1 Veterans Administration
Medical Center, Loss of weight bearing in the growing rat
decreases bone formation, osteoblast numbers, and bone maturation in
unloaded bones. These responses suggest an impairment of osteoblast
proliferation and differentiation. To test this assumption, we assessed
the effects of skeletal unloading using an in vitro model of
osteoprogenitor cell differentiation. Rats were hindlimb elevated for 0 (control), 2, or 5 days, after which their tibial bone marrow stromal
cells (BMSCs) were harvested and cultured. Five days of hindlimb
elevation led to significant decreases in proliferation, alkaline
phosphatase (AP) enzyme activity, and mineralization of BMSC cultures.
Differentiation of BMSCs was analyzed by quantitative competitive
polymerase chain reaction of cDNA after 10, 15, 20, and 28 days of
culture. cDNA pools were analyzed for the expression of
c-fos (an index of proliferation), AP
(an index of early osteoblast differentiation), and osteocalcin (a
marker of late differentiation). BMSCs from 5-day unloaded rats
expressed 50% less c-fos, 61% more
AP, and 35% less osteocalcin mRNA compared with controls. These data
demonstrate that cultured osteoprogenitor cells retain a memory of
their in vivo loading history and indicate that skeletal unloading
inhibits proliferation and differentiation of osteoprogenitor cells in
vitro.
bone; alkaline phosphatase; c-fos; osteocalcin; mineralization
MICROGRAVITY ASSOCIATED with spaceflight leads to a
deficit in bone mass in humans and in animals (15, 18). In rats, the osteopenic response to microgravity is associated with decreased osteoblast numbers, decreased bone formation, and delayed bone maturation (4, 15). Similar changes can be induced in the hindlimbs of
the rat by hindlimb elevation (3, 4, 6, 7, 27). Although the osteoblast
appears to be the principal mediator of osteopenia in these models, the
underlying mechanism for osteoblast inhibition is unclear. Skeletal
unloading may decrease the number of osteoprogenitor cells or inhibit
their proliferation, as suggested by the decrease in osteoblast numbers
in unloaded bone (7) and the decreased proliferation of cultured
osteoblasts isolated from unloaded bones (12, 27). Osteoblast
differentiation may also be inhibited, as suggested by the inhibition
of mineralization and maturation of unloaded bone (3, 6) and by the
altered expression of genes associated with osteoblast differentiation in unloaded bone (4).
Cultured osteoblasts differentiate through phases of proliferation,
organic matrix synthesis, and matrix mineralization, and these phases
are accompanied by the sequential expression of various genes.
C-fos expression is associated with
the proliferative phase of cultured osteoblasts. Alkaline phosphatase
(AP) is a marker of early osteoblast differentiation, and is associated
with organic bone matrix synthesis before its mineralization.
Osteocalcin is expressed by mature osteoblasts in association with
matrix mineralization (17). RNA isolated from whole bone demonstrates
that skeletal unloading due to spaceflight or hindlimb elevation causes
increased AP mRNA expression and decreased expression of osteocalcin
mRNA (4). These changes are consistent with the observed decrease in
calcium-to-hydroxyproline ratio in bones after spaceflight or hindlimb
elevation (6) and suggest a shift toward decreased maturation of bone
(3). It is important to elucidate the response of the osteoblast
population to skeletal unloading to understand the mechanisms by which
bone formation in unloaded bone is inhibited. The effect of skeletal
unloading on the expression of c-fos,
AP, or osteocalcin mRNA at the osteoblast level has not been previously demonstrated. The present study was conducted to determine whether osteoblasts isolated from unloaded bones would recapitulate in vitro
the altered proliferation and differentiation that is suspected to
occur in vivo. This question was addressed by employing quantitative competitive polymerase chain reaction (QC-PCR) to measure the expression of c-fos, AP, and
osteocalcin mRNA in rat bone marrow stromal cell (BMSC) cultures from
unloaded and normally loaded tibiae. Measurements of cell
proliferation, AP activity, and mineralization in vitro further
demonstrate that the observed changes in gene expression translate into
an altered cell phenotype.
Animal protocols and tissue processing.
