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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The theory that bisphosphonates inhibit osteoclast formation through their effects on osteoblastic cells remains controversial. To confirm the inhibitory effect of bisphosphonates on osteoclast formation and gain some insights into the underlying mechanisms, we examined the effect of disodium dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175) on osteoclast-like multinucleated cell (OCL) formation in various mouse coculture systems. When different origins of osteoclast precursors (bone marrow, spleen, or nonspecific esterase-positive cells) were cocultured with the same supporting cells (calvarial osteoblasts), YM175 inhibited OCL formation similarly in all cultures. When the same osteoclast precursors (spleen cells) were cocultured with supporting cells of different origin, the results were variable. YM175 inhibited OCL formation almost completely in cocultures with calvarial osteoblasts or osteoblastic cell line KS4, while it did not, or only slightly, inhibit OCL formation in cocultures with stromal cell lines, ST2 or MC3T3-G2/PA6. Temporal addition of YM175 in cocultures of spleen cells with osteoblastic cells revealed that YM175 was effective when it was present at an early phase of the culture period. Consistent with this observation, YM175 in the presence of osteoblastic cells inhibited proliferation of preosteoclastic cells, but did not inhibit the fusion of mononuclear prefusion osteoclasts. In conclusion, the inhibitory effect of YM175 on OCL formation was confirmed in various murine coculture systems, but the effect was dependent on the types of bone-derived cells supporting osteoclastogenesis. The findings suggest that YM175 inhibits osteoclastogenesis by inhibiting the proliferation of osteoclast precursors through its action on supporting cells of osteoblast lineage rather than acting directly on osteoclast precursors.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

BISPHOSPHONATES ARE pyrophosphate analogs that bind strongly to the bone mineral and inhibit bone resorption.1,2 It is shown that bisphosphonates reduce osteoclastic bone resorption by inhibiting the activity of existing osteoclasts. A major mechanism that has been proposed in this action is that the osteoclast is inactivated directly by taking up bisphosphonates from bone surface where the compounds deposit.2–4 Another possible action of bisphosphonates is the reduction of the osteoclast number, which is determined by the balance between formation and the disappearance of osteoclasts. Concerning the latter, it is suggested that bisphosphonates affect the survival of osteoclasts by inducing apoptosis.5 It is less clear whether and how bisphosphonates affect osteoclast formation.2,4,6–10

Osteoclasts are multinucleated cells (MNCs) formed by the fusion of mononuclear precursors of hematopoietic origin.11 To study osteoclast formation in vitro, bone marrow cultures and various mixed culture systems have been developed.11,12 In these culture systems, osteoclast-like multinucleated cells (OCLs), which have many characteristics of authentic osteoclasts, form in the presence of osteoclast-inducing factors such as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and parathyroid hormone. It is shown that, in addition to soluble factors, osteoblastic/stromal cells are indispensable in the osteoclastogenesis in vitro,11–13 similar to that in vivo.

Using cocultures of mouse bone marrow cells with calvarial osteoblastic cells, we previously reported that disodium dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175) and other bisphosphonates inhibited OCL formation induced by 1,25(OH)2D3.10 Similar findings were reported by other investigators using different culture systems.6,8 While other studies showed no inhibition of OCL formation by bisphosphonates.7,9 Several explanations are possible for the conflicting results among studies. The results may vary depending on the type and dose of bisphosphonates used. It is generally believed that the biological effects and the mechanism of action would not be the same for all bisphosphonates although they share common physicochemical properties.1,2 Of other experimental conditions, influence of species difference is shown in bone marrow cultures of human, mouse, and rat.11 The cell types used might be another contributing factor to the conflicting results among studies since it is not well understood which cell types involved in osteoclastogenesis are affected by bisphosphonates.

