Alteration of bone cell function by RANKL and OPG in different in vitro models

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1 European Journal of Clinical Investigation (2007) 37, Blackwell Publishing Ltd Alteration of bone cell function by RANKL and OPG in different in vitro models J. M. Lin *, K. E. Callon *, C. Q. Lin *, U. Bava *, M. H. Zheng, I. R. Reid * and J. Cornish * * University of Auckland, Auckland, New Zealand, School of Surgery and Pathology, University of Western Australia, Perth, WA, Australia Abstract Background Receptor activator of nuclear factor-κb ligand (RANKL) and osteoprotegerin (OPG) are well-documented potent regulators of osteoclast development. However, their effects in mature bone cells and in organ cultures have not been well studied. It is uncertain whether their activities in different experimental models are comparable. Materials and methods RANKL and OPG were evaluated for their activities in mouse calvarial organ cultures, mouse bone marrow cultures, isolated rat mature osteoclast assays and rat primary osteoblast cultures. Results In murine calvarial organ culture, both murankl ( 10 ng ml 1 ) and rrankl ( 100 ng ml 1 ) significantly stimulated 45 Ca release, while OPG ( 50 ng ml 1 ) was an inhibitor of bone resorption. Meanwhile, [ 3 H]-thymidine incorporation in this assay was also modulated (indicating proliferation increases in the osteoblast lineage of cells) although these peptides had no direct effect on [ 3 H]-thymidine incorporation in isolated osteoblast assays. In mouse bone marrow cultures, murankl ( 1 ng ml 1 ) and rrankl ( 5 ng ml 1 ) significantly stimulated osteoclastogenesis. The number of nuclei per osteoclast was also significantly increased. OPG strongly inhibited this index, with over 90% suppression at 1 ng ml 1. Both murankl (10 ng ml 1 ) and rrankl (100 ng ml 1 ) stimulated, while OPG (10 ng ml 1 ) inhibited osteoclast activity in isolated mature osteoclast assays. Conclusion The current study demonstrated that bone resorption modulated by RANKL and OPG, in murine calvarial organ culture, leads to changes in osteoblast proliferation, suggesting a feedback mechanism from osteoclasts to osteoblasts. In addition, it was found that RANKL and OPG have more potent effects on osteoclastogenesis than on the activity of mature osteoclasts. Keywords Calvariae, OPG, osteoblasts, osteoclasts, RANKL. Eur J Clin Invest 2007; 37 (5): Introduction Osteoclasts are known to differentiate from haematopoietic precursor cells present in bone marrow, spleen and peripheral blood [1]. It was noted that the development of osteoclasts Department of Medicine, University of Auckland, Auckland, New Zealand (J. M. Lin, K. E. Callon, C. Q. Lin, U. Bava, I. R. Reid, J. Cornish), Units of Orthopaedics, School of Surgery and Pathology, University of Western Australia, Perth, WA, Australia (M. H. Zheng). Correspondence to: Professor Jill Cornish, Department of Medicine, University of Auckland, Auckland, New Zealand. Tel.: ext 86255; fax: ; j.cornish@auckland.ac.nz Received 29 September 2006; accepted 23 January 2007 from their precursors usually requires the presence of osteoblasts [2]. The mechanism for this has been revealed by the identification of a trio of peptides, osteoprotegerin (OPG) [3 5], receptor activator of nuclear factor-κb ligand (RANKL) [6,7] and receptor activator of nuclear factor-κb (RANK) [7,8]. RANKL, a product of osteoblasts, stimulates osteoclast formation and function through its cognate receptor RANK, which is a membrane-bound protein present on osteoclasts and their precursors. However, the interaction between RANKL and RANK can be competitively inhibited by the decoy receptor OPG, a soluble protein produced by osteoblasts, and hence the signalling from osteoblasts/rankl to osteoclasts/rank is blocked [9]. The effects of RANKL and OPG on bone remodelling have been demonstrated both in vivo and in vitro. Mice deficient in OPG exhibit severe osteoporosis with loss of trabecular bone and dramatic decreases in bone strength 2007 The Authors. Journal Compilation 2007 Blackwell Publishing Ltd

2 408 J. M. Lin et al. [10]. On the other hand, over-expression of OPG in transgenic mice results in profound osteopetrosis and a decrease in osteoclast differentiation [3]. Systemic administration of OPG to normal mice led to significantly increased bone mineral density and bone volume, and decreased osteoclast number [3,11]. In contrast, severe osteopetrosis was seen in RANKL knockout mice, in which osteoclastogenesis was blocked and tooth eruption was suppressed [12]. Our group has found in humans that a 3 bp inframe deletion in exon 3 of OPG gene is associated with the clinical disorder, idiopathic hyperphosphatasia (also termed as juvenile Paget s disease), which is characterized by deformities of bone and extremely rapid bone turnover [13]. This mutation results in the deletion