SES was administered at two concentrations as a liquid solution and as gel formulations. The low-concentration SES (SES-low) contained 20 parts per million active species of chlorine and oxygen (0.002%), has a pH of 6.5-7.5 and oxidation-reduction potential of ~850 mV (Estericide^® Solución Antiséptica; Esteripharma^® S.A. de C.V.; cat. no. 0412C2016 SSA). Gel formulation is commercially available as Estericide^® Gel Antiséptico (cat. no. 1594C2014 SSA). The high concentration SES (SES-high) contained <80 parts per million (>0.008%) chlorine (Microdacyn^® Solución Antiséptica; Aerobal S.A. de C.V., México; cat. no. 1075C2003 SSA). The hydrogel formulation is commercially available as Microdacyn^® Hydrogel Gel antiséptico and contains ≥40 parts per million of free chlorine (cat. no. 0176C2014 SSA). NF ointment was used at a concentration of 0.2% as the commercially available Furacin^® (Siegfried Rhein^® S.A. de C.V; cat. no. 31258 SSA) and S cream was used at a concentration of 1% as the commercially available Bioargirol-C (Bioresearch de México S.A. de C.V; cat. no. 489M2000 SSA). These products are widely available and frequently used in patients suffering burns covering <15 (adults) or 5% (children) of their total body surface area (19). Physiological saline solution (0.9% NaCl solution; PiSA Pharmaceuticals) was used as a placebo (Pl) in the control group.

Male BALB/c mice (n=252; Inotiv; age, 10-14 weeks; weight, 25-30 g) were used. The duration of the experimental procedure was 32 days. Mice were randomly assigned to seven groups, each comprising 36 animals as follows: Pl, SES-low, SES-low + G, SES-high, SES-high + G, NF and S. All animals were kept at 21±2˚C with 48% humidity in a 12/12-h light/dark cycle, with food and water provided ad libitum. The mice were kept in cages, with a maximum of 6 mice/cage.

The animal experiments were approved by the Research Ethics Committee of the Colima State Institute of Cancerology, Colima, Mexico (approval no. CIIECAN/06/19). Animals were handled in accordance with institutional guidelines (35) and the official Mexican standard for the care and use of laboratory animals (Official Mexican Standard NOM-062-ZOO-1999: Technical specifications for the production, care, and use of laboratory animals) (36-38), in addition to the eighth edition of the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences of the USA (2011) (38). Mice were observed daily to assess for clinical signs of toxicity or distress, and behavioral changes were evaluated by functional observational battery parameters such ass salivation, lacrimation, signs of distress, changes in eating and drinking, activity levels and any signs of infection or discomfort at the wound site (39-41). Humane endpoints were weight loss >20% of body weight, severe illness, infection or necrosis at the wound site or any signs of severe distress, such as lack of grooming, abnormal posture or reduced activity (37,42). No animals met the humane endpoints for euthanasia before the end of the experiment and none were found dead. Pain management included administering paracetamol (200 mg/kg) orally for the first 5 days (1,43-45) and ketamine (120 mg/kg) and xylazine (15 mg/kg) were used during burn induction and prior to euthanasia (45). Death was verified by cessation of heartbeat and respiration, as well as the absence of reflexes (46).

The scald burn model was established as described by Abdullahi et al (47). At 1 day before the intervention, the dorsal area of the mice was shaved and depilated with cream (Nair Sensible, Reckitt Benckiser) for 30 sec and residues were removed with warm water. On the day of the intervention, mice were anesthetized using intraperitoneal ketamine (120 mg/kg) and xylazine (15 mg/kg; PiSA Pharmaceuticals^®, Agropecuaria). Each mouse was placed in a supine position on a template of flame-resistant plastic mold, which included a window exposing the predetermined skin surface area. A test tube with 95˚C water was brought into direct contact with the exposed skin surface of the mouse for 7 sec, resulting in an oval burn with diameters of 1.5-2.0 cm (Fig. 1A).

A total of 12 mice/group was selected to investigate wound size change. Treatment was applied once/day for 32 consecutive days, starting on day 0 of the study (the day of the burn; Fig. 1B). Liquid was directly applied to the burn site, resulting in a total volume of ~1 ml product. For the gel, ~1 g was applied to the burn area using a sterile plastic applicator. On days 6, 9, 18 and 32, 6 mice/group were sacrificed by decapitation after being anesthetized as previously described (48).

Periodic measurements of the wound area were taken on days 3, 6, 9, 18 and 32 as described by Zhang et al (49) with minor modification. The mice were immobilized and the contour of the wound was traced using a transparent graph sheet and marker. The resulting images were analyzed to determine the burn wound area. Wound area reduction was calculated using the following formula: Wound contraction (%)=100-[(wound size x100)/mean value of day 0 wound size]. Changes in morphology were documented by capturing images with a digital camera (Nikon AF-S VR Micro Nikkor; Nikon Corporation) at a constant focusing distance. The resulting images were analyzed using Fiji2.0 software (National Institutes of Health) (50). All images were captured under the same light and exposure.

