The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses

Paindaveine Charlotte C; Bihin Benoit; Lepage Olivier M

doi:10.1371/journal.pone.0299990

. 2024 Mar 7;19(3):e0299990. doi: 10.1371/journal.pone.0299990

The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses

Paindaveine Charlotte C ^1,^*,^#, Bihin Benoit ², Lepage Olivier M ^1,^#

Editor: Carlos Alberto Antunes Viegas³

PMCID: PMC10919598 PMID: 38451976

Abstract

Objectives

To evaluate secondary intention wound healing in the horse’s limbs when treated with the synthetic epidermis spray (Novacika®, Cohesive S.A.S, France) or with a standard bandaging technique.

Methods

Six Standardbred mares were included in the study. Four 2.5 x 2.5 cm full-thickness skin wounds were created on each thoracic limb. Two wounds were located on the dorsoproximal aspect of the cannon bone and the other two at the dorsoproximal aspect of the fetlock. Six hours after creation, wounds were randomly treated with synthetic epidermis spray or standard bandaging. The wounds were assessed every 4 days by gross visual assessment and using a 3D imaging camera. Analysis was performed with a 3D imaging application.

Results

Out of 46 wounds, 22 showed exuberant granulation tissue and were part of the standard bandaging group. Whether the wounds were treated with synthetic epidermis spray or standard bandaging, the time for healing was the same.

Conclusion

The synthetic epidermis spray studied in this model has allowed healing without the production of exuberant granulation tissue but did not reduce the median wound healing time compared to a standard bandaging technique. The synthetic epidermis spray is potentially an interesting alternative for the management of secondary intention wound healing of superficial and non-infected distal limb wounds in adult horses on economical and practical aspects. However, all statistical inference (p-values especially) must be interpreted with caution, given the size of the sample.

Introduction

Skin wounds are among the most common conditions affecting horses [1–3] and have a significant financial impact on the equine industry and caregivers [1,4,5]. A study in the United States revealed that skin wounds are the cause of 16% of euthanasia in adult horses and 23.9% in horses less than 6 months of age [6]. In a retrospective study including 500 injured horses and ponies, only 26% of the wounds in horses were successfully treated with primary closure [7]. Horses often live in highly contaminated environments and secondary intention wound healing is usually predominant [8–10]. However, wounds on the limbs can lead to complications and additional treatment is often required [2,11].

Exuberant granulation tissue (EGT) is the most common complication found on the limbs of a horse and should always be anticipated. The development of EGT is frequent due in particular to prolonged low-grade inflammation, which prevents wound contraction and proper epithelialization, leading to non-healing chronic wounds [4,12]. Wound complications impact the horse’s athletic career and are the reason for prolonged and costly treatments. Therefore, alternative treatments are currently being investigated to improve secondary intention healing in horses [12].

Commonly, distal limb wounds are treated using nonadherent permeable dressing secured with conforming cotton gauze and held in place with a cohesive bandage.

In human medicine, multiple skin sprays have been studied and present numerous benefits such as the ease and short time of application, the possibility to treat large wound areas, and the homogeneous distribution of sprayed material [13]. The ease of application is particularly a major advantage for field practitionners. Acellular skin sprays generally consist of hydrogels that form a thin layer when sprayed over the wound and act as a protective dressing [13]. The synthetic epidermis spray (SES) used in this study is composed of ultraviolet polymerizable methacrylate monomers, comonomers, crosslinker and a photoinitiator. The methacrylate monomers are used as a base in multiple dressings in human medicine and seem to show excellent biocompatibility [14].

It was hypothesized that median wound healing time and prevalence of EGT formation would be lower on experimental wounds treated with a synthetic epidermis spray.

The study’s objectives were to evaluate the median wound healing time and EGT prevalence on experimental open wounds in horses treated with the SES (Novacika®, Cohesive S.A.S., France) or with a standard bandaging technique.

Material & methods

The study protocol was approved by the local animal care ethics committee and following the guidelines of the French Animal Ethics Committee (APAFIS #33883–2021111513453916). It was a prospective, randomized, controlled, cross-over experimental trial.

Animals

Six healthy Standardbred adult mares of varying ages (range: 4 to 13 years old) and body weight (range: 425 to 560 kg), free of any scars or skin disease, were included in the study. Horses were considered healthy based on physical examination, hematology and biochemical panel results.

The horses arrived at the research facilities one week before the start of the study for acclimation. During the study they were housed in box stalls (10 m²) and fed 10 kg of hay twice a day with water available ad libitum. General clinical examinations were performed daily throughout the study.

Wound model

The protocol was based on a standardised wound model previously described [12,15]. The hair of both thoracic limbs was clipped from the dorsal aspect of the cannon (third metacarpal) bone the day before the surgical incision and wound creation.

On day 0, horses were sedated with detomidine chlorhydrate (0.01 mg/kg IV, Somnipron 10 mg/mL, Osalia, France) and butorphanol tartrate (0.02 mg/kg IV, Torbugesic VET 10 mg/mL, Zoetis, France). Additional sedation was administered as required for chemical restraint. Surgical sites were swabbed with a 0.9% NaCl solution prior to manipulation. Surgical scrub was not performed in order to mimic spontaneous trauma with natural contamination.

The skin was infiltrated approximately 2 cm above the surgical site with lidocaine hydrochloride 2% (Lurocaine 20 mg/mL, Vetoquinol, France) on each thoracic limb prior to surgical incision using a hemi-ring anaesthetic block technique. A template was used to standardise the creation of wound area. Using a size 15 blade and Metzenbaum scissors, a total of four 2.5 x 2.5 cm full-thickness skin wounds were created on each thoracic limb. Two wounds were located on the dorsoproximal aspect (one lateral and one medial) of the cannon bone and the other two at the dorsoproximal aspect (one lateral and one medial) of the fetlock. In the case of a hemorrhage with a continuous, pulsating flow of blood, a haemostatic forceps was applied for a period of 2 to 5 minutes. Each wound was numbered according to its location and was left uncovered for 6 hours after wound creation to mimic spontaneous trauma and field contamination.

