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Keywords:

  • microdamage;
  • bone remodeling;
  • bisphosphonate;
  • biomechanics;
  • histomorphometry

Abstract

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

It has been hypothesized that suppression of bone remodeling allows microdamage to accumulate, leading to increased bone fragility. This study evaluated the effects of reduced bone turnover produced by bisphosphonates on microdamage accumulation and biomechanical properties of cortical bone in the dog rib. Thirty-six female beagles, 1–2 years old, were divided into three groups. The control group (CNT) was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with risedronate (RIS) at a dose of 0.5 mg/kg per day or alendronate (ALN) at 1.0 mg/kg per day orally. After sacrifice, the right ninth rib was assigned to cortical histomorphometry or microdamage analysis. The left ninth rib was tested to failure in three-point bending. Total cross-sectional bone area was significantly increased in both RIS and ALN compared with CNT, whereas cortical area did not differ significantly among groups. One-year treatment with RIS or ALN significantly suppressed intracortical remodeling (RIS, 53%; ALN, 68%) without impairment of mineralization and significantly increased microdamage accumulation in both RIS (155%) and ALN (322%) compared with CNT. Although bone strength and stiffness were not significantly affected by the treatments, bone toughness declined significantly in ALN (20%). Regression analysis showed a significant nonlinear relationship between suppressed intracortical bone remodeling and microdamage accumulation as well as a significant linear relationship between microdamage accumulation and reduced toughness. This study showed that suppression of bone turnover by high doses of bisphosphonates is associated with microdamage accumulation and reduced some mechanical properties of bone.


INTRODUCTION

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

Fragility fractures of bone historically have been associated with low bone mass. However, low bone mass is not the only factor contributing to increased fracture incidence in postmenopausal women. There is a component of bone fragility that is independent of bone mass.(1) This component may include the detrimental effect of the accumulation of microdamage. It has been shown that microdamage accumulates in bone as people age.(2–4) This accumulation of damage could contribute, in conjunction with loss of bone mass, to the increased bone fragility that occurs in postmenopausal women. The microdamage burden in bone is a function both of the amount of damage that is produced and the amount of damage that is repaired through normal physiological remodeling processes. Either increased production of damage or suppressed repair can elevate the level of microdamage in bone.(5,6) The reasons for the increased microdamage accumulation with age—whether increased initiation or decreased repair—are not known.

Bisphosphonates increase bone mass and decrease fracture incidence in postmenopausal osteoporotic women.(7,8) Treatment with alendronate or risedronate for 3–4 years decreases the risk of vertebral and nonvertebral fractures in women with osteoporosis.(9–14) They are effective because they inhibit bone remodeling, preventing the loss of bone that occurs through resorption and allowing refilling of the remodeling space. However, in doing so they also may prevent the repair of microdamage. The appropriate balance between suppressing remodeling sufficiently to prevent bone loss and suppressing it so much that it impairs micro-damage repair is unknown.

The purpose of this study is to evaluate the effects of reduced bone turnover for 1 year on microdamage accumulation and biomechanical properties of rib in dogs. We used bisphosphonates that suppress bone remodeling without impairment of mineralization to reduce turnover. The doses of both bisphosphonates were five times higher than the clinical doses used for osteoporosis in humans.

We hypothesized that suppression of bone turnover is associated with accumulation of microdamage and reduced bone mechanical properties.

MATERIALS AND METHODS

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

Experimental animals

Thirty-six female beagle dogs were purchased from L.B.L. Kennels (Redsville, IN, U.S.A.). The dogs were 1–2 years of age at the beginning of the study. All dogs were acclimated for a period of 1 month and housed individually in environmentally controlled rooms and fed standard dog chow containing 1.2% calcium, 1.0% phosphorus, and 851 IU/kg vitamin D3 (Diamond Premium Adult, Diamond Pet Foods, Meta, MO, U.S.A.). The amount of food served and eaten was 350 g/day per animal. Water was available at all times. Drug administration was done approximately 2 h before feeding. All procedures were in accordance with approved National Institutes of Health (NIH) guidelines, under a protocol approved by the Indiana University School of Medicine Animal Care and Use Committee (study MD 1783).

