A Comparative Study of Caerin 1.1/1.9 and Calcium Hydroxide in the Treatment of Apical Periodontitis in Rats

Abstract

Objectives

By establishing a rat model of periapical infection, this study compared the anti‑inflammatory effects of caerin 1.1/1.9 hydrogel and calcium hydroxide paste, aiming to provide a novel therapeutic strategy for refractory apical periodontitis.

Methods

Forty-five male Sprague–Dawley rats were randomly divided: caerin 1.1/1.9 (F1/F3) peptide containing gel group (F1/F3, n = 15), calcium hydroxide group (CH, n = 15) and untreated control group (UC, n = 15). The F1/F3 group and CH group animals received an intracanal application of 5 mg/mL F1/F3-loaded hydrogel or calcium hydroxide paste, respectively, into the ipsilateral mandibular first molar immediately (day 0) and subsequently at 7 and 14 days post apical-foramen enlargement. At the 7th, 14th and 21st days after the operation, the degree of infection was comprehensively evaluated through periapical X-ray films, HE staining, immunohistochemical staining, enzyme histochemical staining and ELISA tests.

Results

At the 7th and 14th day after operation, the expression of IL-17 and the contents of TNF-α and IL-6 in F1/F3 and CH groups were significantly lower than those in UC group. On the 21st day after the operation, the number of osteoclasts in F1/F3 and CH groups decreased and the expression of OPG increased and the difference was statistically significant (P < .05). Compared with the CH group, the number of TNF-α and osteoclasts in F1/F3 group was significantly decreased on the 14th and 21st day after the operation and the expression of OPG was increased and the expression of IL-6 and IL-17 was decreased on the 21st day after the operation in F1/F3 group (P < .05).

Conclusions

The F1/F3 gel showed a more positive trend in regulating the key inflammatory factors and bone immune indicators related to periapical periodontitis compared to the calcium hydroxide paste.

Introduction

Apical periodontitis (AP) is an inflammatory disease of the periapical tissues initiated by the extension of infection from the root canal system.¹ This pathological process is predominantly elicited by a polymicrobial flora in which anaerobic bacteria are the primary contributors.² It typically occurs secondary to pulpal infection or trauma and is histologically characterised by inflammatory cell infiltration accompanied by alveolar bone resorption.³ The dynamic interplay between the resident microbiota and the host defense system leads to a sustained inflammatory response within the periapical tissues.

According to a recent study,¹ IL-17 and TNF-α secreted by Th17 cells promote RANKL expression and osteoclast differentiation. Furthermore, Th17 cells stimulate neutrophil recruitment, triggering an inflammatory response that enhances osteoclast activity, while the clearance of infection by neutrophils conversely suppresses osteoclast function. If the pulpal inflammation progresses apically, it can evolve into chronic periapical lesions such as periapical granulomas and radicular cysts.⁴ Consequently, the early and effective suppression of bacterial growth remains the cornerstone of clinical management for AP, aimed at slowing inflammatory progression, minimising periapical bone loss and ultimately preserving the affected tooth.⁵

Calcium hydroxide paste, valued in clinical practice for its high alkalinity, tissue-dissolving capability, capacity to neutralise endotoxins and potent antimicrobial effect, is frequently employed as an intracanal medicament.^6,7 However, its clinical utility is limited by poor penetration into the apical region,⁸ potential cytotoxicity to periapical tissues, difficulty of removal and postmedication discomfort.⁹ Currently, modified intracanal medications containing compound antibiotics have demonstrated robust antibacterial and anti-inflammatory properties in animal models of AP. Their additional advantage of not causing significant tooth discolouration makes them a viable alternative to most traditional intracanal medicaments in clinical practice.¹⁰ However, their long-term clinical application is constrained by the highly polymicrobial nature of root canal infections and potential adverse effects associated with prolonged antibiotic use.¹¹ Contemporary intracanal disinfectants are further hampered by limited antimicrobial spectra, emerging microbial resistance and an increased risk of root fracture.⁶ Consequently, the development of novel, highly effective intracanal agents capable of attenuating periapical inflammatory progression is of considerable clinical importance.

Caerin 1.1 and 1.9 are host-defense peptides isolated from the cutaneous secretions of Australian amphibians of the genus Litoria. These peptides adopt an α-helical conformation with a cationic amphipathic structure; they destabilise bacterial membranes by forming carpet-like or toroidal pores, leading to membrane disruption, cytoplasmic leakage and bacteriolysis.¹² Their propensity for inducing resistance is low, rendering them attractive candidates for next-generation antimicrobials.^13,14 Extensive in vitro studies have demonstrated that caerin 1.1 and 1.9 peptides possess broad-spectrum antibacterial, antineoplastic and antiviral activities, positioning them as potential antibiotic alternatives.^12,15 Formulating these peptides into hydrogels enhances solubility and provides sustained release,¹⁶ while also conferring antibiofilm activity and promoting tissue repair.¹⁷ In our previous animal study utilising a rabbit mandibular polymicrobial infection model, caerin 1.1 and 1.9 peptides-loaded hydrogels proved effective in eradicating bacteria and facilitating periprosthetic tissue healing.^18,19

