Dental Materials Journal 2011; 30(4): 485–492

Healing of experimental apical periodontitis after apicoectomy using different sealing materials on the resected root end Kaori OTANI1, Tsutomu SUGAYA1, Mahito TOMITA2, Yukiko HASEGAWA3, Hirofumi MIYAJI1, Taichi TENKUMO1, Saori TANAKA1, Youji MOTOKI1, Yasuhiro TAKANAWA1 and Masamitsu KAWANAMI1

1Department of Periodontology and Endodontology, Division of Oral Health Science, Hokkaido University Graduate School of Dental Medicine, N13 W7 Kita-ku, Sapporo 060-8586, Japan 2Dental Office Mahito, 2-2 Kawanacho, Showa-ku, Nagoya 466-0856, Japan 3Kinikyo Sapporo Dental Clinic, 7-25 Kikusui 4-1, Shiroishi-ku, Sapporo 003-0804, Japan Corresponding author, Kaori OTANI; E-mail: [email protected]

This study evaluated apical periodontal healing after root-end sealing using 4-META/MMA-TBB resin (SB), and root-end filling using reinforced cement (EBA) or mineral trioxide aggregate (MTA) when root canal infection persisted. Apical periodontitis was induced in mandibular premolars of beagles by contaminating the root canals with dental plaque. After 1 month, in the SB group, SB was applied to the resected surface following apicoectomy. In the EBA and MTA groups, a root-end cavity was prepared and filled with EBA or MTA. In the control group, the root-end was not filled. Fourteen weeks after surgery, histological and radiographic analyses in a beagle model were performed. The bone defect area in the SB, EBA and MTA groups was significantly smaller than that in the control group. The result indicated that root-end sealing using SB and root-end filling using EBA or MTA are significantly better than control.

Keywords: Root-end sealing, 4-META/MMA-TBB resin, Apicoectomy, Retrograde, Root-end filling

microleakage by sealing both the root canal and dentinal INTRODUCTION tubules13). Rud et al. reported good results using Gluma® The spread of bacteria from the root canal to surrounding (Gluma desensitizer, Heraeus Kulzer G.m.b.H., tissues should be prevented when treating apical lesions. Werheim, Germany) and Retroplast™ (Retroplast When conventional has failed, or Trading, Rønne, Denmark)14-17). However, resin is known when orthograde retreatment is not feasible because the to be sensitive to moisture during application. root canal is obliterated, severely curved, or contains a Contamination of the resected surface with blood and fractured instrument or a large post/core, endodontic humidity in expiration during the application procedure surgical procedures like apicoectomy and intentional causes inferior bonding and resin detachment. reimplantation can be considered. Root-end filling should Since 4-META/MMA-TBB resin adheres to dentin be performed properly for the success of endodontic both in vitro and in vivo18), it has been widely used in surgical procedures, such as apicoectomy. Regrettably, orthopedics and prosthetic . In our previous however, conventional retrograde root filling is not studies, 4-META/MMA-TBB resin showed a number of always successful. advantages, such as high adhesiveness to dentin and A variety of materials have been used as root-end cementum19). According to Tanaka et al.20), dye leakage of filling materials. When infected substances are present root-ends sealed without cavity preparation using in the root canal, it is important that the retrograde 4-META/MMA-TBB resin was significantly less than filling prevents leakage of these substances from the root that of root-end fillings using amalgam and reinforced canal to the periapical tissue. Some reports have zinc oxide eugenol cement. suggested that microleakage is not completely inhibited Some reports have suggested that 4-META/ in vitro with amalgam, zinc oxide eugenol cement or MMA-TBB resin offers good biocompatibility after glass-ionomer cement1-7). Microleakage with mineral complete curing21-24). Furthermore, 4-META/MMA-TBB trioxide aggregate was significantly less than that with resin has a high adhesiveness and biocompatibility to amalgam, gutta-percha and zinc oxide eugenol in a dye periodontal tissue when used for bonding fractured leakage test7-10), and mineral trioxide aggregate showed teeth25,26). Thus, 4-META/MMA-TBB resin might be significantly less leakage than reinforced zinc oxide suitable for retrograde root sealing following apicoectomy eugenol cement when evaluated using a bacterial leakage or intentional replantation27,28). system11). However, microleakage of mineral trioxide In this study, we evaluated apical periodontal aggregate has been reported to increase with time12). healing after root-end sealing using 4-META/MMA-TBB Applying a bonding agent to a resected root end resin and root-end filling using reinforced zinc oxide without cavity preparation has been shown to reduce eugenol cement or mineral trioxide aggregate when root canal infection persisted.

