Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

There are many existing implantable biomaterials which can be used for bone tissue repair without any coatings and side effects. The authors have limited their research on the special poly-ether-ether-ketone (PEEK) with some expected results. The overall level of this article in biomaterials is rudimentary lacking of novelty and attraction. 

   

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Ordinary. Please pay attention to the unit, paragraphing, table and chart formats.

Author Response

Comment 1:  The main question addressed by this research is to enhance the poly-ether-ether-ketone (PEEK) surface bioactivity through phosphate and calcium functionalization. It is a common coating technique which can be used for many different biomaterials. 

Answer 1: We appreciate the reviewer’s comments. We would like to clarify that the methodology does not involve a coating process. Instead, the approach is based on the chemical functionalization of the PEEK surface via the incorporation of calcium and phosphate ions into the activated polymer surface. After oxygen plasma treatment, the oxygen-containing groups created were the basis for anchoring phosphate and calcium ions. This leads to the formation of Ca-O-P and Ca-O bonds as observed by the XPS technique, rather than depositing a separate film. In the revised manuscript version, we have clarified this point in the Discussion section to avoid misunderstanding (Discussion Section in the XPS part).

Comment 2:  Compared with other published biological inert materials, similar results are added to the subject area. How long can the bioactivity remain and is there any improvement after the coatings are detached or dissolved?

Answer 2:   We appreciate the reviewer’s question. Although long-term immersion or degradation studies were beyond the scope of this work, the in vitro results already demonstrate an early and sustained bioactive response. The in vitro results after 24h indicated that the functionalized surfaces effectively promote initial cell adhesion. Then, at 96 h, increased cell attachment and proliferation were observed, suggesting that the bioactive surface remains stable and supports extracellular matrix secretion and cell growth.  Therefore, the observed cellular behavior confirmed that the functionalized PEEK maintains sufficient and persistent bioactivity for its intended purpose.

Comment 3:   Long term in vivo experiments should be added with enough negative and positive controls.

Answer 3:  We appreciate the reviewer’s suggestion. We fully agree that long-term in vivo studies with proper positive and negative controls would provide valuable complementary evidence. However, the present work was focused on the physicochemical modification and in vitro bioactivity evaluation of the functionalized PEEK surfaces, aiming to demonstrate their ability to promote cell attachment and proliferation. The results obtained here provide a foundation for future in vivo investigations, which are already being planned as the next stage of this research. It is important to note that in vivo experiments should be preceded by in vitro studies.  

Comment 4: Though the conclusions are consistent with the evidence, they have addressed only a part of the main questions posed. For biomaterials, the clinical results are the most important contributions.

Answer 4: We appreciate the reviewer’s observation. We agreed that clinical results are an essential part of biomaterials research. However, this study is the basis for clinical studies. We have focused on the bioactivity of the material interface, its characterization, and in vitro studies. We believe these results provide essential mechanistic insight and proof of concept supporting the material’s potential for future in vivo and clinical evaluations. It is important to note that in  biomaterials research  there is an order that must be followed: 1.- Synthesis and Characterization of the biomaterial; 2.-  In vitro studies (in cells); 3.-  In vivo studies (small and then big species); 4.-  Clinical studies in humans.  In our case we are proposing a method for surface bioactivation and characterization of the chemical process, which correspond to stage 1  and also we are presenting  in vitro experiments, corresponding to the stage  2.

Comment 5: The references cannot reflect the overall level of the biomaterials. There are many existing implantable biomaterials which can be used for bone tissue repair without any coatings and side effects. Though the biological performance of the PEEK can be improved to certain extent, both of the materials used and coating techniques lack of novelty and attraction.

Answer 5:  Thanks for this valuable comment. Although several naturally bioactive ceramics, such as hydroxyapatite, bioglass, and β-tricalcium phosphate, have demonstrated excellent osteoconductivity without additional surface modifications, their inherent brittleness and limited mechanical performance limit their use in load-bearing implants. In contrast, the polymer Polyether-ether-ketone (PEEK) offers high mechanical strength,  making it attractive for orthopedic and spinal devices. However, its biological inertness remains a key limitation. In this context, the present study provides a simple, coating-free strategy to enhance the bioactivity of PEEK via a mild chemical functionalization route. We have revised the Introduction section to include additional background on current bioceramic bone substitutes and their mechanical limitations.

