Comparison of Effects of the Bisphosphonate Alendronate Versus the RANKL Inhibitor Denosumab on Murine Fracture Healing

Abstract

The role of osteoclast‐mediated resorption during fracture healing was assessed. The impact of two osteoclast inhibitors with different mechanisms of action, alendronate (ALN) and denosumab (DMAB), were examined during fracture healing. Male human RANKL knock‐in mice that express a chimeric (human/murine) form of RANKL received unilateral transverse femur fractures. Mice were treated biweekly with ALN 0.1 mg/kg, DMAB 10 mg/kg, or PBS (control) 0.1 ml until death at 21 and 42 days after fracture. Treatment efficacy assessed by serum levels of TRACP 5b showed almost a complete elimination of TRACP 5b levels in the DMAB‐treated animals but only ∼25% reduction of serum levels in the ALN‐treated mice. Mechanical testing showed that fractured femurs from both ALN and DMAB groups had significantly increased mechanical properties at day 42 compared with controls. μCT analysis showed that callus tissues from DMAB‐treated mice had significantly greater percent bone volume and BMD than did both control and ALN‐treated tissues at both 21 and 42 days, whereas ALN‐treated bones only had greater percent bone volume and BMC than control at 42 days. Qualitative histological analysis showed that the 21‐and 42‐day ALN and DMAB groups had greater amounts of unresorbed cartilage or mineralized cartilage matrix compared with the controls, whereas unresorbed cartilage could still be seen in the DMAB groups at 42 days after fracture. Although ALN and DMAB delayed the removal of cartilage and the remodeling of the fracture callus, this did not diminish the mechanical integrity of the healing fractures in mice receiving these treatments. In contrast, strength and stiffness were enhanced in these treatment groups compared with control bones.

INTRODUCTION

The vast majority of fractures are treated by cast immobilization, traction, or intramedullary nailing. In all of these treatment modalities, the lack of rigidity of the fixation promotes micromotion that leads to healing in which a periosteal response is elicited and an endochondral component of bone formation takes place. ${}^{\text{((}{}^{\text{1}}\text{))}}$ , ${}^{\text{((}{}^{\text{2}}\text{))}}$ As fracture healing progresses, the cartilage tissues in the callus will mineralize and will subsequently undergo resorption and replacement during the initial period of primary bone formation. This is followed by an extended period of secondary bone formation associated with remodeling of the callus back to the original structure of the injured bone. Therefore, in most situations of fracture healing, two discrete forms of mineralized tissue resorption take place. The first type of resorption occurs during a relatively short period of time when the mineralized cartilage of the callus is removed, whereas the second type takes place over an extended period during secondary bone formation as the callus returns to its prefracture structure. ${}^{\text{((}{}^{\text{3}}\text{))}}$

In previous studies, we have shown that the first period of cartilage resorption is characterized by a peak in macrophage‐colony stimulating factor (M‐CSF) and RANKL expression accompanying parallel increases in osteoprotegerin (OPG) expression. When resorption of the cartilage is initiated, the ratio of OPG to RANKL expression decreases, thereby initiating the resorption process. ${}^{\text{((}{}^{\text{4}}\text{))}}$ The second period of mineralized tissue resorption occurs when the primary trabecular bone that was initially laid down is being remodeled and is characterized by increasing levels of other proresorptive cytokines including interleukin (IL)‐1 and IL‐6. During this second period of resorption, the levels of OPG, M‐CSF, and RANKL are lower than those seen during the period of cartilage resorption but remain elevated compared with those observed in the unfractured bone. ${}^{\text{((}{}^{\text{4}}\text{))}}$ These data suggest that the regulation of the expression of RANKL and OPG plays an essential role in osteoclast recruitment during both the period of cartilage resorption and later during the remodeling of the primary and secondary bone over the time course of fracture healing. Therapies used to treat a variety of metabolic diseases that affect bone loss should be assessed in regard to the effects those therapies might have on bone healing, such as occurs after fractures or orthopedic surgical procedures.

In previous studies, two receptor RANKL inhibitors (OPG‐Fc and RANK‐Fc) were tested in rat and mouse models of fracture repair, respectively. ${}^{\text{((}{}^{\text{5}}\text{))}}$ , ${}^{\text{((}{}^{\text{6}}\text{))}}$ Both of these studies have shown no effect on fracture union, and no significant effects were observed on fracture strength with high‐doses of OPG‐Fc at 3 and 8 wk after fracture. ${}^{\text{((}{}^{\text{5}}\text{))}}$ Denosumab, a fully human monoclonal antibody (IgG2) that specifically inhibits human RANKL, is an investigational agent that has been shown to reduce biochemical markers of bone resorption in postmenopausal women with low bone mass ${}^{\text{((}{}^{\text{7}}\text{))}}$ and in patients with multiple myeloma or with bone metastases from breast cancer. ${}^{\text{((}{}^{\text{8}}\text{))}}$ Denosumab suppresses bone resorption by binding RANKL (also known as OPGL, ODF, or TRANCE), a TNF family member that plays an essential role in bone resorption. ${}^{\text{((}{}^{\text{9}}\text{))}}$

Denosumab has a significantly longer half‐life than OPG‐Fc, ${}^{\text{((}{}^{\text{10}}\text{))}}$ which allows for dosing once every 6 mo, as has been shown in clinical trials with osteoporotic subjects. ${}^{\text{((}{}^{\text{11}}\text{))}}$ Whereas denosumab can remain in the circulation for several months, bisphosphonates also have prolonged effects on bone resorption by virtue of their high‐affinity binding to bone matrix. The skeletal half‐life of alendronate, for example, has been estimated as long as 10 yr. ${}^{\text{((}{}^{\text{12}}\text{))}}$ In the event of a fracture, patients on these therapies might not achieve full recovery of osteoclast function for some time; therefore, it is important to examine the potential consequences of osteoclast suppression with these agents in fracture repair settings. The objectives of this study were to compare the actions of alendronate and denosumab during the time course of fracture healing. Denosumab does not recognize murine RANKL and has no apparent effects in mice or rats, ${}^{\text{((}{}^{\text{13}}\text{))}}$ so genetically modified mice were created that exclusively express a chimeric (human/murine) form of RANKL that can maintain bone resorption in mice in a manner that can be fully inhibited by denosumab. In this study, inhibition of bone resorption by alendronate and denosumab was examined by qualitative histology of the callus and the bone resorption marker serum TRACP 5b, whereas the structural consequence of antiresorptive therapy was examined by callus μCT, biomechanical testing, and ash analysis.

