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Keywords:

  • disuse osteoporosis;
  • bisphosphonates;
  • bone histomorphometry;
  • biomechanics;
  • remodeling

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We sought to determine whether risedronate can preserve cortical bone mass and mechanical properties during long-term disuse in dogs, assessed by histomorphometry and biomechanics on metacarpal diaphyses. Risedronate slowed cortical thinning and partially preserved mechanical properties, but it was unable to suppress bone loss to the degree seen in other osteoporoses.

Introduction: Disuse induces dramatic bone loss resulting from greatly elevated osteoclastic resorption. Targeting osteoclasts with antiresorptive agents, such as bisphosphonates, should be an effective countermeasure for preventing disuse osteoporosis.

Materials and Methods: Single forelimbs from beagles (5–7 years old, n = 28) were immobilized (IM) for 12 months. Age-matched, non-IM dogs served as controls. One-half the animals received either risedronate (RIS, 1 mg/kg) or vehicle daily. Histomorphometry was performed on second metacarpal mid-diaphyses. Cortical mechanical properties were determined by testing third metacarpal diaphyses in four-point bending.

Results: IM caused marked reduction in cortical area (−42%) and cortical thinning (−40%) through endocortical resorption, extensive intracortical tunneling, and periosteal resorption; both bone resorption and formation were significantly elevated over control levels on all envelopes. IM also decreased maximum load and stiffness by ∼80% compared with controls. RIS reduced both periosteal bone loss and marrow cavity expansion; however, cortical area remained significantly lower in RIS-treated IM animals than in untreated non-IM controls (−16%). RIS also increased resorption indices in all envelopes compared with nontreated IM, indicating that RIS suppressed osteoclast activity but not osteoclast recruitment. RIS did not affect bone formation. RIS treatment conserved some whole bone mechanical properties, but they were still significantly lower than in controls. There were no significant differences in tissue level material properties among the groups.

Conclusion: RIS treatment reduces cortical bone loss at periosteal and endocortical surfaces caused by long-term immobilization, thus partially conserving tissue mechanical properties. This modest effect contrasts with more dramatic actions of the bisphosphonate in other osteoporoses. Our results suggest that risedronate impairs osteoclastic function but cannot completely overcome the intense stimulus for osteoclast recruitment during prolonged disuse.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

DISUSE RESULTS IN dramatic bone loss, posing significant problems for areas as diverse as spaceflight(1,2) and rehabilitation medicine, where immobilization after spinal cord injury(3-6) or hemiplegia after stroke(7-9) lead to rapid bone loss and increased fracture risk.(10,11) Both programs have identified effective suppression of the progressive bone loss during disuse as the major countermeasure objective of both spaceflight and rehabilitation programs.

