Therapeutic efficacy of Carboxymethyl Cellulose-based zinc selenite nanoparticles in infected wound healing in a rat model

Therapeutic efficacy of Carboxymethyl Cellulose-based zinc selenite nanoparticles in infected wound healing in a rat model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Therapeutic efficacy of Carboxymethyl Cellulose-based zinc selenite nanoparticles in infected wound healing in a rat model Nermeen- Shereef, Marwa Abass, Emad Tolba, Ekramy Elmorsy, Basma M Hendam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9549669/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 16 You are reading this latest preprint version Abstract Background In the fight against bacterial infections and the growing threat of antibiotic resistance, there is a pressing need to develop safe and effective antimicrobial wound dressings that could be used as alternatives to antibiotics, selectively target bacteria, and overcome the challenges of antibiotic resistance. This study introduced a novel material that meets these criteria: CMC/zinc selenite nanoparticles in the form of a gel. Materials and methods Fifty-four adult male Albino Wistar rats, aged 10–12 weeks with an average body weight of 256.7 ± 13.7 g, were used. A 23 mm diameter full-thickness circular cutaneous defect in the dorsal region between the two scapulae. The CMC/zinc selenite and the infected groups received intradermal inoculation of the wound with 0.3 ml freshly prepared suspension of S. aureus. Rats were divided into three groups (n = 18 per group) after 3 days of surgery according to the infection and treatment protocol ( placebo group : wounds without infection or treatment, infected group : infected wounds without treatment, CMC / zinc selenite group : infected wounds received a topical application of 1 ml of CMC/ZnSeONPs gel twice daily). The wounds were evaluated through macroscopic, immunohistochemical, histopathological, and bacteriological analyses. Results &Conclusion The synthesized advanced gel of CMC/Zinc selenite shows exceptional antimicrobial properties due to its semicrystalline structure and its small nanometric size, promoting faster healing of infected full-thickness skin wounds through exhibiting rapid re-epithelialization by stimulating EGFR, and effective contraction alongside angiogenic properties, tissue regeneration, and could be considered as a protective agent against infection-related weight loss. Biological sciences/Biotechnology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Microbiology Infected wound CMC/zinc selenite nanoparticles Staphylococcus aureus wound healing EGFR α-SMA gel Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1. Introduction The development of strategies to modulate inflammation and enhance tissue regeneration following surgical procedures remains a significant challenge in clinical practice, owing to the complexity of the healing process and the diverse array of cellular and molecular components involved [ 1 ]. The management of exuding wounds is further complicated by microbial contamination, whose prompt eradication is vital to ensure successful wound healing [ 2 ]. The healing of infected wounds has consistently represented a critical global healthcare issue, attributed to its high prevalence, substantial disability burden, and elevated mortality rates [3, 4]. Over the past century, there has been a marked rise in wound infections, contributing significantly to the epidemiology of microbial infection [ 5 ]. Skin injuries, when complicated by infection, can exacerbate trauma and may ultimately lead to fatal outcomes through suppression of the immune system [ 6 ]. Staphylococcus aureus ( S. aureus) is the principal etiological agent of skin and soft tissue infections across all ages, climates, and geographic regions [ 7 – 9 ]. In the absence of treatment, wounds infected with S. aureus exhibited a healing period of approximately 30 to 35 days, as determined by macroscopic evaluation and wound size analysis [ 10 ]. Current approaches for wound healing and infection management primarily utilize antibiotics and analgesics. Despite growing concerns regarding their therapeutic efficacy, antibiotics continue to represent the most commonly employed agents in wound treatment [ 11 ]. Nevertheless, the prolonged use of high doses of multiple drugs not only imposes a significant financial burden on patients but also contributes to the emergence of drug-resistant bacteria, ultimately diminishing therapeutic efficacy [12, 13]. Emerging techniques in wound management, such as photomodulation, skin substitutes, and external tissue expanders, have shown variable efficacy; however, their substantial cost remains a major limitation to their widespread application [ 14 ]. Carboxymethyl cellulose (CMC) is a cellulose derivative that dissolves in water, synthesized by introducing carboxymethyl groups onto the cellulose backbone through a carboxymethylation reaction [ 15 ]. CMC has emerged as a smart wound dressing material with excellent water absorption capabilities due to the polyelectrolytic properties of CMC, along with its sensitivity to pH and ionic strength [ 16 ]. Its hydrophilic nature allows CMC to absorb wound exudate and be easily combined with other polymers or bioactive agents, enhancing its functionality [ 17 ]. Wound dressing materials based on CMC provide biocompatibility, biodegradability, structural similarity to native tissue, affordability, and non-toxicity, positioning them as excellent candidates for infection management and wound healing applications [ 17 ]. Although CMC gels offer an effective approach to wound management by creating an optimal wet environment, promoting cell growth, and potentially separating the wound site, they do not fulfill all requirements for chronic wounds. Thus, new functions and features are highly recommended to assist in the wound healing of chronic wounds. This could be achieved by combining with other biopolymers or bioactive nanoparticles. Nanoparticles (NPs) have emerged as promising novel antibacterial agents, demonstrating significant efficacy against various infectious pathogens [ 18 ]. Nanomaterials can modulate a range of cellular and molecular activities within the wound microenvironment, exerting antibacterial, anti-inflammatory, and angiogenic effects that facilitate the transition from a non-healing to a healing state [ 19 ]. They have demonstrated effectiveness in combating both susceptible and resistant pathogens, as well as tolerant biofilms [ 20 ]. The antimicrobial effects of NPs are thought to arise from a variety of potential mechanisms, including the generation of reactive oxygen species (ROS), disruption of protein and DNA synthesis, activation of metabolic pathways, and alterations in cell wall structure and membrane permeability [ 21 ]. Selenium-based nanoparticles (Se-NPs) have emerged as highly promising bioactive nanoparticles in regenerative medicine due to their unique physicochemical properties, biocompatibility, and multifunctional therapeutic potential with drug delivery capabilities. They are being extensively researched for their anticancer, antioxidant, anti-diabetic, and anti-inflammatory properties, as well as their role in targeted drug delivery. Among them, CMC/zinc selenite (ZnSeO) is an inorganic compound combining zinc (Zn²⁺) and the selenite anion (SeO₃²⁻). While less biologically studied than other zinc or selenium compounds (e.g., zinc selenide (ZnSe), silver selenide (Ag 2 Se), or sodium selenite (Na₂SeO₃)), it has intriguing properties that make it relevant in materials science, catalysis, and biomedicine[ 22 – 25 ]. To the best of our knowledge, CMC / zinc selenite nanoparticles (ZnSeO NPs) have not yet been reported in biomedical literature. The growing interest in zinc- and selenium-based nanomaterials suggests significant untapped potential—particularly in wound healing, antimicrobial therapy, and bioactive coatings. Taking into account the additional properties of ZNO and SeNPs, such as immunomodulatory and anti-inflammatory activities [ 26 ]. Notably, all antimicrobial investigations on SeNPs have reported no bacterial resistance thus far, highlighting their potential as an effective option for treating infectious and chronic wounds [ 27 ]. Thus, this study aimed to assess the effectiveness of topically applied CMC / zinc selenite (CMC/ZnSeONPs) gel for infected full-thickness cutaneous wound healing in rats. The assessment was conducted through macroscopic (body weight, wound size, and contraction), histopathological, and immunohistochemical evaluations, bacterial count, and bacterial viability tests. 2. Materials and methods 2.1. Materials Zinc acetate dihydrate (C 4 H 6 O 4 Zn ⋅ 2H 2 O), sodium selenite (Na 2 SeO 3 ), and sodium hydroxide (NaOH 2 ) were used as chemicals in the synthesis of nanoparticles. All the chemicals were bought from Sigma-Aldrich. The chemicals were used without any further processing. Double-distilled water was used as a solvent for solution preparation and washing. 2.2. Preparation of zinc selenite nanoparticles (Zn SeONPs ) CMC / zinc selenite nanoparticles (ZnSeONPs) were prepared using a chemical reduction approach. Firstly, 0.40 g of Zinc acetate dehydrate (Zn (CH 3 CO 2 ) 2 ·2H 2 O) and 0.25 g of sodium selenite (Na 2 SeO3) were dissolved in 100 ml of DW. The reaction mixture was placed on a magnetic stirrer for 30 min, and then 5 ml of ascorbic acid solution (100 mM) was slowly added while stirring continuously. The mixture color was changed to reddish orange. The reaction mixture was heated up to 80 °C, and the pH was maintained at 10 using NaOH solution (0.1 M). After 2 h, the reaction components were precipitated using an absolute ethanol solution (50 ml) to remove unreacted components and byproducts. The ZnSeONPs were collected by centrifugation at 6,000 rpm for 10 min and washed with water three times. Finally, the precipitate was dried at 80 °C in an air oven and stored at 4 °C in the dark. 2.3. Preparation of CMC/ZnSeONPs gel The dried ZnSeONPs were mixed with sodium CMC solution to obtain CMC gel enriched with ZnSeONPs. First, 2g of sodium CMC were dissolved in 90 mL of distilled water at 80°C for 15 min under constant stirring. After that, the as-prepared ZnSeONPs (0.1g) were dispersed in 10 ml distilled water. The obtained ZnSeONPs were added dropwise to the CMC solution with a magnetic stirrer until the transparent CMC mixture turned milky. The ZnSeO and CMC mixture was subjected to high mixing speed using a high-shear homogenizer at 5000 rpm for 10 min, followed by sonication in a bath sonicator for 30 min. The obtained ZnSeO/CMC gel was stored at 4 o C for further use. 2.4. Ethical approval: 3. The study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code (VM.MS.24.10.166). All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical All procedures were documented following the guidelines specified by ARRIVE [28] 3.1. Animals: In this study, fifty-four adult male Albino Wistar rats, aged 10-12 weeks with an average body weight of 256.7 ± 13.7 g, were used. The animals were obtained from the animal facility at the Faculty of Science, Mansoura University, Mansoura, Egypt. The rats were housed individually in separate polypropylene cages in the laboratory at Mansoura Teaching Hospital. Bedding consisting of wood shavings was provided, and it was changed daily. The lighting cycle was set to 12 hours of light per day. The rats had unrestricted access to food and water. Before the experiment, the rats were acclimatized to the laboratory conditions for two weeks and were thoroughly examined to ensure that only healthy animals were included in the study. 3.2. Preparation of bacterial inoculum: According to [29], S. aureus was cultured in broth at 37°C for 18 hours prior to its use. The culture was then washed with 0.1 M phosphate-buffered saline (PBS) at pH 7.2, and a suspension with a concentration of 2×10⁹ CFU/mL was prepared. The concentration was determined using a colorimetric spectrometer (Biostc) from the Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Mansoura University, Egypt. The freshly prepared S. aureus suspension was then injected into the skin wounds of the infected and CMC / zinc selenite groups. This suspension was obtained from the Bacteriology, Mycology, and Immunology Department at the Faculty of Veterinary Medicine, Mansoura University. 3.3. Microstructure Characterization To confirm the formation of the ZnSeO-NPs ; X-ray diffraction (XRD) was carried out to determine the crystal structure of the as-prepared materials. Also, Transmission electron microscopy (TEM) was performed to study the particle size of the material in nano scale, crystal structure and examine the morphological characteristics of nanoparticles formulas by a high-performance digital imaging transmission electron microscopy (JEOLH-7650, Hitachi High-Technologies Corp., Tokyo, Japan) with an acceleration voltage operating at 200 kV. 3.4. Experimental procedures intraperitoneal injection of 5 mg/kg xylazine HCL (20 mg/ml, Xylaject, ADWIA, Cairo, Egypt), and 60 mg/kg ketamine HCL (50 mg/ml, Aneket ® , NEON Laboratories Ltd, Mumbai, India) for anesthesia of the rats. Aseptic preparation of the dorsal region between the two scapulae. The rats were transported to the surgery room to make a 23 mmdiameter full-thickness circular cutaneous defect by excision of the rat skin. The CMC / zinc selenite and the infected groups received intradermal inoculation of the wound lips with 0.3 ml freshly prepared suspension of 2×10 9 CFU/mL S. aureus [29]. Animals received an intramuscular injection with a single dose of meloxicam (0.4 mg/kg) for analgesic effects. Rats were divided into three groups (n=18 per group) after 3 days of surgery according to the infection and treatment protocol: A. Placebo group: wounds without infection or treatment. B. Infected group: infected wounds without treatment. C. CMC / zinc selenite group : infectedwounds received a topical application of 1 ml of CMC/ZnSeONPs geltwice daily after flushing. All groups undergo flushing twice daily with 5 mL of normal saline (0.9% sodium chloride solution, 500 mL; Ultimate Pharma, Egypt). Neither antiseptics nor antibiotics were utilized throughout the study. All wounds were surgically induced by the same experienced surgeon, followed by standardized bacterial inoculation. 2.9 Evaluation of wound healing: 3.5.1. Macroscopic Examination Wound evaluations were performed by a single experienced surgeon, who was masked to group allocation, on postoperative days 0, 3, 7, 11, 15, and 19. A- Body weight (g) The rats' body weight (g) was measured on postoperative days 0, 3, 7, 11, 15, and 19. B- Wound size (mm 2 ) Wound size was measured using a digital caliper and documented with photographs on postoperative days 0, 3, 7, 11, 15, and 19 C- Wound contraction (%) The extent of wound contraction was evaluated on postoperative days 0, 3, 7, 11, 15, and 19, based on the formula described by [30], and subsequently analyzed statistically. (1) Percentage of wound size at day (x) = wound size at day (x) mm 2 / wound size at day (0) mm 2 x 100. (2) Percentage of wound contraction = 100 - percentage of wound size at day (x) 3.5.2. Microscopic evaluations On postoperative days 7, 14, and 21, six animals from each group were euthanized via intraperitoneal injection of pentobarbital overdose (120 mg/kg, Nembutal®, 50 mg/ml, Akorn Operating Company, Gurnee). Skin samples for histopathological and immunohistochemical analysis were preserved in 10% neutral buffered formalin. All euthanized rats and bedding material were hygienically disposed of. A. Bacterial count Infected wounds were swabbed using a sterile cotton-tipped applicator, which was rotated at the center of the wound. The swabs were then transferred into sterile tubes containing 1 mL of transport solution and subjected to serial dilution with the same medium. The samples were incubated at 37°C for 24 hours after being cultured on Baird-Parker agar plates (Himedia). B. Bacterial viability test Overnight bacterial cultures were grown and adjusted to a density equivalent to 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU/mL) by suspending the cells in sterile PBS. A sterile 24-well plate was prepared, with each well containing 1 mL of nutritive broth with 10 µL of the bacterial suspension for the infected group, and another sterile 24-well plate with each well containing 1 mL of nutritive broth, CMC/ZnSeONPsgel, and 10 µL of the bacterial suspension for the CMC / zinc selenite group. The plates were incubated at 37°C under humid conditions for 1, 6, 12, and 24 hours. To assess bacterial viability, the wound dressing sample (CMC/ZnSeONPsgel) was transferred into sterile Eppendorf tubes containing 1 mL of PBS immediately after incubation. The tubes were vortexed for 20 seconds to release viable cells into the suspension. From each suspension, a 30 µL aliquot was mixed with 270 µL of PBS in a 96-well plate. Serial dilutions were then made, and the resulting samples were cultured in triplicate onto Baird-Parker agar. After 24 hours of incubation at 37°C, colonies displaying a characteristic shiny grey-black appearance with an opaque halo were counted to determine the number of colony-forming units per milliliter (CFU/mL)[31]. C. Histopathological evaluation: Skin samples from the full-thickness wound areas, including sections of adjacent healthy skin, were collected from each group on days 7, 14, and 21. These specimens were immediately fixed in 10% neutral buffered formalin for 24 hours, processed through routine histological procedures, and embedded in paraffin blocks. The resulting 4 µm-thick sections were stained with Hematoxylin & Eosin (H&E) and subsequently examined microscopically[32]. H&E-stained sections were examined for the extent of re-epithelialization, angiogenesis, Bacteria, and collagen deposition according to [33] with modifications. D. Immunohistochemical evaluation Paraffin-embedded tissue samples were sliced into 4 μm-thick sections and mounted on saline-coated glass slides. The sections were deparaffinized using xylene and dehydrated with a series of ethanol solutions. Antigen retrieval was achieved by autoclaving at 120˚C for 10 minutes at pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Primary antibodies targeting Epidermal Growth Factor Receptor (EGFR) and Alpha Smooth Muscle Actin (α-SMA) were applied and incubated at room temperature for one hour. The sections were then washed three times with phosphate-buffered saline. Following a 30-minute incubation with anti-rabbit secondary antibodies, the slides were treated with the 3, 3' diaminobenzidine tetrahydrochloride (DAB) liquid system for 5 minutes at room temperature. Finally, the sections were counterstained with hematoxylin and examined under light microscopy [34]. 2. 10 . Statistical analysis All statistical analyses were performed using GraphPad Prism software (version 10.0; GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SD from at least three independent experiments. Group comparisons were conducted using Two-way analysis of variance (ANOVA) followed by Tukey’s or Šídák's multiple comparisons test, whenever appropriate, to determine statistically significant differences between groups with the effect of both time and treatment, as well as the interaction between the two variables. A p-value of less than 0.05 was considered statistically significant. 3. Results 4.1. Microstructure Characterization: The wet precipitation method is a widely used chemical technique for synthesizing metal and metal oxide nanoparticles due to its simplicity, cost-effectiveness, and ability to control particle size and morphology. In this study, ZnSeONPs were prepared using a reducing agent (e.g., ascorbic acid) to facilitate the reduction of selenium ions and promote ZnSeO formation, as shown in Fig.1. The XRD pattern of the synthesized CMC / zinc selenite via the sol-gel method ( Fig.2A ) displays both amorphous and crystalline features. A broad hump in the 2θ range of 16° to 23° indicates the presence of an amorphous phase in the CMC / zinc selenite sample. Such humps are typically attributed to short-range order and lack of long-range crystallinity, which is common in sol-gel derived materials due to incomplete crystallization or retained organic/inorganic precursors. This feature suggests that the synthesis did not result in a fully crystalline material and that an amorphous CMC / zinc selenite matrix may be present [35, 36]. Superimposed on the hump are several sharp diffraction peaks, which match well with the standard pattern of monoclinic Zn₃Se₄O₁₄, as per the ICDD card 96-156-1697. These peaks confirm the partial crystallization of the material into a monoclinic phase. The good agreement between the experimental peaks and the reference card suggests that the primary crystalline phase is indeed Zn₃Se₄O₁₄. The coexistence of a broad amorphous hump and sharp diffraction peaks indicates that the material is semi-crystalline, containing both crystalline Zn₃Se₄O₁₄ domains and amorphous regions. This dual-phase composition is not uncommon in sol-gel-derived oxides, particularly when calcination or aging conditions are not optimized for complete crystallization [35, 36]. TEM analysis of the obtained ZnSeONPs is presented in Fig. (2-B and C) . As can be seen, the ZnSeONPs presented irregular-shaped nanoparticles. The average diameter of ZnSeONPs was around 51 ± 13 nm. The inserted SAED images reveal a diffraction pattern indicating the crystalline structure of the prepared NPs. 4.2. Macroscopic evaluation: The bacterial inoculation into the wounds led to wound infection after 3 days which was confirmed by Gram stain and bacterial culture and accumulation of pus in wounds. 