- Email: [email protected]
Effect of local delivery of alendronate on bone formation in bioactive glass grafting in rats
Effect of local delivery of alendronate on bone formation in bioactive glass grafting in rats Suthasiny Srisubut,a Arun Teerakapong,a Theparith Vattraphodes,b and Suwimol Taweechaisupapong,c Khon Kaen, Thailand FACULTY OF DENTISTRY, KHON KAEN UNIVERSITY
Objective. The aim of this study was to investigate whether local delivery of alendronate could improve bone formation after bioactive glass grafting in rat mandible. Study design. Twenty-six male Spraque-Dawley rats were divided into control and experimental groups (13 rats/group). A surgical defect was created on the angle of mandible of each animal. In the experimental group, a bioactive glass soaked with the alendronate solution was placed in the bone defect, and in the control group the bioactive glass soaked with saline was used. All animals were killed after 4 weeks. The number of osteoclasts and the amount of new bone formation were evaluated and compared. Results. Four weeks after surgery, the experimental group had significantly more bone formation than the control groups (P ⬍ .05). However, no statistically significant difference was found between the groups when the numbers of osteoclasts were compared. Conclusion. Histologic results showed that a single dose of local delivery of alendronate improves bone formation. However, further studies are required to elucidate the effect of local delivery of alendronate on bone formation in humans. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e11-e16)
Bone grafting to augment skeletal healing has become one of the most common surgical techniques in recent years. However, the morbidity and limited availability associated with autografts, and the potential for disease transmission, immunogenic response, and variable quality associated with allografts, have led to a wide variety of alternative materials. Recently, bioactive glass has been subjected to intensive experimental and clinical use as bone graft substitutes in various types of bone defects, often associated with dental implants, and in sinus lift procedures.1-4 Biogran is a resorbable amorphous bioactive glass of 300-355 m particle size. It is composed of 45% silicon dioxide (SiO2), 24.5% calcium oxide (CaO), 24.5% sodium oxide (Na2O), and 6% phosphorus pentoxide (P2O5). It has been shown to enhance bone repair not only by the osteoconductive properties of the particles but also by their osteostimulative potential, defined as bone formation within internal pouches excavated within the bioglass particles away from the pre-existing bony defect walls.2
a
Department of Periodontology. Department of Oral and Maxillofacial Surgery. c Department of Oral Diagnosis. Received for publication Jan 3, 2007; returned for revision Apr 1, 2007; accepted for publication Apr 17, 2007. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2007.04.022 b
The regeneration of injured or excised bone tissue comprises a complex sequence of events that begins with the recruitment, attachment, and proliferation of progenitor cells, followed by cell differentiation into appropriate phenotypes that are capable of restoring the damaged tissue.5 The improvement of bone regeneration has included the use of biologic mediators to improve the quantity and quality of the bone being regenerated.6,7 One group of bone metabolism mediators is the bisphosphonates, the carbon-substituted pyrophosphate analogs, that are potent inhibitors of bone resorption and have been effectively used to control osteolysis or reduce bone loss in Paget disease, metastatic bone disease, hypercalcemia of malignancy,8 and osteoporosis.9 Bisphosphonates are believed to inhibit osteoclast activity by interfering with the ruffled border membrane of the osteoclasts without destroying the cells.10 This inhibition of resorptive activity is thought to produce a shift in bone turnover equilibrium to more osteoblastic activity. Earlier studies have demonstrated that alendronate and other bisphosphonates reduce bone resorption when administered systemically or locally.11-13 Their biologic effects are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and osteoblasts through a variety of biochemical pathways.10,14,15 Yaffee et al.11 reported that a single dose of locally applied bisphosphonate can give an adequate distribution of bisphosphonate to the bone, because of the high affinity of bisphosphonates to bone mineral. They have e11
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also shown reduced alveolar bone resorption following mucoperiostal surgery in rats after topical application of alendronate.16 In addition, a single dose of topical alendronate can reduce periprosthetic bone resorption in a rat model.17 Since late 2003, an increasing number of reports have suggested a possible association between the use of bisphosphonates and avascular osteonecrosis of the jaw (ONJ).18 Osteonecrosis of the jaw is a complication that is correlated with long-term use of systemic bisphosphonates; this complication has received much publicity and developed considerable controversy recently.19,20 There are several references which reported the successful treatment of osteonecrosis with bisphosphonates.21,22 At present, many issues regarding the pathogenesis of the bisphosphonate-associated osteonecrosis still remain unclear. In addition, although the effect of the bisphosphonates on reducing bone resorption is well documented, their effect on bone formation is still uncertain. Therefore, the aim of the present study was to investigate whether local delivery of alendronate could improve bone formation after bioactive glass grafting in rat mandible. MATERIALS AND METHODS Animals Twenty-six male Spraque-Dawley rats, weighing 240-250 g, obtained from the central animal care unit of the Faculty of Medicine, Khon Kaen University, were used. All rats were 8 weeks old, kept 4-5/cage, and provided ground laboratory food and water ad libitum. The animals were divided into 2 groups (13 rats/group): control group (bioactive glass ⫹ saline) and experimental group (bioactive glass ⫹ alendronate solution). The handling of animals was scrutinized by the Animal Ethics Research Committee, Faculty of Medicine, Khon Kaen University (ref. no. 0501.04/207). Surgical procedures All surgeries considered in this study were carried out by 1 investigator. The rat was anesthesized before surgery using diethyl ether. After the linear incision was made from the ramus to the inferior border of the mandible, and by elevating mucoperiosteal flaps, the lateral aspect of the bone surface around the angle of mandible was exposed. A standardized round throughand-through bone defect (5 mm in diameter)23 was created on the angle of mandible using a round carbide bur. An alendronate solution was prepared by dissolving 20 mg alendronate (Fosamax; MSD, Malmo, Sweden) in 1 mL saline. In the experimental group, a bioactive glass (Biogran; Orthovita, Malvern, PA) soaked with the alendronate solution was placed in the bone defect, and in the control group the bioactive glass soaked with physiologic