Samples of burn areas were surgically excised (1x1 cm) and rinsed with cold PBS following sacrifice and fixed in 10% neutral buffered formalin at room temperature for 24 h, washed, dehydrated with ethanol and embedded in paraffin. The obtained blocks were cut into 5-mm-thick tissue sections, mounted on glass slides, deparaffinized and rehydrated. Slides were stained with hematoxylin-eosin (H&E) at room temperature for 30 min for evaluation of inflammatory infiltration state and epithelial regeneration, and with Masson's trichrome at room temperature for 60 min to analyze collagen fibers (51-55). Each measurement was independently conducted by two qualified scientists in a blinded manner, ensuring unbiased data collection and analysis. Images were captured using a digital camera model Axiocam MRC-5 connected to a t bright-field optical light microscope model AxioPlan 2 M (Zeiss GmbH) with a motorized stage (total magnification, x100, 200 and 400). MosaiX and Autofocus modules were used to scan images of the entire sample surface and the lesions were measured using a calibration line. All images were captured under the same illumination and exposure times using the AxioVs 40 V.4.7.0.0 image software (Carl Zeiss Imaging Solutions GmbH). All histological data were obtained from 30 randomly selected fields of view from 6 mice (5 data/mouse). Counts of total inflammatory cells, polymorphonuclear neutrophils (PMNs) and mononuclear leukocytes (MNC) were manually determined using five randomly selected fields of view (magnification, x10 and 40). The inflammatory infiltration state was determined according to degree of inflammatory infiltrate, by assigning a semi-quantitative and discontinuous score: 1-plenty; 2-moderate and 4-few (51,52,54).

Samples stained with Masson's trichrome were analyzed using a Motic BA310E optical light microscope (Motic China Group Co., Ltd.; magnification, x10). A total of three microphotographs were captured for each tissue sample with a Moticam 1080 digital camera (Motic China Group Co., Ltd.) under the same lighting and exposure. The proportion, shape and type of collagen fibers were analyzed using Fiji 2.0 software. Collagen orientation was classified as follows: 1, vertical; 2 for mixed, and 4 for horizontal. The collagen patterns were categorized as: 1 for reticular, 2, mixed, and 4 for fascicular. The amount of early collagen was qualitatively evaluated as 1, profound; 2, moderate; 3, minimal and 4, absent. Mature collagen was classified as 1, profound; 2, moderate and 4, minimal (52-55).

Epithelial regeneration was evaluated by assessing the migration of cells to the wound edge, defined as the area where epithelial cells meet the edge of the wound, divided by the distance from the wound bed, the base of the wound where new tissue is forming, multiplied by 100% and scored as follows: 0-0; 1- >0 and<50; 2, ≥50 <100; 3-100% and irregular thickness, and 4, 100% and normal thickness (56). Additionally, quantitative (µm) and qualitative (yes/no) measurements of epidermal detachment visualized as separation of wound edges viewed at 2.5X magnification, as well as the thickness of the epidermal lesion (40X magnification), were performed (57). The number of blood vessels and follicles/field was included for evaluation, along with the presence or absence of scar tissue (58). Furthermore, a semi-quantitative assessment of granulation tissue (1, deep; 2, moderate; 3, scant and 4, absent) and a qualitative assessment of presence of the stratum corneum at 2.5X magnification was performed (51,52,59).

Wound healing score and status were determined as described by Gupta and Kumar and Santos et al (51,52). The parameters assessed included granulation tissue amount, inflammatory infiltrate, collagen fiber orientation and pattern and early and mature collagen amount. The total healing score was calculated by adding the scores of individual criteria, with lower scores indicating poorer wound healing. Healing status was graded as follows: 8-11, poor; 12-15, acceptable and 16-19, good (51,52).

Data are presented as the mean and SEM (n=≥6. Normal distribution of data was determined using the Shapiro-Wilk test. Data were analyzed using one-way ANOVA for normally distributed data (parametric) or Kruskal-Wallis test for non-normal or ordinal data (non-parametric). Post hoc analysis was performed using Bonferroni's comparisons or Mann-Whitney U test (non-parametric) and Tukey's multiple comparison test (parametric). The statistical analysis was performed using IBM SPSS version 20 software (IBM Corp.) P<0.05 was considered to indicate a statistically significant difference.

Fig. 2 shows representative pictures of the wound healing process and reduction of burn areas, providing a visual and quantitative assessment of the treatment outcomes.

