Efficacy of Topical Nigella sativa Oil for Oral Wound Healing in Rats

ABSTRACT

Objective

This study aimed to examine the effect of topical Nigella sativa oil (NSO) on intraoral wound healing in rats.

Materials and Methods

Forty‐eight female Wistar albino rats (2.5–3 months old) were assigned to two groups (n = 24 each): A control group receiving no treatment and an NSO group receiving a topical application of 1 cc NSO. A standardized circular full‐thickness wound (3 mm in diameter) was created on the mucoperiosteum of the hard palate. The animals were sacrificed on postoperative Days 3, 7, 14, and 20 to assess wound area macroscopically, wound diameter histologically, and immunohistochemically for anti–transforming growth factor‐β (anti–TGF‐β) and anti‐vascular growth factor (anti–VGF).

Results

No significant differences were observed between groups on days 3 and 20 (p > 0.05). On Days 7 and 14, however, the NSO group exhibited significantly smaller wound areas and wound diameters (p < 0.05). Immunohistochemical staining showed lower TGF‐β expression on Days 7 and 14 in the NSO group compared to control (p < 0.05), whereas VGF staining did not differ significantly (p > 0.05).

Conclusion

Topical NSO accelerated the healing of intraoral wounds, particularly by enhancing re‐epithelialization between postoperative days 7 and 14. This effect appears to be mediated through TGF‐β modulation and NSO's known anti‐inflammatory, antimicrobial, and antioxidant actions. Topical NSO may thus be considered as an adjunct to conventional wound management in the oral cavity.

Level of Evidence

NA.

1. Introduction

Wound healing in the oral cavity differs significantly from healing in other parts of the body, such as the skin, largely due to the specialized environment that exists in the mouth [1]. The presence of a dense microbial flora and constant mechanical stimuli—such as mastication and tongue movement—poses unique challenges for wound healing in oral tissues [2]. Over the past several decades, much research has focused on identifying topical agents that can reduce local bacterial colonization, diminish inflammatory responses, and expedite tissue repair in the mouth [3, 4]. Such agents, when applied locally, have the advantage of concentrating therapeutic effects directly at the wound site, potentially reducing systemic side effects and enhancing healing [5, 6].

1.1. Background of Oral Wound Healing

Wound healing involves a complex cascade of events: hemostasis, inflammation, proliferation, and remodeling [7]. In the early stages, platelet aggregation and blood coagulation collectively establish hemostasis, forming a fibrin clot that provides a provisional matrix. Subsequently, in the inflammatory stage, neutrophils and macrophages migrate to the wound area to clear debris and microbes, releasing various cytokines and growth factors that initiate tissue repair. In the proliferative phase, fibroblasts deposit collagen and other extracellular matrix components, while epithelial cells migrate across the wound to restore mucosal integrity. Finally, in the remodeling phase, collagen fibers become more organized, and the scar (or newly formed tissue) gains tensile strength [8, 9].

In the oral cavity, the bacterial load can be especially high, with many species capable of producing proteases and other metabolic products that interfere with epithelial cell migration and fibroblast function [10]. Although some level of bacterial presence can stimulate a beneficial inflammatory response, an excessive microbial population often results in persistent inflammation and delayed healing [11]. Therefore, controlling bacterial overgrowth, reducing excessive inflammation, and supporting epithelial migration are pivotal in improving oral wound healing outcomes [12].

1.2. Nigella sativa (NS) and Its Traditional Uses

For centuries, medicinal plants have played an essential role in both traditional and modern therapeutic practices [13]. NS, commonly referred to as black cumin or black seed, is a flowering plant of the Ranunculaceae family, widespread in Southwest Asia, North Africa, and the Middle East. Historically, NS seeds have been used to treat various ailments, including respiratory diseases, gastrointestinal disturbances, and infections. The extracted oil from these seeds— Nigella sativa oil (NSO)—has garnered interest due to its broad spectrum of pharmacological properties [14].

1.3. Phytochemistry and Mechanisms of NSO

A major bioactive constituent of NSO is thymoquinone (TQ). Research indicates that TQ exhibits a range of therapeutic properties, including anti‐inflammatory, antimicrobial, antioxidant, and immunomodulatory effects [15, 16]. In inflammatory conditions, TQ can inhibit the lipoxygenase and cyclooxygenase pathways, mitigating the production of proinflammatory mediators such as leukotrienes and prostaglandins. Moreover, TQ scavenges superoxide radicals and hydroxyl ions, thereby reducing oxidative stress—a known barrier to efficient wound healing. Within wound sites, a delicate balance between pro‐oxidant and antioxidant mechanisms is critical; excessive reactive oxygen species (ROS) can disrupt cell membranes, degrade proteins, and damage DNA, while inadequate antioxidant protection can also impair the healing process [17].

