An antioxidant and antibacterial octyl gallate sustained-release ointment for periodontitis treatment

Summary

Periodontitis is a chronic inflammatory disease that leads to periodontal attachment loss and is a major cause of adult tooth loss. Current treatment of mechanical debridement is often ineffective in deep periodontal pockets, and antibiotic adjuncts carry the risk of bacterial resistance. Octyl gallate (OG), a natural plant-derived compound with antibacterial and antioxidant properties, is limited by poor water solubility and stability. This study aimed to develop a sustained-release ointment (OG-Ointment) using Pluronic F127 and ethylcellulose to enhance the delivery and efficacy of OG. The formulated OG-Ointment demonstrated excellent rheological properties, sustained drug release, and potent ABTS⁺ radical scavenging. In vitro, it enhanced bacterial membrane permeability, inhibited P. gingivalis growth, and reduced ROS in macrophages. In a rat periodontitis model, OG-Ointment significantly reduced alveolar bone resorption, decreased ROS and inflammatory cytokines, and exhibited good biocompatibility. These findings suggest that OG-Ointment is a promising non-antibiotic adjunctive therapy for periodontitis, combining dual antioxidant and antibacterial functions with sustained release.

Graphical abstract

Highlights

• •A non-antibiotic ointment (OG-Ointment) is developed for periodontitis treatment
• •Pluronic F127/EC matrix provides sustained drug release and enhances its stability
• •OG-Ointment possesses potent antibacterial and ROS-scavenging efficacy
• •OG-Ointment alleviates alveolar bone resorption and inflammation in vivo

Molecular biology

Introduction

Periodontitis is a chronic inflammatory disease related to microorganisms, mediated by the host, and leads to the loss of periodontal attachment.¹ It has now become the sixth most prevalent epidemic worldwide and is the primary cause of tooth loss among adults.^1,2,3 Research indicates that dental plaque bacteria are the initial causative agents of periodontitis.⁴ Lipopolysaccharides (LPS) on the surface of periodontal pathogens and virulence factors such as gingipains released into the body exhibit strong toxic effects on periodontal tissues and lead to bone resorption. Plaque and its toxic products excessively stimulate the host’s immune response, causing damage to periodontal tissues.⁵ Oxidative stress plays a significant role in this process. At sites of periodontal lesion, neutrophils and macrophages phagocytose bacteria and release lysosomal enzymes or superoxide ions to kill bacteria, producing excess reactive oxygen species (ROS) that disrupts the balance of ROS. Excessive ROS not only sustains a pro-inflammatory microenvironment, perpetuating a vicious cycle of inflammation, but also induces cellular lipid peroxidation, protein/DNA damage, and impaired tissue regeneration.^6,7 Virulence factors released by periodontal pathogens can enter the systemic circulation from the epithelial cells of the periodontal tissues through blood vessels, affecting the host’s overall health. Increasing clinical and experimental evidence suggests that periodontitis is closely associated with the progression of cardiovascular diseases, type II diabetes, Alzheimer’s disease, and oral squamous cell carcinoma.^8,9 Therefore, eliminating pathogenic bacteria, removing excess ROS, and reducing excessive inflammation are key to blocking the progression of periodontitis.

Currently, dental plaque is mainly removed through mechanical means assisted by antibiotics in clinical settings.¹⁰ However, due to the complex physiological structure inside the mouth, instruments cannot reach deep periodontal pockets or furcation areas, leading to poor responses to mechanical treatment in some patients.¹¹ Additionally, the effectiveness of mechanical treatment greatly depends on the clinician’s skill and patient compliance, making treatment outcomes hard to control. To improve these shortcomings, many adjunctive therapies have been introduced into periodontal treatment, such as systemic or local administration of antibiotics and nonsteroidal anti-inflammatory drugs. However, most antibiotics pose issues with bacterial resistance, and the use of anti-inflammatory drugs may carry risks such as gastrointestinal bleeding, with long-term use likely leading to adverse reactions such as liver and kidney damage.¹² These challenges highlight the urgent need for non-antibiotic alternatives that can bypass microbial resistance while providing sustained therapeutic effects. Thus, developing a safe and effective non-antibiotic method for the adjunctive treatment of periodontitis is very important.

Octyl gallate (OG), a derivative of gallic acid, is inexpensive and has low extraction costs. It has become a recognized safe food additive that effectively prevents the deterioration of nutrients in food.^13,14,15 Meanwhile, the unique phenolic hydroxyl groups and fatty chains in its molecular structure endow OG with excellent antioxidant activity, tissue adhesion, and bacterial membrane penetration abilities, suggesting that OG preparations may have broad prospects in the pharmacotherapy of periodontitis. However, OG’s reactive nature makes it susceptible to environmental influences during antibacterial, anti-inflammatory, and antioxidant processes, making it difficult to maintain an effective concentration at the lesion site. Since OG itself is hydrophobic, direct drug loading is challenging, limiting its application range in pharmaceutics and preventing it from fully demonstrating its excellent antibacterial and antioxidant properties.

Pluronic F127, a novel polymeric nonionic surfactant, can enhance the solubility of hydrophobic drugs and rapidly form a gel upon contact with water, adhering to the administration site in the body.^16,17 It slowly releases the drug, enhancing local drug concentration. With its excellent biodegradability, low toxicity to the organism, low irritancy, and good biocompatibility, it is an outstanding sustained-release material.^18,19,20 Ethylcellulose (EC) is non-irritating, insoluble in water but soluble in organic solvents, forming a water-insoluble film when used orally, and serving as a matrix material in sustained-release ointments.^21,22,23 It can increase the stability of the ointment as a hydrophobic polymer plasticizer within the sustained-release ointment system.²⁴ The combination of these two materials could address OG’s limitations, yet no study has integrated OG with Pluronic F127/EC to develop a dual-active sustained-release formulation for periodontitis.