MATERIALS AND METHODS

Study design

All animal research was conducted in conformity with all federal and USDA guidelines with a protocol that was approved by the Boston University Institutional Animal Care and Use Committee. One hundred ten male C57 bib6/129 human RANKL knock‐in mice 8–17 wk of age were provided through a materials transfer agreement with Amgen; they were shipped directly from breeding colonies at Charles River Laboratory, San Diego, CA, USA, to Boston University Laboratory Animal Sciences Center (LASC), Boston, MA, USA. Animals were housed for the duration of the study, plus an additional 3‐day acclimation. The knock‐in mice were created so the coding region of the fifth exon of the RANKL gene was exchanged for the human fifth exon, resulting in expression of a chimeric form of RANKL. This chimeric gene remained under the control of normal endogenous regulatory elements of murine RANKL and was capable of maintaining bone resorption. These mice have no obvious skeletal phenotype except for slightly higher bone mass, modestly lower bone resorption parameters, and the unique ability to respond to denosumab. ${}^{\text{((}{}^{\text{13}}\text{))}}$

All 110 mice were used in the study, with a least 36 mice enrolled into each of the three experimental of groups: vehicle control (Con); alendronate (ALN); and denosumab (DMAB). Each experimental group was further divided into two separate time points for harvest (21 and 42 days after fracture). Because of the wide age distribution of mice used in this study, equal numbers of mice within 2 wk of each other's ages were uniformly divided into each of the three experimental groups (Con, ALN, DMAB) and across both time points of harvest. Thus, each experimental group at both time points contained similar numbers of mice across the 10‐wk distribution of ages. From each of these groups, 15 animals were harvested for each time point, scanned by μCT, and subjected to mechanical testing. Thus, a total of 90 animals were examined by μCT and biomechanical testing. The animals not used for mechanical and μCT testing were used for histological assessments or were excluded because of surgical or postsurgical complications.

The drug doses in this study were (1) denosumab 10 mg/kg (DMAB); (2) alendronate (ALN) 0.1 mg/kg; and (3) PBS (Con), which was also the carrier for both denosumab and alendronate. All drugs were administered by subcutaneous injection twice weekly in the morning between 8:00 and 10:00 a.m. Drug suspensions were formulated based on an average ∼20 g weight per animal, such that that each animal received 0.1 ml of the respective drug at each injection time. The major objectives of this study were as a safety trial of the denosumab. As such, the two dosing regimens were chosen well in excess to normal clinical use. ALN dosing regimen used was chosen to exceed the estimated clinical exposure by roughly 28‐fold. In previous studies, cortical bone remodeling was significantly suppressed in the femurs of adult rats treated with ALN at doses of 0.7–70 μg/kg/wk, SC, ${}^{\text{((}{}^{\text{14}}\text{))}}$ and biochemical and histomorphometric parameters of bone turnover were significantly suppressed in ovariectomized mice treated with ALN at one half the weekly dose administered in this study. The denosumab dosing was chosen to provide a comparable complete inhibition of bone resorption and was twice that of previous use. This higher dosing was used in this study because the longer treatment periods of this study probably resulted in immune‐related clearance of DMAB (a fully human antibody), so essentially the current dose and the frequency of injection was selected with the goal of overwhelming immune responses to DMAB.

Surgery

On day 0, the day of surgery, each animal from the respective time point group was weighed and anesthetized with isoflurane immediately before surgery. Unilateral fractures were produced after insertion of an intramedullary pin in the right femur using a scaled‐down version of the apparatus described by Bonnarens and Einhorn ${}^{\text{((}{}^{\text{15}}\text{))}}$ as previously described. ${}^{\text{((}{}^{\text{16}}\text{))}}$ The location and quality of fractures were assessed by X‐ray analysis (GenDex Dental X‐ray machine) while animals were still anesthetized after surgery. Fractures not occurring in the mid‐diaphysis or that were unduly comminuted were not used in the study. After X‐ray, mice were placed into cages, allowed food and water ad libitum, and allowed unrestricted activity after recovery from anesthesia. Mice were monitored after surgery; any animal showing signs of distress, vocalization, gait guarding, mutilation, fur roughening, failure to eat or drink, pin loosening, or surgical complications and infection were killed.

Serum analysis

Blood was drawn by mandibular (facial vein) bleeding technique before surgery and 7, 14, and 21 days after surgery. Terminal bleeds, which occurred on day 21 or day 42, were collected by cardiac puncture. In‐life bleeds were performed under isoflurane to ensure consistency of data and to reduce stress on animals. Approximately 100 μl of blood was collected from each animal into a labeled 2‐ml microcentrifuge tube. Tubes were placed on ice until blood could be centrifuged to separate serum. Once separated, the serum was removed by a micropipette and transferred to a clean microcentrifuge tube; samples were labeled and stored at −80°C. Serum samples (−80°C) were analyzed for TRACP 5b (an osteoclast marker) by ELISA (SBA Sciences).