Treatments

The treatments were applied to the wounds in a randomised fashion with each horse acting as its own control. All thoracic limb wounds were subjected to one of the two treatments six hours after surgical wound creation. The horses were randomised into two groups (G1 and G2) by coin tossing. In the first group, the left thoracic limb was treated with treatment 1 (T1) and the right thoracic limb with treatment 2 (T2), while the second group received the opposite treatment. All wounds were cleaned with a 0.9% NaCl embedded swab before treatment application. T1 consisted of the application of the commercial SES composed of composed of ultraviolet polymerizable methacrylate monomers, comonomers, crosslinker and a photoinitiator. The spray was applied to the wound and polymerised using a 395 nm, 20 x 30 cm, 40 mW/cm² UV light (Cohesive S.A.S., France) for 60 seconds at a distance of approximately 30 cm (Fig 1). The same procedure was repeated to fix a second layer of the product. The control treatment (T2) consisted of a nonadherent permeable dressing (Aniplast SURGI, Génia, France) secured with conforming cotton gauze (Soffban Synthetic and Ouate Kistler, Alcyon, France) and held in place with a cohesive bandage (Vetrap and Tensoplast B, Alcyon, France) as common treatment of choice for superficial wounds in a field setting. The control treatment was repeated every 4 days until the end of the study to mimic field veterinary follow-up.

Fig 1 — The right thoracic limb has been treated with standard bandaging technique.

Wound assessment

Wounds treated with T1 underwent daily gross visual assessment by the same evaluator (CP) for wound secretion, granulation tissue, contraction and epithelialization during a 60 days-period. T2-treated wounds were evaluated for the same criteria every 4 days at bandage change. EGT was defined as granulation tissue covering at least 3 out of 4 sides of the wound. Wound secretion, contraction and epithelialization were not statistically analysed but used to follow the wound healing.

A 3D digital image of each wound (treated with T1 and T2) was taken every 4 days using a 3D camera (3D LifViz®mini Quantificare, Biot, France), commonly used in human medicine for maxillofacial surgery [16]. This camera uses two laser beams which indicate the optimal distance for image acquisition when the beams converge to a single point. The image was captured in 5 milliseconds and the images were then processed in dedicated 3D software (Lifeviz^TMApp, Biot, France). After manual delineation of the wound contours by CP, the software automatically analysed the delineated area to obtain precise measurements of wound area (cm²), volume (cm³), height (mm) and depth (mm).

Statistical analysis

In order to compare the wound healing kinetics in both treatments, generalized least square models with area, volume, height and depth as the dependent variables and time (in days), treatment (T1 vs T2), body part (cannon vs fetlock) and all interactions as the independent variables were used. Because the outcomes kinetics (area, volume, height and depth) were not linear, we used restricted cubic splines to model the time effects [17,18]. When using restricted cubic splines, it is necessary to specify the number of knots used to approximate the non-linear relationship [17,18]. The choice of the knots number is a compromise between the model’s ability to represent complex kinetics and the need to avoid overfitting. We thus used the Bayesian information criterion to determine the number of knots which are 8, 5, 8 and 6 to model area, volume, height and depth kinetics, respectively.

The use of generalized least square models was necessary because of the data structure. First, it was expected that two successive measurements on the same wound would be more correlated with each other than two measurements further apart in time. The model therefore included an auto-regressive (first-order) correlation structure that allowed for the time correlation. Second, variability in wound sizes was much greater when wounds were still large (during the first two weeks) than when wounds were nearly closed. To account for this heteroscedasticity, the model included a power variance function structure.

Observed values are showed alongside predicted values to describe how close the models fit to the data. Contrasts were used to compute differences between treatments with their 95% confidence intervals and P-values at four arbitrarily chosen timepoints: 0 days, 16 days, 32 days and 48 days.

A sensitivity analysis where the horse is the unit of analysis was performed and is presented in supplement.

In order to compare the EGT occurrence, wounds were first paired by horse and by zone, e.g., each pair comprises one wound treated with T2 and one wound treated with T1 for the same horse and on the same body part (but on the other limb). The wound pairs were then binned in four categories following the presence or absence of EGT. McNemar test was used to compare the EGT frequencies on paired data.

R 4.2.2 (The R Foundation for Statistical Computing, Austria, Vienna, 2022) was used for the statistical analysis with the following packages: ggplot (for graphical representations), Hmisc, nlme and rms (to handle generalized least square models and restricted cubic splines).

Results

Two wounds were excluded from the study of wound healing kinetics because the horses moved during wound creation and their shape did not follow the original template. A total of 46 wounds instead of 48 were analyzed for a period of 60 days. Lameness was not observed in any horse during the study. Only 16 out of 46 wounds achieved a complete healing, defined by a complete epithelialization without scabs, by the end of the study.

The average evolution of wound area, volume, height and depth treated with T1 or T2 according to the anatomical zone is shown in Fig 2. Individual evolution of wound is shown in supporting information. The evolution of wound area treated with T1 or T2 was similar. Regarding cannon wounds, the difference between treatments (T2-T1) at 0, 16, 32 and 48 days were 0,8 cm² [95% confidence interval: -0.4cm² to 2.0 cm²], 0.9 cm² [-0.2cm² to 2.0 cm²], 0.5 cm² [-0.1 cm² to 1.1 cm²] and 0.3 cm² [0.0 cm² to 0.6 cm²], respectively (Table 1).

Table 1. Comparison of wound area, volume, height and depth at different timepoints.

