Reduced RANKL expression impedes osteoclast ... - Springer Link

Cell Tissue Res (2013) 353:79–86 DOI 10.1007/s00441-013-1623-9

REGULAR ARTICLE

Reduced RANKL expression impedes osteoclast activation and tooth eruption in alendronate-treated rats Vivian Bradaschia-Correa & Mariana M. Moreira & Victor E. Arana-Chavez

Received: 17 January 2013 / Accepted: 1 April 2013 / Published online: 1 May 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The creation of the eruption pathway requires the resorption of the occlusal alveolar bone by osteoclasts and signaling events between bone and dental follicle are necessary. The aim of the present study has been to evaluate the effect of alendronate on osteoclastogenesis and the expression of the regulator proteins of osteoclast activation, namely RANK, RANKL and OPG, in the bone that covers the first molar germ. Newborn Wistar rats were treated daily with 2.5 mg/kg alendronate for 4, 8, 14, 21 and 28 days, whereas controls received sterile saline solution. At the time points cited, maxillae were fixed, decalcified and processed for light and electron microscopic analysis. TRAP histochemistry was performed on semi-serial sections and the osteoclasts in the occlusal half of the bony crypt surface were counted. TUNEL analysis was carried out on paraffin sections. The occlusal bone that covers the upper first molar was removed in additional 4- and 8-day-old alendronatetreated and control rats in which the expression of RANK, RANKL and OPG was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. TRAP-positive osteoclasts were more numerous in the alendronate group at all time points, despite their unactivated phenotype and the presence of apoptotic cells. RANKL expression in the alendronate specimens was inhibited at all time points, unlike in controls. Our findings indicate that the expression of RANKL in the occlusal portion of the bony crypt is unrelated to osteoclast recruitment and differentiation but is crucial to their activation during the creation of the eruption pathway. This work was supported by grants from CNPq and Fapesp (grants 06/ 60094-5 and 09/54853-9), Brazil. V. Bradaschia-Correa : M. M. Moreira : V. E. Arana-Chavez (*) Laboratory of Oral Biology, Department of Biomaterials and Oral Biology, School of Dentistry, University of São Paulo, Avenida Prof Lineu Prestes 2227, São Paulo 05508-000, Brazil e-mail: [email protected]

Keywords Tooth eruption . Alendronate . Osteoclasts . RANKL . Eruption pathway . Rat (Wistar)

Introduction Tooth eruption consists in the movement of the developing tooth following an occlusal direction toward the oral cavity and depends on the formation of an eruption pathway in the bony crypt (Marks and Schroeder 1996). The dental follicle that surrounds the tooth germ plays a key role during the resorption of the occlusal portion of the bony crypt that takes place as osteoclast precursors are recruited, fused and activated. Osteoclast recruitment and activation during the various stages of tooth eruption depend on complex signaling mechanisms that have been elucidated during the past few decades (for a review, see Wise and King 2008). The signaling process is initially mediated by the expression of colony stimulating factor-1 (CSF-1), which is highly expressed from the 3rd until the 12th day in rats, when the peak of osteoclastogenesis occurs (Wise and Lin 1995). At this time, the osteoclast plasma membrane expresses the CSF-1 receptor, c-fms (Kawakami et al. 1999). The increased CSF-1 expression elevates the levels of the transcription factor c-fos (Wise et al. 2002) and reduces the expression of osteoprotegerin (OPG) at the 3rd day (Wise et al. 2005). The receptor activator of NFκB ligand (RANKL) increases in the dental follicle (Yao et al. 2004) and its expression peaks from the 9th until the 11th day in rats (Liu et al. 2005). These molecules diffuse and act paracrinally on the dental follicle cells where they stimulate bone resorption by osteoclasts for creating the eruptive pathway (Wise et al. 1995; Wise et al. 2003; Wise and King 2008). Once osteoclasts are recruited to the alveolar crypt bone around the tooth germ, the RANKL/OPG signaling persists along the eruptive process in order to modulate resorptive