Bisphosphonates, the most effective antiresorptive agents currently available, have been shown to strongly inhibit osteoclast activities.(12,13) They are extensively used in prevention of bone loss from metabolic causes, such as postmenopausal(14-18) and steroid-induced osteoporosis,(19,20) as well as Paget's bone disease.(21) Because bone loss in larger mammals deprived of normal mechanical loading results from extremely elevated osteoclastic resorption,(1-9) targeting osteoclasts with bisphosphonate should be an effective countermeasure for preventing disuse osteoporosis. However, the efficacy of bisphosphonates in preventing disuse-type bone loss is less clear. Recent studies suggest that bisphosphonates may not work well in hypodynamic situations. In patients with spinal cord injury(4-6) and hemiplegia after a stroke,(7-9) treatment with high-dose bisphosphonates attenuated BMD loss, but not to the degree seen in other types of osteoporoses.(14-20) Moreover, the bisphosphonate dosages used in these studies were much higher than those used in postmenopausal osteoporosis, suggesting that skeletal sites susceptible to bone loss after spinal cord injury and stroke may be less sensitive to bisphosphonate treatment than more “metabolic” bone. Whether these situations are unique to spinal cord injury and stroke, or reflect a more global difference in the way that disuse osteoporosis responds to bisphosphonate, is unknown. In this study, we sought to test the hypothesis that a new generation pyraminyl bisphosphonate (risedronate) would preserve cortical bone mass and mechanical properties during long-term disuse.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Disuse osteoporosis was induced in the right forelimbs of female retired breeder beagle dogs (5-7 years old, n = 28; Marshall Farms). The canine skeleton was examined because of its high degree of similarity to human bone in overall remodeling physiology. Limbs were immobilized (IM) for 12 months, using a custom formed thermoplastic splint (AquaPlast; Smith & Nephew, Memphis, TN, USA) in which the elbow was flexed at 90°, and the carpal joint volar was flexed slightly as described by Li et al.(22) Synthetic cast padding was used under the splint. The splint was designed with velcro straps to allow easy removal of the splint for skin inspection, cleaning, and change of padding. Age-matched, non-IM dogs served as controls (Con). One-half the animals from each group received risedronate sodium (RIS; Proctor & Gamble Pharmaceuticals, Cincinnati, OH, USA) dissolved in sterile water, at a dose of 1 mg/kg orally daily (Con + RIS and IM + RIS). Risedronate treatment was started 2 days before start of immobilization. The remaining dogs received sterile water vehicle (Con and IM). Risedronate was chosen for use in this experiment because it has the shortest functional life in vivo among the new generation of bisphosphonates.(23) One of the interests that drove this study was the concept that bisphosphonates may play a role in preventing bone loss in long-term spaceflight, reasoning that bisphosphonates that would clear most rapidly from the bone once an astronaut returned to the earth and stopped taking the drug would be advantageous. The dose of RIS in this study is about 7-10 times higher than the clinical dose used in the treatment of postmenopausal osteoporosis.(14-16) RIS at one-half of the current dose has been shown to inhibit cortical and cancellous bone activation frequencies by 50-90% in dogs.(24,25) The high dose of RIS used in this study was chosen to maximize the osteoclastic inhibition with bisphosphonate treatment. Because the drug is poorly absorbed from the gastrointestinal tract, animals received no food or drink for at least 2 h before and after dosing. The absorption characteristics of RIS in dogs are similar to those in humans (Proctor & Gamble, unpublished data, 2003). Animals were group housed in one of two large rooms (∼47 m2 each). Animal health was recorded daily, and body weight was monitored weekly. For IM animals, the splints were removed two to three times per week to assess skin health. Animals were conditioned to handling before the start of the experiment so the splints could be removed and replaced without chemical restraint or sedation. Animals remained healthy and tolerated the IM procedure well throughout the study period. No significant change in feeding behavior or weights loss (<15% of starting body weight) was seen in RIS-treated animals, suggesting good gastrointestinal tolerance of the bisphosphonate. All animals received double bone fluorochrome labels at the end of the experiment. Calcein (10 mg/kg, IV; Sigma) was given following an injection schedule of 2-10-2-5 (2 days on, 10 days off, 2 days on, and 5 days off), after which the animals were killed by overdose of pentobarbital. All procedures were approved by Institutional Animal Care and Use Committees of Mount Sinai School of Medicine and VA Medical Center, Bronx, NY.

Long bones were harvested and cleaned of soft tissues. Bones used for histology were fixed in 10% neutral buffered formalin, whereas those used for biomechanical studies were wrapped in saline-soaked gauze and frozen at −40°C until testing. This study was performed on the second and third metacarpal bones because of their high degree of anatomical similarity to each other.

