Novel Approach to Scar Reduction: Silicone Gel Enriched With Erbium Borate Nanoparticles

Abstract

Background/Aim

Scar formation following skin injuries from trauma or surgery can result in sensory deficits, loss of flexibility, and significant cosmetic concerns. This study evaluates the potential effects of silicone gel combined with erbium borate nanoparticles on wound healing and scar reduction.

Materials and Methods

Sixteen Wistar albino rats, each with four incisional wounds, were divided into four groups: control, silicone gel with vitamin C, silicone gel with erbium borate nanoparticles, and erbium borate hydrogel. Topical treatments were applied daily for 7 or 21 days, and histopathological evaluations were performed on day 14 and day 28, respectively, including Abramov wound healing scoring and scar elevation index measurements. Hematoxylin-eosin and Masson's trichrome staining were used for the assessments.

Results

Histopathological evaluation on day 14 revealed no significant differences between groups; however, the silicone gel + vitamin C group exhibited more immature collagen fibers (p=0.096). On day 28, collagen deposition was significantly higher in the silicone gel + vitamin C group compared to the silicone gel + erbium borate group (p=0.021), whereas the silicone gel + erbium borate group exhibited more organized and mature collagen fibers. Scar elevation index tended to be lower in the silicone gel+erbium borate group than in the control group.

Conclusion

The combination of silicone gel and erbium borate nanoparticles may improve wound healing and may contribute to improved scar-related outcomes compared with untreated wounds, providing a promising therapeutic approach for improving healing outcomes.

Introduction

Injuries often result in scar formation, particularly those caused by trauma or surgery. In many cases, scars lead to decreased tactile sensation, loss of skin elasticity, and reduced mobility. Scars also result in noticeable cosmetic problems, especially on the face and hands (1). Especially after burns, the frequency of wound infections has increased, and serious scars can develop following wound site infections (2). Therefore, there is a need for treatments that accelerate wound healing and minimize scar formation. Discoveries related to cellular pathology, human physiology, molecular biology, microbiology, and polymer chemistry have led to an understanding of the basic mechanisms of wound healing (3). Additionally, various treatment methods have been developed to expedite wound healing and reduce scar formation, including occlusive therapy, scar hydration, vacuum-assisted therapy, hyperbaric oxygen therapy, intralesional injections, laser therapy, topical natural and synthetic polymers, growth factors, stem cell therapy, natural compounds, and silicone dressings and gels (4-6). However, no treatment has achieved perfect success, and there is a continuing need for better treatment methods (7).

Silicone gel treatments are one of the most used treatments for reducing scar formation and treating hypertrophic scars (4, 8, 9). Recent studies have shown that some metal nanoparticles accelerate wound healing in in vitro cell assay models, with one of the most notable studies being the one involving erbium borate nanoparticles (ErB-Nps) (10). The synthesis of ErB-Np relies on the chemical precipitation of erbium ions in an alkaline borate buffer solution. It has been observed that ErB-Nps do not induce cytotoxicity or oxidative stress on damaged cells; instead, they enhance cell proliferation and reduces oxidative stress (10). Building on these findings, the present study was designed to evaluate, in an in vivo animal model, whether the incorporation of ErB-Np into a silicone gel formulation could improvewound healing and scar outcomes compared with conventional treatments.

Materials and Methods

Animals. All in vivo experiments were conducted at the Bezmialem Vakif University Hospital Laboratory Animal Facility after obtaining approval from the Bezmialem Vakif University Ethics Committee (Approval No: 2022-E.86414). All experimental procedures involving animals were conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

A total of 16 female Wistar albino rats, weighing 300-350 g and aged 8 weeks, were obtained from the Laboratory Animal Facility of Bezmialem University. The animals were housed in accordance with the international guidelines for the care and use of laboratory animals. Throughout the experiment, controlled temperature (23±2°C), humidity (55±10%), and automatic lighting (12-h light-dark cycle) were maintained. The rats were provided ad libitum access to food and water. Following surgery, the rats were housed in separate cages with environmental enrichment to prevent biting wounds among cohabitating animals, in a specific pathogen-free room.