Rationale and Objectives: Based on the above gaps, this study aims to: 1) prepare an OG-loaded sustained-release ointment (OG-Ointment) using Pluronic F127 as the gel matrix and EC as the sustained-release barrier; 2) characterize its physicochemical properties, such as rheology, drug release profile, and antioxidant activity; 3) evaluate its in vitro antibacterial efficacy against P. gingivalis and ROS-scavenging capacity in macrophages; and 4) validate its in vivo therapeutic effect and biosafety in a rat periodontitis model, with a focus on alveolar bone protection and inflammation reduction. This work intends to provide a non-antibiotic adjunctive strategy for periodontitis that integrates dual activity and sustained release (Scheme 1).

Scheme 1.

Schematic illustration of the anti-periodontitis mechanism of OG-Ointment

Results and discussion

Construction and characterization of octyl gallate-Ointment

The sustained-release ointment base is prepared by blending F-127 with GT, dissolving them under specific temperatures, and then adding EC for high-temperature processing. OG-Ointment is subsequently formulated by equally mixing this base with a carefully prepared OG solution, followed by thorough grinding and centrifugation to remove air (Figure 1A). As illustrated, the prepared ointment itself does not exhibit any absorption peaks within the range of 200–800 nm (Figure 1B). However, both the UV spectra of OG and OG-Ointment display two similar absorption peaks at 205 nm and 277 nm, which demonstrate that OG was successfully incorporated into the ointment matrix. Persistent infection leads to the excessive local accumulation of ROS, and the activated immune system also generates a substantial amount of ROS. Oxidants convert ABTS to ABTS⁺, while the presence of antioxidants inhibits ABTS⁺ formation.²⁵ Therefore, by detecting the amount of ABTS+ at the OD value of 734 nm, the total antioxidant capacity can be calculated.²⁶ Due to its pyrogallol structure, the OG monomer possesses excellent antioxidant properties. Consequently, the antioxidant efficiency of OG-Ointment was evaluated through the ABTS⁺ radical scavenging experiment in this study. The scavenging rate of ABTS⁺ increases with increasing drug concentration (Figure 1C). When the OG-Ointment concentration reaches 600 μg/mL, it can scavenge 99% of ABTS+ radicals. Rheological experiments were conducted to investigate the changes in G′ and G'' of OG-Ointment under normal conditions. It was found that the storage modulus of OG-Ointment was greater than the loss modulus (Figures 1E and 1F). The same rheological measurements were performed on the OG-Ointment that had been allowed to absorb water and stand for two days. It was observed that the storage modulus of the water-absorbed and swollen OG-Ointment significantly increased and remained stable with increasing amplitude, while the loss modulus gradually decreased with increasing amplitude (Figures 1G and 1H). For the release experiments in this project, a Franz diffusion cell was chosen as the experimental setup. By separating the drug with a semi-permeable membrane of a specific pore size, only small-molecule drugs dissolved in PBS can pass through the membrane, allowing for the calculation of the cumulative drug release (Figure 1I). OG-Ointment exhibited a burst release within the first 12 h, followed by a more gradual release thereafter. By the 9th day, the total release amount reached 96%. Additionally, visual photographs were taken at different release time points to observe the overall changes in the OG-Ointment. Before the release experiment, the drug appeared as a compact and uniform milky white ointment (Figure 1J). The burst release phase is attributed to the rapid dissolution of OG on the ointment surface and its diffusion through the Pluronic F127 gel network, which meets the requirement of “quickly reaching the therapeutic concentration” during the acute phase of periodontitis. In contrast, the sustained slow-release phase relies on the hydrophobic barrier effect of ethylcellulose (EC)—the network formed by EC impedes the further diffusion of OG, enabling continuous drug release.²⁰ This release profile highly matches the therapeutic needs of periodontitis, namely “rapid bacteriostasis in the acute phase plus sustained anti-inflammation in the chronic phase.” Upon the addition of the release medium (PBS), the ointment gradually absorbed water and swelled into a translucent gel. As the release experiment progressed, bubbles were observed within the OG-Ointment, indicating the dissolution of its internal structure with the continuous replacement of the release medium. By the 9th day, it was observed that the OG-Ointment had almost completely dissolved.

Figure 1.

Construction and characterization of OG-Ointment

(A) Schematic illustration for the preparation of OG-Ointment.

(B) UV absorption peaks of OG, ON, and OG-Ointment between 200 and 800 nm.

(C) Radical scavenging capacity of ATBS⁺ at different concentrations of OG-Ointment. Data are mean ± SD (n = 3 independent experiments).

(D) Corresponding color changes of different groups of ATBS⁺ solutions.

(E) OG-Ointment in static state and (F) G′ and G’’.

(G) OG-Ointment after water absorption and swelling, and (H) G′ and G’’.

(I) Release curve of OG-Ointment. Data are mean ± SD (n = 3 independent experiments).

(J) Changes during the drug release process of the OG-Ointment. Scale bars: 0.5 mm.

Evaluation of the cytotoxicity and in vitro hemolytic effect of octyl gallate-Ointment

The excellent compatibility of bioactive materials is crucial for ameliorating the inflammatory environment of periodontal tissues. To investigate the cytocompatibility of the ointment, we conducted CCK8 assays to assess the toxicity of OG-Ointment toward NIH-3T3 and RAW264.7 cells, thereby providing an initial evaluation of its biosafety. The survival rates of both NIH-3T3 and RAW264.7 cells remained close to 100% after 24 and 48 h of coincubation with OG-Ointment at various concentrations (Figures 2A and 2B). Subsequently, we examined the erythrocyte hemolysis induced by different concentrations of both the Ointment and OG-Ointment solutions. Neither the Ointment nor the OG-Ointment solutions exhibited significant hemolysis at any concentration tested, with hemolysis rates consistently below 5% (Figures 2C and 2D). This confirms the good blood compatibility of OG-Ointment.

Figure 2.

Evaluation of the cytotoxicity and in vitro hemolytic effect of OG-Ointment

(A) Viability of RAW264.7 cells after treatment with different concentrations of OG-Ointment for 24 and 48 h. Data are mean ± SD (n = 3). No statistically significant differences were found compared to the control group (p > 0.05, one-way ANOVA).