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saline was used. Then the flaps were carefully repositioned and sutured with resorbable sutures. Histologic procedures Four weeks after surgery, all animals were killed by breaking their necks. The block sections were dissected, fixed in fixative solution (acid phosphatase kit, Sigma, St. Louis, MO) for 24 hours, decalcified in 10% ethylene-diaminetetraacetic acid solution (pH 7.4) at 4°C for 4 weeks and embedded in paraffin. Serial sections, 5 m thick, were prepared in mesiodistal planes and stained with hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP; Sigma), and Masson trichrome24 (4 sections/each stain). All slides were coded and read without prior knowledge (blinded). The sections stained with H&E and Masson trichrome were examined under stereomicroscope to evaluate lesion sizes, and the TRAP-stained sections were evaluated for the number of osteoclasts. Osteoclast counting was performed in the total area of lesion (TA) shown in Fig. 1 for both control and experimental groups, and the mean and standard deviation values were compared. Every third section was included in the total count of osteoclast cells per specimen. The decision to count osteoclast cells in every third section was based on the assumption that the average osteoclast was no more than 15 m in diameter. The amount of new bone formation were evaluated in the H&E- and Masson trichrome–stained sections under compound microscope with Zeiss Vision KS 400 software (Oberkochen, Germany). Histomorphometric analysis The Zeiss Vision KS 400 software was used for the histomorphometric analysis. The most central stained section that represented the maximum diameter of the defect was selected in each block. Each section was initially inspected using a light microscope (Axiostar; Zeiss) and saved as a digital image (Fig. 1, A). A composite digital image was then created by combining 3-4 smaller images, because it was not possible to capture the entire defect in 1 image at the level of magnification that was used in Fig. 1, B. The following criteria were used to standardize the histomorphometric analysis of the composite digital image: 1) the captured images of each histological section were merged on the computer screen to create a single composite image comprising the entire area of the surgical defect, and the TA and the newly formed bone area (NB) to be analyzed were then delineated on the composite image; and 2) the TA calculated in mm2 was considered 100% of the area to be analyzed (Fig. 1, A). The NB was also
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Fig. 1. A, Histologic section of the surgical defect of the experimental group at 4 weeks after treatment, demonstrating the total area (TA) to be analyzed. Masson trichrome stain; scale bar ⫽ 1 mm. B, Greater magnification of A showing newly formed bone tissue (NB) of immature type along the defect border (DB). BG, bioactive glass particles; scale bar ⫽ 200 m.
Table I. Lesion size, osteoclast numbers, and new bone formation of control and experimental groups 4 weeks after treatment Group (n ⫽ 13 each)
Lesion size (mm2)
Number of osteoclasts (cells/mm2)
Amount of new bone formation (mm2)
Percentage of new bone formation
13.18 ⫾ 1.80 14.27 ⫾ 2.87
0.37 ⫾ 0.11 0.37 ⫾ 0.14
4.04 ⫾ 1.39 6.12 ⫾ 2.14*
31.36 ⫾ 11.60 48.14 ⫾ 11.81
Control Experimental Values are mean ⫾ SD. *P ⬍ .05 compared with control.
calculated in mm2. The percentage of NB was calculated according to the following formula: percentage of NB ⫽ 共NB/TA兲 ⫻ 100 The values of NB of each rat were used to calculate the means and standard deviations of the control and experimental groups. Statistical analysis For statistical comparision between osteoclast numbers and amount of new bone formation of control and experimental groups, the independent t test was used, and a P value of ⬍.05 was regarded as significant. RESULTS The average lesion sizes of the control and experimental groups were 13.18 ⫾ 1.80 and 14.27 ⫾ 2.87 mm2, respectively (Table I). There were no statistically significant differences between the groups (P ⬎ .05). The osteoclast counting revealed that the numbers of osteoclasts in the control and the experimental groups were 0.37 ⫾ 0.11 and 0.37 ⫾ 0.14 cells/mm2, respectively (Table I). There were no statistically significant differences between the groups when the numbers of osteoclasts were compared (P ⬎ .05). New bone formation was observed along the borders of the surgical defect in both groups. The new bone trabeculae were immature and poorly organized. Less newly formed
bone was observed in the control group than in the experimental group (Fig. 2). The mean and standard deviation of new bone formation for each group is depicted in Table I. The experimental group showed a significant increase in new bone formation compared with the control group (P ⬍ .05). In the connective tissue adjacent to the bioactive glass particles, moderate numbers of fibroblasts, macrophages, and lymphocytes were present (Fig. 2). DISCUSSION In the present study, we used a bioactive glass as bone graft substitute, because several in vitro studies have shown the nontoxicity of bioactive glass, its positive influence on osteoblast culture,25 and its ability to form calcification foci in periodontal ligament fibroblasts.26 In addition, Zamet et al.27 and Low et al.28 reported good clinical results in intrabony defects in sites treated with a bioactive glass (Biogran) with a wide range of particle sizes compared with debridement. The present results showed that the use of the bioactive glass did not cause any undesirable reaction. No foreign body reactions indicating toxicity were observed, which is in agreement with the findings of several in vitro26 and histologic29-31 investigations in animals. New bone formation was observed along the borders of the surgical defect in both control and experimental groups. The replacement of bioactive glass
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Fig. 2. Photomicrograph showing newly formed bone tissue (NB) along the defect border (DB) of the control (A) and the experimental groups (B) at 4 weeks after treatment. BG, bioactive glass particles; scale bar ⫽ 200 m.