4.2.1. Wound Size: Quantitative assessment of macroscopic wound healing is achieved by measuring wound size at specified intervals (days 0, 3, 7, 11, 15, and 19) across three experimental groups: placebo (non-infected, untreated), infected (infected, treated), and CMC / zinc selenite (infected, treated). Time (Row Factor: p < 0.0001, ε = 0.5244), treatment group (Column Factor: p < 0.0001), and their interaction (p < 0.0001, λ). All groups exhibited statistically equivalent wound diameters at baseline, day 0 (p = 0.87), confirming uniform initial damage. Both the infected (398.3 ± 23.9 mm²) and CMC / zinc selenite (398.7 ± 19.2 mm²) groups exhibited minimal reduction, with a borderline significant difference observed between the placebo and infected groups (Tukey's p = 0.07). In contrast, wound contraction was initiated in the placebo group (332.1 ± 45.5 mm²) by day three. By day 7, notable differences were observed: the placebo group exhibited a significant reduction in wound size (210.0 ± 6.4 mm²), while the infected group retained considerably larger wounds (375.9 ± 17.7 mm²). The CMC / zinc selenite group demonstrated intermediate healing (323.5 ± 46.3 mm²). The differences between the placebo and infected groups (p < 0.0001) and between the CMC / zinc selenite and infected groups (p = 0.004) were statistically significant. On day 11, further healing progression was observed; the infected group exhibited significant impairment (311.2 ± 16.7 mm²), whereas the placebo group demonstrated notable wound closure (76.7 ± 13.1 mm²). Compared to the CMC / zinc selenite group (215.4 ± 48.0 mm²; p = 0.0011), the CMC/ZnSeONPs gel accelerated healing. All intergroup differences were statistically significant (p < 0.0001). On day 15, the placebo group achieved near-complete wound closure (0.6 ± 0.6 mm²), the CMC / zinc selenite group exhibited substantial healing (104.4 ± 15.9 mm²), while the infected group showed minimal healing (228.9 ± 29.0 mm²). On Day 19, the CMC / zinc selenite group exhibited nearly complete healing (2.0 ± 1.8 mm²), while the placebo group achieved complete re-epithelialization, and the infected group showed only partial healing (111.2 ± 11.8 mm²). The results demonstrate a statistically significant improvement over untreated infected controls across all post-baseline periods, indicating the therapeutic efficacy of topical CMC/ZnSeONPs gel in enhancing wound healing under infected conditions ( Table 1, Fig. 3& 5 ). Table 1 . Effect Of Topical CMC/ZnSeONPs Gel On Wound Area Reduction In Rats Over Time. Mean wound area (mm²) ± SD measured on days 0, 3, 7, 11, 15, and 19 in placebo (non-infected untreated), infected (infected untreated), and CMC / zinc selenite (infected treated) groups. Statistical analysis was performed using two-way ANOVA followed by Tukey’s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. *P < 0.05 considered statistically significant. Days Placebo Infected CMC/ Zinc Selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 0 421.5 1.6 422.0 2.0 423.3 1.2 0.87 0.13 0.41 3 332.1 45.5 398.3 23.9 398.7 19.2 0.07 0.05 0.99 7 210.0 6.4 375.9 17.7 323.5 46.3 <0.0001 0.004 0.08 11 76.7 13.1 311.2 16.7 215.4 48.0 <0.0001 0.0011 0.008 15 0.6 0.6 228.9 29.0 104.4 15.9 <0.0001 0.0003 <0.0001 19 0.0 0.0 111.2 11.8 2.0 1.8 <0.0001 <0.0001 <0.0001 4.2.2. Wound contraction: Macroscopic wound contraction was quantitatively measured on days 3, 7, 11, 15, and 19. Two-way ANOVA utilizing the Geisser-Greenhouse correction indicated significant main effects for time (Row Factor: p < 0.0001, ε = 0.7066) and treatment group (Column Factor: p Inter-subject variance was not significant, with a p-value of 0.0513. On day 3, the placebo group exhibited a significantly higher wound contraction (21.2 ± 10.9%) in comparison to the infected group (5.6 ± 5.7%) and the CMC / zinc selenite group (5.8 ± 4.5%). Statistically significant differences were noted between the placebo and infected groups (p = 0.0369) as well as between the placebo and CMC / zinc selenite groups (p = 0.0375), while the CMC / zinc selenite and infected groups showed no significant difference (p = 0.9979). The CMC / zinc selenite group demonstrated a moderate improvement of 23.6 ± 10.8%. The comparison between the placebo and CMC / zinc selenite approached significance (p = 0.0761), with the CMC / zinc selenite group continuing to show enhanced wound contraction by day 7. On day 11, wound contraction was notably enhanced in the placebo group (81.8 ± 3.1%), while the CMC / zinc selenite group (49.1 ± 11.2%) significantly surpassed the infected group (26.2 ± 4.2%), with all intergroup differences reaching statistical significance (p < 0.01). On day 15, the placebo group exhibited nearly complete contraction (99.9 ± 0.2%), while the CMC / zinc selenite group showed a contraction of 75.3 ± 3.7%, and the infected group demonstrated a contraction of 45.8 ± 6.7%. All comparisons were statistically significant (p < 0.0001). On day 19, the CMC / zinc selenite group exhibited nearly complete healing at 99.5 ± 0.4%, while the placebo group achieved full wound closure at 100.0 ± 0.0%. Both groups significantly outperformed the infected group, which recorded a contraction rate of 73.6 ± 2.8%. The difference between placebo and CMC / zinc selenite was not statistically significant (p = 0.0802); however, the differences between infected and CMC / zinc selenite were significant (p < 0.0001) ( Table 2, Figure 4 ). Table 2 . Percentage of Wound Contraction Over Time in different groups. Mean percentage of wound contraction ± SD measured on days 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups. Statistical significance was evaluated using two-way ANOVA followed by Tukey’s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P < 0.05 is considered statistically significant. Days Placebo Infected CMC/ Zinc selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 3 21.2 10.9 5.6 5.7 5.8 4.5 0.0369 0.0375 0.9979 7 50.2 1.4 10.9 4.2 23.6 10.8 <0.0001 0.0039 0.0761 11 81.8 3.1 26.2 4.2 49.1 11.2 <0.0001 0.0013 0.007 15 99.9 0.2 45.8 6.7 75.3 3.7 <0.0001 <0.0001 <0.0001 19 100.0 0.0 73.6 2.8 99.5 0.4 <0.0001 0.0802 <0.0001 4.2.3. Body weight Body weight changes were monitored in the three experimental groups to assess systemic well-being and treatment-related effects. In addition to notable inter-subject variation (p < 0.0001), two-way ANOVA with Geisser-Greenhouse correction indicated significant effects of time and treatment group (p 0.5), indicating no initial differences. On day 3, both the infected group (205.0 ± 8.4 g) and the CMC / zinc selenite group (200.0 ± 12.6 g) exhibited significant weight loss (p = 0.0001 for both), in contrast to the placebo group (249.2 ± 13.9 g; p = 0.0001). The CMC / zinc selenite and infected groups were statistically comparable (p = 0.7508). Beginning on day seven, distinct recovery patterns emerged. The infected group exhibited significant weight loss (171.7 ± 13.3 g), while the placebo group demonstrated a consistent weight increase (258.3 ± 25.8 g). The CMC / zinc selenite group (213.3 ± 8.2 g) demonstrated partial recovery, exhibiting significantly greater weight compared to infected controls (p = 0.0023), although it remained considerably lower than the placebo group (p = 0.0012). On day 11, the infected group exhibited a decline in body weight that may be due to infection, whereas the placebo group continued to gain weight, averaging 281.7 ± 22.3 g. The CMC / zinc selenite rats (242.5 ± 8.8 g) exhibited statistically significant improvements, with changes noted in both the infected (p = 0.0007) and placebo (p < 0.0001) groups. On day 15, the CMC / zinc selenite group exhibited a mean weight of 262.5 ± 7.6 g, which was intermediate between the placebo group at 295.0 ± 24.3 g and the infected group at 154.2 ± 4.9 g. Statistical significance was noted with p = 0.0001 for the comparison between CMC / zinc selenite and infected groups, and p = 0.005 for the placebo versus infected groups. On day 19, the CMC / zinc selenite group exhibited body weight measurements that were statistically similar to those of the placebo group (305.8 ± 26.2 g; p = 0.538), demonstrating nearly complete recovery in body weight (295.8 ± 4.9 g), and significantly higher than the infected group (182.5 ± 7.6 g; p = 0.538) ( Table 3, Figure 5). These findings suggest that CMC/ZnSeONPs gel not only aids in wound healing but also mitigates infection-induced systemic stress, facilitating recovery of body weight comparable to infected controls. Table 3. Changes in Body Weight (g) of Rats During the Wound Healing Period. Mean body weight ± SD recorded on days 0, 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P < 0.05 is considered statistically significant. Days Placebo Infected CMC/ Zinc selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 0 256.7 13.7 250.8 5.8 250.8 3.8 0.5044 0.5044 >0.9999 3 249.2 13.9 205.0 8.4 200.0 12.6 <0.0001 <0.0001 0.7508 7 258.3 25.8 171.7 13.3 213.3 8.2 <0.0001 0.0012 0.0023 11 281.7 22.3 136.7 5.2 242.5 8.8 <0.0001 0.0007 <0.0001 15 295.0 24.3 154.2 4.9 262.5 7.6 <0.0001 0.005 <0.0001 19 305.8 26.2 182.5 7.6 295.8 4.9 <0.0001 0.538 <0.0001 4.3. Microscopic evaluations: 4.3.1. Bacterial viability test The antibacterial activity of CMC/ZnSeONPs gel against S. aureus infection was evaluated over 24 hours. Two-way ANOVA revealed that both treatment and exposure duration significantly influenced bacterial load, with statistically significant effects observed for time (p < 0.0001) and treatment group (p Additionally, subject variability was significant (p 0.99), thereby confirming similar initial contamination at baseline (0 hr.). The CMC / zinc selenite group exhibited a substantial reduction in bacterial load (5.0 × 10⁸ ± 0 CFU), whereas the untreated infected group demonstrated a rapid increase (6.0 × 10⁹ ± 8.94 × 10⁸ CFU) at one hour post-infection, indicating a highly significant difference. The CMC / zinc selenite group exhibited a notable reduction to 9.0 × 10⁴ ± 2.68 × 10⁺ CFU (p < 0.0001), whereas bacterial proliferation continued in the infected group (5.4 × 10⁹ ± 5.37 × 10⁸ CFU) at the six-hour mark. The CMC / zinc selenite group exhibited a further reduction to 3.0 × 10⁴ ± 1.79 × 10¹⁰ CFU, while the infected group achieved 2.4 × 10¹⁰ ± 3.58 × 10⁹ CFU after 12 hours. After 24 hours, the bacterial load in the infected group reached a peak of 4.5 × 10¹⁰ ± 2.68 × 10⁹ CFU, while the CMC / zinc selenite group nearly eradicated the viable bacterial cells, resulting in 1.0 ± 2.0 CFU (p < 0.0001). The findings demonstrate that topical CMC/ZnSeONPs gel exhibits significant and rapid antibacterial efficacy, reducing bacterial viability by over 99.9999% within 24 hours. The strong bactericidal action supports its potential application as a therapeutic agent for managing contaminated wounds ( Table 4, Figure 6 ). Table 4. Changes in the viability of S. aureus bacteria (CFU/ml) recorded on hours (0, 1, 6, 12, and 24) in infected and CMC / zinc selenite groups . Statistical analysis was performed using two-way ANOVA followed by Šídák's multiple comparisons test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P 0.99 1 6.00E+09 8.94E+08 5.00E+08 0.00E+00 <0.0001 6 5.40E+09 5.37E+08 9.00E+04 2.68E+04 <0.0001 12 2.40E+10 3.58E+09 3.00E+04 1.79E+03 <0.0001 24 4.50E+10 2.68E+09 1.00E+00 2.00E+00 <0.0001 4.3.2. Bacterial count On days 2, 7, 14, and 21 post-wounding, the bacterial load in the wound tissue was quantitatively assessed to compare the antimicrobial efficacy of CMC/ZnSeONPs gel against infected and uninfected controls. Subject variability was statistically significant (p = 0.0064). Two-way ANOVA revealed significant effects for treatment group (p = 0.0001), time (p = 0.0001, ε = 0.6210), and their interaction (p = 0.0001). Compared to the placebo (1.10 x 10⁴ ± 2.64 x 10¹ CFU), bacterial counts were significantly elevated in both the infected (1.09 x 10⁴ ± 2.34 x 10¹ CFU) and CMC / zinc selenite groups (9.77 x 10¹ CFU) at day two (p < 0.0001 for both comparisons). There was no significant difference between the infected and CMC / zinc selenite groups (p = 0.5925), indicating similar early bacterial colonization. On day 7, the bacterial burden in the infected group was significantly elevated (1.28 × 10⁴ ± 2.58 × 10³ CFU) compared to the placebo group (1.10 × 10³ ± 0.41 CFU, p < 0.0001). The CMC / zinc selenite group notably decreased the bacterial load to 4.07 × 10³ ± 3.45 × 10³ CFU, representing a significant reduction relative to the infected group (p < 0.0001), although it did not show a statistically significant difference from the placebo (p = 0.1312). On day 14, the infected group exhibited a bacterial count of 5.11 × 10¹ ± 1.83 × 10¹ CFU, while the placebo group demonstrated complete bacterial clearance, recorded at 0 CFU. In comparison to the infected group (p = 0.0004), the CMC / zinc selenite group significantly reduced bacterial counts to 2.76 × 10² ± 5.51 × 10² CFU; this difference was not statistically significant when compared to placebo (p = 0.9338). On day 21, the bacterial load in the infected group was still detectable (2.61 x 10¹ ± 7.08 × 10² CFU), whereas both the placebo and CMC / zinc selenite groups exhibited complete eradication (0 CFU) ( Table 5, Figure 7 ). Table 5. Quantitative Bacterial Load (CFU) in Wound Tissue Over the Study Period. Mean bacterial counts (CFU) ± SD measured on days 2, 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Statistical comparisons were made using two-way ANOVA followed by Tukey’s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P < 0.05 is considered statistically significant. Days Placebo Infected CMC/ Zinc selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 2 1.10E+03 5.16E-01 1.09E+04 2.64E+03 9.77E+03 2.34E+03 <0.0001 <0.0001 0.5925 7 1.10E+03 4.08E-01 1.28E+04 2.58E+03 4.07E+03 3.45E+03 <0.0001 0.1312 0.9999 0.004 4.3.3. Immuno-histochemical evaluation: A. α-SMA expression On days 7, 14, and 21 postoperatively, α-SMA expression was quantified to evaluate myofibroblast activation, serving as an indicator of wound contraction and tissue remodeling. Subject variability was not significant (p = 0.3705), suggesting robust intergroup and temporal effects. The two-way ANOVA revealed significant effects of group (p = 0.0091), time (p = 0.0016, ε = 0.9051), and their interaction (p < 0.0001). There was no significant difference between the CMC / zinc selenite and infected groups (p = 0.1421). However, α-SMA expression was significantly elevated in the placebo group (20.9 ± 0.66) on day 7 compared to both the infected group (4.5 ± 1.02, p = 0.0034) and the CMC / zinc selenite group (7.4 ± 1.64, p = 0.0034). This suggests an initial suppression of myofibroblast differentiation in infected wounds, which is partially restored with CMC/ZnSeONPs gel treatment. On day 14, α-SMA expression in the CMC / zinc selenite group increased to 15.2 ± 2.46, surpassing the levels observed in the placebo group (11.9 ± 3.48) and the infected group (7.9 ± 1.78). While CMC / zinc selenite and placebo (p = 0.3177) or CMC / zinc selenite and infected (p = 0.0355) did not demonstrate statistically significant differences, the elevated expression in the CMC / zinc selenite group indicates a heightened fibroblast-to-myofibroblast transition at this stage. In the infected group, α-SMA levels remained elevated (11.3 ± 2.07), likely indicating persistent inflammation and delayed remodeling. Conversely, in the placebo group, α-SMA levels decreased (4.4 ± 0.51) on day 21, consistent with the expected progression of wound healing. CMC / zinc selenite group exhibited intermediate α-SMA expression (6.9 ± 1.58), which was slightly lower than that of the infected group (p = 0.0438) and not statistically different from the placebo (p = 0.0989). CMC/ZnSeONPs gel application frequently diminished α-SMA dynamics, thereby promoting timely myofibroblast activation during the mid-phase of healing and restricting prolonged expression associated with chronic inflammation, suggesting a beneficial effect on wound maturation and resolution ( Table 6, Figure 8 ). Table 6 . Mean Values and SD Of Immunohistochemical Expression Of (α-SMA) In Wound Tissue. Measured on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Two-way ANOVA followed by Tukey’s post hoc test was used for statistical comparison. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P < 0.05 is considered statistically significant. Days Placebo Infected CMC/ Zinc selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 7 20.9 0.66 4.5 1.02 7.4 1.64 0.0002 0.0034 0.1421 14 11.9 3.48 7.9 1.78 15.2 2.46 0.3177 0.4475 0.0355 21 4.4 0.51 11.3 2.07 6.9 1.58 0.0438 0.1763 0.0989 B. EGFR expression EGFR expression was assessed on days 7, 14, and 21 postoperatively to evaluate epithelial regeneration and proliferative activity during the healing process. Subject-level variability was non-significant (p = 0.2251), supporting the consistency of treatment-related and temporal effects. However, two-way ANOVA revealed significant main effects for treatment group (p = 0.0002) and time. Day 7: EGFR expression in the infected group (1.8 ± 0.24) was significantly lower than in the placebo group (3.4 ± 0.32; p = 0.0065), indicating a reduction in epithelial activation due to infection. CMC / zinc selenite group resulted in EGFR levels of (3.0 ± 0.08), which were significantly higher than those in the infected group (p = 0.0142) and not significantly different from the placebo (p = 0.3008), indicating a potential early reactivation of epithelial repair mechanisms. On day fourteen, EGFR expression reached its maximum level in both CMC / zinc selenite (7.2 ± 0.58) and placebo (9.2 ± 0.77) groups. In the infected group, EGFR slightly elevated from its level on day 7 but remained relatively low at 2.7 ± 0.18. Although CMC / zinc selenite was still lower than the placebo group (p = 0.006), the CMC / zinc selenite group had a significantly increased level of EGFR expression compared to the infected group (p = 0.006), indicating a partial restoration of epithelial proliferative signaling. On day 21, EGFR expression decreased across both CMC / zinc selenite and placebo groups, consistent with the resolution of the proliferative phase. In comparison to placebo (2.8 ± 0.23; p = 0.0091), infected wounds (4.3 ± 0.33) exhibited increased EGFR expression, likely indicating delayed or abnormal healing processes. Although comparisons did not reach statistical significance when compared to infected (p = 0.2716) or placebo (p = 0.0585) groups, CMC / zinc selenite exhibited the highest expression (5.5 ± 0.93)( Table 7, Figure 9 ). These findings indicate that CMC/ZnSeONPs gel enhances EGFR-mediated epithelial responses in the early and mid-phases of healing, potentially mitigating the suppression caused by infection and facilitating wound re-epithelialization. Table 7. Immunohistochemical Expression of EGFR in Wound Tissue. Mean EGFR expression levels ± SD assessed on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Statistical comparisons were made using two-way ANOVA with Tukey’s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. P < 0.05 is considered statistically significant. Days Placebo Infected CMC/ Zinc selenite Tukey’s adjusted p-value M SD M SD M SD P-value1 P-value2 P-value3 7 3.4 0.32 1.8 0.24 3.0 0.08 0.0065 0.3008 0.0142 14 9.2 0.77 2.7 0.18 7.2 0.58 0.006 0.049 0.006 21 2.8 0.23 4.3 0.33 5.5 0.93 0.0091 0.0585 0.2716 4.3.4. Histopathological evaluation On day 7, the CMC / zinc selenite group showed partial loss of epidermal cell layers with granulation tissue, edema, irregularly