In this experiment, the oral mixed bacteria of rats were introduced by exposing the pulp cavity. One week later, the apical foramen was enlarged and the pulp cavity was sealed to simulate the influence of pulp cavity exposure in a complex oral microbial environment on the periapical tissues. During the gradual development of periapical lesions in rats, intracanal application of F1/F3 hydrogel or calcium hydroxide paste was used to observe the effects of different stages of AP formation on inflammatory response and bone destruction. Based on the periapical lesion conditions in the untreated control group, the anti-infective efficacy of these 2 approaches during the progression of periapical lesions was comparatively evaluated, aiming to provide a more comprehensive and effective treatment strategy for early apical infections caused by mixed bacteria.

Materials and methods

Experimental animals

Forty-five male Sprague-Dawley rats, aged 8 weeks and weighing 250 to 300 g, were used in this study (purchased from Changsha Tianqin Biotechnology Co., Ltd., Animal License: SCXK [Xiang] 2019-0014). All experimental procedures were approved by the Institutional Animal Care and Use Committee (Ethical Approval Number: ZMU21-2412-009) and conducted in accordance with relevant guidelines and regulations. Rats were acclimatised for at least 7 days prior to experiments in a standard specific pathogen-free (SPF) animal facility under strictly controlled conditions: temperature 22 ± 2 °C, relative humidity 55 ± 10% and a 12-hour light/12-hour dark cycle (lights on: 7:00 am-7:00 pm) with HEPA-filtered air. Animals were group-housed (2-3 rats per cage) in polycarbonate cages with sterilised wood chip bedding, provided ad libitum access to standard maintenance rodent chow and autoclaved tap water. Bedding was changed regularly and animal health was monitored daily by trained personnel.

Inclusion criteria for experimental animals

Rats had no obvious systemic diseases, no congenital malformations or lesions in the mouth, normal tooth development and no dental caries, periodontitis, tooth loosening and other oral diseases. Exclusion criteria for experimental animals: loss of appetite, reduced activity, failure of experimental operation, poor tolerance to anaesthesia and oral or systemic diseases.

Establishment of periapical periodontitis model with external bacterial infection at the root apex

According to the 'pulp exposure method' established by Wang Lina et al.^20,21 the oral microbiota was allowed to continuously infect the root canal system, thereby simulating a rat model of AP that mimics the natural progression of chronic AP resulting from the spread of pulpal infection in clinical practice. The procedure was conducted as follows: Forty-five rats were intraperitoneally injected with 10% chloral hydrate at a dose of 300 mg/kg. After effective anaesthesia was achieved, the rats were placed in a supine position and secured on a rodent dissection platform (Figure 1A).

Fig. 1.

Establishment of Sprague-Dawley (SD) rats periapical periodontitis model and surgical procedure. A, Exposed left mandibular first molar. B, Opening of the medullary cavity. C, Enlargement of apical foramen to a 15-gauge C-file. D, Hygroscopic paper points were used to dry the canal. E, C-file delivered 5mg/mL F1/F3 gel to the apical foramen. F, Zinc oxide seals the opening pore.

The pulp chamber of the left mandibular first molar was accessed through the central fossa of the occlusal surface near the mesial marginal ridge using a portable dental handpiece equipped with a 1/4 round bur (Figure 1B). A #6 K-file was used to explore the root canal and a #000 barbed broach was employed to completely extirpate the dental pulp. The root canal was dried using sterile absorbent paper points to remove blood, with confirmation of no active bleeding to ensure complete pulp removal. The access cavity was left unsealed to allow continuous ingress of saliva into the pulp chamber and root canal, facilitating colonisation and inducing irreversible pulpal damage.

Experimental grouping and procedures

After 7 days of acclimatisation under standard SPF conditions, the rats were re-anaesthetised using the same protocol described above. The apical foramen of the mesial root was sequentially enlarged using #6 to #15 C-type files until active bleeding was observed (Figure 1C), aiming to mechanically extrude infectious material from the pulp chamber into the periapical region and exacerbate the initial infection. Subsequently, based on empirical practice and literature reference,²⁰ the rats were randomly divided into 3 groups (n = 15 per group):

• 1.F1/F3 Group: Use a C file to introduce 10 μL of F1/F3 gel into the root canal (Figure 1E), repeating this process 3 times until the apical foramen is reached. Repeat the administration every 7 days;
• 2.Calcium hydroxide (CH) group: Use a C file to introduce 36% to 44% injectable hydroxycarbonate paste (Langli) into the root canal, repeating this process 3 times until the apical foramen is reached. Repeat the administration every seven days;
• 3.Untreated control (UC) group: After enlarging the apical foramen, follow the procedures of the F1/F3 group and the CH group simultaneously. No drug intervention is performed to verify the progression of root apex periapical infection in the rats.