Experimental design

The dogs were randomly divided into three groups based on their body weight. Dogs in the control group (CNT, n = 12) were given daily subcutaneous injection of saline vehicle. The remaining two groups of dogs were treated daily with risedronate (Procter and Gamble Pharmaceuticals, Inc., Cincinnati, OH, U.S.A.) orally at a dose of 0.5 mg/kg per day (RIS, n = 12) or alendronate (Merck and Co., Inc., West Point, PA, U.S.A.) orally at 1.0 mg/kg per day (ALN, n = 12). These bisphosphonates were dissolved in saline and the exact amount of the solution was given to each dog orally using a syringe. All dogs were treated for 12 months. At the completion of the experiment, dogs were killed by overdose of a sodium pentobarbital derivative (Beuthanasia-D Special, Schering-Plough Animal Health Co., Kenilworth, NJ, U.S.A., 0.22 ml/kg, intravenously [iv]). Before death, animals in all groups were double-labeled with calcein (5 mg/kg, iv, Sigma, St. Louis, MO, U.S.A.) on a 2–12–2–5 or 2–12–2–6 schedule.

General condition

Physical well being and activity were monitored daily, and body weight was measured weekly. At the end of the experiment, the weight of each animal was recorded.

Radiography

Ventrodorsal and lateral thoracic X-rays were taken of all dogs at baseline and each month from 7 to 12 months to evaluate the occurrence of spontaneous rib fractures. Dogs were anesthetized with thiopental sodium (Pentothal, 10 mg/kg, iv, Abbott Laboratories, N. Chicago, IL, U.S.A.) for each radiography session.

Tissue preparation

The right ninth rib was dissected, fixed for 3 days in cold 10% neutral buffered formalin, and prepared for basic histology or bulk staining in basic fuchsin for microdamage analysis. The sampling site for the rib was a 4-cm portion from the point of greatest curvature of each rib.(15) The sample was divided at the midpoint to provide specimens for basic histology and for microdamage analysis. Specimens for basic histology were transferred to 70% ethyl alcohol (EtOH), processed through a graded series of EtOH, and embedded in methylmethacrylate (MMA; DDK-plast; Delaware Diamond Knives, Wilmington, DE, U.S.A.). Likewise, specimens for microdamage analysis were bulk stained in two changes each of 1% basic fuchsin in 70, 80, 90, and 100% EtOH under vacuum at 20 in Hg for 4 h. After staining, the bone was rinsed in 100% EtOH followed by 100% MMA (Aldrich Chemical Co., Mlwaukee, WI, U.S.A.) for 4 h under vacuum at 20 in Hg and then embedded in DDK-plast (Delaware Diamond Knives).(14) For microdamage analysis, three transverse sections of 80-μm thickness were cut from each block using a diamond wire saw (Histosaw; Delaware Diamond Knives). For basic histology, four transverse sections of 80-μm thickness were cut from each block in the same way. Half were stained with Goldner trichrome stain for static histomorphometry and the remaining sections were left unstained for dynamic histomorphometry.

Histomorphometric measurement

Before measurements, the animal numbers of all slides were masked so that the histomorphometrist could not know the treatment assignment of the dogs. All measurements were carried out by only one histomorphometrist at 150 × using a semiautomatic digitizing system (Bioquant System IV, R & M Biometrics, Inc., Nashville, TN, U.S.A.) attached to a microscope equipped with bright field and UV light sources (Nikon Optihot 2 microscope, Nikon, Tokyo, Japan). Intracortical histomorphometric measurements were made for the entire rib cross-section. Surface measurements were made on periosteal and endocortical bone envelopes. The primary and derived parameters measured or calculated for the ribs are listed in Table 1.

Table Table 1.. Histomorphometric Parameters for the Rib
 Primary parametersDerived parameters
Type of measurementName of parameterAbbreviationName of parameterAbbreviationFormula
  1. * Measured from all single-labeled osteons.

