©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapidly Forming Apatitic Mineral in an Osteoblastic Cell Line (UMR 10601 BSP) (*)

Clark M. Stanford (1), Paul A. Jacobson (2), E. David Eanes (3), Lois A. Lembke (2), Ronald J. Midura (2)(§)

From the (1) Dows Institute for Dental Research, College of Dentistry, (2) Department of Orthopaedic Surgery, College of Medicine, University of Iowa, Iowa City, Iowa 52242 and the (3) Bone Research Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

This study evaluated a rapid biomineralization phenomenon exhibited by an osteoblastic cell line, UMR 106-01 BSP, when treated with either organic phosphates [-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 (EDof 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 Ptreatment 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.


INTRODUCTION

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 Caand 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) .

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,() 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.

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.


EXPERIMENTAL PROCEDURES

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% COatmosphere with routine passage every 3 days. Experimental cultures were created by briefly washing the confluent cell layer with Hank's balanced saline solution without Caor Mgfollowed 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/mminto 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/mmin 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 PPconcentration. Inset, PPinhibits 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 COincubator. 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.

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 Cwas 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 Caby supplementing with CaClto 0.5, 1.0, 2.5, and 5.0 m M Calevels. 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 Por 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) .

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(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 Caby atomic absorption spectrophotometry (see description below) and Pby 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 (Caand Mgfree) 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).


RESULTS

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 Pand 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 Pfor 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 Cato Pratio 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 Psupplementation 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 Pthat 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 Pto 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 Pis 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 Pto 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 (ET7.5 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) Blocks the Mineralization Response

Coaddition of 0.1-1 µ M PPto medium supplemented with 5 m M -GP demonstrated a near 50% decrease in detectable calcium mineral content (Fig. 9). Addition of 100 µ M PPresulted in a near cessation of the mineralization response stimulated by -GP. Even mineralization stimulated by 10 m M Pwas inhibited by as little as 500 µ M PP(Fig. 9, inset). These results suggest that PPmight 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 ETtime 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.

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.

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 Psupplement only formed detectable mineral at a relatively high calcium concentration. Significantly, cultures treated with 1 m M Ca, 5.9 m M Pyielded 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.


DISCUSSION

Numerous groups have reported the addition of organophosphates (typically -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 Pwhich 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.

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 PPblocked apatitic mineral formation initiated by all phosphate supplements including P. PPis 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, PPmay 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) .

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 Pin 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.

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.

  
Table: Effects of actinomycin C, cycloheximide, or brefeldin A on mineral formation in UMR 106-01 BSP cultures

UMR 106-01 BSP cells were inoculated (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 Pconcentrations 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

UMR 106-01 BSP cells were inoculated (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 Pconcentrations 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 Pto 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.



FOOTNOTES

*
This work was funded by the Roy J. Carver Charitable Trust Fund (to R. J. M.), National Institutes of Health Postdoctoral Training Grant AR-0705 to the Department of Orthopaedic Surgery (to P. A. J.), and Public Health Service Grant NCRR 2 SO7 RR05313-31 (to C. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Biomedical Engineering Dept., WB3, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

The abbreviations used are: BSP, bone sialoprotein; PBS, phosphate-buffered saline; SEM, scanning electron microscopy; TEM, transmission electron microscopy; -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.

UMR 106-01 BSP designates that this cell line has been verified to synthesize large amounts of BSP as defined by McQuillian et al. (26, 27) and does not necessarily imply a distinction from the parent stock of UMR 106-01.


ACKNOWLEDGEMENTS

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.


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