1.4. Role of Growth Factors in Oral Wound Repair

Several growth factors modulate wound healing, two of the most pivotal being transforming growth factor‐β (TGF‐β) and vascular growth factor (VGF; often referred to as VEGF in many studies, but here considered generically as vascular growth factors) [18]. TGF‐β orchestrates multiple aspects of wound repair: at low concentrations, it supports epithelial cell proliferation and migration; however, at higher concentrations, TGF‐β can induce apoptosis and excessive collagen deposition, sometimes leading to scar hypertrophy in skin wounds. In the oral cavity, TGF‐β is intricately involved in the re‐epithelialization and connective tissue formation process, but the exact thresholds that distinguish its beneficial versus detrimental roles are not fully understood [19].

Similarly, vascular growth factors are instrumental in angiogenesis—the formation of new blood vessels. Adequate angiogenesis is crucial for delivering nutrients, oxygen, and immune cells to the wound bed. In cutaneous wounds, this process is often robust, but oral mucosal wounds may exhibit different patterns of vascularization that contribute to their tendency to heal with minimal scarring. A better understanding of how angiogenic factors operate in the oral environment could inform therapeutic strategies to enhance healing while minimizing fibrotic tissue formation [20].

1.5. Rationale and Objective of the Present Study

Although NSO has shown promise in cutaneous wound models, little data exist on its efficacy for healing mucosal wounds within the oral cavity. Given the central role that bacterial colonization, cytokine balance, and growth factor levels play in oral wound healing, the reported anti‐inflammatory, antimicrobial, and immunomodulatory effects of NSO make it a compelling candidate [21]. Thus, the purpose of this study was to investigate the effect of topically applied NSO on a rat palatal wound model, focusing on clinical (macroscopic) endpoints—such as wound area—and more refined histopathological parameters, including wound diameter and immunohistochemical staining for TGF‐β and VGF.

2. Materials and Methods

2.1. Ethical Approval and Animal Housing

The study protocol was reviewed and approved by the Bağcılar Experimental Animals Local Ethics Committee (Approval No. 2015/25). All experiments were carried out in accordance with relevant national and international guidelines on the care and use of laboratory animals. Forty‐eight female Wistar albino rats, aged 2.5–3 months and weighing 250–350 g, were selected. These specific inclusion criteria ensured a relatively homogenous sample in terms of age, weight, and sex, thereby minimizing biological variability in wound healing responses.

The animals were housed in standard polypropylene cages containing six rats each. Environmental conditions included a 12‐h light/dark cycle (8 AM–8 PM), a relative humidity of about 50% ± 10%, and a temperature of 23°C ± 4°C. Rats had ad libitum access to standard rodent chow and tap water throughout the study period.

2.2. Experimental Design and Group Allocation

Upon acclimatization, the 48 rats were divided into eight cages of six animals each. Four cages were randomly assigned to the NSO treatment group (NSO group), while the remaining four served as the untreated control group. The animals were further stratified by the intended sacrifice days to yield subgroups of six animals each at postoperative Days 3, 7, 14, and 20. A summary of this arrangement is illustrated in Table 1.

TABLE 1.

Experimental group allocation.

Group number	Group	Time point	Number of rats
1	NSO group	Day 3	6
2	NSO group	Day 7	6
3	NSO group	Day 14	6
4	NSO group	Day 20	6
5	Control group	Day 3	6
6	Control group	Day 7	6
7	Control group	Day 14	6
8	Control group	Day 20	6

Note: This table summarizes the experimental design of the study. The animals were divided into two main groups: the NSO Group, which received topical Nigella sativa oil, and the Control Group, which received no treatment. Each group was further subdivided based on sacrifice time points (Days 3, 7, 14, and 20), with six rats assigned to each subgroup.

2.2.1. NSO Group (n = 24)

Received daily topical NSO, delivered directly onto the palatal wound.

2.2.2. Control Group (n = 24)

Received no topical treatment, allowing for normal wound healing to occur.