(B) Viability of NIH3T3cells after treatment with different concentrations of OG-Ointment for 24 and 48 h. Data are mean ± SD (n = 3). No statistically significant differences were found compared to the control group (p > 0.05, one-way ANOVA). Data are mean ± SD (n = 3).

(C) Hemolytic properties of Ointment base at various concentrations.

(D) Hemolytic properties of OG-Ointment at various concentrations. Data are mean ± SD (n = 3).

In vitro antibacterial effects of octyl gallate-Ointment

P. gingivalis, as a core periodontal pathogen, possesses the ability to disrupt the oral microbiota balance. It can invade gingival epithelial cells, periodontal ligament fibroblasts, osteoblasts, and immune cells,²⁷ and induce the production of pro-inflammatory cytokines, thereby damaging host tissues and interfering with the host’s immune system.²⁸ Additionally, P. gingivalis can utilize inflammatory exudates to provide nutrients for other commensal pathogens, further affecting the oral commensal flora, disrupting the flora balance, and promoting the development of periodontitis, which may ultimately lead to alveolar bone resorption and tooth loss.²⁹ The natural plant-derived Chinese medicinal monomer OG has a unique structure, featuring a long alkyl chain that can penetrate bacterial cell membranes, disrupt their structure, and enhance their permeability to kill pathogenic bacteria. NPN emits blue fluorescence in non-aqueous environments and phospholipids. It cannot cross an intact bacterial outer membrane in its free state, but when the outer membrane is damaged, NPN can enter the phospholipid layer of the bacterial outer membrane, resulting in enhanced blue fluorescence.³⁰ Therefore, changes in fluorescence intensity can be used to verify the bacterial membrane penetration of drugs.³¹ The fluorescence spectra of NPN after the co-incubation of various groups with P. gingivalis indicate that the fluorescence intensity is low in the PBS group, while the OG-Ointment group exhibits a higher NPN fluorescence signal (Figure 3A). This phenomenon can be explained by the molecular structural characteristics of OG: the long alkyl chain (C8) of OG can insert into the hydrophobic core region of the P. gingivalis cell membrane, disrupting the order of lipid bilayer arrangement and increasing membrane permeability. This allows NPN to enter and bind to phospholipids, thereby emitting fluorescence. After 24 h of incubation with P. gingivalis, bacterial activity decreased with increasing concentrations of OG-Ointment. When the concentration of OG-Ointment reached 2.5 mg/mL, the bacterial survival rate was only 21.9% (Figure 3B). The effect of OG-Ointment on bacterial growth density was evaluated through plate inhibition experiments. The blank group exhibited dense and numerous black colonies; the Ointment group was ineffective in inhibiting colony formation, whereas in the OG-Ointment group, colony numbers were almost invisible (Figure 3C). This indicates that OG-Ointment can efficiently inhibit the colony growth of P. gingivalis.

Figure 3.

In vitro antibacterial effects of OG-Ointment

(A) Results of the NPN experiment.

(B) Bactericidal effects of ON and OG-Ointment at different concentrations. Data are mean ± SD (n = 3), ∗∗∗∗p < 0.0001 indicate comparisons between two sets of data, independent samples t test.

(C) Effects of sustained-release ointment containing OG and without OG on P. gingivalis colony formation. Scale bars: 1 cm.

In vitro antioxidant effects of octyl gallate-Ointment

ROS generated in an inflammatory environment can induce intracellular oxidative stress, leading to cell death.^32,33 Macrophages, as the second line of defense against pathogens in the periodontal tissues of the immune system, protect the body by recognizing foreign pathogens, killing target cells, presenting antigens, and regulating tissue immune responses.³⁴ However, excessive activation can lead to local oxidative stress and exacerbate inflammatory responses, creating a vicious cycle that further aggravates tissue damage. Therefore, a periodontal local sustained-release system with antioxidant properties can improve oxidative stress in inflammatory tissues and reduce inflammatory responses. LPS plays a crucial role in inducing inflammatory responses and contributing to various inflammatory diseases.^35,36 It is a compound found in the cell wall of Gram-negative bacteria. When LPS attacks host tissues, receptors on macrophages recognize and bind to it, leading to the activation of NADPH oxidase in macrophages.³⁷ This enzyme transfers electrons from intracellular NADPH to extracellular oxygen molecules, reducing them to superoxide anions and further increasing ROS generation. Excessive release can cause extensive tissue damage and pathological changes.³⁸ Therefore, LPS can be used as an endogenous stimulus to induce RAW 264.7 cells to produce excessive ROS. Furthermore, H₂O₂, as one of the main components of ROS, is also a widely used inducer of oxidative stress. H₂O₂ can easily penetrate various cell membranes, causing significant lipid peroxidation, protein damage, and DNA breakage, ultimately leading to cell death and dysfunction.³⁹ Therefore, in this study, H₂O₂ was directly used as an exogenous stimulus to induce RAW 264.7 cells to produce ROS.

OG, with its pyrogallol structure, exhibits prominent antioxidant effects, effectively improving oxidative stress in the inflammatory environment of periodontitis and regulating inflammation. In this chapter, both exogenous and endogenous stimuli were used to induce oxidative stress in macrophages by stimulating them with H₂O₂ and LPS, respectively. Treatment was then administered using OG-Ointment dissolved in cell culture medium. ROS generation was detected using the DCFH-DA fluorescent probe. Regardless of whether ROS was directly generated by stimulating macrophages with H₂O₂ or through macrophage polarization induced by LPS stimulation, the fluorescence intensity in the OG-Ointment group was significantly lower compared to the positive control group, with statistically significant differences in semi-quantitative analysis (Figures 4A–4D). This demonstrates that OG-Ointment has a significant scavenging effect on excessive ROS in macrophages stimulated by both methods, thereby improving the cellular environment.

Figure 4.

In vitro antioxidant effects of OG-Ointment

(A) Confocal images and (B) corresponding semi-quantitative analysis of the inhibitory effects of different experimental groups on ROS production in RAW264.7 cells stimulated by LPS. Data are means ± SD (n = 3), ∗∗∗∗ indicates p < 0.0001 compared to the control group, #### indicates p < 0.0001 when comparing between two groups, one-way ANOVA. Scale bars: 10 μm.

