From the
This study evaluated a rapid biomineralization phenomenon
exhibited by an osteoblastic cell line, UMR 106-01 BSP, when
treated with either organic phosphates [
The ability to form an extracellular matrix that can undergo
regulated mineralization is the ultimate phenotypic expression of an
osteogenic tissue. Two patterns of mineral deposition on an
extracellular matrix have been described: (i) matrix vesicle-mediated
mineral initiation
(1, 2, 3, 4, 5) and (ii) heterogeneous nucleation of mineral crystals on
collagen
(6, 7, 8, 9, 10) ,
perhaps with the involvement of some noncollagenous glycoproteins that
bind Ca
Understanding the
various stages of mineral formation and subsequent propagation of the
mineral phase is hindered by the inability to describe the initial
events of the organic-inorganic interactions. An osteogenic cell
culture model that initiates biomineralization in a rapid and
reproducible manner would greatly facilitate exploring the variables
that regulate the initiation of mineralization. In this report, we
present data demonstrating such a model system.
The rat osteoblastic
cell line, UMR 106-01 BSP,
The current study demonstrates that cultured UMR cells
rapidly form an apatitic-like, calcium-phosphate mineral associated
with both the cells and their surrounding extracellular matrix. Data
are presented that identify some of the biological requirements for
this mineralization process. Potential mechanisms which may account for
this cell line's ability to form apatitic mineral in vitro are discussed.
All drugs were prepared
as sterile working solutions in growth medium on the day of the
experiment. Ascorbate was prepared at a concentration of 50 µg/ml
in growth medium and was added from freshly prepared stocks at each
medium change. Levamisole was prepared as a 1 m
M stock
solution in growth medium, serially diluted in growth medium, and added
to cultures simultaneously with the phosphate supplements.
Cycloheximide was prepared as a 50 µg/ml stock solution in growth
medium and added to cultures 1 h prior to adding the phosphate
supplement. Preliminary experiments indicated that this dose of
cycloheximide-inhibited protein synthesis to <5% of normal levels
within 10 min of exposure. Actinomycin C
The size of the apatite
crystals along the 002 diffraction vector was calculated from the width
of the 002 peak at half-maximal height using the Scherrer equation
(31) : D = K
Furthermore, the role of
protein processing and secretion in this apatite mineral formation was
explored using brefeldin A. At a concentration of 1 µg/ml, this
agent selectively disrupts the Golgi apparatus and induces a rapid
redistribution of the Golgi into the endoplasmic reticulum
(40, 41, 42) . Protein synthesis is not
significantly inhibited by short exposure to brefeldin A at this dose
(43) . The metabolic consequences of this drug are that newly
synthesized proteins are not properly modified, assembled, and secreted
from the cell. UMR cultures treated with brefeldin A for 13 h revealed
a substantial reduction in the amount of apatitic mineral produced in
the cell layer (16% of the control value, ). Thus, apatite
mineral formation by these osteoblastic cells may also involve protein
processing and/or secretion. The residual mineral formation in the
treated cultures may be the result of a limited amount of crystal
nucleation on an established extracellular matrix, or may suggest the
presence of potential apatite nucleators stored in limited amounts
within the cells which are secreted during the treatment period.
Numerous groups have reported the addition of
organophosphates (typically
The biological relevance of this calcification process is
supported by several critical observations. First, XRD and chemical
analyses confirmed the calcium phosphate mineral produced by UMR cells
is bio-apatitic resembling that of normal bone in terms of its crystal
size and Ca/P ratio. Second, in addition to the phosphate dose- and
time-dependent behavior, the ability of UMR cells to form apatitic
mineral was greatly suppressed by standard agents which block RNA
transcription or inhibit the synthesis, processing, or secretion of
proteins. Thus, this culture system forms most of its apatitic mineral
as a result of a metabolic production and secretion of competent
hydroxyapatite nucleators during the period of phosphate treatment.
However, this study can not exclude the possibility that a portion of
the mineral formed in these cultures might be the result of a direct
heterogeneous nucleation within the established extracellular matrix of
the cell layer. Lastly, morphologic evidence demonstrated a large
number of extracellular mineral deposits on the basal surface of the
cells that contained crystalline structures associated with a fibrous
organic matrix; relatively few deposits were detected on the apical
surface of the cells. Altogether, these data suggest this
mineralization process is not a dystrophic reaction. Rather, it appears
to be a metabolic process involving the production of apatite
nucleators, a vectoral secretion of these nucleators, and the
initiation of apatite crystals in association with these structures.