2.3. Anesthesia and Palatal Wound Model

Surgical procedures were performed on a dedicated bench under aseptic conditions. All rats underwent general anesthesia via intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Ketalar, Pfizer Warner Lambert, USA) and xylazine hydrochloride (5 mg/kg, Rompun, Bayer, Istanbul, Turkey). Once anesthetized, each rat was placed supine, and the mouth was gently propped open using a specialized retractor to enable consistent access to the hard palate.

A dermatological punch biopsy tool (3 mm in diameter) was employed to create a standardized full‐thickness wound on the mucosa of the hard palate. The mucosa was incised circumferentially, and blunt dissection ensured complete exposure down to the periosteum in the central region of the defect (Figure 1a,b). This technique builds upon prior models described in oral surgery and periodontal research, which have validated its reliability for producing uniform defects suitable for evaluating wound healing outcomes.

FIGURE 1.

(a, b) Standardized palatal wound model in rats.

No sutures were placed over the palatal wound to allow healing by secondary intention, thus mirroring the clinical scenario often observed in procedures such as gingival graft donor sites. After the procedure, each rat was placed in a clean cage to recover from anesthesia. Instances of bleeding were minimal, and no additional hemostatic measures were required.

2.4. Topical Application of NSO

A 1 cc volume of pure, cold‐pressed NSO was prepared for daily topical application in the NSO group. The oil was obtained from Purity Products, USA, a manufacturer known for producing high‐potency black seed oil with a TQ concentration of 3%.

To maintain consistency and stability, the NSO was stored at 4°C in a dark glass container to prevent oxidation and light degradation. During administration, each rat was either lightly reanesthetized or gently restrained, depending on the postoperative day, to ensure clear visualization of the palatal wound. A sterile blunt‐tipped syringe was used to apply the oil directly onto the wound surface, ensuring even coverage while avoiding mechanical disturbance.

To minimize ingestion, an oropharyngeal aspirator was positioned at the posterior oral cavity, allowing the NSO to remain in contact with the wound for at least 1 min before aspirator removal. While minor spillage or ingestion was possible, every effort was made to maximize localized retention of the NSO at the wound site.

Rats in the control group received no topical treatment but were subjected to identical handling procedures to control for potential stress or handling‐related effects.

2.5. Sacrifice and Tissue Harvest

On postoperative Days 3, 7, 14, and 20, one subgroup from the NSO group and one subgroup from the control group were sacrificed sequentially by surgical decapitation under anesthesia. Specifically, the 1st and 5th subgroups were sacrificed on postoperative Day 3, the 2nd and 7th subgroups on Day 7, the 3rd and 6th subgroups on Day 14, and the 4th and 8th subgroups on Day 20. These time points were chosen to capture early, intermediate, and later phases of wound healing. Following sacrifice, the hard palate (including the wound area and adjacent tissues) was harvested under a microscope (D.F. Vasconcellos MC‐M22) to ensure standardized excision margins extending at least 2–3 mm beyond the wound edges.

Collected samples were immediately placed in 10% neutral buffered formalin for fixation. Tissues were subsequently decalcified in 20% hydrochloric acid for approximately 24 h, depending on bone thickness, to facilitate microtomy and histological processing.

2.6. Macroscopic Assessment of Wound Area

After tissue harvesting, specimens were carefully oriented and photographed using a Samsung Galaxy camera set to consistent magnification (16:9 aspect ratio) at a fixed distance of 30 cm. The same lighting conditions and camera settings were used for all specimens to reduce variability. Images were transferred to a computer, and wound areas were measured using AutoCAD software (Autodesk Corp., USA). Regions of interest (ROI) outlining the wound margins were manually drawn, and the software calculated the precise area in square millimeters.

2.7. Histopathological Analysis: Wound Diameter

Fixed and decalcified specimens were processed through standard paraffin‐embedding protocols. Serial sections, each about 5 μm thick, were mounted on glass slides and stained with hematoxylin and eosin (H&E). A blinded pathologist, unaware of group allocations, examined the slides under an Olympus BX51 light microscope. The widest gap between the edges of regenerating epithelium was recorded as the “wound diameter,” thus serving as an indicator of re‐epithelialization speed. Larger wound diameters correspond to slower epithelial proliferation, while narrower diameters suggest more advanced epithelial coverage.

2.8. Immunohistochemical Staining for TGF‐β and VGF

To evaluate growth factor expression, sections adjacent to those stained by H&E were processed immunohistochemically for TGF‐β and VGF (both from Abcam, USA). After deparaffinization and rehydration, endogenous peroxidase was quenched with 3% hydrogen peroxide. Antigen retrieval was achieved with citrate buffer (pH 6.0) at 95°C for 20 min. Sections were then incubated with primary antibodies against TGF‐β or VGF, followed by secondary antibodies and a detection system using 3,3′‐diaminobenzidine (DAB) as the chromogen.

