Oral Diseases (2015) 21, 248–256 doi:10.1111/odi.12258 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com

ORIGINAL ARTICLE

Effects of alendronate on the peri-implant bone in rats KC Oh, HS Moon, JH Lee, YB Park, J-H Kim Department of Prosthodontics, Yonsei University College of Dentistry, Seoul, Korea

OBJECTIVES: The purpose of this study was to determine the effects of alendronate on the peri-implant bone in rat maxillae with the aid of micro-computed tomographic, histologic, and biochemical analyses. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were used. After extraction of the maxillary first molars, each rat was given periodic subcutaneous injections of either alendronate (alendronate group) or saline (control group). Customized implants were placed bilaterally 4 weeks after these injections. The rats were sacrificed at either 4, 8, or 12 weeks after implantation (4-, 8-, and 12-week groups, respectively; n = 6 rats per group). Microcomputed tomographic and histologic analyses were conducted for all rats. Biochemical analyses were performed at four time points for the 12-week groups. RESULTS: There were no significant differences between the groups on microcomputed tomographic and histologic analyses. All of the measured biochemical parameters tended to decrease over time, with significant differences among some time points within each group. The serum osteocalcin level was significantly lower in the 12-week alendronate group than in the control group (P < 0.05). CONCLUSIONS: Three approaches were utilized in evaluating the effects of alendronate. It appears serum osteocalcin levels may serve as an adjuvant marker for this purpose, although further studies are required to confirm this. Oral Diseases (2015) 21, 248–256 Keywords: alendronate; peri-implant bone condition; serum carboxy terminal telopeptide of type I collagen; serum osteocalcin; bisphosphonate-related osteonecrosis of the jaws

Correspondence: Jee Hwan Kim, DDS, MSD, PhD, Yonsei University College of Dentistry, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea. Tel.: +82 2 2228 3160, Fax: +82 2 3123 598, E-mails: [email protected]; [email protected] Kyung Chul Oh and Hong Seok Moon contributed equally to the works described in this manuscript. Received 12 December 2013; revised 26 April 2014; accepted 28 April 2014

Introduction The bisphosphonates are chemically stable synthetic analogues of naturally occurring pyrophosphate compounds that are thought to suppress bone resorption by inducing the apoptosis of osteoclasts via disturbance of their intracellular processes (Rogers et al, 1997; Russell, 2011). An alendronate, one of the nitrogen-containing bisphosphonates, has now become one of the most effective agents for treating various bone diseases, including Paget’s disease of bone, metastatic bone diseases, multiple myeloma, osteopenia, and osteoporosis (Drake et al, 2008; Fleisher et al, 2010; Russell, 2011; Kuhl et al, 2012). However, their increased clinical applications have raised concerns about the complications associated with bisphosphonate therapy, such as esophageal irritation/ erosion in patients with known gastroesophageal reflux, atrial fibrillation, frozen bone, severe musculoskeletal pain, hypocalcemia, and flu-like symptoms (Drake et al, 2008). The first study of complications of bisphosphonate therapy in the dental field found that five osseointegrated endosseous implants failed in the anterior mandibular area after bisphosphonate therapy for osteoporosis (Starck and Epker, 1995). Since then, controversial results have been reported dealing with the effects of bisphosphonates. Some clinical studies found that the use of bisphosphonates did not affect successful implant osseointegration (Bell and Bell, 2008; Grant et al, 2008; Martin et al, 2010), while others found that patients receiving bisphosphonate therapy were significantly more prone to failure of implant osseointegration (Kasai et al, 2009; Zahid et al, 2011). Moreover, Martin et al, 2010 reported that late implant failures among patients receiving oral bisphosphonate therapy—defined as implant failure that occurred more than 1 year after placement—were more common than early failures (Martin et al, 2010). In addition to these clinical reports, there have been a few animal studies dealing with the effect of oral bisphosphonates on implant osseointegration (Kurth et al, 2005; Chacon et al, 2006; Eberhardt et al, 2007; VieraNegron et al, 2008; Yildiz et al, 2010; Tsetsenekou et al, 2012). However, most of these animal studies used bisphosphonates other than alendronate or were performed on sites other than jaw bones.