Male Sprague-Dawley rats (Bantam-Kingman, Fremont, CA) weighing 125 g
were fed standard laboratory rat chow ad libitum (Wayne Lab Blox F-6, James Grain, San Jose, CA) containing 1.4% Ca and 0.97%
P and were maintained on a 12:12-h light-dark cycle. A total of 18 rats
were divided into three groups such that the average weights were
similar between groups. Six control rats (normal weight bearing) were
housed in identical cages as the suspended rats. The remaining 12 rats
were hindlimb elevated for 2 (n = 6)
or 5 days (n = 6). Skeletal unloading
was accomplished using the hindlimb elevation model as previously
described (6). To effect unloading, rat tails were cleaned with 70%
ethanol and tincture of benzoin (American Hospital Supply, San
Francisco, CA) was sprayed along the tail and allowed to dry. This
protected the skin from irritation and formed a sticky surface. A
1-cm-wide piece of orthopedic tape (FasTrac, Van Nuys, CA) was attached laterally along each side of the tail to form a loop near the end of
the tail. The tail was then wrapped in a mesh netting (Stockinette, American Hospital Supply) followed by strapping tape. The loop of
orthopedic tape at the end of the tail was attached to a pulley system
allowing the hindquarters of the animal to be lifted off the ground
while permitting the animal free movement about its cage through the
use of its forelimbs. The initiation of hindlimb elevation was
staggered such that all animals were killed on the same day. At the
onset of hindlimb elevation, controls (normal weight-bearing animals)
were switched from ad libitum feeding to a pair-feeding
regimen, wherein the average daily food consumption of the suspended
animals was determined by weighing. This amount was provided to the
control animals to assure similar weight gain between groups throughout
the study. Animals were weighed daily before and during the study.
Hindlimb-elevated animals remained elevated during weighing to prevent
reloading of the hindlimbs. At the end of the hindlimb elevation
period, animals were killed under isofluorane anesthesia by
exsanguination from the dorsal aorta, during which the
hindlimb-elevated rats experienced no more than 1 min of reloading.
Blood was collected in a heparinized tube and immediately assayed for
pH and for serum ionized calcium concentration with a calcium-pH
analyzer (Ciba-Corning 234 Ca2+/pH
Analyzer; Ciba-Corning Diagnostics, Medfield, MA). The right femur from
each animal was removed and cleaned of adherent soft tissue and
defatted in a Soxhlet apparatus (Fisher Scientific, Santa Clara, CA)
using overnight extractions with ether and then with absolute ethanol.
The left femur and both tibiae from each animal were removed, cleaned
of soft tisse, and used for BMSC culture.
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cell culture methods.
To harvest BMSCs, tibiae were briefly immersed in 95% ethanol and then
bisected longitudinally with a razor blade. Marrow cells from the left
and right tibiae were cultured for up to 28 days and used for RNA
isolation, proliferation studies, and AP activity assays. Marrow cells
from the left femur were used to study in vitro mineralization. The
marrow from the left and right tibiae was quantitatively removed with a
curette and pooled for each animal. Marrow was diluted in 20 ml of
-Eagle's minimum essential medium with
L-glutamine and ribonucleosides
and deoxyribonucleosides (GIBCO, Grand Island, NY) containing 15%
fetal bovine serum, fungizone (2.5 µg/ml), and
penicillin-streptomycin (100 U/ml). A single-cell suspension was
obtained by repeated passage through an 18-gauge needle, and cell
yields were determined with a hemacytometer. Trypan blue exclusion
demonstrated that >95% of cells were viable. For tibial cultures,
the six sets of cell suspensions from each group were then reduced to
three pools of cells for each group, with each pool containing the
marrow cells from left and right tibiae from two animals from the same
group. Cell populations isolated from the left femur were cultured
individually (5 plates/animal, 6 animals/group). Cells were then added
to 10-cm tissue culture dishes at 4 × 107/dish for tibial cultures and
at 8 × 106/dish for femoral
cultures and were incubated for 24 h at 37°C in a humidified
incubator with 5% CO2. A media
change at 24 h removed the great majority of nonadherent hematopoietic
cells. The media from this point on also contained ascorbic acid (50 µg/ml) and
-glycerophosphate (10 mM, Sigma, St. Louis, MO) to promote and support bone cell differentiation. Media was changed every
2-3 days for the duration of BMSCs culture, which was extended for
up to 28 days.
RNA isolation. After 10, 15, 20, and 28 days of culture, total RNA was isolated from each pool of tibial cells with an RNA Stat-60 kit (TelTest, Friendswood, TX). RNA was diluted in diethylpyrocarbonate-treated water, and the concentration and purity was assessed with a Uvikon spectrophotometer (Research Instruments International, San Diego, CA). RNA was electrophoresed on a 1% SeaKem agarose gel (FMC Bioproducts, Rockland, ME) containing ethidium bromide and visualized under ultraviolet (UV) light to confirm its integrity. Twenty micrograms of total RNA from each pool were reverse-transcribed into cDNA using oligo(dT) primers and a GIBCO Superscript II Reverse Transcription kit (GIBCO).
