JOURNAL OF BONE A N D MINERAL RESEARCH Volume 7. Number 7, 1992 Mary Ann Liebert, Inc., Publishers

Factors Influencing Synthesis and Mineralization of Bone Matrix from Fetal Bovine Bone Cells Grown In Vitro S. WILLIAM WHITSON,' MARCIA A. WHITSON,' DANIEL E. BOWERS, JR.,' and MICHAEL C . FALK2

ABSTRACT This study of the in vitro synthesis and mineralization of bovine bone demonstrates that sheets of mineralized matrix can be produced consistently within 18-24 days of cell isolation. Mineralization surpasses that achieved by other systems with other species: The deposition of mineral extends beyond nodules to form branching trabeculae and then solid wafers of bone. Comparison of the fetal age of the bone source, enzyme digestion methods, seeding density, culture surface, nutritive media, and concentration of fetal calf serum and other additives, including insulin and ascorbic acid, has yielded a set of optimal culture conditions. I n the presence of ascorbic acid and @-glycerolphosphate, insulin has a dose-dependent effect on the morphology of the mineralized bone matrix produced. Quantitative analysis shows that in these cultures calcium accumulates most rapidly between days 6 and 10 after the introduction of mineralization medium but that mineral accretion continues throughout 14-16 days of culture. Alkaline phosphatase levels rise up to 200-fold, concomitant with a rapid increase in the number of cells per culture during the early mineralization phases: both fall as mineralization proceeds. This system has been used to study the induction of mRNA of type I collagen, alkaline phosphatase, and several noncollagenous bone proteins during the course of mineralization. Because of the degree of mineralization achieved with this system, it has many potential applications.

INTRODUCTION have been characterized by their potential to produce high levels of alkaline phosphatase,'l' their rapid production of 3',5'-cyclic AMP when treated with parathyroid hormone,"] the absence of this cyclic AMP response of bone cells when exposed to calcitonin," 3 1 and their ability to produce type I collagen and several noncollagenous bone proteins. [4-91 Numerous studies have demonstrated the initiation of mineralization of matrix secreted by bone cell^.^'-^^^ Most de novo mineralization studies involve cells isolated from rat and mouse calvariae. In most of these studi e ~ , ( ~ mineralization "~) was not uniform, rather tending to be confined to small, mineralized nodules that appeared only after lengthy periods of time in culture (up to 40 days). Electron microscopy has shown that the tops and

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ONE CELLS I N VITRO

bottoms of these nodules are lined with osteoblasts and that the nodules contain several embedded osteo9) cytochemistry has shown cytes. ( 1 4 , 1 s . 1 8 , 1Ultramicroscopic that the cells associated with these nodules have higher levels of alkaline phosphatase than other cells in the culture1141and that necrosis claims some of the osteocytes in the nodules.1141 Some early investigators(".") stressed the importance of cell seeding density and calcium and phosphorus ion levels. Others showed that when the culture medium was changed several times daily, shorter time periods were sufficient for cultured mesenchymal cells isolated from calvariae to differentiate into bone cells and to produce a mineralized matrix.(12' In a presumptive chick calvarial periosteum organ culture system using a defined medium, @-glycerol phosphate, a substrate for alkaline phosphatase, was shown to stimulate mineralization. ( 2 0 - 2 2 )

'Southern Illinois University School of Dental Medicine, Alton. 'Combat Trauma Research Division, Casualty Care Research Department, Naval Medical Research Institute, Bethesda, Maryland.

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728 Mouse calvarial cells isolated on glass fragments and grown in a medium containing 15% fetal calf serum (FCS) and 10 mM 0-glycerol phosphate were better able to produce and to mineralize bone matrix in much less time.''51 In what is perhaps the best characterized rat bone cell culture system, collagen synthesis, alkaline phosphatase activity, osteopontin and osteocalcin synthesis, and mineralization all follow specific patterns. Alkaline phosphatase activity peaks around day 20, and significant increases in the amount of mineralization occur between days 20 and 30.'81 However, mineralization achieved with this system is limited to discrete nodules. Although bovine bone cells were also shown to be capable of mineralization when cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 20% FCS, 10 mM 0-glycerol phosphate, and 50 pg/ml of ascorbic acid,'*] our earlier method did not consistently produce full mineralization of the cultures. The objectives of this study were therefore (1) to determine how to induce consistent, rapid matrix synthesis and mineralization and (2) to determine how to mineralize the bone matrix well beyond that of the bone nodule, that is, to produce entire sheets of bone.

In this report, we describe the optimal technical procedures and conditions for a viable bovine bone cell culture system that, in fact, consistently produces solid sheets of mineralized bone within 18-24 days of cell isolation from fetal bone. Together with the companion report by Ibaraki et al. (this issue) we discuss the initial results of the quantitative and qualitative evaluation of bovine bone formation using this system. This model of bone formation appears superior to those yet devised, because the amount and uniformity of the mineralization achieved permit in vitro studies of a continuum in fetal bone development, including the possible interactions of growth factors and their carrier proteins with bone cells and the mineralizing matrix.

