Zinc oxide and ellagic acid wound dressing enhances FGF-2 and VEGF expression during early excisional wound healing

Abstract

Introduction:  Zinc oxide-based periodontal dressings are widely used to protect surgical wounds, but conventional formulations have limited anti-inflammatory activity. Ellagic acid is a natural polyphenol with antioxidant and anti-inflammatory properties that may improve local wound-healing response.

Objective:  This study evaluated the effect of a zinc oxide and ellagic acid wound dressing on fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) expression during early excisional wound healing.

Methodology:  Standardized 10×10 mm dorsal excision wounds were created in 20 male Wistar rats. Control wounds received a zinc oxide dressing, whereas treatment wounds received zinc oxide combined with ellagic acid and coconut fatty acids. Tissue samples were collected on days 3 and 7. FGF-2 and VEGF expression were evaluated using immunohistochemistry and quantified as the mean number of positively stained cells in ten microscopic fields. Data were assessed for normality and homogeneity of variance and analyzed using two-way ANOVA to evaluate the effects of dressing type, observation time, and their interaction, followed by Tukey's post hoc test.

Results:  FGF-2 and VEGF expression were higher in the zinc oxide and ellagic acid groups than in the corresponding control groups on both days 3 and 7, with the highest expression observed in the treatment group on day 7. Two-way ANOVA showed significant effects of dressing type and observation time, as well as significant dressing × time interactions for both FGF-2 and VEGF.

Conclusions:  These findings indicate that the treatment-associated increase in growth factor expression was more pronounced on day 7. Zinc oxide and ellagic acid wound dressing increased FGF-2 and VEGF expression during early excisional wound healing, particularly on day 7. These findings suggest that adding ellagic acid to a zinc oxide-based dressing may support fibroblast-related tissue repair and angiogenic activity during the proliferative phase of wound healing.

Keywords:
Zinc oxide; Ellagic acid; Wound dressing; Fibroblast growth factor-2; Vascular endothelial growth factor; Wound healing

Introduction

Wound healing is a coordinated biological process that restores tissue integrity after injury. It involves overlapping phases of hemostasis, inflammation, proliferation, and remodeling, regulated by inflammatory cells, fibroblasts, endothelial cells, cytokines, growth factors, extracellular matrix components, and the vascular system.¹,² In dentistry, tissue injury may occur after periodontal surgical procedures such as gingivectomy, flap surgery, or other interventions involving gingival and periodontal tissues. Therefore, postoperative wound protection and modulation of the early healing response are clinically important.

Periodontal dressings are commonly applied after periodontal surgery to protect the wound surface, reduce mechanical irritation, and improve patient comfort during the initial healing period.³,⁴ Zinc oxide non-eugenol dressings, including Coe-Pak-type materials, are widely used because they provide a protective barrier and exhibit antibacterial effects related to metal oxide and fatty acid components.⁵ However, conventional zinc oxide-based dressings have limited intrinsic anti-inflammatory activity. This limitation may be relevant because excessive or prolonged inflammation can delay the transition to the proliferative phase of wound healing.⁶

Ellagic acid is a polyphenolic compound with antioxidant and anti-inflammatory activities. Previous studies have shown that ellagic acid can suppress pro-inflammatory mediators such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, while supporting anti-inflammatory responses and controlling oxidative stress.⁷,⁸ These biological properties make ellagic acid a promising adjunctive component for wound dressing development, particularly when combined with zinc oxide to balance the protective role of the dressing with a more favorable wound microenvironment.

Growth factor expression is an important indicator of the transition from inflammation to proliferation. Fibroblast growth factor-2 (FGF-2) promotes fibroblast proliferation, keratinocyte migration, extracellular matrix deposition, and tissue remodeling. Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, endothelial cell migration, and new vessel formation, which are essential for oxygen and nutrient delivery during granulation tissue development.⁹–¹³ Increased expression of FGF-2 and VEGF during early healing may indicate enhanced proliferative and angiogenic activity.