particles by new bone in the present study is consistent with the previous study by Villaca et al.31 This indicates that the graft material led to bone repair by an osseoconductive property. However, these studies are preliminary and may not be generalizable to other grafts beyond the limited data presented for bioactive glass. The experimental model was based on that described by Higuchi et al.32 The reasons for choosing this model were: 1) rats are readily available; 2) the surgical procedures on the rat mandibular bone are relatively simple; 3) spontaneous healing would not occur in the control site; 4) observations can be focused on the healing process of bone, because there are not any major nerves or blood vessels around the rat mandibular angles; 5) the preparation of tissue specimen is easy; and 6) the parameters can be simply and accurately measured in each specimen.32 In addition, because the effect of sex steroids, i.e., estrogen, on bone resorption and the development of osteoclasts and osteoblasts has been reported,33 only male rats were used in the present study to minimize those effects. Alendronate sodium is a bisphosphonate that acts as a potent inhibitor of bone resorption. It is now generally accepted that the main cell by which bisphosphonates mediate their action is the osteoclast. Four mechanisms appear to be involved: 1) inhibition of osteoclast recruitment; 2) inhibition of osteoclast adhesion; 3) shortening of osteoclast lifespan (apoptosis); and 4) inhibition of osteoclast activity.34 It has also been proposed that bisphosphonates have osteostimulative properties both in vivo and in vitro, as shown by increase in matrix formation.35,36 Several reports have demonstrated that bisphosphonates not only induce the osteoblasts to secrete inhibitors of osteoclast-mediated resorption but also stimulate the formation of osteoblast precursors and mineralized nodules, thereby promoting early osteoblastogenesis.37,38
Plotkin et al.39 examined the effect of alendronate administration in a murine model of glucocorticoid excess–induced apoptosis of osteocytes and osteoblasts. They reported that bisphosphonates inhibited osteocyte and osteoblast apoptosis. Meraw and Reeve’s study40 in dogs demonstrated that dental implants coated with hydroxyapatite and alendronate resulted in a significant increase in peri-implant bone. Another study revealed that bisphosphonates stimulate osteogenesis in conjuction with regenerative material around osseous defect and endosseous implants.41 The histopathologic results of the alendronate-treated groups in the present experimental study are consistent with those studies reporting the positive effect of bisphosphonates on the induction of osteoblastic activity. However, in the present study, Fosamax rather than pure alendronate was dissolved in saline and mixed with the bioactive glass. Fosamax is formulated with microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, and magnesium stearate. It is possible that these agents may have contributed to the greater stimulation of bone growth at the sites that were grafted with Fosamax combined with the bioactive glass. The local delivery of Fosamax did not appear to have a damaging effect on the osteoclasts 4 weeks after grafting, because no significant difference was found between the control and experimental groups when the numbers of osteoclasts were compared. It is possible that the concentration of the alendronate placed in the defect was not large enough to cause apoptosis. Alendronate may have inhibited the function of the osteoclasts rather than cause cell death, and further studies need to be done to understand the potential mechanism before moving to human studies. Alendronate is available as oral therapy, and the 10 mg daily dose has been approved for the treatment of osteoporosis in postmenopausal women in ⬎80 countries. However, the clinically used 10 mg/day dose of
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alendronate has caused some gastrointestinal side effects and can probably not be exceeded.42 Moreover, oral bisphosphonates are poorly absorbed, with 1% or less of the administered dose being taken up,43 and long-term use of bisphosphonates is correlated with ONJ.44 Because bisphosphonates have a high affinity for bone mineral, topical application of the drug is feasible. The main advantage with topical administration is the possibility of administering a higher dose to the region of interest. The present results suggest that a single dose of topical administration of alendronate combined with bioactive glass was able to induce more bone regeneration, and this might be useful for alveolar ridge augmentation followed by dental implant surgery and for bone regeneration in periodontal defects. Conclusion The results of the present study showed that a single dose of local delivery of alendronate improved bone formation. Alendronate may be considered among the therapeutic options available to improve bone formation process in different bone remodeling cases. But further studies are required to elucidate the effect of local delivery of alendronate on bone formation in humans. This work was supported by a grant from Khon Kaen University. The authors thank Rushmore Co. for their suggestions and assistance with the Zeiss Vision KS 400 software. REFERENCES 1. Leonetti JA, Rambo HM, Throndson RR. Osteotome sinus elevation and implant placement with narrow size bioactive glass. Implant Dent 2000;9:177-82. 2. Schepers EJ, Ducheyne P. Bioactive glass particles of narrow size range for the treatment of oral bone defects: a 1-24 month experiment with several materials and particle sizes and size ranges. J Oral Rehabil 1997;24:171-81. 3. Furusawa T, Mizunuma K, Yamashita S, Takahashi T. Investigation of early bone formation using resorbable bioactive glass in the rat mandible. Int J Oral Maxillofac Implants 1998;13:672-6. 4. Furusawa T, Mizunuma K. Osteoconductive properties and efficacy of resorbable bioactive glass as a bone-grafting material. Implant Dent 1997;6:93-101. 5. Binderman I. Bone and biologically compatible materials in dentistry. Curr Opin Dent 1991;1:836-40. 6. Wozney JM. The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 1992;32:160-7. 7. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res 1993;8:S565-72. 8. Adami S, Zamberlan N, Mian M, Dorizzi R, Rossini M, Braga B, et al. Duration of the effects of intravenous alendronate in postmenopausal women and in patients with primary hyperparathyroidism and Paget’s disease of bone. Bone Miner 1994;25:75-82. 9. Liberman UA, Weiss SR, Broll J, Minne HW, Quan H, Bell NH, et al. Alendronate Phase III Osteoporosis Treatment Study Group. Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N Engl J Med 1995;333:1437-43.