The access cavities of all rats were sealed with a temporary filling material (Shanghai Nifeng; main components: zinc oxide, resin, etc.) (Figure 1F) to prevent salivary recontamination and promote immediate healing, ensuring that subsequent observed differences were attributable solely to the distinct intracanal treatments.

On postoperative days 7, 14 and 21, 5 rats from each group were randomly selected, anaesthetised and subjected to blood collection from the jugular vein using a 1 mL syringe (Figure 2A). The blood samples were centrifuged; the supernatant serum was collected (Figure 2B) and stored at −20 °C for subsequent measurement of serum TNF-α and IL-6 levels. Euthanasia was performed via intracardiac perfusion, followed by complete dissection of the left mandible containing the first molar (Figure 2C). The specimens were then subjected to periapical radiography, haematoxylin-eosin (HE) staining, immunohistochemical staining and osteoclast staining.

Fig. 2.

Experimental sample collection. A, Jugular vein of rats’ blood. B, Rat serum was collected. C, Separate the left mandible of rats. D, Remove the temporary sealing material of the opening hole.

Primary reagents and the preparation of caerin 1.1/1.9 containing gel

Chloral hydrate (Shanghai Biotechnology Co., Ltd.); Calcium hydroxide paste (Langli Biomedical Co., Ltd.); Temporary sealing material (Shanghai Nifeng; main components: zinc oxide, resin, etc.); HE staining kit (Wuhan Servicebio Technology Co., Ltd.); ELISA kit (Wuhan Servicebio Technology Co., Ltd.); Tartrate-resistant acid phosphatase (TRAP) staining kit (Wuhan Servicebio Technology Co., Ltd.).

F1 and F3 peptides were synthesised by Shanghai Qiangyao Biotechnology Co., Ltd. The amino acid sequences of caerin 1.1 (F1) and caerin 1.9 (F3) are as follows:

• F1 is GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH₂

• F3 is GLFGVLGSIAKHVLPHVVPVIAEKL-NH₂.²²

Gel preparation method

The gel was prepared by 46 g of poloxamer 407 (Badische Anilin-und-Soda-Fabrik, WPAK592B) and 10 g of poloxamer 188 (Badische Anilin-und-Soda-Fabrik, WPAK539B) were dissolved in 200 mL of distilled water and F1 and F3 were then added. The solution was mixed thoroughly and filtered through a 0.22 μm membrane filter to prepare a 10 mg/mL gel and stored at 4 °C. When using, 500 μL each of F1 and F3 gels were mixed at a 1:1 molar ratio. The resulting F1/F3 gel working concentration was 5 mg/mL.

Periapical bone destruction analysis by X-ray films

Periapical radiographs of the left mandibular specimens were obtained using a dental X-ray unit (Rayco CS2100, Shanghai Ruike Biotechnology Co., Ltd.) at 7 kV and 8 mA with an exposure time of 0.001 second. The acquired images were imported into Image-J software for analysis. Regions of low-density shadowing in the periapical area were outlined and the corresponding area (mm²) was measured. Periapical bone destruction was quantified based on these measurements. Statistical analysis was performed using SPSS software.

Analysis of periapical histological changes by haematoxylin-eosin (HE) staining

Residual soft tissue was cleared from the left mandibular specimens, which were then fixed in 4% paraformaldehyde for 48 hours in preparation for haematoxylin and eosin (H&E) staining. The detailed procedure was as follows: (1) Tissue fixation and decalcification: Bone specimens were decalcified in 10% EDTA (pH 7.4) at 4 °C for 3 to 4 weeks. The decalcification solution was changed regularly and completion of decalcification was confirmed by needle puncture testing. (2) Tissue dehydration and clearing: Samples were dehydrated through a graded ethanol series (1 hour per step) and cleared in xylene (twice, 1 hour each). (3) Paraffin embedding, sectioning and slide selection: Tissue blocks were embedded in paraffin and serial sections of 4 to 8 μm thickness were cut. Sections containing the apical region of the mandibular first molar were selected for further staining. (4) Deparaffinisation and rehydration: Sections were deparaffinised in xylene for 20 minutes, rehydrated in 100% ethanol (5 minutes) and 75% ethanol (5 minutes) and rinsed under running tap water. (5) Haematoxylin staining: Sections were stained with haematoxylin for 3 to 5 minutes, rinsed under running water to remove excess stain, differentiated in 1% acid alcohol, rinsed again and blued in a bluing solution. (6) Eosin staining and dehydration: Sections were counterstained with eosin for 15 seconds, then dehydrated sequentially in 75%, 95% and absolute ethanol (2 minutes each). (7) Section drying and mounting: After dehydration, sections were air-dried and mounted with neutral resin. (8) Whole-slide scanning and morphological analysis: Stained sections were scanned using a panoramic slide scanner (PANNORAMIC DESK/MIDI/250/1000, 3DHISTECH) and examined for bone tissue morphology using CaseViewer 2.4 software (3DHISTECH).