Cortical bone histomorphometry and geometry

Second metacarpals from right forelimbs were cut into thirds using a low-speed diamond saw, and the pieces were immersion fixed for 48 h. Mid-diaphyseal segments were stained with Villanueva Mineralized Bone Stain (Arizona Histology and Histomorphometry Services, Phoenix, AZ, USA) for 10 days, dehydrated with ethylene glycol monoethyl ether (Fisher Chemicals, Fair Lawn, NJ, USA), cleared in methyl salicylate (J. T. Baker, Phillipsburg, NJ, USA) and embedded with methyl methacrylate with 15% dibutyl phthalate (Fisher Scientific). Undecalcified cross-sections (150 μm thickness) were cut using a Leica 1600 Sawing Microtome (Leica Instruments, Nussloch, Germany), polished to 30 μm thickness, and coverslipped with nonfluorescing mounting medium for histomorphometric analysis.

Cortical bone changes were assessed using brightfield and fluorescent microscopy. Histomorphometry was performed using an OsteoMeasure system (Osteometrics, Atlanta, GA, USA) following standard measures described by Parfitt et al.(26) The structural (static) measures that were quantified include total subperiosteal area (T.Ar; mm2), marrow area (Ma.Ar; mm2), cortical area (Ct.Ar = T.Ar − Ma.Ar; mm2), porosity (diameter ≥ 30 μm) number (Po.N/Ct.Ar; #/mm2) and area (Po.Ar/Ct.Ar; %), net cortical area (Net.Ct.Ar = Ct.Ar − Po.Ar; mm2), and average cortical width (Ave.Ct.Wi; μm). Cellular-based (dynamic) indices included periosteal bone formation (P-sL.Pm/P.Pm; %) and resorption (P-Er.Pm/P.Pm; %), endosteal bone formation (E-sL.Pm/E.Pm; %) and resorption (E-Er.Pm/E.Pm; %), osteonal resorption space number (RON/Ct.Ar; #/mm2), forming osteon number (FON/Ct.Ar = [single-labeled osteon number + double-labeled osteon number]/Ct.Ar; #/mm2), and newly formed osteon number (NON/Ct.Ar; identified by the diffuse bone stain uptake visible under fluorescence microscopy; #/mm2). All measurements were made by a single observer who was blinded to the specimen identity.

Cross-sectional geometry measures (bone area, diameters, and polar moment of inertia) were determined from μCT scans of the mid-diaphysis of the third metacarpal bones. Bones were scanned using a GE MS8X μCT Scanner (GE Medical Systems, London, Ontario, Canada) operated at a 13-μm isotropic voxel resolution. Bone tissue was segment from non-bone tissue using the thresholding algorithm provided by the μCT manufacturer. Cross-sectional properties were determined from ∼200 μCT slices (∼2.6 mm region) of the metacarpal centered about the mid-diaphysis using a custom MATLAB analysis program (Mathworks, Nattik, MA, USA). The mean polar moment of inertia (Jo) of these slices was used in the calculation of tissue properties.

Cortical bone mechanical properties

Bone mechanical properties were evaluated from the third metacarpal bones after μCT scans. Cortical bone structural-mechanical integrity was determined by loading the diaphysis to failure in four-point bending. Bones were positioned in the test apparatus with the dorsal side in compression and the volar side in tension. The midshaft was centered over two cylindrical supports positioned 21 mm (L) apart with upper loading points spaced 7 mm apart (a). Bones were loaded to failure at a displacement rate of 0.1 mm/s. Tests were performed using a servohydraulic testing system (model 8872; Instron, Canton, MA, USA) equipped with a 2000N load cell. The displacements were measured from the system LVDT. Structural properties of whole bones were determined from load-displacement curves as follows: maximum load (the ultimate force that the specimen sustained), stiffness (the slope of linear portion of the load-deformation curve), work-to-failure (the area under the load-deformation curve before failure), and postyield deflection (PYD, as a measure of brittleness). Yield was defined as a 10% reduction in the secant stiffness (load range normalized for deflection range) relative to the initial tangent stiffness. Maximum stress (σ) and tissue modulus (E) were calculated by the formulae as described by Turner and Burr(27): σ = 1/4 maximum load × a × Φxx/Ixx; and E = −1/12 × stiffness × a2 × (4a − 3L)/Ixx, where Φxx and Ixx are the diameter of diaphyses and rectangular moment of inertia in the dorsal-volar direction, respectively.

