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Departments of Pediatrics (T.O.C., K.C.M., B.E.), Orthopaedics and Rehabilitation (M.A., D.B., C.M.G.), and Surgery (T.L.M., M.C.), Yale University School of Medicine, New Haven, Connecticut 06520-8064
Address all correspondence and requests for reprints to: Thomas O. Carpenter, M.D., Department of Pediatrics, Yale University School of Medicine, P.O. Box 208064, New Haven, Connecticut 06520-8064. E-mail: carpenteto{at}maspo3.mas.yale.edu
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Abstract |
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Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response.
Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have demonstrated abnormally elevated circulating levels of the bone protein osteocalcin in Hyp mice (9). Repetitive doses of 1,25(OH)2D3 increase circulating osteocalcin in normal mice, whereas in Hyp mice, abrupt decreases of serum osteocalcin occur with single or repetitive doses. These findings are substantiated by calvarial osteocalcin messenger RNA (mRNA) levels before and after such manipulations and cannot be ascribed to altered clearance or skeletal stores of the protein in the Hyp model (10).
Measurement of osteocalcin serves as a specific marker for the presence of osteoblasts in primary culture and provides a sensitive means of identification of phenotypic changes in these cells (11, 12, 13). Normal rat and chick osteoblasts, isolated by sequential enzyme digestion, have been characterized as having an ordered developmental sequence consisting of three periods where growth, differentiation, and mineralization occur (11). Both stimulatory and inhibitory effects of 1,25(OH)2D3 on osteocalcin expression in primary rat and chick osteoblast cultures have been demonstrated (13, 14), and the magnitude and direction of these effects are dependent upon the developmental phase of the culture at the time of exposure to 1,25(OH)2D3. More recently, clonal murine osteoblastic cell lines have been shown to undergo down-regulation of osteocalcin-secretion by 1,25(OH)2D3 (15). Because of the availability of certain murine models of disease (e.g. Hyp), as well as the development of transgenic (16) and knockout techniques (17), directly isolated mouse cells should provide another useful tool for study. However, regulation of osteocalcin production in isolated primary murine osteoblast cultures has not been described.
With respect to Hyp mice, experiments performed to date have not ascertained whether the described changes in osteocalcin production are caused by intrinsic defects in the osteoblast itself or whether the altered production of the protein reflects an osteoblastic response secondary to a systemic factor. Because our previous studies demonstrated abnormalities of osteocalcin secretion in the in vivo osteoblast of Hyp mice (9, 10), we wished to determine whether these abnormalities persisted in isolated bone cells in primary culture, thereby indicating an intrinsic defect of the Hyp osteoblast. We therefore examined osteocalcin in primary cultures of isolated murine osteoblasts and compared the regulatory effect of 1,25(OH)2D3 in cultures derived from normal and Hyp mice. We describe a down-regulation of osteocalcin production in response to 1,25(OH)2D3 in cells derived from normal mice and demonstrate that Hyp cells behave comparably with normal ones.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because mice generally give birth in the early morning, pregnant mice were checked twice daily to allow earliest identification of delivery. Pups used the day of delivery were called "6- to 12-h old", those used the day after delivery were called "30- to 36-h old," and so forth.
Materials
-MEM (Gibco BRL Products, Grand Island, NY), 10% FBS,
penicillin and streptomycin (Gemini Bio-Products, Calabasas, CA), and
L-glutamine and ß-glycerol phosphate (Sigma Chemical, St.
Louis, MO) were used for the preparation of culture media.
Collagenase CLS2 at 200 U/ml was obtained from Worthington Biochemical
(Freehold, NJ). 1,25(OH)2D3 was obtained from
Biomol Research Lab (Plymouth Meeting, PA). Quantitative colorimetric
determination of alkaline phosphatase and quantitative colorimetric
determination of calcium kits were obtained from Sigma Diagnostics (St.
Louis, MO). Calf thymus DNA was purchased from Calbiochem (La Jolla,
CA). TRIzol reagent for RNA isolation was obtained from Gibco BRL
(Gaithersburg, MD).