Experimental design. A uniform anesthesia protocol was administered to all animals. Anesthesia was induced in the rats by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Each of the 16 rats had four planned incisions on their dorsal side. Incision lines were made on both sides of the vertebral column, totaling two incisions per side. The incision lines were primarily closed with non-absorbable sharp 3/0 polypropylene sutures (Figure 1). On the seventh postoperative day, sutures were removed, and the animals were randomly divided into four groups. Group 1 served as the control group and received no topical treatment. In Group 2, ErB -containing hydrogel was topically applied once daily to the incision lines. In Group 3, silicone gel containing ascorbic acid (Dermatrix™, Menarini Asia-Pacific Holdings Pte Ltd, Singapore, Singapore) was applied once daily to the incision lines. In Group 4, ErB containing silicone gel was applied once daily to the incision lines.

Figure 1.

Macroscopic evaluation of wound healing and scar formation after treatment with silicone-based formulations. A) On postoperative day 0, incision lines were made on both sides of the vertebral column and were closed with primary sutures. B) Macroscopic appearance of incision lines after 7 days of treatment. C) Macroscopic appearance of incision lines after 21 days of treatment. Black arrows indicate representative incision lines. On day 21, wounds treated with silicone-based formulations, particularly silicone gel containing Erbium Borate Nanoparticles (ErB-Nps), demonstrate smoother wound surfaces and reduced scar prominence compared with the control group.

Skin samples were planned to be taken on day 14 (7^th day of suture removal), marking the end of the proliferation phase, and on day 28 (21^st day of suture removal), representing the early remodeling phase. After seven days of topical treatments, two animals were randomly selected from each group. Under general anesthesia, the incision lines were excised as full-thickness skin specimens for histopathological examination. While still under deep anesthesia, the animals were euthanized by cervical dislocation. The remaining animals continued to receive the respective topical treatments for an additional 14 days. After completing the 21-day treatment period, the remaining animals underwent the same procedure, with full-thickness skin samples collected under general anesthesia followed by euthanasia by cervical dislocation while still under deep anesthesia.

Histopathological examination. Tissue samples were fixed in 10% buffered formaldehyde solution. The central part of the wound was sampled in three pieces, each measuring 2 cm in length, 0.5 cm in width. These samples were processed for paraffin embedding using an automated tissue processor (ASP 6026, Leica, Buffalo Grove, IL, USA). Paraffin-embedded samples were sectioned into slices of 3-5 µm thickness. The sections were stained with Hematoxylin and Eosin (HE) (Sigma-Aldrich, Saint Louis, MO, USA) to visualize general tissue structure. Masson’s Trichrome (MT) staining method (Sigma-Aldrich) was utilized to visualize collagen fiber arrangement. Manual staining protocols were applied according to the manufacturer’s instructions. All stained sections were scanned at 40× magnification using a Nikon Eclipse Ti-S microscope (Nikon, Kawaguchi, Saitama, Japan). Histomorphological measurements were performed on the samples using ImageJ 1.48 v. software (National Institutes of Health, Bethesda, MD, USA).

Histological evaluation included assessment of acute inflammatory infiltrate, chronic inflammatory infiltrate, granulation tissue quantity and maturity, collagen deposition, re-epithelialization, and neovascularization using the Abramov histopathological scoring system. Each parameter was evaluated independently. Acute inflammation was defined by the presence of neutrophils, whereas chronic inflammation was defined by the presence of plasma cells and monocytes. Granulation tissue maturity was assessed according to fibroblast maturation, with mature fibroblasts characterized by thin, compact, parallel alignment and immature fibroblasts displaying a stellate morphology with less organized arrangement. In Masson’s trichrome-stained sections, collagen fiber organization, density, diameter, and maturation were additionally evaluated.

In addition to the Abramov scoring system, we also utilized the Scar Elevation Index (SEI) for histological analysis in our study. The SEI was calculated from samples obtained from scar tissues, and intergroup comparisons were made based on the SEI values. The SEI was calculated by comparing the thickness of the scar tissue to the surrounding normal tissue. Measurements were taken from histological sections stained with H&E. Specifically, the maximum vertical height of the scar was measured and divided by the height of the adjacent normal tissue to derive the SEI. A higher SEI value indicates greater scar elevation relative to normal tissue. The SEI measurements and the Abramov scoring were independently performed by two pathologists who were blinded to the treatment groups and had no prior knowledge of the gels applied to each wound.