Similarities exist between the mineralization process in UMR and
primary osteoblastic cultures. First, the amount of apatitic mineral
deposited in the cell layer is dependent on the dose and exposure time
of the phosphate source. Second, alkaline phosphatase is required to
hydrolyze organophosphates in order to stimulate a mineralization
reaction; inhibition of this enzyme's activity by levamisole
totally blocked the stimulatory activity of all organophosphates
tested. Third, low doses of PP
Differences were observed between
the mineralization process in UMR versus normal osteoblastic
cultures. First, the time frame of mineral formation is shorter in UMR
cultures than in primary cultures. This difference is best explained by
the UMR system lacking the extended proliferative and differentiation
stages exhibited by primary culture systems. The UMR cells manifest a
fully differentiated phenotype and appear to be constitutively
competent to produce apatitic mineral when they reach a confluent state
in vitro. Second, UMR cells produce apatitic mineral without
necessarily establishing in vitro ``nodules'' of
multicellularity which are characteristic of primary-derived cells
(52) . In primary osteoblastic culture systems, nodule formation
typically represents the clonal proliferation of osteogenic cells from
within a heterogeneous population of primary cells. The lack of a
requisite nodular morphology for mineralization in UMR cultures is
likely the result of the uniform expression of a mature osteoblast-like
phenotype in this cell line. However, UMR cells do appear to require a
confluent state in order to generate a substantial calcification
response implying a role for cell-cell contact in the mineralization
process. Third, UMR cells are capable of utilizing P
The mineral phase
created by these cells has not been fully evaluated for relevancy to
the collagen-based propagative phase of mineral formation. UMR cells do
not appear to form an extensive collagenous matrix in this short term
culture model. Although mineral crystals were rarely seen attached
directly to collagen, some of the apatitic mineral deposits were
observed in close proximity to collagen fibrils. It is possible the
mineralization phenomenon occurring in these short term cultures
reflects the very early stages of apatite nucleation which have not
progressed sufficiently to transfer or seed mineral crystals onto
collagen fibrils. Therefore, the application of this biomineralization
model for propagation of apatitic mineral onto collagen needs to be
further evaluated using longer incubation periods. Additionally, matrix
vesicles were not readily apparent in these mineralizing cultures.
Indeed, TEM analysis revealed electron dense structures whose
appearance and location within the cells are not characteristic of
matrix vesicles. Thus, further investigation is required to determine
whether any mineral formed in this system involves matrix
vesicle-initiated calcification.
Matrix vesicle- or
collagen-mediated models do not fully account for all aspects of the
apatite mineral formation observed in UMR cultures. Therefore,
alternative mechanisms must be proposed to adequately explain this
biological process. As the metabolic inhibitor experiments
demonstrated, the established extracellular matrix of the cell layer
was a relatively poor initiator of mineral formation even in the
presence of high phosphate concentrations. Thus, a mechanism for this
mineralization process involves the metabolic synthesis and secretion
of competent apatite nucleators during the period of phosphate
exposure. This implies the cells exert a tremendous control over this
process. TEM analyses and brefeldin A treatment suggest the assembly of
these nucleators takes place within intracellular locations. Further,
these structures appear to nucleate apatite crystals soon after they
emerge from the cells. TEM data even suggest the novel possibility that
the nucleation event may occur to some extent within intracellular
locations. In either case, with progressive time outside the cells,
these discrete nucleation structures appear to aggregate and coalesce
into larger mineral deposits which are retained within the
extracellular matrix of the cell layer.
Bone matrix consists of a
collagenous scaffold interlaced with non-collagenous proteins which are
suggested to influence mineral formation
(11, 12, 13, 55) . One of these
glycoproteins, BSP, initiates hydroxyapatite nucleation when the
protein is bound to a solid-phase agarose gel system
(14, 15) . Further, the presence of BSP has been
localized specifically within mineralized tissues
(56) and
osteoid matrix
(16, 57) . At the ultrastructural level,
BSP has been localized within the early mineral accretions deposited in
osteoid and near the basal surface of osteoblasts
(16) . UMR
106-01 BSP cells produce relatively large amounts of BSP in
vitro (28) . Perhaps the rapid mineralization response of
the UMR culture system may be a result of the synthesis and
incorporation of BSP into the proposed nucleator structures described
above. If true, then BSP would be arranged in a conformation competent
to nucleate apatite crystals. This structural arrangement would
contrast with the possible conformations of this protein in solution
because purified BSP did not nucleate detectable apatite crystals when
added to culture medium.
The potential advantage of this system as a
model for a mature osteoblastic cell-type lies in the rapid,
reproducible manner of apatitic mineral formation. This provides a
feasible model to explore the potential regulatory effects of hormones,
growth factors, and other agents (such as mineralization-dependent
ions) on the initiation of this bio-apatitic mineralization process.
Those elements that reveal potent regulation of this calcification
process would then be tested for relevancy to the mineralization in
normal osteoblastic models.
UMR 106-01 BSP cells were inoculated (
UMR 106-01 BSP cells were inoculated
(
We thank Gail Kurriger for histological guidance, Jim
Morgan for SEM, and John Laffoon for TEM analysis. We also express our
gratitude to Drs. Alan Goodridge and James Martin for their reviews of
this manuscript prior to submission.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glycerophosphate
(
-GP), Ser-P, or Thr-P], inorganic phosphate (P
),
or calcium. In a dose-dependent manner, these agents (2-10
m
M) stimulated confluent cultures to deposit mineral in the
cell layer (ED
of
4.6 m
M for
-GP (30
± 2 nmol Ca
/µg DNA) and
3.8
m
M (29 ± 2 nmol Ca
/µg DNA) for
P
) with a plateau in mineral formation by 20 h (ET
12-15 h).
-GP or P
treatment yielded
mineral crystals having an x-ray diffraction pattern similar to normal
human bone. Alizarin red-S histology demonstrated calcium mineral
deposition in the extracellular matrix and what appeared to be
intracellular paranuclear staining. Electron microscopy revealed small,
needle-like crystals associated with fibrillar, extracellular matrix
deposits and intracellular spherical structures. Mineral formation was
inhibited by levamisole (ED
250 µ
M),
pyrophosphate (ED
1-10 µ
M),
actinomycin C
(500 ng/ml), cycloheximide (50 µg/ml), or
brefeldin A (1 µg/ml). These results indicate that UMR 106-01
BSP cells form a bio-apatitic mineralized matrix upon addition of
supplemental phosphate. This process involves alkaline phosphatase
activity, on-going RNA and protein synthesis, as well as Golgi-mediated
processing and secretion.
and collagen
(11, 12, 13) . Recently, attention has been
focused on a bone-specific glycoprotein known as bone sialoprotein
(BSP)
(
)
which nucleates hydroxyapatite formation
in vitro (14, 15) and is localized at sites of
early mineral formation in rat bone
(16) .