Statistical analysis

Results are expressed as mean ± SD. Differences among groups were tested using ANOVA with Fisher's protected least significant difference (PLSD) for posthoc testing. Significance is reported at p < 0.05. Comparison of changes in bone mass and mechanical properties versus polar moment of inertia was performed using regression analyses. Statistical analyses were performed using the StatView program (version 5.0.1; Abacus, Mountview, CA, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cortical bone histomorphometry and geometry

Effects of immobilization:

All comparisons of results are expressed relative to non-IM controls. As expected, 12 months of IM resulted in a marked reduction of net cortical bone area (Net.Ct.Ar, −42%) and dramatic cortical thinning (Ave.Ct.Wi, −40%) (Fig. 1; Table 1). These occurred through endocortical loss (Ma.Ar, +51%; Fig. 1; Table 1), extensive intracortical tunneling (Po.Ar/Ct.Ar, +563%; Figs. 2 and 3; Table 1), and periosteal bone loss (T.Ar, −26%; Fig. 1; Table 1). Loss of periosteal bone resulted in a marked decline of ∼70% from control value in the polar moment of inertia (Table 1).

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Figure FIG. 1. Photomicrographs of metacarpal midshaft cross sections showing (A) control + vehicle, (B) control + RIS, (C) IM + vehicle, and (D) IM + RIS. (C) IM bone showed a smaller subperiosteal area, larger marrow cavity, thinner cortex, and elevated porosity compared with (A) control bone. (D) RIS-treated IM bone showed evidence of significant bone loss, but to a marked lesser degree than IM alone. Bar = 500 μm.20

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Figure FIG. 2. Photomicrographs from metacarpal midshafts showing (A) control + vehicle, (B) control + RIS, (C) IM + vehicle, and (D) IM + RIS. (C) IM bone showed greatly activated bone remodeling, with eroded surface evident on both periosteal (arrow) and endosteal surface (arrowhead) and osteonal canal (thin arrow) compared with (A) the control. (D) RIS-treated IM bone showed thicker cortex with much greater numbers of porosities compared with (C) nontreated IM bone. Bar = 400 μm.20

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Figure FIG. 3. Fluorescent photomicrographs of metacarpal midshafts showing (A) control + vehicle, (B) control + RIS, (C) IM + vehicle, and (D) IM + RIS. (C) IM bone showed that both bone resorption (skinny arrow) and formation (arrow: green color, calcein-labeled surface; red color, osteoid surface; arrowhead, newly formed osteon) were greatly elevated compared with (A) control bone. (D) RIS-treated IM bone showed that bone resorption sites were greatly elevated but bone formation was at the control level. Bar = 100 μm.20

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Table Table 1.. Structural and Remodeling Indices in Right Metacarpal Mid-diaphyses
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In IM animals, resorption surface on both periosteal and endocortical envelopes was significantly elevated (P-Er.Pm/P.Pm, +95%; E-Er.Pm/E.Pm, +216%; Fig. 2; Table 1) over control levels. Surprisingly, there was an increase of bone formation on both periosteal and endocortical surfaces as well (Table 1). Within the intracortical envelope, resorption space number was greatly elevated (Figs. 2 and 3; Table 1). There was also extensive infilling of osteons, with the number of forming osteons (p < 0.006) and newly formed osteons significantly increased (p < 0.0001) in IM bones compared with control bones (Fig. 3; Table 1). Overall, these data indicate that cortical bone loss in long-term disuse is a typical “high bone turnover” process occurring on all cortical bone envelopes.