of aspartate182 in OPG, and impairs the secretion and activity of OPG [14]. The role of OPG and RANKL in regulating osteoclastogenesis have been well established in in vitro assays, such as cultures of bone marrow cells [4,15]. It has been found that RANKL in combination with macrophage colonystimulating factor (M-CSF) is sufficient to induce osteoclast formation from mouse spleen cells [16,17], human peripheral blood monocytes [18,19] without the presence of supporting osteoblastic stromal cells. Osteoclast formation from mouse macrophage-like cell lines RAW can even be induced by RANKL without the presence of M-CSF [16,20]. Thus, many studies have established the importance of OPG and RANKL in regulating osteoclast formation, but the direct action of OPG on mature osteoclasts, and that of OPG and RANKL on osteoblasts have not been well examined. Teitelbaum and Ross have provided preliminary evidence that RANKL is not only a regulator of osteoclasts, but is also a stimulator of osteoblastogenesis [21]. However, no further published evidence appears to be available to support this observation. In the current study, we have used a number of in vitro culture systems, among which the calvarial organ culture is of particular interest because it maintains a tissue microenvironment, which is closer to that in vivo. The present study compares the potency of these peptides on osteoclastic activity in a variety of models, and assesses the peptides direct and indirect actions on osteoblasts. Our data show that neither RANKL nor OPG affect isolated osteoblast proliferation. In addition, we have taken the opportunity to assess the comparative potency of murine RANKL (murankl) and rat RANKL (rrankl) in a number of models for their effects on osteoclastogenesis and mature osteoclast activity. rrankl shares 84% and 96% identity in amino acid sequence with human RANKL and murankl, respectively, but has only been examined for its inductive activity on osteoclastogenesis in one previous study [20]. Materials and methods Reagents, animals and culture conditions Recombinant human OPG (aa22 201) and murankl (aa ) were provided by Amgen Inc. (Thousand Oaks, CA, USA). Recombinant rrankl (aa ) was prepared as previously described [20]. Bovine parathyroid hormone (PTH, directly extracted from parathyroid glands) was kindly provided by Dr Peter Barling, School of Biological Sciences, University of Auckland. 45 CaCl 2 and [ 3 H]-thymidine were purchased from Amersham Pharmacia Biotech Ltd, Auckland. Staining kit (catalogue no. 387-A) for tartrate-resistant acid phosphatase (TRAP) was purchased from Sigma (St. Louis, MO, USA). Foetal bovine serum (FBS) and culture media were from Life Technologies (Grand Island, NY, USA). All media contain 100 units ml 1 of penicillin and 100 µg ml 1 of streptomycin (Gibco, catalogue no , Life Technologies, Grand Island, NY, USA). Bovine serum albumin (BSA) was from Immuno-Chemical Products Ltd, Auckland. The mice (Swiss CD-1) and rats (Wistar) were supplied by the animal resources unit of the School of Medicine, University of Auckland. All cultures were maintained at 37 C in a humidified atmosphere of 5% CO 2 in air. The study had the approval from the local institutional review board. Mouse calvarial organ culture As described previously [22], hemicalvariae were isolated from 1- to 3-day-old mice prelabelled with 45 CaCl 2. The bones were pre-incubated in 0 1% BSA/Medium 199 on supporting metal grids for 24 h. The medium was replaced and test factors or vehicle were added and incubated for 48 h after which an aliquot of media was removed and counted for 45 Ca to determine the amount of calcium released from the bones.[ 3 H]-thymidine was then added to the medium and incubated for a further 4 h. The bones were washed and placed in 5% trichloroacetic acid to extract the remaining 45 Ca from the bone and an aliquot of trichloroacetic acid was taken and counted. The bones were then dehydrated with acetone and ether and weighed before dissolving in KOH and counting for 3 H. Bone resorption was defined as the percentage of 45 Ca in medium/ total 45 Ca. [ 3 H]-thymidine incorporation was calculated as total disintegrations per minute (DPM) per bone/bone weight (µg). Mouse bone marrow culture and histochemical staining of osteoclasts Bone marrow cells were obtained from mouse long bones as previously described [23]. Briefly, cells isolated from the bone marrow were incubated for 2 h and the non-adherent cells collected, washed and seeded into 48-well plates in 15% FBS/α-modified minimum essential medium (αmem). Fresh media was added on days 2 and 4 of culture and 1,25(OH) 2 D 3 (10 8 M final) and test substances or vehicle were added on days 0, 2 and 4. Cultures were maintained for 7 days at which time the cells were fixed, washed and stained for TRAP. TRAP positive cells with three or more nuclei were scored as osteoclasts.