(
)
was isolated
from an induced transplantable osteosarcoma and shares a number of
phenotypic properties with mature osteoblasts. These similarities
include morphological appearance
(17, 18, 19) ,
responsiveness to calciotropic agents such as parathyroid hormone
(17, 19, 20, 21, 22, 23) and 1,25-(OH)
vitamin D
(24) , and a relatively high level of expression of cell
surface alkaline phosphatase activity
(19, 21) .
Additionally, UMR cells synthesize several matrix proteins expressed by
normal osteoblasts including type I collagen
(25) ,
proteoglycans
(26, 27) , and BSP
(28) . UMR cells
are distinguished from primary osteoblasts by their relatively high
expression level of the oncogene H- ras, which is thought to
induce the transformed state of these cells
(29) . When
transplanted into a host animal, UMR cells can form bone mineral
trabeculae in ectopic sites
(18) demonstrating an osteogenic
property.
Materials
Materials and reagents utilized in
this study were of the highest grade commercially available. Tissue
culture media were obtained from either Sigma or Life Technologies,
Inc.; fetal bovine serum was obtained from HyClone Laboratories; type I
collagen, gelatin, fibronectin, 100 nonessential amino acid
solution, 1
M HEPES, pH 7,
-glycerophosphate,
O-phospho-
L-serine,
O-phospho-
L-threonine, levamisole, alizarin red-S,
cetylpyridinium chloride, ascorbate (sodium salt), and tetrasodium
pyrophosphate were obtained from Sigma. Cycloheximide, actinomycin
C
, and brefeldin A were from Calbiochem. BSP was prepared
from UMR 106-01 BSP cultures as described
(28) . All
cultureware was from either Falcon/Becton Dickinson or Costar.
Millicell-HA culture inserts (420 mm
; 0.45 µm porosity)
were from Millipore. Paraffin (Ameraffin) was from Baxter Diagnostics.
EM supplies were from Ted Pella.
Cell Culture
UMR 106-01 BSP cells were
routinely passaged in T-75 culture flasks and cultured in Eagle's
minimum essential medium (EMEM) plus nonessential amino acids, 20
m
M HEPES, pH 7.2, and 10% fetal bovine serum (growth
medium) as described previously
(28) . The normal calcium
and Pconcentrations for EMEM are 1.8 m
M and 1
m
M, respectively. Cultures were grown at 37 °C in a
humidified 5% CO
atmosphere with routine passage every 3
days. Experimental cultures were created by briefly washing the
confluent cell layer with Hank's balanced saline solution without
Ca
or Mg
followed by trypsinization
of the cells (10 ml of 0.05% trypsin + 0.53 m
M EDTA in
Hank's at 37 °C for 10 min). Following formation of a
dispersed cell suspension, cells were counted on a hemacytometer, and
plated at 2000 cells/mm
into 35-mm tissue culture dishes
(960 mm
) or 12-well cluster plates (380 mm
).
Data in Fig. 9were generated using microdot cultures
(30) : UMR cells were plated at 5000 cells/mm
in
10-µl droplet cultures; four microdot cultures were plated per
35-mm dish followed by adding 2 ml of growth medium/plate 1 h later.
Unless otherwise stated, cultures were routinely incubated for 48 h in
growth medium followed by an incubation in fresh medium (with or
without a phosphate supplement) for an additional 24 h. Sources of
organophosphates (
-glycerophosphate (
-GP), phosphoserine
(Ser-P), or phosphothreonine (Thr-P)) or inorganic phosphate
(P
) were prepared as sterile, 0.5
M solutions in
water at neutral pH; pyrophosphate (PP
) was prepared as a
0.1
M solution in water at neutral pH. Aliquots of these
phosphate agents were added directly to medium solutions just prior to
their addition to the cultures.
Figure 9:
PP blocks the mineralization
response. UMR microdot cultures (5000 cell/mm
) were grown
for 48 h prior to changing the medium to one containing 5 m
M
-GP and the respective PP
concentration.
Inset, PP
inhibits mineral formation with exposure
to 10 m
M P
; horizontal axis represents
[PP
] in µ
M. Under these conditions,
cell number was identical in treated and control cultures. Mean and
S.D. ( n = 12/group) representative of two
trials.
A series of control experiments were
performed to determine whether a spontaneous crystallization occurs in
culture medium containing Por organophosphate supplements
at 4 or 10 m
M. Culture dishes were precoated (37 °C for 1
h) with PBS alone or 5 µg/ml solutions of the following proteins in
PBS: type I collagen, gelatin, or fibronectin. Growth medium (with or
without phosphate supplements) was added to these culture dishes in the
absence of cells and stored for 24 h at 37 °C in the CO
incubator. Following incubation, the media were centrifuged (1500
g for 10 min) while the dishes were processed for
alizarin red-S staining to quantify calcium mineral content as
described below. Alizarin red staining of the culture dishes (precoated
or not) was not detected, and crystalline pellets were not observed in
the various media following centrifugation.
was stored
foil-wrapped at -70 °C as a 1 mg/ml solution in absolute
ethanol. This actinomycin stock was diluted in growth medium to a
working concentration of 500 ng/ml and added to cultures 1 h prior to
adding the phosphate supplement. Preliminary experiments indicated that
this dose of actinomycin inhibited RNA synthesis to <10% of normal
levels within 20 min of exposure. Brefeldin A was resuspended in
absolute ethanol at 1 mg/ml and stored at -20 °C. This stock
was diluted in growth medium to a working concentration of 1 µg/ml
and added to cultures 1 h prior to the phosphate supplement.