MATERIALS AND METHODS The bovine bone cell culture (BBCC) system described here has evolved from experiments conducted over a period of several years. The nature and extent of these studies are demonstrated in Table 1 , which lists numerous factors that have been shown to influence BBCC and high-

TABLE 1. FACTORSEXAMINED I N THE SEARCH FOR OPTIMAL CONDITIONS SUPPORTING I N VITRO GROWTH,MATRIX SYNTHESIS, A N D MINERALIZATION OF BOVINEBONECELLS

Variable

Optimal

Notes and observations

Fetal age/crown-rump length 3-4.5 months/25-45 cm 4.5-6.5 months/45-65 cm

X

>6.5 months/>65 cm

X

Maximal cell yield and viability; good mineralization Lower cell yield and slower proliferation than with cells from 45-65 cm fetus; excellent mineralization

Cell isolation medium/time Collagenase (1 mg/ml)/l5, 30, 60, and 90 minutes Dispase (3 mg/ml)/l5, 30, 60,and 90 minutes Collagenase (1 mg/ml), then dispase (3 mg/ml)/20 + 20 and 45 t 45 minutes Collagenase (1 mg/ml) + dispase (3 mg/ m1)/30, 45, and 90 minutes Collagenase (0.5 mg/ml) + dispase (3 mg/ m1)/3 h initial digestion

Tested

x x X

x X

x

Dispase (3 mg/ml)/l6 h after 3 h collagenase + dispase (0.5 and 3 mg/ml, respectively) after 3 h digestion

Growth mediuma M199

x

Cell yield, 'To 80, 25-45 cm fetus 50, 45-65 cm fetus 30, >65 cm fetus Cell yield, Yo 20, 25-45 cm fetus 50, 45-65 cm fetus 70, >65 cm fetus Homogeneous population of mature cells speeds and enhances quality of mineralization Formulation includes 1 .O g/liter of glucose, 1.26 mM Ca, and 0.83 mM P, Glucose (3.5 g/liter) added; total, 4.5 g/liter to support bone cell growth

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MINERALIZATION OF BONE IN VITRO TABLE1. (CONTINUED) Variable

Tested

Optimal X

DMEM

Notes and observations Formulation includes 4.5 g/liter of glucose, I mM pyruvate, 1.8 mM Ca, 0.9 mM Pi

Additives Fetal calf serum, @lo 10

X X

15

20 Ascorbic acid, pg/ml

X

5 50

Triiodothyronine Insulin, 8.2 x

M

Mineralization mediuma MI99

X

DMEM

Supports cell attachment Stimulates matrix synthesis; matrix destroys integrity of culture before multilayering No growth enhancement Causes glycogen and lipid accumulation within cells Formulation includes 1.O g/liter of glucose, 1.26 mM Ca, and 0.83 mM P, Glucose (3.5 g/liter) added; total, 4.5 g/liter to support bone cell growth Formulation includes 4.5 g/liter of glucose, 1 mM pyruvate, 1.8 mM Ca, 0.9 mM P,

Mineralization additives Fetal calf serum, @lo 5

10 X X X

15

20 Ascorbic acid, 50 pg/mI P-Glycerol phosphate None

X

10 mM Insulin None 8.2 x 10-7 M

X X

4.1 x 10-7 M

X

I x 10-7 M

X

X

Calcium, 0.6-1.2 mM

Phosphorus, 1.1 mM

Volume of mineralization medium/area/time 2 m1/22 mm’ coverslip in 35 mm dish/24 h 4 mV22 mm’ coverslip in 60 mm dish/24 h 3 m1/18 mm’ coverslip, 60 mm dish/8 h (daytime, 3 ml; overnight, 6 ml) 25-40 m./T-75 flask/l2 h

X

Supports matrix synthesis Less than optimal mineralization of cultures of cells from 2 6 5 cm fetus Supports maximal mineralization; enhances alkaline phosphatase activity Source: ITS ( 5 pg/ml of insulin, 5 pg/ml of transferrin, 5 ng/ml of selenium) Inadequate matrix synthesis Enhances matrix synthesis at expense of mineralization Supports continued matrix synthesis during maximal mineralization Physiologic level; supports matrix synthesis and mineralization as well as 4.1 x M level Final concentration of 2.4-3.0 mM calcium speeds formation of mineralized nodules and trabeculae Concentration of 2.0 mM P, (1.8 mM Ca) did not improve quantity of mineralized bone matrix

X X X X

apH 7.35. Included in all growth and mineralization media were sodium bicarbonate (3.7 g/liter), HEPES buffer (15 mM), and antibiotics and antimycotics.

730 lights the optimal conditions for cell isolation and growth in primary and secondary cultures, the stimulation of matrix synthesis, and the mineralization of that bone matrix. The hindlimbs of 3- to 8-month-old fetal calves are obtained from a slaughterhouse and transported on ice 10 the laboratory, where within approximately 4 h the skin, muscle, and periosteum are stripped from the bones and the cartilaginous ends of the bones are removed and discarded. Longitudinal strips of the outer cortex, 1-3 mm thick, are excised with a sterile scalpel, and the strips are placed whole in 15-20 ml serum-free medium containing 0.5 mg/ ml of collagenase (Worthington, Freehold, NJ) plus 3 mg/ ml of crude dispase (Boehringer Mannheim, Indianapolis, IN) and incubated in 50 ml centrifuge tubes at 37°C for 3 h. The tubes are then shaken vigorously to release the cells, the cell suspension is removed to fresh tubes, and the bone strips are rinsed with 15% heat-inactivated fetal calf serum (JRH Biosciences, Lenexa, KS) in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) to stop digestion, after which this rinse medium and the cell suspension are combined and filtered through a 40 pm mesh filter. The bone strips are then placed in 15 ml tubes of fresh DMEM containing 5% FCS and 3 mg/ml of dispase and digested at 37°C overnight, after which centrifugation and resuspension proceed as described. The 3 h and overnight digests are centrifuged and resuspended separately for plating in 250 ml culture flasks containing 10 ml “growth medium”: DMEM fortified with 1 mM pyruvate, 15% FCS, and 5 pg/ml of ascorbic acid plus 10 ml/liter of antibioticantimycotic solution (l0,ooO IU penicillin G , 10 mg streptomycin, and 25 pg amphotericin B per ml; Sigma). The cultures are incubated at 37°C in an environment of 95% air and 5 % carbon dioxide. The medium is changed on the first day after seeding and then every 48 h until cultures reach the right stage for passage. After 4-5 days in culture but always before they reach confluence, the cells are passed using 0.25-0.5 mg/ml of trypsin in Tyrode’s solution for 15 minutes and then counted and seeded evenly (approximately 300 cells per mm2) onto 18 mm2 coverslips (Thomas Scientific, Swedesboro, NJ; red label) in 60 mm culture dishes and covered with 3 ml growth medium or directly into 35 or 60 mm dishes ( 2 or 3 ml medium), 6 or 24 well plates (3 or 1 ml), or T-75 flasks (10 ml). Confluence and partial multilayering of the cells occur within 4-5 days of this second seeding, a total of 8 to 10 days from the time of seeding the primary culture. To initiate matrix synthesis and mineralization, the growth medium is supplemented with 10 mM 0-glycerol phosphate, 15 mM HEPES buffer, and ascorbic acid, which is increased to 50 pg/ml. ITS (insulin, transferrin, and selenium; Sigma) is also added to cultures, either at 50% of the manufacturer’s recommended usage level (i.e., half of the 5 &ml of insulin, 5 &ml of transferrin, 5 ng/ ml of selenium standard formulation) or at 12% of full concentration (1 x lo-’ M insulin, physiologic level). The resultant medium containing all additives is identified as “mineralization medium.” In Feparate experiments, the level of calcium in the medium was increased to 2.4-3.0 mM by the addition of calcium chloride and the inorganic

