Technical Feasibility and Histological Analysis of Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube

Round 1

Reviewer 1 Report

The authors investigate the technical feasibility of stent placement and histological changes after stent placement in a porcine ET and evaluate ET stents' optimal size and luminal configuration for porcine models. They use 3 pigs and 6 stents n the experiments. The submitted study is interesting; however, it requires revisions before being accepted as a publication.

The abstract is not clear. Why there is a clinical need for developing a stent to treat obstructive Eustachian tube (ET) dysfunction? Again Why there is a need for an animal model to develop an optimal design and materials of the stent? The abstract also needs to be organized again. The authors can follow a structure to summarize the aim, methods, results, and conclusions in the abstract.

The second paragraph in the Introduction section is not clear. Please revise it.

The authors use the same sentence twice to present different information in the section “2.3. Stent placement under endoscopic and fluoroscopic guidance”.

“All pigs were euthanized immediately prior to stent placement using an overdose of xylazine hydrochloride (Rompun; Bayer, Seoul, Korea).” Page 2, Line 80.

“All pigs were euthanized using an overdose of xylazine hydrochloride (Rompun; Bayer, Seoul, Korea) at 4 weeks after stent placement.” Page 3, Line 98.

So, when the pigs were euthanized, immediately prior to stent placement or at 4 weeks after stent placement?

Why did the authors perform a non-parametric Mann-Whitney U test? Did they test the distribution of the samples?

There are minor grammatical errors in the text. The authors should check the entire manuscript and revise the language.

Author Response

Dear Reviewer,

We are happy to submit our revised version of manuscript (Manuscript ID: applsci-1095762) entitled, “Technical Feasibility and Histological Analysis of Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube” to be considered for publication in Applied Sciences.

We appreciate each reviewer’s insightful suggestions to enhance the quality of our work. In the following pages, we have described our revisions based on the reviewers’ comments.

We hope that you will consider this revised version for publication in Applied Science. If you have any further questions about our manuscript, please do not hesitate to contact us by email. Thank you very much for your time and effort.

On behalf of the authors,

Best regards,

Jung-Hoon Park, Ph.D.

Biomedical Engineering Research Center,

Asan Institute for Life Sciences, Asan Medical Center

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-4123 Fax: 82-2-476-0090

E-mail: [email protected]

Hong Ju Park, M.D., Ph.D.

Otorhinolaryngology-Head & Neck Surgery,

Asan Medical Center, University of Ulsan College of Medicine

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-3700 Fax: 82-2-476-0090

E-mail: [email protected]

Response to the Reviewers’ comments

We first thank the reviewers for their reading of our manuscript and the helpful comments and suggestions. We have revised our manuscript based on these comments and believe that the manuscript is significantly improved. The following are our point-by-point responses to the reviewer’ comments.

Reviewer 1

Comments and Suggestions for Authors

The authors investigate the technical feasibility of stent placement and histological changes after stent placement in a porcine ET and evaluate ET stents' optimal size and luminal configuration for porcine models. They use 3 pigs and 6 stents n the experiments. The submitted study is interesting; however, it requires revisions before being accepted as a publication.

Q1-1) The abstract is not clear. Why there is a clinical need for developing a stent to treat obstructive Eustachian tube (ET) dysfunction? Again Why there is a need for an animal model to develop an optimal design and materials of the stent? The abstract also needs to be organized again. The authors can follow a structure to summarize the aim, methods, results, and conclusions in the abstract.

Response: We have revised the Abstract as follows “There is a clinical need to develop a stent to treat obstructive and refractory Eustachian tube dysfunction (ETD) after balloon Eustachian tuboplasty. An animal model for stent placement in the ET is needed to develop optimal designs and materials as stents for ETD have not been clinically applied. This study evaluated the technical feasibility of stent placement and histological changes in a porcine ET model. Six ETs were evaluated in three pigs. Cobalt-chrome alloy stents with two different diameters were placed in the left and right ET of each animal (right, 3.5 mm; left, 2.5 mm). The outcomes were assessed by endoscopic and fluoroscopic imaging during the procedure, computed tomography after the procedure, and by histological examinations. Stent placement was successful in all specimens after metallic guiding sheaths were located in the nasopharyngeal end of the ET. The mean luminal diameters of the proximal, middle, and distal portions of the larger stents in the right ETs were 3.48 mm, 2.54 mm, and 2.15 mm, respectively. In the left ETs using smaller stents, these values were 2.49 mm, 1.73 mm, and 1.42 mm, respectively. The diameters of the inserted stents differed by stent location and the original diameter. Histological findings showed tissue hyperplasia with severe inflammatory cell infiltration at 4 weeks after stent placement. In conclusion, stent placement into the porcine ET was technically feasible, and stent-induced tissue hyperplasia was significantly evident. The luminal configuration of the placed ET stent changed according to its non-elastic nature and anatomical features of the porcine ET. Using this model, ET stents of various materials and designs with anti-inflammatory or anti-proliferative drugs can be optimized for future treatments of ET dysfunction.”

