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Vol. 280, Issue 2, 1051-1056, 1997
Biomedical Research Center, Leiras Oy, FIN-20101 Turku, Finland
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Abstract |
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 Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Distribution of clodronate in cancellous and cortical bone of the femur and in cancellous bone of lumbar vertebrae in adult rats was examined by means of quantitative autoradiography. In addition, the effects of clodronate on cancellous and cortical bone were evaluated by bone histomorphometry. Six-month-old male rats were given a mixture of unlabeled and 14C-labeled disodium clodronate subcutaneously on 5 consecutive days at cumulative doses of 125 mg/50 µCi/kg or 250 mg/100 µCi/kg and followed up for 2, 23 or 79 days after the last dose. The highest activity of 14C-clodronate was found in the primary spongiosa of the distal femoral metaphysis and in the cortical bone of the femoral diaphysis. Radioactivity in the lumbar vertebra was found to be about half of that in the femur. No marked decrease in radioactivity was found in bone specimens taken after the follow-ups. In these specimens, however, labeled clodronate originally incorporated into the primary spongiosa was situated further away from the growth plate because of longitudinal bone growth. A cross-section of the femoral shaft showed that incorporation of clodronate was more prominent into the periosteal surface than into the endocortical surface. No marked histological effects were seen, except for an increase in the mineralized hard tissue area in the primary spongiosa of the distal femur.
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Introduction |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Bisphosphonates inhibit bone
resorption and are thus beneficial in the treatment of high-turnover
bone disease such as Paget's disease and hypercalcemia of malignancy
(Kanis and McCloskey, 1990
). Because of the high affinity of these
compounds for hydroxyapatite (Jung et al., 1973), they are
rapidly cleared from the blood and incorporated into calcified tissues,
especially bone. The half-life of the skeletal retention of
bisphosphonates is long (Wingen and Schmähl, 1987;
Mönkkönen et al., 1990; Österman and
Laurén, 1991), depending on the turnover rate of the skeleton
itself.
Quantitative analysis of a radioactive drug in removed fragments of
bone is useful for evaluation of radioactivity in the bone as a whole
(Mönkkönen et al., 1990; Österman and
Laurén, 1991), but not for the study of distribution of the drug
in the specimen. In contrast, autoradiography, which is a
well-established radiotracing technique in biomedical research,
provides detailed information on the localization of a labeled
substance in the specimen. Recently, the potential of autoradiographs
as sources of information has increased considerably with the advent of
computer-assisted image analysis systems (Ramm et al., 1984;
d`Argy et al., 1990).
The effect of bisphosphonates on the skeleton generally appears to
depend on the dose and duration of treatment, as well as on the age and
species of experimental animals (Russell and Fleisch, 1975). The rat
skeleton, mainly the long bones and lumbar vertebrae, has been used as
a model of the human skeleton to study bone loss and its treatment
(Kalu, 1991; Frost and Jee, 1992; Österman et al.,
1994; Kippo et al., 1995). The development of rat femur and
lumbar vertebrae can be divided into two stages according to the age of
the animals (Sontag, 1992, 1994). In young growing rats, from birth to
an age of about 6 months, the bones grow rapidly in length and width,
and progressively more cancellous bone appears in the metaphysis. In
adult rats older than 6 months, the increase in width and length is
diminished and the epiphyseal growth plate becomes narrower.
Administration of clodronate to young growing rats dose-dependently
increases metaphyseal mineralized tissue mass of long bones (Schenk
et al., 1973; Miller and Jee, 1977; Shinoda et
al., 1983) as a result of a marked decrease in bone resorption
rate. In addition to age, the effects and distribution of bone-seeking
agents, such as bisphosphonates, may also depend on the site in the
skeleton, because bone turnover rate has been shown to vary between
different bones and between different parts of the same bone (Sontag,
1986, 1992, 1994).
The aim of this study was to examine by means of quantitative autoradiography the distribution of clodronate in cancellous and cortical bone of the femur and in cancellous bone of lumbar vertebrae in adult 6-month-old rats. In addition, the effects of clodronate on cancellous and cortical bone were evaluated by bone histomorphometry.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals. Forty-four male Sprague-Dawley rats (Han:SPRD, ZFV Hannover, Germany), about 6 months old at the start of the study and with a mean (S.E.M.) weight of 535 (7) g, were used. The animals were fed a standard pellet diet (RM3 E SQC, Special Diets Services Ltd, Witham, U.K.) and had free access to tap water during the study.