On day 3, NF, SES-low + G, and S treatments exhibited the smallest wound areas, with closure of 59.7±5.9, 48.7±4.0 and 44.3±7.1%, respectively (Table SI). The NF group exhibited significantly greater wound closure compared with all other groups. SES-low + G showed better wound healing compared to SES-high, though not significantly different from the S group. Pl and SES-high groups had the poorest closure (Table SII).

On day 6, wound closure was highest in the SES-low + G (62.6±2.5%), NF (58.2±10.5%), and SES-high + G (59.2±9.2%) groups (Tables SI and SII). SES-low +G treatment demonstrated significantly better closure compared to the SES-low and SES-high groups.

At day 9, the SES-low + G (72.4±1.4%) and NF (67.8±12.0%) groups again exhibited the highest wound closure rates (Table SII). These were statistically different from the Pl and SES-high + G groups, showing superior wound healing outcomes.

From day 18 to 32, all treatments groups demonstrated similar wound healing progress, eventually reaching full wound closure (Fig. 2A). However, the SES-low + G and NF groups continued to exhibit the smallest final wound areas (Fig. 2B, Table SI), with the highest overall closure rates (Table SII). While SES-low + G outperformed the other treatments on days 6 and 9, the differences were not statistically significant (Table SII).

To assess the inflammatory response number of polymorphonuclear cells (PMNs) (Table SIII) and monocytes (Table SIV) at the days 6, 9, 18 and 32 of the wound healing process. The total inflammatory infiltrate was also measured, and multiple comparison tests were conducted to evaluate the significance of these counts across treatments (Table SV). As expected, abundant inflammatory infiltrate was observed in the early stages of wound healing as part of the typical course of the re-epithelization process (52,60-62) and reached maximum values on day 9 (Fig. 3A). On day 6, the S, NF and SES-high + G groups exhibited the highest cell/field values (92.90±7.19, 72.80±5.35 and 54.30±5.87, respectively). S showed the most abundant infiltrate, being significantly different from the rest of the groups except with NF. On the other hand, SES-low (29.33±3.20), SES-low + G (33.10±3.16) and SES-high (35.86±2.87) groups had similar effects to Pl (26.06±4.43).

On day 9, a general and significant increase in inflammatory infiltrate was observed (Table SV). However, SES-low (63.30±4.87) and SES-low + G (77.10±5.04 cells/field) groups exhibited significantly lower cell counts compared with all other groups. S (134.75±5.58) and NF (131.50±5.19 cells/field) groups had the highest levels of inflammatory infiltrate, followed by SES-high (120.93±5.77 cells/field), SES-high + G (119.93±5.00 cells/field) and Pl (110.90±6.47 cells/field) groups. No statistical differences were observed between SES-high, SES-high + G, NF, S and Pl groups. Fig. 3B shows histological images on day 9, demonstrating the differences in inflammatory infiltrate abundance, primarily macrophages (▲). SES-low and SES-low + G produced less infiltration in the tissue, indicating an anti-inflammatory.

On day 18, the inflammatory infiltrate all groups became similar, without no significant differences observed-The average cell count was 41.44±3.01 cells/field. By day 32, the counts of total inflammatory cells decreased in all groups. The S group exhibited the highest inflammatory infiltrate, with a mean value of 33.04±2.76 cells/field, which was significantly higher than the rest of the groups. SES-low and SES-low + G exhibited the lowest cell counts, with mean values of 16.53±1.20 and 18.00±1.01 cells/field, respectively. The cell counts in the SES-low group were significantly lower compared with those of the S and SES-high + G group (24.10±1.3). SES-low was better than SES-high at modulating the inflammatory process. Additionally, S and NF groups exhibited inflammatory infiltrate, suggesting an irritant effect. In the specifics counts of PMNs, the SES-low group had consistently lower cell counts throughout the study, maintaining modest values compared to the S and NF groups, particularly on day 32 where SES-low recorded 3.0±0.49 cells/field compared to S at 6.6±0.38 (Table SIII). Similarly, for monocytes, the SES-low group showed reduced counts at all time points, especially at day 32, with 13.5±1.54 cells/field compared to S, with 26.4±2.26 cells/field (Table SIV). This suggests that the treatment with SES-low and SES-low + G, may induce a regulated and balanced inflammatory response at the early and late stages of the healing process. S group induced a stronger pro-inflammatory reaction, particularly evident at later stages of wound healing.