Erbium borate containing silicone gel preparation. ErB-Nps were synthesized using a chemical precipitation technique optimized for their potential application in wound healing. Initially, erbium nitrate was dissolved in polyethylene glycol (PEG) with a molecular weight of 2,000 g/mol at a stoichiometric ratio of 1:1.5. This solution was then gradually introduced into an alkaline borate buffer (pH 9.2) while being stirred mechanically at 2,000 rpm at room temperature. After 30 min of continuous mixing, the suspension was centrifuged at 7,000 rpm, corresponding to approximately 5,500×g based on a 10-cm rotor radius, for 10 min to separate the precipitate. The precipitate was thoroughly washed three times with deionized water to eliminate residual impurities. The resulting nanoparticles exhibited a high surface charge (+41.9±8.8 mV), which ensured their dispersion stability in aqueous solutions for over two months. Nanoparticle tracking analysis (NTA) confirmed that the synthesized ErB-Nps had a size distribution ranging from 80 to 200 nm. Water-based, and silicone-based gels containing ErB-Nps were prepared separately. In both gels, ErB was prepared in aqueous phase for the concentration of 3% (w/w). A conventional silicone oil concentration of 40% (w/w) was used for the silicone-based gel. The ErB-Np suspension was homogenously mixed with the silicone oil to ensure uniform distribution. In the preparation of the water-based gel, the aqueous solution of ErB-Nps was directly incorporated into the gel matrix to form a stable hydrogel system. To ensure the homogeneous distribution of ErB-Nps within the prepared gels, the formulations were visually inspected for any signs of aggregation or phase separation. The uniformity of the nanoparticle distribution was further verified by preparing thin sections of the gel and examining them under a standard optical microscope. The absence of visible aggregates or irregularities confirmed the even dispersion of ErB-Nps throughout the gel matrix. The synthesis and physicochemical characterization of the ErB-Nps, including XRD, FTIR, and elemental analyses, have been reported previously by our research group (10). The ErB-Nps used in the present study were obtained from the same laboratory and prepared using the same synthesis protocol described in that report.

Statistical analysis. The data obtained from the study were analyzed using SPSS version 26.0 software (IBM Corp., Armonk, NY, USA). The normal distribution of variables was assessed using the Kolmogorov-Smirnov test. Data demonstrating normal distribution are presented as mean±standard deviation (SD), whereas data not showing normal distribution are presented as median-interquartile range (IQR) values. One-way analysis of variance (ANOVA) was conducted for numerical data showing homogeneous distribution. Categorical ordinal data and numerical data that did not show homogeneous distribution were compared using the Kruskal-Wallis test. For pairwise comparisons between subgroups, post-hoc Tukey test was used for data showing homogeneous distribution, while the Mann-Whitney U test was used for data not showing homogeneous distribution. Because each animal contributed multiple wounds, scar elevation index (SEI) measurements were analyzed using a linear mixed-effects model with treatment group as a fixed effect and animal as a random effect to account for clustering within animals. Degrees of freedom were estimated using the Kenward–Roger method, and Sidak adjustment was applied for pairwise comparisons. SEI was considered the primary outcome. Histopathological parameters were analyzed as secondary exploratory outcomes, and no adjustment for multiple comparisons was applied to these analyses. A significance level of p<0.05 was set for all tests.

Results

A total of eight rats including two for each group were sacrificed by cervical dislocation on day 14 following the surgical procedure, while the remaining 8 rats were sacrificed by cervical dislocation on day 28 post-surgery. No adverse effects were observed following topical treatment. At the end of the 14-day period, a total of 32 incision lines, with a total of 4 incision lines per rat, were evaluated. The macroscopic and microscopic views of the incision lines after 7 days of treatment are illustrated in Figure 1 and Figure 2, respectively. At the end of the 14-day period, histopathological examination based on Abramov scoring revealed no significant difference between the groups in any parameter. Although not statistically significant, the group treated with topical silicone gel + vitamin C exhibited more immature collagen fibers compared to the other groups (Table I). There was no significant difference observed between the groups in scar elevation index measurements when analyzed using a linear mixed-effects model accounting for clustering of wounds within animals (p=0.169).

Figure 2.

Representative histological sections of incisional wounds stained with hematoxylin and eosin (H&E). Upper row shows sections obtained after 7 days of treatment, and lower row shows sections obtained after 21 days of treatment in the control group, Erbium Borate Nanoparticles (ErB-Nps) hydrogel group, silicone gel+ErB-Np group, and silicone gel+vitamin C group. Arrows indicate areas of re-epithelialization, collagen organization, and fibroblast-rich granulation tissue. All images were obtained at ×40 magnification.

Table I. The median(IQR) values of Abramov scores after 7 days of treatment under Hematoxylin Eosin(HE) and Masson Trichrome staining.

