Arch. Histol. Cytol., Vol. 55, No. 4 (1992) p. 415-422
Morphological
and Immunocytochemical
Osteoblast
Cultures
Rong-Sen
Tang-Kue
YANG,
Departments of Orthopaedics', University, Taipei, Taiwan
from LIU1, Keh-Sung
Long
Bones
TSAI2,
Shoei-Yn
Characterization of Neonatal LIN-SHIAU3
and
of
Rats* Kuo-Shyan
LU4
Clinical Pathology2, Pharmacology3 and Anatomy4, College of Medicine, National Taiwan
Received July 13, 1992
Summary. The present study reports a successful culture system of rat bone cells obtained from the bare long bone fragments of neonatal Wistar rats. The morphology, histochemical and immunocytochemical properties of the cultured cells were characterized by phase contrast microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), histochemical staining with alkaline phosphatase (ALP), and immunocytochemical staining with the anti-osteocalcin antibodies. Under phase contrast microscopy, the cultured rat bone cells changed their morphology from being slender with relatively-short cytoplasmic processes to becoming polygonal with longer processes, and subsequently flattened. These cultured bone cells formed bone nodules after 3 weeks. Under SEM, the cultured bone cell appeared polygonal with some microvilli. The TEM showed that their cytoplasm contained abundant endoplasmic reticulum and well-developed Golgi apparatus. Positive histochemical staining of ALP was also detected as a blue coloration and granular appearance in the cultured bone cells. Immunocytochemistry with the polyclonal and monoclonal anti-osteocalcin antibodies showed the localization of osteocalcin in both the cytoplasm and nucleus. All these data lead us to consider that the cultured bone cells in our system were probably osteoblasts from growing bone tissue in vitro. This provides a convenient test system for further studies on the regulation of the growth and metabolism of rat bone cells.
AUF'MKOLKet al. 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIER et al., 1990). Once the culture of bone cells is established, the characterization of the bone cells becomes essential for further studies. However, the exact nature of the cultured cells remains conjectural, since these cultures probably represent mixtures of cell types of a mesenchymal origin. By the morphology of cultured cells alone, it is very difficult to differentiate among various mesenchymal cells, e.g., osteoblasts, chondrocytes, and fibroblasts. Therefore, identification by morphological distinctions with phase microscopy, SEM and TEM, histochemical staining with ALP, and responsiveness to parathyroid hormone etc., have been employed to establish the exact nature of the cultured bone cells (WILLIAMS et al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983; AUF'MKOLK et al. 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIER et al., 1990). Moreover, osteocalcin was determined to be produced only by osteoblasts and osteocytes; therefore, immunocytochemical staining with a monoclonal or polyclonal antibody against osteocalcin provided a specific method of identification (BIANCOet al., 1985; GROOTet al., 1986; THAVARAJAHet al., 1986; CAMARDA et al., 1987; MARK et al., 1987; WANG et al., 1987, 1991; VRIES et al., 1988; OHTA et al., 1989; BOIVIN et al., 1990). The present study describes a successful explant culture system of rat bone cells from long bone segments of the neonatal Wistar rat. The data on the characterization of these bone cells obtained from SEM, TEM, histochemical staining with ALP and immunocytochemical staining with anti-osteocalcin
Successful bone cell cultures, initiated by PECK et al. (1964), have enabled us to investigate the regulation of the growth and metabolism of the bone cells. Many sources of bone cells have been described, including rat, mice, chicks, human, feline and human osteosarcoma cell lines (WILLIAMS et al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983;
This work was supported in part by the National Science Council of the Republic of China, NSC80-0412-B002-159 and by the National Taiwan University Hospital, NTUH-81016-A11. 415
NSC80-0412-B002-210
and
416
R.-S. YANGet al.:
antibody have led us to consider these cultured cells to be osteoblasts.
then ion sputter coated with a thin layer (about 10 nm) of gold and examined in a JOEL T330A scanning electron microscope at 25 kV.
MATERIALS
TEM
AND METHODS
Cell culture The long bones of wistar rat neonates, including the femur, tibia, humerus, radius, and the ulna, were dissected with aseptic techniques. After defleshing, the bones were diced into bare bone fragments and were washed in phosphate buffered saline (PBS) to remove marrow cells. The primarily separated rat bone cells obtained from these explants were grown in Dulbecco's modified calcium free Eagle's medium (DMEM) (Biofluids, USA) supplemented with 7% calcium free F12 (Gibco, USA) and 10% fetal calf serum (Gibco, USA), pH 7.6 containing glutamine (58.5,ug/ ml), MgSO4 (200 jig/ml), and antibiotics (100 U penicillin and 100ug streptomycin sulfate per ml). They were kept in a humidified atmosphere containing 95% air and 5% C02 at 3TC (ROBEY and TERMINE, 1985). The viability of the cells was monitored by the trypanblue exclusion test. Using the original explant or the cultured bone cells, the cells were subcultured twice a week. The culture medium was changed twice weekly. Cell passage was carried out by incubating a monolayer for 5-10 min in calcium- and magnesium-free Tyrode's solution containing 0.25% trypsin (Sigma, USA). Explant pieces were removed when the resulting monolayer approached confluence and the cells were replated in fresh medium at about one third their confluent density. The morphology of cultured bone cells was observed under a phase contrast microscope, and the population doubling time was calculated by counting the cell numbers at regular intervals. For the mineralization features of these cultured cells, the culture medium was supplemented with 50,ug/ml ascorbic acid (Sigma) and 10 mM R-glycerophosphate (Sigma). The conditioned medium was changed every other day for 12 days. The cultured cells and bone nodules were stained with the von Kossa method and 0.5% safranin 0 for light microscopy. SEM For SEM studies, the osteoblasts in the culture dishes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate for 20 min. At fixation, the cells were rinsed in phosphate buffer for 5 min, postfixed in 1 % osmium tetroxide for 20 min, then dehydrated in a graded series of alcohol, and subsequently critical-point dried with liquid carbon dioxide. The specimens were