We have added the following:  "… there also exist intrinsically bioactive materials, such as bioceramics, in which calcium and phosphate are part of their bulk composition. Hydroxyapatite, tricalcium phosphate, and bioactive glasses are widely used because they bond directly to bone and support both osteoconduction and osteogenesis. Nonetheless, several reviews have noted that their main mechanical drawback is that they are brittle, exhibit low fracture toughness, and that their strength decreases further when high porosity is required for bone ingrowth, which limits their application in load-bearing sites [28,29,30]."

Comment 6: The formats of units, tables and figures should be accord to the international unified standards. There are too many errors and typos in the content.

Aswer 6: We thank the reviewer for pointing this out. We have utilized the Coating Journal service to address it. 

We appreciate your good comments and the time you took for reviewing , definitely makes us better.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors are commended for the systematic approach to the experiments and quality data representation. A few suggestions to improve the scientific soundness are proposed.

Minor comments

In each Discussion-sub-section, the authors offer a clear explanation of their observations. However, they are encouraged to propose and reference potential hypotheses and the validation of these observations. For example, starting with the hydrophilic response, the authors merely state that SD is due to heterogeneity of ion distribution. I would suggest they start by addressing how the presence of Ca, Ca-P and pre-treatment affect surface groups on PEEK and the binding, the preferential adsorption in a mix Ca-P solution and so on, result in binding and only at a later stage talk about the distribution. Or similarly, just a lack of mechanistic explanation is absent throughout the text. Here for example, the mechanism by which oxygen plasma induces surface etching, including the role of reactive species and bond scission. Additionally, a brief discussion of plasma penetration depth would clarify why bulk crystallinity remains unaffected, supporting the FT-IR findings. In the XPS analysis, is P-P phosphate treated after plasma or the control? It would be ideal to have the control data explicitly stated as such in the table followed by the modification. The authors should briefly explain the chemical basis for this interaction for example, the formation of calcium phosphate complexes or the affinity of phosphate groups for divalent cations like Ca²⁺. Without this context, the interpretation remains speculative. I would request the authors to carefully go beyond these observational influences described and offer mechanistic clarity. How does the surface modification translate to preferential cellular response? Also, what does metabolically active but not dividing' contexually imply here? Overall, the discussion would benefit from a more rigorous connection between surface chemistry and biological outcomes

 

Author Response

Comments 1:  In each Discussion-sub-section, the authors offer a clear explanation of their observations. However, they are encouraged to propose and reference potential hypotheses and the validation of these observations. For example, starting with the hydrophilic response, the authors merely state that SD is due to heterogeneity of ion distribution. I would suggest they start by addressing how the presence of Ca, Ca-P and pre-treatment affect surface groups on PEEK and the binding, the preferential adsorption in a mix Ca-P solution and so on, result in binding and only at a later stage talk about the distribution. Or similarly, just a lack of mechanistic explanation is absent throughout the text. Here for example, the mechanism by which oxygen plasma induces surface etching, including the role of reactive species and bond scission.

Response 1:  We thank the reviewer for this insightful suggestion. The Discussion section has been revised to include mechanistic hypotheses and supporting references that explain how plasma activation, phosphate and calcium functionalization collectively modify PEEK surface chemistry and hydrophilicity.    This was added to the discussion section:

"Plasma contains ions, electrons, free radicals, and photons generated by ultraviolet radiation. This radiation generates enough energy to break chemical bonds on the surface of the material and then oxygen binds to reactive sites, creating polar groups (–OH, –COOH, –C=O) [36,37]. These polar functionalities increase surface energy and hydrophilicity. Similar mechanisms have been reported by other authors [38,39], who demonstrated that the extent of oxidation and surface activation depends strongly on plasma parameters, including species flux, exposure time, and gas composition."

In the same discussion section, we also added an explanation of the functionalization.

