Purified Clinoptilolite-Tuff as a Trap for Amines Associated with Chronic Wounds: Binding of Cadaverine, Putrescine, Histamines and Polyamines
, Stephane Nizet 2, Cornelius Tschegg 2 and Michael Freissmuth 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript presents an innovative approach to managing malodorous discharges in chronic wounds using clinoptilolite-tuff, addressing a critical gap in wound care treatment. However, major revisions are necessary to enhance the scientific rigor, clarity, and reproducibility of the study. The authors should be encouraged to carefully address the following points to improve the overall quality and impact of the manuscript:
- The abstract effectively summarizes the study but could briefly mention the limitations to provide a balanced perspective.
- The introduction provides sufficient context but would benefit from elaboration on alternative existing treatments for malodorous wounds and their limitations to better justify the significance of the proposed approach.
- While the methodology is detailed, the inclusion of visual or schematic diagrams of experimental setups could significantly improve comprehension.
- The criteria for selecting the concentration ranges of radioligands in the experiments should be clarified.
- The section discussing competitive displacement experiments (Figures 3 and 4) should explicitly compare results to highlight differences in binding affinities among the tested compounds.
- The discussion is thorough but could be strengthened by comparing the findings with other studies involving clinoptilolite or similar materials in wound care.
- Further elaboration on the potential mechanisms underlying the binding preferences for diamines and polyamines would provide deeper insights into the observed results.
- The scalability and cost-effectiveness of integrating clinoptilolite into wound dressings should be highlighted to underline the translational potential of this approach.
- Abbreviations (e.g., PCT for purified clinoptilolite-tuff) should be defined upon their first use, and units and terms should be standardized throughout the manuscript to maintain consistency.
- The conclusion could be expanded to explicitly state the next steps or future research directions, such as testing on different wound types or conducting larger-scale clinical trials.
Author Response
Reviewer 1:
The manuscript presents an innovative approach to managing malodorous discharges in chronic wounds using clinoptilolite-tuff, addressing a critical gap in wound care treatment. However, major revisions are necessary to enhance the scientific rigor, clarity, and reproducibility of the study. The authors should be encouraged to carefully address the following points to improve the overall quality and impact of the manuscript:
1) The abstract effectively summarizes the study but could briefly mention the limitations to provide a balanced perspective.
Reply:
The abstract is currently comprised of 211 words. Hence, its length is actually beyond the limit of the allowed word count. Accordingly, we did not include an additional sentence. However, we added a paragraph to the Discussion, which points out the major limitations of our study, which are difficult to address in a comprehensive way, namely (i) the heterogeneity of wound fluids, (ii) the impact of variation in pH (and in ionic strength) on the adsorptive capacity, (iii) the possibility that adsorption of wound fluid constituents by PCT may have detrimental effects on wound healing. The pertinent paragraph (on p. 13/p. 14, lines 426-454) reads:
“Our study has several limitations: (i) Wound fluids are heterogenous. We did not have access to exudate from fumigating wounds, but used wound exudates collected from patients, who had undergone breast cancer surgery. We cannot formally rule out that constituents of fungating wound exudates interferes with binding of cadaverine and putrescine. However, it is worth pointing out that the binding capacity of PCT is large. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is evident from the large discrepancy, which we observed in the binding affinity determined in saturation experiments and the IC50 estimated from the competition experiments. Under the realistic conditions where 10 ml of fungating wound exudate contain 1 nmol to 10 nmol of cadaverine (that is 100 µM to 1 mM) [16], 1 mg of PCT is predicted to readily suffice for sorbing the cadaverine molecules and a large fraction of binding sites remain unoccupied and thus available to bind additional compounds. In the current prototype, 1.3 cm2 of PCT-doped wound dressing provides 1 mg of PCT and thus suffices to so adsorb a substantially larger amount than 10 nmol of cadaverine (or putrescine), but a wound of this size is unlikely to be covered with 10 ml of fluid. (ii) We also did not test the effect of varying pH and ionic strength on the adsorptive capacity. We consider ionic strength of modest interest, because wound exudates have the same ionic composition as plasma [45]. There is a large variation in the pH of wound exudates [46]. Bacterially contamination results in wound exudate, which are alkaline [47,48]. The wound exudates, which were collected used were alkaline (average pH = 8.0). This did not affect the ability of PCT to bind cadaverine (cf. Figures 5 & 6). This was to be expected, because the pKa of the amine groups of cadaverine is 9.1 and 10.2. Thus, variation in pH only have a modest effect on the ionization of cadaverine. (iii) We cannot rule out that removal of solutes, which are present in wound fluids, may have a negative impact on wound healing. However, in cell culture, PCT did not affect the viability and the ohmic resistance of an epithelial cell layer [35]. Importantly, in a phase I trial with artificial wounds, the current preparation of micronized purified PCT did not affect wound closure [26].“
2) The introduction provides sufficient context but would benefit from elaboration on alternative existing treatments for malodorous wounds and their limitations to better justify the significance of the proposed approach.
Reply:
Two alternative approaches were mentioned on p. 2, lines 56-62, namely wound dressings containing charcoal and aldehyde-functionalized cellulose. We expanded this section by referring to a review (Akhmetova et al., new ref. 18), which systematically examines the other options: these are based on silver particles, iodine-release, hyperosmotic sugar solutions, (manuka) honey and topically applied antibacterial agents (e.g. metronidazole). The pertinent sentences (on p. 2, lines 62 - 71) read as follows:
“Earlier approaches to reduce malodor aimed at reducing bacterial growth by incorporating silver, iodine, metronidazole and plant-derived oils (from eucalyptus, thyme, lavender etc.) into the wound dressing [18]. Similarly, bacterial growth may be reduced by applying hyperosmolar sucrose or manuka honey. However, the effectiveness of these approaches is considered modest in the management of malignant fungating wounds: in a systematic survey, charcoal-based dressings and metronidazole-containing gels were rated as being highly effective in about 50% of the cases. Silver- and iodine-based containg dressings were considered to be highly effective in only 23 and 17% of the cases, respectively; the ratings for all other approaches were even lower [14]. Thus, there is a large unmet medical need [14,18].”
3) While the methodology is detailed, the inclusion of visual or schematic diagrams of experimental setups could significantly improve comprehension.
Reply:
We now include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
4) The criteria for selecting the concentration ranges of radioligands in the experiments should be clarified.
Reply:
We added a statement on preliminary experiments, which had been carried out to identify the range, where competition occurred. The pertinent sentences on p.16, lines 553-558 reads:
“Preliminary experiments (with total concentrations increasing by one order magnitude from 1 nM to 10 mM) were carried out to identify the concentration range, where the compounds competed effectively with the radiolabeled tracers. Because total concentrations ≤ 0.1 mM failed to displace the bound radiotracers, logarithmically concentrations covering the range of 0.1 to 10 mM were subsequently to determine the inhibitory potency of competitors.”
5) The section discussing competitive displacement experiments (Figures 3 and 4) should explicitly compare results to highlight differences in binding affinities among the tested compounds.
Reply:
We expanded the section on p. 11/p.12, to highlight the differences in IC50-values. In response to the last point (“Figures and Tables”) raised by reviewer 3, we also created a graphic representation of these differences. The pertinent text (on p. 11/p. 12, lines 343-362) reads as follows:
“The difference in binding site density is illustrated in Figure 8A: it is evident that the [3H]histamine binding capacity (43 nmol/mg) was lower than the binding capacity for [3H]spermidine (sum of Bmax,1 and Bmax,2 = 153 nmol/mg) and for [3H]cadaverine (sum of Bmax,1 and Bmax,2 = 273 nmol/mg). (ii) [3H]Histamine was displaced by all compounds tested. In contrast, unlabeled histamine was ineffective in displacing [3H]spermidine and only poorly displaced [3H]cadaverine. (iii) Similarly, the structure-activity relation of diamines and polyamines differed substantially in their ability to displace [3H]spermidine and [3H]cadaverine: the difference in inhibitory potency can be illustrated by calculating the pairwise ratio of IC50 for all compounds other than histamine (for which IC50-values in displacing [3H]spermidine and [3H]cadaverine could not be determined). As shown in Figure 8B, unlabeled cadaverine was 11.3- and 5.2-fold more potent in self-competition than in displacing [3H]spermidine (red bar indicating S/C-ratio in Figure 8B). Unlabeled cadaverine was also 5.2-fold more potent in competing with [3H]histamine than with [3H]spermidine (green bar indicating S/H-ratio in Figure 8B) and two-fold more potent in self-competition than in displacing [3H]histamine (blue bar indicating S/H-ratio in Figure 8B). Similarly, large differences in potency ratios were observed with putrescine, which was e.g. 3.2-fold in competing for binding of [3H]histamine than for that of [3H]spermidine (green bar indicating S/H-ratio in Figure 8B). In contrast, for both, spermidine and spermine, the IC50-ratios were less pronounced, i.e ranging from 1.7 to 0.6-fold (cf. also Table 1) and thus not substantially different from unity (indicated by the black line in Figure 8B).“
6) The discussion is thorough but could be strengthened by comparing the findings with other studies involving clinoptilolite or similar materials in wound care.
Reply:
The binding of cadaverine to charcoal-coated and aldehyde-functionalized cellulose was determined in ref. 17. As suggested by the reviewer, we compared the data provided in this reference with our binding data and inserted a paragraph in the discussion (on p. 13, lines 396-404) summarizing this comparison, which reads as follows:
“The adsorption kinetics of cadaverine to charcoal-coated or aldehyde-functionalized cellulose and their combination were recently determined [17]: the binding equilibrium was reached after ≥4 hours. Consistent with these slow binding kinetics, the affinity, which can be estimated from the available data, is in the range of 32 mM, >100 mM and 65 mM for charcoal-coated cellulose, for aldehyde functionalized cellulose and their combination, respectively [17]. Thus, the indirect comparison of the data of Wen et al. [17] and of our observations indicates that clinoptilolite depletes cadaverine from the solution substantially more rapidly and with higher affinity than charcoal-coated cellulose, aldehyde-functionalized cellulose or a combination thereof. “
7) Further elaboration on the potential mechanisms underlying the binding preferences for diamines and polyamines would provide deeper insights into the observed results.
Reply:
We agree that it is desirable to understand the structural basis for differences in binding preferences and for binding site heterogeneity.
We added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
In our opinion, the required experiments are beyond the scope of the current work and certainly cannot be accomplished within the time limit imposed (of ten days, extended to 20 days due to the holiday season) by the editorial recommendation.
