Dental Traumatology 2014; 30: 415–422; doi: 10.1111/edt.12116
Effects of the bisphosphonate alendronate on molars of young rats after lateral luxation udia Pires Rothbarth1,2, Vivian Cla Bradaschia-Correa1, Lorraine Braga Ferreira1, Victor Elias Arana-Chavez1 1 Department of Biomaterials and Oral Biology, ~o Paulo School of Dentistry, University of Sa ~o Paulo; 2Department of Dentistry, Federal Sa , Bele m, Brazil University of Para
Key words: lateral luxation; alendronate; root formation; osteopontin Correspondence to: Victor Elias Arana-Chavez, Department of Biomaterials ~o Paulo and Oral Biology, University of Sa School of Dentistry, Av Prof Lineu Prestes ~o Paulo, SP, Brazil 2227, 05508-000 Sa Tel.: +55 11 30917840 Fax: +55 11 30917840 e-mail: [email protected] Accepted 17 April, 2014
Abstract – Background and aim: The bisphosphonate alendronate (ALN) was employed with the aim of investigating its effects on dental and periodontal tissues after lateral luxation of developing molars. Material and methods: Twenty-one-day-old Wistar rats had their second upper molars laterally luxated. Daily 2.5 mg kg 1 ALN injections started at the day of the luxation; controls received sterile saline solution. The teeth were analyzed 7, 14, and 21 days after the procedure. On the days cited, the maxillae were fixed, decalcified, and embedded in paraffin or Spurr resin. The paraffin sections were stained with H&E, incubated for TRAP histochemistry or immunolabeled for osteopontin (OPN). Spurr ultrathin sections were examined in a transmission electron microscope. Results: After 21 days, the root apex of luxated molars without ALN was wide open and disorganized and also covered by an irregular layer of cellular cementum, which was not observed in ALN-treated animals. Ankylosis sites were observed in ALN rats in both luxated and non-luxated teeth. The TRAP-positive osteoclasts were more numerous in ALN group, despite their latent ultrastructural appearance without the presence of resorption apparatus compared to controls. OPN immunolabeling revealed a thick immunopositive line in the dentin that must be resultant from the moment of the luxation, while ALN-treated specimens did not present alterations in dentin. Conclusion: The present findings indicate that alendronate inhibits some alterations in dentin and cementum formation induced by dental trauma.
Lateral luxation is one of the most common injuries in dental practice. It results in the buccal, lingual, mesial or distal tooth dislocation that usually accomplishes an apical root displacement (1) and is more frequent in young patients with erupting teeth (1–3). During root formation, cells of the cervical loop proliferate apically and give rise to the Hertwig’s epithelial root sheath (HERS) and epithelial diaphragm, which cells induce the differentiation of dental papilla ectomesenchymal cells into odontoblasts (4). Ectomesenchymal cells from the dental follicle differentiate into fibroblasts and cementoblasts that form the cementum over the root dentin, as well as into fibroblasts and osteoblasts that form the collagen fibers of the periodontal ligament and the alveolar bone, respectively (5). Disruption of such tissues may alter the root and periodontium development, as well as promote root resorption (1). The most reported effects on tooth development resulting from dental luxation involve disruption of the periodontal fibers and periapical blood vessels and nerve fibers, which can culminate in failed dentinogenesis, narrow pulp chamber, disturbed root development, odontoma formation, ankylosis, and failed eruption (6). External root resorption has also been reported as © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
a consequence of dental trauma and occurs by recruitment and activation of clastic cells that resorb dental mineralized tissues by a mechanism similar to that of bone resorption (7). Tooth resorption may be inhibited by bisphosphonate treatment that targets the clastic cells. Indeed, as bisphosphonates are antiresorptive drugs widely employed in several bone disorders (8), their employment could be beneficial to the prognosis of luxated teeth. Alendronate is a nitrogenated bisphosphonate that has been employed in previous studies in which it clearly inhibited clastic activation, inducing these cells to present a latent phenotype (9). It was also showed that these effects on the clastic cells were occasioned because this bisphosphonate reduces the expression of the protein receptor activator of NFjB ligand (RANKL) in alveolar bone (10). As alendronate is currently the most prescribed bisphosphonate, it was elected to test this hypothesis in the present study. As the cellular effects of lateral luxation of incompletely formed teeth are not completely understood, and the possibility of modifying its repercussion on dental and periodontal mineralized tissues with alendronate treatment, the present study aimed to clarify 415
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these aspects structurally and immunohistochemically. The tissue responses after the lateral luxation of upper second molars undergoing the root stage of tooth development in young alendronate-treated rats were evaluated by light and transmission electron microscopy. It was also analyzed the presence of clastic cells by tartrate-resistant acid phosphatase histochemistry (TRAP) histochemistry. The immunolocalization of osteopontin, a non-collagenous protein associated with the mineralization of collagen fibrils in bone, cementum, and dentin, as well as with the resorption of mineralized tissues (11, 12), was also carried out in order to verify how the lateral luxation and the alendronate treatment influenced on the dental mineralized tissues. Material and methods
solution overnight at 4°C (14). The specimens were decalcified in 4.13% EDTA during 4 weeks and embedded in paraplast. Five-mm thick sections were obtained in a Micron HM360 microtome and stained with hematoxylin and eosin. The slides were examined in an Olympus BX60 light microscope equipped with an Olympus DP72 CCD camera. Specimens destined to ultrastructural analysis were postfixed in osmium tetroxide and embedded in Spurr epoxy resin (EMS, Hatfield, PA, USA). Sections 80-nm thick were obtained with a diamond knife on a Leica Ultracut R ultramicrotome (Leica, Buffalo, NY, USA), collected onto 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1010 transmission electron microscope operated at 80 kV. The images were digitally obtained with the GATAN imaging platform equipped with a SC1000 Orius CCD camera.