The OCL formation in vitro is a complex process including proliferation and differentiation of precursors to mononuclear preosteoclasts and subsequent fusion into OCLs. Previous studies have shown that some osteotropic factors act selectively on a certain phase of OCL formation, while others act rather continuously.11 Concerning bisphosphonates, it is suggested that they inhibit proliferation of osteoclast precursors as well as the fusion of preosteoclasts.6,8 To our knowledge, however, no studies have systematically examined in cell cultures whether bisphosphonates inhibit any or all of the process of OCL formation.

In an effort to better understand the cellular mechanisms and the sequence of events involved in the inhibition of OCL formation by bisphosphonates, we conducted the present study with two aims. First, to elucidate the influence of experimental conditions, particularly of cellular components on the results, we have examined the effects of YM175 on OCL formation in various murine cocultures using three different sources of osteoclast precursors and four different sources of osteoblastic/stromal cells. Second, we have investigated if there is a crucial stage of osteoclast formation at which YM175 exerts its inhibitory effect most efficiently.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Materials

YM175 was kindly provided by Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan), recombinant human macrophage colony-stimulating factor (M-CSF) by Morinaga Milk Industry Co., Ltd. (Tokyo, Japan), and echistatin by Merck & Co., Inc. (Rahway, NJ, U.S.A.). Collagenase and dexamethasone were purchased from Wako Pure Chemical Co. (Osaka, Japan); 1,25(OH)2D3 from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, U.S.A.); dispase from Godo Shusei Co. (Tokyo, Japan); reagents for nonspecific esterase (NSE) staining and for DNA measurements from Sigma Chemical Co. (St. Louis, MO, U.S.A.); fetal calf serum from CSL Ltd. (Victoria, Australia); α-MEM from Flow Laboratories (McLean, VA, U.S.A.), and culture plates from Becton Dickinson Labware (Lincoln Park, NJ, U.S.A.). Other chemicals and reagents were of analytical grade and obtained from local suppliers.

Cells and culture conditions

Ddy mice were purchased from Japan SLC (Shizuoka, Japan). Bone marrow and spleen cells were freshly prepared from 6- to 9-week-old male mice immediately before experiments as described elsewhere.13 Osteoblastic cells were prepared from 10–20 newborn mouse calvariae by enzymatic digestion as reported13 and cultured to confluence in 10-cm culture dishes. Harvested cells were kept frozen in aliquots until use. Mouse osteoblastic cell line KS414 were kindly provided by Kirin Brewery Co. (Gunma, Japan). Preadipocytic stromal cell lines, ST2 and MC3T3-G2/PA6 (PA6), were obtained from Riken Cell Bank (Ibaraki, Japan). ST2 was established from long-term mouse marrow cultures15 and PA6 from newborn mouse calvariae.16 It is shown that the OCLs formed in cocultures of spleen cells with any of these cell lines make resorption pits on dentine slices,14,17 as in cocultures with calvarial osteoblasts and bone marrow cells or spleen cells.13

Before the start of experiments, the calvarial osteoblastic cells were thawed, precultured in 10-cm dishes to confluence, and then seeded in experimental plates, at 2 × 104 cells/ml unless otherwise stated. Clonal cells were similarly seeded after two successive precultures in 25-cm2 culture flasks to expand the cell stocks. All cultures were maintained in α-MEM supplemented with heat-inactivated 10% fetal calf serum at 37°C in a humidified atomosphere of 5% CO2 and 95% air. Cultures lasted for 6–11 days, depending on the study protocols with the medium change every 2–4 days. In experiments where YM175 was added for only part of the culture period, cultures were washed with 0.5 ml of α-MEM three times at the time of medium change to minimize the remaining YM175.

Colony proliferation assay in methylcellulose

Methylcellulose culture was conducted according to the method described by Takahashi et al.18 In short, bone marrow cells obtained from tibiae were suspended (105/ml) in α-MEM containing 0.88% methylcellulose and 50 U/ml M-CSF and cultured on the osteoblastic cell layer prepared 1 day before in 6-well plates (2 ml/well). After 6 days of culture with or without YM175, the number of colonies was counted under a microscope. To confirm that the colonies were composed of monocyte-macrophages, cytocentrifuged preparations of recovered cells were fixed and stained for NSE using a commercially available kit. More than 90% of these cells were NSE positive.

