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hr. The EDTA extracts are desalted and freed of residual unincorporated radioactivity by gel filtration on 1 × 10-cm columns of Sephadex G-25 equilibrated in Tris/inhibitor solution. Fractions of 1.0 ml are collected, and radioactivity is measured on aliquots of the fractions. The high-molecular-weight fraction eluting in the void of the column contains proteins presumably synthesized by odontoblasts and secreted into the calcified dentin matrix. Characterization of the proteins synthesized by the organ cultures can follow several routines. We have used chromatographc and electrophoretic separations, immunoprecipitation, and CaC12 precipitation to identify the products. 19-21 The cultures may also prove valuable in studies on stimulation or inhibition of the synthesis of dentin matrix proteins by certain drugs or hormones. 21

[16] B o n e C e l l C u l t u r e s

By

J. SODEK

and F. A.

BERKMAN

Cells in culture provide relatively simple systems in which basic biological phenomena can be studied in detail. Culture of bone cells can be extremely valuable, therefore, in analyzing those biological features that are peculiar to bone. In particular, cell culture will continue to be instrumental in studies on the origin and differentiation of bone cells, in the determination of how a mineralizing connective tissue matrix is formed under the control of osteoblastic cells, and how bone formation and resorption are influenced by the many and various hormones, physiologically active agents, and growth factors. Contrasting the relative simplicity of cell culture systems are difficulties in isolating homogeneous cell populations that will retain their characteristic phenotype under in vitro conditions. Most of the cells surviving isolation procedures and proliferating in vitro are likely to be largely undifferentiated, and the characteristics they display will be influenced by the culture environment. In particular, the presence in the culture medium of serum, which induces a "wound healing" type of response in cultured cells, the nature of the substratum to which cells attach, and the activity of neighboring cells can have profound effects on the phenotype of the cells as they are maintained in vitro. It is unlikely that a single methodology for isolating and culturing bone cells exists or will be developed that is ideal for studying all aspects of bone biology. Rather, specific systems that can be adapted to answer specific questions will be utilized. A more comprehensive review on bone METHODS IN ENZYMOLOGY, VOL. 145

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cell culture and its history has been published recently, l In this chapter, only a few selected methodologies for culturing normal 2 bone cells with osteoblast-like properties will be detailed with emphasis placed on the importance of biochemical characterization of the osteoblastic phenotype. Cells of Bone Tissues Although developmentally bone tissues are formed by either intramembranous or endochondral processes, the mechanism of bone formation is considered to be similar) The mineralized tissue in developing bone and in healing fractures is characterized by a loose random weave of collagen fibers within and between which hydroxyapatite crystals are formed. This tissue, known as woven bone, is usually replaced by the more mature lamellar and cancellous types of bone. In addition to the mineralized connective tissue, fibrous tissue is found covering the endosteal and periosteal surfaces of bone, and soft tissue is present in endosteal spaces and in the marrow cavities. Thus, in addition to osteoblasts and osteoclasts, which are involved in the formation and resorption of mineralized bone tissue, fibroblasts, reticulocytes, and adipocytes are to be found in the associated fibrous, reticular, and marrow tissues, and cells of the chondrocyte lineage are present in cartilage patches found in endochondral bone. Also residing in the marrow spaces are the hematopoietic cells, and endothelial cells are found as an integral part of the sinusoidal blood vessels. Origin and Differentiation of Bone Cells Two principal cellular systems are recognized as existing within the bone and associated marrow tissues. The stromal cell system, from which the osteoblastic cells arise, generates the cells of the fibroblast, reticulocyte, and adipocyte lineages. The hematopoietic system, from which the bone-resorbing osteoclasts develop, generates the many and various cell types found in the blood under the influence of the stromal tissue matrix. In contrast to the extensively studied hematopoietic system, however, the relationships between cells of the stromal system are poorly characterM. Silbermann and G. Maor, in "Methods of Calcified Tissue Preparation" (G. R. Dickson, ed.), pp. 467-529. Elsevier, New York, 1984. 2 "Normal" is used here to describe cells of finite life span obtained from healthy bone tissues. 3 A. W. Ham and D. H. Cormack, in "Histophysiology of Cartilage, Bone and Joints." Lippincott, Toronto, Ontario, Canada, 1979.

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ized. In a recent review of this topic, Owen 4 has prepared a lineage diagram describing possible relationships between the cells of the stromal system. This provides a foundation upon which studies of stromal cell differentiation can be based and from which the complexities of cellular heterogeneity can be appreciated. Bone Cell Heterogeneity

When cells are released from bone tissues, cells from the different cell systems and from the various lineages outlined above will be obtained. Also, within a particular lineage, cells are likely to exist at different stages of differentiation, ranging from stem cells or progenitor forms to more highly differentiated cells that have more limited replicative capacities. The final composition of cells released from bone will depend on the type and age of the bone, the species of origin, the degree of tissue dissection, and the methods used to isolate the cells. On subsequent culture, stromal cells will attach to the tissue culture surface, allowing removal of the nonadherent hematopoietic cells. Growth in culture will further select for those cells with the greatest replicative rates and replicative capacities, and the culture environment will also affect phenotypic expression. Osteoblast Phenotype Differentiating and differentiated cells can be identified by their ability to express cell-type specific molecules, which may be components of the cell or structural molecules of the tissue matrix. Despite considerable effort being expended in this area, such specific molecules have yet to be identified unambiguously for osteoblastic cells. Consequently, the osteoblast phenotype has generally been characterized by using a combination of morphological, biochemical, and endocrinological features.l,5,6 However, since the principle function of osteoblasts is to form bone, osteogenic capacity expressed by bone tissue formation in vitro provides the best evidence of osteoblastic activity. In vivo, osteoblasts are cuboidal in shape, form a contiguous layer, and are polarized, secreting extracellular matrix on one side only. The cells on the bone surface and those that have become embedded in the 4 M. Owen, in "Bone and Mineral Research Annual" (W. A. Peck, ed.), Vol. 3, pp. 1-25. Elsevier, New York, 1985. 5 G. A. Rodan and S. B. Rodan, in "Bone and Mineral Research Annual" (W. A. Peck, ed.), Vol. 2, pp. 244-285. Elsevier, New York, 1983. 6 R. J. Majeska, S. B. Rodan, and G. A. Rodan, in "The Chemistry and Biology of Mineralized Tissues" (W. T. Butler, ed.), pp. 279-285. EBSCO, Birmingham, Alabama, 1985.