Osteoblast isolation and culture
Murine osteoblasts were isolated from pups at 30–36 h old,
unless otherwise specified. The animals were sedated by hypothermia and
then killed by decapitation. Calvariae were removed and bathed in
-MEM. The fibrous tissue surrounding the bone was gently scraped,
the calvariae were divided in two, and sutures were removed. Because of
the small size of the mouse pups, the entire calvaria was used from
each animal. The trimmed calvariae were transferred to a 50-ml
Erlenmeyer flask and washed with PBS three times, at 37 C for 10 min
per wash, in an oscillating water bath. Calvariae were then subjected
to a series of collagenase digestions in an oscillating 37-C water
bath. The first two digests were discarded. Digests 3, 4, and 5 (15 min
each, which was sufficient to release cells from these small calvariae)
were neutralized with -MEM, pooled, and filtered through sterile
polypropylene mesh of 200–297 µ. The filtrate was centrifuged for 6
min at 1500 rpm, the supernatant was removed, and cells were
resuspended in 3–5 ml -MEM containing 10% FCS. Cells were counted
via hemocytometer using trypan blue to exclude nonviable cells. Cells
were then diluted to 2 x 104 per ml and plated onto
6-well plates, 2.5 ml per well. Medium was changed twice weekly, and
the cultures reached confluence at 7–9 days. Collection of medium for
analysis occurred 1–3 days after the previous medium change.
B-glycerol phosphate was not used in any of these studies, to avoid the
confounding effect of excess phosphate in the Hyp
cultures.
For rat osteoblast cultures, fetuses were removed via cesarian section
from 22-day-old gestation mothers, and osteoblasts were isolated as
described (18). Cells were subjected to five sequential 20-min
collagenase digestions, and the last three digests were quantitated and
pooled. Cells were plated in 6-well plates at 3 x
104/ml in DMEM containing HEPES, ascorbic acid, penicillin,
streptomycin, and 10% FBS. Confluence was reached at 7–8 days, and
thereafter, cells were maintained in -MEM with 10% FCS, which was
changed twice a week. Collection of medium for analysis occurred 2–4
days after the previous medium change.
1,25(OH)2D3
Purchased 1,25(OH)2D3 was repurified by
isocratic straight-phase HPLC on a silica column (Microsorb, Rainin,
Woburn, MA) using methylene chloride:isopropyl alcohol (96:4) as the
mobile phase. A 1-ng/µl solution of purified
1,25(OH)2D3 was prepared in -MEM:ethanol
(85:15), which was added to individual wells so that the final
concentration of 1,25(OH)2D3 was 2.4 x
10-8 M. For dose-response experiments, a dose
range of 1,25(OH)2D3 from 2.4 x
10-9 M to 4.8 x 10-7
M was used. With murine cells, cultures were exposed to
1,25(OH)2D3 containing medium for 24, 48, or
72 h in short-term experiments or continuously with each medium
change from time of exposure through completion of the experiment. With
rat osteoblasts, cells were exposed to
1,25(OH)2D3 containing medium for either
48 h in short-term experiments or continuously with each medium
change beginning on day 9, 16, 23, or 30, and continuing through day
37.
Analytical methodology
Osteocalcin. For osteocalcin in media, 100-µl aliquots
were collected at each time point, 1–3 days after the last medium
change, and determined using a previously described RIA (9). Briefly,
for each species, iodinated rat or murine osteocalcin was used as
tracer, species-specific purified protein was used as standards, and
specific (to the rat or murine protein) goat antibody was employed. For
cell layer osteocalcin, cultures were incubated at 37 C with 1 ml of
0.5 M HCl for 30 min, scraped, and centrifuged for 1 min in
a microfuge. The resultant supernatant was neutralized and then
immediately analyzed for osteocalcin by RIA, as described.