WHITSON ET AL. phosphorus level was elevated to 2.0 mM with sodium phosphate dibasic. Matrix production and mineralization are monitored daily by both visual observation and inverted phase-contrast microscopy for the duration of each experiment. Selected cultures grown on coverslips are processed for light and electron microscopy by removing the growth medium from the culture dishes, replacing it with 0.2 M phosphatesucrose buffer (pH 7.3; 425 mOsmol), followed by fixation in 1% glutaraldehyde in the same buffer for 0.5 h at room temperature and 1 h postfixation in 1% osmium tetroxide in 0.1 M phosphate-sucrose buffer at 4°C. The tissue is dehydrated with a graded series of ethanol rinses followed by several 100% acetone rinses before infiltration with Polybed 812 epoxy resin (Polysciences, Warrington, PA). During dehydration, selected areas of tissue may be removed from the surface with a 4 mm diameter Baker-Cummins skin biopsy punch. The circular tissue wafers are subsequently embedded in flat molds. Thick sections of plastic-embedded tissue are cut and stained with toluidine blue and alizarin red(*3’for orientation and detection of calcified matrix. Thin sections are obtained with a diamond knife on an LKB Ultratome 111, stained with uranyl acetate and lead citrate, and examined with a Philips 300 electron microscope at 60 kV. Quantitation of the total calcium present in cultures grown on coverslips is accomplished by removing the medium, rinsing the cultures in phosphate-buffered saline, and transferring them to six-well dishes. To each culture dish or well containing a coverslip is added 0.5 mi concentrated nitric acid, and this is incubated at room temperature overnight. The calcium dissolved in acid is then transferred to a 1.5 ml vial, and the sample is rinsed with an additional 0.5 ml nitric acid, which when added to the vial gives a final volume of 1.0 ml. Aliquots of sample are diluted ] : I 0 0 with 0.38% potassium chloride. Calcium analysis is performed on 2 ml of each diluted sample using flame atomic absorption spectrophotometry (Perkin-Elmer model 500 equipped with a P / E lntensitron lamp for elemental calcium) with an acetylene-nitrous oxide flame. Alkaline phosphatase is determined spectrophotometrically at 410 nm wavelength by increasing the conversion of p-nitrophenylphosphate to p-nitrophenol (kit 104, Sigma, St. Louis). Cultured cells are treated for 20 minutes with 0.5 mg/ml of trypsin in Tyrode’s solution, pelleted, resuspended in medium, and counted. Standard aliquots of cells (25,000-100,000) are placed in 1 ml of 10 mM magnesium sulfate in saline to activate the enzyme and then pelleted, after which the magnesium sulfate solution is discarded. The alkaline phosphatase substrate is added to the pellet, resuspended, and incubated for 5-15 minutes. To stop the reaction, 10 ml of 0.05 N sodium hydroxide is added to each test tube. Readings are obtained on a Gilford spectrophotometer.

RESULTS AND DISCUSSION The focus of this report is on optimal conditions contributing to the growth, matrix synthesis, and mineralization of bovine bone cell cultures like those shown in Figs. 1

MINERALIZATION OF BONE IN VITRO and 2. Because the emphasis is on a system that clearly produces and mineralizes bone more rapidly and in greater quantity than other in vitro systems r e p ~ r t e d , ( ~ -the ’ ~ )results presented here exclude comparative data from experiments that did not produce optimal results. However, several categories of factors investigated are listed in Table 1 and discussed as necessary to demonstrate the evolution of this BBCC system and its potential for use in future studies of bone formation and resorption. Mineralization in this BBCC system commences as small nodules (Figs. la, 3a, and 3b) that are quickly transformed into trabeculae (Figs. l a and 3c), which in turn fuse as

731 bone formation continues (Fig. lb), finally forming solid sheets of mineralized matrix that cover surfaces ranging from coverslips (18 and 22 mm’) to culture dishes (35 and 60 mm and 6- and 24-well) to T-75 flasks (Figs. Ic and 2). The osteocytes that form within this mineralizing matrix are sandwiched between the single layer of cells that adhere to the glass or plastic surface and the growing layer(s) of newly proliferated cells that cover the new bone and interface with the medium (Figs. 4, 5 , and 6). This rapid and progressive pattern of matrix synthesis and mineralization appears to mimic closely the embryonic bone formation that occurs in vivo.

FIG. 1. Unretouched photographs of bovine bone cells growing and synthesizing bone matrix on coverslips, demonstrating typical progressive mineralization after (a) 4-7 days, (b) 10-12 days, and (c) 16 days of maintenance on DMEM containing 20% fetal calf serum, 1 mM pyruvate, 10 mM &glycerol phosphate, 1 x lo-’ M ITS, and 50 pg/ml of ascorbic acid. Note limited outgrowth of mineralized matrix and intermittent spots of mineralization on the surface of the culture dishes ( x 1.75).

FIG. 2. A T-75 flask containing a sheet of mineralized bovine bone over its entire surface after 16 days on mineralization medium ( x 0.91).

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WHITSON ET AL.

FIG. 3. Inverted phase microscopy of bovine bone cell cultures showing the progressive mineralization occurring 2-6 days from stimulation of matrix synthesis: (a) a diffuse nodule formed at the initiation of mineralization; (b) a fully formed nodule; (c) trabeculae formed from fusion of nodules or independent of nodule formation; (d) trabeculae fused to form a solid sheet of bone (typically by day 10-12). ( ~ 4 4 0 ) .

Bovine bone cell culture consists of two distinct sequential stages: (1) an initial stage of proliferation during which the seeded cells reach confluence and then a significant degree of multilayering, followed by (2) the production of bone matrix and its subsequent stepwise mineralization. These two sequential events in bone cell culture are influenced by the interrelationship of a number of factors, including the age of fetus from which the cells are derived, the method of isolating cells from bone, seeding density in primary and secondary culture, the timing of cell passage, and the composition as well as the volume and the frequency of replenishing growth and mineralization media.

Age of fetus and bone cell isolation Mineralized bone can be produced from cells isolated from 3- to 8-month bovine fetuses, arbitrarily classified as

young (gestational age 5 4 months, crown-rump length 24-45 cm), midterm (4.5-6 months, 45-65 cm), or mature (6.5-8 months, >65 cm). However, the midterm fetus is optimal for use in this bone cell culture system because of ease of dissection and of obtaining bone slices, bone size, and high cell yield. Bone cells are more difficult to isolate from mature fetuses, and they proliferate somewhat more slowly; however, when cells from more mature fetuses are stimulated to synthesize and mineralize bone matrix, the result is a slightly more rapid onset of mineralization (2-3 days instead of 4), particularly when the cultures are grown from cells isolated via overnight digestion in dispase. Cultures of cells isolated from young fetuses are slower to produce mineralized matrix (6-8 days), and the initial mineralization is not as uniform across the entire surface of the dish as when cultures are grown from the cells of older fetuses.

733

MINERALIZATION OF BONE IN VITRO

c

z

FIG. 4. A toluidine blue-stained cross-section through a culture of bovine bone cells after 7 days on mineralization medium. The bone cells on the surface of the culture are multilayered and separated from the heavily mineralized matrix by a partially mineralized layer of bone matrix.

Sa

5b

FIG. 5. Light microscopy of representative cross-sections of bovine bone cell cultures grown for 12 days on DMEM containing 20% fetal calf serum, 1 mM pyruvate, 10 mM @-glycerolphosphate, 50 pg/ml of ascorbic acid, and 8.2 x lo-’ M ITS (insulin, 5 pg/ml; transferrin, 5 pg/ml; and selenium, 5 ng/ml). The bone matrix is very cellular and mineralizes to varying degrees. Sections stained with alizarin red and toluidine blue ( x 512).

Our early use of the enzymes collagenase or collagenase and trypsin (1 and 0.5 mg/ml, respectively) to isolate bone cells from fetal bovine bone was disappointing. Cell yields were poor, partially because of difficulty getting the cells to release from the bovine bone, but equally because cell

viability was poor. Substituting various concentrations of crude dispase far trypsin and reducing the collagenase concentration, we established 0.5 mg/ml of collagenase and 3 mg/ml of dispase in serum-free DMEM as optimal for isolating fetal bovine bone cells. Bone slices treated up to 6 h

134

c - r..

WHITSON ET AL.

.

. x

6a

6b

t\

FIG. 6. Light microscopy of representative cross-sections of bovine cell cultures grown for 12 days in medium identical to that used for the cultures shown in Fig. 5, except that the concentration of ITS was halved to 4.1 x lo-’ M (i.e., final concentration of insulin, 2.5 pg/ml; transferrin, 2.5 pg/ml; and selenium, 2.5 ng/ml). Culture thickness is decreased but mineralization is increased compared with that of high-ITS cultures shown in Fig. 5. Sections stained with alizarin red and toluidine blue ( x 512). suffer no apparent loss in cell viability. This method has allowed the use of a much wider age range of fetal calves, selected sizes of which are often in short supply. For the longer digestions required for more mature specimens, dispase, which is not inhibited by fetal calf serum, is used overnight in 5% serum to increase cell yields even further. Also dependent upon the age of the fetus is the cell yield from equal amounts of bone strips. The 3 h digestion with collagenase and dispase releases approximately 80% of the cells from a young fetus, with about 20% released only after overnight digestion in dispase and 5% FCS. In contrast, in a midterm fetus, the cell release is nearer 5050 for the two isolation times and about 30:70 for the near-term fetus. Primary cultures of bone cells from 3 h digests have very different growth characteristics that correspond to different fetal ages. The cell isolates from young fetuses include many large plaques of from 5 to 100 cells that appear fibroblastic in primary culture. Filtering (10 pm filters) eliminates most of these fibroblastic plaques as well as partially digested matrix and bone fragments in the digests. In contrast, cells isolated from midterm or mature fetuses after 3 h contain fewer fibroblastic plaques and cells collected after overnight digestion in dispase appear very homogeneous and virtually fibroblast free. This homogeneity likely contributes significantly to the uniformity of mineralization observed when such cells are stimulated to synthesize and mineralize bone matrix.

Growth medium Whether in primary or secondary culture, the time a culture requires to reach the fully multilayered stage at which ii is capable of matrix synthesis and mineralization depends to a great extent upon the medium used (see Table 1). DMEM plus 15% fetal calf serum is optimal throughout growth and mineralization in the BBCC system. We

found that adding ITS (manufacturer’s recommended level) to the growth medium from the outset of culture slowed the rate at which cells became confluent and formed rnultilayers; electron microscopy demonstrated that in the presence of ITS, the cells are filled with glycogen and fat droplets (data not shown). The addition of 5 pg/ml of ascorbic acid to DMEM (which contains none) promotes cell adhesion to the culture surface during the growth phase of BBCC; however, at 50 pg/ml, ascorbic acid in growth medium stimulated matrix synthesis and the cells and matrix detached from the surface and rolled up.

Seeding density, passage of cells, and multilayering Previous studies in this laboratory have demonstrated that a minimum seeding efficiency of 1 x lo4 bone cells per 22 mm2 coverslip in primary culture is necessary to support a rate of growth that promotes good matrix production and mineralization. Our early experiments indicated that optimal timing for cell confluence was 7-10 days in primary culture; multilayering of cells sufficient to support matrix synthesis routinely occurred in 10-14 days. In later experiments, improved cell yields, better cell viability, and the use of 250 ml flasks for growing primary cultures (5 x lo5 cells) facilitated seeding secondary cultures of cells at concentrations of 100,ooO-150,OOOcells per 18 mm2 coverslip. After several days in culture but before confluence, primary cultures are digested with trypsin in Tyrode’s solution and passed. Trypsin separates the cultures into individual cells or clusters of no more than five cells, which allows dense, even seeding onto the coverslip, ensuring the forrnation of an even multilayer of osteoblasts. The most consistent mineralization is achieved when the cells in primary culture are passed after 4-5 days’ growth in flasks and then grown in secondary culture on coverslips or culture dishes for another 4-5 days, that is, a total of 8-10 days from cell

MINERALIZATION OF BONE IN VITRO isolation at a standard seeding density of 300 cells per mm’. (However, our experience with culturing cells of other species indicates that effective seeding density varies in the culture of cells isolated from nonbovine fetuses or nonfetal bones.) Passage and high-density seeding apparently allow growth in a nonconfluent condition such that the cells maintain a rate of mitosis that favors multilayering. “Significant” multilayering is defined as the stage at which the bone cell layers covering most of the culture surface are at least two cells thick. Inverted phase-contrast micrographs show, in Fig. 7a, confluent cells that are beginning to multilayer and, in Fig. 7b, a more fully multilayered cell culture. After the first 2-3 days on mineralization medium, an even greater extent of multilayering can be observed (Figs. 7c and d). The multilayering achieved before the introduction of mineralization medium is critical.

73s Formation of a continuous sheet of bone matrix depends upon the presence of a continuous sheet of cells across the entire culture surface. Any large imperfections or breaks in the cellular sheet that cannot be filled rapidly by cells undergoing division leads to failure to attain complete mineralization. Our studies have shown that the syntheses of matrix and subsequent mineralization are drastically delayed or impeded when any of the following occurs: seeding at such a low density in primary or secondary culture that confluence and multilayering take longer than 10 days, growing the primary cultures in flasks past confluence, and/or delaying the introduction of mineralization medium once the cultures are rnultilayered. The addition of mineralization medium should not slow cell division (Figs. 4 and 8). Rather, the multilayering begun on the coverslips before the introduction of mineralization r edium ideally should

FIG. 7. Inverted phase microscopy of (a) confluent bovine bone cells beginning to multilayer, (b) multilayered bone cells (note the rounded or beaded nature of some of the cells in the surface layer that occurs when more than two cell layers are present), (c) cells and matrix after 2 days on mineralization medium, and (d) after 3 days on mineralization medium (note highly beaded appearance of areas of future mineralization). ( x 440).

736

WHITSON ET AL.

I ,

/ I

I

1 ~

i4

1 12

FIG. 8. Alkaline phosphatase activity (circles) as average units of activity per cell for three 18 mmz coverslips peaks on day 6 after mineralization medium is introduced. Trypsinized cells released per culture, which also peak at day 6, number approximately 1.5 million cells per 18 mmz coverslip (triangles).

be maintained for as long as possible during matrix synthesis and mineralization. Learning how to provide an environment that fosters the ongoing renewal of the cell multilayer, simultaneous with producing and mineralizing new matrix, may be the key to achieving unlimited bone formation in vitro.

Culture surface and cell adhesion Although cell adhesion cannot be considered apart from seeding density, cell homogeneity, and multilayering, i t is an important factor in the formation of a uniform sheet of mineralized bone. There is a distinct difference between the adhesion of bovine bone cells to glass and to plastic. Cells adhere to plastic better than to glass, and cells from midterm or older fetuses adhere better than cells from young fetuses. Furthermore, cells d o not adhere to all kinds of coverslips equally. However, growing bone cells on coverslips despite their relatively poor ability to adhere to glass facilitates both electron microscopy and immunocytochemistry. We recently demonstrated that, handled with care, mineralized sheets of bone can be removed essentially intact from coverslip surfaces, enhancing the potential for the use of partially mineralized sheets of bone for transplantation studies. I z 4 )

BBCC differ from cultures of chick and rat bone cells, however, forming not only mineralized nodules (Figs. l a , 3a, and 3b) but fine, branching, mineralizing trabeculae that radiate from the nodules as well as developing independently (Figs. l a and 3c). Because of continuous cell proliferation and matrix synthesis, the trabeculae increase in size, fusing to form continuous sheets (Figs. Ib, Ic, and 3d). Thus, mineralization in the BBCC is not limited to the discrete nodules described by other investigators. Contributing to full mineralization of the sheet of cultured bovine bone is the time interval between matrix synthesis and the first visible nodules. When the time interval is too long (generally more than 6 days), mineralization either never occurs or occurs very slowly. It is likely that cell maturation and cell homogeneity are extremely important, but providing adequate levels of nutrients, including calcium, and preventing acidification by frequent replenishment of the medium are also important in maintaining the integrity and viability of cultures on coverslips or culture dishes. In this BBCC system, insulin (ITS), calcium, and 0-glycerol phosphate in the culture medium are associated with enhanced mineralization after 10 days (Fig. 9). It should be noted that throughout mineralization, the level of ascorbic acid is maintained at 50 pg/ml in this BBCC system, in contrast to the higher levels (100 pg/ml) used by some other investigators in cell(6-B’and organ culture.1z0~z1~z5’ Insulin: Like cultured rat bone calvariae,1z5,’61 bovine bone cell cultures are sensitive to insulin. Our histologic comparison of bovine bone cells receiving media fortified with two different levels of ITS (8.2 x lo-’ and 4.1 x M) revealed differences in the morphology of the bone formed. At the higher concentration of ITS, the cells generally secreted more matrix, but this matrix mineralized poorly and was very cellular (Fig. 5 ) . Conversely, in cultures receiving half this concentration of ITS, the matrix thickness and cellularity were decreased and mineralization increased (Fig. 6). After 12-14 days on mineralization me-

Mineralization medium When properly multilayered bovine bone cell cultures are placed on mineralization medium, matrix synthesis begins immediately; cell proliferation continues. During this premineralization period, morphologic changes occur in cell arrangements. As matrix is synthesized in the BBCC, plaques of cells and associated matrix appear to aggregate, thickening to form nonmineralized nodules. Nodule development and subsequent mineralization have been described for both chick and rat osteoblast culture^.(^^^^^'^'^^''^'^^ Our

f 7111

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FIG. 9. The accumulation of mineral on coverslips after 10 days of stimulation, showing the synergistic effect of adding 0-glycerol phosphate (10 mM) and insulin (1 x M ITS) to DMEM mineralization formula. Each determination is based on the average amount of calcium found on three 18 mm’ coverslips.

MINERALIZATION OF BONE IN VITRO dium, the cultures maintained on the lower levels of ITS routinely had only a single layer of cells on the surface of the mineralized bone; no osteoid seam was apparent (Fig. 6). Since that comparative study, which was conducted on the basis of the manufacturer's recommended usage, we have found that the physiologic level of insulin (1 x lo-' M) is adequate to support BBCC matrix synthesis and mineralization. Electron microscopy of the bovine bone cell cultures reM ITS shows typical bone cell ultraceiving 8.2 x structural features and mineralized bundles of collagen at 12 days (Fig. 10). The mineral in 4.1 x M ITS-treated cultures was so dense that cutting undemineralized sections for electron microscopy was impossible.

Calcium: The standard high-glucose formulation of DMEM, without additional calcium, has produced the consistent rapid mineralization shown in Figs. 1-6 and 11. However, the level of calcium contained in D M E M ' (1.8 mM) is substantially below that in BGJ medium (2.5 mM), which is used in many rat and chick bone cell culture systems. ( 6 - 8 ) Although we have not systematically measured calcium levels in the mineralization medium used in this BBCC system, as others have done for the rat bone culture system,'9' we routinely change the mineralization medium twice daily, and when maintaining large numbers of cells directly on culture dishes, we double the amount of medium overnight to ensure adequate calcium levels for the continuous support of mineralization. In T-75 flasks, 25-40 ml mineralization medium twice a day for 16 days has proven adequate to produce the large, evenly mineralized sheets of bone. In recent BBCC studies, we have found that the addition of calcium to elevate the concentration to 2.4 or 3.0 mM in the mineralization medium clearly enhances the speed with which visible mineralizing nodules and trabeculae appear in culture. The recent report that fetal ovine plasma calcium is around 3.2 mM (i.e., significantly higher than the adult mammalian circulating calcium level, 2.5 mM) and that a plasma calcium level of 1.95 mM does not support normal matrix synthesi~"~' suggests the value of increasing the BBCC calcium level, if indeed fetal bovine plasma calcium is found to be similarly high. 0-Glycerol Phosphate: The addition of 0-glycerol phosphate to BBCC medium may be subject to controversy because it has been shown to cause ectopic mineralization in cultured rat calvariae.'25' However, the culture conditions associated with ectopic mineralization in the presence of 0glycerol phosphate differed substantially from those of this BBCC system. Those investigators' modification of FittonJackson BGJ medium is much higher in both inorganic phosphorus (3 mM) and calcium (approximately 2.5 mM) than DMEM, which has 0.9 mM phosphorus and 1.8 mM calcium. The calcium x phosphate ion product of BGJ medium is thus 7.5, almost double the 2 4 . 0 product reported (R.E. Wuthier, personal communication, 1991) to stimulate spontaneous precipitation of calcium phosphate in cartilage cell culture. ( 2 1 . 2 8 ) Whereas the addition of &glycerol phosphate, which in-

737 duces increased alkaline phosphatase activity in bone cells'4 and leads to further increases in phosphorus, could only add to the 7.5 BGJ ion product, it is not surprising that, driven by high phosphorus, ectopic mineralization occurred in the rat calvarial cell In the BBCC system, we d o not supplement the 0.9 mM phosphorus level in DMEM because trials with higher levels of phosphorus failed to produce fully mineralized sheets of bone. In vivo, the level of inorganic phosphorus that bathes mineralizing cartilage is about 2 rnMl2''; however, full mineralization of isolated matrix vesicles from cartilage cells grown in culture requires organically bound phosphate (ATP or AMP).(22)Conceding that the level of organically bound phosphorus bathing cells in vivo is extremely low and that 0-glycerol phosphate is not a normal cell constituent, we nonetheless include 0-glycerol phosphate in our BBCC mineralization medium. Increasing phosphorus naturally via the biologic action of alkaline phosphatase on @-glycerol phosphate seems to us preferable to risking spontaneous precipitation by adding large amounts of phosphorus to the medium. Our BBCC studies have also confirmed rat"8' and chickl6 ') bone cell culture studies showing that mineralization can occur without the addition of 0-glycerol phosphate. Mineralization in the absence of 0-glycerol phosphate occurs most readily in cultures of cells isolated from the bones of calves of fetal age 7-8 months. DMEM containing 2.4 mM calcium, as well as ascorbic acid (50 pg/ml but not higher) and ITS ( 1 x M), produces a complete sheet of mineral on the surface of 35 mm dishes in 10 days; however, the bone appears to be less dense than that of cultures stimulated with 10 mM 0-glycerol phosphate. Neither ectopic mineralization nor other toxicity has been observed with the BBCC regimen; nonetheless, further testing of the stimulatory effects of increased levels of both calcium, which is underway, and of phosphorus as well, is warranted. The high blood calcium level reported for fetal lambs is startling, considering the high alkaline phosphatase levels present; however, the investigators did not measure organic phosphorus levels. ( I 7 ) Clearly, the results of that study indicate the need to compare fetal circulatory levels of both calcium and phosphorus in each species used for calvarial or isolated bone cell culture studies. The continuing enigma of how high fetal calcium and phosphorus levels relate to alkaline phosphatase levels, particularly the peak levels of alkaline phosphatase reported in most studies at the outset of mineralization, remains to be clarified. We speculate that homogeneous bovine cell populations isolated by overnight enzyme digestion of bone slices obtained from mature fetuses have great potential to answer many of these questions because pglycerol phosphate is not required to stimulate mineralization.

Alkaline phosphatase When stimulated to produce mineralized bone matrix, the cultured bone cells from bovine fetuses of all ages show increased alkaline phosphatase activity and cell proliferation. The number of cells peaks at approximately day

FIG. 10. Electron micrographs of undemineralized thin cross-sections taken of cultures on day 12 of maintenance on mineralization medium (8 x lo-’ M ITS; see Fig. 5 ) : (a) surface cells ( x 25,000); (b) buried osteocytes ( x 9900).

739

MINERALIZATION OF BONE IN VITRO

The expression of alkaline phosphatase and the production of mineralized matrix by cells obtained from young bovine fetuses compare best to data reported for cultured fetal rat cell^,{^.^) whereas values for the midterm fetuses compare more favorably to those of embryonic chick bone c ~ l t u r e s . ‘The ~ . ~ pattern ~ of alkaline phosphatase activity and the onset of mineralization in the BBCC system appear to be closely linked in time, but we are unable to determine whether they occur concurrently, as reported for the chick.l71

Histology and RNA expression 0

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6

8

10

12

14

TIME ( d a y s )

FIG. 11. Mineral accumulated on the surface of the coverslips over time. Each bar represents the daily average of calcium in three coverslip cultures of cells isolated and grown from a 3 h digest of a young fetus. Compared with day 2 of mineralization, there is a significant increase in mineral accumulation at day 8 (p 5 0.05) and even more significant increases by days 10, 12, and 14 (p 5 0.01).

6 and decreases by day 10 following the addition of mineralization factors to the culture medium, as shown in Fig. 8, which illustrates the approximate 200-fold increase in alkaline phosphatase level of cells obtained from younger fetuses. In contrast, alkaline phosphatase levels in cells from midterm or older fetuses increase only 20- to 100-fold (data not shown). However, the mineralization of matrix by bone cells from the young fetuses is generally not as uniform as the mineralization by cells from more mature fetuses. When the BBCC contains a complete sheet of mineral, alkaline phosphatase activity declines slowly, concomitant with a decrease in the total number of nonmineralized bone cells on the surface of the culture (Fig. 8). Our interpretation of the decrease in cell number is that cell division is slowing, that the multilayer is not being maintained (compare Fig. 4 with Fig. 6), and that alkaline phosphatase activity, being primarily associated with the partially differentiated osteoblast, is decreasing because there are fewer preosteoblasts. The difference in alkaline phosphatase activity and mineralization in BBCC derived from fetuses of different ages may be attributed t o the fact that the celk isolated from older fetuses are functionally more mature and homogeneous. The isolation of bone cells from mature fetuses is more difficult, requiring overnight enzyme digestion, and the cells obtained proliferate somewhat more slowly than those from younger fetuses. When the homogeneous cell cultures from more mature fetuses are stimulated to synthesize and mineralize bone matrix, however, the result is a slightly more rapid onset of mineralization (2 days versus 4-6 days for those from young fetuses). Despite slight differences in the time of onset of mineralization in this BBCC system, the accumulation of mineral during the first 2 weeks of bone formation follows a similar pattern in all cultures, no matter what the age of the fetus (Fig. 11).

Our companion study of RNA induction in mineralizing BBCC revealed simultaneous peaks in mRNA expression for alkaline phosphatase, type I collagen, osteonectin, osteopontin, and biglycan on day 6 after the introduction of mineralization medium (see Ibaraki et al., this volume), in striking contrast to the sequential predominance of first type I collagen, then alkaline phosphatase, followed by osteopontin, and then osteocalcin over the extended period (at least 30 days) required for rat bone cell cultures to minerali~e.(’.~’ Figure 4, a BBCC cross-section obtained after 7 days’ culture in mineralization medium, demonstrates the histology typical of a rapidly growing bone in vivo, confirming the difference between mRNA expression in rat and bovine bone cell cultures. Rather than occurring sequentially, as reported for bone formation in rat cells,‘*) BBCC matrix synthesis and mineralization appear to proceed simultaneously. The surface layer of bovine bone cells, which is more than one cell thick and lies on top of the nonmineralized bone matrix (osteoid), continues to proliferate. Although full sheets of mineralized bovine bone matrix are usually formed within 10 days of transferring the cultures to mineralization medium (Fig. Ib), mineral continues to accumulate beyond day 10 (Figs. Ic, 2, and 11). As described in the companion paper, mRNA levels are high for bone sialoprotein, osteopontin, and decorin as late as day 20 on mineralization medium, suggesting these matrix proteins’ association with fully differentiated osteoblasts. The relatively high bone sialoprotein and mRNA expression at day 20 in BBCC is consistent with the report of sialoprotein secretion by rat osteoblasts during nodule mineralization. ( 2 9 )

Growth factors and mineralization The differences observed in the thickness, cellularity, and mineralization of bone produced when BBCC are treated with higher concentrations of insulin (Figs. 5 and 6) could be attributed to an insulin-like growth factor (IGF) effect when insulin is above physiologic levels. I 3 O 1 Our data suggest that there may be an important relationship between growth factor titers and matrix synthesis and mineralization. We have observed that when bone cells are grown on coverslips and the culture medium (without calcium supplementation) is changed only once every 24 h, matrix synthesis continues unimpeded; however, mineralization is

WHITSON ET AL.

740

The unique elements of the BBCC system are being apslowed and generally occurs on only a portion of the culture surface. In lieu of mineralization of the existing ma- plied in our laboratory to studies involving nonembryonic trix, there is an explosive proliferation of cells beyond the bone cells from other mammalian species, including huedge of the coverslip onto the dish. The new cell growth is man. The BBCC thus serves as a standard in the search for rapid, covering the entire 60 mm culture dish by day 10 on ways to induce bone cells isolated from other species to mineralization medium. If mineralization medium is then mimic the phenotypic expression of the mature fetal boprovided twice daily for an additional 10 days, the out- vine cell. The high level of mineralization achieved with growth of cells on the dish produces a thin matrix and be- this BBCC system in a relatively short culture time also gins to mineralize much of it, whereas only about 50% of lends itself to the development of an improved bone rethe original matrix on the coverslip ever mineralizes sorption assay. (probably due in part to a time-dependent linkage between ACKNOWLEDGMENTS osteoid synthesis and mineralization). Conversely, when, under optimal conditions, the medium is changed twice This work was supported by Southern Illinois School of daily from the outset, mineralization begins within 2-6 days and progresses rapidly, forming a solid sheet that en- Dental Medicine, by the Graduate School, Southern Illicompasses the coverslip and the limited outgrowth of cells nois University at Edwardsville, and by the Naval Medical Research and Development Command, Work Unit 61 152N from it. An explanation of these phenomena is that growth fac- MROOOOl.001-1366. The opinions and assertions contained tors such as IGF, fibroblastic growth factor (FGF), and here are those of the writers and are not to be construed as transforming growth factor (TGF-P), all of which are official at large. The authors thank Sharon Gilmore, HMI known to be synthesized by bone cells or their precur- Rolando Estrada, HM3 Mark Palka, HM3 Annette Wright, Robert Ide, and Sandra Sawyer for their technical SOTS, (31-34) are sequestered in mineralizing matrix, effectively slowing the proliferation of bone cells on the cover- assistance and Donna Young for typing the manuscript. slip. The incorporation of FGF and IGF into mineralized This work was presented in part at meetings of the Ameribone m a t ~ i x ‘ ~ ’suggests . ~ ~ ) that once matrix synthesis is ini- can Society for Bone and Mineral Research. The abstracts tiated, the rate and perhaps the degree of mineralization were published as follows: Calcified Tissue International may partially control cell proliferation. In a current appli- 37(suppI l):A88, 1985; Journal of Bone and Mineral Recation of the BBCC system, cells at different stages of cul- search 5(suppl 2):S217, 1990; and Journal of Bone and ture are being probed for their relative expression of these Mineral Research 6(suppl 1):S255, 1991. and other growth factors.

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CONCLUSION Compared with the culture of bone cells from other species (chick and rat), the BBCC system offers a much wider window of opportunity for investigation of the complex interrelationships of the factors involved in bone growth and mineralization, that is, bone formation. The development of the bovine skeleton and skeletal support structures clearly exceeds that of the fetal or neonatal rat: Its strength is sufficient that the newborn calf can stand alone at birth. Furthermore, bone maturation during the 9 month bovine gestation period may parallel that of the human, albeit superseding it during the last months. Thus the relative speed and ease with which bovine bone cells synthesize and mineralize bone matrix may reflect the cells’ maturity and homogeneity . The use of the bovine cell culture system provides the opportunity to study in one species the events and multiple factors that control the development of bone and the maturation of the skeleton along a continuum of fetal age. The system’s potential comprises a range of applications, from demonstrating the effects of growth factors, hormones, and nutrition in normal bone to isolating and determining environmental and hereditary factors in disease states, and, ultimately, to pharmacologic or environmental manipulation of growth and maturation. Underlying all these applications is the study of the possible plasticity in phenotypic expression of the preosteoblast and osteoblast and the relationship of this expression to the aging of bone cells.

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Address reprint requests to: S. William Whitson, Ph. D. Department of Biomedical Sciences Southern Illinois University School of Dental Medicine 2800 College Avenue Alton, IL 62002

Received for publication February 14, 1991; in revised form January 27, 1992; accepted January 28, 1992.