Effective Modalities of Periodontitis Induction in Rat Model

The ligature method is one of the more commonly used procedures for the experimental induction of periodontal disease in rats.²³ The placement of a ligature, preferably using a suture or wire, acts as a local irritant, which contributes to the accumulation of dental plaque and ulceration of the sulcular epithelium, which then enables the invasion of periodontal pathogens into the connective tissue.²⁴ Bacterial plaque formation due to ligature placement initiates gingival inflammation, which leads to periodontal tissue destruction and initiates alveolar bone resorption, which is similar to what happens in humans.²⁴ However, some researchers claimed that the absence of bacteria in the ligature precludes periodontal disease, and in such cases, bone loss has been observed as a result of direct mechanical trauma.^23,25 In response to this argument, studies have shown that the orthodontic wire works as a plaque retentive factor to initiate gingival inflammation.²¹ Recently, a study showed that the placement of orthodontic wire causes the accumulation of endogenous oral microbiota, which then leads to gingival inflammation and alveolar bone loss.²⁶ However, in another study, it was shown that no gingival tissue or bone damage was observed in the micro-CT image after 24 h of ligature wire placement, which indicates low possibility of mechanical trauma induction.²⁶ Nevertheless, in the ligature model, the bone destruction process is rapid, severe, and usually observed to be greater than in other areas. Thus, several researchers have considered the ligature technique to be an acute model of periodontal disease induction.^25,27

Non-absorbable sterile 2/0^a, 3/0^b, 5/0^c cotton, 4/0 silk^d and nylon sutures are usually used, and these are placed around the maxillary or mandibular teeth in the suture-ligation technique. Upon placement, the suture is applied and tied gently to prevent damage to the periodontal tissue.²¹ Usually, a ligature is placed through the proximal contacts of the selected tooth (Figure 1a). The suture is passed around the crown of the tooth sub gingivally (Figure 1b) and tied firmly with a double knot on the buccal side, labial side, or mesial side.²⁸ The ligature method is the most popular for experimental periodontitis induction because it is inexpensive and suture materials are easily available. Moreover, periodontitis can be promptly induced in a specific site of the oral cavity.²⁹ However, special skill is needed to apply this method on rats as inserting the ligature into the interproximal spaces between rat molars can be challenging due to the small size of the tooth, limitation in viewing the sulcus region, and the tiny oral space in which to perform knotting and insertion. Additional instruments are also needed, for example a mouth gag, to keep the mouth cavity open for ligature placement. A major drawback of this technique is that the suture is easily torn due to the force applied to place the ligatures. Moreover, frequent monitoring is needed because it could be easily displaced due to tongue movement during mastication. While the ligature method by suture is still commonly used, several studies have recently focused on using orthodontic wire instead of suture. Orthodontic wire (0.2 mm^e, 0.4 mm^f, 0.5 mm^f) can be placed into the interdental space by using a needle holder and forceps (Figure 2). Before insertion of orthodontic wire, it is necessary to make a space in the interdental area to prevent damage to the gingival sulcus. A study has demonstrated a modified method for wire placement, where #8 and #10 endodontic files^g are bent 90° from the tip and inserted carefully into the interdental area by push and pull movements to make a space for wire insertion. Compared to passing the sutures into the interdental space, the wire can be easily inserted into a similar site.²⁶ Orthodontic ligature wire is also economical, feasible and can be placed by a single person within a short period of time. Moreover, the wire can be clamped with the tip of the needle holder properly and is less likely to be displaced by tongue movement. Also, less technical skill is required for wire placement, and maintaining position of the wire is much easier compared to using a suture. However, the wire needs to be carefully inserted, as any excessive force could damage the gingival tissue and lead to bleeding. Table 1 shows the different ligation techniques to induce experimental periodontal disease in rats.

