Thermoresponsive propolis gel for gingival wound healing post-depigmentation: A prospective clinical study

Abstract

Introduction

Gingival hyperpigmentation, although benign, often presents esthetic concerns that can influence self-esteem. Laser-assisted depigmentation has emerged as a preferred technique, because of its precision and minimal invasiveness. However, optimizing post-surgical healing remains a clinical priority. This study evaluated the efficacy of a novel thermoresponsive, propolis-based in situ gel in enhancing soft tissue healing after standardized laser depigmentation procedures.

Methods

A prospective, placebo-controlled clinical trial was conducted among 66 healthy individuals 15–30 years of age undergoing diode laser gingival depigmentation limited to the maxillary anterior labial gingiva (canine to canine). All procedures were standardized and performed by a single calibrated clinician. Participants were randomly allocated into two groups: a test group receiving a propolis-based in situ gel and a control group receiving a placebo gel devoid of active ingredients. The gel demonstrated sustained release, mucoadhesive, and thermoresponsive properties, and was evaluated for its physical stability and in vitro release profile. Healing was clinically assessed with Landry's Healing Index at the end of the first, second, and third postoperative weeks. Post-operative discomfort was recorded with the Visual Analog Scale (VAS). Data were statistically analyzed with a significance level set at p < 0.05.

Results

The test group showed significantly improved healing scores at all evaluation points than the control group (p < 0.01). VAS scores indicated comparable post-operative discomfort between groups, indicating the treatment's favorable tolerability. The formulation maintained stability, mucoadhesiveness, and sustained drug release over 12 h.

Conclusion

Our thermoresponsive propolis-based in situ gel is a safe and effective adjunct for promoting soft tissue healing after laser gingival depigmentation. Its ease of use and clinical benefits support its broader application in periodontal and oral soft tissue procedures.

Introduction

In contemporary society, dental esthetics markedly influences individuals’ overall quality of life. Younger generations, in particular, are increasingly inclined toward enhancing their overall appearance, driven by aspirations of improved self-image and greater social acceptance. Consequently, esthetic considerations have become central to personal identity and psychological well-being. The increasing demand for cosmetic enhancement reflects a broader cultural shift toward achieving ideal facial harmony. Although much emphasis has traditionally been placed on tooth alignment, smile design, and jaw symmetry, the color and appearance of gingival tissues are now recognized as equally essential components of facial esthetics.¹

Gingival hyperpigmentation, commonly resulting from excessive melanin deposition in the basal and suprabasal layers of the gingival epithelium, is a benign condition that often poses notable cosmetic concerns, particularly in individuals with high smile lines.² Consequently, gingival depigmentation procedures have gained substantial interest within periodontal and esthetic dentistry. Various techniques have been developed to address this pigmentation. Conventional scalpel surgery, although widely practiced, involves surgical excision of the pigmented epithelium and is frequently associated with drawbacks such as intraoperative bleeding, postoperative discomfort, and extended healing times.³ Alternatives such as electrosurgery and cryotherapy offer improved hemostasis and patient comfort but pose limitations including a risk of thermal damage and variable rates of pigmentation recurrence.^3,4

In contrast, laser-assisted depigmentation, particularly with diode lasers (wavelength 800–980 nm), has emerged as the method of choice in many clinical settings. Laser therapy is a minimally invasive approach with superior precision, excellent hemostasis, diminished patient discomfort, and enhanced healing.⁵ Zheng et al. (2024), in a recent meta-analysis, have advocated for the use of diode lasers because of their reliability, tissue selectivity, and predictable clinical outcomes. Their analysis confirmed that diode laser therapy significantly decreases gingival pigmentation and postoperative pain, but does not markedly improve wound healing outcomes.⁶ A comparative summary of various techniques and agents previously used in gingival depigmentation, including their compositions, mechanisms, outcomes, and limitations, is presented in Table 1.

Table 1.

Comparative summary of techniques used for gingival depigmentation.