Q1-2) The second paragraph in the Introduction section is not clear. Please revise it.

Response: We have revised the second paragraph in the Introduction section with additional reference as follows “Until recently, otolaryngologists’ understanding of ET dysfunction (ETD) was limited and few treatment options were available [4-8]. Currently, the most common surgical approach is myringotomy with or without the insertion of a ventilation tube with reported success rate of 79% after 4 months follow up period [6]. However, this is only a temporary management and it does not address the ET directly. Other conservative managements of ETD also include the Valsalva maneuver for pressure equalization, use of nasal steroids and decongestants [9]. With the introduction of the balloon Eustachian tuboplasty (BET), however, surgical management of ETD is now possible [10]. The proposed mechanisms underlying the effects of BET include microtears in the cartilaginous part of the ET [10], decreased mucosal inflammation and reduced biofilm infection load [11]. Since 2010, studies have reported the use of BET to treat ETD, with success rates ranging between 36% and 80% [8, 12-14]. Although these prior studies reported that BET is superior to conventional medical management, some patients with ETD do not respond to this dilation treatment.”

Q1-3) The authors use the same sentence twice to present different information in the section “2.3. Stent placement under endoscopic and fluoroscopic guidance”.

“All pigs were euthanized immediately prior to stent placement using an overdose of xylazine hydrochloride (Rompun; Bayer, Seoul, Korea).” Page 2, Line 80.

“All pigs were euthanized using an overdose of xylazine hydrochloride (Rompun; Bayer, Seoul, Korea) at 4 weeks after stent placement.” Page 3, Line 98.

So, when the pigs were euthanized, immediately prior to stent placement or at 4 weeks after stent placement?

Response: We have revised the anesthesia method and sacrifice time point (4 weeks after stent placement) in the Materials and Methods section as follows, “Anesthesia was induced by intramuscular injection of 50mg of ketamine under the supervision of a veterinarian. An endotracheal tube was placed, and anesthesia was maintained by inhalation (0.5–2% isoflurane [Ifran; Hana Pharm. Co., Seoul, Korea] with oxygen [510 mL/kg per min] at 1:1).”

Q1-4) Why did the authors perform a non-parametric Mann-Whitney U test? Did they test the distribution of the samples?

Response: The Mann-Whitney U test is usually used to compare differences between two independent groups when the dependent variable is either ordinal or continuous, but not normally distributed and the sample size is small.

Q1-5) There are minor grammatical errors in the text. The authors should check the entire manuscript and revise the language.

Response: We already received the English editing as indicated in the Acknowledgements “Acknowledgments: The authors thank Enago (http://www.enago.co.kr) for the English language review.” The English language and grammar have been re-reviewed.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript by Yehree Kim and Woo Seok Kang et al. entitled Technical Feasibility and Histological Analysis with Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube deals with the problem of introducing a stent for the treatment of obstruction-related Eustachian tube disorders. The paper is very concise, rather well written and fits the scope of the Applied Sciences journal.

However, before it could be accepted, some drawbacks have to be addressed by the authors:

My biggest concern regarding the presented study is the lack of a control group. Stents were placed in the ETs of three pigs, but there were no non-treated animals in the study. What is more, there were no specimens that underwent the same procedure, but without the stent placement. This is a serious limitation, as the observed histological changes cannot be unambiguously associated with the application of the stents. Formation of microtears, that are connected with the balloon-procedure, could be also the effect of the stent placement procedure itself. In such situation, it is difficult to discuss the results obtained, and thus the discussion revolves mostly on possible further experiments that can be conducted.

There are also some inconsistencies in the text, as in lines 80 and 98 there is information that “All pigs were euthanized immediately prior to stent placement using an overdose of xylazine”. I suppose that there is a mistake in line 80 and the anesthesia procedure should be described.

It seems weird to provide the range and median value of porcine weight, as there are only three specimens – wouldn’t it be more intuitive to present the weight of the three pigs?

The conventional treatment options should be elaborated more in the introduction section. Also, their estimated success rates should be given if possible.

Some minor mistakes with English and style: with (should be “of”, line 2); prostate (line 123).

Author Response

Dear Reviewer,

We are happy to submit our revised version of manuscript (Manuscript ID: applsci-1095762) entitled, “Technical Feasibility and Histological Analysis of Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube” to be considered for publication in Applied Sciences.

We appreciate each reviewer’s insightful suggestions to enhance the quality of our work. In the following pages, we have described our revisions based on the reviewers’ comments.