8) The scalability and cost-effectiveness of integrating clinoptilolite into wound dressings should be highlighted to underline the translational potential of this approach.
Reply:
We addressed this point by adding the following sentences (on p. 14, lines 466-475):
“The scalability of incorporating PCT into wound dressings is promising. It can be integrated to wound care materials using standard production methods, such as mixing, impregnation or coating of fibers or the finished dressing. This allows production of large quantities of functional wound dressings at a large scale and with consistent quality.
PCT is manufactured from a natural clinoptilolite-bearing high-grade raw material, which can be sourced sustainably and cost-effectively. PCT, derived through a quality-controlled refinement process, has undergone clinical evaluation in a phase I trial [26] and imparts enhanced properties to wound dressings. These properties expand their functionality, making them applicable in a broader range of advanced wound care solutions, including the adsorption of toxins, pathogens, inflammatory factors, and excess fluids.”
9) Abbreviations (e.g., PCT for purified clinoptilolite-tuff) should be defined upon their first use, and units and terms should be standardized throughout the manuscript to maintain consistency.
Reply:
The abbreviation PCT is explained upon its first use (in the abstract) and on p. 2, line 88. We scrutinized the manuscript and did not find any other non-standard abbreviation. We assume that S.D., cpm (counts per minute) and Ci (Curie) are standard abbreviations, which need not be explained. In the legend to Fig. 2, the radionuclide tritium was not indicated by a superscript: this was corrected – throughout the manuscript, tritiated compounds are now indicated as “[3H]”.
10) The conclusion could be expanded to explicitly state the next steps or future research directions, such as testing on different wound types or conducting larger-scale clinical trials.
Reply:
This point was addressed by adding the following sentences (on. p. 14, lines 475-478):
“Future research will investigate the potential of PCT-doped alginate dressings to bind and inactivate bacterial and viral pathogens. In addition, the focus will be on clinical evaluation in both chronic and acute wound settings.”
Reviewer 2:
1) Wound fluids are complex mixtures containing proteins, lipids, and other charged molecules. How does PCT selectively adsorb these target molecules without significant nonspecific binding?
Reply:
(i) Experiments were done with seven different wound fluids, which differed substantially in composition. However, binding of cadaverine was comparable in these different wound exudates. (ii) We do not intend to differentiate between specific and non-specific binding. From the perspective of wound management, the only aspect that is relevant is removal of potentially detrimental compounds. (iii) The binding capacity is large; accordingly, binding sites are in excess of potential ligands.
We addressed these points in the paragraph (on p. 13/p. 14, lines 426-454), which describes the limitations of our study (see reply to point 1 of reviewer1).
2) While the binding assays demonstrate efficacy in vitro, the clinical setting involves dynamic conditions such as changes in pH, fluid flow, and microbial activity. How do these factors influence the binding efficiency of PCT?
Reply:
See reply to point 1 (and point 1 of reviewer 2) – paragraph on p. 13/p. 14, lines 426-454.
3) PCT’s binding of histamine and spermidine could disrupt their physiological roles in chronic wounds. Have potential negative consequences of prolonged depletion of these molecules been considered, especially in patients with comorbidities?
Reply:
This was already addressed in detail in the Discussion of the previous version (the pertinent paragraph is now on p. 13, lines 405 - 425).
4) The binding heterogeneity of PCT is attributed to non-equivalent sites, but this is presented as circumstantial evidence. Without molecular or structural validation, how can the authors definitively conclude this heterogeneity?
Reply:
We agree that we only have circumstantial evidence for binding site heretogeneity (and this was explicitly stated in the text). In response to the reviewer’s point, we (i) down-toned the wording by replacing “conclusion” with “conjecture” (p. 11, lines 339-340) reads
“Three arguments support the conjecture that the binding sites are heterogenous and non-equivalent.”).
(ii) In addition, we added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
5) The study uses drainage fluid from breast cancer surgeries, which may not represent the composition of exudates from other chronic wounds, such as diabetic foot ulcers or venous ulcers. How generalizable are these findings?
Reply:
We explicitly mention this in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
6) The manuscript asserts that PCT retains binding capacity even in the presence of wound fluid. However, the displacement of binding by other molecules in the fluid, particularly proteins and ions, was not fully investigated. Could these competing interactions diminish PCT’s real-world efficacy?
Reply:
We address this issue in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
7) The manuscript highlights rapid binding in vitro but does not address whether this is maintained over the typical usage period of wound dressings (e.g., 24-48 hours). Could saturation or leaching of adsorbed molecules occur, reducing efficacy?
Reply:
We should like to point out that we carried out comparative binding assays at 2h and 24 h: binding efficacy was maintained at 24 h. The pertinent data are shown in Figure 5 and Figure 6.
Reviewer 3:
This research investigates the potential of micronized purified clinoptilolite-tuff (PCT) as an effective adsorbent for malodor-causing amines (cadaverine, putrescine, histamine, and polyamines) associated with chronic wounds. The study demonstrates rapid, stable, and high-capacity binding of these amines, both in controlled experiments and in the presence of wound drainage fluids, supporting the integration of PCT into wound dressings for malodor control. The topic is significantly important considering emerging applications of new materials in chronic wound medicine and clinics, however, there are major concerns which needs to be addressed before a decision can be made. My comments are given below-
Introduction and Background- The background section lacks a clear and detailed explanation of the unique physicochemical properties of purified clinoptilolite-tuff (PCT) that make it suitable for amine binding. Adding information on its microporous structure and prior applications in adsorption would provide a stronger foundation for the study. The role of specific amines (cadaverine, putrescine, histamine, etc.) in contributing to wound malodor and their biological significance could be expanded. This would help contextualize why their adsorption is crucial for chronic wound management.
Reply:
(i) We added a paragraph to the introduction (on p. 2, lines 73-88), which reads as follows:
“Clinoptilolite, is a heulandite-type zeolite, a hydrated aluminosilicate characterized by a unique framework of interconnected SiO4 and AlO4 tetrahedra. The negatively charged crystal framework, resulting from the isomorphous substitution of Si4+ by Al3+, enables the mineral to absorb and exchange mono- and divalent cations within the crystal lattice [19], and to adsorb larger ions and/or molecules onto the surface [20-22]. Nizet et al. [23] and Sarabi et al. [24] recently also demonstrated that virions of various species were adsorbed to and effectively neutralized by clinopitolite. Here, we explored the hypothesis that clinoptilolite bound diamines and polyamines with a capacity, which sufficed to effectively deplete these compounds in wound exudates. The microporous structure of clinoptilolite provides a large surface area and numerous adsorption sites, its framework harbors an overall negative net charge. Hence, it is reasonable to posit that clinoptilolite avidly interacts with the amine groups of polyamines, which – based on their pKa in the range of 9.1 to 10.8 are almost completely ionized at physiological pH [25]. We examined this hypothesis by using a preparation of micronized purified clinoptilolite-tuff (PCT), which has been shown to be well tolerated in a phase I trial with artificial wounds [26].”
(ii) The role of cadaverine and of putrescine in wound malodor (and in periwound dermatitis) was stated on p. 2, lines 48 to 56. The higher abundance of cadaverine (than that of putrescine) was stated on p. 6, lines 195 to 197. In our opinion, this suffices to contextualize the importance of adsorbing cadaverine and putrescine and to provide a rationale for the experiments.
Study Objectives- While the aim of the study is clear, it would be beneficial to explicitly define the hypothesis being tested and the novelty of using PCT for binding amines in chronic wound settings. This would distinguish the research from prior studies on odor control in medical applications.
Reply:
The working hypothesis is now explicitly stated on p. 2, lines 79-88 (see also above, reply to the first point)
Methods - Radiolabeled Tracer Experiments- The methodology for radiolabeling tracers and quantifying binding is not described in sufficient detail. A brief description of the techniques, including validation and potential limitations, would improve reproducibility and transparency. The rationale for selecting the specific tracers ([3H]cadaverine, [3H]histamine, and [3H]spermidine) should be provided. Were these selected based on their relative abundance or significance in wound malodor?
Reply:
(i) We include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
(ii) Reviewer 1 found our description of the methodology to be detailed (see point 3 of reviewer 1). The reproducibility is evident from the error bars; our data presentation is also transparent, because we show data points from individual experiments in the saturation experiments (i.e. Figure 2C and D, Figures 5 and 6, Figure 7) and because we show the original signal – i.e. the radioactivity, which was measured in cpm (in Figure 3A and 3B, Figure 4D) – or the original signal is readily accessible from the specific activity of the radioligand, which explicitly stated in the other figures.
(iii) The rationale for selecting histamine and spermidine was stated on p. 2/p. 3, lines 94 – 97. The rationale for selecting cadaverine was stated on p. 6, lines 195 – 197. We do not see why this explanation does not suffice.
Binding Affinity Studies- Clarify whether the binding affinities in the low µM range are competitive with other known adsorbents. A comparative analysis or reference to standard materials for amine adsorption would contextualize the performance of PCT. Include details on how binding site heterogeneity was assessed during the displacement experiments. Was this determined through binding kinetics or equilibrium modeling?
Reply:
(i) The binding capacity is large; accordingly, binding sites are in excess of potential ligands. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is explained on p. 13/ p. 14 (lines 426-454; see also reply to point 1 of reviewer 1).
(ii) We are not aware of any “standard materials” for amine absorption, which can be used as a reference. All dressings, which have been tested in fungating wounds, were found to be not poor in odor control, see refs. 14 and 18. The poor performance of these materials is stated in the introduction. The comparison of our results to those published in ref. 17 is described on p. 13, lines 396-404 (see reply to point 6 of reviewer 1).
(ii) Binding site heterogeneity was assessed under equilibrium binding conditions. This is now explicitly stated on p. 4, lines 115-116 and p. 5, lines 153-154.
Wound Drainage Fluid Experiments- The study mentions the use of wound drainage fluids, but details about their source, preparation, and composition are absent. Specify whether they were collected from clinical samples and if so, ethical considerations should be discussed. Discuss the potential variability in binding efficiency due to differences in drainage fluid composition (e.g., pH, ionic strength, or presence of proteins) and how this variability was accounted for.
Reply:
(i) We do not understand the first part of the comment: The source of the wound drainage fluid was mentioned under subheading 4.2. in the Materials and Methods section. The pertinent section also (on p. 15, lines 501 - 504) mentioned the approval by the University Ethics Committee (including the submission number 2132/2013) and explicitly stated that the patients gave their written consent.
(ii) The variability of the drainage fluid was explicitly mentioned on p. 9, lines 263 – 266. Figures 5 and 6 show that this variability did not affect the ability of PCT to bind cadaverine.
