Localization of Minodronate in Mouse Femora Through Isotope Microscopy

Abstract

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate’s effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([¹⁵N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [¹⁵N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [¹⁵N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase–positive osteoclasts and alkaline phosphatase–reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.

Introduction

Bisphosphonates are mainstream drugs for the treatment of osteoporosis. Drugs of this class have high affinity for crystalline calcium phosphates,^1–3 thus binding to calcium phosphates in the bone matrix upon administration. When osteoclasts degrade bone tissue bound to bisphosphonates, bone resorption is inhibited as the drug induces osteoclast apoptosis.^4,5

Among the several commercially available bisphosphonates, alendronate is the most commonly used one. In general, nitrogen-containing bisphosphonates (such as pamidronate, alendronate, ibandronate, zoledronate, and risedronate) act by inhibiting farnesyl pyrophosphate synthase, an enzyme involved in the mevalonate pathway,^6,7 which then impairs protein prenylation, especially that of the small GTPase of the Ras family.⁸ These small GTPase proteins are crucial for vesicular trafficking and cell survival and seem to be involved in the cytoskeletal organization of bone-resorbing osteoclasts.^9–11 In short, the cellular events that lead to osteoclastic inhibition seem to be shared by all nitrogen-containing bisphosphonates. On the other hand, bisphosphonate-driven osteoclastic inhibition appears to reduce osteoblastic population and bone formation, which can negatively affect bone turnover as demonstrated by decreased bone formation and mineral apposition rates in rats,^12,13 mice,¹⁴ and dogs.¹⁵

Minodronate, a third-generation, nitrogen-containing bisphosphonate, is an approved drug for treatment of osteoporosis in the Japanese market.^16–18 Studies showed that minodronate suppressed bone resorption in ovariectomized rats and cynomolgus monkeys^19–21 and affected cortical bone response to mechanical loading in rats.²² In addition, other in vivo and in vitro studies reported on the high potency of minodronate compared with alendronate regarding the inhibition of bone resorption,²³ with an intermediate mineral-binding affinity.²⁴

As patients seem to prefer weekly and monthly dosing regimens,²⁵ long-acting bisphosphonates such as minodronate are gradually becoming the first line of treatment for many prescribing physicians. Still, a pervasive question among medical professionals and researchers is how can a single dose of bisphosphonate sustain its effects for over a month? Therefore, we postulated that the localization of minodronate may offer new insights that might help address this issue. For instance, the use of radioisotope-labeled minodronate may assist on determining the localization and distribution of minodronate in vivo; however, animal studies involving radioisotopes are ethically questionable. We have explored the idea of using minodronate’s own high affinity for binding crystalline calcium phosphates in the bone matrix to circumvent that limitation by substituting ¹⁵N (very rare in nature) for ¹⁴N (naturally abundant) in the minodronate molecule. [¹⁵N]-minodronate was then injected into mice to determine the localization of [¹⁵N]-minodronate using isotope microscopy. Isotope microscopy is a mode of secondary ion mass spectrometry and two-dimensional isotope detection technique.^26–28 Recently, isotope microscope systems have been used to analyze living matter specimens.²⁹

To contribute for a better understanding on the mechanism of the sustained minodronate effects, we have examined the localization of [¹⁵N]-minodronate in murine bone through isotope microscopy complemented by histochemistry and transmission electron microscopy (TEM) observations.

Materials and Methods

Preparation of ¹⁵N- and ¹³C-Labeled Minodronate

The ¹⁴N and ¹²C in the minodronate molecule were substituted with the stable isotopes ¹⁵N and ¹³C to generate {1-hydroxy-2-[(1-¹⁵N)imidazo[1,2-a]pyridin-3-yl](¹³C2)ethane-1,1-diyl}bis(phosphonic acid) hydrate (Fig. 1), which was kindly provided by Astellas Pharma Inc., Tokyo, Japan. The isotope microscope of the Creative Research Institution, Hokkaido University,²⁶ was used for identification and localization of ¹⁵N of ¹⁵N- and ¹³C-labeled minodronate. For the sake of convenience, ¹⁵N- and ¹³C-labeled minodronate will be referred to as [¹⁵N]-minodronate from here on.

Figure 1.