Effects of risedronate treatment:

RIS treatment significantly reduced, but did not completely prevent cortical bone loss in IM animals. Overall bone loss (Net.Ct.Ar) in RIS-treated IM animals was reduced nearly 60% relative to nontreated IM animals, representing the reduction of 16% (p < 0.003) and 42% (p < 0.0001) from the control level, respectively. RIS treatment in IM significantly reduced both periosteal bone loss and marrow cavity expansion. Changes of Jo and in overall bone area in RIS-treated IM were similar (Fig. 1; Table 1).

Surprisingly, indices of bone resorption (intracortical resorption space number, eroded surface at periosteal and endocortical surfaces) were elevated in RIS-treated IM bone, more so in fact than in IM alone (Figs. 2 and 3; Table 1). Bone formation indices (labeled periosteal and endocortical surfaces, forming and newly form osteons) were at control levels in the RIS-treated IM group (Figs. 2 and 3; Table 1), indicating that the high dose of risedronate used in this study did not inhibit osteoblastic activity. Together, these data suggest that bisphosphonate treatment did not prevent the activation of osteoclasts during disuse, but it did blunt the activity of osteoclasts so that bone loss was slowed after the bisphosphonate treatment.

Cortical bone mechanical properties

Maximum load, stiffness, and work to failure in IM bone decreased by ∼80% compared with control levels (Table 2). Loss of these mechanical properties was almost double the loss of cortical bone mass (Net.Ct.Ar, −42%; Table 1) but was closely related to the decrease in the average diaphyseal polar moment of inertia (Table 3). RIS treatment in IM conserved some of the mechanical properties, but maximum load and stiffness were still significantly lower than that in controls (−29% and −24%, respectively; Table 2). Postyield displacement was unchanged in IM- and RIS-treated bones, indicating no change in bone brittleness with either long-term IM or bisphosphonate treatment. Maximum stress in IM bone was reduced by 45% compared with the control level, but in RIS-treated IM, was nearly identical to the control level. Tissue modulus was unaffected by either IM or RIS treatment (Table 2). The relationships between declines in Jo and loss of bone mass and strength (maximum load and stiffness) for all groups were essentially identical, showing that diaphyseal strength was closely correlated with cortical bone mass and diaphyseal polar moment of inertia (Table 3).

Table Table 2.. Mechanical Properties of Four-Point Bending Test in Right Metacarpal Mid-Diaphyses
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Table Table 3.. Regression Analysis of Polar Moment of Inertial vs. Bone Mass and Mechanical Properties in Right Metacarpal Mid-Diaphyses
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study shows that RIS treatment attenuated cortical bone loss in long-term disuse osteoporosis but not to the degree expected from studies of bisphosphonates in other type of osteoporoses. Dramatic bone loss in disuse is caused by extremely elevated bone resorption. Thus, targeting osteoclasts to reduce bone loss would be expected to be an effective strategy in disuse. Bisphosphonates are the most potent antiresorptive agents currently available. They have been shown to stop bone loss after menopause(14-18) and in glucocorticoid treatment.(19,20) So effective are the bisphosphonates in preventing bone loss in these metabolic osteoporoses that increases of BMD are seen from a combination of infilling of the remodeling space, increased BMC, and perhaps uncoupled bone formation.(14,17) Thus, it has been shown that alendronate at 5-10 mg daily effectively prevented bone loss in postmenopausal osteoporosis(17,18) and in patients on steroid therapy.(19) Risedronate at 5 mg daily prevented bone loss and led to increases of BMD in both early postmenopausal women(15) and those with established osteoporosis.(16)

