Proliferating cells in the primary spongiosa express osteoblastic phenotype in vitro

Bone Vol. 20, No. 2 February 1997:93-100 ELSEVIER

Proliferating Cells in the Primary Spongiosa Express Osteoblastic Phenotype In Vitro J. E. O N Y I A , 1'2 B. M I L L E R , 2 J. H U L M A N , 1 J. L I A N G , 1 R. G A L V I N , l C. F R O L I K , 1 S. C H A N D R A S E K H A R , l A. K. H A R V E Y , 1 J. B I D W E L L , 2 J. H E R R I N G , l and J. M. H O C K 1'2 J Endocrine Division, Lilly Research Labs and 2 Indiana University School of Dentistry, Indianapolis, IN, USA

these in vitro osteoblast-like cultures poorly model the properties that characterize osteoblasts, such as high alkaline phosphatase activity, production of cyclic AMP in response to PTH, production of type I collagen, and in vitro mineralization. 22 One major discrepancy is that, while cells in these in vitro models have been obtained from bone, their precise origin and location within bone in vivo is not known. Multipotential cells capable of differentiating into osteoblasts, fibroblasts, chondrocytes, adipocytes or muscle cells, can be identified in marrow-cell cultures as adherent colonies. 4'35'36'38 These cultures have provided the basis on which osteoblast differentiation and function have been modeled.41,42 It has not been established if any of these cell types are the target osteoprogenitor or source of osteoblasts in vivo. Direct evidence to demonstrate that these cells are osteoprogenitors in vivo is needed. We, and others, have shown that, in young rats, the proliferating osteoprogenitor cells in long bone and vertebrae are located in the metaphyseal primary spongiosa, in a 1-2 mm band subjacent to the growth plate. 22"23"25'47 These proliferating primary spongiosa (PPS) cells respond to PTH by differentiating to mature osteoblasts. 22'23 Because the primary spongiosa cells are sensitive to PTH in vivo and since PTH is an anabolic agent in vivo, PPS cells may be a useful source of osteoprogenitor cells to investigate the function and regulation of osteoblasts in vitro. In the present study, we describe a procedure to isolate and culture PPS cells from the distal femur in young growing rats. BrdUrd was incorporated in vivo, as a marker to identify these PPS cells for in vitro studies and characterization. In vitro, the cultured cells proliferated rapidly and expressed the osteoblast phenotype, but differed in several respects from other in vitro osteoblast differentiation models. 9-11'17'27'28'4° The cells aggregated within hours of isolation, did not require dexamethasone (dex) or 13-glycerol phosphate to express the osteoblast phenotype and spontaneously formed nodules within 4-7 days of isolation. Since these cells recapitulated osteoblast differentiation in an accelerated fashion, it may serve as a useful model to study the regulation of osteoprogenitor differentiation.

We have shown that intermittent parathyroid hormone (PTH) treatment targets proliferating cells in the primary spongiosa of trabecular bone of young rats, resulting in an increased number of osteoblasts. To further characterize these proliferating osteoprogenitor cells, bromodeoxyuridine (BrdUrd) incorporated in vivo, was used as a marker to identify and isolate cells for in vitro studies. Proliferating cells were labeled in vivo in young rats with BrdUrd and 24 h later were isolated by trypsinization of sections of the primary spongiosa of the distal femur metaphysis. Within 12 h of isolation, BrdUrd+ cells formed distinct foci containing 20500 cells with fibroblast morphology. Stimulation of proliferation as determined by [3H]-thymidine incorporation was observed for these cells in response to fetal bovine serum, platelet derived growth factor, and transforming growth factor 13-1. Neither insulin-like growth factor-1 (IGF-1) nor insulin stimulated proliferation PTH (1-34) and dexamethasone inhibited proliferation. The effects of PTH and dexamethasone were additive. Cells expressed the osteoblast phenotype as evidenced by synthesis of type I collagen, expression of high alkaline phosphatase activity, and production of increased intracellular cAMP in response to PTH (1-34). Confluent cell aggregates spontaneously formed mineralized nodules within 4-7 days, in the absence of inducers. These observations suggest that the primary spongiosa cells recapitulates the differentiation process in vitro in an accelerated fashion and may serve as a useful model to study osteoblast differentiation. (Bone 20:93-100; 1997) © 1997 by Elsevier Science Inc.