¹⁵N- and ¹³C-labeled minodronate. As shown here, ¹⁵N- and ¹³C-labeled minodronate [{1-hydroxy-2-[(1-¹⁵N)imidazo[1,2-a]pyridin-3-yl](¹³C2)ethane-1,1-diyl}bis(phosphonic acid) hydrate] was generated by substitution of ¹⁴N and ¹²C with the stable isotopes ¹⁵N and ¹³C.

Animals and Tissue Preparation

All animal experiments were approved by Hokkaido University and were conducted in accordance with the standards of humane animal care (No. 15-0030). Mice were anesthetized with an intraperitoneal injection of chloral hydrate and then were injected with [¹⁵N]-minodronate (1 mg/kg) through the external jugular vein. Three hours, 24 hr, 1 week, and 1 month after the injection, mice (n=6 for each) were perfused with 4% paraformaldehyde diluted in 0.1-M phosphate buffer (pH 7.4, for histochemistry) or a mixture of 2% paraformaldehyde + 2.5% glutaraldehyde diluted in 0.067-M cacodylate buffer (pH 7.4, for isotope microscopy and TEM) through the heart’s left ventricle. Femora were removed and immersed in the respective fixatives for 18 hr (for histochemistry) or 3 days (for isotope microscopy and TEM) at 4C. Specimens for histochemistry were decalcified with 10% EDTA disodium salt (EDTA-2Na) and dehydrated in ascending ethanol solutions before paraffin embedding. Samples for isotope microscopy were not decalcified, but instead, they were immediately dehydrated and then embedded in epoxy resin (Epon 812; TAAB Laboratories Equipment Ltd, Berkshire, UK). Some TEM specimens were decalcified with 5% EDTA-2Na, but others were not decalcified. All TEM specimens were postfixed with 1% osmium tetroxide in 0.1-M cacodylate buffer for 4 hr at 4C and dehydrated in ascending acetone solutions before embedding in epoxy resin. Semithin sections were placed on Si wafers for isotope microscope observation. Ultrathin sections were stained with uranyl acetate and lead citrate for TEM observations (Hitachi H-7100; Hitachi Co. Ltd, Tokyo, Japan) at 80 kV.

Isotope Microscopy

The sections placed on Si wafers were coated with a 30-nm layer of gold to prevent the accumulation of positive charges generated by the primary beam of the isotope microscopy. Hokkaido University’s isotope microscope system (Cameca IMS 1270 + SCAPS; Sapporo, Hokkaido, Japan) was used to visualize isotope distribution in the bone tissue, a technique known as isotopography.^26–30

Frequency Histogram for Osteoblast and Osteoclast Numbers and ¹⁵N/¹⁴N Intensity Ratio

To understand how much [¹⁵N]-minodronate was deposited beneath osteoblasts (bone-forming surfaces) or osteoclasts (bone-resorbing surfaces), we counted the osteoblasts and osteoclasts seen on bone surfaces labeled with [¹⁵N]-minodronate. Semithin sections of metaphyseal areas were used to generate data for the histogram. The intensity of ¹⁵N ([¹⁵N]-minodronate) lines seen underneath osteoblasts or osteoclasts was divided by the intensity of ¹⁴N labeling in the same region. A series of ¹⁵N/¹⁴N ratios is shown on the horizontal axis of the histogram, whereas the vertical axis shows the numbers of osteoblasts or osteoclasts located on the bone surfaces with the corresponding ¹⁵N/¹⁴N ratio, thus indicating the volume of deposited [¹⁵N]-minodronate per surface type (bone forming vs. bone resorbing).

Double Staining for Tissue-Nonspecific ALP and TRAP

Dewaxed paraffin sections were treated for endogenous peroxidase inhibition with 0.3% hydrogen peroxide in PBS for 20 min and for nonspecific staining blocking with 1% BSA in PBS (1% BSA-PBS) for 30 min at room temperature. Sections were incubated with rabbit polyclonal antisera against tissue-nonspecific alkaline phosphatase (ALP)³¹ for 2 hr at room temperature and then incubated with horseradish peroxidase–conjugated antirabbit IgG for 1 hr at room temperature. Immunoreactions were detected with 3.3′-diaminobenzidine tetrahydrochloride (Dojindo Laboratories, Kumamoto, Japan). Following immunostaining, tartrate-resistant acid phosphatase (TRAP) was detected as previously described.³² In short, slides were rinsed with PBS and incubated in a mixture of 2.5 mg of naphthol AS-BI phosphate (Sigma-Aldrich, St. Louis, MO), 18 mg of red violet LB (Sigma-Aldrich) salt, and 100-mM l(+)-tartaric acid (0.76 g) diluted in 30 ml of a 0.1-M sodium acetate buffer (pH 5.0) for 15 min at 37C.