(C) Confocal images and (D) corresponding semi-quantitative analysis of the inhibitory effects of different experimental groups on ROS production in RAW264.7 cells stimulated by H₂O₂. Data are means ± SD (n = 3), ∗∗∗∗ indicates p < 0.0001 compared to the control group, #### indicates p < 0.0001 when comparing between two groups, one-way ANOVA. Scale bars: 10 μm.

The pivotal role of oxidative stress in periodontitis extends beyond a mere consequence of inflammation, and it represents a core driver of the disease’s progression. The evolution of oxidative stress in periodontitis follows a vicious cycle: 1) Initiation: Bacterial pathogens and their metabolites activate host immune cells such as neutrophils and macrophages, triggering a respiratory burst and generating a surge of ROS as a primary defense mechanism.⁴⁰ 2) Amplification and Sustenance: In a dysregulated inflammatory environment, this ROS production becomes excessive and uncontrolled. The overproduced ROS not only fails to eliminate pathogens effectively but also damages surrounding periodontal tissues—including gingival fibroblasts, periodontal ligament cells, and osteoblasts—by inducing lipid peroxidation, protein dysfunction, and DNA damage.⁴¹ This tissue damage, in turn, releases more damage-associated molecular patterns, further priming immune cells and perpetuating a pro-inflammatory feedback loop that sustains high ROS levels. 3) Progression and Systemic Linkage: The persistent oxidative stress contributes significantly to the degradation of collagen fibers, activation of osteoclasts leading to alveolar bone resorption, and ultimately, the clinical manifestation of periodontitis.^42,43 Furthermore, the systemic spillover of inflammatory and oxidative mediators from the periodontal pocket is a proposed mechanism linking periodontitis to other chronic conditions such as diabetes and cardiovascular diseases.^44,45 Therefore, an effective therapeutic strategy must break this vicious cycle. Our findings that OG-Ointment potently scavenges ROS in both exogenously and endogenously stimulated macrophages position it as a promising agent to intervene at the “Amplification” stage of this oxidative stress evolution. By neutralizing excessive ROS, OG-Ointment is expected to mitigate subsequent tissue damage and dampen the pro-inflammatory feedback, thereby arresting the disease’s progression.

In vivo therapeutic effect of octyl gallate-Ointment against periodontitis

Reactive oxygen species (ROS) play a crucial role in cell signaling, gene regulation, and antibacterial defense mechanisms within the periodontal inflammatory environment.^46,47 However, when ROS levels exceed the normal range, they trigger oxidative stress within the tissues. This stress state further promotes pathological changes, leading to the increased destruction of periodontal tissues.⁴⁸ Studies have confirmed that patients with more severe periodontitis exhibit higher levels of ROS in their gingival crevicular fluid and saliva. To evaluate the efficacy of OG-Ointment in scavenging ROS and treating inflammation in vivo, we established a rat periodontitis model using ligature wire and assessed periodontal damage through visual inspection and Micro-CT imaging.⁴⁹ After four weeks of modeling, the bilateral second molar gingiva exhibited redness, swelling, and mild bleeding (Figure 5A). Collectively, these findings confirm the successful establishment of the rat periodontitis model involving the bilateral second molars in the maxilla.

Figure 5.

In vivo therapeutic effect of OG-Ointment on periodontitis Evaluation

(A) Schematic diagram of the in vivo experimental design and procedure.

(B) Three-dimensional and two-dimensional Micro-CT images of rat alveolar bone after different treatments. Scale bars: 1 mm (C) Results of ROS immunofluorescence staining in each group. Scale bars: 200 μm (D). Corresponding semi-quantitative fluorescence analysis, (E) CEJ-ABC distances in rats from different treatment groups obtained using CT An software, and (F) BV/TV values in rats from different treatment groups obtained using CT Vox software. Data are means ± SD (n = 5), ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 compared to the periodontitis group, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 indicate comparisons between two sets of data, one-way ANOVA.

(G) H&E staining of alveolar bone and gingiva in each group. Scale bars: 100 μm.

(H) Immunohistochemical staining of TNF-α and IL-1β in periodontal tissues in each group. Scale bars: 100 μm.

Using CT Vox software to reconstruct three-dimensional images and Data Viewer software to reconstruct alveolar bone tomographic images, significant alveolar bone resorption and widened periodontal ligament spaces were observed at the site of the second molar in the periodontitis group (Figure 5B). In contrast, rats in the OG-Ointment group exhibited less alveolar bone resorption, comparable to that in the minocycline group, indicating its efficacy in treating rat periodontitis and inhibiting alveolar bone resorption.

In previous experiments, we demonstrated the excellent in vitro ROS scavenging performance of OG-Ointment. In this part, we further investigate its ROS inhibitory capability in vivo. Compared to the normal control group, the ROS fluorescence intensity was markedly enhanced in the periodontitis group, and there was no significant difference in ROS fluorescence intensity between the group treated with the ointment base alone and the periodontitis group, indicating that the ointment base alone does not reduce ROS levels in animal models of periodontitis (Figures 5C and 5D). Conversely, the OG-Ointment group exhibited a significant reduction in red ROS fluorescence intensity, which was even lower than that observed in the minocycline group, with statistical significance. These results suggest that OG-Ointment effectively reduces ROS levels in the gingival tissues of rats with periodontitis, outperforming minocycline ointment.

To further quantify alveolar bone resorption across different groups, we measured the alveolar bone crest-cementoenamel junction (ABC-CEJ) distance. Compared to the periodontitis group, treatment with OG-Ointment significantly reduced alveolar bone resorption, outperforming the ointment base alone group with statistical significance (Figure 5E). The effect of OG-Ointment was comparable to that of minocycline, with no statistically significant difference between the two groups. These measurement results are consistent with the 3D imaging findings. Additionally, assessing changes in bone volume fraction (BV/TV) in rats provides further insights into the therapeutic effects of each group (Figure 5F). The trend in therapeutic efficacy aligns with the results. Rats with periodontitis treated with OG-Ointment exhibited the highest BV/TV values in their alveolar bone, approaching normal levels.