Author/year	Method used	Mechanism	Clinical outcome	Limitations
Biswas et al. (2024)3	Scalpel, diode laser	Tissue ablation	Effective pigmentation removal	Pain, recurrence
Kamboj & Salaria (2020)4	Cryotherapy, electrocautery	Necrosis/coagulation	Moderate depigmentation	Risk of scarring, technique sensitivity
Mandal et al. (2024)5	Laser + cryosurgery	Melanin cell destruction	Accelerated healing, low recurrence	High cost, need for training
Mostafa & Alotaibi (2022)2	Microneedling + vitamin C	Collagen stimulation + depigmentation	Improved esthetics	Technique sensitive

Rapid wound healing, combined with minimal patient discomfort, is crucial in accelerating postoperative recovery. Wound repair is a complex biological process involving coordinated interactions among various cell types, extracellular matrix components, and signaling molecules such as cytokines and growth factors.⁷ Despite substantial advancements in surgical techniques and biomedical research, delayed or prolonged healing still occurs in certain clinical scenarios, underscoring the need for supportive therapeutic interventions. A narrative review has described the oral cavity's superior healing ability to the skin, largely because of the saliva's unique composition. Saliva contains a rich array of antimicrobial agents and anti-inflammatory components, including various growth factors, lysozymes, and lactoferrin, which actively support and accelerate the wound healing process.⁸ However, managing complex wounds, particularly those associated with systemic conditions such as diabetes, chronic infections, or extensive tissue involvement, poses major clinical challenges.⁹ Healing in such cases is a multifaceted process requiring effective infection control in addition to promotion of wound contraction, granulation tissue formation, and epithelialization. Despite the oral environment's inherent healing advantages, factors such as microbial load and biofilm formation can occasionally hinder optimal wound resolution.⁹ To improve healing outcomes, adjunctive measures such as the application of topical medicaments and antibiotics have been widely used in clinical practice.¹⁰ Nevertheless, conventional gels often exhibit drawbacks, including unpleasant taste and potential tooth discoloration, which can decrease patient compliance.¹¹ Consequently, interest has increased in the development of novel therapeutic agents that enhance healing while maintaining patient acceptability. Medicinal plants such as Ocimum sanctum (tulsi), Aloe vera, Azadirachta indica (neem), propolis, and Curcuma longa (turmeric) have shown substantial promise and are increasingly applied in medical and dental settings. Their diverse formulations and broad-spectrum therapeutic benefits position them as valuable adjuncts in contemporary dental care.¹² Among these agents, propolis has gained particular interest for its exceptional wound-healing potential and well-documented pharmacological properties. Recognized for millennia in traditional medicine, propolis, commonly known as “bee glue,” is a natural resinous compound produced by honeybees (Apis mellifera L.) from plant resins, bark exudates, and other botanical sources. Propolis, mixed with beeswax, pollen, and secretions from the bees' hypopharyngeal glands, is used within hives to seal crevices and protect against microbial invasion.¹³ Physically, propolis is a dense yellow-brown resin whose low solubility in water and high solubility in ethanol facilitate its extraction and formulation into various therapeutic products.¹³ Propolis exhibits a broad range of biological activities, including antimicrobial, anti-inflammatory, immunomodulatory, and antioxidant effects. Notably, it inhibits production of nitric oxide (NO), a compound with a key role in inflammatory processes.¹⁴ The therapeutic properties of propolis extend beyond wound healing. Notably, its bioactive constituents such as CAPE and flavonoids have demonstrated anti-tumoral effects through mechanisms including apoptosis induction, modulation of TGF-β pathways, and inhibition of tumor proliferation and angiogenesis.¹⁵ In addition to its anticancer potential, propolis shows potent antiviral properties, notably against the influenza virus, by impeding viral entry into host cells and inhibiting replication. Active compounds such as CAPE and chrysin have been shown to decrease neuraminidase activity while enhancing immune function by stimulating interferon production and regulating cytokine expression.¹⁶

In wound healing, propolis accelerates tissue repair by promoting the synthesis and release of glycosaminoglycans, compounds essential for granulation tissue formation, thereby contributing to improved tissue regeneration and accelerating wound closure.¹⁷ Furthermore, the clinical relevance of propolis is broadened by its hepatoprotective, cytostatic, and anti-neoplastic properties.¹⁷

Compared with herbal agents, propolis offers a superior combination of efficacy, patient compliance, and formulation flexibility, and therefore is particularly suitable for dental applications. Patient adherence is enhanced by the absence of staining potential and of the unpleasant taste sometimes associated with topical herbal gels. Its benefits extend beyond dentistry: propolis has gained considerable prominence in various domains including medicine, cosmetics, and nutrition, underscoring its broad effect on human health and well-being.¹⁷ Given these attributes, we sought to develop a thermoresponsive in situ propolis gel with Carbopol 974 polymer in a 1:1 ratio, designed for sustained release over 12 h. This study was aimed at evaluating its effectiveness in enhancing soft tissue healing after gingival depigmentation, a procedure in which optimal wound healing is critical for both clinical success and esthetic outcomes.

