Animals.
Two adult M. mulatta monkeys with full permanent dentitions were used. The animals were born and kept in a monkey colony of the Institute of Psychology, Hungarian Academy of Sciences (Budapest). A few weeks before the experiments, the animals were transported and kept in Pecs in the animal house of the Department of Physiology, Faculty of Medicine, Pecs University. The facility is in compliance with applicable US and Hungarian regulations and has a complete 24 h veterinary service. Animal housing, care and the application of experimental procedures were in accordance with the‘Principles of Laboratory Animal Care’ (NIH publication No. 86-23, revised 1985) and the institutional guidelines under an approved protocol. These animals were otherwise experimentally na|ve and were used for neurologic studies. The modified ‘Endodontic Usage Test’ (ISO/TR 7405) (Crstavik & Mjor 1992) was used where the anterior teeth, maxillary premolars and the palatal roots of maxillary molars and the distal roots of mandibular molars were included. Maxillary second premolars were used for over instrumentation and overfilling. Maxillary first premolars were perforated and overfilled at the furcation area. Thus, there were 60 root canals and 4 perforations treated in this study.

Root canal preparation.
The animals were treated under general anaesthesia. The teeth were isolated with rubber dam and the operation area was disinfected with 30% hydrogen peroxide and 5%tincture of iodine. The pulp chamber was opened and the working length was determined radiographically (Trophy Oramatic 708, Vincennes, France). Root canal enlargement was completed using an instrument two sizes greater than the first one that was engaged at length. During instrumentation, each canal was rinsed repeatedly with 0.5% sodium hypochlorite solution. Preparation was completed by ultrasonic cleaning (Cavi- Endo, Dentsply, Milford, DE, USA) for 2 min using 0.5% sodium hypochlorite solution and flushed with sterile physiological saline solution. Overinstrumentation of second premolars was taken 2 mm beyond the radiological apex with Flex-o-files (Maillefer, Ballaigues, Switzerland). The furcal perforations of the first premolars were carried out with sterile 0.8 mm diameter tungsten carbide fissure burs (Maillefer, Ballaigues, Switzerland) extended through the floor of the pulp chamber.

Root canal filling.
The root canals were dried with sterile paper points. The lateral condensation technique was used to fill the canals with standardized and accessory gutta-percha points and AH26 (Dentsply De Trey, Zurich, Switzerland), Apexit (Vivadent, Schaan, Liechtenstein), Endomethasone (Septodont, Saint-Maur, France) or Grossman’s (Sultan Chemists, Englewood, NJ, USA) sealer. The sealers were placed in the canals with sterile K-files. Master gutta-percha points were inserted into the canals to the predetermined length and were condensed with finger spreaders (Kerr Manufacturing Company, Detroit, MI, USA). Sufficient number of accessory gutta-percha points were used to obliterate the canal. In over-instrumented canals, master gutta-percha points were inserted to the radiographically determined working length, and lateral condensation was carried out. No gutta-percha points were inserted into the apical perforations. In cases with furcal perforations, the lateral condensation technique was used to fill the canals. No gutta-percha points were inserted into the perforations. Sealers were used to fill the perforations, and a small portion of zinc-oxide-eugenol cement (Caryosan, Spofa Dental, Praha, Czech Republic) was placed over the sealers. In all cases, the crowns were filled with amalgam (Goodfill 430 NG2,Ogussa Dental, Wien, Austria).

Histology.
Animals were observed over a 6-month period. On sacrifice, fixation in buffered neutral formaldehyde was completed by perfusion of the ascending aorta. The mandible and maxilla were stored in buffered neutral formaldehyde following the removal of soft tissues. Tissue blocks were demineralized, embedded in paraffin and axially oriented serial sections were made. Slices were alternatively stained by haematoxilin and eosin, and haematoxilin and Gram.

Histopathology.
The type of tissue reaction was determined based on the infiltration of inflammatory cells within the periapical tissue. Four types of tissue reactions were determined including suppurative (polymorphonucleic leucocytes), chronic lymphocytic/plasmocytic (lymphocytes and plasma cells), granulomatous (epitheloid and multinucleated giant cells) and necrotic (tissue necrosis). The degree of inflammation was rated according to the criteria described previously (Crstavik &Mjor1992) as mild (few scattered inflammatory cells), moderate (focal accumulation of inflammatory cells without tissue necrosis) and severe (dense infiltration of inflammatory cells, external root resorptionand/orosteolysis with necrosis).

Results.
Sixty root canals and four furcal perforations with associated tissues were histologically evaluated. Thirty-one were excluded because the histologic sections failed to provide a minimum 2.0 mm of periapical tissue or were not longitudinally sectioned. A further case was also excluded from the study because Gramstaining revealed microbial infection in the periapical tissue. The histological scoring system was applied on 24 correctly filled root canals, on eight perforated and overfilled canals and teeth with furcal perforations. The results of histological reactions are summarized in Table 1 and representative examples are demonstrated in Figs 1 and 2.

Table 1. Periapical tissue reactions in Macaca mulatta initiated by four different root canal sealers.

AH26.
Seven root canals were filled within the canal, and two were overfilledwithAH26. In five of the root fillings con fined to the canal, no inflammatory reaction was detected, and in two cases mild lymphocytic/plasmocytic infiltration was seen. In the two overfilled cases, moderate lymphocytic/plasmocytic and granulomatous infiltration was observed around the sealer. Extracellular sealer particles were detected at the centre of the inflammatory reaction. Sealer particles were also seen in the peripheral zones of the inflammation, but in this region all the sealer particles were in the cytoplasm of macrophages. The inflammatory reaction contained no foreign body giant cells (Fig.1).

Figure 1. Periapical tissue reaction in root canal over filled with AH26.
(A) Lymphocyte, plasmacell and macrophage infiltration around sealer particles.
(B) Surplus sealer particles are surrounded by lymphocytes, plasmacells and macrophages.
(C) Peripheral parts of the inflammatory.

Apexit.
Four root canals were filled within the canal system and two were overfilled with Apexit. No inflammatory reaction was observed in the periapical tissues of the cases with no overfilling. In one of the overfilled cases, mild lymphocytic/plasmocytic reaction was detected around the sealer without foreign body giant cells (not shown).

Endomethasone.
Nine root canals were filled with in the root canal system and two were overfilled with Endomethasone. In six of the nine cases without overfilling, no inflammatory tissue reaction was detected. In three additional cases, mild lymphocytic/plasmocytic infiltration was observed. In both the cases that were overfilled with Endomethasone, moderate to severe lymphocytic/plasmocytic infiltration and granulomatous reaction around the sealer particles were detected. In the granulomatous reaction, epitheloid and foreign body-type giant cells sharply demarcated the sealer particles from the periapical tissue (Fig. 2).

Figure 2. Periapical tissue reaction in root canal over filled with Endomethason.
(A) Lymphocytic/plasmocytic and giant cell infiltration around sealer particles.
(B, C) Foreign body giant cells and epitheloid cells separate the sealer from the periapical tissue.

Grossman’s sealer.
Four root canals were filled within the canal system and two root canals were overfilled with Grossman’s sealer. In the cases without overfilling, no inflammatory reaction was detected. In both the overfilled cases, mild to moderate lymphocytic/plasmocytic reaction was observed around the sealer (not shown).