Electromechanical Reshaping of Porcine Eustachian Tube Cartilage Ex Vivo

Byungwoo Yoon; Soo-Kweon Koo; Moo-Jin Baek; Il-Woo Lee; Se-Joon Oh; Soo-Keun Kong

doi:10.7874/jao.2025.00157

J Audiol Otol > Volume 29(3); 2025 > Article

Yoon, Koo, Baek, Lee, Oh, and Kong: Electromechanical Reshaping of Porcine Eustachian Tube Cartilage Ex Vivo

Original Article

Journal of Audiology and Otology 2025;29(3):219-225.

Published online: July 18, 2025

DOI: https://doi.org/10.7874/jao.2025.00157

Electromechanical Reshaping of Porcine Eustachian Tube Cartilage Ex Vivo

Byungwoo Yoon¹

, Soo-Kweon Koo¹

, Moo-Jin Baek²

, Il-Woo Lee³

, Se-Joon Oh⁴

, Soo-Keun Kong⁴

¹Department of Otorhinolaryngology, Busan St. Mary’s Medical Center, Busan, Korea

²Department of Otorhinolaryngology-Head and Neck Surgery, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Korea

³Department of Otorhinolaryngology and Biomedical Research Institute, Pusan National University Yangsan Hospital, Yangsan, Korea

⁴Department of Otorhinolaryngology and Biomedical Research Institute, Pusan National University Hospital, Busan, Korea

Address for correspondence Soo-Keun Kong, MD, PhD Department of Otorhinolaryngology, Pusan National University School of Medicine, Pusan National University Hospital, 179 Gudeok-ro, Seo-gu, Busan 49241, Korea Tel +82-51-240-7335 Fax +82-51-246-8668 E-mail entkong@gmail.com

Received March 11, 2025 Revised April 14, 2025 Accepted April 25, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background and Objectives

The Eustachian tube cartilage (ETC) is the cartilaginous frame of the Eustachian tube (ET), which plays a pivotal role in its function. Existing treatments for ET dysfunction (ETD) are primarily based on mechanical deformation. Electromechanical reshaping (EMR) is a novel method used to reshape the cartilage into the desired form by applying an electric current using strategically placed needles for mechanical deformation. In this study, EMR was evaluated for its ability to reshape the ETC.

Materials and Methods

A total of 84 ETCs were harvested from 42 post-mortem porcine crania and randomly divided into control and experimental (EMR) groups. To reshape the ETC, the control ETCs were subjected to a mechanical force, whereas the experimental group was subjected to the same force and current of 3, 4, or 5 V for 2 or 3 min. Changes in shape were assessed by measuring the contour angles using digital photographs.

Results

In the analysis of the contour angle, ETC post-EMR for 2 min at ≥4 V demonstrated significant reshaping relative to the control group (p<0.05).

Conclusions

EMR can be used to reshape the ETCs ex vivo. Future experiments with well-designed ex vivo and in vivo conditions are required to develop novel therapeutic options for ETD.

Keywords: Electromechanical reshaping · Eustachian tube · Eustachian tube cartilage.

Introduction

The function of the Eustachian tube (ET) is crucial to equalize pressure, clear mucociliary secretions, and protect the middle ear [1]. It is well known that the cartilaginous portion is functionally more significant compared to the osseous portion, and the ET cartilage (ETC) forms the keyframe [2]. Numerous trials have been conducted to treat ET dysfunction (ETD) by changing the shape of ETC [3-5]. Balloon Eustachian tuboplasty (BET) is a recent, more promising treatment mode because of its simplicity and evidence of clinically favorable results [6]. This technique involves inducing local dilation through microfractures in the ETC [6].

