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Originally published In Press as doi:10.1074/jbc.M312409200 on March 5, 2004

J. Biol. Chem., Vol. 279, Issue 24, 25464-25473, June 11, 2004
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Bone Acidic Glycoprotein-75 Delineates the Extracellular Sites of Future Bone Sialoprotein Accumulation and Apatite Nucleation in Osteoblastic Cultures*

Ronald J. Midura{ddagger}§, Aimin Wang{ddagger}, Dinah Lovitch¶, Douglas Law||, Kimerly Powell{ddagger}, and Jeff P. Gorski¶

From the {ddagger}Department of Biomedical Engineering and the Orthopaedic Research Center, Lerner Research Institute, The Cleveland Clinic and Foundation, Cleveland, Ohio 44195, the Division of Biochemistry and Molecular Biology, School of Biological Sciences, and Department of Oral Biology, School of Dentistry, University of Missouri-Kansas City, Kansas City, Missouri 64108, and the ||Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110

Received for publication, November 12, 2003 , and in revised form, March 5, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Addition of an organophosphate source to UMR osteoblastic cultures activates a mineralization program in which BSP localizes to extracellular matrix sites where hydroxyapatite crystals are subsequently nucleated (Wang, A., Martin, J. A., Lembke, L. A., and Midura, R. J. (2000) J. Biol. Chem. 275, 11082–11091). This study identifies for the first time novel extracellular spherical structures, termed biomineralization foci (BMF), containing bone acidic glycoprotein-75 (BAG-75), bone sialoprotein (BSP), and alkaline phosphatase that are the exclusive sites of initial nucleation of hydroxyapatite crystals in the UMR model. Importantly, in the absence of added phosphate, UMR cultures after reaching confluency contain two size populations of morphologically identifiable BMF precursors enriched in BAG-75 (15–25 and 150–250 µm in diameter). The shape and size of the smaller population are similar to structures assembled in vitro through self-association of purified BAG-75 protein (Gorski, J. P., Kremer, E. A., Chen, Y., Ryan, S., Fullenkamp, C., Delviscio, J., Jensen, K., and McKee, M. D. (1997) J. Cell. Biochem. 64, 547–564). After organophosphate addition, BSP accumulates within these BAG-75-containing BMF precursors, with hydroxyapatite crystal nucleation occurring subsequently. In summary, BAG-75 is the earliest detectable biomarker that accurately predicts the extracellular sites of de novo biomineralization in UMR cultures. We hypothesize that BAG-75 may perform a key structural role in the assembly of BMF precursors and the recruitment of other proteins such as alkaline phosphatase and BSP. Furthermore, we propose a hypothetical mechanism in which BAG-75 and BSP function actively in nucleation of apatite within BMF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms responsible for biomineralization of mammalian bone remain unresolved (1–8). One theory proposes that biomineralization reactions are initiated within phospholipid membrane-delimited vesicles (matrix vesicles) released into the extracellular matrix by osteoblasts (1, 3, 5, 8). Another proposes that mineralization is initiated within the "hole zone" of collagen fibrils at the mineralization front of osteoid in a process requiring noncollagenous phosphoproteins (1, 2, 4). In vitro studies have shown that some of these phosphoproteins can nucleate the formation of apatite crystals when located within collagen or agarose gels (6, 9, 10). However, most prior studies of biomineralization have not seriously considered whether their models were consistent with the kinetics or spatial patterning with which bone mineralizes in vivo.

Several noncollagenous bone matrix proteins are believed to play an active role in the biochemical reactions involved in bone biomineralization. One of these proteins, bone sialoprotein (BSP),1 is a sulfated, phosphorylated matrix glycoprotein containing an RGD integrin receptor-binding site (11). BSP has been proposed to function as an apatite nucleator because it is found in the mineralizing boundaries of bone, dentin, and calcifying cartilage tissues (12–17) co-localizing with the smallest detectable foci of newly forming mineralized matrix in osteoid (18), and is expressed prior to and during the active mineralization periods of osteoblastic cultures that produce apatite crystals of biological proportions (19, 20). Furthermore, purified BSP nucleates a small amount of apatite in vitro (9) likely mediated by its polyglutamate domains (10, 21).

Another noncollagenous bone matrix protein, bone acidic glycoprotein-75 (BAG-75), is an acidic glycoprotein of 75 kDa in apparent size that contains elevated contents of Asp/Glu (29%), sialic acid, and covalently bound phosphate (44 residues/mol) (22). Research to date indicates that BAG-75 is restricted in its expression to actively forming primary or woven bone and dentin (23–26). Purified BAG-75 displays a strong propensity to self-associate into supramolecular spherical complexes (10–20 µm diameter) composed of 10–12-nm diameter microfibrils (24, 27). Electronegative complexes composed of BAG-75 can sequester millimolar quantities of phosphate ions that are available for crystal nucleation reactions (24); BAG-75 also binds up to 135 atoms of Ca2+/molecule at saturation (28). Multimeric BAG-75 complexes exist in vivo at sites of new primary bone formation as demonstrated by staining frozen tissue sections with a BAG-75 aggregation-specific monoclonal antibody (24).

Whereas conclusions from in vitro models require validation in vivo (see Gorski et al. (29)), culture systems are readily controlled and reproducible. The UMR 106-01 BSP cell line constitutively expresses a mature osteoblastic phenotype and is able to deposit ample amounts of apatite crystals within discrete focal sites over a 24-h incubation period after addition of an organophosphate source (19, 30). Several findings from our laboratory support the physiological relevancy of the UMR culture model. Most notably, Wang et al. (30) have shown that the biomineralization processes in both UMR and primary osteoblast cultures are similarly regulated by parathyroid hormone treatment. Furthermore, the UMR biomineralization process is an active metabolic process requiring continuous protein synthesis and secretion (19). Lastly, x-ray diffraction analyses demonstrate that mineral crystals formed in mineralizing UMR cultures are apatite and have a similar C-axis length as that of apatite crystals isolated from pediatric bone samples (19). Altogether, our prior work indicates that UMR cultures deposit apatite crystals at focal sites in a physiologically relevant manner.