This study evaluated the effect of a zinc oxide and ellagic acid wound dressing on FGF-2 and VEGF expression in a standardized rat excisional wound model at days 3 and 7. The working hypothesis was that adding ellagic acid to a zinc oxide dressing would increase FGF-2 and VEGF expression compared with zinc oxide dressing alone, especially during the proliferative phase of wound healing.

Methodology

Study design and animals

This in vivo laboratory experimental study used a post-test-only control group design. The study animals consisted of male Wistar rats (Rattus norvegicus) obtained from the Biochemistry Laboratory, Faculty of Medicine, Airlangga University. The study was approved by the institutional ethics committee (No. 1114/HRECC.FODM/X/2025).

The sample size was calculated using the Lemeshow formula, resulting in a minimum of five animals per group. Twenty healthy male Wistar rats, aged 3 months and weighing 200–300 g, were included. Animals were excluded if they showed clinical signs of illness, physical abnormalities, or impaired general condition before intervention.

The animals were housed under standard laboratory conditions at a controlled temperature of 22 °C, relative humidity of 50–60%, and a 12-hour light/12-hour dark cycle. Standard laboratory feed and drinking water were provided ad libitum. The animals were acclimatized for seven days before the experimental procedures and monitored daily for general health, food and water intake, behavior, and wound condition.

The animals were allocated to control and treatment groups, with each group evaluated on days 3 and 7 after treatment, resulting in four subgroups of five animals each. Control groups received zinc oxide mixed with coconut fatty acids, whereas treatment groups received zinc oxide combined with ellagic acid and coconut fatty acids.

Wound creation and dressing application

Animals were anesthetized by intramuscular administration of ketamine at 80 mg/kg body weight and xylazine at 10 mg/kg body weight. Anesthetic depth was confirmed by the absence of a withdrawal response to toe pinch before wound creation. The dorsal skin was shaved and disinfected with 70% ethanol. A standardized 10×10 mm excision wound with an approximate depth of 2 mm was created using a scalpel, followed by irrigation with sterile normal saline. No silicone splint or other device was applied around the wound margin to restrict wound contraction.

The treatment dressing was prepared by mixing zinc oxide powder (87.5%; Sigma-Aldrich, Cat. No. 1088490500) with ellagic acid powder (12.5%; ellagic acid 90%, Xi'an Biof-Technology Co., Ltd., China) until a homogeneous mixture was obtained. The resulting powder mixture was then combined with coconut fatty acids at a 1:1 weight ratio. The 12.5% proportion of ellagic acid in the powder mixture was selected based on a previously published study using the same formulation, which demonstrated increased macrophage infiltration, angiogenesis, and collagen deposition during excisional wound healing.¹⁴ Therefore, the complete paste applied to the wound contained 43.75% zinc oxide, 6.25% ellagic acid, and 50% coconut fatty acids by weight. The control dressing consisted of zinc oxide powder mixed with coconut fatty acids at a 1:1 weight ratio.¹⁴ The assigned dressing was applied directly to the wound surface and secured with hypoallergenic tape.

Tissue collection and immunohistochemistry

Animals were euthanized on days 3 and 7 according to group allocation using CO₂ inhalation. Tissue specimens containing the wound bed and surrounding wound-margin tissue were excised using a standardized 10x10 mm sampling area. Skin tissues were fixed in 10% buffered formalin for 24 hours, processed for paraffin embedding, sectioned at 4 μm, and mounted on glass slides.

Immunohistochemical staining was performed using an indirect immunohistochemical method. The sections were deparaffinized in two changes of xylene for 10 minutes each and rehydrated through a graded ethanol series of 100%, 95%, 80%, and 70% ethanol for 5 minutes each, followed by rinsing in distilled water. Heat-induced antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 95–98 °C for 20 minutes, after which the sections were allowed to cool at room temperature for 20 minutes. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 10 minutes at room temperature. Nonspecific antibody binding was blocked by incubating the sections with 5% normal serum from the host species of the secondary antibody in phosphate-buffered saline for 30 minutes at room temperature.