Hematoxylin and eosin (H&E) staining of periodontal tissues in the normal control group revealed no significant inflammation, bleeding, or abnormal fiber distribution, with adequate alveolar bone height (Figure 5G). In contrast, rats in the periodontitis group exhibited mild bleeding, loosely and disordered fiber distribution, inflammatory cell infiltration, and significant alveolar bone resorption, indicating acute and widespread inflammation in the gingival tissues. After various treatments, rats in the ointment base alone group exhibited incomplete gingival epithelial tissue, a prominent keratinized layer, and localized bleeding, suggesting severe periodontal inflammation with no significant improvement. The OG-Ointment group demonstrated the best inhibitory effect on periodontitis, with relatively normal gingival layers and no significant inflammatory cell infiltration, similar to the positive control group.

Immunohistochemical staining was used to detect the expression of inflammatory cytokines IL-1β and TNF-α in the periodontal tissues of rats across different groups.^37,50 the periodontitis group exhibited extensive brown or dark brown staining in the periodontal tissues, indicating high expression levels of TNF-α and IL-1β and a high degree of inflammation (Figure 5H). Similar results were observed in the ointment base alone group, with abundant dark brown staining for TNF-α and IL-1β, suggesting that the ointment base alone does not alleviate inflammation. In contrast, the OG-Ointment group exhibited minimal dark brown staining, indicating a significant decrease in the expression levels of both inflammatory cytokines, comparable to the minocycline group. These findings suggest that OG-Ointment effectively controls inflammation in the periodontal tissues of rats. Finally, to further investigate the in vivo biosafety of the ointment, we collected the major organs (heart, liver, spleen, lungs, and kidneys) from mice in each group for H&E staining (Figure 6). The major organs in all treated groups were comparable to those in the normal control group, with no apparent pathological changes observed. Moreover, the hematological and blood biochemical analysis of SD rats after receiving various treatments revealed the absence of significant differences (Figures S1A–S1F). The observations validated the overall safety of OG-Ointment.

Figure 6.

In vivo biocompatibility evaluation of OG-Ointment

H&E staining images of the heart, liver, spleen, lungs, and kidneys in rats from each group after in vivo experiments. Scale bars: 100 μm.

Collectively, the promising results of OG-Ointment have enriched and improved the current landscape of non-antibiotic adjunctive therapies for periodontitis. Current strategies largely focus on single-pathway interventions, such as the use of metal nanoparticles (e.g., silver, zinc oxide) for their broad-spectrum antibacterial properties with potential cytotoxicity concerns,^51,52 host modulation therapies (e.g., sub-antimicrobial dose doxycycline) that primarily inhibit matrix metalloproteinases to control tissue destruction but lack direct antibacterial efficacy,⁵³ and natural plant extracts (e.g., curcumin and catechins) which possess antioxidant and anti-inflammatory activities but often suffer from poor stability and limited sustained-release profiles.⁵⁴ In contrast, the OG-Ointment platform developed here offers a multi-faceted and synergistic approach. It uniquely combines several functions. Firstly, it exerts direct antibacterial action against keystone pathogens such as P. gingivalis through membrane permeabilization, which is a mechanism distinct from antibiotics and minimizes resistance risk. In addition, the OG-Ointment platform has potent antioxidant activity that directly quenches excess ROS in the inflammatory microenvironment, thereby breaking the cycle of oxidative stress and tissue damage. Moreover, a sustained-release profile engineered through the Pluronic F127/EC matrix ensures prolonged therapeutic presence at the disease site, preventing the limitation of rapid clearance seen with many rinse-based or injectable agents. This dual-activity (antibacterial/antioxidant) and sustained-release design positions OG-Ointment as a comprehensive and advanced alternative within the non-antibiotic arsenal, potentially offering superior efficacy by simultaneously targeting both the microbial insult and the host’s destructive inflammatory response.

In this study, we successfully developed a sustained-release ointment (OG-Ointment) using a simple and scalable method, incorporating OG into a Pluronic F127/EC matrix. The formulation exhibited excellent stability, sustained drug release, and potent antioxidant and antibacterial activities. In vitro, OG-Ointment enhanced bacterial membrane permeability, inhibited the growth of P. gingivalis, and scavenged excessive ROS in macrophages. In vivo, it effectively alleviated periodontal inflammation, reduced alveolar bone loss, and demonstrated good biocompatibility in a rat model of periodontitis. These results highlight the potential of OG-Ointment as a non-antibiotic, dual-functional adjunctive therapy for periodontitis, addressing both microbial infection and oxidative stress. Future studies should focus on clinical translation and long-term safety evaluation.

Limitations of the study

Despite the promising results, this study has several limitations that should be acknowledged. First, the antibacterial assessment was focused solely on P. gingivalis as a keystone pathogen. The effect of OG-Ointment on other periodontal pathogenic bacteria and the complex oral microbiome remains to be elucidated. Second, although we demonstrated excellent ROS-scavenging capacity and anti-inflammatory effects, the precise intracellular molecular mechanisms underlying these actions, such as the involvement of the Nrf2 signaling pathway or the regulation of inflammasome activation, were not deeply investigated. Finally, the long-term stability in vivo (e.g., over weeks) and the precise pharmacokinetic profile of the ointment within the periodontal pocket require further optimization and analysis to pave the way for clinical translation.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mingxin Cao (mingxincao@tmu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •All data reported in this article will be shared by the lead contact upon request.
• •This article does not report original code.
• •Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

Acknowledgments

This work was supported by the Tianjin Medical University Integrated Traditional Chinese and Western Medicine Discipline Promotion Plan (2024XKZXY20).