Specimen harvesting

On the day of termination (day 21 or day 42), animals were placed in a CO₂ chamber for primary euthanasia by asphyxiation; secondary euthanasia was by cardiac puncture. The right fractured femora, as well as the left contralateral femora, were excised and dissected free of soft tissues, and the pin was removed after which they were wrapped in sterile isotonic saline‐soaked gauze and put into labeled tubes kept on ice. Bone samples were kept in a −20°C freezer until testing.

μCT

μCT measurement was performed using a Scanco MicroCT 40 system (Scanco Medical, Basserdorf, Switzerland). Bones were thawed at room temperature, and excess tissue was removed. Femurs were vertically aligned inside a plastic μCT scanning tube, and styrofoam was used to keep the bones stabilized and separated. The tube was filled with PBS and sealed with parafilm, specimens were scanned, and images were reconstructed to a nominal 12‐μm voxel size. The scan area was manually adjusted to include only the callus and cortical bone immediately adjacent to the callus, resulting in scans with varying numbers of slices (∼500 to ∼800). Contralateral femora were similarly scanned, with 100 slices taken at the mid‐diaphysis of each bone. After μCT analysis, specimens were wrapped in saline soaked gauze and refrozen at −20°C.

3D evaluation

The volume of interest in the μCT image data was the callus or, in the case of the contralateral bones, a 1.2‐mm‐long section of the mid‐diaphysis. The callus volume of interest was defined using an image segmentation process within the Scanco software. For 2D tomograms (transverse callus slices) spaced 12 μm apart, the outside of the callus was manually delineated and refined through an automated intensity matching procedure such that it matched the outer boundary of the callus. The periosteal surface of the original cortex was manually delineated, and linear interpolation was used to define the callus and cortical boundaries in the intervening tomograms. The callus volume of interest was defined as the region enclosed by these two contours over all serial tomograms. This procedure thus excluded the preexisting cortical bone and the medullary canal volume from the analysis. Abbreviations as presented in the figures for each of the parameters are as presented below. The following were quantified for the volume of interest: total volume (TV), bone volume (BV), bone volume fraction percent (BV/TV), BMC, and average cross‐sectional area (CsAr). Before calculating the values of these parameters, Gaussian smoothing was applied (sigma = 0.8, support = 1). Only nonthresholded values are presented for BMC determinations because these values are more representative of the total callus mineral content as seen by X‐ray opacity. Grayscale threshold values were set to 250 mil on a 16‐bit scale (gray level of 8192 on 2², or 25% maximum intensity level). Contralateral specimens were outlined and evaluated by a midshaft analysis in the system software, which provided additional data including cortical thickness and polar moment of inertia (pMOI).

Mechanical testing

After μCT scans were performed on all samples, femora were subjected to torsion testing using an Instron 55MT1 MicroTorsion testing system (Instron, Norwood, MA, USA). Immediately before testing, each bone was thawed, and any remaining soft tissue was removed. A total of 157 bones (84 fracture bones and 73 contralateral bones) were included in the data collected from torsion testing. Ten fractured bones were excluded because of physical damage to the bone during handling before testing, whereas 10 contralateral bones were excluded because of technical reasons. The proximal and distal ends were potted in polymethylmethacrylate (PMMA) in 1/32‐in‐thick square aluminum casing containing a vertically oriented V‐shaped channel. After the PMMA set, the casing with the bone was inverted and lowered into the center and potted in the second casing. The gauge length of each specimen was calculated as the average of measurements taken on each of the four sides of the test sample. Torsional rotation was applied to the distal end of the specimen at a rate of 0.5°/s to failure. For fractured femora, torque and displacement data were recorded at a rate of 10 Hz, whereas intact femur data were recorded at a rate of 20 Hz using Partner Software (Instron, Norwood, MA, USA). After testing, each bone was viewed and photographed using a Nikon SMZ800 Zoom Stereomicroscope (Nikon Instruments, Melville, NY, USA), and the location of the post‐torsion refracture was recorded. Maximum torque, torsional rigidity, twist to failure (normalized by gage length), and toughness were calculated using a custom routine in MATLAB (The MathWorks, Natick, MA, USA). Torsional rigidity was defined as the slope of the initial portion of the torque‐twist curve, where twist was normalized by gage length, and toughness was defined as the area beneath the curve up until the maximum torque was reached.

Ash analysis

A total of 68 samples after mechanical testing were cut flush to their surfaces and potted in PMMA blocks, and the two halves were refrozen and subjected to ash analysis to assess callus mineralization. Fractured samples were aligned, and an approximate specimen length was measured with calipers. Samples were placed in crucibles and heated in a muffled furnace (type 48000; Barnstead International) to 100°C for 24 h. Crucibles were cooled to room temperature in a desiccator, and dry weight was measured with a digital scale (resolution ± 0.5 mg). Dried samples were heated to 800°C for 24 h, and resulting ash weights were recorded. Tissue mineralization (%) was calculated as the ratio of ash weight to dry weight.

Histology

For histological assessments, a subset of bones from days 21 and 42 after fracture with a small amount of surrounding muscle and soft tissues were fixed for 2 days in 4% paraformaldehyde in PBS at 4°C. Specimens were completely decalcified in 14% wt/vol EDTA changed three times per week for ∼2–3 wk while shaking at 4°C. Specimens were rinsed and placed in ice cold PBS for final processing. After decalcification, the intramedullary pin was removed, and the anatomic center of the callus was determined from the X‐ray measurements as the point of the fracture. Embedding and sectioning was carried out as described in Gerstenfeld et al. ${}^{\text{((}{}^{\text{17}}\text{))}}$ Sections were stained with Safranin O‐Fast Green as previously described to discriminate mature cartilage from bone and noncartilage connective tissues, and TRACP, a marker for osteoclasts, was detected using an azo‐dye coupling method with fast red violet LB salt (F‐3381; Sigma Chemicals, St Louis, MO, USA) replacing the fast red TR salt. ${}^{\text{((}{}^{\text{18}}\text{))}}$ Each section was photographed with an Olympus BX51 light microscope attached to a digital camera at ×10.25 and downloaded into an Image‐Pro Plus Version 4.1.0.0 for Windows program.