In contrast, bisphosphonates seem to be much less effective in preventing mechanically driven bone loss. Pamidronate at 30 mg per every 4 weeks (the similar dose for postmenopausal osteoporosis) for 6 months did not improve BMD in the paretic side of complete spinal cord injury patients.(4) Etidronate at high dose did not decrease the rate of bone loss in complete spinal cord injury patients(6) and in hemiplegia patients after a stroke.(8) Sniger and Garshick recently reported that high-dose alendronate treatment in spinal cord injury patient attenuated BMD loss but not to the degree seen in other osteoporoses.(5) Given that the bisphosphonate dosages used in these studies(5, 6, 8) were typically much higher than those used in postmenopausal osteoporosis, bone loss after spinal cord injury and stroke may be less sensitive to bisphosphonate treatment than more metabolically driven bone loss. Whether these situations are unique to spinal cord injury and stroke, or reflect a more global difference in the way that disuse osteoporosis responds to bisphosphonate, is unclear. In view of these studies, these data suggest that bone loss that follows unloading, that is, disuse osteoporosis, is less sensitive than other, more metabolically driven bone loss processes to suppression of resorption with bisphosphonates.

Disuse in adult dogs leads to a very rapidly evolving, dramatic, and severe bone loss.(28-31) In this study, cortical bone loss in canine metacarpal bones after 1 year of IM was on the order of 40%, similar to the 50% bone loss reported in dog metacarpals by Jaworski and Uhthoff and Uhthoff et al. in their 1+ year long-term studies of cortical bone loss.(28,29) Thus, the cortical bone loss rates over 1 year of immobilization in adult dogs are in the range of 3-4% per month. Because cortical bone loss (∼5.8 mm2) occurred principally at the periosteal (∼4 mm2), followed by endosteal surface (∼1.3 mm2) and intracortical envelope (∼0.5 mm2) in IM animals, this in agreement with the finding of Jaworski and Uhthoff in the young dogs.(28) Jaworski and Uhthoff and Uhthoff et al. also found that bone loss in disuse is not uniform. They showed that bone loss was more pronounced in more distally located bones, with the metacarpals on the high end of bone loss range for this model.(28,29) Nevertheless, profound bone loss occurs with disuse at all forelimb bones with this immobilization model. In this study, we examined only the metacarpals to allow us to correlate bone histomorphometric and biomechanical changes in two anatomically comparable bones within the same animal. Moreover, the cortical bone loss rates in these immobilized limbs are comparable to loss rates in long bones after spinal cord injury and in bed rest. Wilmet et al. found that BMD loss occurred at ∼2% per month at cortical bone sites (4% in cancellous bone) in paralyzed legs of spinal cord injury patients within the first year after IM.(32) Other studies of spinal cord injury reported similar monthly BMD loss rates, typically ranging 3-6% per month.(33) Minaire et al. reported that cortical bone of iliac crests in spinal cord injury patients thinned by almost 50% over 10 months (5% per month).(34) Bone loss in bed rest study has been reported in the range of 2-4% per month at some sites.(35) In contrast to disuse-type bone loss, BMD decline in postmenopausal osteoporosis is on the order of 0.5-1.5% per year,(36) but may reach as high as 3% per year in the most rapid bone loss period after menopause.(37) Thus, these data indicate that bone loss caused by unloading is much greater and faster than that in postmenopausal osteoporosis.