BSP quantitatively accumulates within biomineralization foci (BMF) only after treatment of UMR cultures with organophosphates (30). BSP is first detected within BMF after 4–8 h, well before the first appearance of apatite crystals within BMF (30). Thus, in this model system, BSP fulfills the minimal necessary temporal-spatial requirements of a protein involved in apatite nucleation. However, it is not known how BSP is targeted quantitatively to BMF. The present study was undertaken to determine whether mineralizing UMR cultures, like initial sites of mineral nucleation in primary bone (see Gorski et al. (29)), accumulate BSP and then hydroxyapatite within BAG-75 containing BMF precursors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The sources of antibodies used in the study were as follows: monoclonal anti-BSP, WV1D1(9C5) antibody was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA); anti-BAG-75 protein antibodies (number 504) were provided by Jeff Gorski (23). Monospecific polyclonal antiserum raised against rat BSP (LF-87) was obtained from Larry Fisher (NICDR, National Institutes of Health) (31). The 504 polyclonal antiserum for BAG-75 (23, 24, 26, 27), and the WVIDI (9C5) monoclonal antibody for BSP (30) have been shown to be monospecific in light level immunolocalization, electron microscopic ultrastructural studies, and Western blot analyses.

Cell Culture and Treatment with {beta}-Glycerophosphate—MC3T3-E1 (subclone M4) osteoblastic cells were cultured and treated with 5 mM {beta}-glycerophosphate ({beta}-GP) as previously described (32, 33). UMR 106-01 BSP cells were passaged and cultured as described by Wang et al. (30). Prior to immunochemical analyses, cells were seeded onto either 25-mm Aclar® 33C .005 thick plastic coverslips (Allied Signal's medical grade) in 6-well dishes or serum-coated Fisher Plus glass slides in Petri dishes (both for confocal analyses), or Permanox® tissue culture dishes (for electron microscopy analyses, Fisher Scientific), at a density of 1000 cells/mm2 in growth medium (Eagle's minimum essential medium plus nonessential amino acids, 2 mM L-glutamine, 20 mM HEPES (pH 7.2), and 10% fetal bovine serum). At 64 h of incubation, the medium was replaced with fresh growth medium for an additional 24 h (biomineralization period) supplemented with or without {beta}-GP at a final concentration of 7 mM. {beta}-GP was prepared as sterile 0.7 M stocks in Nanopure water adjusted to a final pH of 7.0; aliquots were added directly to fresh medium immediately before initiating biomineralization. Unless otherwise stated, cells were fixed by immersion in cold 70% ethanol and stored as such at 4 °C until processed further.

Wang et al. (30) have previously demonstrated that UMR cultures treated with as little as 1 nM parathyroid hormone expressed high alkaline phosphatase activity levels, and a near complete conversion of {beta}-GP into inorganic phosphate, yet did not precipitate calcium phosphate crystals. Chang et al. (34) also reported that spontaneous crystallization of calcium and phosphate did not occur in UMR culture medium even when the concentrations of these ions were elevated. Therefore, chemical precipitation is not the primary mechanism governing mineral formation in this model system.

Culture of Fetal Rat Calvarial Cells and Treatment with {beta}-GP—As described previously (35, 36), calvaria were removed from 19-day-old fetal rats and sequentially digested with 0.2% collagenase, 0.05% trypsin in Hanks' balanced salt solution 6 times for 20 min each. The supernatants from the first two digests were discarded and the supernatants from the remaining digests, rich in osteoblast precursor cells, were pooled for culture. Cells were plated at 2–3 x 108 cells per T75 flasks in {alpha}-minimal essential media supplemented with glutamine and nucleosides (Mediatech, Inc.), 10% fetal bovine serum, and 0.5% penicillin/streptomycin antibiotic. Cells were grown to confluence (3–4 days), trypsinized, and then plated for experiments in chamber slides at 8000 cells/chamber. After reaching confluency, first passage cultures were re-fed every 3 days with medium supplemented with 5% fetal bovine serum, 0.5% penicillin/streptomycin, 100 µg/ml ascorbic acid, and with 5 mM {beta}-GP to promote the formation and mineralization of bone mineralization foci. Controls did not receive {beta}-GP. At 7 and 17 days after reaching confluence, culture slides were fixed in 4% paraformaldehyde for 2.5 h at room temperature and were then stored in 70% ethanol at 4 °C until stained with alizarin red S.

Visualization of Apatite Mineral using Alizarin Red S—After removal of medium, UMR cultures were briefly rinsed with Tris-buffered saline followed by fixation in ice-cold 70% ethanol for 1 h. Fixed samples were washed three times with Tris-buffered saline, rinsed with deionized water, and then stained for 10 min with either 40 or 4 mM alizarin red S dye, pH 4.2, at room temperature for brightfield (19) or epifluorescent microscopy (30), respectively. Cultures were then rinsed five times with water followed by a 15-min wash with Tris-buffered saline to reduce nonspecific alizarin red S stain. When necessary, fixed mineralized cultures were decalcified using one of three methods: 1) treatment with Tris-buffered saline, pH 8.0, containing 50 mM EDTA overnight at 4 °C; 2) treatment with 10% trichloroacetic acid for 10 min at 4 °C; or 3) treatment with 0.1 M HCl in 70% ethanol for 30 min at 4 °C (30).

Fluorescent Immunolabeling—Fixed cells on glass slides (FisherPlus slides, Fisher Scientific Co.), chamber slides (Lab-Tek, Inc.), or Aclar® coverslips were briefly rehydrated in Tris-buffered saline, pH 7.5, and then blocked by incubation in Tris-buffered saline, pH 7.5, containing 1% (w/v) bovine serum albumin (Sigma) for 60 min. Slides were then rinsed five times with Tris-buffered saline, pH 7.5, and incubated for 1–3 h with primary antibodies diluted in the above blocking buffer (typically 1:100 to 1:200 dilutions). Slides were then washed five times as before and treated with either anti-mouse or anti-rabbit IgG Fc domain secondary antibodies conjugated to fluorescein isothiocyanate or TRITC fluorochromes diluted 1:1000 in blocking buffer (Jackson ImmunoResearch and Molecular Probes, Inc.). After washing off unbound secondary antibody, specimens were stained with 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI), mounted with VectaShield (Vector Labs), and slides were cover slipped prior to imaging with a Leica TCS-SP AOBS (True Confocal Scanner-SpectroPhotometer Acousto-Optic Beam Splitter) equipped with 4 lasers (Krypton, Argon, UV, and HeNe), or a Nikon Microphot Fx epifluorescence microscope outfitted with a SPOT-rt CCD digital camera (Diagnostic Instruments). Digital images were post-processed with Adobe Photoshop (version 6.0).