Animals and surgical procedure
Principles of laboratory animal care (NIH publication 85–23, 1985) and national laws on animal use were observed for the present study, which was authorized by the Ethical Committee for Animal Research of the University of S~ ao Paulo, Brazil. Fifty-four 21-day-old Wistar rats (Rattus norvegicus albinus) from both sexes were utilized in the present sudy. Previously to the luxation, the animals were anesthetized with 2% chloridrate 2-(6,6-xilidine)-5,6-dihydro-4-H-1,3-tiazine (Rompunâ, Bayer Animal Health, Leverkusen, Germany) diluted 1:1 in ketamine (Francotarâ, Virbac, Roseira, Brazil), 1 ml kg 1 body weight. The luxation was executed by utilizing a discoid cleoid carver instrument, which was inserted in the gingival sulcus facing the palatal surface of the upper right second molar; lever movements were exerted until a slight buccal dislocation had occurred, and discrete bleeding was noted in the palatal gingival sulcus. The left second molar was not luxated. Alendronate treatment
Twenty-seven animals were daily weighed, and a dose of 2.5 mg kg 1 body weight of sodium alendronate (ALN) was freshly prepared in 0.01 M phosphate saline buffer, pH 7.2, (PBS) and injected subcutaneously. The remaining animals were used as controls and received daily PBS injections (13). The injections with alendronate or PBS solutions started at the same day of the luxation procedure until 7, 14, and 21 days. The non-luxated second molars were also evaluated in the same time points for alendronate-treated and control animals. Specimen obtaining, fixation, decalcification and embedding
On the time points cited, the animals were anesthetized as described and euthanized. The maxillae were fixed in 0.1% glutaraldehyde and 4% formaldehyde buffered in 0.1 M sodium cacodylate, pH 7.4 under microwave irradiation in a Pelco 3440 laboratory microwave oven (Ted Pella, Redding, CA, USA) during three cycles with 100% potency and maximum temperature of 37°C. They then remained immersed in fresh fixative
Tartrate-resistant acid phosphatase histochemistry
Three-lm thick paraffin sections were collected onto glass slides and submitted to TRAP histochemistry. The slides containing the sections were incubated during 2 h in Burstone complete medium containing 50 mM D( ) tartaric acid (Sigma Chemical Co., St. Louis, MO, USA). After that, the slides were washed in tap water and then counterstained with Harris’ hematoxylin for 10 min (15). Immunohistochemical detection of osteopontin
Some sections were collected onto silane-coated glass slides and submitted to immunohistochemical detection of osteopontin. After dewaxing, the sections were heated to 60°C for 15 min and treated with H2O2/ methanol solution (1:1). The non-specific binding sites were blocked during 1 h with 30% non-immune swine serum (Dako, Carpinteria, CA, USA) in 1% BSA. Then, they were incubated with the primary antibody (LF175 1:1200; Larry Fisher, NIH, Bethesda, MD, USA) during 90 min, at room temperature within a humid chamber. After rinsing with buffer, detection was achieved using DAB as substrate (Dako), and nuclei were stained with Harris’s hematoxylin. Negative controls were incubated in the absence of primary antibody (16). Results Hematoxylin and eosin staining
None of the specimens analyzed presented signs of pulp necrosis in all time points. On the first time point, the non-luxated molars of CON and ALN groups presented normal root development; the epithelial diaphragm and HERS were intact (Fig. 1a,b), while the luxated specimens from CON group presented a cement line in the dentin. Extensive and deep resorption lacunae were observed on the root dentin at the periodontal ligament side from the cervical to the central portion of the root (Fig. 1c). The apical portion was disrupted, and a thick layer of dentin matrix © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Fig. 1. Light micrographs of rat second molars. Non-luxated (N LUX) specimens of CON (a) and ALN (b) groups at the first time point present typical features of root development, with odontoblasts (ob) forming root dentin, cementoblasts (cb), HERS cells (hers), and epithelial diaphragm (ed). b, bone. LUX CON specimens 7 days after luxation present a cement line (cl) in crown (cd) and root dentin (rd). A large resorption lacuna (rl) is observed in the root surface in (c). In (d), numerous cells are situated inside lacunae (arrows) in the apical root dentin. LUX ALN specimens at this time point present some clastic cells (cc) adjacent to resoption lacunae in root dentin in (e). The N LUX CON (f) on the next time point present cellular cementum (cem) formed on the apical portion of the root. The N LUX ALN (g) presents a thin layer of cellular cementum. In the CON LUX 14 days after luxation (h), the acellular cementum (ac) present irregularities and, at the apical portion, dentin, and cellular cementum are disorganized. pl, periodontal ligament. At this time pont, LUX ALN (i) shows cellular cementum and apical root dentin more organized than LUX CON. At the last time point, the second molar root is almost completely formed, and its apical portion is covered by cellular cementum in N LUX CON (j). In N LUX ALN (k) specimens, thin bone trabeculae invade the periodontal ligament space near the thin cellular cementum. The LUX CON (l) 21 days after luxation shows a disrupted root apex. P, pulp. The LUX ALN (m) at this time point present an ankylosis site between alveolar bone and cellular cementum (arrowhead). Note the irregular shape of the cementum. Bars: a–g, i, k = 50 lm; h, j = 100 lm.
containing numerous embedded cells was observed (Fig. 1d). The ALN luxated specimens also presented some resorption lacunae at the root surface facing the periodontal ligament (Fig. 1e). On the second time point, the non-luxated molars on CON and ALN groups still presented normal morphology (Fig. 1f,g). The luxated CON 14 days after the luxation presented disorganized root dentin matrix with lacunae containing cells at the apical portion (Fig. 1h), which was not observed in the ALN group (Fig. 1i). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
On the last time point, the non-luxated CON specimens presented typical features (Fig. 1j). The non-luxated ALN specimens, however, presented a thin layer of cellular cementum over the apical portion of the root and disorganized alveolar bone trabeculae (Fig. 1k). The CON specimens 21 days after the luxation presented a severely disorganized root apex (Fig. 1l). ALN specimens 21 days after luxation present deformities in the root and cementum surface and some ankylosis sites characterized by the contact of
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alveolar bone trabeculae with the cellular cementum (Fig. 1m). Tartrate-resistant acid phosphatase histochemistry
Osteoclasts were evidenced by TRAP histochemistry in light micrographs and were also ultrastructurally examined by transmission electron microscopy. The non-luxated (Fig. 2a) and luxated (Fig. 2b) CON specimens presented numerous TRAP-positive osteoclasts attached to the alveolar bone surface, adjacent to Howship lacunae. The utrastructural analysis confirmed the activated phenotype of osteoclasts, which were adhered to the bone matrix surface and presented clear zone and exuberant ruffled border (Fig. 2c). Both ALN nonluxated (Fig. 2d) and luxated (Fig. 2e) presented numerous TRAP-positive osteoclasts but they were mostly rounded-shaped and not adhered to the bone surfaces; these features were confirmed by transmission electron micrographs where clastic cells presented short contact areas of the plasma membrane with the bone matrix (Fig. 2f). They did not present the resorptive apparatus as described for the CON specimens.