  2. Measured from all double-labeled osteons.

  3. Measured from labeled osteons without osteoid.

IntracorticalTotal areaTtArCortical areaCtArTtAr — MeAr
 Medullary areaMeArPercent cortical area%CtArCtAr/TtAr
 Osteoid areaOArResorption cavity numberRsN/CtArRsN/CtAr
 Osteoid perimeterOPmLabeled osteon numberLOnN/CtAr(sLOnN + dLOnN)/CtAr
 Resorption cavity numberRsNMineral apposition rateMAR2 · (outdLAr — inndLAr)/(outdLPm + inndLPm)/labeling interval
 Single-labeled osteon numbersLOnNOsteoid areaOAr/CtArOAr/CtAr
 Double-labeled osteon numberdLOnNOsteoid thicknessOThOAr/OPm
 Single-label perimeter*sLPmOsteoid maturation timeOmtOTh/MAR
 Outer label perimeteroutdLPmMean wall thicknessWTh2 · (LOnAr — HCaAr)/(LOnPm + HCaPm)
 Inner label perimeterinndLPmFormation periodFPWTh/MAR
 Outer label areaoutdLArActivation frequencyAcf365 · LOnN/CtAr/FP
 Inner label areainndLArBone formation rateBFR/BV365 · MAR · (sLPm + outdLPm + inndLPm)/2/CtAr
 Labeled osteon perimeterLOnPm   
 Area of labeled osteonLOnAr   
 Haversian canal perimeterHCaPm   
 Area of Haversian canalHCaAr   
Cortical surfaceBone surfaceBSMineralizing surfaceMS/BS(sLS/2 + dLS)/BS
 Single-labeled surfacesLSMineral apposition rateMARLTh/labeling interval
 Double-labeled surfacedLSBone formation rateBFR/BSMS/BS · MAR
 Label thicknessLTh   

Microdamage measurement: Microdamage was assessed from two sections per dog. The entire rib cross-sectional area was evaluated. Stained microcracks were defined by sharp edges, some depth of field, and permeation of stain into the crack walls (Fig. 1). Measurements included crack density (CrDn = crack number [CrN]/cortical area [CtAr], #/mm2), mean crack length (CrLe, μm), and crack surface density (CrSDn = CrN*CrLe/CtAr, μm/mm2) in cortical bone.

Biomechanical testing

The left ninth ribs were dissected at death and a 4-cm portion from the point of greatest curvature of each rib was used as a test specimen. Bones were wrapped in gauze soaked in isotonic saline and frozen at −20°C until testing. After thawing at room temperature, samples were tested to failure in three-point bending using an MTS 810 servohy-draulic testing machine (MTS Corp., Minneapolis, MN, U.S.A.). The midpoint of the convex side served as the loading point during the testing. To ensure the same moment for all specimens, the lower supports were placed 12.5 mm from the midpoint. The actuator was displaced at a rate of 1 mm/s until failure occurred. Load versus displacement data were recorded using the HP 7090A measurement plotting system (Hewlett Packard, Camas, WA, U.S.A.), and then ultimate force (maximum force that the specimen sustained), stiffness (the slope of the linear portion of the load-deformation curve), and work to failure (the area under the load-deformation curve before failure) were measured using a digitizer (Jandel Scientific, Corte Madera, CA, U.S.A.). These parameters are structural parameters that depend on both intrinsic material properties and geometry.(16)

The load-displacement data were normalized to obtain material properties, such as ultimate stress, elastic modulus, and toughness, which are independent of cross-sectional size and shape.(16)

Ultimate stress (σ) was calculated from the ultimate force (Fu) by

  • equation image(1)

where L is the length between supports, b is the width of the rib in the anteroposterior direction, and I is the cross-sectional moment of inertia. Elastic modulus (E) was calculated as

  • equation image(2)

Toughness was calculated from work to failure (W) using

  • equation image(3)

Cross-sectional moment of inertia (I) was calculated using the assumption that rib cross-sections were elliptically shaped:

  • equation image(4)

where a is the width of the cross-section in the mediolateral direction, and t is the average cortical thickness. Average cortical thickness was calculated from the histomorphometric thickness measurements made in each of four quadrants of the right ninth rib section using a semiautomatic digitizing system.

thumbnail image

Figure FIG. 1.. (A) In vivo microcrack and (B) artifactual microcrack in a rib section from an ALN-treated beagle. Stained en bloc with basic fuchsin. Original magnification, ×98.

