Fatigue Testing of Human Flexor Tendons Using a Customized 3D-Printed Clamping System
, Sarah Safavi 3,4, Maziar Ramezani 5, Benjamin Ondruschka 6 and Niels Hammer 2,7,8,*
Round 1
Reviewer 1 Report
I recommend the publication of the article
Author Response
Point-by-Point Response
The authors would like to thank the Editor and the Reviewers for their suggestions to improve the quality of the manuscript. We have accordingly changed and extended the manuscript, with each point outlined below in this Point-by-Point Response. Changes in the manuscript were tracked by using the “Track Changes” function.
Reviewer comments
Reviewer #1
I recommend the publication of the article
Answer: Thank you very much for the recommendation.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript describes the new tailored clamps designed to examine the fatigue strength of natural tissues like tendons. Cadaveric flexor digitorum tendons were harvested and mounted for tensile testing with no tapering being made, using 3D-printed clamps and holder arms, while ensuring a consistent testing length.
The manuscript is interesting because describes the detailed device to try to solve the big issue regarding how to generate a consistent methodology for mechanical characterization of biological tissue intended for tissue engineering approaches. However, some issues are found in the manuscript. Detailed as follow:
1. I strongly recommend changing the order of some sections in the manuscript: The materials and methods should be after the introduction and before the results.
2. The big concern in the results is most failures were observed on the edge of the clamps. Out of these failures, 78.3% were longitudinal and 21.7% transverse. Only 0.6% of the clamps failed longitudinal in the central area. This is a big concern due to this is an indicator that the clamps are affecting the experimental procedure by acting as a stress concentration, and this lead to lower peak stress than real as a result, please explain this in the manuscript.
3. You should cite other research related to mechanical testing in other biological tissues as: http://dx.doi.org/10.1016/j.msec.2015.10.094 and https://doi.org/10.1016/j.msec.2019.110142
Author Response
Point-by-Point Response
The authors would like to thank the Editor and the Reviewers for their suggestions to improve the quality of the manuscript. We have accordingly changed and extended the manuscript, with each point outlined below in this Point-by-Point Response. Changes in the manuscript were tracked by using the “Track Changes” function.
Reviever #2
The manuscript describes the new tailored clamps designed to examine the fatigue strength of natural tissues like tendons. Cadaveric flexor digitorum tendons were harvested and mounted for tensile testing with no tapering being made, using 3D-printed clamps and holder arms, while ensuring a consistent testing length.
The manuscript is interesting because describes the detailed device to try to solve the big issue regarding how to generate a consistent methodology for mechanical characterization of biological tissue intended for tissue engineering approaches. However, some issues are found in the manuscript. Detailed as follow:
- I strongly recommend changing the order of some sections in the manuscript: The materials and methods should be after the introduction and before the results.
Answer: Thank you for this suggestion, this has been changed accordingly.
- The big concern in the results is most failures were observed on the edge of the clamps. Out of these failures, 78.3% were longitudinal and 21.7% transverse. Only 0.6% of the clamps failed longitudinal in the central area. This is a big concern due to this is an indicator that the clamps are affecting the experimental procedure by acting as a stress concentration, and this lead to lower peak stress than real as a result, please explain this in the manuscript.
Answer: Many thanks for this comment and constructive criticism. In pilot tests with sandpaper and metal clamps, cyclic testing of the tendons was impossible and resulted in a high number of direct failures of the tendons in the clamping area (for high clamping pressures), or excessive specimen slippage (for low clamping pressures). Our new approach using 3D printed clamps allowed for cyclic testing of these tendons. To keep the natural fiber orientation intact without creating a likely failure site, the tendon specimens were left completely intact and thus in their natural shape. This as a result led to unintended failure sites, but was necessary to allow for fatigue testing without premature failure. In future studies we will improve the clamping quality by using other/stronger materials for printing. As the clamping failures can potentially influence the results by acting as a stress concentration and leading to lower tolerable maximum forces of such tissues, further analyses to quantify these effects, e.g., using image correlation techniques, is recommended for future studies. The explanations and limitations in the manuscript have been extended accordingly. Please refer to page 6, lines 163 to 165; page 7, lines 181 to 186 and page 9, lines 288 to 291.
