Dental Traumatology 2015; 31: 465–470; doi: 10.1111/edt.12202
Could zoledronic acid prevent root resorption in replanted rat molar? Jung Eun Yoo1, Mi Sun Kim1, Yong–dae Kwon2, Eun-Cheol Kim3, Kwang Chul Kim4, Sung Chul Choi1 1 Department of Pediatric Dentistry, School of Dentistry, Kyung Hee University; 2Department of Maxillofacial Surgery, School of dentistry, Kyung Hee University; 3Department of Maxillofacial Tissue Regeneration and Research Center for Tooth & Periodontal Regeneration (MRC), School of Dentistry, Kyung Hee University, Seoul; 4Department of Pediatric Dentistry, Kyung Hee University Dental Hospital, Gangdong, Korea
Key words: zoledronic acid; zoledronate; avulsion; replacement resorption; soft X-ray; replantation Correspondence to: Sung Chul Choi, Hoegidong, Dong dae moon-gu, Department of Pediatric Dentistry, School of Dentistry, Kyung Hee University, 130-702 Seoul, Korea Tel.: +82 2 958 9440 Fax: +82 2 966 4572 e-mail: [email protected]
Abstract – Background/Aim: In this study, we evaluated whether zoledronate could suppress the progression of external root resorption in rat due to delayed replantation by inhibiting osteoclastic activity. Also, we estimated the optimal dosage of zoledronate in root treatment of the rat model for a maximum effect of zoledronate. Material and methods: Maxillary first molars in Sprague Dawley rats (N = 84) were extracted, dried for 60 min, and then replanted. The rats were divided into 6 groups (1 mM alendronate, and 1, 5, 10, 20, 40 lM zoledronate). At 4 and 8 weeks postreplantation, the animals were sacrificed and evaluated by radiographic and histological analysis. Results and Conclusion: There were no significant differences at 4 weeks. However, at 8 weeks, 10, 20, and 40 lM ZOL showed more increased radiopaque and smaller periapical lesion in radiographic analysis. In histological analysis, all groups showed similar inflammatory root resorption rate at 4 weeks. However, at 8 weeks, 20 and 40 lM ZOL showed lower rate than those of other groups (P < 0.05). In concerning of replacement resorption, there were no significant differences statistically. In this animal experiment, zoledronate was capable of limiting the occurrence of root resorption in delayed replantation model. In particular, 20 lM dosage of zoledronate solution showed the most effective dose in long-term follow up and might be suitable for inhibition of root resorption in delayed tooth replantation.
Accepted 28 May, 2015
The main concern of delayed tooth replantation is the prevention of replacement root resorption and reduction of inflammation to save the time for the connective tissue arranged parallel to the root surface to fill and repair the periodontal ligament space. There are many studies to inhibit or reduce root resorption and promote repair of the replanted area using sodium fluoride which leads the root surface to become more resistant to root resorption (1–3). EmdogainÒ (BIORA AB, Malmo, Sweden) which stimulates the generation of extrinsic cementum through the basic process is similar to the generation of cementum (4), and bisphosphonate compounds that are used to inhibit the proliferation and circulation of the osteoclast precursor cells (5). Bisphosphonates (BPs) are widely used as drugs for patients with bone disorders showing increase in osteoclastic activity (6–8). The main mechanism of these drugs has been attributed to inhibit osteoclast by decreasing the osteoclast activity, differentiating monocytes and macrophages into osteoclasts (9, 10). Levin et al. (5) reported that topical treatment of root surfaces with alendronate (ALN) resulted in better © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
healing than treatment with saline in replanted dog teeth. Since then many in vitro studies showed that ALN has been employed for root surface treatment before replantation of avulsed teeth to prevent the occurrence of inflammatory resorption (11–13). Zoledronate (ZOL), which is most frequently administered intravenously, has the highest binding affinity with hydroxyapatite (14). It has the most rapid onset time, the longest-lasting action and an action potential only in osteoclast without action of other cells (15). However, there are a few studies of ZOL for dental purpose. Our previous study showed that ZOL might prevent the root resorption and allow time for regeneration of periodontal tissues after delayed replantation in rat model (16). Also Mori et al. (17) showed that zoledronic acid showed better results than those of sodium fluoride in 30-min extra-oral dried teeth. However, the problems in previous study are as follows: the dosage of ZOL has not reached to their maximum effect for topical use compared with that of ALN; not enough follow-up period for the longest-lasting drug although the rat model showed fast turn-over 465
466
Yoo et al.