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Bisphosphonate-related osteonecrosis of the jaws (BRONJ), a bone-related disorder, is thought to be associated with invasive dental procedures including extraction and implantation (Marx et al, 2007). Although the pathogenic mechanisms underlying BRONJ remain to be clarified, several hypotheses have been suggested (Allen and Burr, 2009). One of the most popular hypotheses is that BRONJ is caused by bisphosphonateinduced suppression of bone remodeling. Evidence for this comes from the presence of non-viable bone that lacks osteocytes in the lacunae, so-called empty lacunae, which can be observed by histologic examination with hematoxylin–eosin (H&E) staining (Futami et al, 2000; Hansen et al, 2006; Allen and Burr, 2009). In addition, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells is known to be related to the negative effects of bisphosphonate application (Hikita et al, 2009; Kim et al, 2011). However, histologic assessment alone is not sufficient to reveal the effects of alendronate on the peri-implant bone condition, making a multilateral analytical approach necessary. This study selected microcomputed tomographic and biochemical analyses as well as histologic analysis to achieve this purpose. Several biochemical indicators have been tested as tools for assessing the risk of future BRONJ development, but their efficacies remain a matter of debate. These indicators are known to be correlated with bone turnover rates and include serum carboxy terminal telopeptide of type I collagen (s-CTX), which is a bone resorption marker, and serum osteocalcin (s-OC), which is a bone formation marker. Some studies (Kwon et al, 2009; Marx et al, 2007) suggest that s-CTX and BRONJ risk are correlated, while others throw doubt on such a relationship (Kwon et al, 2011; O’Connell et al, 2012). Meanwhile, some studies have found that the s-OC level is slightly decreased in patients on bisphosphonate therapy (Kim et al, 2005; Raisz et al, 2000), while another found no abnormal change in s-OC level in five patients with BRONJ (Lehrer et al, 2008). Kwon et al, 2011 performed a retrospective study of the utility of both of these markers in clinical situations and concluded that a set of these markers might be used as indicators of risk prediction for BRONJ (Kwon et al, 2011). Schlemmer and Hassager reported that these markers are influenced by many pre-analytical factors that are encountered in various clinical conditions (Schlemmer and Hassager, 1999). To the best of our knowledge, no previous animal studies have compared the serum bone turnover marker levels between bisphosphonate-treated and control groups under controlled conditions.

This situation prompted this study to use various tools to determine the effects of alendronate on the peri-implant bone condition in rat maxillae. The results of microcomputed tomographic, histologic, and biochemical analyses were evaluated. The null hypotheses were that (i) alendronate does not negatively affect peri-implant bone condition in the rat maxilla and (ii) the levels of s-CTX and s-OC in rats treated with alendronate do not differ from those of untreated rats.

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Materials and methods Classification of the experimental animals Thirty-six 4-week-old male Sprague-Dawley rats (typical body weight, 130–140 g) were used in the study. The maxillary first molars of all rats were extracted bilaterally at the age of 4 weeks. After a healing period of 4 weeks, the rats received subcutaneous injections of either alendronate sodium salt (alendronate group, n = 18; Merial, Parramatta, New South Wales, Australia) or saline (control group, n = 18) twice per week for a minimum of 8 weeks. At 4 weeks after the commencement of the injection protocol, the animals were provided with dental implants into each extraction socket. Implantation was performed after having 4 weeks of injection period. The two animal groups were then subdivided further into three groups according to the time between implant placement and sacrifice: 4-, 8-, and 12-week groups (n = 6 per group). To clarify, the rats in the 4-week groups were sacrificed 4 weeks after implant placement, which corresponded to 8 weeks after the starting point of either alendronate or saline injection. The injection protocol (for both alendronate and saline) was continued after implant placement until sacrifice.

Experimental procedures A schematic of the overall experimental design is shown in Figure 1. All of the surgical interventions were performed under general anesthesia by intramuscular injection of an anesthetic mixture comprising Rompunâ (xylazine, 20 mg ml 1, 0.5 ml kg 1 body weight; Bayer, Leverkusen, Germany) and Zoletilâ (tiletamine and zolazepam, 100 mg ml 1, 0.5 ml kg 1 body weight; Virbac Lab., Carros, France) (Del Signore et al, 2006). All rats were housed with food pellets and tap water available ad libitum during the experimental period.

