Periodontitis animal models tend to involve rodents and are used to study the etiology of periodontitis and to evaluate treatment modalities [1]. Rodents have incisor and molars in each quadrant [2]. Rodents molar structure is similar to that of human molars and is often used as an animal model for periodontitis. In addition, rodents are used in periodontitis research because they are relatively easy to handle, inexpensive to acquire and maintain, and have extensively immune system [1,3]. In periodontitis research, the ligature-induced periodontitis (LIP) mice model in which periodontitis is induced by ligation is frequently used in research [4-10]. The LIP, which induces plaque accumulation by the ligation of suture materials on molars, occurs alveolar bone loss within a few days after ligation [11,12]. And the LIP destroys gingival epithelium, increases osteoclast and alveolar bone loss [2].

The LIP mice model is a commonly used model, but some challenges remain, including difficulties associated with ligation, insufficient tissue samples due to the size of mouse oral cavity, and differences among mouse strains. The mice of ICR, C57BL/6, or BALB/C strains have been widely used in ligature-induced periodontitis models [3,13,14], which involved animals aged 8–12 weeks and an induction period of 1–45 days [13,15,16]. However, standardized mouse strain and age, and induction period criteria remain lacking.

Examining the impact of mouse strain and age, and ligature thickness and period is required to standardize the tooth ligature-induced periodontitis mouse model. Therefore, this study aims to compare the alveolar bone loss between mouse strain for the tooth ligature-induced periodontitis mouse model.

Experimental animals

The ICR, BALB/C, and C57BL/6 mice (n=20 per group), aged 4 weeks, were purchased from Orient Bio (Seongnam, Korea), and were used in this study at the age of 5 weeks after an acclimatization period of 1 week. In the case of ICR mice, the contact point of the molar was more compact than that of other mice. In order to use the same silk in all animals, a 5 weeks old mouse was used based on the contact point of the ICR mouse.

All mice were maintained at 22℃ and 50% humidity with free access to food and water in a 12-hr light/dark cycle. The study procedures were performed according to the applicable guidelines. The study protocol was permitted by the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University (GWNU-2020-31).

Mice were randomly allocated into six subgroups: (1) ICR-control (CTL) group (n=10), (2) ICR-LIP group (n=10), (3) BALB/C-CTL group (n=10), (4) BALB/C-LIP group (n=10), (5) C57BL/6-CTL group (n=10), and (6) C57BL/6-LIP group (n=10).

Induction of periodontitis

Periodontitis was induced by ligation around the cervical region of the maxilla left second molar using 6-0 black silk (AILEE, Busan, Korea). The suture was knotted gently to avoid injury to the periodontal tissue. All mice stayed healthy throughout the experimental period (14 days). The ligature site was evaluated daily. No specific adverse events occurred.

According to Zebrowitz et al. [17] (2021), significant alveolar bone loss began to appear on the 5 days, and alveolar bone loss were clearly visible on the 10 days. Therefore, in this study, ligation was maintained for 14 days to cause clearly alveolar bone loss due to induction of periodontitis.

Micro-computed tomography analysis

Micro-computed tomography scans were performed to estimate alveolar bone level (SkyScan1173, Bruker-CT, Kontich, Belgium) at a resolution of 14 µm (achieved using 90 kV and 88 µA, with an exposure time of 500 ms). All scan images were re-oriented before the examination, to uniformly align the scan axes and anatomical position, using NRecon software (ver 1.7.04), and Dataviewer (Bruker-CT). To assess the alveolar bone level, the distances between the cemento-enamel junction (CEJ) and alveolar bone crest (ABC) were measured (mm) alongside the mesial and distal aspects of the ligated tooth. Every image was aligned, so that both the CEJ and apex of the root appeared in the same micro-computed tomography scanning slice [14,18].

Tissue processing for histology

At 2 weeks after ligation, animals were sacrificed. Subsequently, the mice were perfused intracardially with 0.9% saline and 4% paraformaldehyde in phosphate-buffered saline (pH 7.5). The left maxillary tissues were extracted and post-fixed in 4% paraformaldehyde in phosphate-buffered saline, and fixed in paraffin and sectioned at 5 µm thickness using microtome (Leica, Wetzlar, Germany).