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activity, which occurs at various regions of the bony crypt during the successive stages of eruptive movement. Examination of the bone from the basal and occlusal portions of the crypt isolated by laser microdissection has revealed that the occlusal bone has high levels of RANKL during tooth eruption, whereas the basal bone presents elevated rates of bone morphogenetic protein 2 (BMP-2), which is associated with bone formation, from the 1st until the 10th day in rats (Wise and Yao 2006). The bisphosphonates are drugs with a known capacity of inhibiting bone resorption by osteoclasts; this feature is increased in nitrogen-containing bisphosphonates such as sodium alendronate. Previous experiments have demonstrated that the administration of high daily doses of alendronate or other nitrogenated bisphosphonates is capable of inhibiting tooth eruption and the formation of molar roots in rats (Bradaschia-Correa et al. 2007; Hiraga et al. 2010) and also disturbs the ossification of their growing mandible condyles (Bradaschia-Correa et al. 2012). The molars remain covered by alveolar bone, despite the presence of numerous osteoclasts, which remain latent. Since bisphosphonates are employed in therapies for bone disorders of young patients (Maasalu et al. 2003; Shaw 2009; Arana-Chavez and Bradaschia-Correa 2009; Castillo and Samson-Fang 2009; Bachrach and Ward 2009) and since reports of delayed tooth eruption of children treated with these drugs have been presented (Kamoun-Goldrat et al. 2008), the evaluation of signaling molecules that could be disturbed by alendronate during the eruptive process is necessary. Thus, the aim of the present study has been to evaluate the protein expression of RANK, RANKL and OPG and correlate it with osteoclastogenesis at the occlusal portion of the alveolar crypt during the intraosseous phase of the eruption of the upper first molars of young rats treated with alendronate. The presence of tartrate-resistant acid phosphatase (TRAP)-stained multinucleated osteoclasts has also been examined.

Cell Tissue Res (2013) 353:79–86

rats were weaned during the entire study in order that their nutrition was only provided maternally. On the days cited, 16 alendronate-treated and eight control rats were anesthetized with 2% chloridrate 2-(6,6-xilidine)-5,6-dihydro-4-H-1,3tiazine (Rompun) diluted 1:1 in ketamine (Francotar) at 1 ml/kg body wt and decapitated. Their maxillary alveolar processes and mandibles were dissected out and quickly processed as follows. TRAP histochemistry

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 São Paulo, Brazil.

The maxillary alveolar processes of four alendronate-treated and two control rats from each group were fixed in 0.1% glutaraldehyde and 4% formaldehyde buffered in 0.1 M sodium cacodylate, pH 7.4. Specimens were immersed in a beaker that contained 40 ml fixative solution at room temperature, which was subsequently placed in a 20×20 cm glass container that was filled with ice and transferred to a Pelco 3440 laboratory microwave oven (Ted Pella, Redding, Calif., USA). The temperature probe of the oven was submersed into the fixative and the specimens were then exposed to microwave irradiation at the 100% setting for three cycles of 5 min, with the temperature programmed to a maximum of 37°C. After microwave irradiation, specimens were transferred into fresh fixative solution and left submersed in the fixative overnight at 4°C (Massa and Arana-Chavez 2000). Decalcification was carried out with standard protocols. The specimens were dehydrated in a graded ethanol series and embedded in JB-4 historesin (Polysciences, Warrington, Pa., USA). Sections (3 μm thick) were collected onto glass slides and submitted to TRAP histochemistry. Briefly, Burstone complete medium was prepared by dissolving 4 mg naphthol As-Bi phosphate substrate (Sigma Chemical, St. Louis, Mo., USA) in 0.25 ml N-N-dimethylformamide, followed by the addition of 25 ml 0.2 M acetate buffer pH 5.0, 35 mg Fast Red Violet LB as the coupling agent and 60 μl 10% MgCl. Next, 25 ml of the medium was filtered into a Coplin jar and warmed to 37°C, followed by the addition of 50 mM D(−) tartaric acid. The sections were incubated in this solution for 2 h, following which they were washed in running water, dried at 25°C and then counterstained with Harris’ hematoxylin for 10 min. Coverslips were mounted with Entellan (Merck, Germany) and the sections examined in an Olympus BX-60 light microscope (Bradaschia-Correa et al. 2012).

Sodium alendronate treatment

TRAP-positive cell counting and statistic analysis

A total of 66 newborn Wistar albino rats were used, of which 44 were subjected to daily subcutaneous injections of 2.5 mg/kg/day sodium alendronate (Massa et al. 2006) from the day of birth to 4, 8, 14, 21 and 28 days of age. An additional 22 rats were daily injected with sterile saline solution during the same period. None of the alendronate-treated

A microscopist blinded to the groups was trained to identify TRAP-positive cells. Cells positively stained for TRAP situated on the half occlusal surface of the alveolar crypt were counted. They were classified according to their activity as activated (attached to bone surface) or latent (rounded-shaped and lying loosely between the bone trabeculae). Results were

