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Effects of long-term suppression of bone remodeling by bisphosphonate were investigated in cortical bone of dog rib. Although microdamage was accumulated, BMD was increased without increasing cortical bone area. Consequently, the intrinsic material properties were not reduced.
Introduction: Recently, we have reported that long-term suppression of bone remodeling increases microdamage accumulation but is not necessarily associated with vertebral fragility because of compensated increase of bone mass and improved microarchitecture. This study aimed to investigate the effect of long-term suppression of bone remodeling by bisphosphonate on the degree of mineralization, accumulation of microdamage, and mechanical properties of cortical bone in the same dogs.
Materials and Methods: Twenty-nine 1-year-old beagles (15 males, 14 females) were divided into three groups and treated daily with vehicle (CNT) or with incadronate at a dose of 0.3 (LOW) or 0.6 mg/kg/day (HIGH) orally for 3 years. After death, pQCT, histomorphometry, microdamage measurements, and three-point bending mechanical test were performed using the ninth rib.
Results: Cortical BMD was increased in the incadronate-treated groups. Cortical activation frequency was suppressed by 82% and 70% in HIGH and LOW, respectively, compared with CNT, without impairment of mineralization. Microdamage accumulation was increased in both incadronate-treated groups. Although there were no significant differences in total and cortical area among the three groups, structural mechanical properties were significantly increased after incadronate treatment while intrinsic material properties were not changed in the incadronate-treated groups.
Conclusion: This study suggests that long-term suppression of bone remodeling by bisphosphonate increases microdamage accumulation. However, this was not necessarily associated with a reduction of intrinsic material properties probably because of an increased degree of mineralization.
BISPHOSPHONATES ARE POTENT inhibitors of bone resorption and have been used as effective therapeutic agents for a variety of bone diseases associated with high bone resorption. In osteoporotic patients, many studies had demonstrated that bisphosphonates increase bone mass and decrease fracture risk.(1–7) On the other hand, several investigators reported that the main reason for decreased fracture incidence in bisphosphonate-treated osteoporotic women might be associated with an increased degree of mineralization rather than increased bone mass.(8,9) Because the bisphosphonate suppresses bone turnover, long-term bisphosphonate treatment elongates the lifespan of bone structure units (BSUs) and prolongs the duration of secondary mineralization.(8)
Previous studies have postulated that accumulation of microdamage in bone underlies the development of stress fractures and plays a role in bone fragility associated with aging and osteoporosis.(10) Microdamage burden in bone is a function of the amount of damage produced and the amount repaired. Therefore, either increased production of microdamage or suppressed repair can induce microdamage accumulation in bone.(11,12) Microdamage is presumably repaired by physiologic bone remodeling. Suppressed bone remodeling caused by bisphosphonate may accumulate microdamage and consequently decrease the biomechanical properties of bone. Previously, Mashiba et al.(13,14) demonstrated that suppression of bone remodeling by 1-year treatment with risedronate or alendronate increased microdamage accumulation and decreased some intrinsic mechanical properties in dog rib and lumbar vertebra. Osteoporotic patients are usually treated with therapeutic agents for longer periods. Because of the time-dependent accumulation and continuous action, it is more important to determine the longer-term effects of bisphosphonate on bone. Recently, we have reported that suppression of bone remodeling by 3-year incadronate treatment increases microdamage accumulation in the vertebral trabecular bone, but not necessarily associated with vertebral fragility because of compensated increase of bone mass and improved microarchitecture.(15)
In this study, using the same animals as in the previous report, we evaluated the effects of long-term suppression of bone remodeling by 3-year bisphosphonate treatment on densitometry, microdamage accumulation, and mechanical properties in the cortical bone of dog rib. Incadronate has almost the same in vitro antiresorptive effect as alendronate(16) and has been shown to have beneficial effects in various animal models of osteoporosis induced by ovariectomy and immobilization.(17–19) Because the measurement of incadronate concentration in bone has already been established,(20) the relationship between concentration of incadronate in bone and histomorphometric parameters may provide important information about the dose-dependent effects of this agent on bone.
MATERIALS AND METHODS
Twenty-nine beagles (15 males and 14 females; Marshall Farms, N Rose, New York, USA), 73-77 weeks of age, were used in this experiment. They weighed 7.3-13.0 kg at the start of the study, and closure of the vertebral growth plate was confirmed by X-ray. The animals were acclimated for at least 1 month and housed individually in environmentally controlled rooms. A standard dry diet (Diet A: Special Diet Services, Withman, Essex, UK) was given to each animal at 40 g/day. Water was supplied ad libitum.