Srisubut et al. e15 10. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, et al. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 1991;88:2095-105. 11. Yaffe A, Iztkovich M, Earon Y, Alt I, Lilov R, Binderman I. Local delivery of an amino bisphosphonate prevents the resorptive phase of alveolar bone following mucoperiosteal flap surgery in rats. J Periodontol 1997;68:884-9. 12. Yaffe A, Fine N, Alt I, Binderman I. The effect of bisphosphonate on alveolar bone resorption following mucoperiosteal flap surgery in the mandible of rats. J Periodontol 1995;66:999-1003. 13. Schenk R, Eggli P, Fleisch H, Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int 1986; 38:342-9. 14. Kaynak D, Meffert R, Gunhan M, Gunhan O, Ozkaya O. A histopathological investigation on the effects of the bisphosphonate alendronate on resorptive phase following mucoperiosteal flap surgery in the mandible of rats. J Periodontol 2000;71:790-6. 15. Masarachia P, Weinreb M, Balena R, Rodan GA. Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996;19:281-90. 16. Yaffe A, Binderman I, Breuer E, Pinto T, Golomb G. Disposition of alendronate following local delivery in a rat jaw. J Periodontol 1999;70:893-5. 17. Astrand J, Aspenberg P. Topical, single dose bisphosphonate treatment reduced bone resorption in a rat model for prosthetic loosening. J Orthop Res 2004;22:244-9. 18. Leite AF, Figueiredo PT, Melo NS, Acevedo AC, Cavalcanti MG, Paula LM, et al. Bisphosphonate-associated osteonecrosis of the jaws. Report of a case and literature review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:14-21. 19. Robinson NA, Yeo JF. Bisphosphonates—a word of caution. Ann Acad Med Singapore 2004;33:48-9. 20. Tarassoff P, Csermak K. Avascular necrosis of the jaws: risk factors in metastatic cancer patients. J Oral Maxillofac Surg 2003;61:1238-9. 21. Agarwala S, Sule A, Pai BU, Joshi VR. Alendronate in the treatment of avascular necrosis of the hip. Rheumatology (Oxf) 2002;41:346-7. 22. Astrand J, Aspenberg P. Systemic alendronate prevents resorption of necrotic bone during revascularization. A bone chamber study in rats. BMC Musculoskelet Disord 2002;3:19. 23. Kaban LB, Glowacki J. Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 1981;60: 1356-64. 24. Luna L. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw; 1968. p. 94 –5. 25. Matsuda T, Davies JE. The in vitro response of osteoblasts to bioactive glass. Biomaterials 1987;8:275-84. 26. Hench LL, Paschall HA. Histochemical responses at a biomaterial’s interface. J Biomed Mater Res 1974;8:49-64. 27. Zamet JS, Darbar UR, Griffiths GS, Bulman JS, Bragger U, Burgin W, et al. Particulate bioglass as a grafting material in the treatment of periodontal intrabony defects. J Clin Periodontol 1997;24:410-8. 28. Low SB, King CJ, Krieger J. An evaluation of bioactive ceramic in the treatment of periodontal osseous defects. Int J Periodontics Restor Dent 1997;17:358-67. 29. Hench LL, Paschall HA. Direct chemical bond of bioactive glass-ceramic materials to bone and muscle. J Biomed Mater Res 1973;7:25-42.
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30. Wilson J, Clark AE, Hall M, Hench LL. Tissue response to Bioglass endosseous ridge maintenance implants. J Oral Implantol 1993;19:295-302. 31. Villaca JH, Novaes AB Jr, Souza SL, Taba M Jr, Molina GO, Carvalho TL. Bioactive glass efficacy in the periodontal healing of intrabony defects in monkeys. Braz Dent J 2005;16:67-74. 32. Higuchi T, Kinoshita A, Takahashi K, Oda S, Ishikawa I. Bone regeneration by recombinant human bone morphogenetic protein-2 in rat mandibular defects. An experimental model of defect filling. J Periodontol 1999;70:1026-31. 33. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 2000;21:115-37. 34. Fleisch H. Bisphosphonates: mechanisms of action. Endocr Rev 1998;19:80-100. 35. Tenenbaum HC, Torontali M, Sukhu B. Effects of bisphosphonates and inorganic pyrophosphate on osteogenesis in vitro. Bone 1992;13:249-55. 36. Goziotis A, Sukhu B, Torontali M, Dowhaniuk M, Tenenbaum HC. Effects of bisphosphonates APD and HEBP on bone metabolism in vitro. Bone 1995;16:317S-27S. 37. Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G. Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone 1998;22:455-61. 38. Vitte C, Fleisch H, Guenther HL. Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology 1996;137:2324-33.
39. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999;104: 1363-74. 40. Meraw SJ, Reeve CM. Qualitative analysis of peripheral periimplant bone and influence of alendronate sodium on early bone regeneration. J Periodontol 1999;70:1228-33. 41. Tenenbaum HC, Shelemay A, Girard B, Zohar R, Fritz PC. Bisphosphonates and periodontics: potential applications for regulation of bone mass in the periodontium and other therapeutic/ diagnostic uses. J Periodontol 2002;73:813-22. 42. Bone HG, Adami S, Rizzoli R, Favus M, Ross PD, Santora A, et al. Weekly administration of alendronate: rationale and plan for clinical assessment. Clin Ther 2000;22:15-28. 43. Vasikaran SD. Bisphosphonates: an overview with special reference to alendronate. Ann Clin Biochem 2001;38:608-23. 44. Ruggiero SL, Mehrotra B, Rosenberg TJ, Engroff SL. Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases. J Oral Maxillofac Surg 2004; 62:527-34. Reprint requests: Assosiate Professor Dr. Suwimol Taweechaisupapong Department of Oral Diagnosis, Faculty of Dentistry Khon Kaen University Khon Kaen, 40002 Thailand [email protected]
Objective. The aim of this study was to investigate whether local delivery of alendronate could improve bone formation after bioactive glass grafting in rat mandible. Study design. Twenty-six male Spraque-Dawley rats were divided into control and experimental groups (13 rats/group). A surgical defect was created on the angle of mandible of each animal. In the experimental group, a bioactive glass soaked with the alendronate solution was placed in the bone defect, and in the control group the bioactive glass soaked with saline was used. All animals were killed after 4 weeks. The number of osteoclasts and the amount of new bone formation were evaluated and compared. Results. Four weeks after surgery, the experimental group had significantly more bone formation than the control groups (P ⬍ .05). However, no statistically significant difference was found between the groups when the numbers of osteoclasts were compared. Conclusion. Histologic results showed that a single dose of local delivery of alendronate improves bone formation. However, further studies are required to elucidate the effect of local delivery of alendronate on bone formation in humans. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e11-e16)
Bone grafting to augment skeletal healing has become one of the most common surgical techniques in recent years. However, the morbidity and limited availability associated with autografts, and the potential for disease transmission, immunogenic response, and variable quality associated with allografts, have led to a wide variety of alternative materials. Recently, bioactive glass has been subjected to intensive experimental and clinical use as bone graft substitutes in various types of bone defects, often associated with dental implants, and in sinus lift procedures.1-4 Biogran is a resorbable amorphous bioactive glass of 300-355 m particle size. It is composed of 45% silicon dioxide (SiO2), 24.5% calcium oxide (CaO), 24.5% sodium oxide (Na2O), and 6% phosphorus pentoxide (P2O5). It has been shown to enhance bone repair not only by the osteoconductive properties of the particles but also by their osteostimulative potential, defined as bone formation within internal pouches excavated within the bioglass particles away from the pre-existing bony defect walls.2
a
Department of Periodontology. Department of Oral and Maxillofacial Surgery. c Department of Oral Diagnosis. Received for publication Jan 3, 2007; returned for revision Apr 1, 2007; accepted for publication Apr 17, 2007. 1079-2104/$ - see front matter © 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2007.04.022 b
The regeneration of injured or excised bone tissue comprises a complex sequence of events that begins with the recruitment, attachment, and proliferation of progenitor cells, followed by cell differentiation into appropriate phenotypes that are capable of restoring the damaged tissue.5 The improvement of bone regeneration has included the use of biologic mediators to improve the quantity and quality of the bone being regenerated.6,7 One group of bone metabolism mediators is the bisphosphonates, the carbon-substituted pyrophosphate analogs, that are potent inhibitors of bone resorption and have been effectively used to control osteolysis or reduce bone loss in Paget disease, metastatic bone disease, hypercalcemia of malignancy,8 and osteoporosis.9 Bisphosphonates are believed to inhibit osteoclast activity by interfering with the ruffled border membrane of the osteoclasts without destroying the cells.10 This inhibition of resorptive activity is thought to produce a shift in bone turnover equilibrium to more osteoblastic activity. Earlier studies have demonstrated that alendronate and other bisphosphonates reduce bone resorption when administered systemically or locally.11-13 Their biologic effects are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and osteoblasts through a variety of biochemical pathways.10,14,15 Yaffee et al.11 reported that a single dose of locally applied bisphosphonate can give an adequate distribution of bisphosphonate to the bone, because of the high affinity of bisphosphonates to bone mineral. They have e11
e12
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also shown reduced alveolar bone resorption following mucoperiostal surgery in rats after topical application of alendronate.16 In addition, a single dose of topical alendronate can reduce periprosthetic bone resorption in a rat model.17 Since late 2003, an increasing number of reports have suggested a possible association between the use of bisphosphonates and avascular osteonecrosis of the jaw (ONJ).18 Osteonecrosis of the jaw is a complication that is correlated with long-term use of systemic bisphosphonates; this complication has received much publicity and developed considerable controversy recently.19,20 There are several references which reported the successful treatment of osteonecrosis with bisphosphonates.21,22 At present, many issues regarding the pathogenesis of the bisphosphonate-associated osteonecrosis still remain unclear. In addition, although the effect of the bisphosphonates on reducing bone resorption is well documented, their effect on bone formation is still uncertain. Therefore, the aim of the present study was to investigate whether local delivery of alendronate could improve bone formation after bioactive glass grafting in rat mandible. MATERIALS AND METHODS Animals Twenty-six male Spraque-Dawley rats, weighing 240-250 g, obtained from the central animal care unit of the Faculty of Medicine, Khon Kaen University, were used. All rats were 8 weeks old, kept 4-5/cage, and provided ground laboratory food and water ad libitum. The animals were divided into 2 groups (13 rats/group): control group (bioactive glass ⫹ saline) and experimental group (bioactive glass ⫹ alendronate solution). The handling of animals was scrutinized by the Animal Ethics Research Committee, Faculty of Medicine, Khon Kaen University (ref. no. 0501.04/207). Surgical procedures All surgeries considered in this study were carried out by 1 investigator. The rat was anesthesized before surgery using diethyl ether. After the linear incision was made from the ramus to the inferior border of the mandible, and by elevating mucoperiosteal flaps, the lateral aspect of the bone surface around the angle of mandible was exposed. A standardized round throughand-through bone defect (5 mm in diameter)23 was created on the angle of mandible using a round carbide bur. An alendronate solution was prepared by dissolving 20 mg alendronate (Fosamax; MSD, Malmo, Sweden) in 1 mL saline. In the experimental group, a bioactive glass (Biogran; Orthovita, Malvern, PA) soaked with the alendronate solution was placed in the bone defect, and in the control group the bioactive glass soaked with physiologic