Figure 1 Schematic of the experimental design (ADN, alendronate; extraction, extraction of maxillary first molars) Oral Diseases

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The maxillary first molars on both sides of the 4-weekold rats were extracted carefully using dental explorers and forceps under further local infiltrative anesthesia (2% lidocaine with 1:100 000 epinephrine). The extraction sockets were allowed to heal for 4 weeks, after which the rats were commenced on the subcutaneous alendronate or saline injection regimen (i.e., 1.0 mg kg 1 injected twice per week until sacrifice: a total of 8, 12, and 16 weeks for the 4-, 8-, and 12-week groups, respectively). Implantation was performed 4 weeks after the start of the injection regimen (alendronate or saline). In brief, a 1.5-mm-deep osteotomy was performed with Ø1.3-mm low-speed, round and fissure burs under additional local infiltrative anesthesia (2% lidocaine with 1:100 000 epinephrine) at the extraction sites after flap elevation. Sufficient saline irrigation was implemented during this process. The custom-made sterile, machined-surface implants (Ø1.5 mm 9 1.5 mm; Figure 2), which were manufactured from grade IV titanium, were embedded into the drilled cavities on each side of the maxilla by tapping them with a mallet. The superior-most surfaces of the implants were planned to be flush with the level of the cortical surface of the adjacent bone. The flaps were carefully approximated without suturing. Blood samples were taken from the 12-week alendronate and control groups every 4 week from the time point of the commencement of the subcutaneous injection regimen (i.e., 4 weeks after the extractions). Four blood samples were therefore taken from each rat in these groups; the rats were euthanized on the day of their final blood collection. The rats were sacrificed at the end of their predetermined survival period (i.e., 4, 8, and 12 weeks following implantation) by transcardial perfusion of the fixative containing 4% paraformaldehyde under general anesthesia (Futami et al, 2000). Their maxillae including the implants were removed en bloc and immersed in 4% paraformaldehyde solution for an additional 24 h. Microcomputed tomographic, histologic, and biochemical analyses were carried out as described below. All rats in this study were housed at the animal experimental laboratory of Yonsei University College of Dentistry, Seoul, Republic of Korea. All experimental procedures were approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea (approval no. 09-224) and performed under guidelines for animal experiments at Yonsei University College of Dentistry.

Figure 2 Dimensions of the custom-made implant Oral Diseases

Microcomputed tomographic analyses After fixing the specimens in 4% paraformaldehyde solution, 3D microcomputed tomographic images were made using a microcomputed tomography scanner (Skyscan 1076, Kontich, Belgium) at a tube voltage of 100 kV and a tube current of 100 lA over 360° with rotation steps of 0.5°. The images were reconstructed using the NRecon software (version 1.6.1.4, Skyscan). The region of interest (ROI) was set as an annular area around the implant (Figure 3). Specifically, the area was defined as a hollow cylinder with an outer diameter of 2.1 mm (1.5 mm + 0.3 mm 9 2) and an inner diameter of 1.5 mm over the 1.5-mm length of each implant. Image-analysis programs (CTAn version 1.12.0.0 and CTVol version 2.2.1.0, Skyscan) were used for morphometric analysis and 3D rendering. The ratio of bone volume to tissue volume (BV/TV, %) and the bone mineral density (BMD, mg ml 1) were calculated for each ROI and each implant. Histologic analyses The specimens were decalcified with 10% EDTA solution at 4°C for 2 months after they had been analyzed with the aid of microcomputed tomography. A series of 3-lm-thick sections was prepared from the specimens using standard protocols. Two slides were selected for each tissue block and subjected to one of two kinds of stain: H&E and TRAP. An acid phosphatase leukocyte kit (Sigma, St. Louis, MO, USA) was used for the TRAP stain. The slides were observed with the aid of a light microscope (Leica DM LB, Wetzlar, Germany), and images of each sample were captured at two magnifications (912.5 and 9100) and saved in TIFF format. Computer-based histologic analysis was performed using IMT i-Solution Lite software (version 8.1, IMT i-Solution Inc., Vancouver, BC, Canada). The numbers of empty lacunae and TRAP-positive cells were counted in H&E- and TRAP-stained sections, respectively, within the pre-established ROI located at the distal surface of the implant. The ROI was set from the surface of the vertical part of the implant to a distance of 300 lm from this surface horizontally and from the bottom of the horizontal