For TEM study, cultured cells were fixed with 2.5 glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min. After post-fixation in 1% aqueous osmium tetroxide for 1 h, the specimens were stained en bloc with 0.5% uranyl acetate, dehydrated in alcohol and then embedded in Epon-araldite mixture. Thin section were cut and stained with uranyl acetate before examination in a JOEL 2000EXII electron microscope at 100 kV. Histochemical
study of ALP
Cells grown on cover slips for 2 weeks after primary explant culture were fixed in buffered acetone fixative (0.03 M Na-citrate, 0.03 M citric acid, 60% acetone) for 30 sec. The cover slips were rinsed in distilled water for 10 sec, air-dried, and stained in incubation solution at room temperature for 1 h. The incubation solution contained 6 mg Naphthol AS phosphate (Sigma), 0.1 ml N, N dimethyl f ormamide (Sigma), and 20 mg Fast blue BB salt (Sigma) in 20 ml 0.5 M Tris buffer, pH 10.2. The mixture was vigorously agitated for 30 sec and filltered directly onto the cover slips. The slips were then rinsed in distilled water for 10 sec, air-dried, counterstained with 0.5 or 0.05% safranin 0 solution, mounted in gelatinglycerin jelly and observed under oil immersion. For the control, the cells were heat-inactivated by dipping the cover slips in boiled water for 1 min before incubation. Immunocytochemistry
of the osteocalcin
The monoclonal anti-osteocalcin antibody was a gift from Dr. K. MANN and B. L. RIGGS, Mayo Clinic and Foundation (TRACY et al., 1987, 1990). Analysis with synthetic peptides revealed an epitopic specificity residing in residues 1-19 of osteocalcin. This monoclonal antibody requires Ca++ in order to bind normally carboxylated osteocalcin, but will also bind to decarboxylated osteocalcin (TRACY et al., 1987). The polyclonal anti-bovine bone osteocalcin antiserum, purchased from Immuno Nuclear Corp., USA, was reported to crossreact with rat osteocalcin (VRIES et al., 1988). Both biotinylated goat anti-rabbit IgG and horse anti-mouse IgG immunoglobulins were obtained from Vector Lab., USA. For the immunocytochemical staining, the cultured cells were washed
Characterization
of Rat
Osteoblasts
417
Fig. 1. Phase contrast photomicrographs of cultured bone cells. A. Bone cells grow initially from the explant cultures. B. Bone cells in the second passage display a polygonal shape and longer cell process. C. Bone cells at confluence become smaller and packed. D. Some cells flatten with time. The cytoplasm spreads out. E. Bone nodules form after 3 weeks of incubation. F. The cells cultured in the medium supplemented with ascorbic acid and fl-glycerophosphate are embedded in a mineralized matrix (bone nodule) stained by the von Kossa method.
418
R.-S. YANGet al.:
with PBS and then fixed in methanol or 4 % paraformaldehyde for 10 min. After washing in PBS and treating with 10% normal goat albumin, the cells were incubated consecutively with: 1) 1:50 primary antibody (anti-osteocalcin serum) for 1 h at 37°C; 2) a secondary antibody (1:100 diluted biotinylated horse anti-mouse IgG for monoclonal anti-osteocalcin antibody, or biotinylated goat anti-rabbit IgG for polyclonal antibody) at 37°C for 1 h; and 3) avidin-biotin peroxidase (1:100 diluted with PBS) for 1 h at 37°C. After coloration with diaminobenzidine (DAB) solution (0.5 mg/ml in 0.1 M Tris buffer, pH 7.4, containing 0.05% H2O2), for 20 min, the cells were mounted with gel/mount (Biomeda) and examined in an inverted phase contrast microscope. The cells incubated in anti-osteocalcin serum free medium served as controls.
RESULTS Cell culture
and light microscopy
The outgrowth in explant cultures resulted in layers of sparse cells which proliferated slowly and spread over the culture dishes, reaching a confluence at a density of 7x10 cells/cm' within 7 days. The population doubling time from the initial cell outgrowth in explant pieces was found to be 114h. Immediately after culturing, rat bone cells appeared slightly slender in shape and displayed sparse and relatively-short cytoplasmic processes (2-5 um in length) (Fig. 1A). They became polygonal in shape and developed longer cell processes (5-30 um) after 3 days (Fig. 1B). Upon reaching confluence, they
Fig. 2. Scanning electron micrograph and transmission electron micrograph of bone cells. A. SEM of a cultured bone cell shows a polygonal shape and displays many short cellular processes on the cell surface. B. TEM of the bone cells shows a cytoplasm rich in rough endoplasmic reticulum (r) and Golgi apparatus (G) near the nucleus (n). Moderate numbers of small mitochondria (m), vesicles (v) and microvilli (mv) are found in the periphery.
Characterization
of Rat Osteoblasts
419
Fig. 3. Histochemical demonstration of alkaline phosphatase (ALP) activity in the cytoplasm. A. The control cells show no ALP activity (0.5% safranin 0 counterstained). B and C. The ALP-positive cells are characterized by a blue coloration of varying density and occasionally by a light and deep discrete blue granular appearance (0.05% safranin 0 counterstained).
Fig. 4.
Immunocytochemical
localization
of endogenous
show no reaction product. B. The immunocytochemical shows brown granules in the cytoplasm. C. The osteocalcin
antibody
shows
staining
in both
osteocalcin
in the cultured
staining with the monoclonal immunocytochemical staining
the cytoplasm
and nucleus.
bone
cells.