Dulbecco's modification of Eagle's medium (DMEM) without
CaCl
(Life Technologies, Inc.) was used for the experiments
analyzing the role of Ca
by supplementing with
CaCl
to 0.5, 1.0, 2.5, and 5.0 m
M Ca
levels. Unless otherwise indicated, experimental data are
reported as mean ± S.D.; n = 3/trial; data
representative of at least two trials.
Alizarin Red-S Histology
UMR cells were inoculated
into Millicell HA culture inserts at 2000 cells/mmand
cultured for 48 h in growth medium. At this time, fresh growth medium
in the presence or absence of an additional 5 or 10 m
M P
(or
-GP) was added to the cultures which were incubated for
a further 24 h. At the end of the incubation, the medium was removed
and the culture inserts were briefly washed with phosphate-buffered
saline (PBS) followed by fixation in ice-cold 70% ethanol (minimum
fixation time of 1 h). After fixation, the ethanol was removed, and the
fixed cell layers (attached to the filter membranes) were rinsed with
Nanopure water (Bioresearch Nanopure System, Barnstead/Thermolyne) then
stained with 40 m
M alizarin red-S (AR-S), pH 4.2, for 10 min
at room temperature. Stained cell layers were further processed by five
rinses with Nanopure water followed by a 15-min wash with PBS (which
removes most of the nonspecific stain not associated with calcium
mineral deposits). Stained cell layers were then dehydrated with a
series of ethanol solutions followed by xylene. Dehydrated cell layers
on the filter membranes were embedded in paraffin, sectioned (7 µm)
and mounted on glass slides for brightfield microscopy.
Scanning Electron Microscopy
Cultures were fixed
(3% (v/v) glutaraldehyde-formaldehyde in 0.1
M cacodylate
buffer, 10 min) followed by dehydration with graded acetone and
critical point drying using a COgas/liquid technique
(Balzer CPD 030). Samples were then sputter-coated with 25-nm Au/Pd
(Balzer SCD 040) prior to viewing in an Amray 1820D SEM.
Transmission Electron Microscopy
Cultures were
fixed (3% (v/v) glutaraldehyde-formaldehyde in 0.1
M
cacodylate buffer) for 1 h. Following rinsing in buffer and dehydration
in a graded ethanol solution series, cultures were removed from the
surface of the plastic dish with propylene oxide and embedded in
Spurr's plastic. Following trimming, sections were placed on
copper grids, stained with uranyl acetate, and examined in a Zeiss EM
10 TEM.
X-ray Diffraction
Cultures were established in
150-mm culture plates (2000 cells/mm), grown for 48 h in
growth medium, and then switched to growth medium supplemented with
either 8 m
M P
or 8 m
M
-GP. Cultures
were incubated for a further 72 h with fresh media changes every 24 h.
After aspirating the medium and washing with PBS, the cell layers were
scraped, washed in ice-cold ammoniated Nanopure water, and pelleted by
centrifugation (1500
g, 10 min). The pellets were
washed three times with 70% ethanol and lyophilized prior to analysis
(each sample had a dry weight of
30 mg). X-ray diffraction
patterns were recorded with a Rigaku x-ray Diffractometer equipped with
a graphite monochromator calibrated to CuK
radiation (
= 0.154 nm). A scintillation counter detector coupled to a
linear ratemeter was used for data collection. Recordings were made in
a continuous scan mode between 23 and 37° 2
(where 2
= the scattering angle) at an angular velocity of 0.25°
2
/min. The modulated (10 s time constant) analog output signal
from the ratemeter was converted at 0.01° intervals for plotting.
The 002 apatite peak (between 24.7 and 27° 2
) was recorded a
minimum of four times at an angular velocity of 0.125° 2
for
x-ray line broadening analysis
(31) .
(57.3)/
cos
= 8.15/
, where D is the crystal size, K is a geometric shape factor with a value of 0.9 for half-maximum
line breadths,
is the x-ray wavelength (0.154 nm), 57.3 is the
radian-degree conversion factor,
is half the diffraction angle
for the 002 peak (12.94°), and
is the line width corrected
for instrumental effects using Warren's relation
(31) :
= ( B
-
b
)
. In the latter equation, B is the measured width and b is the instrumental width as
measured from the 002 peak of a well-crystallized hydroxyapatite having
no intrinsic broadening. After x-ray diffraction analysis, the pellets
were analyzed for Ca
by atomic absorption
spectrophotometry (see description below) and P
by the UV
spectrophotometric method of Murphy and Riley
(32) .
Alizarin Red-S Assay to Quantify Calcium Mineral
Content
Alizarin red-S (AR-S) is a dye which binds selectively
to calcium salts and is widely used for calcium mineral histochemistry
(33) . AR-S binds 2 mol of Ca
/mol of dye
in solution
(34) . At the end of each experiment, cultures were
briefly rinsed with PBS followed by fixation (ice-cold 70% ethanol, 1
h). Cultures were rinsed with Nanopure water and stained for 10 min
with 40 m
M AR-S, pH 4.2, at room temperature with rotation (1
ml/35-mm dish). Cultures were then rinsed five times with water
followed by a 15-min wash with PBS (with rotation) to reduce
nonspecific AR-S stain. Stained cultures were photographed followed by
a quantitative destaining procedure using 10% (w/v) cetylpyridinium
chloride (CPC) in 10 m
M sodium phosphate, pH 7.0, for 15 min
at room temperature. Aliquots of these AR-S extracts were diluted
10-fold in the 10% CPC solution, and the AR-S concentration determined
by absorbance measurement at 562 nm on a multiplate reader
( V
; Molecular Devices) using an AR-S standard
curve in the same solution. Values were normalized to total DNA as
described below.