			T1		T2		Difference between T1 and T2
		Day	Mean	SD	Mean	SD	T1-T2	[95% CI]	P
Area	Cannon	0	6.2	1.1	5.4	0.8	0.8	[-0.4; 2.0]	0.19
		16	5.7	1.9	5,0	1.8	0.9	[-0.2; 2.0]	0.09
		32	2.2	1.3	1.6	0.6	0.5	[-0.1; 1.1]	0.09
		48	0.9	0.2	0.6	0.3	0.3	[0.0; 0.6]	0.09
	Fetlock	0	6.4	1.2	5.3	0.9	1.1	[-0.1; 2.3]	0.08
		16	5.0	1.2	4.7	0.8	0.5	[-0.6; 1.5]	0.37
		32	1.3	0.7	1.8	0.8	-0.5	[-1.0; 0.1]	0.10
		48	0.7	0.4	0.8	0.6	0.0	[-0.4; 0.3]	0.80
Volume	Cannon	0	0.41	0.20	0.39	0.28	0.01	[-0.24; 0.26]	0.92
		16	1.16	0.70	0.57	0.60	0.44	[0.14; 0.74]	0.004
		32	0.19	0.29	0.10	0.09	0.07	[-0.01; 0.15]	0.08
		48	0.02	0.02	0.02	0.02	0.01	[-0.01; 0.03]	0.33
	Fetlock	0	0.50	0.42	0.25	0.16	0.20	[-0.04; 0.43]	0.10
		16	0.80	0.30	0.36	0.13	0.24	[0.02; 0.45]	0.03
		32	0.06	0.06	0.09	0.10	-0.01	[-0.07; 0.06]	0.88
		48	0.03	0.04	0.03	0.05	0.01	[-0.02; 0.03]	0.58
Height	Cannon	0	0.78	0.30	0.81	0.44	-0.04	[-0.35; 0.27]	0.79
		16	2.13	0.65	1.09	0.56	1.22	[0.81; 1.64]	<0.0001
		32	0.73	0.46	0.56	0.31	0.17	[-0.09; 0.43]	0.19
		48	0.30	0.14	0.22	0.16	0.10	[-0.03; 0.23]	0.12
	Fetlock	0	0.88	0.55	0.58	0.26	0.20	[-0.10; 0.50]	0.20
		16	1.72	0.32	0.81	0.26	1.13	[0.75; 1.51]	<0.0001
		32	0.42	0.20	0.44	0.26	0.02	[-0.20; 0.25]	0.84
		48	0.30	0.31	0.25	0.24	0.08	[-0.06; 0.23]	0.27
Depth	Cannon	0	0.094	0.063	0.073	0.068	0.006	[-0.039; 0.051]	0.80
		16	0.008	0.008	0.021	0.018	-0.016	[-0.027; -0.006]	0.002
		32	0.014	0.018	0.012	0.009	0.006	[-0.002; 0.014]	0.15
		48	0.017	0.017	0.013	0.013	0.002	[-0.007; 0.010]	0.71
	Fetlock	0	0.083	0.080	0.104	0.046	-0.008	[-0.091; 0.075]	0.85
		16	0.011	0.008	0.016	0.011	-0.013	[-0.026; 0.001]	0.06
		32	0.009	0.009	0.024	0.026	-0.014	[-0.026; -0.003]	0.02
		48	0.022	0.031	0.020	0.016	0.004	[-0.010; 0.017]	0.60

Open in a new tab

Mean and SD (standard deviation) describe the observed values while the differences between wounds treated by T1 and by T2 (T1-T2), the 95% confidence interval (CI) and the P-values are computed from the generalized least square models.

Fetlock wounds showed similar evolution (Fig 2 and Table 1).

The evolution of wound volume differed significantly between treatments for cannon and fetlock anatomical zone. After 16 days the T2 treated cannon and fetlock wounds volume were 0.44 cm³ ([0.14 cm³ to 0.74 cm³], P = 0.004) and 0.24 cm³ ([0.02 cm³ to 0.45 cm³], P = 0.03) higher than the T1 treated cannon wounds which represented relative increases in volume of 77% and 67%, respectively (Fig 2 and Table 1).

This volume difference is to be explained by differences in wound heights which showed a large peak around 16 days for T2-treated wounds but not for T1-treated wounds. The difference between treatment was +1.22 cm ([0.81 cm to 1.64 cm], P<0.0001), which represent a 124% increase, for cannon wounds and +1.13 cm ([0.75 cm to 1.51 cm], P<0.0001), which represent a 150% increase, for fetlock wounds (Fig 2 and Table 1).

The wounds depths showed a similar evolution for both treatments. The fetlock wounds were 0.07 cm ([0.01 cm to 0.13 cm], P = 0.03) deeper at start than cannon wounds.

Gross evaluation of secretion, contraction and epithelialization was only used as a follow-up for evolution of wound healing and not analyzed.

Wounds were assessed for EGT in 46 wounds for a period of 60 days (Fig 3). Wounds were paired by horse and by anatomical zone (1 to 4) and each pair consisted of one wound treated with T2 and one treated with T1. Out of 22 wounds treated with T1 and 24 treated with T2, 22 out of 24 treated with T2 showed EGT and none for T1-treated wounds. The EGT was far more frequent for T2-treated wounds than T1-treated wounds (20/22 vs 0/22 p<0.0001).

Discussion

The results of this study suggest that the SES (T1) had a significant effect on the prevalence of EGT development in experimentally induced limb wounds in adult horses compared with a standard bandaging technique (T2). This was consistent with our hypothesis that the prevalence of EGT formation would be reduced in wounds treated with the SES compared to standard bandaging. However, a previous study showed that covering a wound with a non-occlusive dressing in a 3-layer bandage modulated the rate of wound contraction and promoted EGT [19]. It is therefore difficult to conclude that the absence of EGT on T1 treated wounds is related to the application of SES and/or the lack of covering bandage. A study comparing T1 treated wounds with non-treated wounds (naked wounds) could give further information on the matter.

It should be noted, that in our study, the EGT observed in the T2 group that did not receive the SES decreased without any trimming procedure during the 60-day period. Young oedematous granulation tissue protruding just above the wound margins has been described and generally does not require special treatment. The EGT may be characterized and differentiated by a firm and granular appearance [20]. In this study, the granulation tissue was considered exuberant. However, a histological study of this tissue at different moments of the healing process would be necessary to assess whether we are in the presence of a temporary increase in cell volume, an increase in the number of cells followed by a decrease in their plasma volume or another interstitial phenomenon and therefore differentiate young oedematous granulation tissue or EGT. The authors chose to not perform biopsy to avoid any interference with the wound healing considering the small surface of the wound.

