A modified method for inducing periodontitis in dogs using a silk-wire twisted ligature

Periodontal inflammatory disease is one of the most prevalent disorders affecting both humans and small animals [1, 7]. Therefore, several studies have examined the occurrence and treatment of periodontal disease using models of experimentally induced periodontitis. This condition has been induced in rats using a range of methods including pathogen intake, endotoxin injection, high carbohydrate feeding, or ligature placement around the tooth cervix [12, 13, 20]. Compared to rat models, ligature induction of periodontitis is used more frequently in dogs [3, 17, 21].

Ligation materials used to induce periodontitis including silk, cotton, and nylon are placed between the tooth and periodontium to mechanically widen the periodontal pocket and to facilitate plaque accumulation in the dento-gingival region [5]. Several studies have induced experimental periodontitis using a more invasive method in which a full-thickness gingival flap is made and the alveolar bone is destroyed prior to ligation [3, 18]. These methods take several months to promote advanced periodontitis, and thread-type ligatures might be lost due to loosening or wearing during this period. In general, ligatures that are lost during the periodontitis induction phase are immediately replaced to maintain the same induction period and environment [3, 13]. These reparative procedures require redundant general anesthesia. Furthermore, the induction site can be affected by additional manipulations that may act as variables in the experimental procedure. Therefore, the present study was designed to identify more effective methods for periodontitis induction using modified ligatures, which can promote cases of more advanced periodontitis using a less invasive approach and reduce unnecessary interventions compared to previously established techniques.

The protocol used for this study was approved by the Institutional Animal Care and Use Committee (SNU-100208-3; Seoul National University, Korea). Twenty beagle dogs (13 females and 7 males) approximately 1.5 years old were used in this study. All experimental operations and examinations were performed with dogs under general anesthesia induced by a combination of medetomidine (0.01 mg/kg; Orion Pharma, Finland), tramadol (2 mg/kg; Samsung Pharm, Korea), and a commercial solution of zolazepam and tiletamine (2.5 mg/kg; Virbac, France) administered via intramuscular injection. To prepare the healthy gingiva, all teeth were scaled and polished using a piezo-type ultrasonic scaler (BonArt, USA), and tooth brushing was performed once daily without anesthesia for the following 2 weeks. During these periods of prophylactic care, the beagles were fed a hard pellet diet to reduce plaque formation [11].

Experimentally induced periodontitis

Two weeks after scaling, experimental periodontitis was induced on the left upper second premolar (PM2), third premolar (PM3), and fourth premolar (PM4) as well as the left lower PM3, PM4, and first molar (M1). Prior to ligation, the gingival attachment was incised slightly by inserting a number 11 scalpel blade (Ailee, Korea), and the periodontal ligaments were undermined until a periodontal pocket depth reached up to 3 mm with a straight elevator (Osung MND, Korea). After undermining, shallow notches for ligature retention were made in the mesial and distal cervical region of each tooth with a round bur (#1; Komet, Germany). An ancillary notch was placed in the mesial surface of the upper PM4.

The dogs were randomly assigned to four groups. Periodontitis was induced in three groups (Fig. 1) by tying the ligatures around the cervical region of the tooth using dental ligature wire [wire ligature (WL), n = 5; ClassOne Orthodontics, USA)], 2-0 braided silk [silk ligature (SL), n = 5; Ailee, Korea] or a twisted wire with 2-0 silk [silk-wire twisted ligature (SWL), n = 5]. The remaining group did not undergo any surgical intervention but was fed only soft-moistened food as a control (SF, n = 5). For pain control, tramadol (4 mg/kg, intramuscular injection) was administered twice a day for 3 days after placing the ligatures. To promote plaque formation, soft-moistened food was given to all groups for the following 60 days. The ligatures were checked daily and the day the ligature fell out was recorded.

Fig. 1
Ligature materials. (A) 0.012-inch ligature wire, (B) 2-0 braided silk, (C) twisted ligature with silk and wire.