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Statistical analysis

Statistical computation of data was performed using the statistical package Statview (SAS Institute, Inc., Cary, NC, U.S.A.). Differences among treatment groups were tested by one-way analysis of variance (ANOVA). If significant differences were indicated, comparison between group means was tested by Fisher's protected least significant difference (PLSD) tests for post hoc analysis. Simple linear or polynomial regression analysis was performed to test the relationship between a histomorphometric parameter of bone turnover (activation frequency, Acf) with microdamage parameters and with biomechanical data. The relationship between microdamage parameters and toughness also was tested in the same manner. The significance of fits was determined using ANOVA. A p value of less than 0.05 was considered significant.

RESULTS

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

General condition

One animal in the RIS group was excluded because of distemper at the beginning of treatment. The other 35 animals remained healthy and were used for the analysis. They had normal body weight gains and there were no significant differences among groups either at the beginning or at the end of the treatment (data are not shown.). There were no treatment-related physical signs.

Radiography

No fractures were observed in any group.

Bone histomorphometry

Total area (TtAr) of the right ninth rib was significantly higher in both RIS and ALN than in CNT (p < 0.05; Table 2). No significant differences were observed among groups in either medullary area (MeAr) or CtAr.

Intracortical remodeling was suppressed by bisphosphonate treatment (Table 2). Acf was 53% less in RIS (p < 0.001) and 68% less in ALN (p < 0.0001) than in CNT. Bone formation rate (BFR/BV) was 72% less in ALN (p < 0.001) and 32% less in RIS (p = NS) than in CNT. These changes in bone turnover are reflected in the significantly fewer resorbing sites (resorption cavity number [RsN]/CtAr) in both RIS and ALN when compared with CNT (p < 0.05), and in the smaller amount of osteoid (OAr/CtAr; p < 0.05 and 0.01 for RIS and ALN respectively). There was no evidence that these bisphohsphonates inhibited mineralization of bone. Osteoid thickness was lower in both RIS and ALN than in CNT, although this only reached statistical significance for ALN (p < 0.05). Osteoid maturation time was well within normal ranges for all groups, and no significant differences were detected among groups. There were no significant differences between RIS and ALN for any of the histomorphometric variables.

Table Table 2.. Structural and Intracortical Remodeling Indices in Right Ninth Rib (Means ± SEM)
 Group
ParametersCNTRISALN
  1. * P < 0.05 versus CNT.

  2. P < 0.01 versus CNT.

  3. P < 0.001 versus CNT.

  4. §P < 0.0001 versus CNT.

TtAr (mm2)12.01 ± 0.6213.90 ± 0.33*14.02 ± 0.36*
MeAr (mm2)4.64 ± 0.505.54 ± 0.355.67 ± 0.66
CtAr (mm2)7.37 ± 0.298.36 ± 0.278.30 ± 0.45
RsN/CtAr (#/mm2)0.750 ± 0.1370.372 ± 0.069*0.420 ± 0.078*
OAr/CtAr (μm2/mm2)1278.8 ± 352.5421.3 ± 139.7*195.4 ± 70.0
OTh (μm)4.037 ± 0.3482.826 ± 0.3862.230 ± 0.533*
Omt (day)6.835 ± 0.7905.055 ± 0.7794.003 ± 1.328
LOnN/CtAr (#/mm2)3.878 ± 0.3892.049 ± 0.5841.375 ± 0.240
MAR (μm/day)1.104 ± 0.0451.086 ± 0.1030.926 ± 0.101
WTh (μm)50.218 ± 1.92048.455 ± 3.25953.309 ± 1.921
FP (day)48.252 ± 1.65747.752 ± 3.52951.705 ± 7.068
Acf (#/mm2 per year)29.397 ± 2.96713.697 ± 3.7839.436 ± 2.089§
BFR/BV (%/year)19.745 ± 3.86014.369 ± 4.0945.521 ± 1.206

On the periosteal surface, there were no significant differences among groups in any parameter (Table 3). On the endosteal surface, mineralizing surface (MS/BS) and bone formation rate (BFR/BS) were significantly less in both RIS and ALN compared with CNT (p < 0.01). Mineral apposition rate was unchanged by the treatments.

