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Institute of Physiology, Physiological Chemistry and Animal Nutrition (R.G.E.), Ludwig Maximilians University, 80539 Munich, Germany; University of Sheffield Medical School (A.M.S., D.M.), Sheffield, United Kingdom S10 2RX; and Schering Research Laboratories (U.K., M.H.), Schering Ltd., 13342 Berlin, Germany
Address all correspondence and requests for reprints to: Dr. Reinhold G. Erben, Institute of Animal Physiology, University of Munich, Veterinaerstrasse 13, D-80539 Munich, Germany. E-mail: R.Erben{at}lrz.uni-muenchen.de
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast to this, there is ample evidence that 1,25-(OH)2D3 can directly influence bone cell function. 1,25-(OH)2D3 is a potent stimulator of bone resorption in vitro (4), and acute administration of pharmacological amounts of 1,25-(OH)2D3 to rats results in a transient augmentation of osteoclast activity and recruitment (5, 6). The initial increase in osteoclast number and activity after high dose 1,25-(OH)2D3 is thought to be caused by enhanced monocytic differentiation of immature hematopoietic cells and subsequent commitment of monocytic cells into preosteoclasts and by augmented cell activity of mature osteoclasts (7). Mature osteoclasts do not possess receptors for 1,25-(OH)2D3, and the latter effect is mediated through cells of the osteoblastic lineage (7), which have been shown to contain intracellular 1,25-(OH)2D3 receptors (8). However, the direct stimulating effects of high dose 1,25-(OH)2D3 on bone resorption are counteracted in vivo by a suppression of PTH secretion by both a direct inhibitory effect on the parathyroid glands (9) and an indirect effect via stimulation of intestinal calcium absorption and a subsequent rise in serum calcium. It is likely that the PTH suppression induced by external administration of 1,25-(OH)2D3 overrides the direct stimulating effects of 1,25-(OH)2D3 on bone resorption, thereby depressing osteoclast activity within a few days after acute 1,25-(OH)2D3 administration. This hypothetical sequence of events is supported by a histomorphometric study in rats in which daily injections of high doses of 1,25-(OH)2D3 increased osteoclast numbers in tibial cancellous bone on day 1, but diminished osteoclast numbers by day 6 of the study (6). Furthermore, chronic administration of active vitamin D metabolites to humans and rats results in a suppression of bone resorption (10, 11, 12).
It is also well established that 1,25-(OH)2D3 has distinct effects on the activity of bone-forming cells. In vitro studies have shown that 1,25-(OH)2D3 can modulate osteoblast proliferation and osteoblast production of type I collagen, alkaline phosphatase, and osteocalcin (1). In vivo, high doses of 1,25-(OH)2D3 administered to rats up-regulate tibial osteocalcin messenger RNA levels within hours (13) and profoundly increase osteoblastic matrix formation, with subsequent accumulation of osteoid in cancellous bone within days (6). Thus, although 1,25-(OH)2D3 may have only a minor direct physiological role in bone turnover, its pharmacological administration results in profound effects on bone cells in vivo.
Remodeling is a cyclical repair and renewal process in which bone resorption is followed by bone formation in the same site. Remodeling is the prevailing bone turnover activity in cancellous bone of adult humans (14) and also in cancellous bone of the axial skeleton in rats (15). It has been proposed that cancellous bone mass could be increased by pharmacological modulation of synchronized bone-remodeling units. Such a treatment regimen has been named ADFR (activate, depress, free, repeat) or coherence therapy (16). The central idea behind ADFR therapy is that after creation of a number of synchronized cancellous bone-remodeling units (activate) a temporary suppression of bone resorption (depress) followed by a therapy-free interval (free) would eventually result in a positive bone balance for each of these newly created remodeling units. From the above-mentioned temporal sequence of in vivo effects of 1,25-(OH)2D3 on bone resorption and bone formation, it is evident that a short-term application of high dose 1,25-(OH)2D3 may combine all components necessary for ADFR therapy. Using an experimental rat model, this study was undertaken to test this hypothesis. The results of the current experiment provide evidence that it may be possible to conduct an ADFR-like treatment regimen by short-term application of active vitamin D metabolites.