In the case of a distal limb wound in a horse, it is generally accepted that after cleaning the wound, a dressing should be applied and covered with a bandage to decrease contamination and oedema, absorb exudate, minimise movement and protect against more trauma [19]. It is recommended to change bandages every 3 to 5 days depending on the discharge and evolution of the wound healing [21]. To uniformize the method, the authors chose to change the bandages every 4 days.

Contrary to our hypothesis that wounds treated with the synthetic epidermis spray would heal faster, no significant effect on the kinetics of the healing time was observed after day 32. The transient increase in wound volume that occurred in wounds treated with standard bandaging did not affect the healing time. This was supported by a previous report [19].

The SES was only applied once on day 0. No further application was made. The manufacturer describes a progressive resorption of its product. The application technique of the synthetic epidermis is easy and only requires the horse to be immobilised for approximately three minutes to allow polymerisation. The minimal material requirement also has a significant impact on the overall cost of treatment compared to standard bandaging changed every 4 days. In addition, this treatment could be an alternative for dangerous horses that do not tolerate bandages and could have an impact on the safety of veterinarians and owners. However, itching, external trauma or self-trauma to the wounds would not be prevented by this type of dressing.

There were several limitations associated to the present study, including the small number of horses. The first limitation relates to the animal species studied. The study protocol used and well described for horses is always carried out on a small number of animals [15]. The horses were all adult and healthy and the results cannot be extrapolated to foals, elderly horses or individuals with a systemic disease. Although we deliberately left the wounds open for 6 hours before treatment to mimic normal contamination, for ethical reasons we had to create the wounds with a surgical technique that met the standards of cleanliness and sterilisation of the material. Similarly, our wounds only affect the skin tissue, whereas very often the deeper layers are also affected to varying degrees. The type of wounds studied here are similar to, naturally occurring, acute, poorly contaminated superficial degloving injuries and cannot be extrapolated to wounds presented at a more chronic stage, highly contaminated or infected, or affecting deeper structures such as bone or synovial cavities. The authors believe that deeper wounds would necessitate different care and therefore would not be treated in the first instance with SES. In addition, the study was conducted in winter excluding the presence of insects (e.g. flies).

Conclusion

The synthetic epidermis spray (Novacika®, Cohesive S.A.S, France) studied in this wound model has allowed healing without the production of EGT but did not reduce the median wound healing time compared to a standard bandaging technique. The SES is potentially an interesting alternative for the management of secondary intention wound healing of superficial and non-infected distal limb wounds in adult horses because of its economical and practical aspects. However, all statistical inference (p-values especially) must be interpreted with caution, given the size of the sample.

Supporting information

S1 Table. Data of the wound assessment through the study period.

(XLSX)

pone.0299990.s001.xlsx^{(70.4KB, xlsx)}

Acknowledgments

The authors wish to acknowledge Maud Roux and Claire Forray for their technical help during the study.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The study was funded by Cohesives SAS, Dijon, France. The funders did not play any role in the study design, data collection and analysis. The contract with the funders however specified that they have to accept the publication, which they did. The authors declare that there were no other conflicts of interest. https://www.societe.com/societe/cohesives-807497771.html https://novacika.com/ The funders had no role in study design, data collection and analysis, or preparation of the manuscript. However, the funders had the right to refuse publication of the study data and results.

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PLoS One. doi: 10.1371/journal.pone.0299990.r001

Decision Letter 0

Carlos Alberto Antunes Viegas

2 Jul 2023

PONE-D-23-16094The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.PLOS ONE

Dear Dr. Paindaveine,

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https://www.societe.com/societe/cohesives-807497771.html

" ext-link-type="uri" xlink:type="simple">https://novacika.com/"

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Thanks for this manuscript.

I read this work with some attention and believe that this is a valid clinical research work. However, there are some obvious limitations to your study that you recognise yourself: Low numbers, non-standardised conditions for each animal, minor control of age range and animals acting as their own control.

I would like recommend some improvements if PlosOne are willing to take this on as a manuscript:

1. There is no introduction to the merits or reasoning for using the components in this wound dressing. There should some, although minimal intro of why this might be helpful: “Therefore, alternative treatments are currently being investigated to improve secondary intention healing in horses” is not enough. The reader needs to know why this type of dressing deserves a study of this nature.

I understand this work is sponsored by the wound dressing company. Fair enough but we need to know a bit more about the components of this material. Also, the type of lamp (size and power) are important aspects.

Other specific aspects:

Lines 83-85 This step introduces extremely high variability in the wound environment. Was there any type of microbiological understanding (ie. Culture) of the wound environment prior to treatment?

Lines 126-127 Area, height, depth and volume are related measurements and should be treated as such statistically. Also, Can you explain the difference between the measurements: depth and height? Figure 6 should detail this but seems to only be an illustration of the software interface.

Lines 136 “The numbers of knots used, respectively 8, 5, 8 and 6”, please explain this part of the text. I cannot relate to this.

Line 246: add itching, trauma and self trauma of the wounds as potential aspects that are not protected by this dressing

Lines 248-251 This conclusion is biased. If there is no advantage in healing of time of the wounds the advantages might be of economic and logistical order. This must be stated.

There are too many figures in the manuscript. I would recommend you reduce these to maximum 3.

Reviewer #2: PONE-D-23-16094

The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.

General comments

The authors report a prospective study on the effect of a synthetic spray on secondary intention wound healing in adult horses. The study presents the results of an original research that I believe has not been published elsewhere. Some details in the material and methods should be clarified to understand better the wound model (see specific comments). The paper’s conclusion seems appropriate, but some statements in the discussion are too strong for the results presented. I believe some major improvements could be achieved with the wording and how the figures and tables are presented. Most of the figures could be enhanced with better legend, annotations, and composition for better clarity for the reader. This paper could benefit from a more detailed and nuanced discussion. The research meets most applicable standards for the ethics of experimentation and research integrity. However, the study is funded by a company (Cohesive SAS), and it is not mentioned anywhere in the core of the paper that it is the company owning Novacika®. I believe the company’s name should be at least mentioned each time the product name is used in the paper.