Key Words: Proliferating cells; Primary spongiosa; Osteoblast; Rat.

Introduction Proliferating osteoprogenitor cells from which mature osteoblasts originate, are believed to be located in specific areas of the bone marrow stroma, t2"18'35'36 A number of approaches have been used to isolate and culture osteoblast-like cells from b o n e . 9'13'17'32"33'4°'46 Aside from being laborious, the efficiency and reproducibility of these methods differ remarkably. Many of

Materials and Methods Materials Animals. Young virus-antibody-free, male Sprague-Dawley rats, 70-100g, were purchased from Harlan Laboratories (Indianapolis, IN) and fed Purina chow [(calcium I%, phosphate 0.61%): PMI Feeds, Inc., St. Louis, MO] and water ad libitum. Animal protocols were approved by the Lilly Animal Care

Address for correspondence and reprints: Dr. J. E. Onyia, Skeletal Disease Research Group, 0403, Endocrine Division, Lilly Research Labs, Indianapolis, IN 46285.

© 1997 by ElsevierScience Inc. All rights reserved.

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8756-3282/97/$17.00 PII $8756-3282(96)00350-X

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and Use Committee. Animals were 3-5 weeks old when used in experiment.

Cells and Cell Culture Isolation of primary spongiosa cells from distal femurs. Rats were injected with BrdUrd (Sigma, St Louis, MO) at 50 mg/kg sc, to label cells in S-phase. After 24 h, rats were euthanized. Femurs were resected, and all exterior connective tissue, including periosteum, completely removed before rinsing the bone in ethanol. The distal epiphysis was removed, and a subjacent 2-3 mm band of the primary spongiosa resected. Bands from two to four femurs were pooled, minced and digested for 1 h at 37°C in 0.25% trypsin (Sigma, St. Louis, MO). After neutralization of trypsin with 1 mg/mL trypsin inhibitor (Sigma, St. Louis, MO), cells were filtered through a 70 txm pore cell strainer (Becton Dickinson and Co., Lincoln Park, N J) and counted. Cells were plated at 1-5 × 10 6 cells/150 cm 2 tissue culture plate in a - M E M (GIBCO BRL, Grand Island, NY), with 20% fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT) and 1% penicillinstreptomycin (GIBCO BRL, Grand Island, NY), and maintained at 37°C in 95% humidity with 5% CO2. The medium was replaced every 3-4 days, with o~-MEM containing 10% FBS and 1% penicillin-streptomycin. Cells used in experiments were either primary cultures or in first passage. Marrow stromal culture. The diaphyseal middle third of the femurs was cut out and the marrow flushed with fresh a-MEM, containing 10% FBS and 1% penicillin-streptomycin, and prepared as single cell suspensions. Cells were counted and viable cells were plated at 1-2 × 107 cells/150 cm 2 tissue culture plate and maintained at 37°C in 95% humidity with 5% CO 2. As described by Owen et al., 35 cells were allowed to adhere in the absence of media changes for 6 days, and then media was changed every 3--4 days. Other cells lines. The human osteosarcoma cell SaOS-2, was maintained in DMEM/Ham's F-12 (3:1) containing 10% fetal bovine serum plus glutamine. The immortalized human stromal cell, F-12, was grown in DMEM containing 10% fetal bovine serum, while the immortalized mouse calverial derived osteoblast-like cell, MC3T3-E-1, was maintained in MEM supplemented with 10% FBS, 10 mmol/L HEPES and 1 mmol/L sodium pyruvate. All cultures were maintained at 37°C in 95% humidity with 5% CO2. Immunocytochemistry of BrdUrd. To confirm that proliferating cells had been isolated, adherent cells were cultured for 1216 h, then fixed in 10% neutral buffered formalin and processed for immunocytochemistry. 43 After three washes with phosphate buffered saline (PBS)/Tween buffer (1 x PBS, 1% Tween 20), the cells were immediately immersed in a graded ethanol series (50%-70%-80%-95%-100%) for 1 min. Endogenous peroxidase activity was quenched by incubating the cells in 1.5% hydrogen peroxide in absolute methanol for 20 min. Cells were then rinsed in PBS/Tween buffer, and nuclear chromatin (DNA) denatured in 4N HC1 for 30 min at room temperature. The cells were covered with blocking buffer [1.52% (vol/vol) normal horse serum in 1 × PBS] for 20 min and then incubated for 1 h at room temperature in mouse anti-BrdUrd monoclonal antibody (Amersbam International, Arlington Heights) diluted 1:23 in PBS. After a 10 min wash in PBS/Tween buffer, detection of primary antibody was accomplished by using a biotinylated secondary antibody [horse antimouse immunogloblin G (IgG)] and the avidin-biotinperoxidase system (Vectastain ABC Kit, Vector Laboratories,