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mineralized matrix (osteocytes) communicate through intercellular gap junctions and cytoplasmic extensions. Cytoskeletal structures, comprising microfilaments and microtubules, are similar to those in other connective tissue-forming cells; they function in the maintenance of cellular integrity and secretion of matrix components, and they provide continuity between the cell, the cell surface, and the extracellular matrix. The synthesis of large quantities of type I collagen, which forms the structural framework of the mineralized matrix, is characteristic of the osteoblast phenotype. A number of other glycoproteins present in mineralized bone have been isolated and characterized. These include osteonectin, osteocalcin, sialoproteins, phosphoproteins, and proteoglycans, proteins which tend to remain bound to the hydroxyapatite crystal surface in the presence of 4 M guanidine-HCl and which are known to be synthesized by osteoblastic cells. Whether any of these glycoproteins are specific to the osteoblast phenotype has yet to be determined. Osteonectin is known to be synthesized by fibroblastic cells7 and is present in various connective tissues. 8 The proteoglycan9 and sialoprotein 1° exist in several forms which need to be analyzed individually before specificity to bone tissue and the osteoblastic phenotype can be clearly assessed. Osteocalcin, however, has been demonstrated only in mineralized tissues ~l and may prove to be specific to mineralized tissue-forming cells, since the synthesis of osteocalcin has been demonstrated only in bone cells. In osteosarcoma cells 12 and in cells derived from human trabecular bone, 13 synthesis of osteocalcin is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3 ]. Active osteoblastic cells characteristically demonstrate high levels of alkaline phosphatase activity which is localized largely to the plasma membrane. Of several isoenzyme forms of alkaline phosphatase found in vertebrate tissues, the osteoblast enzyme is identical to the bone-liver7 S. Wasi, K. Otsuka, K.-L. Yao, P. S. Tung, J. E. Aubin, J. Sodek, and J. D. Termine, Can. J. Biochem. Cell Biol. 62, 470 (1984). s p. S. Tung, C. Domenicucci, S. Wasi, and J. Sodek, J. Histochem. Cytochem. 33, 531 (1985). 9 A. Franz6n and D. Heinegh'd, Biochem. J. 224, 47 (1984). 10 L. W. Fisher, in "The Chemistry and Biology of Mineralized Tissues" (W. T. Butler, ed.), pp. 188-196. EBSCO, Birmingham, Alabama, 1985. 11 A. L. J. J. Bronckers, S. Gay, M. T. DiMuzio, and W. T. Butler, Collagen Rel. Res. 5, 273 (1985). 12 p. A. Price and S. A Baukol, J. Biol. Chem. 255, 11660 (1980). 13 H. Skjodt, J. A. Gallagher, J. N. Beresford, M. Couch, J. W. Poser, and R. G. G. Russell, J. Endocrinol. 105, 391 (1985).

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kidney enzyme. ~4 However, despite the apparent relationship between elevated alkaline phosphatase levels and the formation of mineralized tissue and the capability of the enzyme to generate phosphate ions, a direct role for alkaline phosphatase in the process of mineralization has yet to be established. Consistent with the ability of fibroblasts to both form and degrade the organic components of the connective tissue matrix and to regulate these processes, osteoblastic cells have been shown to produce collagenolytic enzymes, including a collagenase with characteristics of the fibroblast enzyme, and specific inhibitors of these enzymes. 15Osteoblasts could conceivably degrade organic matrix following osteoclastic resorption, but it appears more likely that their role is to remove osteoid and thereby expose the mineralized bone surface to osteoclasts) 6 In this regard, osteoblasts usually have receptors for and respond to parathyroid hormone (PTH), glucocorticoids, prostaglandin Ez (PGE2), prostacyclin (PGI2), vitamin D3, interleukin-1, and retinoic acid, factors that are believed to stimulate bone resorption. The presence of cell-surface receptors for PTH is typical of osteoblastic cells which respond to the hormone by an increase in membrane permeability to Ca 2÷ and by increased adenylate cyclase activity. Intracellular receptors for vitamin D3 metabolites and glucocorticoids are also found in osteoblastic cells. The hormonal responses of bone cells are detailed elsewhere. Although each of the hormones and physiological agents described above are known to affect the activity of osteoblasts, variable and contrasting effects have been observed even when well-characterized cells in the relatively simple confines of an in oitro environment have been studied. Clearly, the cellular response depends on the nature of the cell, for example, whether it is normal, established, or tumorigenic, its stage of differentiation, whether it is rapidly dividing or quiescent, and the type and number of receptors. As well, the cell's environment, the substratum, the presence of serum or serum components, other hormones and agents, and the level of these factors can also modulate the cellular response. In general, however, it has been found that PTH, vitamin D3 metabolites, PGE2, PGI2, and retinoic acid tend to promote the "resorptive" phenotype in osteoblasts. This may involve a secondary effect on osteoclast activity and may also be accompanied by a decrease in the production of I4 D. J. Goldstein, C. E. Roger, and H. Harris, Proc. Natl. Acad. Sci. U.S.A. 77, 2857 (1980). ~5 K. Otsuka, J. Sodek, and H. Limeback, Eur. J. Biochem. 145, 123 (1980). i6 G. A. Rodan and T. J. Martin, Calcif. Tissue Int. 33, 349 (1981).