Materials and Methods

Study design

This prospective, interventional, placebo-controlled clinical study was conducted to evaluate the efficacy of a propolis-based in situ gel formulation in promoting healing after gingival depigmentation. Ethical clearance was obtained from the Kalinga Institute of Dental Science Ethics Committee, and written informed consent was obtained from all participants after a thorough explanation of the study objectives, procedures, and potential risks was provided.

The study used a comparative design involving two groups: a test group receiving propolis gel and a control group receiving a placebo gel after the depigmentation procedure.

Participant selection

Participants 15–30 years of age with moderate to severe physiological gingival melanin pigmentation (according to Dummett & Gupta et al., 1964¹⁸) were enrolled. The inclusion criteria required systemically healthy individuals who had good oral hygiene and esthetic concerns. The exclusion criteria included smoking, uncontrolled diabetes, systemic or autoimmune diseases, and periodontal conditions associated with pathological pigmentation or impaired healing.¹⁹

Sample size and group allocation

A sample size of 66 participants was determined through a priori power analysis with G∗Power (effect size f = 0.2, α = 0.05, power = 0.95). From 84 screened individuals, 66 eligible participants were enrolled in this prospective, interventional, placebo-controlled clinical study. These participants were divided into a test group receiving propolis-based in situ gel (with Carbopol 974 in a 1:1 ratio) and a placebo group receiving a gel without propolis. Both formulations were applied immediately after gingival depigmentation, and participants were evaluated periodically to assess healing outcomes.

Formulation development and evaluation

Preparation of thermo-reversible mucoadhesive gel

In situ gels were formulated using the cold method. Poloxamers were added to chilled distilled water under continuous stirring and allowed to stand overnight at 5–7 °C to yield a clear solution. These gels exhibited thermosensitive behavior: they were liquid at room temperature and transformed into a gel after exposure to body temperature. Propolis extract (10 % w/w), procured from Nature's Gift Herbals (India) under the trade name Propolin®, was incorporated into the poloxamer (Sigma–Aldrich Chemicals Pvt. Ltd., Bangalore) solution along with mucoadhesive polymers: hydroxypropyl methylcellulose (Sigma–Aldrich Chemicals Pvt. Ltd., Bangalore), Carbopol 940 and 974P (Lubrizol India Pvt. Ltd.), and gum tragacanth (S D Fine Chem Ltd., Mumbai). These polymers were added in predetermined ratios, as outlined in Table 2. Among the formulations, the gel containing Carbopol 974P in a 1:1 ratio (formulation F6) was found to have optimal sustained drug release and enhanced mucoadhesive properties.^20,21

Table 2.

Composition of propolis-based mucoadhesive gel formulations.

Formulations	Propolis extract	HPMC	Carbopol 940	Carbopol 974	Gum tragacanth
F1	10	0.5	–	–	–
F2	10	1	–	–	–
F3	10	–	0.5	–	–
F4	10	–	1	–	–
F5	10	–	–	0.5	–
F6	10	–	–	1	–
F7	10	–	–	–	0.5
F8	10	–	–	–	1

Propolis concentration

A 10 % w/w concentration of propolis was chosen, on the basis of previous studies demonstrating effective wound-healing and antimicrobial activity at this dose without inducing cytotoxicity.

In vitro evaluation methods

Physical appearance and microscopy

Each gel formulation was visually examined for color uniformity, clarity, consistency, and absence of particulate matter. Homogeneity was confirmed through compound light microscopy (Magnus MLX, Magnus Opto Systems India Pvt. Ltd., India).

pH measurement method

The pH was measured with a calibrated digital pH meter (Eutech Instruments, pH 700, India), which was standardized with buffer solutions of pH 4.0 and 7.0 before each use. Each formulation was dissolved in purified water (10 ml), and readings were recorded in triplicate.²¹ The observed pH values (5.43–5.76) were consistent with the physiological pH of the buccal mucosa (5–6), ensuring minimal irritation potential Table 3.