rate. Therefore, the purpose of this study was to estimate the effective dosage of ZOL in root treatment of the rat model for a maximum effect and evaluate a long-term effect of ZOL possibilities.
20 000 IU of G penicillin via intramuscular injection and supplied with a soft diet for 7 days. If any of rats died or the root was broken during the surgical procedure, it was excluded from this study.
Materials and methods
Radiography and image analysis
Preparing animals and defining experimental groups
This study proposal was reviewed and approved by the Ethics in Institutional Animal Care and Use Committee of Kyung Hee Medical Center, Kyung Hee University, Seoul, Korea (KHMC-IACUC: 10-054). Eightyfour five- to six-week old, male Sprague Dawley rats (Semtaco, Kyungkido, Korea) weighing 150–200 g were used in this study. During the experimental period, the animals were fed a powdered diet and given water. They were divided into seven groups according to the root treatment dosage of ZOL after extra-alveolar 60min dry, as follows: Group I (A)—10-min soaking in 1 mM ALN solution (Sigma-Aldrich Corporation, St. Louis, MO, USA) in HBSS and replanted; Group II (Z01)—10-min soaking in 1 lM ZOL solution (Novatis Pharma AG., Basel, Switzerland) in HBSS before replantation; Group III (Z05)—10-min soaking in 5 lM ZOL solution in HBSS before replantation; Group IV (Z10)—10-min soaking in 10 lM ZOL solution in HBSS before replantation; Group V (Z20)—10min soaking in 20 lM ZOL solution in HBSS before replantation; and Group VI (Z40)—10-min soaking in 40 lM ZOL solution in HBSS before replantation. All groups sacrificed at 4 and 8 weeks post-treatment. Surgical procedures and root treatments
For all procedures, animals were anesthetized with an intramuscular injection of Zoletil 50 (100–150 mg kg 1; Virbac Lab, Carros, France). They were given a 5-day supply of 0.4% b-aminopropionitrile (b-APN: SigmaAldrich Corporation) to avoid traumatic extraction. Then, a clean operational field was obtained with a 2% chlorhexidine solution and the left maxillary first molars were atraumatically extracted using extraction forceps, and the molars were placed in a pink wax sheet to be dried out at the room temperature. The animals with root fractures were excluded, and experimental animals were added to reach five animals in each group. To minimize pulpal infection as stimulus for external root resorption, all teeth were accessed, instrumented with K-type files to the apical stop, and irrigated with sterile saline. Canals were dried with paper points and filled with calcium hydroxide paste (Metapaste: Meta Biomed Co., Chungcheongbukdo, Korea), and occlusal accesses were obturated with Caviton (GC co., Tokyo, Japan). In all groups, teeth were dried at the room temperature (20–24°C) for 60 min and treated root canal during dry time. After drying at the room temperature, the root was placed in citric acid (pH 1) for 3 min, curetted, and cleaned with saline for 2 min to remove the impaired and dead periodontal tissues. All avulsed sockets were cleaned with enough saline prior to replantation. The animals received a single dose of
All animals were sacrificed at 4 and 8 weeks postoperation by means of flowing fixation with 10% formalin under anesthesia with Zoletil 50. The relevant maxillae were dissected, and the samples were fixed in 10% buffered formalin for 2 weeks. After fixation, the jaws were processed for gross radiographic examination. Radiographs were taken by soft X-ray film shooting apparatus (CMS-2; Softex Co., Ltd., Tokyo, Japan) for lateral projections on HS Fuji Softex film (Fuji Film, Co., Ltd., Tokyo, Japan) and analyzed by PIVIEW STAR DICOM (Digital Imaging and Communications in Medicine) viewer software (version 5.0.6.1; INFINITT healthcare Co., Ltd., Seoul, Korea). The areas of periapical lesions at the mesiobuccal root apices of the maxillary first molars were quantified in pixels. Resolution of the images was 3200 dpi, and the area of one pixel was 645.16 (3200 9 3200) mm2. Then, the data were able to be converted into mm2 from using the equation. Histological analysis