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Materials and Methods |
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It was the aim of Exp 2 to assess the effects of short-term, high dose 1,25-(OH)2D3 on bone formation and serum PTH levels. Twenty-four 6-month-old female Wistar rats (Schering) with a mean initial body weight of 268 g were used for this study. The animals were allowed free access to a pelleted standard rat chow (Altromin 1320, with 0.9% calcium and 0.75% phosphorus) and tap water. The rats were allocated into two weight-matched groups (n = 12 each) and were sc injected with either 0.2 µg 1,25-(OH)2D3/kg BW·day or vehicle on days 1, 2, and 3 of the study. The fluorochromes (all obtained from Sigma, Deisenhofen, Germany) oxytetracycline (25 mg/kg BW), xylenol orange (90 mg/kg BW), demeclocycline (20 mg/kg BW), alizarin complexone (30 mg/kg BW), and calcein (20 mg/kg BW) were administered ip on days 0, 5, 10, 15, and 20 of the study, respectively. Blood was always collected at the same time of day (and before administration of fluorochromes) on days 0, 4, 15, and 24 by tail vein puncture and analyzed for PTH. The serum samples were stored at -40 C until assayed. Six rats from each group were killed on day 7 by exsanguination from the abdominal aorta under ether anesthesia, and the rest of the rats were killed on day 24. Lumbar vertebrae of these rats were used for bone histomorphometry.
The purpose of Exp 3 was to study the effects 1,25-(OH)2D3 on the number of osteoblast precursor cells in bone marrow using bone marrow cultures. For this study, male rats were used to exclude possible effects of the stage of the estrous cycle on the growth of bone marrow stromal cells in ex vivo cell cultures. Sixty 6-month-old male Wistar rats (University of Sheffield, Sheffield, UK), with a mean initial body weight of 285 g, were used for this study. The animals were allowed free access to a pelleted standard rat chow (Altromin 1320) and tap water. The rats were allocated into two weight-matched groups (n = 30 each) and were sc injected with either 0.2 µg 1,25-(OH)2D3/kg BW or vehicle on days 1, 2, and 3 of the study. Five rats of each group were killed on days 4, 6, 8, 10, 12, and 14 by cervical dislocation, and the tibiae of these rats were excised, freed of soft tissue, and subsequently used for bone marrow cultures.
Blood and urine analysis
Serum and urine samples in Exp 1 were analyzed for total calcium
using flame photometry. To dissolve crystalline calcium salts, the
urine samples were acidified in 10 ml 2 N HCl before
analysis. Serum PTH for Exp 2 was measured with a rat-specific
immunoradiometric assay reacting with both N-terminal and intact PTH
(Immutopics, San Clemente, CA). The intra- and interassay variabilities
of this assay in our laboratory were 4.5% and 8.3%, respectively.
Histology
At autopsy of the rats in Exp 2, the first lumbar vertebrae were
defleshed and fixed immediately in 40% ethanol at 4 C for 48 h.
After fixation, the bones were embedded undecalcified in
methylmethacrylate, as described previously (17). Five-micron thick
undecalcified sections were prepared in the median plane of the
vertebrae with a Polycut E sledge microtome (Reichert-Jung, Nussloch,
Germany) and stained with toluidine blue at acid pH (18) and, after
etching with formic acid and methanol, with toluidine blue for
demonstration of cement lines (19).
Histomorphometry
All histomorphometric parameters are presented as
two-dimensional terms and were calculated and expressed according to
the suggestions made by the American Society of Bone and Mineral
Research nomenclature committee (20). All measurements were performed
in a blind fashion.
Histomorphometric measurements were made using a semiautomatic system (Videoplan, C. Zeiss, Oberkochen, Germany) and a Zeiss Axioskop microscope with a drawing attachment. In the centrally located cancellous bone of the first lumbar vertebral body, about 2 mm2 of tissue area were evaluated in each section, corresponding to about 15–20 mm of trabecular bone surface. The area within 0.5 mm from the cranial and caudal growth plates was excluded from the measurements. Static histomorphometric parameters were measured in sections stained with toluidine blue, and dynamic, fluorochrome-based parameters were measured in unstained sections. Polarized light was used to analyze for woven bone formation in sections stained with toluidine blue.