Cell Death Assay—Apoptotic cells were detected in UMR cultures using a cell death detection kit (Roche Applied Science). Cultures were fixed in 2% paraformaldehyde in phosphate-buffered saline and processed according to the manufacturer's instructions. Samples were incubated with a Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) reaction mixture that adds fluorescein-conjugated dUTP to the free 3'-OH groups in single- and double-strained DNA fragments within apoptotic nuclei. After washing, the specimens were stained with alizarin red S (see above) and then 1 µg/ml DAPI, followed by mounting in VectaShield. All fluorochromes were detected using confocal microscopy. TUNEL specificity was determined by omitting the terminal deoxynucleotidyl transferase necessary for adding dUTP to the free 3'-OH ends of DNA strands.

Electron Microscopy—For conventional electron microscopy, cultures were fixed for 24 h with 2% glutaraldehyde in 150 mM sodium cacodylate, pH 7.2, at 4 °C. Samples were post-fixed in 1% OsO4, dehydrated, embedded, sectioned, and stained according to published protocols (19, 30). For ultrastructural immunocytochemistry, tissue samples were fixed with 2% formaldehyde in phosphate-buffered saline, overnight at 4 °C. Samples were cryo-protected, frozen in liquid nitrogen, and sectioned as described previously (37), using a method adapted from that of Tokuyasu (38). Frozen sections 80–100-nm thick were processed for indirect immunolabeling using secondary antibodies coupled to 6- or 12-nm colloidal gold (Jackson ImmunoResearch, West Grove, PA), under conditions designed to minimize cross-reactivity and nonspecific binding of antibodies (37). In some cases, sections were decalcified by floating them on a drop of ice-cold 10% trichloroacetic acid for 60 min prior to immunolabeling. Sections were examined using a JEOL 1200EX transmission electron microscope at an accelerating voltage of 100 kV.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mineralizing UMR Cultures Produce Focal Deposits of Apatite That Also Contain BSP and Alkaline Phosphatase Activity—When given an organophosphate stimulant like {beta}-GP, UMR cultures deposit an apatite mineral phase by a utilization of phosphate ions released from {beta}-GP by alkaline phosphatase (19, 30). Fig. 1A demonstrates the focal nature of the biomineralization process in UMR cultures after {beta}-GP exposure, and also reveals the close spatial relationship between BSP and apatite deposits in this biomineralization model system. Fig. 1B shows that these focal areas of apatite in UMR cultures also stain positive for alkaline phosphatase activity. BSP has been reported to accumulate quantitatively in these focal areas prior to the detection of an apatite phase, thereby meeting the necessary temporal-spatial requirements of a matrix protein involved in apatite formation (30). However, it is not clear how BSP is targeted to these BMF prior to mineral deposition.



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  		FIG. 1.
Mineralizing UMR cultures deposit BSP and calcium mineral in extracellular matrix complexes referred to as BMF. Panel A shows a UMR culture that had been treated with {beta}-GP for 24 h, and immunostained with anti-BSP monoclonal WV1D1(9C5) followed by alizarin red S staining. BSP is denoted by the green color, whereas calcium mineral is denoted by the red color (alizarin red S). DIC, differential interference contrast view of the same field of view. Color overlay panel shows both the green and red color bands represented in a full RGB color spectrum. Panel B shows a UMR culture that had been treated with {beta}-GP for 24 h, and immunostained with anti-BSP monoclonal WV1D1(9C5) followed by Vector Red alkaline phosphatase (ALP) activity staining. BSP is denoted by the green color, whereas alkaline phosphatase activity is denoted by the red color. The white arrowheads point to BMF that stain positive for both BSP and alkaline phosphatase activity. Panel C depicts a three-dimensional confocal analysis of a mineralizing UMR culture. BSP is denoted by the green color, hyaluronan is denoted by the red color, and DAPI-stained nuclei are indicated by the blue color. The X-Y view is a single 0.5-µm slice at the indicated depth in the X-Z view panel. Panel D shows the results of a fluorescein isothiocyanate-TUNEL assay on a 24-h mineralized UMR culture. Top panel shows a color overlay of the TUNEL (green) and alizarin red S-stained calcium mineral (red) fluorescence. Bottom panel shows a color overlay of the TUNEL (green), calcium mineral (red), and DAPI-stained nuclei (blue). Note that calcium mineral deposits do not co-localize with either DAPI-stained or TUNEL-stained nuclei, and that only about 1–2% of the nuclei are TUNEL-positive in this field of view.

 
BMF Are Located within the Extracellular Matrix between UMR Cells—Fig. 1C shows X-Y and X-Z views from a three-dimensional stack of confocal images each representing an optical slice of 0.5 µm depth. BMF are identified by their strong immunostaining for BSP, whereas nuclei are identified by their strong staining with DAPI. These three-dimensional images demonstrate that BMF are located in the extracellular matrix space between cells, and that nearly all BMF are devoid of staining for the presence of double-stranded DNA. Furthermore, BMF neither stain positively with the TUNEL reaction nor exhibit evidence of DAPI-stained apoptotic bodies (Fig. 1D). Taken together, the absence of DNA remnants, of TUNEL-positive signal, and of apoptotic bodies indicates that BMF are not apoptotic or necrotic cell debris. These results strengthen the conclusions from our previous studies (19, 30) that the biomineralization process in UMR cultures is not dystrophic.

