Morphological features of osteoclasts derived from a co-culture system

J Mol Hist (2006) 37:171–177 DOI 10.1007/s10735-006-9058-1

ORIGINAL PAPER

Morphological features of osteoclasts derived from a co-culture system Vanessa Nicolin Æ Giovanna Baldini Æ Renato Bareggi Æ Marina Zweyer Æ Giorgio Zauli Æ Mauro Vaccarezza Æ Paola Narducci

Received: 25 April 2006 / Accepted: 24 August 2006 / Published online: 15 September 2006 Ó Springer Science + Business Media B.V. 2006

Abstract The interaction between the receptor activator of NfKB (RANK) and its ligand receptor activator of NfKB ligand (RANKL) has recently been proven to be pivotal for osteoclast differentiation and activation. The influence of RANK-RANKL signaling on osteoclast formation was established by co-culturing murine osteoblasts (type CRL-12257) and murine mononuclear monocytes (RAW 264.7). The aim of the present study was to examine, by means of morphological techniques, the interaction between these two cell lines grown in the absolute absence of exogenous cytokines and other stimulating factors. Moreover, we wanted to show that our model could provide a system to analyze the bone resorption process. Mineralized matrix induced morphological changes of osteoclasts (OC) by the formation of organized ruffled-border and a large number of secondary lysosomal vesicles. On the contrary, OC grown on glass coverslips without dentin showed no organized ruffled border or secondary lysosomes. The study of the relationship between these two cell types could establish new approaches for a potential pharmacological control of these cell types and tissues in health and disease.

V. Nicolin (&) Æ G. Baldini Æ R. Bareggi Æ M. Zweyer Æ G. Zauli Æ P. Narducci Department of Biomedicine, Section of Human Morphology and Molecular Biology, University of Trieste, Via manzoni,16, Trieste 34138, Italy e-mail: [email protected] M. Vaccarezza Department of Health and Human Movement Sciences, University of Cassino, Viale Bonomi, 03043 Cassino (FR), Italy

Keywords Osteoclast Æ Pre-osteoclast Æ Receptor activator of NfKB ligand Æ Transmission electron microscopy Æ Scanning electron microscopy

Introduction Bone tissue is dynamically regulated organ system responsible for the structural/mechanical integrity of the human body and serves as the main reservoir of calcium in humans. Maintaining stable bone mass and homeostasis relies on a constant process of bone remodeling that involves the resorption of bone by osteoclasts (OC) and the synthesis of bone matrix by osteoblasts. OC are specialized members of the monocyte/macrophage family that are derived from hematopoietic precursors (Suda et al. 1999). The differentiation of OC is regulated by osteoblasts that are essential regulators of osteoclast function. OC are regulated by different types of signals that include endocrine hormones, cell–cell interactions, cell contact with bone matrix, and neighboring cell types (Boyle et al. 2003; Husheem et al. 2005). The pathway involved in osteoclast differentiation and activation requires two key elements: macrophage colony stimulating factor (M-CSF) and receptor activator of NfKB ligand (RANKL), that is homologous to other TNFR family ligands such as TNF, fasL, and TRAIL (Zauli et al. 2004). When expressed on the osteoblast surface, RANKL binds to its receptor activator of NfKB (RANK) on the surface of hematopoietic precursor cells, and in the presence of M-CSF stimulates differentiation, fusion, activation, and survival of OC. Consequently, osteoblasts support two distinct functions: bone formation and the promotion

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of osteoclast formation (Atkins et al. 2003). Given this, we assessed by morphological analyses the relationship between osteoblasts and monocytes during the process of osteoclastogenesis driven in vitro in the absence of osteotropic stimuli.