The following primary parameters were determined at x200: bone area, bone perimeter (B.Pm), osteoid area, osteoid perimeter, osteoblast perimeter, number of osteoclasts, osteoclast perimeter, and fluorochrome-labeled bone perimeter for each of the 5 different fluorochromes. With the exception of the measurement of mineral apposition rate (MAR) for determination of osteoid maturation time (Omt) in animals killed on day 7, fluorochrome-based parameters were measured only in the animals labeled with all 5 fluorochromes and killed on day 24 of the study. From the primary data, the structural parameters bone area (bone area/tissue area), bone perimeter, trabecular width, trabecular number, and trabecular separation were calculated. For the calculation of trabecular number and trabecular separation, the parallel plate model was used (20). Osteoid width was determined directly at x400 by sampling each osteoid seam every 50 µm. Osteoclasts were defined as large, irregularly shaped cells with a foamy, slightly metachromatic cytoplasma containing 1 or more nuclei. Osteoclast numbers were expressed using the mineralized bone perimeter as referent. The MAR between 2 adjacent fluorochrome labels was measured at x400, sampling each site showing both labels every 50 µm. Values for MAR were not corrected for obliquity of the plane of section. The labeled perimeter was defined as the percentage of fluorochrome-labeled bone perimeter for each fluorochrome label. The bone formation rate (bone formation rate/B.Pm) for the time interval between 2 fluorochrome labels was calculated by multiplying the arithmetic mean of the 2 individual values for labeled perimeter with the respective MAR. The Omt was defined as osteoid width divided by the respective MAR and was expressed in days. For determination of Omt in animals killed on day 7, MAR was additionally measured in these animals. The wall width of completed cancellous bone structural units was measured in animals killed on day 24 of the study on sections stained with toluidine blue for demonstration of cement lines at x200. Each completed remodeling site (at least 15/animal) was sampled at 3–4 equidistant points.
Bone marrow cultures
Preparation of total bone marrow cells.
Total bone marrow
cells were obtained from the tibiae of the rats in Exp 3. The distal
end of the bones was removed, a hole was made in the proximal end, and
the cells were flushed out with 10 ml DMEM containing 12% FCS,
antibiotics, and 50 µg/ml ascorbic acid. All chemicals for the bone
marrow cultures were obtained from Sigma. The cells were dispersed by
repeated pipetting, and a single cell suspension was achieved by
forcefully expelling the cells through a 20-gauge syringe needle.
Fibroblastic colony-forming units-f (CFU-f).
To analyze for
CFU-f, 106 nucleated cells from each tibia were plated out
in triplicate in 55 cm2 petri dishes in the above-mentioned
medium containing 10-8 M dexamethasone. The
medium was first changed after 5 days for DMEM containing 12% FCS, 50
µg/ml ascorbic acid, 10 mM ß-glycerophosphate, and
10-8 M dexamethasone and thereafter twice
weekly. The cultures were maintained for 18 days, after which the cells
were washed with PBS and fixed by the addition of cold ethanol. After
fixation, the cultures were stained sequentially for alkaline
phosphatase (APase)-positive colonies, calcified colonies,
collagen-positive colonies, and total colonies as described previously
(21). After each staining procedure, the culture dishes were
photographed, destained, and then restained as required. The number of
colonies were counted macroscopically by hand. Colonies were defined as
methylene blue-positive clusters of cells with a diameter of at least 1
mm. Total fibroblastic colonies, i.e. colonies predominantly
containing cells with a mesenchymal morphology, were considered to
represent CFU-f. Colonies that also stained positive for APase,
calcium, and/or collagen were considered to be derived from CFU-f with
the ability to express APase, calcify, and/or synthesize collagen and
were termed CFU-fAP, CFU-fCa, and
CFU-fCO.