In the oral inoculation method, monomicrobial or polymicrobial mediums are used for periodontal pathogen inoculation.³⁸ In this method, individual isolates of P. gingivalis^h,i,j, A. actinomycetemcomitans^k,l, F. nucleatum^m, T. forsythiaⁿ, and T. denticola^o or a combination of different bacterial strains are commonly used for bacterial inoculation.⁶ Among these bacterial species, inoculation of P. gingivalis alone or in combination with other bacteria is considered a significant causative agent for the oral dysbiosis process because of its strong ability to initiate periodontal bone destruction.³⁹ This bacterium can locally invade periodontal tissue and is able to affect the host cell, immune cells and the cells responsible for bone remodelling.⁴⁰ It can survive in the oral environment via multiple virulence factors, which are fimbriae, gingipains, LPSs, capsules, and hemagglutinins.⁴⁰

In the polymicrobial inoculation model, greater bone loss is observed compared to the monomicrobial inoculation model. A previous study demonstrated that polymicrobial infection by P. gingivalis and T. forsythia significantly increased maxillary, mandibular, and total vertical alveolar bone loss compared to monomicrobial infection.⁴¹ Moreover, marked apical migration of junctional epithelium and enhanced alveolar bone destruction were observed in that previous study.⁴² Another study reported that mixed inoculation with P. gingivalis and T. denticola induces marked interproximal as well as horizontal alveolar bone resorption compared to monomicrobial infection.⁴³ Meanwhile, a mixed oral infection model with P. gingivalis, T. forsythia and T. denticola demonstrates a lower serum IgG level compared to oral monomicrobial infection, which suggests alteration of host responses, which is due to the evasion of protective immune responses. In addition, a significant amount of maxillary and mandibular bone resorption was reported by this bacterial consortium.⁴⁴ As shown in Table 2, high bacterial concentrations were used, which varied from 10⁸ to 10¹² cells/ml, depending on the preparation route, either mixed with 2% carboxymethylcellulose or phosphate buffered saline and administered via food and drinking water. This induction method does not require general anesthesia or a special operating procedure, which allows it to be performed by a single operator. In addition, minimal observation of the induction site is needed. However, one of the significant issues with this method is the complex bacterial interaction that can occur. For example, in the P. gingivalis monomicrobial infection, it was shown that this bacterium collaborates with other endogenous bacterial species and may modify the oral biofilm, which contributes to the periodontal destruction process rather than producing a direct effect on its own.²⁴ In addition, the induction period by this method requires a minimum of 4 weeks to produce some significant results.

In oral bacterial inoculation, the bacteria contributed to leukocyte migration into the gingival connective tissue. A study has reported that inoculation of bacteria induces migration of leukocytes (mononuclear cells and neutrophils) into the gingival connective tissue and apical migration of the junctional epithelium, as shown by histological examination (H&E staining).⁴⁹ An increased level of inflammatory cytokines, such as TNF-α, IL-12, IFN-γ, within the gingival tissue in response to oral bacterial inoculation was detected by another study via a real time PCR assay.⁵⁰ Moreover, research has shown that mono and mixed bacterial inoculation induces the secretion of IL-1beta, IL-6, IL-8, PGE2, MMP-9 in response to a bacterial challenge.⁴⁴

LPS is a major constituent of the outer membrane of Gram-negative bacteria that is recognized by the immune system as a marker for bacterial pathogen invasion.⁵¹ Several methods have been used for the isolation and purification of bacterial LPS, including trichloroacetic acid extraction at 4 °C, extraction in aqueous butanol, triton/Mg+2, cold ethanol, extraction in water 100 °C, phenol, chloroform, petroleum ether, and methanol.⁵² Among these methods, hot phenol water and phenol chloroform extraction methods are widely used to extract LPS from various bacterial strains.⁵¹ The LPS from periodontal pathogens is a potential endotoxin that provides persistent inflammatory stimulation.⁵³ It is a significant stimulus that triggers a natural immune response when injected into the gingival tissue.²⁴ Many studies have demonstrated that bacterial LPS induces the expression of inflammatory cytokines and initiates osteoclastic activity, thus leading to bone destruction, which is the key feature of periodontitis.²²

In this technique, a micro syringe^p (28-33G) was used to inject a measured amount of purified bacterial LPS suspended in microvolumes (1-10 μl) into the gingival tissue (Figure 3).²⁴ Preferred injection sites can be the palatal or lingual area of the molar region by approaching from the opposite direction for better access. The injection can be administered 1 to 3 times per week, depending on the experimental design. Bacterial LPS can be injected at single or multiple sites into the sulcus area based on the experiment protocol.