(iii) pH, ionic strength and other constituents are discussed in the paragraph on the limitations of the study (on p. 13/p. 14, lines 426-454 – see reply to comment 1 of reviewer 1)
Incorporation into Wound Dressings- The integration of PCT into wound dressings is briefly mentioned but lacks technical details. Include a description of the preparation process, material compatibility, and any modifications made to ensure sustained adsorption. Evaluate the potential mechanical or functional impact of PCT incorporation on the wound dressing. Does it affect absorbency, flexibility, or ease of use?
Reply:
We added (on p. 14/p. 15, lines 493-499) the following sentences under the subheading Materials:
“PCT in particulate form was mixed into the alginate solution before extrusion, leading to a homogeneous distribution of PCT within the resulting calcium-alginate fibres. In the subsequent production steps, the PCT-doped alginate fibre was combined with cellulose-type fibres, enhancing the mechanical strength as well as the absorbency characteristics of the final dressing. The integration of PCT does not adversely affect the ease of use, flexibility, weight, strength or integrity of the dressing.”
Binding Capacity and Competitive Adsorption- The observation that the wound dressing's binding capacity matches the PCT content is intriguing but requires quantitative backing. Include a table or figure summarizing these findings. Explain how competitive adsorption among different amines was quantitatively assessed and how this impacts the real-world efficacy of the dressing in clinical scenarios.
Reply:
We do not understand the comment: Figure 7 shows a comparative quantification of the binding of cadaverine binding over the relevant concentration range.
Discussion - Binding Mechanism- The discussion of non-equivalent binding sites is underexplored. Elaborate on the chemical or structural basis for this observation. For instance, are specific functional groups on the PCT surface responsible? Consider addressing whether binding is reversible or if the adsorption is influenced by environmental factors like pH changes in chronic wounds.
Reply:
(i) We expanded the discussion on non-equivalent sites on p. 11/p. 12, lines 343-362 (see reply to point 5 of reviewer 1).
(ii) The effect of pH is now explicitly discussed in the paragraph on the limitations of the study (p. 14, lines 442 – 450).
Clarity and Language- The abstract and main text occasionally use non-technical language (e.g., “carrion-like smell”, “justified to posit”). Opt for precise scientific terms to maintain professionalism and consistency. Ensure the text is free from redundant phrases, such as the repetitive mention of "binding was rapid, stable, and of high capacity."
Reply:
“Carrion-like smell” was replaced by “carrion odor”; I do not see anything inappropriate or non-scientific in using the wording “justify to posit”. The same is true about the statement "binding was rapid, stable, and of high capacity" regardless of whether it used once or an several occasions. I should like to point out that, over the past 40+ years, I have written more than 200 original articles and more than 40 reviews, commentaries and book chapters. Thus, I need not be lectured on how to write a scientific text.
Broader Implications and Comparisons- The paper does not discuss alternative malodor control strategies or how PCT compares in terms of efficacy, cost, and safety. Adding this comparison would strengthen the argument for its clinical adoption. The potential for PCT to adsorb other harmful wound-related compounds (e.g., toxins or inflammatory mediators) could be hypothesized, suggesting broader applications.
Reply:
(i) Alternative measures to control malodor include – as mentioned in the previous version of the manuscript – charcoal and functionalized cellulose. The low efficacy of charcoal was pointed out. Safety issues may arise from aldehyde-functionalized cellulose. These were also mentioned. We expanded this section in the introduction (on p. 2, lines 62 - 71 – see reply to point 2 of reviewer 1).
(ii) The binding of toxins was mentioned on p. 11, line 315, that of viruses on p. 11, line 316. The introduction now also mentions the neutralization of viruses by binding to clinoptilolite (p. 2, lines 77 - 79).
Limitations and Challenges- Address potential challenges in scaling up PCT production and ensuring consistency in its properties for clinical use. Discuss any known or potential adverse effects of PCT on wound healing or its interaction with biological tissues.
Reply:
(i) For scalability, see reply to point 8 of reviewer 1 and text on p. 14, lines 466-475.
(ii) Potential adverse effects are discussed in the paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
Future Directions- The conclusion should propose specific avenues for future research, such as testing PCT in vivo in animal models or controlled clinical trials to evaluate efficacy and safety. Investigate the possibility of functionalizing PCT to enhance selectivity or binding efficiency for specific amines.
Reply:
See reply to point 10 of reviewer 1 and text on p. 14, lines 475-478.
References and Citations- Cite previous studies that have explored the use of zeolites or similar materials in medical or environmental applications to provide a broader context. Ensure all cited studies are up to date and relevant to the topic, especially in the discussion of clinical applications. For example, cite https://dx.doi.org/10.4081/vl.2018.7196 with the sentence ´…the initial inflammatory response is dependent on the recruitment of leucocytes (macrophages, neutrophils, mast cells) and the exudation of blood constituents´, a seminal work on the topic.
Reply:
(i) We provided numerous citations for the potential application of clinoptilolite, e.g. refs. 20-24; 26, 34-36, and refer to reviews for its current use (refs. 30 & 31)
(ii) The reference Singh et al. is now cited as ref. 38 on p. 13, line 408.
Figures and Tables- Consider including graphical representations of the adsorption kinetics and binding affinity results. Visual data can significantly enhance reader comprehension. A schematic diagram of the experimental setup for tracer binding or wound drainage fluid experiments would clarify the methodology.
Reply:
(i) We now include a scheme to explain the binding assay (on p. 15).
(ii) We also included a graphical representation of the binding results as Figure 8 with panel A showing the binding capacity calculated from the saturation experiments and panel B showing the pairwise comparison of potency ratios, that is the IC50 ratios for cadaverine, putrescine, spermidine and putrescine (see also reply to point 5 of reviewer 1 and description on p. 11/p. 12, lines 343-362).
Reviewer 2 Report
Comments and Suggestions for AuthorsWound fluids are complex mixtures containing proteins, lipids, and other charged molecules. How does PCT selectively adsorb these target molecules without significant nonspecific binding?
While the binding assays demonstrate efficacy in vitro, the clinical setting involves dynamic conditions such as changes in pH, fluid flow, and microbial activity. How do these factors influence the binding efficiency of PCT?
PCT’s binding of histamine and spermidine could disrupt their physiological roles in chronic wounds. Have potential negative consequences of prolonged depletion of these molecules been considered, especially in patients with comorbidities?
The binding heterogeneity of PCT is attributed to non-equivalent sites, but this is presented as circumstantial evidence. Without molecular or structural validation, how can the authors definitively conclude this heterogeneity?
The study uses drainage fluid from breast cancer surgeries, which may not represent the composition of exudates from other chronic wounds, such as diabetic foot ulcers or venous ulcers. How generalizable are these findings?
The manuscript asserts that PCT retains binding capacity even in the presence of wound fluid. However, the displacement of binding by other molecules in the fluid, particularly proteins and ions, was not fully investigated. Could these competing interactions diminish PCT’s real-world efficacy?
The manuscript highlights rapid binding in vitro but does not address whether this is maintained over the typical usage period of wound dressings (e.g., 24-48 hours). Could saturation or leaching of adsorbed molecules occur, reducing efficacy?
Comments on the Quality of English Languagetypo-grammatical check
Author Response
Reviewer 1:
The manuscript presents an innovative approach to managing malodorous discharges in chronic wounds using clinoptilolite-tuff, addressing a critical gap in wound care treatment. However, major revisions are necessary to enhance the scientific rigor, clarity, and reproducibility of the study. The authors should be encouraged to carefully address the following points to improve the overall quality and impact of the manuscript:
1) The abstract effectively summarizes the study but could briefly mention the limitations to provide a balanced perspective.
Reply:
The abstract is currently comprised of 211 words. Hence, its length is actually beyond the limit of the allowed word count. Accordingly, we did not include an additional sentence. However, we added a paragraph to the Discussion, which points out the major limitations of our study, which are difficult to address in a comprehensive way, namely (i) the heterogeneity of wound fluids, (ii) the impact of variation in pH (and in ionic strength) on the adsorptive capacity, (iii) the possibility that adsorption of wound fluid constituents by PCT may have detrimental effects on wound healing. The pertinent paragraph (on p. 13/p. 14, lines 426-454) reads:
“Our study has several limitations: (i) Wound fluids are heterogenous. We did not have access to exudate from fumigating wounds, but used wound exudates collected from patients, who had undergone breast cancer surgery. We cannot formally rule out that constituents of fungating wound exudates interferes with binding of cadaverine and putrescine. However, it is worth pointing out that the binding capacity of PCT is large. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is evident from the large discrepancy, which we observed in the binding affinity determined in saturation experiments and the IC50 estimated from the competition experiments. Under the realistic conditions where 10 ml of fungating wound exudate contain 1 nmol to 10 nmol of cadaverine (that is 100 µM to 1 mM) [16], 1 mg of PCT is predicted to readily suffice for sorbing the cadaverine molecules and a large fraction of binding sites remain unoccupied and thus available to bind additional compounds. In the current prototype, 1.3 cm2 of PCT-doped wound dressing provides 1 mg of PCT and thus suffices to so adsorb a substantially larger amount than 10 nmol of cadaverine (or putrescine), but a wound of this size is unlikely to be covered with 10 ml of fluid. (ii) We also did not test the effect of varying pH and ionic strength on the adsorptive capacity. We consider ionic strength of modest interest, because wound exudates have the same ionic composition as plasma [45]. There is a large variation in the pH of wound exudates [46]. Bacterially contamination results in wound exudate, which are alkaline [47,48]. The wound exudates, which were collected used were alkaline (average pH = 8.0). This did not affect the ability of PCT to bind cadaverine (cf. Figures 5 & 6). This was to be expected, because the pKa of the amine groups of cadaverine is 9.1 and 10.2. Thus, variation in pH only have a modest effect on the ionization of cadaverine. (iii) We cannot rule out that removal of solutes, which are present in wound fluids, may have a negative impact on wound healing. However, in cell culture, PCT did not affect the viability and the ohmic resistance of an epithelial cell layer [35]. Importantly, in a phase I trial with artificial wounds, the current preparation of micronized purified PCT did not affect wound closure [26].“
2) The introduction provides sufficient context but would benefit from elaboration on alternative existing treatments for malodorous wounds and their limitations to better justify the significance of the proposed approach.