HE: Hematoxylin and eosin; MT: Masson’s trichrome; IQR: interquartile range; ErB-Np: erbium borate nanoparticle; Silicone gel + Vitamin C: silicone gel containing vitamin C; Silicone gel + ErB-Np: silicone gel containing erbium borate nanoparticles; ErB-Np hydrogel: hydrogel containing erbium borate nanoparticles. p-Values are reported for exploratory analyses.

At the end of the 28-day period, a total of 32 incision lines, with a total of 4 incision lines per rat, were evaluated. The macroscopic and microscopic views of the incision lines after 21 days of treatment are illustrated in Figure 1 and Figure 2, respectively. At the end of the 28-day period, subgroup analysis revealed that in the group treated with silicone gel+ErB, the amount of collagen deposition was significantly lower compared to the group treated with silicone gel+vitamin C (p=0.021). Although not statistically significant, it was noticeable that collagen fibers were more organized, homogeneous, and mature in this group. When compared to the group treated with ErB hydrogel, the group treated with silicone gel+ErB did not show a significant difference in terms of granulation tissue amount, collagen deposition, and neovascularization. Although not statistically significant, when comparing median values, it was observed that reepithelialization was more mature and collagen fibers were more organized and homogeneous in the group treated with silicone gel+ErB compared to the control group (Table II).

Table II. The median (IQR) values of Abramov scores after 21 days of treatment under Hematoxylin Eosin(HE) and Masson Trichrome staining.

HE: Hematoxylin and eosin; MT: Masson’s trichrome; IQR: interquartile range; ErB-Np: erbium borate nanoparticle; Silicone gel + Vitamin C: silicone gel containing vitamin C; Silicone gel + ErB-Np: silicone gel containing erbium borate nanoparticles; ErB-Np hydrogel: hydrogel containing erbium borate nanoparticles. p-Values are reported for exploratory analyses.

The organization of collagen structures, density of collagen fibers, and maturation of collagen were evaluated in samples stained with Masson's Trichrome, and no statistically significant difference was observed between the groups (Table III). The appearance of incision lines after 21 days of treatment under Masson Trichrome staining is illustrated in Figure 3. When scar elevation index measurements were analyzed using a linear mixed-effects model accounting for clustering of wounds within animals, no statistically significant overall difference was observed among the treatment groups (F(3, 4)=6.04, p=0.057). Although the silicone gel+ErB-Np group showed the lowest estimated scar elevation index compared with the control group, pairwise comparisons were not statistically significant after Sidak adjustment (Table III).

Table III. Scar elevation index values after 21 days of treatment.

Overall comparison (linear mixed-effects model): F(3,4)=6.04, p=0.057. ErB-Np: Erbium borate nanoparticle; Silicone gel + Vitamin C: silicone gel containing vitamin C; Silicone gel + ErB-Np: silicone gel containing erbium borate nanoparticles; ErB-Np hydrogel: hydrogel containing erbium borate nanoparticles; CI: confidence interval; SD: standard deviation.

Figure 3.

Representative histological sections of incisional wounds stained with Masson’s trichrome after 21 days of treatment. Collagen fibers are stained blue. Sections are shown from the control group, Erbium Borate Nanoparticle (ErB-Np) hydrogel group, silicone gel+ErB-Np group, and silicone gel+vitamin C group. Arrows indicate areas of increased collagen deposition and improved collagen organization in the dermis. Images were obtained at ×40 magnification.