The three-dimensional confocal dataset in Fig. 1C shows that an average BMF is spherical in shape with typical dimensions of 10–20 µm in X-Y and 10–15 µm in the X-Z viewing planes. Notably, not all negatively charged macromolecules secreted into the extracellular space are deposited in BMF as indicated by the lack of overlap between the alizarin red/BSP signals and hyaluronan staining (Fig. 1C). Thus, the incorporation of BSP into BMF is unlikely to result from nonspecific electrostatic interactions. Rather, the data suggest that the strict BSP deposition in BMF is because of more specific interactions.

Bone Acidic Glycoprotein-75 Is a Biomarker for BMF Precursors Prior to Active Mineralization—Figs. 2 and 3 demonstrate for the first time that spherical BMF precursors are recognizable as BAG-75 containing supramolecular complexes prior to the addition of {beta}-GP into UMR cultures. Briefly, Fig. 2, B and D, depicts the representative brightfield appearance of UMR monolayer cultures at 64 h of incubation, just after confluence has been reached, but prior to organophosphate addition ("zero time"). At this time, the cultures consist of a confluent monolayer of UMR cells containing two size populations of refractile, roughly spherical BMF precursors (15–25 m and 150–250 µm in diameter), which project upward from the cell layer at apparently random intervals. After immunostaining, BAG-75 was detected in both size populations of BMF precursors prior to initiating mineralization (Fig. 2, A and C). The immunofluorescent signal for BAG-75 within the larger BMF precursors consisted of several bright focal areas each 10–20 µm in diameter embedded within a slightly lower level of diffuse staining (Fig. 2C). This appearance suggests that the larger sized BMF precursors may represent a cluster of smaller ones.



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  		FIG. 2.
BAG-75 is a biomarker for precursors of BMF prior to nucleation of mineral crystals. Panels A–D are representative of confluent UMR cultures at 0 h prior to the addition of {beta}-GP; E and F are representative of cultures 24 h after the addition of {beta}-GP. A and B, C and D, and E and F, respectively, represent images of the same field of view. Panel A, anti-BAG-75 antibodies specifically label small 15–25-µm diameter populations of BMF precursors (white arrows). Panel B, brightfield view of small BMF precursors (black arrows). Panel C, anti-BAG-75 antibodies immunostain a large 150–250-µm diameter BMF precursor (white arrow). Panel D, brightfield image of a large 150–250-µm diameter BMF precursor (black arrow). Panels E and F, large mineralized BMF stained with alizarin red S dye (white arrows) still stains positive for BAG-75 on its surface and within its structure (white arrowheads). Scale bars, 200 µm for A–D and 100 µm for E and F.

 



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  		FIG. 3.
BAG-75-enriched focal deposits represent the initial sites of mineralization in osteoblastic cultures from primary fetal rat calvarial cells and MC3T3-E1 cells. Refer to the "Materials and Methods" for culture details. Panel A, phase-contrast view of fetal rat calvarial culture after 7 days of {beta}-GP treatment; no alizarin red S staining was detected at this time. Panel B, BAG-75 immunostaining of fetal rat calvarial culture after 7 days of {beta}-GP treatment; same field of view as shown in panel A. Panel C, non-immune IgG control staining of FRC culture after 7 days of {beta}-GP treatment. Panels D and E, alizarin red S staining of apatite deposits in fetal rat calvarial culture after 17 days of {beta}-GP treatment. Panels F and G, BAG-75-immunostained FRC culture after 17 days of {beta}-GP treatment after decalcification using EDTA; the same fields of view as shown in panels D and E, respectively. Panel H, brightfield view of alizarin red S-stained MC3T3-E1 (subclone M4) culture after 12 days of {beta}-GP treatment. Panel I, higher magnification brightfield view of an individual alizarin red S-stained foci from panel H after partial decalcification. Panel J, BAG-75 immunostaining of MC3T3-E1 culture (subclone M4) after 12 days of {beta}-GP treatment; same field of view as shown in panel I. Scale bars in panels A–H are 100 µm, whereas those shown in panels I and J are 2 µm.

 
Both sizes of BMF precursors were able to nucleate calcium phosphate crystals when UMR cultures were incubated for an additional 24 h with medium containing {beta}-GP (i.e. 64 to 88 h after plating). This is evident by the strong staining obtained with alizarin red S dye binding to large (Figs. 2, E and F, and 5E) and small BMF (Fig. 5E). Several points can be made regarding the relative staining patterns observed when BAG-75 immunofluorescence and alizarin red S-stained mineral crystals are imaged together. At low magnification, the stronger alizarin red S signal displays a punctate appearance comprised of numerous 10–20-µm diameter round deposits within the interior domain of the larger BMF (Fig. 2E). In contrast, the BAG-75 immunofluorescence signal appears as a layer surrounding the alizarin red S-stained deposits and, internally, in junctional areas separating individual alizarin red S-stained deposits (Fig. 2F). In these undecalcified cultures, the regions of strongest alizarin red S staining are seemingly devoid of apparent BAG-75 staining, whereas BAG-75 staining is readily visible in the outer layer surrounding the mineral deposits and within the internal areas physically separating alizarin red S-stained round deposits. This apparent lack of BAG-75 immunogenicity deeper within these larger BMF may be because of the coverage of antigenic sites by the hydroxyapatite crystalline phase. This point is documented upon decalcification of mineralized cultures as seen in the next section.



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  		FIG. 5.
BSP co-localizes to large and small BMF complexes containing BAG-75. Confluent UMR cultures were incubated for 24 h in the presence of {beta}-GP, fixed in ethanol, and then processed for immunofluorescence microscopy with anti-BSP monoclonal WV1D1(9C5) (red) and anti-BAG-75 (green) antibodies as described under "Materials and Methods." Panels A–D represent images of the same field of view containing small BMF. White and black arrows mark BMF that stain strongly for both BAG-75 and BSP antigens. Panel D, color overlay image for both BSP (red) and BAG-75 (green). Panel E, brightfield view of an alizarin red S-stained culture showing the presence of many small BMF (black arrowheads) and two large BMF (black arrows). Panels F–H are images from the same field of view containing a large and several small BMF. Upper (panel G) and lower (panel H) confocal optical planes of anti-BSP stained large BMF. Approximate displacement of images in G and H is about 15 µm (4 versus 19 µm from the basal aspect of the culture, respectively). Note the presence of BSP-stained small BMF (white arrows) and intracellular BSP staining of individual cells within the surrounding monolayer (white arrowheads) in this lower optical plane only. Scale bars, panel D, 50 µm; panels E–G, 200 µm.