Author contributions

Conceptualization, H.-P.W., Y.-S.W., and Y.W.; methodology, H.-P.W., C.-Y.X., and Y.-S.W.; validation, C.-Y.X. and W.S.; investigation, H.-P.W., C.-Y.X., and W.S.; data curation, H.-P.W., C.-Y.X., and W.S.; writing – original draft, H.-P.W., Y.-S.W., Y.W., and M.-X.C.; writing – review and editing, H.-P.W., C.-Y.X., W.S., Y.-S.W., Y.W., and M.-X.C.; visualization, H.-P.W. and C.-Y.X.; supervision, Y.-S.W., Y.W., and M.-X.C.; funding acquisition, Y.W.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Ethylcellulose (EC)	Solarbio	Cat#IE9000
Octyl gallate (OG)	Sigma-Aldrich	Cat#LEYH9ACECE2D
Pluronic F127	Solarbio	Cat#S7071
Triacetin (GT)	Baiao Leibo	Cat# M07985
Glycerol (GLY)	Solarbio	Cat#G8190
Carbomer 794P (CP)	Solarbio	Cat#C6751
ABTS+	Aladdin	Cat#A755512
2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA)	Sigma-Aldrich	CAS#4091-99-0
N-phenyl-1-naphthylamine (NPN)	Sigma-Aldrich	Cat#104043
4′, 6-diamidine-2-phenylindole dihydrochloride (DAPI)	Sigma-Aldrich	Cat#10236276001
Phosphate Buffer Saline (PBS)	Biosharp	Cat#BL601A
Brain Heart Infusion (BHI) broth	Solarbio	Cat#LA0360
Cell Counting Kit-8 (CCK-8)	Sparkjade	Cat# CT0001
Hydrogen Peroxide (H2O2)	Aike Reagent	Cat#124945
Dulbecco’s modified Eagle’s medium (DMEM)	Hyclone	Cat#SH30243.02
Fetal bovine serum (FBS)	Hyclone	Cat#SH30396.02
Penicillin/streptomycin solution	Hyclone	Cat#SV30010
Dimethyl sulfoxide (DMSO)	Meilun Biotechnology	Cat#PWL064
Trypsin	Thermo Fisher	Cat#15050065
Critical commercial assays
H&E staining kit	Beyotime	Cat#C0105M
DHE staining kit	Beyotime	Cat#S0064S
Anti-IL-1β Rabbit pAb	Servicebio	Cat#GB11113
Anti-TNF-α Rabbit pAb	Servicebio	Cat#GB11188
Experimental models: Cell lines
Mouse: NIH-3T3	BioVector NTCC	Cat#ACC59
Mouse: RAW264.7	BioVector NTCC	Cat#3735430
Experimental models: Organisms/strains
P. gingivalis	Bena Culture	BNCC 337441
SPF rats	Huafukang Biotechnology	Sprague-Dawley
Software and algorithms
CTVox	Bruker	N/A
CTAn	Bruker	N/A
Data Viewer	Bruker	N/A
GraphPad prism 9	GraphPad Software	N/A
ImageJ	NIH	N/A
Origin 2021	Originlab Company	N/A

Experimental model and study participant details

Cell culture

Mouse RAW264.7 macrophage cell line and mouse embryonic fibroblast cell line NIH-3T3 were obtained from BioVector NTCC (Beijing, China) and authenticated using Short Tandem Repeat (STR) analysis technology. They were tested not having mycoplasma contamination. RAW264.7 cells were selected for their relevance in studying immune responses and oxidative stress, while NIH3T3cells were used as a model for general cytocompatibility assessment. All cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM, Life Technologies, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixed solution in a humidified atmosphere with 5% CO₂ at 37°C. Cells within 10 passages and exhibiting normal morphology (e.g., adherent, non-granular, without signs of contamination) under microscopic examination were included in the experiments. Cells showing abnormal growth, contamination, or passage number exceeding 15 were excluded.

The strain of P. gingivalis (code BNCC 337441) was obtained from BeNa Culture Collection (Beijing, China). P. gingivalis was specifically selected as it is a well-established keystone pathogen in periodontitis. It was cultured in brain heart infusion (BHI) broth or Columbia blood agar plates (Solarbio, Beijing, China) under an anaerobic atmosphere at 37°C. Only bacterial cultures in the logarithmic growth phase, confirmed by optical density measurement, were used for the experiments. Cultures that failed to grow or showed inconsistent colony morphology on blood agar plates were excluded.

Animals

Male SD rats with an average body weight of 180 g were provided by Huafukang Biotechnology (Beijing, China) and housed in plastic cages with access to food and water. Male SD rats were chosen due to their established use and reliability in ligature-induced periodontitis models, and no evident influences of sex on the periodontitis-related animal studies have been reported. The ligature-induced periodontitis animal model was established as previously reported.²⁴ In detail, the rats were anesthetized and ligated at the gingival sulcus of the left maxillary second molar with orthodontic steel wires for 4 weeks to induce the experimental periodontitis. The success of the periodontitis model was confirmed by both visual inspection (presence of redness, swelling, and bleeding) and Micro-CT imaging (observable alveolar bone loss) after 4 weeks. Rats that failed to show these clear signs of periodontitis were excluded from the subsequent treatment groups. All rats were adaptively fed for 7 days before treatment, and all the animal experiments were performed in accordance with the guidelines approved by the Animal Care and Use Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College (IRM-DWLL-2022262).

Method details

Synthesis of OG-Ointment

To achieve the effective loading and sustained release of octyl gallate (OG), this study used Pluronic F127 as the gel matrix (responsible for gelling upon contact with water to adhere to periodontal pockets), ethyl cellulose (EC) as the sustained-release barrier (delaying the diffusion of OG), and Carbomer 794P (CP) as the thickener (enhancing ointment stability). The total mass of each OG-Ointment batch was 8.42 g, with the following component composition (w/w): Pluronic F127 (23.8%), triacetin (GT, 23.8%), EC (1.2%), OG (2.4%, active ingredient), glycerol (GLY, 47.5%), and CP (1.3%). The OG-Ointment was prepared via a three-step process of “heating and dissolving - dispersing and mixing - degassing and shaping,” with the specific steps outlined below:

Mix 2 g of F-127 with 2 g of GT and dissolve in a water bath at 80°C for 0.5 h, then add 100 mg of EC, increase the temperature to 95°C and continue the water bath for 2 h before stopping the heating. After cooling, a sustained-release ointment base is obtained. Under photoprotection, take 200 mg of OG and disperse it in a small amount of ethanol before dissolving it in 4 g of GLY. Separately, take 120 mg of CP and mix it with the above liquid in a mortar, and grind at room temperature to fully mix, resulting in an OG solution. Mix the ointment base with the OG solution in equal proportions in a mortar, and grind at room temperature for 20 min, then centrifuge at 1000 rad/min for 10 min to degas, and the OG-Ointment is prepared.

