Dynamic Mechanical and Biological Characterization of New 3D-Printed Polymeric Dental Materials: A Preliminary Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.     Dynamic mechanical analysis (DMA) has not been used much for denture base resins, but has received attention with resilient denture liners.  There are no references to this field of denture prostheses.

2.     DMA testing is appropriate for rigid materials like denture base resins.  But running the test to 100 Hz is inappropriate since human chewing rates do not come close to 100 Hz.  The data beyond ~5 Hz is interesting, but is of little consequence. 

3.     The Mettler DMA apparatus can provide the range of temperatures experienced in the human mouth, 5 to 70 degrees Celsius (cold and hot food and liquids).  It would have been more appropriate to test the resins over the 5-70 degree temperature range at no more than 1-5 Hz.

4.     As mentioned in the conclusions, these resins in service in the mouth are continuously wet.  Hydration can have a powerful effect on denture base resins, affecting their strength and dimensional stability.  The tests should be revised under wet conditions.

5.     Rather than plotting E*, it might be important to see tan delta values displayed because they relate to deformation of the denture base materials to respond to occlusal forces and return to its original shape (viscoelasticity).

6.     Why were keratocytes chosen for biocompatibility tests?  Denture base resins accumulate a biofilm, mostly from Candida species.  It would have been more appropriate to test how well denture bases resist attachment of microbes, not keratocytes.  Incorporation of antifungal and antibacterial agents into denture base resins and denture liners has been done for decades.

7.     Since there is some chemistry involved with these denture base materials, it would have been appropriate to test these materials days or weeks in flowing water after fabrication/curing, since residual resins and small molecules slowly leach out.  This is especially true for the keratocyte tests.  The presence of moisture can affect tests of materials, as pointed out in Mettler literature.

8.     Page 4, the symbol “©” for copyright appears in my copy on line 135 and 137.  Should this be “μ” or “micro?”  Also, “liters” are abbreviated with the upper case “L,” not the lower case “l,”

9.     Page 6, line 214, change “ascending” to “descending.”

10.           Page 7, line 262, “accuracy” was not measured in this study and should not be mentioned with other advantages of these methods.

11.           Authors use “better,” “higher,” and “worse” throughout the text.  It would be better in a scientific report to use words such as “higher” or “lower.”

12.       Table 1.  Change “Mpa” to “MPa.”

Author Response

2. Thank you for your thoughtful insight on the frequency range utilized in the study. While we agree with the reviewer that frequency higher than 5 Hz are not physiologically possible, we’d like to provide further insight into our rationale for extending the testing frequency to 100 Hz.

In our study, we aimed to simulate the aging process of the resins, particularly with a focus on their potential application in definitive dental prostheses. Unlike temporary prostheses, which are typically worn for a shorter duration of 4 months, definitive prostheses are expected to withstand mechanical stresses over a longer period. Therefore, we sought to subject the materials to more rigorous testing conditions to better understand their long-term performance.

By increasing the frequency at which the mechanical tests were conducted, we aimed to accelerate the aging process and assess how the materials would behave under more demanding conditions. While we acknowledge that human chewing rates do not typically reach 100 Hz, we believe that this approach provides valuable insights into the durability and reliability of the denture base resins, especially in the context of long-term clinical use.

We appreciate your concern regarding the relevance of data beyond ~5 Hz. In a future study, we’d like to explore more in detail the data below 5Hz.

To better explain the authors' intention we added the following sentence in the discussion section, line 290-297: “An aspect to consider within our study is the apparent discrepancy between the physiological frequencies of the masticatory cycle and those used in the mechanical tests, the authors' intention was to simulate and accelerate the aging process and assess how the materials would behave under more demanding conditions, considering that this approach provides valuable insights into the durability and reliability of the denture base resins, especially in the context of long-term performance and clinical use. A more in-depth analysis of what happens at frequencies lower than 5 Hz will be the purpose of a future scientific paper.”

 

3. Your feedback serves as a valuable reminder of the importance of methodological rigor in research, and we are committed to addressing this aspect in future studies. Since this work was a preliminary study following a literature review on 3D-printed prosthetic materials [Valenti C, et al. Mechanical properties of 3D-printed prosthetic materials compared with milled and conventional processing: A systematic review and meta-analysis of in vitro studies. J Prosthet Dent. 2022, S0022-3913(22)00415-2. doi:10.1016/j.prosdent.2022.06.008].