Masson trichrome staining for histological analysis

The histological changes and the degree of inflammatory infiltration was assessed with Masson trichrome staining using commercial kit (Abcam, Cambridge, UK) to evaluate. The gingiva tissues were washed twice with xylene for 10 minutes to remove the paraffin. The general hydration process was performed. The tissues were treated with the Bouin fluid for 60 minutes and cooled for 10 minutes, followed by treatment with the Weigert hematoxylin for 5 minutes. The tissues were washed in tap water for 2 minutes. Then, Biebrich scarlet-acid fuchsin solution was sprayed on the tissues for 15 minutes and rinsed in distilled water. The tissue samples were treated with a phosphomolybdic-phosphotungstic acid solution for 10–15 minutes and transferred into aniline blue solution for 5–10 minutes; then, the tissue samples were washed. Finally, 1% acetic acid solution was sprayed on the tissues. Afterward, the dehydration and clarification processes were performed and sealed with the Canada balsam (Kanto, Tokyo, Japan). The samples were observed using a microscope (Axio Imager A2, Carl Zeiss, Oberkochen, Germany) and images were captured using a digital camera (EOS 100D, Canon, Tokyo, Japan).

Statistical analysis

All analyses were achieved in SPSS version 21.0 (IBM Co., Armonk, NY, USA). The values are expressed as mean±standard deviation. The CEJ-ABC distance comparisons between the CTL and LIP groups were performed with the Mann-Whitney U-test. One-way analysis of variance (ANOVA) with the Bonferroni was used to compare the distances between CEJ and ABC in the mice strain. The level of significance was set at p-values of <0.05.

Micro-computed tomography analysis

The distance between the CEJ and ABC was measured along the mesial and distal sides of the tooth. Alveolar bone loss was observed in all LIP groups, in contrast to the CTL groups (Fig. 1). In ICR-CTL group was 0.085±0.02 mm at mesial and 0.135±0.02 mm at distal aspect. In ICR-LIP group was 0.308±0.06 mm at mesial and 0.378±0.06 mm at distal aspect. There were significant differences in the extent of bone loss between the ICR-LIP and CTL groups (Fig. 1A, a, and D). In BALB/C-CTL groups were 0.148±0.01 mm at mesial and 0.165±0.03 mm at distal aspect. In BALB/C-LIP group was 0.318±0.08 mm at mesial and 0.333±0.07 mm at distal aspect. There were significant differences in the extent of alveolar bone loss between the BALB/C-LIP and CTL groups (Fig. 1B, b, and D). In C57BL/6-CTL group was 0.115±0.01 mm at mesial and 0.1±0.01 mm at distal aspect. In C57BL/6-LIP group was 0.360±0.07 mm at mesial and 0.445±0.21 mm at distal aspect. There were significant differences in the extent of alveolar bone loss between the C57BL/6-LIP and CTL groups (Fig. 1C, c, and D). In addition, there was no significant difference in the difference in alveolar bone loss according to mouse strain.

Fig. 1. Representative micro-computed tomography (micro-CT) images. The CTL groups (A-C) show significantly lower alveolar bone loss than the ligature-induced periodontitis (LIP) groups (a-c). The amount of alveolar bone loss in LIP groups was significantly higher than CTL groups (D). CEJ, cemento-enamel junction; ABC, alveolar bone crest; CTL, control. N=10 per group; *p<0.05, significantly different from the CTL group (The bars indicate the mean±standard error of the mean).

Masson trichrome staining for histological analysis

The tissue stained with Masson trichrome was observed at a low magnification (Fig. 2A). After that, it was observed at a high magnification to confirm in detail the changes in the periodontal tissue (Fig. 2C).

Fig. 2. Masson trichrome staining of periodontal tissue of the maxilla left second molar. (A) 10× scale bar=200 µm. (B) 40× scale bar=50 µm. The periodontal tissue at the maxilla left second molar was observed 100× magnification (A). The marked region in the periodontal tissue schematic diagram (B) was observed 400× magnification. The CTL groups (Ca-Cc) show connective tissue papillae and normal epithelium of gingiva. The ligature-induced periodontitis (LIP) groups (Ca’-Cc’) show a thick epithelium of gingiva, some vessels and inflammatory infiltration; no connective tissue papillae were observed. Ep, epithelium of gingiva; P, connective tissue papillae; CTL, control; arrow, inflammatory infiltration. *Blood vessel.

Connective tissue papillae were present and connective tissue fibers were regular and tight in the CTL group of ICR, BALB/C, and C57BL/6 mice (Fig. 2Ca-Cc). In contrast, connective tissue papillae were not observed, while thickened gingiva epithelium was observed in the LIP group of the ICR, BALB/C, and C57BL/6 mice (Fig. 2Ca’-Cc’). In the connective tissue area, irregular and relatively loose fibers were detected in the LIP group than in the CTL group. And, infiltration of inflammatory cells was observed in the epithelial tissue, and many blood vessels were observed between the gingiva epithelium and connective tissue (Fig. 2Ca’-Cc’).