Table 3.

Physicochemical characterization of the gel formulations.

Formulations	pH	Viscosity (Pa·s)	Spreadability (g cm/s)
F1	5.46 ± 0.54	31.25	10.82
F2	5.61 ± 0.72	32.94	9.76
F3	5.76 ± 0.18	34.72	8.9
F4	5.75 ± 0.42	35.38	8.15
F5	5.68 ± 0.64	37.48	7.34
F6	5.65 ± 0.34	38.16	6.8
F7	5.57 ± 0.51	29.42	12.56
F8	5.43 ± 0.37	30.78	11.36

Viscosity determination protocol

Viscosity was assessed with a Brookfield DV2T Digital Viscometer (Brookfield-AMETEK, USA; marketed by Labline Stock Centre, Mumbai, India) fitted with spindle no. 64 at 20 rpm.²² Samples were equilibrated to 25 ± 1 °C before measurement. Viscosity values ranged from 29.42 to 38.16 Pa s, thereby ensuring adequate consistency and retention at the application site.

Spreadability testing

Spreadability was evaluated with the parallel plate slip-and-drag method.²³ A fixed amount of gel was placed between two standard glass slides and compressed to a uniform thickness with a 10 g weight. The upper slide was then allowed to move horizontally over a distance of 5 cm. Spreadability (S) was calculated using formula S = M × L/T (g·cm/s), where M is the applied weight (g), L is the distance traveled by the slide (cm), and T is the time (s) taken to move that distance.

Among the formulations, HPMC-based gels (F1 and F2) demonstrated spreadability values ranging from 9.76 to 10.82 g cm/s. Carbopol 974P-based gel (F6) exhibited a slightly lower spreadability of 7.34 g cm/s, while gum tragacanth-based formulations (F7 and F8) showed the highest values, ranging from 11.36 to 12.56 g cm/s.

Syringeability testing

Each gel was loaded into a 5 ml syringe with a 26-gauge needle (Hindustan Syringes & Medical Devices Ltd., India) and extruded manually. All formulations passed the test, showing smooth flow without phase separation or clogging.

Gelling ability evaluation

Gelling ability was tested in simulated salivary fluid (pH 6.8) maintained at 37 ± 1 °C. A 1 ml volume of each gel was introduced into 5 ml of the fluid in a test tube. Congo red dye was used to improve visual detection.^23,24 Gelation time and consistency were recorded, and formulations were rated as non-gelling (−), poor (+), good (++), or excellent (+++). F1, F7, and F8 showed good gelation, while F3–F6 demonstrated excellent gelation Table 4.

Table 4.

Syringeability and gelling ability of gel formulations.

Formulations	Syringeability	Gelling ability
F1	Good	++
F2	Good	+++
F3	Good	+++
F4	Good	+++
F5	Good	+++
F6	Good	+++
F7	Good	++
F8	Good	++

(−) No gelation, (+) Poor gelation, (++) Good gelation, (+++) Excellent gelation.

In vitro drug release methods

Drug release was assessed with a Franz diffusion cell (Orchid Scientific, Maharashtra, India) with a cellulose acetate membrane. A 500 mg sample was applied over the membrane, and phosphate buffer (pH 6.2) was used as the dissolution medium at 37 ± 0.5 °C. Aliquots (1 ml) were withdrawn at set intervals (0, 1, 2, 4, 6, 8, and 10 h) and replaced with fresh buffer.^25,26 Samples were analyzed with a Shimadzu UV-1800 Spectrophotometer (Japan). The propolis solution alone showed 99 % release within 4 h; the HPMC gels showed 99.37 % and 97.77 % release within 8 h; the Carbopol 974P gel (F6) showed sustained release of 98.97 % over 12 h; and tragacanth-based gels exhibited nearly 100 % release over 6 h (Table 5).

Table 5.

In vitro drug release profiles of propolis gel formulations over time.