arranged fibroblasts and many newly formed blood vessels admixed with cellular infiltrates, While the infected group showed focal to complete loss of epidermal layers with desquamated epidermal cells infiltrated with many neutrophils, the dermal layers showing granulation tissue with dermal hemorrhage or leaky immature formed blood vessels and immature fibroblasts admixed with low numbers of lymphocytes, plasma cells and macrophages. The placebo group showed complete epithelization with regularly arranged collagen ( Figure.10 ). Fourteen days postoperatively, the CMC / zinc selenite group showed a decreased wound gap with eosinophilic debris infiltrated with many inflammatory cells and dermal granulation tissue with many angiogenesis. The infected group showed diffuse loss of epidermal layers with extensive dermal infiltrations with abundant neutrophils, lymphocytes, plasma cells, and macrophages, besides abundant angiogenesis (immature blood vessels) with immature, irregularly and randomly arranged fibroblasts. The placebo group showed normal epithelium and dermal collagen with hair follicles ( Figure.11 ). Day 21, the CMC / zinc selenite group showed complete reepithelialization with many well-developed hair follicles, dense collagen bundles, and few subepidermal fibroblast aggregations, while the infected group showed focal incomplete loss of the stratum basalis of epidermal layers. multifocal to coalescing partial or complete loss of epidermal cell layers (partial migration of epidermal cells to close the wound gap), besides the presence of randomly arranged collagen admixed with scattered few inflammatory aggregates. The placebo group showed normal architecture of skin layers ( Figure.12 ). · Histopathology scoring Semi-quantitative histopathological scoring was conducted to assess angiogenesis, collagen deposition, bacterial presence, and re-epithelialization in wound tissue sections collected on days 7, 14, and 21. Group comparisons were analyzed using appropriate non-parametric tests ( Table.8). Angiogenesis : On day 7, a trend toward increased angiogenesis was noted in the CMC / zinc selenite and placebo groups (score 2 in most of the animals), though not statistically significant ( p = 0.08). By day 21, the CMC / zinc selenite group demonstrated significantly higher angiogenesis scores ( p = 0.0286), with 2 out of 3 rats scoring 3, compared to the infected group. These findings indicate enhanced neovascularization with CMC/ZnSeONPs gel, which supports granulation tissue formation and tissue regeneration. Collagen Deposition : Collagen fiber deposition improved progressively in all groups. On day 21, the CMC / zinc selenite group showed significantly enhanced collagen deposition ( p = 0.0214), with two rats scoring 3, indicative of wound maturation and remodeling. While early differences were not statistically significant (Day 7: p > 0.99; Day 14: p = 0.08), trends favored the CMC / zinc selenite group, highlighting its role in matrix remodeling. Bacterial Presence: Bacterial load scores differed significantly on day 7 ( p = 0.02) and day 21 ( p = 0.0357), with placebo and CMC / zinc selenite groups showing lower scores compared to the infected control. CMC / zinc selenite consistently showed superior bacterial clearance, with all animals scoring 0 by day 21, underscoring its antimicrobial efficacy in vivo, these results were in line with the results of the results of bacterial count. Re-epithelialization: The CMC / zinc selenite group achieved complete epithelial coverage by day 21 (score 3 in all rats), significantly outperforming the infected group (p = 0.0357). Early trends were noted as well, particularly at day 14 (p = 0.02), suggesting accelerated epithelial regeneration. The infected group consistently lagged in this parameter, indicating prolonged wound exposure and impaired barrier restoration. Collectively, these results demonstrate that CMC/ZnSeONPs gel facilitates wound healing through enhanced angiogenesis, collagen maturation, microbial clearance, and epithelial regeneration. (Table 8). Semi-Quantitative Histopathological Scores for Angiogenesis, Collagen Deposition, Bacterial Presence, and Re-epithelialization . Histopathological parameters were scored on a scale of 0 (none), 1 (mild), 2 (moderate), and 3 (extensive) in skin wound samples collected on days 7, 14, and 21 from placebo, infected, and CMC / zinc selenite groups (n = 3 per time point). Statistical analysis was performed using Fisher’s exact test or chi-square test as appropriate. P < 0.05 is considered statistically significan t . Groups 7 days 14 days 21 days 0 1 2 3 p-value 0 1 2 3 p-value 0 1 2 3 p-value Angiogenesis Placebo 0 1 2 0 0.08 0 0 2 1 0.44 0 0 0 3 0.029* Infected 3 0 0 0 2 1 0 0 1 2 0 0 CMC / zinc selenite 0 1 2 0 0 1 1 1 0 0 2 1 Collagen Placebo 2 1 0 0 >0.99 0 1 2 0 0.08 0 0 0 3 0.021* Infected 3 0 0 0 3 0 0 0 2 1 0 0 CMC / zinc selenite 3 0 0 0 1 2 0 0 0 1 2 0 Bacteria Placebo 2 1 0 0 0.02* 2 1 0 0 0.16 3 0 0 0 0.035* Infected 0 0 0 3 0 0 1 2 0 2 1 0 CMC / zinc selenite 0 2 1 0 1 2 0 0 3 0 0 0 Epithelization Placebo 1 2 0 0 0.68 0 0 1 2 0.02* 0 0 0 3 0.035* Infected 3 0 0 0 3 0 0 0 1 2 0 0 CMC / zinc selenite 2 1 0 0 0 1 2 0 0 0 0 3 4. Discussion The healing of infected wounds has long been a major global healthcare challenge, owing to its high prevalence, the potential for disability, and considerable mortality rates [37]. A study on dogs with infected wounds reported a mortality rate of around 33%, with bacteremia being a major contributing factor strongly associated with the high mortality rate [38]. The severity and mortality associated with wounds are expected to rise due to increased exposure to antimicrobial-resistant pathogens. Currently, wound infections are responsible for approximately 75% of post-operative deaths [39]. Therefore, the aim of this study is to address this ongoing challenge by exploring antimicrobial alternatives to conventional antibiotics to minimize their previously reported adverse effects. The selection of S. aureus as the focus for wound infection studies is the leading pathogen associated with wound infections, responsible for 30.3% of reported cases. Notably, S. aureus is a common commensal organism residing on human skin; however, when the skin barrier is broken, such as through wounds, it can transition from a harmless inhabitant to a pathogenic agent [40] . The topical treatment with CMC/zinc selenite gel was applied following thorough flushing of the wound with normal saline 0.9%, as the S. aureus can develop resistance to topical treatments by forming biofilms at the wound site, which impede drug penetration and interfere with host immune signaling. Therefore, the physical removal of colonizing bacteria is considered a crucial step in wound management, as it helps reduce bacterial load, enhances drug efficacy and penetration, and minimizes required dosages [10] . SeNPs are considered a promising alternative to antibiotics for treating hard-to-heal skin infections, offering excellent stability, favorable toxicological profiles, and strong antibacterial efficacy [41]. SeNPs exhibit minimal or no toxicity toward mammalian cells, primarily due to their selective targeting capability, which allows them to differentiate between bacteria and healthy mammalian cells [42] . Also, all antimicrobial studies conducted to date have reported no evidence of bacterial resistance to SeNPs, highlighting their strong potential as an effective treatment option for infectious and chronic wounds [27]. ZnONPs possess notable antimicrobial properties and are recognized for their biocompatibility, cost-effectiveness, non-toxicity, and environmental safety. By releasing zinc ions at the wound site, ZnONPs stimulate keratinocyte activity, thereby accelerating the wound healing process [43], and these were the reasons for selecting the combination of these materials. The current research demonstrates that S. aureus infection significantly impacts body weight. The infected group exhibited weight loss until day 11 post-infection, after which weight gain resumed. This result is in line with [44, 45]. In contrast, CMC / zinc selenite group started to gain weight after 3 days of infection. These findings highlight that CMC / zinc selenite has protective effects against infection-induced weight loss. The infection-related weight loss likely results from the breakdown of muscle protein, a process influenced by an imbalance among key factors such as glucocorticoids, proinflammatory cytokines, and catecholamines. These mediators typically rise during infection, reflecting an inflammatory and stress response that promotes catabolism and muscle wasting [46], or may be attributed to that S.aureus can invade adipose tissue or infect adipocytes, triggering a localized inflammatory response. This inflammation, along with pathogen-derived signals, promotes lipolysis, the breakdown of stored triglycerides into free fatty acids, these liberated fatty acids serve to meet the increased energy requirements of the host and support immune system activity during infection [47]. The bacterial viability test revealed that CMC/CMC / zinc selenite gel led to a total inhibition of bacterial growth up to 24 hr. These results highlight that in the case of CMC/CMC / zinc selenite gel, only one application daily is effective to reach the total bacterial inhibition, these results may indicate that the addition of zinc selenite to the CMC potentiates its antibacterial effect as CMC alone is weak antibacterial agent [2]. A study by [48] on ZnONPs revealed that the effect of ZnONPs as an antibacterial agent was significant after 6 hours of contact with S.aureus, and caused significant reduction of bacterial viability after 24 hours but not total inhibition, while in the current study, CMC / zinc selenite gel showed significant reduction of bacterial viability after only one hour of contact with S.aureus, and caused significant total inhibition after 24 hours highlighting the potent antibacterial effect of CMC / zinc selenite gel. The results of bacterial count revealed that on day 2, infected and CMC / zinc selenite groups showed similarly high bacterial counts with no significant differences. This is because the treatment protocol started after 3 days of surgery, following confirmation of bacterial infection. While on day 7, notable reductions in bacterial count were observed in the CMC / zinc selenite groups compared to the infected control. On day 14, the bacterial count in the CMC / zinc selenite group had nearly reached baseline levels, with no significant differences between the placebo, suggesting the strong antibacterial effect of CMC / zinc selenite gel. These results were in agreement with [49], who reported that CMC in the control group without NPs exhibited no inhibition of S.aureus growth, and also reported that antibacterial activity due to the presence of ZnONPs reduced the growth of S. aureus by 75% but not total inhibition. However, in our study, we reached total inhibition of bacterial growth by CMC / zinc selenite, which suggests that CMC / zinc selenite is a better antibacterial agent than ZnONPs or CMC alone. The potential antibacterial activity of CMC/ zinc selenite may be attributed to its semi-crystalline structure, which contains both crystalline domains and amorphous regions. The amorphous regions typically allow for a more rapid release of ions (Zn and Se), which can enhance the initial antibacterial efficacy upon application to the wound. In contrast, the crystalline domains enable a slower, more sustained release, ensuring prolonged protection against infection and allowing the material to persist longer at the wound site. Or maybe due to the small diameter of CMC / zinc selenite (Zn₃Se₄O₁₄), around 51 nm, which facilitates its internalization into the bacterial cells for more destruction. Or as mentioned by [50] . The extended inflammatory phase disrupts fibroblast differentiation, slowing wound contraction and potentially contributing to scar formation [51, 52] . The histopathological analysis revealed that the infected group exhibited a marked inflammatory response, characterized by severe infiltration of the skin layers with inflammatory cells persisting up to day 21. Additionally, the infected group displayed signs of irritation and scratching, further indicating sustained inflammation, which may be attributed to the bacterial infection and its associated released toxins into the tissues, and the weak anti-inflammatory effect of CMC. In contrast, the CMC / zinc selenite group demonstrated a notably milder inflammatory reaction. This superior outcome of the CMC / zinc selenite group may be explained by a synergistic interaction between the anti-inflammatory mechanisms of both ZnONPs and SeNPs. These findings agreed with Alnomasy (2024) and Khosravian et al. (2025) Excessive inflammation often results from the imbalance in macrophage polarization toward either the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which promotes skin tissue regeneration and facilitates revascularization. This imbalance disrupts the immune homeostasis within the wound microenvironment, potentially impairing the healing process [53] . Zinc ions released from the gel play a role in immune regulation within the wound microenvironment, promoting macrophage polarization toward the M2 phenotype. This shift enhances the expression of anti-inflammatory cytokines, effectively reducing inflammation [54]. The anti-inflammatory properties of SeNPs are attributed to Selenium itself, which is a critical component of various selenoproteins, which play pivotal roles in regulating inflammatory responses, as well as phagocytic and chemotactic functions. specific selenoproteins such as glutathione peroxidase 1 and 4 are responsible for the suppression of pro-inflammatory cytokines, antioxidant defense mechanisms, and the detoxification of reactive nitrogen species like peroxy nitrite during the inflammatory phase of tissue repair [55]. The second mechanism of suppressing the inflammation is their ability to suppress the mRNA expression of key pro-inflammatory cytokines, such as interleukin-1 (IL-1), TNF-α, and inducible nitric oxide synthase (iNOS). Additionally, SeNPs inhibit the production of pro-inflammatory mediators by preventing the nuclear translocation of the transcription factor NF-κB, thereby modulating the inflammatory signaling pathways involved in the wound healing process [56]. Wounds are frequently associated with the damage or loss of dermal vascular tissues. These vascular networks supply oxygen and nutrients needed for cell growth, proliferation, and migration; they also enhance the transport of vital nutrients and waste. Their regeneration is of significant importance. Therefore, promoting vascular network formation is a promising approach to enhance wound healing [57]. In our histopathological analysis, the CMC / zinc selenite group exhibited a robust angiogenic response, as evidenced by the presence of numerous newly formed blood vessels at all examined time points from day 7 onward. Also, the histopathological score for angiogenesis was significantly higher in the CMC / zinc selenite group compared to the infected one. This may be due to The angiogenic capacity of CMC / zinc selenite gel may be attributed to the internalization of ZnONPs, which upregulate the expression of VEGF, triggering downstream signaling pathways, including PI3K/Akt and MAPK/ERK, which promote endothelial cell survival, motility, and new blood vessel formation. Furthermore, ZnONPs activate endothelial nitric oxide synthase, leading to increased nitric oxide production, which facilitates vasodilation and vascular remodeling for effective angiogenesis [58]. This explains the early angiogenic effect demonstrated in the CMC / zinc selenite group; the addition of SeNPs has a synergistic effect and potentiates the ZnONPs effect in neovascularization, which promotes tissue regeneration. Histopathological findings revealed that the infected group on day 21 had partial closure of wound, and thinning of the epidermal layer with a scanty immature epithelialization, with the results of IHC that showed low expression of EGFR on day 7, which started to increase significantly after that, reaching the peak on day 21 but still low level compared to the other groups, suggesting improper epithelialization and weak stimulation of keratinocyte migration, likely causing significantly larger wounds on day 21, which may be attributed to the infection that alters the wound microenvironment. The CMC / zinc selenite group showed complete closure of the wound with complete epithelialization in the histopathological section. These results were in line with IHC, which may be primarily attributed to the zinc ions. As Zn serves as a cofactor for various enzymes, including zinc-dependent matrix metalloproteinases, which enhance keratinocyte migration, facilitate autodebridement, and promote epithelialization during wound healing [59]. Also, Zinc regulates keratinization and keratinocyte migration by influencing the expression of integrins α2β1, α3β1, α6β4, and αvβ5, which facilitate intercellular adhesion, attachment to the basement membrane, and keratinocyte motility. Each integrin plays a role in epithelialization during skin wound healing. However, these integrins can be altered due to inflammation or tissue injury [60]. These results were in line with [49], who reported that the sponge containing undoped ZnONPs exhibited the highest cell attachment and proliferation, and [61], who reported that ZnONPs had outperforming results on wound closure time, contraction rate, histopathological changes, and cell regeneration, and [62] who made a study on chitosan-ZnO/SeNPs composite scaffolds demonstrated enhanced keratinocyte migration in cell lines in vitro. Moreover, in vivo experiments confirmed accelerated wound closure and improved re-epithelialization. The EGFR level was the highest in the placebo group on day 7, then markedly decreased onwards, which may suggest the early complete closure and re-epithelialization of the wounds in the placebo group, as there was no challenge of infection. Immunohistochemical analysis of α-SMA revealed that the infected group exhibited the lowest expression of α-SMA on day 7, increased moderately by day 14, and peaked by day 21, which may be attributed to persistent inflammation caused by the infection and delayed remodeling with scar formation. Notably, persistent elevation of α-SMA beyond the proliferative phase may contribute to pathological healing due to abnormal collagen synthesis, resulting in excessive scar formation [63]. That explains the lower contraction rate of wounds in the infected group, which caused significantly larger wound areas compared to the CMC / zinc selenite and placebo groups. In contrast, CMC / zinc selenite groups displayed moderate α-SMA expression on day 7 that markedly increased by day 14, followed by a significant reduction on day 21. This pattern likely reflects the activation of fibroblasts, as indicated by α-SMA expression, which enables their differentiation into myofibroblasts that are essential for effective wound contraction and proliferation during the healing process [51]. Therefore, the significant decrease in α-SMA levels after day 14 in the CMC / zinc selenite groups likely indicates the transition from the proliferative phase into the remodeling phase and explains the accelerated wound contraction and the anti-scarring properties of the CMC / zinc selenite and CMC/ZnONPs gel. Similar trends were coincided with [64]. Wound size reduction in both the placebo and CMC / zinc selenite groups consistently demonstrated superior healing outcomes compared to the infected control groups throughout the study period, which may be attributed to the high level of α-SMA that caused effective wound contraction and significantly higher levels of EGFR compared to the infected group, which led to complete epithelialization. In contrast, wounds in the infected group remained significantly larger, reflecting delayed wound closure likely attributable to inadequate infection control. [65] found that selenium 5% ointment in uninfected Wistar rats caused a healing rate of up to 85% within 18 days. Also, a study by [66] on 10 mm diameter S. aureus -infected wounds, which closed totally after 21 days with Se-chitosan as a wound dressing. Meanwhile, in our study, the 23 mm diameter S. aureus -infected wounds closed totally on 19 days, which indicates that CMC/zinc selenite is more effective in infected wounds than Selenium alone or Se-chitosan. 