The primary antibodies used were anti-FGF basic/FGF-2 antibody (Abcam, ab64207; sheep polyclonal; 1:300 dilution) and anti-VEGFA antibody (Abcam, ab1316; mouse monoclonal; 5 μg/mL). After incubation with the primary antibodies, sections were treated with an appropriate HRP-conjugated secondary antibody. The antigen–antibody reaction was visualized using 3,3′-diaminobenzidine (DAB), producing brown staining in positive cells. Sections were counterstained with hematoxylin, dehydrated, cleared, and mounted.

Positive staining was identified by brown cytoplasmic immunoreactivity in cells within the wound area. The wound area was initially examined at 100× magnification to assess tissue orientation and select the region of interest. Final quantitative counting was performed at a total magnification of 1000× to allow clear identification of individual positively stained cells. For each sample, positively stained cells were counted in ten randomly selected, non-overlapping microscopic fields within the wound area, and the mean value was used for statistical analysis.

Statistical analysis

Statistical analysis was performed using SPSS version 25.0. Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene's test. The effects of dressing type and observation time, as well as the interaction between these factors, were analyzed using a two-way analysis of variance (ANOVA), followed by Tukey's honestly significant difference post hoc test for pairwise comparisons. A p-value < 0.05 was considered statistically significant.

Results

FGF-2 expression

FGF-2 expression was evaluated by immunohistochemical staining (Figure 1A). Positive expression was identified by brown staining in the cytoplasm of cells within the wound area. Quantitative analysis showed that FGF-2 expression increased from day 3 to day 7 in both groups and was higher in the ZnO-E group than in the control group at both time points (Figure 1B). On day 3, the FGF-2 expression values were 3.80±0.84 in the control group and 6.80±0.84 in the ZnO-E group, whereas on day 7, the corresponding values were 5.40±0.55 and 10.80±0.84, respectively. The highest FGF-2 expression was observed in the ZnO-E group on day 7. These findings indicate that ZnO-E dressing enhanced fibroblast-related reparative activity, particularly during the proliferative phase of wound healing.

Figure 1
FGF-2 expression in excision wounds treated with zinc oxide and ellagic acid. (A) Representative immunohistochemical staining of FGF-2 in the control and zinc oxide and ellagic acid groups on days 3 and 7. Brown staining indicates positive FGF-2 expression. Insets show low-magnification views, and red boxes indicate the analyzed areas. (B) Quantitative analysis of FGF-2 expression. Zinc oxide and ellagic acid increased FGF-2 expression compared with the control group at both time points, with the highest expression observed on day 7. Data are presented as mean ± SD. ***p<0.001; ****p<0.0001. ZnO-E: zinc oxide and ellagic acid.

VEGF expression

VEGF expression was evaluated by immunohistochemical staining (Figure 2A). Positive expression was identified by brown staining in the cytoplasm of cells within the wound area. Quantitative analysis showed that VEGF expression increased from day 3 to day 7 in both groups and was higher in the ZnO-E group than in the control group at both time points (Figure 2B). On day 3, the VEGF expression values were 4.20±0.45 in the control group and 6.40±0.55 in the ZnO-E group, whereas on day 7, the corresponding values were 7.40±0.55 and 10.60±0.55, respectively. The highest VEGF expression was observed in the ZnO-E group on day 7. These findings indicate that ZnO-E dressing promoted angiogenic activity, which may support blood vessel formation during early wound repair.