Cloning of competitor cDNA templates for QC-PCR. Oligonucleotide primers were designed to produce PCR fragments of the rat genes for c-fos, AP, osteocalcin, and glyceraldehyde phosphate dehydrogenase (GAPDH). Primer sets were chosen to flank a unique restriction site within each gene of interest. Each upper strand primer was designed to include a 5' EcoR I restriction site, and each lower strand primer contained a 3' Hind III site. Upper strand primer for c-fos: 5'-TGCATGAATTCCCCAGCCGACTCCTTCTCCA-3'; lower strand primer for c-fos: 5'-TGCATAAGCTTCAGCTCCCTCCTCCGATTCC-3'; upper strand primer for AP: 5'-TGCATGAATTCCCTGCCTTACCAACTCATTT-3'; lower strand primer for AP: 5'-TGCATAAGCTTGAGAGCCACAAAGGGGAACT-3'; upper strand primer for osteocalcin: 5'-TGCATGAATTCGACCTAGCAGACACCATGAG-3'; lower strand primer for osteocalcin: 5'-TGCATAAGCTTGCTCCAAGTCCATTGTTGA-3'; upper strand primer for GAPDH: 5'-TGCATGAATTCTGATTCTACCCACGGCAAGT-3'; lower strand primer for GAPDH: 5'-TGCATAAGCTTGTCATGAGCCCTTCCACGAT-3'. Bone cell cDNA was amplified via PCR using these primer sets to produce the following PCR products: c-fos from nucleotide 230-617 [388 base pair (bp) product], AP from nucleotide 187-443 (256 bp), osteocalcin from nucleotide 15-485 (470 bp), and GAPDH from nucleotide 171-551 (380 bp). These products were gel-purified using a Qiaex II Gel Extraction Kit (Qiagen, Chatsworth, CA) and were then digested with EcoR I and Hind III. All restriction endonucleases were purchased from New England Biolabs (Beverley, MA). Digested PCR products were ligated into EcoR I/Hind III-digested pGEM-4Z plasmid (Promega, Madison, WI) with T4 DNA ligase (New England Biolabs). Ligation products were transfected into competent XL1-Blue cells (Stratagene, La Jolla, CA). Individual bacterial clones containing each PCR product within the pGEM-4Z vector were identified by EcoR I/Hind III restriction digests, and vector DNAs containing c-fos, AP, osteocalcin, and GAPDH inserts were purified with a Qiaprep Spin Plasmid Miniprep Kit (Qiagen, Chatsworth, CA). These vectors were then linearized with restriction endonucleases, which recognized unique sites within each PCR product insert. C-fos was linearized with Bgl II, AP and GAPDH were linearized with Msc I, and osteocalcin was linearized with BsaM I. A 25-bp DNA oligo with compatible ends was then ligated into the linearized vectors. This oligo, which serves as spacer DNA, contains an internal EcoR I recognition sequence and has either Bgl II, Msc I, or BsaM I sites on both ends for ligation into the appropriately digested vector. Oligo for c-fos: 5'-AGATCTATCGATGAATTCATGCATGCGATCT-3'; oligo for AP and for GAPDH: 5'-TGGCCAATGCATGAATTCATCGATGCTGGCCA-3'; oligo for osteocalcin: 5'-CATTCTGCATCCAAGGATCGATGCATT-3'. Ligation products were transfected into XL1-Blue cells as described above. Bacterial clones were identified that contained these spacer DNA oligos within the original PCR insert. Identification of these clones was facilitated by digesting the vector DNA with EcoR I/Hind III, which cuts the PCR insert out of the vector and also cleaves the PCR insert into two pieces due to the nested EcoR I site within the spacer DNA oligo. These competitor vectors were purified as described above, quantified spectrophotometrically, and diluted to 1 ng/µl.