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components involved in bone tissue formation. The observed anabolic effects of PTH 17 and the stimulation of alkaline phosphatase levels by 1,25(OH)2D318 may reflect the role of osteoblasts in maintaining the delicate balance between bone formation and resorption. This balance appears to be mediated, at least in part, by TGF-fl, a growth factor whose synthesis is stimulated in osteoblastic cells by vitamin D3 and PTH. Osteoblasts in culture also respond to other growth factors and, as found with TGF-fl, the responses appear to be dependent on the cell type and its environment. Those growth factors, such as PDGF, FGF, EGF, and TGF-fl, that have been classified as "competence factors" promote cellular proliferation, but tend to inhibit the expression of the differentiated phenotype. In contrast, "progression factors," such as IGF-I, IGF-II, insulin, and somatomedins, generally stimulate both growth and the expression of the differentiated phenotype. Source of Osteoblasts Bone tissues, primarily calvariae, from rat and mouse have been used most frequently as an economical and convenient source of bone cells. Bone cells from chicken and rabbit and from porcine and bovine sources have also been used. However, sufficient differences exist between bones of birds, rodents, and higher mammals to warrant careful consideration of the source of osteoblasts for particular studies. Consideration should also be given to the age of the animals used. Although cells from adult tissue will generally have lower proliferative capacity than cells from the more commonly used fetal tissues, they are more appropriate to studies investigating the formation and regulation of mature bone. Cells from rodent tissues are known to establish to culture with relatively high frequencies. 19As a consequence, a number of clonal cell lines with osteoblastic properties have been isolated. These include the MMB1f2°and MC3T3-E121 cells from fetal mouse bones and the RCB and RCJ 22 cells lines from fetal rat calvariae. The combination of immortality and stability in culture has allowed more detailed characterization of the phe17 M. P. M. Herrmann-Erlee, J. N. M. Heersche, J. W. Hekkelman, P. J. Gaillard, G. W. Tregaar, J. A. Parsons, and J. T. Potts, Endocrinol. Res. Commun. 3, 21 (1976). is j. W. Dietrich, E. M. Canalis, M. Maina, and L. G. Raisz, Endocrinology 98, 943 (1976). 19 j. Ponten, Biochim. Biophys. Acta 458, 397 (1976). 20 M. R. Waiters, D. M. Rosen, A. W. Norman, and R. A. Luben, J. Biol. Chem. 257, 7481 (1982). 2t H. Sudo, H. A. Kodama, Y. Amagai, S. Yamamoto, and S. Kasai, J. Cell Biol. 96, 191 (1983). 22 j. E. Aubin, J. N. M. Heersche, M. J. Merrilees, and J. Sodek, J. Cell Biol. 92, 452 (1982).

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notype of these cells than has been possible with normal cells. The RCJ cell lines display phenotypes that range from fibroblastic to osteoblastic, but so far only the MC3T3-E1 cells have been shown to form a mineralizing matrix in vitro. Clonal cell lines have also been established from rat osteosarcomaY ,24 Similar to the RCJ clonal populations, a range of phenotypes has been described for clonal ROS cell populations. 5 The most osteoblastic phenotype is expressed by ROS 17/2 and its subclone ROS 17/2.8. These cells express high alkaline phosphatase levels, have a good cAMP response to PTH, show vitamin D3-regulated synthesis of osteocalcin, and synthesize type I collagen almost exclusively. 5 These cells have been used extensively for analyzing the osteoblastic phenotype and its regulation by hormones and physiologically active agents. The range of phenotypes expressed by the various clonal populations of permanent cells may represent a combination of subspecialization in osteoblasts, multipotentiality of progenitors, and cells at different stages of differentiation. The relatively stable phenotype of these cells may facilitate analysis of these differences. However, the stability of phenotype could be a disadvantage for investigations of phenotypic changes that are associated with differentiation, such as occurs in osteogenic induction, and for studies of environmental influences on cell expression. In particular, permanent cell lines would not be appropriate for studies on the mitogenic effects of growth factors. Bone Cell Isolation and Culture Techniques The presence of various cell types in bone tissues requires that an isolation procedure will not only release cells from the matrix, but will facilitate the separation of the osteoblastic cells. Mechanical 25,26and enzymatic procedures 27-29 have been developed to achieve this. Modified methodologies utilizing each approach are described below for systems in which osteogenic capacity is expressed. Both procedures use tissue from 23 R. J. Majeska, S. B. Rodan, and G. A. Rodan, Endocrinology 107, 1494 (1980). 24 N. C. Partridge, R. J. Frampton, J. A. Eisman, V. P. Michelangeli, E. Elms, T. R. Bradley, and T. J. Martin, FEBS Lett. 115, 139 (1980). 25 p. j. Nijweide, A. S. van Iperen-van Gent, E. W. M. Kawilarang-de Haas, A. van der Plas, and A. M. Wassenaar, J. Cell Biol. 93, 318 (1982). 26 D. C. Williams, G. B. Boder, R. E. Toomey, D. C. Paul, C. C. Hillman, K. L. King, R. M. van Frank, and C. C. Johnson, Calcif. Tissue Int. 30, 233 (1980). 27 W. A. Peck, S. J. Birge, and S. A. Fedak, Science 146, 1476 (1964). 2s G. L. Wong and D. V. Cohn, Nature (London) 252, 713 (1974). 29 L. G. Rao, B. Ng, D. M. Brunette, and J. N. M. Heersche, Endocrinology 100, 1233

(1977).