After imaging, the samples were decalcified in 0.1 M EDTA for 4 weeks. Thereafter, the maxillae were washed, dehydrated, embedded in paraffin, and sectioned serially at 5–8 lm in sagittal orientation by microtome. At each sectioning level, three sections were mounted and the section that was technically the best was stained with hematoxylin and eosin. Histomorphometric analysis was performed under light microscope. The histological assessment was performed as shown in Table 1. For the exact evaluation, we assessed histological score on each anterior and posterior side of mesiobuccal root. Statistical analysis
The data of each group were expressed as mean of standard deviation (SD). Comparison was analyzed
Table 1. Categories for scoring the statues of estimated point Score
Inflammatory root resorption
Replacement root resorption
0 1
None Mild: cementum only
2
4
Moderate: cementum and dentin resorption under 1/2 of the root area Severe: cementum and dentin resorption over 1/2 of the root area Tooth perforation
None No PDL space: only bone contact Mild resorption: under cementum Moderate resorption: between dentin Severe resorption: replaced with bone
5
Tooth exfoliation
3
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Zoledronic acid prevent root resorption statistically from the groups using Kruskal–Wallis test with post hoc analysis by Duncan’s multiple range test and Mann–Whitney test. P-values
Could zoledronic acid prevent root resorption in replanted rat molar? Jung Eun Yoo1, Mi Sun Kim1, Yong–dae Kwon2, Eun-Cheol Kim3, Kwang Chul Kim4, Sung Chul Choi1 1 Department of Pediatric Dentistry, School of Dentistry, Kyung Hee University; 2Department of Maxillofacial Surgery, School of dentistry, Kyung Hee University; 3Department of Maxillofacial Tissue Regeneration and Research Center for Tooth & Periodontal Regeneration (MRC), School of Dentistry, Kyung Hee University, Seoul; 4Department of Pediatric Dentistry, Kyung Hee University Dental Hospital, Gangdong, Korea
Key words: zoledronic acid; zoledronate; avulsion; replacement resorption; soft X-ray; replantation Correspondence to: Sung Chul Choi, Hoegidong, Dong dae moon-gu, Department of Pediatric Dentistry, School of Dentistry, Kyung Hee University, 130-702 Seoul, Korea Tel.: +82 2 958 9440 Fax: +82 2 966 4572 e-mail: [email protected]
Abstract – Background/Aim: In this study, we evaluated whether zoledronate could suppress the progression of external root resorption in rat due to delayed replantation by inhibiting osteoclastic activity. Also, we estimated the optimal dosage of zoledronate in root treatment of the rat model for a maximum effect of zoledronate. Material and methods: Maxillary first molars in Sprague Dawley rats (N = 84) were extracted, dried for 60 min, and then replanted. The rats were divided into 6 groups (1 mM alendronate, and 1, 5, 10, 20, 40 lM zoledronate). At 4 and 8 weeks postreplantation, the animals were sacrificed and evaluated by radiographic and histological analysis. Results and Conclusion: There were no significant differences at 4 weeks. However, at 8 weeks, 10, 20, and 40 lM ZOL showed more increased radiopaque and smaller periapical lesion in radiographic analysis. In histological analysis, all groups showed similar inflammatory root resorption rate at 4 weeks. However, at 8 weeks, 20 and 40 lM ZOL showed lower rate than those of other groups (P < 0.05). In concerning of replacement resorption, there were no significant differences statistically. In this animal experiment, zoledronate was capable of limiting the occurrence of root resorption in delayed replantation model. In particular, 20 lM dosage of zoledronate solution showed the most effective dose in long-term follow up and might be suitable for inhibition of root resorption in delayed tooth replantation.
