Biofilm Removal from In Vitro Narrow Geometries Using Single and Dual Pulse Er:YAG Laser Photoacoustic Irrigation

Round 1

Reviewer 1 Report

Dear Authors,

The topic of the manuscript entitled "Single and Dual Pulse Er:YAG Laser Photoacoustic Irrigation removal of biofilms from narrow geometries" might be of interest to the readers, but before considering it for publication it has to be corrected extensively.

Comments:

Title:

-        The title: unclear information for the readers; “biofilms from narrow geometries”- I suggest to change for more clear title where should be add that study is done on the titanium disks grade 2 and that is in vitro study

Introduction:

-        Very interesting is physical part which describe differences between super short pulse and shock wave enhanced emission photoacoustic streaming in the introduction part;

-        lack information about clinical modalities perio- implant complication treatment with lasers, including parameters and different pulse duration;

-        the authors claimed that “although titanium grade 4 has the highest mechanical strength titanium grade 2 is often used for dental implants due to lower brittleness, its elasticity and flexibility that are beneficial in clinical situations”- I would prefer to stay straight in the position based on the literature review, that the most popular and high quality implant has grade IV CpTi , which “presents the highest oxygen content (0.4%) and consequently, excellent mechanical strength, which is why it is the most widely used type of titanium for dental implants”

Material and Methods:

-        the study involves only one spp. E. faecalis and on the clinical inflamed implant surface we have all biofilm, which has been covered by many bacteria. So, the authors are using in the manuscript wrong wording “E.faecalis biofilms” which should be corrected for E. faecalis strain.

-        It should be clear comment that Enterococcus faecalis strain, which was incubated in the disk is not even similar for the natural condition, where in clinical scenario we have usually more difficulties to remove sticky biofilm from the implant surface because it is more consist and diverse. It should be discussion part where the authors will share with all limitation of this study;

-        It should be clearly written division and description of groups and how many disks were used in each group? the authors states that “9 for the irrigation model A and 7 for the irrigation model B with 4 - 7 discs per treatment group” Why is so huge differences with the disks number?  how many repetitions in each group was done (for SSP and SWEEPS) in each model? Is the group A with SSP mode and group B for SWEEPS? Please rewrite manuscript with more clear statements.

Discussion:

-        Usually in the literature we can observe that the main recommendation for perio-implant treatment is using different mode than the authors recommendation. The authors should include paragraph of the discussion why they used SSP mode not SP, which is less popular in the perio field;

-        We can find in the literature that some studies were used combination Er:YAG laser with irrigation of antioxidant, which should be mark in the discussion too (eg. doi: 10.3390/microorganisms7120612)

-        In the discussion section the authors only describe one or two studies do not discuss with all literature and with all published laser methods,

-        Discussion section is too short and not developed well, should be more elaborate, should be extensively rewritten

 

All together, the manuscript requires major changes and revision as describe above.

Moderate editing of English language required

Author Response

 

Reviewer No.1

I’ve revied the paper titled "Efficacy of Single and Dual Pulse Er:YAG Photoacoustic Irrigation Modalities for Biofilm Decontamination from Titanium Surfaces in Different Narrow Model Geometries" submitted to “microorganisms” having ID microorganisms-2532238 with interest. I have some critical comments that I believe would strengthen the paper and enhance its scientific impact. Please find my suggestions bellow:

 

1. General comments: The presents a study focused on addressing the challenge of disinfection and removal of biofilm from dental implants. The authors evaluated and compared the efficacy of two photoacoustic irrigation modalities, namely Er:YAG-SSP and Er:YAG-AutoSWEEPS, for biofilm decontamination in narrow model geometries. The study design involved treating mature Enterococcus faecalis biofilms on titanium discs using the SSP and AutoSWEEPS Er:YAG photoacoustic irrigation techniques in saline solution in both narrow cylindrical and square gap geometries. The density of bacteria and the number of live bacteria were assessed before and after the photoacoustic treatments.

 

2. Title: It is unclear from your title about the nature of this study, please consider rephrasing it by including ‘the in vitro study,  grown biofilms model, etc’

As suggested by reviewer we have rephrase the title to indicate that this is an in vitro study. The new title is: Biofilm removal from in vitro narrow geometries using single and dual pulse Er:YAG laser photoacoustic irrigation

 

3. Methodology: I suggest providing more comprehensive details regarding the specific parameters used for the Er:YAG-SSP and Er:YAG-AutoSWEEPS photoacoustic irrigation techniques. Information such as laser settings, energy levels, and pulse durations would greatly aid in reproducibility and facilitate future research in this area.