Bone Vol. 20, No. 2 February 1997:93-100 Burlingame, CA). The cells were counter stained briefly with Gill's hematoxylin (10 sec). The negative controls, omitting BrdUrd or the primary antibody, were completely unstained. The percentage of BrdUrd+ cells in the aggregates was calculated as the percent of cells with nuclei stained for BrdUrd divided by the total number of cells in an aggregate.

Bone histomorphometry. These procedures were done using conventional techniques on formalin-fixed, decalcified, waxembedded sections of the distal femur as described. ~°'2j'23'47 After sacrifice, the distal femur were removed and fixed in formalin solution for 3 days. Decalcification was completed in 10% EDTA and tissue were embedded in paraffin, sectioned at 6 Ix and prepared for qualitative immunocytochemistry to identify the location of cells labeled by BrdUrd in S-phase. DNA synthesis. Primary cells were seeded in 96 well plates (12,000 cells per well) in c~-MEM supplemented with 10% FBS. After a 24 h attachment and recovery period the cells were transferred to serum-free medium for 16 h, and then stimulated with FBS or indicated hormone or growth factors for 24 h, in the presence of 10 ixCi/ml [3H]-thymidine (82.7 Ci/mmol; Amersham International, Arlington Heights). After 24 h, the medium was removed and the cell layer was trypsinized (0.25%) for 10 mins, followed by two freeze thaw cycles at -70°C. The harvesting of the plates onto a filter paper and the subsequent five washes of the filter were done on an automated 96 well cell harvester (Tomtec, Orange, CT) in accordance with the manufacturer's instructions. The filters were dried in a 100°C oven for 20 min and subsequently immersed in 10 mL of scintillation fluid in a heat sealed bag. Samples were counted in a Beckman LS 1801 [3 counter and [3H]-thymidine incorporated into DNA expressed as percentage increase over control cells without growth factor addition. The results were calculated as the mean + SEM of five to eight experiments. Alkaline phosphatase activity. Alkaline phosphatase (ALPase) activity was detected by cytochemistry using sigma kit 86-R (Sigma, St. Louis, MO). In confluent cultures, cellassociated (ALPase) activity was determined using the method of Galvin et al. 15 Briefly, the confluent cell layers were washed with Ca2+ and Mg2+-free Hanks balanced salt solution and scraped in lysis buffer (1 mmol/L NaC1, 0.1% Triton X-100, and 0.1% type 1-s trypsin inhibitor). Cells were subjected to three cycles of freezing in a dry ice: ethanol slurry followed by a rapid thawing at 37°C. ALPase activity in an aliquot of the cell lysate was determined using the Sigma Kit 245. Following analysis of the cell-associated ALPase activity, NaOH was added to the remaining cell lysate so that the final concentration was 200 mmol/L and the samples were incubated at 37°C for 18 h. The protein content of the solubilized cell layers was determined using the BCA kit (Pierce Chemical Co., Rockford, IL) and ALPase activity was expressed in enzyme units as ~ m o l e of pnitrophenol phosphate (PNP) cleaved/h/mg protein. Collagen biosynthesis and identification. Confluent monolayers of cells in 24 well plates were labeled with 100 IXCi/mL [3H]-proline (ICN; specific activity, 100 Ci/mmol) for 4 h in serum-free media containing ascorbic acid (50 Ixg/mL). The conditioned media and matrix (extracted with 0.5 mmol/L acetic acid for 48 h at 4°C) were dialyzed against 0.5 mol/L acetic acid, pH 2 and an aliquot from each fraction after dialysis, was digested with 100 Ixg]mL pepsin (Sigma) for 16 h at 4°C. The samples