Figure 3 ROI for microcomputed tomographic analyses

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Figure 4 ROI for histologic analyses

part of the implant to the end of the vertical portion of the implant vertically (Figure 4). Biochemical analyses: s-CTX and s-OC Blood samples (approximately 5 ml) were obtained from the cardium of the 12-week rats using an injection syringe at four time points (Figure 1). These samples were transferred to Eppendorf tubes, and the serum of each sample was separated by centrifugation (10 min at 715.52 g at 4°C). The tubes containing the serum were stored at 200°C in a freezer until they were used in laboratory assays. The levels of s-CTX and s-OC in the serum samples were measured with the SpectraMax190 (Molecular Devices, Sunnyvale, CA, USA) using a Rat Laps enzyme immunoassay (EIA) kit (Immunodiagnostic Systems, Boldon, UK) and a Rat Osteocalcin EIA kit (Biomedical Technologies Inc., Stoughton, MA, USA), respectively. Statistical analyses A repeated-measures, single-factor analysis of variance model was applied to the data gleaned from microcomputed tomographic, histologic, and biochemical analyses. All statistical analyses were performed using SPSS version 20.0 software (IBM SPSS Statistics, IBM, Somers, NY, USA). The data are presented as mean  s.d values, and the level of statistical significance was set at P < 0.05.

Results Five of the 36 rats died of unknown causes (4 in the alendronate group and 1 in the control group). Of the 62 implants in the remaining 31 rats, 43 well-fixed implants were available for analysis (Table 1). Implants were excluded for the following reasons: retained roots (n = 9), injury during the implantation procedure (n = 5), and inappropriate implant stability (n = 5). Table 1 Numbers of survived implants 4-week group Control 10

8-week group

12-week group

Alendronate

Control

Alendronate

Control

Alendronate

6

5

10

6

6

The BV/TV appeared to be higher in the alendronate groups than in the control groups at each time point, whereas there was no such tendency for BMD. However, the differences between the alendronate and control groups for both BV/TV and BMD at each time point were not significantly different (Figure 5, Table 2). The numbers of empty lacunae and TRAP-positive cells did not differ significantly between the two groups (Figure 6, Table 2). Furthermore, there were no statistically significant differences over time in both the groups with respect to microcomputed tomographic and histologic findings. The results of the biochemical analysis are summarized in Table 3. Both the control and alendronate groups exhibited overall decreasing tendencies with respect to s-CTX and s-OC values over time. Significant differences over time were found for both parameters between some time points within each group (asterisks in Figures 7 and 8), with the exception of the s-OC values in the control group (P < 0.05). No statistically significant differences were found between the control group and alendronate group for s-CTX, while a statistically significant difference was identified at 12 weeks after implantation for s-OC, with the control group having a higher mean value than the alendronate group (P < 0.05). There were no significant differences at the three other time points.

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Discussion The rat maxilla was used as the site of implant placement in this study, as it is known that bone turnover rates differ between the jaw and other areas. Male rats were used to minimize or eliminate the potentially confounding effects of estrogen. The dose of alendronate used (1.0 mg kg 1) and the time of implant placement (i.e., 28 days after extraction) followed protocols employed in previous studies (Karimbux et al, 1995; Fujii et al, 1998; Hikita et al, 2009; Aguirre et al, 2010). The dose used in the present study was much higher than that for the treatment of osteoporotic human patients. Aguirre et al (2010) stated that the subcutaneous injection of 15 lg kg 1 twice weekly in rats corresponds to the clinically relevant dose for the treatment of osteoporotic human patients. The reason to choose the higher dose was based on the lower incidence of BRONJ. According to the review paper by Kuhl et al (2012), the mean incidence of BRONJ after taking oral bisphosphonates was about 0.12% (0.0~4.3%) and that of BRONJ after receiving intravenous bisphosphonates was around 7% (0.0~27.5%). Fujii et al (1998) found that new bone formed at the extraction site 1 month after extraction. In addition, osseointegration was accomplished around the implant at about 30 days after implantation in most specimens. On the other hand, Karimbux et al (1995) reported that the initial osseointegration was seen at 28 days after implantation, and it was not until 56 days after implantation that the mature type of osseointegration was noticed. Some researchers might consider a 12-week period to be excessive for assessing whether implant osseointegration has occurred, and so that including this time point in our study was unnecessary. However, there is a clinical report Oral Diseases

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(a)

(d)

(b)

(e)

(c)

(f)

Figure 5 Three-dimensionally reconstructed images of the ROI for microcomputed tomographic analyses: (a, d) 4-week groups, (b, e) 8-week groups, and (c, f) 12-week groups Table 2 Microcomputed tomographic and histologic findings in ROI 4-week group Control 1

BMD (mg ml ) BV/TV (%) Empty lacunae (n) TRAP-positive cells (n)

1.53 13.05 19.17 3.50

   

0.04 4.32 8.13 2.17

8-week group

Alendronate 1.54 13.85 21.00 5.90

   

0.02 3.23 11.52 3.96

Control 1.61 17.64 18.70 3.90

   

0.06 3.68 13.29 2.03

12-week group

Alendronate 1.57 18.69 13.83 4.33

   

0.06 6.74 8.13 2.50

Control 1.58 16.04 13.67 3.83

   

0.05 2.41 6.71 2.04

Alendronate 1.60 19.72 18.50 3.00

   

0.08 4.47 9.48 1.41

ROI, region of interest; BMD, bone mineral density; BV/TV, the ratio of bone volume to tissue volume; TRAP, tartrate-resistant acid phosphatase.

of the late failure of the implants or BRONJ occurrence (Lazarovici et al, 2010b). According to the report, such complications occurred as a late complication (mean, 16.2 months; median, 11 months) in 21 patients of the 27 patients involved in the study. Hence, the present study considered both the short- and long-term effects of alendronate. As microcomputed tomographic analysis has been validated as a method for the 3D evaluation of trabecular bone micro-architecture relative to conventional histologic analysis, it has become employed in 3D assessments around peri-implant areas (Kuhn et al, 1990; Muller et al, 1998; Jiang et al, 2005). However, this procedure requires relatively complex image-processing procedures. In contrast, histologic slides provide clear-cut information with Oral Diseases

high resolution and image contrast and allow the evaluation of many structures, including cells (Rebaudi et al, 2004; Park et al, 2005). However, these slides may only provide information about a small part of a sample, and so, any differences in the degree of osseointegration within the same sample may or may not be observed depending on the location of the sections that are being observed histologically (Sennerby et al, 2001; Sarve et al, 2011). This situation prompted the decision in the present study to perform both histologic and microcomputed tomographic analyses—these complementary methods would yield more reliable results. BV/TV appeared to be higher in the alendronate groups than in control groups at each time point, but it seems that

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(a)

Figure 6 Histologic images: (a–c) 4-week alendronate group, (d–f) 12-week control group, and (a, d) H&E-stained images at lower magnification (912.5). Scale bars = 500 lm. (b, e) H&E-stained images involving the ROI at higher magnification (9100). Scale bars = 200 lm. Black arrows indicate empty lacunae. (c, f) TRAP-stained images involving the ROI at higher magnification (9100). Scale bars = 200 lm. White arrows indicate TRAPpositive cells

(d)

(b)

(e)

(c)

(f)

Table 3 Biochemical findings (ng ml 1) 4 weeks before implantation

s-CTX (ng ml 1) s-OC (ng ml 1)

4 weeks after implantation

8 weeks after implantation

12 weeks after implantation

Control

Alendronate

Control

Alendronate

Control

Alendronate

Control

Alendronate

53.81  15.42 18.95  4.50

65.32  25.43 23.20  6.58

38.57  8.04 24.81  10.32

27.55  12.86 15.59  7.47

23.40  11.31 15.66  8.29

20.41  7.60 9.81  0.60

21.76  5.28 15.65  2.32

23.70  9.00 8.71  1.95

s-CTX, serum carboxy terminal telopeptide; s-OC, serum osteocalcin.

alendronate did not alter the peri-implant bone structure significantly, given that statistically significant differences were found for neither BV/TV nor BMD. This finding may be attributable to microcomputed tomography being able to measure mineralized tissues only beyond a certain threshold of mineralization (Rebaudi et al, 2004). Reduced bone resorption may result in dead bones as a result of traumatic procedures. According to some previous studies, empty lacunae can appear after dental extraction or implantation; the number of empty lacunae reduces as the healing process progresses (Shimizu et al, 1998; Futami et al, 2000). However, the numbers of empty lacunae did not decrease when bone remodeling was suppressed by bisphosphonates (Bi et al, 2010; Kikuiri et al, 2010). There are diverse findings regarding the association between the use of bisphosphonate and TRAP-positive cell counts (Hikita et al, 2009; Bi et al, 2010). The present study found that the numbers of TRAP-positive cells