Discussion

The present study suggested that topical application of silicone gel enriched with ErB-Nps resulted in improved wound healing outcomes and was associated with lower scar elevation indices, particularly at later stages of healing. While no statistically significant differences were observed during the early healing phase, histopathological evaluation at 21 days showed a lower scar elevation index in wounds treated with ErB-Np–containing silicone gel compared to the control group; however, this difference did not reach statistical significance in the clustered analysis. These findings suggest that the beneficial effects of this combined formulation may become more apparent during the remodeling phase of wound healing, which is consistent with the delayed but progressive nature of scar modulation. Recent evidence has continued to support the use of silicone- and hydrogel-based formulations in scar modulation and wound repair (11). In a randomized placebo-controlled clinical study, Jahnavi et al. showed that early topical silicone gel application improved several clinically relevant scar characteristics, including pigmentation, vascularity, pliability, and scar height, in head and neck skin wounds. This finding is in line with the established role of silicone-based products as simple and non-invasive options in postoperative scar management (12). Likewise, a recent systematic review evaluating randomized controlled trials on postoperative scar treatment modalities reported that silicone-based topical products remain among the commonly used and supported interventions, although differences in treatment protocols, timing of application, follow-up periods, and outcome measures make direct comparison between studies difficult (11). At the same time, current research on nanohybrid and collagen-based hydrogels has drawn attention to the potential value of nanoparticle-integrated wound dressings. These systems may contribute to wound healing through several complementary mechanisms, including regulation of inflammation and oxidative stress, stimulation of angiogenesis, modulation of fibroblast activity, and improvement of collagen deposition and extracellular matrix remodeling (13). In particular, preclinical studies with metal oxide nanoparticle-containing hydrogels, such as collagen-cerium oxide nanohydrogels, have reported favorable regenerative, antioxidant, and anti-inflammatory effects (14). These observations are compatible with the findings of the present study. In our model, silicone-based formulations, especially silicone gel containing ErB-Nps, were associated with a smoother macroscopic wound appearance and more favorable histopathological features, including collagen organization and maturation.

In recent years, especially metallic-based nanoparticles such as silver and gold derivatives have been shown to have an accelerating effect on chronic and contaminated wound healing. In this context, an in vitro study conducted by Kırbaş et al., ErB-Nps were also demonstrated to be a potential agent for scarless wound healing. According to this study, treatment with ErB-Nps showed no significant difference in cell line proliferation compared to the control group at 24 h in endothelial cell lines (HUVEC), but a significant increase in cell proliferation rate was observed at 48 and 72 h. Additionally, it was determined that ErB-Np treatment increased angiogenesis potential in cells. In the same study, in a keratinocyte cell model (HaCaT), significant reduction in wound distance and area was observed in the experimental group incubated with ErB-Np solution, and the wound closure rate was found to be twice as high compared to the control group. Furthermore, it was observed that ErB-Nps exhibited antioxidant properties by reducing intracellular reactive oxygen species levels in HUVEC and HaCaT cell lines (10). These findings formed the scientific rationale for the present study, which aimed to translate the biological effects of ErB-Nps into a clinically applicable silicone-based formulation for scar modulation.

To achieve this goal, we created an incisional wound model in rats as described by Paskal et al. and topically applied our in-house-produced silicone gel containing ErB-Np(15). After seven days of treatment, histopathological examination of the samples revealed no significant differences in Abramov wound healing scores and scar elevation index compared to the control group. Although statistically significant differences were not observed, it was noted that neovascularization and reepithelialization scores were higher in samples treated with ErB-Np-containing treatment compared to the control group during this early phase of healing. This result is consistent with the cell study conducted by Kırbaş et al., suggesting that ErB-Nps may promote angiogenesis and epithelialization (10).

In vitro studies have shown that in wound cell models treated with silicone gel, basic fibroblast growth factor (bFGF) is expressed at a higher level compared to the control group (16). Although not statistically significant in our study, it is noteworthy that the degree of fibroblast maturation in the granulation tissue was relatively higher in the groups treated with silicone-containing therapies compared to the control group. The lack of significant difference in scar elevation indices after seven days of treatment is consistent with the literature, supporting the necessity of long-term treatment for scarless wound healing. In a study conducted by Choi et al., biopsies were taken from scar tissues at four months postoperatively and immunohistochemically examined for TGF-β1, PDGF, and bFGF levels. In this study, it was observed that in the group treated with silicone gel, the levels of TGF-β1 and PDGF were significantly lower compared to the control group, while the bFGF levels were higher than the control group (17). TGF-β stimulates the synthesis of collagen, elastin, and fibronectin while inhibiting extracellular matrix degradation. It is believed that the increased expression of TGF-β1 and PDGF plays a role in the formation of hypertrophic scars (17, 18). According to the histological evaluations performed after 21 days of treatment in our study, both groups treated with silicone showed a higher amount of collagen deposition compared to the control group. In the group treated with silicone gel containing vitamin C, a statistically significant increase in collagen deposition was observed compared to the control group. In the group treated with silicone gel containing ErB-Nps, although not statistically significant, the amount of collagen deposition was higher compared to the control group. The obtained results do not align with the hypothesis that silicone gel application reduces TGF-β1 expression, thereby suppressing collagen synthesis. It is possible that the silicone gel treatments used in our study containing either vitamin C or ErB-Nps may have yielded incongruent results with the literature. Vitamin C acts as a cofactor in collagen synthesis, and the study by Kırbaş et al. on ErB-Nps showed a decrease in the expression of genes synthesizing matrix metalloproteinase-1 and -2 (MMP) enzymes and an increase in MMP-9 gene expression in the cell groups treated with ErB-Nps (6, 10). This finding supports our hypothesis. Additionally, in this study, while the expression of the TGF-β1, IL-6, and IL1a genes remained unchanged in the treated group, the expression of the TGF-β3 and IL-10 genes was found to have significantly increased (10). After 21 days of treatment, histological evaluation revealed that wounds treated with silicone gel containing vitamin C and silicone gel containing ErB-Nps showed lower scar elevation indices compared to the control group. In wounds treated with ErB-Np-containing hydrogel, scar elevation indices were lower compared to the control group; however the difference did not reach statistical significance. Our study suggests that ErB-Nps and silicone gel may improve wound healing and result in less scarring.