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Transmission electron microscopy
The CON specimens presented cellular cementum where lacunae containing two cells were observed (Fig. 3a). The epithelial diaphragm and HERS were severely disrupted (Fig. 3b). The ALN specimens presented some resorpion lacunae along the root dentin (Fig. 3c). Some areas of collagen-based matrix surrounded by connective cells were seen at the apical region of the dental follicle adjacent to the HERS cells (Fig 3d). Some areas were detected where bone trabeculae had invaded the periodontal ligament and were localized near the forming cellular cementum (Fig. 3e). The apical portion of the root presented inversion of the epithelial diaphragm, which resulted in dilacerations of the apical portion of the root (Fig. 3f). Immunodetection of osteopontin
At the first time point, non-luxated CON and ALN specimens presented immunolabeling for osteopontin on dentin, cementum, and on the forming alveolar bone surface (Fig. 4a,b). The luxated CON at the same
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Fig. 2. Non-luxated (N LUX) and luxated (LUX) CON specimens (a and b, respectively) present activated TRAPpositive osteoclasts (stained in red) resorbing the alveolar (AB) bone during the root and periodontium formation. D, dentin, and PL, periodontal ligament. The ultrastructural examination, in c, confirmed the activated phenotype of the osteoclasts (Oc), showing typical clear zone (cz) and ruffled border (RB) and are attached to the bone matrix (B). ALNtreated N LUX (d) and LUX (e) specimens present numerous TRAPpositive osteoclasts in the marrow spaces, but most of them are not adhered to the bone matrix. Ultrastructurally, an osteoclast with latent phenotype (LOc), without the resorption apparatus is observed in f. Bars: a, b, d, e = 50 lm; c = 5 lm; f = 10 lm. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lateral luxation and alendronate in developing rat molars
Fig. 3. Representative transmission electron micrographs of luxated specimens from CON and ALN groups. In a, CON specimen the lacunae observed in the cellular cementum (cem) are occupied by two cells (arrows). In b, the epithelial diaphragm cells (ed) are disorganized. The ALN specimens, in c, show a resorption lacune (rl) in cementum (c) filled by cementoblasts that are probably going to repair the resorbed matrix. In d, a fibrous matrix appeared surrounded by dental follicle cells (M). In e, the structures of the periodontal ligament (pl) are being compressed by a bone trabecule (bt). In f, the disorganized structure of the apical portion of the root, with atypical arrangement of cementoblasts (cb). do, differentiating odontoblasts. Bars: a, b = 20 lm; c, e, f = 10 lm; d = 5 lm.
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time point, after 7 days of luxation, presented strongly immunoreactive cement lines on the root dentin and a diffuse labeling in the osteoid tissue formed in the apical area (Fig. 4c). The luxated ALN specimens presented strong immunodetection in the surface of Howship’s lacunae in root dentin (Fig. 4d). On the following time point, non-luxated CON specimen presented positive immunolabeling on the cementum matrix and on cement lines and surfaces of the alveolar bone (Fig 4e). The non-luxated ALN specimen showed cement lines and cementum immunopositive for osteopontin, as well as some points of ankylosis (Fig. 4f). Fourteen days after the luxation, CON specimens presented strongly immunopositive cementum matrix, which appeared to be covering the root dentin in a discontinuous pattern (Fig. 4g). The cellular cementum of the luxated ALN specimens appeared © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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thinner than CON and weakly immunolabeled. The bone matrix presented cement lines strongly immunolabeled for osteopontin (Fig. 4h). On the last time point, the root dentin, cellular cementum, and alveolar bone of non-luxated CON specimens were strongly immunopositive (Fig. 4i). In contrast, non-luxated ALN specimens at this time point presented weak immunolabeing in the cellular cementum, which was thinner than observed in CON (Fig. 4j). The CON specimens 21 days after luxation presented a cement line in all the extension of the crown dentin. Large resorption lacunae with the surface immunolabeled for osteopontin were observed on the root dentin facing the periodontal ligament (Fig. 4k). In the luxated ALN specimen 21 days after luxation, the immunolabeling for osteopontin on the alveolar bone surface was more intense than CON (Fig. 4l).
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Fig. 4. Immunolabeling for OPN in brown. Non-luxated (N LUX) specimens of CON (a) and ALN (b) groups at the first time point present cementum (cem) and the alveolar bone (ab) surface positively immunolabeled. LUX CON specimens 7 days after luxation (c) present immunolabeling in the cement lines (cl) in root dentin. LUX ALN specimens at this time point, in d, present resorption lacunae (rl) in root dentin (rd) strongly positive for OPN. The N LUX CON (e) on the next time point presents OPN immunolabeling in cellular cementum (cem) and alveolar bone (ab) surface, as well as N LUX ALN in f. In g, a LUX CON specimen 14 days after luxation presents positively immunolabeled cellular cementum irregularly deposited onto the root dentin. The LUX ALN (h) at this time point shows strong immunolabeling in the alveolar bone surface and some ankylosis points (arrows). On the last time point, N LUX CON (i) presents a typical thick cellular cementum over the root dentin immunopositive for OPN. Differently, the cellular cementum in N LUX ALN (j) is thin and weakly immunoreactive. The CON LUX 21 days after luxation (k) shows an extensive resorbed area on the root dentin immunopositive on its surface (arrowhead). Note the presence of an increment of dentin strongly immunolabeled for OPN. p, dental pulp. The LUX ALN in l, like the N LUX ALN in j, presents a discrete cellular cementum. Note that this specimen has less deformities than the CON LUX in k. Bars: a, b = 50 lm, e–g = 100 lm; c, d = 50 lm.