Analysis of IL-17 and OPG levels by immunohistochemical (IHC) staining

The paraffin-embedded mandibular tissue sections were subjected to standard dewaxing and rehydration procedures. The specific steps are as follows: The sections were first washed in environmentally friendly dehydrating agents I and II (each for 20 minutes), then dehydrated through a series of ethanol concentrations and finally rinsed with distilled water. During antigen retrieval, the sections were heated in an EDTA buffer solution (pH 9.0) in a microwave oven (medium power heating for 5 minutes, rest for 5 minutes, then low power heating for 10 minutes). To inhibit endogenous peroxidase activity, the sections were incubated with 3% hydrogen peroxide at room temperature for 25 minutes. After 3 5-minute PBS washes, the sections were blocked at room temperature for 30 minutes with 3% bovine serum albumin (BSA) to prevent nonspecific binding.

Subsequently, the sections were incubated overnight at 4 °C, using the following specific antibodies: anti-IL-17 (Cebus Company, GB11110-1; dilution ratio 1:100) or anti-OPG (Cebus Company, GB115706; dilution ratio 1:100). After washing with PBS, the sections were incubated at room temperature for 50 minutes with a secondary antibody of goat anti-rabbit IgG linked to HRP (Cebus Company, GB23303; dilution ratio 1:200). After another PBS wash, the sections were stained with DAB kit, then counterstained with haematoxylin.

Finally, the sections were dehydrated using a series of ethanol gradients, cleared with xylene and sealed with neutral paraffin. The stained sections were observed under an optical microscope (product of Nikon). Digital images were captured and the number of positive cells and the total number of cells in each field of view were quantified using Aipathwell digital pathology image analysis software. The positive cell rate was calculated as: (positive cell number/total cell number) × 100%.

Analysis of osteoclasts by enzyme histochemical staining

The paraffin-embedded mandibular tissue sections were deparaffinised, rehydrated and stained with tartrate-resistant acid phosphatase (TRAP) to detect osteoclast activity. The staining solution was prepared according to the manufacturer’s instructions (Wuhan Saiwei Biotechnology Co., Ltd.). The sections were incubated with the TRAP working solution in a dark room at 37 °C for 20 minutes, washed with PBS to terminate the staining reaction and then briefly stained with haematoxylin (15 seconds), followed by rinsing with running water.

The sections were dehydrated with a series of gradient alcohols and transparentised in xylene (Guohuang Group Chemical Reagents Co., Ltd.) and then sealed with neutral resin. The stained sections were observed under an optical microscope (product of Nikon). Positive TRAP expression could be identified by the purple-red cytoplasmic staining and blue nuclear staining. In each sample, 3 random regions were selected from the root apex area and the number of TRAP-positive osteoclasts was counted for quantitative analysis.

Analysis of TNF-α and IL-6 levels in serum by ELISA

After general anaesthesia in rats, 1 μL of blood was collected from the jugular vein of each rat. The blood was left to coagulate at room temperature for 30 minutes, then centrifuged at 4000 revolutions per minute for 15 minutes. The resulting supernatant (serum) was collected and stored at −80 °C until it was used again.

The commercial rabbit-specific enzyme-linked immunosorbent assay kit (Rixin Biotechnology Company) was used to measure the levels of TNF-α and IL-6 in the serum. About 50 μL of the standard (with different concentrations) or the diluted serum sample was added to the designated wells on the microplate. About 100 μL of horseradish peroxidase-labelled detection antibody (except the blank well) was added to each well and then the plate was incubated in the dark at 37 °C for 60 minutes. The wells were washed 5 times with the washing buffer. Then, a second incubation was performed by adding the substrate solution. After the reaction was completed, the reaction was terminated with 50 μL of stop solution.

The absorbance was measured using an enzyme reader (RT-6100, Shenzhen Leidu Life Science Co., Ltd.) at 450 nm. A standard curve was generated by fitting the absorbance values of the standard samples to a 4-parameter logistic curve. The sample concentrations of TNF-α and IL-6 were determined by interpolation from the standard curve.

Statistical analysis

All quantitative data were analysed by SPSS 29.0 (IBM Corp, Version 29.0). Quantitative data are presented as mean ± standard deviation and normal distribution was tested by Shapiro-Wilk test. One-way analysis of variance (ANOVA) was used for normal data and Kruskal–Wallis rank sum test (H-test) was used for non-normal data. P values were corrected by Bonferroni and P < .05 was considered statistically significant. Statistical plots were made using GraphPad Prism 9.5 (GraphPad Software).

Results

Imaging manifestations

The X-ray films showed that there was a low-density shadow in the root apex of the mandibular first molar in each group (Figure 3A). The bone destruction gradually enlarged in the UC group (Figure 3a-c) and the trend of bone destruction in the CH group (Figure 3d-f) and the F1/F3 group (Figure 3g-i) was basically the same (Figure 3B). There was no significant difference between the 2 treatment groups (P > .05). After 14 and 21 days of treatment, the F1/F3 group and the CH group had significantly less apical bone destruction than the UC group, indicating that both groups could effectively inhibit periapical infection (Table 1 and Figure 3B).