"Building on this activated surface, in the Ca-S sample, calcium interacted mainly with the oxygenated groups. Under the alkaline conditions of the Ca(OH)₂ treatment, these oxygen atoms become partially deprotonated, acquiring negative charges that enable coordination with calcium ions to form stable Ca–O bonds. Similar Ca²⁺–oxygen coordination has been reported for carboxylate and hydroxyl ligands [41]. In our Ca-only sample, this mechanism explains the Ca–O binding observed by XPS, Figure 4b. In the PCa-S sample, the interaction between calcium and phosphate species is strongly influenced by the protonation state of phosphate groups, which depends on the pH of the medium. During the first step with sodium dihydrogen phosphate (NaH₂PO₄, pH≈5), the main phosphate form is H₂PO₄⁻, which interacts with the oxygenated groups generated during plasma activation through hydrogen bonding and electrostatic attraction. When calcium hydroxide is added, the pH rises and the phosphate species partly deprotonate to HPO₄²⁻. Under these alkaline conditions, calcium associates not only with phosphate oxygen, forming CaHPO₄, but also with oxygenated surface sites on PEEK, leading to additional Ca–O bonds observed in the XPS spectra, Figure 4c. These interactions likely involve both electrostatic attraction and coordination bonding. This behavior is consistent with Chen et al. (2022), who reported that deprotonated phosphate groups coordinate Ca²⁺ through charge-transfer bonding under alkaline conditions [42]."

Comments 2:   Additionally, a brief discussion of plasma penetration depth would clarify why bulk crystallinity remains unaffected, supporting the FT-IR findings.

Response 2:   We appreciate the reviewer’s comments regarding the plasma penetration depth. Although this study did not include direct depth profiling, the literature consistently indicates that oxygen plasma modification of PEEK is restricted to the outermost molecular layers.

We have added some explanations in the Discussion Section

"The plasma process used in this study is a low-pressure, non-thermal treatment in which energetic species interact primarily with the outermost molecular layers of the polymer. In this regime, the kinetic energy of the ions and radicals, reported by Versel et al. to be between 6 and 12 eV, is insufficient to penetrate beyond the near-surface region. This behavior is consistent with previous studies reporting that plasma activation modifies the surface chemistry while preserving the bulk properties of PEEK [8,36,40]."

Comments 3:  In the XPS analysis, is P-P phosphate treated after plasma or the control? It would be ideal to have the control data explicitly stated as such in the table followed by the modification.

Response 3: Thanks for the observation. Our mistake, we apologize. The table had the wrong name; P-P should be the Control sample. We corrected it.

Comments 4:  The authors should briefly explain the chemical basis for this interaction for example, the formation of calcium phosphate complexes or the affinity of phosphate groups for divalent cations like Ca²⁺. Without this context, the interpretation remains speculative. I would request the authors to carefully go beyond these observational influences described and offer mechanistic clarity.

Response 4: You are right  , we have already discussed the chemical basis of the interactions in the Discussion Section, as we mentioned above.

Comments 5:   How does the surface modification translate to preferential cellular response? Also, what does metabolically active but not dividing' contexually imply here? Overall, the discussion would benefit from a more rigorous connection between surface chemistry and biological outcomes. 

Response 5:   We appreciate the reviewer’s insightful comment. We have revised the Discussion to better explain how the observed surface modifications correlate with the cellular response.    Regarding the reviewer’s question on the expression “metabolically active but not dividing,” this phrase refers to cells that maintain mitochondrial metabolic activity while showing a reduced proliferation rate. Such behavior indicates that the cells remain metabolically active and viable, although their proliferation appears to be reduced under the evaluated conditions.   Accordingly, we have expanded the discussion to include these mechanistic links between surface chemistry and cell response to provide a clearer biological interpretation of the results.

We appreciate your valuable comments and the time you took for reviewing , definitely makes us better.

 

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article is scientifically interesting, but requires some corrections:

1) Several methods for activating polymer surfaces to deposit ceramic layers in biomedical applications have been described in the literature. For example:

- electrodeposition (https://doi.org/10.1016/j.actbio.2013.08.041),

- laser activation (https://doi.org/10.1016/j.compscitech.2020.108279),

- chemical etching (https://doi.org/10.1021/am100972r).

It would be good to refer to such methods in the introduction. Consequently, to compare the obtained results with those in other works. It should be possible to indicate why the method proposed by the authors is superior and perhaps what it allows for compared to other methods.

