Egg yolk immunoglobulins’ impact on experimental periodontitis caused by Porphyromonas gingivalis

Abstract

BACKGROUND:

Periodontitis is a chronic inflammatory disease caused by Porphyromonas gingivalis that leads to a series of periodontal tissue injuries. Egg yolk immunoglobulins (IgY) is procuded in egg yolk and inhibits P. gingivalis.

OBJECTIVE:

The aim was to evaluate the effect of IgY on experimental periodontitis caused by P. gingivalis.

METHODS:

The second molars of rats were ligatured using medical 5-0 silk and smeared with P. gingivalis to induce experimental periodontitis. Then, the rats were smeared with 2 mL IgY solutions or 0.9% NaCl in the oral cavity for up to 4 wk. The scores for gingival index, plaque index and probe on bleeding, the levels of IL-6 and TNF- $\alpha$ , X-ray radiography and histology were used to determine the efficacy of the IgY on experimental periodontitis.

RESULTS:

The clinical indices improved; the levels of IL-6 and TNF- $\alpha$ were significantly ( $p<$ 0.05) decreased; the X-rays and histomorphological observations suggested that the periodontal inflammation and periodontitis were alleviated compared to the control.

CONCLUSIONS:

IgY showed significant effects on anti-inflammatory, anti-coaggregation activity, and protected against alveolar bone loss. Therefore, it had a beneficial effect on preventing experimental periodontitis caused by P. gingivalis.

Keywords

Periodontitis egg yolk immunoglobulins IgY Porphyromonas gingivalis IL-6 TNF-α

1. Introduction

Periodontitis is ranked among the 10 most prevalent chronic diseases worldwide, and is considered a major public health problem [1]. It is a chronic non-specific inflammatory disease that compromises the integrity of the periodontium, periodontal ligament, alveolar bone, tooth cementum and other supporting tissues, and originates from microbial infection [2, 3]. Clinical manifestations of periodontitis include swollen and bleeding gums, and damage to the gums and teeth including periodontal pockets, periodontal atrophy, root exposures, alveolar bone loss and loose teeth [4].

Using the human oral microbe identification micro-array (HOMIM), subgingival biofilms showed the presence of Porphyromonas endodontalis/Porphyromonas spp. and Tannerella forsythia, and the absence of Neisseria polysaccharea and Prevotella denticola [5]. Porphyromonas gingivalis was considered to be responsible for more than 95% of chronic periodontitis lesions. It is one of the main flora in the mouth [6]. It acts as a keystone pathogen at low colonization levels, and specifically induces the conversion from a benign symbiotic community structure to a symbiotic community capable of causing destructive inflammations and periodontal bone loss [7, 8, 9]. It was reported that P. gingivalis induced cellular necroptosis through its effect on the receptor-interacting protein 1 (RIP1) and protein 3 (RIP3), which affected the mixed lineage kinase domain-like (MLKL) signaling pathway in animal models. Animals pre-treated with an MLKL inhibitor showed significantly enhanced P. gingivalis clearance [10]. These findings may suggested that P. gingivalis has an essential role in necroptosis process during periodontitis.

Therefore, P. gingivalis has become the main target of microbiology and immunology research on periodontitis. The most common treatment to inhibit the growth of P. gingivalis has been to use antibiotics, such as metronidazole and tetracycline, in patients with chronic periodontitis [11]. However, this has become inconsistent with the concerns about antibiotic resistance [12]. Schmuch et al. reported that a proanthocyanidin-enriched extract from Rumex acetosa that has been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue inhibited the activity of Arg-gingipain and hemaglutinin from P. gingivalis and decreased P. gingivalis adhesion to KB cells [13]. McIntosh et al. also reported that the CXC-chemokine receptor-4 (CXCR4) of P. gingivalis instigated a subversive crosstalk with the Toll-like receptor 2 that inhibited leukocyte killing of this periodontal pathogen. Thus, a CXCR4 antagonist should inhibit P. gingivalis-induced periodontitis [14].