Bone Vol. 20, No. 2 February 1997:93-100 were dialyzed against water, lyophilized, and resolved on an SDS-polyacrylamide gel (3% stacking gel/3%-15% linear gradient running gel), under reducing conditions. Rat tail tendon collagen and high molecular weight globular protein standards (Bio-Rad Laboratories, Melville, NY) were used for comparison. The gels were stained with Coomassie Blue for visualization of standards, equilibrated with "Entensify," dried onto filter paper and subjected to fluorography. To demonstrate the presence of type III collagen, the media samples were subjected to a delayed reduction protocol, in which samples were initially electrophoresed for 30 rain under nonreducing conditions, followed by in situ reduction of samples in the gel. 44

In vitro mineralization. Mineralized matrix was identified morphologically as a nodule overlying a cell cluster. Mineralization was confirmed by Von Kossa staining. 33"46 Cyclic AMP Production. Confluent primary cells derived from the primary spongiosa, or SOAS-2 cells, were plated at 1 x 105/ well in a 24 well plate, cultured for 8 h, serum starved overnight and then treated with hPTH 1-34 (Bachem, Torrence, CA) at 0-10 nmol/L in DMEM/F-12 (3:1) containing 20 mmol/L HEPES, 0.5% BSA and 0.2 mmol/L 3-isobutyl-l-methylxanthine [IBMX; (Sigma, St. Louis, MO)] for 60 min. Cells were rinsed, extracted with cold ethanol and then assayed for adenylate cyclase activity by measuring c A M P using a radioimmunoassay (Amersham, Arlington Heights, IL). Note the PTH incubation for 60 min was chosen for convenience. Statistical analysis. Results were analyzed by the Student's t-test or one-way analysis of variance.

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Results In Situ Location of BrdUrd+ Cells in the Primary Spongiosa of Distal Femur of Young Rats We, and others, have demonstrated that, in the metaphysis of growing long bone and vertebrae, the proliferating "osteoprogenitor" cells that provide a source of osteoblasts are found within the primary s p o n g i o s a subjacent to the growth plate. 22'23'25'47 To label proliferating cells in the primary spongiosa in S-phase, male Sprague-Dawley 3-5 week old rats were injected with BrdUrd, an analog of thymidine, for 24 h and BrdUrd immunocytochemistry was done on formalin-fixed, decalcified, wax-embedded sections of distal femur. As shown in Figure 1, the proliferating cells (BrdUrd+) that give rise to osteoblasts 22"23"25'47 are located primarily within the primary spongiosa, in a band immediately subjacent to the growth plate. In subsequent studies we used the BrdUrd incorporated in vivo into PPS, as a marker to identify PPS cells for in vitro studies and characterization.

BrdUrd+ Cells from the Primary Spongiosa Aggregate within 12 h in Culture PPS cells were cultured from the distal femur of young rats, given BrdUrd in vivo as described in Materials and Methods. Twelve hours after the initiation of the cultures, the adherent cells formed distinct foci or aggregates containing 20-500 mononuclear cells with fibroblastic morphology (Figure 2A). The nonadherent cell population was removed by replacing the medium with fresh medium. The adherent cells in these aggregates consisted of less than 1% of the total number of cells plated (data

A

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Growth~Plate

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Pdmary Spongloaa

Primary Spongiosa

Figure 1. Photomicrograph of formalin-fixed, decalcified, wax-embedded sagittal section of distal femur showing regional distribution of BrdUrd+ cells (cells with brown nuclei) within the marrow of the primary spongiosa subjacent to the growth plate of rats killed 24 h after vehicle (A) or BrdUrd injection in vivo (B). The femurs were processed for immunocytochemistry of BrdUrd. Original magnification x275. Note growth plate and primary spongiosa. The arrows show BrdUrd+ cells. The negative controls, omitting BrdUrd, were completely unstained.