The osteoclast numbers in the three groups were assessed by histomorphometry. In the TRACP histomorphometry studies, a total of six slides with three slides per animal were examined, and the number of stained cells in each section (8–15 fields) encompassing an entire cross‐sectional area of a callus was determined. Sections were taken at center of the callus and increments at ∼1000 μm proximal and distal from the center.

Statistical analysis

All statistical analysis was performed using JMP 6 (SAS Institute, Cary, NC, USA). For each outcome measure from μCT and torsion testing, two‐way ANOVA (treatment, time postfracture) followed by a Tukey HSD posthoc test was carried out. Statistical significance was set at p < 0.05. Trends denote significance levels under p < 0.1. The analysis of the patterns of refracture was carried out by contingency analysis (χ² test for equal response rates). Because of the absence of an available posthoc test for the contingency analysis, pairwise comparisons of patterns of refracture were carried out by Fisher exact tests. Statistical analyses for ash values were based on a one‐way ANOVA with Tukey's HSD post‐test (GraphPad Prism v. 5.00), within separate analyses for the day 21 and day 42 time points. The Kruskal‐Wallis test of ranks was performed to evaluate differences in osteoclast number among treatments. A significance level of 0.05 was used for all analyses.

RESULTS

Systemic and local osteoclast activity

Serum TRACP 5b was assayed to provide an assessment of osteoclast activity with fracture and with ALN and DMAB treatment (Fig. 1). Over the first 2 wk, the control mice exhibited modestly increasing levels of TRACP 5b in the serum. After reaching a peak in observed serum levels at 14 days after fracture, the levels fell to below control prefracture levels by day 21 after fracture. Thereafter, the serum levels began to again slowly rise to their original prefracture quantities by the end of experimental period. ALN treatment was associated with modest reductions in serum TRACP 5b that reached significance only at week 6. Almost a complete and statistically significant inhibition of the appearance of TRACP5b in the serum was seen across the entire postfracture time course in the DMAB‐treated mice. To assess the effects of the two treatments at the local levels of osteoclast activity within skeletal tissues, total TRACP 5b enzyme levels were histologically examined in day 21 postfracture calluses (Fig. 1B). The day 21 time point after fracture was chosen for this analysis because our previous studies examining osteoclast activity within the fracture callus of C57/B6 mice had shown that osteoclast activity peaked at ∼14 days after fracture when mineralized cartilage was being resorbed. ${}^{\text{((}{}^{\text{4}}\text{))}}$ , ${}^{\text{((}{}^{\text{13}}\text{))}}$ However, to assess if these drugs delayed cartilage removal, we chose to examine a slightly later period after this peak had passed. It should be noted that osteoclast levels are still much higher than resting bone because it is still in a very active phase of remodeling. Global levels of osteoclast within the callus can be appreciated by examining low magnification images of the total cross‐sectional areas of the callus (Fig. 1B, top panels). Whereas callus tissues from both the control and ALN‐treated animals had observable amounts of TRACP⁺ cells present within the callus tissues, the animals treated with DMAB showed almost no detectable areas of activity. Higher magnifications showed individual TRACP⁺ multinucleated cells both on surfaces of new primary bone as well as on the surfaces of mineralized cartilage in the control specimens and ALN‐treated specimens. The osteoclast numbers in the three groups were assessed by histomorphometry (Fig. 1C). This analysis showed that callus tissues from the ALN‐treated animals had statistically larger numbers of TRACP⁺ cells than the controls, whereas the DMAB samples had essentially no positive cells.

Figure Figure 1.

Osteoclast activity across the time course of fracture in responses to alendronate or denosumab treatments. (A) Measured systemic activities based on serum levels of averaged TRACP 5b for each drug group across the time of treatment. Error bars are depicted in the figure are for SD, and levels of significance between individual drug groups to the control are denoted by asterisks. Significance was between all groups at 6 wk of treatment and is denoted by the asterisk above the control. (B) Panoramic histological assessments of local osteoclast activity within callus tissues based on TRACP 5b staining within 21‐day callus tissues. Composite images of fracture calluses (magnification, ×40). Bottom panel shows osteoclasts morphology on bone/cartilage surface fro each of the treatment groups. (magnification, ×600). (C) Osteoclast counts per unit total callus area. Osteoclast counts were determined. Letters denote individual groups (C, control; D, denosumab) that show statistical significance to the group that is denoted.

μCT analysis

μCT analysis was carried out to determine how the two therapies would affect tissue mineralization and the progression of structural changes that accompany fracture healing. Fugure 2 presents representative images from which the quantitative data were derived. Qualitative examination of the images from coronal cutaways of 3D reconstructions of the entire callus and from single transverse slices from the μCT scans taken from the center of the callus showed readily observable differences in the callus structures between the treatment groups. Qualitatively, the first apparent difference was the greater callus size of the DMAB‐ and ALN‐treated animals compared with the control, which was most apparent in day 42 postfracture groups. Another feature that was observable for the 21‐day specimens was the areas of increased X‐ray translucency in the ALN and DMAB calluses as seen primarily in the transverse slices and the central region of the reconstructed images. As will be shown below, this is primarily caused by the decreased amount of cartilage resorption at day 21 in both the ALN and DMAB treatment groups. These regions of higher X‐ray translucency ALN‐ and DMAB‐treated animals at 21 days may be contrasted to the most striking feature of this analysis, which was the intense increase in X‐ray opacity seen for these two groups at 42 days after fracture. This was most dramatic for the DMAB‐treated animals in which there was almost no discernible space between nascent trabecular elements that formed within the callus.