Egg yolk immunoglobulin (IgY) has been shown to inhibit P. gingivalis in vitro. Purified IgY significantly inhibited the hemagglutinating activity of P. gingivalis [15], and the co-aggregation of P. gingivalis with Streptococcus gordonii [16]. Hens that have developed the appropriate immunity could produce specific antibodies against P. gingivalis. Egg yolk can concentrate and aggregate IgY antibodies leading to higher titers [17]. IgY has been shown to be safe with no acute or chronic toxicities [18]. It is also effective and easy to prepare so it could be a candidate for clinical prevention and treatment of periodontitis [19, 20].

The purpose of this study was to build an experimental periodontitis model using Sprague-Dawley (SD) rats induced by P. gingivalis, and to evaluate the effects of IgY using the gingival index (GI), the plaque index (PI) and bleeding on probing (BOP), the levels of IL-6 and TNF- $\alpha$ , X-ray radiography and histomorphological observations.

2. Materials and methods

2.1 Materials

IgY specific for P. gingivalis (25600 titer, 370 mmol/L, according to the manufacturer) was purchased from Maxam Ltd. (Shanghai, China), and P. gingivalis (ATCC33277) was purchased from the American Type Culture Collection (Manassas, VA, USA). All other chemicals were analytical reagent grade or better from Chinese suppliers, including phosphate, agar, pentobarbital sodium, sucrose, ethylenediaminetetraacetic acid, paraformaldehyde, hematoxylin, eosin, etc.

2.2 Preparation of P. gingivalis

P. gingivalis was streaked onto blood agar plates (Sangon Biotech Co., Ltd., Shanghai, China) and cultured at 37 ${}^{\circ}$ C for 48 h using an anaerobic package (Anaero Pack, Mitsubishi Gas Chemical Co., Tokyo, Japan). A single colony was inoculated into 5 mL of brain/heart infusion broth (Shandong Haibo Technology Information System Co., Ltd., Qingdao, Shandong, China) and grown for 24 h with anaerobic conditions. The bacteria were counted using a hemocytometer (Shanghai Refined Biochemical Reagent Instrument Co., Ltd., Shanghai, China). The bacterial culture solution was diluted with sterile sodium phosphate buffer saline (50 mmol/L, pH 7.4) and supplemented with carboxymethocel to give a suspension with 10 ${}^{8}$ CFU/mL.

2.3 Animal models

Healthy male SD rats ( $n=$ 26, weight 160 $\pm$ 10 g) were purchased from Slac Laboratory Animal Co., Ltd. (Shanghai, China) and raised in a specific pathogen free (SPF) animal room. The use of animals, ethical clearance, and the study protocol were approved by the Ethics Committee of Shanghai Ocean University (Shanghai, China). After intraperitoneal injection of 3% sodium pentobarbital anesthesia (1 mg/kg), maxillary second molars of the rats were ligatured with 5-0 silk thread (Sangon Biotech Co., Ltd., Shanghai, China) and inoculated with freshly prepared P. gingivalis (10 ${}^{8}$ CFU/mL). The liquid was switched to tap water instead of the 10% sugar water used prior to ligation. The rats were challenged with P. gingivalis for 4 wk every two days [21]. Two rats were randomly sacrificed using spinal dislocation after 4 wk. maxillas were measured using X-ray photography and pathological sections to determine whether the rats had become infected.

2.4 Animal experiments

The infected rats were randomly divided into three groups ( $n=$ 8). After 4 wk the oral cavity was smeared using a syringe with 0.2 mL specific IgY (370 mmol/L) in high-dose rats, 0.2 mL specific IgY (3.7 mmol/L) in low-dose rats, and 0.2 mL non-specific IgY (Sangon Biotech Co., Ltd., Shanghai, China) each day in control rats for up to 4 wk.