Atomic Absorption
Following exposure to the
respective reagents, cultures were washed twice with Saline G (with
Caand Mg
), followed by extraction
of cell layer mineral with a 24-h exposure to 0.6
N HCl in
0.02
M PBS (Ca
and Mg
free) at room temperature. PBS was used as the diluent for the
HCl because it provided an iso-osmolar solution to prevent cellular
lysis; the final pH of this solution was
2.0, and it
quantitatively removed extracellular calcium from the cultures as
assayed by AR-S histochemistry. Aliquots were then added to a solution
of 2.5% (w/v) lanthanum oxide in 25% (v/v) HCl followed by atomic
absorption analysis of calcium content using a Perkin Elmer model 2380
atomic absorption spectrophotometer optimized to 422.7 nm, slit width
= 0.7 nm. The AA Lamp (0.125 nmol/ml detection limit) was
optimized with a calcium standard curve (2.5-1200 nmol/ml) using
a serial dilution of a calcium atomic absorption standard (Sigma,
C-5649). Values were normalized to total DNA as described below.
DNA Assay
DNA was assayed in cultures separate
from those processed for Camineral determination.
Ethanol-fixed cultures were rinsed with Nanopure water and then
solubilized with a solution of 10
M formamide, 1% (w/v) sodium
dodecyl sulfate, 50 m
M sodium acetate, pH 6.0 (60 °C, 1.5
h). After cooling, lysates were sonicated briefly on ice (150 watts, 20
s) to reduce the viscosity of the samples. This solubilization
procedure did not affect the double-stranded nature of the DNA
necessary for fluorometric detection. DNA content was determined from
replicate cultures using the high salt (2
M NaCl)
Tris-NaCl-EDTA fluorometric approach with the Hoechst 33258 dye binding
assay in a TKO 100 minifluorometer (Hoefer Scientific).
Cell Density Dependence of Mineral Formation Stimulated
by Phosphate
UMR 106-01 BSP cells were plated at low
density ( 420 cells/mm
) and allowed to proliferate up
to 5 days with media changes every 48 h. These cultures exhibited a
confluent state by 96 h as demonstrated by phase contrast microscopy
(data not shown) and a near-plateau in DNA content (Fig. 1 b).
Selected cultures on each day were provided with an additional 24-h
exposure to medium with or without a phosphate supplement (10
m
M
-GP or P
) and were assayed for calcium
mineral content using a quantitative AR-S staining procedure. A
positive staining for calcium mineral was observed in cultures exposed
to
-GP or P
and increased in a linear fashion through
the entire culture period (Fig. 1 a). Mineral formation
was not dependent on the presence of ascorbate addition to the medium
(data not shown). When normalized to DNA content, a plateau in the
amount of calcium mineral associated with the cell layer was obtained
by 96 h of incubation (Fig. 1 c). Thus, mineral formation
was maximal when cultures reached a confluent state. For all subsequent
experiments, cells were plated at subconfluent density, allowed to grow
to confluence, and then assessed for mineral formation within a
subsequent 24-h period. Given this time frame, this model emphasizes
the initiation of mineral formation rather than mineral propagation and
maturation.
Figure 1:
Effect of phosphate and
cell density on alizarin red-S staining. UMR cultures were grown in
EMEM growth medium for various periods of time. Cultures at 24-h
intervals were supplemented with either 10 m
M P,
10 m
M
-GP, or no phosphate supplement ( control).
AR-S content in the cell layer was quantitatively determined by a
method described under ``Experimental Procedures.'' Data are
normalized to DNA content/culture with mean and S.D. ( n = 3).
Morphological Analysis of the Mineral
Deposits
Histological analysis of cross-sections from cultures
stained with AR-S demonstrated a fairly uniform positive staining
across the culture upon exposure to Por
-GP. Cultures
(
2000 cells/mm
) were grown for 2 days in standard EMEM
growth medium, then changed to growth medium containing either no
phosphate supplement, 5 m
M P
, or 5 m
M
-GP for an additional 24 h prior to fixation. Only cultures
exposed to the supplemental phosphate demonstrated a positive AR-S
stain for calcium mineral (Fig. 2). Cultures exposed to 10 m
M
supplemental phosphate were more difficult to section than those
treated with the 5 m
M concentration because of the higher
amount of mineral content (data not shown). In addition to staining the
extracellular matrix, AR-S staining was demonstrated within the
cytoplasm and pericellular matrix (Fig. 2 c). Occasional
regions of local nodular multicellularity were observed in both control
and phosphate-supplemented cultures which were not the result of
phosphate treatment. AR-S-positive accretions were observed to be
deposited on the basal side of the cells within the supporting culture
membrane. Ultrastructural analysis demonstrated mineral-like structures
in the pericellular environment associated with UMR 106-01 BSP
cells upon addition of 5 m
M
-GP for the terminal 24 h of
a 72 h culture period (Fig. 3 a). Mineral deposition was
observed to be localized to specific basal areas of the cultured cells
with multiple cell processes making intimate contact with the superior
surface of the mineral structures. Mineral deposits appeared to have a
fibrillar component suggesting the association of organic matrix
molecules in the calcium accretions. Transmission electron microscopy
indicated the confluent cultures of UMR 106-01 BSP cells exhibit
morphological features similar to normal osteoblastic cells: numerous
cellular contacts involving multiple junctional complexes, large
euchromatic nuclei, extensive rough endoplasmic reticulum, a
well-developed Golgi apparatus, and multiple mitochondrial figures
(Fig. 3 b). Extracellular mineral deposits of varying
size were observed beneath and between cells (Fig. 3 , b and d). Many of these mineral deposits had small,
needle-like crystals (Fig. 3 d) , and some were located
near, but rarely on, collagen fibrils (Fig. 3 c).