With some improvements, this research has the potential to be a good fit for the journal.

Specific comments

Title

The title is appropriate, short, descriptive, and interesting for the reader.

Short title

The short title is the same as the long title.

Keywords

Keywords seem appropriate.

Abstract

L.15 (Novacika®)

L.21 remove “with”

L.23 evaluation?

L.29 “synthetic epidermis spray (Novacika®)”? o

Introduction

L34. Usually, in-text reference numbers should be inserted to the left or inside colons/semi-colons and placed outside or after full stops and commas. Please apply to the entire document.

L.40 “wounds on the limbs” or “limb wound” or “distal limb wounds”

L.42 “on the limbs”`

L.43 remove “in fact”

L.44 “inflammation, which”

L.46 “complications impact the horse’s…”

L.46 “horse’s athletic career”

L.49-53 I usually place the hypothesis before your objective as it describes how you plan to challenge your hypothesis, but it is up to debate…

L.51 “synthetic epidermis spray (Novacika®)”? Also, there are guidelines for citing trademarks as you did with detomidine: “detomidine chlorhydrate (0.01 mg/kg IV, Somnipron, 10 mg/mL, Osalia, France)”. Please be consistent throughout the paper when you add a trademark or a company/drug name. You could also use an abbreviation: SE or SES to simplify.

L.51 “or a standard bandaging…”

L.53 “hypothesized”

Having some data or comments to introduce the synthetic epidermis spray would be interesting. Are there any studies on similar products on other species, on humans? Description of the product, reported use, or effects? Also, maybe a comment on the other treatment is warranted. Why is it the comparative as opposed to no bandage at all?

Material and Methods

L.60 How do you justify the small number of horses in your study?

L.65 “acclimation”

L.71 Could you comment on the reason why you did not clip the hair on the same day as the surgery? As for our veterinary patients, clipping is recommended immediately before surgery.

L.75 Could you comment on the reason why you elected not to perform a surgical scrub before surgery? I assume it was to try to mimic a traumatic injury and/or to avoid skin flora disruption…it may be worth mentioning it.

L.77-78 Maybe specify where the regional bloc was performed

L.80-82 Based on your figures, it seems that wounds were more located on the dorsolateral and dorsomedial aspects of the limb, which does not fit your description in the text.

L.83 “hemostatic forceps”

See comments for Figure 1-3. I am convinced there is a better way of illustrating your wound model.

L.94-95 How did you randomize your horses? This is important to mention the method used.

L.102 How did you standardize the distance of UV exposition?

L.103-106 Why is your control to bandage wounds? Why not let the wounds heal without any bandages? You need to justify it or at least discuss it later on. As you mentioned in your discussion, bandage use promotes EGT, so this complication is to be expected down the line, so you need to justify your choice of control treatment. Otherwise, it will be difficult to compare the prevalence of EGT in both of your groups because if the wounds treated with the spray did not develop EGT, is that because they were not bandaged? Would naked wounds have developed EGT?

L.107 Why 4 days? Knowing what is reported in the literature about EGT and bandages, would a more frequent bandage change have been more appropriate to avoid EGT development?

L.114 “scoring system”

L.115 What were your criteria for each category of your contraction and epithelialization grading system? I understand it is a subjective grading system, but how could you visually assess contraction or epithelialization properly to classify it as mild, moderate, or severe?

L.117-118 What do you mean by “EGT aspect”? Do you mean “EGT was defined…”?

L.131-134 “…both treatments, a generalized…independent variable was used.”

Results

L.162-163 It would be interesting to know how long it took for the wounds to heal completely between both groups.

L.166 I am not able to see or download the supplementary material that is referenced in the paper

L164-199 This section is challenging to follow as the nomenclature does not match the one used in the table… P-values should be mentioned in the text for significant results

L183 Synthetic epidermis spray (Novacika,….) To simplify, you could use an abbreviation for it (SES) throughout the text.

L.191 Did you compare statistically treatments between locations? I do not see any p-value related to that

Discussion

L.206-208 I think it is an overstatement as the prevalence of EGT could be more related to the bandage application and the absence of it in the other group related to the lack of bandage… It is difficult to discriminate what decrease the occurrence of EGT between the spray and the absence of a bandage. Then the hypothesis could not be verified by the methods used in the study. There is probably a way to reformulate the conclusion.

L.210 Again, synthetic epidermis spray (Novacika….) or SES. Please apply to the rest of the manuscript.

L.216-217 This could be because it was just edematous granulation tissue that usually does not require any treatment. There is mention of this type of edema in the textbook Equine Wound Management by Dre Theoret et al. 3rd ed. (p378-379)

L.236 You need to justify the number of horses used. Maybe based on other studies using the same model?

L.239-240 In your MMs, it is not mentioned if the skin was surgically prepared before surgery. I assume it was to mimic an injury, but here it seems you did use a surgical technique to create the wounds… It is confusing; please clarify.

L.248 The conclusion is more appropriate to the study results than the beginning of the discussion.

The discussion seems a little bit short. Maybe you could talk about the pertinence of having a bandage group in your study vs. a non-bandaged group? Discuss the effect of the bandage (type, duration) on the production of EGT. Discuss the specific of the spray product, is it considered occlusive? Why do wounds not develop EGT under it? What is the evidence in other species that supports its use in horses? Why not biopsy the wounds throughout the healing to see if there is any difference histologically? Discuss the model used and the number of horses used. Could you have gone deeper into the tissues with your model to mimic classic trauma? Etc.

References

Reference 1 seems incomplete. Same for reference 4. There are missing pieces of information in many references, and the format does not fit the requirements of the journal… Please refer to the journal author guidelines to format your references…

Figures and Tables

Figure 1.

It does not represent your template well. You could try to have a picture of the template on a horse to help the reader understand how the template was used. If it is not possible, I would not keep the figure 1. Also, it seems that you used a permanent marker, but your protocol does not mention it. Was it a surgical

marker?

Also, the legend of a figure should always allow the reader to understand its significance without reading the text. Depending on whether or not you decide to keep the figure, you need to add more to your legend.

Figure 2.