Figure 2
VEGF expression in excision wounds treated with zinc oxide and ellagic acid. (A) Representative immunohistochemical staining of VEGF in the control and zinc oxide and ellagic acid groups on days 3 and 7. Brown staining indicates positive VEGF expression. Insets show low-magnification views, and red boxes indicate the analyzed areas. (B) Quantitative analysis of VEGF expression. Zinc oxide and ellagic acid increased VEGF expression compared with the control group at both time points, with the highest expression observed on day 7. Data are presented as mean ± SD. ***p<0.001; ****p<0.0001. ZnO-E: zinc oxide and ellagic acid.

Discussion

This study demonstrated that zinc oxide and ellagic acid (ZnO-E) dressing increased FGF-2 and VEGF expression during early excisional wound healing. Expression of both markers was higher in the ZnO-E groups than in the corresponding control groups on days 3 and 7, with the highest values observed in the ZnO-E group on day 7. Two-way ANOVA showed significant effects of dressing type and observation time, as well as significant treatment × time interactions for both FGF-2 and VEGF. These interactions indicate that the effect of the ZnO-E dressing varied according to the observation period, with a greater treatment-associated increase on day 7.

The concurrent increase in FGF-2 and VEGF suggests that the ZnO-E dressing supported complementary aspects of the proliferative phase of wound healing. FGF-2 contributes to fibroblast proliferation and migration, extracellular matrix production, keratinocyte activity, and granulation tissue formation. VEGF promotes endothelial cell migration and proliferation and new blood vessel formation, thereby supporting oxygen and nutrient delivery to the healing tissue.⁹–¹³ Therefore, the increase in both markers may reflect coordinated fibroblast-related tissue repair and angiogenic activity rather than two independent biological responses.

The enhanced expression observed after ZnO-E application may be explained by the complementary biological properties of zinc oxide and ellagic acid. Zinc oxide may contribute to wound healing through its protective barrier function, antimicrobial activity, and zinc ion release. Zinc is required for enzymes involved in DNA synthesis, RNA transcription, cellular proliferation, and extracellular matrix repair.¹⁵ However, excessive zinc ion release or reactive oxygen species generation may impair fibroblast and endothelial cell viability, particularly when zinc oxide is present at high concentrations or in nanoparticle form.¹⁶,¹⁷ The biological effects of zinc oxide may therefore depend on maintaining a balance between reparative cellular signaling and oxidative injury.

Ellagic acid may complement the effects of zinc oxide with its antioxidant and anti-inflammatory activities. Excessive reactive oxygen species and prolonged inflammatory signaling can impair fibroblast viability, endothelial cell function, and growth factor activity. Ellagic acid has been reported to suppress pro-inflammatory mediators, including tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6, while supporting oxidative stress control.⁷,⁸ By reducing excessive oxidative stress and inflammatory signaling, including NF-κB-related pathways, ellagic acid may create a more favorable wound microenvironment for fibroblast and endothelial cell activity.⁷,⁸,¹⁰

A plausible complementary mechanism is therefore that zinc oxide provides wound protection, antimicrobial activity, and zinc-dependent support for cellular repair, whereas ellagic acid limits excessive oxidative and inflammatory stress. This balance may preserve fibroblast and endothelial cell function and support FGF-2 and VEGF expression. The stronger response observed on day 7 is consistent with the increasing importance of fibroblast activity, granulation tissue development, and angiogenesis during the proliferative phase of wound healing.

The present findings are also consistent with a previous study using the same ZnO-E formulation in an excisional wound model. That study demonstrated increased macrophage infiltration, newly formed blood vessels, and collagen deposition after treatment with ZnO-E compared with zinc oxide alone. The higher VEGF expression observed in this study is consistent with the increased angiogenesis reported previously, whereas the increase in FGF-2 may support fibroblast activity, extracellular matrix production, and granulation tissue development. Together, these findings provide additional biological evidence that adding ellagic acid may improve the tissue response produced by a zinc oxide-based dressing.