Alkaline phosphatase, calcium, and DNA. Cell layers from alternate wells were lysed in 20 mM Tris, 0.5 mM MgCl2, 0.1 mM ZnCl2, and 0.1% Triton X. One milliliter of this lysis buffer was added to each well and incubated for 30 min at room temperature, scraped, and centrifuged for 1 min in a microfuge. Supernatant was immediately assayed for alkaline phosphatase determination by colorimetric kit methods using p-nitrophenyl phosphate as substrate. Cell layers from alternate wells were lysed by incubation for 1 h with 0.5 M HCl at room temperature, scraped, and centrifuged for 1 min in a microfuge. Supernatant was immediately assayed for calcium by colorimetric kit methods. Total DNA in cell layers was determined by fluorometry after incubation with perchloric acid and m-diaminobenzoic acid dihydrochloride, using calf thymus DNA as a standard (19). Mineralized nodules were visualized after application of von Kossa’s stain, as described (20).
Osteocalcin mRNA isolation and analysis. mRNA was obtained using a single-step isolation method kit, according to the manufacturer’s instructions (Life Technologies, Gaithersburg, MD). Cells were lysed directly in the culture dish with TRIzol reagent, transferred to Corex glass tubes, and incubated for 5 min at room temperature. After addition of chloroform, samples were shaken and centrifuged. The aqueous phase was transferred to a fresh tube, and RNA was precipitated after addition of isopropyl alcohol and incubation at room temperature for 10 min. After centrifugation and removal of supernatant, the RNA pellet was washed with 75% ethanol and centrifugation repeated. After air drying, the RNA was dissolved in 0.5% SDS solution, and a Northern blot was performed. Ten micrograms of total RNA, as determined by optical density measurement at 260 nm, were denatured with glyoxal/dimethylsulfoxide and loaded onto 1.5% agarose gels (21). Hybridizations were performed using Zetabind nylon membranes (Cuno, Inc., Meriden, CT) and mouse cDNA for osteocalcin (22). Integrity of mRNA was assessed by ethidium bromide staining before hybridization. Uniformity of gel loading was analyzed using a synthetic 18S ribosomal RNA sequence (Ambion Inc, Austin, TX). Probes were radiolabeled by random priming (BioPrime, Gibco BRL), and RNA was quantitated via densitometry.
Statistical analysis. Results are reported as mean ± SEM. Data are representative of three-to-four experiments, with each experimental point comprised of three or six wells/group. Data were analyzed using t tests for two sample comparisons and, otherwise, ANOVA and Fisher’s least squares difference (LSD) for post hoc multiple comparison testing. The Systat for Windows (SPSS, Chicago, IL) software program for IBM-compatible personal computers was employed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Cultures from "30-
to 36-h" pups demonstrated slight increases in osteocalcin by 2
weeks, with marked increases in production by 3 and 4 weeks (Fig. 1).
Low levels of osteocalcin were observed in "78- to 84-h"-derived
cells, but higher levels were obtained when cells were plated at an
initial plating density considerably greater (1 x 105
cells/ml) than that routinely used (2 x 104
cells/ml). Mineral deposition was evident by 3 weeks in culture and
seemed denser by 4 weeks (Fig. 2).
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shows the time frame in which
osteocalcin, alkaline phosphatase, and calcium appeared in these
cultures. Alkaline phosphatase activity in the cell layer (Table 1) was
found in substantial amounts by 2 weeks in culture, increased by 3
weeks, and remained at maximal levels at 4 weeks. Osteocalcin in the
medium was evident by 3 weeks in culture, coincident with the
appearance of calcium in the matrix. Osteocalcin content of the cell
layer rose in concert with media osteocalcin and was maximal at 3 weeks
(data not shown).
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). Inhibition of
osteocalcin production occurred when
1,25(OH)2D3 was added early or relatively late
in culture. Cultures exposed to 1,25(OH)2D3
from just after confluence (day 9) onward produced markedly less medium
osteocalcin than cultures not exposed to
1,25(OH)2D3. This was also true for alkaline
phosphatase content of the cell layer (Table 1
). This effect persisted
as medium was sampled throughout the life of the culture. Exposure to
1,25(OH)2D3 from day 17 onward or day 25 onward
markedly attenuated the time-dependent increase in medium osteocalcin
seen in control cultures. Commensurate with the findings in the medium,
deposition of osteocalcin in the cell layer was significantly reduced
by exposure to 1,25(OH)2D3 (Fig. 5). Incubation with
1,25(OH)2D3 from day 9 onward prevented
mineralization and markedly reduced cell matrix osteocalcin.