The dogs were randomly divided into three groups based on body weight. Dogs in the control group (CNT; 5 males and 5 females) were given lactose at 12 mg/kg/day. The remaining two groups were treated daily with incadronate disodium (YM-175; Yamanouchi Pharmaceutical Co., Tokyo, Japan) at a dose of 0.3 (LOW; 5 males and 5 females) or 0.6 mg/kg/day (HIGH; 5 males and 4 females). These doses are 2.5 or 5 times higher, respectively, than the proposed clinical dose for osteoporotic patients. Incadronate was packed in gelatin capsules (prefit gelatin capsules size 000; Parke-Davis, Morris Plains, NJ, USA) and administrated orally. Drug was administered daily 4 h before feeding in the morning. All animals were treated for 3 years. All dogs were double-labeled with 20 mg/kg oxytetracycline hydrochloride (Pfizer, Tokyo, Japan) by intravenous injection 18 and 7 days before death. At the completion of drug administration, the dogs were killed with an overdose of sodium pentobarbital derivative (Abbott Laboratories, North Chicago, IL, USA), and the left eighth rib and bilateral ninth ribs were dissected and cleared of soft tissue. From the right ninth rib, a 3-cm sample taken 2 cm proximal to the osteochondral junction was frozen at −80°C until pQCT measurement, and the adjacent 4-cm sample was fixed for 3 days in cold 10% neutral buffered formalin for basic histology or bulk staining with basic fuchsin for microdamage analysis. The latter sample was divided at the midpoint; the proximal specimen was assigned for microdamage measurement and the distal specimen for cortical histomorphometry. From the left ninth rib, a 4-cm sample taken from the great curvature was wrapped in gauze soaked in isotonic saline and frozen at −20°C until mechanical test. From the left eighth rib, the proximal 6-cm portion from the osteochondral junction was frozen at −20°C until measurement of incadronate concentration in bone.
pQCT and data acquisition
After thawing at room temperature, each specimen was scanned by pQCT (Norland/Stratec XCT Research SA; Stratec Medizintechnic GmbH, Pforzheim, Germany). The bones were placed horizontally inside a glass tube and scanned using a voxel size of 0.12 mm. The scan line was adjusted using the scout view of the pQCT system. For analysis, a threshold 169 mg/cm3 at contour mode 2 was used to separate the bone area from the marrow regions. To separate the cortical area from trabecular area, we used a constant threshold of 690 mg/cm3. The total BMC (Tt.BMC, mg/mm), volumetric total BMD (Tt.BMD, mg/cm3), cortical BMC (Ct. BMC, mg/mm), and volumetric cortical BMD (Ct.BMD; mg/cm3) were calculated.
The specimen for cortical histomorphometry was fixed in 70% ethanol, stained with Villanueva bone stain (Maruto Ins. Co., Tokyo, Japan), dehydrated in increasing series of ethanol, defatted in xylene, and embedded undecalcified in methyl methacrylate (Wako Pure Chemicals, Osaka, Japan). For each specimen, a 150-μm-thick section was cut using a band saw (Exakt; Otto Herrmann Co., Nordestedt, Germany) at 1 mm distal to the divided surface and ground to 80-μm thickness for cortical histomorphometry. The specimen for microdamage measurement was fixed and stained en block with 1% basic fuchsin in 70% ethanol, dehydrated in increasing series of ethanol, defatted in xylene, and embedded undecalcified in methyl methacrylate.(21,22) A 150-μm-thick section was cut at 1 mm proximal to the divided surface and further ground to 80-μm thickness for microdamage analysis.
Histomorphometric measurement was performed using a semiautomated digitizing image analyzer; consisting of a light or epifluorescent microscope and a digitizing pad connected to a computer installed with a histomorphometric software (System Supply Co., Nagano, Japan).