Fig. 3.

Intracanal application of F1/F3 gel attenuates periapical bone destruction in rats. A, Periapical radiographs of the left mandibular first molar at 7th, 14th and 21st day after apical foramen enlargement. Progressive enlargement of the periapical radiolucency was observed in the UC group. The areas of bone destruction in the CH and F1/F3 groups were comparable and consistently smaller than those in the UC group. B, Quantitative comparison of periapical bone destruction. At all time-points, the F1/F3 group exhibited significantly smaller periapicular radiolucent areas than the UC group (P < .05). The difference between the F1/F3 gel and CH paste was not statistically significant, indicating equivalent efficacy in limiting periapical bone loss.

Table 1.

Comparison of the apical shadow area (mm², x̅ ± s) in each group at each time point after operation.

Groups	7 d	14 d	21 d
UC group	1.26 ± 0.25	1.50 ± 0.15	1.46 ± 0.47
CH group	0.86 ± 0.20	0.83 ± 0.50*	0.74 ± 0.78*
F1/F3 group	0.75 ± 0.19*	0.74 ± 0.55*	0.65 ± 0.17*

Compared with the UC group,

^⁎P < .05.

Histopathological analysis (H&E staining)

At 7 days postoperatively, the UC group exhibited dense infiltration of inflammatory cells in the periapical region, accompanied by irregular bone resorption margins (Figure 4a, a1). In the CH group, periapical exudation was predominantly composed of plasma cells, with a noticeable reduction in inflammatory response (Figure 4d, d1). The F1/F3 group showed only scattered infiltration of neutrophils, along with dilated small vessels containing neutrophils and localised bone resorption (Figure 4g, g1).

Fig. 4.

F1/F3 gel attenuates inflammatory cell infiltration in periapical bone tissue. a–i, show H&E‑stained sections of the root canal morphology at 40× magnification. a1–i1, show the periapical tissue morphology at 400× magnification, where T denotes root tissue. a1, Dense infiltration of neutrophils (red arrows) accompanied by abscess formation, focal necrosis and liquefaction (blue arrows) and partial resorption of periapical bone tissue are observed in the apical region. b1, Extensive neutrophil infiltration (red arrows), histiocyte reaction, abscess formation and localised bone resorption are evident in the periapical area. c1, Fibroblast/myofibroblast proliferation and fibrous hyperplasia are present, with scant perivascular lymphocyte infiltration (black arrows) around small vessels in the apical region. d1, Scattered neutrophils (red arrows) along with lymphocytes and plasma cells (blue arrows) are seen, accompanied by marked fibroblast/myofibroblast hyperplasia. e1, Scattered neutrophils, lymphocytes (green arrows) and plasma cells are observed, with focal micro‑abscess formation (black arrow). f1, Sparse plasma cell infiltration (blue arrows) and bone resorption are noted, associated with surrounding tissue necrosis (black arrow). g1, Mild scattered neutrophil infiltration (red arrows), fibroblast proliferation and dilated small vessels containing neutrophils are visible, together with localised bone resorption. h1, A small number of lymphocytes (green arrows) are distributed in the apical region, accompanied by fibroblast hyperplasia (yellow arrows) and no significant tissue edema. i1, Abundant fibrous tissue formation (yellow arrows) is present without notable inflammatory cell infiltration.

At 14 days postoperatively, the UC group demonstrated progressive inflammation characterised by extensive neutrophil infiltration, histiocytic reaction, formation of small periapical abscesses and focal bone destruction (Figure 4b, b1). In the CH group, scattered inflammatory cells were observed around the apex, with localised small abscesses indicative of transition from acute to chronic inflammation (Figure 4e, e1). The F1/F3 group exhibited minimal lymphocyte infiltration, accompanied by fibrous band formation and absence of significant tissue edema, suggesting gradual resolution of inflammation (Figure 4h, h1).

At 21 days postoperatively, the UC group displayed focal perivascular lymphocyte infiltration, fibrous hyperplasia, expanded bone destruction and formation of inflammatory granulation tissue (Figure 4c, c1). The CH group showed sparse plasma cell infiltration and surrounding tissue necrosis, consistent with chronic inflammatory progression (Figure 4f, f1). In the F1/F3 group, prominent fibrous tissue formation was observed without notable inflammatory cell infiltration, indicating initiation of the repair phase (Figure 4i, i1).

In summary, both F1/F3 hydrogel and calcium hydroxide effectively reduced periapical inflammatory cell infiltration in the early stages, resulting in relatively stable progression of inflammation.

Expression of IL-17 and OPG

The positive expression of IL-17 in the apical bone tissue of the UC group increased first and then tended to increase and reached the peak on the 14th day after operation. The IL-17 expression in the CH group did not change much in the first 2 weeks, but increased at the 21st day, but the overall expression was lower than that in the UC group. The expression of IL-17 in the F1/F3 group was lower than that in the UC group and the difference was significant at 14 days before administration (P < .05). On the 21st day after administration, the expression of IL-17 in the F1/F3 group was significantly lower than that in the CH group (P < .05). These results suggest that simultaneous administration of F1/F3 gel and calcium hydroxide to enlarge the apical foramen can significantly reduce the expression of IL-17 and inhibit apical bone resorption (Figure 5).