On day 6, all groups exhibited epidermal detachment (Fig. 4). However, SES-low and SES-low + G groups showed a more defined lesion with indications of dermal recovery and greater differentiation of cutaneous layers, while Pl showed deeper burn damage. Subsequently, on day 9, a serohemorrhagic crust was present in all groups, indicating an ongoing repair process (+). By day 18, the repair and re-epithelization was indicated by epithelial edge junctions and the hair follicle presence (▲). The Pl, SES-high and NF groups did not show epithelial edge union, the process where wound edges come together as new skin forms, while SES-low, SES-low + G, S and SES-high + G-treated groups exhibited complete junction of epithelial edges. Additionally, traces of serohemorrhagic crust were observed in the Pl, SES-high + G and S groups. The presence of hair follicles in SES-low, SES-low + G and NF groups indicated a more advanced repair process (63-65). Furthermore, on day 32, complete healing and re-epithelization of the burn was observed in all groups, as evidenced by the union of edges, indicating full closure of the wound, and presence of a stratum corneum and hair follicles. The SES-low, SES-low + G, SES-high + G and NF groups showed a thicker stratum corneum (*), compared with Pl, SES-high, and S groups. Therefore, NF, SES-low + G and SES-low groups demonstrated a more advanced progression towards re-epithelization, characterized by a compact and well-defined serohemorrhagic crust.

SES-low + G, SES-low and Nitrofurazone generate better collagen matrix reorganization. The analysis of collagen parameters at days 6, 9, 18, and 32, was performed using Mason's staining (Fig. 5). The scores for orientation and amount of early or mature collagen at day 32 are presented in Fig. 6. Scores for days 6, 9 and 18 are shown in Fig. S1, Fig. S2 and Fig. S3, respectively while Table SVI, Table SVII and Table SVIII provide the statistical analysis of these parameters. The collagen pattern showed no significant differences between groups on any of the days.

Granulation tissue and inflammatory infiltrate were predominant at the early stages of wound healing with collagen deposition mainly early collagen (light blue) observed (Fig. 5). As wound healing progressed, increased collagen deposition was noted, with agglomeration of mature collagen, stained as deep blue, in SES-low, SES-low + G, SES-high, SES-high + G and NF groups, alongside a gradual decrease in inflammatory infiltrate and granulation tissue. By day 32 of follow-up, the collagen matrix reached its maximum in all groups, with statistical differences noted for minimal or absent deposition of young collagen in NF, SES-low and SES-low + G groups (Fig. 6) (66,67). Clear differences in the aggregation and organization of mature collagen were observed among treatments. In comparison with SES-low, S, NF and Pl showed thicker and more irregular deposition of mature collagen, with greater collagen matrix deposition. Vascularization and newly formed hair follicles were also noted in the SES-low group (Fig. 5) (66). The semi-quantitative analysis of collagen matrix deposition is shown in Fig. S1, Fig. S2 and Fig. S3.

At day 32, SES-low + G and SES-low exhibited the most uniform and horizontal orientation of collagen fibers, followed by SES-high (Fig. 5). This indicated better collagen matrix reorganization and improved final healing process (Fig. S4). Orientation analysis of collagen fibers at day 32 revealed significant differences between SES-low, SES-low + G and S, NF, Pl, and SES-high (Fig. 6). According to semiquantitative scores, NF was the third best treatment for inducing organized collagen matrix deposition, though no significant difference was observed compared to SES-high (Fig. S1, Fig. S2 and Fig. S3).

At day 32, granulation tissue was absent and an uniform collagen pattern across the groups. This indicates that the healing process was complete and comparable in terms of collagen deposition, so all groups were rated with the highest score for this parameter. Additionally, scores for the amount of inflammatory infiltrate and type/abundance of deposited collagen were assigned. The highest scores were assigned to treatments that induced the lowest inflammatory infiltrate (Fig. 3; Table SIII, Table SIV and Table SV). For quality of collagen deposition, the highest values were registered for treatments that produced an organized matrix, composed by horizontal deposition (Fig. 6; Table SVI, Table SVII and Table SVIII). The higher the healing score, the more favorable outcome in terms of tissue repair. A high healing score suggested that the healing process progressed well and achieved the desired results. At day 32, SES-low + G and SES-low demonstrated the highest healing scores (20.85±0.36 and 20.03±0.19, respectively) compared with Pl with 17.50±0.22 and S with (17.11±0.20 (Fig. 7; Table SIX). SES-high, SES-high + G and NF had similar performance (~18 points) and without significant differences between them or the rest of the groups. It is interesting to notice that on day 6, treatments with the highest healing scores were NF, SES-low and SES-low + G; NF was significantly different compared with S. At day 9, SES-low + G, SES-low and Pl groups had the highest values; only SES-low + G was significantly different compared with S. These results partially coincide with the speed of wound closure observed in Fig. 2, which demonstrates that proper wound healing is not only matter of fast wound closure.

There was a significant difference in healing status at day 32 between Pl and SES-low and SES-low + G (P<0.01), as well as between S and SES-Low + Gel. At this day, SES-low + G yielded the best healing status (Fig. 8; Table SX). This effect can be attributable to the low inflammatory infiltrate observed in such groups, during wound healing evolution and particularly at day 9. On the contrary, treatments with Pl and S had the worst healing status, with no significant difference between them. No significant differences in healing status were observed between groups at days 6, 9, or 18 (Fig. 8; Table SX).