The information about laser settings, energy levels, and pulse durations, and other laser settings, energy levels, and pulse durations has been provided in the text. However, to make this clearer we have rewritten the text to:

The photoacoustic irrigation was performed with Fotona laser system LightWalker (Fotona d.o.o) with the following parameters for the Super Short Pulse SSP (50 microseconds) experiments; wavelength: 2940 nm; handpiece: contact H14; fiber tip: FlatSweeps 400/14; energy: 20 mJ; frequency: 15 Hz; water off; air off. For AutoSWEEPS (2 times Ultra Short Pulse USP: 2 x 25 microseconds) experiments the parameters were; wavelength: 2940 nm; handpiece: contact H14; fiber tip: FlatSweeps 400/14, energy: 2 x 20 mJ; frequency: 15 Hz; water off; air off.

 

4. The study focuses solely on Enterococcus faecalis biofilms, which may limit the generalizability of your findings to other bacterial species commonly associated with peri-implant diseases. It would be valuable to discuss the implications and potential applicability of your results to a broader range of pathogens encountered in clinical practice if possible or at least compare your model with current available evidence from the literature. 

Thank you for the comment. Enterococcus faecalis represents the main cause of persistent bacterial infections in oral cavity. The results of our study are comparable to already published studies on the efficacy of photoacoustic irrigation in biofilm removal from the root canal system such as:

Afrasiabi S, Parker S, Chiniforush N. Synergistic Antimicrobial Effect of Photodynamic Inactivation and SWEEPS in Combined Treatment against Enterococcus faecalis in a Root Canal Biofilm Model: An In Vitro Study. Applied Sciences. 2023; 13(9):5668.

Rostami G, Afrasiabi S, Benedicenti S, Signore A, Chiniforush N. The Evaluation of SWEEPS Plus Antimicrobial Photodynamic Therapy with Indocyanine Green in Eliminating Enterococcus faecalis Biofilm from Infected Root Canals: An In Vitro Study. Biomedicines. 2023; 11(7):1850.

Seghayer I, Lee AHC, Cheung GSP, Zhang C. Effect of Passive Ultrasonic Irrigation, Er,Cr:YSGG Laser, and Photon-Induced Photoacoustic Streaming against Enterococcus faecalis Biofilms in the Apical Third of Root Canals. Bioengineering (Basel). 2023 Apr 20;10(4):490.

Wen C, Yan L, Kong Y, Zhao J, Li Y, Jiang Q. The antibacterial efficacy of photon-initiated photoacoustic streaming in root canals with different diameters or tapers. BMC Oral Health. 2021 Oct 21;21(1):542. doi: 10.1186/s12903-021-01903-7. PMID: 34674671; PMCID: PMC8532288.

We have included these references in the new manuscript and improve the introduction as suggested by reviewer No.2.

 

5. I am concerned about the practical applicability of the narrow square gap geometry utilized in your study. It would be worthwhile to discuss whether this geometry accurately represents clinical scenarios and if the results can be extrapolated to different implant designs and anatomical situations. Please explain this further.

We have improved the Discussion section to explain this further.

The geometries that we have studied represent only two of many diverse anatomical situations in clinical scenarios [55]. Walls of the model systems were solid which is different from the soft functional tissue of the peri-implant pocket [56-57]. Nevertheless, we envisage that in the clinical practice, a solid polymer barrier (i.e. cylindrical or square tube) could be employed to surround the implant for an effective surgical cleaning. This will separate implant from the soft tissue and enable higher efficiency of photoacoustic treatment due to the significant increase in fluid flow rate facilitated by interaction with the solid walls and enhanced cleaning action at a distance [51].

 

6. It would be beneficial to include a section on future research directions. Suggestions such as exploring combination therapies, evaluating the long-term effects on implant osseointegration, or conducting clinical trials in real patient populations would help guide further investigations in this field. The potential adverse effects or limitations associated with the Er:YAG photoacoustic irrigation techniques should be addressed. Discussing aspects such as thermal damage to surrounding tissues or potential alterations in implant surface characteristics would provide a more comprehensive evaluation of the techniques.

Thank you for the suggestions. In the new manuscript we have added a new paragraph in the Discussion section.