This picture is blurry. Maybe a picture of a distal limb with all the fresh wounds would be more appropriate for the reader to understand the localization of the wounds, and you could potentially add your nomenclature (from Figure 3) to such a picture. Otherwise, I do not think that this picture is necessary.

Figure 3.

If you decide to keep this figure, maybe you could add little squares on your drawing to locate each wound. You could also combine that figure with a picture of your template, a picture of one wound, and a picture of each treatment. You add your protocol timeframe, giving you a nice study design figure to help any reader understand your protocol quickly.

Also, the legend does not correspond with what is shown in the figure…different nomenclature?

Figure 4.

I suggest adding the distance and the duration of the application in your legend.

Figure 5.

Figure 6.

The figure is extremely blurry. It may not be relevant. Maybe a figure showing the 3D camera in use and the results of the software?

Figure 7.

The figure is really blurry; you should use vector images for your graphics. It may be the pdf format that changes the resolution. It would be helpful to add some legends on the figure itself (colors, axis) and maybe consider a vertical organization with two columns of graphics (fetlock and metacarpus)

Table 1

Why are the groups named differently in the table (B, N)? Also, the table legend does not explain that, confusing the reader…

Figure 8

The background is blurry, so it is difficult to really appreciate the wounds on the left front limb…

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2024 Mar 7;19(3):e0299990. doi: 10.1371/journal.pone.0299990.r002

Author response to Decision Letter 0

8 Sep 2023

Dear Reviewers,

Here are the answers to your comments and questions:

Reviewer #1 :

- Comment: Lines 83-85 This step introduces extremely high variability in the wound environment. Was there any type of microbiological understanding (ie. Culture) of the wound environment prior to treatment?

Answer: No there wasn’t. We believe that the environment contained multiple bacteria as it is on the field. We did not focus on the infection risk as it is rarely a problem in superficial wounds on horse’s limb. Culture is usually not performed on superficial wounds in clinical cases.

- Comment: Lines 126-127 Area, height, depth and volume are related measurements and should be treated as such statistically. Also, Can you explain the difference between the measurements: depth and height? Figure 6 should detail this but seems to only be an illustration of the software interface.

Answer: The wounds were analysed for area, volume, height and depth because wound healing on horse’s limb appears irregular with the granulation tissue being more prominent on a side. The height and depth values are giving information about the “landforms” of the granulation tissue. For example: the higher height would be where the granulation tissue was the more prominent and depth where there was poor granulation tissue.

- Comment: Lines 136 “The numbers of knots used, respectively 8, 5, 8 and 6”, please explain this part of the text. I cannot relate to this.

Answer: This part of the text was adapted accordingly.

Reviewer #2:

- Comment: Usually, in-text reference numbers should be inserted to the left or inside colons/semi-colons and placed outside or after full stops and commas. Please apply to the entire document.

Answer: I am not sure to understand this comment. I followed the journal requirement concerning the reference https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

- L.49-53 I usually place the hypothesis before your objective as it describes how you plan to challenge your hypothesis, but it is up to debate...

Answer: we followed your advice

- Comment: L.60 How do you justify the small number of horses in your study?

Answer: Above all, it's to respect the 3Rs (Replace, Reduce, and Refine) of animal research considered a systematic approach to animal experimentation that puts the well-being of the animal’s in front and by the fact that the experimental model we have used has already been validated many times in this large, expensive animal.

- Comment: L.71 Could you comment on the reason why you did not clip the hair on the same day as the surgery? As for our veterinary patients, clipping is recommended immediately before surgery.

Answer: To facilitate the ease of the next day. It is true however that we should have done it the same day if we want to mimic natural occurring trauma.

- Comment: L.75 Could you comment on the reason why you elected not to perform a surgical scrub before surgery? I assume it was to try to mimic a traumatic injury and/or to avoid skin flora disruption...it may be worth mentioning it.

Answer: indeed, we wanted to be as close as possible of the reality for natural occurring wound. We will add an explanation in the manuscript.

- Comment: L.102 How did you standardize the distance of UV exposition?

Answer: Subjectively, it was the width of the manipulator's open hand (about 30 cm). A distance that could vary by a few centimetres if the horse moved its opposite limb.

- Comment: L.103-106 Why is your control to bandage wounds? Why not let the wounds heal without any bandages? You need to justify it or at least discuss it later on. As you mentioned in your discussion, bandage use promotes EGT, so this complication is to be expected down the line, so you need to justify your choice of control treatment. Otherwise, it will be difficult to compare the prevalence of EGT in both of your groups because if the wounds treated with the spray did not develop EGT, is that because they were not bandaged? Would naked wounds have developed EGT?

Answer: we believe that another study should compare the product to naked wounds. However, in this study, we chose to compare to standard bandaging technique as most of the clinician cover wounds using this technique when facing superficial wounds. We explain this more clearly in the text.

- Comment: L.107 Why 4 days? Knowing what is reported in the literature about EGT and bandages, would a more frequent bandage change have been more appropriate to avoid EGT development?

Answer: We chose to change it every 4 days as we believe veterinarian on the fields commonly change bandages every 3 to 5 days.

- Comment: L.115 What were your criteria for each category of your contraction and epithelialization grading system? I understand it is a subjective grading system, but how could you visually assess contraction or epithelialization properly to classify it as mild, moderate, or severe?

Answer: As it is always the same assessor who inspects the wounds, he could more easily assess whether a smooth whitish-pink edge was developing around the periphery (epithelialization) and, or whether a reduction in the granulation surface was observed (contraction).

- Comment: L.162-163 It would be interesting to know how long it took for the wounds to heal completely between both groups.

Answer: Regarding the area, only 16 wounds out of 46 achieve a complete healing (defined by a null wound area) during the follow-up. This is why we compared the wound kinetics instead of the time to complete healing.

- Comment: L164-199 P-values should be mentioned in the text for significant results

Answer: P-values were added in the text.