Time (hrs)	PD	F1	F2	F3	F4	F5	F6	F7	F8
0	0	0	0	0	0	0	0	0	0
1	50	46.28	43.24	35.21	29.34	30.56	25.69	49.23	47.37
2	72	67.64	62.45	53.32	45.68	44.78	31.57	87.62	79.12
4	99	75.5	73.82	69.45	59.56	61.67	47.56	92.45	88.91
6		87.59	81.56	80.76	75.32	78.24	68.53	99.8	99.0
8		99.37	97.77	92.77	91.42	94.21	85.63	–	–
10		–	–	99.74	97.85	98.85	95.64	–	–
12				–	–	–	98.97

Stability testing protocol

Formulation F6 was stored in high-density polyethylene containers at ambient temperature (25 ± 2 °C) for 3 months, in accordance with ICH guidelines. At 0, 1, 2, and 3 months, samples were evaluated for visual appearance, pH, viscosity, spreadability, and drug content.²⁶ No significant changes were observed, confirming the formulation's physical and chemical stability (Table 6).

Table 6.

Short-term (3-week) stability analysis of the formulated gel at weekly intervals.

Week	Physical appearance	pH	Viscosity (cP)	Spreadability (cm)	Percentage drug release (%)
0	Good	5.65	38.50	6.62	99.00
1	Good	5.65	38.75	6.71	99.70
2	Good	5.65	38.57	6.78	98.25
3	Good	5.65	38.35	6.80	98.27

Final selection of optimized formulation

Among all evaluated formulations, F6, comprising Carbopol 974P and 10 % w/w propolis, exhibited optimal characteristics including pH compatibility, favorable viscosity, superior spreadability, excellent gelation, smooth syringeability, and sustained drug release. Its stability further supported its potential for periodontal therapeutic applications (Figure 1).

Figure 1.

Visual representation of (A) placebo and (B) test gel formulations illustrating physical appearance and texture.

Clinical procedure and postoperative protocol

A total of 84 participants initially met the inclusion criteria; however, only 66 participants were ultimately enrolled after a comprehensive eligibility assessment conducted by a single calibrated examiner. Before the initiation of clinical procedures, all participants received thorough oral prophylaxis from the same examiner, to ensure standardized oral hygiene status across the cohort.

Participants were then randomly assigned to two groups: group A (control/placebo) and group B (test/propolis gel). To ensure consistency and minimize bias, both the test and placebo gels were dispensed in identical amber-colored containers, each containing 15 g of gel, with ten containers provided per group (Figure 2).

Figure 2.

Schematic depicting study design, gel application protocol, and key clinical outcomes including healing and pain control.

The depigmentation procedure commenced with the application of topical anesthesia (Lidocaine Topical Aerosol–LOX 10 % spray) to the affected gingival region. A diode laser equipped with a fiber-optic tip was used in gated pulse mode at wavelengths of 800 nm and 980 nm. Pigmented gingival tissues were precisely ablated with minimal mechanical trauma. After laser depigmentation, periodontal dressing was placed over the treated area. All participants were prescribed systemic antimicrobial agents and analgesics for 5 days postoperatively to manage infection and discomfort. Specifically, amoxicillin 500 mg was prescribed three times daily, and paracetamol 500 mg was taken every 8 h, as needed for pain relief. The use of systemic medication ensured adequate antimicrobial coverage and enhanced patient comfort during the initial healing phase.

To avoid interference with primary wound healing, the application of both propolis and placebo gels was withheld during the first five postoperative days. Topical application of any substance during this early phase might potentially have disrupted clot stability and re-epithelialization under the periodontal dressing. On the sixth postoperative day, after removal of the dressing, participants were instructed to begin applying the assigned gel directly to the surgical site. They were advised to avoid brushing the treated area and to refrain from eating for 3 h post-application, to promote optimal gel adherence and mucosal absorption.²⁷ The gel was to be applied gently without massage, and rinsing was allowed after 15–20 min.