Color figures can be viewed in the online issue, which is avail- able at J-STAGE. Received Sep 28, 2010: Accepted Mar 29, 2011 doi:10.4012/dmj.2010-158 JOI JST.JSTAGE/dmj/2010-158 486 Dent Mater J 2011; 30(4): 485–492

After the surgery, the apical morphology and MATERIALS AND METHODS completely filled apical cavity were checked ona Animals radiograph. Five healthy female beagle dogs, 27–28 months old, were The animals received ampicillin sodium (300 mg/kg, used in this experiment. Seventy roots from forty Viccillin®, Meiji Seika) daily for 3 days after the surgery. mandibular premolars were used. The experimental protocol (No. 06013) followed the guidelines for the care Classification of the healing results and use of laboratory animals of the Graduate School of The area of periradicular radiolucency was measured Medicine, Hokkaido University. using Scion Image® (National Institute of Health). Two-by-two analyses were performed by Wilcoxon test Surgical procedure and Mann-Whitney U test with Bonferroni correction. P Surgical procedures were performed under general values were considered statistically significant at <0.01 anesthesia with medetomidine hydrochloride (0.1 mL/ and <0.05. All statistical procedures were performed kg, Domitor® Meiji Seika, Tokyo, Japan) and ketamine using SPSS 10.0 J. hydrochloride (0.1 mL/kg, Ketaral 50® Sankyo, Tokyo, Healing was also evaluated clinically and Japan), and local anesthesia with lidocaine hydrochloride radiographically 14 weeks after the surgery. The area of (2% with 1:80,000 epinephrine, Xylocaine®, DENTSPLY periradicular radiolucency at 14 weeks was compared SANKIN, Tokyo, Japan). with that at baseline. Periapical healing of the roots was The pulp tissue was removed and dental plaque was evaluated according to the criteria by Rud et al.16,29) as placed into the canals and sealed with glass ionomer follows. Complete healing: Complete bone regeneration cement (Fuji IXGP® GC, Tokyo, Japan). Apical around the apex with or without a recognizable periodontitis developed in 1 month and was verified periodontal space. Incomplete healing: A periapical radiographically (baseline). The area of periradicular rarefaction with an irregular, often asymmetrical outline radiolucency was evaluated using Scion Image® (National and an angular connection to the periodontal ligament. Institute of Health, Frederick, MD, USA). Uncertain healing: Uncertain healing or cases After reflecting a mucoperiosteal flap on the buccal undergoing phases of healing. A rarefaction located side, the root ends were resected and inflammatory symmetrically around the apex with funnel-shaped tissue was removed. To access the apical lesion, the connection to the periodontal ligament space and which alveolar bone on the buccal side with a diameter of 5 mm gradually decreased compared to postoperative was removed using a rotating round bur under water radiograph. Unsatisfactory healing: The same irrigation. The teeth were randomly divided into 4 radiographic signs as for uncertain healing, except that groups based on the periapical treatment: 4-META/ the rarefaction is either enlarged or unchanged compared MMA-TBB resin (SB), reinforced zinc oxide eugenol to the postoperative or a previously taken follow-up cement (EBA), mineral trioxide aggregate (MTA), and radiograph. It is impossible to distinguish between bone control groups. resorption and sealed 4-META/MMA-TBB resin because In the SB group, the root-end surface was made 4-META/MMA-TBB resin appears radiolucent. Although slightly concave with a round bur under water irrigation. the thickness of 4-META/MMA-TBB resin is less than According to the manufacturer’s instructions, an aqueous 0.1 mm. Then, in the SB group, the area of periodontal solution of 10% citric acid and 3% ferric chloride radiolucency is measured as the bone resorption. (activator Green®, Sun Medical Co. Ltd., Shiga, Japan) Statistical differences were analyzed using Chi-square was applied to the apical surface with a Benda® Brush test with SPSS 10.0 J (S.P.S.S. Co. Ltd., Tokyo, Japan). (Centrix, Shelton, CT, USA) for 5 s. After the apical surface was washed with distilled water and air-dried, Histological procedure 4-META/MMA-TBB resin (Super Bond C&B®, clear type, The animals were euthanized with an overdose of sodium Sun Medical) was applied to the apical resected surface pentobarbital (0.5 mL/kg, Nembutal® injection, Abbott using the brush dip technique. After curing, the excess Laboratories, Abbott Park, IL, USA) following general resin and the surface layer were removed with a hand anesthesia 14 weeks after surgery. The tissue blocks scaler. Finally, the flap was sutured back. were fixed in 10% buffered formalin, decalcified in 10% In the EBA and MTA groups, a 3-mm-deep root-end formic-citric acid, and embedded in paraffin wax. Six- cavity was prepared with an ultrasonic tip (ST37-90 micrometer-thick sections were serially prepared along ENAC-10®, OSADA, Tokyo, Japan). Cavities were the long axis of the tooth and the sections close to the retrofilled with reinforced zinc oxide eugenol cement center of the root were stained with hematoxylin and (Super-EBA™, Harry J Bosworth Co., Skokie, IL, USA) eosin (H–E). or mineral trioxide aggregate (ProRoot® MTA, Three HE-stained sections were taken; one DENTSPLY Tulsa Dental, Johnson City, TN, USA). approximately from the center of the root, and the other Finally, the flap was sutured back. two 100 µm from either side of the center. The mean In the control group, the root end did not receive any value of these three sections was established as a retrograde root filling. The flap was sutured back. measured value. The following measurements were All preparations were performed without root performed under light microscope: debridement and disinfection. 1. The area of the bone defect, which was the area Dent Mater J 2011; 30(4): 485–492 487