2) In my opinion, the biological and surface analyses are very thorough and add significant value to the work. However, I have some reservations about the bulk measurements.

- The authors examined the polymer's crystallinity using only FTIR techniques. What mode were the studies conducted in? ATR?

- Such studies are highly unreliable in quantitatively determining crystallinity (band intensity depends on the porosity/roughness of the material). Secondly, determining a baseline for such a narrow range of wavenumbers is subject to significant error. Thirdly, there are many more precise techniques for studying crystallinity, such as DSC or WAXD.

- On what basis do the authors claim: ‘It can be assumed that the oxygen plasma applied was a  surface treatment that did not affect the bulk properties of the material’? This is not confirmed by the research conducted, and FTIR tests, as I have already mentioned, are insufficient to confirm this. Besides crystallinity, there are many other factors that influence bulk properties: chemical structure, porosity, and macromolecular entanglement... In addition to ATR, it would be beneficial to support such hypotheses with, for example, mechanical or density/porosity studies.

Author Response

Comments 1:  The article is scientifically interesting, but requires some corrections:

1. Several methods for activating polymer surfaces to deposit ceramic layers in biomedical applications have been described in the literature. For example:

- electrodeposition (https://doi.org/10.1016/j.actbio.2013.08.041),

- laser activation (https://doi.org/10.1016/j.compscitech.2020.108279),

- chemical etching (https://doi.org/10.1021/am100972r).

It would be good to refer to such methods in the introduction. Consequently, to compare the obtained results with those in other works. It should be possible to indicate why the method proposed by the authors is superior and perhaps what it allows for compared to other methods.

Response 1:  We sincerely appreciate the reviewer’s constructive suggestion. We have expanded the Introduction to incorporate the recommended references about polymer surface activation techniques (electrodeposition, laser activation, and chemical etching/functionalization).

In addition, we have clarified how our method differs from previously reported methods. Unlike electrodeposition or laser-based coatings that deposit a Ca/P layer, our approach achieves surface functionalization via ionic immobilization after plasma activation. This distinction is relevant because the process does not produce a detachable coating but rather establishes chemical coordination of Ca²⁺ and phosphate groups onto the plasma-activated PEEK surface. Such an interaction is consistent with the mechanism described by Sunarso et al. (2016 and 2019), in which Ca and P ions are co-immobilized on titanium and PEEK surfaces, enhancing bioactivity without forming a bulk ceramic layer. Therefore, our method combines the chemical stability of functionalization with the bioactivity of calcium and phosphate, while avoiding the adhesion issues typically associated with conventional coatings.

To clarify, the following was added to the introduction: 

 “In addition to bioactive coatings, various surface activation methods have been explored to enhance polymer bioactivity and promote calcium phosphate deposition. Electrodeposition enables rapid, controllable mineralization of polymer scaffolds, followed by immersion in simulated body fluid (SBF) to enhance apatite formation [25]. Laser activation has also been employed to induce selective mineralization on PLLA films, facilitated apatite nucleation after subsequent SBF incubation [26]. Furthermore, chemical etching and functionalization have been employed to introduce reactive groups that promote Ca/P nucleation on polymeric scaffolds, leading to the formation of uniform hydroxyapatite-like coatings after immersion in SBF [27].”

[25]       C. He, X. Jin, P.X. Ma, …..https://doi.org/10.1016/j.actbio.2013.08.041.

[26]       K. Szustakiewicz, B. Kryszak, M. Gazińska, J. Chęcmanowski, B. Stępak, M. Grzymajło, A. Antończak,….. https://doi.org/10.1016/j.compscitech.2020.108279.

[27]       K. Rodríguez, S. Renneckar, P. Gatenholm…..https://doi.org/10.1021/am100972r.

 

Comments 2:   In my opinion, the biological and surface analyses are very thorough and add significant value to the work. However, I have some reservations about the bulk measurements.  - The authors examined the polymer's crystallinity using only FTIR techniques. What mode were the studies conducted in? ATR?

Response 2.  Thank you for your comment. The FTIR analyses were conducted in ATR mode (FTIR-ATR). This information has been added and clarified in the Methods section of the revised manuscript.