A semi‐quantitative scale (0–3+) was used to classify staining intensity within the inflamed submucosal area beneath the wound edges. Briefly, “0” indicated no detectable staining, and “3+” indicated intense staining in the majority of cells. Intermediate scores (1+ or 2+) captured mild and moderate intensities, respectively. This methodology follows standard immunohistochemical practices for semi‐quantitative evaluations of protein expression in wound healing studies.

2.9. Statistical Analysis

All data were analyzed using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics (mean, standard deviation, median, and range) were generated for each parameter. Normal distribution was assessed via the Kolmogorov–Smirnov test. Since most variables did not conform to normality, the Mann–Whitney U test was utilized to compare the NSO and control groups within each time point. A p‐value < 0.05 was considered statistically significant.

3. Results

All animals recovered uneventfully from the surgical procedure, except for one mortality in subgroup 2 and one in subgroup 4, likely due to anesthesia‐related complications. These animals were replaced per protocol, ensuring that the planned sample size of six rats per subgroup was maintained. No signs of infection, excessive bleeding, or abnormal feeding behavior were noted in the surviving animals. The numerical values for wound area, wound diameter, and immunohistochemical evaluations are presented in detail in Table 2.

TABLE 2.

Comparison of wound healing parameters between NSO and control groups.

	NSO groups		Control groups		p
	Mean. ± S.D.	Median	Mean. ± S.D.	Median
Wound area evaluation (mm2)
3nd day	5.54 ± 0.18	5.49	5.45 ± 0.33	5.33	0.361m
7th day	0.37 ± 0.11	0.36	0.65 ± 0.14	0.69	0.009 m
14th day	0.02 ± 0.01	0.02	0.07 ± 0.02	0.08	0.008 m
20th day	0.0 ± 0.00	0.00	0.00 ± 0.00	0.00	1000m
Wound diameter evaluation (μm)
3nd day	2303 ± 89	2295	2400 ± 187	2460	0.273m
7th day	681.7 ± 95.4	665	784.0 ± 87.3	760	0.021 m
14th day	30.0 ± 7.1	30.0	58.0 ± 8.4	60.0	0.008 m
20th day	0.00 ± 0.00	0.00	0.00 ± 0.00	0.00	1000m
Anti‐TGF‐β immunohistochemical evaluation
3nd day	1.00 ± 0.00	1.00	1.00 ± 0.00	1.00	1000m
7th day	1.40 ± 0.55	1.00	2.40 ± 0.55	2.00	0.031 m
14th day	1.33 ± 0.52	1.00	2.40 ± 0.55	2.00	0.018 m
20th day	1.33 ± 0.52	1.00	1.20 ± 0.45	1.00	0.637m
Anti‐VGF immunohistochemical evaluation
3nd day	1.50 ± 0.55	1.50	1.40 ± 0.55	1.00	0.752m
7th day	2.17 ± 0.75	2.00	2.40 ± 0.55	2.00	0.609m
14th day	1.60 ± 0.55	2.00	1.60 ± 0.55	2.00	1000m
20th day	1.50 ± 0.55	1.50	1.40 ± 0.55	1.00	0.752m

Note: Bold italics = significant vs. control (Mann–Whitney U, p < 0.05).

Abbreviation: m, Mann–whitney U test.

3.1. Macroscopic Wound Area

1. Day 3: The wound area did not differ significantly between the NSO group and the control group (p > 0.05). Both groups showed an expected inflammatory response with evidence of fibrinous exudate but relatively minimal size reduction from baseline.

2. Day 7: The NSO‐treated wounds displayed a significantly smaller area compared to the control group (p < 0.05). Visual inspection showed that the NSO group had more advanced epithelial coverage at the wound margins, with reduced fibrin coverage (Figure 2a,b).

3. Day 14: Consistent with the Day 7 findings, the NSO group exhibited statistically smaller wound areas than controls (p < 0.05). Clinical observation confirmed robust epithelial in‐growth.

4. Day 20: By this time, wounds in both groups showed significant contraction and epithelial bridging. No statistically significant difference was observed (p > 0.05), reflecting convergence in late‐stage healing (Figure 3a,b).

FIGURE 2.

(a, b) Macroscopic comparison of wound healing on postoperative Day 7.

FIGURE 3.

(a, b) Macroscopic comparison of wound healing on postoperative Day 20.