The rats (n = 4/group) were given a mixture of unlabeled (Bonefos, Leiras Oy, Turku, Finland) and 14C-labeled (specific activity, 5.15 µCi/mg, Leiras Oy) disodium clodronate subcutaneously at a dose of 25 mg/10 µCi/kg or 50 mg/20 µCi/kg on 5 consecutive days (cumulative doses, 125 mg/50 µCi/kg or 250 mg/100 µCi/kg, respectively). The rats were sacrificed after a follow-up of either 2, 23 or 79 days (i.e., 2, 23 or 79 days after the last dose). A physiological sodium chloride solution was used as a vehicle of 14C-labeled clodronate and also for the dilution of Bonefos infusion concentrate to adjust the injection volume to 1 ml/kg. The age-matched control rats, receiving 1 ml/kg of 0.9% NaCl, were sacrificed at the same times and used as controls for histomorphometric analysis. Double-fluorochrome labeling of bone was carried out with intraperitoneal injections of calcein (25 mg/kg) (Sigma Chemie GmbH, Deisenhofen, Germany) on the same day as the administration of clodronate began and 7 days before sacrifice of the animals. Labeling was used only in the groups sacrificed after a follow-up of 23 or 79 days.Histology.
The distal end of the right femur and the fourth
lumbar vertebral body were processed for autoradiography and
histomorphometry without decalcification (Schenk et al.,
1984), after ascertaining that no radioactivity was released during
fixation and polymerization. Longitudinal 5-µm-thick sections were
cut at the frontal plane with a Polycut S heavy-duty microtome
(Reichert-Jung, Leica Instruments GmbH, Germany). The sections were
stained after autoradiography by the von Kossa method (Schenk et
al., 1984) with toluidine blue counterstain (Baron et
al., 1983).
. Five-millimeter-high cylinders of the
femoral diaphysis were cut proximal to the middiaphyseal area so that
the lower part of the third trocanther was included in the bone
cylinder with use of a precision bone saw (Exakt-cutting grinding
system, Exakt Apparatebau, Norderstedt, Germany). The bone cylinders
were dehydrated in ethanol, infiltrated and embedded in photocuring
resin Technovit 7200 VLC (Heraeus Kulzer GmbH, Wehrheim, Germany), and
polymerized with blue light. Thin slices of about 200-µm thickness
were cut from the proximal end of the block and thinned to a final
thickness of about 20 µm with the Exakt microgrinding system. After
autoradiography and dynamic histomorphometric measurements, the
cross-sections were surface stained with toluidine blue (Baron et
al., 1983) without removing the plastic.
Macroautoradiography and densitometry. The unstained bone sections were placed in x-ray cassettes and apposed to Ektascan NMB1 film (Kodak, France). After 6-week exposure at 17°C, the films were developed in Kodak LX-24 developer, fixed in Kodak AL-4 fixer and dried.
Autoradiographs were quantified by densitometry with a digital image analyzer (MCID, model M1) with a Northern Light desktop illuminator (Imaging Research Inc., St. Catharines, Ontario, Canada). The MCID system digitizes a continuous range of image gray shades into 256 discrete gray levels, which are transformed to optical densities. The mean densities of the radioactivity zone in the primary spongiosa of the distal femoral metaphysis and of the fourth lumbar vertebral body were measured. The optical density units, obtained from autoradiographs, were converted into radioactivities (nanoCurie per gram) with reference to the carbon-14-labeled microscales (Amersham, Buckinghamshire, U.K.) coexposed with the radiolabeled sections. By aligning (MCID, densitometry program) the autoradiograph with the corresponding histological image, the dislocation of the activity zone was determined in the metaphysis of the distal femur and lumbar vertebra. The distance of the activity zone from the growth plate-epiphyseal junction was measured 2 days after discontinuation of drug administration and after follow-ups of 23 and 79 days. The radioactivity of the microground cross-sections of the femoral diaphysis was measured both at periosteal and endosteal surfaces. The aligning method was used to show the location of radioactivity in the bone.Histomorphometry. Static histomorphometric measurement of the distal femoral metaphysis and the lumbar vertebral body was carried out using the MCID digital image analyzer with M1 morphometry software. Measurement was carried out both on the primary and the secondary spongiosa at 4× objective magnification. In the primary spongiosa, an area that is situated within 1.1 mm from the growth plate-epiphyseal junction was digitized (i.e., the area where the activity zone of labeled clodronate was located). In the secondary spongiosa, measurement was carried out at distances greater than 1.1 mm from the growth plate-epiphyseal junction. For the primary and secondary spongiosa, Tt.T.Ar and Tt.B.Ar were measured fully automatically for calculation of the B.Ar/T.Ar.