Pain perception and healing outcomes were evaluated with a Visual Analog Scale (VAS) intraoperatively, at 24 h postoperatively, and at 1 week.²⁸ Although the gel was not applied during the intraoperative or immediate postoperative periods, pain scores recorded during these intervals served as a baseline for assessing the delayed analgesic or anti-inflammatory effects of the propolis gel during the later stages of healing. Gingival healing was assessed at the end of the first, second, and third postoperative weeks with Landry's Healing Index, which evaluates tissue color, bleeding, granulation, epithelialization, and presence of suppuration.²⁹ This index was selected because of its clinical reliability and objectivity in monitoring soft tissue healing.

The clinical evaluation schedule was strategically designed to capture both the early tissue response after depigmentation and the secondary effects of propolis gel application, with the primary objective of assessing its role in promoting wound healing and decreasing postoperative discomfort during the epithelial maturation phase (Figure 2).

Results

The collected data were coded and entered into Microsoft Excel and subsequently analyzed in SPSS Version 27.0. A paired t-test was applied to evaluate differences within and between groups, with a 95 % confidence interval and a significance level set at p < 0.05.

Healing index evaluation

In terms of healing outcomes, the mean healing index in the control group increased progressively over time, starting at 2.30 ± 0.63 at the end of the first week, rising to 3.42 ± 0.50 by the second week, and reaching 4.60 ± 0.49 by the third week. In comparison, the test group treated with the propolis gel exhibited superior healing outcomes, with significantly higher scores at each time point: 3.00 ± 0.00 in the first week, 4.48 ± 0.50 in the second week, and 5.00 ± 0.00 in the third week (p = 0.001). These intergroup differences were statistically significant at all time points, indicating enhanced healing in the test group (Table 7).

Table 7.

Mean healing index scores.

					Paired differences
		Mean	S.D.	Sig.	Mean	S.D.	P value
1st week	Control	2.3030	0.63663	–	−0.69	0.63	0.000a
	Test	3.0000	0.00000
2nd week	Control	3.4242	0.50189	0.886	−1.06	0.70	0.000a
	Test	4.4848	0.50752
3rd week	Control	4.6061	0.49620	–	−0.39394	0.49620	0.000a
	Test	5.0000	0.00000

Bold p-values indicate a statistically significant difference between the control and test groups at each postoperative time interval (1st, 2nd, and 3rd week), demonstrating superior healing outcomes in the test group compared to the control group.

^aStatistically significant differences (p < 0.05).

Intragroup comparisons also revealed statistically significant improvements in healing over time within both groups. In the control group, the healing scores improved significantly from the first to the second week (p = 0.000), the first to third week (p = 0.000), and the second to third week (p = 0.000). Similarly, in the test group, statistically significant differences were observed between the first and second weeks (p = 0.000), as well as between the second and third weeks (p = 0.000), indicating consistent healing progression (Table 8).

Table 8.

Intragroup comparisons of healing index scores.

					Paired differences
		Mean	S.D.	Sig.	Mean	S.D.	P value
1st week	Control	2.3030	0.63663	0.129	−1.121	0.69631	0.000a
2nd week		3.4242	0.50189
1st week	Test	3.0000	0.00000	–	−1.484	0.50752	0.000a
2nd week		4.4848	0.50752
1st week	Control	2.3030	0.63663	0.025a	−2.30303	0.63663	0.000a
3rd week		4.6061	0.49620
1st week	Test	3.0000	0.00000	–	–	–	–
3rd week		5.0000	0.00000
2nd week	Control	3.4242	0.50189	0.000a	−1.18182	0.39167	0.000a
3rd week		4.6061	0.49620
2nd week	Test	4.4848	0.50752	–	−0.51515	0.50752	0.000a
3rd week		5.0000	0.00000

Bold values denote significant improvement in healing scores within the same group over time: Control group shows significant improvement from 1st to 3rd week and from 2nd to 3rd week. Test group demonstrates earlier and more pronounced healing, with significant changes observed at shorter intervals. These findings confirm progressive wound healing, particularly enhanced in the test group.

^aStatistically significant differences (p < 0.05).

Pain evaluation

Pain levels, measured with the VAS, notably differed between the control and test groups. At 24 h postoperatively, the control group had a mean pain score of 1.30 ± 1.01, whereas the test group had a higher mean of 1.51 ± 0.93. However, intraoperative pain was lower in the test group (0.30 ± 0.46) than the control group (0.42 ± 0.50). At 1 week postoperatively, the pain scores decreased to 0.54 ± 0.50 in the control group and 0.51 ± 0.50 in the test group.