Reply:
Two alternative approaches were mentioned on p. 2, lines 56-62, namely wound dressings containing charcoal and aldehyde-functionalized cellulose. We expanded this section by referring to a review (Akhmetova et al., new ref. 18), which systematically examines the other options: these are based on silver particles, iodine-release, hyperosmotic sugar solutions, (manuka) honey and topically applied antibacterial agents (e.g. metronidazole). The pertinent sentences (on p. 2, lines 62 - 71) read as follows:
“Earlier approaches to reduce malodor aimed at reducing bacterial growth by incorporating silver, iodine, metronidazole and plant-derived oils (from eucalyptus, thyme, lavender etc.) into the wound dressing [18]. Similarly, bacterial growth may be reduced by applying hyperosmolar sucrose or manuka honey. However, the effectiveness of these approaches is considered modest in the management of malignant fungating wounds: in a systematic survey, charcoal-based dressings and metronidazole-containing gels were rated as being highly effective in about 50% of the cases. Silver- and iodine-based containg dressings were considered to be highly effective in only 23 and 17% of the cases, respectively; the ratings for all other approaches were even lower [14]. Thus, there is a large unmet medical need [14,18].”
3) While the methodology is detailed, the inclusion of visual or schematic diagrams of experimental setups could significantly improve comprehension.
Reply:
We now include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
4) The criteria for selecting the concentration ranges of radioligands in the experiments should be clarified.
Reply:
We added a statement on preliminary experiments, which had been carried out to identify the range, where competition occurred. The pertinent sentences on p.16, lines 553-558 reads:
“Preliminary experiments (with total concentrations increasing by one order magnitude from 1 nM to 10 mM) were carried out to identify the concentration range, where the compounds competed effectively with the radiolabeled tracers. Because total concentrations ≤ 0.1 mM failed to displace the bound radiotracers, logarithmically concentrations covering the range of 0.1 to 10 mM were subsequently to determine the inhibitory potency of competitors.”
5) The section discussing competitive displacement experiments (Figures 3 and 4) should explicitly compare results to highlight differences in binding affinities among the tested compounds.
Reply:
We expanded the section on p. 11/p.12, to highlight the differences in IC50-values. In response to the last point (“Figures and Tables”) raised by reviewer 3, we also created a graphic representation of these differences. The pertinent text (on p. 11/p. 12, lines 343-362) reads as follows:
“The difference in binding site density is illustrated in Figure 8A: it is evident that the [3H]histamine binding capacity (43 nmol/mg) was lower than the binding capacity for [3H]spermidine (sum of Bmax,1 and Bmax,2 = 153 nmol/mg) and for [3H]cadaverine (sum of Bmax,1 and Bmax,2 = 273 nmol/mg). (ii) [3H]Histamine was displaced by all compounds tested. In contrast, unlabeled histamine was ineffective in displacing [3H]spermidine and only poorly displaced [3H]cadaverine. (iii) Similarly, the structure-activity relation of diamines and polyamines differed substantially in their ability to displace [3H]spermidine and [3H]cadaverine: the difference in inhibitory potency can be illustrated by calculating the pairwise ratio of IC50 for all compounds other than histamine (for which IC50-values in displacing [3H]spermidine and [3H]cadaverine could not be determined). As shown in Figure 8B, unlabeled cadaverine was 11.3- and 5.2-fold more potent in self-competition than in displacing [3H]spermidine (red bar indicating S/C-ratio in Figure 8B). Unlabeled cadaverine was also 5.2-fold more potent in competing with [3H]histamine than with [3H]spermidine (green bar indicating S/H-ratio in Figure 8B) and two-fold more potent in self-competition than in displacing [3H]histamine (blue bar indicating S/H-ratio in Figure 8B). Similarly, large differences in potency ratios were observed with putrescine, which was e.g. 3.2-fold in competing for binding of [3H]histamine than for that of [3H]spermidine (green bar indicating S/H-ratio in Figure 8B). In contrast, for both, spermidine and spermine, the IC50-ratios were less pronounced, i.e ranging from 1.7 to 0.6-fold (cf. also Table 1) and thus not substantially different from unity (indicated by the black line in Figure 8B).“
6) The discussion is thorough but could be strengthened by comparing the findings with other studies involving clinoptilolite or similar materials in wound care.
Reply:
The binding of cadaverine to charcoal-coated and aldehyde-functionalized cellulose was determined in ref. 17. As suggested by the reviewer, we compared the data provided in this reference with our binding data and inserted a paragraph in the discussion (on p. 13, lines 396-404) summarizing this comparison, which reads as follows:
“The adsorption kinetics of cadaverine to charcoal-coated or aldehyde-functionalized cellulose and their combination were recently determined [17]: the binding equilibrium was reached after ≥4 hours. Consistent with these slow binding kinetics, the affinity, which can be estimated from the available data, is in the range of 32 mM, >100 mM and 65 mM for charcoal-coated cellulose, for aldehyde functionalized cellulose and their combination, respectively [17]. Thus, the indirect comparison of the data of Wen et al. [17] and of our observations indicates that clinoptilolite depletes cadaverine from the solution substantially more rapidly and with higher affinity than charcoal-coated cellulose, aldehyde-functionalized cellulose or a combination thereof. “
7) Further elaboration on the potential mechanisms underlying the binding preferences for diamines and polyamines would provide deeper insights into the observed results.
Reply:
We agree that it is desirable to understand the structural basis for differences in binding preferences and for binding site heterogeneity.
We added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
In our opinion, the required experiments are beyond the scope of the current work and certainly cannot be accomplished within the time limit imposed (of ten days, extended to 20 days due to the holiday season) by the editorial recommendation.
8) The scalability and cost-effectiveness of integrating clinoptilolite into wound dressings should be highlighted to underline the translational potential of this approach.
Reply:
We addressed this point by adding the following sentences (on p. 14, lines 466-475):
“The scalability of incorporating PCT into wound dressings is promising. It can be integrated to wound care materials using standard production methods, such as mixing, impregnation or coating of fibers or the finished dressing. This allows production of large quantities of functional wound dressings at a large scale and with consistent quality.
PCT is manufactured from a natural clinoptilolite-bearing high-grade raw material, which can be sourced sustainably and cost-effectively. PCT, derived through a quality-controlled refinement process, has undergone clinical evaluation in a phase I trial [26] and imparts enhanced properties to wound dressings. These properties expand their functionality, making them applicable in a broader range of advanced wound care solutions, including the adsorption of toxins, pathogens, inflammatory factors, and excess fluids.”
9) Abbreviations (e.g., PCT for purified clinoptilolite-tuff) should be defined upon their first use, and units and terms should be standardized throughout the manuscript to maintain consistency.
Reply:
The abbreviation PCT is explained upon its first use (in the abstract) and on p. 2, line 88. We scrutinized the manuscript and did not find any other non-standard abbreviation. We assume that S.D., cpm (counts per minute) and Ci (Curie) are standard abbreviations, which need not be explained. In the legend to Fig. 2, the radionuclide tritium was not indicated by a superscript: this was corrected – throughout the manuscript, tritiated compounds are now indicated as “[3H]”.
10) The conclusion could be expanded to explicitly state the next steps or future research directions, such as testing on different wound types or conducting larger-scale clinical trials.
Reply:
This point was addressed by adding the following sentences (on. p. 14, lines 475-478):
“Future research will investigate the potential of PCT-doped alginate dressings to bind and inactivate bacterial and viral pathogens. In addition, the focus will be on clinical evaluation in both chronic and acute wound settings.”
Reviewer 2:
1) Wound fluids are complex mixtures containing proteins, lipids, and other charged molecules. How does PCT selectively adsorb these target molecules without significant nonspecific binding?
Reply:
(i) Experiments were done with seven different wound fluids, which differed substantially in composition. However, binding of cadaverine was comparable in these different wound exudates. (ii) We do not intend to differentiate between specific and non-specific binding. From the perspective of wound management, the only aspect that is relevant is removal of potentially detrimental compounds. (iii) The binding capacity is large; accordingly, binding sites are in excess of potential ligands.
We addressed these points in the paragraph (on p. 13/p. 14, lines 426-454), which describes the limitations of our study (see reply to point 1 of reviewer1).
2) While the binding assays demonstrate efficacy in vitro, the clinical setting involves dynamic conditions such as changes in pH, fluid flow, and microbial activity. How do these factors influence the binding efficiency of PCT?
Reply:
See reply to point 1 (and point 1 of reviewer 2) – paragraph on p. 13/p. 14, lines 426-454.
3) PCT’s binding of histamine and spermidine could disrupt their physiological roles in chronic wounds. Have potential negative consequences of prolonged depletion of these molecules been considered, especially in patients with comorbidities?
Reply:
This was already addressed in detail in the Discussion of the previous version (the pertinent paragraph is now on p. 13, lines 405 - 425).
4) The binding heterogeneity of PCT is attributed to non-equivalent sites, but this is presented as circumstantial evidence. Without molecular or structural validation, how can the authors definitively conclude this heterogeneity?
Reply:
We agree that we only have circumstantial evidence for binding site heretogeneity (and this was explicitly stated in the text). In response to the reviewer’s point, we (i) down-toned the wording by replacing “conclusion” with “conjecture” (p. 11, lines 339-340) reads
“Three arguments support the conjecture that the binding sites are heterogenous and non-equivalent.”).
(ii) In addition, we added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
5) The study uses drainage fluid from breast cancer surgeries, which may not represent the composition of exudates from other chronic wounds, such as diabetic foot ulcers or venous ulcers. How generalizable are these findings?
Reply:
We explicitly mention this in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
6) The manuscript asserts that PCT retains binding capacity even in the presence of wound fluid. However, the displacement of binding by other molecules in the fluid, particularly proteins and ions, was not fully investigated. Could these competing interactions diminish PCT’s real-world efficacy?
Reply:
We address this issue in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
7) The manuscript highlights rapid binding in vitro but does not address whether this is maintained over the typical usage period of wound dressings (e.g., 24-48 hours). Could saturation or leaching of adsorbed molecules occur, reducing efficacy?
Reply:
We should like to point out that we carried out comparative binding assays at 2h and 24 h: binding efficacy was maintained at 24 h. The pertinent data are shown in Figure 5 and Figure 6.
Reviewer 3:
This research investigates the potential of micronized purified clinoptilolite-tuff (PCT) as an effective adsorbent for malodor-causing amines (cadaverine, putrescine, histamine, and polyamines) associated with chronic wounds. The study demonstrates rapid, stable, and high-capacity binding of these amines, both in controlled experiments and in the presence of wound drainage fluids, supporting the integration of PCT into wound dressings for malodor control. The topic is significantly important considering emerging applications of new materials in chronic wound medicine and clinics, however, there are major concerns which needs to be addressed before a decision can be made. My comments are given below-
Introduction and Background- The background section lacks a clear and detailed explanation of the unique physicochemical properties of purified clinoptilolite-tuff (PCT) that make it suitable for amine binding. Adding information on its microporous structure and prior applications in adsorption would provide a stronger foundation for the study. The role of specific amines (cadaverine, putrescine, histamine, etc.) in contributing to wound malodor and their biological significance could be expanded. This would help contextualize why their adsorption is crucial for chronic wound management.
Reply:
(i) We added a paragraph to the introduction (on p. 2, lines 73-88), which reads as follows:
“Clinoptilolite, is a heulandite-type zeolite, a hydrated aluminosilicate characterized by a unique framework of interconnected SiO4 and AlO4 tetrahedra. The negatively charged crystal framework, resulting from the isomorphous substitution of Si4+ by Al3+, enables the mineral to absorb and exchange mono- and divalent cations within the crystal lattice [19], and to adsorb larger ions and/or molecules onto the surface [20-22]. Nizet et al. [23] and Sarabi et al. [24] recently also demonstrated that virions of various species were adsorbed to and effectively neutralized by clinopitolite. Here, we explored the hypothesis that clinoptilolite bound diamines and polyamines with a capacity, which sufficed to effectively deplete these compounds in wound exudates. The microporous structure of clinoptilolite provides a large surface area and numerous adsorption sites, its framework harbors an overall negative net charge. Hence, it is reasonable to posit that clinoptilolite avidly interacts with the amine groups of polyamines, which – based on their pKa in the range of 9.1 to 10.8 are almost completely ionized at physiological pH [25]. We examined this hypothesis by using a preparation of micronized purified clinoptilolite-tuff (PCT), which has been shown to be well tolerated in a phase I trial with artificial wounds [26].”
(ii) The role of cadaverine and of putrescine in wound malodor (and in periwound dermatitis) was stated on p. 2, lines 48 to 56. The higher abundance of cadaverine (than that of putrescine) was stated on p. 6, lines 195 to 197. In our opinion, this suffices to contextualize the importance of adsorbing cadaverine and putrescine and to provide a rationale for the experiments.
Study Objectives- While the aim of the study is clear, it would be beneficial to explicitly define the hypothesis being tested and the novelty of using PCT for binding amines in chronic wound settings. This would distinguish the research from prior studies on odor control in medical applications.
Reply:
The working hypothesis is now explicitly stated on p. 2, lines 79-88 (see also above, reply to the first point)
Methods - Radiolabeled Tracer Experiments- The methodology for radiolabeling tracers and quantifying binding is not described in sufficient detail. A brief description of the techniques, including validation and potential limitations, would improve reproducibility and transparency. The rationale for selecting the specific tracers ([3H]cadaverine, [3H]histamine, and [3H]spermidine) should be provided. Were these selected based on their relative abundance or significance in wound malodor?
Reply:
(i) We include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
(ii) Reviewer 1 found our description of the methodology to be detailed (see point 3 of reviewer 1). The reproducibility is evident from the error bars; our data presentation is also transparent, because we show data points from individual experiments in the saturation experiments (i.e. Figure 2C and D, Figures 5 and 6, Figure 7) and because we show the original signal – i.e. the radioactivity, which was measured in cpm (in Figure 3A and 3B, Figure 4D) – or the original signal is readily accessible from the specific activity of the radioligand, which explicitly stated in the other figures.
(iii) The rationale for selecting histamine and spermidine was stated on p. 2/p. 3, lines 94 – 97. The rationale for selecting cadaverine was stated on p. 6, lines 195 – 197. We do not see why this explanation does not suffice.
Binding Affinity Studies- Clarify whether the binding affinities in the low µM range are competitive with other known adsorbents. A comparative analysis or reference to standard materials for amine adsorption would contextualize the performance of PCT. Include details on how binding site heterogeneity was assessed during the displacement experiments. Was this determined through binding kinetics or equilibrium modeling?
Reply:
(i) The binding capacity is large; accordingly, binding sites are in excess of potential ligands. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is explained on p. 13/ p. 14 (lines 426-454; see also reply to point 1 of reviewer 1).
(ii) We are not aware of any “standard materials” for amine absorption, which can be used as a reference. All dressings, which have been tested in fungating wounds, were found to be not poor in odor control, see refs. 14 and 18. The poor performance of these materials is stated in the introduction. The comparison of our results to those published in ref. 17 is described on p. 13, lines 396-404 (see reply to point 6 of reviewer 1).
(ii) Binding site heterogeneity was assessed under equilibrium binding conditions. This is now explicitly stated on p. 4, lines 115-116 and p. 5, lines 153-154.
Wound Drainage Fluid Experiments- The study mentions the use of wound drainage fluids, but details about their source, preparation, and composition are absent. Specify whether they were collected from clinical samples and if so, ethical considerations should be discussed. Discuss the potential variability in binding efficiency due to differences in drainage fluid composition (e.g., pH, ionic strength, or presence of proteins) and how this variability was accounted for.
Reply:
(i) We do not understand the first part of the comment: The source of the wound drainage fluid was mentioned under subheading 4.2. in the Materials and Methods section. The pertinent section also (on p. 15, lines 501 - 504) mentioned the approval by the University Ethics Committee (including the submission number 2132/2013) and explicitly stated that the patients gave their written consent.
(ii) The variability of the drainage fluid was explicitly mentioned on p. 9, lines 263 – 266. Figures 5 and 6 show that this variability did not affect the ability of PCT to bind cadaverine.
(iii) pH, ionic strength and other constituents are discussed in the paragraph on the limitations of the study (on p. 13/p. 14, lines 426-454 – see reply to comment 1 of reviewer 1)
Incorporation into Wound Dressings- The integration of PCT into wound dressings is briefly mentioned but lacks technical details. Include a description of the preparation process, material compatibility, and any modifications made to ensure sustained adsorption. Evaluate the potential mechanical or functional impact of PCT incorporation on the wound dressing. Does it affect absorbency, flexibility, or ease of use?
Reply:
We added (on p. 14/p. 15, lines 493-499) the following sentences under the subheading Materials:
“PCT in particulate form was mixed into the alginate solution before extrusion, leading to a homogeneous distribution of PCT within the resulting calcium-alginate fibres. In the subsequent production steps, the PCT-doped alginate fibre was combined with cellulose-type fibres, enhancing the mechanical strength as well as the absorbency characteristics of the final dressing. The integration of PCT does not adversely affect the ease of use, flexibility, weight, strength or integrity of the dressing.”
Binding Capacity and Competitive Adsorption- The observation that the wound dressing's binding capacity matches the PCT content is intriguing but requires quantitative backing. Include a table or figure summarizing these findings. Explain how competitive adsorption among different amines was quantitatively assessed and how this impacts the real-world efficacy of the dressing in clinical scenarios.
Reply:
We do not understand the comment: Figure 7 shows a comparative quantification of the binding of cadaverine binding over the relevant concentration range.
Discussion - Binding Mechanism- The discussion of non-equivalent binding sites is underexplored. Elaborate on the chemical or structural basis for this observation. For instance, are specific functional groups on the PCT surface responsible? Consider addressing whether binding is reversible or if the adsorption is influenced by environmental factors like pH changes in chronic wounds.
Reply:
(i) We expanded the discussion on non-equivalent sites on p. 11/p. 12, lines 343-362 (see reply to point 5 of reviewer 1).
(ii) The effect of pH is now explicitly discussed in the paragraph on the limitations of the study (p. 14, lines 442 – 450).
Clarity and Language- The abstract and main text occasionally use non-technical language (e.g., “carrion-like smell”, “justified to posit”). Opt for precise scientific terms to maintain professionalism and consistency. Ensure the text is free from redundant phrases, such as the repetitive mention of "binding was rapid, stable, and of high capacity."
Reply:
“Carrion-like smell” was replaced by “carrion odor”; I do not see anything inappropriate or non-scientific in using the wording “justify to posit”. The same is true about the statement "binding was rapid, stable, and of high capacity" regardless of whether it used once or an several occasions. I should like to point out that, over the past 40+ years, I have written more than 200 original articles and more than 40 reviews, commentaries and book chapters. Thus, I need not be lectured on how to write a scientific text.
Broader Implications and Comparisons- The paper does not discuss alternative malodor control strategies or how PCT compares in terms of efficacy, cost, and safety. Adding this comparison would strengthen the argument for its clinical adoption. The potential for PCT to adsorb other harmful wound-related compounds (e.g., toxins or inflammatory mediators) could be hypothesized, suggesting broader applications.
Reply:
(i) Alternative measures to control malodor include – as mentioned in the previous version of the manuscript – charcoal and functionalized cellulose. The low efficacy of charcoal was pointed out. Safety issues may arise from aldehyde-functionalized cellulose. These were also mentioned. We expanded this section in the introduction (on p. 2, lines 62 - 71 – see reply to point 2 of reviewer 1).
(ii) The binding of toxins was mentioned on p. 11, line 315, that of viruses on p. 11, line 316. The introduction now also mentions the neutralization of viruses by binding to clinoptilolite (p. 2, lines 77 - 79).
Limitations and Challenges- Address potential challenges in scaling up PCT production and ensuring consistency in its properties for clinical use. Discuss any known or potential adverse effects of PCT on wound healing or its interaction with biological tissues.
Reply:
(i) For scalability, see reply to point 8 of reviewer 1 and text on p. 14, lines 466-475.
(ii) Potential adverse effects are discussed in the paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
Future Directions- The conclusion should propose specific avenues for future research, such as testing PCT in vivo in animal models or controlled clinical trials to evaluate efficacy and safety. Investigate the possibility of functionalizing PCT to enhance selectivity or binding efficiency for specific amines.
Reply:
See reply to point 10 of reviewer 1 and text on p. 14, lines 475-478.
References and Citations- Cite previous studies that have explored the use of zeolites or similar materials in medical or environmental applications to provide a broader context. Ensure all cited studies are up to date and relevant to the topic, especially in the discussion of clinical applications. For example, cite https://dx.doi.org/10.4081/vl.2018.7196 with the sentence ´…the initial inflammatory response is dependent on the recruitment of leucocytes (macrophages, neutrophils, mast cells) and the exudation of blood constituents´, a seminal work on the topic.
Reply:
(i) We provided numerous citations for the potential application of clinoptilolite, e.g. refs. 20-24; 26, 34-36, and refer to reviews for its current use (refs. 30 & 31)
(ii) The reference Singh et al. is now cited as ref. 38 on p. 13, line 408.
Figures and Tables- Consider including graphical representations of the adsorption kinetics and binding affinity results. Visual data can significantly enhance reader comprehension. A schematic diagram of the experimental setup for tracer binding or wound drainage fluid experiments would clarify the methodology.
Reply:
(i) We now include a scheme to explain the binding assay (on p. 15).
(ii) We also included a graphical representation of the binding results as Figure 8 with panel A showing the binding capacity calculated from the saturation experiments and panel B showing the pairwise comparison of potency ratios, that is the IC50 ratios for cadaverine, putrescine, spermidine and putrescine (see also reply to point 5 of reviewer 1 and description on p. 11/p. 12, lines 343-362).
Reviewer 3 Report
Comments and Suggestions for Authors
This research investigates the potential of micronized purified clinoptilolite-tuff (PCT) as an effective adsorbent for malodor-causing amines (cadaverine, putrescine, histamine, and polyamines) associated with chronic wounds. The study demonstrates rapid, stable, and high-capacity binding of these amines, both in controlled experiments and in the presence of wound drainage fluids, supporting the integration of PCT into wound dressings for malodor control. The topic is significantly important considering emerging applications of new materials in chronic wound medicine and clinics, however, there are major concerns which needs to be addressed before a decision can be made. My comments are given below-
Introduction and Background- The background section lacks a clear and detailed explanation of the unique physicochemical properties of purified clinoptilolite-tuff (PCT) that make it suitable for amine binding. Adding information on its microporous structure and prior applications in adsorption would provide a stronger foundation for the study. The role of specific amines (cadaverine, putrescine, histamine, etc.) in contributing to wound malodor and their biological significance could be expanded. This would help contextualize why their adsorption is crucial for chronic wound management.
Study Objectives- While the aim of the study is clear, it would be beneficial to explicitly define the hypothesis being tested and the novelty of using PCT for binding amines in chronic wound settings. This would distinguish the research from prior studies on odor control in medical applications.
Methods - Radiolabeled Tracer Experiments- The methodology for radiolabeling tracers and quantifying binding is not described in sufficient detail. A brief description of the techniques, including validation and potential limitations, would improve reproducibility and transparency. The rationale for selecting the specific tracers ([3H]cadaverine, [3H]histamine, and [3H]spermidine) should be provided. Were these selected based on their relative abundance or significance in wound malodor?
Binding Affinity Studies- Clarify whether the binding affinities in the low µM range are competitive with other known adsorbents. A comparative analysis or reference to standard materials for amine adsorption would contextualize the performance of PCT. Include details on how binding site heterogeneity was assessed during the displacement experiments. Was this determined through binding kinetics or equilibrium modeling?
Wound Drainage Fluid Experiments- The study mentions the use of wound drainage fluids, but details about their source, preparation, and composition are absent. Specify whether they were collected from clinical samples and if so, ethical considerations should be discussed. Discuss the potential variability in binding efficiency due to differences in drainage fluid composition (e.g., pH, ionic strength, or presence of proteins) and how this variability was accounted for.
Incorporation into Wound Dressings- The integration of PCT into wound dressings is briefly mentioned but lacks technical details. Include a description of the preparation process, material compatibility, and any modifications made to ensure sustained adsorption. Evaluate the potential mechanical or functional impact of PCT incorporation on the wound dressing. Does it affect absorbency, flexibility, or ease of use?
Binding Capacity and Competitive Adsorption- The observation that the wound dressing's binding capacity matches the PCT content is intriguing but requires quantitative backing. Include a table or figure summarizing these findings. Explain how competitive adsorption among different amines was quantitatively assessed and how this impacts the real-world efficacy of the dressing in clinical scenarios.
Discussion - Binding Mechanism- The discussion of non-equivalent binding sites is underexplored. Elaborate on the chemical or structural basis for this observation. For instance, are specific functional groups on the PCT surface responsible? Consider addressing whether binding is reversible or if the adsorption is influenced by environmental factors like pH changes in chronic wounds.
Clarity and Language- The abstract and main text occasionally use non-technical language (e.g., “carrion-like smell”, “justified to posit”). Opt for precise scientific terms to maintain professionalism and consistency. Ensure the text is free from redundant phrases, such as the repetitive mention of "binding was rapid, stable, and of high capacity."
Broader Implications and Comparisons- The paper does not discuss alternative malodor control strategies or how PCT compares in terms of efficacy, cost, and safety. Adding this comparison would strengthen the argument for its clinical adoption. The potential for PCT to adsorb other harmful wound-related compounds (e.g., toxins or inflammatory mediators) could be hypothesized, suggesting broader applications.
Limitations and Challenges- Address potential challenges in scaling up PCT production and ensuring consistency in its properties for clinical use. Discuss any known or potential adverse effects of PCT on wound healing or its interaction with biological tissues.
Future Directions- The conclusion should propose specific avenues for future research, such as testing PCT in vivo in animal models or controlled clinical trials to evaluate efficacy and safety. Investigate the possibility of functionalizing PCT to enhance selectivity or binding efficiency for specific amines.
References and Citations- Cite previous studies that have explored the use of zeolites or similar materials in medical or environmental applications to provide a broader context. Ensure all cited studies are up to date and relevant to the topic, especially in the discussion of clinical applications. For example, cite https://dx.doi.org/10.4081/vl.2018.7196 with the sentence ´…the initial inflammatory response is dependent on the recruitment of leucocytes (macrophages, neutrophils, mast cells) and the exudation of blood constituents´, a seminal work on the topic.
Figures and Tables- Consider including graphical representations of the adsorption kinetics and binding affinity results. Visual data can significantly enhance reader comprehension. A schematic diagram of the experimental setup for tracer binding or wound drainage fluid experiments would clarify the methodology.
Comments on the Quality of English Language
see detail report
Author Response
Reviewer 1:
The manuscript presents an innovative approach to managing malodorous discharges in chronic wounds using clinoptilolite-tuff, addressing a critical gap in wound care treatment. However, major revisions are necessary to enhance the scientific rigor, clarity, and reproducibility of the study. The authors should be encouraged to carefully address the following points to improve the overall quality and impact of the manuscript:
1) The abstract effectively summarizes the study but could briefly mention the limitations to provide a balanced perspective.
Reply:
The abstract is currently comprised of 211 words. Hence, its length is actually beyond the limit of the allowed word count. Accordingly, we did not include an additional sentence. However, we added a paragraph to the Discussion, which points out the major limitations of our study, which are difficult to address in a comprehensive way, namely (i) the heterogeneity of wound fluids, (ii) the impact of variation in pH (and in ionic strength) on the adsorptive capacity, (iii) the possibility that adsorption of wound fluid constituents by PCT may have detrimental effects on wound healing. The pertinent paragraph (on p. 13/p. 14, lines 426-454) reads:
“Our study has several limitations: (i) Wound fluids are heterogenous. We did not have access to exudate from fumigating wounds, but used wound exudates collected from patients, who had undergone breast cancer surgery. We cannot formally rule out that constituents of fungating wound exudates interferes with binding of cadaverine and putrescine. However, it is worth pointing out that the binding capacity of PCT is large. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is evident from the large discrepancy, which we observed in the binding affinity determined in saturation experiments and the IC50 estimated from the competition experiments. Under the realistic conditions where 10 ml of fungating wound exudate contain 1 nmol to 10 nmol of cadaverine (that is 100 µM to 1 mM) [16], 1 mg of PCT is predicted to readily suffice for sorbing the cadaverine molecules and a large fraction of binding sites remain unoccupied and thus available to bind additional compounds. In the current prototype, 1.3 cm2 of PCT-doped wound dressing provides 1 mg of PCT and thus suffices to so adsorb a substantially larger amount than 10 nmol of cadaverine (or putrescine), but a wound of this size is unlikely to be covered with 10 ml of fluid. (ii) We also did not test the effect of varying pH and ionic strength on the adsorptive capacity. We consider ionic strength of modest interest, because wound exudates have the same ionic composition as plasma [45]. There is a large variation in the pH of wound exudates [46]. Bacterially contamination results in wound exudate, which are alkaline [47,48]. The wound exudates, which were collected used were alkaline (average pH = 8.0). This did not affect the ability of PCT to bind cadaverine (cf. Figures 5 & 6). This was to be expected, because the pKa of the amine groups of cadaverine is 9.1 and 10.2. Thus, variation in pH only have a modest effect on the ionization of cadaverine. (iii) We cannot rule out that removal of solutes, which are present in wound fluids, may have a negative impact on wound healing. However, in cell culture, PCT did not affect the viability and the ohmic resistance of an epithelial cell layer [35]. Importantly, in a phase I trial with artificial wounds, the current preparation of micronized purified PCT did not affect wound closure [26].“
2) The introduction provides sufficient context but would benefit from elaboration on alternative existing treatments for malodorous wounds and their limitations to better justify the significance of the proposed approach.
Reply:
Two alternative approaches were mentioned on p. 2, lines 56-62, namely wound dressings containing charcoal and aldehyde-functionalized cellulose. We expanded this section by referring to a review (Akhmetova et al., new ref. 18), which systematically examines the other options: these are based on silver particles, iodine-release, hyperosmotic sugar solutions, (manuka) honey and topically applied antibacterial agents (e.g. metronidazole). The pertinent sentences (on p. 2, lines 62 - 71) read as follows:
“Earlier approaches to reduce malodor aimed at reducing bacterial growth by incorporating silver, iodine, metronidazole and plant-derived oils (from eucalyptus, thyme, lavender etc.) into the wound dressing [18]. Similarly, bacterial growth may be reduced by applying hyperosmolar sucrose or manuka honey. However, the effectiveness of these approaches is considered modest in the management of malignant fungating wounds: in a systematic survey, charcoal-based dressings and metronidazole-containing gels were rated as being highly effective in about 50% of the cases. Silver- and iodine-based containg dressings were considered to be highly effective in only 23 and 17% of the cases, respectively; the ratings for all other approaches were even lower [14]. Thus, there is a large unmet medical need [14,18].”
3) While the methodology is detailed, the inclusion of visual or schematic diagrams of experimental setups could significantly improve comprehension.
Reply:
We now include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
4) The criteria for selecting the concentration ranges of radioligands in the experiments should be clarified.
Reply:
We added a statement on preliminary experiments, which had been carried out to identify the range, where competition occurred. The pertinent sentences on p.16, lines 553-558 reads:
“Preliminary experiments (with total concentrations increasing by one order magnitude from 1 nM to 10 mM) were carried out to identify the concentration range, where the compounds competed effectively with the radiolabeled tracers. Because total concentrations ≤ 0.1 mM failed to displace the bound radiotracers, logarithmically concentrations covering the range of 0.1 to 10 mM were subsequently to determine the inhibitory potency of competitors.”
5) The section discussing competitive displacement experiments (Figures 3 and 4) should explicitly compare results to highlight differences in binding affinities among the tested compounds.
Reply:
We expanded the section on p. 11/p.12, to highlight the differences in IC50-values. In response to the last point (“Figures and Tables”) raised by reviewer 3, we also created a graphic representation of these differences. The pertinent text (on p. 11/p. 12, lines 343-362) reads as follows:
“The difference in binding site density is illustrated in Figure 8A: it is evident that the [3H]histamine binding capacity (43 nmol/mg) was lower than the binding capacity for [3H]spermidine (sum of Bmax,1 and Bmax,2 = 153 nmol/mg) and for [3H]cadaverine (sum of Bmax,1 and Bmax,2 = 273 nmol/mg). (ii) [3H]Histamine was displaced by all compounds tested. In contrast, unlabeled histamine was ineffective in displacing [3H]spermidine and only poorly displaced [3H]cadaverine. (iii) Similarly, the structure-activity relation of diamines and polyamines differed substantially in their ability to displace [3H]spermidine and [3H]cadaverine: the difference in inhibitory potency can be illustrated by calculating the pairwise ratio of IC50 for all compounds other than histamine (for which IC50-values in displacing [3H]spermidine and [3H]cadaverine could not be determined). As shown in Figure 8B, unlabeled cadaverine was 11.3- and 5.2-fold more potent in self-competition than in displacing [3H]spermidine (red bar indicating S/C-ratio in Figure 8B). Unlabeled cadaverine was also 5.2-fold more potent in competing with [3H]histamine than with [3H]spermidine (green bar indicating S/H-ratio in Figure 8B) and two-fold more potent in self-competition than in displacing [3H]histamine (blue bar indicating S/H-ratio in Figure 8B). Similarly, large differences in potency ratios were observed with putrescine, which was e.g. 3.2-fold in competing for binding of [3H]histamine than for that of [3H]spermidine (green bar indicating S/H-ratio in Figure 8B). In contrast, for both, spermidine and spermine, the IC50-ratios were less pronounced, i.e ranging from 1.7 to 0.6-fold (cf. also Table 1) and thus not substantially different from unity (indicated by the black line in Figure 8B).“
6) The discussion is thorough but could be strengthened by comparing the findings with other studies involving clinoptilolite or similar materials in wound care.
Reply:
The binding of cadaverine to charcoal-coated and aldehyde-functionalized cellulose was determined in ref. 17. As suggested by the reviewer, we compared the data provided in this reference with our binding data and inserted a paragraph in the discussion (on p. 13, lines 396-404) summarizing this comparison, which reads as follows:
“The adsorption kinetics of cadaverine to charcoal-coated or aldehyde-functionalized cellulose and their combination were recently determined [17]: the binding equilibrium was reached after ≥4 hours. Consistent with these slow binding kinetics, the affinity, which can be estimated from the available data, is in the range of 32 mM, >100 mM and 65 mM for charcoal-coated cellulose, for aldehyde functionalized cellulose and their combination, respectively [17]. Thus, the indirect comparison of the data of Wen et al. [17] and of our observations indicates that clinoptilolite depletes cadaverine from the solution substantially more rapidly and with higher affinity than charcoal-coated cellulose, aldehyde-functionalized cellulose or a combination thereof. “
7) Further elaboration on the potential mechanisms underlying the binding preferences for diamines and polyamines would provide deeper insights into the observed results.
Reply:
We agree that it is desirable to understand the structural basis for differences in binding preferences and for binding site heterogeneity.
We added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
In our opinion, the required experiments are beyond the scope of the current work and certainly cannot be accomplished within the time limit imposed (of ten days, extended to 20 days due to the holiday season) by the editorial recommendation.
8) The scalability and cost-effectiveness of integrating clinoptilolite into wound dressings should be highlighted to underline the translational potential of this approach.
Reply:
We addressed this point by adding the following sentences (on p. 14, lines 466-475):
“The scalability of incorporating PCT into wound dressings is promising. It can be integrated to wound care materials using standard production methods, such as mixing, impregnation or coating of fibers or the finished dressing. This allows production of large quantities of functional wound dressings at a large scale and with consistent quality.
PCT is manufactured from a natural clinoptilolite-bearing high-grade raw material, which can be sourced sustainably and cost-effectively. PCT, derived through a quality-controlled refinement process, has undergone clinical evaluation in a phase I trial [26] and imparts enhanced properties to wound dressings. These properties expand their functionality, making them applicable in a broader range of advanced wound care solutions, including the adsorption of toxins, pathogens, inflammatory factors, and excess fluids.”
9) Abbreviations (e.g., PCT for purified clinoptilolite-tuff) should be defined upon their first use, and units and terms should be standardized throughout the manuscript to maintain consistency.
Reply:
The abbreviation PCT is explained upon its first use (in the abstract) and on p. 2, line 88. We scrutinized the manuscript and did not find any other non-standard abbreviation. We assume that S.D., cpm (counts per minute) and Ci (Curie) are standard abbreviations, which need not be explained. In the legend to Fig. 2, the radionuclide tritium was not indicated by a superscript: this was corrected – throughout the manuscript, tritiated compounds are now indicated as “[3H]”.
10) The conclusion could be expanded to explicitly state the next steps or future research directions, such as testing on different wound types or conducting larger-scale clinical trials.
Reply:
This point was addressed by adding the following sentences (on. p. 14, lines 475-478):
“Future research will investigate the potential of PCT-doped alginate dressings to bind and inactivate bacterial and viral pathogens. In addition, the focus will be on clinical evaluation in both chronic and acute wound settings.”
Reviewer 2:
1) Wound fluids are complex mixtures containing proteins, lipids, and other charged molecules. How does PCT selectively adsorb these target molecules without significant nonspecific binding?
Reply:
(i) Experiments were done with seven different wound fluids, which differed substantially in composition. However, binding of cadaverine was comparable in these different wound exudates. (ii) We do not intend to differentiate between specific and non-specific binding. From the perspective of wound management, the only aspect that is relevant is removal of potentially detrimental compounds. (iii) The binding capacity is large; accordingly, binding sites are in excess of potential ligands.
We addressed these points in the paragraph (on p. 13/p. 14, lines 426-454), which describes the limitations of our study (see reply to point 1 of reviewer1).
2) While the binding assays demonstrate efficacy in vitro, the clinical setting involves dynamic conditions such as changes in pH, fluid flow, and microbial activity. How do these factors influence the binding efficiency of PCT?
Reply:
See reply to point 1 (and point 1 of reviewer 2) – paragraph on p. 13/p. 14, lines 426-454.
3) PCT’s binding of histamine and spermidine could disrupt their physiological roles in chronic wounds. Have potential negative consequences of prolonged depletion of these molecules been considered, especially in patients with comorbidities?
Reply:
This was already addressed in detail in the Discussion of the previous version (the pertinent paragraph is now on p. 13, lines 405 - 425).
4) The binding heterogeneity of PCT is attributed to non-equivalent sites, but this is presented as circumstantial evidence. Without molecular or structural validation, how can the authors definitively conclude this heterogeneity?
Reply:
We agree that we only have circumstantial evidence for binding site heretogeneity (and this was explicitly stated in the text). In response to the reviewer’s point, we (i) down-toned the wording by replacing “conclusion” with “conjecture” (p. 11, lines 339-340) reads
“Three arguments support the conjecture that the binding sites are heterogenous and non-equivalent.”).
(ii) In addition, we added two sentences (on p. 13, lines 392-395):
“We stress that our conjecture about binding heterogeneity remains a hypothesis based on indirect evidence: further studies employing advanced techniques such as X-ray powder diffraction are required to verify the binding properties and the heterogeneity of binding sites in PCT at the molecular or structural level.”
5) The study uses drainage fluid from breast cancer surgeries, which may not represent the composition of exudates from other chronic wounds, such as diabetic foot ulcers or venous ulcers. How generalizable are these findings?
Reply:
We explicitly mention this in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
6) The manuscript asserts that PCT retains binding capacity even in the presence of wound fluid. However, the displacement of binding by other molecules in the fluid, particularly proteins and ions, was not fully investigated. Could these competing interactions diminish PCT’s real-world efficacy?
Reply:
We address this issue in our paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
7) The manuscript highlights rapid binding in vitro but does not address whether this is maintained over the typical usage period of wound dressings (e.g., 24-48 hours). Could saturation or leaching of adsorbed molecules occur, reducing efficacy?
Reply:
We should like to point out that we carried out comparative binding assays at 2h and 24 h: binding efficacy was maintained at 24 h. The pertinent data are shown in Figure 5 and Figure 6.
Reviewer 3:
This research investigates the potential of micronized purified clinoptilolite-tuff (PCT) as an effective adsorbent for malodor-causing amines (cadaverine, putrescine, histamine, and polyamines) associated with chronic wounds. The study demonstrates rapid, stable, and high-capacity binding of these amines, both in controlled experiments and in the presence of wound drainage fluids, supporting the integration of PCT into wound dressings for malodor control. The topic is significantly important considering emerging applications of new materials in chronic wound medicine and clinics, however, there are major concerns which needs to be addressed before a decision can be made. My comments are given below-
Introduction and Background- The background section lacks a clear and detailed explanation of the unique physicochemical properties of purified clinoptilolite-tuff (PCT) that make it suitable for amine binding. Adding information on its microporous structure and prior applications in adsorption would provide a stronger foundation for the study. The role of specific amines (cadaverine, putrescine, histamine, etc.) in contributing to wound malodor and their biological significance could be expanded. This would help contextualize why their adsorption is crucial for chronic wound management.
Reply:
(i) We added a paragraph to the introduction (on p. 2, lines 73-88), which reads as follows:
“Clinoptilolite, is a heulandite-type zeolite, a hydrated aluminosilicate characterized by a unique framework of interconnected SiO4 and AlO4 tetrahedra. The negatively charged crystal framework, resulting from the isomorphous substitution of Si4+ by Al3+, enables the mineral to absorb and exchange mono- and divalent cations within the crystal lattice [19], and to adsorb larger ions and/or molecules onto the surface [20-22]. Nizet et al. [23] and Sarabi et al. [24] recently also demonstrated that virions of various species were adsorbed to and effectively neutralized by clinopitolite. Here, we explored the hypothesis that clinoptilolite bound diamines and polyamines with a capacity, which sufficed to effectively deplete these compounds in wound exudates. The microporous structure of clinoptilolite provides a large surface area and numerous adsorption sites, its framework harbors an overall negative net charge. Hence, it is reasonable to posit that clinoptilolite avidly interacts with the amine groups of polyamines, which – based on their pKa in the range of 9.1 to 10.8 are almost completely ionized at physiological pH [25]. We examined this hypothesis by using a preparation of micronized purified clinoptilolite-tuff (PCT), which has been shown to be well tolerated in a phase I trial with artificial wounds [26].”
(ii) The role of cadaverine and of putrescine in wound malodor (and in periwound dermatitis) was stated on p. 2, lines 48 to 56. The higher abundance of cadaverine (than that of putrescine) was stated on p. 6, lines 195 to 197. In our opinion, this suffices to contextualize the importance of adsorbing cadaverine and putrescine and to provide a rationale for the experiments.
Study Objectives- While the aim of the study is clear, it would be beneficial to explicitly define the hypothesis being tested and the novelty of using PCT for binding amines in chronic wound settings. This would distinguish the research from prior studies on odor control in medical applications.
Reply:
The working hypothesis is now explicitly stated on p. 2, lines 79-88 (see also above, reply to the first point)
Methods - Radiolabeled Tracer Experiments- The methodology for radiolabeling tracers and quantifying binding is not described in sufficient detail. A brief description of the techniques, including validation and potential limitations, would improve reproducibility and transparency. The rationale for selecting the specific tracers ([3H]cadaverine, [3H]histamine, and [3H]spermidine) should be provided. Were these selected based on their relative abundance or significance in wound malodor?
Reply:
(i) We include a schematic diagram on p. 15 to illustrate the steps involved in the binding assay.
(ii) Reviewer 1 found our description of the methodology to be detailed (see point 3 of reviewer 1). The reproducibility is evident from the error bars; our data presentation is also transparent, because we show data points from individual experiments in the saturation experiments (i.e. Figure 2C and D, Figures 5 and 6, Figure 7) and because we show the original signal – i.e. the radioactivity, which was measured in cpm (in Figure 3A and 3B, Figure 4D) – or the original signal is readily accessible from the specific activity of the radioligand, which explicitly stated in the other figures.
(iii) The rationale for selecting histamine and spermidine was stated on p. 2/p. 3, lines 94 – 97. The rationale for selecting cadaverine was stated on p. 6, lines 195 – 197. We do not see why this explanation does not suffice.
Binding Affinity Studies- Clarify whether the binding affinities in the low µM range are competitive with other known adsorbents. A comparative analysis or reference to standard materials for amine adsorption would contextualize the performance of PCT. Include details on how binding site heterogeneity was assessed during the displacement experiments. Was this determined through binding kinetics or equilibrium modeling?
Reply:
(i) The binding capacity is large; accordingly, binding sites are in excess of potential ligands. In fact, the goal is to provide a wound dressing, where the amount of available binding sites exceeds the mass of potential ligands. Under these conditions, a possible competition for common binding sites becomes irrelevant. In fact, our assay conditions mimic this situation. This is explained on p. 13/ p. 14 (lines 426-454; see also reply to point 1 of reviewer 1).
(ii) We are not aware of any “standard materials” for amine absorption, which can be used as a reference. All dressings, which have been tested in fungating wounds, were found to be not poor in odor control, see refs. 14 and 18. The poor performance of these materials is stated in the introduction. The comparison of our results to those published in ref. 17 is described on p. 13, lines 396-404 (see reply to point 6 of reviewer 1).
(ii) Binding site heterogeneity was assessed under equilibrium binding conditions. This is now explicitly stated on p. 4, lines 115-116 and p. 5, lines 153-154.
Wound Drainage Fluid Experiments- The study mentions the use of wound drainage fluids, but details about their source, preparation, and composition are absent. Specify whether they were collected from clinical samples and if so, ethical considerations should be discussed. Discuss the potential variability in binding efficiency due to differences in drainage fluid composition (e.g., pH, ionic strength, or presence of proteins) and how this variability was accounted for.
Reply:
(i) We do not understand the first part of the comment: The source of the wound drainage fluid was mentioned under subheading 4.2. in the Materials and Methods section. The pertinent section also (on p. 15, lines 501 - 504) mentioned the approval by the University Ethics Committee (including the submission number 2132/2013) and explicitly stated that the patients gave their written consent.
(ii) The variability of the drainage fluid was explicitly mentioned on p. 9, lines 263 – 266. Figures 5 and 6 show that this variability did not affect the ability of PCT to bind cadaverine.
(iii) pH, ionic strength and other constituents are discussed in the paragraph on the limitations of the study (on p. 13/p. 14, lines 426-454 – see reply to comment 1 of reviewer 1)
Incorporation into Wound Dressings- The integration of PCT into wound dressings is briefly mentioned but lacks technical details. Include a description of the preparation process, material compatibility, and any modifications made to ensure sustained adsorption. Evaluate the potential mechanical or functional impact of PCT incorporation on the wound dressing. Does it affect absorbency, flexibility, or ease of use?
Reply:
We added (on p. 14/p. 15, lines 493-499) the following sentences under the subheading Materials:
“PCT in particulate form was mixed into the alginate solution before extrusion, leading to a homogeneous distribution of PCT within the resulting calcium-alginate fibres. In the subsequent production steps, the PCT-doped alginate fibre was combined with cellulose-type fibres, enhancing the mechanical strength as well as the absorbency characteristics of the final dressing. The integration of PCT does not adversely affect the ease of use, flexibility, weight, strength or integrity of the dressing.”
Binding Capacity and Competitive Adsorption- The observation that the wound dressing's binding capacity matches the PCT content is intriguing but requires quantitative backing. Include a table or figure summarizing these findings. Explain how competitive adsorption among different amines was quantitatively assessed and how this impacts the real-world efficacy of the dressing in clinical scenarios.
Reply:
We do not understand the comment: Figure 7 shows a comparative quantification of the binding of cadaverine binding over the relevant concentration range.
Discussion - Binding Mechanism- The discussion of non-equivalent binding sites is underexplored. Elaborate on the chemical or structural basis for this observation. For instance, are specific functional groups on the PCT surface responsible? Consider addressing whether binding is reversible or if the adsorption is influenced by environmental factors like pH changes in chronic wounds.
Reply:
(i) We expanded the discussion on non-equivalent sites on p. 11/p. 12, lines 343-362 (see reply to point 5 of reviewer 1).
(ii) The effect of pH is now explicitly discussed in the paragraph on the limitations of the study (p. 14, lines 442 – 450).
Clarity and Language- The abstract and main text occasionally use non-technical language (e.g., “carrion-like smell”, “justified to posit”). Opt for precise scientific terms to maintain professionalism and consistency. Ensure the text is free from redundant phrases, such as the repetitive mention of "binding was rapid, stable, and of high capacity."
Reply:
“Carrion-like smell” was replaced by “carrion odor”; I do not see anything inappropriate or non-scientific in using the wording “justify to posit”. The same is true about the statement "binding was rapid, stable, and of high capacity" regardless of whether it used once or an several occasions. I should like to point out that, over the past 40+ years, I have written more than 200 original articles and more than 40 reviews, commentaries and book chapters. Thus, I need not be lectured on how to write a scientific text.
Broader Implications and Comparisons- The paper does not discuss alternative malodor control strategies or how PCT compares in terms of efficacy, cost, and safety. Adding this comparison would strengthen the argument for its clinical adoption. The potential for PCT to adsorb other harmful wound-related compounds (e.g., toxins or inflammatory mediators) could be hypothesized, suggesting broader applications.
Reply:
(i) Alternative measures to control malodor include – as mentioned in the previous version of the manuscript – charcoal and functionalized cellulose. The low efficacy of charcoal was pointed out. Safety issues may arise from aldehyde-functionalized cellulose. These were also mentioned. We expanded this section in the introduction (on p. 2, lines 62 - 71 – see reply to point 2 of reviewer 1).
(ii) The binding of toxins was mentioned on p. 11, line 315, that of viruses on p. 11, line 316. The introduction now also mentions the neutralization of viruses by binding to clinoptilolite (p. 2, lines 77 - 79).
Limitations and Challenges- Address potential challenges in scaling up PCT production and ensuring consistency in its properties for clinical use. Discuss any known or potential adverse effects of PCT on wound healing or its interaction with biological tissues.
Reply:
(i) For scalability, see reply to point 8 of reviewer 1 and text on p. 14, lines 466-475.
(ii) Potential adverse effects are discussed in the paragraph on the limitations of the study on p. 13/p. 14, lines 426-454 (see reply to point 1 of reviewer 1).
Future Directions- The conclusion should propose specific avenues for future research, such as testing PCT in vivo in animal models or controlled clinical trials to evaluate efficacy and safety. Investigate the possibility of functionalizing PCT to enhance selectivity or binding efficiency for specific amines.
Reply:
See reply to point 10 of reviewer 1 and text on p. 14, lines 475-478.
References and Citations- Cite previous studies that have explored the use of zeolites or similar materials in medical or environmental applications to provide a broader context. Ensure all cited studies are up to date and relevant to the topic, especially in the discussion of clinical applications. For example, cite https://dx.doi.org/10.4081/vl.2018.7196 with the sentence ´…the initial inflammatory response is dependent on the recruitment of leucocytes (macrophages, neutrophils, mast cells) and the exudation of blood constituents´, a seminal work on the topic.
Reply:
(i) We provided numerous citations for the potential application of clinoptilolite, e.g. refs. 20-24; 26, 34-36, and refer to reviews for its current use (refs. 30 & 31)
(ii) The reference Singh et al. is now cited as ref. 38 on p. 13, line 408.
Figures and Tables- Consider including graphical representations of the adsorption kinetics and binding affinity results. Visual data can significantly enhance reader comprehension. A schematic diagram of the experimental setup for tracer binding or wound drainage fluid experiments would clarify the methodology.
Reply:
(i) We now include a scheme to explain the binding assay (on p. 15).
(ii) We also included a graphical representation of the binding results as Figure 8 with panel A showing the binding capacity calculated from the saturation experiments and panel B showing the pairwise comparison of potency ratios, that is the IC50 ratios for cadaverine, putrescine, spermidine and putrescine (see also reply to point 5 of reviewer 1 and description on p. 11/p. 12, lines 343-362).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have responded to my comments and suggestions; therefore, the manuscript can be accepted
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors addressed all comments and I recommend publishing the article.
Comments on the Quality of English Language-
Reviewer 3 Report
Comments and Suggestions for Authorsaccept