Fig. 5.

F1/F3 gel suppresses IL-17‑positive cell expression in periapical bone. A, Representative immunohistochemical staining (×200) showing IL‑17‑positive expression. a-c, (UC group): Dense and high-level distribution of IL‑17‑positive cells in the periapical region. d-f, (CH group) and g-i, (F1/F3 group): Scattered and lower-level IL‑17‑positive cells in the periapical area, with the lowest expression observed in the F1/F3 group on day 21. B, Quantitative analysis of the percentage of IL‑17‑positive cells in the periapical region on postoperative days 7, 14 and 21. On days 7 and 14, both the CH and F1/F3 groups showed significantly lower percentages compared to the UC group (P < .05). On day 21, the F1/F3 group exhibited a markedly lower percentage than the CH group (P < .05).

In the UC group, OPG-positive expression in the periapical region progressively declined over time. In contrast, both the F1/F3 and CH groups exhibited a gradual increase in OPG expression. By day 21, the F1/F3 group demonstrated the highest level of OPG-positive expression, with a statistically significant difference compared to the other groups (P < .05) (Figure 6B). These results indicate that prolonged administration of either F1/F3 hydrogel or calcium hydroxide effectively upregulates OPG expression, with the F1/F3 hydrogel providing a more favourable immune microenvironment for bone formation.

Fig. 6.

F1/F3 gel enhances OPG protein expression in periapical bone tissue. A, OPG immunopositivity in IHC sections (×200). a-c, (UC group): OPG is detectable only intermittently between bone trabeculae at 7th day and remains sparse at later intervals. d-f, (CH group) and g-i, (F1/F3 group): abundant OPG surrounds bone trabeculae at every time-point; expression becomes progressively denser and more confluent with prolonged treatment, reaching maximal intensity in the F1/F3 group at 21st day. B, Quantitative analysis of OPG-positive cell ratios in periapical bone at 7th, 14th and 21st day post-operatively. At 14th day, the F1/F3 group already exhibits a significantly higher ratio than the UC group (P < .05). At 21st day, both CH and F1/F3 groups display markedly elevated ratios relative to the UC group, with the F1/F3 group achieving the highest value; all intergroup differences are statistically significant (P < .05).

Osteoclast count

In the UC group, the number of osteoclasts in the periapical region increased initially and subsequently declined, peaking on day 14 post-treatment (Figure 7). Both the CH and F1/F3 groups exhibited a gradual reduction in osteoclast counts, which were significantly lower than those in the UC group on days 14 and 21 (P < .05) (Figure 7A). Moreover, on days 14 and 21, the F1/F3 group showed significantly fewer osteoclasts compared to the CH group (P < .05) (Figure 7B). Although neither F1/F3 hydrogel nor calcium hydroxide significantly altered osteoclast numbers on day 7, both treatments led to a marked decrease by day 14, with the effect being more pronounced in the F1/F3 group. These findings suggest that both F1/F3 hydrogel and calcium hydroxide require a certain duration of action to effectively suppress osteoclast formation.

Fig. 7.

F1/F3 gel suppresses osteoclast differentiation in periapical bone. A, TRAP-stained sections (×400) show osteoclast number and distribution (black arrows indicate osteoclasts). a-c, (UC group), d-f, (CH group) and g-i, (F1/F3 group) illustrate osteoclast formation and distribution at 7th, 14th and 21st day after apical foramen enlargement. Osteoclast counts are comparable amongst groups at 7th day, but are markedly reduced in the F1/F3 group at 14th and 21st day compared with the other 2 groups. B, Quantitative analysis of osteoclast numbers in periapical bone at 7th, 14th and 21st day postoperatively. At 14th day, the F1/F3 group exhibits significantly fewer osteoclasts than both the CH and UC groups (P < .05). At 21st day, both CH and F1/F3 groups display lower osteoclast counts than the UC group, with the F1/F3 group showing the lowest value; all intergroup differences are statistically significant (P < .05).

Serum expression of TNF-α and IL-6

In the UC group, serum IL‑6 levels increased progressively each week postsurgery, while TNF‑α levels rose gradually on days 7 and 14, followed by a slight decline on day 21. In contrast, both the CH and F1/F3 groups showed a steady decline in both IL‑6 and TNF‑α levels over the postoperative period (Figure 8). On days 14 and 21 after treatment, serum IL‑6 and TNF‑α levels in both treatment groups were significantly lower than those in the UC group (P < .05), indicating that F1/F3 hydrogel and CH paste effectively suppressed periapical infection. Compared with the CH group, serum TNF‑α levels in the F1/F3 group were significantly reduced from day 14 onward (P < .05). Moreover, by day 21, IL‑6 levels were also markedly lower in the F1/F3 group (P < .05). These results suggest that F1/F3 hydrogel exerts a more potent anti‑inflammatory effect beginning 14 days after administration (Table 2 and Figure 8).