3 Effects of RANKL and OPG on bone cells 409 Figure 1 Effects of murankl and OPG on 45 Ca release (a and c) and [ 3 H]- thymidine incorporation (b and d) in mouse calvarial organ cultures. Data in each figure are the combination of the results from two experiments and are presented as the mean ± SE, n = 5 7/group. Significantly different from control: **P < Osteoblast proliferation assay Osteoblasts were prepared from foetal rat calvariae as previously described [24]. Briefly, cells isolated from sequential collagenase digestion were grown to semiconfluence at which time they were subcultured into 24-well plates in 5% FBS/ minimum essential medium (MEM) and incubated for 24 h. Cells were serum starved (in 0 1% BSA/MEM) for 24 h followed by a media change and the addition of test substances or vehicle and incubated for a further 24 h. Four hours prior to the end of the incubation time [ 3 H]-thymidine was added and at the termination of the experiment the cells were processed and [ 3 H]-thymidine incorporation was measured. Mature osteoclast activity assay Osteoclasts were isolated from the long bones of 1-day-old neonatal rats as previously described [25]. Briefly, rat long bones were minced with a scalpel blade and the cell suspension collected and placed onto bone slices. After a brief incubation the bone slices, with adherent osteoclasts, were washed vigorously to remove contaminating cells and placed into 12-well plates containing acidified 10% FBS/Earle s MEM. Test substances or vehicle were added and the bone slices were incubated for 24 h. Bone slices were then fixed washed and stained for TRAP. Multinucleated TRAP-fixed positive cells were counted and then the bone slices were cleaned of cells, stained with toluidine blue and the resorption pits assessed using reflective light microscopy. Bone-resorbing activity of the osteoclasts was expressed as the number of pits produced per osteoclast. Statistical analysis Data analysis was performed using one-way analysis of variance with post-hoc examination of significant main effects using the method of Dunnett. Data are presented as the mean ± SE. All tests were two-tailed and a 5% significance level was maintained throughout these analyses. All experiments were repeated at least three times with similar results. Results Mouse calvarial organ cultures In this assay, murankl stimulated the release of prelabeled 45 Ca, with significant effects seen at concentrations of 10 ng ml 1 and greater. A maximum increase in bone resorption, approximately 100% higher than control, was reached at a concentration of 100 ng ml 1 (Fig. 1a). At the same time, [ 3 H]-thymidine incorporation into the calvariae, which generally reflects the rate of DNA synthesis and cell (osteoblast) proliferation [26], was also increased in a dose-dependent manner (Fig. 1b). OPG significantly inhibited both 45 Ca release and [ 3 H]- thymidine incorporation by about 35% at a concentration of 50 ng ml 1 (Fig. 1c,d). The results showed that the inhibitory effect on 45 Ca release by OPG at maximal concentration (50 ng ml 1 ) was completely reversed by murankl at 50 ng ml 1 or greater (Fig. 2). rrankl was also tested in the calvarial organ culture. Recombinant rrankl( ), which contains the full

4 410 J. M. Lin et al. Figure 2 Interaction of OPG and murankl in regulating 45 Ca release in mouse calvarial organ cultures. Both OPG and murankl were added at the same time and cultures were continued for 48 h. Data are from a representative experiment and presented as the mean ± SE, n = 4 6/group. Significantly different from control: *P < Figure 4 murankl (a) and OPG (b) have no effect on the proliferation of rat primary osteoblasts in culture. Data in each figure are from a representative experiment and presented as the mean ± SE, n = 5 6/group. Figure 3 rrankl enhances 45 Ca release in mouse calvarial organ cultures. Parathyroid hormone (PTH) is included for comparison. Data are from a representative experiment and presented as the mean ± SE, n = 5 7/group for rrankl and n = 3/group for PTH. Significantly different from control: **P < osteoblasts are thought to be the major contributing cell type for [ 3 H]-thymidine incorporation in these cultures [26]. Both murankl and OPG were added to the cultures of primary rat osteoblasts to assess the direct actions of these factors on the cells. Neither murankl nor OPG at 10, 50 and 100 ng ml 1 produced any detectable effects (Fig. 4). TNF-like core region and covers the equivalent sequence for recombinant murankl( ), significantly stimulated 45 Ca release at concentrations of 100 ng ml 1 and greater (Fig. 3). However, compared to murankl and PTH, rrankl produced a smaller maximal effect and was less potent. The half-maximally effective concentration of murankl was between 1 and 10 ng ml 1, whereas that for rrankl was between 50 and 100 ng ml 1. Rat osteoblast cultures In light of the increases in [ 