Electromechanical reshaping (EMR) is a technique employed for altering the shape of cartilage by simultaneous application of mechanical deformation and electric current [7]. In the EMR procedure, the target cartilage is mechanically shaped into the desired form using a preformed jig, while surface or needle electrodes make contact with the tissue, and voltage is applied for several minutes. Since its initial report by Ho, et al. [7] in 2003, which demonstrated EMR’s efficacy in reshaping porcine septal cartilage ex vivo, several studies have shown that EMR can successfully reshape the auricle, nasal septum, costal, and tracheal cartilage ex vivo in animals [8-10]. However, to the best of our knowledge, there have been no reports on EMR of the ETC. Reshaping and measuring the change in shape of the ETC is challenging because of its small size and shape complexity compared to other types of head and neck cartilage.

This study aimed to evaluate the feasibility of EMR for ex vivo animal ETC. This was the first step in evaluating the potential of this technology for treating ETD.

Materials and Methods

Harvesting porcine ETC and control and experimental pairing

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pusan National University Hospital (Approval number: PNUH-2017-113). A total of 84 ETCs were harvested from 42 porcine skulls. Fresh porcine material from 6-month-old male and female pigs was sourced from a local abattoir. The ETC from both sides was obtained within 48 hours of death, and their gross symmetry was checked (Fig. 1A). The ETC was immersed in normal saline and stored at 5°C prior to use.

The cartilage was randomly assigned to control and experimental groups. The control group was subjected only to mechanical force, whereas the experimental group was subjected to both the mechanical force and electrical current, with current levels set at 3, 4, or 5 V for 2 or 3 minutes.

The proximal pharyngeal end portion of the ETC, although thick, stable, and easy to handle, is highly variable and asymmetric. Therefore, we selected the mid-portion closest to the pharyngeal end after removing the asymmetric regions. The distance between the pharyngeal end and the first cut was not controlled by the cranium. However, for each cranium, the control and experimental pairs were obtained from the same position at the pharyngeal end. Both cartilages were cut obliquely at 5 and 7 mm from the point at which bilateral symmetry first emerged (Fig. 1A). Digital images of the straight sections were obtained using a digital camera (Canon EOS 500D; Canon). The minimum and maximum cartilage thicknesses were measured using ImageJ 1.48v (National Institutes of Health).

Electromechanical reshaping

The initial step in the EMR process involved mechanical deformation to flatten both the control and experimental specimens. The ETC has a rolled spiral structure. A 1-mm-thick, conventional steel ruler was used to gently flatten the ETC without fracturing the cartilage.

Next, we designed a straight-needle electrode configuration for the procedure. In this configuration, the cathode is centrally positioned at a distance of 2 mm, with the anode placed 3 mm lateral to each cathode (Fig. 1B). Flattened cartilage was serially placed under a flat acrylic jig. Four platinum needle electrodes (PT007115; Goodfellow) were inserted into the specimens through holes in the jig (Fig. 1C). The length of each electrode was 25 mm, with an outer and inner diameter of 0.48 mm and 0.16 mm, respectively, completely penetrating the cartilage. We designed a hand-held, level electric energy device, as previously reported (Fig. 1D) [11]. This device could apply direct current (DC) voltage to the anode, feed current from the cathode, and display current flow. The EMR parameters were 3, 4, and 5 V for 2 or 3 minutes, as described by Hussain, et al. [10].

We obtained an exclusive reference value for mechanical effects in the control and experimental groups by harvesting four ETC from two crania. The cartilage was pressed at the same weight without a current transfer (n=2, parameter group “A”). The parameters of each group were set as follows: 3 V for 3 minutes for Group “B,” 4 V for 2 minutes for Group “C,” 4 V for 3 minutes for Group “D,” 5 V for 2 minutes for Group “E,” and 5 V for 3 minutes in Group“ F.” Each EMR parameter group consisted of 8 samples (n=8, parameter group B-F).