5. Conclusion We synthesized CMC/zinc selenite gel, which has demonstrated excellent antibacterial efficacy on staphylococcus aureus infected wounds, due to its unique physicochemical properties represented as a semicrystalline structure and smaller nanometric size. This advanced gel not only enhanced the angiogenesis but also led to rapid epithelialization and promoted fibroblast differentiation that resulted in effective wound contraction, thereby accelerating infected wound healing and regeneration. CMC/zinc selenite gel could also be considered as a protective agent against weight loss caused by infection. Declarations Author Contributions Nermeen -Shereef: conceptualization, data curation, methodology, writing – original draft, writing – review and editing. Marwa Abass: conceptualization, formal analysis, investigation, writing – original draft, and editing. Emad Tolba preparation and characterization of the materials used and Methodology. Ekramy Elmorsy contributed to the statistical analysis of this study. Basma Hendam : Methodology and Immunohistochemical evaluation Awad Rizk: conceptualization, data curation, writing – original draft, and supervision. Gamal Karrouf: conceptualization, data curation, supervision, writing – review and editing and final revision of the manuscript. All authors have read and approved the final version of the manuscript. Data availability: All data generated or analyzed during this study are included in this published article Funding: T his research did not receive funding. Acknowledgment The authors acknowledge the Science, Technology & Innovation Funding Authority (STDF), in cooperation with the Egyptian Knowledge Bank (EKB), for covering the article processing charges. No funding, grants, or other financial support were received for the design, execution, or reporting of this study. Ethics approval and consent to participate 5.1. Ethical approval: The study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code (VM.MS.24.10.166). All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical All procedures were documented following the guidelines specified by ARRIVE Consent for publication: All authors confirm their consent for publication. Competing interests: The authors declare that no competing interests. References Gonzalez AC de O, Costa TF, Andrade Z de A, Medrado ARAP. Wound healing - A literature review. An Bras Dermatol. 2016;91:614–20. Das A, Kumar A, Patil NB, Viswanathan C, Ghosh D. 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Skin wound healing: an update on the current knowledge and concepts. Eur Surg Res. 2017;58:81–94. Ahtzaz S, Nasir M, Shahzadi L, Amir W, Anjum A, Arshad R, et al. A study on the effect of zinc oxide and zinc peroxide nanoparticles to enhance angiogenesis-pro-angiogenic grafts for tissue regeneration applications. Mater Des. 2017;132:409–18. Lansdown ABG, Mirastschijski U, Stubbs N, Scanlon E, Agren MS. Zinc in wound healing: theoretical, experimental, and clinical aspects. Wound Repair Regen. 2007;15:2–16. Tenaud I, Leroy S, Chebassier N, Dreno B. Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins. Exp Dermatol. 2000;9:407–16. Asif M, Chaudhry AS, Ashar A, Rashid HB, Saleem MH, Aslam HB, et al. Zinc oxide nanoparticles accelerate the healing of methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds in rabbits. Asian Pac J Trop Biomed. 2023;13:488–96. Ruan Q, Yuan L, Gao S, Ji X, Shao W, Ma J, et al. 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2","display":"","copyAsset":false,"role":"figure","size":112302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eshows (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) XRD pattern, (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eB\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) TEM image, and (\u003c/em\u003e\u003cem\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e) selected area electron diffraction (SAED) pattern of the synthesized ZnSeONPs.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/670cadb4d9ed5ffd16f8d9dc.jpg"},{"id":109170561,"identity":"e111bcb4-eb2a-4e76-9938-34c8e456aa6e","added_by":"auto","created_at":"2026-05-13 08:50:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEffect of Topical CMC/ZnSeONPs gel on Wound Area Reduction in Rats Over Time\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cbr\u003e\nMean wound area (mm²) ± SD is shown on days 0, 3, 7, 11, 15, and 19 in placebo (non-infected untreated), infected (infected untreated), and CMC / zinc selenite (infected treated) groups.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/886ab12e229eaaa96c37be8a.jpg"},{"id":109205392,"identity":"62592866-8ba5-4ecd-83a2-6de6653d3b26","added_by":"auto","created_at":"2026-05-13 15:04:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePercentage of Wound Contraction Over Time in different groups.\u003c/em\u003e\u003cbr\u003e\nMean percentage of wound contraction ± SD measured on days 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/1ccfda02409f9141b77c183f.jpg"},{"id":109205368,"identity":"77391bc0-b269-4a38-b167-d538208195ad","added_by":"auto","created_at":"2026-05-13 15:04:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43173,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChanges in Body Weight (g) of Rats During the Wound Healing Period.\u003c/em\u003e\u003cbr\u003e\nMean body weight ± SD recorded on days 0, 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups .\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/a77a06950998396472e3c277.jpg"},{"id":109205158,"identity":"a747f2dd-5ba4-4e61-a4e0-9b7c203c00dd","added_by":"auto","created_at":"2026-05-13 15:03:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChanges in the viability of S. aureus bacteria (CFU/ml) recorded on hours (0, 1, 6, 12, and 24) in infected and CMC / zinc selenite groups.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/f91de74b85ccb7b82dbd64f9.jpg"},{"id":109170565,"identity":"6b6ca01f-63c3-44c3-ae93-7faa98fcb9a2","added_by":"auto","created_at":"2026-05-13 08:50:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":46402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eQuantitative Bacterial Load (CFU) in Wound Tissue Over the Study Period on days 2, 7, 14, and 21, Mean bacterial counts (CFU/ ml) ± SD measured in placebo, infected, and CMC / zinc selenite groups\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/8f0e83395ae6ce992a47f744.jpg"},{"id":109205067,"identity":"518e25ca-bd13-4e3b-a3e3-9f7c36bf3b19","added_by":"auto","created_at":"2026-05-13 15:03:15","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":225348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003e(A\u0026amp;B)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003eshows the immunohistochemical Expression of (α-SMA) in Wound Tissue.\u003c/em\u003eMean α-SMA expression levels ± SD measured on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/c7168623e5d4a2a6eecb8822.jpg"},{"id":109170570,"identity":"cb3ba0ab-3135-4705-99e2-e5860ccfc97e","added_by":"auto","created_at":"2026-05-13 08:50:09","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":148379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eA and B show immunohistochemical Expression of EGFR in Wound Tissue.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMean EGFR expression levels ± SD assessed on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/b12bdaa453bf8c9b782cda6f.jpg"},{"id":109170566,"identity":"b539ecd6-4f13-4013-b759-5a506079b87e","added_by":"auto","created_at":"2026-05-13 08:50:08","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":181157,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRepresentative photomicrograph of skin wounds from different groups on day 7 at 100x magnification.\u003c/strong\u003e\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) the placebo group, (\u003cstrong\u003eB\u003c/strong\u003e) the infected group, (\u003cstrong\u003eC\u003c/strong\u003e) CMC / zinc selenite group.\u003c/p\u003e\n\u003cp\u003eThick arrow= focal loss of epidermal layer, twisted arrow= immature granulation tissue, E= epithelium, c= collagen, G= granulation tissue, bl = newly formed blood vessels, B= bacteria, star= inflammation\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/6a3836dc0c2c079ae08c7cd6.jpg"},{"id":109170567,"identity":"f28090c3-699c-4760-9221-c052f4cbbbf6","added_by":"auto","created_at":"2026-05-13 08:50:09","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":202633,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRepresentative photomicrograph of skin wounds from different groups on day 14 at 100x magnification\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e(\u003cstrong\u003eA\u003c/strong\u003e) the placebo group, (\u003cstrong\u003eB\u003c/strong\u003e) the infected group, (\u003cstrong\u003eC\u003c/strong\u003e) CMC / zinc selenite group\u003c/p\u003e\n\u003cp\u003eE= epithelium, c= collagen, star= inflammation, square= lymphoplasmacytic infiltration, arrowhead= angiogenesis, twisted arrow= immature fibroblasts.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/ba87a6b8b21bd322f0f4d84a.jpg"},{"id":109170564,"identity":"1b41f750-4ee3-4b33-943b-0470b0613852","added_by":"auto","created_at":"2026-05-13 08:50:08","extension":"jpg","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":165179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRepresentative photomicrograph of skin wounds from different groups on day 21 at 100x magnification\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e (\u003cstrong\u003eA\u003c/strong\u003e) the placebo group, (\u003cstrong\u003eB\u003c/strong\u003e) the infected group, (\u003cstrong\u003eC\u003c/strong\u003e) CMC / zinc selenite group.Thin arrow= dermal inflammation, arrowhead= incomplete dermal closure, partial loss of epidermal cell layer, c= collagen, h= hair follicle, F= fibroblast proliferation.\u003c/p\u003e","description":"","filename":"12.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/023200d4a823e0df83c6e6fe.jpg"},{"id":109207269,"identity":"e962b49a-9da4-4b96-98ac-a3c7d92e3f39","added_by":"auto","created_at":"2026-05-13 15:19:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1905610,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9549669/v1/0673ace2-304a-4ae5-9c65-a44993120e64.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic efficacy of Carboxymethyl Cellulose-based zinc selenite nanoparticles in infected wound healing in a rat model","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe development of strategies to modulate inflammation and enhance tissue regeneration following surgical procedures remains a significant challenge in clinical practice, owing to the complexity of the healing process and the diverse array of cellular and molecular components involved [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The management of exuding wounds is further complicated by microbial contamination, whose prompt eradication is vital to ensure successful wound healing [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The healing of infected wounds has consistently represented a critical global healthcare issue, attributed to its high prevalence, substantial disability burden, and elevated mortality rates [3, 4].\u003c/p\u003e \u003cp\u003eOver the past century, there has been a marked rise in wound infections, contributing significantly to the epidemiology of microbial infection [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Skin injuries, when complicated by infection, can exacerbate trauma and may ultimately lead to fatal outcomes through suppression of the immune system [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eStaphylococcus aureus (\u003c/em\u003eS. aureus) is the principal etiological agent of skin and soft tissue infections across all ages, climates, and geographic regions [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the absence of treatment, wounds infected with S. aureus exhibited a healing period of approximately 30 to 35 days, as determined by macroscopic evaluation and wound size analysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent approaches for wound healing and infection management primarily utilize antibiotics and analgesics. Despite growing concerns regarding their therapeutic efficacy, antibiotics continue to represent the most commonly employed agents in wound treatment [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Nevertheless, the prolonged use of high doses of multiple drugs not only imposes a significant financial burden on patients but also contributes to the emergence of drug-resistant bacteria, ultimately diminishing therapeutic efficacy [12, 13]. Emerging techniques in wound management, such as photomodulation, skin substitutes, and external tissue expanders, have shown variable efficacy; however, their substantial cost remains a major limitation to their widespread application [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCarboxymethyl cellulose (CMC) is a cellulose derivative that dissolves in water, synthesized by introducing carboxymethyl groups onto the cellulose backbone through a carboxymethylation reaction [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. CMC has emerged as a smart wound dressing material with excellent water absorption capabilities due to the polyelectrolytic properties of CMC, along with its sensitivity to pH and ionic strength [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Its hydrophilic nature allows CMC to absorb wound exudate and be easily combined with other polymers or bioactive agents, enhancing its functionality [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Wound dressing materials based on CMC provide biocompatibility, biodegradability, structural similarity to native tissue, affordability, and non-toxicity, positioning them as excellent candidates for infection management and wound healing applications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although CMC gels offer an effective approach to wound management by creating an optimal wet environment, promoting cell growth, and potentially separating the wound site, they do not fulfill all requirements for chronic wounds. Thus, new functions and features are highly recommended to assist in the wound healing of chronic wounds. This could be achieved by combining with other biopolymers or bioactive nanoparticles.\u003c/p\u003e \u003cp\u003eNanoparticles (NPs) have emerged as promising novel antibacterial agents, demonstrating significant efficacy against various infectious pathogens [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nanomaterials can modulate a range of cellular and molecular activities within the wound microenvironment, exerting antibacterial, anti-inflammatory, and angiogenic effects that facilitate the transition from a non-healing to a healing state [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. They have demonstrated effectiveness in combating both susceptible and resistant pathogens, as well as tolerant biofilms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The antimicrobial effects of NPs are thought to arise from a variety of potential mechanisms, including the generation of reactive oxygen species (ROS), disruption of protein and DNA synthesis, activation of metabolic pathways, and alterations in cell wall structure and membrane permeability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Selenium-based nanoparticles (Se-NPs) have emerged as highly promising bioactive nanoparticles in regenerative medicine due to their unique physicochemical properties, biocompatibility, and multifunctional therapeutic potential with drug delivery capabilities. They are being extensively researched for their anticancer, antioxidant, anti-diabetic, and anti-inflammatory properties, as well as their role in targeted drug delivery. Among them, CMC/zinc selenite (ZnSeO) is an inorganic compound combining zinc (Zn\u0026sup2;⁺) and the selenite anion (SeO₃\u0026sup2;⁻). While less biologically studied than other zinc or selenium compounds (e.g., zinc selenide (ZnSe), silver selenide (Ag\u003csub\u003e2\u003c/sub\u003eSe), or sodium selenite (Na₂SeO₃)), it has intriguing properties that make it relevant in materials science, catalysis, and biomedicine[\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To the best of our knowledge, CMC / zinc selenite nanoparticles (ZnSeO NPs) have not yet been reported in biomedical literature. The growing interest in zinc- and selenium-based nanomaterials suggests significant untapped potential\u0026mdash;particularly in wound healing, antimicrobial therapy, and bioactive coatings.\u003c/p\u003e \u003cp\u003eTaking into account the additional properties of ZNO and SeNPs, such as immunomodulatory and anti-inflammatory activities [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Notably, all antimicrobial investigations on SeNPs have reported no bacterial resistance thus far, highlighting their potential as an effective option for treating infectious and chronic wounds [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thus, this study aimed to assess the effectiveness of topically applied CMC / zinc selenite (CMC/ZnSeONPs) gel for infected full-thickness cutaneous wound healing in rats. The assessment was conducted through macroscopic (body weight, wound size, and contraction), histopathological, and immunohistochemical evaluations, bacterial count, and bacterial viability tests.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e2.1.\u0026nbsp;Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZinc acetate dihydrate (C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eZn\u0026nbsp;\u0026sdot;\u0026nbsp;2H\u003csub\u003e2\u003c/sub\u003eO),\u0026nbsp;sodium selenite (Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e), and sodium hydroxide (NaOH\u003csub\u003e2\u003c/sub\u003e) were used as chemicals in the synthesis of nanoparticles. All the chemicals were bought from Sigma-Aldrich. The chemicals were used without any further processing. Double-distilled water was used as a solvent for solution preparation and washing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.\u0026nbsp;Preparation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ezinc selenite\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003enanoparticles (Zn\u003c/strong\u003e\u003cstrong\u003eSeONPs\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCMC / zinc selenite nanoparticles (ZnSeONPs) were prepared using a chemical reduction approach. Firstly, 0.40\u0026nbsp;g of Zinc acetate dehydrate (Zn (CH\u003csub\u003e3\u003c/sub\u003eCO\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO) and 0.25\u0026nbsp;g of sodium selenite (Na\u003csub\u003e2\u003c/sub\u003eSeO3) were dissolved in 100\u0026nbsp;ml of DW. The reaction mixture was placed on a magnetic stirrer for 30 min, and then 5 ml of ascorbic acid solution (100 mM) was slowly added while stirring continuously. The mixture color was changed to reddish orange. The reaction mixture was heated up to 80 \u0026deg;C, and the pH was maintained at 10 using NaOH solution (0.1 M). After 2 h, the reaction components were precipitated using an absolute ethanol solution (50 ml) to remove unreacted components and byproducts. The ZnSeONPs were collected by centrifugation at 6,000\u0026nbsp;rpm for 10\u0026nbsp;min and washed with water three times. Finally, the precipitate was dried at 80\u0026nbsp;\u0026deg;C in an air oven and stored at 4 \u0026deg;C in the dark.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3.\u0026nbsp;Preparation of CMC/ZnSeONPs gel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dried ZnSeONPs were mixed with sodium CMC solution to obtain CMC gel enriched with ZnSeONPs. First, 2g of sodium CMC were dissolved in 90 mL of distilled water at 80\u0026deg;C for 15 min under constant stirring. After that, the as-prepared ZnSeONPs (0.1g) were dispersed in 10 ml distilled water. The obtained ZnSeONPs were added dropwise to the CMC solution with a magnetic stirrer until the transparent CMC mixture turned milky.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe ZnSeO and CMC mixture was subjected to high mixing speed using a high-shear homogenizer at 5000 rpm for 10 min, followed by\u0026nbsp;sonication in a bath sonicator for 30 min.\u0026nbsp;The obtained ZnSeO/CMC gel was stored at 4 \u003csup\u003eo\u003c/sup\u003eC for further use.