surrounded by the resected dentinal surface and bone (Fig. 1). 2. The percentage of cementum-like tissue on the resected dentinal surface, which was the percentage of the length of newly deposited cementum-like tissue on the resected dentinal surface (a) /the length of the resected dentinal surface (b+c) (Fig. 2). 3. The percentage of cementum-like tissue on the root-end filling materials, which was the percentage of the length of newly formed cementum-like tissue on the surface of the root-end filling materials (a) /the length of the root-end material (b) (Fig. 3). Fig. 1 The figure shows the area of the bone defect in Histological measurements were taken using Scion measurement. Image® (National Institute of Health). Means and standard deviation of the histomorphometric parameters were calculated for each group. Two-by-two analyses were performed by Mann-Whitney U test with Bonferroni correction. P values were considered statistically significant at <0.01 and <0.05. All statistical procedures were performed using SPSS 10.0 J.

RESULTS Two SB specimens were excluded from the study because of technical problems that occurred during histological preparation. Three EBA specimens were excluded because perforations were made when root-end filling cavities were prepared. One EBA and two MTA specimens were excluded because failures in the root-end filling material were detected on the radiographs after surgery. Consequently, a total of 17 SB, 14 EBA, 17 MTA Fig. 2 The percentage of cementum-like tissue on the and 14 control specimens were available for radiographic resected dentinal surface. and histological examination. a: the length of newly deposited cementum-like tissue on the resected dentinal surface, b+c: the Observations of periradicular radiolucency length of the resected dentinal surface. The areas of preoperative periradicular radiolucency in the SB, EBA, MTA and control groups were 2.24±1.51 mm2, 2.10±0.98 mm2, 2.12±1.00 mm2, and 2.15±1.67 mm2, respectively. There were no significant differences in the area of preoperative periradicular radiolucency among the 4 groups. The area of postoperative periradicular radioluency in the SB, EBA, MTA and control groups was 8.55±4.01 mm2, 8.92±3.15 mm2, 8.05±3.39 mm2, and 8.41±3.86 mm2, respectively. There were no significant differences in the area of postoperative periradicular radiolucency among the 4 groups. Healing was evaluated radiographically 14 weeks after the surgery. The areas of periradicular radiolucency in the SB, EBA, MTA and control groups were 1.22±1.07 mm2, 1.37±1.07 mm2, 1.27±1.29 mm2, and 6.17±2.18 mm2, respectively (Fig. 4 & Table 1). The healing in the SB, EBA and MTA groups was significantly better than that in the control group (p<0.01). The periapical lesion in the Fig. 3 The percentage of cementum-like tissue on the 14 weeks after the surgery of the SB, EBA and MTA root-end filling materials. groups had become significantly better than the a: the length of newly formed cementum-like preoperative lesion (p<0.05), whereas the healing in the tissue on the surface of the root-end filling control group was significantly worse p( <0.01). materials, b: the length of the root-end material. The healing was also evaluated clinically and radiographically 14 weeks after the surgery. In the SB group, complete and incomplete healing was observed in 488 Dent Mater J 2011; 30(4): 485–492

Fig. 4 Observations of periradicular radiolucency. There were no significant differences in all groups at immediately after surgery. The SB, EBA and MTA groups had became significantly better than the control group (p<0.01) and the preoperative lesion (p<0.05).

Table 1 Observations of periradicular radiolucency Area of preoperative Area of postoperative Area of periradicular radiolucency [mm2] radiolucency [mm2] radiolucency at 14 weeks [mm2] mean (SD) mean (SD) mean (SD) SB 2.24 (1.51) 8.55 (4.01) 1.22 (1.07)*† EBA 2.10 (0.98) 8.92 (3.15) 1.37 (1.07)*† MTA 2.12 (1.00) 8.05 (3.39) 1.27 (1.29)*† Control 2.15 (1.67) 8.41 (3.86) 6.17 (2.18)** * Significantly different from the area of preoperative radiolucency (p<0.05) ** Significantly different from the area of preoperative radiolucency (p<0.01) † Significantly different from control group (p<0.01)

Table 2 Radiographic classification of healing Complete Incomplete Uncertain Unsatisfactory Total SB* 12 5 0 0 17 EBA* 9 5 0 0 14 MTA* 12 5 0 0 17 Control 0 0 4 10 14 * Significantly different from control group (p<0.01) Dent Mater J 2011; 30(4): 485–492 489

12 and 5 of the 17 roots, respectively (Table 2). In the Histological observations EBA group, complete and incomplete healing was In the control group, infiltrating inflammatory cells, observed in 9 and 5 of the 14 roots, respectively. In the composed of leukocytes, lymphocytes and some MTA group, complete and incomplete healing was macrophages, were observed in the apical periodontal observed in 12 and 5 of the 17 roots, respectively. In the tissue (Fig. 5). control group, uncertain and unsatisfactory healing was In the SB group, few inflammatory cells were observed in 4 and 10 of the 14 roots, respectively. In the observed in the apical periodontal tissue facing 4-META/ SB, EBA and MTA groups, the healing 14 weeks after MMA-TBB resin. It was observed that 4-META/ apicoectomy was classified as “almost good”. MMA-TBB resin was encapsulated with thin fibrous The healing in the SB, EBA and MTA groups was tissue. Alveolar bone had formed close to 4-META/ significantly better than that in the control group MMA-TBB resin (Fig. 6). (p<0.01). There were no significant differences among In the EBA group, the tissue facing reinforced zinc the SB, EBA, and MTA groups. oxide eugenol cement contained a few inflammatory

Fig. 5 Control group at 14 weeks (H–E staining). Fig. 7 EBA group at 14 weeks (H–E staining). EBA: a) Bone defect and marked inflammatory cells reinforced zinc oxide eugenol cement. (arrow) are shown. Scale bar, 500 µm. a) Bone regeneration was observed. Scale bar, 500 b) Higher magnification of framed area (b) in a. µm. Scale bar, 50 µm. b) Higher magnification of framed area (b) in a. Cementum-like tissue (arrow) on the dentin surface and a few inflammatory cells on reinforced zinc oxide eugenol cement are shown. Scale bar, 50 µm.

Fig. 6 SB group at 14 weeks (H–E staining). SB: Fig. 8 MTA group at 14 weeks (H–E staining). MTA: 4-META/MMA-TBB resin. mineral trioxide aggregate. a) Well-marked bone regeneration was observed. a) Bone regeneration was observed. Scale bar, 500 4-META/MMA-TBB resin was dissolved during µm. the preparation of specimens. Scale bar, 500 µm. b) Higher magnification of framed area (b) in a. b) Higher magnification of framed area (b) in a. Cementum-like tissue (arrow) on the dentin Thin fibrous tissue (arrow) on the surface of surface and mineral trioxide aggregate and few 4-META/MMA-TBB resin and few inflammatory inflammatory cells are shown. Scale bar, 50 µm. cells are shown. Scale bar, 50 µm. 490 Dent Mater J 2011; 30(4): 485–492

Table 3 Area of bone defect and percentage of cementum-like tissue on resected dentinal surface and materials Cementum-like tissue on Cementum-like tissue on the Bone defect [mm2] resected dentinal surface [%] root-end filling materials [%] mean (SD) mean (SD) mean (SD) SB 1.23 (1.06)* 5.8 (9.2) 0 EBA 1.37 (1.03)* 55.1 (19.5)† 0 MTA 1.00 (0.87)* 74.8 (25.3)† ‡ 73.1 (38.1)§ Control 6.45 (1.04) 4.0 (10.0) 0 * Significantly different from control group (p<0.01) † Significantly different from SB and control group (p<0.01) ‡ Significantly different from EBA group (p<0.05) § Significantly different from SB, EBA and control group (p<0.01)

cells, such as lymphocytes and plasma cells. Cementum- DISCUSSION like tissue formation was only observed on the resected dentin surface (Fig. 7). In this study, pulp tissues were removed from the canals In the MTA group, inflammatory cells were rarely and apical periodontitis was induced by contaminating seen in the apical periodontal tissue facing mineral the root canals with dental plaque. Periradicular trioxide aggregate. Cementum-like tissue formation was radiolucency was verified. No significant difference was observed on both the resected dentin surface and mineral observed in the area of preoperative periradicular trioxide aggregate in 14 of the 17 sections. Mostly, radiolucency among the 4 groups. It was therefore cementum-like tissue was formed in succession to the assumed that the preoperative condition of the 4 groups original cementum at the root ends (Fig. 8). was almost identical. The radiographic and histological findings of the Area of bone defect present study indicate that apical periodontal healing The areas of bone resorption in the SB, EBA, MTA and was stimulated by root-end treatment with reinforced control groups were 1.23±1.06 mm2, 1.37±1.03 mm2, zinc oxide eugenol cement, mineral trioxide aggregate 1.00±0.87 mm2, and 6.45±1.04 mm2, respectively (Table and 4-META/MMA-TBB resin. These results suggest 3). Bone resorption in the SB, EBA and MTA groups was that reinforced zinc oxide eugenol cement, mineral significantly less than that in the control group (p<0.01). trioxide aggregate and 4-META/MMA-TBB resin may There were no significant differences among the SB, prevent leakage of contamination from the root canals EBA, and MTA groups. to the surrounding apical tissue. Upon histological observation, the morphological characteristics of the Percentage of cementum-like tissue on resected dentinal healing in each of the groups were different. surface The tissue facing reinforced zinc oxide eugenol The percentages of cementum-like tissue deposition on cement contained a few inflammatory cells and this the resected dentinal surface in the SB, EBA, MTA and result was in agreement with those of Baek et al.30). control groups were 5.8±9.2%, 55.1±19.5%, 74.8±25.3%, However, the degree of inflammation was extremely 4.0±10.0%, respectively (Table 3). The percentages of mild and the bone resorption was not significantly cementum-like tissue deposition on the resected dentinal different among the SB, EBA, and MTA groups. As surface in the EBA and MTA groups were significantly Rubinstein et al.31) reported long-term success when higher than those in the SB and control groups (p<0.01). using reinforced zinc oxide eugenol cement as the The MTA group showed significantly more deposition root-end filling material, a few inflammatory cells facing than the EBA group (p<0.05) (Table 3). reinforced zinc oxide eugenol cement might be clinically acceptable32). In contrast, reinforced zinc oxide eugenol Percentage of cementum-like tissue on root-end filling cement has been reported to have inadequate long-term materials sealing ability6,10). In the EBA group, cementum-like The percentage of cementum-like tissue deposition on tissue deposition was observed on the resected dentinal the root-end filling materials in the MTA group was surface, but not on reinforced zinc oxide eugenol cement. 73.1±38.1% (Table 3). This was significantly greater Thus, newly formed cementum-like tissue may not than that in the SB, EBA and control groups (p<0.01). prevent leakage, and inflammation of apical periodontal Cementum-like tissue deposition on the root-end filling tissues might recur over the long term when materials was not observed with 4-META/MMA-TBB contamination is present in the root canal. resin or reinforced zinc oxide eugenol cement. Cementum-like tissue formation on the resected dentinal surface and mineral trioxide aggregate has been reported30,33-36). In the present study, cementum-like tissue deposition was observed on the resected dentinal Dent Mater J 2011; 30(4): 485–492 491 surface in all sections and on the mineral trioxide histological analysis and radiography. Only root-end aggregate in 14 of the 17 sections. 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