We hope that you will consider this revised version for publication in Applied Science. If you have any further questions about our manuscript, please do not hesitate to contact us by email. Thank you very much for your time and effort.

On behalf of the authors,

Best regards,

Jung-Hoon Park, Ph.D.

Biomedical Engineering Research Center,

Asan Institute for Life Sciences, Asan Medical Center

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-4123 Fax: 82-2-476-0090

E-mail: [email protected]

Hong Ju Park, M.D., Ph.D.

Otorhinolaryngology-Head & Neck Surgery,

Asan Medical Center, University of Ulsan College of Medicine

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-3700 Fax: 82-2-476-0090

E-mail: [email protected]

Response to the Reviewers’ comments

We first thank the reviewers for their reading of our manuscript and the helpful comments and suggestions. We have revised our manuscript based on these comments and believe that the manuscript is significantly improved. The following are our point-by-point responses to the reviewer’ comments.

Reviewer 2

Comments and Suggestions for Authors

The manuscript by Yehree Kim and Woo Seok Kang et al. entitled Technical Feasibility and Histological Analysis with Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube deals with the problem of introducing a stent for the treatment of obstruction-related Eustachian tube disorders. The paper is very concise, rather well written and fits the scope of the Applied Sciences journal. However, before it could be accepted, some drawbacks have to be addressed by the authors:

Q2-1) My biggest concern regarding the presented study is the lack of a control group. Stents were placed in the ETs of three pigs, but there were no non-treated animals in the study. What is more, there were no specimens that underwent the same procedure, but without the stent placement. This is a serious limitation, as the observed histological changes cannot be unambiguously associated with the application of the stents. Formation of microtears, that are connected with the balloon-procedure, could be also the effect of the stent placement procedure itself. In such situation, it is difficult to discuss the results obtained, and thus the discussion revolves mostly on possible further experiments that can be conducted.

Response: We agree with your opinion and have included this study limitation in the revised Discussion section as follows “This study had several limitations. First, the total number of specimens was relatively small to perform a robust statistical analysis. Second was the lack of a control group, namely non-treated animals, because of the limited number of animals. Additional studies are required to compare to an untreated control group in the porcine ET and to evaluate the histological changes and tissue reaction after stent placement with long-term follow-up.”

Based on the normal porcine ET histological findings as below reference, we analyzed and interpreted our histological results after stent placement.

An FW, Yuan H, Guo W, Hou ZH, Cai JM, Luo CC, Yu N, Jiang QQ, Cheng W, Liu W, Yang SM. Establishment of a Large Animal Model for Eustachian Tube Functional Study in Miniature Pigs. Anat Rec (Hoboken). 2019 Jun;302(6):1024-1038. doi: 10.1002/ar.24098. Epub 2019 Mar 15. PMID: 30779320.

Q2-2) There are also some inconsistencies in the text, as in lines 80 and 98 there is information that “All pigs were euthanized immediately prior to stent placement using an overdose of xylazine”. I suppose that there is a mistake in line 80 and the anesthesia procedure should be described.

Response: We have revised the anesthesia method and sacrifice time point (4 weeks after stent placement) in the Materials and Methods section as follows, “Anesthesia was induced by intramuscular injection of 50 mg of ketamine under the supervision of a veterinarian. An endotracheal tube was placed, and anesthesia was maintained by inhalation (0.5–2% isoflurane [Ifran; Hana Pharm. Co., Seoul, Korea] with oxygen [510 mL/kg per min] at 1:1).”

Q2-3) It seems weird to provide the range and median value of porcine weight, as there are only three specimens – wouldn’t it be more intuitive to present the weight of the three pigs?

Response: We agree with your opinion and have revised the sentence as follows “This study tested both ETs in three pigs weighing 33.7, 37.2, and 35.4 kg, respectively; i.e., six total ETs.”

Q2-4) The conventional treatment options should be elaborated more in the introduction section. Also, their estimated success rates should be given if possible.

Response: We have revised the second paragraph in the Introduction section with additional reference as follows “Until recently, otolaryngologists’ understanding of ET dysfunction (ETD) was limited and few treatment options were available [4-8]. Currently, the most common surgical approach is myringotomy with or without the insertion of a ventilation tube with reported success rate of 79% after 4 months follow up period [6]. However, this is only a temporary management and it does not address the ET directly. Other conservative managements of ETD also include the Valsalva maneuver for pressure equalization, use of nasal steroids and decongestants [9]. With the introduction of the balloon Eustachian tuboplasty (BET), however, surgical management of ETD is now possible [10]. The proposed mechanisms underlying the effects of BET include microtears in the cartilaginous part of the ET [10], decreased mucosal inflammation and reduced biofilm infection load [11]. Since 2010, studies have reported the use of BET to treat ETD, with success rates ranging between 36% and 80% [8, 12-14]. Although these prior studies reported that BET is superior to conventional medical management, some patients with ETD do not respond to this dilation treatment.”