NSE-positive cell proliferation in cocultures with osteoblastic cells

NSE-positive cells were prepared in semisolid methylcellulose as described above in the absence of osteoblastic cells and without YM175. After 6 days of culture, the cells were recovered by centrifugation. NSE-positive cells thus obtained were cultured at 102/well with osteoblastic cells prepared 1 day before in 24-well plates. After 8 days of culture in the presence of 10 nM 1,25(OH)2D3 with or without YM175, the number of colonies containing 20 or more NSE-positive cells was counted.

OCL formation in various cocultures

Osteoblastic or clonal cells were seeded in 24-well plates 24 h prior to the addition of bone marrow cells, spleen cells, or NSE-positive cells. All cocultures were conducted in the presence of 10 nM 1,25(OH)2D3 with or without YM175. In cocultures with ST2 or PA6 cells, dexamethasone at 100 nM was added as reported.17 To make culture conditions identical, dexamethasone was also added to some cocultures with osteoblastic cells or KS4 cells even though they did not require this treatment to support OCL formation as ST2 or PA6 cells did. At the end of each coculture, cells were stained for tartrate-resistant acid phosphatase (TRAP) as reported previously.19 TRAP-positive cells containing three or more nuclei (TRAP(+)MNC) were counted as OCL under a microscope.

Cocultures of marrow cells with osteoblastic cells:

Bone marrow cells (5 × 103/well) and calvarial osteoblastic cells were cocultured for 6 days with the medium change on the fourth day.

Cocultures of NSE-positive cells with osteoblastic cells:

According to the method described by Takahashi et al. as the two-step culture system,18 bone marrow-derived NSE-positive cells were collected from the colonies formed in the methylcellulose culture in the presence of M-CSF. The recovered cells (3 × 102/well) were cocultured with osteoblastic cells for 7 days with the medium change on the fourth day.

Cocultures of spleen cells with various supporting cells:

Spleen cells (5 × 105/well) were cocultured with calvarial osteoblastic cells or clonal cells (KS4, ST2, or PA6). Cultures lasted for 7 days (with calvarial osteoblastic cells, KS4, or ST2) or 11 days (with PA6) according to the previous observations.14,17 YM175 was present for the entire or specified periods as shown in the Results with the medium change every 2 or 3 days.

Fusion of preosteoclasts

Prefusion cells were isolated according to the methods of Wesolowski et al.,20 with a slight modification. Marrow cells (107) and calvarial osteoblastic cells (5 × 105) were cocultured in a 10-cm dish in the presence of 10 nM 1,25(OH)2D3. After 3 days, when many mononuclear TRAP-positive cells were formed, culture dishes were washed twice with phosphate-buffered saline (PBS) and treated with collagenase/dispase (1 mg/ml each in PBS) at 37°C for 30 minutes. Released cells, mostly osteoblasts, were removed with a pipette, and the dishes were washed three times with PBS. The remaining cells were incubated with 30 nM echistatin, an RGD containing snake venom,21 in α-MEM containing 1% bovine serum albumin for 30 minutes at 37°C. The cells released by this treatment were collected and plated at high density in 24-well plates. After 24 h of culture in the presence of 10 nM 1,25(OH)2D3 with or without YM175, OCLs were counted as described above.

Proliferation of osteoblastic or stromal cells

To examine the effect of YM175 on the proliferation of supporting cells, calvarial osteoblastic cells or clonal cell lines were cultured separately under exactly the same culture conditions as cocultures, except for the absence of osteoclast precursors. The time course of cell proliferation was assessed by measurement of DNA content. After the indicated number of days of culture with or without YM175, cells were washed with PBS and sonicated in a high-salt buffer (2 M NaCl, 0.05 M sodium phosphate buffer, pH 7.4). The DNA content of cell lysate was measured by the method of fluorescence enhancement using Hoechst 33258 binding (Hoechst Marion Roussel Corp., Cincinnati, OH, U.S.A.) to DNA.22 Calf thymus DNA was used as a standard.