Statistical analysis
Statistical comparisons were generally made using the
nonparametric Wilcoxon-Mann-Whitney test. For statistical analysis of
paired values, the nonparametric Wilcoxon signed rank test was used.
P < 0.05 was considered significant. The data are
presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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that a 3-day
administration of 1,25-(OH)2D3 at the dose of
0.2 µg/kg BW·day elicited a pronounced hypercalcemic and
hypercalciuric response during the treatment period and the days
thereafter. The elevated urinary excretion of calcium in
1,25-(OH)2D3-treated rats showed a more delayed
decline to normal compared to serum calcium and had returned to
baseline levels by about 7 days after discontinuation of
1,25-(OH)2D3 administration. As shown in Fig. 2, a 65% decrease in serum PTH levels
was observed in Exp 2 in the
1,25-(OH)2D3-treated group on day 4 compared
with day 0 (P < 0.005). On days 15 and 24, however,
serum PTH concentrations did not differ between the two groups of
animals.
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and 2)
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Exp 2: dynamic bone histomorphometry (Fig. 3)
In lumbar vertebral cancellous bone,
1,25-(OH)2D3 administration caused a 108%
increase (P < 0.005) in percent labeled perimeter for
the demeclocycline label administered on day 10 of the study.
Furthermore, compared with vehicle controls, short-term
1,25-(OH)2D3 resulted in 34% and 39%
increases in MAR (P < 0.005) and 128% and 139%
increases in bone formation rate (P < 0.005) in the
time intervals between days 5 and 10 and days 10 and 15, respectively.
The increase in MAR was seen in remodeling sites created both before
(bearing an oxytetracycline label) and after (without oxytetracycline
label) the 1,25-(OH)2D3 treatment. A comparison
of fluorochrome labeling with adjacent cement line-stained sections
revealed that the majority of new bone formation sites induced by
1,25-(OH)2D3 were remodeling sites.
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Discussion |
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However, in clinical studies assessing the effects of short- term 1,25-(OH)2D3 treatment on calcium and bone metabolism, such an initial resorptive phase could not be demonstrated (22, 23, 24). It is possible that the biochemical markers of bone resorption used in these studies (urinary hydroxyproline and serum C-terminal telopeptide of type I collagen) were too insensitive to measure a transient increase in bone resorption or that the response of bone metabolism to short- term 1,25-(OH)2D3 treatment may differ between rodents and humans. On the other hand, the doses used in humans may not be high enough to unequivocally show an early increase in bone resorption after acute 1,25-(OH)2D3 administration. All of these clinical studies, however, were able to demonstrate an increase in biochemical markers of bone formation after short-term treatment with 1,25-(OH)2D3. In the study by Geusens et al. (22) a rise in serum osteocalcin was found after treatment of postmenopausal women with 4 µg 1,25-(OH)2D3/day for 4 days. Gram et al. (24) administered 1,25-(OH)2D3 in a dose range of 1–2 µg/day to healthy male volunteers for 7 days and could show a dose-dependent increase in serum levels of osteocalcin and procollagen type I C-terminal propeptide within 24 h after start of the treatment. The rapidity of the effects of 1,25-(OH)2D3 on osteocalcin secretion by osteoblasts in humans is supported by animal experimental data demonstrating a rise in messenger RNA levels for osteocalcin in tibiae of rats within 3–6 h after a single dose of 1,25-(OH)2D3 (13).
The stimulating effects of 1,25-(OH)2D3 on bone formation were seen in the current experiment as a severalfold increase in static parameters of bone formation (osteoid area, osteoid perimeter, and osteoblast perimeter) by day 7. With a time lag of a few days, the newly formed osteoid began to mineralize, as documented by a profound increase in labeled perimeter by day 10 and an increase in MAR between days 5–10 of the study. One of the positive aspects of the multiple fluorochrome labeling schedule used in the present study is that changes in bone formation can be followed in one animal over the time period between the first and the last in vivo fluorochrome label. Using this technique, a change in MAR was not demonstrable in 1,25-(OH)2D3-treated animals within days 0–5 after the start of treatment. Thus, the immediate effect of 1,25-(OH)2D3 on osteoblasts is an increase in matrix production, followed by a later increase in MAR. Although some of the fluorochrome labels were broadened in 1,25-(OH)2D3-treated rats, the dose of 0.2 µg 1,25-(OH)2D3/kg BW·day administered over 3 days in this study did not significantly impair bone mineralization. Higher doses of 1,25-(OH)2D3, especially if given over a longer time period, inhibit bone mineralization and induce woven bone formation in rats (25). As elevated serum levels of PTH were not observed in the 1,25-(OH)2D3-treated groups during the course of the experiment, it is unlikely that the increase in bone formation caused by 1,25-(OH)2D3 administration was due to a PTH rebound phenomenon.