Microcracks were observed more frequently in the bisphosphonate-treated animals than in CNT animals (Fig. 2). Cracks were significantly longer in RIS (p < 0.01) and ALN (p < 0.01) than in CNT. CrDn was 155% greater in RIS (NS) and 322% greater in ALN (p < 0.01) than in CNT. The total microdamage burden, which can be estimated by the crack surface density (CrSDn) of all cracks, was five to seven times greater in RIS (p < 0.05) and ALN (p < 0.01) than in CNT. There was a significant association (r2 = 0.35; p < 0.01) between reduced Acf with more microcracks (Table 4).

Biomechanical tests

The ultimate force, stiffness, and work to failure in the left ninth rib did not differ significantly among the three groups (Table 5). When these data were normalized by cross-sectional moment of inertia, the intrinsic material properties, such as ultimate stress, elastic modulus, or toughness, tended to decline in both bisphosphonate-treated groups although most of them did not achieve statistical significance. However, toughness was significantly lower in ALN than in CNT (p < 0.05). There were no differences between RIS and ALN for any mechanical parameter.

A weak but significant association was observed between CrDn or CrSDn and tissue toughness (r2 = 0.13; p < 0.05; Table 4). Elastic modulus (r2 = 0.15;p < 0.02) and toughness (r2 = 0.31; p < 0.001) decreased significantly with suppressed Acf but ultimate stress did not change significantly with reduced Acf.

DISCUSSION

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

Bisphosphonates are compounds that significantly reduce the activation frequency for bone remodeling. Histologically, bisphosphonates reduce resorption depth and reduce activation frequency.(17–20) Depending on dosage, activation frequency with the newer generation bisphosphonates can be reduced as much as 93%, but the effect can be titrated by lower doses and shorter treatment periods.(20–23) Although earlier bisphosphonates such as etidronate inhibited mineralization of new bone and led to spontaneous fractures of ribs and spinous processes in animal models, the newer more potent bisphosphonates have an antiresorptive effect at lower doses without preventing normal mineralization.(15,20,22–26) In this study, we evaluated the effects of suppressed bone turnover on microdamage accumulation and mechanical properties in the rib of dogs. We used RIS or ALN only as agents to suppress bone turnover without impairment of mineralization and not to evaluate their safety or efficacy, both of which have been shown previously. As expected, neither RIS nor ALN caused osteoid accumulation in the cortical bone of the ninth rib of the beagle dogs used in this study, even at doses much higher than the projected clinical dose.

After 12 months of treatment no spontaneous fractures were observed in any dog. Our data showed that intracortical remodeling in the rib was suppressed by 53% with RIS (p < 0.001) and by 68% with ALN (p < 0.0001). This suppression of remodeling was associated with an increased microdamage burden (CrSDn) of 5- to 7-fold. Although there were no significant differences in extrinsic bone strength, such as ultimate force, stiffness, or work to failure, with either treatment, one of the intrinsic material properties, tissue toughness, significantly decreased by about 20% in the dogs with higher levels of remodeling suppression (p < 0.05). No significant reduction was found after RIS treatment. The differential effects on mechanical properties between RIS and ALN are attributed to the dosage and amount of remodeling suppression that was caused by each, rather than to any innate difference in their activity.

Table Table 3.. Dynamic Parameters of Cortical Surfaces in Right Ninth Rib (Means ± SEM)
  Group
 ParametersCNTRISALN
  1. * P < 0.01 versus CNT.