Discussion
The present study revealed that sodium alendronate attenuated the alterations on root and periodontal tissues after the lateral luxation of rat molars without impairing the root development; however, the ALN treatment provoked ankylosis sites in either luxated or non-luxated specimens. Although lateral luxations are highly common in anterior teeth (1), the present study utilized the rat molar because it is comparable in many aspects to
human teeth. Despite some studies have used the rat incisor model for studying dental trauma (17–19), they erupt continuously and therefore amelogenesis and dentinogenesis take place continuously as well. Most important, while their lingual alveolar wall faces a periodontal ligament, their buccal alveolar wall presents a dental follicle-like tissue rather than a periodontal ligament proper (20). The present study involved the lateral luxation on 21-day-old rats in which the second molar roots are not completely formed and still present embryonic structures at their apical end, such as HERS © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lateral luxation and alendronate in developing rat molars and epithelial diaphragm. By this experimental design, it was possible to verify whether the complete root formation was altered and how alendronate would potentially interfere in these alterations. Previous reports proposed experimental models in which calibrated apparatus are employed to reproduce the same dental trauma among the specimens (21, 22); nevertheless, establishing an accurate dental trauma experimental model is difficult, and clinical situations present extremely diverse etiologies, with variable intensities of forces applied over teeth and rates of dislocation. Thus, the luxation procedure employed in the present study proved an appropriate model for evaluating the completion of root formation after trauma on rat molars. The lateral luxation did not cause dental ankylosis in controls, but it occurred in alendronate-treated specimens. In contrast, previous reports have shown that alendronate pretreatment of roots prevented the shortterm ankylosis after replantation of avulsed rat molars (23). Ankylosis was described in replanted alendronatetreated rat incisors (24) and molar roots (25) after long periods. In the present study, the trauma occasioned morphological alterations in the dentin and cellular cementum, as well as in the immunolocalization of osteopontin in control specimens. The apical portion of root dentin did not present the typical tubular organization in all time points. Instead, it presented numerous cells inside lacunae, resembling a primary bone aspect. Similarly, previous studies describe the presence of cells entrapped into the tertiary dentin of rat incisors after extrusive luxation (19). The dentin presented a cement line intensely labeled for osteopontin in its complete extension at both their coronal and root portions, suggesting that the secretion of dentin matrix by odontoblasts was temporarily interrupted after the trauma. Osteopontin is synthesized by odontoblasts during dentinogenesis but only small amounts which are not immunodetected are present in the dentin matrix after its mineralization (26–28). The intense immunodetection of this protein in dentin suggests that the lateral luxation stimulated an increase of its synthesis and secretion into the matrix, which presented stronger labeling in the deeper portion of dentin compared with the superficial dentin. Previous reports also demonstrated that reactionary dentin after luxation of rat incisors induces odontoblasts to secrete dentin matrix with primary bone appearance, highly immunopositive for osteopontin (19). Regarding the alendronate-treated luxated specimens, they presented discrete alterations in the root morphology, but the appearance of the dentin matrix was similar to the non-luxated molars, as well as the distribution of osteopontin. ALN did not impede the differentiation of cementoblasts, despite the impairment of root elongation of erupting molars described in previous studies (29); in fact, less cellular cementum was formed in both luxated and non-luxated ALN-treated specimens. The non-luxated specimens without ALN treatment presented acellular and cellular cementum strongly immunopositive to osteopontin, while both luxated and non-luxated ALN-treated specimens showed weak immunolabeling in the cementum. As ALN impaired the resorption © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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activity of osteoclasts, which remained in a latent stage, the alveolar bone trabeculae were not remodeled and grew in a disorganized pattern toward the dental follicle/periodontal ligament space. As the dental follicle is a source of ectomesenchymal cells that differentiate into fibroblasts and cementoblasts (5), the disruption of these structures in luxated and non-luxated ALN-treated molars may be related to the reduced thickness of cellular cementum in all time points. The differentiation of cementum forming cells was demonstrated to occur in the dental follicle of developing molars from ALN-treated rats, but the compression of dental follicle cells by bone trabeculae affect their secretory activity and root elongation (29). Although ALN is often related to increased risk of complications like jaw osteonecrosis (8, 16) and impairment of tooth eruption (9, 10), the current results demonstrate that ALN treatment was able to attenuate the severe alterations that were observed in luxated teeth with incomplete roots. Acknowledgements
The authors would like to thank Eloiza Rezende for her help with the animal handling. C. P. Rothbarth was the recipient of a research fellowship from the Special PostGraduate Program CAPES/DINTER, Brazil. This work was partially supported by FAPESP and CNPq, Brazil. References 1. Andreasen JO, Andreasen FM, Andersson L. Textbook and color atlas of traumatic injuries to the teeth, 4th edn. Oxford: Blackwell Munksgaard; 2007. 2. Caldas AF Jr, Burgos MEA. A retrospective study of traumatic dental injuries in a Brazilian dental trauma clinic. Dent Traumatol 2001;17:250–3. 3. Hecova H, Tzigkounakis V, Merglova V, Netolicky J. A retrospective study of 889 injured permanent teeth. Dent Traumatol 2010;26:466–75. 4. Janones DS, Massa LF, Arana-Chavez VE. Immunocytochemical examination of the presence of amelogenin during the root development of rat molars. Arch Oral Biol 2005;50:527–32. 5. Zeichner-David M. Regeneration of periodontal tissues: cementogenesis revisited. Periodontol 2000 2006;41:196–217. 6. Andreasen JO, Raven JJ. Epidemiology of traumatic dental injuries to primary and permanent teeth in a Danish population sample. Int J Oral Surg 1972;1:235–9. 7. Arana-Chavez VE, Bradaschia-Correa V. Clastic cells: mineralized tissue resorption in health and disease. Int J Biochem Cell Biol 2009;41:446–50. 8. Russell RG. Bisphosphonates: the first 40 years. Bone 2011;49:34–41. 9. Bradaschia-Correa V, Massa LF, Arana-Chavez VE. Effects of alendronate on tooth eruption and molar root formation in young growing rats. Cell Tissue Res 2007;330:475–85. 10. Bradaschia-Correa V, Moreira MM, Arana-Chavez VE. Reduced RANKL expression impedes osteoclast activation and tooth eruption in alendronate-treated rats. Cell Tissue Res 2013;353:79–86. 11. McKee MD, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover and repair. Microsc Res Tech 1996;33:141–64.