- You should cite other research related to mechanical testing in other biological tissues as: http://dx.doi.org/10.1016/j.msec.2015.10.094 and https://doi.org/10.1016/j.msec.2019.110142
Answer: Thank you, the references have been added. Please refer to page 8, lines 229 to 231.
Author Response File: Author Response.docx
Reviewer 3 Report
Thank you for conducting this research using innovative tools and methods.
Results are original and will be useful in both engineering and medical community. The Wohler diagrams look "ideal" separating low/high cycle areas, S-N curve then needed to be below 20MPa, testing at 20, 15, 10, 5MPa.
Introduction seems short, yet discussion reviews the literature. Move the reviews in introduction.
Authors needed to discuss individual specimens, length and other dimensions in a table. How the samples were gripped, which end faced the actuator and load-cell. Was it same for all samples. You also needed to discuss the asymmetry, anisotropy for each end of the samples. Perhaps literature showing some imaging data of the tendons and how Hounsfield unit varied that presented mechanical properties of the tendons. Did you x-ray them, before testing, to see if there were any defects? (may be not necessary)
Authors needed to comment on how samples prepared, how far away the bony end of the tendons were? Did priming change displacement of grips?
Do you have any load-displacement curves? Have you considered the low cycle fatigue possibility instead of Wohler, stress- no of cycle testing. I will be interested if you comment on this. My intent is not to add to your project but for my own curiosity controlling the displacement than load. May be next project.
Overall good effort and data presented by the authors.
Author Response
Point-by-Point Response
The authors would like to thank the Editor and the Reviewers for their suggestions to improve the quality of the manuscript. We have accordingly changed and extended the manuscript, with each point outlined below in this Point-by-Point Response. Changes in the manuscript were tracked by using the “Track Changes” function.
Reviever #3
Thank you for conducting this research using innovative tools and methods.
Results are original and will be useful in both engineering and medical community. The Wohler diagrams look "ideal" separating low/high cycle areas, S-N curve then needed to be below 20MPa, testing at 20, 15, 10, 5MPa.
- Introduction seems short, yet discussion reviews the literature. Move the reviews in introduction.
Answer: Many thanks for this constructive criticism. We agree that moving the literature to the introduction would be equally suitable, however, decided leaving our discussion on published work on the topic presented by others in the last section of the paper to underline that this yet seems an emerging area in soft tissue testing, and to also discuss our findings from the given paper with those of others. If Reviewer #3, in spite of this clarification, still feels we should bring the reviews to the front we are of course happy to do so.
- Authors needed to discuss individual specimens, length and other dimensions in a table. How the samples were gripped, which end faced the actuator and load-cell. Was it same for all samples. You also needed to discuss the asymmetry, anisotropy for each end of the samples. Perhaps literature showing some imaging data of the tendons and how Hounsfield unit varied that presented mechanical properties of the tendons. Did you x-ray them, before testing, to see if there were any defects? (may be not necessary)
Answer: Thank you very much for this advice. Unfortunately, it is no longer possible to reconstruct how every single sample was clamped regarding the natural tapering. This was honestly not done systematically, nevertheless we are quite sure that at these low amplitudes such influences are not present. We have nonetheless included this as a limitation in the manuscript. Please refer to page 9, lines 285 to 287. X-ray analysis was not performed because the study mainly focused on developing a method for cyclic biomechanical testing of human tendons.
- Authors needed to comment on how samples prepared, how far away the bony end of the tendons were? Did priming change displacement of grips?
Answer: Thank you, we are happy to do so and have added a section in the methods section of the paper draft accordingly. Please refer to page 2, lines 79 to 83. Priming, i.e., further processing beyond the here described techniques has not been done to assure maximum integrity of the specimens.