The first objective of this study was to try to understand the effect of aging of resins, considering we can apply these materials in the oral cavity for longer periods of time than temporary prostheses for which they are designed, but we understand the importance of testing materials under conditions that closely mimic the oral cavity when in contact with food of different temperatures. We conducted the mechanical tests in room temperature, and we acknowledge that this is a limitation. Moving forward, we will explore alternative methods or equipment that can reliably provide the desired temperature control to better simulate oral conditions, and certainly temperature variation is an important aspect to be considered, we have included our intentions for future studies, line 369-370, as follows: "…, and the effects in a temperature range between 5 and 70 degrees Celsius, to simulate the ingestion of cold or hot food in the oral cavity …" 

 

4.  We would like to thank the reviewer for this consideration, this aspect was taken very much into account in the continuation of our research; due to the timing of the submission of this preliminary work, the work following this study has already been published: Zara T et al. Effects of saliva on additive manufacturing materials for dentistry applications: Experimental research using flexural strength analysis. 2023, Acta IMEKO, doi: https://doi.org/10.21014/actaimeko.v12i2.1519.

We have added details regarding the results drawn from this work in the discussion section, line 298-302: “Another aspect to be considered is the possible change in the behavior of these resins under wet conditions. In a parallel study conducted by our working group, evaluating the effect of saliva on mechanical performance by analyzing flexural modulus loss, it was observed that surface material properties and manufacturing techniques were the main factors influencing the properties of the resins exposed to artificial saliva for 72 hours [24].”

 

5.  Thank you for your insightful suggestion regarding the presentation of our data. We appreciate your perspective on the importance of tan delta values in assessing the viscoelastic behavior of denture base materials, particularly in understanding their response to occlusal forces and ability to return to their original shape. Tan delta values indeed provide valuable insights into the material's viscoelastic properties, which are critical considerations in dental prosthesis design and functionality.

However, I would like to clarify that the raw data obtained from the DMA Mettler Toledo apparatus provided us with the Young modulus rather than the separate loss and storage modulus. As you rightly pointed out, tan delta is the ratio of the loss modulus to the storage modulus. Since we did not have access to the separate loss and storage modulus data from the DMA output, we were unable to directly calculate the tan delta values from the raw data.

While we recognize the significance of tan delta values in characterizing viscoelastic materials, the limitations of the available data prevented us from including this parameter in our analysis and presentation. We apologize for any inconvenience this may have caused and acknowledge the potential value in exploring alternative methods to obtain the necessary data for calculating tan delta values in future studies, in addition to temperature. This will be important in creating a tan delta vs. temperature plot, which is a very valuable analysis when comparing the different resins.

 

6.  We thank the reviewer for the comment, we will certainly consider it for future studies on the subject; we have added this in the 'future perspectives', line 370-372, as follows: “…and it would be important to evaluate how these resins manufactures can resist the attack of microbes and the accumulation of biofilms, especially Candida species.”

We selected keratinocytes to assess biocompatibility in particular, not attachment, mainly because there are no studies in the literature on the cytotoxicity of such resinous materials on oral mucosa cells, and especially because not all the materials selected were for prosthetic denture bases; we modified Table 1 according with this information and added a sentence detailing this in the materials and methods section: “ …11 new AM polymeric materials, designed for crowns and denture bases, were selected for mechanical and biological characterization…”

 

7.  We thank the reviewer for drawing attention to this aspect; All the materials selected are commonly used in clinical practice by dental laboratories for the fabrication of crowns and denture bases and are validated by the manufacturer, but in the conclusions section we assessed how it was indispensable to have research follow-ups with longer timescales in order to be able to assess the long-term effects of resinous materials, even though for dental purposes they are not intended to remain in the oral cavity for too long, because they are generally intended for the fabrication of temporary prosthetic restorations. Conclusions, line 356-362: “Not having conducted experiments for more than 24 h, but assuming that in the oral cavity such materials should remain for a longer time, it would be appropriate to perform further investigations under more realistic conditions, especially considering the fact that from a biological point of view there is some recovery in cell viability after 24 hours. In fact, this research is proposed as a preliminary study, and it might be interesting to evaluate the trend of viability for extended periods to assess whether the reduction in cytotoxicity is confirmed.”.

 

8. We are sorry, we corrected all the mistakes.

 

9. We thank the reviewer and corrected the wrong word.

 

10. We are sorry, we corrected the word, because we assessed the measurement uncertainty in relation to repeatability.

 

11. We apologise, we have corrected all improper or ambiguous terms as advised.

 

12. We apologise for the error, we have corrected the unit of measurement.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article by Chiara Valenti et al. presents the results of a study of the mechanical properties and characteristics of new dental materials in the process of dynamic loading. The materials are printed on a 3D printer. Cytotoxicity was also investigated 24 hours after mechanical testing. The presented results indicate high uncertainty in the frequency range of 1-11 Ghz, and require additional research. The paper provides an adequate and up-to-date review of the literature data. The research methods are described in full. The results of the work are relevant for dentistry in terms of quickly obtaining data on new materials and the possibility of their use in dentistry. There are comments on the work:

1) Why is this particular frequency range chosen for research? Is this the standard range for dental materials research?

2)The work does not provide photographs or any confirmation of the changes that occurred in the materials under study after the tests.

3) The conclusion should be expanded and the main results obtained should be presented.