 
Thus, in UMR cultures, BAG-75 is a biomarker both for BMF precursors prior to initiating mineralization, and for mature BMF containing mineral crystals regardless of size. Within the larger BAG-75-enriched mature BMF, the apatite mineral deposits seem to be organized into small domains (10–20 µm) having sizes similar to those of the smaller BMF. Again, this appearance suggests that the larger sized BMF precursors may represent a cluster of smaller ones.

BAG-75-enriched Focal Deposits Represent the Initial Sites of Mineralization in Primary Calvarial Osteoblastic Cultures— The presence of BMF in the UMR osteoblastic culture system raises the possibility that structures like these may also exist in primary osteoblastic cultures. Fig. 3, D and E, indicate that initial focal mineral deposits in fetal rat calvarial osteoblastic cell cultures have structural features and dimensions similar to those of UMR BMF. When decalcified by EDTA, these focal sites stain positively for the presence of BAG-75 (Fig. 3, F and G). Two other characteristics of mineralization in primary osteoblastic cultures are consistent with that occurring in BMF in UMR cultures (Fig. 2): (a) the focal area for BAG-75 immunostaining is larger than the focal area measurement for alizarin red S staining (compare Fig. 3, D with F, and E with G), and (b) focal BAG-75 staining temporally precedes that of apatite deposition (for example, compare Fig. 3, A and B with D and F). Additional support for the concept that BMF are the sites of mineral nucleation in many osteoblastic model systems is shown in Fig. 3, H–J. Non-transformed osteoblastic cell line MC3T3-E1 exhibits focal alizarin red S-stained mineral deposits similar to those observed in UMR cultures (Fig. 3, H and I) that also stain positively for the presence of BAG-75 after partial decalcification using EDTA (Fig. 3J).

Decalcification Reveals That the BAG-75 Content Increases within BMF during Active Mineralization—Because the results above suggest that BAG-75 epitopes might be partially blocked by hydroxyapatite mineral deposits, it was important to show that this antigenicity could be recovered upon decalcification. To this end, replicate UMR cultures at 64 h of incubation were treated with or without {beta}-GP supplementation for 24 h. Mineralized cultures were fixed and then decalcified using EDTA chelation (Fig. 4) or other decalcification methods (30). Atomic absorption measurement of elemental calcium determined that these treatments released more than 98% of the original calcium content of fully mineralized cultures (30). In the absence of confounding mineral crystals, a large increase in BAG-75 signal is observed indicating that the BAG-75 content of BMF is substantially increased over the 24-h mineralization period (Fig. 4). This accumulation of BAG-75 occurs only after addition of {beta}-GP to the UMR cultures, as control cultures not exposed to {beta}-GP over the same time frame do not exhibit this increase in BAG-75 immunofluorescence (data not shown). Thus, as shown previously for BSP (30), the content of the BAG-75 epitope within BMF increases during active mineralization.



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  		FIG. 4.
BAG-75 content of BMF increases during mineralization. Fully mineralized cultures (24 h {beta}-GP) were decalcified overnight with Tris-buffered saline, pH 8.0, containing 50 mM EDTA. Cultures were then processed for immunofluorescence microscopy with anti-BAG-75 and then with Alexa green-conjugated anti-rabbit IgG antibody. For comparison, a parallel fully mineralized culture without decalcification is shown in Fig. 2, E and F. Left panels, paired anti-BAG-75 and brightfield images of zero time ({beta}-GP minus) culture. Right panels, paired anti-BAG-75 and brightfield images of 24-h ({beta}-GP plus) cultures after decalcification. Scale bar, 20 µm.

 
When decalcification solutions were recovered and processed for Western blotting with anti-BAG-75 (data not shown) or BSP antibodies (30), very little BAG-75 or BSP protein was detectable in these fractions. These findings indicate that most of the BAG-75 and BSP epitopes are not solubilized by decalcification of mineralized cultures. This suggests that most of the BAG-75 and BSP accumulated within BMF is not the result of adsorption to pre-existing hydroxyapatite crystals. A similar conclusion was made with respect to the BAG-75 present at sites of initial mineral nucleation in primary bone (see Gorski et al. (29)).

BSP Co-localizes to Large and Small BMF Containing BAG-75—Without {beta}-GP, BSP is predominantly secreted into the media by UMR cells, although after {beta}-GP is added, BSP rapidly accumulates within supramolecular complexes that subsequently mineralize (30). We demonstrate in Fig. 5 that these extracellular sites in mineralizing UMR cultures seem to be the same as BMF precursors enriched in BAG-75 and recognizable in zero time cultures without {beta}-GP (Figs. 2 and 4). In fully mineralized cultures, BSP was localized within both large (Fig. 5, G and H) and small (Fig. 5, C and H) mature BMF, which also stained strongly for BAG-75 (Figs. 4 and 5A). Note that the shape and boundaries of the areas of BSP staining corresponded exactly with those delimited by BAG-75 immunostaining (Fig. 5D, overlay image). The physical characteristics and BAG-75 content of the small BMF precursors seen in Figs. 2 and 4, A–D, identify them as similar structures. Interestingly, although many BMF exhibited a strong immunofluorescence signal for both proteins (Fig. 5D, arrows), some small BMF structures displayed noticeably lower levels of co-staining. These findings could be because of differences in mineral content, which may lead to blocking of antigens. Altogether, these results demonstrate that BSP begins to accumulate in BAG-75-enriched BMF after the addition of {beta}-GP, but prior to nucleation of hydroxyapatite crystals (30).