Comments 3.  Such studies are highly unreliable in quantitatively determining crystallinity (band intensity depends on the porosity/roughness of the material). Secondly, determining a baseline for such a narrow range of wavenumbers is subject to significant error. Thirdly, there are many more precise techniques for studying crystallinity, such as DSC or WAXD.

Response 3.  We appreciate the reviewer’s valuable observation. We fully agree that FTIR is a semi-quantitative technique and that band intensity can be influenced by surface roughness or porosity. In our case, the FTIR-ATR analysis was used only to compare the relative surface crystallinity between the control and plasma-treated samples, not as an absolute quantitative method. The evaluation was performed according to ASTM F277-09 and the commonly reported ratio between the 1305 and 1280 cm⁻¹ bands, which has been widely applied in the literature for PEEK surfaces. The full FTIR spectra for the samples are provided in the Raw Data to support transparency and reproducibility of the analysis.

As the plasma treatment employed here is a low-pressure, non-thermal process that modifies only the outermost molecular layers, more bulk-sensitive techniques such as DSC or WAXD were not expected to reveal significant differences. This behavior is consistent with previous reports showing that oxygen plasma induces chemical modification confined to the surface without affecting the bulk crystallinity of PEEK. We have clarified this rationale and the methodological limitation in the revised Discussion section as follows:

“Although this technique is not a fully quantitative method for determining crystallinity, it offers a comparative indication of surface structural changes. The plasma process used in this study is a low-pressure, non-thermal treatment in which energetic species interact primarily with the outermost molecular layers of the polymer. In this regime, the kinetic energy of the ions and radicals, reported by Versel et al. to be between 6 and 12 eV, is insufficient to penetrate beyond the near-surface region. This behavior is consistent with previous studies reporting that plasma activation modifies the surface chemistry while preserving the bulk properties of PEEK [8,36,40]”

Comments 4:  On what basis do the authors claim: ‘It can be assumed that the oxygen plasma applied was a  surface treatment that did not affect the bulk properties of the material’? This is not confirmed by the research conducted, and FTIR tests, as I have already mentioned, are insufficient to confirm this. Besides crystallinity, there are many other factors that influence bulk properties: chemical structure, porosity, and macromolecular entanglement... In addition to ATR, it would be beneficial to support such hypotheses with, for example, mechanical or density/porosity studies.

Response 4. We appreciate the reviewer’s insightful comment. We agree that FTIR-ATR alone is not sufficient to confirm that the bulk properties remain unaffected. To address this concern, we have removed the sentence claiming this assumption from the Discussion.  Instead, we now clarify that the conclusion is based on the nature of the plasma process itself, which is a low-pressure, non-thermal treatment whose energetic species (6–12 eV, according to Versel et al.) primarily interact with the outermost molecular layers of the polymer. This interpretation is supported by previous studies reporting that oxygen plasma induces chemical modifications confined to the surface while preserving the bulk structure of PEEK. The revised text reflects this clarification and avoids overstatement.

This was added to the discussion:

 “The FTIR-ATR results indicated a crystallinity index value of around 1.00 for the Control and PL-S sample, respectively, with a percentage of Crystallinity of 17.88% and 18.03%, suggesting that the oxygen plasma process caused only minor structural changes at the surface level [8]. Although this technique is not a fully quantitative method for determining crystallinity, it offers a comparative indication of surface structural changes. The plasma process used in this study is a low-pressure, non-thermal treatment in which energetic species interact primarily with the outermost molecular layers of the polymer. In this regime, the kinetic energy of the ions and radicals, reported by Versel et al. to be between 6 and 12 eV, is insufficient to penetrate beyond the near-surface region. This behavior is consistent with previous studies reporting that plasma activation modifies the surface chemistry while preserving the bulk properties of PEEK [8,36,40].”

We appreciate your valuable comments and the time you took for reviewing , definitely makes us better.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The quality of the manuscript has been improved.

Comments on the Quality of English Language

The table format is still wrong.

Reviewer 3 Report

Comments and Suggestions for Authors

In my opinion, the authors did a great job responding to comments. I am satisfied and believe the article in the present form is suitable for publication.