Fig. 8.

F1/F3 gel markedly attenuates systemic release of IL-6 and TNF-α in rats. A, Serum IL-6 levels at 7th, 14th and 21st day post apical foramen enlargement were significantly lower in the F1/F3 group than in the UC group (P < .05); at 21st day, the F1/F3 group also differed significantly from the CH group (P < .05). B, Similarly, serum TNF-α concentrations at 7th, 14th and 21st day were significantly reduced in the F1/F3 group compared with the UC group (P < .05) and a significant difference vs the CH group was observed at 21st day (P < .05).

Table 2.

Comparison of serum IL-6 and TNF-α levels in each group (pg/mL, x̅ ± s).

Groups	IL-6			TNF-α
	7 d	14 d	21 d	7 d	14 d	21 d
UC	48.87 ± 1.75	81.65 ± 12.98	103.49 ± 9.07	342.55 ± 28.80	450.72 ± 39.36	409.77 ± 51.40
CH	26.21 ± 11.09	24.81 ± 3.98*	45.76 ± 16.86*	265.16 ± 36.76*	220.51 ± 9.92*	158.61 ± 18.36*
F1/F3	31.16 ± 3.00*	23.12 ± 4.09*	24.25 ± 5.39*,⁎⁎	255.03 ± 16.82*	140.23 ± 14.10*,⁎⁎	87.27 ± 11.75*,⁎⁎

Compared with the UC group,

^⁎P < .05.

Compared with the CH group,

^⁎⁎P < .05.

Discussion

The F1/F3 has been extensively validated in prior experimental studies for its broad-spectrum antibacterial activity and immunomodulatory properties against multiple bacterial species.^14,17,22 Although the Poloxamer carrier in the F1/F3 hydrogel itself exhibits limited antibacterial effects, our group previously conducted in vitro experiments in which different concentrations of F1/F3 hydrogel were cocultured with purulent exudate from rat skin MARS infections and subjected to colony counting. The results demonstrated that antibacterial efficacy increased significantly with higher F1/F3 concentrations, whereas at concentrations below 1.5625 mg/mL, no significant difference was observed compared to blank Poloxamer gel. This confirms that the antibacterial activity of the F1/F3 hydrogel originates primarily from the F1/F3 peptides rather than the Poloxamer gel. Beyond retaining the inherent antibacterial efficacy of Caerin peptides, the gel formulation further enhances antimicrobial performance while enabling sustained drug release,¹⁷ thereby overcoming the limitation posed by salivary hydrolases in the complex oral microenvironment that would otherwise compromise the bioactive antibacterial function of F1/F3 peptides. Building on the demonstrated effectiveness and favourable biosafety of Caerin 1.9 in a multibacterial mandibular infection model in New Zealand rabbits reported by You et al. and Long et al.^18,19 the present study extends the application of Caerin 1.1/Caerin 1.9 hydrogel to a novel clinical domain – root canal infection.

Wang et al.²⁰ inoculated Enterococcus faecalis into the exposed pulp chambers of rat first molars (5 rats per time point) and sacrificed groups at 0, 7, 14, 21 and 28 days. Histological examination via H&E staining revealed slight widening of the periodontal ligament and initial bone resorption at day 7, with peak bone loss occurring at day 21, confirming that the pulp exposure method successfully establishes a rat model of AP. confirming that the pulp exposure method successfully establishes a rat model of AP. In the present study, an experimental rat model of AP was established. This model aligns with the findings of Park et al.²¹ who used Micro-CT imaging to demonstrate gradually enlarging periapical radiolucencies at 7-, 14- and 21-days postpulp exposure, further validating the successful construction of the experimental AP model. These radiographic observations are consistent with our own X-ray findings and support the efficacy of both F1/F3 hydrogel and calcium hydroxide paste. Our results also reinforce the therapeutic potential of host defense peptides (HDPs) in root canal therapy, as initially demonstrated by Lima et al.²³ While their study focused on treating established AP, our investigation examines pharmacological intervention during the dynamic progression of chronic AP, thereby exploring the influence of F1/F3 on the developmental course of periapical lesions. However, it should be noted that the current evidence supporting the use of HDPs in endodontics is primarily derived from in vitro and animal studies. Moreover, there is currently no standardised protocol for establishing animal models of chronic AP, which limits the translational potential of existing findings.