Materials and methods


compared by Student’s t-test by using the program GraphPad Prism 5 (GraphPad Software, La Jolla, Calif., USA) and Pvalues less than 0.05 were considered significant. Terminal deoxynucleotidyl transferase dUTP nick end labeling Specimens from the alendronate group at 28 days were fixed, decalcified as described above and embedded in Paraplast xtra (Sigma). Sections (4 μm thick) were collected onto silane-coated glass slides. For the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method, we used the Apop Tag-Plus Kit (Millipore). The deparaffinated slides were pretreated in 20 μg/ml proteinase K (Millipore) for 15 min at 37°C, rinsed in distilled water and immersed in 3% hydrogen peroxide in phosphate-buffered saline (PBS; 50 mM sodium phosphate, pH 7.4, 200 mM NaCl) for 15 min; they were then immersed in the equilibration buffer. After incubation in TdT enzyme (terminal deoxynucleotidyl transferase) at 37°C for 2 h in a humidified chamber, the reaction was stopped by immersion in the stop/wash buffer for 15 min; the sections were then rinsed in PBS for 10 min. The sections were subsequently incubated in anti-digoxigenin-peroxidase at room temperature for 30 min, in a humidified chamber, rinsed in PBS and then treated with diaminobenzidine tetrahydrochloride (DAB) for 3–6 min, at room temperature. The sections were counterstained with Harris’ hematoxylin for 3 min, dehydrated in 100% butanol, rinsed in xylene and mounted in Entellan medium. Involuting mammary gland sections were used as positive controls for the TUNEL method. Negative controls were incubated in medium lacking TdT enzyme. The specimens were examined and photographed in an OLYMPUS BX-60 microscope. Quantification was not carried out in TUNEL-labeled sections, because this methodology is known to produce uneven results attributable to the variable number of apoptotic bodies present in a given tissue (Boabaid et al. 2001). Specimens from the control group at the same time point were not incubated because their molars had erupted. Transmission electron microscopy Upper alveolar processes from four alendronate-treated and two control rats from each group were fixed and decalcified as described for histochemistry. They were then post-fixed in 1% osmium tetroxide in 0.1 M cacodylate-buffered for 2 h at room temperature, dehydrated in a graded series of alcohol and embedded in Spurr resin (Electron Microscopy Sciences, USA). Toluidine-blue-stained 1-μm-thick sections were examined in a light microscope and cervical and

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occlusal regions of the tooth germ/alveolar bony crypt were selected for ultrathin sectioning. Sections (80 nm thick) were obtained with a diamond knife on a Leica Ultracut R ultramicrotome (Leica, Buffalo, N.Y., 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 charge-coupled device camera. Gel electrophoresis and Western blotting Protein extracts were prepared from frozen samples of the occlusal portion of the upper first molar bony crypt of 4- and 8-day-old control rats and 4-, 8-, 14-, 21- and 28-day-old alendronate-treated rats. The samples were crushed in a mortar with 1 ml extraction buffer (MgCl2 + KCl+HEPES+EDTA+Glycerol+dithiothreitole+SDS) and 10 μl proteinase inhibitor (Sigma), centrifuged at 1000 rpm at 4°C for 10 min and the supernatant was aliquoted. The protein concentration was measured by BCA assay (Pierce, Rockford, Ill., USA). From each sample, 50 μg total protein was loaded onto the gels. The protein extracts were then resolved on 10% gradient SDS-acrylamide gels, followed by transference. The protein levels of RANK, RANKL, OPG and α-actin were determined with relevant antibodies (1:200, Santa Cruz Biotechnology, Santa Cruz, Calif., USA). After incubation in primary antibodies, followed by incubation in horseradish-peroxidase-conjugated secondary antibody (peroxidase-conjugated anti-rabbit IgG; Amersham Pharmacia Biotech, Aylesbury, UK), specific bands were visualized by enzymatic chemiluminescence (ECL Plus system, Amersham Biosciences, Piscataway, N.J., USA).