Inflammation and bone resorption have been observed after different induction times depending on the volume of bacterial suspension injected into the site, as shown in Table 3. Several studies reported bone loss, which can be observed as early as 7 days after the first injection.²⁴ The most important advantage of the LPS injection method is that periodontitis can be induced within a short time due to the direct delivery of virulent pathogenic stimuli into the tissue. The method also allows greater control over induction. Moreover, inflammation and bone resorption in the targeted area can be achieved due to specific site administration of LPS.

Periodontal disease is the most common disease in cats and dogs, with incidence rates of 70% and 80%, respectively, in 2-year-old animals.⁵⁸ Gram-negative bacteria, such as A. actinomycetemcomitans, T. forsythia, Campylobacter spp., Capnocytophoga spp., E. corrodens, P. gingivalis, P. intermedia, and T. denticola, can contribute to subgingival plaque formation.⁵⁹ Although there are many bacterial species found in both dog and human dental plaque, there are considerable differences. While humans are known to carry the catalase-negative bacteria P. gingivalis, dogs are thought to have catalase-positive P. gingivalis-like species, for example P. canoris, P. salivosa, P. denticanis, and P. gulae.⁶⁰ Interestingly, a study using next generation sequencing (NGS) has found that Gram-negative bacteria are the most frequent in dogs with healthy periodontal status, whereas Gram-positive bacteria are mostly abundant in periodontitis, which is the opposite of the finding in humans.⁶¹ Moreover, although dogs may be an excellent model for studying experimental periodontitis due to their similar histological traits to humans,⁶¹ their cost and animal care requirements make their use in periodontal research less feasible.¹³ In cats, anaerobic bacteria, mostly from the genera Bacteroides and Fusobacterium, dominate the typical oral microflora. However, Porphyromonas spp. have been isolated from the dental plaque of cats with periodontal disease.⁵⁸ Furthermore, similar to dogs, certain studies have found that around 75% of subgingival bacteria in cats with good oral health are Gram-negative bacteria. The percent of Gram-positive bacteria only increased when periodontal health deteriorated to the disease stage.⁶² Another study has reported that Prevotella intermedia, A. actinomycetemcomitans, Porphyromonas gulae, and Prevotella nigrescens were shown to be substantially more common in cats than in dogs.⁵⁹

In horses, the naturally occurring oral diseases include buccal abrasion, calculus, gingival recession, and periodontal pockets.¹³ According to a recent equine survey, the prevalence of periodontal pockets and gingival recession is higher in older horses and mostly associated with other dental disorders and tooth loss.⁶³ Horses are not a feasible model for basic science research on periodontitis or the testing of prospective therapeutics due to husbandry constraints. Meanwhile, periodontitis has been induced in minipigs within 4 to 8 weeks by ligatures in combination with bacterial inoculations of P. gingivalis, S. mutans, and A. actinomycetemcomitans.⁶⁴ Minipigs can be suitable for periodontal as well as oromaxillo-facial investigations. However, minipigs are rather costly, and again, husbandry concerns make this model less suitable. Nonhuman primates have human-like oral structures and teeth, as well as naturally occurring dental plaque, calculus, oral microbial infections (eg, P. gingivalis), and periodontal disease.⁶⁵ Although periodontitis in primates most closely resembles the human disease, the expense and special husbandry requirements for these animals limit their use in periodontal studies.