Nevertheless, the proposed mechanism should be interpreted cautiously. This study evaluated FGF-2 and VEGF expression but did not directly assess reactive oxygen species, antioxidant capacity, zinc ion release, inflammatory cytokines, or intracellular signaling pathways. Furthermore, because an ellagic acid-only group was not included, the independent effect of ellagic acid and a true synergistic interaction between zinc oxide and ellagic acid cannot be established. Therefore, the findings demonstrate an enhanced effect after adding ellagic acid to the zinc oxide dressing but do not confirm a specific molecular pathway or pharmacological synergy.

This study has several limitations. First, the biological assessment was limited to immunohistochemical quantification of FGF-2 and VEGF. Although these growth factors are associated with fibroblast-related tissue repair and angiogenesis, their increased expression alone does not directly demonstrate enhanced wound closure, re-epithelialization, collagen deposition, or new blood vessel formation. The model also used dorsal skin excision wounds rather than intraoral or periodontal wounds. Although this model provides a standardized approach to evaluate early tissue repair, oral wounds have distinct microbial, salivary, epithelial, and mechanical environments. Additionally, no silicone splint was used to restrict wound contraction. Because rat dorsal wounds heal substantially through contraction, this may have influenced the wound microenvironment and the observed expression of FGF-2 and VEGF. Although wound closure was not evaluated as an outcome in this study, future studies should use a splinted wound model to minimize contraction and enable a more specific evaluation of granulation tissue formation and re-epithelialization. Second, the observation period was limited to days 3 and 7; therefore, the effect of the dressing on later stages of remodeling remains unknown. Third, additional outcomes, such as wound closure rate, collagen deposition, inflammatory cytokines, oxidative stress markers, histomorphometry, and cytotoxicity, would strengthen the biological interpretation of the findings. Finally, the sample size was modest, and the absence of an ellagic acid-only group prevents determination of the independent effect of ellagic acid or confirmation of a synergistic interaction between zinc oxide and ellagic acid. Future studies should therefore include larger cohorts, oral or periodontal wound models, detailed material characterization, an ellagic acid-only group, and direct evaluation of the proposed biological mechanisms.

Overall, the findings indicate that adding ellagic acid to a zinc oxide dressing increased FGF-2 and VEGF expression during early wound healing, particularly on day 7. The concurrent increase in these markers may reflect enhanced fibroblast-related reparative activity and angiogenesis. Although the underlying molecular mechanisms require direct confirmation, the antioxidant and inflammation-modulating properties of ellagic acid may improve the local biological response produced by zinc oxide dressing.

Conclusions

Zinc oxide and ellagic acid wound dressing increased FGF-2 and VEGF expression compared with zinc oxide dressing alone on both day 3 and day 7 of early excisional wound healing, with the highest levels observed on day 7. These findings suggest that adding ellagic acid to a zinc oxide-based dressing may support growth factor expression associated with fibroblast-related tissue repair and angiogenic activity. However, because this study was limited to immunohistochemical assessment of FGF-2 and VEGF, further studies evaluating functional wound-healing outcomes and using oral or periodontal wound models are needed before clinical application can be recommended.

Funding

This research was funded by Universitas Airlangga in the schema Airlangga Research Fund (ARF) Batch I Skema Penelitian Dasar Unggulan number 1760/UN3.LPPM/PT.01.03. 2025.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Correspondence

Prof. Dr. Intan Nirwana - Universitas Airlangga - Faculty of Dental Medicine - Department of Dental Materials - Jalan Prof. Dr. Moestopo Street 47 - Surabaya 60132 – Indonesia - Email: [e-mail]

Editor:

Linda Wang

Associate Editor:

Ana Carolina Morandini Ramos

Conflict of interest

The authors declare no conflict of interest.

Publication Dates

• Publication in this collection
  26 Oct 2026
• Date of issue
  2026

History

• Received
  15 June 2026
• Reviewed
  27 Aug 2026
• Accepted
  07 Sept 2026