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~v

Figure 2. (A) Phase contrast photomicrograph showing aggregation of cells from the primary spongiosa of a distal femur 12 h after isolation. Original magnification ×40. (B) Photomicrograph of formalin-fixed cells stained for BrdUrd. BrdUrd was given 24 h prior to euthanasia. Original magnification x40. The nuclei of 70%-85% cells within an aggregate were BrdUrd+ (u). The small round cells (h) are mononuclear hematopoetic (marrow) cells that disappear when medium was changed. (C) Phase contrast microscopy showing confluent culture of cells derived from the primary spongiosa after 5-6 days. Original magnification x40.

not shown). BrdUrd immunocytochemistry showed that these cell aggregates were BrdUrd+ as demonstrated by the dark brown stained nuclei in 7 0 % - 8 5 % of the cells in the aggregate (Figure 2B). The PPS cells proliferated and reached confluency within 4 - 7 days (Figure 2C). In the confluent cultures, the cells maintained a fibroblastic morphology.

(131%). The peak in DNA synthesis for all the factors occurred within 24 h and thereafter declined gradually over 72 h examined (data not shown). Neither IGF-1 at 10 ng/mL nor insulin at 60 ng/mL stimulated proliferation. In contrast, PTH at 10 nmol/L and dex at 100 nmol/L inhibited proliferation to 73% and 62% of control values (Figure 3B). The effects of PTH and dex were additive.

Primary Spongiosa Cells Respond to Serum, PDGF and TGFf3 by Increased DNA Synthesis and to PTH and Dex by Decreased DNA Synthesis

Primary Spongiosa Cells Show Markedly Higher Alkaline Phosphatase Activity Than Other Bone-Derived Cells

To further characterize these cells, the proliferative capacity of quiescent (serum deprived) PPS cells was evaluated in response to stimulation by selected growth factors. As illustrated by [3H]thymidine incorporation studies (Figure 3A), the cells proliferated rapidly in serum-free cultures containing either 10% FBS (645%) or PDGF at 10 ng/mL (206%). TGF[3-1 at 5 ng/mL, a concentration that has been shown to be mitogenic for osteoblastlike cells ~'29 had a small but significant effect on DNA synthesis

Cytochemical and biochemical analysis of PPS cells showed a high alkaline phosphatase activity (Figures 4 and 5). All cell aggregates formed within 12 h after the initiation of primary culture were cytochemically positive for alkaline phosphatase (Figure 4A). In confluent primary cultures, more than 85% of the cells were cytochemically positive for alkaline phosphatase (Figure 4B). The enzymatic activity in confluent cultures was found to be as high as 285 units/mg protein in the cell layer (Figure 5).

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SaOS - 2 with PTH (1-34) caused dose dependent accumulation of intracellular cAMP. The optimal increase in cAMP occurred at doses ranging from 1 to 10 nmol/L PTH in the SaOS-2 cells. At doses ranging from 1 to 10 nmol/L PTH, a significant accumulation of cAMP (>50 fold higher than that exhibited by control cultures) was detected in the PPS cells. However, the increase in cAMP production at 10 nm PTH was variable. These results indicate a phenotypic osteoblast response to PTH.

7

0 0

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Figure 3. Comparative effect of serum and serum growth factors on DNA synthesis in quiescent cultures of cells derived from the primary spongiosa, Cells were seeded in 96 well plates (12,000 cells per well) in alpha-MEM supplemented with 10% FBS. After a 24 h attachment and recovery period the cells were switched to serum-free medium for 16 h and then stimulated with FBS or indicated hormone for 24 h; 3[H]thymidine was added at 2 uCi per well (10 uCi/mL). DNA synthesis was evaluated by 3[H]-thymidine incorporation into DNA after 24 h and expressed as percentage increase over control cells without growth factor addition. (A) Primary Spongiosa cells respond to serum, PDGF and TGFI3 by increased DNA synthesis and (B) to PTH and dex by decreased DNA synthesis. The results represent the mean + SEM of five to eight experiments. Similar alkaline phosphatase activity persisted in passaged cultures [up to 14 passages tested so far; (data not shown)]. Cells from the primary spongiosa showed a sevenfold higher alkaline phosphatase activity than marrow stromal cells obtained from the same animal, and approximately 80-fold higher activity than either F12 or MC3T3-E1 osteoblastic cells.