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Effects of the bisphosphonate alendronate on molars of young rats after lateral luxation udia Pires Rothbarth1,2, Vivian Cla Bradaschia-Correa1, Lorraine Braga Ferreira1, Victor Elias Arana-Chavez1 1 Department of Biomaterials and Oral Biology, ~o Paulo School of Dentistry, University of Sa ~o Paulo; 2Department of Dentistry, Federal Sa , Bele m, Brazil University of Para
Key words: lateral luxation; alendronate; root formation; osteopontin Correspondence to: Victor Elias Arana-Chavez, Department of Biomaterials ~o Paulo and Oral Biology, University of Sa School of Dentistry, Av Prof Lineu Prestes ~o Paulo, SP, Brazil 2227, 05508-000 Sa Tel.: +55 11 30917840 Fax: +55 11 30917840 e-mail: [email protected] Accepted 17 April, 2014
Abstract – Background and aim: The bisphosphonate alendronate (ALN) was employed with the aim of investigating its effects on dental and periodontal tissues after lateral luxation of developing molars. Material and methods: Twenty-one-day-old Wistar rats had their second upper molars laterally luxated. Daily 2.5 mg kg 1 ALN injections started at the day of the luxation; controls received sterile saline solution. The teeth were analyzed 7, 14, and 21 days after the procedure. On the days cited, the maxillae were fixed, decalcified, and embedded in paraffin or Spurr resin. The paraffin sections were stained with H&E, incubated for TRAP histochemistry or immunolabeled for osteopontin (OPN). Spurr ultrathin sections were examined in a transmission electron microscope. Results: After 21 days, the root apex of luxated molars without ALN was wide open and disorganized and also covered by an irregular layer of cellular cementum, which was not observed in ALN-treated animals. Ankylosis sites were observed in ALN rats in both luxated and non-luxated teeth. The TRAP-positive osteoclasts were more numerous in ALN group, despite their latent ultrastructural appearance without the presence of resorption apparatus compared to controls. OPN immunolabeling revealed a thick immunopositive line in the dentin that must be resultant from the moment of the luxation, while ALN-treated specimens did not present alterations in dentin. Conclusion: The present findings indicate that alendronate inhibits some alterations in dentin and cementum formation induced by dental trauma.
Lateral luxation is one of the most common injuries in dental practice. It results in the buccal, lingual, mesial or distal tooth dislocation that usually accomplishes an apical root displacement (1) and is more frequent in young patients with erupting teeth (1–3). During root formation, cells of the cervical loop proliferate apically and give rise to the Hertwig’s epithelial root sheath (HERS) and epithelial diaphragm, which cells induce the differentiation of dental papilla ectomesenchymal cells into odontoblasts (4). Ectomesenchymal cells from the dental follicle differentiate into fibroblasts and cementoblasts that form the cementum over the root dentin, as well as into fibroblasts and osteoblasts that form the collagen fibers of the periodontal ligament and the alveolar bone, respectively (5). Disruption of such tissues may alter the root and periodontium development, as well as promote root resorption (1). The most reported effects on tooth development resulting from dental luxation involve disruption of the periodontal fibers and periapical blood vessels and nerve fibers, which can culminate in failed dentinogenesis, narrow pulp chamber, disturbed root development, odontoma formation, ankylosis, and failed eruption (6). External root resorption has also been reported as © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
a consequence of dental trauma and occurs by recruitment and activation of clastic cells that resorb dental mineralized tissues by a mechanism similar to that of bone resorption (7). Tooth resorption may be inhibited by bisphosphonate treatment that targets the clastic cells. Indeed, as bisphosphonates are antiresorptive drugs widely employed in several bone disorders (8), their employment could be beneficial to the prognosis of luxated teeth. Alendronate is a nitrogenated bisphosphonate that has been employed in previous studies in which it clearly inhibited clastic activation, inducing these cells to present a latent phenotype (9). It was also showed that these effects on the clastic cells were occasioned because this bisphosphonate reduces the expression of the protein receptor activator of NFjB ligand (RANKL) in alveolar bone (10). As alendronate is currently the most prescribed bisphosphonate, it was elected to test this hypothesis in the present study. As the cellular effects of lateral luxation of incompletely formed teeth are not completely understood, and the possibility of modifying its repercussion on dental and periodontal mineralized tissues with alendronate treatment, the present study aimed to clarify 415
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these aspects structurally and immunohistochemically. The tissue responses after the lateral luxation of upper second molars undergoing the root stage of tooth development in young alendronate-treated rats were evaluated by light and transmission electron microscopy. It was also analyzed the presence of clastic cells by tartrate-resistant acid phosphatase histochemistry (TRAP) histochemistry. The immunolocalization of osteopontin, a non-collagenous protein associated with the mineralization of collagen fibrils in bone, cementum, and dentin, as well as with the resorption of mineralized tissues (11, 12), was also carried out in order to verify how the lateral luxation and the alendronate treatment influenced on the dental mineralized tissues. Material and methods
solution overnight at 4°C (14). The specimens were decalcified in 4.13% EDTA during 4 weeks and embedded in paraplast. Five-mm thick sections were obtained in a Micron HM360 microtome and stained with hematoxylin and eosin. The slides were examined in an Olympus BX60 light microscope equipped with an Olympus DP72 CCD camera. Specimens destined to ultrastructural analysis were postfixed in osmium tetroxide and embedded in Spurr epoxy resin (EMS, Hatfield, PA, USA). Sections 80-nm thick were obtained with a diamond knife on a Leica Ultracut R ultramicrotome (Leica, Buffalo, NY, USA), collected onto 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1010 transmission electron microscope operated at 80 kV. The images were digitally obtained with the GATAN imaging platform equipped with a SC1000 Orius CCD camera.