Von Kossa Staining

Undecalcified semithin sections were incubated with an aqueous solution of 5% silver nitrate (Wako Pure Chemical Industries, Ltd, Tokyo, Japan) for 60 min at room temperature under sunlight until taking on a dark brown color, as previously described.³³

Statistical Analyses for Osteoclast Number, Osteoblastic Area, and Percentage of Apoptotic Osteoclasts

Sagittal femora sections from samples of all time points (3 hr, 24 hr, 1 week, and 1 month; n=6 for each) were cut as shown in Fig. 2. The region of interest (ROI) for counting TRAP-positive osteoclasts and measuring the area of ALP-reactive osteoblastic cells, as well as the determination of percentage of apoptotic osteoclasts, was set as a boxed area neighboring the chondro-osseous junction and the endosteal surfaces of the cortical bone up to a horizontal line 2 mm distant from the chondro-osseous junction. As reported previously,³³ ALP-positive cells located on the bone surface were considered osteoblasts, whereas multinucleated (more than two nuclei) TRAP-reactive cells were regarded as osteoclasts. TRAP-positive osteoclasts with nuclear pyknosis were categorized as apoptotic osteoclasts. We counted the numbers of TRAP-positive osteoclasts and also measured the ALP-reactive area in the ROI by using the Image-Pro Plus 6.2 software (Media Cybernetics, Silver Spring, MD). The percentage of apoptotic osteoclasts was obtained after dividing the number of pyknotic, TRAP-positive osteoclasts by the total osteoclast number.

Figure 2.

Femoral metaphyseal trabeculae after minodronate administration. Femora treated with single administration of minodronate showed an increase in metaphyseal trabeculae as time goes on. Note many trabeculae at 1 week (C) and 1 month (D) when compared with those at 3 (A) and 24 (B) hr after injection. Panels E to H show highly magnified images of metaphyseal trabeculae. Osteocytes (arrows) seemed intact in all time points (see insets). Panels I to L show the statistical analyses on bone histomorphometry. Note the significant increases in bone volume/tissue volume (BV/TV) at 1 week and 1 month (I) and trabecular number (Tb.N) at 1 month (K) when compared with those at 3 hr after the administration, as well as the absence of statistical significance in trabecular thickness (Tb.Th; L). Trabecular separation (Tb.Sp) was significantly reduced at 1 week and 1 month after minodronate administration (J). Abbreviations: meta, metaphysis; N.S, not significant. Bars: A–D = 500 μm; E–H: 100 μm.

Bone Histomorphometry of BV/TV, Tb.N, Tb.Th, and Tb.Sp

Bone histomorphometric static parameters were assessed as presented in a recent report from our group.³⁴ In brief, a 400-µm × 600-µm ROI located 150 µm below the growth plate of the femoral metaphysis (n=6 for each) was used for assessing the following static parameters: bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Whenever possible, abbreviations and calculations followed the guidelines of the American Society for Bone and Mineral Research (ASBMR) Histomorphometry Nomenclature Committee.³⁵

Statistical Analysis

One-way ANOVA and the Tukey–Kramer multiple comparisons test were used to assess the presence of statistical differences among groups. All values are presented as mean ± standard deviation. The level of significance was set to p<0.01.