Ultrastructural Analysis of BMF during Active Mineralization of UMR Cultures—The X-Z view from the confocal imaging shown in Fig. 1C indicates that small BMF are roughly spherical in shape with a diameter range of 10–20 µm. The X-Z ultrastructural image of a BMF in a mineralizing UMR culture (Fig. 6A) indicates that these confocal estimates are quite accurate. The inset scanning EM image in Fig. 6A also shows that the overall dimension and shape of supramolecular complexes formed by purified BAG-75 protein are similar to those of small BMF observed in mineralized UMR cultures.



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  		FIG. 6.
Ultrastructural views of BMF. Panel A shows a view cut perpendicular to the culture surface through a single BMF from a mineralizing UMR culture incubated for 12 h in the presence of {beta}-GP, then fixed and processed for electron microscopy as described under "Materials and Methods." The black line shown roughly 2 µm above the basal aspect of the culture represents the relative position of a 100-nm thin section along a plane parallel to the culture surface representing the viewing planes shown in panels B–D. Inset shows a scanning EM view of two supramolecular complexes formed spontaneously from purified BAG-75 (24). Panel B shows a view of small and large spherical structures within a mineralizing BMF. The arrowheads indicate the locations of 50–200-nm diameter opaque structures having an ultrastructural appearance similar to matrix vesicles. The asterisks indicate the locations of 300–600-nm diameter translucent spheres that we refer to as bioapatite vesicles. The inset in the right panel is a higher magnification view of the small and large vesicles indicated by the arrowhead and asterisk at the top of the right panel. Note how both vesicular structures appear to each touch the surface of the other. Panels C and D show views cut parallel to the culture surface through a mineralizing BMF (panel C), and a BMF precursor from a non-mineralizing control (panel D); note the healthy looking cells near the BMF areas. Boxes in panels C (left) and D (left) are areas shown at higher magnification in their respective right panels. Arrows in panel C (left) indicate positions of other BMF not shown at higher magnification in panel C (right). Insets are higher magnification views of the boxed areas in panels C (right) and D (right). Note how both vesicular structures appear to each touch the surface of the other. Panels C and D show views cut parallel to the culture surface through a mineralizing BMF (panel C), and a BMF precursor from a non-mineralizing control (panel D); note the healthy looking cells near the BMF areas. Boxes in panels C (left) and D (left) are areas shown at higher magnification in their respective right panels. Arrows in panel C (left) indicate positions of other BMF not shown at higher magnification in panel C (right). Insets are higher magnification views of the boxed areas in panels C (right) and D (right).

 
BMF are filled with both thin fibrillar and granular substances (Fig. 6B). In addition, BMF contain what appear as translucent round structures of 300–800 nm diameter, and electron opaque round structures of 50–200 nm in diameter (Fig. 6, A–C). BMF did not, however, exhibit evidence of opaque, rounded structures having a classic appearance of condensed chromatin or apoptotic bodies (39, 40). This finding is consistent with DAPI and TUNEL staining analyses shown in Fig. 1, C and D, respectively. A comparison of BMF during mineralization (Fig. 6C) as compared with unmineralized controls (Fig. 6D) indicates that the only visible differences are that the former are completely filled with fibrillar, granular, and vesicular structures with little void space (Fig. 6C, inset), whereas the latter exhibit more void space and are less granular in appearance (Fig. 6D, inset).

Figs. 6B and 7C (arrowhead) show clearly that there are two distinct size classes of spherical shaped structures within mineralized BMF. One population, ranging in diameters of 50–200 nm, has a highly opaque core with a discernable 6–8-nm thick trilaminar-delimiting boundary (i.e. "dark-light-dark") consistent with the ultrastructural characteristics of a phospholipid membrane (Fig. 7C, arrowhead). These smaller sized structures resemble the ultrastructural appearance of matrix vesicles (1, 3, 5, 8). Another larger sized population (Figs. 6B, asterisks, and 7C), ranging in diameters from 300 to 800 nm, has a relatively translucent core and a 10–20-nm thick boundary surrounding the entire structure. Within mineralizing BMF, both the small and large spherical structures appear to come in close contact with each other at their surfaces (Figs. 6B, inset, and Fig. 7C).



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  		FIG. 7.
Ultrastructural view of a mineralizing BMF stained with anti-BSP antibody LF-87. Panel A, the arrows indicate the positions of matrix areas containing clusters of more than 10 12-nm gold particles. The immunogold particles appear associated with thin filament fibrils and not banded fibrillar collagen structures. Panel B, the arrows indicate the positions of clusters of immunogold particles associated with the larger translucent bioapatite vesicles. Arrowheads indicate the positions of structures that resemble matrix vesicles, and do not contain associated gold particles. Panel C, a higher magnification view of both large and small vesicular structures. Arrow indicates a region of the larger structure containing a cluster of immunogold particles that appears to be at its surface. Arrowhead indicates a clear view of the trilaminar-delimiting boundary surrounding the smaller vesicle. Note how both larger and smaller vesicles appear to touch the surface of the other.

 
Localization of BAG-75 and BSP within BMF at the Ultrastructural Level—Fig. 2, E and F, demonstrate that the inner core of the BMF contains more apatite mineral than its outer boundaries. Within the inner core of BMF, the immunogold particles indicating the locations of BSP were generally distributed on 10-nm thin filament fibrils (Fig. 7A, arrows). Toward the outer surface of BMF, BSP immunogold particles were generally associated with larger vesicles, but not the smaller ones (Fig. 7, B and C, arrows versus arrowheads, respectively). Close inspection of the gold particle staining pattern on these larger structures gives the impression that BSP epitopes are on the surface. This is particularly evident for the structure shown in Fig. 7C (arrow) where gold particles were only observed in a location that appeared to cut tangentially across the vesicle surface. Non-immune antibody control sections typically contained a few immunogold particles, usually present in groups of one or two, but never in clusters as large as those shown here (see Fig. 3C in this paper, and Fig. 7E in Gorski et al. (29) for images of non-immune antibody staining).