QC-PCR. QC-PCR is a sensitive and accurate method for quantifying gene expression and is based on the coamplification of an unknown amount of wild-type cDNA with a known amount of competitor cDNA template (19). A constant amount of cDNA from each cDNA pool was added to mastermixes containing PCR Supermix (GIBCO), various known amounts of individual competitor cDNA templates, and the corresponding gene-specific primers that were used to clone the competitor cDNA templates. These primers recognize both the competitor cDNA template and the corresponding wild-type gene within the mixed cDNA pool and thus lead to the production of PCR products that differ in size by 25 bp. The ratio of the larger competitor PCR product to the smaller wild-type product reflects the relative concentration of each template in the PCR reaction. This relationship is linear within at least one order of magnitude surrounding the point of equivalency of competitor and wild-type products (unpublished data and Ref. 19). PCR amplification was performed with an EriComp Twinblock thermal cycler (EriComp, San Diego, CA), and the resulting reactions were resolved over a 3.5% NuSieve Agarose gel (FMC Bioproducts) containing 1 µg/ml ethidium bromide. For each gene, all cDNA pools were analyzed together and resolved on the same gel. Gels were photographed under UV light and scanned into an Adobe Photoshop file (Mountain View, CA) using a UC630 Max Color Scanner (UMAX Data System, Hsinchu, Taiwan). Scanned images were analyzed densitometrically using NIH Image 1.59 software, and the ratios of optical densities of the competitor and wild-type PCR products were calculated. These ratios were plotted against the amount of competitor cDNA template added. Linear regression analysis was used to calculate the concentration of wild-type product, which represents the amount of competitor cDNA template that would give a competitor-to-wild type ratio of 1.00.
The GAPDH gene was used to control for potential variations in the efficiency of reverse transcription between the various cDNA pools. GAPDH levels were determined in triplicate for all cDNA pools, and the data for c-fos, AP, and osteocalcin gene expression were then normalized to GAPDH levels and expressed as nanograms per micrograms GAPDH. Before reverse transcription, RNA aliquots were subjected to Northern blot analysis with a GAPDH probe (data not shown). This analysis demonstrated that GAPDH gene expression was not influenced by hindlimb elevation or by duration in culture and was thus a valid housekeeping gene to control for potential variations in reverse transcription efficiency between the 36 independent cDNA pools.Cell proliferation and AP activity assays. BMSCs were isolated from the tibiae of a separate group of rats (n = 12/group) that had been hindlimb elevated for 0 (control) or 5 days. Cells were combined to produce four pools of cells per group, with each pool including the left and right marrow populations from three control or hindlimb-elevated rats. Cells were added to six-well tissue culture plates at 4 × 106 cells/well under the conditions described above and cultured for up to 28 days. After various durations of culture, cultures were fixed for 1 h with 10% Formalin and rinsed with distilled water. Cell number was determined by staining with crystal violet as previously described (5). Briefly, fixed cultures were incubated at room temperature with 0.2% crystal violet in 2% ethanol for 20 min. The staining solution was then aspirated, and cultures were rinsed four times with distilled water to remove unbound stain. Specifically bound nuclear stain was then eluted with 0.2% Triton X-100, the intensity of which was measured with a spectrophotometer at 590 nm. The same destained cultures were then rinsed again with distilled water and incubated for 15 min at 37°C with 1 ml of a solution containing equal parts p-nitrophenol phosphate (1× Sigma 104 phosphatase substrate) and alkaline buffer solution (Sigma 221). The reaction was stopped by adding 0.05 N NaOH, and the AP activity was measured with a spectrophotometer at 410 nm.
In vitro mineralization. After 28 days of culture, 10-cm dishes containing femoral BMSCs were rinsed with phosphate-buffered saline and fixed for 1 h with 10% Formalin. After fixed cultures were rinsed with distilled water, they were stained for 5 min with 1% alizarin red in 2% ethanol to reveal mineral. The cultures were then rinsed five times with distilled water to remove loosely bound stain. The resulting stained nodules were too numerous to accurately quantify, so the stain was solubilized for 30 min at room temperature with 0.5 N HCl-5% sodium dodecyl sulfate (SDS). The solubilized stain was removed from the plates, and the absorbance was measured in a spectrophotometer at 415 nm. For each animal, five replicate 10-cm dishes were analyzed and averaged, and the mean values for six animals were averaged for each group. Preliminary experiments demonstrated that the absorbance was linearly related to the amount of alizarin red eluted over the range measured in these experiments.
Statistical analysis. Differences between hindlimb-elevated and normally loaded animals were determined with a two-way analysis of variance (ANOVA) using SigmaStat (Jandel Scientific Software, San Rafael, CA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Five days of hindlimb elevation led to a significant decrease in the fat-free weight of the femur (P < 0.01) and a small but significant increase in serum ionized calcium concentration (Table 1). The average body weight of each group was not different before or after hindlimb elevation. There was no significant effect of 2 days of hindlimb elevation on these parameters, although serum ionized calcium tended to be elevated.
|
QC-PCR was used to measure the levels of mRNA expression of GAPDH, c-fos, AP, and osteocalcin in each cDNA pool. The data obtained represents the average of triplicate measures of each gene in each cDNA pool. Each suspension condition consisted of three independent cDNA pools, each of which represented RNA obtained from bone marrow cells isolated from two rats. GAPDH expression, which was used to normalize data as a control for potential differences in the reverse transcription of RNA, was similar among all groups, as assessed by Northern blot analysis and by QC-PCR (data not shown). QC-PCR was then used to determine the GAPDH levels for each cDNA pool, and the expression of c-fos, AP, and osteocalcin was normalized to the GAPDH level.