Characterization

Dissolve 1 mg of OG in 1 mL of anhydrous ethanol, dilute to 100-fold with anhydrous ethanol to a final concentration of 10 μg/mL, and then perform an ultraviolet spectrum scan between 200 nm and 1000 nm. Next, dissolve 1 mg of Ointment base (ON) and 1 mg of ON-OG separately in 1 mL of DDW, dilute to diluted 5-fold with DDW to a final concentration of 200 μg/mL, and perform an ultraviolet spectrum scan within the same wavelength range to detect whether OG has changed from hydrophobic to hydrophilic after being added to ON. Add OG-Ointment solutions with concentrations of 100, 200, 300, 400, 500, and 600 μg/mL respectively to the ABTS⁺ solution. After mixing thoroughly, keep them in the dark for 20 min. Measure the OD value at a wavelength of 734 nm and calculate the antioxidant activity. A Franz diffusion cell was used for the release experiment. 2 g of OG periodontal sustained-release ointment was weighed and placed in the donor pool, with PBS as the receiver pool. A semi-permeable membrane with a pore size of 0.45 μm was positioned between the donor and receiver pools. The temperature was maintained at 37°C. Centrifugation at 1000 rad/min for 10 min was employed as an effective degassing method prior to the release study to remove entrapped air bubbles, which was confirmed by visual inspection of a bubble-free, homogeneous ointment surface. At scheduled intervals, all the release medium from the receiver pool was removed and replaced with an equal volume of fresh blank release medium. Over 10 days, the OD values of the medium at different time points were measured, the concentration was calculated using the standard curve, and the total percentage released was determined to evaluate the in vitro release profile of the ointment. Place the sample in the center of the sample stage and perform an amplitude sweep at 37°C with a frequency of 1Hz, measuring the storage modulus (G′) and loss modulus (G'') within a strain range of 10⁻¹ to 10². Observe the changes in the sample’s moduli within an angular frequency range of 0–150 rad/s. Observe the viscosity changes of the two samples within a shear rate range from 10⁻³/s to 10²/s. Then, add 500 μL of DDW to the surface of 1.5 g of OG-Ointment. The sample was then placed in a temperature-controlled incubator at 37°C for 48 h until the ointment absorbs water and transforms into a gel-like state, after which repeat the above experiments to compare the rheological properties of OG-Ointment in different states.

In vitro evaluation of the bactericidal effect of OG-Ointment

To evaluate the changes in bacterial biofilm permeability caused by OG-Ointment. Take a bacterial suspension, centrifuge and discard the supernatant, wash twice, and dilute the bacterial concentration to 1 × 10⁴ CFU/mL. Add PBS, ON, and OG-Ointment solutions (1.5 mg/mL) respectively to 1 mL of bacterial suspension, and incubate anaerobically at 37°C for 2 h. Add NPN to a final concentration of 20 μM to each group of bacterial suspensions and incubate for 30 min, then use a fluorescence spectrophotometer to detect the fluorescence intensity of each group. Analyze the changes in bacterial cell wall membrane permeability through changes in fluorescence intensity.

Take P. gingivalis bacterial suspension, centrifuge and discard the supernatant, resuspend in BHI, and dilute the bacterial concentration to 1 × 10⁵ CFU/mL. Treat the strains with OG-Ointment at concentrations of 0.5, 1, 1.5, and 2 mg/mL respectively. After co-incubation for 24 h, measure the OD value of the bacterial suspension at a wavelength of 600 nm using an enzyme labeling instrument to calculate the antibacterial effect of the drug.

The plate inhibition experiment evaluates the inhibitory effect of OG-Ointment on the growth of P. gingivalis. Take a P. gingivalis bacterial suspension, centrifuge and discard the supernatant, and dilute the bacterial concentration to 1 × 10⁶ CFU/mL. Add BHI, ON, and OG-Ointment (2.5 mg/mL) solutions, and evenly spread 20 μL of the diluted bacterial suspension 100 times on the surface of a blood agar plate. Incubate anaerobically at 37°C for 7 days and observe the growth of colonies.

In vitro ROS inhibitory capacity of OG-Ointment

The effects of OG-Ointment in inhibiting ROS were verified using exogenous and endogenous ROS stimuli with H₂O₂ and LPS, respectively. The groups were divided into: blank control group, negative control group, Ointment group, and OG-Ointment group. For exogenous stimulation: Macrophages in the logarithmic growth phase were digested and resuspended, then seeded into a 48-well plate and cultured for 24 h. Afterward, the supernatant was removed, and the cells were washed with PBS. Apart from the blank control group, each group was treated with 200 μm/mL of H₂O₂ to stimulate the cells for 1 h. The cells were then resuspended, the supernatant was removed, and the cells were washed with PBS. Each group was treated with 1 mL of Ointment or OG-Ointment (300 μg/mL) resuspended in culture medium, while the positive control group received fresh culture medium. After 24 h of incubation in a culture box, DCFH-DA probe was added, and fluorescence production of ROS was observed under a microscope to detect the inhibitory effect of OG periodontal sustained-release ointment on cellular oxidative stress. For endogenous stimulation: The experiment was repeated with H₂O₂ replaced by 1 μg/mL LPS and observed under a microscope.

In vitro cytotoxicity assay of OG-Ointment

Digest NIH-3T3 and RAW264.7 cells, centrifuge, discard the supernatant, count the cells, and seed the cell suspension at a density of 1×10⁴ cells/well in a 96-well plate, culturing for 24 h. Dilute OG-Ointment with culture medium to prepare suspensions of 5, 10, 50, 100, 500 μg/mL. Discard the cell supernatant, add an equal amount of culture medium to the control group, and add different concentrations of OG-Ointment suspension to the experimental groups, incubating for 24 h. Then, add 10 μL of CCK8 solution to each well and incubate for 4 h. Measure the absorbance at a wavelength of 450 nm using an enzyme labeling instrument. Calculate the cell survival rate: Cell survival rate = (OD experimental group - OD blank group/OD control group - OD blank group) × 100%.