3 H]-thymidine incorporation observed in the calvarial organ cultures, we examined whether the peptides affected osteoblasts directly. The Mouse bone marrow cultures The potencies of murankl, rrankl and OPG were assessed in mouse bone marrow cultures. These peptides showed much greater effects on osteoclast formation in this model than the bone resorption effects seen in mouse calvariae. murankl at 1 ng ml 1 and 50 ng ml 1 enhanced the osteoclast-like cell number by 2 4- and 5-fold, respectively. No further stimulation was seen when the concentration was increased to 100 ng ml 1 (Fig. 5a). The number of nuclei per osteoclast was also significantly increased, by about 1 5-fold, at concentrations of 50 and 100 ng ml 1 (Fig. 5b). In addition, rrankl( ) enhanced osteoclast-like cell formation by 2- and 3 8-fold at concentrations of 5 and 50 ng ml 1, respectively (Fig. 5c), again more potent than its effect in calvarial organ cultures.

5 Effects of RANKL and OPG on bone cells 411 Figure 5 Effects of RANKL and OPG on osteoclast formation from mouse bone marrow cultures. Stimulation of murankl on osteoclast number (a) and degree of multinucleation (b); stimulatory effect of rrankl (c) and inhibitory effect of OPG (d) on osteoclast formation. The number of osteoclasts and nuclei were scored as osteoclasts/well and nuclei/ osteoclast, respectively. Figures (a) and (b) show the combined results from two experiments while Figures (c) and (d) each show the results from a representative experiment. Data are presented as the mean ± SE, n = 8 10/group. Significantly different from control: *P < 0 05; **P < In contrast, OPG strongly inhibited osteoclastogenesis in bone marrow cell cultures. It reduced the osteoclast-like cell number by over 90% at a concentration of 1 ng ml 1. When the OPG concentration was increased to 10 ng ml 1 or greater, the osteoclast-like cell formation was almost completely blocked (Fig. 5d). Mature rat osteoclast assays Since bone resorption in calvarial organ cultures mainly reflects the activity of mature osteoclasts because of the limited bone marrow present in calvariae, the effects and potency of RANKL and OPG on bone resorption in calvarial organ cultures should be comparable to their action in isolated mature osteoclast assays. Indeed, the results from the two assay systems were consistent. With murankl at concentrations of 10 and 50 ng ml 1, osteoclast activity (expressed as pits/osteoclast) was increased by about 1 8- and 2 0-fold, respectively. However, there was no significant effect on osteoclast number, consistent with the absence of osteoclast formation in this culture system (Fig. 6a). Similar to murankl, rrankl( ) stimulated isolated osteoclast activity without affecting osteoclast number. Figure 6(b) shows that pit formation was significantly increased following treatment with rrankl at 100 ng ml 1 (but not at 10 ng ml 1 ). This concentration (100 ng ml 1 ) was the same as that required to produce a significant effect in calvarial organ cultures, but was higher than that (5 ng ml 1 ) needed in bone marrow cultures. It is also noted that the magnitude of the effect of rrankl at 100 ng ml 1 was close to that produced by murankl at 50 ng ml 1 as shown in Fig. 6(a). Based on the generally recognized mode of action, OPG would only be expected to act via interaction with RANKL. It is not known whether OPG can directly act on osteoclasts without the involvement of RANKL. Therefore, OPG was tested in the isolated osteoclast assay system. It was found that OPG strongly inhibited pit formation at concentrations of 10 ng ml 1 and greater in the absence of added RANKL (Fig. 6c). Comparison of effects in different models By comparing the results above, it can be seen that the concentrations of the peptides required for significant effects varied greatly between the different culture systems. Overall, the concentrations needed in bone marrow cultures were 10- to 50-fold lower than in either calvarial organ cultures or isolated mature osteoclast cultures. To compare the difference between these models, the lowest concentrations required for the significant effects are summarized in Table 1. Discussion Osteoclasts are multinucleated cells developed from the haematopoietic lineage and formed by fusion of mononuclear precursors [27,28]. The development of osteoclasts from their precursors and the activation of existing mature osteoclasts determine the level of bone resorption. The differences in minimally effective concentrations among the culture systems used in the current study may reflect