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saline was used. Then the flaps were carefully repositioned and sutured with resorbable sutures. Histologic procedures Four weeks after surgery, all animals were killed by breaking their necks. The block sections were dissected, fixed in fixative solution (acid phosphatase kit, Sigma, St. Louis, MO) for 24 hours, decalcified in 10% ethylene-diaminetetraacetic acid solution (pH 7.4) at 4°C for 4 weeks and embedded in paraffin. Serial sections, 5 m thick, were prepared in mesiodistal planes and stained with hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP; Sigma), and Masson trichrome24 (4 sections/each stain). All slides were coded and read without prior knowledge (blinded). The sections stained with H&E and Masson trichrome were examined under stereomicroscope to evaluate lesion sizes, and the TRAP-stained sections were evaluated for the number of osteoclasts. Osteoclast counting was performed in the total area of lesion (TA) shown in Fig. 1 for both control and experimental groups, and the mean and standard deviation values were compared. Every third section was included in the total count of osteoclast cells per specimen. The decision to count osteoclast cells in every third section was based on the assumption that the average osteoclast was no more than 15 m in diameter. The amount of new bone formation were evaluated in the H&E- and Masson trichrome–stained sections under compound microscope with Zeiss Vision KS 400 software (Oberkochen, Germany). Histomorphometric analysis The Zeiss Vision KS 400 software was used for the histomorphometric analysis. The most central stained section that represented the maximum diameter of the defect was selected in each block. Each section was initially inspected using a light microscope (Axiostar; Zeiss) and saved as a digital image (Fig. 1, A). A composite digital image was then created by combining 3-4 smaller images, because it was not possible to capture the entire defect in 1 image at the level of magnification that was used in Fig. 1, B. The following criteria were used to standardize the histomorphometric analysis of the composite digital image: 1) the captured images of each histological section were merged on the computer screen to create a single composite image comprising the entire area of the surgical defect, and the TA and the newly formed bone area (NB) to be analyzed were then delineated on the composite image; and 2) the TA calculated in mm2 was considered 100% of the area to be analyzed (Fig. 1, A). The NB was also
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Fig. 1. A, Histologic section of the surgical defect of the experimental group at 4 weeks after treatment, demonstrating the total area (TA) to be analyzed. Masson trichrome stain; scale bar ⫽ 1 mm. B, Greater magnification of A showing newly formed bone tissue (NB) of immature type along the defect border (DB). BG, bioactive glass particles; scale bar ⫽ 200 m.
Table I. Lesion size, osteoclast numbers, and new bone formation of control and experimental groups 4 weeks after treatment Group (n ⫽ 13 each)
Lesion size (mm2)
Number of osteoclasts (cells/mm2)
Amount of new bone formation (mm2)
Percentage of new bone formation
13.18 ⫾ 1.80 14.27 ⫾ 2.87
0.37 ⫾ 0.11 0.37 ⫾ 0.14
4.04 ⫾ 1.39 6.12 ⫾ 2.14*
31.36 ⫾ 11.60 48.14 ⫾ 11.81
Control Experimental Values are mean ⫾ SD. *P ⬍ .05 compared with control.
calculated in mm2. The percentage of NB was calculated according to the following formula: percentage of NB ⫽ 共NB/TA兲 ⫻ 100 The values of NB of each rat were used to calculate the means and standard deviations of the control and experimental groups. Statistical analysis For statistical comparision between osteoclast numbers and amount of new bone formation of control and experimental groups, the independent t test was used, and a P value of ⬍.05 was regarded as significant. RESULTS The average lesion sizes of the control and experimental groups were 13.18 ⫾ 1.80 and 14.27 ⫾ 2.87 mm2, respectively (Table I). There were no statistically significant differences between the groups (P ⬎ .05). The osteoclast counting revealed that the numbers of osteoclasts in the control and the experimental groups were 0.37 ⫾ 0.11 and 0.37 ⫾ 0.14 cells/mm2, respectively (Table I). There were no statistically significant differences between the groups when the numbers of osteoclasts were compared (P ⬎ .05). New bone formation was observed along the borders of the surgical defect in both groups. The new bone trabeculae were immature and poorly organized. Less newly formed
bone was observed in the control group than in the experimental group (Fig. 2). The mean and standard deviation of new bone formation for each group is depicted in Table I. The experimental group showed a significant increase in new bone formation compared with the control group (P ⬍ .05). In the connective tissue adjacent to the bioactive glass particles, moderate numbers of fibroblasts, macrophages, and lymphocytes were present (Fig. 2). DISCUSSION In the present study, we used a bioactive glass as bone graft substitute, because several in vitro studies have shown the nontoxicity of bioactive glass, its positive influence on osteoblast culture,25 and its ability to form calcification foci in periodontal ligament fibroblasts.26 In addition, Zamet et al.27 and Low et al.28 reported good clinical results in intrabony defects in sites treated with a bioactive glass (Biogran) with a wide range of particle sizes compared with debridement. The present results showed that the use of the bioactive glass did not cause any undesirable reaction. No foreign body reactions indicating toxicity were observed, which is in agreement with the findings of several in vitro26 and histologic29-31 investigations in animals. New bone formation was observed along the borders of the surgical defect in both control and experimental groups. The replacement of bioactive glass
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Fig. 2. Photomicrograph showing newly formed bone tissue (NB) along the defect border (DB) of the control (A) and the experimental groups (B) at 4 weeks after treatment. BG, bioactive glass particles; scale bar ⫽ 200 m.
