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Bone turnover suppression in sites that already have a low surface-based remodeling rate may lead to oversuppression that could have negative effects on the biomechanical properties of bone. The goal was to determine how alendronate suppresses bone turnover at sites with different surface-based remodeling rates.
Dynamic histomorphometric parameters were assessed in trabecular bone of the femoral neck and lumbar vertebrae obtained from skeletally mature beagles treated with saline (1 ml/kg/day) or alendronate (ALN 0.2 or 1.0 mg/kg/day). The ALN0.2 and ALN1.0 doses approximate, on a milligram per kilogram basis, the clinical doses used for the treatment of postmenopausal osteoporosis and Paget’s disease, respectively.
Alendronate treatment resulted in similar absolute levels of bone turnover in the femoral neck and vertebrae, although the femoral neck had 33% lower pre-treatment surface-based remodeling rate than the vertebra (p < 0.05). Additionally, the high dose of alendronate (ALN 1.0) suppressed bone turnover to similar absolute levels as the low dose of alendronate (ALN 0.2) in both sites.
Alendronate treatment may result in a lower limit of trabecular bone turnover suppression, suggesting that sites of low pre-treatment remodeling rate are not more susceptible to oversuppression than those of high pre-treatment remodeling rate.
The hallmark of osteoporosis is an increase in activation frequency and marked imbalance in bone remodeling at the cellular level resulting in bone loss, with consequent increased susceptibility to fracture [1-5]. Numerous studies have shown that bisphosphonates are effective in preventing bone loss and reducing the incidence of osteoporotic bone fractures [6-11]. For example, Black et al.  have found that three years of treatment with alendronate increases lumbar spine BMD by about 6% and reduces vertebral fracture rate by about 50% . Interestingly, alendronate treatment results in a 32% greater BMD increment in the lumbar spine than the hip, but reduces fracture risk at both sites by roughly the same degree . This suggests that factors other than BMD play an important role in dictating the effects of bisphosphonates on preventing bone fractures. These factors may include the site specific biomechanical milieu, the propensity to fall or alterations in the bone tissue matrix properties. It has also been proposed that the pre-treatment rate of bone turnover contributes to the ability of bone to resist fracture [12-15].
Clinical studies have found that after adjusting for BMD, the biochemical markers of bone turnover can still predict fracture risk [12-15]. This finding is consistent with Riggs et al.  who demonstrated that suppression of bone turnover and an increase in vertebral BMD contribute equally to the propensity of bone to resist a vertebral fracture. Riggs et al.  proposed that the efficacy of the suppression of bone turnover rate in reducing the incidence of bone fractures could be explained by considering the pre-treatment turnover rate. The high turnover rate in the vertebra may exacerbate the effects of bone loss by accelerating the loss of trabecular connectivity and thickness [1, 16, 17, 18, 19]. Normalization of the bone turnover rate in the vertebra, therefore, reduces the effects of high turnover rate on the microarchitectural deterioration and improve bone’s biomechanical properties even more than expected based on BMD alone [16, 17, 20]. However, the effects of turnover suppression on sites with lower pre-treatment rates may lead to oversuppression that could compromise the bone tissue matrix properties. As treatment with alendronate is extending beyond the first decade in some patients, long term suppression at sites that already have low turnover could have negative effects that may override the beneficial effects of bone turnover suppression on BMD and bone microarchitecture.
The aim of this study was to determine whether alendronate treatment suppresses bone turnover more, on either an absolute or percentage basis, in sites with lower surface-based pre-treatment remodeling rates. Greater suppression in sites of low pre-treatment remodeling rate could suggest that these sites are more susceptible to oversuppression than those of high pre-treatment remodeling rate. The turnover rates in the trabecular bone of the vertebra and femoral neck of beagles treated with alendronate for 3 years were compared, as previous data have shown that, in trabecular bone, the femoral neck has a lower pre-treatment remodeling rate than the vertebra [21, 22].
Thirty-six intact female beagles (~ 1 year old) were purchased from Marshall Farms USA (North Rose, NY) and LBL (Reelsville, IN). On arrival, skeletal maturity was confirmed using radiographs to assess closure of the proximal tibia and lumbar vertebra growth plates. Dogs were housed two per cage at the Indiana University School of Medicine’s AALAC-accredited facility, under environmentally controlled conditions, with free access to dry canine chow and water. All procedures were in accordance with NIH guidelines and approved by the Indiana University School of Medicine Animal Care and Use Committee.