Animals and surgical procedure
Principles of laboratory animal care (NIH publication 85–23, 1985) and national laws on animal use were observed for the present study, which was authorized by the Ethical Committee for Animal Research of the University of S~ ao Paulo, Brazil. Fifty-four 21-day-old Wistar rats (Rattus norvegicus albinus) from both sexes were utilized in the present sudy. Previously to the luxation, the animals were anesthetized with 2% chloridrate 2-(6,6-xilidine)-5,6-dihydro-4-H-1,3-tiazine (Rompunâ, Bayer Animal Health, Leverkusen, Germany) diluted 1:1 in ketamine (Francotarâ, Virbac, Roseira, Brazil), 1 ml kg 1 body weight. The luxation was executed by utilizing a discoid cleoid carver instrument, which was inserted in the gingival sulcus facing the palatal surface of the upper right second molar; lever movements were exerted until a slight buccal dislocation had occurred, and discrete bleeding was noted in the palatal gingival sulcus. The left second molar was not luxated. Alendronate treatment
Twenty-seven animals were daily weighed, and a dose of 2.5 mg kg 1 body weight of sodium alendronate (ALN) was freshly prepared in 0.01 M phosphate saline buffer, pH 7.2, (PBS) and injected subcutaneously. The remaining animals were used as controls and received daily PBS injections (13). The injections with alendronate or PBS solutions started at the same day of the luxation procedure until 7, 14, and 21 days. The non-luxated second molars were also evaluated in the same time points for alendronate-treated and control animals. Specimen obtaining, fixation, decalcification and embedding
On the time points cited, the animals were anesthetized as described and euthanized. The maxillae were fixed in 0.1% glutaraldehyde and 4% formaldehyde buffered in 0.1 M sodium cacodylate, pH 7.4 under microwave irradiation in a Pelco 3440 laboratory microwave oven (Ted Pella, Redding, CA, USA) during three cycles with 100% potency and maximum temperature of 37°C. They then remained immersed in fresh fixative
Tartrate-resistant acid phosphatase histochemistry
Three-lm thick paraffin sections were collected onto glass slides and submitted to TRAP histochemistry. The slides containing the sections were incubated during 2 h in Burstone complete medium containing 50 mM D( ) tartaric acid (Sigma Chemical Co., St. Louis, MO, USA). After that, the slides were washed in tap water and then counterstained with Harris’ hematoxylin for 10 min (15). Immunohistochemical detection of osteopontin
Some sections were collected onto silane-coated glass slides and submitted to immunohistochemical detection of osteopontin. After dewaxing, the sections were heated to 60°C for 15 min and treated with H2O2/ methanol solution (1:1). The non-specific binding sites were blocked during 1 h with 30% non-immune swine serum (Dako, Carpinteria, CA, USA) in 1% BSA. Then, they were incubated with the primary antibody (LF175 1:1200; Larry Fisher, NIH, Bethesda, MD, USA) during 90 min, at room temperature within a humid chamber. After rinsing with buffer, detection was achieved using DAB as substrate (Dako), and nuclei were stained with Harris’s hematoxylin. Negative controls were incubated in the absence of primary antibody (16). Results Hematoxylin and eosin staining
None of the specimens analyzed presented signs of pulp necrosis in all time points. On the first time point, the non-luxated molars of CON and ALN groups presented normal root development; the epithelial diaphragm and HERS were intact (Fig. 1a,b), while the luxated specimens from CON group presented a cement line in the dentin. Extensive and deep resorption lacunae were observed on the root dentin at the periodontal ligament side from the cervical to the central portion of the root (Fig. 1c). The apical portion was disrupted, and a thick layer of dentin matrix © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Fig. 1. Light micrographs of rat second molars. Non-luxated (N LUX) specimens of CON (a) and ALN (b) groups at the first time point present typical features of root development, with odontoblasts (ob) forming root dentin, cementoblasts (cb), HERS cells (hers), and epithelial diaphragm (ed). b, bone. LUX CON specimens 7 days after luxation present a cement line (cl) in crown (cd) and root dentin (rd). A large resorption lacuna (rl) is observed in the root surface in (c). In (d), numerous cells are situated inside lacunae (arrows) in the apical root dentin. LUX ALN specimens at this time point present some clastic cells (cc) adjacent to resoption lacunae in root dentin in (e). The N LUX CON (f) on the next time point present cellular cementum (cem) formed on the apical portion of the root. The N LUX ALN (g) presents a thin layer of cellular cementum. In the CON LUX 14 days after luxation (h), the acellular cementum (ac) present irregularities and, at the apical portion, dentin, and cellular cementum are disorganized. pl, periodontal ligament. At this time pont, LUX ALN (i) shows cellular cementum and apical root dentin more organized than LUX CON. At the last time point, the second molar root is almost completely formed, and its apical portion is covered by cellular cementum in N LUX CON (j). In N LUX ALN (k) specimens, thin bone trabeculae invade the periodontal ligament space near the thin cellular cementum. The LUX CON (l) 21 days after luxation shows a disrupted root apex. P, pulp. The LUX ALN (m) at this time point present an ankylosis site between alveolar bone and cellular cementum (arrowhead). Note the irregular shape of the cementum. Bars: a–g, i, k = 50 lm; h, j = 100 lm.
containing numerous embedded cells was observed (Fig. 1d). The ALN luxated specimens also presented some resorption lacunae at the root surface facing the periodontal ligament (Fig. 1e). On the second time point, the non-luxated molars on CON and ALN groups still presented normal morphology (Fig. 1f,g). The luxated CON 14 days after the luxation presented disorganized root dentin matrix with lacunae containing cells at the apical portion (Fig. 1h), which was not observed in the ALN group (Fig. 1i). © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
On the last time point, the non-luxated CON specimens presented typical features (Fig. 1j). The non-luxated ALN specimens, however, presented a thin layer of cellular cementum over the apical portion of the root and disorganized alveolar bone trabeculae (Fig. 1k). The CON specimens 21 days after the luxation presented a severely disorganized root apex (Fig. 1l). ALN specimens 21 days after luxation present deformities in the root and cementum surface and some ankylosis sites characterized by the contact of
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alveolar bone trabeculae with the cellular cementum (Fig. 1m). Tartrate-resistant acid phosphatase histochemistry
Osteoclasts were evidenced by TRAP histochemistry in light micrographs and were also ultrastructurally examined by transmission electron microscopy. The non-luxated (Fig. 2a) and luxated (Fig. 2b) CON specimens presented numerous TRAP-positive osteoclasts attached to the alveolar bone surface, adjacent to Howship lacunae. The utrastructural analysis confirmed the activated phenotype of osteoclasts, which were adhered to the bone matrix surface and presented clear zone and exuberant ruffled border (Fig. 2c). Both ALN nonluxated (Fig. 2d) and luxated (Fig. 2e) presented numerous TRAP-positive osteoclasts but they were mostly rounded-shaped and not adhered to the bone surfaces; these features were confirmed by transmission electron micrographs where clastic cells presented short contact areas of the plasma membrane with the bone matrix (Fig. 2f). They did not present the resorptive apparatus as described for the CON specimens.