Results TRAP histochemistry At the 4th day, the alveolar crypt bone presented few TRAP-positive cells in both control and alendronatetreated specimens. The cells were mostly detected at the occlusal and basal portions of the alveolar crypt. At this time point, the alendronate specimens exhibited bone trabeculae contacting the dental follicle and molar germ structures (Fig. 1a–d). On the 8th day, the control specimens presented numerous activated TRAP-positive osteoclasts attached to the bone surfaces at the basal and lateral portions of the alveolar crypt. The activated osteoclasts were also observed at the occlusal portion of the crypt at which the eruption pathway was being

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Fig. 1 a, b Control specimens at 4 days (d). c, d Alendronate group at the same time point. Few tartrate-resistant acid phosphatase (TRAP)positive cells (arrows) can be seen at the alveolar crypt walls in both groups (df dental follicle). The alendronate molar germ is contacted by some bone trabeculae at the cervical portion (arrowheads). e, f Control specimens at 8 days. g, h Alendronate specimens at 8 days. Numerous osteoclasts are found at the lateral portion of the crypt and the occlusal portion is partially resorbed in the controls. The TRAP-positive cells examined at higher magnification are attached to the bone surfaces and seem activated. TRAP-positive cells lie at the occlusal and lateral walls of the crypt in the alendronate specimens. At higher magnification (h), the cells appear multinucleated and adjacent to bone trabeculae (dp dental papilla). i, j Control specimens at 14 days. k, l Alendronate specimens at the same time point. The eruptive pathway is seen in the controls and numerous-TRAP positive osteoclasts are localized in the


occlusal and lateral walls of the crypt (oe oral epithelium). k Occlusal portion of the molar germ is still covered with bone trabeculae and numerous TRAP-positive osteoclasts are observed throughout the alveolar crypt. l Bone trabeculae contact the epithelial diaphragm (ed epithelial diaphragm, bt bone trabeculae). m, n At 21 days, the control first molar erupts, the root is being formed and osteoclasts are seen in the alveolar septum (dr developing root). o Bone trabeculae cover the occlusal portion of the first molar of an alendronate specimen at 21 days. p Alveolar bone with numerous TRAP-positive osteoclasts (star). q, r At 28 days, the control specimens contain few osteoclasts in the forming alveolar bone at the apical portion of the alveolus. s At the same time point, the alendronate specimen does not possess an established eruptive pathway and numerous osteoclasts (star) are observed in the alveolar crypt bone (t). Bars 200 μm (a, c, e, g, i, k, m, q, s), 40 μm (b, d, f, h, j, l, n, p, r, t)


created. At the same time point, the alendronate specimens contained many TRAP-positive osteoclasts throughout the alveolar crypt. The occlusal portion of the molar germ was still covered by bone trabeculae. At higher magnification, the osteoclasts appeared inactivated (Fig. 1e–h). At the 14th day, numerous osteoclasts were observed in the occlusal and lateral portions of the crypt and the eruptive pathway was established. TRAP-positive osteoclasts were observed in the developing alveolar bone and root formation was underway. The alendronate specimens exhibited many osteoclasts all over the alveolar crypt walls but the eruption pathway was not created. The osteoclasts appeared to be inactivated (Fig. 1i–l). At the 28th day, the controls presented the first molar crown almost completely erupted and the root was almost completely formed. Some osteoclasts were resorbing the alveolar bone at the apical region. In the alendronate group, the molar crown did not erupt. It remained surrounded by bone in the alveolar crypt, which contained numerous inactivated TRAP-positive osteoclasts ((Fig. 1q–t).

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TRAP-positive cell counting The alendronate group presented more TRAP-positive cells than controls at 14 and 28 days (Fig. 2a). At 4 days, alendronate-treated specimens presented more cells unattached to the bone surfaces than the controls. At 14 and 28 days, the number of attached and unattached TRAPpositive cells was significantly higher in the alendronate group (Fig. 2b). At 28 days, the TRAP-positive cells were seen only in the alendronate group at the area analyzed; these cells were not seen at the control group in which the molar crown had erupted (Fig. 2b). TUNEL analysis On the alendronate specimens, several cells with TUNELpositive nuclei were detected at the occlusal alveolar crypt bone. Multinucleated cells with TUNEL-positive nuclei were also observed at the same portion of the alveolar crypt. Some of the multinucleated TUNEL-positive cells were adjacent to bone trabeculae, whereas others lay loose in the intertrabecular spaces (Fig. 3). Transmission electron microscopy The ultrastructural examination of osteoclasts at the occlusal portion of the alveolar crypt of rats at 8 days revealed that, in controls, typical activated multinucleated osteoclasts were attached to bone trabeculae and presented a resorptive apparatus such as sealing zones, a ruffled border and abundant vacuoles (Fig. 4a, b). In the alendronate specimens,

Fig. 2 Frequency of TRAP-positive osteoclasts in the surface of the occlusal alveolar crypt wall. In a, at 14 days (d), the number of total osteoclasts was higher in the alendronate group (ALN). At 28 days, the control group (CON) did not present osteoclasts, because the occlusal portion of the bony crypt was totally resorbed. *P