The model presented in this study can be enhanced to better represent clinical situations. We have recently demonstrated that cavitation dynamics is significantly affected when cavitation bubbles interact with soft tissue [52]. In the future it would be important to test the effect of soft walls in contact with the implant on biofilm removal as well as on the removal of multi-species biofilm. Customized mouth tray with titanium discs exposed to microbial community in the oral cavity which will allow the accumulation of a natural biofilm plaque [53] could be tried. Exploring combination therapies and evaluating the long-term effects on implant osseointegration will further help to guide investigations in the field [58-60]. Although laser energies used in this study were below the ablation threshold care must be taken not to exceed laser energies of more than 80 mJ/pulse for direct implant treatment, and 50 to 80 mJ/pulse for dental root treatment, as this may cause material damage and possibly delayed growth and adhesion of gingival cells [54].

Author Response File: Author Response.docx

Reviewer 2 Report

I’ve revied the paper titled "Efficacy of Single and Dual Pulse Er:YAG Photoacoustic Irrigation Modalities for Biofilm Decontamination from Titanium Surfaces in Different Narrow Model Geometries" submitted to “microorganisms” having ID microorganisms-2532238 with interest. I have some critical comments that I believe would strengthen the paper and enhance its scientific impact. Please find my suggestions bellow:

 

1.     General commnets: The presents a study focused on addressing the challenge of disinfection and removal of biofilm from dental implants. The authors evaluated and compared the efficacy of two photoacoustic irrigation modalities, namely Er:YAG-SSP and Er:YAG-AutoSWEEPS, for biofilm decontamination in narrow model geometries. The study design involved treating mature Enterococcus faecalis biofilms on titanium discs using the SSP and AutoSWEEPS Er:YAG photoacoustic irrigation techniques in saline solution in both narrow cylindrical and square gap geometries. The density of bacteria and the number of live bacteria were assessed before and after the photoacoustic treatments.

2.     Title: It is unclear from your title about the nature of this study, please consider rephrasing it by including ‘the in vitro study,  grown biofilms model, etc’

3.     Methodology: I suggest providing more comprehensive details regarding the specific parameters used for the Er:YAG-SSP and Er:YAG-AutoSWEEPS photoacoustic irrigation techniques. Information such as laser settings, energy levels, and pulse durations would greatly aid in reproducibility and facilitate future research in this area.

4.     The study focuses solely on Enterococcus faecalis biofilms, which may limit the generalizability of your findings to other bacterial species commonly associated with peri-implant diseases. It would be valuable to discuss the implications and potential applicability of your results to a broader range of pathogens encountered in clinical practice if possible or at least compare your model with current available evidence from the literature. 

5.     I am concerned about the practical applicability of the narrow square gap geometry utilized in your study. It would be worthwhile to discuss whether this geometry accurately represents clinical scenarios and if the results can be extrapolated to different implant designs and anatomical situations. Please explain this further

 

6.     It would be beneficial to include a section on future research directions. Suggestions such as exploring combination therapies, evaluating the long-term effects on implant osseointegration, or conducting clinical trials in real patient populations would help guide further investigations in this field. The potential adverse effects or limitations associated with the Er:YAG photoacoustic irrigation techniques should be addressed. Discussing aspects such as thermal damage to surrounding tissues or potential alterations in implant surface characteristics would provide a more comprehensive evaluation of the techniques.

Author Response

Reviewer No.2

The subject is of interest in the dental field. The manuscript presents some deficiencies that need to be resolved or clarified:

The introduction is extremely long and difficult to follow, the main concepts are mixed up and do not have a logical order. It is suggested to redo this section with 3 relevant paragraphs and conclude with the objective and the proposed hypothesis. Some sentences lack references. It is also important to check the reference format according to the authors' instructions. The methodology must indicate the design of the experiment carried out.

As suggested by the reviewer we have shorten the introduction, make three relevant paragraphs, clearly stated objective and proposed hypothesis. We have indicated the design of the two model systems in the Material and method section. In addition, we have more clearly stated the laser experiment parameters.

According to the journal recommendations the references may be in any style, provided that one uses the consistent formatting throughout. It is essential to include author(s) name(s), journal or book title, article or chapter title (where required), year of publication, volume and issue (where appropriate) and pagination. DOI numbers (Digital Object Identifier) are not mandatory but highly encouraged. We have included more references in the text and used consistent formatting throughout the text.

Like the introduction, the discussion section requires work, as it's pretty much a continuation of the introduction itself. A better structure, greater depth, comparison with other studies, relevance and originality of the proposed study, main strengths, limitations and proposal for future studies are required. The conclusions must adequately and specifically respond to the stated objectives.

We have sharpened the discussion and either removed parts that were a continuum of the introduction or merged them in the introduction. We have included two new paragraphs to better discussed relevance, originality, strength and limitations of our approach and suggested improvements of our model approach for future studies. In addition, we have included several new relevant references. The text has been modified on several places in the Discussion.