  2. P < 0.001 versus CNT.

PeriostealMS/BS (%)15.879 ± 4.18712.757 ± 3.56711.908 ± 2.109
 MAR (μm/day)0.449 ± 0.1620.454 ± 0.1070.467 ± 0.095
 BFR/BS (μm3/μm2 per day)10.559 ± 7.2207.801 ± 2.7546.711 ± 1.501
EndostealMS/BS (%)17.300 ± 3.5543.860 ± 1.0113.815 ± 0.969
 MAR (μm/day)0.695 ± 0.1160.320 ± 0.0980.451 ± 0.139
 BFR/BS (μm3/μm2 per day)15.007 ± 3.7831.923 ± 0.8313.165 ± 1.418*
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Figure FIG. 2.. Microcrack measurement of cortical bone in right ninth rib. (A) Mean crack length was significantly higher in both RIS and ALN than in CNT. (B) Microcrack density was significantly higher in ALN than in CNT. (C) Microcarack surface density was significantly higher in both RIS and ALN than in CNT. Data are expressed as mean ± SEM. *p < 0.5 and **p < 0.01 compared with CNT.

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Table Table 4.. Regression Analysis of Acf and Microdamage Parameters and Normalized Biomechanical Properties in Dog Ninth Rib
XYEquationR2P value
AcfCrLeY = 47.50 — 0.65 · X0.170.05 (NS)
AcfCrDnY = 0.62 — 0.049 · X + 0.002 · X2 – 0.00018 · X30.35<0.01
AcfCrSDnY = 24.7 — 1.59 · X + 0.039 · X2 – 0.0036 · X30.36<0.01
AcfUltimate stressY = 21.39 + 0.088 · X0.10.06 (NS)
AcfElastic modulusY = 8630.92 + 63.24 · X0.15<0.05
AcfToughnessY = 23.27 + 0.26 · X0.31<0.001
CrLeToughnessY = 31.02 — 0.09 · X0.060.27 (NS)
CrDnToughnessY = 30.21 — 9.46 · X0.13<0.05
CrSDnToughnessY = 30.05 — 0.21 · X0.13<0.05

The extrinsic bone strength depends on both geometry and intrinsic material properties. However, in this study extrinsic bone strength was not changed by the bisphosphonate treatment even though TtAr was significantly increased, suggesting a reduction of intrinsic material properties in the ribs of dogs. Work to failure, an extrinsic structural property, is a measure of the amount of energy a bone specimen can absorb before it breaks. The smaller the work to failure, the less energy required to cause fracture; thus the bone is more prone to fracture. Toughness is normalized work to failure, and it quantified the amount of energy the tissue can absorb per cubic millimeter before failure independent of bone size or shape. The combination of a nonsignificant decrease in work to failure and increased TtAr may be responsible for the significant reduction of toughness in ALN-treated animals.

After bisphosphonate treatment TtAr of the bone was increased (p < 0.05), although cortical area was unchanged, indicating expansion of the periosteal surface and resorption on the endosteal surface of the rib. However, we were not able to detect a significant change in periosteal MS or BFR. There are two possible explanations for the apparent inconsistency between unchanged MS but greater area. First, modeling drifts, defined as an uncoupled sequence with bone formation on some surfaces and bone resorption on other surfaces occur in dog ribs. There was evidence for this in CNT dogs, in which some periosteal surfaces were undergoing resorption, while others were forming. It is possible that bisphosphonates suppress only bone resorption on the periosteal surface without affecting direct apposition of bone to the periosteal surface via modeling processes. A second possible explanation is that new bone was added to the periosteal surface in the early phases of treatment, but that after 12 months of treatment (at the time the fluorochrome labels were given) new bone formation had returned to baseline values.

Table Table 5.. Biomechanical Test of Left Ninth Rib (Means ± SEM)
 Group
ParametersCNTRISALN
  1. * P < 0.05 versus CNT.

Ultimate force (N)184.4 ± 10.5196.6 ± 6.5190.6 ± 11.3
Stiffness (N/mm)278.0 ± 22.6285.0 ± 15.4287.9 ± 19.1
Work to failure (Nmm)693.4 ± 63.7680.9 ± 35.0611.9 ± 44.7
Ultimate stress (MPa)24.56 ± 1.4422.28 ± 0.7421.95 ± 0.71
Elastic modulus (MPa)10,643 ± 7269262 ± 6979287 ± 391
Toughness (MJ/m3)31.16 ± 2.2527.21 ± 1.2925.16 ± 1.32*

Data from this study suggest that suppression of bone turnover over a continuous 12-month period can allow microdamage to accumulate and can lead to increased skeletal fragility but not to a reduction in bone strength or stiffness. It will be important to determine the result of microdamage accumulation caused by suppressed remodeling over a longer time period. Osteoporotic women generally will be treated for longer than 1 year. The balance between microdamage accumulation and repair and the effect of this balance on mechanical properties over these longer time periods is still unclear.