Results

Histology of Femoral Metaphyseal Trabeculae and TRAP/ALP Double Histochemistry After Minodronate Administration

Femora treated with minodronate showed time-from-administration-dependent increases in meta-physeal bone (see Fig. 2A–D and 2I–L). The histo-logical observations demonstrated that trabeculae were more robust at 1 week and 1 month after minodronate administration than at 3 and 24 hr after the injections (Fig. 2A–D). At higher magnification, osteocytes seemed intact, and no signs of inflammation or microdamage were identified at all time points (Fig. 2E–H). Consistently, BV/TV at 1 week and 1 month after minodronate administration increased significantly when compared with that seen 3 hr after the drug administration (Fig. 2I). Conversely, Tb.Sp was significantly reduced at 1 week and 1 month after minodronate administration (Fig. 2J). Despite the absence of statistical sig-nificance concerning Tb.Th (Fig. 2L), there was a significant increase in Tb.N 1 month after the administration when compared with values seen 3 hr after the injections (Fig. 2K).

At earlier time points (3 and 24 hr), the distribution of TRAP-positive osteoclasts and ALP-reactive osteoblasts was similar (Fig. 3A, B, E, and F), which is consistent with the absence of statistically significant differences in the number of osteoclasts and in ALP-reactive area (Fig. 3I and J). Several TRAP-positive osteoclasts and ALP-reactive areas were seen at 1 week and 1 month postinjection despite the obvious increase in trabecular volume (Fig. 3G–J). At higher magnification, apoptotic osteoclasts with nuclear pyknosis began to appear in 1-week and 1-month samples (Fig. 3G–I), but several osteoclasts could still be identified on the trabecular surfaces. In contrast, ALP reactivity seemed to be persistent though it slightly tended to be decreased (Fig. 3E–H, I). Although the index of ALP-reactive area was significantly lower at 1 week, it recovered to be increased at 1 month later, being similar to that of 3 hr after minodronate administration (Fig. 3I).

Figure 3.

Double staining of TRAP and ALP on femoral metaphyses after minodronate administration. At all time points, there seemed the distribution of TRAP-positive osteoclasts (red color) and ALP-reactive osteoblasts (brown color) (A–D). When observing at a higher magnification, several TRAP-positive osteoclasts were seen throughout the experimental period despite the obvious increase in trabecular volume at 1 week and 1 month postinjection (E–H). Note that apoptotic osteoclasts can be seen at 1 week and 1 month (insets in G and H). There were no significant differences in the numbers of TRAP-positive osteoclasts in all the experimental periods (I). However, notice apoptotic osteoclasts significantly increased not at the early stage but at the later stage such as 1 week and 1 month of the minodronate injection. Although ALP-reactive area is decreased at 1 week later, it recovered to be increased similar to that at 3 hr (J). Abbreviations: TRAP, tartrate-resistant acid phosphatase; ALP, alkaline phosphatase; meta, metaphysis; tb, trabecular bone. Bars: A–D = 200 µm; E–H = 50 µm.

Localization of [¹⁵N]-Minodronate Assessed by Isotope Microscope

At 3 hr after minodronate administration, most trabecular surfaces showed traces of [¹⁵N]-minodronate with various intensities of [¹⁵N]-minodronate labeling (Fig. 4A–G). After 1 month, [¹⁵N]-minodronate labeling was seen only on trabeculae that were distant from the growth plate cartilage (Fig. 4H–N). Serial semithin sections stained with toluidine blue and isotope microscopic images at 3 hr after minodronate injection showed a faint [¹⁵N]-minodronate labeling underneath osteoclasts, whereas an intense labeling of minodronate was seen adjacent to mature, cuboidal osteoblasts (Fig. 5A–D). The frequency histogram for osteoblast and osteoclast numbers and the ¹⁵N/¹⁴N intensity ratio demonstrated that most osteoclasts were found on bone surfaces with low ¹⁵N/¹⁴N ratios, whereas many osteoblasts were seen on bone surfaces with various ¹⁵N/¹⁴N intensity ratios. Thus, the histogram suggests that minodronate seems to be deposited predominantly on bone-forming surfaces (beneath osteoblasts) rather than on bone-resorbing surfaces (beneath osteoclasts) (see Fig. 5E and F).

Figure 4.