and empty lacunae did not differ significantly between the alendronate and control groups. s-CTX specifically measures a particular cross-link peptide of type I collagen in bone. Type I collagen constitutes 98% of the total protein in bone. During the bone resorption phase, the telopeptide fragment of type I collagen is cleaved from its main chain via the action of osteoclasts. Hence, Marx et al (2007) insisted that s-CTX levels would be proportionally related to the osteoclast activity and the amount of the osteoclastic resorption at the time the blood is drawn. In other words, lower levels of s-CTX are suggestive of some degree of suppression of bone turnover rates. Kunchur et al (2009) and Lazarovici et al (2010a) reported that the s-CTX level could aid in the identification of patients who are at risk of BRONJ, in agreement with Marx et al (2007), but they believed that this value is not predictive of the occurrence of BRONJ for individual patients. Some authors (Edwards et al, Oral Diseases

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Figure 7 Mean s-CTX values according to the time points. The asterisks indicate statistically significant differences (P < 0.05)

Figure 8 Mean s-OC values according to the time points. The asterisks indicate statistically significant differences (P < 0.05)

2008; Fleisher et al, 2010; Lee and Suzuki, 2009; O’Connell et al, 2012) considered that it was not possible to determine predictive threshold values of s-CTX when managing patients undergoing bisphosphonate therapy. The level of another bone turnover marker, s-OC, is an indicator of bone formation. Osteocalcin, also known as bone Gla protein, is the most abundant non-collagenous matrix protein in bone (Brown et al, 2009). This protein appears to affect osteoid mineralization, although its precise function remains to be determined (Lee et al, 2000). It is postulated that a fraction of newly synthesized Oral Diseases

osteocalcin is released into the circulation (Lee et al, 2000). Hence, s-OC can serve as a marker of bone formation, as indicated by studies of calcium kinetics and bone histomorphometry (Charles et al, 1985; Delmas et al, 1985). Decreasing tendencies for both s-CTX and s-OC were found in the present study, with generally lower values being observed in the alendronate groups than in the control groups and statistically significant differences being found among some time points. In addition, a statistically significant difference was found between the two groups for s-OC at 12 weeks after implantation. Decreasing tendencies of both values and the significant difference being only observed in the s-OC are partially in agreement with the previous findings of Kwon et al (2011). Furthermore, these results may partially reflect that the alendronate affected the bone remodeling process and subsequently magnified the osteonecrosis-related risks. In addition, the tendency toward a change in the biochemical markers observed in this study parallels the histologic findings of Kim et al (2011). The main feature of the present study was the use of biochemical analyses to evaluate the effects of alendronate. Nevertheless, this study was subject to several limitations: (i) a few samples were studied from each group and were not uniformly distributed, (ii) the implants were not given the chance to function within the oral cavities, (iii) male rats were used in the present study although the major population receiving the bisphosphonate therapy is women, and (iv) rats of the age used in this study are in a growing period during which they exhibit quite variable biochemical bone markers (Duque et al, 2009). In conclusion, the null hypotheses were partially accepted. Implant osseointegration was generally achieved well in both the groups, based on microcomputed tomographic and histologic analyses, at least for the alendro-

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nate dose and predetermined healing periods used in this study. The s-OC value differed significantly between the groups at the final blood sampling time point. It was significantly lower in the 12-week alendronate group than in the control group (P < 0.05). Hence, it appears that s-OC levels may serve as an adjuvant marker for evaluating the effects of alendronate, although further studies are required to confirm this. Given the difficulty of establishing a BRONJ model and the low incidence of BRONJ, especially when using lower bisphosphonate doses, the combination of microcomputed tomographic, histologic, and biochemical assessment may aid in studying the effects of bisphosphonates on the periimplant bone condition. Further researches that address the limitations of the present study are necessary to identify the apoptotic structures inside the osteoclasts by combining the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method with TRAP staining (Cerri et al, 2003) and to elucidate whether s-OC data can be used as practical references in clinical situations. Acknowledgements This study was supported by a faculty research grant of Yonsei University College of Dentistry (6-2013-0084).

Author contributions KC Oh performed the experiments and drafted the paper. J-H. Kim involved in the research design and performed the experiments. JH. Lee and YB. Park interpreted the data. HS. Moon involved in the research design and revised the paper.

Conflict of Interest None.

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