Red blood cell hemolysis assay

Rat arterial blood was centrifuged at 1500 rpm for 10 min, washed and clarified. Ointment and OG-Ointment were dissolved in PBS at different concentrations (50, 100, 500, 1000 μg/mL) and each mixed with 50 μL of red blood cells to examine the hemolytic properties of ON and OG-Ointment at various concentrations. Deionized water and PBS solution served as positive and negative controls, respectively. After incubation at 37°C for 30 min, the samples were centrifuged, and the OD value of the supernatant was measured at a wavelength of 541 nm.

In vivo evaluation of anti-periodontitis efficacy

SD rats were kept at room temperature, with 5 rats per cage. They were provided with regular feed and water and allowed to adapt to the environment for one week. Each rat was anesthetized with isoflurane. Once fully anesthetized without response, the rat was secured, and a 0.25 mm diameter orthodontic ligature wire was threaded through the mesial interproximal space on the palatal side of the rat’s maxillary second molar at one end, and through the distal palatal interproximal space at the other end. The ligature wire was then tightly tied within the buccal gingival sulcus using a needle holder. Four weeks later, under general anesthesia, the ligature wires were removed, and the local inflammation at the modeling site was observed with the naked eye. Photos were taken, and the modeling site of the rats was scanned with Micro-CT to determine whether the modeling was successful. After verifying the success of the modeling, rats without modeling were set as the normal control group. The remaining rats with successful periodontitis modeling were randomly divided into four groups of 5 each, and minocycline hydrochloride ointment available on the market was selected as the positive control drug for the study. The final groups were normal control group, periodontitis group, Ointment group, OG-Ointment group, and minocycline group. The method of administration was that the latter three groups were injected with 150 μL of the respective drugs into the periodontal pockets on both sides of the rats at the modeling site, once a week for two weeks, ending the treatment after two weeks. After the treatment ended, all rats in each group were subjected to general anesthesia and euthanized for material collection.

Fresh gingival tissue was taken from each sample, and the surface moisture of the tissue was gently absorbed with filter paper. The tissue was then quickly frozen in liquid nitrogen for approximately 15 s. Subsequently, the tissue was transferred to a freezer maintained at a temperature of −80 °C for storage. The rapidly frozen tissues were quickly embedded and then sectioned rapidly to ensure that the thickness of the slices remained within the range of 8–10 μm. The prepared sections were stored at −20 °C for subsequent use. Before staining, the sections needed to be brought to room temperature. The sections were treated with an autofluorescence quencher for about 5 min, followed by a 10-min rinse with water. At 37 °C and under light-free conditions, the sections were stained with DHE staining solution for 30 min. Then, the nuclei were stained with DAPI dye for 10 min. Afterward, three washes were performed to remove excess dye, and the sections were sealed for observation.

Small animal Micro-CT was used to scan the maxilla of rats with preserved gingiva on both sides. The three-dimensional image of the maxilla was observed using CT Vox software, according to the method by Ni et al.⁵⁵ The distances between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) at the mesial, middle, and distal aspects of the buccal and palatal sides of the second molars in rats were measured using Data Viewer and CTan software, and the average values were calculated. Additionally, the bone volume fraction (BV/TV) was determined.

After CT scanning, the maxilla and attached soft tissues of the rats were paraffin-embedded and stained with H&E to compare the pathological conditions of the periodontal tissues. The maxilla was rinsed with PBS twice and decalcified with EDTA decalcifying solution for 30 days before sagittal sectioning. The sections were rinsed with deionized water, dehydrated through an ethanol series, embedded in paraffin, baked on a paraffin slide warmer for 2 h, and then deparaffinized in xylene, followed by dehydration through an ethanol series. The sections were then rinsed, stained with hematoxylin solution for 3.5 min, rinsed with tap water for 2 min, stained with eosin solution for 4 min, rinsed with tap water for 2 min, and rinsed with distilled water for 2 s; dehydrated through an ethanol series, treated with xylene for 5 min twice, and finally sealed with neutral resin. After drying, the sections were observed under a microscope and photographed. The decalcification, embedding, and sectioning steps for the samples were the same as above. Sodium citrate repair solution was heated at high temperature for 30 min and then allowed to cool naturally. The sections were rinsed with PBS for 5 min three times. For primary antibody incubation, PBS containing 10% goat serum was used as the antibody diluent. Diluted rabbit anti-TNF-α antibody or rabbit anti-IL-1β antibody was added dropwise, left at room temperature for 30 min, and then incubated overnight at 4°C. The sections were rinsed with PBS for 2 min three times; biotinylated goat anti-rabbit IgG was added, left at room temperature for 1.5 h, and then rinsed with PBS for 2 min three times. DAB solution was added for 5 min followed by PBS rinsing; the sections were negatively stained for 2 min, dehydrated, and sealed with neutral resin. The sections were then observed under a microscope and photographed.

In vivo biosafety assessment

To assess in vivo biological safety of OG-Ointment, organs including the heart, liver, spleen, lungs, and kidneys were collected from rats in each group of the aforementioned in vivo experiment. Different organs from various groups were separately stained with H&E and observed under a light microscope for analysis. In addition, prior to euthanization, blood was taken from periodontitis rats that had undergone various treatment regimens, with the aim of performing hematological and biochemical analyses.

Quantifications and statistical analysis

All in vitro experiments were conducted independently at least three times. Quantitative data are presented as mean ± standard deviation (SD). Statistical differences between multiple groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for pairwise comparisons. For all analyses, a p-value of less than 0.05 was considered statistically significant. GraphPad Prism 9 software was used for all statistical analyses and graph generation. The exact value of n (number of independent replicates) and the statistical test used are provided in the corresponding figure legends.

Published: December 31, 2025