Histomorphometric measurements were performed for the whole cross-sectional area of the specimen at a magnification of ×100. Surface measurements were also made on periosteal and endocortical bone envelopes. We used the nomenclature, symbols, and units described in the Report of the American Society of Bone and Mineral Research Committee.(23) Some derived parameters measured or calculated for cortical bone have been described by Mashiba et al.(14): total area (Tt.Ar, mm2), medullary area (Me.Ar, mm2), cortical area (Ct.Ar, mm2), resorption cavity number (Rs.N/Ct.Ar, #/mm2), labeled osteon number (L.On.N/Ct.Ar, #/mm2), mineral apposition rate (MAR, μm/day), osteoid thickness (O.Th, μm), osteoid maturation time (Omt, day), mean wall thickness (W.Th, μm), formation period (FP, day), activation frequency (Ac.f, #/ mm2/year), and bone formation rate (BFR/BV, %/year). For cortical surface measurement, we calculated mineralizing surface (MS/BS, %), MAR (μm/day), BFR/BS (mm3/mm2/year) as Foldes et al.(24) reported. All measurements were carried out in a blind manner by one histomorphometrist.
Microdamage measurement was performed for the whole cross-sectional area at a magnification of ×200. A stained microcrack was defined as a typical crack morphology, with a certain depth of field and a halo of increased basic fuchsin staining surrounding it (Fig. 1).(13,25–27) Measurements included crack density (Cr.Dn = Cr.N/B.Ar; #/mm2), mean crack length (Cr.Le; μm), and crack surface density (Cr.S.Dn = Cr.N × Cr.Le/B.Ar; μm/mm2) in cortical bone.
After thawing at room temperature, all samples were tested by the three-point bending method using a mechanical testing machine (Model TK-252C; Muromachi Kikai Co., Tokyo, Japan). The specimen was placed on the two lower support bars (20 mm apart) with the convex side facing toward the loading bar. The loading bar was positioned at the midpoint of the specimen. The load was applied at a strain rate of 0.5 mm/minute until break. Ultimate load, stiffness, and work to ultimate load were automatically determined from the load-displacement curve by a connected computer.
As previously described, the load-displacement data were normalized to obtain intrinsic material properties such as ultimate stress, elastic modulus, and modified-toughness, which are independent of cross-sectional size and shape.(28) The formulae for calculation are as follows:
where L is the length between supports, b is the width of the rib in ventro-dorsal direction, and I is the cross-sectional moment of inertia. Elastic modulus was calculated as
Modified-toughness was calculated from work to ultimate load (W) using
Cross-sectional moment of inertia (I) was calculated using the assumption that the rib cross-section was elliptically shaped:
where a is the width of the cross-section in the cranio-caudal direction, and t is the average cortical thickness calculated from the histomorphometric thickness measured in each of four quadrants of the right ninth rib cross-section using a semiautomatic digitizing system.
Incadronate concentration in bone
After thawing at room temperature, incadronate concentration in the eighth rib was measured using high performance liquid chromatography (HPLC) following the procedures described previously.(20) Briefly, bone specimens were digested in HCl at 50°C, and 10 M NaOH was added to the digest to obtain a transparent liquid phase. A 2-ml aliquot of the liquid phase was spiked with 50 μl of the internal standard (IS; cyclo-octylaminomethylene-bisphosphonic acid) for incadronate. The mixture was filtered and applied to a Sep-Pak C18 cartridge. The contents were eluted with 6.5 ml of 0.01 M NaOH. The first 0.5-ml fraction was discarded. The next 6 ml was spiked with 0.25 ml calcium phosphate and 0.4 ml of 10 M NaOH. After centrifugation, the precipitate was washed using water and acetonitrile and dissolved in 0.2 ml of 0.2 M orthophosphoric acid. To remove excess calcium ions in the sample, 0.1 ml of AG 50 W-X8 resin (BioRad, Richmond, CA, USA) was added. After centrifugation, the supernatant was filtered and alkalinized with 30 μl of 2 M NaOH. A standard solution of incadronate was used to construct a calibration curve by plotting the peak-height ratios of incadronate to IS versus concentrations of incadronate. Then, an 80-μl aliquot of the sample was injected into the HPLC column for measurement, and the concentration of incadronate was obtained from the calibration curve.
Statistical computation of data was performed using the statistical package Stat View 5.0 (SAS Institute, Cary, NC, USA). Although each group consisted of male and female animals, they were pooled for analysis because there were no significant differences between male and female in all evaluated data. Differences among treatment groups were tested by one-way ANOVA. If significant differences were indicated, differences between pairs of group means were tested by Fisher's protected least significant difference (PLSD). Simple linear regression analyses were performed to test the relationship between histomorphometric data for bone turnover, activation frequency (Ac.f) and microdamage accumulation, incadronate concentration in bone, or BMD. Goodness of fit and statistical significance of fit were determined by the r2 value and ANOVA, respectively. A p value <0.05 was considered significant.