Fracture risk comparisons after 3 years of ALN or RIS treatment show a significant reduction in fracture risk in the vertebrae, hip, and forearm.(9–11,13,14) Four years of ALN treatment significantly decreased vertebral fracture risk for women with low bone mineral density.(12) However, 3 years of ALN treatment does not seem to prevent the rib fractures. Data from the study reported by Karpf et al. showed that the fracture prevention effect of ALN was less in the rib than at any other site examined.(11) Thus, physiological effects of these bisphosphonates differ by site of evaluation.

In human clinical trials, ALN at 10 mg/day has been shown to reduce activation frequency by 88% after 2 years and by 93% after 3 years.(20) These data were obtained from the iliac crest, a cancellous bone site without particular clinical relevance. However, our data show that if remodeling was suppressed to this degree at more clinically relevant sites, this suppression could cause the accumulation of microdamage and reduce tissue toughness.

The microdamage burden in the rib is higher than that at other skeletal sites.(21) The reasons for this are not entirely clear, but some speculate that the frequent occurrence of microdamage in the ribs might be a result of the frequency with which they are loaded on a daily basis. Therefore, the relationship between bone remodeling suppression and the rate at which microdamage accumulates is probably location dependent. Data from other regions, such as the vertebrae, ilium, femoral neck, or thoracic spinous processes, will provide important regional information about the relationship of activation frequency, microdamage accumulation, and the mechanical properties of these bones.

Our results are consistent with the findings reported by Norman et al. in which microdamage parameters were measured from the human femoral shaft and correlated with fracture toughness tested in the same bone.(27) Their results showed a significant inverse relationship between fracture toughness and microdamage parameters for tension loading of the femur. Because toughness is a measure of how much energy the bone tissue can absorb before fracture, this suggests that a significant accumulation of microdamage potentially will hasten the onset of fracture.

One question is how much bone remodeling can be suppressed without causing a significant reduction of mechanical properties secondary to the accumulation of microdamage. An earlier study evaluated the accumulation of bone microdamage in the femoral necks of dogs treated for 2 years with doses of RIS that were between 5 and 20 times the anticipated clinical dose of 0.1 mg/kg per day.(21) Activation of new bone remodeling was reduced by 80% in both cortical (p < 0.05) and trabecular (p < 0.005) bone of the femoral neck. Although cortical and trabecular bone areas both increased with treatment (p < 0.05), no statistical differences were observed among groups for CrDn or CrLe, suggesting that bone microdamage does not accumulate in the femoral neck after treatment with high doses of RIS. However, in the rib our results showed that 53% suppression of remodeling did not reduce bone mechanical properties significantly even though microdamage accumulated, whereas 68% suppression led to reduced bone tissue energy absorbing capability.

Based on the histomorphometric and biomechanical evaluation of the ninth rib in dogs treated with RIS or ALN for 12 months, we conclude that intracortical bone remodeling can be suppressed by bisphosphonates without deficit of bone mineralization, suppression of bone remodeling to these levels is associated with accumulation of microdamage, and bone toughness of the rib may decline significantly with suppression of remodeling.(1–3) The hypothesis that suppressed bone turnover can lead to reduction in bone mechanical properties by microdamage accumulation is supported if the suppression of remodeling is great enough.

Acknowledgements

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

The authors thank Mary Hooser, Diana Jacob, and Thurman Alvey for histological preparation and Kunihiko Tokunaga, M.D., for surgical assistance. This work was supported by an NIH grant 2 PO1 AG05793. Merck and Co., Inc., and Procter and Gamble Pharmaceuticals, Inc., kindly supplied the bisphosphonates.

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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