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fetal or newborn rodents. A third method describes the isolation of osteoblastic cells from adult human bone. These methods should be adaptable to other bone tissues in other species. Standard Cell Culture Solutions

Phosphate-buffered saline (PBS) PBS supplemented with 100/xg/ml penicillin G, 50/zg/ml gentamycin sulfate, and 0.3/.~g/ml fungizone Fetal bovine serum (FBS) Minimum essential medium (MEM), a-MEM with antibiotics (as in PBS above), or Dulbecco's modified Eagle's medium (DMEM) buffered with 15 mM HEPES, pH 7.4, with antibiotics Sodium ascorbate (5 mg/ml in water, stored at - 2 0 ° in the dark) Sodium/3-glycerophosphate (fl-GP), 1.0 M in PBS (stored frozen) All solutions are obtained sterile or are sterilized by filtration through 0.2/zm Millipore filters. Mechanical Isolation

The following method is modified from Ecarot-Charrier et al. 3° Principle. The isolation technique is based on the ability of osteoblasts to migrate from bone onto glass fragments. 3~ Procedure. Frontal and parietal bones from calvariae (four) of 5- to 6day-old mice (C57BL/6J strain) are dissected under sterile conditions. Periosteal and endosteal fibrous tissues are carefully removed by stripping with fine forceps, while keeping the bone tissue immersed in DMEM. The calvariae are then placed (four calvariae per dish) in 60-mm Petri dishes (Lux Scientific) containing 6 ml DMEM supplemented with 10% FBS and 50/xg/ml ascorbic acid. Glass coverslips (Corning Glass Co., Ithaca, New York), 0.18 mm thick, are fragmented, and pieces with a surface area of approximately 1 mm z are sterilized and placed in the endocranial surface, avoiding suture areas. After 24 hr of culture in the same medium at 37° in 5% CO2/95% air, cells migrate onto the glass fragments and by day 4-5 form multilayers. Glass fragments are subsequently removed and cell multilayers are scraped off. Occasionally, pieces of multilayered cells remain attached to the bone surface from which they can be removed with fine forceps. Cell clusters obtained from the four calvariae are transferred to a tissue culture dish (35 mm) where they reattach within 2 hr. Cells in the center of the clusters have a polygo3o B. Ecarot-Charrier, F. H. Glorieux, M. v a n der Rest, and G. J. Periera, J. Cell Biol. 96, 639 (1983). 3i S. J. J o n e s and A. Boyde, Cell Tissue Res. 184, 179 (1977).

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nal shape, whereas cells at the periphery are more fibroblastic in appearance. After 9 days in culture (with medium changes every 2-3 days), 1 × 106 cells/dish are obtained. When 10 mM fl-GP is added after 5 days in culture, mineral deposits are visible within the cell cluster within 24 hr. Subcultured cells obtained by collagenase treatment of the primary cultures are also able to initiate mineral deposition in the presence of fl-GP when seeded at very high density (1 × 106 cells/cm2). Cells in culture for 1-2 weeks devote 11.2 -+ 2.9% of total protein synthesis to collagen, - 2 0 % of which is estimated to be in the cell layer. By comparison, mouse fibroblasts synthesize 7.1 +- 1.0% collagen. Analysis of pepsin-digested radiolabeled collagen o~-chains by SDS-PAGE and fluorography has shown the collagen to be predominantly type I (97% of the total). Of the remaining 3%, small amounts of type III and V collagens can be identified together with polymeric forms of collagen t~-chains. 3°

Enzymatic Isolation The following method is modified from the procedure of Rao et al. 29 and is similar to a procedure described by Wong and Cohn 28 for isolating bone cell populations from mouse calvariae. Principle. The method utilizes a bacterial collagenase-rich protease mixture to perform a timed release of cells from the bone surface; fibroblastic cells in the outer layers are released first with the osteoclasts, whereas osteoblasts in the inner layer are released later.

Special Solutions Enzyme cocktail: Bacterial collagenase 3 mg/ml, 6.25 U/ml elastase, 18.22 mg/ml o-sorbitol (D-glucitol), 6 mg/ml chondroitin sulfate (all from Sigma) dissolved in Kreb's IIA buffer (111.2 mM NaC1, 21.3 mM Tris base, 13.0 mM glucose, 5.4 mM KC1, 1.3 mM MgCI2, 0.5 mM ZnCI2, pH 7.4) and sterilized by filtration. Procedure. The following procedures are carried out under sterile conditions. The frontal and parietal bones of calvariae are dissected from 21-day-old Wistar rat fetuses and are washed thoroughly in ice-cold PBS supplemented with antibiotics. The periostea and loosely attached soft tissue are removed, and the calvariae minced with scissors into small fragments. Fragments from approximately 30 calvariae are digested at 37 ° with 4 ml enzyme cocktail stirred with a magnetic stirrer in a water bath. After 10 min, the supernatant containing released cells is removed and immediately mixed with an equal volume of cold FBS to inhibit further enzymatic activity. A second 4 ml of enzyme cocktail is added to the calvarial fragments, and the procedure is repeated at 20, 30, 50, and 70

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min after the start of the digestion, producing five populations (populations I-V) of cells. The cell suspensions are individually filtered through a sterilized stainless-steel sieve (200 mesh), and an aliquot taken for cell counting. The remaining cells are gently pelleted by centrifugation at 400 g for 5-8 min, and after removing the supernatant, the cells are resuspended in ot-MEM containing 15% FBS and antibiotics. Cells from each population are plated at 5 × 105 cells/disk in 60-mm tissue culture dishes or in T-25 tissue culture flasks and are placed in an incubator at 37° in a humidified atmosphere of 95% air/5% CO2. Media are changed after 24 hr and again after 3-4 days. The cells reach confluence - 7 days after plating. Primary cells from individually plated or pooled populations I - V exhibit a range of morphologies from spindle-shaped (fibroblast-like) to polygonal (more osteoblast-like). Consistent with results obtained with mouse calvarial cells prepared in a similar manner, z8,32 the cells released in the later populations have a greater cAMP response to PTH, higher alkaline phosphatase levels, and synthesize a relatively low proportion of type III collagen (1-3%). In addition, these cells synthesize osteocalcin and osteonectin and can be stimulated to synthesize collagenase) 5 Subculturing the later eluting cells is generally accompanied by a loss in osteoblastic characteristics including decreased cAMP response to PTH, decreased alkaline phosphatase activity, and an increase in type III collagen synthesis. When cells from some of these populations are maintained in longterm culture in the presence of added ascorbic acid (50/zg/ml), nodules - 7 5 /zm thick and covered with polygonal cells resembling osteoblasts begin to form about 3 days after confluency ( - 1 0 days in vitro). These increase in size to - 3 mm over the next 3-4 days. In the additional presence of 10 mM Na fl-GP, 33 the nodules will mineralize and histologically resemble woven bone. 34 The lining cells have high alkaline phosphatase levels, whereas cells embedded within the mineralized matrix have the appearance of osteocytes. A definite osteoid seam, devoid of demonstrable mineral, lies between the lining cells and the mineralized matrix. Immunohistochemical studies show that type I collagen is prevalent throughout the nodule and is mineral associated, whereas a much weaker type III collagen staining is observed which is more diffuse and is not mineral associated. Osteonectin appears to be bound to the mineral phase. These mineralizing nodules, which provide the best evidence of 32 D. M. Scott, G. N. Kent, and D. V. Cohn, Arch. Biochem. Biophys. 201, 384 (1980). 33 H. C. Tenenbaum and J. N. M. Heersche, Calcif. Tissue Int. 34, 70 (1982). 34 C. G. Bellows, J. E. Aubin, J. N. M. Heersche, and M. E. Antosz, Calcif. Tissue Int. 38, 143 (1986).

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the osteoblastic phenotype, are not formed in population I cultures, and their number increases through populations I I - V ) 4 Dexamethasone at concentrations of 10 -9 M to 10 -7 M increases the number of nodules) 5 Because only a small proportion of the isolated cells appear to express an osteogenic phenotype and because individual nodules are difficult to isolate, biochemical studies of bone formation and hormonal responses are difficult in this system. Based on the assumption that the majority of cells in the later eluting populations are of the osteoblastic lineage, we have plated cells at high density onto bone disks (3 mm in diameter, prepared from adult rat calvariae). Preliminary analyses of this system have revealed that the cells form a multilayer and, in the presence of ascorbate, synthesize a matrix beneath them on the disk surface which will mineralize in media supplemented with 10 mM Na fl-GP. Notably, essentially all the cells that attach to the disk surface show high alkaline phosphatase activity which is evident at 5 days and remains high through 35 days in culture.

Explant Procedure Principle. Bone cells allowed to grow out from bone explants obtained from adult tissues will retain osteoblastic properties on subsequent culture. 36 Procedure. To isolate osteoblastic cells from adult human bone, trabecular fragments obtained as biopsies or at surgery are washed in sterile PBS to remove blood and marrow components. Particles 3-5 mm in diameter are prepared by dissection and placed in 90-mm tissue culture dishes with 0.2-0.6 g of bone/dish. Treatment with bacterial collagenase as described for rat calvariae can be used at this stage to remove any fibroblastic cells and undifferentiated osteoblastic cells. The bone explants in 5-10 ml a-MEM supplemented with 10% FBS and containing 50 U/ml penicillin, 15 ~g/ml streptomycin, and 2 mM glutamine are cultured at 37° in 95% air/5% CO2. Medium is changed first after 24 hr and subsequently at 5-day intervals. Outgrowths of cells from the bone fragments appear within 1 week and these form a confluent monolayer at 3-4 weeks. After removing the bone explants, the confluent cells are trypsinized by the addition of 5 ml 0.01% w/v trypsin in citrate-saline. Within 5 min at 37°, the cells are detached by shaking and combined with an equal volume of medium supplemented with 10% FBS. After pelleting by centrifugation at 400 g, the cells are washed in the above medium, dispersed by repeated aspira35 C. G. Bellows, J. E. Aubin, and J. N. M. Heersche, unpublished (1986). 36 j. N. Beresford, J. A. Gallagher, M. Gowen, M. K. B. McGuire, J. W. Poser, and R. G. G. Russell, Clin. Sci. 64, 38 (1982).

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tion through a 19-gauge needle, and plated in 35-mm tissue culture dishes at 5 × 104 cells/dish. First subcultures of the cells from various types of bone respond to PTH, express high alkaline phosphatase levels, and synthesize high levels of collagen and osteocalcin, the production of which is regulated by 1,25(OH)2D3. 37

Cloning Osteoblastic Cells Preparation of isogenic cell populations is useful for studies of bonecell heterogeneity and osteoblast differentiation and to determine specific cell types that respond to particular hormones and physiologically active agents. Principle. The procedure described combines the probability of initiating cultures from single cells using a low plating number with a visual check for single colonies. 22 Procedure. Although cells can be cloned from freshly explanted cells, much better results have been obtained using cells from early subcultures. Subconfluent cultures in 60-mm dishes are washed once with PBS and then incubated with 3 ml trypsin (0.01% w/v trypsin) in citrate-saline at 37° until the cells become rounded and can be detached by shaking (5-10 min). The protease activity is stopped by the addition of an equal volume of a-MEM containing 15% v/v FBS and antibiotics, and cells are flushed from the surface of the dish with a Pasteur pipette. The cells are transferred to a sterile polypropylene tube (Falcon 2070), and an aliquot of the cell suspension is taken to determine the cell number. Using serial dilutions that are no greater than 1 : 100, the suspension is diluted to 5 cells/ml (1 cell/200/xl) with supplemented medium. From the diluted cell suspension, 200-/xl aliquots are transferred into individual wells of 96-well microwell tissue culture plates and left undisturbed for 7-10 days in an incubator at 37 ° in 95% humidified air with 5% CO2. The wells are screened using a phase-contrast microscope, and those wells containing single colonies scored. Such colonies are retained and the cell medium changed by carefully aspirating off most of the old medium without disturbing the cells and replacing it with fresh medium. The cells are refed in this way every 7 days until the cells begin to multilayer (which often occurs before the cells reach confluency) at which point they are subcultured. The cells are carefully rinsed in PBS to remove serum and one to two drops of trypsin solution are added. When the cells become rounded, two to three drops of a-MEM containing 15% FBS are added, and the cells are brought into 37 j. N. Beresford, J. A. Gallagher, J. W. Poser, and R. G. G. Russell, Metab. Bone Dis. Relat. Res. 5, 229 (1984).

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suspension by gently pipetting up and down. The cells are replated into fresh 24-well plates and fresh medium is added. In the early stages of cloning, the split ratio of the subculture should be kept between l : 2 and 1 : 4, gradually changing to larger sized wells (in 24- and 12-well plates) before proceeding to dishes and flasks. Using this procedure, a number of isogenic populations of embryonic rat calvarial cells that differ in cell morphology, hormone response, and matrix protein synthesis have been prepared. 22 Many of these rat bone cell clones have become established to culture, and on long-term subculture, these populations tend to lose osteoblastic characteristics as found for mixed populations. 38 Recloning of some of these populations 39 and ROS 17/24° cells has shown that heterogeneity also exists within isogenic populations of established and tumorigenic cells. Characterization of Cells To characterize the isolated bone cells properly, morphological characteristics in culture, alkaline phosphatase activity, hormone responsiveness, and matrix expression should be determined. Even though the ultimate criterion for osteoblastic cells is the ability to form a bonelike tissue, this more general characterization will eventually assist in the determination of factors that are important for osteogenesis. Only characterization with respect to biochemical aspects of bone matrix production will be detailed here as hormonal responses are dealt with elsewhere. The morphological appearance of cells in culture is routinely recorded by phase-contrast microscopy together with determinations of population doublings and replicative rates. The formation of a mineralizing tissue matrix can be analyzed with the following histochemical stains; hematoxylin and eosin for cells and collagenous matrix, alcian blue for acidic glycoproteins and proteoglycans in the matrix, the von Kossa technique or alizarin red staining for mineral deposition, 4~ and naphthol AS MX phosphate (Sigma) coupled with a diazonium salt such as fast red violet for cellular alkaline phosphatase activity. With the availability of specific antibodies to matrix components, cells can also be readily evaluated for 38 j. N. M. Heersche, J. E. Aubin, A. E. Grigoriadis, and Y. Moriya, in "The Chemistry and Biology of Mineralized Tissues" (W. T. Butler, ed.), pp. 286-295. EBSCO, Birmingham, Alabama, 1985. 39 C. G. Bellows, J. Sodek, K.-L. Yao, and J. E. Aubin, J. Cell. Biochem. 31, 153 (1986). 40 A. E. Grigoriadis, P. M. Petkovich, R. Ber, J. E. Aubin, and J. N. M. Heersche, Bone 6, 249 (1985). 41 R. A. B. Drury and E. A. Wallington, "Carleton's Histological Technique." Oxford Univ. Press, London, 1967.

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the synthesis of bone proteins. As shown in Fig. 1, secreted proteins located by indirect fluorescence give a characteristic perinuclear punctate pattern. Antibodies will also aid in the analysis of the mineralizing tissue matrix formed in culture. Although these approaches can be highly sensitive, they are limited to qualitative assessments. In the following section, procedures for the quantitative analysis of matrix protein production are detailed.

Protein Synthesis To study protein synthesis by bone cells in culture, it is convenient to radiolabel the newly synthesized proteins metabolically using [35S]methionine. The advantages of using [35S]methionine as a precursor include its availability at high specific radioactivities, the high-energy/3radiation, economy, and the fairly uniform distribution of methionine residues in proteins. However, the possibility that methionine is absent in a particular protein of interest should not be overlooked. The high specific

I

FIG. 1. Indirect immunofluorescent staining of human bone cells. Specific antibodies to type I collagen (A), osteonectin (B), a 25-kDa collagenous bone protein (C), and bone proteoglycan have been used to localize matrix proteins in bone cells (D). A perinuclear punctate pattern, reflecting the concentration of these proteins in the Golgi and secretory vesicles, is characteristically observed. (Bone cells kindly provided by Drs. P. Grant and H. C. Tenenbaum.)

[16]

BONE CELL CULTURES

317

radioactivity of [35S]methionine permits the analysis of low numbers of cells (studies can be performed on less than 1000 cells) and is ideal for studying the biosynthesis and processing of bone proteins using pulsechase and mRNA translation protocols. Principle. Protein synthesis, representing the cellular phenotype, is analyzed at a defined period by a pulse-chase procedure. Using the high specific radioactivity of [35S]methionine, several types of analysis can be performed on relatively few cells.

Materials [35S]Methionine (specific activity > 800 Ci/mmol, NEG-009T New England Nuclear Corp.) DMEM-Met (Dulbecco's modified Eagle's medium minus methionine) Dialyzed FBS Sodium fl-aminopropionitrile (fl-APN, 1 mg/ml) in water stored at -20 ° Proteolytic enzyme inhibitors, 10× stock solution containing 250 mM tetrasodium ethylenediaminetetraacetic acid, I0 mM benzamidineHC1, 100 mM N-ethylmaleimide in water Labeling Procedure. Triplicate 35-mm dishes of cells are washed three times by incubating with 2.0-ml aliquots of DMEM-Met, containing 0.1% v/v dialyzed FBS, over a 15-min period at 37° to deplete endogenous methionine levels. The cells are then pulse-labeled for 20-30 min at 37° in 1.0 ml of the same medium supplemented with 50-100 /zCi [35S]methionine, 50/zg/ml ascorbic acid, and 50/~g/ml fl-APN. Following the pulse period, the cells are washed twice in complete DMEM containing 0-1% v/v FBS and incubated in 1.0 ml of the same medium for 4 hr. The media from the cultures are collected separately and, after adding proteolytic enzyme inhibitors, dialyzed against at least three changes of water at 4 °. The cells are washed six times with 2-ml volumes of ice-cold PBS to remove free [35S]methionine. The cells are then extracted with 1.0 ml of 0.5 N NH4OH for 2-3 min which solubilizes the cellular proteins and leaves the underlying matrix intact. 4z The cell extract is sonicated on ice for 2 × l0 sec using a Branson 185 sonifier (setting #5). The matrix is scraped from the dishes into 1.0 ml ice-cold PBS using a rubber policeman. The efficiency of this transfer is improved by including two additional washes with 0.5 ml aliquots of PBS. Using this procedure, sufficient chase time is given to allow essentially all of the radiolabeled proteins that are to be secreted by the cell to be deposited either into the culture medium or into the tissue matrix forming 42 G. Greenburg and D. Gospodarowicz, Exp. Cell Res. 140, 1 (1982).

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beneath the cells. Thus, the secreted proteins in these compartments can be analyzed separately from each other and from the cellular proteins in the NH4OH extract. The amount of radiolabeled protein in each compartment can be determined by taking aliquots for scintillation counting. Additional information on protein synthesis can be derived from the electrophoretic separation of the radiolabeled proteins by SDS-PAGE 43 using 5-20% gradient gels and fluorography. 44 The fluorographs, which can be further refined with two-dimensional separations using isoelectric focusing in the first dimension, provide a fingerprint (see Fig. 2) characteristic of the cellular phenotype. Densitometric tracing of the fluorographic tracks can provide an objective analysis of qualitative and quantitative differences between cell populations.

Collagen Synthesis The proportion of radioactivity that has been incorporated into collagens and the type of collagens synthesized can be determined as follows. Principle. Highly purified bacterial collagenase is used to specifically degrade collagenous proteins to small peptides which can be separated from undigested proteins by precipitation with trichloroacetic acid ( T C A ) . 45 Pepsin is used to degrade noncollagenous proteins, and the constituent a-chains of the undegraded collagen molecules can then be identified by SDS-PAGE.

Solutions Assay buffer (0.05 M Tris-HC1, 5 mM CaCIE, pH 7.6) Bacterial collagenase (Advance Biofactures, or Worthington, CLSPA further purified by Sephacryl S-200 filtration) 1 mg/ml stored at - 2 0 ° in 1.0-ml aliquots N-ethylmaleimide (NEM) (0.5 mM in assay buffer) Trichloroacetic acid (TCA), 70% w/v, 10% w/v tannic acid in water (10× stock) Bovine serum albumin (BSA), 2.5 mg/ml in assay buffer Pepsin (3 × crystallized, Sigma) Procedure. Freeze-dry - 2 × l 0 4 dpm of dialyzed protein sample in a 1.5-ml plastic centrifuge tube and redissolve in 1.0 ml assay buffer. To 0.5 ml, add 25 /xl NEM and 25 /zl bacterial collagenase, mix gently, and incubate at 37° for 2 hr. To the second 0.5-ml aliquot, add 25 txl buffer instead of collagenase and treat identically as a control. Chill sample on 4~ U. K. Laemmli, Nature (London) 277, 680 (1970). 44 W. M. B o n n e r and R. A. L a s k e y , Eur. J. Biochem. 46, 83 (1974). 45 B. Peterkofsky, this series, Vol. 82, p. 453.

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BONE CELL CULTURES

Cell DP

212 K~,-

116 KJ,92.5 K j" 66 K~-

319

Chase Matrix DP D P

~ronectin ocollagen fllagen

43 K ~" 31 K ~ 22.1 K~.14.4 K ~

FIG. 2. S D S - P A G E analysis of [35S]methionine-labeled proteins synthesized by rat calvarial bone cells. Cells were grown on bone disks (D) and tissue culture plastic (P). Radiolabeled proteins secreted by the cells into the culture medium (Chase), those incorporated into the tissue matrix and extracted by 4 M guanindine-HC1 (Matrix), and those associated with the cells and extracted with 0.5 N NH4OH (Cell) are compared. Note differences between D and P in Chase and Matrix protein profiles, indicating the effect of the substratum on the cellular phenotype.

320

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B I O C H E M I S T ROF Y THE EXTRACELLULAR MATRIX

ice, add 20/.d BSA then 55/zl ice-cold TCA to precipitate the noncollagenous proteins (NCP). After 30 min, centrifuge for l0 min at 10,000 g on a microfuge and transfer the supernatant (collagen peptides) to a clean tube. Reextract the pellet with 0.5 ml of 7% TCA. Pool the supernatants and extract three times with equal volumes of diethyl ether to remove the TCA. After removing the last traces of ether under a gentle air stream, transfer the aqueous phase quantitatively to a scintillation vial, add scintillation cocktail, and determine radioactivity. Subtract the radioactivity from the control to give the amount of radioactivity specifically associated with the degraded collagen (A). To the NCP pellet, add 0.5 ml 7% TCA, heat to 90° for 20 min to extract any remaining collagen. Centrifuge for 5 min, transfer the supernatant, and extract with ether to remove TCA. Determine radioactivity (B) as described above. Add 100/xl of 70% formic acid to dissolve the NCP pellet and transfer quantitatively for scintillation counting (C). If the collagenase digestion is efficient B should be less than 10% of the total protein radioactivity. Percentage of radioactivity in collagen -

A A+B+C

x 100

To analyze for any collagen associated with the cell layers, a slightly different approach must be used to prevent digestion of the noncollagenous protein by endogenous proteinases. The following procedure is modified from Flaherty and Chojkier. 46After washing the cell layers with PBS until the free isotope has been removed, the cells are quantitatively scraped from the dishes in 1-2 ml PBS (for a 35-mm dish), and the cells are lysed by sonication for 20 sec as described in the section on Labeling Procedure. TCA is added to a 10% w/v final concentration, and the resultant precipitate is collected by centrifugation at 10,000 g for 5 min on a microfuge. The pellet is redissolved in 0.2 N NaOH and is dialyzed against assay buffer. Collagen radioactivity can then be determined using the collagenase assay as described above. To determine the types of collagen synthesized, approximately 1 × 10 4 dpm of radiolabeled protein is freeze-dried and redissolved in 0.5 ml of 0.5 N acetic acid adjusted to pH 2.2 with HCI. After adding 50/~g pepsin, digestion is carried out for 4 hr at 15°. The digest is freeze-dried, redissolved in electrophoresis sample buffer, and separated by SDS-PAGE on 7.5% cross-linked polyacrylamide gels using the delayed reduction procedure 47 (see Fig. 3). Collagens I, III, and V which are likely to be synthe46 M. Flaherty and M. Chojkier, J. Biol. Chem. 261, 12060 (1986). 47 B. Sykes, B. Puddle, M. Francis, and R. Smith, Biochem. Biophys. Res. Commun. 72, 1472 (1976).