A. Control
cells
anti-osteocalcin antibody with the polyclonal anti-
420
R.-S. YANGet al.:
became smaller and packed (Fig. 1C). Some were multipolar and became flattened with time in culture (Fig. 1D). The cultured bone cells formed bone nodules after incubation for longer than 3 weeks (Fig. iE). The cells and bone nodules cultured in the medium supplemented with ascorbic acid and 3-glycerophosphate developed a mineralized matrix which could be stained with von Kossa method (Fig. iF).
calcin was mainly present in the cytoplasm of cultured bone cells although weak nuclear staining was also observed (Fig. 4B). The polyclonal anti-osteocalcin antibody brought about more clear staining of the nucleus (Fig. 4C). Since cultured cells displayed the same pattern of immunostaining, the results showed that these cultured rat bone cells were osteoblast-like, and that the cell population is highly homogeneous.
SEM Under the SEM, the cultured cell displayed a large, polygonal soma, but few long cytoplasmic processes. A protrusion of the nucleus at the center of soma was prominent, and at high magnification, the cell surfaces of the soma were equipped with some microvilli (Fig. 2A). TEM TEM observation revealed that the cytoplasm of cultured bone cells contained abundant free ribosomes and rough endoplasmic reticulum which were occasionally arranged in parallel. A well-developed Golgi apparatus occupied the space adjacent to the nucleus. A moderate number of mitochondria was distributed throughout the cytoplasm. In addition, a few vesicles containing a finely granular material, probably lysosomes, were located in the periphery of the cytoplasm (Fig. 2B). Histochemical
study of ALP
ALP activity is considered a marker of the osteoblast phenotype. Positive ALP activity was demonstrated by brilliant blue coloration with the aforementioned histoenzymatic method. The control cells did not show any staining (Fig. 3A). The positive ALP activity in cultured cells was characterized by a blue coloration of varying intensity (Fig. 3B), and occasionally by the discrete blue granular appearance of the cells (Fig. 3C), indicating that these cells were osteoblast-like cells. About 78% of the cell population demonstrated positive reactions, reflecting a high degree of homogeneity. Immunocytochemistry
of the osteocalcin
In our systems, the DAB staining caused a light to deep brown color for the positively-stained cells. The control cells did not show any immunoreactivity to osteocalcin (Fig. 4A). The results of the immunocytochemical staining with monoclonal anti-osteocalcin antibody indicated that immunoreactivity of osteo-
DISCUSSION Bone cells play a fundamental role in bone tissue remodeling throughout life; their function is under the regulation of many systemic and local growth factors (HAGEL-BRADWAYand DZIAK, 1989; MARTIN et al., 1989; ARNETT,1990). The isolation and culture of the bone cells remain the basis for the investigation of the bone metabolism and therefore, the identification of cultured bone cells is a cornerstone for further studies. The cultured rat bone cells in the present study were identified by histochemical staining with ALP, SEM, TEM and immunocytochemical staining with polyclonal and monoclonal anti-osteocalcin antibodies. Although the spindle-shaped cells prevailed in fibroblast cultures and osteoblasts were characterized by their polygonal shape and in forming condensed bands of cells, the latter also assumed a spindle shape. It is thus difficult to distinguish cultured cells from mesenchymal origins by their morphology. However, the morphological features of the cultured bone cells under the phase microscope reported in this paper resembled those described for the osteoblasts cultured from other animals (WILLIAMSet al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983; AUF'MKOLK et al., 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIERet al., 1990). In addition, the osteoblasts normally did not divide in vivo. The osteoblasts regained their mitotic activity when placed into suitable culture conditions. The present study's data are similar to those of the others (SHAW et al., 1989; MASQUELIERet al., 1990). The observed growth rate for bone cells was slower than that of fibroblasts (AUF'MKOLKet al., 1985). Besides, the bone cells in this study were characterized by bone nodule formation with calcification. Both SEM and TEM studies revealed the morphological characterization of the separated individual and confluent cultured osteoblasts. The sparse osteoblasts of the culture exhibited a polygonal shape with some microvilli on the smooth surface. In the confluent cell culture, the intercellular space was greatly
Characterization
reduced and the intercellular connections increased abundantly. TEM studies revealed rich rough endoplasmic reticulum and prominent Golgi apparatus in the cytoplasm. This may reflect the active function of protein formation and transportation in the cultured bone cells. The presence of ALP is an early feature and marker of osteoblasts (BARD et al.,1972; FORREST et al., 1985; SHAW et al., 1989; MASQUEKIERet al., 1990). ALP is considered to be in an intimate relationship with mineralization. Electron microscopic localization of ALP showed that ALP was associated with the plasma membrane and matrix vesicles (ECAROTCHARRIER et al., 1988). Since the major portion of ALP activity is located on the cell membrane with a small portion possibly in the Golgi complex, the granular apperance in our cultured cells may be due to the overlapping effect of both reactive portions. The more differentiated osteoblast developed a higher activity of the ALP (MASQUELIERet al., 1990; COLLIN et al., 1992). Positive histochemical staining for ALP was demonstrated in the present study, indicating that our cultured cells were osteoblast-like cells. Variation in staining intensity of ALP in our cultured bone cells may reflect different stages of differentiation or different levels of metabolism in the cultured cells. Positive immunoreactivity for osteocalcin is a highly sensitive and specific criterion of osteoblasts. The existence of osteocalcin was demonstrated in the cultured rat osteosarcoma cell clone ROS 17/2, osteoblasts and bone matrix in the mineralization front (NISHIMOTO and PRICE, 1980; PRICE et al., 1981; BIANCOet al., 1985; GROOTet al., 1986; THAVARAJAH et al., 1986; CAMARDAet al., 1987; OHTA et al., 1989; BOIvIN et al., 1990). Osteocalcin was also detected in osteocytes. BIANCO et al. (1985) showed that endogenous osteocalcin was present in the cytoplasm and nucleus of osteoblasts and in the collagenous matrix. Moreover, CAMARDAet al. (1987), OHTA et al. (1989) and BoIvIN et al. (1990) demonstrated that osteocalcin was present in the rough endoplamic reticulum and Golgi apparatus of osteocytes and osteoblasts by immunoelectron microscopy, and suggested that osteoblasts and osteocytes are the cells responsible for its synthesis. In our systems, osteocalcin was demonstrated in both the nucleus and cytoplasm of these cells. Although the presence of osteocalcin antigenicity in the nucleus appears very difficult to explain, we are inclined to believe that cultured cells obtained in our system were osteoblasts. The presence of ALP and osteocalcin in our cultured bone cells and the absence of any osteocalcin synthesis in fibroblasts, osteoclasts, and chondrocytes (BoIvIN et
of Rat Osteoblasts
421
al., 1990) provided rationales for justification of this osteoblast phenotypic expression. The present study demonstrated that the cultured bone cells in our system obtained from explant cultures of the rat endosteal bone fragments consistently displayed osteoblastic properties. Their morphological characterizations, histochemical staining of ALP activity and immunocytochemical localization of endogenous osteocalcin demonstrated the specific features of osteoblasts. These data strongly support the view that our cultured rat bone cells are a homogenous culture of functional osteoblast-like cells. These cells are expected to provide a good experimental model system for further investigation of the osteoblast functions and their regulation. Acknowledgements. We thank Dr. S. M. WANG(Department of Anatomy, NTU) for her advice in immunocytochemistry. We thank Ms. C. M. TONG, H. L. LIou, M. H. KUo, S. H. YANGand Mr. B. N. HUANGfor their technical assistance.
REFERENCES ARNETT, T. R.: Update of bone cell biology. Eur. J. Orthodont. 12: 81-90 (1990). AUF'MKOLK, B., P. V. HAUSCHKA and E. R. SCHWARTZ: Characterization of human bone cells in culture. Calcif. Tiss. Int. 37: 228-235 (1985). BARD, P. R., M. J. DICKENS,A. U. SMITH and J. M. ZAREK: Isolation of living cells from mature mammalian cells. Nature 236: 314-315 (1972). BIANCO, P., Y. HAYASHI, G. SILVERSTRINI, J. D. TERMINE and E. BoNUCCI:Osteonectin and GLA-protein in calf bone. Calcif. Tiss. Int. 37: 684-686 (1985). BOIVIN,G., G. MOREL,J. B. LIAN, C. ANTHOINE-TERRIER, P. M. DUBoIS and P. J. MEUNIER: Localization of endogenous osteocalcin in neonatal rat bone and its absence in articular cartilage. Virchows Arch. A 417: 505-512 (1990). CAMARDA, A. J., W. T. BUTLER, R. FINKELMAN and A. NANCI: Immunocytochemical localization of osteocalcin in rat bone and dentin. Calcif. Tiss. Int. 40: 349-355 (1987). COLLIN, P., J. R. NEFUSSI, A. WETTERWALD, V. NICOLAS, M. BOY-LEFEVRE, H. FLEISCH and N. FOREST: Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture. Calcif. Tiss. Int. 50: 175-183 (1992). ECAROT-CHARRIER, B., N. SHEPARD, G. CHARETTE, M. GRYNPAS and F. H. GLORIEUX: Mineralization in osteoblast cultures: A light and electron microscopic study. Bone 9: 147-154 (1988). FORREST,S. M., K. W. NG, D. M. FINDLAY, V. P. MICHELANGELI, S. A. LIVERSY, N. C. PATRIDGE, J. D. ZAJAI and T. J. MARTIN: Characterization of an osteo-
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blast-like clone cell line which responds to both parathyroid hormone and calcitonin. Calcif. Tiss. Int. 37: 5156 (1985). GROOT, C. G., J. K. DANES, J. BLOK and A. HOOGENDIJK: Light and electron microscopical immunocytochemistry of osteocalcin in plastic sections of rat bone and bone cells. Calcif. Tiss. Int., -Suppl. 38: 16 (1986). HAGEL-BRADWAY, S. and R. DZIAK: Regulation of bone cell metabolism. J. Oral Pathol. Med. 18: 344-351(1989). MARIE, P. J., A. LOMRI, A. SABBAGH and M. BASLE: Culture and behavior of osteoblastic cells isolated from normal trabecular bone surfaces. In Vitro Cell Devel. Biol. 25: 373-380 (1989). MARK, M. P., C. W. PRINCE, S. GAY, R. L. AUSTIN, M. BHOWN, R. D. FINKELMAN and W. T. BUTLER: A comparative Immunocytochemical study on the subcellular distributions of 44 KDa bone phosphoprotein and bone y-carboxyglutamic acid (Gla)-containing protein in osteoblasts. J. Bone Mineral Res. 4: 337-346 (1987). MARTIN, T. J., K. W. NG and T. SUDA: Bone cell physiology. Endocrinol. Metabol. Clin. North. Amer. 18: 833858 (1989). MASQUELIER, D., B. HERBERT, N. HAUSER, P. MERMILLOD, E. SCHONNE and C. REMACLE: Morphological characterization of osteoblast-like cell cultures isolated from newborn rat calvaria. Calcif. Tiss. Int. 47: 92-104 (1990). NIJWEIDE, P. J., A. VAN DER PLAS and J. P. SCHERFT: Biochemical and histological studies on various bone cell preparations. Calcif. Tiss. Int. 33: 529-540 (1981). NISHIM0T0, S. K, and P. A. PRICE: Secretion of vitamin K-dependent protein of bone by rat osteosarcoma cells. J. Biol. Chem. 255: 6579-6583 (1980). OHTA, T., M. MORI, K. OGAWA, T. MATSUYAMA and S. ISHII: Immunocytochemical localization of BGP in human bones in various developmental stages and pathological conditions. Virchows Arch. A 415: 459-466 (1989). PARTRIDGE, N. C., D. ALCORN, V. P. MICHELANGELI, G. RYAN and T. J. MARTIN: Morphological and biochemical characterization of four clonal osteogenic sarcoma cell lines of rat origin. Cancer Res. 43: 4308-4314 (1983). PECK, W. A., S. J. BINGE and S. A. FEDAK: Bone cells: biochemical and biological studies after enzymatic isolation. Science 146: 1476-1477 (1964). PRICE, P. A., J. W. LOTHRINGER, S. A. BANKOL and A. H. REDDI: Developmental appearance of the vitamin K. dependent protein of bone during calcification. J. Biol. Chem. 256: 3781-3784 (1981). ROBEY, P. G. and J. D. TERMINE: Human bone cells in vitro. Calcif. Tiss. Int. 37: 453-460 (1985).
SHAW, K. Y., J. S. TANG and C. F. CHAO: Functional evaluation of cultured rabbit osteoblast-like cells. Proc. Nat. Sci. Counc. (ROC) 13: 192-200 (1989). THAVARAJAH, M., D. B. EVANS, R. G. G. RUSSELL and J. A. KANIS: Immunocytochemical demonstration of osteocalcin in human bone derived cells. Calcif. Tiss. Int., Suppl. 38: 16 (1986). TRACY, R. P., M. L. LAMPHERE, B. L. RIGGS and K. G. MANN: Monoclonal antibody-based immunoassay for bone Gla-protein (BGP, osteocalcin). (ed. by) C. CHRISTIANSEN, J. S. JOHANSEM and B. J. RIIS: Osteoporosis 1987. Osteopress APS, Copenhagen, 1987 (p. 688-690). TRACY, R. P., A. ANDRIANORIVO, B. L. RIGGS and K. G. MANN: Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin. J. Bone Mineral Res. 5: 451-461 (1990). VRIES, I. G., D. COOMANSand E. WISSE: Immunocytochemical localization of osteocalcin in human and bovine teeth. Calcif. Tiss. Int. 43: 128-130 (1988). WANG, S. M., C. M. LUE and H. S. LIN: Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster. Histol. Histopathol. 2: 163-171 (1987). WANG, S. M., C. L. LIU and H. S. LIN: An Immunocytochemical study of effects of light deprivation on prolactin cells in the adenohypophysis of the golden hamster. Histol. Histopathol. 6: 287-293 (1991). WILLIAMS, D. C., G. B. BORER, R. E. TOOMEY, D. C. PAUL, C. C. HILLMAN, K. L. KING, R. M. VAN FRANK and C. C. JOHNSTON: Mineralization and metabolic response in serially passaged adult rat bone cells. Calcif. Tiss. Int. 30: 233-246 (1980). WONG, G. L.: Bone cells cultures as an experimental model. Arthritis Rheum. 23: 1081-1086 (1980).
Dr. Kuo-Shyan LU Department of Anatomy College of Medicine National Taiwan University 1-1, Jen-Ai Road, Taipei 100 Taiwan, Republic of China 盧 國 賢 中華民 國墓 濁省 國立 墓濁大 學 讐 學院 解剖學 科
Morphological
and Immunocytochemical
Osteoblast
Cultures
Rong-Sen
Tang-Kue
YANG,
Departments of Orthopaedics', University, Taipei, Taiwan
from LIU1, Keh-Sung
Long
Bones
TSAI2,
Shoei-Yn
Characterization of Neonatal LIN-SHIAU3
and
of
Rats* Kuo-Shyan
LU4
Clinical Pathology2, Pharmacology3 and Anatomy4, College of Medicine, National Taiwan
Received July 13, 1992
Summary. The present study reports a successful culture system of rat bone cells obtained from the bare long bone fragments of neonatal Wistar rats. The morphology, histochemical and immunocytochemical properties of the cultured cells were characterized by phase contrast microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), histochemical staining with alkaline phosphatase (ALP), and immunocytochemical staining with the anti-osteocalcin antibodies. Under phase contrast microscopy, the cultured rat bone cells changed their morphology from being slender with relatively-short cytoplasmic processes to becoming polygonal with longer processes, and subsequently flattened. These cultured bone cells formed bone nodules after 3 weeks. Under SEM, the cultured bone cell appeared polygonal with some microvilli. The TEM showed that their cytoplasm contained abundant endoplasmic reticulum and well-developed Golgi apparatus. Positive histochemical staining of ALP was also detected as a blue coloration and granular appearance in the cultured bone cells. Immunocytochemistry with the polyclonal and monoclonal anti-osteocalcin antibodies showed the localization of osteocalcin in both the cytoplasm and nucleus. All these data lead us to consider that the cultured bone cells in our system were probably osteoblasts from growing bone tissue in vitro. This provides a convenient test system for further studies on the regulation of the growth and metabolism of rat bone cells.
AUF'MKOLKet al. 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIER et al., 1990). Once the culture of bone cells is established, the characterization of the bone cells becomes essential for further studies. However, the exact nature of the cultured cells remains conjectural, since these cultures probably represent mixtures of cell types of a mesenchymal origin. By the morphology of cultured cells alone, it is very difficult to differentiate among various mesenchymal cells, e.g., osteoblasts, chondrocytes, and fibroblasts. Therefore, identification by morphological distinctions with phase microscopy, SEM and TEM, histochemical staining with ALP, and responsiveness to parathyroid hormone etc., have been employed to establish the exact nature of the cultured bone cells (WILLIAMS et al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983; AUF'MKOLK et al. 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIER et al., 1990). Moreover, osteocalcin was determined to be produced only by osteoblasts and osteocytes; therefore, immunocytochemical staining with a monoclonal or polyclonal antibody against osteocalcin provided a specific method of identification (BIANCOet al., 1985; GROOTet al., 1986; THAVARAJAHet al., 1986; CAMARDA et al., 1987; MARK et al., 1987; WANG et al., 1987, 1991; VRIES et al., 1988; OHTA et al., 1989; BOIVIN et al., 1990). The present study describes a successful explant culture system of rat bone cells from long bone segments of the neonatal Wistar rat. The data on the characterization of these bone cells obtained from SEM, TEM, histochemical staining with ALP and immunocytochemical staining with anti-osteocalcin
Successful bone cell cultures, initiated by PECK et al. (1964), have enabled us to investigate the regulation of the growth and metabolism of the bone cells. Many sources of bone cells have been described, including rat, mice, chicks, human, feline and human osteosarcoma cell lines (WILLIAMS et al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983;
This work was supported in part by the National Science Council of the Republic of China, NSC80-0412-B002-159 and by the National Taiwan University Hospital, NTUH-81016-A11. 415
NSC80-0412-B002-210
and
416
R.-S. YANGet al.:
antibody have led us to consider these cultured cells to be osteoblasts.
then ion sputter coated with a thin layer (about 10 nm) of gold and examined in a JOEL T330A scanning electron microscope at 25 kV.
MATERIALS
TEM
AND METHODS
Cell culture The long bones of wistar rat neonates, including the femur, tibia, humerus, radius, and the ulna, were dissected with aseptic techniques. After defleshing, the bones were diced into bare bone fragments and were washed in phosphate buffered saline (PBS) to remove marrow cells. The primarily separated rat bone cells obtained from these explants were grown in Dulbecco's modified calcium free Eagle's medium (DMEM) (Biofluids, USA) supplemented with 7% calcium free F12 (Gibco, USA) and 10% fetal calf serum (Gibco, USA), pH 7.6 containing glutamine (58.5,ug/ ml), MgSO4 (200 jig/ml), and antibiotics (100 U penicillin and 100ug streptomycin sulfate per ml). They were kept in a humidified atmosphere containing 95% air and 5% C02 at 3TC (ROBEY and TERMINE, 1985). The viability of the cells was monitored by the trypanblue exclusion test. Using the original explant or the cultured bone cells, the cells were subcultured twice a week. The culture medium was changed twice weekly. Cell passage was carried out by incubating a monolayer for 5-10 min in calcium- and magnesium-free Tyrode's solution containing 0.25% trypsin (Sigma, USA). Explant pieces were removed when the resulting monolayer approached confluence and the cells were replated in fresh medium at about one third their confluent density. The morphology of cultured bone cells was observed under a phase contrast microscope, and the population doubling time was calculated by counting the cell numbers at regular intervals. For the mineralization features of these cultured cells, the culture medium was supplemented with 50,ug/ml ascorbic acid (Sigma) and 10 mM R-glycerophosphate (Sigma). The conditioned medium was changed every other day for 12 days. The cultured cells and bone nodules were stained with the von Kossa method and 0.5% safranin 0 for light microscopy. SEM For SEM studies, the osteoblasts in the culture dishes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate for 20 min. At fixation, the cells were rinsed in phosphate buffer for 5 min, postfixed in 1 % osmium tetroxide for 20 min, then dehydrated in a graded series of alcohol, and subsequently critical-point dried with liquid carbon dioxide. The specimens were
For TEM study, cultured cells were fixed with 2.5 glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 30 min. After post-fixation in 1% aqueous osmium tetroxide for 1 h, the specimens were stained en bloc with 0.5% uranyl acetate, dehydrated in alcohol and then embedded in Epon-araldite mixture. Thin section were cut and stained with uranyl acetate before examination in a JOEL 2000EXII electron microscope at 100 kV. Histochemical
study of ALP
Cells grown on cover slips for 2 weeks after primary explant culture were fixed in buffered acetone fixative (0.03 M Na-citrate, 0.03 M citric acid, 60% acetone) for 30 sec. The cover slips were rinsed in distilled water for 10 sec, air-dried, and stained in incubation solution at room temperature for 1 h. The incubation solution contained 6 mg Naphthol AS phosphate (Sigma), 0.1 ml N, N dimethyl f ormamide (Sigma), and 20 mg Fast blue BB salt (Sigma) in 20 ml 0.5 M Tris buffer, pH 10.2. The mixture was vigorously agitated for 30 sec and filltered directly onto the cover slips. The slips were then rinsed in distilled water for 10 sec, air-dried, counterstained with 0.5 or 0.05% safranin 0 solution, mounted in gelatinglycerin jelly and observed under oil immersion. For the control, the cells were heat-inactivated by dipping the cover slips in boiled water for 1 min before incubation. Immunocytochemistry
of the osteocalcin
The monoclonal anti-osteocalcin antibody was a gift from Dr. K. MANN and B. L. RIGGS, Mayo Clinic and Foundation (TRACY et al., 1987, 1990). Analysis with synthetic peptides revealed an epitopic specificity residing in residues 1-19 of osteocalcin. This monoclonal antibody requires Ca++ in order to bind normally carboxylated osteocalcin, but will also bind to decarboxylated osteocalcin (TRACY et al., 1987). The polyclonal anti-bovine bone osteocalcin antiserum, purchased from Immuno Nuclear Corp., USA, was reported to crossreact with rat osteocalcin (VRIES et al., 1988). Both biotinylated goat anti-rabbit IgG and horse anti-mouse IgG immunoglobulins were obtained from Vector Lab., USA. For the immunocytochemical staining, the cultured cells were washed
Characterization
of Rat
Osteoblasts
417
Fig. 1. Phase contrast photomicrographs of cultured bone cells. A. Bone cells grow initially from the explant cultures. B. Bone cells in the second passage display a polygonal shape and longer cell process. C. Bone cells at confluence become smaller and packed. D. Some cells flatten with time. The cytoplasm spreads out. E. Bone nodules form after 3 weeks of incubation. F. The cells cultured in the medium supplemented with ascorbic acid and fl-glycerophosphate are embedded in a mineralized matrix (bone nodule) stained by the von Kossa method.
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with PBS and then fixed in methanol or 4 % paraformaldehyde for 10 min. After washing in PBS and treating with 10% normal goat albumin, the cells were incubated consecutively with: 1) 1:50 primary antibody (anti-osteocalcin serum) for 1 h at 37°C; 2) a secondary antibody (1:100 diluted biotinylated horse anti-mouse IgG for monoclonal anti-osteocalcin antibody, or biotinylated goat anti-rabbit IgG for polyclonal antibody) at 37°C for 1 h; and 3) avidin-biotin peroxidase (1:100 diluted with PBS) for 1 h at 37°C. After coloration with diaminobenzidine (DAB) solution (0.5 mg/ml in 0.1 M Tris buffer, pH 7.4, containing 0.05% H2O2), for 20 min, the cells were mounted with gel/mount (Biomeda) and examined in an inverted phase contrast microscope. The cells incubated in anti-osteocalcin serum free medium served as controls.
RESULTS Cell culture
and light microscopy
The outgrowth in explant cultures resulted in layers of sparse cells which proliferated slowly and spread over the culture dishes, reaching a confluence at a density of 7x10 cells/cm' within 7 days. The population doubling time from the initial cell outgrowth in explant pieces was found to be 114h. Immediately after culturing, rat bone cells appeared slightly slender in shape and displayed sparse and relatively-short cytoplasmic processes (2-5 um in length) (Fig. 1A). They became polygonal in shape and developed longer cell processes (5-30 um) after 3 days (Fig. 1B). Upon reaching confluence, they
Fig. 2. Scanning electron micrograph and transmission electron micrograph of bone cells. A. SEM of a cultured bone cell shows a polygonal shape and displays many short cellular processes on the cell surface. B. TEM of the bone cells shows a cytoplasm rich in rough endoplasmic reticulum (r) and Golgi apparatus (G) near the nucleus (n). Moderate numbers of small mitochondria (m), vesicles (v) and microvilli (mv) are found in the periphery.
Characterization
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Fig. 3. Histochemical demonstration of alkaline phosphatase (ALP) activity in the cytoplasm. A. The control cells show no ALP activity (0.5% safranin 0 counterstained). B and C. The ALP-positive cells are characterized by a blue coloration of varying density and occasionally by a light and deep discrete blue granular appearance (0.05% safranin 0 counterstained).
Fig. 4.
Immunocytochemical
localization
of endogenous
show no reaction product. B. The immunocytochemical shows brown granules in the cytoplasm. C. The osteocalcin
antibody
shows
staining
in both
osteocalcin
in the cultured
staining with the monoclonal immunocytochemical staining
the cytoplasm
and nucleus.
bone
cells.
A. Control
cells
anti-osteocalcin antibody with the polyclonal anti-
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became smaller and packed (Fig. 1C). Some were multipolar and became flattened with time in culture (Fig. 1D). The cultured bone cells formed bone nodules after incubation for longer than 3 weeks (Fig. iE). The cells and bone nodules cultured in the medium supplemented with ascorbic acid and 3-glycerophosphate developed a mineralized matrix which could be stained with von Kossa method (Fig. iF).
calcin was mainly present in the cytoplasm of cultured bone cells although weak nuclear staining was also observed (Fig. 4B). The polyclonal anti-osteocalcin antibody brought about more clear staining of the nucleus (Fig. 4C). Since cultured cells displayed the same pattern of immunostaining, the results showed that these cultured rat bone cells were osteoblast-like, and that the cell population is highly homogeneous.
SEM Under the SEM, the cultured cell displayed a large, polygonal soma, but few long cytoplasmic processes. A protrusion of the nucleus at the center of soma was prominent, and at high magnification, the cell surfaces of the soma were equipped with some microvilli (Fig. 2A). TEM TEM observation revealed that the cytoplasm of cultured bone cells contained abundant free ribosomes and rough endoplasmic reticulum which were occasionally arranged in parallel. A well-developed Golgi apparatus occupied the space adjacent to the nucleus. A moderate number of mitochondria was distributed throughout the cytoplasm. In addition, a few vesicles containing a finely granular material, probably lysosomes, were located in the periphery of the cytoplasm (Fig. 2B). Histochemical
study of ALP
ALP activity is considered a marker of the osteoblast phenotype. Positive ALP activity was demonstrated by brilliant blue coloration with the aforementioned histoenzymatic method. The control cells did not show any staining (Fig. 3A). The positive ALP activity in cultured cells was characterized by a blue coloration of varying intensity (Fig. 3B), and occasionally by the discrete blue granular appearance of the cells (Fig. 3C), indicating that these cells were osteoblast-like cells. About 78% of the cell population demonstrated positive reactions, reflecting a high degree of homogeneity. Immunocytochemistry
of the osteocalcin
In our systems, the DAB staining caused a light to deep brown color for the positively-stained cells. The control cells did not show any immunoreactivity to osteocalcin (Fig. 4A). The results of the immunocytochemical staining with monoclonal anti-osteocalcin antibody indicated that immunoreactivity of osteo-
DISCUSSION Bone cells play a fundamental role in bone tissue remodeling throughout life; their function is under the regulation of many systemic and local growth factors (HAGEL-BRADWAYand DZIAK, 1989; MARTIN et al., 1989; ARNETT,1990). The isolation and culture of the bone cells remain the basis for the investigation of the bone metabolism and therefore, the identification of cultured bone cells is a cornerstone for further studies. The cultured rat bone cells in the present study were identified by histochemical staining with ALP, SEM, TEM and immunocytochemical staining with polyclonal and monoclonal anti-osteocalcin antibodies. Although the spindle-shaped cells prevailed in fibroblast cultures and osteoblasts were characterized by their polygonal shape and in forming condensed bands of cells, the latter also assumed a spindle shape. It is thus difficult to distinguish cultured cells from mesenchymal origins by their morphology. However, the morphological features of the cultured bone cells under the phase microscope reported in this paper resembled those described for the osteoblasts cultured from other animals (WILLIAMSet al., 1980; WONG, 1980; NIJWEIDE et al., 1981; PARTRIDGE et al., 1983; AUF'MKOLK et al., 1985; ROBEY and TERMINE, 1985; MARIE et al., 1989; SHAW et al., 1989; MASQUELIERet al., 1990). In addition, the osteoblasts normally did not divide in vivo. The osteoblasts regained their mitotic activity when placed into suitable culture conditions. The present study's data are similar to those of the others (SHAW et al., 1989; MASQUELIERet al., 1990). The observed growth rate for bone cells was slower than that of fibroblasts (AUF'MKOLKet al., 1985). Besides, the bone cells in this study were characterized by bone nodule formation with calcification. Both SEM and TEM studies revealed the morphological characterization of the separated individual and confluent cultured osteoblasts. The sparse osteoblasts of the culture exhibited a polygonal shape with some microvilli on the smooth surface. In the confluent cell culture, the intercellular space was greatly
Characterization
reduced and the intercellular connections increased abundantly. TEM studies revealed rich rough endoplasmic reticulum and prominent Golgi apparatus in the cytoplasm. This may reflect the active function of protein formation and transportation in the cultured bone cells. The presence of ALP is an early feature and marker of osteoblasts (BARD et al.,1972; FORREST et al., 1985; SHAW et al., 1989; MASQUEKIERet al., 1990). ALP is considered to be in an intimate relationship with mineralization. Electron microscopic localization of ALP showed that ALP was associated with the plasma membrane and matrix vesicles (ECAROTCHARRIER et al., 1988). Since the major portion of ALP activity is located on the cell membrane with a small portion possibly in the Golgi complex, the granular apperance in our cultured cells may be due to the overlapping effect of both reactive portions. The more differentiated osteoblast developed a higher activity of the ALP (MASQUELIERet al., 1990; COLLIN et al., 1992). Positive histochemical staining for ALP was demonstrated in the present study, indicating that our cultured cells were osteoblast-like cells. Variation in staining intensity of ALP in our cultured bone cells may reflect different stages of differentiation or different levels of metabolism in the cultured cells. Positive immunoreactivity for osteocalcin is a highly sensitive and specific criterion of osteoblasts. The existence of osteocalcin was demonstrated in the cultured rat osteosarcoma cell clone ROS 17/2, osteoblasts and bone matrix in the mineralization front (NISHIMOTO and PRICE, 1980; PRICE et al., 1981; BIANCOet al., 1985; GROOTet al., 1986; THAVARAJAH et al., 1986; CAMARDAet al., 1987; OHTA et al., 1989; BOIvIN et al., 1990). Osteocalcin was also detected in osteocytes. BIANCO et al. (1985) showed that endogenous osteocalcin was present in the cytoplasm and nucleus of osteoblasts and in the collagenous matrix. Moreover, CAMARDAet al. (1987), OHTA et al. (1989) and BoIvIN et al. (1990) demonstrated that osteocalcin was present in the rough endoplamic reticulum and Golgi apparatus of osteocytes and osteoblasts by immunoelectron microscopy, and suggested that osteoblasts and osteocytes are the cells responsible for its synthesis. In our systems, osteocalcin was demonstrated in both the nucleus and cytoplasm of these cells. Although the presence of osteocalcin antigenicity in the nucleus appears very difficult to explain, we are inclined to believe that cultured cells obtained in our system were osteoblasts. The presence of ALP and osteocalcin in our cultured bone cells and the absence of any osteocalcin synthesis in fibroblasts, osteoclasts, and chondrocytes (BoIvIN et
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al., 1990) provided rationales for justification of this osteoblast phenotypic expression. The present study demonstrated that the cultured bone cells in our system obtained from explant cultures of the rat endosteal bone fragments consistently displayed osteoblastic properties. Their morphological characterizations, histochemical staining of ALP activity and immunocytochemical localization of endogenous osteocalcin demonstrated the specific features of osteoblasts. These data strongly support the view that our cultured rat bone cells are a homogenous culture of functional osteoblast-like cells. These cells are expected to provide a good experimental model system for further investigation of the osteoblast functions and their regulation. Acknowledgements. We thank Dr. S. M. WANG(Department of Anatomy, NTU) for her advice in immunocytochemistry. We thank Ms. C. M. TONG, H. L. LIou, M. H. KUo, S. H. YANGand Mr. B. N. HUANGfor their technical assistance.
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Dr. Kuo-Shyan LU Department of Anatomy College of Medicine National Taiwan University 1-1, Jen-Ai Road, Taipei 100 Taiwan, Republic of China 盧 國 賢 中華民 國墓 濁省 國立 墓濁大 學 讐 學院 解剖學 科