2.5 Oral test

The gingival index (GI), plaque index (PI), and bleeding on probing (BOP) were measured using a periodontal probe [22]. Briefly, the periodontal pockets or gingival crevices of the ligated second molar were measured using a probe and graded as follows [23]: Score 0: Gingiva is healthy; no dental plaque; no bleeding after a slight probe. Score 1: Gingiva is mildly inflamed; small amounts of dental plaques are observed after scraping using a probe; occurrence of punctate hemorrhages after a slight probe. Score 2: Gingiva is moderately inflamed; a number of dental plaques are observed after scraping using a probe; bleeding after a slight probe. Score 3: Gingival are severely inflamed; a number of dental plaques are visually observed without using a probe; spontaneous bleeding.

2.6 IL-6 and TNF- $\alpha$ assay

Blood sampling from the heart was done immediately after the rats were put down. Then, the serum was obtained using centrifugation at 500 g (2500 rpm, 5424R, Eppendorf, Mittelsachsen, Saxony, Germany) for 10 min and stored at $-$ 20 ${}^{\circ}$ C until use, a maximum of 4 wk. The levels of IL-6 and TNF- $\alpha$ were quantified using an enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China).

2.7 Radiology

The maxillas were dissected from the rats after 4 wk and fixed in 4% paraformaldehyde sodium phosphate buffer for 48 h and analyzed using a medical X-ray radiograph (Faxitron MX-20DC12 system; Faxitron Bioptics, Lincolnshire, IL, USA).

2.8 Histomorphology

The dissected samples were fixed in the 4% paraformaldehyde phosphate buffer for 48 h. The fixed samples were then demineralized in 8% EDTA containing sucrose (1.8 mol/L, pH 7.4) at 4 ${}^{\circ}$ C for 2 wk. The tissues were embedded in paraffin and serial 4 $\mu$ m sections were prepared using a microtome (Leica Biosystems Inc., Buffalo Grove, IL, USA). Sections were stained with hematoxylin and eosin (HE) (Sangon Biotech Co., Ltd., Shanghai, China). Then sections were observed with a microscopethey (Olympus Corp., Shinjuku, Tokyo) to evaluate bone destruction and soft tissue changes compares with control.

2.9 Statistical analysis

Statistical analysis was done using SPSS 18.0 (SPSS Inc., Chicago, IL, USA) for Windows. Data are shown as mean $\pm$ SD and level of significance was evaluated using one-way ANOVA with a two-tailed Student’s test. The differences were considered significant at $p<$ 0.05.

3. Results and discussion

3.1 Evaluating the animal models

In normal SD rats, the gum was pink, and the gingival margin was thin and tightly affixed to the tooth surface. After 4 wk of infection, a large amounts of chyme surrounded the upper second molars, and periodontium in the upper jaw showed erosion and atrophy. The X-ray photographs showed a larger space in the interdental region (Fig. 1). The pathological sections showed a loss in alveolar bone and an increase in the number of inflammatory cells in the second molars (Fig. 1).

Figure 1.

X-ray photographs and pathological sections of maxilla in experiment periodontitis rats. The upper picture is the X-ray photograph; the left lower side is the pathological section of the periodontium; the right lower side is the pathological section of an alveolar bone. The white arrows point to radiolucent space in the interdental region; the black arrows point to inflammatory cell infiltration; *points to the cemento-enamel junction (CEJ). Sections were stained with haematoxylin and eosin (H&E), observed with a microscope ( $\times$ 200).

Rats are often used in models of experimental periodontitis because periodontal anatomy in the molar region shares some similarities with that of humans. Furthermore, rats are easy to handle and can be obtained with different genomes and microbial status [24]. Inoculations or injections of periodontal pathogens P. gingivalis can lead to periodontal lesions [25]. In this study, the results of the X-ray photographs and pathological sections showed a remarkable loss of the alveolar bone and disruption of the periodontal ligaments in the experimental periodontitis rats. They reflected the success of the experimental periodontitis model to show that P. gingivalis can lead to periodontitis [22].