Figure Figure 2.

Callus structures at 21 and 42 days after fracture as assessed by μCT. (A) Cutaway views of representative μCT 3D reconstructions of fracture calluses at 21 and 42 days after fracture (coronal plane cutaway). (B) Representative μCT images of single transverse slices of fracture calluses at 21 and 42 days after fracture. Images of these individual slices were taken from the central region of each callus. Experimental groups and times after fracture are indicated in the figure.

The quantitative features of the callus structures that were obtained from the μCT analysis are summarized in Fig. 3. In all groups, the total volumes of the calluses were greater at 21 days than at 42 days, and the calluses of both treatment groups (ALN and DMAB) were greater than the control at both 21 and 42 days, although none of these differences was statistically significant (Fig. 3A). Bone volume was significantly different between control and DMAB‐treated animals at both 21 and 42 days. In contrast, the ALN treatment group only showed a significantly greater bone volume than the control calluses at 42 days (Fig. 3B). Comparing the two treatment groups, the callus from the DMAB‐treated animals also had a significantly greater bone volume than those from the ALN‐treated animals at both 21 and 42 days.

Figure Figure 3.

Graphical analysis of μCT measurements of fracture calluses. (A) Total bone volume (TV). (B) Total bone volume (BV). (C) Mean cross‐sectional area (CsAr). (D) Percent bone volume (BV/TV). (E) BMC based on calibration to a standard quantity of hydroxyapatite. For all graphs, error bars are ±SD. Cross indicates significance relative to the same treatment group at day 21. Dots indicate significance between ALN and DMAB treatment groups at the same time point. Asterisks indicate significance compared with the control at the same time point. Crosses indicate significance between time points for an individual experimental group. Significance is at p < 0.05.

The next structural feature assessed was the average cross‐sectional areas of the calluses. In general, this was greater for all groups at 21 days than at 42 days; however, these differences were not significant. At both time points, the ALN and DMAB bones were larger than control bones, but the only statistically significant results were between bones from DMAB treated animals at day 42 and bones from a control group at day 42 (Fig. 3C). The mineralized volume fraction is shown in Fig. 3D. These values largely paralleled those that were observed for bone volume with the ALN‐treated bones showing a significant difference to the control at 42 days (Fig. 3D). The DMAB specimens showed significantly greater percentage of mineralized tissue than both the control and the ALN treatment groups at both 21 and 42 days. It is interesting to note that between days 21 and 42, the amount of mineralized tissue increased in a significant manner for the DMAB group. The continuing increases in total bone volume, percent bone volume, as well as the smaller diminishment in cross sectional areas of callus for DMAB group, suggests that there is less resorption occurring in these callus tissues in comparison with those from animals treated with ALN. The last data that was assessed from the μCT analyses were total BMC. Differences in BMC were similar to those found in bone volume and percent bone volume, with the DMAB mean exceeding that of both ALN and control specimens at both time points. The total BMC also showed a significant increase over time with DMAB treatment, whereas it did not show this temporal pattern in either control or ALN‐treated animals. Whereas ALN treatment also showed an elevated BMC level at both time points compared with controls, significance was only reached 42 days after fracture (Fig. 3E).

Ash analysis

As a means of assessing the ratio of matrix to inorganic material, ash analysis was next performed (Fig. 4). This analysis showed that significant increases were found in the dry and ash weights of the day 21 and 42 DMAB and ALN treatment groups in comparison with their time‐matched control (Fig. 4A). Comparing the DMAB to ALN groups further showed that the DMAB was significantly greater in both dry and ash weight compared with ALN treatment. Tissue mineralization was not significantly affected by either treatment at day 21 in comparison with the control, although it was highest in the DMAB group. At day 42, however, tissue mineralization for the DMAB group was significantly higher (70.9%) than both the control (66.6%) and the ALN treatment group (66.6%). Across time, tissue mineralization increased for all groups in a significant manner from 21 to 42 days. Linear regression analysis was used to examine the correlation of the ash results to the BMC values obtained from the μCT assessments of the day 42 femurs (Fig. 4B). These results showed that femur ash weight was strongly and positively correlated with μCT‐derived callus BMC, both within each group individually and across all groups (Fig. 4B).

Figure Figure 4.

Results from ash analysis of fractured femurs after 21 and 42 days of treatment. (A) Ash analysis: left, dry weight; middle, ash weight; right, tissue mineralization as the ratio of ash weight to dry weight. *p < 0.05 vs. control. ^○ p < 0.05 vs. ALN. (B) Correlations between results from ash and μCT analyses in day 42 femurs. Femur ash weight was strongly correlated to callus vBMC as measured by μCT, both within and across all groups (r ² = 0.556).

Mechanical testing

Torsional testing over the time course of the fracture healing was used to provide a means of assessing the functional regain of mechanical strength by fractured bones, and these data are summarized in Fig. 5. These analyses showed that the fractured bones from all treatment groups and time points exhibited a higher maximum torque than their contralateral bones, with both the ALN and DMAB groups showing significantly greater mechanical strength compared with the matched contralateral bones at 42 days (Fig. 5A). When group comparisons were made for the fractured bones only, both ALN and DMAB treatment groups showed higher maximum torque than controls, with the ALN group significantly greater at 42 days. It is also interesting to note that the strength of the ALN group also significantly increased over time from 21 to 42 days (Fig. 5B).

Figure Figure 5.