3.2. Histopathological Wound Diameter

1. Day 3: There was minimal re‐epithelialization in either group, with no statistically significant difference (p > 0.05) (Figure 4a,b).

2. Day 7: The NSO group demonstrated a notable decrease in epithelial gap width compared to the control group (p < 0.05), correlating with the macroscopic findings (Figure 4c,d).

3. Day 14: This reduced epithelial gap persisted in the NSO group, remaining significantly smaller than in controls (p < 0.05). Representative micrographs showed complete or near‐complete bridging by epithelial cells in several NSO‐treated samples (Figure 4e,f).

4. Day 20: Epithelial continuity was nearly restored in both groups, and no significant difference was detected (p > 0.05) (Figure 4g,h).

FIGURE 4.

(a–h): Histopathological comparison of wound diameter across postoperative day.

3.3. Immunohistochemical Staining: TGF‐β and VGF

• TGF‐β: On days 7 and 14, sections from the NSO group revealed lower overall staining intensity (generally 0–1+ or 1–2+) compared to control specimens (commonly 2+). Statistical analysis confirmed a significant difference between groups (p < 0.05). On days 3 and 20, however, the difference was not significant (p > 0.05). Lower TGF‐β expression in the mid‐proliferative phase may have facilitated more efficient epithelial migration and minimized unnecessary fibroblast hyperplasia.

• VGF: No significant differences in staining intensity were observed at any time (p > 0.05). Mild to moderate staining (1–2+) was detected in both groups, reflective of ongoing angiogenesis common to wound sites. Oral tissues often feature rapid revascularization that proceeds relatively independently of exogenous influences, which may explain the lack of a measurable difference in VGF activity (Figure 5).

FIGURE 5.

Comparison of Anti‐TGF and Anti‐VGF expression over time.

4. Discussion

This experimental study sought to elucidate the healing properties of NSO when applied topically to intraoral defects in rats. The palatal wound model provided a reproducible platform for comparing spontaneous healing (control) to healing enhanced by NSO (treatment), with a multi‐dimensional analysis spanning macro‐level wound area, histological re‐epithelialization, and immunohistochemical markers of inflammation and angiogenesis.

4.1. Interpretation of Key Findings

The study's most noteworthy outcome was the significantly reduced wound area and epithelial gap in the NSO group at postoperative Days 7 and 14. Mechanistically, these observations align with the established anti‐inflammatory and antioxidant roles of NSO and TQ, which likely reduced the local inflammatory load [22]. By modulating the expression of TGF‐β, NSO may have supported epithelial proliferation and migration. Prior research underscores that TGF‐β plays a dichotomous role: while crucial for initiating granulation tissue and epithelial cell proliferation at optimal levels, excessive TGF‐β can lead to apoptotic phenomena and overzealous extracellular matrix deposition, as documented in cutaneous scarring scenarios [23]. Our findings suggest that NSO helps maintain TGF‐β levels within a beneficial therapeutic window.

Meanwhile, the lack of significant differences in VGF expression, although somewhat surprising, is consistent with data illustrating that oral wounds naturally exhibit rapid angiogenesis [20]. The high vascular network in oral tissues and exposure to saliva might already optimize conditions for vascular regeneration [24]. Hence, an exogenous factor like NSO might not substantially alter VGF‐related pathways under these experimental conditions. Nonetheless, improved blood flow associated with lower inflammation could have indirectly contributed to faster epithelial closure [25].

4.2. Comparison With Previous Studies

Multiple reports on cutaneous burn or excisional wound models have demonstrated the beneficial effects of NSO in accelerating wound closure. For instance, Selcuk et al. showed that thymoquinone treatment significantly reduced healing times and improved histological parameters in a rat burn model, potentially through its capacity to suppress proinflammatory cytokines and mitigate oxidative damage [26]. Similar findings in other studies emphasize how NSO's bioactive components can modulate the inflammatory phase, enhance collagen deposition, and support faster wound contraction in skin wounds [17, 22].