The initial current and subsequent currents at 1, 2, and 3 minutes were recorded. Immediately following the experiment, PBS (pH 7.4) was added through the holes of the jig to the cartilages at 21°C for 15 minutes, for rehydration and establishment of ionic equilibrium. Throughout both the EMR procedure and rehydration, a consistent pressure of 240 g was applied to the jig to act as a mechanical deformation force. The same weight was applied for the same duration as the control. All cartilages were removed from the jig and allowed to stand until no further change in shape occurred, typically taking approximately 5 minutes, and were photographed as “post-EMR.”

Measurement of shape changes

We measured the “contour angles” of ETC on pre- and post-EMR photographs. The contour angle was defined as the total rotated angle on the outer circle from one terminal point to another. This was used to calculate the difference between the two terminal indicator arrow point angles using Power-Point (Microsoft Corp.). Fig. 2 depicts the contour angle measurement method.

The primary objective during EMR was to flatten the ETC, resulting in cartilages with smaller contour angles. We calculated both the pre-EMR and post-EMR contour angles. Regarding the ETC cross-sections obtained at various levels, unique controls were used, unlike previous reports [8-10]. where the contralateral cartilage served as a control in each experiment. We compared pre-EMR and post-EMR contour angles between the experimental and control groups.

Statistical analysis

IBM SPSS Statistics for Windows (version 22.0; IBM Corp.) was used for the statistical analysis. Values are expressed as the mean±standard error. Statistical comparisons between the control and experimental groups for contour angles were performed using the nonparametric Mann–Whitney U test. Statistical significance was set at p<0.05.

Tissue histology

For histological analysis, control and experimental groups were formalin-fixed and paraffin-embedded. A microtome was used to serially section the paraffin blocks, which were then deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E; Sigma Aldrich) to visualize the fracture state and tissue characteristics without prior decalcification. Slices measuring 3 μm in thickness, cut from the most distal portion (i.e., near the pharyngeal end), were used to make the slides. The slide was scanned using slide scanner (Aperio CS2, Leica Biosystems). Using Aperio Imagescope version 12.1.0.5029 (Leica), the slide was magnified by 24 times and by 200 times. The number of fractures was counted and compared between the controls and the group F (n=5).

Results

EMR findings

During EMR, subtle color changes and gas bubbles were observed near the electrodes, principally in group “F.” However, after rehydration, the color returned to the pre-EMR state. Fig. 3 illustrates the cartilage thickness values and the initial cutting position, represented by the distance from the pharyngeal end.

The minimum and maximum total ETC thickness measured at 0.68±0.02 mm and 1.15±0.04 mm, respectively. Distance from the pharyngeal end was 7.06±0.34 mm.

In groups “B,” “D,” and “F,” the current intensity after 3 min was recorded as 0.31±0.01 mA, 1.72±0.15 mA, and 4.53±0.64, respectively. An increase in the applied voltage resulted in higher current intensity.

Shape change

We observed a marked contrast between the experimental and the control group, principally at higher voltages and longer durations of treatment. Fig. 4 shows serial representative images of the ETC pairs that underwent mechanical deformation only (control) or EMR (experiment). The mean and standard errors of the contour angle changes are shown in Fig. 5.

Groups “C,” “D,” “E,” and “F” show significantly decreased contour angle compared to the control group (p<0.05). Specifically, in group “F,” the change in contour angle compared to control was 183.9 (p=0.016) degrees. The change in value decreased by the current intensity in group “D,” 156.25 (p=0.006), group “E,” 140.53 (p=0.002), group “C,” 121.3 (p=0.005), whereas group “B” did not show statistical significance (p=0.916).

Histological findings

H&E staining revealed that EMR induced shape changes (Fig. 6). Fractures were noted in the hinge and stressed regions. The mean fracture number was 2±1.58 in the control group and 1.2±0.84 in the group F. The number differences between the two groups did not show statistical significance (p=0.390).

Discussion

This study was the first trial of the use of EMR in ETC. It demonstrates that combining a low-voltage electric field with mechanical deformation induces a more effective shape change than mechanical deformation alone. This suggests that EMR holds promise for reshaping the ETC.