Accepted 28 May, 2015
The main concern of delayed tooth replantation is the prevention of replacement root resorption and reduction of inflammation to save the time for the connective tissue arranged parallel to the root surface to fill and repair the periodontal ligament space. There are many studies to inhibit or reduce root resorption and promote repair of the replanted area using sodium fluoride which leads the root surface to become more resistant to root resorption (1–3). EmdogainÒ (BIORA AB, Malmo, Sweden) which stimulates the generation of extrinsic cementum through the basic process is similar to the generation of cementum (4), and bisphosphonate compounds that are used to inhibit the proliferation and circulation of the osteoclast precursor cells (5). Bisphosphonates (BPs) are widely used as drugs for patients with bone disorders showing increase in osteoclastic activity (6–8). The main mechanism of these drugs has been attributed to inhibit osteoclast by decreasing the osteoclast activity, differentiating monocytes and macrophages into osteoclasts (9, 10). Levin et al. (5) reported that topical treatment of root surfaces with alendronate (ALN) resulted in better © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
healing than treatment with saline in replanted dog teeth. Since then many in vitro studies showed that ALN has been employed for root surface treatment before replantation of avulsed teeth to prevent the occurrence of inflammatory resorption (11–13). Zoledronate (ZOL), which is most frequently administered intravenously, has the highest binding affinity with hydroxyapatite (14). It has the most rapid onset time, the longest-lasting action and an action potential only in osteoclast without action of other cells (15). However, there are a few studies of ZOL for dental purpose. Our previous study showed that ZOL might prevent the root resorption and allow time for regeneration of periodontal tissues after delayed replantation in rat model (16). Also Mori et al. (17) showed that zoledronic acid showed better results than those of sodium fluoride in 30-min extra-oral dried teeth. However, the problems in previous study are as follows: the dosage of ZOL has not reached to their maximum effect for topical use compared with that of ALN; not enough follow-up period for the longest-lasting drug although the rat model showed fast turn-over 465
466
Yoo et al.
rate. Therefore, the purpose of this study was to estimate the effective dosage of ZOL in root treatment of the rat model for a maximum effect and evaluate a long-term effect of ZOL possibilities.
20 000 IU of G penicillin via intramuscular injection and supplied with a soft diet for 7 days. If any of rats died or the root was broken during the surgical procedure, it was excluded from this study.