QC-PCR analysis demonstrated that the expression of c-fos mRNA was significantly decreased in osteoprogenitor cells isolated from animals that were hindlimb elevated for 5 days (Fig. 1). Decreased c-fos mRNA expression was not apparent at day 10 in cultures from unloaded rats, but, at days 15 and 20, the cells from the 5-day hindlimb-elevated group had 50% lower c-fos levels. At day 28, c-fos mRNA levels in these cultures were reduced by 80% vs. controls. Two days of hindlimb elevation caused a consistent decrease in c-fos mRNA expression compared with controls, but the overall 42% decrease throughout the culture period did not reach statistical significance (P = 0.09).
|
AP mRNA expression was significantly elevated in osteoprogenitor cells isolated from 5-day hindlimb-elevated rats compared with controls (Fig. 2). Most of this increased expression was manifest during the early time points in culture (10 and 15 days), and the overall increase throughout the culture period was 61% compared with controls (P < 0.05). Two days of hindlimb elevation led to an insignificant 26% increase in AP mRNA expression compared with controls. Osteocalcin mRNA expression was decreased overall by 35% in cultures from the 5-day hindlimb group, whereas the 2-day hindlimb-elevated group showed nonsignificant reductions in osteocalcin mRNA compared with controls (Fig. 3). Because osteocalcin mRNA expression is associated with mineralization, we used an extra set of cultures in 10-cm dishes to examine in vitro mineralization. Alizarin red staining binds precipitated calcium salts and was used to reveal mineral in 28-day-old cultures. The mean absorbance of solubilized stain in the 5-day unloaded group was 40% lower than in the control group (P < 0.05), whereas the 2-day unloaded group was not significantly lower than controls (20%) (Fig. 4).
|
|
|
Five days of hindlimb elevation were sufficient to cause significant changes in the differentiation and mineralization of cultured osteoprogenitor cells. We therefore examined the effects of 5 days of hindlimb elevation on the proliferation and AP activity of these cells. Cell number was determined by crystal violet staining, and the initial plating density of the different groups (at days 3, 4, and 5 of culture) was shown to be the same in the two groups (see Fig. 5, inset). By day 7 there was a small but consistent decrease in cell number in the cultures from hindlimb-elevated rats compared with controls, and this difference tended to get larger over time. The cultures from hindlimb-elevated rats reached quiescence by day 21, as evidenced by a plateau of the growth curve, whereas, in the control cultures, cell number was still increasing at day 28. The decreased cell number in cultures from hindlimb-elevated rats was significant (P < 0.05, 2-way ANOVA). These same cultures were then assayed for AP activity in a novel adaptation of the standard Sigma colorimetric enzyme activity assay. The standard assay involves solubilizing the cells with detergent, measuring enzyme activity, and expressing activity as a function of total protein or another variable. The cell membrane-associated enzyme activity was found to be stable in fixed cultures before and after crystal violet staining and destaining, so fixed cultures were incubated with AP substrate without solubilizing the cells. AP activity was then expressed by dividing the absorbance of liberated phenol (410 nm) by the previously determined absorbance of crystal violet (590 nm). Hindlimb elevation led to a significant decrease in AP activity at all time points compared with control cultures (Fig. 6A). When AP activity is expressed on a per cell basis, the cultures from hindlimb-elevated rats are still significantly lower than controls (Fig. 6B).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gravity induces mechanical loading in weight-bearing bones, which is necessary for the long-term maintenance of normal skeletal architecture. The nature of the biological signal that mediates mechanotransduction in bone is poorly understood, but it is clear that skeletal unloading significantly alters bone metabolism. Skeletal unloading decreases osteoblast number, bone formation rate, bone mass, bone maturation, and mechanical strength (3, 6, 7, 15, 22, 25, 26). Cells of the osteoblast lineage are responsible for bone formation and are known to respond to mechanical loading (8, 14). These cells, which include osteoprogenitor cells, osteoblasts, and osteocytes, are the most conspicuous candidates for mediating the skeletal response to unloading. It is important to elucidate the biological response of these cells to skeletal unloading before one can understand the nature of the mechanical signals to which they respond.