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e2.4. Ethical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. \u003c/strong\u003eThe study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code (VM.MS.24.10.166). All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical All procedures were documented following the guidelines specified by ARRIVE [28]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1. \u003c/strong\u003e\u003cstrong\u003eAnimals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, fifty-four adult male Albino Wistar rats, aged 10-12 weeks with an average body weight of 256.7 \u0026plusmn; 13.7 g, were used. The animals were obtained from the animal facility at the Faculty of Science, Mansoura University, Mansoura, Egypt. The rats were housed individually in separate polypropylene cages in the laboratory at Mansoura Teaching Hospital. Bedding consisting of wood shavings was provided, and it was changed daily. The lighting cycle was set to 12 hours of light per day. The rats had unrestricted access to food and water. Before the experiment, the rats were acclimatized to the laboratory conditions for two weeks and were thoroughly examined to ensure that only healthy animals were included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Preparation of bacterial inoculum:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to [29], \u003cem\u003eS. aureus\u003c/em\u003e was cultured in broth at 37\u0026deg;C for 18 hours prior to its use. The culture was then washed with 0.1 M phosphate-buffered saline (PBS) at pH 7.2, and a suspension with a concentration of 2\u0026times;10⁹ CFU/mL was prepared. The concentration was determined using a colorimetric spectrometer (Biostc) from the Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Mansoura University, Egypt. The freshly prepared \u003cem\u003eS. aureus\u003c/em\u003e suspension was then injected into the skin wounds of the infected and CMC / zinc selenite groups. This suspension was obtained from the Bacteriology, Mycology, and Immunology Department at the Faculty of Veterinary Medicine, Mansoura University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Microstructure Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the formation of the ZnSeO-NPs ; X-ray diffraction (XRD) was carried out to determine the crystal structure of the as-prepared materials. Also, Transmission electron microscopy (TEM) was performed to study the particle size of the material in nano scale, crystal structure and examine the morphological characteristics of nanoparticles formulas by a high-performance digital imaging transmission electron microscopy (JEOLH-7650, Hitachi High-Technologies Corp., Tokyo, Japan) with an acceleration voltage operating at 200 kV.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Experimental procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eintraperitoneal injection of 5 mg/kg xylazine HCL (20 mg/ml, Xylaject, ADWIA, Cairo, Egypt), and 60 mg/kg ketamine HCL (50 mg/ml, Aneket\u003csup\u003e\u0026reg;\u003c/sup\u003e, NEON Laboratories Ltd, Mumbai, India) for anesthesia of the rats. Aseptic preparation of the dorsal region between the two scapulae. The rats were transported to the surgery room to make a 23 mmdiameter full-thickness circular cutaneous defect by excision of the rat skin. The CMC / zinc selenite and the infected groups received intradermal inoculation of the wound lips with 0.3 ml freshly prepared suspension of 2\u0026times;10\u003csup\u003e9 \u003c/sup\u003eCFU/mL \u003cem\u003eS. aureus\u003c/em\u003e [29]. Animals received an intramuscular injection with a single dose of meloxicam (0.4 mg/kg) for analgesic effects.\u003c/p\u003e\n\u003cp\u003eRats were divided into three groups (n=18 per group) after 3 days of surgery according to the infection and treatment protocol:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Placebo group: \u003c/strong\u003ewounds without infection or treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Infected group: \u003c/strong\u003einfected wounds without treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003e\u003cstrong\u003eCMC / zinc selenite group\u003c/strong\u003e: infectedwounds received a topical application of 1 ml of CMC/ZnSeONPs geltwice daily after flushing.\u003c/p\u003e\n\u003cp\u003eAll groups undergo flushing twice daily with 5 mL of normal saline (0.9% sodium chloride solution, 500 mL; Ultimate Pharma, Egypt). Neither antiseptics nor antibiotics were utilized throughout the study. All wounds were surgically induced by the same experienced surgeon, followed by standardized bacterial inoculation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Evaluation of wound healing:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.1. Macroscopic Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWound evaluations were performed by a single experienced surgeon, who was masked to group allocation, on postoperative days 0, 3, 7, 11, 15, and 19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA- Body weight (g)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats\u0026apos; body weight (g) was measured on postoperative days 0, 3, 7, 11, 15, and 19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB- Wound size (mm\u003csup\u003e2\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWound size was measured using a digital caliper and documented with photographs on postoperative days 0, 3, 7, 11, 15, and 19\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC- Wound contraction (%)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extent of wound contraction was evaluated on postoperative days 0, 3, 7, 11, 15, and 19, based on the formula described by [30], and subsequently analyzed statistically.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e(1)\u003c/u\u003e Percentage of wound size at day (x) = wound size at day (x) mm\u003csup\u003e2\u003c/sup\u003e / wound size at day (0) mm\u003csup\u003e2\u003c/sup\u003e x 100.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003e(2)\u003c/u\u003e Percentage of wound contraction = 100 - percentage of wound size at day (x)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.2. Microscopic evaluations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn postoperative days 7, 14, and 21, six animals from each group were euthanized via intraperitoneal injection of pentobarbital overdose (120 mg/kg, Nembutal\u0026reg;, 50 mg/ml, Akorn Operating Company, Gurnee). Skin samples for histopathological and immunohistochemical analysis were preserved in 10% neutral buffered formalin. All euthanized rats and bedding material were hygienically disposed of.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. Bacterial count\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfected wounds were swabbed using a sterile cotton-tipped applicator, which was rotated at the center of the wound. The swabs were then transferred into sterile tubes containing 1 mL of transport solution and subjected to serial dilution with the same medium. The samples were incubated at 37\u0026deg;C for 24 hours after being cultured on Baird-Parker agar plates (Himedia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. Bacterial viability test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvernight bacterial cultures were grown and adjusted to a density equivalent to 0.5 McFarland standard (approximately 1.5 \u0026times; 10⁸ CFU/mL) by suspending the cells in sterile PBS. A sterile 24-well plate was prepared, with each well containing 1 mL of nutritive broth with 10 \u0026micro;L of the bacterial suspension for the infected group, and another sterile 24-well plate with each well containing 1 mL of nutritive broth, CMC/ZnSeONPsgel, and 10 \u0026micro;L of the bacterial suspension for the CMC / zinc selenite group. The plates were incubated at 37\u0026deg;C under humid conditions for 1, 6, 12, and 24 hours. \u003c/p\u003e\n\u003cp\u003eTo assess bacterial viability, the wound dressing sample (CMC/ZnSeONPsgel) was transferred into sterile Eppendorf tubes containing 1 mL of PBS immediately after incubation. The tubes were vortexed for 20 seconds to release viable cells into the suspension. From each suspension, a 30 \u0026micro;L aliquot was mixed with 270 \u0026micro;L of PBS in a 96-well plate. Serial dilutions were then made, and the resulting samples were cultured in triplicate onto Baird-Parker agar. After 24 hours of incubation at 37\u0026deg;C, colonies displaying a characteristic shiny grey-black appearance with an opaque halo were counted to determine the number of colony-forming units per milliliter (CFU/mL)[31].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. Histopathological evaluation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkin samples from the full-thickness wound areas, including sections of adjacent healthy skin, were collected from each group on days 7, 14, and 21. These specimens were immediately fixed in 10% neutral buffered formalin for 24 hours, processed through routine histological procedures, and embedded in paraffin blocks. The resulting 4 \u0026micro;m-thick sections were stained with Hematoxylin \u0026amp; Eosin (H\u0026amp;E) and subsequently examined microscopically[32]. H\u0026amp;E-stained sections were examined for the extent of re-epithelialization, angiogenesis, Bacteria, and collagen deposition according to [33] with modifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. Immunohistochemical evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded tissue samples were sliced into 4 \u0026mu;m-thick sections and mounted on saline-coated glass slides. The sections were deparaffinized using xylene and dehydrated with a series of ethanol solutions. Antigen retrieval was achieved by autoclaving at 120˚C for 10 minutes at pH 6.0. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Primary antibodies targeting Epidermal Growth Factor Receptor (EGFR) and Alpha Smooth Muscle Actin (\u0026alpha;-SMA) were applied and incubated at room temperature for one hour. The sections were then washed three times with phosphate-buffered saline. Following a 30-minute incubation with anti-rabbit secondary antibodies, the slides were treated with the 3, 3\u0026apos; diaminobenzidine tetrahydrochloride (DAB) liquid system for 5 minutes at room temperature. Finally, the sections were counterstained with hematoxylin and examined under light microscopy [34].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003cstrong\u003e10\u003c/strong\u003e.\u003cstrong\u003e Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed using GraphPad Prism software (version 10.0; GraphPad Software, San Diego, CA, USA). Data are presented as mean \u0026plusmn; SD from at least three independent experiments. Group comparisons were conducted using Two-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s or \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test, whenever appropriate, to determine statistically significant differences between groups with the effect of both time and treatment, as well as the interaction between the two variables. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003cp\u003e\u003cstrong\u003e4.1.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMicrostructure Characterization:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wet precipitation method is a widely used chemical technique for synthesizing \u003cstrong\u003emetal and metal oxide nanoparticles\u003c/strong\u003e due to its simplicity, cost-effectiveness, and ability to control particle size and morphology. In this study, ZnSeONPs were prepared using a reducing agent (e.g., ascorbic acid) to facilitate the reduction of selenium ions and promote ZnSeO formation, as shown in Fig.1.\u003c/p\u003e\n\u003cp\u003eThe XRD pattern of the synthesized CMC / zinc selenite via the sol-gel method (\u003cstrong\u003eFig.2A\u003c/strong\u003e) displays both amorphous and crystalline features. A broad hump in the 2\u0026theta; range of 16\u0026deg; to 23\u0026deg; indicates the presence of an amorphous phase in the CMC / zinc selenite sample. Such humps are typically attributed to short-range order and lack of long-range crystallinity, which is common in sol-gel derived materials due to incomplete crystallization or retained organic/inorganic precursors. This feature suggests that the synthesis did not result in a fully crystalline material and that an amorphous CMC / zinc selenite matrix may be present [35, 36]. Superimposed on the hump are several sharp diffraction peaks, which match well with the standard pattern of monoclinic Zn₃Se₄O₁₄, as per the ICDD card 96-156-1697. These peaks confirm the partial crystallization of the material into a monoclinic phase. The good agreement between the experimental peaks and the reference card suggests that the primary crystalline phase is indeed Zn₃Se₄O₁₄. The coexistence of a broad amorphous hump and sharp diffraction peaks indicates that the material is semi-crystalline, containing both crystalline Zn₃Se₄O₁₄ domains and amorphous regions. This dual-phase composition is not uncommon in sol-gel-derived oxides, particularly when calcination or aging conditions are not optimized for complete crystallization\u0026nbsp;[35, 36].\u003c/p\u003e\n\u003cp\u003eTEM analysis of the obtained ZnSeONPs is presented in \u003cstrong\u003eFig.\u0026nbsp;(2-B and C)\u003c/strong\u003e. As can be seen, the ZnSeONPs presented irregular-shaped nanoparticles. \u0026nbsp;The average diameter of ZnSeONPs was around 51\u0026thinsp;\u0026plusmn;\u0026thinsp;13 nm. The inserted SAED images reveal a diffraction pattern indicating the crystalline structure of the prepared NPs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.\u0026nbsp;Macroscopic evaluation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial inoculation into the wounds led to wound infection after 3 days which was confirmed by Gram stain and bacterial culture and accumulation of pus in wounds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.1. \u0026nbsp; Wound Size:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantitative assessment of macroscopic wound healing is achieved by measuring wound size at specified intervals (days 0, 3, 7, 11, 15, and 19) across three experimental groups: placebo (non-infected, untreated), infected (infected, treated), and CMC / zinc selenite (infected, treated). Time (Row Factor: p \u0026lt; 0.0001, \u0026epsilon; = 0.5244), treatment group (Column Factor: p \u0026lt; 0.0001), and their interaction (p \u0026lt; 0.0001, \u0026lambda;). All groups exhibited statistically equivalent wound diameters at baseline, day 0 (p = 0.87), confirming uniform initial damage. Both the infected (398.3 \u0026plusmn; 23.9 mm\u0026sup2;) and CMC / zinc selenite (398.7 \u0026plusmn; 19.2 mm\u0026sup2;) groups exhibited minimal reduction, with a borderline significant difference observed between the placebo and infected groups (Tukey\u0026apos;s p = 0.07). In contrast, wound contraction was initiated in the placebo group (332.1 \u0026plusmn; 45.5 mm\u0026sup2;) by day three. By day 7, notable differences were observed: the placebo group exhibited a significant reduction in wound size (210.0 \u0026plusmn; 6.4 mm\u0026sup2;), while the infected group retained considerably larger wounds (375.9 \u0026plusmn; 17.7 mm\u0026sup2;). The CMC / zinc selenite group demonstrated intermediate healing (323.5 \u0026plusmn; 46.3 mm\u0026sup2;). The differences between the placebo and infected groups (p \u0026lt; 0.0001) and between the CMC / zinc selenite and infected groups (p = 0.004) were statistically significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;On day 11, further healing progression was observed; the infected group exhibited significant impairment (311.2 \u0026plusmn; 16.7 mm\u0026sup2;), whereas the placebo group demonstrated notable wound closure (76.7 \u0026plusmn; 13.1 mm\u0026sup2;). Compared to the CMC / zinc selenite group (215.4 \u0026plusmn; 48.0 mm\u0026sup2;; p = 0.0011), the CMC/ZnSeONPs gel accelerated healing. All intergroup differences were statistically significant (p \u0026lt; 0.0001). On day 15, the placebo group achieved near-complete wound closure (0.6 \u0026plusmn; 0.6 mm\u0026sup2;), the CMC / zinc selenite group exhibited substantial healing (104.4 \u0026plusmn; 15.9 mm\u0026sup2;), while the infected group showed minimal healing (228.9 \u0026plusmn; 29.0 mm\u0026sup2;). On Day 19, the CMC / zinc selenite group exhibited nearly complete healing (2.0 \u0026plusmn; 1.8 mm\u0026sup2;), while the placebo group achieved complete re-epithelialization, and the infected group showed only partial healing (111.2 \u0026plusmn; 11.8 mm\u0026sup2;). The results demonstrate a statistically significant improvement over untreated infected controls across all post-baseline periods, indicating the therapeutic efficacy of topical CMC/ZnSeONPs gel in enhancing wound healing under infected conditions (\u003cstrong\u003eTable 1, Fig. 3\u0026amp; 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 1\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Effect Of Topical CMC/ZnSeONPs Gel On Wound Area Reduction In Rats Over Time.\u003c/em\u003e\u003cbr\u003e\u0026nbsp;Mean wound area (mm\u0026sup2;) \u0026plusmn; SD measured on days 0, 3, 7, 11, 15, and 19 in placebo (non-infected untreated), infected (infected untreated), and CMC / zinc selenite (infected treated) groups. Statistical analysis was performed using two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. *P \u0026lt; 0.05 considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSelenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e421.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e422.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e423.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e332.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e45.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e398.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e23.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e398.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e210.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e6.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e375.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e323.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e46.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e76.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e311.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e16.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e215.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e48.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e228.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e104.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e15.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e111.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2. \u0026nbsp; Wound contraction:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMacroscopic wound contraction was quantitatively measured on days 3, 7, 11, 15, and 19. Two-way ANOVA utilizing the Geisser-Greenhouse correction indicated significant main effects for time (Row Factor: p \u0026lt; 0.0001, \u0026epsilon; = 0.7066) and treatment group (Column Factor: p Inter-subject variance was not significant, with a p-value of 0.0513. \u0026nbsp;On day 3, the placebo group exhibited a significantly higher wound contraction (21.2 \u0026plusmn; 10.9%) in comparison to the infected group (5.6 \u0026plusmn; 5.7%) and the CMC / zinc selenite group (5.8 \u0026plusmn; 4.5%). Statistically significant differences were noted between the placebo and infected groups (p = 0.0369) as well as between the placebo and CMC / zinc selenite groups (p = 0.0375), while the CMC / zinc selenite and infected groups showed no significant difference (p = 0.9979). The CMC / zinc selenite group demonstrated a moderate improvement of 23.6 \u0026plusmn; 10.8%. The comparison between the placebo and CMC / zinc selenite approached significance (p = 0.0761), with the CMC / zinc selenite group continuing to show enhanced wound contraction by day 7. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day 11, wound contraction was notably enhanced in the placebo group (81.8 \u0026plusmn; 3.1%), while the CMC / zinc selenite group (49.1 \u0026plusmn; 11.2%) significantly surpassed the infected group (26.2 \u0026plusmn; 4.2%), with all intergroup differences reaching statistical significance (p \u0026lt; 0.01). On day 15, the placebo group exhibited nearly complete contraction (99.9 \u0026plusmn; 0.2%), while the CMC / zinc selenite group showed a contraction of 75.3 \u0026plusmn; 3.7%, and the infected group demonstrated a contraction of 45.8 \u0026plusmn; 6.7%. All comparisons were statistically significant (p \u0026lt; 0.0001). On day 19, the CMC / zinc selenite group exhibited nearly complete healing at 99.5 \u0026plusmn; 0.4%, while the placebo group achieved full wound closure at 100.0 \u0026plusmn; 0.0%. Both groups significantly outperformed the infected group, which recorded a contraction rate of 73.6 \u0026plusmn; 2.8%. The difference between placebo and CMC / zinc selenite was not statistically significant (p = 0.0802); however, the differences between infected and CMC / zinc selenite were significant (p \u0026lt; 0.0001) (\u003cstrong\u003eTable 2, Figure 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 2\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Percentage of Wound Contraction Over Time in different groups.