The pathological hallmark of AP is periapical inflammation and bone destruction, which triggers an immuno-inflammatory response. Within the complex microenvironment of periapical lesions, tissue destruction and remodelling are governed by interactive factors including TNF-α, IL-6 and osteoclasts, which play crucial roles in modulating the inflammatory balance.24, 25, 26 In this experiment, the UC group exhibited elevated serum TNF-α levels on day 14, a trend consistent with IL-17 expression and osteoclast counts. The possible reason for this is that after the root apex canal was enlarged, the infectious substances accumulated in the root apex, leading to a continuous aggravation of root apex infection and causing a strong systemic inflammatory response. This phenomenon may be attributed to the accumulation of infectious material in the periapical region following apical foramen enlargement, which likely exacerbated persistent apical infection²⁷ and triggered a pronounced systemic inflammatory response. Conversely, local application of F1/F3 hydrogel and CH was associated with milder inflammatory cell infiltration, enhanced fibrous tissue formation and significantly reduced serum TNF-α and IL-6 levels by day 14. This suggests that both agents may²⁸ provide robust initial immunomodulation or activate extracellular signalling pathways during the formative phase of AP, thereby attenuating inflammatory cytokine release.

In the local immune microenvironment of periapical bone, untreated animals showed an initial sharp rise in IL-17 expression around the apex, followed by a plateau, mirroring the temporal trend in osteoclast numbers. In contrast, intervention with F1/F3 hydrogel or CH paste gradually reduced osteoclast counts, progressively increased OPG expression and significantly lowered IL-17 levels by day 14, indicating gradual control of the apical infection and a shift towards an immune milieu favourable for bone formation. While these cytokine and cellular trends are consistent with the efficacy of modified triple antibiotic paste reported by Meng et al. the F1/F3 hydrogel – as a non-antibiotic peptide – exerts its effects primarily through immunomodulation and bacterial membrane disruption,^12,29 potentially offering an advantage in circumventing bacterial resistance.¹⁴ The precise immunomodulatory mechanism of F1/F3 in AP remains unclear but may involve regulation of the RANKL/RANK/OPG pathway to inhibit periapical bone destruction.^14,28,30 By establishing more effective immune regulation, F1/F3 could create a more conducive microenvironment for tissue repair, although its efficacy in promoting bone regeneration requires longer-term investigation.

In this study, both the F1/F3 and CH groups exhibited smaller periapical radiolucent areas on radiographs compared to the UC group, but no statistically significant difference was detected between the 2 treatment groups. This may be attributed to the fact that histopathological inflammation reflects immediate biochemical cascades following pharmacological intervention, whereas radiographic changes in bone require substantial mineral alteration before becoming evident. The superior performance of the F1/F3 group in osteoclast counts and OPG-positive expression supports this interpretation. Thus, a 21-day observation period may be insufficient to demonstrate statistically significant differences in radiographic bone-defect area.

In clinically early AP cases presenting with pronounced acute inflammation and considerable exudation, dentinal tubular fluid can buffer the alkaline environment of CH paste, thereby limiting its efficacy.³¹ In contrast, the immunomodulatory advantages of F1/F3 hydrogel may enable faster control of inflammation and symptom relief. Through its sustained-release properties in vivo, F1/F3 hydrogel could serve as a superior intracanal medicament, creating favourable biological conditions for periapical tissue repair and regeneration.

This study has certain limitations. First, preliminary attempts to collect intraradicular bacteria for bacterial load quantification were hindered by the limited space within rat root canals, yielding insufficient bacterial RNA for reliable assessment postintervention. Second, the 21-day observation period may be inadequate to fully evaluate the resolution of apical infection or bone regeneration. Future studies will expand sample sizes and employ Micro-CT imaging to better monitor periapical tissue repair over time. Finally, although the rat model is widely used in chronic periapical periodontitis-related research, given the significant differences in its anatomical structure and microbiological characteristics compared to that of humans, this study found that there is a relatively high risk of bias in the clinical translational evidence of the F1/F3 gel. In the future, further experimental designs are still needed to verify the effectiveness and safety of HDP application in root canals in clinical practice, in order to clarify its broader clinical applicability in dental and periodontal inflammatory diseases.

Conclusion

The F1/F3 gel showed a more positive trend in regulating the key inflammatory factors and bone immune indicators related to periapical periodontitis compared to the calcium hydroxide paste.

Author contributions

Wei Yang, Jiangling Sun, Xiaosong Liu and Guoying Ni, contributed to conception, design, analysis and interpretation and drafted and critically revised the manuscript. Li Qin, Hang You and Quanlan Fu, Zhijun Lin and Meiyan Rong performed the experiments and analysed the results and provided materials and resources. Zhuxue Zhang corrected the operation steps and results of the entire tissue sectioning process of the experiment. Li Qin and Hang You wrote the first draft of the manuscript. Wei Yang, Tianfang Wang and Hejie Li revised the final draft. Li Qin and Hang You contributed equally to this work. All authors read and approve the manuscript.

Ethics approval

This study was approved by the Laboratory Animal Welfare and Ethics Committee of Zunyi Medical University (EAE- ZMU21-2412-009). This study was performed in line with the principles of the Declaration of Helsinki.

Funding

This project was supported by Guizhou Provincial Department of Science and Technology (Qiankehe support normal [2022] No. 196 and [2022] No. 272).

Conflict of interest

All authors declare no competing of interest.