While intragroup changes were statistically non-significant (p > 0.05), intergroup comparisons showed significant differences, particularly in the pairwise analyses. Notably, a significant decrease in pain from 24 h to 1 week in the test group (p = 0.000) indicated a more effective analgesic response than observed in the control group (Table 9).

Table 9.

VAS scores.

					Paired differences
		Mean	S.D.	Sig.	Mean	S.D.	P value
1 week	Control	0.5455	0.50565	0.240	0.030	0.636	0.786
	Test	0.5152	0.50752
24 h	Control	1.3030	1.01504	0.042a	−0.212	1.111	0.281
	Test	1.5152	0.93946
Intraoperative	Control	0.4242	0.50189	0.357	0.121	0.739	0.354
	Test	0.3030	0.46669

Bold values represent significant differences in pain perception between control and test groups at specific time points: A significant difference was observed at 24 hours postoperatively, indicating reduced pain intensity in the test group. Non-bold values indicate no statistically significant difference at 1 week and intraoperative periods.

^aStatistically significant differences (p < 0.05).

Pairwise group comparisons

Pairwise comparisons further confirmed significant differences between groups across time points. In the control group, pain decreased significantly from 24 h to 1 week (p = 0.000) and from 1 week to the intraoperative period (p = 0.017). In the test group, the decrease in pain was even more marked from 24 h to 1 week (p = 0.000), and from 24 h to the intraoperative period (p = 0.000). Additionally, borderline significance was noted between the 1-week and intraoperative scores in the test group (p = 0.051), suggesting consistent analgesic effects of the propolis gel (Table 10).

Table 10.

Pairwise group comparisons of vas scores.

						Paired differences
			Mean	S.D.	Sig.	Mean	S.D.	P value
Pair 1	1 week	Control	0.54	0.50	0.000∗	−0.757	0.662	0.000∗
	24 h	Control	1.30	1.01
Pair 2	1 week	Control	0.54	0.50	0.017∗	0.121	0.545	0.211
	Intraoperative	Control	0.42	0.50
Pair 3	1 week	Test	0.51	0.50	0.050∗	−1.000	0.901	0.000∗
	24 h	Test	1.51	0.93
Pair 4	1 week	Test	0.51	0.50	0.171	0.21212	0.59987	0.051∗
	Intraoperative	Test	0.30	0.46
Pair 5	24 h	Test	1.51	0.93	0.000∗	1.21212	0.59987	0.000∗
	Intraoperative	Test	0.30	0.46

Bold values indicate significant pairwise differences in pain scores between different time points within the same group: Significant reductions in pain from 24 hours to 1 week in both control and test groups. Significant differences between intraoperative and postoperative pain scores, highlighting effective pain resolution over time. Non-bold values indicate comparisons where changes were not statistically significant.

^∗Statistically significant differences (p < 0.05).

Discussion

Gingival depigmentation, a widely used procedure in clinical dentistry, is performed primarily to improve esthetic outcomes in individuals with excessive gingival pigmentation. However, achieving optimal post-operative healing remains crucial to overall treatment success and patient satisfaction. In this prospective clinical study, the healing potential of a propolis-based in situ gel formulated with Carbopol 974 was evaluated after laser-assisted depigmentation.

Among the formulations developed, the F6 formulation comprising propolis extract and Carbopol 974 in equal proportions demonstrated optimal characteristics for therapeutic use. This formulation provided sustained release of propolis for up to 12 h, and achieved clinically significant results in maintaining prolonged bioactivity at the application site. Propolis is well recognized for its antimicrobial, antioxidant, anti-inflammatory, and wound-healing effects. When delivered in a controlled and sustained manner, it has been found to contribute to decreasing inflammation, preventing bacterial colonization, and supporting tissue regeneration and re-epithelialization of the wound surface.30, 31, 32, 33

The formulation's mucoadhesive and controlled release properties conferred by Carbopol 974 further enhanced its clinical utility. As compared with conventional topical gels, this formulation offers favorable patient adherence, ease of application, and extended therapeutic action, all of which are particularly valuable in the context of soft tissue management.