particles by new bone in the present study is consistent with the previous study by Villaca et al.31 This indicates that the graft material led to bone repair by an osseoconductive property. However, these studies are preliminary and may not be generalizable to other grafts beyond the limited data presented for bioactive glass. The experimental model was based on that described by Higuchi et al.32 The reasons for choosing this model were: 1) rats are readily available; 2) the surgical procedures on the rat mandibular bone are relatively simple; 3) spontaneous healing would not occur in the control site; 4) observations can be focused on the healing process of bone, because there are not any major nerves or blood vessels around the rat mandibular angles; 5) the preparation of tissue specimen is easy; and 6) the parameters can be simply and accurately measured in each specimen.32 In addition, because the effect of sex steroids, i.e., estrogen, on bone resorption and the development of osteoclasts and osteoblasts has been reported,33 only male rats were used in the present study to minimize those effects. Alendronate sodium is a bisphosphonate that acts as a potent inhibitor of bone resorption. It is now generally accepted that the main cell by which bisphosphonates mediate their action is the osteoclast. Four mechanisms appear to be involved: 1) inhibition of osteoclast recruitment; 2) inhibition of osteoclast adhesion; 3) shortening of osteoclast lifespan (apoptosis); and 4) inhibition of osteoclast activity.34 It has also been proposed that bisphosphonates have osteostimulative properties both in vivo and in vitro, as shown by increase in matrix formation.35,36 Several reports have demonstrated that bisphosphonates not only induce the osteoblasts to secrete inhibitors of osteoclast-mediated resorption but also stimulate the formation of osteoblast precursors and mineralized nodules, thereby promoting early osteoblastogenesis.37,38
Plotkin et al.39 examined the effect of alendronate administration in a murine model of glucocorticoid excess–induced apoptosis of osteocytes and osteoblasts. They reported that bisphosphonates inhibited osteocyte and osteoblast apoptosis. Meraw and Reeve’s study40 in dogs demonstrated that dental implants coated with hydroxyapatite and alendronate resulted in a significant increase in peri-implant bone. Another study revealed that bisphosphonates stimulate osteogenesis in conjuction with regenerative material around osseous defect and endosseous implants.41 The histopathologic results of the alendronate-treated groups in the present experimental study are consistent with those studies reporting the positive effect of bisphosphonates on the induction of osteoblastic activity. However, in the present study, Fosamax rather than pure alendronate was dissolved in saline and mixed with the bioactive glass. Fosamax is formulated with microcrystalline cellulose, anhydrous lactose, croscarmellose sodium, and magnesium stearate. It is possible that these agents may have contributed to the greater stimulation of bone growth at the sites that were grafted with Fosamax combined with the bioactive glass. The local delivery of Fosamax did not appear to have a damaging effect on the osteoclasts 4 weeks after grafting, because no significant difference was found between the control and experimental groups when the numbers of osteoclasts were compared. It is possible that the concentration of the alendronate placed in the defect was not large enough to cause apoptosis. Alendronate may have inhibited the function of the osteoclasts rather than cause cell death, and further studies need to be done to understand the potential mechanism before moving to human studies. Alendronate is available as oral therapy, and the 10 mg daily dose has been approved for the treatment of osteoporosis in postmenopausal women in ⬎80 countries. However, the clinically used 10 mg/day dose of
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alendronate has caused some gastrointestinal side effects and can probably not be exceeded.42 Moreover, oral bisphosphonates are poorly absorbed, with 1% or less of the administered dose being taken up,43 and long-term use of bisphosphonates is correlated with ONJ.44 Because bisphosphonates have a high affinity for bone mineral, topical application of the drug is feasible. The main advantage with topical administration is the possibility of administering a higher dose to the region of interest. The present results suggest that a single dose of topical administration of alendronate combined with bioactive glass was able to induce more bone regeneration, and this might be useful for alveolar ridge augmentation followed by dental implant surgery and for bone regeneration in periodontal defects. Conclusion The results of the present study showed that a single dose of local delivery of alendronate improved bone formation. Alendronate may be considered among the therapeutic options available to improve bone formation process in different bone remodeling cases. But further studies are required to elucidate the effect of local delivery of alendronate on bone formation in humans. This work was supported by a grant from Khon Kaen University. The authors thank Rushmore Co. for their suggestions and assistance with the Zeiss Vision KS 400 software. REFERENCES 1. Leonetti JA, Rambo HM, Throndson RR. Osteotome sinus elevation and implant placement with narrow size bioactive glass. Implant Dent 2000;9:177-82. 2. Schepers EJ, Ducheyne P. Bioactive glass particles of narrow size range for the treatment of oral bone defects: a 1-24 month experiment with several materials and particle sizes and size ranges. J Oral Rehabil 1997;24:171-81. 3. Furusawa T, Mizunuma K, Yamashita S, Takahashi T. Investigation of early bone formation using resorbable bioactive glass in the rat mandible. Int J Oral Maxillofac Implants 1998;13:672-6. 4. Furusawa T, Mizunuma K. Osteoconductive properties and efficacy of resorbable bioactive glass as a bone-grafting material. Implant Dent 1997;6:93-101. 5. Binderman I. Bone and biologically compatible materials in dentistry. Curr Opin Dent 1991;1:836-40. 6. Wozney JM. The bone morphogenetic protein family and osteogenesis. Mol Reprod Dev 1992;32:160-7. 7. Baylink DJ, Finkelman RD, Mohan S. Growth factors to stimulate bone formation. J Bone Miner Res 1993;8:S565-72. 8. Adami S, Zamberlan N, Mian M, Dorizzi R, Rossini M, Braga B, et al. Duration of the effects of intravenous alendronate in postmenopausal women and in patients with primary hyperparathyroidism and Paget’s disease of bone. Bone Miner 1994;25:75-82. 9. Liberman UA, Weiss SR, Broll J, Minne HW, Quan H, Bell NH, et al. Alendronate Phase III Osteoporosis Treatment Study Group. Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. N Engl J Med 1995;333:1437-43.