Our understanding of the bone cellular components affected by disuse is still far from complete. Whereas there is an agreement that disuse in the adult skeleton activates high levels of bone resorption, the precise osteoclast kinetics in disuse is not well understood. It has been argued that the unusually large resorption spaces seen in immobilized bone are the result of extremely aggressive osteoclasts (i.e., increased cellular activity).(38) However, recent studies suggest that early disuse in dog cortical bone is characterized by very high activation of normal-sized resorption sites, indicating normal cellular activity and a high birthrate of new resorption foci. These foci later coalesce into large resorption spaces eventually join with the marrow cavity.(39) Studies on spinal cord injury(6) or paraplegic patients(40,41) showed that increase of bone resorption in iliac crest bone occurs in conjunction with decrease of bone formation. Similarly, in healthy subjects after 12 weeks of bed rest, osteoclast number and eroded surface increased, but osteoblast surface and mineral apposition rate decreased.(35) Study of humans during spaceflight showed that bone resorption markers tend to increase, whereas formation markers tend to decrease.(2) These studies have led to the idea that disuse uncouples of resorption from formation during bone remodeling. However, several lines of evidence suggest that this is not the case in larger mammals. This study on dogs showed that both bone formation and resorption markedly increased in disuse after 1 year of IM. A number of others studies in dogs and primates showed similar results. Lane et al.(30) and Grynpas et al.(31) determined from the level of newly created bone mineral that significant new bone must form during disuse. Study of monkeys showed that long-term immobilization significantly increased level of immature collagen cross-links in bone, which results from new bone formation.(42) Schaffler and Pan(43) found that bone formation resumes on previously resorbed bone surfaces during long-term disuse, but the onset of this formation is delayed compared with normal bone remodeling. Together, these data suggest that the bone loss pattern in disuse in dogs is a typical “high bone turnover” process, similar to postmenopausal osteoporosis, even though the former is a remodeling-based osteoporosis in which there may be a temporal delay in the onset of bone formation processes.

It has been widely accepted that bisphosphonates inhibit both osteoclast recruitment and activity.(12,13) However, in this study, we found that the resorption indices on all envelopes of immobilized cortical bone under RIS treatment at the end of 1 year were highly elevated and in fact greater than that in nontreated IM animals. RIS-treated IM bone had more eroded surface at the periosteal and endocortical envelopes and more numerous intracortical resorption spaces than IM bones. However, RIS treatment during long-term IM suppressed bone loss, suggesting that the effectiveness of osteoclastic bone removal is diminished by bisphosphonate treatment. RIS suppression of bone loss appeared to differ in each bone envelope, ranging from 29% at the endocortical surface, 21% at periosteal, and eventually unchanged at the intracortical envelope. Such differences could reflect site-specific cellular responsiveness to bisphosphonate treatment or difference in drug variability. However, because of variability particularly in endocortical area measurement in IM animals, it is not clear of the difference is significant. While seemingly paradoxical, these observations are consistent with the previous data from the iliac crest in bed rest and spinal cord injury. Etidronate treatment of bed rest subjects for 120 days led to increases in eroded surface in the iliac crest, but decreases in osteoclast number.(44) In paraplegic patients treated with high-dose tiludronate for 3 months, Chappard et al. found that the eroded surface was at the same level as in placebo patients, whereas osteoclast number was reduced to normal level.(9) Minaire et al.(41) reported similar results in spinal cord injury patients treated with clodronate. The reasons for this high level of eroded surface and diminished osteoclast number in immobilization patients treated with bisphosphonate are unclear. In this study, the dose of RIS used was quite high. High doses of bisphosphonates can suppress bone formation(45); however, in our study, bone formation indices in the RIS-treated IM group remained at control levels, indicating that the high dose of RIS we used in this study did not inhibit osteoblastic activity. Thus, the high levels of eroded surface and intracortical resorption spaces in RIS-treated IM animals was not a result of failure to form bone. Alternatively, the effect of bisphosphonates on osteoclasts in long-term immobilization may differ from other circumstances.

These data, as well as those from previous studies of bisphosphonates in bed rest and paralysis, suggest a scenario wherein bisphosphonate treatment may not prevent the activation of osteoclasts during long-term disuse. However, once recruited, bisphosphonates seem to be able to diminish the osteoclastic activity such that bone loss is reduced after the treatment. Why the recruitment of osteoclast in unloading would be especially high is not known, but disuse can cause a number of physiological changes that lead to increased bone resorption (e.g., osteocyte injury, hypoxia and apoptosis in bone, changes in limb blood flow, venous stasis, tissue perfusion, and changes in local tissue pH).(46,47) Interestingly, recent studies indicate that an elevation of resorption indices also occurs with very-long-term bisphosphonate treatment of postmenopausal osteoporosis. With both long-term RIS(14) and alendronate(17) treatment, bone resorption indices (eroded surface and osteoclast number) were not suppressed by the treatment, despite long-term cessation of bone loss. Taken together, these results suggest that the effects of bisphosphonates on osteoclast recruitment versus those on cell activity (vigor and lifespan) can be uncoupled in vivo. Thus, osteoclast recruitment will occur in the presence of bisphosphonates if the resorption signal is very intense (as in immobilization) or perhaps, less intense signal of sufficient duration (as in post menopausal bone loss). However, once recruited, the effectiveness of these cells in removing bone is diminished by the presence of bisphosphonate.