Static histomorphometric measurements of the femoral diaphyseal cross-sections were carried out with the MCID M1 system with use of the Northern Light desktop illuminator. T.Ar, Ct.Ar and Ma.Ar were measured. Dynamic histomorphometric measurements of the femoral cross-sections were carried out with a MCID system with use of morphometry software of model M4 (Imaging Research Inc.). The Ir.L.Wi between the calcein labels was measured at the periosteal surface at 20× objective magnification. Two microscopical fields were analyzed for lateral, posterior, medial and anterior site of each cross-section. The periosteal MAR was obtained by dividing the distance between the two calcein labels by the time interval between the administration of these two labels (21 or 77 days).Statistical analyses. The statistical analyses were carried out with the SAS system (SAS Institute Inc., Cary, NC). The results were analyzed by two-way analysis of variance with two between factors, "dose" and "time." If any differences occurred between the effects of doses or times or significant dose-time interaction, comparisons were made with contrasts. P values lower than 0.05 were considered statistically significant.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Distribution of labeled clodronate in the bone. The results showed that 14C-clodronate incorporated more readily in the femur than in the lumbar vertebra (table 1). High uptake of labeled clodronate was seen particularly below the epiphyseal growth plate in the femoral primary spongiosa and at the periosteal surface of the femoral diaphysis. Labeled clodronate was also found at the surfaces of trabeculae in the secondary spongiosa of the metaphysis and in the subchondral and cancellous bone of the epiphysis, but the activity levels were below the lowest standard value (<32.40 nCi/g) and could thus not be measured reliably.
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Effect of clodronate on cancellous bone. After a follow-up of 2 days, clodronate treatment did not increase the percentage of mineralized hard tissue area (B.Ar/T.Ar) in the primary spongiosa of the distal femur; whereas, after a follow-up of 23 days, it was significantly increased by both doses of clodronate (table 2). The same phenomenon was seen after a follow-up of 79 days, but only with the higher dose (P < .05). In contrast to findings in the femoral primary spongiosa, mineralized hard tissue area was not increased by clodronate treatment in the primary spongiosa of the lumbar vertebra (data not shown).
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Effect of clodronate on cortical bone. Histomorphometric analysis of femoral diaphyseal cross-sections showed substantial bone apposition at the periosteal surface between the follow-ups of 23 and 79 days, as evidenced by a significant increase in cross-sectional tissue area and cortical area (table 3). Clodronate treatment at cumulative doses of 125 mg/50 µCi/kg and 250 mg/100 µCi/kg had no effect on any of the static histomorphometric variables after a follow-up of 23 or 79 days.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Quantitative analysis of labeled clodronate and other
bisphosphonates has been carried out previously with removed bone
fragments, and radioactivity in the bone as a whole has been determined
(Mönkkönen et al., 1990; Österman and
Laurén, 1991; Lin et al., 1991). In the present study,
labeled clodronate was quantified in macroautoradiographs. This method
showed the distribution of 14C-clodronate in the bone,
measured radioactivity in various regions of the femur and lumbar
vertebrae and determined the dislocation of the activity zone during
the observation period.
High uptake of labeled clodronate was seen particularly below the
growth plate in the primary spongiosa of the distal femur. Similar
macrodistribution has been shown previously for technetium-labeled methylene bisphosphonate in rat long bones (Khan et al.,
1979; Christensen and Krogsgaard, 1981) and for alendronate in tibiae of neonatal rats (Azuma et al., 1995). The longer the
follow-up after the last dose, the further away from the growth plate
the activity zone of 14C-clodronate was located in the
distal femoral and lumbar vertebral metaphyses because of longitudinal
bone growth, which indicated that the 6-month-old male rats used in the
present study were still growing.
Autoradiographs also showed minor amounts of 14C-clodronate
locally on the surfaces of trabeculae in the secondary spongiosa of the
distal femur and lumbar vertebra, which reflected direct incorporation
of the radiolabeled compound into the secondary spongiosa, presumably
at sites of bone remodeling activity. However, the activity level of
labeled clodronate in the secondary spongiosa was too low to be
measured reliably. A difference in bone turnover may explain the fact
that incorporation of clodronate is greater into the primary spongiosa
than into the secondary spongiosa (Kimmel and Jee, 1980).