Editor’s Highlight

Localization of [¹⁵N]-minodronate assessed by isotope microscope. Panels A to G show the localization of [¹⁵N]-minodronate at 3 hr after minodronate administration, whereas panels H to N demonstrate [¹⁵N]-minodronate at 1 month. Panels A and H show semithin sections stained with toluidine blue, which represent the areas of isotope microscopy observation. At 3 hr, trabecular surfaces showed white lines indicative of [¹⁵N]-minodronate in all the areas (yellow-colored arrows, B–G). However, after 1 month later, the regions close to the growth plate do not show the labeling of [¹⁵N]-minodronate, though the distant regions revealed [¹⁵N]-minodronate (see white lines indicated by yellow arrows in L–N). Abbreviations: meta, metaphysis; GP, growth plate; dis, diaphysis; bm, bone marrow. Bars: A, H = 100 µm; B–G, I–N: 10 µm.

Figure 5.

Localization of [¹⁵N]-minodronate on bone-forming surface and bone-resorbing surface. Panels A to D are serial semithin sections stained with toluidine blue (A, C) and isotope microscopic (IM) images (B, D) at 3 hr after minodronate injection. Note a faint [¹⁵N]-minodronate labeling underneath osteoclasts (A, white arrows in B), whereas an intense labeling of minodronate adjacent to mature osteoblasts (C, white arrows in D). Panels E and F are the histogram demonstrating the number of osteoblasts (bone-forming surfaces) and osteoclasts (bone-resorbing surfaces) located on regions of various ¹⁵N/¹⁴N intensity ratios. Abbreviation: bm, bone marrow. Bars: A = 10 µm; B, D = 20 µm; C = 5 µm.

High Resolution and Ultrastructural Observation of Osteoclasts After Minodronate Administration

At 3 hr after minodronate administration, osteoclasts featured the characteristic ruffled border attached to the underlying bone surfaces, whereas cuboidal, mature osteoblasts were lying on the trabecular surfaces (Fig. 6A, B). However, 24 hr after minodronate injection, osteoclasts were partially detached from the bone surfaces and had lost their ruffled borders (Fig. 6C). Von Kossa staining showed that osteoclasts not treated with minodronate incorporate fragments of mineralized bone matrix (Fig. 6D), whereas minodronate-treated osteoclasts failed to do so (Fig. 6E). At 1 week after minodronate administration, some osteoclasts began to show signs of apoptosis, such as nuclear condensation and spherical cell bodies, whereas others were still partially attached to the bone surfaces with defective ruffled borders (Fig. 7A, B). TEM images showed osteoclasts with collapsed nuclei and without ruffled borders. On the other hand, these defective osteoclasts extended short cytoplasmic processes toward the bone matrix (Fig. 7C). One month postinjection, the region close to the chondro-osseous junction featured several osteoclasts with well-developed ruffled borders and numerous cuboidal mature osteoblasts on the trabecular surfaces (Fig. 8A–D). Away from the chondro-osseous junction, most osteoclasts lacked ruffled borders, but there were many mature osteoblasts on the nearby trabeculae (Fig. 8E, F).

Figure 6.

Osteoclasts and osteoblasts at 3 and 24 hr after minodronate administration. Panels A to E show semithin sections stained with toluidine blue. At 3 hr after minodronate administration, both osteoclasts and osteoblasts seem intact: Osteoclasts have ruffled border (RB, white arrows in A), whereas cuboidal, mature osteoblasts were localized on the trabeculae (tb) (B). In contrast, at 24 hr after minodronate injection, osteoclasts are shown to be partially detached from the bone surfaces without their RBs (white arrows, C). Von Kossa staining visualizes that control osteoclasts without minodronate treatment incorporate fragments of mineralized bone matrix (brown color, yellow arrows, D). However, after minodronate administration, no osteoclasts are shown to engulf mineralized bone matrices inside (E). Abbreviations: oc, osteoclasts; ob, osteoblasts; BM, mineralized bone matrix. Bars: A–E = 10 µm.

Figure 7.

Osteoclasts at 1 week after minodronate administration. Toluidine blue staining of semithin sections (A, B) showing that some osteoclasts were partially attached to the bone surfaces despite the lack of ruffled borders (an arrow, A), and several were apoptotic (double arrows, B) at 1 week after minodronate administration. TEM observation demonstrates osteoclasts with collapsed nuclei (an asterisk) and without ruffled borders (see arrowheads, C). Such defective osteoclasts extended short cytoplasmic processes toward the BM (arrows, an inset of C). TEM, transmission electron microscopy; BM, bone matrix. Bars: A, B = 10 µm; C = 5 µm.