Double staining for BAG-75 and BSP using different sized immunogold particle antibodies (12 and 6 nm, respectively) revealed that these two bone matrix proteins are co-localized in the outer boundary region of BMF (Fig. 8). BAG-75 epitopes were enriched in the outermost layer while BSP epitopes were underneath this layer within submicron spatial resolution of each other. What is interesting about this outer region of BMF is that it represents the boundary between unmineralized and mineralized matrix areas (Fig. 2, E and F). BAG-75 immunogold particles were difficult to detect within the core of BMF possibly reflecting the effect of protein-protein interactions on epitope accessibility.



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  		FIG. 8.
Ultrastructural view of a mineralized BMF stained with both anti-BAG-75 and anti-BSP WV1D1(9C5) antibodies. UMR culture was allowed to mineralize for 24 h and then decalcified prior to immunostaining as described under "Materials and Methods." Top panel, the box indicates an area of a BMF near its outer boundary shown at higher magnification in the bottom panel. Bottom panel, the area labeled BAG-75 indicates the position of a large cluster of 12-nm immunogold particles identifying BAG-75 epitopes associated with thin filament fibrils. The box labeled BSP indicates the position of a cluster of 6-nm immunogold particles identifying BSP epitopes. Inset shows a higher magnification view of the 6-nm gold particles. Note that submicron distances separate both 12- and 6-nm gold particles.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study has revealed that BAG-75 is expressed very early during the biomineralization process of UMR 106-01 BSP osteoblastic cultures. Furthermore, this study provides evidence that focal extracellular deposition sites enriched in BAG-75 accurately predict the location of subsequent active mineral nucleation in mineralizing osteoblastic cultures. This finding may be a general feature of all osteoblastic cultures undergoing in vitro biomineralization because UMR, primary fetal calvarial, and MC3T3-E1 osteoblastic model systems share this property. While sharing a common enrichment in BAG-75, mineralized BMF sites in these models display some structural differences in overall size (smaller in MC3T3-E1 than in UMR or FRC cultures) and shape (more three-dimensional in UMR than in MC3T3-E1 and FRC cultures). We hypothesize that relative differences in secretion rates of individual BMF components (vesicles, BSP, and BAG-75) or in the local distribution of cells committed to osteogenic differentiation may contribute to such distinctions.

BAG-75 is a biomarker for locating the future positions of BMF, and we refer to these BAG-75-enriched areas prior to mineral formation as BMF precursors. Two size populations of these spherical supramolecular extracellular complexes were recognized morphologically and by virtue of their enrichment in BAG-75. Shortly after the addition of {beta}-GP, an inducer of mineralization, UMR cultures begin to quantitatively accumulate BSP within these BAG-75-enriched BMF precursors (30). A few hours later, these BAG-75- and BSP-enriched BMF then deposit hydroxyapatite within, thereby converting them into mature BMF. Regardless of mineral content, BAG-75 is consistently present within the outer layer of mineralized BMF suggesting a role in defining the dimensions of the forming mineralized matrix. A similar expression pattern of BAG-75 in developing bone implies it participates in setting the boundary limits of new mineralized trabeculae (see Gorski et al. (29)).

As the earliest recognized biomarker of BMF, we hypothesize that BAG-75 serves two roles, one as a structural scaffold and the other as a source of phosphate ions for apatite nucleation. With regard to its first function, we propose that BAG-75 plays a key role in the initial assembly of the structural scaffold of BMF precursors. This is supported by evidence that purified BAG-75 protein alone is able to self-assemble into thin linear strands of up to 1 µm in length (24, 27, 41). These BAG-75 microfibrils can coalesce to form supramolecular spherical structures of up to 10–15 µm in diameter (Fig. 6A, inset), which have the capability to retain large quantities of phosphate ions (24). These structures are remarkably similar in shape and dimension to the smaller BAG-75-enriched BMF identified here in UMR cultures.

We further rationalize that such a BAG-75 framework could provide key secondary binding sites to recruit other proteins, such as BSP or alkaline phosphatase, required for subsequent nucleation of hydroxyapatite. Despite excellent morphological descriptions of membranous bone formation during embryogenesis (42), few matrix protein biomarkers have been identified that uniquely define future sites of osteogenesis. Zhu et al. (43) found that the BSP message was weakly expressed in the center of mesenchymal cartilaginous condensations at 14 days of gestation in the mouse. In the current study, we demonstrate that BAG-75 accumulation in the extracellular matrix precedes the deposition of even BSP, and represents one of the earliest matrix protein markers for sites of biomineralization.

Alkaline phosphatase also appears to be expressed very early during osteogenesis (44–46), and an elevated expression of alkaline phosphatase has been used to define committed proliferating osteogenic cells (14). As described in our BMF model of mineralization below, alkaline phosphatase is an important component of matrix vesicles (47, 48), where it is believed to produce inorganic phosphate ions from complex phosphate sources to feed the process of crystal nucleation. Addition of alkaline phosphatase inhibitors such as levamisole blocks UMR biomineralization reactions initiated by {beta}-GP (19). With regard to its second functional role in UMR biomineralization, we hypothesize that the de-phosphorylation of BAG-75 protein itself could also provide a potential source of phosphate ions for apatite nucleation reactions because this protein contains an estimated 44 phosphoryl residues/mole (22).

Debate continues regarding the underlying mechanism(s) of vertebrate biomineralization. One view proposes that phospholipid membrane-delimited vesicles (matrix vesicles) released into the extracellular matrix by osteoblasts mediate biomineralization reactions (1, 3, 5, 8, 49). They are reported to exist in nearly all mineralizing tissues, but are best described in calcifying cartilage. They are small (50–200 nm diameter) vesicles containing high levels of phosphatidylserine and cardiolipin (50, 51). They contain several enzymes including alkaline phosphatase and pyrophosphatase (52), and have cytoskeletal (53) and matrix proteins (54) associated with their inner and outer membrane surfaces, respectively. Another biomineralization theory proposes that noncollagenous protein structures assembled within the type I collagen-rich, extracellular matrix of osteoid directly mediates the heterogeneous nucleation of apatite crystals (2, 7, 55). Based on the presence of polyacidic domains and a capacity to nucleate hydroxyapatite in vitro, BSP has been proposed to act as such a nucleator in bone and cartilage (21).