Tt.BMC in HIGH was 30.0% and 15.7% higher compared with CNT (p < 0.05) and LOW (p < 0.05), respectively (Table 1). The volumetric Tt.BMD in HIGH was 15.8% and 8.5% higher compared with CNT (p < 0.001) and LOW (p < 0.05), respectively. Cortical bone mineral parameter was increased after high-dose incadronate treatment. The cortical BMC (Ct.BMC) in HIGH was 35.4% and 20.5% higher compared with CNT (p < 0.05) and LOW (p < 0.05), respectively. Similarly, the volumetric cortical BMD was higher in HIGH than in CNT and LOW (p < 0.001 and p < 0.05, respectively). No significant differences in these parameters were observed between CNT and LOW.
Table Table 1.. pQCT Indices in Right Ninth Rib of Beagle
No significant differences in Tt.Ar, Me.Ar, and Ct.Ar were observed among the groups (Table 2).
Table Table 2.. Structural and Intracortical Remodeling Indices in Right Ninth Rib of Beagle
Intracortical remodeling was suppressed dose-dependently by incadronate. Ac.f was 40% lower in LOW (not significant [NS]) and 82% lower in HIGH (p < 0.01) than in CNT. This dose-dependent suppression was also reflected in the lower values for L.On.N/Ct.Ar, MAR, FP, and BFR/BV, although the difference of these parameters between CNT and LOW did not reach statistical significance.
On the periosteal surface, there were no significant differences among groups in all the dynamic parameters (Table 3). In contrast, on the endosteal surface, MS/BS, MAR, and BFR/BS were significantly lower in both LOW and HIGH compared with CNT.
Table Table 3.. Dynamic Parameters of Cortical Surfaces in Right Ninth Rib of Beagles
The mean crack length (Cr.Le) was 29% higher in LOW (p < 0.05) and 62% higher in HIGH (p < 0.0001) compared with CNT and 25% higher in HIGH than in LOW (p < 0.05; Table 4). Cr.Dn was 1.6-fold greater in LOW (p < 0.01) and 1.9-fold greater in HIGH (p < 0.0001) compared with CNT. Cr.S.Dn was 2.1-fold greater in LOW (p < 0.01) and 3.0-fold greater in HIGH (p < 0.0001) compared with CNT. There was a simple linear relationship between Ac.f and Cr.Dn (r2 = 0.46, p < 0.0001; Table 5).
Table Table 4.. Microdamage Measurement of Cortical Bone in Right Ninth Rib of Beagles
Table Table 5.. Regression Analysis of Activation Frequency in Bone vs. Microdamage Parameter, Incadronate Concentration, and BMD in Canine Ninth Rib
Incadronate treatment increased some structural properties dose-dependently. Ultimate load was 24% higher in LOW (p < 0.05) and 46% higher in HIGH (p < 0.001) compared with CNT (Table 6). Stiffness was 33% higher in LOW (p < 0.05) and 39% higher in HIGH (p < 0.001) than in CNT. No significant difference in CSMI was observed (Table 2). No significant differences in intrinsic material properties (ultimate stress, elastic modulus, and Modified-toughness) were observed among the three groups.
Table Table 6.. Mechanical Test of Left Ninth Rib of Beagle
Incadronate concentration in bone
Incadronate accumulation in the cortical bone increased dose-dependently. Incadronate concentration in the eighth rib was significantly higher in HIGH (10.6 ± 1.0 μg/g) than in LOW (5.9 ± 0.8 μg/g; p < 0.0001). There was a significant linear relationship between increased incadronate concentration in bone and reduced Ac.f (Table 5). Also, there was a significant linear relationship between reduced Ac.f and increased BMD (Table 5).
Recently several studies have focused on the relationship between bone remodeling and microdamage accumulation or mechanical properties of bone.(12–14) They have demonstrated that suppressed bone remodeling caused by bisphosphonate increased microdamage accumulation through inhibiting damage repair and decreased some mechanical properties of bone. As for the cortical bone of dog rib, Mashiba et al.(14) reported that 1-year treatment with risedronate or alendronate suppressed intracortical bone remodeling by 57-68%, increased microdamage accumulation by 2- to 3-fold, and decreased toughness by 20%. In our study, 82% suppression of intracortical remodeling after 3-year incadronate treatment was also associated with 1.6- to 1.9-fold increase of damage accumulation. These two studies clearly indicate that prolonged suppression of bone remodeling by bisphosphonate allows microdamage to accumulate in bone.