- Do you have any load-displacement curves? Have you considered the low cycle fatigue possibility instead of Wohler, stress- no of cycle testing. I will be interested if you comment on this. My intent is not to add to your project but for my own curiosity controlling the displacement than load. May be next project.
Answer: Thanks for the comment. Unfortunately, we have only recorded force-controlled data as a function of cycles. A displacement-controlled measurement of the number of cycles is a very good idea but will probably lead very quickly to a sharp drop in the maximum forces due to the viscoelastic biomechanical properties of the tendons. Nevertheless, we will continue to adopt this approach. A further approach for research could be the relaxation behavior at constant load with different load-free recovery times...perhaps also a maximum number of cycles that can be sustained could then be investigated. All in all, thanks again for the advice.
- Overall good effort and data presented by the authors.
Answer: Thank you very much for your review.
Author Response File: Author Response.docx
Reviewer 4 Report
The article presents the use of a 3D printing clamping system in medical applications.
The article has a low degree of novelty.
The article is written in an unclear manner and should be rearranged.
Certain elements must be detailed or added: the measurement method, the chosen shape and the customization of the 3D printing clamping system, the measured parameters, etc.
The chapter 2. Results must be moved towards the end of the article.
Table 1 and figure 1 what is the meaning of 0813-5, 0814-4, 0801-4, 0822-4. In the text, it says that it is about 50 tendons. What is the importance of the results presented in table 1 and figure 1? I was expecting results to appear for all the 50 tendons, in a manner that illustrates the description in the text.
In chapter 3. Discussion is a description of the methodology and should be renamed. Also, the chapter should be moved after chapter 1. Introduction
The text from rows 113 -128 must be moved to conclusions.
Regarding the clamp system, I think it needs to be described in more detail. Why does it have this shape? What are the advantages and disadvantages of other mechanical solutions?
Equations 1.1 -1.4 have notations that are not explained.
The importance of the Correlation coefficient R2 is not explained enough.
I understand that the measurement is done with a digital caliper in three areas of a tendon. It is not clear when the digital caliper is used: at the beginning of the measurement, after each cycle, at the end of the test, ...
Why was not used another method of measurement? For example, an optical method. What is the error and measurement accuracy?
There is no comparison of the results with those in the literature.
The text and the figures between lines 244-282 should be moved to line 176. The chapter 5. Conclusions is far too short.
Author Response
Point-by-Point Response
The authors would like to thank the Editor and the Reviewers for their suggestions to improve the quality of the manuscript. We have accordingly changed and extended the manuscript, with each point outlined below in this Point-by-Point Response. Changes in the manuscript were tracked by using the “Track Changes” function.
Reviewer #4
The article presents the use of a 3D printing clamping system in medical applications.
The article has a low degree of novelty.
The article is written in an unclear manner and should be rearranged.
Answer: Thank you for the constructive criticism. Dynamic and especially cyclic characteristics of soft tissues are relatively unexplored. The main challenge is the availability of tissues for extensive testing as well as problems such as slippage and damage to the tissues due to the clamping. These effects have a negative impact on the reliability of data even for quasistatic tests as well as for the number of cycles that can be sustained in fatigue tests. In the current study, our group presents a clamping method for fatigue testing of tendons, which is easy to manufacture by FDM and first results by means of S-N plots. A novelty is in particular the extensive fatigue testing of tendons and the acquisition of Woehler-curves, which was achieved by standardizing the testing with 3D-printed frames and clamps.
The chapters have been rearranged to the suggested order and the article has been extensively revised.
- Certain elements must be detailed or added: the measurement method, the chosen shape and the customization of the 3D printing clamping system, the measured parameters, etc.
Answer: Thank you for your comment. In fact, we have thoroughly considered the idea of adding in-depth methods. Further detail has been added in line with this request. However, the some of the underlying data has already been published elsewhere by our group and these references have been cited throughout the methods section. Our aim was not to expend the paper unreasonably, and to minimize redundance. We have added further explanation of the references used in the methods section (page 3, lines 95 to 104).