We have hypothesized that skeletal unloading causes decreases in osteoblast proliferation and differentiation, which leads to a decrease in osteoblast numbers and a shift within the osteoblast population to cells that are less capable of producing mature, well-mineralized bone matrix. Several studies have suggested that skeletal unloading decreases osteoblast proliferation. The decreased number of osteoblasts observed in unloaded bone (7) may indicate a decrease in the proliferation or recruitment of osteoblast precursors from bone marrow or from the periosteum. In fact, BMSCs isolated from unloaded bone have been shown to proliferate more slowly than BMSCs from normally loaded bone (Refs. 12 and 27 and present study). These results could reflect deficits in the absolute number or recruitment of osteoblast precursors or decreased proliferation of committed osteoprogenitor cells. A previous study reported that skeletal unloading led to a significant decrease in osteoprogenitor cell number as early as day 6 of culture, but that thymidine incorporation was not different from controls at this time point (9). The present data suggest that the number of potential osteoprogenitor cells is not different in unloaded bone, because the number of adherent stromal cells measured in culture immediately after isolation from unloaded bone is virtually identical to control bone. The sensitivity of the crystal violet assay permits the quantification of as few as 500 cells (5), and as such we are able to accurately measure cell number before they begin to proliferate. This is demonstrated by a lack of change in cell number from day 3 to day 5 in culture. Proliferation is evident by day 7, which coincides with the onset of decreased cell number in cultures of hindlimb-elevated rats. In addition to a decrease in proliferation rate, the total proliferative potential of cells from unloaded bone may also be reduced, as evidenced by an earlier plateau of the growth curve (at 21 days in cultures from hindlimb-elevated rats, and >28 days in control cultures).
The decreased proliferation of osteoprogenitor cells isolated from unloaded bone is consistent with the observed decrease in the expression of c-fos, a gene that is associated with osteoblast proliferation (11, 17). The unloading-induced decrease in c-fos expression suggests that the decreased proliferative activity of BMSCs occurs throughout their differentiation pathway and is not restricted to the earliest stages of osteoprogenitor recruitment. The 80% decrease in c-fos expression at day 28 in cultures from 5-day hindlimb-elevated rats is further evidence that these cells may have reduced proliferative potential and may reach quiescence earlier than controls.
Immature osteoblasts synthesize organic bone matrix, which is then mineralized as differentiation progresses. Immature osteoblasts are characterized by their high levels of type I collagen production and high AP gene and protein expression (1, 13, 17, 24). Inhibition of osteoblast differentiation may be manifest by a relative increase in the population of these immature cells. Weightlessness has been shown to inhibit the differentiation of osteoblasts in vivo (20, 21), and this inhibition may explain changes that occur at the whole bone level after skeletal unloading. These changes include increased collagen concentration (16), decreased mineralization (6, 15, 25), decreased calcium-to-hydroxyproline ratio (6, 23), and increased AP mRNA in unloaded bone (4). In the present study, we have demonstrated a specific increase in level of AP mRNA expression in cultured BMSCs isolated from tibiae that were unloaded for 5 days. This result is consistent with the increased level of AP mRNA observed in whole rat tibiae unloaded by spaceflight and by hindlimb elevation (4).
AP enzyme activity was reduced by hindlimb elevation, both at the level of the whole culture and on a per-cell basis. AP activity has been previously reported to be reduced in cells isolated from femurs that were unloaded by sciatic neurectomy (9). In the present study, the apparent discepancy between AP mRNA expression and AP enzyme activity has several potential explanations. The increased AP mRNA induced by unloading may not be quantitatively translated into protein, or the catalytic activity of the AP protein may be inhibited after unloading. Furthermore, AP mRNA levels have been previously shown to be influenced by an inducible mRNA-stabilizing factor (10), indicating a complex system of posttransciptional regulation of AP. It is tempting to speculate that the regulation of AP enzyme activity may contribute to the osteoblast response to unloading.
The ability to form mineralized matrix in culture is perhaps the most important index of well-differentiated osteoblasts. The increased expression of AP mRNA and decreased expression of osteocalcin mRNA in unloaded bones suggests that a greater proportion of osteoblasts remain in the early stage of matrix synthesis and are delayed in their normal progression to active mineralization. We addressed this question by staining 28-day femoral BMSC cultures with alizarin red, which reveals mineralized bone cell nodules. BMSCs isolated from 5-day unloaded femurs formed 40% less mineral compared with controls, which indicates that skeletal unloading in vivo leads to impaired mineralization in vitro. This result is consistent with in vivo data demonstrating that skeletal unloading causes a decreased mineralization and an increased calcium-to-hydroxyproline ratio (6, 23, 25). These data support the hypothesis that skeletal unloading decreases osteoblast differentiation and demonstrate the utility of this model for studying the effects of weightlessness on osteoblast differentiation.