- Comment: L.191 Did you compare statistically treatments between locations? I do not see any p-value related to that

Answer: We added the detail about the comparison between locations.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s002.docx^{(19.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0299990.r003

Decision Letter 1

Carlos Alberto Antunes Viegas

20 Oct 2023

PONE-D-23-16094R1The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.PLOS ONE

Dear Dr. Paindaveine,

Please submit your revised manuscript by Dec 04 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Carlos Alberto Antunes Viegas, DVM; MSc; PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

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6. Review Comments to the Author

Reviewer #1: Dear Authors,

Thanks for your reviews to the manuscript. I am overal happy with them.

Kind regards

Reviewer #2: L57 ‘, such as the ease and short time of application’

L57 ‘the short time of application’

L58 ‘areas, and the homogeneous’

L58-59 ‘The ease of application is particularly a major advantage for field practitioners.’

L61 ‘and act as a protective’

L64 ‘human’

L66-67 ‘It was hypothesized that median wound healing time and prevalence of EGT formation would be lower on experimental wounds treated with a synthetic epidermis spray.’

L69 ‘The study's objectives were to evaluate the median wound healing time and EGT prevalence on experimental open wounds in horses treated with the SES (Novika®, Cohesive S.A.S., France) or with a standard bandaging technique.’

L122 ‘as a common treatment of choice for superficial wounds in a field setting.’

L123 ‘field veterinary’

''- Comment: L.115 What were your criteria for each category of your contraction and epithelialization grading system? I understand it is a subjective grading system, but how could you visually assess contraction or epithelialization properly to classify it as mild, moderate, or severe?

Reviewer answer: That does not answer the question. For example, what is the difference between mild epithelialization, moderate epithelialization and severe epithelization? Same question for contraction. You used a subjective grading system without a reference atlas or measurements describing each category. That questions the pertinence of such assessment and its value in the paper. Later in the results section, you even mention that these assessments are not analyzed, so why do you bother explaining this subjective assessment? You could just mention that wounds were evaluated at each time point for the presence or absence of epithelization, contraction, EGT, etc.

L181 ‘defined by a complete epithelialization without scabs’

L185 Do you mean ‘supporting information’?

L185-225 Please use past tense when you report your results.

L221 Do you mean ‘Macroscopic aspect of healing thoracic limb wound’? These wounds are already granulating.

L222 ‘observed’

L224-225 I do not think this comment should be in the figure legend as it is not seen in the picture.

L227-232 The first paragraph of your discussion seems out of place. Maybe you could integrate it when you discuss the reported effect of bandages on the occurrence of EGT

L269 I already made a comment on the number of horses used. This has not be addressed. If you mention that one of your limitations is ‘the small number of horses’ you need to justify it by referring to other work/studies published with significant data with a similar model.

L283 ‘in this wound model’

L287-288 ‘…adult horses because of its economical and practical aspects.’?

References

There are still inconsistencies in the references...:

For example, AJVR is not a proper abbreviation…

Please use proper abbreviations for each journal (example of a database with journal abbreviations = https://images.webofknowledge.com/images/help/WOS/A_abrvjt.html)

Some references are incomplete (9, 12)

When you cite books, you usually have to specify the chapter with authors and pages. Do not cite an entire book.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2024 Mar 7;19(3):e0299990. doi: 10.1371/journal.pone.0299990.r004

Author response to Decision Letter 1

28 Nov 2023

Response to Reviewer 2,

Comment: L.115 What were your criteria for each category of your contraction and epithelialization grading system? I understand it is a subjective grading system, but how could you visually assess contraction or epithelialization properly to classify it as mild, moderate, or severe?

Answer: As it is always the same assessor who inspects the wounds, he could more easily assess whether a smooth whitish- pink edge was developing around the periphery (epithelialization) and, or whether a reduction in the granulation surface was observed (contraction).''

Answer: please find a table in the file " Response to Reviewers" with the criteria for the grading system. However, as it was not analyzed, we will follow what you suggested.

Comment: L269 I already made a comment on the number of horses used. This has not be addressed. If you mention that one of your limitations is ‘the small number of horses’ you need to justify it by referring to other work/studies published with significant data with a similar model.

Answer: this protocol was used in a previous article (ref 15 in the manuscript) and showed that they could see a statistical difference with 6 horses. We added it on the text.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s003.docx^{(19.5KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0299990.r005

Decision Letter 2

Carlos Alberto Antunes Viegas

12 Jan 2024

PONE-D-23-16094R2The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.PLOS ONE

Dear Dr. Paindaveine,

Please submit your revised manuscript by Feb 26 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Academic Editor

PLOS ONE

Additional Editor Comments:

Dear author,

One of the initial reviewers asked pertinent questions about the statistical analysis of your work. In this sense, we requested support for the review in this particular aspect from a statistics specialist. This recommends a major revision in order to redo some aspects of the statistical treatment.

Best regards

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

Reviewer #3: No

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #2: Dear authors,

Thank you for your work on the manuscript ! I am satisfied with its actual form.

Kind regards

Reviewer #3: The authors set out to evaluate the secondary intention wound healing in the horse's limbs when treated with the synthetic epidermis spray or with a standard bandaging technique. The design and analysis appears to be simple using the generalized least square models with area, volume, height and depth as the dependent variables and time (in days), treatment (T1 vs T2), body part (cannon vs fetlock) and all interactions as the independent variables . The interpretation, as noted below, is not simple. Also the investigators claim that six horses are adequate for the sample size as noted from previous publications. However, the sample size is not discussed and certainly not justified with respect to any of the variables in this presentation.

Simply stated the overall result is that whether the wounds were treated with synthetic epidermis spray or standard bandaging, the time for healing was the same. This is not obvious in light of the time and location effect. The presentation of the results are seen primarily in Table 2. It appears like a multivariate issue with the major variables of interest being time, treatment and location. There are no gross p-values where the authors state in the overall experiment whether there is an independent time, treatment or location effect adjusted for the others. The attempt at explaining the interactions may be reasonable. Table 2 is a summary univariate analysis for the dependent variables in five tables. The organization of the analysis is confusing. There are many analyses and many p-values, resulting in possible multiple comparisons and no adjustment for such.