EMR is an electrochemical reaction [7]. Although the precise molecular mechanisms behind EMR is currently unknown, a previous EMR study has proposed it to be comprising hydrolysis and protein electrophoresis [7]. Because cartilage tissue is approximately 75% water, the predominant redox reactions that occur in the structure of matrix molecules during EMR are understood to be the reduction of water to hydrogen gas and hydroxide ions at the cathode and the oxidation of water to oxygen gas and hydrogen ions at the anode. These reactions result in transient localized changes in tissue pH, which alter the local tissue water content, and subsequently modify the mechanical properties of cartilage tissue without heat production [12]. Additionally, redistribution of fixed electric charge associated with proteoglycan aggregates embedded in the collagen matrix and alteration of mobile ion concentration change the stress relaxation and shape of the cartilage [13].

This proposal is based on two characteristics from EMR experimental data. First, the extent of shape alteration is correlated with the intensity of the electrical field. Second, the extent of shape changes saturation at some point. An increase in voltage creates a stronger electric field that in turn accelerates hydrolysis, thus enhancing shape change. Likewise, an increase in the application time allows further loss of water from the cartilage matrix, leading to greater reshaping [7]. Saturation of shape change can be attributed to the consumption of reagents in the tissue (free water), which is limited by the diffusion of water or transport of charged moieties. An accumulation of EMR products also prevents redox reactions.

Our data elucidate that the extent of shape alteration is correlated with the intensity of electrical current, although they are not strictly proportional. This is consistent with previous research on EMR in other head and neck cartilages and the proposed mechanisms of EMR [7,8,10]. However, we could not determine the trend of saturation of shape change from our data because of issue of chondrocyte viability decrement. It is caused by accumulation of the toxic byproducts of electrochemical reaction [10]. According to previous research on chondrocyte EMR performed with porcine tracheal cartilage [10], treatment with 5 V for 3 minutes was found to induce chondrocyte injury at a depth of 2.52 mm from the anode in porcine tracheal cartilage [10]. In our study, the minimum needle-to-needle distance was 2 mm, and the needle-to-end distance was 2.5 mm. Future experiments will evaluate the saturation trend of EMR in ETC by setting a dosimeter stronger than 5 V for 3 minutes, which causes minimal ETC chondrocyte viability.

Precisely sectioning the ETC into uniform shapes and dimensions poses a challenge due to its small and fragile nature. The ETC is a more complex and twisted structure compared to tracheal cartilage, exhibiting substantial differences in cross-sectional shape. In this study, we employed cartilage from the same level as the contralateral cartilage as a control. Variations in distance from the pharyngeal end and thickness may contribute to differences in the outcomes of shape change. While specimen thickness and cutting position were not controlled, we ensured that the current flow remained consistent and reproducible.

In our study, unintended cartilage fractures were identified through histological examination. This could be induced by the flattening force exerted by the steel ruler and weight pressure during the EMR and rehydration processes. Despite our efforts to avoid fractures during the flattening process by using equal weight and pressure during the entire EMR process, the fractures markedly contributed to the variation in the outcome of the shape change. However, since there was no statistical significance between control and group F, we anticipated fracture is not main factor for our induced shape change.

In clinical practice, such as in BET treatment, cartilage fractures occur because the primary mechanism of BET is microfractures in the ETC. Our EMR results for fractured cartilage were similar to those observed in clinical practice. Future studies are needed to examine the power of EMR irrespective of cartilage fractures.

This study has some limitations that should be addressed. First, we did not assess the viability of ETC chondrocytes affected by the EMR due to technical difficulties. Establishing biologically noninvasive and effective dosimetric parameters is essential before clinical application. Second, our metrics for measuring shape change were limited, as we relied on manual determination of angle indicators and lumen shape. Moreover, although we intended to remove the perichondrium, H&E staining was unsuccessful. This may have resulted in errors in measured values. Third, our needle insertion points should have been geographically and biologically controlled based on a hinge region abundant in elastic cartilage to allow for efficient opening of the ET. Matsune, et al. [14] assessed the quantity of elastin in the hinge portion of the ET and postulated that the elastin-rich hinge portion is crucial for the function of the ET, compared to the elastin non-rich portion. Fourth, we performed EMR after harvesting the ETC. The interplay between the ET and the surrounding muscles and ligaments is important for the ETC. Performing EMR within the cranium before harvesting may better replicate clinical conditions. Future ex vivo experiments with well-designed EMR within the cranium are required. To date, the pathophysiology of ETD has not been elucidated and a simple standardized test to objectify ETD is lacking [6]. To apply EMR in clinical practice for ETD, extensive in vivo animal trials must be performed to understand dosimetric parameters.

In conclusion, based on the results of this study, EMR can be used to reshape porcine ETC, consistent with prior research on head and neck cartilage. Our findings will aid in planning well-designed ex vivo ETC experiments prior to in vivo ETC experiments and in the development of novel therapies for treating ETD.

Notes

Conflicts of Interest

The authors have no financial conflicts of interest.

Author Contributions

Conceptualization: Soo-Keun Kong. Data curation: Byungwoo Yoon. Formal analysis: Byungwoo Yoon. Investigation: Byungwoo Yoon. Methodology: Byungwoo Yoon. Project administration: Soo-Keun Kong. Resources: Soo-Keun Kong. Software: Byungwoo Yoon. Supervision: Soo-Kweon Koo, Moo-Jin Baek, Il-Woo Lee, Se-Joon Oh, Soo-Keun Kong. Validation: all authors. Visualization: Byungwoo Yoon. Writing—original draft: Byungwoo Yoon. Writing—review & editing: all authors. Approval of final manuscript: all authors.

Funding Statement

None

Acknowledgments

None

Fig. 1.

Schematic of the experiment. A: ETC specimen removed from the cranium. The entire connective tissue (except for the perichondrium) was removed. For EMR, the specimens were excised at the same distance from the pharyngeal orifice. At 5 and 7 mm from the point at which bilateral symmetry first appeared, the specimens were cut obliquely to create different cross-sectional planes. B: Right, flattened mid-portion of the ETC and four needle electrodes (two pairs). A conventional steel ruler was used to gently flatten the ETC. The plane that represented the cross-section of pre-EMR is illustrated. Left, cut midportion of the ETC (pre-EMR). Pre-EMR image before flattening of the ETC. C: Schematic of charge transfer. The acrylic jig featured 9×3 mm rectangular holes and two 120 g weights. The tissues were placed in contact with PBS (pH 7.4) for rehydration through the holes in the jig. D: Device for delivering direct current. The magnitude and duration of the voltage were set, and the current flow (mA) was recorded. ETC, Eustachian tube cartilage; EMR, electromechanical reshaping.

Fig. 2.

Measurement of contour angle on pre- (A) and post-EMR (B) photographs (left: control; right: experiment). i) An arrow indicator, which directs the “end of cartilage” direction, was set. ii) A circle was drawn around the entire cartilage. iii) The arrow indicator was moved on the circle. iv) An arc was made between the two points, with the center of the circle and “central angle of arc, alpha” was calculated. v) Proper calculation (add or reduce “central angle of arc” from 360°). The end of each cartilage was marked with a red indicator (i). Differences in the angle between red indicators were calculated in the Microsoft PowerPoint program as alpha (iv). The blue circle represents the contour angle of the ETC (v) (A: pre-EMR images. Control (left): contour angle=360°+alpha. Experiment (right): contour angle=360°+alpha. B: post-EMR images. Control (left): contour angle=360°-alpha. Experiment (right): contour angle=alpha). ETC, Eustachian tube cartilage; EMR, electromechanical reshaping.

Fig. 3.

ETC thicknesses, the initial cutting position from the pharyngeal end, and electric currents obtained during EMR. The thickness value was represented by the average value measured in maximal point, mid-point, and minimum point of the ETC. ETC, Eustachian tube cartilage; EMR, electromechanical reshaping.

Fig. 4.

Representative images of control (left) and experimental (right) ETC pairs. ETC, Eustachian tube cartilage.

Fig. 5.

Changes in contour angle following EMR. Data is presented as mean±standard error. Asterisks indicate significant differences between the control and experiment (Mann–Whitney test). EMR, electromechanical reshaping.

Fig. 6.

Hematoxylin and eosin staining findings of the ETC after EMR. Red arrow indicates the microfracture. A: Control (×24). B: Group B (×24). C: Group D (×24). D: Group F (×24). E: Control (×200). F: Group F (×200). ETC, Eustachian tube cartilage; EMR, electromechanical reshaping.

REFERENCES

1. Bluestone CD, Hebda PA, Alper CM, Sando I, Buchman CA, Stangerup SE, et al. Recent advances in otitis media. 2. Eustachian tube, middle ear, and mastoid anatomy; physiology, pathophysiology, and pathogenesis. Ann Otol Rhinol Laryngol Suppl 2005;194:16–30.

2. Oshima T, Kikuchi T, Hori Y, Kawase T, Kobayashi T. Magnetic resonance imaging of the Eustachian tube cartilage. Acta Otolaryngol 2008;128:510–4.

3. Wullstein H. Eustachian tube in tympanoplasty. AMA Arch Otolaryngol 1960;71:408–11.

4. House WF, Glasscock ME 3rd, Miles J. Eustachian tuboplasty. Laryngoscope 1969;79:1765–82.

5. Ockermann T, Reineke U, Upile T, Ebmeyer J, Sudhoff HH. Balloon dilatation Eustachian tuboplasty: a clinical study. Laryngoscope 2010;120:1411–6.

6. Huisman JML, Verdam FJ, Stegeman I, de Ru JA. Treatment of Eustachian tube dysfunction with balloon dilation: a systematic review. Laryngoscope 2018;128:237–47.

7. Ho KH, Diaz Valdes SH, Protsenko DE, Aguilar G, Wong BJ. Electromechanical reshaping of septal cartilage. Laryngoscope 2003;113:1916–21.

8. Manuel CT, Foulad A, Protsenko DE, Sepehr A, Wong BJ. Needle electrode-based electromechanical reshaping of cartilage. Ann Biomed Eng 2010;38:3389–97.

9. Manuel CT, Foulad A, Protsenko DE, Hamamoto A, Wong BJ. Electromechanical reshaping of costal cartilage grafts: a new surgical treatment modality. Laryngoscope 2011;121:1839–42.

10. Hussain S, Manuel CT, Protsenko DE, Wong BJ. Electromechanical reshaping of ex vivo porcine trachea. Laryngoscope 2015;125:1628–32.

11. Kim S, Manuel CT, Wong BJ, Chung PS, Mo JH. Handheld-level electromechanical cartilage reshaping device. Facial Plast Surg 2015;31:295–300.

12. Nguyen TD, Hu AC, Protsenko DE, Wong BJF. Effects of electromechanical reshaping on mechanical behavior of exvivo bovine tendon. Clin Biomech (Bristol) 2020;73:92–100.

13. Protsenko DE, Ho K, Wong BJ. Stress relaxation in porcine septal cartilage during electromechanical reshaping: mechanical and electrical responses. Ann Biomed Eng 2006;34:455–64.

14. Matsune S, Sando I, Takahashi H. Elastin at the hinge portion of the Eustachian tube cartilage in specimens from normal subjects and those with cleft palate. Ann Otol Rhinol Laryngol 1992;101(2 Pt 1):163–7.