Graphical analysis of torsion test results. One set of comparisons measure the differences between the contralateral bones and fractured bones (A and C), thereby providing a direct comparison of the regain of biomechanical competence to its unfractured state. The second set of measurements is the comparisons between the different drug groups and control across the two times within the fractured bones (B and D). This provides a direct comparison of the varying efficiencies of the two treatments vs. the control on the mechanical properties within the fractured bones themselves. The measure of the overall mechanical strength of the healing bones is presented in A and B. (A) Comparison of torque to failure between fractured bones and intact contralateral bones. (B) Comparison of fractured bones at between times and between experimental groups. Crosses indicate significance relative to the same treatment group at day 21. Asterisks indicate significance relative to the control at the same time point. (C) Comparison of torsional rigidity between fractured bones and intact contralateral bones. (D) Comparison of torsional rigidity between times and between experimental groups. Crosses indicate significance relative to the same treatment group at day 21. Asterisks indicate significance relative to the control at the same time point. For all graphs, error bars are ±SD. All p < 0.5.

The second parameter that was obtained from the torsional test was the overall torsional rigidity of the callus (Figs. 5C and 5D). Fractured bones from all groups and both time points exhibited significantly higher torsional rigidity than their contralateral bones with the exception of the ALN treatment group at day 21 (Fig. 5C). Among the fractured bones, all groups showed an increasing level of stiffness over time, with both drug treatment groups showing significant differences to the control at 42 days (Fig. 5D).

As part of the mechanical assessment, the location of refracture, or post‐torsion testing pattern, was documented. Each bone was categorized as having the break from testing located within the callus or outside the callus (Fig. 6). The post‐torsion testing patterns are summarized in Tables 1 and 2. For day 21 fractured bones, the percentages of specimens that broke through the callus were 60%, 50%, and 84.6% for the ALN, DMAB, and control specimens, respectively. For day 42 fractured bones, 57.1% of ALN specimens broke through the callus, 28.6% of DMAB specimens broke through the callus, and 86.7% of control specimens broke through the callus. Contingency analysis yielded significant results for day 42 bones at p = 0.0045. Pairwise comparisons of these proportions by Fishers exact test showed that both ALN and DMAB groups rebroke outside of the callus region to a significantly greater extent than the controls. The mechanical test results were reanalyzed to determine whether fracture location affected these results in each group. No significant differences in maximum torque or torsional rigidity were found between specimens that broke within the callus and those that broke outside the callus.

Figure Figure 6.

Representative photographs showing postmechanical testing fracture location. (Left panel) Break through the callus typical of what is seen in the control group. (Right panel) Break outside the callus typical of the groups treated with DMAB.

Table Table 1.

Post‐Torsional Testing Fracture Location

Table Table 2.

Individual Analysis of Groups

Qualitative histological assessments

Whereas osteoclastic activity has been associated with resorption of the mineralized cartilage of the callus ${}^{\text{((}{}^{\text{4}}\text{))}}$ , ${}^{\text{((}{}^{\text{13}}\text{))}}$ and it is presumed that this is necessary for healing to progress, the current data suggested that neither antiresorptive treatment affected the progression of the regain in biomechanical competency of the fractured bone. To assess how these treatments affected the resorption of the cartilage tissue, histological analysis of both day 21 and day 42 postfracture calluses was carried out. These results are shown in Fig. 7. Data from the day 21 postfracture calluses are shown in Fig. 7A. Lower ×40 magnifications of the entire transverse cross‐sectional areas showed that both ALN and DMAB treatments grossly inhibited cartilage resorption as seen by the large areas of red safranin O staining. The higher magnifications of these specimens showed that normal remodeling is evident in the control group, characterized by abundant woven bone and initial formation of trabecular bone, minimal mineralized cartilage, and almost no hypertrophic chondrocytes. In comparison, bones from both the ALN and DMAB treatment groups exhibited increased amounts of hypertrophic chondrocytes at the 21‐day time point, particularly in the tissues from the DMAB‐treated animals. The DMAB treatment specimens also seemed to have the greatest amount of residual cartilage, which is consistent with the greater X‐ray translucency seen in these samples at 21 days. In addition, large areas of mesenchymal and connective tissue were seen adjacent to the areas of unresorbed cartilage matrix. It is also noteworthy that the control group calluses were clearly smaller than the calluses of the ALN and DMAB treatment groups. Another interesting aspect of the bones from the DMAB treatment groups was the observation of rings of osteoid within the empty lacunae where the hypertrophic chondrocytes had resided. One such ring is highlighted by the boxed region in the micrograph.

Figure Figure 7.

Representative histological samples of fracture calluses at days 21 and 42 after fracture (safranin‐O and fast green). (A) Representative micrographs from 21 days after fracture. (Top panel) Composite images of fracture calluses (magnification, ×40). (Middle panel) Magnification, ×100. (Bottom panel) Magnification, ×400. Boxed areas show areas that were selected for each set of progressive magnifications in the three panels. Single boxed area in the panel showing the ×400 micrograph from the DMAB‐treated group highlights an individual ring of osteoid within the empty lacunae where a hypertrophic chondrocyte had resided. Also of note are distinct areas of hypertrophic chondrocytes and mixed bone and cartilage trabeculae that are still present in ALN and DMAB bones. (B) Representative micrographs from 42 days after fracture. (Top panel) Composite images of fracture calluses (magnification, ×40). (Middle panel) Magnification, ×100. (Right panels) Micrographs from two separate DMAB‐treated specimens.