Also, the unit of analysis is the wound (it appears). The horse may also be a unit of analysis which gets into the issue of intraclass correlation of the treatments across these units. The variance of the estimates could be

inflated in this situation.

The unit of analysis issue and multiple tests could possibly be addressed by the authors already as seen on line 157 in which they state that the model therefore included an auto-regressive (first-order) correlation structure that allowed for this or is this addressing the spatial (location) and time correlation? All of this has to be better clarified by the investigators.

**********

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Reviewer #2: No

Reviewer #3: No

**********

PLoS One. 2024 Mar 7;19(3):e0299990. doi: 10.1371/journal.pone.0299990.r006

Author response to Decision Letter 2

1 Feb 2024

Response to Reviewer #3:

Comment: The authors set out to evaluate the secondary intention wound healing in the horse's limbs when treated with the synthetic epidermis spray or with a standard bandaging technique. The design and analysis appears to be simple using the generalized least square models with area, volume, height and depth as the dependent variables and time (in days), treatment (T1 vs T2), body part (cannon vs fetlock) and all interactions as the independent variables. The interpretation, as noted below, is not simple. Also the investigators claim that six horses are adequate for the sample size as noted from previous publications. However, the sample size is not discussed and certainly not justified with respect to any of the variables in this presentation.

Answer: Thank you for your remarks.

1. We clarified the generalized least square model concerning the auto-regressive correlation structure.

2. We included an additional table presenting a simplified version of the analysis, which can be considered a sensitivity analysis, with the horse as the unit of analysis. We computed and averaged the area under the curve for each wound kinetics, grouped by horse and treatment (refer to the table below). The conclusions mirror those of the complex model: the height is observed to decrease with SES bandaging, while the area and volume remain unaffected.

Correction for multiple comparisons does not seem necessary, as observations on the various outcomes at different times are highly dependent.

Concerning the sample size, it was predefined based on material constraints. A posteriori, this sample size proved adequate to highlight the treatment effect on wound height evolution and convincingly demonstrate that the healing duration is not strongly influenced by the type of bandage.

Area under the curve 95% CI

Horse Outcome B N Difference Mean lower limit upper limit P

A Area 177 234 -57 8 -45 61 0,70

C 166 202 -36

H 172 141 31

I 218 172 46

V 183 191 -8

Y 233 158 75

A Depth 1,9 2,0 -0,1 -0,3 -0,6 0,0 0,07

C 1,4 1,9 -0,5

H 1,2 1,9 -0,7

I 1,6 1,6 0,0

V 1,3 1,5 -0,2

Y 2,2 2,3 -0,1

A Height 49 51 -2 16 1 31 0,04

C 48 42 6

H 44 24 20

I 55 29 26

V 50 40 11

Y 60 24 37

A Volume 16 21 -5 8 -3 19 0,13

C 17 17 0

H 17 7 10

I 27 13 14

V 19 16 3

Y 31 7 24

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s004.docx^{(25.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0299990.r007

Decision Letter 3

Carlos Alberto Antunes Viegas

7 Feb 2024

PONE-D-23-16094R3The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.PLOS ONE

Dear Dr. Paindaveine,

Please submit your revised manuscript by Mar 23 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Carlos Alberto Antunes Viegas, DVM; MSc; PhD

Academic Editor

PLOS ONE

Journal Requirements:

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: The limitations are listed. However, given the small sample size, the authors should put a qualification in the conclusions that, "All statistical inference (p-values especially) must be interpreted with caution, given the size of the sample.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #3: No

**********

PLoS One. 2024 Mar 7;19(3):e0299990. doi: 10.1371/journal.pone.0299990.r008

Author response to Decision Letter 3

13 Feb 2024

Response to Reviewer #3:

Comment: The limitations are listed. However, given the small sample size, the authors should put a qualification in the conclusions that, "All statistical inference (p-values especially) must be interpreted with caution, given the size of the sample.”

Answer: Thank you for your remarks. We added the sentence to the manuscript.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s005.docx^{(18.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0299990.r009

Decision Letter 4

Carlos Alberto Antunes Viegas

20 Feb 2024

The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses.

PONE-D-23-16094R4

Dear Dr. Charlotte Paindaveine,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Carlos Alberto Antunes Viegas, DVM; MSc; PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: (No Response)

**********

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Reviewer #3: (No Response)

**********

6. Review Comments to the Author

Reviewer #3: (No Response)

**********

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Reviewer #3: No

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PLoS One. doi: 10.1371/journal.pone.0299990.r010

Acceptance letter

Carlos Alberto Antunes Viegas

26 Feb 2024

PONE-D-23-16094R4

PLOS ONE

Dear Dr. Charlotte C,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Data of the wound assessment through the study period.

(XLSX)

pone.0299990.s001.xlsx^{(70.4KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s002.docx^{(19.9KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s003.docx^{(19.5KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s004.docx^{(25.6KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0299990.s005.docx^{(18.2KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

The effects of a synthetic epidermis spray on secondary intention wound healing in adult horses

Paindaveine Charlotte C

Bihin Benoit

Lepage Olivier M

Roles

Abstract

Objectives

Methods

Results

Conclusion

Introduction

Material & methods

Animals

Wound model

Treatments

Fig 1. Polymerization of the synthetic epidermis spray on a left thoracic limb using a 395 nm UV light for 60 seconds at a distance of approximately 30 cm.

Wound assessment

Statistical analysis

Results

Fig 2. Evolution of wound area, volume, height and depth.

Table 1. Comparison of wound area, volume, height and depth at different timepoints.

Fig 3. Macroscopic aspect of healing thoracic limb wounds.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Carlos Alberto Antunes Viegas

Roles

Author response to Decision Letter 0

Decision Letter 1

Carlos Alberto Antunes Viegas

Roles

Author response to Decision Letter 1

Decision Letter 2

Carlos Alberto Antunes Viegas

Roles

Author response to Decision Letter 2

Decision Letter 3

Carlos Alberto Antunes Viegas

Roles

Author response to Decision Letter 3

Decision Letter 4

Carlos Alberto Antunes Viegas

Roles

Acceptance letter

Carlos Alberto Antunes Viegas

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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