Q2-5) Some minor mistakes with English and style: with (should be “of”, line 2); prostate (line 123).

Response: We apologize for this error and have deleted “of the prostate.”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors respond to the comments from the reviewer sufficiently. The submitted manuscript can be accepted as a publication.

Reviewer 2 Report

The manuscript improved significantly and all the comments were fully answered. The language used in the paper was checked throughout and changed where necessary. With the limitations of the study clearly highlighted in the revised version, I recommend the article to be accepted for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Summary: This paper used a porcine model post mortem for establishing feasibility for ET stenting with commercially available stents.

This is an interesting paper to show the expanded possible use of a metal stent in the ET for ET dysfunction. There are some major issues with the paper that can be addressed primarily the statistical analysis and discussion improvement.

 Materials and Methods:

Page 2

Line 62- What is the rationale for using metal sheath for delivering stent? Some other types of J-catheters are rigid plastics.

Lines 80-86:

What is the name of the commercially available stent used in the study?

What is the target size, diameter (mm) x length (cm), of the commercially available stent?

What are the baseline measurements of ET prior to stenting?

How long was the balloon inflated before deflating?

Page 4

Lines 116-121:

Where is the location of the proximal and distal region of the stent in the ET? Please label on Figure 1 or change notation to cranial and caudal for better clarification.

Table 1: What is the standard deviation of the mean diameters? Add them to the table.

Is the decrease rate (%) an average? If so, add the standard deviation to the table.

For example, stent mean lumen diameter: 3.48±0.06

Data on proximal 3.5mm stent does not match value on page 6 Line 135-138.

What is the percent in diameter decrease that is noted as a failure of the stent? For example cardiovascular stents fail at 20-30% diameter reduction.

What is the expected elastic recoil of these commercial stents?

Where is the statistical methods section? The author shows statistical analysis in Figure 2 but no mention of the methods used for the analysis in the paper.

What is the hypothesis testing criterion and null hypothesis?

If comparing left to right ET, then explain why? Most stents are compared to similar vessels. These ET tubes seem to be very different in sizing. It doesn’t seem to make sense to statistically compare left to right unless, baseline measurements are compared to show statistical difference in ET sizes.

Stent ET sizing should be compared to baseline measurements of that particular size of the ET. Did the stent increase the lumen size of the ET or have any effect on the lumen before and after stenting?

Page 7:

Lines 160-169

The discussion touches upon tissue hyperplasia and some methods for mitigation the stent by adding a drug or surface modifications. It is weak on the following points.

Discuss what are the risks associated to having a stent implanted in the ET with it open long term?  Inner ear infections and tissue sensitivity to the metal implanted into the ET?

Discuss how to mitigate infections, biofilm formations using an ET stent or what has been done in other fields such as airway stenting? See reference below.

Lee HJ, Labaki W, Yu DH, Salwen B, Gilbert C, Schneider AL, Ortiz R, Feller-Kopman D, Arias S, Yarmus L. Airway stent complications: the role of follow-up bronchoscopy as a surveillance method. J Thorac Dis 2017;9(11):4651-4659. doi: 10.21037/jtd.2017.09.139

Lines 183-188

Why do the authors think the stent collapsed? For example, what are the underlying tissue components applying stress onto the stent to cause it to collapse? Is the ET a rigid structure, a soft structure with muscles pushing on it to cause the stent to collapse?

What are the limitation and advantages to using a stent for the ET compared to other treatment options such as BET?

Author Response

Dear Editors of Applied Sciences,

Here we are gladly submitting a revised version of manuscript (Manuscript ID: applsci-1017036) entitled, “Technical Feasibility of Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube” to be considered for publication in Applied Sciences.

We very much appreciate each reviewer’s insightful suggestions directed to enhancing the quality of our work. In the following pages, we provided all the items revised according to the reviewers’ comments.

We hope that our revised version will be considered for publication in your journal. If you have any further questions about our manuscript, please do not hesitate to contact us by email. Thank you very much for your time and efforts that go into the publication of this paper.

On behalf of authors,

Best regards,

Jung-Hoon Park, Ph.D.

Biomedical Engineering Research Center,

Asan Institute for Life Sciences, Asan Medical Center

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-4123 Fax: 82-2-476-0090

E-mail: [email protected]

Hong Ju Park, M.D., Ph.D.

Otorhinolaryngology-Head & Neck Surgery,

Asan Medical Center, University of Ulsan College of Medicine

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-3700 Fax: 82-2-476-0090

E-mail: [email protected]

Response to the Reviewers’ comments

First of all, we would like to thank the reviewers for a thorough reading of the manuscript and helpful comments and suggestions. We have revised our manuscript based on the comments made by the reviewer, and we believe the manuscript is now significantly improved. The followings are the point-by-point responses to the reviewer’s comments.