A previous study found that pamidronate treatment in short-term disuse (12 weeks) in dogs preserved diaphyseal mechanical properties.(31) This differs from the results of this study, in which we found that RIS treatment during a 1-year immobilization conserved a significant amount of diaphyseal strength and stiffness, but the RIS-treated bones were still much weaker (∼30% lower) than normal bones. Given that studies from the spinal cord injury or stroke patients showed that bone loss was characteristically sustained for all bisphosphonates examined, it seems likely that the differences in biomechanical results between this long-term study (12 months) and that of Grynpas et al. (3 months)(31) reflect the difference in experimental duration rather than a fundamental difference in the action of different bisphosphonates.

Long-duration treatment with bisphosphonates is associated with altered material properties in bone. Treatment with either alendronate or risedronate for 1 year in young adult dogs(24,25) has recently shown that bisphosphonate treatment results in a significant increase in bone brittleness and a decrease in the energy needed to fracture a bone; these properties were attributed to both bone microdamage accumulation and increased matrix mineralization resulting from inhibition of bone turnover. In this study, we did not find any change in bone brittleness or work-to-fracture. The possible reason for this discrepancy could be attributed to the animal age and the different bone sites studied. Bone turnover rates are high in young adult dogs at the vertebral cancellous bone (BFR/BV, ∼80 μm3/μm2/yr) and the rib cortical bone (BFR/BV, ∼20%/yr in intracortical site; BFR/BS on periosteal and endocortical sites, ∼11-15 μm3/μm2/yr)(24,25) compared with 5- to 7-year-old dogs, where bone turnover in diaphyses is very low (BFR/B.Ar in cancellous bone, ∼2%/yr [data not shown], BFR/BS cannot be determined for periosteal and endocortical envelopes in control bones from this study because of lack of double labeling). Thus, it seems reasonable to expect that remodeling suppression in a high turnover bone will show a greater relative effect than a similar degree of suppression in a low turnover bone. This latter possibility may have important implications for understanding how remodeling suppression differentially affects bone sites, and in turn, bone fragility.

In conclusion, the results of this study in combination with data from a range of other bisphosphonate studies in paralysis patients suggest that bisphosphonates cannot fully overcome the stimulus to recruit osteoclast during long duration of immobilization. However, once osteoclasts are recruited, their activity seems to be diminished by bisphosphonates, and they remove less bone. Thus, RIS treatment can attenuate cortical bone loss in long-term disuse osteoporosis, but can not inhibit bone loss to the degree expected based on the action of bisphosphonate in other, more “metabolic” types of osteoporosis. Nevertheless, the results of this study show that RIS treatment can reduce cortical bone loss by more than one-half and was particularly effective in reducing the periosteal and endocortical bone loss that can disproportionately weaken long bones diaphyses.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This study was supported by the National Space Biomedical Research Institute (BL00203) and NIH AR41210 and AR48699. The authors thank Dr Robert Majeska for the English editing, Gregory Bouyer for expert technical assistance, and Richard Mann, DVM, from Bronx VA Medical Center for the veterinary guidance. The risedronate used in this study was generously provided by Procter & Gamble Pharmaceuticals; we also thank Roger Phipps, PhD, and Mark Lundy, PhD, from Proctor & Gamble for helpful comments.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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