Figure 8.

Osteoclasts and osteoblasts at 1 month after minodronate administration. Panels A, B, E, and F are semithin sections stained with toluidine blue, whereas panels C and D are TEM images. Panels A to D are images of osteoclasts (A, C) and osteoblasts (B, D) in the close region to the chondro-osseous junction, whereas panels E and F are those from the distant region. The region close to the chondro-osseous junction reveals osteoclasts with well-developed ruffled borders (RBs) and numerous cuboidal mature osteoblasts on the trabeculae when observed under light microscopy (A, B) and TEM (C, D). In the distant region from the chondro-osseous junction, most osteoclasts are fattened and still lack ruffled borders (E), whereas many osteoblasts are seen (F). oc, osteoclasts; ob, osteoblasts; TEM, transmission electron microscopy. Bars: A, B, E, F = 10 µm; C, D = 5 µm.

Discussion

To our knowledge, this is the first report to show the localization of minodronate in vivo in bone tissue. Isotope microscopy demonstrated that [¹⁵N]-minodronate could be found underneath osteoblasts, that is, on bone formation surfaces, rather than near bone-resorbing osteoclasts. This finding suggests that the bone-forming trabecular surfaces are coated by minodronate, and we postulated that osteoclasts might somehow fail to resorb the minodronate-coated bone matrix. If osteoclasts do not degrade the minodronate-coated bone matrix, they would not be exposed to minodronate and would not, therefore, enter apoptosis—at least immediately upon administration of the drug. Our histochemical findings demonstrated that many TRAP-positive osteoclasts are seen at all time points studied here, whereas apoptotic osteoclasts were not clearly identified until the later time points of 1 week and 1 month postadministration. Taken together, a single injection of minodronate seems to produce a long-lasting effect that avoids osteoclastic bone resorption by coating the bone surface and rendering it somewhat “resorption-proof.”

However, this particular tissue distribution seems to produce a “useful” side effect because maintaining osteoclast presence instead of forcing osteoclastic cells into apoptosis may guarantee osteoblast activation through cell coupling.³⁶ As shown with the TRAP/ALP double staining, not only TRAP-positive cell numbers were stable but also many ALP-reactive osteoblasts were persistent even at a later time point (1 month) following minodronate administration, though they temporally decreased at 1 week. Then again, bisphosphonates have been reported to reduce bone formation and bone turnover.^12–15 An autoradiography study showed that alendronate accumulated on resorption surfaces,³⁷ which suggests that alendronate would be readily incorporated by bone-resorbing osteoclasts and immediately halt bone resorption. While alendronate seems to target bone-resorbing osteoclasts directly, minodronate’s distribution and localization on bone formation surfaces indicates that the latter does not inhibit bone-resorbing osteoclasts immediately but instead protects the bone matrix from osteoclastic bone resorption. In addition, the dysfunctional, non-resorbing osteoclasts may allow nearby osteoblasts somehow to be activated through cell coupling. Retaining osteoblastic activity may favor combined therapy with an anabolic drug such as teriparatide [hPTH(1-34)]. We have demonstrated that cell coupling with osteoclasts during preosteoblastic differentiation into mature osteoblasts is necessary for parathyroid hormone–driven anabolic effect to occur,³⁸ as others have reported elsewhere.^39,40 Indeed, daily alendronate administration appears to reduce the anabolic effect of PTH(1-84) on hip bone mineral density (BMD) and cortical volume⁴¹; on the other hand, combining teriparatide and long-term zoledronate or denosumab treatment produced positive effects on lumbar spine and hip BMD.^42,43 Likewise, the association of risedronate with teriparatide increased cancellous bone mass in orchidectomized rats,⁴⁴ as well as lumbar spine, total hip, and femoral neck BMD in men.⁴⁵ If minodronate sustains osteoblastic activities, as suggested by our findings, it could also produce better results when combined with teriparatide. In fact, one recent study described that a combination of minodronate and teriparatide resulted in increased bone volume and Tb.N, while reducing Tb.Sp compared with teriparatide alone.²⁰ To date, unfortunately, reports focusing on the cumulative effects of once-monthly minodronate and other anabolic drugs are scarce.

In conclusion, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect.