ARTICLE |
Correspondence to: Antonio Nanci, Laboratory for the Study of Calcified Tissues and Biomaterials, Dept. of Stomatology, Faculty of Dentistry, Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal, QC, Canada. E-mail: antonio.nanci@umontreal.ca
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Summary |
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Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro. (J Histochem Cytochem 51:633641, 2003)
Key Words: rat, bone, calvarial cell culture, immunofluorescence, bone sialoprotein, collagen, fibronectin, osteopontin
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Introduction |
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OSTEOGENIC CELL CULTURE SYSTEMS are routinely used to study the tightly regulated cellular and extracellular matrix events of bone formation in a controlled environment. A commonly used system to study bone formation in vitro is primary culture of rat calvaria osteogenic cells. Cell isolation procedures, culture conditions, the temporal sequence of osteoblastic differentiation, and expression of matrix proteins have been well defined for this system (reviewed in
The expression pattern of matrix proteins in vitro has been correlated with acquisition and maturation of the osteoblast phenotype (
In culture, ideally, one would want to have a single cell population that evolves synchronously over time. However, cell isolates used for osteogenic cultures usually comprise a mixture of populations, ranging from precursor cells to fully differentiated osteoblasts (
Immunolabeling was selected as the protein detection method because molecular signals do not always correlate with the timing of protein synthesis and secretion and do not provide any indication of localization. The results obtained indicate that a significant proportion of differentiated osteoblasts in cell isolates already produce matrix proteins and, most likely, secrete them as soon as they are placed in culture.
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Materials and Methods |
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Cell Isolation and Primary Culture of Osteogenic Cells
Cells were isolated by sequential trypsin/collagenase digestion of calvarial bone from newborn (24 days) Wistar rats (Charles River Canada; St-Constant, QC, Canada) as previously described (
Cytocentrifuge Preparations of Isolated Cells
For preparation of cytocentrifuge slides, 60-µl aliquots of isolated cells suspended in MEM (2.5 x 105 cells/ml) were placed in each Cytofunnel of a Cytospin 3 centrifuge (Thermo Shandon; Pittsburgh, PA) and spun onto polylysine-coated microscope slides (Baxter Diagnostics; Deerfield, IL) at 900 rpm for 5 min. All slides were allowed to dry for 1 hr before fixation.
Immunofluorescence
Cells grown on coverslips and cytocentrifuge preparations of cells used for plating (time 0) were fixed for 15 min at room temperature (RT) in 4% paraformaldehyde buffered with 0.1 M PBS, pH 7.2. After washing in PBS, cells were then routinely processed for immunofluorescence labeling. Briefly, they were permeabilized with 0.5% Triton X-100 in PBS for 15 min, followed by blocking with 1% ovalbumin in PBS for 30 min. For extracellular labeling, cells were not permeabilized. Primary monoclonal and/or polyclonal antibodies to BSP, OPN, FN, type I pro-collagen, and tubulin (for detecting cell outlines) were used (Table 1), followed by corresponding Alexa Fluor 488 (green fluorescence)- or 594 (red fluorescence)-conjugated goat secondary antibodies (Molecular Probes; Eugene, OR), or by rhodamine (TRITC)-conjugated secondary antibodies (Jackson Immunoresearch Laboratories; West Grove, PA), diluted 1:200 in PBS. All incubations were performed in a humidified environment for 60 min at RT, followed by three 5-min washes in PBS. Replacement of the primary antibody with PBS was used as control. For type I pro-collagen labeling, incubations with pre-immune IgG were also carried out. Dual labeling was done with a 1:1 mixture of two primary antibodies followed by a 1:1 mixture of corresponding secondary antibodies. Mixtures were prepared to yield the same working dilutions as the ones used in single labeling (Table 1).
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Coverslips were mounted on microscope slides with Airvol 205 (Airproducts; Allentown, PA) and examined by epiluminescence under a conventional fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany), using Plan-Neofluar objectives (x40, NA 0.75, and x100, NA 1.30). For quantitative analysis, approximately 20 microscopic fields at x100 were randomly selected for a total of 350450 cells. Proportions of immunoreactive cells were expressed as the mean percentage per microscopic field ± standard deviation and comparisons were carried out using the non-parametric MannWhitney U-test for independent samples. Photographic recordings were made on Kodak Elite Chrome 400 reversal film (Eastman Kodak; Rochester, NY) with different exposure times (515 sec), depending on the intensity of fluorescence labeling. Double photographic exposure was done for the simultaneous visualization of dual labeling. Slides were then digitally recorded at 1200 dpi using an Epson Expression 1600 scanner and processed with Adobe Photoshop software. The results described below are representative of at least three different sets of primary cultures, except for cytocentrifuge preparations, which were prepared from two different digests only.
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Results |
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Cell Morphology
Phase-contrast visualization of enzymatically released calvarial cell preparations (time 0) fixed after cytocentrifugation revealed partially spread, rounded cells with variable dimensions, nucleus-to-cytoplasm ratios, and vacuolation (Fig 1). Fixation of cell suspensions before cytocentrifugation resulted in refractile, rounded cells (data not shown). Although the latter preparation permitted immunolabeling of cells with different antibodies, it was not used in this study because of the difficulty in assessing nuclear and cytoplasmic morphology by phase-contrast.
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Calvarial cells in primary cultures exhibited variable phenotypes and dimensions (Fig 2 and Fig 3), and, as expected, became progressively larger and more spread over the 3-day culture period. By 6 hr, cells attached and partially spread on the coverslip surface, showing predominantly a polygonal morphology with relatively short cytoplasmic extensions (Fig 2). By days 1 and 3, morphology was variable and cells were polygonal, stellate, or fusiform (Fig 3). Occasionally some of them had long cytoplasmic extensions, which frequently contacted other cells. Mitotic figures were also found at all time points.
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Antibodies
The various primary antibodies used labeled different proportions of cells and yielded different fluorescence intensities. Dual labeling with monoclonal and polyclonal antibodies against BSP or OPN showed similar labeling patterns, as confirmed by the presence of yellow fluorescence with double photographic exposure. However, immunoreactivity appeared more defined with monoclonal antibodies (compare Fig 1D and Fig 1E). Polyclonal antibodies against chicken tubulin and against recombinant N-propeptide of the 1 (I) human collagen chain recognized epitopes in rat osteogenic cells and resulted in characteristic labeling patterns. No significant labeling was observed in control incubations.
Distribution of Labeling
In a majority of cases, OPN was localized in an extensive perinuclear tubular network, vesicular structures, and punctate deposits throughout the cytoplasm (Fig 1B, Fig 2A, Fig 2C, and Fig 3A, Fig 3B, Fig 3D, Fig 3F, and Fig 3I). Some cells showed labeling of a large, juxtanuclear region reminiscent of the Golgi area in active osteoblasts (Fig 2A, Fig 3D, and Fig 3F). A small proportion of OPN-positive cells (see below) also contained a peripheral cytoplasmic labeling (so-called "perimembranous intracellular OPN";
Anti-BSP fluorescence was distinctively and mainly detected in the juxtanuclear Golgi area and as punctate deposits throughout the cytoplasm (Fig 1D, Fig 1E, Fig 2B, Fig 3C, and Fig 3H). Some rare cells showed perinuclear labeling. Extracellular accumulations of BSP-reactive matrix were sporadically detected at day 3, mainly in areas with relatively higher cell density.
At all time points, type I pro-collagen labeling appeared diffuse around the nucleus (Fig 2F, Fig 3E, and Fig 3H). Near the periphery of the cells, the labeling assumed an almost reticular pattern and, at day 3, fluorescent globular structures were sometimes found in this region. In addition, at 6 hr, a subset of immunoreactive cells exhibited labeling of the Golgi area and some granules. Both labeling patterns of type I pro-collagen were not observed when the primary antibody was replaced with pre-immune IgG.
In permeabilized and non-permeabilized cells, FN labeling revealed an extracellular fibrillar network extending from one cell to another, but not exclusively associated with the cell surface. These findings occurred in areas of high cell density at all time points (Fig 3G). In areas of low cell density, only small amounts of FN could be observed and appeared as small dots or short fibrillar structures associated with the cell surface. In permeabilized cells, strong diffuse perinuclear labeling was occasionally observed (Fig 2E). Cells showing such labeling were frequently associated with trails of FN on the substrate adjacent to them.
It is generally accepted that cell fixation with paraformaldehyde does not preserve microtubules very well. Nevertheless, tubulin labeling was detected throughout the entire cytoplasm (Fig 2D), revealing cell outlines that closely matched the ones observed by phase-contrast.
Quantitative Analysis of Labeled Cells
Quantitative analysis of immunoreactive cells was carried out at two time points (time 0 and day 1). Different proportions of osteogenic cells were immunoreactive for BSP, OPN, FN, type I pro-collagen, and tubulin (Table 2). Cytocentrifuge preparations (time 0) contained about two times more OPN- than BSP-positive cells (p<0.0001). Almost all BSP-positive cells (97%) were also immunoreactive for OPN. Tubulin labeling was detected in all isolated cells.
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After 1 day in culture, almost half the cell population was OPN- and type I pro-collagen-positive (p=0.3192) but only 27% of cells were dual-labeled with these antibodies (Fig 3E and Fig 3F). The proportion of cells labeled with BSP antibodies was significantly lower than that of OPN- and type I pro-collagen-positive cells (p<0.0001 for both). About 20% of cells were immunoreactive for BSP and virtually all of these cells also expressed OPN (Fig 3C and Fig 3D) and type I pro-collagen. Cells with cytoplasmic FN labeling occurred in almost the same proportion as BSP-positive cells (p=0.0427), whereas extracellular FN was associated with almost all cultured cells. Peripheral accumulations of OPN were found in about 7% of cells. All cells in the culture were immunoreactive for tubulin.
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Discussion |
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In the present study, we demonstrated that a significant proportion of cells enzymatically released from newborn rat calvarial bone are immunoreactive for some major non-collagenous matrix proteins (NCPs). In addition, at early time points, different proportions of osteogenic cells express collagenous and NCPs. Therefore, the early expression of some matrix proteins by cultured osteogenic cells does not necessarily represent de novo gene expression but may also reflect pre-existent synthetic activity taking place during bone matrix formation and mineralization in vivo, before sacrifice of the animal.
Trypsin/collagenase digestion of fetal or newborn rat calvarial bone yields a heterogeneous cell population, which comprises osteoprogenitors, pre-osteoblasts, differentiated osteoblasts, and fibroblasts (
In vivo and in vitro studies have demonstrated that differentiated osteoblasts are phenotypically diverse. Expression of OPN, BSP, and osteocalcin by these cells is extremely variable (
Although OPN and BSP are generally regarded as multifunctional extracellular matrix molecules involved in cell adhesion and migration and in the regulation of mineral deposition (
Even though only 15% of cells showed intracellular FN labeling, the association of almost all cells with extracellular FN at early time points is not unexpected because (a) cultured cells are capable of assembling FN fibrillar matrix in the presence of soluble FN molecules (
In conclusion, although it is generally agreed that enzymatically isolated cells comprise mixed populations, we have analyzed quantitatively the percentage of cells in the isolate and after short-term culture that express matrix proteins. A non-negligible proportion of cells isolated from newborn rat calvaria were found to already contain, at least, BSP and OPN. Most likely, they also express other matrix proteins such as FN and type I pro-collagen. Based on the labeling pattern in the Golgi area (
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Footnotes |
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1 Present address: Faculty of Dentistry, University of São Paulo at Ribeirão Preto, Brazil.
2 Present address: Health Sciences University of Hokkaido, Hokkaido, Japan.
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Acknowledgments |
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Supported by the Canadian Institutes of Health Research (CIHR). Paulo Tambasco de Oliveira is the recipient of a postdoctoral fellowship (00/11604-4) from FAPESP (Brazil) and is also supported by the University of São Paulo (Brazil).
We thank Ana Carina da Paula (Department of Immunology and Microbiology, Université de Montréal) for technical assistance in cytocentrifuge preparations, Dr Larry W. Fisher (NIH) for providing anti-BSP (LF-87 and LF-100) and anti-OPN (LF-123) antibodies, Drs M.C. FarachCarson and W.T. Butler (University of Texas at Houston, TX) for the anti-rat OPN antibody, and Drs Malcolm Collins and Paul Bornstein (University of Washington at Seattle, WA) for an antibody to recombinant N-propeptide of the 1 (I) human collagen chain. The mouse monoclonal anti-rat osteopontin (MPIIIB10-1) and anti-rat bone sialoprotein (WVID1-9C5) antibodies, developed by Michael Solursh and Ahnders Franzen, were obtained from the Developmental Studies Hybridoma Bank formed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA).
Received for publication August 22, 2002; accepted December 11, 2002.
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