 

The geometries that we have studied represent only two of many diverse anatomical situations in clinical scenarios [55]. Walls of the model systems were solid which is different from the soft functional tissue of the peri-implant pocket [56-57]. Nevertheless, we envisage that in the clinical practice, a solid polymer barrier (i.e. cylindrical or square tube) could be employed to surround the implant for an effective surgical cleaning. This will separate implant from the soft tissue and enable higher efficiency of photoacoustic treatment due to the significant increase in fluid flow rate facilitated by interaction with the solid walls and enhanced cleaning action at a distance [51].

The model presented in this study can be enhanced to better represent clinical situations. We have recently demonstrated that cavitation dynamics is significantly affected when cavitation bubbles interact with soft tissue [52]. In the future it would be important to test the effect of soft walls in contact with the implant on biofilm removal as well as on the removal of multi-species biofilm. Customized mouth tray with titanium discs exposed to microbial community in the oral cavity which will allow the accumulation of a natural biofilm plaque [53] could be tried. Exploring combination therapies and evaluating the long-term effects on implant osseointegration will further help to guide investigations in the field [58-60]. Although laser energies used in this study were below the ablation threshold care must be taken not to exceed laser energies of more than 80 mJ/pulse for direct implant treatment, and 50 to 80 mJ/pulse for dental root treatment, as this may cause material damage and possibly delayed growth and adhesion of gingival cells [54].

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Single and Dual Pulse Er:YAG Laser Photoacoustic Irrigation removal of biofilms from narrow geometries

 

The subject is of interest in the dental field. The manuscript presents some deficiencies that need to be resolved or clarified:

The introduction is extremely long and difficult to follow, the main concepts are mixed up and do not have a logical order. It is suggested to redo this section with 3 relevant paragraphs and conclude with the objective and the proposed hypothesis. Some sentences lack references. It is also important to check the reference format according to the authors' instructions. The methodology must indicate the design of the experiment carried out.

Like the introduction, the discussion section requires work, as it's pretty much a continuation of the introduction itself. A better structure, greater depth, comparison with other studies, relevance and originality of the proposed study, main strengths, limitations and proposal for future studies are required.

The conclusions must adequately and specifically respond to the stated objectives.

Minor editing of English language required

Author Response

Reviewer No.2

The subject is of interest in the dental field. The manuscript presents some deficiencies that need to be resolved or clarified:

The introduction is extremely long and difficult to follow, the main concepts are mixed up and do not have a logical order. It is suggested to redo this section with 3 relevant paragraphs and conclude with the objective and the proposed hypothesis. Some sentences lack references. It is also important to check the reference format according to the authors' instructions. The methodology must indicate the design of the experiment carried out.

As suggested by the reviewer we have shorten the introduction, make three relevant paragraphs, clearly stated objective and proposed hypothesis. We have indicated the design of the two model systems in the Material and method section. In addition, we have more clearly stated the laser experiment parameters.

According to the journal recommendations the references may be in any style, provided that one uses the consistent formatting throughout. It is essential to include author(s) name(s), journal or book title, article or chapter title (where required), year of publication, volume and issue (where appropriate) and pagination. DOI numbers (Digital Object Identifier) are not mandatory but highly encouraged. We have included more references in the text and used consistent formatting throughout the text.

Like the introduction, the discussion section requires work, as it's pretty much a continuation of the introduction itself. A better structure, greater depth, comparison with other studies, relevance and originality of the proposed study, main strengths, limitations and proposal for future studies are required. The conclusions must adequately and specifically respond to the stated objectives.

We have sharpened the discussion and either removed parts that were a continuum of the introduction or merged them in the introduction. We have included two new paragraphs to better discussed relevance, originality, strength and limitations of our approach and suggested improvements of our model approach for future studies. In addition, we have included several new relevant references. The text has been modified on several places in the Discussion.

 

The geometries that we have studied represent only two of many diverse anatomical situations in clinical scenarios [55]. Walls of the model systems were solid which is different from the soft functional tissue of the peri-implant pocket [56-57]. Nevertheless, we envisage that in the clinical practice, a solid polymer barrier (i.e. cylindrical or square tube) could be employed to surround the implant for an effective surgical cleaning. This will separate implant from the soft tissue and enable higher efficiency of photoacoustic treatment due to the significant increase in fluid flow rate facilitated by interaction with the solid walls and enhanced cleaning action at a distance [51].