The radioactivity in the lumbar vertebra was about half that found in
the femur; and, after the follow-ups of 23 and 79 days, the activity
zone was nearer to the growth plate in the metaphysis of the lumbar
vertebra than in the femur, reflecting the slower bone turnover rate
(Sontag, 1994) and longitudinal growth rate of vertebrae (Baron
et al., 1984; Wronski et al., 1986).
Static histomorphometry of cancellous bone showed no increase in mineralized hard tissue area in the primary spongiosa of the lumbar vertebra after clodronate treatment. In contrast, in the primary spongiosa of the distal femur, where the highest activity of labeled clodronate occurred, an increase in mineralized hard tissue area was found after follow-up periods of 23 and 79 days. The results of the present study show that in adult rats no increase in trabecular bone mass was seen in the secondary spongiosa because, as a result of decreased longitudinal bone growth, labeled clodronate originally incorporated into the primary spongiosa did not reach the region of secondary spongiosa even during the follow-up period of 79 days, and because the amount of clodronate directly incorporated into the secondary spongiosa was very low.
A larger amount of labeled clodronate was incorporated into the periosteal surface, the site of modeling-dependent bone formation. Autoradiographs of a cross-section of the femoral shaft also showed that after the follow-up periods of 23 and 79 days radioactive clodronate was buried in the bone, which indicated bone formation on top of the clodronate-containing surface.
Histomorphometric analysis of the femoral cross-sections showed that active modeling of the femoral diaphysis occurred during the study, evidenced by increases in both cross-sectional tissue area and cortical bone area. Clodronate treatment had no effect on any of the static histomorphometric variables of cortical bone after the follow-up time of 23 or 79 days, which indicated that it did not disturb the radial growth of the femoral diaphysis and the related increase in cortical bone mass. In addition, clodronate treatment had no statistically significant effect on the periosteal MAR measured at the lateral site of the cross-sections.
In clodronate-treated groups, the first calcein label on the periosteal
surface was very weak and totally absent at some sites, especially in
the high-dose (250 mg/100 µCi/kg) clodronate group. Because
clodronate and the first calcein label were given on the same day,
clodronate before calcein, the reduction in the length and intensity of
the first calcein label in femoral cross-sections may have been caused
by competition of clodronate with fluorochrome labels for binding sites
on mineralizing bone surface. This possibility is supported by
microautoradiographic studies, in which the uptake of
technetium-labeled bisphosphonates coincided with the interphase between osteoid and mineralized bone (Fogelman, 1980) and the fluorescence of tetracycline labels (Christensen and Krogsgaard, 1981).
No remarkable decrease in radioactivity was found in bone specimens
taken after a follow-up of 23 or 79 days compared with specimens taken
immediately after discontinuation of administration, which is
consistent with the long half-life of bisphosphonates in bone (Wingen
and Schmähl, 1987; Mönkkönen et al., 1990; Österman and Laurén, 1991).
In conclusion, our results show that the distribution of 14C-clodronate throughout different parts of bones, or among various bones, was uneven depending on the prevailing rate of bone turnover. The highest activity of labeled clodronate was seen in the primary spongiosa of the distal femur and on the periosteal surface of the femoral diaphysis. In spite of the accumulation of clodronate in the bone of normal adult rats, no marked histological effects were seen, except for an increase in the mineralized hard tissue area in the primary spongiosa of the distal femur.
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Acknowledgments |
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The authors thank the staff of Preclinical Research in Leiras Oy for skillful technical assistance.
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Footnotes |
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Accepted for publication October 10, 1996.
Received for publication May 6, 1996.
Send reprint requests to: Thua Österman, Leiras Oy, Biomedical Research Center, P.O. Box 415, FIN-20101 Turku, Finland.
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Abbreviations |
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MCID, microcomputer imaging device; Tt.T.Ar, total tissue area; Tt.B.Ar, total mineralized trabecular bone area; B.Ar/T.Ar, percentage of total trabecular bone area; T.Ar, cross-sectional tissue area (bone + bone marrow); Ct.Ar, cortical bone area; Ma.Ar, marrow area; Ir.Li.Wi, interlabel width; MAR, mineral apposition rate.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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