Figure 3:
Ultrastructural morphology of mineralizing
cultures. UMR cultures were grown for 2 days in growth medium followed
by a 24-h exposure to 5 m
M -GP. SEM ultrastructure
( panel a) demonstrated a fibrillar appearance of mineral
deposits ( white arrow). TEM sections ( panel b)
demonstrated mineral deposits on the bottom of the culture dish
( black arrow), collagenous extra-cellular matrix ( panel
c; asterisk denotes a small mineral deposit), and
localized areas of needle-like spicule clusters ( panel d).
SEM: panel a, 2,000
(final), bar = 10
µm; TEM: panel b, 10,000
(final), bar = 1 µm; panel c, 59,000
(final),
bar = 0.5 µm; and panel d, 118,000
(final), bar = 0.1 µm.
In
phosphate-supplemented cultures, small electron-dense foci were
observed inside several cells (Fig. 4). In general, they appeared
spherical in shape with an electron-dense core and a less dense
periphery. They numbered from a few to many within a cell and typically
ranged in size from 0.2-0.5 µm in diameter (Fig.
4 a). Irregular-shaped, intracellular structures of 1-2
µm in size were occasionally observed and small, needle-like
crystals detected within these structures (Fig. 4 b).
Often, these electron-dense foci were observed near the cell surface
and just outside the cell membrane suggestive of a secretory process
(Fig. 4 a). The appearance and intracellular location of these
structures are not consistent with the characteristics of matrix
vesicles.
Figure 4:
Nascent electron-dense structures
appearing within and around UMR cells as a result of phosphate
treatment. Panel a demonstrates intracellular ( white
arrow) and cell surface ( black arrows) structures
suggestive of a secretory process. Panel b depicts small,
needle-like crystals associated with two intracellular structures in a
paranuclear location ( black arrow). Panel a, 9000
(final), bar = 2 µm; panel b,
17,000
(final), bar = 1
µm.
X-ray Diffraction Analysis of the Mineral
Phase
Biological mineralization is a complex process resulting
in the deposition of poorly crystalline solids of calcium and
phosphate, principally bioapatites, on an extracellular matrix
(11) . X-ray diffraction (XRD) analysis of the mineral phase
from UMR 106-01 BSP cultures was performed in order to assess the
nature and crystalline quality of the solids formed in vitro.
In order to accumulate enough material for XRD analysis, cultures were
treated with 8 m
M -GP or 8 m
M P
for
a period of 72 h with daily medium changes. The XRD patterns of the
mineral phase showed two distinct peaks at 26 and 32° 2
with
a less distinct series of peaks at 28-29° 2
(Fig. 5,
c and d). Comparison to a reference standard of
well -crystallized hydroxyapatite (Fig. 5 a) and a
sample of human bone obtained from a 5-year-old patient
(Fig. 5 b; courtesy Dr. U. Vetter) indicates the mineral
phase from UMR 106-01 BSP is a poorly crystalline apatite with no
evidence of additional crystalline phases. Scherrer's analysis
(31) of the width at half-maximal height of the 002 diffraction
peak at 26° 2
indicated that the mean size along the c axis or length direction of the crystal was 15.7 ± 0.6 and
14.8 ± 0.2 nm for the
-GP- and P
-treated UMR
cultures, respectively, and 19.4 ± 0.9 nm for the human bone
sample. The molar Ca
to P
ratio of the
mineral phase from the UMR cultures ranged from 1.53 to 1.74.
Figure 5:
X-ray
diffractograms. Diffraction patterns of a highly crystalline synthetic
hydroxyapatite ( a), human bone apatite ( b), and UMR
cultures treated for 3 continuous days with 8 m
M
-glycerophosphate ( c) or 8 m
M inorganic
phosphate ( d). The UMR profiles are consistent with the full
profile of a bio-apatitic mineral phase.
Dose-response Relationship of Phosphate to Calcium
Mineral Content
Calcium mineralization within the cell layer
increased as a function of increasing -GP or P
supplementation in confluent cultures of UMR 106-01 BSP
cells exposed to a supplemental phosphate source for 24 h (Fig. 6). At
the normal medium concentration of calcium (1.8 m
M), the doses
of
-GP and P
that achieved a half-maximal response
(ED
) were 4.6 m
M and 3.8 m
M,
respectively. Mineral formation did not seem to increase substantially
with supplemental phosphate above 8-10 m
M;
concentrations at or above 15 m
M were occasionally toxic. In
comparison to a direct measurement of calcium by atomic absorption, a
quantitative AR-S procedure for assaying apatitic mineral demonstrated
a relatively constant stoichiometry of 1-2 mol of
Ca
/mol of AR-S (Fig. 6). Thus, the AR-S
staining approach is capable of quantitatively evaluating the apatitic
mineral content of cell cultures when atomic absorption measurements
are not available. The addition of P
to UMR cultures
demonstrates a stimulation of apatitic mineral production/molar dose
similar to that measured for
-GP. Organophosphates require
alkaline phosphatase activity to stimulate biomineralization
(35) , whereas P
is the final hydrolysis product of
alkaline phosphatase (see discussion below). Thus, the role of alkaline
phosphatase in the mineralization process can be addressed by comparing
the efficacy of these phosphate supplements.