3.2 Effect of IgY on PI, GI, and BOP

A significant ( $p<$ 0.05) decrease was observed in the indices in IgY-treated groups compared to control (Fig. 2). More specifically, the score for PI in control was 2.75, which decreased to 0.50 and 0.75 in the high-dose and low-dose group, with mean percentage reductions of 82 and 73%, respectively. The score for GI in control was 2.5, which decreased to 0.25 and 0.5 in the high-dose and low-dose group, with mean percentage reductions of 90 and 80%, respectively. The score for BOP in control was 2, which decreased to 0 and 0.25 in the high-dose and low-dose group, with mean percentage reductions of 100 and 88%, respectively.

Figure 2.

The changes of gingival index (GI), plaque index (PI), and bleeding on probing (BOP). The scores for gingival index (GI), plaque index (PI), and bleeding on probing (BOP) are shown as mean $\pm$ SD ( $n=$ 8). *means $p<$ 0.05 compared to control.

Figure 3.

The levels of IL-6 and TNF- $\alpha$ . The levels of IL-6 and TNF- $\alpha$ was measured using an ELISA Kit. Data are shown as mean $\pm$ SD ( $n=$ 8). *means $p<$ 0.05 compared to control.

The PI, GI and BOP were used to diagnose periodontitis and monitor traditional parameters [26]. PI measured the number and size of dental plaque biofilm on teeth. It suggested the occurrence of co-aggregation of microorganism. GI measures the color and degree of swelling in the gingival, which suggested that the inflammation response was induced by the bacteria. BOP measures whether the gingival bleeds when a probe presses on it, which can be used to grade gingival destruction [27]. These indicators reflected the success of P. gingivalis infection in inducing periodontitis. The scores for PI, GI and BOP were significantly ( $p<$ 0.05) decreased compared to control. It suggested that IgY had a positive effect on periodontitis recovery. Hou et al. reported that gingivitis rats treated with the IgY specific to P. intermedia showed significantly decreased GI, PI and BOP, similar to this study [28].

3.3 Effect of IgY on IL-6 and TNF-

\alpha

Abnormally high levels of inflammatory factors were seen in controls. After treatment with IgY for 4 wk, the levels of IL-6 and TNF- $\alpha$ in the low-dose group decreased significantly ( $p<$ 0.05) compared to control. IL-6 and TNF- $\alpha$ in rat serum promote the production of inflammatory factors [22]. These markers can be studied both in vitro and in vivo to learn more about the mechanism. The experimental rats’ serum stimulated a pro-inflammatory response induced by infection of P. gingivalis, while the treatment with IgY decreased the levels of IL-6 and TNF- $\alpha$ and shifted the immune response towards an anti-inflammatory response [29]. The levels of IL-6 and TNF- $\alpha$ were increased with the formation of periodontitis, and reduced when periodontitis was mitigated [30]. Thus, the levels of IL-6 and TNF- $\alpha$ were chosen to evaluate the protective effect of IgY on periodontitis.

3.4 X-ray radiography of maxilla in experimental induced periodontitis rats

The X-ray photographs showed the destruction of alveolar bone and widening of the interdental space with periodontitis rats [31]. After treatment with IgY for 4 wk, the teeth that were arranged closely were asymptomatic compared to the controls (Fig. 4). Specifically, there was no space or little space in the high-dose or low-dose groups, respectively.

Figure 4.

X-ray photographs of maxilla in experiment periodontitis rats. The images from left to right are the control, the high-dose, and the low-dose groups. The arrows point to radiolucent space in the interdental region.

Figure 5.

Pathological sections of periodontium in experiment periodontitis rats. The left side is the control group; the middle is the high-dose (370 mmol/L) group; and the right side is the low-dose (3.7 mmol/L) group. The black arrows point to inflammatory cell infiltration. Sections were stained with H&E, observed with a microscope ( $\times$ 200).