Statistical analysis

Results are given as means ± SEM for n = 4 or 6 wells per experimental group. Although data from representative studies were shown in the results, each study was repeated at least twice, mostly three times or more, to confirm the findings. Statistical analyses were performed using Macintosh Statview software (Abacus Concepts, Berkeley, CA, U.S.A.). The difference between two groups was tested by unpaired t-test. Multiple-group comparisons were made by one-way analysis of variance and Fisher's protected least significant difference. p values less than 0.05 were considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Effects of YM175 on OCL formation from different precursor cells cultured with calvarial osteoblastic cells

In cocultures of calvarial osteoblastic cells with bone marrow cells or spleen cells, YM175 at 10−8 M did not inhibit OCL formation (data not shown). YM175 at 10−7 M inhibited OCL formation by 20–25%. However, the inhibition was not always statistically significant as reported previously.10 In contrast, YM175 at 10−6 M inhibited OCL formation significantly and consistently. As shown in Fig. 1, the inhibitory effects of YM175 at 10−6 M were comparable in three cocultures in which different osteoclast precursors were cultured with calvarial osteoblastic cells as the common supporting cells. The finding was further confirmed by repeating experiments four to six times for each coculture. The magnitude of inhibition of OCL formation ranged from 83.6–96.5% (mean 91.0%) with bone marrow cells, 80.2–95.0% (mean 85.5%) with spleen cells, and 69.3–98.2% (mean 88.1%) with NSE-positive cells.

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Figure FIG. 1. Effects of YM175 on OCL formation from three different sources of precursor cells cocultured with the same supporting cells. Bone marrow cells (5 × 103/well), spleen cells (5 × 105/well), or NSE-positive cells (3 × 102/well) were plated on the calvarial osteoblastic cell layers and cultured without or with YM175. TRAP(+) MNCs were counted after 6 days (with bone marrow cells) or 7 days (with spleen cells and NSE-positive cells) of culture. C, control cultures without YM175; YM175, cultures treated with 10−6 M YM175.

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Effects of YM175 on OCL formation in cocultures of spleen cells with various supporting cells

Dose–response study again showed that YM175 at 10−7 and 10−8 M did not significantly inhibit OCL formation in any cocultures (Table 1). Therefore, only the effects of YM175 at 10−6 M were examined in the following studies. As shown in Fig. 2, YM175 markedly inhibited OCL formation from spleen cells cocultured with calvarial osteoblastic cells. Similarly, YM175 inhibited OCL formation in cocultures with KS4, a osteoblastic cell line. Although data are not shown, YM175 also decreased the number of TRAP(+) mononuclear cells in these cocultures. YM175 was much less effective at decreasing the number of OCL and TRAP(+) mononuclear cells in cocultures with stromal cell line ST2 or PA6 (Fig. 2).

Table Table 1. Dose-Response Study of YM175 on OCL Formation in Cocultures of Spleen Cells with Various Supporting Cells
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Figure FIG. 2. Effects of YM175 on OCL formation in cocultures of spleen cells with four different supporting cells. Spleen cells (5 × 105/well) were plated on the osteoblastic (OB or KS4) or stromal cell (ST2 or PA6) layers and cultured without or with YM175. In experiments with ST2 and PA6 cells, 100 nM dexamethasone was added to culture media. TRAP(+) MNCs were counted after 7 days (with OB, KS4, and ST2 cells) or 11 days (with PA6 cells) of culture. C, control cultures without YM175; YM175, cultures treated with 10−6M YM175; OB, calvarial osteoblastic cells. Data are representative of five to six repeated experiments as listed in Table 2.

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Repeated experiments confirmed the marked difference in inhibitory effects of YM175 between cocultures with calvarial osteoblastic cells or KS4 and those with ST2 or PA6. As shown in Table 2, the difference between groups with and without YM175 was significant in all cocultures with calvarial osteoblastic cells or KS4 cells. In contrast, statistical significance was achieved in only one of six experiments with ST2 cells. The mean inhibition of OCL formation by YM175 calculated from five to six separate experiments was by 85.5% for calvarial osteoblastic cells, 77.1% for KS4, 18.0% for ST2, and 22.3% for PA6.