Srisubut et al. e15 10. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, et al. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 1991;88:2095-105. 11. Yaffe A, Iztkovich M, Earon Y, Alt I, Lilov R, Binderman I. Local delivery of an amino bisphosphonate prevents the resorptive phase of alveolar bone following mucoperiosteal flap surgery in rats. J Periodontol 1997;68:884-9. 12. Yaffe A, Fine N, Alt I, Binderman I. The effect of bisphosphonate on alveolar bone resorption following mucoperiosteal flap surgery in the mandible of rats. J Periodontol 1995;66:999-1003. 13. Schenk R, Eggli P, Fleisch H, Rosini S. Quantitative morphometric evaluation of the inhibitory activity of new aminobisphosphonates on bone resorption in the rat. Calcif Tissue Int 1986; 38:342-9. 14. Kaynak D, Meffert R, Gunhan M, Gunhan O, Ozkaya O. A histopathological investigation on the effects of the bisphosphonate alendronate on resorptive phase following mucoperiosteal flap surgery in the mandible of rats. J Periodontol 2000;71:790-6. 15. Masarachia P, Weinreb M, Balena R, Rodan GA. Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996;19:281-90. 16. Yaffe A, Binderman I, Breuer E, Pinto T, Golomb G. Disposition of alendronate following local delivery in a rat jaw. J Periodontol 1999;70:893-5. 17. Astrand J, Aspenberg P. Topical, single dose bisphosphonate treatment reduced bone resorption in a rat model for prosthetic loosening. J Orthop Res 2004;22:244-9. 18. Leite AF, Figueiredo PT, Melo NS, Acevedo AC, Cavalcanti MG, Paula LM, et al. Bisphosphonate-associated osteonecrosis of the jaws. Report of a case and literature review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:14-21. 19. Robinson NA, Yeo JF. Bisphosphonates—a word of caution. Ann Acad Med Singapore 2004;33:48-9. 20. Tarassoff P, Csermak K. Avascular necrosis of the jaws: risk factors in metastatic cancer patients. J Oral Maxillofac Surg 2003;61:1238-9. 21. Agarwala S, Sule A, Pai BU, Joshi VR. Alendronate in the treatment of avascular necrosis of the hip. Rheumatology (Oxf) 2002;41:346-7. 22. Astrand J, Aspenberg P. Systemic alendronate prevents resorption of necrotic bone during revascularization. A bone chamber study in rats. BMC Musculoskelet Disord 2002;3:19. 23. Kaban LB, Glowacki J. Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 1981;60: 1356-64. 24. Luna L. Manual of histologic staining methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw; 1968. p. 94 –5. 25. Matsuda T, Davies JE. The in vitro response of osteoblasts to bioactive glass. Biomaterials 1987;8:275-84. 26. Hench LL, Paschall HA. Histochemical responses at a biomaterial’s interface. J Biomed Mater Res 1974;8:49-64. 27. Zamet JS, Darbar UR, Griffiths GS, Bulman JS, Bragger U, Burgin W, et al. Particulate bioglass as a grafting material in the treatment of periodontal intrabony defects. J Clin Periodontol 1997;24:410-8. 28. Low SB, King CJ, Krieger J. An evaluation of bioactive ceramic in the treatment of periodontal osseous defects. Int J Periodontics Restor Dent 1997;17:358-67. 29. Hench LL, Paschall HA. Direct chemical bond of bioactive glass-ceramic materials to bone and muscle. J Biomed Mater Res 1973;7:25-42.
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30. Wilson J, Clark AE, Hall M, Hench LL. Tissue response to Bioglass endosseous ridge maintenance implants. J Oral Implantol 1993;19:295-302. 31. Villaca JH, Novaes AB Jr, Souza SL, Taba M Jr, Molina GO, Carvalho TL. Bioactive glass efficacy in the periodontal healing of intrabony defects in monkeys. Braz Dent J 2005;16:67-74. 32. Higuchi T, Kinoshita A, Takahashi K, Oda S, Ishikawa I. Bone regeneration by recombinant human bone morphogenetic protein-2 in rat mandibular defects. An experimental model of defect filling. J Periodontol 1999;70:1026-31. 33. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr Rev 2000;21:115-37. 34. Fleisch H. Bisphosphonates: mechanisms of action. Endocr Rev 1998;19:80-100. 35. Tenenbaum HC, Torontali M, Sukhu B. Effects of bisphosphonates and inorganic pyrophosphate on osteogenesis in vitro. Bone 1992;13:249-55. 36. Goziotis A, Sukhu B, Torontali M, Dowhaniuk M, Tenenbaum HC. Effects of bisphosphonates APD and HEBP on bone metabolism in vitro. Bone 1995;16:317S-27S. 37. Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G. Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone 1998;22:455-61. 38. Vitte C, Fleisch H, Guenther HL. Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclast-mediated resorption. Endocrinology 1996;137:2324-33.
39. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 1999;104: 1363-74. 40. Meraw SJ, Reeve CM. Qualitative analysis of peripheral periimplant bone and influence of alendronate sodium on early bone regeneration. J Periodontol 1999;70:1228-33. 41. Tenenbaum HC, Shelemay A, Girard B, Zohar R, Fritz PC. Bisphosphonates and periodontics: potential applications for regulation of bone mass in the periodontium and other therapeutic/ diagnostic uses. J Periodontol 2002;73:813-22. 42. Bone HG, Adami S, Rizzoli R, Favus M, Ross PD, Santora A, et al. Weekly administration of alendronate: rationale and plan for clinical assessment. Clin Ther 2000;22:15-28. 43. Vasikaran SD. Bisphosphonates: an overview with special reference to alendronate. Ann Clin Biochem 2001;38:608-23. 44. Ruggiero SL, Mehrotra B, Rosenberg TJ, Engroff SL. Osteonecrosis of the jaws associated with the use of bisphosphonates: a review of 63 cases. J Oral Maxillofac Surg 2004; 62:527-34. Reprint requests: Assosiate Professor Dr. Suwimol Taweechaisupapong Department of Oral Diagnosis, Faculty of Dentistry Khon Kaen University Khon Kaen, 40002 Thailand [email protected]