Yet another biomineralization theory proposes that calcified spherically shaped structures with a size range of 300–600 nm in diameter, termed crystal ghost aggregates, nucleate the initial apatite crystals within osteoid and then subsequently seed a heterogeneous nucleation reaction within the fibrillar collagen matrix (3, 4, 56–59). These mineralized structures in bone tissue stain positive with acridine orange or ruthenium red suggesting the presence of sulfate groups (4, 60). This staining was originally interpreted as evidence for sulfated proteoglycans in these structures (61). However, it is possible that a sulfated glycoprotein could also contribute to these sulfate groups. BSP has been shown to co-localize with crystal ghost aggregates in osteoid (18) and in osteoblastic cultures (20, 30); it also contains several sulfate groups bound to oligosaccharides and tyrosine residues (62, 63). Interestingly, BSP has not yet been reported to be a component of matrix vesicles (1, 47, 49, 64). Our findings indicate that BSP is associated with a population of large vesicle-like structures, but not with the smaller matrix vesicles. In this way, BSP may define a biochemically distinct population of vesicles, which are larger than matrix vesicles. Because apatite mineral forms in association with these "bioapatite vesicles" (65), we hypothesize that these larger vesicles may be related to, and likely an early structural stage of, the fully mineralized BSP-enriched particles referred to as crystal ghost aggregates (3, 4).

It is suggested that the large size, amorphous character, and prior lack of an authentic biomarker for BMF precursor and mature BMF complexes may have prevented their routine detection with ultrastructural methods, which tend to emphasize discrete electron-opaque structures of uniform appearance (e.g. matrix vesicles). Despite this limitation, similar spherical, interfibrillar regions containing crystal ghost aggregates enriched in BSP and an amorphous extracellular matrix have been identified in vitro (66, 67) and in vivo (68) as sites of subsequent initial mineral nucleation. In addition, Irie et al. (67) have noted the apparent similarity between these nucleation sites in vitro and those present in membranous or primary bone. The above mentioned studies suggest that BMF-like structures have been observed before, but seem to have been considered obscure entities lacking a clear context with respect to mineralization reactions. It is our contention that only with data such as that provided by our current study can one properly interpret the significance of these structures to the biomineralization process.

We propose a five step biomineralization model (Fig. 9) in which BMF, typically 15–25 µm in diameter, serves as a supramolecular domain that facilitates the initial sequestration of calcium and phosphate ions in an extracellular matrix environment. Step 1 involves the determination of the boundaries, shape, and size of future mineralized matrix areas by BAG-75 expressing cells. Step 2 involves the assembly of extracellular BMF precursor structures via BAG-75 self-assembly reactions. Step 3 involves the accumulation of matrix vesicles and bioapatite vesicles within the BMF precursor matrix. We envision that the large amount of membrane surface area within BMF packed with vesicles could facilitate heterogeneous nucleation reactions through adsorption and concentration of required proteins and ions. Upon the introduction of an organophosphate stimulant, step 4 involves the recruitment of required accessory proteins such as BSP, and the continued accumulation of BAG-75 into the BMF. We suggest that BMF-bound BSP would function to sequester calcium ions in proximity to copious amounts of phosphate ions associated with BAG-75. Together these events could lead to the supersaturation conditions necessary to nucleate hydroxyapatite crystals on the vesicle surfaces within BMF. Finally, step 5 involves the growth of these seed crystals and the formation of mineralized aggregates. Continued self-assembly of BAG-75 at the outer boundaries could foster new matrix growth within individual BMF. Future studies will investigate potential protein-protein interactions of monomeric and oligomeric BAG-75 complexes with BSP. In addition, identification, isolation, and characterization of the structure and function of BMF precursors are now feasible by following their BAG-75 biomarker.



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  		FIG. 9.
Diagram depicting the UMR biomineralization model. Times shown in steps 4 and 5 refer to organophosphate exposure times. Note that BSP is not retained within the BMF precursor containing BAG-75 (steps 2 and 3) until an organophosphate stimulant is added to the culture (step 4), at which time BSP is then retained in the BMF matrix. Key: large BAG-75 containing spherical structure; cross-hatches, 15–25-µm BMF and BMF precursor; small dark spheres, 50–300-nm matrix vesicle-like structures; vesicles with white interior, 500–800-nm bioapatite vesicles frequently observed with BSP on their surface; vesicles with dark outer ring, products of apparent fusion of matrix vesicles with 500–800-nm vesicles; granular dark material, associated with vesicles at step 5 represents initial mineral crystals.

 

   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant AR-45171 and The Lerner Research Institute of The Cleveland Clinic Foundation (to R. J. M.) and National Institutes of Health Grants DE-14619 and DE-11197 and the University of Missouri Research Board (to J. P. G). This work was presented in preliminary form at the 25th Annual Meeting of the American Society for Bone and Mineral Research, September 19–23, 2003, Minneapolis, MN (69). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. Biomedical Engineering, ND20, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-3212; Fax: 216-445-4383; E-mail: midura{at}bme.ri.ccf.org.

1 The abbreviations used are: BSP, bone sialoprotein; {beta}-GP, {beta}-glycerophosphate; BMF, biomineralization foci; BAG-75, bone acidic glycoprotein-75; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4',6-diamidino-2-phenylindole; bioapatite vesicles, 300–600 nm diameter translucent vesicular structures within BMF; matrix vesicles, 50–200 nm diameter opaque vesicular structures exhibiting a trilaminar membrane and accumulating within BMF; TRITC, tetramethylrhodamine isothiocyanate. Back


   ACKNOWLEDGMENTS
 
We acknowledge the expert assistance with confocal imaging by Dr. Judith Drazba, Imaging Facility, The Lerner Research Institute, and electron microscopy by Jessica Kueker, University of Missouri-Kansas City. We thank Dr. Mark McKee, McGill University, and Dr. Sarah Dallas, University of Missouri-Kansas City School of Dentistry, for providing the MC3T3-E1 and fetal rat calvarial cell cultures, respectively, and Dr. Bjorn R. Olsen, Harvard University, for careful critique of the manuscript.



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