- The chapter 2. Results must be moved towards the end of the article.
Answer: Thank you for this feedback, this has been changed accordingly and the article has been rearranged to the suggested order in the template of Applied Sciences (Introduction, Materials and Methods, Results, Discussion, Conclusions).
- Table 1 and figure 1 what is the meaning of 0813-5, 0814-4, 0801-4, 0822-4. In the text, it says that it is about 50 tendons. What is the importance of the results presented in table 1 and figure 1? I was expecting results to appear for all the 50 tendons, in a manner that illustrates the description in the text.
Answer: Thank you for this suggestion. The tissue ID numbers are distinctive identifiers for various types of tissues. Table 1 has been removed as it added unnecessary detail and sample numbers moved to table 2.
- In chapter 3. Discussion is a description of the methodology and should be renamed. Also, the chapter should be moved after chapter 1. Introduction
Answer: Thank you for this comment. Due to the rearrangement this has been changed accordingly and the article has been rearranged to the suggested order in the template of Applied Sciences (Introduction, Materials and Methods, Results, Discussion, Conclusions).
- The text from rows 113 -128 must be moved to conclusions.
Answer: This has been done in line with Reviewer #4’s suggestion.
- Regarding the clamp system, I think it needs to be described in more detail. Why does it have this shape? What are the advantages and disadvantages of other mechanical solutions?
Answer: Please refer to our response of question 1.
- Equations 1.1 -1.4 have notations that are not explained.
Answer: Thank you for commenting, this has been added. Please refer to page 4. lines 137 to page 5 to 146.
- The importance of the Correlation coefficient R2 is not explained enough.
Answer: Thank you for this comment. Detail can now be found on page 7, lines 185 to 195. We have furthermore included the equations for the fitted power regression.
- I understand that the measurement is done with a digital caliper in three areas of a tendon. It is not clear when the digital caliper is used: at the beginning of the measurement, after each cycle, at the end of the test, ...
Why was not used another method of measurement? For example, an optical method. What is the error and measurement accuracy?
Answer: Thank you for this comment. The cross-sectional area was measured prior to fatigue testing under an axial pre-load of 10 N on the specimen, which allowed for stabilized conditions. This has been clarified in the manuscript, please refer to page 4, lines 124 to 146. An optical method or even an impression method could not be used for this study because the entire tissue would have to be analyzed in cross-section along its entire length. Such methodology was not available and would have been very complex even for the number of 50 single experiments. The cross-sectional area has been calculated by assuming a rectangular shape of the tendons and this forms a clear limitation which has been added to the manuscript. Please refer to page 8, lines 282 to page 9 line 290.
- There is no comparison of the results with those in the literature.
Answer: Thank you for this constructive criticism. To the best of our knowledge, we have included the available literature on fatigue tests on the tested tissues (human flexor tendons). A further intensive literature search did not yield any additional results for comparison. If Reviewer #4, has additional literature on fatigue testing of human tendons, we would be happy to include it in our manuscript.
- The text and the figures between lines 244-282 should be moved to line 176. The chapter 5. Conclusions is far too short.
Answer: Thank you for this comment. Due to the rearrangement these figures and text now directly appear after the introduction in the materials and methods section, as suggested by Reviewer #4. The figures described show CAD data as well as specimen data and methodological details such as testing machine and details and therefore fit ideally into the materials and methods section. Further, in line with the recommendation of Reviewer #4, the entry part (paragraph) of the Discussion section has been moved to the Conclusions, thereby extending this section.
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The suggestion and previous requirements were addressed
Author Response
Thank you very much for these comments. It seems the comments made by Reviewer #2 have been fully incorporated to his suggestions.
Reviewer 4 Report
I don't have other comments. The authors have modified it according to the reviewing suggestions.
Author Response
Thank you very much for these comments. It seems the comments made by Reviewer #4 have been fully incorporated to his suggestions.