Experimental periodontitis model can be induced by infection of bacterial pathogens such as Porphyromonas gingivalis, injection of P. gingivalis lipopolysaccharide (Pg-LPS), or ligation of teeth in animals. The BALB/C and C57BL/6 mice are particularly susceptible to infection with P. gingivalis and the injection of Pg-LPS, respectively [3,13]. However, it remains unclear which mice strain is particularly susceptible to the tooth ligature-induced periodontitis model. Also many studies used ligation of teeth of achieving a periodontitis mouse model, but the impact of mouse strain has not been examined. Therefore, this study aimed to evaluate the difference in the extent of ligature-induced periodontitis among different mouse strain models for 2 weeks.

Previous periodontitis research has mainly used C57BL/6 mice. It has been suggested that C57BL/6 mice provide ideal conditions for inducing periodontal disease because bone loss and inflammatory response occur within a short period [16]. In the studies of Aung et al. [19] (2020), Zebrowitz et al. [17] (2021), and Suh et al. [20] (2020), periodontitis was induced using the C57BL/6 mouse. BALB/C mice are susceptible to periodontal disease and are often used as a periodontal disease model [1]. In the studies of Greene et al. [21] (2022) and Candeo et al. [22] (2017), periodontitis was induced using the BALB/C mouse. BALB/C and C57BL/6 mice have been used in various periodontitis-induced studies. And, we have induced periodontitis using ICR mice in previous studies [14,23].

Based on the results of previous studies, periodontitis was induced in three mouse strain and compared. Alveolar bone loss occurred significantly in all LIP group than in the CTL group of ICR, BALB/C and C57BL/6 mice. The rates of alveolar bone loss on the mesial sides in the ICR-LIP, BALB/C-LIP and C57BL/6-LIP group compared to the CTL group were 72.36%, 53.54%, and 68.06%, respectively. The rates of alveolar bone loss on the distal sides in the ICR-LIP, BALB/C-LIP and C57BL/6-LIP group compared to the CTL group were 64.24%, 50.38%, and 77.53%, respectively. However, there was no statistically significant difference in alveolar bone loss according to mice strain.

And, in previous studies, periodontitis was induced using mice of various age. Abe and Hajishengallis [13] (2013) were used C57BL/6 mice of 9–10 weeks and Suh et al. [20] (2020) were used C57BL/6 mice of 7 weeks. Greene et al. [21] (2022) were used BALB/C mice of 6–8 weeks and Candeo et al. [22] (2022) were used BALB/C mice of 2–8 months. Kim et al. [14] (2020) and Jung et al. [23] (2021) were used ICR mice of 5 weeks. In this study, ICR, BALB/C, and C57BL/6 mice of 5 weeks old. Although there are differences in age of ICR, BALB/C, and C57BL/6 mice in this study and previous study, alveolar bone loss occurred in all mice in this study. And in previous studies, the thickness of silk used in the ligation of mice teeth was varied [13,17,24]. This study used 6-0 silk, which is relatively easily inserted into the interdental space of ICR mice.

When comparing this study with previous studies, alveolar bone loss occurred regardless of mouse strain, age, and silk thickness. It shows that ligature induced periodontitis couldn’t affect by mouse strain, age and silk thickness.

Tissue staining results were similar in ICR, BALB/C, and C57BL/6 mice. In previous study, damage of gingival epithelium and infiltration of inflammatory cells were reported due to periodontitis-induced [25]. In addition, our previous study also reported damage of gingival epithelium, infiltration of inflammatory cells, unclear connective tissue papillae and vascular changes between the epithelium and the lamina propria in the LIP mouse model [23]. In this study, it was observed such damage around the periodontal tissue in the LIP group of ICR, BALB/C, and C57BL/6 mice. Similar changes in periodontal tissue in the LIP group of ICR, BALB/C, and C57BL/6 mice suggest that all three mice strain could be used as experimental animals in LIP experimental animal model.

In this study, the extent of alveolar bone loss was compared among mouse strains commonly used as a periodontitis model. However, this study could not examine the extent of alveolar bone loss in all mice strain that could be used in this context. Nevertheless, the results of this study suggest that there is no difference in alveolar bone loss between mice when periodontitis-induced study. Therefore, the LIP mice model can be used by selecting a mouse according to the aim of a study, the content of a study and the economic.

This study was supported by the 2020 Scientific Research Program (SR2002) of the Gangneung-Wonju National University Dental Hospital.

The authors declare that they have no competing interests.