of three observers blinded to the study.
Statistical analysis
The Mann–Whitney U test was used to analyze the differences between the groups as appropriate. A value of p < 0.05 was considered statistically significant. A Bonferroni-corrected Mann–Whitney U test was performed for p values < 0.05 to detect group differences (p < 0.008 as statistically significant). Statistical analyses were performed using the SPSS software (version 27.0; SPSS, IBM, Chicago, IL, USA).
Ethics declarations
All experiments were performed in accordance with relevant ARRIVE guidelines and regulations.
Ethical approval
All experiments were approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Sciences (IACUC-2020-12-189).