Materials and methods
Radiography and image analysis
Preparing animals and defining experimental groups
This study proposal was reviewed and approved by the Ethics in Institutional Animal Care and Use Committee of Kyung Hee Medical Center, Kyung Hee University, Seoul, Korea (KHMC-IACUC: 10-054). Eightyfour five- to six-week old, male Sprague Dawley rats (Semtaco, Kyungkido, Korea) weighing 150–200 g were used in this study. During the experimental period, the animals were fed a powdered diet and given water. They were divided into seven groups according to the root treatment dosage of ZOL after extra-alveolar 60min dry, as follows: Group I (A)—10-min soaking in 1 mM ALN solution (Sigma-Aldrich Corporation, St. Louis, MO, USA) in HBSS and replanted; Group II (Z01)—10-min soaking in 1 lM ZOL solution (Novatis Pharma AG., Basel, Switzerland) in HBSS before replantation; Group III (Z05)—10-min soaking in 5 lM ZOL solution in HBSS before replantation; Group IV (Z10)—10-min soaking in 10 lM ZOL solution in HBSS before replantation; Group V (Z20)—10min soaking in 20 lM ZOL solution in HBSS before replantation; and Group VI (Z40)—10-min soaking in 40 lM ZOL solution in HBSS before replantation. All groups sacrificed at 4 and 8 weeks post-treatment. Surgical procedures and root treatments
For all procedures, animals were anesthetized with an intramuscular injection of Zoletil 50 (100–150 mg kg 1; Virbac Lab, Carros, France). They were given a 5-day supply of 0.4% b-aminopropionitrile (b-APN: SigmaAldrich Corporation) to avoid traumatic extraction. Then, a clean operational field was obtained with a 2% chlorhexidine solution and the left maxillary first molars were atraumatically extracted using extraction forceps, and the molars were placed in a pink wax sheet to be dried out at the room temperature. The animals with root fractures were excluded, and experimental animals were added to reach five animals in each group. To minimize pulpal infection as stimulus for external root resorption, all teeth were accessed, instrumented with K-type files to the apical stop, and irrigated with sterile saline. Canals were dried with paper points and filled with calcium hydroxide paste (Metapaste: Meta Biomed Co., Chungcheongbukdo, Korea), and occlusal accesses were obturated with Caviton (GC co., Tokyo, Japan). In all groups, teeth were dried at the room temperature (20–24°C) for 60 min and treated root canal during dry time. After drying at the room temperature, the root was placed in citric acid (pH 1) for 3 min, curetted, and cleaned with saline for 2 min to remove the impaired and dead periodontal tissues. All avulsed sockets were cleaned with enough saline prior to replantation. The animals received a single dose of
All animals were sacrificed at 4 and 8 weeks postoperation by means of flowing fixation with 10% formalin under anesthesia with Zoletil 50. The relevant maxillae were dissected, and the samples were fixed in 10% buffered formalin for 2 weeks. After fixation, the jaws were processed for gross radiographic examination. Radiographs were taken by soft X-ray film shooting apparatus (CMS-2; Softex Co., Ltd., Tokyo, Japan) for lateral projections on HS Fuji Softex film (Fuji Film, Co., Ltd., Tokyo, Japan) and analyzed by PIVIEW STAR DICOM (Digital Imaging and Communications in Medicine) viewer software (version 5.0.6.1; INFINITT healthcare Co., Ltd., Seoul, Korea). The areas of periapical lesions at the mesiobuccal root apices of the maxillary first molars were quantified in pixels. Resolution of the images was 3200 dpi, and the area of one pixel was 645.16 (3200 9 3200) mm2. Then, the data were able to be converted into mm2 from using the equation. Histological analysis
After imaging, the samples were decalcified in 0.1 M EDTA for 4 weeks. Thereafter, the maxillae were washed, dehydrated, embedded in paraffin, and sectioned serially at 5–8 lm in sagittal orientation by microtome. At each sectioning level, three sections were mounted and the section that was technically the best was stained with hematoxylin and eosin. Histomorphometric analysis was performed under light microscope. The histological assessment was performed as shown in Table 1. For the exact evaluation, we assessed histological score on each anterior and posterior side of mesiobuccal root. Statistical analysis
The data of each group were expressed as mean of standard deviation (SD). Comparison was analyzed
Table 1. Categories for scoring the statues of estimated point Score
Inflammatory root resorption
Replacement root resorption
0 1
None Mild: cementum only
2
4
Moderate: cementum and dentin resorption under 1/2 of the root area Severe: cementum and dentin resorption over 1/2 of the root area Tooth perforation
None No PDL space: only bone contact Mild resorption: under cementum Moderate resorption: between dentin Severe resorption: replaced with bone
5
Tooth exfoliation
3
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Zoledronic acid prevent root resorption statistically from the groups using Kruskal–Wallis test with post hoc analysis by Duncan’s multiple range test and Mann–Whitney test. P-values