Primary Spongiosa Cells Synthesized Collagen 1 Since type I collagen represents the major phenotypic marker for osteoblasts,l 1.32,33,4o.46 we analyzed the newly synthesized collagens of the PPS cells. Cells were labeled with [3H]-proline in serum-free media containing ascorbic acid for 4 h and the proteins released into the conditioned media and matrix (acid soluble) were analyzed by SDS-gel/Fluorography. A majority of the collagen synthesized was present in unprocessed form (Figure 6, lanes 1 and 3). The collagenous nature was further established by pepsin digestion that resulted in the appearance of two pepsin-resistant bands, that comigrated with authentic rat tail tendon collagen (Figure 6, lanes 2 and 4). Further evaluation of the pepsinized media by delayed reduction analysis revealed that it was primarily type I collagen with little detectable levels of type III collagen (data not shown).

Rapid and Spontaneous Formation of Mineralized Nodules in Primary Spongiosa Derived Cell Cultures Confluent cell aggregates of the PPS cells spontaneously formed nodules within 4-7 days in the absence of inducers of bone cell mineralization, [3-glycerol phosphate and dex (Figure 7A). After a few days of initial growth, cells in the center of aggregates appear condensed followed by the appearance of small nodules in several places in the cell culture dish. These nodules grew rapidly in size and showed strong Von Kossa staining demonstrating that these nodules are accumulating calcium phosphate (Figure 7B).

hPTH 1-34 Induces cAMP in Primary Spongiosa Cells

Figure 8 shows the dose-response effect of PTH on cAMP accumulation. Treatment of PPS cells and human osteosarcoma cell

Discussion Bone formation is a complex process dependent on the proliferation of osteoprogenitor cells, the differentiation of preosteoblasts into mature osteoblasts, and the functional activity of differentiated osteoblasts. 37 At the tissue level, the rate of bone formation is more dependent on the number of osteoblasts than on their activity. 31'39 The cellular mechanisms involved in regulating osteoblast number remain largely unknown. We and others have previously shown that, in young growing rats, the proliferating osteoprogenitor cells that provide a major source of osteoblasts in vivo are localized in the primary metaphyseal spongiosa. 22'23"25'47 In the present study, we have isolated and characterized the PPS cells in vitro. BrdUrd was used to label and localize the PPS cells in situ within the marrow of the primary spongiosa, in a region immediately subadjacent to the growth plate. The BrdUrd+ PPS cells were isolated from the primary spongiosa by trypsin digestion. In culture, the adherent cell population was positive for BrdUrd suggesting they represent the in vivo BrdUrd+ PPS. Cell cycle kinetics of bone cells in the metaphysis of growing long bone in vivo and in vitro has been previously investigated with [3H]thymidine. 24'25'47 Incorporation of the nucleoside analog, (precursor of DNA) is restricted to cells undergoing DNA synthesis. Consequently, the fate of labeled cells may be traced by examination of specimens (with immunochemical detection) at intervals after BrdUrd injection. This obviates the need for timeconsuming autoradiography and other problems inherent in using radioactive thymidine. 24"25 Thus, cells isolated 24 h post BrdUrd injection represented the in vivo population of cycling osteoprogenitor cells. On initiation of culture, adherent cells formed distinct foci or aggregates with fibroblastic morphology. These aggregates were not mere cell clumps, as they arose after trypsin digestion yielded single cells. The cell suspension after trypsin had also been filtered through a 70 pum cell strainer to further ensure generation of a single cell suspension. Interestingly, while the number and size of aggregates varied over a number of experiments, the percentage of cells recovered as aggregates remained uniform over a number of experiments (data not shown). These findings suggest that these aggregates arise as a result of specific cellular event(s). We speculate that this may involve adhesion molecules and/or may arise due to chemotactic attraction. Similar cellular aggregates of stromal cells and hematopoietic cells have been demonstrated in marrow cell suspension in vitro, and these are thought to reflect normal cell associations in the marrow environment in vivo. 14 On the bone surfaces in vivo, osteoblasts never appear or function individually, but are always found in clusters of cells, 37 The PPS cell clusters may represent normal osteoblast characteristics and function. Another unique feature of the PPS cells is that, when cultured in 10% FBS, they proliferated rapidly and attained confluence in a few days. Other osteoblast-like bone derived primary cells require media supplements and weeks of culture. 13"36 Previous studies have suggested a stimulatory effect by serum, ~'t3