Following two weeks of acclimatization, the animals were treated for either 1 year (N = 12) with saline (1ml/kg/day) or 3 years (N = 24) with alendronate. The group of dogs treated with saline was designated as the pre-treatment control group. The dogs treated with alendronate were assigned to one of two treatment groups (N =12/group): 1) Alendronate (ALN0.2, 0.2 mg/kg/day); or 2) Alendronate (ALN1.0, 1.0 mg/kg/day). The ALN0.2 and ALN1.0 doses approximate, on a milligram per kilogram basis, the clinical doses used for the treatment of postmenopausal osteoporosis and Paget’s disease, respectively. Alendronate sodium (Merck and Co., Inc) was dissolved in saline and administered orally, by a syringe, each morning after an overnight fast and at least 2 h prior to feeding. Prior to sacrifice, the animals were injected intravenously with calcein (0.20 ml/kg) using a 2–12–2–5 day labeling schedule. Due to a scheduling error, a 2–5–2–5 day labeling schedule was used for some of the pre-treatment control animals (N = 3). Two animals in the three-year ALN0.2 group developed hernias, one of which progressed to the point that the animal was euthanized early (month 34 of treatment); the data obtained from this dog were included in all analyses. All other animals completed the study without complication. Animals were euthanized by an overdose of sodium pentobarbital (0.22 mg/kg Beuthanasia-D Special, IV). After death, the right femoral neck and the second lumbar vertebrae were dissected and fixed in 10% neutral buffered formalin for histology.
Section preparation procedure and histomorphometric data of the second lumbar vertebrae were previously published [23, 24]. The femoral neck specimens were stained in basic fuchsin (1 %) dissolved in increasing concentrations and embedded in methyl metacrylate . Two transverse sections (80 - 100 μm) from each specimen were cut using a diamond wire saw (Histosaw; Delaware Diamond Knives). The basic fuchsin staining was used to identify microdamage for a separate investigation.
Histological measurements were performed in trabecular bone using a semiautomatic analysis system (Bioquant OSTEO 7.20.10, Bioquant Image Analysis Co.) attached to a microscope equipped with an ultraviolet light source (Nikon Optiphot 2 microscope, Nikon). An approximately 20 mm2 region of interest was examined from each specimen. Primary variables of interest included single- and double label perimeter (sL.Pm, dL.Pm), bone perimeter (B.Pm) and interlabel width (Ir.L.Wi). From these variables, the following dynamic histomorphometric parameters were calculated: mineralizing surface (MS/BS = 100 × [0.5 × sL.Pm + dL.Pm]/B.Pm; %), mineral apposition rate (MAR = Ir.L.Wi/d; μm/day; d is the labeling period in days), and bone formation rate (BFR/BS = MAR × MS/BS × 3.65; μm3/ μm2/year). One femoral neck specimen in the ALN1.0 group did not have double label and was assigned a value of 0.3 μm/day for MAR . All variables were measured and calculated in accordance with ASBMR recommended standards .
The differences in the histomorphometric parameters among the groups (Pre-treatment control; ALN0.2; ALN1.0) within each site were examined using one-way analysis of variance (ANOVA) tests following Anderson-Darling normality tests. When a significant overall F value (p < 0.05) was present, differences between individual group means were compared using Fisher’s protected least-significant difference (PLSD) posthoc tests. To compare the differences in the histomorphometric parameters between sites within each group, paired t-tests were used following Anderson-Darling normality tests. For those variables failing the normality test, nonparametric tests (Kruskal-Wallis or Wilcoxon signed rank test) were used. For all tests, p < 0.05 was considered statistically significant. MINITAB 15 software (Minitab, Inc.) was used for all the statistical analyses.
Bone formation rate (surface-based remodeling rate) in the pre-treatment control animals was significantly lower in the femoral neck (p < 0.05; Fig. 1) compared to the vertebra, due to both lower MAR (p < 0.05; Table 1) and MS/BS (p < 0.05; Table 1) in the femoral neck.
Following three years of treatment, BFR/BS was significantly lower in both the femoral neck and vertebra compared to pre-treatment control (p < 0.05; Fig. 1). The absolute levels of BFR/BS after alendronate treatment were similar between the femoral neck and vertebra (Fig. 1). This suppression of bone formation rate was achieved by lower MAR (p < 0.05; Table 1) and MS/BS (p < 0.05; Table 1) compared to pre-treatment control. However, the similarity in BFR/BS between sites was a result of different trends in MAR and MS/BS between the femoral neck and vertebra. The MAR at the femoral neck was significantly less than the vertebra after treatment with either dose of alendronate (p < 0.05; Table 1). MS/BS was, however, lower (non-significantly) in the vertebra (Table 1) such that there were no differences in BFR/BS between the femoral neck and vertebra. Also, there was no difference in any bone formation parameter between the two doses of ALN at either site (Fig. 1 and Table 1).