These data, however, largely pertain to skin wounds, which possess distinct adnexal structures (e.g., hair follicles, sweat glands) that can aid re‐epithelialization—a process not directly comparable to oral mucosal healing [7, 8]. By contrast, intraoral wound models are more susceptible to high bacterial loads and mechanical irritation, underscoring the importance of anti‐inflammatory and antimicrobial strategies [1, 10]. Prior research on NS in oral wounds is relatively sparse, and existing studies seldom include detailed immunohistochemical analyses of growth factors such as TGF‐β [27, 28]. Thus, the present study not only complements the broader evidence on NSO's anti‐inflammatory and antioxidant potential but also highlights its capacity to modulate TGF‐β expression specifically in the oral cavity. This mechanism is especially relevant in balancing necessary tissue repair with the risk of excessive scarring [19, 23]. Meanwhile, the lack of significant differences in VGF staining aligns with the concept that oral tissues often exhibit intrinsically robust vascularization and angiogenic responses [24, 25].

4.3. Potential Mechanisms and Clinical Implications

Beyond TGF‐β modulation, other mechanisms may be at play, including direct antibacterial effects against oral flora and synergy with the host immune response [11, 12]. NSO has been shown to inhibit a variety of Gram‐positive and Gram‐negative bacteria, which could reduce the microbial burden and subsequent local inflammation at the wound site [21]. Additionally, the rich composition of essential fatty acids (like linoleic and oleic acids) in NSO might bolster the integrity of cell membranes in regenerating mucosal tissue [29]. Essential fatty acids are recognized for their role in regulating inflammatory cell function, facilitating fibroblast activity, and stimulating cytokine release patterns conducive to tissue repair [30].

In addition to thymoquinone, NSO contains a rich composition of bioactive constituents such as p‐cymene, carvacrol, 4‐terpineol, t‐anethole, and long‐chain fatty acids like linoleic acid, oleic acid, and palmitic acid. These compounds may synergistically enhance wound contraction. For instance, linoleic acid is known to promote keratinocyte migration and modulate inflammatory responses, while oleic acid supports membrane fluidity and fibroblast proliferation [31]. Furthermore, NSO's phytosterols and flavonoids possess antioxidant activity, which may reduce ROS that otherwise impair wound contraction [32]. Recent in vivo and in vitro studies suggest that these constituents can modulate fibroblast activity, enhance collagen matrix deposition, and accelerate granulation tissue formation—key events underlying effective wound contraction [33].

From a clinical standpoint, topical NSO might be beneficial for postoperative wounds such as palatal donor sites, oral mucosal biopsies, or alveolar ridge augmentation procedures where secondary healing is allowed or unavoidable. In the setting of oral surgeries involving large donor areas (e.g., free gingival grafts), additional discomfort and delayed healing can hinder patient recovery and affect surgical outcomes. NSO application could potentially reduce postoperative morbidity by accelerating epithelial closure, thereby minimizing patient discomfort and risk of secondary infection.

4.4. Study Limitations

While the study design was robust in terms of randomization, control, and the number of animals, a few limitations must be acknowledged:

1. Systemic Absorption: Despite our precautions, some NSO might have been ingested or absorbed into the bloodstream, especially given the well‐vascularized oral mucosa. Future studies could employ advanced local delivery systems (e.g., NSO‐infused gels or membranes) to minimize systemic distribution.

2. Quantification of Inflammatory Markers: We focused on TGF‐β and VGF due to their established relevance in wound healing; however, a broader analysis including markers such as TNF‐α, IL‐6, and IL‐1β could further elucidate the anti‐inflammatory properties of NSO in oral tissues.

3. Long‐Term Scar Quality: Although we assessed healing up to Day 20, oral wounds can continue remodeling beyond this time frame. Evaluations at extended intervals (e.g., 30 or 45 days) might reveal differences in scar tissue quality or mucosal integrity.

4. Translational Outlook: Rodent oral anatomy diverges somewhat from human oral anatomy; thus, clinical trials in human subjects are ultimately needed to confirm the safety, efficacy, and cost‐effectiveness of NSO‐based topical therapies for intraoral wounds.

5. Conclusion

In this study, topical NSO accelerated intraoral wound healing by reducing wound area and epithelial gap on postoperative Days 7 and 14. This effect appears to stem primarily from modulating TGF‐β expression and leveraging NSO's anti‐inflammatory, antimicrobial, and antioxidant properties. Although no significant changes were observed in VGF levels, the findings suggest that NSO promotes re‐epithelialization rather than directly affecting angiogenesis. Future clinical research is warranted to confirm these results and to optimize NSO application methods for oral wound management.

Ethics Statement

The study was approved by the Bağcılar Experimental Animals Local Ethics Committee with the approval number 2015/25. After obtaining ethical approval, surgical procedures, follow‐up, and sacrification procedures were conducted at Bağcılar Experimental Research Center. All procedures adhered to institutional and national guidelines for the care and use of laboratory animals.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.