Compared with the previous study reported by Mashiba et al.,(14) an important observation was found regarding the relationship between Ac.f and microdamage accumulation. Ac.f in the control group of our study was lower than that in the alendronate-treated group that exhibited the greatest suppression of intracortical remodeling in Mashiba et al.(14) study, suggesting that an age increase of 2 years in beagles (killed at 4 years of age in our study and at 2 years of age in Mashiba et al.(14) study) has a greater effect than 1-year alendronate treatment in reducing intracortical bone remodeling. This was reflected in greater accumulation of microdamage in our control group than in their alendronate-treated group. Thus, if bone remodeling was suppressed by either aging or bisphosphonate, microdamage accumulation increased in response to the extent of remodeling suppression.
The bone density can increase with no actual changes in the bone structure or mass if the mineralization density increases.(29) Using pQCT in this study, we found that Tt.BMC, Ct.BMC, Tt.BMD, and Ct.BMD were increased in the high-dose group. There were no differences among the three groups in histomorphometric structural parameters of cortical bone, such as Tt.Ar, Ct.Ar, or Me.Ar. These data may suggest that 3-year treatment of incadronate increased the degree of mineralization rather than cortical bone mass. Because intracortical bone turnover was suppressed dose-dependently after 3-year treatment of incadronate, severe suppression of osteonal bone turnover could prolong the duration of secondary mineralization and increase the degree of mineralization in the cortical bone.
Mashiba et al.(14) reported that 1-year treatment of alendronate in 1-year-old beagles increased microdamage accumulation and reduced toughness, intrinsic energy-absorbing capacity of cortical bone in the ninth rib. In this study, however, there was no decrease in intrinsic material properties in the incadronate-treated groups, although microdamage accumulation was significantly increased. Although it is difficult to directly compare the results of these two studies because of the differences in agent, duration of treatment, and animal age, there are several possible explanations for this difference. First, in this study, we derived energy as work to ultimate load instead of work to failure. Therefore, the mechanical phase after yielding point until failure of the specimen was not included in our analysis, which may cause the considerable differences especially for the energy absorption parameters. Second, there were also some differences in the conditions of the mechanical tests, such as loading rate and unsupported length. This may also cause the different results of these two studies. Finally, the microdamage accumulation was higher in controls of this study compared with the Mashiba et al.(14) study, indicating an already negative effect on some intrinsic material properties in control animals of our study. This may also explain the failure to find the difference among groups in toughness.
Because of its high affinity to bone mineral, bisphosphonates deposit rapidly in bone, both in areas of bone formation and resorption, and skeletal retention of bisphosphonates lasts a long period of time.(30,31) In this study, the concentration of incadronate in the eighth rib was increased in a dose-dependent manner. In our previous study, 3-year treatment of incadronate increased the incadronate concentration in the 11th thoracic vertebra, with a significant linear relationship between reduced Ac.f and increased incadronate concentration in the 11th thoracic vertebra.(15) In this study, as shown in Table 5, there was a significant linear relationship between reduced Ac.f and increased microdamage accumulation. Also, there were significant linear relationships between reduced Ac.f and incadronate concentration and between reduced Ac.f and total BMD or cortical BMD. Although these regression analyses, pooling different dosing groups, may not be the best method and have limitations, these results suggested that bisphosphonate in bone reduced Ac.f, followed by increased BMD and accumulation of microdamage.
In this study, evaluating cortical bone, there was no significant difference between LOW and CNT in BMD, structural, or most remodeling parameters. However, in our previous study, evaluating trabecular bone using the same animals, we reported that even the lower-dose treatment with incadronate significantly suppressed bone remodeling and increased bone mass.(15) This difference may be caused by the difference of responsiveness to the agent between trabecular and cortical bone. Less surface-to-volume ratio in the cortical bone compared with trabecular bone may be one possible explanation.
Based on pQCT, histomorphometric, and mechanical evaluations of rib cortical bone in dogs treated for 3 years with incadronate disodium at a dose 2.5 or 5 times higher than the proposed human clinical dose, we conclude that suppression of intracortical remodeling increases microdamage accumulation but also increases the bending strength, probably because of an increased degree of mineralization.
The authors thank Mika Kawada and Yoshiko Fukuda for histological preparation and Yamanouchi Pharmaceuticals Co. for kindly supplying the bisphosphonate.