6 412 J. M. Lin et al. Figure 6 Effects of murankl (a), rrankl (b) and OPG (c) on mature osteoclast activity. Data in figure (a) are from a representative experiment; data in figure (b) and (c) each are the combination of the results from two experiments. Data are presented as the mean ± SE, n = 6 8/group. Each bone slice contains at least 40 osteoclasts. Significantly different from control: *P < 0 05; **P < Table 1 Comparison of the minimal concentration (ng ml 1 ) required for the significant effects in different culture systems Mouse calvariae Mouse bone marrow murankl OPG rrankl Isolated rat osteoclasts differences in sensitivities of these cells to OPG and RANKL in vivo. We confirmed that the peptides regulated osteoclastogenesis in bone marrow cultures, while mature osteoclast activity was assessed in isolated osteoclast cultures and neonatal calvarial organ culture. The calvariae contain very little bone marrow, so little osteoclastogenesis occurs in this model. In support of this, it was reported that hydroxyurea, a DNA synthesis inhibitor, did not inhibit the rate of bone resorption in neonatal mouse calvariae [29], suggesting that bone resorption in calvariae is independent of cell proliferation, which is a major process leading to osteoclast recruitment and formation. In addition, our group demonstrated that in calvarial bones [ 3 H]-thymidine was taken up predominately by osteoblasts rather than osteoclasts, further indicating the absence of proliferation in osteoclast lineage and hence the absence of osteoclast formation [26]. In the current study, the effective concentrations in calvarial organ culture were comparable to those in the isolated osteoclast assay, further supporting the similar actions of the peptides in these two models. The current data show that the lowest effective concentrations were seen in bone marrow cultures compared to calvarial organ cultures and isolated osteoclast assays. These results are in agreement with a previous report, which showed that about 10-fold higher concentrations of OPG were required to inhibit bone resorption in mature osteoclast assays and long bone organ cultures than to inhibit osteoclast formation from bone marrow cells [5]. The data from the current study show that rrankl is obviously less effective than PTH on 45 Ca release in calvarial organ culture and has much lower potency than murankl in all the models used. This difference between rrankl and murankl may arise from either the different recombinant systems employed or the difference in the amino acid sequence or both. It is known that there is only 4% difference in amino acid identities between the two sources of RANKL [20]. If this small difference has led to the marked difference in potency, then these different amino acids in murankl could be critical to the activity of the peptide. In addition to the direct effect on the functional activity of mature osteoclasts, the impact of RANKL and OPG on mature osteoclast viability might also have contributed to the bone resorptive activity in calvarial bones. It has been reported that RANKL increases the survival of osteoclasts [30 33] in association with the suppression of Fas (a death receptor) expression and Fas-mediated apoptosis [34], while OPG has the opposite effect [31,35]. Therefore, it is likely that two mechanisms are involved in the modulation of bone resorption by RANKL and OPG in the calvarial organ cultures: regulation of osteoclast activity and an effect on osteoclast survival. In isolated osteoclast cultures, no change in osteoclast number was observed following treatment with RANKL or OPG. This indicates that neither osteoclastogenesis nor osteoclast survival was affected by the peptides. In this mature osteoclast-enriched preparation, contamination with osteoclast precursors might be present. However, they do not seem to have contributed to the number of functional osteoclasts, so are also unlikely to have contributed to resorption pit formation. This is probably because of the unfavourable incubation conditions. First, the incubation duration was so short (24 h) that any osteoclastogenesis was unlikely to produce functional osteoclasts. Second, the low ph ( 7 0) medium does not favour osteoclast development [36]. Lastly, M-CSF was not added to the cultures, and RANKL alone is not sufficient to induce osteoclast formation in this system [18,19]. In calvarial organ cultures, RANKL stimulated while OPG inhibited 45 Ca release from the bones. Interestingly, [ 3 H]-thymidine incorporation was also changed in the same way as that for 45 Ca release, indicating that these peptides also modulated the proliferation of osteoblasts, which has been shown previously to be the predominant proliferating