These data contrast with those obtained by Machwate et al. (12), who reported that 14 days of hindlimb elevation did not alter the osteoblast phenotype in vitro. In our study, animals were hindlimb elevated for 2 or 5 days. Previous studies indicate that the effects of skeletal unloading on bone formation in the growing rat are transient. After 5 days of hindlimb elevation, bone formation, calcium accumulation in bone, and serum 1,25(OH)2D3 concentration are decreased. However, these parameters all return toward normal levels after 12-14 days of continuous hindlimb elevation, indicating a recovery of bone formation despite continued unloading in these young animals (6, 7). Therefore, osteoblasts isolated from tibiae after 14 days of hindlimb elevation might not be expected to differ substantially from control cells.
In conclusion, we have demonstrated that the osteoprogenitor cells isolated from the tibiae of rats that were hindlimb elevated for 5 days expressed significantly less c-fos mRNA, more AP mRNA, and less osteocalcin mRNA than did cells from normally loaded tibiae. Changes in gene expression with 5 days of unloading were also accompanied by decreases in proliferation, AP activity, and mineralization compared with control cultures. The changes in osteoblast gene expression and phenotype after unloading are consistent with a shift toward a less mature osteoblast population, suggesting an inhibition of osteoprogenitor cell differentiation with skeletal unloading. These data also demonstrate that cultured osteoprogenitor cells retain a "memory" of their prior in vivo loading history, indicating that this model is valuable for studying the effects of skeletal unloading on osteoblast function in vitro.
![]() |
ACKNOWLEDGEMENTS |
---|
This study was supported by National Aeronautics and Space Administration Grant NAGW-4460.
![]() |
FOOTNOTES |
---|
Address for reprint requests: D. D. Bikle, Veterans Administration Medical Center (111N), 4150 Clement St., San Francisco, CA 94121.
Received 13 December 1996; accepted in final form 12 August 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aronow, M.,
L. Gertenfeld,
T. Owen,
M. Tassinari,
G. Stein,
and
J. Lian.
Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvarial cells.
J. Cell. Physiol.
143:
213-221,
1990[Medline].
2.
Barengolts, E.,
T. Kouznetsova,
A. Segalene,
P. Lathon,
C. Odvina,
S. Kukreja,
and
T. Unterman.
Effects of progesterone on serum levels of IGF-I and on femur IGF-I mRNA in ovariectomized rats.
J. Bone Miner. Res.
11:
1406-1412,
1996[Medline].
3.
Bikle, D.,
B. Halloran,
C. Cone,
R. Globus,
and
E. Morey-Holton.
The effects of simulated weightlessness on bone maturation.
Endocrinology
120:
678-684,
1987[Abstract].
4.
Bikle, D.,
J. Harris,
B. Halloran,
and
E. Morey-Holton.
Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation.
Am. J. Physiol.
267 (Endocrinol. Metab. 30):
E822-E827,
1994
5.
Gillies, R.,
N. Didier,
and
M. Denton.
Determination of cell number in monolayer cultures.
Anal. Biochem.
159:
109-113,
1986[Medline].
6.
Globus, R.,
D. Bikle,
and
E. Morey-Holton.
The temporal response of bone to unloading.
Endocrinology
118:
733-742,
1986[Abstract].
7.
Halloran, B.,
D. Bikle,
T. Wronski,
R. Globus,
M. Levens,
and
E. Morey-Holton.
The role of 1,25-dihydroxyvitamin D in the inhibition of bone formation induced by skeletal unloading.
Endocrinology
118:
948-954,
1986[Abstract].
8.
Harter, L.,
K. Hruska,
and
R. Duncan.
Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation.
Endocrinology
136:
528-535,
1995[Abstract].
9.
Keila, S.,
S. Pitaru,
A. Grosskopf,
and
M. Weinreb.
Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro.
J. Bone Miner. Res.
9:
321-327,
1994[Medline].
10.
Kyeyune-Nyombi, E.,
K. W. Lau,
D. Baylink,
and
D. Strong.
1,25-Dihydroxyvitamin D3 stimulates both alkaline phosphatase gene transcription and mRNA stability in human bone cells.
Arch. Biochem. Biophys.
291:
316-325,
1991[Medline].
11.