To determine to what extent the loss of osteoclast function affected mineralized cartilage resorption over more prolonged time periods, specimens from 42 days after fracture were assessed. These data are shown in Fig. 7B. The lower power micrographs at the edge of the callus from each of the three treatment groups showed that control bone had well‐developed trabecular bone with distinctly formed marrow. Whereas the specimens from the ALN groups also showed the formation of individual trabeculae, they were much thicker in size and much less marrow was observed. In contrast, the tissues from the DMAB groups showed no distinct trabeculae had formed, consistent with the extremely dense appearing μCTs shown in Fig. 2 that demonstrated no visible marrow spaces. One of the two specimens that were examined also contained large areas of unresorbed cartilage tissues. Higher magnification of the three specimens showed that, whereas very small strands of red staining matrix were observed in the control trabecular tissue, both the tissues from alendronate‐ and denosumab‐treated animals showed that the osteoid matrix appeared to lay down on top of the underlying mineralized cartilage tissues that had remained unresorbed. This is exemplified by the light pink staining in the central elements of each of the trabeculae in the samples from the alendronate‐treated animals and the appearance of this staining pattern throughout the entire matrix from the denosumab‐treated mice.

DISCUSSION

The purpose of this study was to compare the effects of denosumab, a fully human monoclonal antibody to RANKL, and alendronate, a nitrogen‐containing bisphosphonate, on fracture healing in a mouse closed fracture model. Previous studies with both bisphosphonates and RANKL inhibitors have shown mixed results in regard to their effects on the regain of the mechanical properties during fracture healing. Studies examining osteoprogerin treatment on fracture healing in rats showed increased callus size over an 8‐wk period after fracture; however, no differences in mechanical properties were found. ${}^{\text{((}{}^{\text{5}}\text{))}}$ Studies using a mouse model and high doses of RANK:Fc also showed increases in callus size, but mechanical properties were not assessed. ${}^{\text{((}{}^{\text{6}}\text{))}}$ In a more recent study, in wildtype mice and mice with osteogenesis imperfecta mice (oim/oim), RANK‐Fc treatment initiated 2 wk before fracture and continued for 6 wk after fracture showed increased radiographic intensity in oim/oim mice and mechanical testing showed a greater work to failure. In wildtype mice, RANK‐Fc treatment was associated with significant increases in bending stiffness in the healing fractures. ${}^{\text{((}{}^{\text{19}}\text{))}}$ Numerous studies with different types of bisphosphonates have also been carried out in a wide variety of animal models. Using a canine model, ALN treatment initiated either before or during fracture healing produced larger calluses but did not result in any significant differences in ultimate strength or stiffness. ${}^{\text{((}{}^{\text{20}}\text{))}}$ Similar results were noted in a study comparing ALN, raloxifene, and estrogen in ovariectomized rats. ${}^{\text{((}{}^{\text{21}}\text{))}}$ Studies by Li et al. ${}^{\text{((}{}^{\text{22}}\text{))}}$ , ${}^{\text{((}{}^{\text{23}}\text{))}}$ showed that larger callus size and increased strength and stiffness was seen with incardronate treatment in rats. Treatment with a single systemic dose of pamidronate in rats ${}^{\text{((}{}^{\text{24}}\text{))}}$ and continuous administration of pamidronate (pre‐ and postoperatively) in sheep ${}^{\text{((}{}^{\text{25}}\text{))}}$ also produced increased callus size, and this study did show increased mechanical strength. Finally, zoledronic acid increased callus Cs.Ar and peak load in a rabbit fracture model ${}^{\text{((}{}^{\text{26}}\text{))}}$ , ${}^{\text{((}{}^{\text{27}}\text{))}}$ and increased callus volume, polar moment of inertia, peak load, and stiffness in a closed rat fracture model. ${}^{\text{((}{}^{\text{28}}\text{))}}$

The results of this study showed that both ALN and DMAB treatment produced larger callus size at both 21 and 42 days after fracture, although these differences in size were not statistically significant. The improved mechanical integrity of both treatment groups, however, is contributed to in aggregate, by the greater callus volumes, cross‐sectional areas and higher mineral contents of these callus tissues compared with the controls. Whereas the biomechanical assessment showed that both ALN and DMAB treatment groups had statically greater torsional rigidity (stiffness) after 42 days of treatment, only the ALN treatment group showed statistically greater torsional strength. It is noteworthy, however, that the majority of tested bones in the DMAB group failed outside the actual callus tissue. Because this test will lead to failure at the weakest structural point in the whole bone, these results would suggest that the calluses in this test group were in fact stronger than those in either the control or the ALN treatment groups because, in these groups, failure occurred through the callus. These results are consistent with previous studies showing that antiresorptive therapeutics administered during fracture healing lead to larger calluses that display increased load‐carrying capacity. The greater incidence of statistical significance in our structural and biomechanical data may have resulted from differences in the techniques and study design. The high‐resolution μCT analysis provided greater insight into callus specific changes which could not be assessed from plain X‐ray film or DXA. Torsion testing provided a quantitative assessment of strength that avoids applying loads to the callus region directly as is done in three‐point bend tests. Finally, the large number of animals that were used in this study provided sufficient statistical power to examine differences in bone strength.

A number of previous studies have suggested that the larger callus sizes observed after treatment with antiresorptive therapies was caused by delayed remodeling of the callus. However, the lack of detailed analysis of early time points and histological assessments has made it difficult to determine to what degree the larger callus size is caused by delayed resorption of the mineralized cartilage rather than the subsequent remodeling of primary spongiosa and woven bone. Delayed cartilage removal has been reported after RANKL inhibition by RANK:Fc in a murine fracture model. ${}^{\text{((}{}^{\text{6}}\text{))}}$ In this study, the large areas of the calluses in the ALN and DMAB groups that, by μCT at 21 days were devoid of definable trabecular bone, were consistent with the presence of cartilage. Histological studies also support this conclusion. It is also important to note that, with the threshold used in this study, at least some of the mineralized cartilage will have been counted as mineralized tissue. Thus, we propose that the higher BMC and bone volume found in the DMAB and, to a lesser extent, ALN groups compared with the control group reflect increased callus size and larger volumes of mineralized cartilage in the treatment groups. The increased BV/TV or percent bone, found in the treatment groups compared with controls, is also consistent with increased volumes of mineralized cartilage. Alternatively, because the callus is analyzed as a whole by μCT, the higher BMC and bone volume in the ALN and DMAB groups could be caused by differences in the quantity of new bone that is accumulated but not remodeled in the proximal and distal regions of the callus. Indeed, both these possibilities are supported by the histological studies from day 42 after fracture in which cartilage and underlying proteoglycan matrix were still observed in the tissues from the animals treated with ALN and DMAB.