Table Table 2. Effects of YM175 on OCL Formation in Cocultures of Spleen Cells with Various Supporting Cells
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Effect of dexamethasone on YM175-induced inhibition of OCL formation

To examine the possible influence of dexamethasone on the results, spleen cells were cocultured with calvarial osteoblastic cells or KS4 cells with or without dexamethasone, although these cocultures did not require dexamethasone for OCL formation. In cocultures of either cell types, YM175 significantly inhibited OCL formation both in the absence and the presence of dexamethasone. The mean inhibition of OCL formation by YM175 calculated from four separate experiments was by 86.7% and 95.2%, with and without dexamethasone, respectively. As for cocultures with ST2 or PA6 cells, comparison was not made between cultures with and without dexamethasone since no OCLs were formed in the absence of dexamethasone in these cultures.

Influence of the time of YM175 addition on OCL formation in cocultures of spleen cells with various supporting cells

Figure 3 shows the effects of YM175 added for different intervals in cocultures of spleen cells with various supporting cells. In cocultures with calvarial osteoblastic cells, OCL formation from spleen cells was inhibited by 80.2% when YM175 was present throughout 7 days (group B). YM175 was similarly effective when it was present during the first 4 days but absent during the last 3 days (group C). Addition of YM175 during the last 3 days (group D) was without effect. Even when YM175 was present only for 2 days, days 1–2 (group E) or days 3–4 (group G), the inhibitory effects were still significant. In a separate experiment, the culture period was extended to 9 days to exclude the possibility that YM175 added for the first 2 days did not inhibit but merely delayed OCL formation. In cocultures treated with YM175 for the first 2 days, the OCL number was significantly smaller than in control cocultures not only at day 7 but also at day 9 (data not shown).

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Figure FIG. 3. Influence of the time of YM175 addition on OCL formation in cocultures of spleen cells with various supporting cells. Spleen cells were plated on the osteoblastic (OB or KS4) or stromal cell (ST2) layers. Medium was changed on days 2 and 4 as shown by thick horizonal lines. Shaded area shows the interval when 10−6 M YM175 was added to the culture. TRAP(+) MNCs were counted 7 days later. All the data shown here derived from one set of representative experiments performed at the same time. *Different (p < 0.01) from group A; **different (p < 0.01) from groups A and B.

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The findings with KS4 cells were similar, although not identical, to those with calvarial osteoblastic cells. OCL formation was almost equally inhibited by YM175 whether it was present throughout 7 days (group B), during the first 4 days (group C), or during the first 2 days (group E). Unlike the results with calvarial osteoblastic cells, OCL formation was significantly inhibited by treatment with YM175 for only 1 day (group F), while no inhibition was seen in group G. However, these small differences between calvarial osteoblastic cells and KS4 cells were not always observed in repeated experiments. In cocultures with ST2 cells, the presence of YM175 for any interval did not affect OCL formation.

Effects of YM175 on the proliferation of NSE-positive cells in cocultures with calvarial osteoblastic cells

YM175 did not inhibit the colony formation of NSE-positive cells from bone marrow cells cultured with osteoblastic cells in methylcellulose in the presence of M-CSF (Fig. 4A). In contrast, when the NSE-positive cells were harvested and cultured with osteoblastic cells in the presence of 1,25(OH)2D3 for a further 8 days, YM175 significantly reduced the number of NSE-positive colonies (Fig. 4B) as well as the number of TRAP(+) cells formed within the colonies (data not shown).

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Figure FIG. 4. Effects of YM175 on the proliferation of NSE-positive osteoclast progenitor/precursor cells. (A) Bone marrow cells (105/ml) were cultured with calvarial osteoblastic cells in semisolid methylcellulose in the presence of 50 U/ml of M-CSF without or with YM175. After 6 days, the number of NSE-positive colonies was counted. (B) NSE-positive cells recovered from methylcellulose culture were further cultured with osteoblastic cells (2 × 104/ml) in the presence of 1,25(OH)2D3, without or with YM175. After 8 days, the number of NSE-positive colonies was counted. C, control cultures without YM175; YM175, cultures treated with 10−6 M YM175. *Different (p < 0.01) from control.

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Effects of YM175 on the final step of OCL formation

As reported by Wesolowski et al.,20 OCL were formed after 24 h incubation of TRAP(+) mononuclear cells prepared by echistatin treatment (Fig. 5). Although no osteoblastic cells were added during the 24 h culture, there were many remaining osteoblastic cells from the preceding coculture. YM175 did not inhibit the process of cell fusion to form OCLs (the number of OCLs per well: 535.5 ± 10.0 without YM175 vs. 537.7 ± 36.1 with YM175).

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Figure FIG. 5. Effect of YM175 on the final step of OCL formation. TRAP(+) mononuclear cells formed after 3 days of culture of marrow cells with calvarial osteoblastic cells were plated at high density in the presence of 1,25(OH)2D3 and cultured for 24 h. Photomicrographs show the results of TRAP staining at the time of plating (left, 0 h) and after 24 h culture without (middle, control 24 h) or with 10−6 M YM175 (right, YM175 24 h).

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Effects of YM175 on the proliferation of osteoblastic/stromal cells

In all cultures, the DNA contents increased in a time-dependent manner from day 1 to day 3. Judging from the DNA contents, the proliferation rate was fastest in KS4 cells, followed by ST2, PA6, and calvarial osteoblastic cells, in descending order. YM175 exerted no inhibitory effect on cell proliferation during these periods (Fig. 6). YM175 also did not affect the DNA contents from day 5 to day 7, during which they reached plateau (data not shown).

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Figure FIG. 6. Effects of YM175 on the DNA contents in cultures of supporting cells. Calvarial osteoblastic cells (OB) and three clonal cell lines (KS4, ST2, and PA6) were plated at 104/well and cultured without osteoclast precursors under the same culture conditions as cocultures. After the indicated days of culture without (open circle) or with (closed circle) 10−6 M YM175, the DNA content was measured by the method of the enhancement of fluorescence using Hoechst 33258 binding to DNA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The inhibitory effect of bisphosphonates on osteoclast recruitment in vitro was first reported by Boonekamp et al. using the bone explants.23 However, studies later conducted have reported conflicting data on the effects of bisphosphonates on OCL formation.6–10 Similarly, in vivo studies have shown that the number of osteoclasts either increases or decreases after treatment with bisphosphonates.24–26 Thus, it is still controversial whether bisphosphonates interfere with the formation of osteoclasts, and if so, through what mechanisms. It is especially unclear why results are conflicting among similar cell culture studies, in which experimental conditions are not as diverse as in the in vivo studies. To answer these questions, we conducted the present study and found that YM175, one of the third-generation bisphosphonates, inhibited OCL formation in various cocultures, but the inhibitory effect of YM175 was dependent on the type of bone-derived cells supporting this process.

Using various combinations of osteoclast precursors and stromal/osteoblastic cells, we first examined whether the inhibitory effect of YM175 was influenced by the cell types cocultured. We thought that the findings would give us some clue to help define which cells were mainly affected by bisphosphonates, either osteoclast precursors, stromal/osteoblastic cells, or both. Of three sources of osteoclast precursors, NSE-positive cells are nearly homogeneous cell populations derived from colony forming unit for monocyte macrophages.18 Marrow cells are heterogenous, and only a fraction of them represents osteoclast progenitors and precursors. Spleen cells contain an even smaller fraction of osteoclast precursors than marrow cells.27 The cell populations other than osteoclast lineage cells are also different between marrow and spleen cells. Despite these differences, OCL formation was inhibited similarly by YM175 in all three cocultures to which supporting cells were common.