Periodontal ultrasonography [32] and $\mu$ -computed tomography (CT) [31] have also been used to evaluate and monitor periodontal tissues, as they give information similar to that obtained with X-ray photography. Furthermore, the correlations, which are obtained between the alveolar bone loss and the gingival size, could suggest that there was an accelerated bone resorption when periodontal inflammation persisted [33]. Thereby reducing space in the interdental region suggested that the periodontal inflammation was being alleviated and periodontitis was decreasing.

3.5 Histomorphological observations of periodontium and alveolar bones in experimental periodontitis rats

Severe inflammatory cell infiltration was observed in periodontal tissues as well as a disordered arrangement of collagen fiber bundle and severe interdental alveolar bone loss in the control group. X-ray photographs and pathological sections were used to evaluate the gingival inflammation and destruction [32]. Biju et al. also reported connective tissue breakdown, loss of attachment, and alveolar bone resorption in periodontal disease patients [34]. After 4 wk of treatment, the inflammatory infiltration and periodontal fiber disorganization improved in pathological sections (Fig. 5). Compared to the low-dose group, the maxillas in the high-dose group showed fewer inflammatory cells and less alveolar bone loss (Fig. 6). In vivo attenuation of bone loss and accretion of osteocalcin suggested an increase in the presence of mature osteoblasts and a reduction of osteoclasts numbers [35]. The results in this study showed that alveolar bone was increasing bone mass and bone mineralization. Finally, experimental periodontitis recovered gradually.

4. Conclusion

P. gingivalis is one of the major pathogens that triggers innate, inflammatory, and adaptive immune responses. These processes result in the destruction of the tissues surrounding and supporting the teeth, and eventually result in tissue, bone, and, finally, tooth loss with periodontitis [36, 37]. Based on the pathogenesis of periodontitis, a successful experiment periodontitis model was developed using SD rats. Treatment with IgY for 4 wk led to improved clinical indices. The levels of IL-6 and TNF- $\alpha$ were significantly ( $p<$ 0.05) decreased. The X-ray photographs showed the teeth were arranged closely with no or little space in the interdental region. Histomorphological observation showed inflammatory infiltrates and periodontal fiber disorganization improved compared to the control, which suggested that the experimental periodontitis was gradually being reversed.

Figure 6.

Pathological sections of alveolar bone in experiment periodontitis rats. The left side is the control group; the middle is the high-dose (370 mmol/L) group; and the right is the low-dose (3.7 mmol/L) group. *points to the cemento-enamel junction (CEJ). Sections were stained with H&E, observed with a microscope ( $\times$ 200).

In summary, IgY had a positive effect on recovery from periodontitis. Treatment with IgY for 4 wk showed the remarkable effects on anti-inflammatory and anti-coaggregation activity of microorganisms, and the protection against alveolar bone loss. It can be hypothesized that IgY combined with the surface antigen receptors inducible P. gingivalis apoptosis pathway [10, 38] decreased the infectivity of P. gingivalis in the experimental periodontitis. Thus, specific antibody containing IgY applications in the prevention and treatment of periodontitis may be possible after further research.

Footnotes

Conflict of interest

None to report.

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Egg yolk immunoglobulins’ impact on experimental periodontitis caused by Porphyromonas gingivalis

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Materials

2.2 Preparation of P. gingivalis

2.3 Animal models

2.4 Animal experiments

2.5 Oral test

2.6 IL-6 and TNF- α assay

2.7 Radiology

2.8 Histomorphology

2.9 Statistical analysis

3. Results and discussion

3.1 Evaluating the animal models

3.4 X-ray radiography of maxilla in experimental induced periodontitis rats

4. Conclusion

Footnotes

Conflict of interest

References

2.6 IL-6 and TNF- $\alpha$ assay