1,25(OH)2D3 added at day 17, 25, or 33 also
reduced cell matrix osteocalcin, and reduction was progressively less
with later initiation of 1,25(OH)2D3
treatment.
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). This inhibitory pattern was most
evident at later points during maximum osteocalcin production in
control cultures. Furthermore, dose-related responsivity to
1,25(OH)2D3 was examined at 3 weeks in culture.
Accumulation of osteocalcin in medium was examined at 24, 48, and
72 h after addition of 2.4 x 10-9
M, 2.4 x 10-8 M, 2.4 x
10-7 M, or 4.8 x 10-7
M 1,25(OH)2D3 to the cultures and
compared with wells receiving no 1,25(OH)2D3
(Fig. 7). A significant dose-related
inhibition of osteocalcin accumulation occurred after 48 and 72 h
of incubation. A slight (but statistically insignificant) inhibition of
osteocalcin accumulation occurred at 24 h of incubation.
Osteocalcin mRNA was reduced in parallel in cultures receiving 2.4
x 10-8 M 1,25(OH)2D3
at 24, 48, and 72 h of incubation.
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Continuous exposure to 2.4 x 10-8 M
1,25(OH)2D3, beginning on day 9 of culture,
reduced osteocalcin levels in the medium, such that a constant (but
relatively low) level of the protein was evident throughout the
remaining life of the culture, much like that seen in the murine
osteoblasts. In addition, the accumulated osteocalcin deposited in the
cell matrix of these cultures was decreased after the introduction of
1,25(OH)2D3 at day 9 (data not shown). However,
exposure to 2.4 x 10-8 M
1,25(OH)2D3 at later times (16–30 days in
culture) resulted in up to 9-fold increases in osteocalcin levels in
the medium (Fig. 8). Thus, in our hands,
1,25(OH)2D3 affected osteocalcin production in
primary rat osteoblasts, dependent upon the developmental stage of
the culture, consistent with the experience of other investigators
(13).
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). Two-way ANOVA revealed significant
reduction in osteocalcin production with addition of
1,25(OH)2D3, and a significant effect of time
in culture on media osteocalcin levels, but there was no difference
between Hyp-derived cultures and those from their normal
litter mates.
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). These high-density cultures
consistently demonstrated a significant reduction in osteocalcin
accumulation with addition of 1,25(OH)2D3, and
again, Hyp-derived cultures were no different from
normals.
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. When normal
cultures were treated with 1,25(OH)2D3 from day
20 to day 23, inhibition to undetectable levels of osteocalcin mRNA
occurred, consistent with the down-regulation of medium osteocalcin
concentration. Similarly, in Hyp-derived cultures, treatment
with 1,25(OH)2D3 from day 20 to day 23 resulted
in a significant inhibition of osteocalcin mRNA, consistent with medium
osteocalcin concentrations. This pattern of mRNA down-regulation was
minimally evident by 6 h and substantially evident by 24 h of
treatment with 1,25(OH)2D3, as well (Fig. 11, lower panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Characterization of murine primary osteoblast cultures
The present studies demonstrate that in some respects, osteoblasts
derived from mice exhibit characteristics similar to other species.
Patterns of tissue organization similar to rats and chick were noted in
murine cultures (13, 14, 29). Before 1 week in culture, when the cells
are actively proliferating, there is no production of osteocalcin or
alkaline phosphatase. Just after the cultures reach confluence, a rapid
increase in alkaline phosphatase activity occurs, which is followed by
the initiation of mineralization, coincident with osteocalcin
expression.