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Transmission electron microscopy
The CON specimens presented cellular cementum where lacunae containing two cells were observed (Fig. 3a). The epithelial diaphragm and HERS were severely disrupted (Fig. 3b). The ALN specimens presented some resorpion lacunae along the root dentin (Fig. 3c). Some areas of collagen-based matrix surrounded by connective cells were seen at the apical region of the dental follicle adjacent to the HERS cells (Fig 3d). Some areas were detected where bone trabeculae had invaded the periodontal ligament and were localized near the forming cellular cementum (Fig. 3e). The apical portion of the root presented inversion of the epithelial diaphragm, which resulted in dilacerations of the apical portion of the root (Fig. 3f). Immunodetection of osteopontin
At the first time point, non-luxated CON and ALN specimens presented immunolabeling for osteopontin on dentin, cementum, and on the forming alveolar bone surface (Fig. 4a,b). The luxated CON at the same
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Fig. 2. Non-luxated (N LUX) and luxated (LUX) CON specimens (a and b, respectively) present activated TRAPpositive osteoclasts (stained in red) resorbing the alveolar (AB) bone during the root and periodontium formation. D, dentin, and PL, periodontal ligament. The ultrastructural examination, in c, confirmed the activated phenotype of the osteoclasts (Oc), showing typical clear zone (cz) and ruffled border (RB) and are attached to the bone matrix (B). ALNtreated N LUX (d) and LUX (e) specimens present numerous TRAPpositive osteoclasts in the marrow spaces, but most of them are not adhered to the bone matrix. Ultrastructurally, an osteoclast with latent phenotype (LOc), without the resorption apparatus is observed in f. Bars: a, b, d, e = 50 lm; c = 5 lm; f = 10 lm. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lateral luxation and alendronate in developing rat molars
Fig. 3. Representative transmission electron micrographs of luxated specimens from CON and ALN groups. In a, CON specimen the lacunae observed in the cellular cementum (cem) are occupied by two cells (arrows). In b, the epithelial diaphragm cells (ed) are disorganized. The ALN specimens, in c, show a resorption lacune (rl) in cementum (c) filled by cementoblasts that are probably going to repair the resorbed matrix. In d, a fibrous matrix appeared surrounded by dental follicle cells (M). In e, the structures of the periodontal ligament (pl) are being compressed by a bone trabecule (bt). In f, the disorganized structure of the apical portion of the root, with atypical arrangement of cementoblasts (cb). do, differentiating odontoblasts. Bars: a, b = 20 lm; c, e, f = 10 lm; d = 5 lm.
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time point, after 7 days of luxation, presented strongly immunoreactive cement lines on the root dentin and a diffuse labeling in the osteoid tissue formed in the apical area (Fig. 4c). The luxated ALN specimens presented strong immunodetection in the surface of Howship’s lacunae in root dentin (Fig. 4d). On the following time point, non-luxated CON specimen presented positive immunolabeling on the cementum matrix and on cement lines and surfaces of the alveolar bone (Fig 4e). The non-luxated ALN specimen showed cement lines and cementum immunopositive for osteopontin, as well as some points of ankylosis (Fig. 4f). Fourteen days after the luxation, CON specimens presented strongly immunopositive cementum matrix, which appeared to be covering the root dentin in a discontinuous pattern (Fig. 4g). The cellular cementum of the luxated ALN specimens appeared © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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thinner than CON and weakly immunolabeled. The bone matrix presented cement lines strongly immunolabeled for osteopontin (Fig. 4h). On the last time point, the root dentin, cellular cementum, and alveolar bone of non-luxated CON specimens were strongly immunopositive (Fig. 4i). In contrast, non-luxated ALN specimens at this time point presented weak immunolabeing in the cellular cementum, which was thinner than observed in CON (Fig. 4j). The CON specimens 21 days after luxation presented a cement line in all the extension of the crown dentin. Large resorption lacunae with the surface immunolabeled for osteopontin were observed on the root dentin facing the periodontal ligament (Fig. 4k). In the luxated ALN specimen 21 days after luxation, the immunolabeling for osteopontin on the alveolar bone surface was more intense than CON (Fig. 4l).
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Fig. 4. Immunolabeling for OPN in brown. Non-luxated (N LUX) specimens of CON (a) and ALN (b) groups at the first time point present cementum (cem) and the alveolar bone (ab) surface positively immunolabeled. LUX CON specimens 7 days after luxation (c) present immunolabeling in the cement lines (cl) in root dentin. LUX ALN specimens at this time point, in d, present resorption lacunae (rl) in root dentin (rd) strongly positive for OPN. The N LUX CON (e) on the next time point presents OPN immunolabeling in cellular cementum (cem) and alveolar bone (ab) surface, as well as N LUX ALN in f. In g, a LUX CON specimen 14 days after luxation presents positively immunolabeled cellular cementum irregularly deposited onto the root dentin. The LUX ALN (h) at this time point shows strong immunolabeling in the alveolar bone surface and some ankylosis points (arrows). On the last time point, N LUX CON (i) presents a typical thick cellular cementum over the root dentin immunopositive for OPN. Differently, the cellular cementum in N LUX ALN (j) is thin and weakly immunoreactive. The CON LUX 21 days after luxation (k) shows an extensive resorbed area on the root dentin immunopositive on its surface (arrowhead). Note the presence of an increment of dentin strongly immunolabeled for OPN. p, dental pulp. The LUX ALN in l, like the N LUX ALN in j, presents a discrete cellular cementum. Note that this specimen has less deformities than the CON LUX in k. Bars: a, b = 50 lm, e–g = 100 lm; c, d = 50 lm.