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BONE CELL CULTURES

2

lp

321

2p

FN

pro al (I) (11 pro a2 (I)

al (111) al (I) a'2

FIG. 3. Analysis of collagens synthesized by rat calvarial bone cells. [35S]Methioninelabeled proteins from culture media of two clonal populations of rat calvarial bone cells have been analyzed by SDS-PAGE (7.5% cross-linked gel) and fluorography. Aliquots were digested with pepsin (p) which converts procollagens to collagen a-chains. The collagens were separated by SDS-PAGE with a delayed reduction. Prolonged exposure of fluorographs are often necessary to reveal type V collagen and low amounts of type III collagen.

sized by bone cells in culture have been successfully analyzed in this and have been quantitated by densitometric scanning of fluorographs .48 w a y 22,39

Immunoprecipitation of Bone Proteins Although [35S]methionine is the best general label for the bone proteins, preferential labeling of phosphoproteins with 32po4 and proteogly48 H. F. Limeback and J. Sodek, Eur. J. Biochem. 100, 541 (1979).

322

BIOCHEMISTRY OF THE EXTRACELLULAR MATRIX

1234

ON

[16]

[16]

BONE CELL CULTURES

323

cans with either Na35SO4 or [14C]glucosamine can be advantageous for studies of these proteins. Some bone-matrix proteins, such as osteonectin, can also be selectively adsorbed onto synthetic hydroxyapatite in the presence of 4 M guanidine-HC149 and can be subsequently identified by SDS-PAGE and fluorography (Fig. 4). However, it is more satisfactory to use specific antibodies, whenever these are available, for either radioimmunoassay, as has been described for osteocalcin, or for immunoprecipitation of biosynthetically radiolabeled protein, as has been described for osteonectin. 49,5°Immunoprecipitation can be used to study the biosynthesis and processing of bone proteins, as well as for quantitating production. Principle. The affinity of IgG antibodies for protein A is used to insolubilize complexes formed between specific antibodies and the radiolabeled antigen.

Materials Protein A-Sepharose (Pharmacia), protein A-agarose (Sigma), or MAPS (BioRad). Immunoprecipitation buffer (0.3% Nonidet P-40, 0.3% sodium deoxycholate, 0.1% w/v BSA in Tris-saline, 0.02% sodium azide) Procedure. Radiolabeled proteins (1-10 × 104 dpm) in 0.5 ml buffer are incubated with 10/xl preimmune or normal serum for 2 hr at 4°. One hundred microliters protein A-Sepharose is then added for a further 1 hr to provide a nonspecific precipitate. After centrifugation at 10,000 g for 5 min, the supernatant is incubated with 5-10 p,g specific antibodies (or - 5 /~1 of specific antiserum) overnight. A second 100-/zl aliquot of protein A Sepharose is added, and incubation is continued at 4° for a further 1 hr. The specific immunoprecipitate is pelleted by centrifugation and washed at least six times with buffer before analysis by SDS-PAGE and fluorography. For antibodies that do not bind well to protein A (or MAPS), antibodies can be reacted with avidin and subsequently linked to biotin resin before proceeding with the immunoprecipitation. 49 K, Otsuka, K.-L. Yao, S. Wasi, P. S. Tung, J. E. Aubin, J. Sodek, and J. D. Termine, J. Biol. Chem. 259, 9805 (1984). ~0 F. Kuwata, K.-L. Yao, J. Sodek, S. Ives, and D. Pulleyblank, J. Biol. Chem. 260, 6993 (1985). FIG. 4. Analysis of matrix proteins synthesized by porcine calvarial bone cells in culture. [35S]Methionine-labeled proteins from culture media (1) were incubated with hydroxyapatite in the presence of 4 M guanidine-HCl. Selective adsorption of several proteins onto hydroxyapatite is observed (2). Specific immunoprecipitation with antiosteonectin antibodies has been used to identify the M~ 39,000 protein as osteonection (ON) (4). Collagenous protein is observed to bind to protein A in the nonspecific immunoprecipitation (3).

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Assay for Alkaline Phosphatase The expression of high levels of alkaline phosphatase activity is typical of osteoblastic cells. A simple method for measuring this activity in multiple samples is described. Principle. Alkaline phosphatase activity in cell extracts is determined using a modification of the Lowry 51 assay in which p-nitrophenyl phosphate (P-NPP) is used as the substrate.

Solution Substrate in assay buffer [60 mM P-NPP (Sigma), 10 mM MgC12 6HzO in 0.375 M 2-amino-2-methyl-l-propanol, pH 10.3 (buffer #221, Sigma)] Procedure. Cells in a 35-mm dish are washed in PBS and are scraped into 0.5 ml ice-cold 50 mM Tris-HCl buffer, pH 7.4. The cells are transferred together with a second wash of the culture dish to a test tube and are sonicated for 2 x 10 sec (Branson Sonifier, setting #5). After centrifuging for 5 min at 10,000 g, 100/zl of substrate is added to 25/A of the supernatant in individual wells of 96-well tissue culture plates. Incubation is carried out lbr 30-60 min at 30°, stopping the reaction with 100/zl of 0.5 N NaOH. The absorbance is read at 405 nm, most conveniently in a Multiscan (Titertek), and is compared to a standard curve obtained using commercially available alkaline phosphatase (Sigma). 5I O. H. Lowry, this series, Vol. 4, p. 371.

[17] H o r m o n a l I n f l u e n c e s on B o n e Cells

By T. J. MARTIN, K. W. NG, N. C. PARTRIDGE,and S. A. LIVESEY Hormone receptors and responses have been studied in osteoblastrich cultures derived from newborn rodent bones L2 and in osteogenic i R. A. Luben, G. L. Wong, and D. V. Cohn, Endocrinology 92, 526 (1976). 2 N. C. Partridge, D. Alcom, V. P. Michelangeli, B. E. Kemp, G. B. Ryan, and T. J. Martin, Endocrinology 108, 213 (1981).

METHODS IN ENZYMOLOGY,VOL. 145

Copyright © 1987by Academic Press, Inc. All rights of reproductionin any formreserved.