In contrast, however, murine-derived osteoblasts have certain features that differ from osteoblasts isolated from other species. Basal osteocalcin production is 10- to 20-fold greater in mouse cultures than in rat cultures at comparable time points. Moreover, the age of the mouse pups was critical for optimal production of osteocalcin, and animals over 72 h old were capable of expressing osteocalcin only when plated at a high density. There seems to be a small window of time in which isolation by this method will yield mouse cells expressing the osteoblast phenotype in vitro. Nonenzymatic methods have been previously used for the isolation of osteoblasts from 7-day-old mice (30). These cells express alkaline phosphatase and are capable of elaborating a calcified matrix. However, osteocalcin production has not been studied in osteoblasts derived by this method.
The most striking distinction of the mouse cultures, however, is their response to 1,25(OH)2D3. Studies in normal rat osteoblast cultures demonstrate that vitamin D can both positively and negatively regulate gene expression, depending upon the developmental stage of the cell. As described elsewhere (13) and confirmed here, when 1,25(OH)2D3 is added to rat osteoblast cultures before the onset of mineralization, osteocalcin biosynthesis is suppressed throughout the life-span of the culture, as a result of an arrested stage of cell differentiation. Once the cultures begin to mineralize and the cells are terminally differentiated, however, media osteocalcin concentrations are increased by either chronic or acute application of the vitamin. Thus, in our hands, 1,25(OH)2D3 affected osteocalcin production in primary rat osteoblasts, dependent upon the developmental stage of the culture, consistent with the experience of other investigators (13) and verified our methodology using primary rat osteoblast cultures.
In contrast to these patterns observed in rat cell cultures, 1,25(OH)2D3 fails to stimulate osteocalcin at any time in murine cultures, either acutely or chronically. Rather the medium concentration of osteocalcin is suppressed when vitamin D is introduced. This response seems to be specific to osteocalcin, because alkaline phosphatase behaves in the mouse cultures as described in rat cultures treated with 1,25(OH)2D3 (13). These results are consistent with a recent study by Lian et al., who showed that in the murine osteoblastic cell line, MC3T3-E1, osteocalcin production is down-regulated by 1,25(OH)2D3 at both early and late time points (15). In collaboration with Clemens et al., we also have confirmed these findings (31). Furthermore, Zhang et al. (32) have reported differences in the vitamin D response elements in the mouse gene, compared with the human and rat genes, and further demonstrated down-regulation of osteocalcin mRNA in primary murine osteoblast cultures after 1,25(OH)2D3 addition.
These data also are consistent with our earlier in vivo studies that demonstrate that normal mice are relatively resistant to 1,25(OH)2D3 stimulation of osteocalcin (10). To elicit an increase in serum osteocalcin levels in normal mice, relatively large and multiple doses of the vitamin must be administered. This also differs from our findings in rats (33) and humans (34), in which a significant increase in circulating osteocalcin occurs after injection with 1,25(OH)2D3. Increases in serum osteocalcin after prolonged stimulation with 1,25(OH)2D3 are most likely the result of secondary effects of the hormone on other factors that influence osteocalcin regulation.
Interestingly, in murine osteoblasts immortalized by transgenic expression of simian virus 40 T antigen, 1,25(OH)2D3 stimulated osteocalcin mRNA, but there are alterations in growth regulation and differentiation in these cells (35). Whether the differentiation stage of the osteoblast plays a role in the phenomenon is unclear. Previously, Gerstenfeld et al. (26) reported that stimulation of osteocalcin by 1,25(OH)2D3 in chicks is increased when cultures are derived from 12-day embryos, but decreased in 17-day embryos. We are currently investigating the effect of differentiation stage by isolating cells from mouse embryos of varying developmental ages.
Comparison of primary osteoblast cultures derived from normal and
Hyp mice
Mating of normal female and Hyp male mice resulted in
litters exclusively consisting of affected females and normal males. By
demonstrating that no differences were apparent in cultures derived
from normal male and female mice, we could identify mice carrying the
mutation at birth, thereby allowing for rapid and simultaneous
harvesting of normal and Hyp cells. There are significant
advantages to this: First, postnatal age was critical for osteocalcin
production; therefore variability introduced by time of delivery
between normal and control litters is eliminated. Second, other
potential variables related to litter differences is minimized. Third,
the use of a normal mother eliminates the confounding variable of
maternal hypophosphatemia.