Discussion
The present study revealed that sodium alendronate attenuated the alterations on root and periodontal tissues after the lateral luxation of rat molars without impairing the root development; however, the ALN treatment provoked ankylosis sites in either luxated or non-luxated specimens. Although lateral luxations are highly common in anterior teeth (1), the present study utilized the rat molar because it is comparable in many aspects to
human teeth. Despite some studies have used the rat incisor model for studying dental trauma (17–19), they erupt continuously and therefore amelogenesis and dentinogenesis take place continuously as well. Most important, while their lingual alveolar wall faces a periodontal ligament, their buccal alveolar wall presents a dental follicle-like tissue rather than a periodontal ligament proper (20). The present study involved the lateral luxation on 21-day-old rats in which the second molar roots are not completely formed and still present embryonic structures at their apical end, such as HERS © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lateral luxation and alendronate in developing rat molars and epithelial diaphragm. By this experimental design, it was possible to verify whether the complete root formation was altered and how alendronate would potentially interfere in these alterations. Previous reports proposed experimental models in which calibrated apparatus are employed to reproduce the same dental trauma among the specimens (21, 22); nevertheless, establishing an accurate dental trauma experimental model is difficult, and clinical situations present extremely diverse etiologies, with variable intensities of forces applied over teeth and rates of dislocation. Thus, the luxation procedure employed in the present study proved an appropriate model for evaluating the completion of root formation after trauma on rat molars. The lateral luxation did not cause dental ankylosis in controls, but it occurred in alendronate-treated specimens. In contrast, previous reports have shown that alendronate pretreatment of roots prevented the shortterm ankylosis after replantation of avulsed rat molars (23). Ankylosis was described in replanted alendronatetreated rat incisors (24) and molar roots (25) after long periods. In the present study, the trauma occasioned morphological alterations in the dentin and cellular cementum, as well as in the immunolocalization of osteopontin in control specimens. The apical portion of root dentin did not present the typical tubular organization in all time points. Instead, it presented numerous cells inside lacunae, resembling a primary bone aspect. Similarly, previous studies describe the presence of cells entrapped into the tertiary dentin of rat incisors after extrusive luxation (19). The dentin presented a cement line intensely labeled for osteopontin in its complete extension at both their coronal and root portions, suggesting that the secretion of dentin matrix by odontoblasts was temporarily interrupted after the trauma. Osteopontin is synthesized by odontoblasts during dentinogenesis but only small amounts which are not immunodetected are present in the dentin matrix after its mineralization (26–28). The intense immunodetection of this protein in dentin suggests that the lateral luxation stimulated an increase of its synthesis and secretion into the matrix, which presented stronger labeling in the deeper portion of dentin compared with the superficial dentin. Previous reports also demonstrated that reactionary dentin after luxation of rat incisors induces odontoblasts to secrete dentin matrix with primary bone appearance, highly immunopositive for osteopontin (19). Regarding the alendronate-treated luxated specimens, they presented discrete alterations in the root morphology, but the appearance of the dentin matrix was similar to the non-luxated molars, as well as the distribution of osteopontin. ALN did not impede the differentiation of cementoblasts, despite the impairment of root elongation of erupting molars described in previous studies (29); in fact, less cellular cementum was formed in both luxated and non-luxated ALN-treated specimens. The non-luxated specimens without ALN treatment presented acellular and cellular cementum strongly immunopositive to osteopontin, while both luxated and non-luxated ALN-treated specimens showed weak immunolabeling in the cementum. As ALN impaired the resorption © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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activity of osteoclasts, which remained in a latent stage, the alveolar bone trabeculae were not remodeled and grew in a disorganized pattern toward the dental follicle/periodontal ligament space. As the dental follicle is a source of ectomesenchymal cells that differentiate into fibroblasts and cementoblasts (5), the disruption of these structures in luxated and non-luxated ALN-treated molars may be related to the reduced thickness of cellular cementum in all time points. The differentiation of cementum forming cells was demonstrated to occur in the dental follicle of developing molars from ALN-treated rats, but the compression of dental follicle cells by bone trabeculae affect their secretory activity and root elongation (29). Although ALN is often related to increased risk of complications like jaw osteonecrosis (8, 16) and impairment of tooth eruption (9, 10), the current results demonstrate that ALN treatment was able to attenuate the severe alterations that were observed in luxated teeth with incomplete roots. Acknowledgements
The authors would like to thank Eloiza Rezende for her help with the animal handling. C. P. Rothbarth was the recipient of a research fellowship from the Special PostGraduate Program CAPES/DINTER, Brazil. This work was partially supported by FAPESP and CNPq, Brazil. References 1. Andreasen JO, Andreasen FM, Andersson L. Textbook and color atlas of traumatic injuries to the teeth, 4th edn. Oxford: Blackwell Munksgaard; 2007. 2. Caldas AF Jr, Burgos MEA. A retrospective study of traumatic dental injuries in a Brazilian dental trauma clinic. Dent Traumatol 2001;17:250–3. 3. Hecova H, Tzigkounakis V, Merglova V, Netolicky J. A retrospective study of 889 injured permanent teeth. Dent Traumatol 2010;26:466–75. 4. Janones DS, Massa LF, Arana-Chavez VE. Immunocytochemical examination of the presence of amelogenin during the root development of rat molars. Arch Oral Biol 2005;50:527–32. 5. Zeichner-David M. Regeneration of periodontal tissues: cementogenesis revisited. Periodontol 2000 2006;41:196–217. 6. Andreasen JO, Raven JJ. Epidemiology of traumatic dental injuries to primary and permanent teeth in a Danish population sample. Int J Oral Surg 1972;1:235–9. 7. Arana-Chavez VE, Bradaschia-Correa V. Clastic cells: mineralized tissue resorption in health and disease. Int J Biochem Cell Biol 2009;41:446–50. 8. Russell RG. Bisphosphonates: the first 40 years. Bone 2011;49:34–41. 9. Bradaschia-Correa V, Massa LF, Arana-Chavez VE. Effects of alendronate on tooth eruption and molar root formation in young growing rats. Cell Tissue Res 2007;330:475–85. 10. Bradaschia-Correa V, Moreira MM, Arana-Chavez VE. Reduced RANKL expression impedes osteoclast activation and tooth eruption in alendronate-treated rats. Cell Tissue Res 2013;353:79–86. 11. McKee MD, Nanci A. Osteopontin at mineralized tissue interfaces in bone, teeth, and osseointegrated implants: ultrastructural distribution and implications for mineralized tissue formation, turnover and repair. Microsc Res Tech 1996;33:141–64.
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