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Figure 4. Photomicrographs show intense histochemical staining for alkaline phosphatase activity in cells derived from the primary spongiosa. (A) Cell aggregate 12 h after isolation. The arrows show alkaline phosphatase positive + cells in the cell aggregate. Original magnification x40. (B) Confluent cells. Original magnification x40. Positive stain was not dependent on cell con fluency.

PDGF, ~8 TGF [3s,1'29 iGFs,5,19 and insulin, 26 and an inhibitory effect of PTH 16 and d e x 9'27 o n DNA synthesis in osteoblast-like bone derived cells. As assessed by thymidine incorporation, the PPS cells proliferated rapidly in serum-free cultures containing either FBS or PDGF. TGF[3-1, at a concentration that has been shown to be mitogenic in other bone cell cultures,l"29 had a small but significant effect on DNA synthesis. Neither IGF-1 at 10 ng/mL, nor insulin at 60 ng/mL, stimulated proliferation. A combination of PDGF, TGF-131, IGF-1, and insulin did not equal the magnitude of induction by serum (data not shown), indicating that other factors present in serum are required for optimal stimulation of proliferation. In contrast, PTH (10 nmol/L) and dex (100 nmol/L) inhibited proliferation. Antiproliferative effects of PTH have been shown in several osteoblast-like cell lines in culture. 16 Similarly, we recently demonstrated that inhibition of proliferation in cells of the primary spongiosa in vivo may be one of the early events in PTH-induced cell differentiation to mature osteoblast. 34

The extensive proliferative capacity was accompanied with expression of specific osteoblastic differentiation markers. PPS cells in culture exhibited high levels of alkaline phosphatase activity. In vivo, alkaline phosphatase is expressed at all stages of osteoblast differentiation39 and is an accepted characteristic of osteoblast phenotype. Compared to bone marrow derived osteogenic stromal cells from the same animal and to other immor-

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Figure 5. Alkaline phosphatase activity in confluent primary cells from the primary spongiosa compared to that of other bone derived stromal and osteoblastic cells. Marrow stromal cells: diaphyseal marrow stromal cells obtained from the same animal as the primary spongiosa cells; F 12: immortalized human bone marrow stromal cells (F12) and MC3T3-E1 osteoblastic cells were grown to confluency in T75 cm2 flasks and activity determined as described in Materials and Methods. Significant difference from F12 cells, ap < 0.05. The results represent the mean -+ SEM of three replicate experiments.

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Figure 6. SDS polyacrylamide gel electrophoresis of 3H-proline pulse labeled proteins synthesized by primary spongiosa derived cells cultured for 7 days. Confluent primary cultures of cells derived from the primary spongiosa were pulse labeled for 4 h with 3H-proline as described in experimental procedure. Both pepsin (+) and nonpepsin-digested (-) proteins from the cell layer and media were extracted and samples were lyophilized and analyzed by SDS-PAGE (3% stacking gel / 3%-15% linear gradient running gel) under reducing conditions. High molecular weight protein standards from Bio-rad and 14C-labeled rat tail tendon type I collagen were included for comparison of electrophoretic mobility. The bands designated al (I) and ct2(I) represent collagen type I, al and ~x7 chains.