Reviewer 1

Comments and Suggestions for Authors

Summary: This paper used a porcine model post mortem for establishing feasibility for ET stenting with commercially available stents.

This is an interesting paper to show the expanded possible use of a metal stent in the ET for ET dysfunction. There are some major issues with the paper that can be addressed primarily the statistical analysis and discussion improvement.

Q1-1) Page 2 Line 62- What is the rationale for using metal sheath for delivering stent? Some other types of J-catheters are rigid plastics.

Response: Thank you very much for your comment. There are two reasons for usage of the sheath. We have revised in the Materials and Methods section as follows “The angled distal tip of the sheath easily was located in the nasopharyngeal orifice of the ET and the sheath served as a stent delivery system.”

Q1-2) Lines 80-86: What is the name of the commercially available stent used in the study?

Response: The stent (Genoss BMS) is commercially available in Korea. We have added the name of stent used in the study in the Materials and Methods section.

Q1-3) What is the target size, diameter (mm) x length (cm), of the commercially available stent?

Response: The stent was developed for the coronary arterial stenosis within the range of 2 – 4 mm in diameter and 8-24 mm length.

Q1-4) What are the baseline measurements of ET prior to stenting?

Response: Unfortunately, we did not measure the diameter or length of the porcine ET before stent placement. The rationale for choosing stent size has added in the Materials and Methods section as follows “The stent size was chosen considering the diameter (2.3 to 3.4 mm) and length (24.2 to 36.4 mm) of the normal ET in a porcine model [14, 15].”

Q1-5) How long was the balloon inflated before deflating?

Response: We have added the balloon inflating time in the Materials and Methods section as follows “After maintaining the fully expanded balloon for 30 seconds, the balloon catheter was deflated under continuous fluoroscopic monitoring.”

Q1-6) Page 4 Lines 116-121: Where is the location of the proximal and distal region of the stent in the ET? Please label on Figure 1 or change notation to cranial and caudal for better clarification.

Response: We have revised in Figure 1.

Q1-7) Table 1: What is the standard deviation of the mean diameters? Add them to the table.

Response: We have added the standard deviation of the all values in the table 1.

Q1-8) Is the decrease rate (%) an average? If so, add the standard deviation to the table. For example, stent mean lumen diameter: 3.48±0.06

Response: We have added the standard deviation of the all values in the table 1.

Q1-9) Data on proximal 3.5mm stent does not match value on page 6 Line 135-138.

Response: We have revised the values in the table 1.

Q1-10) What is the percent in diameter decrease that is noted as a failure of the stent? For example cardiovascular stents fail at 20-30% diameter reduction.

Response: Thank you very much for your comment. However, ET stents have not yet been applied clinically. It is difficult to provide an accurate answer.

Q1-11) What is the expected elastic recoil of these commercial stents?

Response: According to the data received from the Genoss Co., the elastic recoil rate of the used stent in this study is 0.65% and shortening rate is 0.50%.

Q1-12) Where is the statistical methods section? The author shows statistical analysis in Figure 2 but no mention of the methods used for the analysis in the paper.

Response: We have added the statistical methods in the Materials and Methods section as follows “2.6. Statistical analysis: Data are expressed as the mean ± standard deviation (SD). The differences between the two specimens were analyzed using the Mann–Whitney U test. A p value of <0.05 was considered statistically significant. Statistical analyses were performed using SPSS software (version 24.0; SPSS, IBM, Chicago, IL, USA).”

Q1-13) What is the hypothesis testing criterion and null hypothesis?

Response: We have added the study hypothesis and purpose in the Introduction section as follows “The purpose of this study was to investigate the technical feasibility of stent placement in a porcine ET and to evaluate the optimal size and luminal configuration of ET stents for porcine model.

Q1-14) If comparing left to right ET, then explain why? Most stents are compared to similar vessels. These ET tubes seem to be very different in sizing. It doesn’t seem to make sense to statistically compare left to right unless, baseline measurements are compared to show statistical difference in ET sizes.

Response: On this comment, we placed a cobalt-chrome alloy stent with two different diameters into the left and right ET (right-side, 3.5 mm; left-side, 2.5 mm). We assumed that the diameter or length of the both ET in an animal would be similar to each other and we wanted to compare the difference of the dilated diameters of the ET by the different diameters of the inserted stents.

Q1-15) Stent ET sizing should be compared to baseline measurements of that particular size of the ET. Did the stent increase the lumen size of the ET or have any effect on the lumen before and after stenting?

Response: We did not measure the diameter or length of the porcine ET before stent placement. The rationale for choosing stent size has added in the Materials and Methods section as follows “The stent size was chosen considering the diameter (2.3 to 3.4 mm) and length (24.2 to 36.4 mm) of the normal ET in a porcine model [14, 15].”

Q1-16) Page 7:Lines 160-169 The discussion touches upon tissue hyperplasia and some methods for mitigation the stent by adding a drug or surface modifications. It is weak on the following points.

Response: We totally agreed with your opinion and have added the limitation section in the Discussion section as follows “This study had several limitations of note. First, the total number of specimens was relatively small to perform a robust statistical analysis. Second, the stented ET was not histologically analyzed to confirm the mucosal tissue response to the placed stent. Third, this study was only to evaluate technical feasibility of stent placement and all enrolled pigs were immediately sacrificed. Additional studies are required to evaluate the histological changes and tissue reaction after stent placement with long-term follow-up and to investigate the safety and efficacy of stent placement in the ET. Furthermore, stent modification of the length and shape for the ET should be considered based on our findings. Although further studies are needed, our results support the basic concept of stent placement in a porcine ET model.”

Q1-17) Discuss what are the risks associated to having a stent implanted in the ET with it open long term?  Inner ear infections and tissue sensitivity to the metal implanted into the ET?

Response: That is what we are going to figure out in the near future. We want to evaluate the difference of the tissue reaction to the inserted stents of different materials and sizes in further studies. Because open (patulous) ET would result in many symptoms, such as voice autophony (93%), breath autophony (92%), and aural fullness (Ward BK), modification of the shape and length of the stent would be needed. This study is a feasibility study of inserting a stent in a pig model, suggesting that stents with two diameters (2.5 and 3.5 mm) are feasible in a pig model and the further modification of the length and shape of the stent can be performed based on our findings. We have added the study limitations as follows “This study had several limitations of note. First, the total number of specimens was relatively small to perform a robust statistical analysis. Second, the stented ET was not histologically analyzed to confirm the mucosal tissue response to the placed stent. Third, this study was only to evaluate technical feasibility of stent placement and all enrolled pigs were immediately sacrificed. Additional studies are required to evaluate the histological changes and tissue reaction after stent placement with long-term follow-up and to investigate the safety and efficacy of stent placement in the ET. Furthermore, stent modification of the length and shape for the ET should be considered based on our findings. Although further studies are needed, our results support the basic concept of stent placement in a porcine ET model.”

Ward BK, Ashry Y, Poe DS. Patulous Eustachian Tube Dysfunction: Patient Demographics and Comorbidities. Otol Neurotol. 2017 Oct;38(9):1362-1369.

Q1-18) Discuss how to mitigate infections, biofilm formations using an ET stent or what has been done in other fields such as airway stenting? See reference below.

Lee HJ, Labaki W, Yu DH, Salwen B, Gilbert C, Schneider AL, Ortiz R, Feller-Kopman D, Arias S, Yarmus L. Airway stent complications: the role of follow-up bronchoscopy as a surveillance method. J Thorac Dis 2017;9(11):4651-4659. doi: 10.21037/jtd.2017.09.139

Response: On this comment, we have revised the Discussion section with additional four references as follows “Another cause of stent re-obstruction is biofilm formation with infections on the stent surfaces [21-23]. Various functional stents such as drug-eluting biodegradable or non-biodegradable and nano-functionalized stents using gold or silver nanoparticles have been investigated using various stented animal models to overcome these problems [24-27], but therapeutic strategies for the treatment of obstructive ETD that could be tested in human clinical trials have yet to be developed.”

21. Familiari, P.; Bulajic, M.; Mutignani, M.; Lee, L.S.; Spera, G.; Spada, C.; Tringali, A.; Costamagna, G.

Endoscopic removal of malfunctioning biliary self-expandable metallic stents. Gastrointest Endosc 2005, 62, 903–10, doi: 10.1016/j.gie.2005.08.051.

22. Faigel, D.O. Preventing biliary stent occlusion. Gastrointest Endosc 2000, 51, 104–7, 10.1016/s0016-5107(00)70403-2.
23. Lee, H.J.; Labaki, W.; Yu, D.H.; Salwen, B.; Gilbert, C.; Schneider, A.L.; Ortiz, R.; Feller-Kopman, D.; Arias, S.; Yarmus, L. Airway stent complications: the role of follow-up bronchoscopy as a surveillance method. J Thorac Dis 2017, 9, 4651-9, doi: 10.21037/jtd.2017.09.139.
24. Park, W.; Kim, K.Y.; Kang, J.M.; Ryu, D.S.; Kim, D.-H.; Song, H.-Y.; Kim, S.-H.; Lee, S.O.; Park, J.-H. Metallic stent mesh coated with silver nanoparticles suppresses stent-induced tissue hyperplasia and biliary sludge in the rabbit extrahepatic bile duct. Pharmaceutics 2020, 12, 563, doi: 10.3390/pharmaceutics12060563.

Q1-19) Lines 183-188: Why do the authors think the stent collapsed? For example, what are the underlying tissue components applying stress onto the stent to cause it to collapse? Is the ET a rigid structure, a soft structure with muscles pushing on it to cause the stent to collapse?

Response: Because a cobalt chrome stent does not have self-expandable nature, the inserted stent would be collapsed by the surrounding soft tissue and the degree of the collapse was different according to the location of the stent. The medial part of the cartilaginous ET was wider than the lateral part of the ET, thus the luminal configuration of the placed ET stent changes according to its non-elastic nature and anatomical features of the porcine ET.

Q1-20) What are the limitation and advantages to using a stent for the ET compared to other treatment options such as BET?

Response: Recently, ET balloon dilation has emerged as a new minimally invasive intervention in the treatment of ET dysfunction [Huisman JML,]; however, success rates ranged from 64% to 97% and additional treatment modality is needed for the refractory cases to ET balloon dilation. Though balloon dilation has been an accepted treatment for a variety of benign strictures involving other nonvascular organs for more than 20 years, temporary stent placement has been an accepted therapeutic option for a variety of refractory, benign, nonvascular strictures (Fuccio L; Kim JH; Song HY).

Huisman JML, Verdam FJ, Stegeman I, de Ru JA. Treatment of Eustachian tube dysfunction with balloon dilation: A systematic review. Laryngoscope. 2018 Jan;128(1):237-247.

Fuccio L, Hassan C, Frazzoni L, et al. Clinical outcomes following stent placement in refractory benign esophageal stricture: a systematic review and meta-analysis. Endoscopy 2016; 48:141–148.

Kim JH, Shin JH, Song HY, et al. Benign tracheobronchial strictures: longterm results and factors affecting airway patency after temporary stent placement. AJR Am J Roentgenol 2007; 188:1033–1038.

Song HY, Jin YH, Kim JH, et al. Nonsurgical placement of a nasolacrimal polyurethane stent: long-term effectiveness. Radiology 1996; 200:759–763.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper aims at investigating the technical feasibility of placing a stent into ET. Although the team presented data to support that they placed stents in porcine ET tissue, the study lack scientific aspects.  

1. Whether the stent can work for a disease is not clear. There is no clear matrix to evaluate the success of the technique placement of a stent.
2. There is no data to support the authors’ claim that the stenting did not cause the damage of mucosal layer.
3. There is no biological analysis data to indicate how the mucosal tissue responded to the insertion of the stent with time.
4. It is not clear about how long the stent was placed in the ET. Did the stent migrate? If the stent collapsed, how will it play the role in the future treatment? What is the collapse rate?

Author Response

Dear Editors of Applied Sciences,

Here we are gladly submitting a revised version of manuscript (Manuscript ID: applsci-1017036) entitled, “Technical Feasibility of Balloon-Expandable Metallic Stent Placement in a Porcine Eustachian Tube” to be considered for publication in Applied Sciences.

We very much appreciate each reviewer’s insightful suggestions directed to enhancing the quality of our work. In the following pages, we provided all the items revised according to the reviewers’ comments.

We hope that our revised version will be considered for publication in your journal. If you have any further questions about our manuscript, please do not hesitate to contact us by email. Thank you very much for your time and efforts that go into the publication of this paper.

On behalf of authors,

Best regards,

Jung-Hoon Park, Ph.D.

Biomedical Engineering Research Center,

Asan Institute for Life Sciences, Asan Medical Center

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-4123 Fax: 82-2-476-0090

E-mail: [email protected]

Hong Ju Park, M.D., Ph.D.

Otorhinolaryngology-Head & Neck Surgery,

Asan Medical Center, University of Ulsan College of Medicine

88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Republic of Korea

Tel: 82-2-3010-3700 Fax: 82-2-476-0090

E-mail: [email protected]

Response to the Reviewers’ comments

First of all, we would like to thank the reviewers for a thorough reading of the manuscript and helpful comments and suggestions. We have revised our manuscript based on the comments made by the reviewer, and we believe the manuscript is now significantly improved. The followings are the point-by-point responses to the reviewer’s comments.

Reviewer 2

Comments and Suggestions for Authors

This paper aims at investigating the technical feasibility of placing a stent into ET. Although the team presented data to support that they placed stents in porcine ET tissue, the study lack scientific aspects.  

Q2-1) Whether the stent can work for a disease is not clear. There is no clear matrix to evaluate the success of the technique placement of a stent.

Response: We agreed with your comment. We have added the definition of technical success in this study in the Materials and Methods section as follows “Technical success was defined as successful stent placement in the cartilaginous portion of the porcine ET” After stent placement, CT examination was performed to evaluate the exact position of the stent, but our results could not be provided clinical effectiveness or evidence of stent placement in the ET.

Recently, ET balloon dilation has emerged as a new minimally invasive intervention in the treatment of ET dysfunction [Huisman JML,]; however, success rates ranged from 64% to 97% and additional treatment modality is needed for the refractory cases to ET balloon dilation. Though balloon dilation has been an accepted treatment for a variety of benign strictures involving other nonvascular organs for more than 20 years, temporary stent placement has been an accepted therapeutic option for a variety of refractory, benign, nonvascular strictures (Fuccio L; Kim JH; Song HY).

Huisman JML, Verdam FJ, Stegeman I, de Ru JA. Treatment of Eustachian tube dysfunction with balloon dilation: A systematic review. Laryngoscope. 2018 Jan;128(1):237-247.

Fuccio L, Hassan C, Frazzoni L, et al. Clinical outcomes following stent placement in refractory benign esophageal stricture: a systematic review and meta-analysis. Endoscopy 2016; 48:141–148.

Kim JH, Shin JH, Song HY, et al. Benign tracheobronchial strictures: longterm results and factors affecting airway patency after temporary stent placement. AJR Am J Roentgenol 2007; 188:1033–1038.

Song HY, Jin YH, Kim JH, et al. Nonsurgical placement of a nasolacrimal polyurethane stent: long-term effectiveness. Radiology 1996; 200:759–763.

Q2-2) There is no data to support the authors’ claim that the stenting did not cause the damage of mucosal layer.

Response: On this comment, we performed endoscopic examination during and immediately after stent placement and gross examination during surgical exploration of the ET with the placed stent to evaluate the mucosal injuries with or without touch bleeding. We also have additionally described the study limitation in the Discussion section as follows “This study had several limitations of note. First the total number of specimens was relatively small to perform a robust statistical analysis. Second, the stented ET was not histologically analyzed to confirm the mucosal tissue response to the placed stent. Third, this study was only to evaluate technical feasibility of stent placement and all enrolled pigs were immediately sacrificed. Additional studies are required to evaluate the histological changes and tissue reaction after stent placement with long-term follow-up and to investigate the safety and efficacy of stent placement in the ET. Furthermore, stent modification of the length and shape for the ET should be considered based on our findings. Although further studies are needed, our results support the basic concept of stent placement in a porcine ET model.”

That is what we are going to figure out in the near future. We want to evaluate the difference of the tissue reaction to the inserted stents of different materials and sizes in further studies. Because open (patulous) ET would result in many symptoms, such as voice autophony (93%), breath autophony (92%), and aural fullness (Ward BK), modification of the shape and length of the stent would be needed. This study is a feasibility study of inserting a stent in a pig model, suggesting that stents with two diameters (2.5 and 3.5 mm) are feasible in a pig model and the further modification of the length and shape of the stent can be performed based on our findings.

Q2-3) There is no biological analysis data to indicate how the mucosal tissue responded to the insertion of the stent with time.

Response: We totally agreed with your opinion and have added the limitation section in the Discussion section as follows “This study had several limitations of note. First the total number of specimens was relatively small to perform a robust statistical analysis. Second, the stented ET was not histologically analyzed to confirm the mucosal tissue response to the placed stent. Third, this study was only to evaluate technical feasibility of stent placement and all enrolled pigs were immediately sacrificed. Additional studies are required to evaluate the histological changes and tissue reaction after stent placement with long-term follow-up and to investigate the safety and efficacy of stent placement in the ET. Furthermore, stent modification of the length and shape for the ET should be considered based on our findings. Although further studies are needed, our results support the basic concept of stent placement in a porcine ET model.”

Q2-4) It is not clear about how long the stent was placed in the ET. Did the stent migrate? If the stent collapsed, how will it play the role in the future treatment? What is the collapse rate?

Response: On this comment, we have added the sentence regarding time point of sacrifice in the Materials and Methods section as follows “All pigs were euthanized using an overdose of xylazine hydrochloride (Rompun; Bayer, Seoul, Korea) immediately after stent placement”. The distal portion of the all stents were severely collapsed when the inserted stent was observed grossly because of anatomic configuration of the ET. This finding can be an evidence when designing the stent to be used in ET in the future.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

This paper has been significantly improved. It would be very interesting to see survival studies in the future on this technique for stenting ET.

Reviewer 2 Report

Although the authors made corresponding response to the comments of the reviewers, the essential problems have not addressed. There is no new data added to support the claim. The reviewer understood that it may take more time to perform some experiments to address those questions. However, a technique report may not bring interest to readers in this field. To prove the success of the technique, only the successful insertion may not be sufficient. The feasibility of a technique needs to show not only the success of insertion but also the biological reaction.