\u003c/em\u003e\u003cbr\u003eMean percentage of wound contraction \u0026plusmn; SD measured on days 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups. Statistical significance was evaluated using two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eselenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e21.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0375\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.9979\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e50.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e23.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0761\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e81.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e26.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e49.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.007\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e99.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e45.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e75.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e100.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e73.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3. \u0026nbsp; Body weight\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody weight changes were monitored in the three experimental groups to assess systemic well-being and treatment-related effects. In addition to notable inter-subject variation (p \u0026lt; 0.0001), two-way ANOVA with Geisser-Greenhouse correction indicated significant effects of time and treatment group (p \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003eAll groups exhibited comparable body weights at baseline (day 0; mean range: 250.8\u0026ndash;256.7 g; p \u0026gt; 0.5), indicating no initial differences. On day 3, both the infected group (205.0 \u0026plusmn; 8.4 g) and the CMC / zinc selenite group (200.0 \u0026plusmn; 12.6 g) exhibited significant weight loss (p = 0.0001 for both), in contrast to the placebo group (249.2 \u0026plusmn; 13.9 g; p = 0.0001). The CMC / zinc selenite and infected groups were statistically comparable (p = 0.7508). Beginning on day seven, distinct recovery patterns emerged. The infected group exhibited significant weight loss (171.7 \u0026plusmn; 13.3 g), while the placebo group demonstrated a consistent weight increase (258.3 \u0026plusmn; 25.8 g). The CMC / zinc selenite group (213.3 \u0026plusmn; 8.2 g) demonstrated partial recovery, exhibiting significantly greater weight compared to infected controls (p = 0.0023), although it remained considerably lower than the placebo group (p = 0.0012). On day 11, the infected group exhibited a decline in body weight that may be due to infection, whereas the placebo group continued to gain weight, averaging 281.7 \u0026plusmn; 22.3 g. The CMC / zinc selenite rats (242.5 \u0026plusmn; 8.8 g) exhibited statistically significant improvements, with changes noted in both the infected (p = 0.0007) and placebo (p \u0026lt; 0.0001) groups.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;On day 15, the CMC / zinc selenite group exhibited a mean weight of 262.5 \u0026plusmn; 7.6 g, which was intermediate between the placebo group at 295.0 \u0026plusmn; 24.3 g and the infected group at 154.2 \u0026plusmn; 4.9 g. Statistical significance was noted with p = 0.0001 for the comparison between CMC / zinc selenite and infected groups, and p = 0.005 for the placebo versus infected groups. On day 19, the CMC / zinc selenite group exhibited body weight measurements that were statistically similar to those of the placebo group (305.8 \u0026plusmn; 26.2 g; p = 0.538), demonstrating nearly complete recovery in body weight (295.8 \u0026plusmn; 4.9 g), and significantly higher than the infected group (182.5 \u0026plusmn; 7.6 g; p = 0.538) (\u003cstrong\u003eTable 3, Figure 5).\u003c/strong\u003e These findings suggest that CMC/ZnSeONPs gel not only aids in wound healing but also mitigates infection-induced systemic stress, facilitating recovery of body weight comparable to infected controls.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 3.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Changes in Body Weight (g) of Rats During the Wound Healing Period.\u003c/em\u003e\u003cbr\u003eMean body weight \u0026plusmn; SD recorded on days 0, 3, 7, 11, 15, and 19 in placebo, infected, and CMC / zinc selenite groups. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eselenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e256.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e13.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e250.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e250.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.5044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.5044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e249.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e13.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e205.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e200.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.7508\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e258.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e25.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e171.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e213.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e281.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e136.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e242.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0007\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e295.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e24.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e154.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e262.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.005\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e305.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e26.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e182.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e295.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.\u0026nbsp;Microscopic evaluations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e4.3.1. \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003eBacterial viability test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibacterial activity of CMC/ZnSeONPs gel against S. aureus infection was evaluated over 24 hours. Two-way ANOVA revealed that both treatment and exposure duration significantly influenced bacterial load, with statistically significant effects observed for time (p \u0026lt; 0.0001) and treatment group (p Additionally, subject variability was significant (p \u0026lt; 0.0001), thereby reinforcing the robustness of the findings. Both CMC / zinc selenite and infected groups exhibited comparable bacterial burdens (1.5 \u0026times; 10⁸ CFU; p \u0026gt; 0.99), thereby confirming similar initial contamination at baseline (0 hr.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe CMC / zinc selenite group exhibited a substantial reduction in bacterial load (5.0 \u0026times; 10⁸ \u0026plusmn; 0 CFU), whereas the untreated infected group demonstrated a rapid increase (6.0 \u0026times; 10⁹ \u0026plusmn; 8.94 \u0026times; 10⁸ CFU) at one hour post-infection, indicating a highly significant difference. The CMC / zinc selenite group exhibited a notable reduction to 9.0 \u0026times; 10⁴ \u0026plusmn; 2.68 \u0026times; 10⁺ CFU (p \u0026lt; 0.0001), whereas bacterial proliferation continued in the infected group (5.4 \u0026times; 10⁹ \u0026plusmn; 5.37 \u0026times; 10⁸ CFU) at the six-hour mark. The CMC / zinc selenite group exhibited a further reduction to 3.0 \u0026times; 10⁴ \u0026plusmn; 1.79 \u0026times; 10\u0026sup1;⁰ CFU, while the infected group achieved 2.4 \u0026times; 10\u0026sup1;⁰ \u0026plusmn; 3.58 \u0026times; 10⁹ CFU after 12 hours. \u0026nbsp; After 24 hours, the bacterial load in the infected group reached a peak of 4.5 \u0026times; 10\u0026sup1;⁰ \u0026plusmn; 2.68 \u0026times; 10⁹ CFU, while the CMC / zinc selenite group nearly eradicated the viable bacterial cells, resulting in 1.0 \u0026plusmn; 2.0 CFU (p \u0026lt; 0.0001). \u0026nbsp;The findings demonstrate that topical CMC/ZnSeONPs gel exhibits significant and rapid antibacterial efficacy, reducing bacterial viability by over 99.9999% within 24 hours. The strong bactericidal action supports its potential application as a therapeutic agent for managing contaminated wounds (\u003cstrong\u003eTable 4, Figure 6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 4.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Changes in the viability of S. aureus bacteria (CFU/ml) recorded on hours (0, 1, 6, 12, and 24) in infected and CMC / zinc selenite groups\u003c/em\u003e. Statistical analysis was performed using two-way ANOVA followed by \u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s multiple comparisons test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003eHours\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" style=\"width: 31.5921%;\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eSelenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Scaron;\u0026iacute;d\u0026aacute;k\u0026apos;s\u0026nbsp;multiple\u003c/p\u003e\n \u003cp\u003ecomparisons\u0026nbsp;test\u003c/p\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003e1.50E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.50E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u0026gt;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003e6.00E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e8.94E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.00E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003e5.40E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.37E+08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e9.00E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.68E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003e2.40E+10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.58E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.00E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.79E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" style=\"width: 9.9502%;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 15.9204%;\"\u003e\n \u003cp\u003e4.50E+10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.68E+09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.2. \u0026nbsp; Bacterial count\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn days 2, 7, 14, and 21 post-wounding, the bacterial load in the wound tissue was quantitatively assessed to compare the antimicrobial efficacy of CMC/ZnSeONPs gel against infected and uninfected controls. Subject variability was statistically significant (p = 0.0064). Two-way ANOVA revealed significant effects for treatment group (p = 0.0001), time (p = 0.0001, \u0026epsilon; = 0.6210), and their interaction (p = 0.0001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Compared to the placebo (1.10 x 10⁴ \u0026plusmn; 2.64 x 10\u0026sup1; CFU), bacterial counts were significantly elevated in both the infected (1.09 x 10⁴ \u0026plusmn; 2.34 x 10\u0026sup1; CFU) and CMC / zinc selenite groups (9.77 x 10\u0026sup1; CFU) at day two (p \u0026lt; 0.0001 for both comparisons). There was no significant difference between the infected and CMC / zinc selenite groups (p = 0.5925), indicating similar early bacterial colonization.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;On day 7, the bacterial burden in the infected group was significantly elevated (1.28 \u0026times; 10⁴ \u0026plusmn; 2.58 \u0026times; 10\u0026sup3; CFU) compared to the placebo group (1.10 \u0026times; 10\u0026sup3; \u0026plusmn; 0.41 CFU, p \u0026lt; 0.0001). The CMC / zinc selenite group notably decreased the bacterial load to 4.07 \u0026times; 10\u0026sup3; \u0026plusmn; 3.45 \u0026times; 10\u0026sup3; CFU, representing a significant reduction relative to the infected group (p \u0026lt; 0.0001), although it did not show a statistically significant difference from the placebo (p = 0.1312).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day 14, the infected group exhibited a bacterial count of 5.11 \u0026times; 10\u0026sup1; \u0026plusmn; 1.83 \u0026times; 10\u0026sup1; CFU, while the placebo group demonstrated complete bacterial clearance, recorded at 0 CFU. In comparison to the infected group (p = 0.0004), the CMC / zinc selenite group significantly reduced bacterial counts to 2.76 \u0026times; 10\u0026sup2; \u0026plusmn; 5.51 \u0026times; 10\u0026sup2; CFU; this difference was not statistically significant when compared to placebo (p = 0.9338). On day 21, the bacterial load in the infected group was still detectable (2.61 x 10\u0026sup1; \u0026plusmn; 7.08 \u0026times; 10\u0026sup2; CFU), whereas both the placebo and CMC / zinc selenite groups exhibited complete eradication (0 CFU) (\u003cstrong\u003eTable 5, Figure 7\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 5.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Quantitative Bacterial Load (CFU) in Wound Tissue Over the Study Period.\u003c/em\u003e\u003cbr\u003eMean bacterial counts (CFU) \u0026plusmn; SD measured on days 2, 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Statistical comparisons were made using two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"587\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u003c/p\u003e\n \u003cp\u003eselenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.10E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.16E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.09E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.64E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e9.77E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.34E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.5925\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.10E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.08E-01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.28E+04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.58E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.07E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.45E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.1312\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026lt;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.11E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.83E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.76E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.51E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.9338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.61E+03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.08E+02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.00E+00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.004\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.3. \u0026nbsp; Immuno-histochemical evaluation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u0026nbsp;\u0026nbsp;\u0026alpha;-SMA expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn days 7, 14, and 21 postoperatively, \u0026alpha;-SMA expression was quantified to evaluate myofibroblast activation, serving as an indicator of wound contraction and tissue remodeling. Subject variability was not significant (p = 0.3705), suggesting robust intergroup and temporal effects. The two-way ANOVA revealed significant effects of group (p = 0.0091), time (p = 0.0016, \u0026epsilon; = 0.9051), and their interaction (p \u0026lt; 0.0001). \u0026nbsp;There was no significant difference between the CMC / zinc selenite and infected groups (p = 0.1421). However, \u0026alpha;-SMA expression was significantly elevated in the placebo group (20.9 \u0026plusmn; 0.66) on day 7 compared to both the infected group (4.5 \u0026plusmn; 1.02, p = 0.0034) and the CMC / zinc selenite group (7.4 \u0026plusmn; 1.64, p = 0.0034). This suggests an initial suppression of myofibroblast differentiation in infected wounds, which is partially restored with CMC/ZnSeONPs gel treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day 14, \u0026alpha;-SMA expression in the CMC / zinc selenite group increased to 15.2 \u0026plusmn; 2.46, surpassing the levels observed in the placebo group (11.9 \u0026plusmn; 3.48) and the infected group (7.9 \u0026plusmn; 1.78). While CMC / zinc selenite and placebo (p = 0.3177) or CMC / zinc selenite and infected (p = 0.0355) did not demonstrate statistically significant differences, the elevated expression in the CMC / zinc selenite group indicates a heightened fibroblast-to-myofibroblast transition at this stage.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In the infected group, \u0026alpha;-SMA levels remained elevated (11.3 \u0026plusmn; 2.07), likely indicating persistent inflammation and delayed remodeling. Conversely, in the placebo group, \u0026alpha;-SMA levels decreased (4.4 \u0026plusmn; 0.51) on day 21, consistent with the expected progression of wound healing. CMC / zinc selenite group exhibited intermediate \u0026alpha;-SMA expression (6.9 \u0026plusmn; 1.58), which was slightly lower than that of the infected group (p = 0.0438) and not statistically different from the placebo (p = 0.0989).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;CMC/ZnSeONPs gel application frequently diminished \u0026alpha;-SMA dynamics, thereby promoting timely myofibroblast activation during the mid-phase of healing and restricting prolonged expression associated with chronic inflammation, suggesting a beneficial effect on wound maturation and resolution (\u003cstrong\u003eTable 6, Figure 8\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 6\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Mean Values and SD Of Immunohistochemical Expression Of (\u0026alpha;-SMA) In Wound Tissue.\u003c/em\u003e Measured on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test was used for statistical comparison. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"568\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u003c/p\u003e\n \u003cp\u003eselenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0034\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.1421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e15.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.3177\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.4475\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0355\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e1.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.1763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0989\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u0026nbsp;EGFR expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEGFR expression was assessed on days 7, 14, and 21 postoperatively to evaluate epithelial regeneration and proliferative activity during the healing process. Subject-level variability was non-significant (p = 0.2251), supporting the consistency of treatment-related and temporal effects. However, two-way ANOVA revealed significant main effects for treatment group (p = 0.0002) and time.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDay 7: EGFR expression in the infected group (1.8 \u0026plusmn; 0.24) was significantly lower than in the placebo group (3.4 \u0026plusmn; 0.32; p = 0.0065), indicating a reduction in epithelial activation due to infection. CMC / zinc selenite group resulted in EGFR levels of (3.0 \u0026plusmn; 0.08), which were significantly higher than those in the infected group (p = 0.0142) and not significantly different from the placebo (p = 0.3008), indicating a potential early reactivation of epithelial repair mechanisms. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn day fourteen, EGFR expression reached its maximum level in both CMC / zinc selenite (7.2 \u0026plusmn; 0.58) and placebo (9.2 \u0026plusmn; 0.77) groups. In the infected group, EGFR slightly elevated from its level on day 7 but remained relatively low at 2.7 \u0026plusmn; 0.18. Although CMC / zinc selenite was still lower than the placebo group (p = 0.006), the CMC / zinc selenite group had a significantly increased level of EGFR expression compared to the infected group (p = 0.006), indicating a partial restoration of epithelial proliferative signaling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;On day 21, EGFR expression decreased across both CMC / zinc selenite and placebo groups, consistent with the resolution of the proliferative phase. In comparison to placebo (2.8 \u0026plusmn; 0.23; p = 0.0091), infected wounds (4.3 \u0026plusmn; 0.33) exhibited increased EGFR expression, likely indicating delayed or abnormal healing processes. Although comparisons did not reach statistical significance when compared to infected (p = 0.2716) or placebo (p = 0.0585) groups, CMC / zinc selenite exhibited the highest expression (5.5 \u0026plusmn; 0.93)(\u003cstrong\u003eTable 7, Figure 9\u003c/strong\u003e). These findings indicate that CMC/ZnSeONPs gel enhances EGFR-mediated epithelial responses in the early and mid-phases of healing, potentially mitigating the suppression caused by infection and facilitating wound re-epithelialization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 7.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Immunohistochemical Expression of EGFR in Wound Tissue.\u003c/em\u003e Mean EGFR expression levels \u0026plusmn; SD assessed on days 7, 14, and 21 in placebo, infected, and CMC / zinc selenite groups. Statistical comparisons were made using two-way ANOVA with Tukey\u0026rsquo;s post hoc test. P-value1: placebo vs. infected; P-value2: placebo vs. CMC / zinc selenite; P-value3: infected vs. CMC / zinc selenite. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significant.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"572\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eDays\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" style=\"width: 13.2867%;\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\" style=\"width: 13.8112%;\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"2\"\u003e\n \u003cp\u003eCMC/ Zinc\u003c/p\u003e\n \u003cp\u003eselenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"3\"\u003e\n \u003cp\u003eTukey\u0026rsquo;s adjusted p-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 7.6923%;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" style=\"width: 5.7692%;\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eP-value3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 7.6923%;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 5.7692%;\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.3008\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0142\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 7.6923%;\"\u003e\n \u003cp\u003e0.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 5.7692%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.049\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.006\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 7.6923%;\"\u003e\n \u003cp\u003e0.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\" style=\"width: 5.7692%;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0091\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.0585\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" valign=\"bottom\"\u003e\n \u003cp\u003e0.2716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e4.3.4. \u0026nbsp; Histopathological evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOn day 7, the CMC / zinc selenite group showed partial loss of epidermal cell layers with granulation tissue, edema, irregularly arranged fibroblasts and many newly formed blood vessels admixed with cellular infiltrates, While the infected group showed focal to complete loss of epidermal layers with desquamated epidermal cells infiltrated with many neutrophils, the dermal layers showing granulation tissue with dermal hemorrhage or leaky immature formed blood vessels and immature fibroblasts admixed with low numbers of lymphocytes, plasma cells and macrophages. The placebo group showed complete epithelization with regularly arranged collagen (\u003cstrong\u003eFigure.10\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFourteen days postoperatively, the CMC / zinc selenite group showed a decreased wound gap with eosinophilic debris infiltrated with many inflammatory cells and dermal granulation tissue with many angiogenesis. The infected group showed diffuse loss of epidermal layers with extensive dermal infiltrations with abundant neutrophils, lymphocytes, plasma cells, and macrophages, besides abundant angiogenesis (immature blood vessels) with immature, irregularly and randomly arranged fibroblasts. The placebo group showed normal epithelium and dermal collagen with hair follicles (\u003cstrong\u003eFigure.11\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eDay 21, the CMC / zinc selenite group showed complete reepithelialization with many well-developed hair follicles, dense collagen bundles, and few subepidermal fibroblast aggregations, while the infected group showed focal incomplete loss of the stratum basalis of epidermal layers. multifocal to coalescing partial or complete loss of epidermal cell layers (partial migration of epidermal cells to close the wound gap), besides the presence of randomly arranged collagen admixed with scattered few inflammatory aggregates. The placebo group showed normal architecture of skin layers (\u003cstrong\u003eFigure.12\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026middot; \u003cstrong\u003e\u003cu\u003eHistopathology scoring\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSemi-quantitative histopathological scoring was conducted to assess angiogenesis, collagen deposition, bacterial presence, and re-epithelialization in wound tissue sections collected on days 7, 14, and 21. Group comparisons were analyzed using appropriate non-parametric tests (\u003cstrong\u003eTable.8).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAngiogenesis\u003c/u\u003e\u003c/strong\u003e\u003cu\u003e:\u003c/u\u003e\u003cbr\u003eOn day 7, a trend toward increased angiogenesis was noted in the CMC / zinc selenite and placebo groups (score 2 in most of the animals), though not statistically significant (\u003cem\u003ep\u003c/em\u003e = 0.08). By day 21, the CMC / zinc selenite group demonstrated significantly higher angiogenesis scores (\u003cem\u003ep\u003c/em\u003e = 0.0286), with 2 out of 3 rats scoring 3, compared to the infected group. These findings indicate enhanced neovascularization with CMC/ZnSeONPs gel, which supports granulation tissue formation and tissue regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eCollagen Deposition\u003c/u\u003e\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eCollagen fiber deposition improved progressively in all groups. On day 21, the CMC / zinc selenite group showed significantly enhanced collagen deposition (\u003cem\u003ep\u003c/em\u003e = 0.0214), with two rats scoring 3, indicative of wound maturation and remodeling. While early differences were not statistically significant (Day 7: \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.99; Day 14: \u003cem\u003ep\u003c/em\u003e = 0.08), trends favored the CMC / zinc selenite group, highlighting its role in matrix remodeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eBacterial Presence:\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial load scores differed significantly on day 7 (\u003cem\u003ep\u003c/em\u003e = 0.02) and day 21 (\u003cem\u003ep\u003c/em\u003e = 0.0357), with placebo and CMC / zinc selenite groups showing lower scores compared to the infected control. CMC / zinc selenite consistently showed superior bacterial clearance, with all animals scoring 0 by day 21, underscoring its antimicrobial efficacy in vivo, these results were in line with the results of the results of bacterial count.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eRe-epithelialization:\u003c/u\u003e\u003c/strong\u003e\u003cu\u003e\u003cbr\u003e\u003c/u\u003eThe CMC / zinc selenite group achieved complete epithelial coverage by day 21 (score 3 in all rats), significantly outperforming the infected group (p = 0.0357). Early trends were noted as well, particularly at day 14 (p = 0.02), suggesting accelerated epithelial regeneration. The infected group consistently lagged in this parameter, indicating prolonged wound exposure and impaired barrier restoration.\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that CMC/ZnSeONPs gel facilitates wound healing through enhanced angiogenesis, collagen maturation, microbial clearance, and epithelial regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e(Table 8).\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003cstrong\u003eSemi-Quantitative Histopathological Scores for Angiogenesis, Collagen Deposition, Bacterial Presence, and Re-epithelialization\u003c/strong\u003e.\u003c/em\u003e Histopathological parameters were scored on a scale of 0 (none), 1 (mild), 2 (moderate), and 3 (extensive) in skin wound samples collected on days 7, 14, and 21 from placebo, infected, and CMC / zinc selenite groups (n = 3 per time point). Statistical analysis was performed using Fisher\u0026rsquo;s exact test or chi-square test as appropriate. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 is considered statistically significan\u003cu\u003et\u003cstrong\u003e.\u003c/strong\u003e\u003c/u\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"695\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\" rowspan=\"2\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e7 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e14 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003e21 days\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"16\"\u003e\n \u003cp\u003eAngiogenesis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.029*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eCMC / zinc selenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"16\"\u003e\n \u003cp\u003eCollagen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u0026gt;0.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.021*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eCMC / zinc selenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"16\"\u003e\n \u003cp\u003eBacteria\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.02*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.035*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eCMC / zinc selenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" colspan=\"16\"\u003e\n \u003cp\u003eEpithelization\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003ePlacebo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.02*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.035*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eInfected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003eCMC / zinc selenite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd nowrap=\"\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"4.\tDiscussion","content":"\u003cp\u003eThe healing of infected wounds has long been a major global healthcare challenge, owing to its high prevalence, the potential for disability, and considerable mortality rates [37]. A study on dogs with infected wounds reported a mortality rate of around 33%, with bacteremia being a major contributing factor strongly associated with the high mortality rate [38]. The severity and mortality associated with wounds are expected to rise due to increased exposure to antimicrobial-resistant pathogens. Currently, wound infections are responsible for approximately 75% of post-operative deaths [39]. \u0026nbsp; Therefore, the aim of this study is to address this ongoing challenge by exploring antimicrobial alternatives to conventional antibiotics to minimize their previously reported adverse effects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe selection of \u003cem\u003eS. aureus\u003c/em\u003e as the focus for wound infection studies is the leading pathogen associated with wound infections, responsible for 30.3% of reported cases. Notably, \u003cem\u003eS. aureus\u003c/em\u003e is a common commensal organism residing on human skin; however, when the skin barrier is broken, such as through wounds, it can transition from a harmless inhabitant to a pathogenic agent [40]\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe topical treatment with CMC/zinc selenite gel was applied following thorough flushing of the wound with normal saline 0.9%, as the \u003cem\u003eS. aureus\u003c/em\u003e can develop resistance to topical treatments by forming biofilms at the wound site, which impede drug penetration and interfere with host immune signaling. Therefore, the physical removal of colonizing bacteria is considered a crucial step in wound management, as it helps reduce bacterial load, enhances drug efficacy and penetration, and minimizes required dosages [10]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeNPs are considered a promising alternative to antibiotics for treating hard-to-heal skin infections, offering excellent stability, favorable toxicological profiles, and strong antibacterial efficacy\u0026nbsp;[41]. SeNPs exhibit minimal or no toxicity toward mammalian cells, primarily due to their selective targeting capability, which allows them to differentiate between bacteria and healthy mammalian cells [42]\u003cstrong\u003e.\u003c/strong\u003e Also, all antimicrobial studies conducted to date have reported no evidence of bacterial resistance to SeNPs, highlighting their strong potential as an effective treatment option for infectious and chronic wounds [27]. ZnONPs possess notable antimicrobial properties and are recognized for their biocompatibility, cost-effectiveness, non-toxicity, and environmental safety. By releasing zinc ions at the wound site, ZnONPs stimulate keratinocyte activity, thereby accelerating the wound healing process [43], and these were the reasons for selecting the combination of these materials.\u003c/p\u003e\n\u003cp\u003eThe current research demonstrates that \u003cem\u003eS. aureus\u003c/em\u003e infection significantly impacts body weight. The infected group exhibited weight loss until day 11 post-infection, after which weight gain resumed. This result is in line with\u0026nbsp;[44, 45]. In contrast, CMC / zinc selenite group started to gain weight after 3 days of infection. These findings highlight that CMC / zinc selenite has protective effects against infection-induced weight loss. The infection-related weight loss likely results from the breakdown of muscle protein, a process influenced by an imbalance among key factors such as glucocorticoids, proinflammatory cytokines, and catecholamines. These mediators typically rise during infection, reflecting an inflammatory and stress response that promotes catabolism and muscle wasting [46], or may be attributed to that \u003cem\u003eS.aureus\u003c/em\u003e can invade adipose tissue or infect adipocytes, triggering a localized inflammatory response. This inflammation, along with pathogen-derived signals, promotes lipolysis, the breakdown of stored triglycerides into free fatty acids, these liberated fatty acids serve to meet the increased energy requirements of the host and support immune system activity during infection [47].\u003c/p\u003e\n\u003cp\u003eThe bacterial viability test revealed that CMC/CMC / zinc selenite gel led to a total inhibition of bacterial growth up to 24 hr. These results highlight that in the case of CMC/CMC / zinc selenite gel, only one application daily is effective to reach the total bacterial inhibition, these results may indicate that the addition of zinc selenite to the CMC potentiates its antibacterial effect as CMC alone is weak antibacterial agent [2]. A study by [48] on ZnONPs revealed that the effect of ZnONPs as an antibacterial agent was significant after 6 hours of contact with \u003cem\u003eS.aureus,\u0026nbsp;\u003c/em\u003eand caused significant reduction of bacterial viability after 24 hours but not total inhibition, while in the current study, CMC / zinc selenite gel showed significant reduction of bacterial viability after only one hour of contact with \u003cem\u003eS.aureus,\u0026nbsp;\u003c/em\u003eand caused significant total inhibition after 24 hours highlighting the potent antibacterial effect of CMC / zinc selenite gel.\u003c/p\u003e\n\u003cp\u003eThe results of bacterial count revealed that on day 2, infected and CMC / zinc selenite groups showed similarly high bacterial counts with no significant differences. This is because the treatment protocol started after 3 days of surgery, following confirmation of bacterial infection. While on day 7, notable reductions in bacterial count were observed in the CMC / zinc selenite groups compared to the infected control.\u0026nbsp;On day 14, the bacterial count in the CMC / zinc selenite group had nearly reached baseline levels, with no significant differences between the placebo, suggesting the strong antibacterial effect of CMC / zinc selenite gel. These results were in agreement with [49], who reported that CMC in the control group without NPs exhibited no inhibition of \u003cem\u003eS.aureus\u003c/em\u003e growth, and also reported that antibacterial activity due to the presence of ZnONPs reduced the growth of \u003cem\u003eS. aureus\u003c/em\u003e by 75% but not total inhibition. However, in our study, we reached total inhibition of bacterial growth by CMC / zinc selenite, which suggests that CMC / zinc selenite is a better antibacterial agent than ZnONPs or CMC alone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe potential antibacterial activity of CMC/ zinc selenite may be attributed to its semi-crystalline structure, which\u0026nbsp;contains both crystalline domains and amorphous regions. The amorphous regions typically allow for a more rapid release of ions (Zn and Se), which can enhance the initial antibacterial efficacy upon application to the wound. In contrast, the crystalline domains enable a slower, more sustained release, ensuring prolonged protection against infection and allowing the material to persist longer at the wound site. Or maybe due to the small diameter of CMC / zinc selenite (Zn₃Se₄O₁₄), around 51\u0026nbsp;nm, which facilitates its internalization into the bacterial cells for more destruction. Or as mentioned by [50]\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extended inflammatory phase disrupts fibroblast differentiation, slowing wound contraction and potentially contributing to scar formation [51, 52]\u003cstrong\u003e.\u003c/strong\u003e The histopathological analysis revealed that the infected group exhibited a marked inflammatory response, characterized by severe infiltration of the skin layers with inflammatory cells persisting up to day 21. Additionally, the infected group displayed signs of irritation and scratching, further indicating sustained inflammation, which may be attributed to the bacterial infection and its associated released toxins into the tissues, and the weak anti-inflammatory effect of CMC. \u0026nbsp; In contrast, the CMC / zinc selenite group demonstrated a notably milder inflammatory reaction. This superior outcome of the CMC / zinc selenite group may be explained by a synergistic interaction between the anti-inflammatory mechanisms of both ZnONPs and SeNPs. These findings agreed with\u0026nbsp;\u003cstrong\u003eAlnomasy (2024) and Khosravian et al. (2025)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExcessive inflammation often results from the imbalance in macrophage polarization toward either the pro-inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which promotes skin tissue regeneration and facilitates revascularization. This imbalance disrupts the immune homeostasis within the wound microenvironment, potentially impairing the healing process [53]\u003cstrong\u003e.\u003c/strong\u003e Zinc ions released from the gel play a role in immune regulation within the wound microenvironment, promoting macrophage polarization toward the M2 phenotype. This shift enhances the expression of anti-inflammatory cytokines, effectively reducing inflammation\u0026nbsp;[54].\u003c/p\u003e\n\u003cp\u003eThe anti-inflammatory properties of SeNPs are attributed to Selenium itself, which is a critical component of various selenoproteins, which play pivotal roles in regulating inflammatory responses, as well as phagocytic and chemotactic functions. specific selenoproteins such as glutathione peroxidase 1 and 4 are responsible for the suppression of pro-inflammatory cytokines, antioxidant defense mechanisms, and the detoxification of reactive nitrogen species like peroxy nitrite during the inflammatory phase of tissue repair [55]. The second mechanism of suppressing the inflammation is their ability to suppress the mRNA expression of key pro-inflammatory cytokines, such as interleukin-1 (IL-1), TNF-\u0026alpha;, and inducible nitric oxide synthase (iNOS). Additionally, SeNPs inhibit the production of pro-inflammatory mediators by preventing the nuclear translocation of the transcription factor NF-\u0026kappa;B, thereby modulating the inflammatory signaling pathways involved in the wound healing process [56].