Bone Vol. 20, No. 2 February 1997:93-100

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Figure 7. Primary cells derived from primary spongiosa were cultured for 4-7 days in the absence of inducers of mineralization. (A) Phase photomicrograph of nodule overlying a cell cluster. Original magnification ×40. (B) Photomicrograph of mineralized nodule stained by the Von Kossa procedure. Original magnification xl00. talized bone derived stromal cells, (F12) and MC3T3-E1 osteoblastic cells, PPS cells produced significantly larger amounts of alkaline phosphatase. The high alkaline phosphatase activity suggested that PPS cells are committed to the osteoblast cell lineage. The predominant synthesis of type I collagen by the PPS cells in culture also indicated the cells are committed to the osteoblast cell lineage. Previous studies have shown that osteoblast-like c e l l s in c u l t u r e p r o d u c e p r e d o m i n a n t l y t y p e I c o l l a gen. 11,32.33.4o,46 The PPS cells elaborated a collagen I rich matrix, which was capable of mineralizing in vitro, as evidenced by Von Kossa staining of the nodule. Also as demonstrated by molecular analysis, the PPS cells express other osteoblast lineage specific markers such as osteocalcin and osteopontin. 2'3° The PPS cells differ from most of the other bone derived osteoblastic cells, in that in vitro mineralization occurred in the absence of an inducer of bone cell mineralization, such as [3-glycerol phosphate and dex. Another unique feature of these cells was that their

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hPTH(1-34) (nM)

Figure 8. Serum-starved primary spongiosa derived cells, cultured for 24 h, showed a dose dependent increase in intracellular cAMP when treated with hPTH 1-34 at 0.01-10 nmol/L. Cells (PPS cells and human osteosarcoma cell SaOS-2) were incubated for 60 min in the presence of indicated concentrations of human PTH (1-34) and adenylate cyclase activity determined as described in Materials and Methods. Significant difference from control, ap < 0.05.

nodule formation occurred very rapidly. This is similar to the temporal pattern of rapid bone formation that occurs in vivo after surgical marrow ablation 3"43 and is indicative of the strong osteogenic potential of these cells. PTH is known to act on osteoblastic cells at nanomolar concentrations. The physiological actions of PTH are multiple, and may be mediated, in part, via cAMP production. The PPS cells responded to PTH with an increase in intracellular cAMP accumulation. The effect of hPTH (1-34) was dose dependent and occurred at doses ranging from 1 to 10 nmol/L PTH, indicating a normal response to PTH. The increase in cAMP occurred at a high concentration of PTH when compared to osteoblastic SaOS cells (present study) and UMR 106 (Onyia, J.E. et al., unpublished data). The correlation between the magnitude of cAMP produced and PTH actions remains to be determined. The PPS have well established osteoblast-like features, as demonstrated in the present study in vitro and previously in v i v o . 22'23 Relatively little is known about the number of steps leading from mesenchymal stem cell through committed osteoprogenitor to fully differentiated osteoblast, nor is it clear how many discrete stages can he identified in the osteoblast differentiation pathway, or how cells in each stage differ. The mitotic capability of the PPS in vivo and in vitro would suggest that they represent an osteoblast progenitor or preosteoblast. Cell kinetic studies in vivo using BrdUrd, demonstrated the PPS differentiated into osteoblast and consequently contributed to over 50% of BrdUrd+ osteocytes, corroborating that these cells are osteoblast progenitors. 22'23 In summary, we have demonstrated that the proliferating cells of the primary spongiosa from which osteoblasts are derived in vivo, provide a readily available and reproducible source of osteoblasts. In vitro, these cells retain the ability to concurrently proliferate and express the osteoblast phenotype, but differed remarkably from other in vitro osteoblast differentiation models in their constitutive and immediate high alkaline phosphatase activity upon isolation, and the rapid and spontaneous formation of mineralized matrix. We suggest that the use of osteoblastic cells of the primary spongiosa will be an important cell culture model for studying mechanisms involved in the regulation of trabecular bone in normal and pathologic conditions. Acknowledgments: This work was supported in part by Eli Lilly & Co. and in part by USPHS DE07272 Grant award to Dr. J. M. Hock.

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Date Received: June 21, 1996 Date Revised: October 2, 1996 Date Accepted: October 30, 1996