In contrast, the results varied when the same osteoclast precursors were cocultured with different supporting cells. OCL formation from spleen cells was markedly inhibited by YM175 in cocultures with calvarial osteoblasts or KS4, while it was not at all or only slightly inhibited in cocultures with ST2 or PA6. These findings seemed to support the view that YM175 inhibited OCL formation through its effects on stromal/osteoblastic cells whose sensitivity to YM175 was different depending on the cell types. An alternative explanation might be that YM175 exerted its inhibitory effect directly on osteoclast precursors, but the stimulating activity of supporting cells opposed the effect of YM175 variably depending on the cell types. However, our data were not supportive, although not entirely exclusive, of this view because the number of OCLs formed in the absence of YM175 was not greater in cocultures with ST2 or PA6 cells compared with those with calvarial osteoblasts or KS4 cells. It also seemed unlikely that the OCL populations formed in different cocultures were not the same and therefore their formation could be affected by YM175 variably. As reported by other investigators,13,14,17 we confirmed that the OCLs being formed in each coculture with different supporting cells resorbed dentine slices in proportion to the number of OCLs (unpublished observation).

It is unknown what mechanisms or factors made the effects of YM175 on OCL formation apparently dependent on supporting cell types. A noticeable feature common to the two cell types considered to be sensitive to YM175 was osteoblastic phenotype, while the other two cell types little affected by YM175 had preadipocytic phenotype.15,16 Other cellular characteristics such as the tissue origin of cells and their proliferation rates did not correlate with the different effects of YM175 among cocultures. Of three cell lines, KS4 and PA6 derived from calvaria, while ST2 derived from bone marrow. Calvarial osteoblasts and KS4, both of which were similarly sensitive to YM175, showed the slowest and the fastest proliferation rates, respectively. Among other possibilities, we ruled out dexamethasone as a contributing factor. It is well known that glucocorticoids affect the differentiation of osteoblastic and stromal cells28 and that they influence OCL formation in cocultures through various mechanisms.29 Therefore, dexamethasone, added in cocultures with ST2 or PA6 cells but not in cocultures with calvarial osteoblasts or KS4 cells, might have antagonized the inhibitory effects of YM175. However, it was found that YM175 significantly inhibited OCL formation in cocultures with calvarial osteoblasts or KS4 cells not only in the absence but also in the presence of dexamethasone.

Stromal/osteoblastic cells have crucial roles in osteoclastogenesis from hematopoietic stem cells.12,13,30 However, not all bone-derived cell lines are able to promote OCL formation, and little is known about the cellular characteristics that determine the ability of individual cell lines. Therefore, it was beyond the scope of the present study to explore how YM175 made osteoblastic cells incapable of supporting osteoclastogenesis. We only confirmed that YM175 at the concentration effective to inhibit OCL formation did not inhibit the proliferation of osteoblastic cells. It remains to be elucidated in the future study whether YM175 affects cell–cell interaction or induces a change in the production of stimulators or inhibitors of osteoclastogenesis. In this regard, previous studies have shown that osteoblastic cells produce unknown inhibitors of OCL formation in the presence of bisphosphonates.10,31 However, a question remains whether the inhibitors contained in the media of osteoblastic cells treated with bisphosphonates and cultured under serum-free conditions actually represent the inhibitory activity observed when osteoblastic cells are cocultured with osteoclast precursors in serum-containing medium.

The next issue we investigated was which phase of OCL formation was mainly inhibited by YM175. Although we did not show the actual number of TRAP(+) mononuclear cells in the results, it was reduced significantly in cultures where the formation of OCL was inhibited by YM175. This microscopic observation led us to hypothesize that the major action of YM175 was to inhibit proliferation of osteoclast precursors and reduce the number of prefusion osteoclasts. The hypothesis was supported by the findings that YM175 effectively inhibited OCL formation in cocultures of spleen cells with osteoblastic cells when it was present for the first 2–4 days, which corresponded to the proliferation phase of osteoclast precursors. The inhibitory effect of YM175 on the proliferation of osteoclast precursors was further confirmed in cocultures of NSE-positive cells with osteoblastic cells. The number of osteoclast precursors that were easily identified by NSE staining decreased significantly in the presence of YM175.