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWounds are frequently associated with the damage or loss of dermal vascular tissues. These vascular networks supply oxygen and nutrients needed for cell growth, proliferation, and migration; they also enhance the transport of vital nutrients and waste. Their regeneration is of significant importance. Therefore, promoting vascular network formation is a promising approach to enhance wound healing [57]. In our histopathological analysis, the CMC / zinc selenite group exhibited a robust angiogenic response, as evidenced by the presence of numerous newly formed blood vessels at all examined time points from day 7 onward. Also, the histopathological score for angiogenesis was significantly higher in the CMC / zinc selenite group compared to the infected one. This may be due to\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe angiogenic capacity of CMC / zinc selenite gel may be attributed to the internalization of ZnONPs, which upregulate the expression of VEGF, triggering downstream signaling pathways, including PI3K/Akt and MAPK/ERK, which promote endothelial cell survival, motility, and new blood vessel formation. Furthermore, ZnONPs activate endothelial nitric oxide synthase, leading to increased nitric oxide production, which facilitates vasodilation and vascular remodeling for effective angiogenesis [58]. This explains the early angiogenic effect demonstrated in the CMC / zinc selenite group; the addition of SeNPs has a synergistic effect and potentiates the ZnONPs effect in neovascularization, which promotes tissue regeneration. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistopathological findings revealed that the infected group on day 21 had partial closure of wound, and thinning of the epidermal layer with a scanty immature epithelialization, with the results of IHC that showed low expression of EGFR on day 7, which started to increase significantly after that, reaching the peak on day 21 but still low level compared to the other groups, suggesting improper epithelialization and weak stimulation of keratinocyte migration, likely causing significantly larger wounds on day 21, which may be attributed to the infection that alters the wound microenvironment.\u003c/p\u003e\n\u003cp\u003eThe CMC / zinc selenite group showed complete closure of the wound with complete epithelialization in the histopathological section. These results were in line with IHC, which may be primarily attributed to the zinc ions. As Zn serves as a cofactor for various enzymes, including zinc-dependent matrix metalloproteinases, which enhance keratinocyte migration, facilitate autodebridement, and promote epithelialization during wound healing [59]. Also, Zinc regulates keratinization and keratinocyte migration by influencing the expression of integrins \u0026alpha;2\u0026beta;1, \u0026alpha;3\u0026beta;1, \u0026alpha;6\u0026beta;4, and \u0026alpha;v\u0026beta;5, which facilitate intercellular adhesion, attachment to the basement membrane, and keratinocyte motility. Each integrin plays a role in epithelialization during skin wound healing. However, these integrins can be altered due to inflammation or tissue injury [60]. These results were in line with [49], who reported that the sponge containing undoped ZnONPs exhibited the highest cell attachment and proliferation, and [61], who reported that ZnONPs had outperforming results on wound closure time, contraction rate, histopathological changes, and cell regeneration, and [62]\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewho made a study\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eon\u0026nbsp;chitosan-ZnO/SeNPs composite scaffolds\u0026nbsp;demonstrated enhanced keratinocyte migration in cell lines\u0026nbsp;in vitro. Moreover, in vivo experiments confirmed accelerated wound closure and improved re-epithelialization. The EGFR level was\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethe highest in the placebo group on day 7, then markedly decreased onwards, which may suggest the early complete closure and re-epithelialization of the wounds in the placebo group, as there was no challenge of infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis of \u0026alpha;-SMA revealed that the infected group exhibited the lowest expression of \u0026alpha;-SMA on day 7, increased moderately by day 14, and peaked by day 21, which may be attributed to persistent inflammation caused by the infection and delayed remodeling with scar formation. Notably, persistent elevation of \u0026alpha;-SMA beyond the proliferative phase may contribute to pathological healing due to abnormal collagen synthesis, resulting in excessive scar formation [63]. That explains the lower contraction rate of wounds in the infected group, which caused significantly larger wound areas compared to the CMC / zinc selenite and placebo groups. In contrast, CMC / zinc selenite groups displayed moderate \u0026alpha;-SMA expression on day 7 that markedly increased by day 14, followed by a significant reduction on day 21. This pattern likely reflects the activation of fibroblasts, as indicated by \u0026alpha;-SMA expression, which enables their differentiation into myofibroblasts that are essential for effective wound contraction and proliferation during the healing process [51]. \u0026nbsp;Therefore, the significant decrease in \u0026alpha;-SMA levels after day 14 in the CMC / zinc selenite groups likely indicates the transition from the proliferative phase into the remodeling phase and explains the accelerated wound contraction and the anti-scarring properties of the CMC / zinc selenite and CMC/ZnONPs gel. Similar trends were coincided with [64].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWound size reduction in both the placebo and CMC / zinc selenite groups consistently demonstrated superior healing outcomes compared to the infected control groups throughout the study period, which may be attributed to the high level of \u0026alpha;-SMA\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethat caused effective wound contraction and significantly higher levels of EGFR compared to the infected group, which led to complete epithelialization. In contrast, wounds in the infected group remained significantly larger, reflecting delayed wound closure likely attributable to inadequate infection control. [65] found that selenium 5% ointment in uninfected Wistar rats caused a healing rate of up to 85% within 18 days. Also, a study by [66] on 10 mm diameter \u003cem\u003eS. aureus\u003c/em\u003e-infected wounds, which closed totally after 21 days with Se-chitosan as a wound dressing. Meanwhile, in our study, the 23 mm diameter \u003cem\u003eS. aureus\u003c/em\u003e-infected wounds closed totally on 19 days, which indicates that CMC/zinc selenite is more effective in infected wounds than Selenium alone or Se-chitosan.\u003c/p\u003e"},{"header":"5.\tConclusion","content":"\u003cp\u003eWe synthesized CMC/zinc selenite gel, which has demonstrated excellent antibacterial efficacy on staphylococcus aureus infected wounds, due to its unique physicochemical properties represented as a semicrystalline structure and smaller nanometric size. This advanced gel not only enhanced the angiogenesis but also led to rapid epithelialization and promoted fibroblast differentiation that resulted in effective wound contraction, thereby accelerating infected wound healing and regeneration. CMC/zinc selenite gel could also be considered as a protective agent against weight loss caused by infection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNermeen -Shereef:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, methodology,\u003c/p\u003e\n\u003cp\u003ewriting \u0026ndash; original draft, writing \u0026ndash; review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarwa Abass:\u0026nbsp;\u003c/strong\u003econceptualization, formal analysis, investigation, writing\u0026nbsp;\u0026ndash;\u0026nbsp;original\u003c/p\u003e\n\u003cp\u003edraft, and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmad Tolba\u0026nbsp;\u003c/strong\u003epreparation and characterization of the materials used and Methodology.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEkramy Elmorsy\u0026nbsp;\u003c/strong\u003econtributed to the statistical analysis of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBasma Hendam :\u0026nbsp;\u003c/strong\u003eMethodology and\u0026nbsp;Immunohistochemical evaluation\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAwad Rizk:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, writing \u0026ndash; original draft, and supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGamal Karrouf:\u0026nbsp;\u003c/strong\u003econceptualization, data curation, supervision, writing\u0026nbsp;\u0026ndash;\u0026nbsp;review and editing and\u003c/p\u003e\n\u003cp\u003efinal revision of the manuscript. All authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e \u003cstrong\u003eT\u003c/strong\u003ehis research did not receive funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Science, Technology \u0026amp; Innovation Funding Authority (STDF), in cooperation with the Egyptian Knowledge Bank (EKB), for covering the article processing charges. No funding, grants, or other financial support were received for the design, execution, or reporting of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.1.\u0026nbsp;Ethical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol, including the research objectives, design, and procedures, was reviewed and approved by the Medical Research Ethics Committee of the Faculty of Veterinary Medicine, Mansoura University, under approval code (VM.MS.24.10.166). \u0026nbsp;All animal care and experimental procedures were conducted in accordance with institutional and national guidelines for the ethical All procedures were documented following the guidelines specified by ARRIVE\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors confirm their consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGonzalez AC de O, Costa TF, Andrade Z de A, Medrado ARAP. 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Elsevier Health Sciences; 2008.\u003c/li\u003e\n\u003cli\u003eWu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25:2648\u0026ndash;59.\u003c/li\u003e\n\u003cli\u003ede Souza MV, Silva MB, Pinto J de O, Lima MB de S, Crepaldi J, Lopes GFM, et al. Immunohistochemical Expression of Collagens in the Skin of Horses Treated with Leukocyte-Poor Platelet-Rich Plasma. Biomed Res Int. 2015;2015:893485.\u003c/li\u003e\n\u003cli\u003eBrinker CJ, Scherer GW. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing. illustrated, reprint edition. Elsevier Science; 1990.\u003c/li\u003e\n\u003cli\u003eHench LL, West JK. The sol-gel process. Chem Rev. 1990;90:33\u0026ndash;72.\u003c/li\u003e\n\u003cli\u003eJ\u0026auml;rbrink K, Ni G, S\u0026ouml;nnergren H, Schmidtchen A, Pang C, Bajpai R, et al. Prevalence and incidence of chronic wounds and related complications: a protocol for a systematic review. Syst Rev. 2016;5:152.\u003c/li\u003e\n\u003cli\u003eArias MVB, Padilha FN, Perugini MRE. Deep tissue culture and hemoculture in dogs with wounds and sepsis. Pesq Vet Bras. 2017;37:1483\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eSen CK. Human wounds and its burden: an updated compendium of estimates. Adv Wound Care (New Rochelle). 2019;8:39\u0026ndash;48.\u003c/li\u003e\n\u003cli\u003eMarimuthu K, Eisenring M-C, Harbarth S, Troillet N. Epidemiology of Staphylococcus aureus Surgical Site Infections. Surg Infect (Larchmt). 2016;17:229\u0026ndash;35.\u003c/li\u003e\n\u003cli\u003eDagmar H, Kristyna C, Pavel K, Vojtěch A, Ren\u0026eacute; K. SELENIUM NANOPARTICLES AND EVALUATION OF THEIR ANTIMICROBIAL ACTIVITY ON BACTERIAL ISOLATES OBTAINED FROM CLINICAL SPECIMENS. 2015.\u003c/li\u003e\n\u003cli\u003eChung S, Ercan B, Roy AK, Webster TJ. Addition of selenium nanoparticles to electrospun silk scaffold improves the mammalian cell activity while reducing bacterial growth. Front Physiol. 2016;7:297.\u003c/li\u003e\n\u003cli\u003eAsif N, Amir M, Fatma T. Recent advances in the synthesis, characterization and biomedical applications of zinc oxide nanoparticles. Bioprocess Biosyst Eng. 2023;46:1377\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eHriouech S, Akhmouch AA, Mzabi A, Chefchaou H, Tanghort M, Oumokhtar B, et al. The Antistaphylococcal Activity of Amoxicillin/Clavulanic Acid, Gentamicin, and 1,8-Cineole Alone or in Combination and Their Efficacy through a Rabbit Model of Methicillin-Resistant Staphylococcus aureus Osteomyelitis. Evid Based Complement Alternat Med. 2020;2020:4271017.\u003c/li\u003e\n\u003cli\u003eR\u0026ouml;hrig C, Huemer M, Lorg\u0026eacute; D, Luterbacher S, Phothaworn P, Schefer C, et al. Targeting Hidden Pathogens: Cell-Penetrating Enzybiotics Eradicate Intracellular Drug-Resistant Staphylococcus aureus. MBio. 2020;11.\u003c/li\u003e\n\u003cli\u003eChang HR, Bistrian B. The role of cytokines in the catabolic consequences of infection and injury. JPEN J Parenter Enteral Nutr. 1998;22:156\u0026ndash;66.\u003c/li\u003e\n\u003cli\u003eAmin A, Ferreira DV, Figueiredo LM. How pathogens drive adipose tissue loss in the host. Curr Opin Microbiol. 2025;85:102597.\u003c/li\u003e\n\u003cli\u003eRayyif SMI, Mohammed HB, Curuțiu C, B\u0026icirc;rcă AC, Grumezescu AM, Vasile B Ștefan, et al. ZnO Nanoparticles-Modified Dressings to Inhibit Wound Pathogens. Materials (Basel). 2021;14.\u003c/li\u003e\n\u003cli\u003eNaserian F, Mesgar AS. Development of antibacterial and superabsorbent wound composite sponges containing carboxymethyl cellulose/gelatin/Cu-doped ZnO nanoparticles. Colloids Surf B Biointerfaces. 2022;218:112729.\u003c/li\u003e\n\u003cli\u003eSerov DA, Khabatova VV, Vodeneev V, Li R, Gudkov SV. A review of the antibacterial, fungicidal and antiviral properties of selenium nanoparticles. Materials (Basel). 2023;16.\u003c/li\u003e\n\u003cli\u003eDarby IA, Laverdet B, Bont\u0026eacute; F, Desmouli\u0026egrave;re A. Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol. 2014;7:301\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003eLand\u0026eacute;n NX, Li D, St\u0026aring;hle M. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci. 2016;73:3861\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eTu Z, Chen M, Wang M, Shao Z, Jiang X, Wang K, et al. Engineering bioactive M2 macrophage‐polarized anti‐inflammatory, antioxidant, and antibacterial scaffolds for rapid angiogenesis and diabetic wound repair. Adv Funct Mater. 2021;31:2100924.\u003c/li\u003e\n\u003cli\u003eHussien NA, Khalil MAEF, Schagerl M, Ali SS. Green Synthesis of Zinc Oxide Nanoparticles as a Promising Nanomedicine Approach for Anticancer, Antibacterial, and Anti-Inflammatory Therapies. Int J Nanomedicine. 2025;20:4299\u0026ndash;317.\u003c/li\u003e\n\u003cli\u003eHariharan S, Dharmaraj S. Selenium and selenoproteins: it\u0026rsquo;s role in regulation of inflammation. Inflammopharmacology. 2020;28:667\u0026ndash;95.\u003c/li\u003e\n\u003cli\u003eAnsari JA, Malik JA, Ahmed S, Manzoor M, Ahemad N, Anwar S. Recent advances in the therapeutic applications of selenium nanoparticles. Mol Biol Rep. 2024;51:688.\u003c/li\u003e\n\u003cli\u003eSorg H, Tilkorn DJ, Hager S, Hauser J, Mirastschijski U. Skin wound healing: an update on the current knowledge and concepts. Eur Surg Res. 2017;58:81\u0026ndash;94.\u003c/li\u003e\n\u003cli\u003eAhtzaz S, Nasir M, Shahzadi L, Amir W, Anjum A, Arshad R, et al. A study on the effect of zinc oxide and zinc peroxide nanoparticles to enhance angiogenesis-pro-angiogenic grafts for tissue regeneration applications. Mater Des. 2017;132:409\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eLansdown ABG, Mirastschijski U, Stubbs N, Scanlon E, Agren MS. Zinc in wound healing: theoretical, experimental, and clinical aspects. Wound Repair Regen. 2007;15:2\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eTenaud I, Leroy S, Chebassier N, Dreno B. Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins. Exp Dermatol. 2000;9:407\u0026ndash;16.\u003c/li\u003e\n\u003cli\u003eAsif M, Chaudhry AS, Ashar A, Rashid HB, Saleem MH, Aslam HB, et al. Zinc oxide nanoparticles accelerate the healing of methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds in rabbits. Asian Pac J Trop Biomed. 2023;13:488\u0026ndash;96.\u003c/li\u003e\n\u003cli\u003eRuan Q, Yuan L, Gao S, Ji X, Shao W, Ma J, et al. Development of ZnO/selenium nanoparticles embedded chitosan-based anti-bacterial wound dressing for potential healing ability and nursing care after paediatric fracture surgery. Int Wound J. 2023;20:1819\u0026ndash;31.\u003c/li\u003e\n\u003cli\u003eXue M, Jackson CJ. Extracellular matrix reorganization during wound healing and its impact on abnormal scarring. Adv Wound Care (New Rochelle). 2015;4:119\u0026ndash;36.\u003c/li\u003e\n\u003cli\u003eMao L, Wang L, Zhang M, Ullah MW, Liu L, Zhao W, et al. In Situ Synthesized Selenium Nanoparticles-Decorated Bacterial Cellulose/Gelatin Hydrogel with Enhanced Antibacterial, Antioxidant, and Anti-Inflammatory Capabilities for Facilitating Skin Wound Healing. Adv Healthc Mater. 2021;10:e2100402.\u003c/li\u003e\n\u003cli\u003eRamya S, Shanmugasundaram T, Balagurunathan R. Biomedical potential of actinobacterially synthesized selenium nanoparticles with special reference to anti-biofilm, anti-oxidant, wound healing, cytotoxic and anti-viral activities. J Trace Elem Med Biol. 2015;32:30\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eAbbaszadeh A, Tehmasebi-Foolad A, Rajabzadeh A, Beigi-Brojeni N, Zarei L. Effects of chitosan/nano selenium biofilm on infected wound healing in rats; an experimental study. Bull Emerg Trauma. 2019;7:284\u0026ndash;91.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Infected wound, CMC/zinc selenite, nanoparticles, Staphylococcus aureus, wound healing, EGFR, α-SMA, gel","lastPublishedDoi":"10.21203/rs.3.rs-9549669/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9549669/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn the fight against bacterial infections and the growing threat of antibiotic resistance, there is a pressing need to develop safe and effective antimicrobial wound dressings that could be used as alternatives to antibiotics, selectively target bacteria, and overcome the challenges of antibiotic resistance. This study introduced a novel material that meets these criteria: CMC/zinc selenite nanoparticles in the form of a gel.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eFifty-four adult male Albino Wistar rats, aged 10\u0026ndash;12 weeks with an average body weight of 256.7\u0026thinsp;\u0026plusmn;\u0026thinsp;13.7 g, were used. A 23 mm diameter full-thickness circular cutaneous defect in the dorsal region between the two scapulae. The CMC/zinc selenite and the infected groups received intradermal inoculation of the wound with 0.3 ml freshly prepared suspension of \u003cem\u003eS. aureus.\u003c/em\u003e Rats were divided into three groups (n\u0026thinsp;=\u0026thinsp;18 per group) after 3 days of surgery according to the infection and treatment protocol (\u003cb\u003eplacebo group\u003c/b\u003e: wounds without infection or treatment, \u003cb\u003einfected group\u003c/b\u003e: infected wounds without treatment, \u003cb\u003eCMC / zinc selenite group\u003c/b\u003e: infected wounds received a topical application of 1 ml of CMC/ZnSeONPs gel twice daily). The wounds were evaluated through macroscopic, immunohistochemical, histopathological, and bacteriological analyses.\u003c/p\u003e\u003ch2\u003eResults \u0026amp;Conclusion\u003c/h2\u003e \u003cp\u003eThe synthesized advanced gel of CMC/Zinc selenite shows exceptional antimicrobial properties due to its semicrystalline structure and its small nanometric size, promoting faster healing of infected full-thickness skin wounds through exhibiting rapid re-epithelialization by stimulating EGFR, and effective contraction alongside angiogenic properties, tissue regeneration, and could be considered as a protective agent against infection-related weight loss.\u003c/p\u003e","manuscriptTitle":"Therapeutic efficacy of Carboxymethyl Cellulose-based zinc selenite nanoparticles in infected wound healing in a rat model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-13 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