7 Effects of RANKL and OPG on bone cells 413 cell in calvariae [26]. To see if the changes in [ 3 H]-thymidine incorporation in the calvariae were the direct effects of RANKL and OPG, proliferation assays with isolated osteoblasts from foetal calvariae were conducted. The results showed that neither RANKL nor OPG had any impact on the proliferation of osteoblasts in culture, contrary to the previous observation that RANKL stimulated osteoblastogenesis [21]. The finding that RANKL and OPG have no direct effect on osteoblasts suggests that the changes in [ 3 H]-thymidine incorporation, associated with changes in bone resorption in calvarial cultures, reflect indirect effects on osteoblasts. It has been well documented that bone matrix contains various kinds of osteoblast-stimulating factors such as insulin-like growth factors (IGF)-1 and -II, and transforming growth factor (TGF)-β. When bone matrix is resorbed by osteoclasts, these factors are released, and in turn stimulate osteoblast proliferation and bone formation [37 41]. This process has been recognized to contribute to the coupling of bone resorption and formation. In the current assays, it is likely that RANKL stimulated bone resorption and led to higher levels of the factors released and hence higher levels of osteoblast proliferation, while OPG inhibited these processes. The coupling between bone resorption and formation has also been noted in OPG-deficient mice (OPG / ), in which increased bone resorption was associated with increased bone formation, and both of the processes were down-regulated by risedronate, an inhibitor of bone resorption [42]. It has been speculated that osteoclasts may also talk to osteoblasts through a more direct pathway, and the evidence for this has emerged in recent years. RAW cells, in response to stimulation with RANKL, express plateletderived growth factor BB, which is stimulatory to osteoblast proliferation [43]. A novel factor has been identified from RAW cell-derived osteoclasts. This factor binds specifically to osteoblasts and stimulates osteoblast proliferation [44]. Most recently, it was found that ephrinb2 ligand expressed by osteoclasts and its receptor EphB4 expressed by osteoblasts display bidirectional signalling for osteoclatogenesis and osteoblastogenesis: inhibiting osteoclastogenesis by interaction between ephrinb2 ligand and c-fos-nfatc1 pathway (reverse signalling) and stimulating osteoblastogenesis through interaction between ephrinb2 ligand and its receptor EphB4 (forward signalling) [45 47]. It is possible in the current study that the observed osteoblast proliferation in organ culture involves factors released from osteoclasts acting on osteoblasts, due to the actions of OPG and RANKL. OPG significantly inhibited bone resorption in calvarial organ cultures and this could be through the blockade of endogenous RANKL. However, since it has been found to inhibit pit formation in isolated osteoclast assays in the current investigation as well as in a previous study [48], it is likely that OPG also directly regulated the activity of the mature osteoclasts in the calvarial bones. The direct effect of OPG on mature osteoclasts has not been well investigated and the results have been controversial. Previous reports showed that OPG affected mature osteoclast activity only through the interaction with RANKL, and its direct action on mature osteoclasts was excluded [15,25]. However, Kwon et al. found that OPG inhibited bone resorption in crude isolated osteoclast cultures and in foetal mouse long bone cultures [5]. Moreover, Hakeda et al. reported that OPG directly inhibited mature osteoclast activity with the identification of an OPG binding protein on the surface of the osteoclasts [48]. In their isolated osteoclast preparation with the purity over 95%, no RANKL mrna was detected by Northern blots. In the present study, the data from the isolated osteoclast assays with OPG were consistent with the results from both Kwon et al. and Hakeda et al. However, since it is very difficult to purify osteoclasts to a satisfactory degree, the existence of very trace amounts of endogenous RANKL produced by the contaminated osteoblasts still could not be completely ruled out. In addition, it is uncertain whether mature osteoclasts themselves express RANKL. RANKL expression in osteoclasts in situ in bone tissues was related to its resorptive capabilities [49], and in pure osteoclast preparations its expression was only detected in three of the 12 samples [50]. Furthermore, in all of these assays, the culture media contain FBS, in which there may be a small amount of RANKL that can interact with the OPG added. For these reasons, the present data do not definitively establish that there is a direct interaction between OPG and mature osteoclasts. Further studies using culture systems free of RANKL and with the isolated osteoclasts from RANKL-null animals will be required to further address this question. In conclusion, the current study has investigated the effects of murankl, rrankl and OPG on bone resorption (calvarial organ cultures and isolated osteoclast assays), on osteoclast formation (bone marrow cultures) and on osteoblast proliferation (calvarial model and primary osteoblast cultures). The results indicate that bone resorption in response to these peptides is coupled with osteoblast proliferation as seen in calvarial cultures. 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