Lean, J. M.,
A. G. MacKay,
J. W. M. Chow,
and
T. J. Chambers.
Osteocytic expression of mRNA for c-fos and IGF-I: an immediate early gene response to an osteogenic stimulus.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E937-E945,
1996[Medline].
12.
Machwate, M.,
E. Zerath,
X. Holy,
M. Hott,
D. Modrowski,
A. Malouvier,
and
P. J. Marie.
Skeletal unloading in rat decreases proliferation of rat bone and marrow-derived osteoblastic cells.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E790-E799,
1993
13.
Malaval, L., D. Modrowski, A. Gupta, and J. Aubin. Cellular
expression of bone-related proteins during in vitro osteogenesis in rat
bone marrow stromal cell cultures. J. Cell. Physiol.
555-572, 1994.
14.
Mikuni-Takagaki, Y.,
Y. Suzuki,
T. Kawase,
and
S. Saito.
Distinct responses of different populations of bone cells to mechanical stress.
Endocrinology
137:
2028-2035,
1996[Abstract].
15.
Morey, E.,
and
D. Baylink.
Inhibition of bone formation during space flight.
Science
201:
1138-1141,
1978[Medline].
16.
Patterson-Buckendahl, P.,
S. B. Arnaud,
G. L. Mechanic,
R. B. Martin,
R. E. Grindeland,
and
C. E. Cann.
Fragility and composition of growing rat bone after one week in spaceflight.
Am. J. Physiol.
252 (Regulatory Integrative Comp. Physiol. 21):
R240-R246,
1987
17.
Pockwinse, S.,
L. Wilming,
D. Conlon,
G. Stein,
and
J. Lian.
Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue-like organization in primary osteoblast cultures.
J. Cell. Biochem.
49:
310-323,
1992[Medline].
18.
Rambaut, P.,
C. Leach,
and
G. Whedon.
A study of metabolic balance in crewmembers of Skylab IV.
Acta Astronaut.
6:
1313-1322,
1979.[Medline]
19.
Reiner, S.,
S. Zheng,
D. Corry,
and
R. Locksley.
Constructing polycompetitor cDNAs for quantitative PCR.
J. Immunol. Methods
165:
37-46,
1993[Medline].
20.
Roberts, W. E.,
P. J. Fielder,
L. M. L. Rosenoer,
A. C. Maese,
M. R. Gonsalves,
and
E. R. Morey.
Nuclear morphometric analysis of osteoblast precursor cells in periodontal ligament, SL-3 rats.
Am. J. Physiol.
252 (Regulatory Integrative Comp. Physiol. 21):
R247-R251,
1987
21.
Roberts, W., P. Mozsary, and E. Morey. Suppression of
osteoblast differentiation during weightlessness.
Physiologist 24(6) Suppl.:
S75-S76, 1981.
22.
Shaw, S. R.,
A. C. Vailas,
R. E. Grindeland,
and
R. F. Zernicke.
Effects of a 1-wk spaceflight on morphological and mechanical properties of growing bone.
Am. J. Physiol.
254 (Regulatory Integrative Comp. Physiol. 23):
R78-R83,
1988
23.
Simmons, D.,
J. Russell,
and
M. Grynpas.
Bone maturation and quality of bone material in rats flown on the space shuttle "Spacelab-3 Mission".
Bone Miner.
1:
485-493,
1986[Medline].
24.
Strauss, P.,
E. Closs,
J. Schmidt,
and
V. Erfle.
Gene expression during osteogenic differentiation in mandibular condyles in vitro.
J. Cell Biol.
110:
1369-1378,
1990[Abstract].
25.
Van Loon, J.,
D. Bervoets,
E. Burger,
S. Dieudonne,
J. Hagen,
C. Semeins,
E. Doulabi,
and
J. Veldhuijzen.
Decreased mineralization and increased calcium release in isolated fetal mouse long bones under near weightlessness.
J. Bone Miner. Res.
10:
550-557,
1995[Medline].
26.
Westerlind, K.,
and
R. Turner.
The skeletal effects of spaceflight in growth rats: tissue-specific alterations in mRNA levels for TGF-.
J. Bone Miner. Res.
10:
843-848,
1995[Medline].
27.
Zhang, R.,
S. Supowit,
G. Klein,
Z. Lu,
M. Christensen,
R. Lozano,
and
D. Simmons.
Rat tail suspension reduces messenger RNA level for growth factors and osteopontin and decreases the osteoblastic differentiation of bone marrow stromal cells.
J. Bone Miner. Res.
10:
415-423,
1995[Medline].