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Periodontitis is a chronic inflammatory reaction that occurs as a consequence of interactions between the host immune system and oral pathogens [8, 14]. Therefore, previous experimental studies investigating periodontal disease have generally used the animal models of periodontitis gradually induced by promoting plaque accumulation [3-5, 9, 11, 15, 18]. The model most similar to naturally occurring cases of periodontitis is one in which this condition is induced by a carbonate-enriched diet consisting of soft food. Nevertheless, the induction period for this method is greater than 5 to 7 month in dogs after clinical gingivitis has developed [9]. In our study, the control group (SF) showed no significant changes in CAL or BoP (factors that can represent destructive periodontal alterations) up to 60 days after the initial SF feeding.

Compared to the previous studies, we found that the use of hygroscopic and braided-type ligatures to promote periodontitis had a shorter induction period in dogs. In some studies, cotton floss with or without wire, which can provoke significant tissue reactions within the periodontal tissue, has been used to induce periodontitis [4, 5, 15, 17, 18]. In contrast to cotton, the degree of tissue reaction to a SL is quite low whereas wire has been shown to produce no reaction [2, 6]. Although tissue reactions resulting from cotton do not directly cause an inflammatory response, we used silk with wire ligatures in the present study to reduce unnecessary environmental changes caused by the ligature.

For ligature-induced periodontitis, the ligature material not only forms a periodontal pocket subject to plaque accumulation but also serves as a constant irritant that can harbor a multitude of oral pathogens [18]. Considering these characteristics, the loss of ligatures that enhance plaque formation might result in the failure to induce experimental periodontitis. For these reasons, other groups performed studies in which shallow notches were created on the mesial and distal cervix of the tooth using a round bur and act as retentive grooves for the ligature [3, 13]. Another study employed a more invasive method in which a full-thickness gingival flap was created to secure the ligature to the alveolar bone crest [18]. In the present study, retentive grooves were made at two points of each tooth cervix to fix the ligature onto the initial location without loosening whereas a gingival flap was not made. Instead of using a relatively invasive method, we performed a closed undermining of the pocket up to a depth of 3 mm to expose the cement-enamel junction.

The period of ligature maintenance was significantly decreased in the SL group whereas all ligatures in the SWL group were maintained for 60 days. These results could be due in part by the fact that the tensile strength of silk sutures in tissue was reduced within 14 days and the relative security of the knot was also poor, whereas stainless steel ligatures were stable in the tissue [6]. The ligature maintenance period of the WL group also decreased compared to the SWL group, although this difference was not significant. The smooth surface of the wire might have cause the ligature to slip and fall out because the wire had been loosened by cervical connective tissue breakdown in the WL group.

Differences in clinical periodontal status varied significantly according to the ligature materials used. The SL and SWL groups, in which ligatures containing silk were used, showed significantly more advanced periodontal destructive changes than the other groups. Furthermore, most periodontal indices, including PI, GI, CAL, BoP, and ABL, were significantly worsened in the SWL group compared to the SL group. Considering the ability of ligatures to harbor oral pathogens while inducing periodontitis as mentioned above [18], these differences between the SL and SWL groups were attributed to the stability and surface area of silk. In the SWL group, the twisted ligature made with silk and wire could not only be firmly secured around the cervix of tooth due to the tension of the wire, but the coiling silk also provided a wide surface that housed a multitude of pathogenic micro-organisms.

Two cases of oral mucosal ulceration were encountered in both of the WL and SWL groups. The ligature used for both these animals included wires, and ulceration was attributed to a twisted knot of wire. Nevertheless, oral ulceration was not observed in all animals with ligatures containing wire. These results indicate that care should be taken when making the knot so as not to directly disturb the oral mucosa.

In summary, we found that ligatures composed of twisted silk and wire were maintained for a significantly longer time than ones made of silk. Furthermore, the SWL induced more significantly advanced cases of experimental periodontitis compared to the other types of ligatures over the same period of time. According to these results, twisted silk and wire ligatures can be an effective method for studying periodontitis in dogs.