The model presented in this study can be enhanced to better represent clinical situations. We have recently demonstrated that cavitation dynamics is significantly affected when cavitation bubbles interact with soft tissue [52]. In the future it would be important to test the effect of soft walls in contact with the implant on biofilm removal as well as on the removal of multi-species biofilm. Customized mouth tray with titanium discs exposed to microbial community in the oral cavity which will allow the accumulation of a natural biofilm plaque [53] could be tried. Exploring combination therapies and evaluating the long-term effects on implant osseointegration will further help to guide investigations in the field [58-60]. Although laser energies used in this study were below the ablation threshold care must be taken not to exceed laser energies of more than 80 mJ/pulse for direct implant treatment, and 50 to 80 mJ/pulse for dental root treatment, as this may cause material damage and possibly delayed growth and adhesion of gingival cells [54].

 

 

Author Response File: Author Response.docx

Reviewer 4 Report

 Comments

1.     How the Stocks of Enterococcus faecalis Symbioflor were maintained?

2.     Abstract should be written in a more constructive way.

3.     Provide the conclusions section.

4.     Clear statements of the novelty of the work should also appear briefly in the Abstract and Conclusions sections.

5.     Key findings of the performed study must be included in the revised version.

6.     Materials section is not sound enough.

 

no

Author Response

Reviewer No. 4

1. How the Stocks of Enterococcus faecalis Symbioflor were maintained?

The master stock of Enterococcus faecalis Symbioflor was stored at -80 °C. From the master stock working stocks were prepared by subculturing the culture on Trypticase soy agar. We have improved text in the new manuscript:

The master stock of Enterococcus faecalis Symbioflor was stored at -80 °C. Working stocks of Enterococcus faecalis Symbioflor 1 (kindly donated by SymbioGruppe GmbH & CoKG SymbioPharm GmbH) were maintained by weekly subculture on Trypticase soy agar (TSA). Before the biofilm experiment, a single colony of E. faecalis strain was transferred from TSA and grown overnight in 5 ml Trypticase soy broth (TSB) in the dark under aerobic conditions at 37 °C, 200 rpm.

 

2. Abstract should be written in a more constructive way.

Abstract has been rewritten.

The disinfection and removal of biofilm from titanium dental implants remains a great challenge in oral medicine. Here we present results of a novel photoacoustic irrigation laser modalities for biofilm removal in model geometries mimicking peri-implant pocket. The efficacy of single pulse (Er:YAG-SSP) and dual pulse (Er:YAG-AutoSWEEPS) photoacoustic irrigation modalities were determined for Enterococcus faecalis biofilms decontamination from titanium surfaces in the narrow cylindrical and square gap geometries. The density of bacteria as well as the number of live bacteria were determined prior and after different photoacoustic treatments. Both SSP and AutoSWEEPS photoacoustic irrigation techniques removed at least 92 % of biofilm bacteria during the 10 s photoacoustic treatment. The effectiveness of cleaning was better in the narrow square gap geometry compared to cylindrical geometry. The dual pulse Er:YAG-AutoSWEEPS photoacoustic irrigation showed better results compared to SSP modality. No chemical adjuvants were needed to boost the effectiveness of the photoacoustic irrigation in the saline solution. The results imply that photoacoustic irrigation is an efficient cleaning method for debridement and decontamination in narrow geometries and should be considered as new therapeutic options for the treatment of peri-implant diseases.

 

3. Provide the conclusions section.

As suggested we are providing a conclusion section.

CONCLUSIONS

Here we present results of a novel photoacoustic irrigation laser modalities for biofilm removal in model geometries mimicking peri-implant pocket. The results demonstrate that both the Er:YAG-SSP and Er:YAG-SWEEPS laser modalities have very good cleaning efficiency in the model narrow geometries, better than reported in the literature. Overall the dual pulse Er:YAG-AutoSWEEPS photoacoustic irrigation modality showed improved results compared to the SSP modality. It is important to note that no chemical adjuvants were needed to boost the effectiveness of the photoacoustic irrigation in the saline solution.The effectiveness of cleaning was better in the fabricated narrow square gap geometry compared to the cylindrical geometry. The new results support the introduction of both SSP and AutoSWEEPS laser modalities in clinical practice as the new tools for the treatment of biofilm related peri-implant diseases.

 

4. Clear statements of the novelty of the work should also appear briefly in the Abstract and Conclusions sections.

The statement of novelty has been added to the abstract and conclusions.

 

5. Key findings of the performed study must be included in the revised version.

The key findings have been accentuated in the new manuscript.

 

6. Materials section is not sound enough.

We have improved the Material section in several places.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

 Accept after minor revision (corrections to English language and references format)

Minor editing of English language required