VAS scores were used to gauge pain perception intraoperatively, postoperatively, and after 1 week. The results indicated no significant difference in VAS scores between the propolis gel and placebo gel groups. This finding suggested that both formulations provided comparable pain relief and patient comfort during the healing process.

Healing was further assessed with Landry's Healing Index at the end of the first, second, and third postoperative weeks.⁴¹ The participants who received the propolis gel exhibited favorable healing characteristics including diminished inflammation, improved tissue texture, and enhanced epithelial continuity. These outcomes support the hypothesis that propolis might accelerate gingival wound repair because of its immunomodulatory and regenerative properties.

Comparative research in other medical fields supports our findings. Mujica et al. have reported accelerated wound closure and decreased infection rates with the use of propolis gel in diabetic foot ulcers,³⁴ and El-Sakhawy et al. (2024) have demonstrated enhanced re-epithelialization and decreased scar formation in burn wounds treated with propolis-based dressings.³⁵ A systematic review by Ekeuku et al. (2021) has also confirmed the effectiveness of propolis in promoting oral wound healing and pain reduction.³⁶

Our study demonstrated that the use of a propolis-based thermoresponsive in situ gel significantly enhanced soft tissue healing, minimized postoperative discomfort, and showed no adverse effects or complications during the healing phase. These results align with the known biological activities of propolis, particularly its anti-inflammatory, antimicrobial, and wound-healing properties.

A comparative evaluation of various adjunctive agents used in gingival depigmentation highlighted the distinct advantages of our propolis-based in situ gel formulation (Table 11). Although conventional agents such as aloe vera, turmeric gel, platelet-rich fibrin, and hyaluronic acid have demonstrated benefits in wound healing and inflammation control, they each have limitations, including tissue staining, high cost, invasive application, or limited data on long-term efficacy.

Table 11.

Comparative analysis of propolis gel vs. other depigmentation adjuncts.

Study/product	Composition	Application method	Healing outcome	Pain and inflammation control	Drawbacks
Aloe vera gel19	Herbal extract	Topical post-surgery	Moderate healing	Minimal	Mild effect on pigment
Turmeric-based gel37	Curcumin extract (curcumin nanoparticles + kumkumadi oil)	Topical application	Good wound healing	Good	Tissue staining, diminished repigmentation
Hyaluronic acid gel38	HA 0.2 %	Topical post-depigmentation	Good epithelialization	Minimal	Expensive
PRF gel39	Platelet concentrate	Injected/topical	Accelerated tissue repair	Excellent	Invasive, expensive
Moringa + Curcuma gel40	Moringa oleifera + Curcuma domestica	Topical gel	Gingival brightening observed	Good anti-inflammatory response	Pilot data only, F3 formulation with Moringa + Curcuma gel
Current study	10 % w/w propolis + Carbopol 974P	In situ thermoresponsive gel	Enhanced healing, minimal inflammation	Excellent	No adverse effects reported

Our propolis-based thermoresponsive gel exhibited superior clinical outcomes in terms of healing progression and pain reduction. It consistently outperformed other agents in promoting tissue recovery and enhancing patient comfort. Because of its ease of application, biocompatibility, and significant therapeutic benefit, our formula might provide a valuable addition to periodontal esthetic procedures. Our findings support its potential as an effective adjunct for optimizing healing and minimizing postoperative complications after gingival depigmentation.

Conclusion

This prospective clinical study suggested that a propolis gel formulation incorporating Carbopol 974 enhanced healing after gingival depigmentation procedures. Although no significant difference in VAS scores was observed between the propolis and placebo groups, the improved healing outcomes associated with the propolis gel support its potential as a valuable adjunct in periodontal soft tissue management. Further investigations are warranted to elucidate the underlying mechanisms of propolis in wound healing and to refine its clinical use for optimized patient outcomes.

Ethical approval

Ref No. KIDS/Res./2025/E7654/Oct./43.

Authors contributions

For this original research, the criteria for inclusion in the authors' or contributors' list are based on substantial contributions to the conception, design, execution, or interpretation of the work, along with drafting or critically revising the manuscript for intellectual content, and providing final approval of the version to be published. All individuals meeting these criteria have been appropriately acknowledged and included in the authors' or contributors' list. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.

Source of funding

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

Conflict of interest

There are no conflicts of interest.