Following three years of alendronate treatment, the absolute levels of bone turnover in the trabecular bone of the femoral neck and vertebra were similar, even though the pre-treatment surface-based remodeling rate was significantly lower (-33%) in the femoral neck than the vertebra. In addition, the high dose of alendronate (ALN 1.0) suppressed bone turnover to similar absolute levels as the low dose of alendronate (ALN 0.2) in both sites. These findings imply that a lower limit for bone turnover may exist, beyond which bone turnover cannot be suppressed further with the clinical doses of alendronate. This in turn suggests that compared to sites of high pre-treatment remodeling rate, sites of low pre-treatment remodeling rate are no more susceptible to oversuppression.
When an anti-resorptive agent is administered, there is an increase in bone density due to suppression of the activation of new remodeling sites, but with continued bone formation in resorptive cavities excavated by pre-treatment remodeling cycles . Here, we show that irrespective of the pre-treatment remodeling rate, the end-point point of bone turnover may be similar suggesting that those sites with higher pre-treatment rates will experience a greater percentage reduction in bone turnover, but not a greater absolute suppression. A greater percentage suppression of bone turnover would in turn lead to a greater absolute increase in BMD. This is consistent with Gonnelli et al.  who found that treatment with alendronate results in a greater BMD increment in patients with high pre-treatment turnover rate compared to patients with low pre-treatment turnover rate.
The data reported here indicate that treatment with alendronate will suppress but will not abolish bone turnover. Bone turnover will be reduced to a non-zero limit and it will reach this limit regardless of the pre-treatment remodeling rate or the treatment dose. In agreement with these observations, clinical trials have demonstrated that in postmenopausal women treated up to 10 years with 5 mg or 10 mg of alendronate, bone turnover reaches a non-zero steady state after about 12 months of treatment [29, 9]. More importantly, both doses suppress bone turnover to a similar limit [29, 9]. Our findings also suggest that no oversuppression of bone turnover will be expected in patients on alendronate with low pre-treatment turnover rate because bone turnover will reach the same lower limit regardless of the pre-treatment turnover rate, or the drug dosage.
A study by Odvina et al  showed the absence of single or double-tetracycline labels in cancellous bone biopsies obtained from four patients treated with alendronate. Three of these patients were, however, also administered estrogen or glucocorticoids. The co-treatment of alendronate with estrogen or glucocorticoids may have augmented the effects of alendronate on bone turnover leading to the complete absence of bone formation.
Two different approaches have been proposed in the literature to calculate mineral apposition rate (MAR) in the absence of double-labeled surfaces. Foldes et al.  proposed that in patients with missing double labels MAR be given a low value, reflecting the fact that formation was occurring, but very slowly. They suggested a lower biological limit of 0.3 μm/day. On the other hand, to be consistent with other bisphosphonate clinical trials [31-33], Recker et al  chose not to assign a specific missing value for MAR when no double-label was observed, and to consider these values as missing values (i.e., no value was assigned and BFR were not calculated from these data, reducing sample size for both MAR and BFR). This has the potential to lower the average turnover rate in the bisphosphonate treated group, and represents a bias. In the current study, only one specimen did not show the presence of double labeling. Using either approach for analyzing this specimen yielded the same conclusions. We elected to assign the value of 0.3 μm/day to this specimen.
Our findings should be interpreted with various limitations in mind. We considered measurements made from placebo-treated 2 year old dogs to represent the pre-treatment control values, even though alendronate treatment was initiated in 1 year old dogs. The reason for this is that this investigation was not part of the original experimental design of our dog study [23, 24] and so no baseline control measurements were made in 1 year dogs. This only affects the calculated percentage reduction in turnover rate in the treated groups compared to the pre-treatment controls and does not affect the absolute calculated values for turnover in any way. Moreover, it reduces the normal age-related decline in activation frequency, providing a more stable baseline value. Another limitation of this study is the use of intact, non-ovariectomized, beagle dogs. It is also important to note that a large dose of alendronate, higher than the clinical doses, could suppress bone turnover beyond the lower limit observed here. However, there is no clinical rationale for such large doses of alendronate, and so those were not investigated here.
In conclusion, this study demonstrates that both doses of alendronate result in similar absolute levels of bone turnover in the trabecular bone of the femoral neck and vertebra, even though the femoral neck has significantly lower pre-treatment surface-based remodeling rate than the vertebra. This implies that sites with low pre-treatment remodeling rate are no more susceptible to oversuppression than those with higher pre-treatment remodeling rate. A better understanding of the relationship between bisphosphonates and bone turnover can provide better insight into how these agents can be most effectively and safely used in different patient populations.
The authors thank Keith Condon and Diana Jacob for histological preparation. This work was supported by NIH Grants AR047838 and AR007581 and utilized an animal facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR10601-01 from the National Center for Research Resources, National Institutes of Health. Merck and Co. kindly provided alendronate.