Cultures derived from the Hyp mouse model of XLH exhibited characteristics that were no different from cultures of their normal litter mates. These cultures reached confluence at 7–9 days in culture, mineralized, and produced osteocalcin in a time-dependent manner throughout the life of the cultures. Furthermore, a down-regulatory effect of 1,25(OH)2D3 on media osteocalcin production was seen in the Hyp cultures, and the magnitude of this effect was comparable with that seen in cultures derived from normal litter mates. Moreover, these 1,25(OH)2D3 treatments of both normal and Hyp cultures resulted in comparable down-regulation of osteocalcin mRNA levels. Finally, plating cells at higher density did not result in any significant differences between normal and Hyp cultures. Earlier studies also found no differences in collagen synthesis or alkaline phosphatase activity in proliferating cultures of normal and Hyp osteoblasts (36).
These findings are at variance with the aberrant regulation of osteocalcin in the Hyp mouse in vivo. In studies of the intact animals, circulating osteocalcin and calvarial mRNA for osteocalcin increase in normal mice with repetitive dosing (daily, for 3–6 days) of 1,25(OH)2D3, whereas Hyp mice show a decrease in these parameters (9, 10). Given that current evidence indicates systemic mediation of the renal defect of phosphate transport in Hyp mice (1, 2), these findings suggest that the skeletal abnormalities of osteocalcin secretion and 1,25(OH)2D3 regulation also may involve systemic factors. Indeed, a recent study by Tsuji et al. (37) suggests that ambient calcium and PTH levels may play a role in modulating the osteocalcin response in Hyp mice.
It is of particular note that the discrepancy between the acute in vivo and in vitro 1,25(OH)2D3 regulation of osteocalcin levels is observed in the normal animals, whereas the down-regulatory effect of 1,25(OH)2D3 on osteocalcin in Hyp mice is consistent in vivo and in vitro. One interpretation of this observation, i.e. that the in vivo effect in normal animals is not recapitulated in vitro, again suggests the possibility that additional systemic factors are required to generate in vivo differences. Furthermore, the consistency between the in vivo and in vitro findings in the Hyp strain suggest that these factors may be absent in the Hyp systemic milieu, thereby resulting in regulation that differs from the normal in vivo but not in vitro.
The mutated gene in Hyp mice (Pex) encodes an endopeptidase that is abundantly expressed in murine bone. Human and murine studies suggest that a humoral factor mediates the defect in renal phosphate transport. It seems likely that the PEX product is involved in the processing of such a factor and may have variable functions at various target tissues. Alternatively the endopeptidase may act on a variety of substrates. Either of these suggestions would accommodate the observations that bone lesions seem to be independent of disease expression at the kidney. The tissue origin of this factor, however, has not been definitely established, although liver and bone have been suggested as potential natural sources (38). Nevertheless, a putative phosphate-regulating substance may be rendered abnormal in XLH and Hyp by mutations in the PEX gene, such that normal regulation of the Pi homeostatic system is disrupted. The fact that regulation of osteocalcin biosynthesis in Hyp mice is abberant in vivo, but apparently normal in vitro, suggests that mechanisms governing osteocalcin regulation are intact in the Hyp osteoblast. Interruption of normal osteoblastic function may result from a direct interaction with systemic factors that are not present in vitro. Alternatively, the synthesis, processing, or secretion of a putative humoral factor that is abnormal in XLH and Hyp may alter the calcium/PTH/vitamin D system, resulting in a secondary effect on osteocalcin biosynthesis.
In summary, we show that primary mouse osteoblasts display certain features, previously described for rat and chick cultures, but that important species differences are present, with respect to 1,25(OH)2D3 induction of osteocalcin synthesis. Furthermore the differences between normal and Hyp mice seen in vivo, in both circulating osteocalcin and its regulation by 1,25(OH)2D3, are not recapitulated in primary osteoblast cultures. The findings suggest that mediation of the differences observed in vivo require mediation by factors not present in the primary osteoblast culture system.
Received June 27, 1997.
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