Although it is hard to determine the degree to which the areas of mineralized cartilage have been replaced by bone by 42 days, the extreme X‐ray opacity of the calluses in the treatment groups at 42 days suggests that new bone formation continues to take place even in the absence of either mineralized cartilage or primary bone tissue resorption. The strong correlation between BMC and ash weight lends confidence to the μCT quantification of the BMC. A final explanation for the high mineral contents that are observed in the treatment groups in comparison with control callus tissues come from previous studies that have shown that total calcium to phosphorous ratios in cultured chondrocytes undergoing hypertrophy and mineralization are four times those seen in normal apatite, ${}^{\text{((}{}^{\text{29}}\text{))}}$ whereas other studies have shown that the mean calcium content of mineralized cartilage is ∼15–20% greater than mineralized bone. ${}^{\text{((}{}^{\text{30}}\text{))}}$

The observation that the serum levels of TRACP 5b peaked at 14 days after fracture are consistent with two previous studies from our laboratory that showed that RANKL, M‐CSF, and TRACP 5b mRNA expression levels in the calluses also peaked at 14 days after fracture. ${}^{\text{((}{}^{\text{4}}\text{))}}$ , ${}^{\text{((}{}^{\text{17}}\text{))}}$ In this regard, both the serum assay and the histological assessment of TRACP⁺ multinucleated cells in the calluses at a slightly later time point at 21 days suggested that DMAB inhibition of RANKL led to almost a complete loss of osteoclasts, whereas ALN was not inhibitory to osteoclast numbers. Indeed, in the limited samples that were tested, the ALN treatment groups showed statistically higher numbers of TRACP⁺ cells. These data would suggest that DMAB was able to virtually abolish osteoclastogenesis, whereas ALN does not affect osteoclastogenesis, indicating that ALN primarily acts by inhibiting mature osteoclast function. Whereas TRACP staining does not provide a means of quantifying total production of the enzyme by the individual cells, serum TRACP 5b findings would suggest that the cells that are present are less active. Indeed, using TRACP 5b measurements alone tends to underestimate the antiresorptive effects of alendronate relative to results obtained with the type I collagen degradation product serum carboxy‐terminal cross‐linking telopeptide (CTX). ${}^{\text{((}{}^{\text{31}}\text{))}}$ Therefore, these results might underestimate the antiresorptive impact of ALN in these mice. Other data that also suggest that ALN has no effect on either osteoclast number or the development of TRACP⁺ cells comes from studies of alendronate effects in growing rats during skeletal unloading. In these studies, alendronate treatment failed to diminish osteoclast number based in histomorphometric measurements of TRACP⁺ multinucleated cells while inhibiting calcified epiphyseal cartilage resorption. ${}^{\text{((}{}^{\text{32}}\text{))}}$

The final important aspect of these data is related to the role of osteoclast activity in the mediation of calcified cartilage resorption and the relationship between cartilage tissue removal and the regulation of new bone formation. The histological analysis clearly showed that the absence of osteoclast activity led to a considerable delay in the resorption of the cartilage. However, the μCT results showed that callus size decreased from day 21 to 42, suggesting that some form of resorptive activity was able to continue, albeit at a slower pace. Previous studies examining the function of MMP 9, MMP13, and MMP1433, 34, 35, 36, 37 have shown that osteoclastic cells are not the sole producers of proteolytic activities that remove the calcified cartilage. These enzymes are also produced by many other cell types including chondrocytes that mediate both proteolytic and proteoglycolytic activities, as well by the invading vascular endothelia, and mesenchymal cells that give rise to the stromal and osteogenic components during endochondral bone formation. Thus, our current data suggest that, whereas the complete absence of osteoclast activity greatly impairs this process, it can still progress but at much slower pace. These data suggest that chondrocytes autonomously mediate many aspects of their own development, whereas osteoclastic activities help to provide redundant functions and signals that increase the temporal pace of these processes. This conclusion is consistent with our previous studies that showed increased inflammatory mediators associated with diabetic fracture healing are related to the premature resorption of cartilage and increased osteoclast activity. ${}^{\text{((}{}^{\text{38}}\text{))}}$ These studies further showed that the premature loss of the cartilage tissues led to diminished bone formation. This latter finding also provides evidence to suggest that chondrocytes provide paracrine signals that promote osteogenesis. ${}^{\text{((}{}^{\text{39}}\text{))}}$ This suggestion would be consistent with the diminished amount of bone that is formed when the cartilage anlage is prematurely removed (as in the case during diabetic healing) and with the massive increase in new bone formation seen when the removal of cartilage tissue is delayed as in this study.

In summary, the results presented here show that, although both DMAB and ALN delayed callus remodeling, neither drug impeded the regain in mechanical strength. In fact, after 42 days, both DMAB and ALN treatments increased strength and stiffness of the fractured bones. Longer‐term studies will need to be conducted to assess the impact of these antiresorptive agents on the remodeling of callus back to the original bone structure and whether the prolonged delay in this remodeling has any negative consequences. This study suggests that neither denosumab nor alendronate has negative implications for short‐term aspects of fracture repair. Finally, they provide a demonstration that the absence of osteoclasts as seen with denosumab treatment does not lead to impairment in the initial regain of mechanical function during fracture healing.