Author Response

1. We’d like to thank you for your interest in understanding the motivation behind the frequency range. We agree that frequency higher than 5 Hz are not physiologically possible, we’d like to provide further insight into our rationale for extending the testing frequency to 100 Hz.

In our study, we aimed to simulate the aging process of the resins, particularly with a focus on their potential application in definitive dental prostheses. Unlike temporary prostheses, which are typically worn for a shorter duration of 4 months, definitive prostheses are expected to withstand mechanical stresses over a longer period. Therefore, we sought to subject the materials to more rigorous testing conditions to better understand their long-term performance.

By increasing the frequency at which the mechanical tests were conducted, we aimed to accelerate the aging process and assess how the materials would behave under more demanding conditions. While we acknowledge that human chewing rates do not typically reach 100 Hz, we believe that this approach provides valuable insights into the durability and reliability of the denture base resins, especially in the context of long-term clinical use.

We appreciate your concern regarding the relevance of data beyond ~5 Hz. In a future study, we’d like to explore more in detail the data below 5Hz.

To better explain the authors' intention we added the following sentence in the discussions, line 290-297: “An aspect to consider within our study is the apparent discrepancy between the physiological frequencies of the masticatory cycle and those used in the mechanical tests, the authors' intention was to simulate and accelerate the aging process and assess how the materials would behave under more demanding conditions, considering that this approach provides valuable insights into the durability and reliability of the denture base resins, especially in the context of long-term performance and clinical use. A more in-depth analysis of what happens at frequencies lower than 5 Hz will be the purpose of a future scientific paper.”

 

2.  We are sorry we did not provide any photographs of the sample after the tests. We have now attached below the photographs of the specimen after both the mechanical tests and the biological tests (available in the Word document).

While no discernible deformation or morphological changes were observed with the naked eye during our mechanical characterization, we recognize the importance of diving deeper into the microstructure of the materials under study. In our future studies, we intend to utilize scanning electron microscopy (SEM) to obtain images of the samples. This will allow for a more detailed examination of any potential microfractures or structural alterations that may not be visible to the naked eye. By incorporating SEM imaging into our methodology, we aim to provide a comprehensive analysis of the materials' morphology. We reported our intentions to conduct further microscopic tests later on in future perspectives, line 372-374: “…and perform further analysis with scanning electron microscopy to assess the presence of any potential microfractures or structural alterations following the mechanical tests. ”

We have also included photographs of the biological experimentations in the supplementary materials.

 

3. We thank the reviewer and have made the recommended changes, which we report below (line 352-360): “ In conclusion, the choice of the best materials for clinical application thus depends on both the mechanical and biological behavior. At low frequencies, such as 1 to 11 Hz, the materials show less fragility and lower strength, with high uncertainty, accompanied by significantly reduced oral keratinocyte cell viability both after 3 and after 24 hours of treatment. Not having conducted experiments for more than 24 hours, but assuming that in the oral cavity such materials should remain for a longer time, it would be appropriate to perform further investigations under more realistic conditions, especially considering the fact that from a biological point of view there is some recovery in cell viability after 24 hours.”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am gratified that the authors have accepted all of my suggestions and criticisms for future research using their techniques.  But this paper should probably add the word "preliminary" in the title due to several flaws or useless measurements that were made.  Overall, I guess that it should be published in hopes that they will expand the work to a) keep frequencies 5 Hz or less, b) test the materials after much longer time periods (when water/saliva reaches equilibrium in the resins) and any soluble or volatile moieties are washed out, and c) microbe (bacterial and mycological) is tested, rather than mammalian karyocytes.  Finally, the apparatus used apparently cannot provide tan delta data on the viscoelasticity of the denture base resins, so another device should be used for the DMA tests.

Further suggestions: Explain what the FDM, Polyjet, and SLS processes are in material fabrication.  Unknown is misspelled in line 100, and Supplementary material could be explained in lines 378-80. 

Author Response

We thank the reviewer again for his opinion and comments; we apologize for the errors, we have corrected the incorrect word, changed the title as suggested, and made the requested additions: a detailed description of the figures added in the supplementary materials (lines 386-390) has been included; and descriptive paragraphs have been inserted in the introduction on the printing techniques mentioned in the specimen production (FDM, PJ and SLS), with new related references, as follows "FDM is a useful technique thanks to its geometric flexibility and low cost; with this technique it is possible to produce polymer prostheses with hollow, semi-hollow and solid structures, easily fabricating various dental applications [5]. The SLS technique, on the other hand, can also be used to cast various metal alloys, for metal frameworks for dentures, crowns and dental implants; while the polymers used in SLS are usually applied in the manufacture of surgical guides and dental models [6].…. Polyjet printers can be used to produce items of varying density, hardness and flexibility, at high resolution, and commonly employed devices include customized dental models, surgical guides and scaffolds [6].”