It is known that MAR in remodeling sites is higher in the early phases of bone formation than in the later phases (26). Thus, the increase in MAR found in this investigation may in part be due to the creation of new remodeling sites by short- term 1,25-(OH)2D3 treatment. However, this phenomenon cannot account for the observation that 1,25-(OH)2D3 also augmented MAR in preexisting remodeling sites. This finding can only be explained by either a stimulating effect of 1,25-(OH)2D3 on existing osteoblasts or an increased supply of new osteoblasts to preexisting remodeling sites (27). It is thought that osteoblast perimeter and fluorochrome-labeled perimeter reflect the birth rate of new teams of osteoblasts, whereas MAR and wall width reflect the average collective performance of individual teams of osteoblasts (27). The histomorphometric results of this study suggest, therefore, that short-term, high dose 1,25-(OH)2D3 not only creates new cancellous bone-remodeling sites, but also stimulates existing ones by a direct effect of 1,25-(OH)2D3 on osteoblasts already engaged in bone formation and/or by an increased supply of new osteoblasts through enhanced proliferation and differentiation of osteoblast precursor cells in bone marrow. The idea that 1,25-(OH)2D3 treatment has positive effects on osteoblast team performance is further supported by the augmented values for wall width in 1,25-(OH)2D3-treated rats on day 24 of Exp 2.
The finding of an increased number of APase-, calcium-, and collagen-positive CFU-f in ex vivo bone marrow cultures on day 4 after a 3-day administration of 1,25-(OH)2D3 in Exp 3 strongly supports the idea that enhanced osteoblast recruitment is a major component of the stimulating effects of 1,25-(OH)2D3 on bone formation. In agreement with this, it has been shown in vitro that 1,25-(OH)2D3 augments the proliferation of stromal cells and their differentiation into APase-positive preosteoblastic cells in rat bone marrow stromal cell cultures (28). The observation in the present study that the percentage of APase-, calcium-, and collagen-positive CFU-f relative to the number of total CFU-f in bone marrow cultures of 1,25-(OH)2D3-treated rats remained unchanged suggests that short-term 1,25-(OH)2D3 in vivo increased the total number of mesenchymal precursor cells together with a proportional increase in the number of mesenchymal precursor cells with osteoblastic potential, but had no specific effect on their differentiation. Between days 6 and 14 in Exp 3, the numbers of CFU-f, CFU-fAP, CFU-fCa, and CFU-fCO remained unchanged relative to control levels. The histomorphometric measurements in Exp 2, however, suggest that the increased number of osteoblast precursor cells made available in the early phase of short-term 1,25-(OH)2D3 treatment translates into a rise in osteoblastic matrix and bone formation that lasts for 2–3 weeks. It may be hypothesized in this context that osteoblast precursor cells once delivered to bone-forming surfaces remain active for 2–3 weeks in the rat.
Therefore, in the context of the ADFR hypothesis, short- term 1,25-(OH)2D3 not only behaves like a pure activator of bone remodeling, but additionally suppresses bone resorption shortly after its administration and enhances bone formation and osteoblast team performance, possibly through its stimulating effects on preexisting osteoblasts and osteoblast recruitment. It is currently under investigation in our laboratory whether repeated cycles of short-term 1,25-(OH)2D3 would increase cancellous bone mass in an experimental rat model in a manner predicted by the ADFR hypothesis.
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Acknowledgments |
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Footnotes |
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Received March 7, 1997.
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