Figure 6:
Dose-response relationship of phosphate to
calcium mineral content. Cultures were grown for 48 h in growth medium
followed by an incubation in medium supplemented with the respective
phosphate concentrations ( panel a, -GP; panel b,
P
) for a 24-h period. Cell layer calcium mineral was
determined by either atomic absorption ( open circle) or AR-S
spectrophometry ( dark circle). Analysis was performed on
separate cultures. Mean and S.D. ( n = 3),
representative of two trials.
Temporal Progression of the Mineralization
Response
Upon the addition of a phosphate source, calcium
mineral formation in the UMR 106-01 BSP cell line is a
time-dependent process displaying a kinetic relationship reminiscent of
a biological process. Confluent cultures were exposed to -GP
supplements at either 4 or 10 m
M concentrations for various
lengths of time up to 48 h (Fig. 7 a). Under these conditions,
little or no mineral was formed by 6-8 h after treatment. This
was followed by a rapid elevation in the mineral content of the cell
layer over the next 12-16 h of treatment with a half-maximal time
(ET
) of 12-15 h relative to a plateau in calcium
mineral formation by 20-24 h. The initial rate of calcium mineral
deposition appeared to be directly proportional to the concentration of
-GP suggesting that the rate of hydrolysis of
-GP by alkaline
phosphatase is a controlling factor for mineral formation stimulated by
organophosphates. Addition of P
to parallel cultures (Fig.
7 b) demonstrated a somewhat faster rate of mineral formation
relative to
-GP that appeared to be the result of a shorter lag
period and a slightly faster initial rate of mineral formation
(ET
7.5 h for 10 m
M P
versus
12 h for 10 m
M
-GP). The addition of higher
levels of either source of phosphate appeared to shorten the lag
period, increase the initial rate of mineral formation, and yield a
higher level of calcium mineral within the cell layer by 15-24-h
exposure.
Alkaline Phosphatase Inhibition Blocks the Mineralization
Response Stimulated by Organophosphates
Confluent cultures of
UMR 106-01 BSP cells were exposed to various
phosphate-supplemented media in the presence or absence of the alkaline
phosphatase inhibitor levamisole. The stimulatory effects of either
-GP, phosphoserine, or phosphothreonine at concentrations up to 10
m
M were inhibited by simultaneous levamisole treatment in a
dose-dependent manner (Fig. 8); similar results were obtained for each
organophosphate at lower concentrations (data not shown). A
half-maximal inhibitory dose (ED
) of 200-300
µ
M was determined for all organophosphate supplements.
Treatment with up to 500 µ
M levamisole did not alter the
amount of calcium mineral formed when P
, the hydrolysis
product of alkaline phosphatase, was used to stimulate mineralization
(Fig. 8). Thus, the drug did not appear to act as a general
metabolic inhibitor. UMR cells appear to be less sensitive to
levamisole than primary calvarial-derived osteoblastic cells
(35) in which levamisole completely inhibited mineral deposition
at 100 µ
M. UMR cells constitutively express a more
uniform, slightly higher level of alkaline phosphatase activity
compared to primary osteoblastic cell cultures
(36) . This may,
in part, explain the need for a 3-5-fold higher concentration of
levamisole for complete inhibition. However, UMR cells are more
sensitive to levamisole than calcifying chrondrogenic model systems
which require concentrations of 1 m
M or higher for complete
inhibition
(37, 38, 39) .
Figure 8:
Alkaline phosphatase inhibition will block
the mineralization response stimulated by organophosphates. Cultures
were grown for 48 h in growth medium followed by a change to medium
supplemented with 10 m
M P,
-GP, phosphoserine
(Ser-P) or phosphothreonine (Thr-P) for 24 h. Levamisole was added at
the time of phosphate supplementation. Control cultures were not
exposed to phosphate supplementation. Cultures exposed to phosphate
supplements at 4 m
M showed similar data as shown here. Under
these conditions, cell number was identical in treated and control
cultures. Mean and S.D. ( n = 3), representative of two
trials.
Pyrophosphate (PP
Coaddition of 0.1-1 µ
M PP) Blocks the Mineralization
Response
to medium supplemented with 5 m
M
-GP demonstrated a
near 50% decrease in detectable calcium mineral content (Fig. 9).
Addition of 100 µ
M PP
resulted in a near
cessation of the mineralization response stimulated by
-GP. Even
mineralization stimulated by 10 m
M P
was inhibited
by as little as 500 µ
M PP
(Fig. 9,
inset). These results suggest that PP
might block
mineral formation by multiple inhibiting processes which would include
alkaline phosphatase activity, crystal nucleation, growth and
propagation, and possibly cellular transport of P
.
Metabolic Inhibitors Block the Mineralization
Response
The dependence of the rapid mineralization response of
UMR 106-01 BSP cells on RNA transcription as well as protein
synthesis, processing, and secretion was explored by the addition of
selective inhibitors just prior to the addition of the phosphate source
(Table I). Phosphate-stimulated cultures were exposed to actinomycin
Cor cycloheximide (in the presence or absence of exogenous
BSP) at concentrations and durations that rapidly inhibit RNA
transcription or protein synthesis, respectively. When cultures were
exposed to these agents (13-h treatment) and then assayed for
mineral-bound AR-S stain at the ET
time point for
mineralization, a pronounced reduction in the amount of calcium mineral
associated with the cell layer was determined for either drug
(12-16% of the control value). These data suggest that mineral
formation by these osteoblastic cells is a metabolic process involving
on-going protein and RNA synthesis. In addition, cultures exposed to
cycloheximide in the presence of purified BSP, a potential nucleator of
hydroxyapatite, did not elicit a mineralization response (6% of the
control value, ). Therefore, the presence of exogenous BSP
was not sufficient to stimulate apatite mineral formation in cultures
whose protein synthesis was inhibited.