Study limitations. The sample size was determined based on an a priori power analysis while carefully adhering to ethical principles aimed at minimizing animal use, in accordance with the principle of reduction in experimental animal research. Because the power calculation was based on wound-level observations, whereas the experimental intervention was assigned at the animal level, the effective sample size for between-group comparisons was limited, potentially reducing the power to detect modest treatment effects. Although multiple standardized incisional wounds were created per animal to allow comparative evaluation between treatment groups, the relatively small sample size and the use of an animal model may limit the generalizability of the findings to human wound healing. Therefore, the results should be interpreted as preliminary in vivo evidence rather than definitive proof of efficacy. Furthermore, the biological effects of ErB-Nps have been predominantly demonstrated in vitro, and data regarding their in vivo behavior remain limited. In this context, the present study should be considered a proof-of-concept investigation designed to explore the translational potential of ErB-Nps when incorporated into a silicone gel formulation. Wound healing was assessed using semi-quantitative histopathological scoring systems, including the Abramov scoring system and the scar elevation index. While these methods are widely accepted and commonly employed in experimental wound-healing research, they may not fully capture the molecular and biomechanical complexity of scar formation. The absence of quantitative molecular analyses, such as the evaluation of pro-fibrotic cytokines, angiogenic markers, collagen subtypes, or myofibroblast activity, limits the mechanistic interpretation of the observed effects. Finally, the proposed mechanisms related to cytokine modulation, angiogenesis, and oxidative stress were inferred from existing literature rather than directly investigated, highlighting the need for future experimental and clinical studies to further elucidate these pathways.

Conclusion

This animal study demonstrates the potential positive effects of treatment with ErB-Np-containing silicone gel on wound healing. The more favorable outcomes observed with the ErB-Np-containing silicone gel compared to the ErB-Np-containing hydrogel suggest that silicone may be a promising option for wound healing. According to the scar elevation indices, the similar results obtained with ErB-Np-containing silicone gel and silicone gel + vitamin C compound, together with lower observed scar elevation indices than the control group, indicate that ErB-Np-containing silicone gel could be a promising alternative for scar modulation. Further studies and clinical trials are warranted to confirm these findings and elucidate the mechanism by which it affects wound healing.

Conflicts of Interest

MG, MMS, RU, HS, OA, AD, EA, EY, FS, and UOI declare that they have no conflicts of interest.

Authors’ Contributions

Mert Guler: Conceptualization, Methodology, Formal analysis, Investigation, Funding acquisition, Writing-original draft. Mert Mahsuni Sevinc: Conceptualization, Writing-review & editing. Husnu Sevik: Investigation, Data curation, Writing-review & editing. Omer Akay: Investigation, Data curation, Writing-review & editing. Anil Demir: Investigation, Data curation, Writing-review & editing. Ramazan Ucak: Investigation, Resources, Writing-review & editing. Elcin Yenigul: Resources, Data curation, Investigation. Erhan Aysan: Supervision, Writing-review & editing. Fikrettin Sahin: Supervision, Resources, Writing-review & editing. Ufuk Oguz Idiz: Conceptualization, Methodology, Formal analysis, Writing-review & editing. All Authors read and approved the final version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Artificial Intelligence (AI) Disclosure

The Authors declare that no artificial intelligence (AI)-assisted technologies, including generative AI tools or large language models, were used in the design of the study, data collection, data analysis, interpretation of findings, manuscript drafting, revision, or preparation of figures and tables.