As reported previously,10 YM175 did not affect the colony formation of monocyte-macrophages from marrow cells cultured in the presence of M-CSF in methylcellulose. Thus, it can be summarized that YM175 inhibited the proliferation and differentiation of osteoclast precursors stimulated by 1,25(OH)2D3 but did not inhibit the earlier proliferation of osteoclast progenitors stimulated by M-CSF. These observations suggest two possibilities: one is the stimulator-related difference in the sensitivity to the inhibitory effect of YM175 and the other is the difference between cells at varying differentiation stages. Previously, Cecchini et al. reported that bisphosphonates inhibited the proliferation of both marrow cells and marrow-derived macrophages, but the dose-responsiveness was slightly different between the two.32 It is very likely, therefore, that the dose responsiveness of the inhibitory effects of YM175 was different between the proliferation of osteoclast progenitors and that of more differentiated osteoclast precursors.

The observation that OCL formation was not inhibited when YM175 was added only for the last 3 days of culture suggested that the later phase of differentiation and fusion of preosteoclasts were unaffected by YM175. To make this point clear, we examined directly the effect of YM175 on the final step of OCL formation using the method of Wesolowski et al.20 with a slight modification. As expected, YM175 had no inhibitory effect on the fusion of mononuclear preosteoclasts. This finding was at variance with that of Schmidt et al., who recently suggested, although the data were not shown, that alendronate inhibited OCL formation by inhibiting the fusion of preosteoclasts.33 The different results could be explained by the difference in the bisphosphonates used and/or the difference in the dose of bisphosphonates. With YM175 at 10−5 M, a dose that was ten times higher than that used in the present study and probably within cytotoxic range, the fusion of preosteoclasts was inhibited in our culture also (unpublished observation).

The question remained whether the apparent discrepancies among studies were due to the true difference in the mechanism of action of individual bisphosphonates or to differences in their dose responsiveness for similar actions. Differences in experimental methods may also contribute to the inconsistent results. In this regard, the difference between our results and those of Murakami et al. is of interest.9 Using cocultures of mouse bone marrow cells with osteoblastic cells, they reported that tiludronate did not inhibit OCL formation. Aside from the difference in bisphosphonates used, their cocultures were different from ours in terms of marrow cell number, plating ∼100–200 times more marrow cells than ours. Based on the findings of the present study, the discrepancy between their results and ours can be explained in two ways. Assuming that the stromal cells are relatively insensitive to bisphosphonates, a considerable number of marrow stromal cells present in cocultures of Murakami et al.9 might promote OCL formation by themselves even when osteoblastic cells did not support it in the presence of tiludronate. In contrast, in our cocultures with a much smaller number of marrow cells, there might not be enough stromal cells to promote OCL formation when osteoblastic cells did not support osteoclastogenesis in the presence of YM175. Alternatively, if the inhibitory effect of bisphosphonates is limited to the early proliferation of osteoclast precursors, a certain fraction of differentiated precursor cells could escape from the inhibition by bisphosphonates. With a large number of total marrow cells, as in cocultures of Murakami et al.,9 the eventual number of preosteoclasts would narrowly be plenty enough to form OCL, but not in ours.

In summary, the present study showed that YM175 inhibited OCL formation in mouse cocultures by inhibiting the proliferation of osteoclast precursors, most likely through its action on cells of osteoblast lineage. Although we did not conduct a detailed dose–response study for each coculture, it was found that the inhibitory effect of YM175 at the same dose was different in cocultures of different cellular components, even under identical or similar experimental conditions. Whatever the underlying mechanisms may be, these observations suggest that minor differences in experimental conditions could lead to conflicting data and different conclusions on the effects of bisphosphonates.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This work was supported in part by a grant-in-aid for Cancer Research (9–22) from the Ministry of Health and Welfare of Japan.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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