Calcium Supplementation Enhances the Mineralization
Response
The mineralization response of UMR 106-01 BSP
cells depends on phosphate supplementation in a time- and
dose-dependent fashion at the standard concentration of calcium present
in EMEM. In order to evaluate the role of calcium in the mineralization
response, a commercially available calcium-free DMEM was utilized which
contained a basal concentration of phosphate similar to EMEM. Confluent
cultures in DMEM growth medium at different concentrations of calcium
(0.5-5 m
M) were exposed for 24 h to an identical medium
with or without an additional 5 m
M Psupplement.
These cultures exhibited a calcium-dependent increase in mineral
formation (Table II). However, cultures without an additional P
supplement only formed detectable mineral at a relatively high
calcium concentration. Significantly, cultures treated with 1
m
M Ca
, 5.9 m
M P
yielded
nearly twice as much calcium mineral as those exposed to 5 m
M
Ca
, 0.9 m
M P
. Thus, phosphate
supplementation, in this culture system, appears to initiate a mineral
formation response which is subsequently enhanced by calcium levels.
-GP) to cultures of primary
osteoblastic cells results in the formation of calcium mineral
(35, 37, 44, 45) . The UMR 106-01
BSP osteoblastic cell line demonstrates a rapid and highly reproducible
biologic formation of hydroxyapatitic mineral phases that are
stimulated by various phosphate agents in a dose- and time-dependent
manner. Similar to primary osteoblastic systems, this UMR
mineralization process requires alkaline phosphatase to hydrolyze
organophosphates thereby releasing P
which appears to be
the actual initiator of mineral formation. This mineralization process
is tightly controlled by phosphate supplements in a dose range
(ED
4-5 m
M) which is similar to that
reported in primary osteoblastic systems
(35, 37, 46, 47) . Interestingly,
calcium appears to function in this system by regulating the amount of
mineral deposited in the cell layer after prior initiation by
phosphate.
blocked apatitic mineral
formation initiated by all phosphate supplements including
P
. PP
is thought to inhibit calcium mineral
formation by either a physical-chemical blocking of hydroxyapatite
propagation
(48) or through an alteration of alkaline
phosphatase activity
(49) . In the UMR culture system, PP
may function in both capacities because it inhibited even
P
-stimulated mineral formation. Lastly, the ultrastructural
appearance of the apatitic mineral in UMR cultures bears a resemblance
to that observed in the early stages of mineral formation in primary
osteoblastic systems
(44, 50, 51) and normal
rat bone sections
(16) .
in the
mineralization process in contrast to previous reports in other systems
(35, 45) . Lastly, in vitro mineralization in
primary osteoblastic cultures has been argued to require ascorbic acid
to stimulate greater collagen production and cross-linking
(53, 54) . The mineralization process observed in UMR
cultures occurs in the absence of additional ascorbate. Possible
explanations for this discrepancy are that (i) UMR cells could
constitutively produce a large amount of type I collagen
(25) in an ascorbate-independent manner, or (ii) the
mineralization conditions used in this study might emphasize the
initial stages of apatitic mineral deposition that precede the steps
which propagate mineral crystals onto collagen.
Table:
Effects of actinomycin C,
cycloheximide, or brefeldin A on mineral formation in UMR 106-01
BSP cultures
2000
cells/mm
) into 12-well cluster dishes (
380
mm
/well) and cultured for 48 h in EMEM growth medium. At
this time, the medium was replaced with growth medium containing either
no additives, 500 ng/ml actinomycin C
, 50 µg/ml
cycloheximide (in the presence or absence of 1 µg/ml BSP), or 1
µg/ml brefeldin A; cultures were preincubated for 1 h. After this
incubation, the medium was replaced with an identical medium containing
P
concentrations either at normal (1 m
M) or
elevated (11 m
M) levels. Cultures were incubated for an
additional 12 h and then assayed for either mineral-bound AR-S or DNA
content as described under ``Experimental Procedures.'' The
cell number/dish was at control levels for brefeldin A-treated, and at
70-80% of control levels for actinomycin- and
cycloheximide-treated cultures. Data represent mean ± S.D. from
three independent trials containing three cultures for each assay.
Table:
Effect of calcium on mineral formation in UMR
106-01 BSP cultures
2000 cells/mm
) into 12-well cluster dishes (
380
mm
/well) and cultured for 48 h in DMEM growth medium
containing calcium at final concentrations of either 0.5, 1.0, 2.5, or
5.0 m
M. At 48-h incubation, the culture medium was replaced
with a medium of identical calcium content and P
concentrations either at normal (0.9 m
M) or elevated
(5.9 m
M) levels. Cultures were incubated for an additional 24
h and then assayed for either mineral-bound AR-S or DNA content as
described under ``Experimental Procedures.'' At no time was
cloudiness noted in the media during these experiments ( i.e. no spontaneous precipitation). Media was made fresh the day of
each trial and phosphate supplements made from 100
stock
solutions in water to avoid extended exposure periods of P
to the high levels of Ca
. Under these
conditions, cell number was identical in treated and control cultures.
Data represent mean ± S.D. from one trial containing triplicate
cultures for each assay.
-GP,
-glycerophosphate; XRD, x-ray diffraction; AR-S,
alizarin red-S; CPC, cetylpyridinium chloride; EMEM, Eagle's
minimal essential medium; DMEM, Dulbecco's modification of
Eagle's medium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.