Materials and methods Cell cultures As a model system of osteoclastogenesis, we used RAW 264.7 type CRL 2278 murine monocytic/macrophagic cell line, purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) cocultured with murine osteoblasts type CRL 12257 (ATCC). CRL-2278 and CRL-12257 were cultured in a 24-well plate at a density of 105 cells/ml for each cell line in RPMI-1640 medium with 2 mM L-glutamine and modified to a final concentration of 4.5 g/l glucose, 1.5 g/l sodium bicarbonate, 10 mM Hepes, 1 mM sodium piruvate, and 10% fetal bovine serum (FBS). The culture medium was replaced every 48 h. Cells were grown for 4 days then washed once in PBS (Phosphate Buffer Saline, Sigma, St. Louis, MO, USA) and fixed for 30 min in a solution of 4% paraformaldehyde. Experiments were always performed in triplicate.

Phase contrast microscopy A Zeiss Axiovert 25 inverted microscope was used with positive phase-contrast objectives 32/0.40, 20/0.39, 10/0.25 with an ultra-long working distance NA 0.20 phase-contrast condenser supplied of Canon Power Shot G3 digital camera. TRAP assay For cytochemical tartrate-resistant acid phosphatase (TRAP) analysis, cells were stained using a leukocyte acid phosphatase kit, according to the manufacturer’s instructions (387-A, Sigma). Briefly, cells were washed once in PBS and fixed for 30 min in a solution of 4% paraformaldehyde. Fixed cells were then washed in PBS and incubated for 1 h at 37°C in TRAP staining solution. After TRAP reaction, slides were rinsed in deionized water and counterstained with hematoxylin solution for 1–2 min, then dried on air and evaluated microscopically by Zeiss Axiophot microscope supplied by Photometrics Cool Snap camera (Roper Scientific, Duluth, GA, USA).

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Immunocytochemistry The cells cultured on coverslips were air-dried and fixed in 4% paraformaldehyde in PBS, washed in PBS (pH 7.2) and blocked by incubation with 5% Normal Goat Serum (NGS), 4% Bovine Serum Albumin (BSA) in PBS. Specimens were incubated overnight at 4°C with mouse anti-actin monoclonal antibody (Sigma) diluted 1:50 in blocking buffer. After three washes in PBS, they were incubated with an antimouse fluorescein-isothiocyanate-conjugated (FITC) secondary antibody diluted 1:100 (Sigma) for 1 h at 37°C. Slides were reacted with 0.1 mg/mL 4-6-diamidino-2 phenylindole diluted in 1% Tween 20 (DAPI, Sigma) to detect cell nuclei. The slides were washed three times in PBS (pH 7.4), dehydrated in a graded series of ethanol and finally were mounted with a solution containing 2.3% 1,4-diazobicyclo [2.2.2] octane (DABCO; Sigma) to delay fading. Samples were photographed using a Zeiss Axiophot epifluorescence microscope. Digital images were obtained using a Photometrics Cool SNAP camera (Roper Scientific). Semi-thin sections Semi-thin or ultra-thin sections (50 samples) of the cell monolayers were cut with a Reichert OM ultramicrotome. Semi-thin sections (0.5 lm thick) were collected on slides, stained with 1% toluidine blue in 0.5% sodium carbonate, dried, mounted with Canada balsam and photographed using a Zeiss Axiophot microscope supplied by Photometrics Cool Snap camera (Roper Scientific). Transmission electron microscopy Cells were grown on coverslips in the same conditions previously described for optical microscopy analysis. The coverslip was removed from the cell layer by immersion in liquid nitrogen after resin polymerization at warm temperature (60°C). After a brief rinse in PBS, cells attached to the glass surface were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 10 min at room temperature and then for 20 min at 4°C. Cells were then rinsed in 0.1 M phosphate buffer, pH 7.3, post-fixed with 1% osmium tetroxide in the same buffer for 1 h at 4°C then dehydrated in ascending alcohols, and finally treated with propylene oxide. After embedding in Araldite (Electron Microscopy Sciences, Ft. Washington, PA, USA), ultra-thin sections were cut with a Reichert OM ultramicrotome. Sections were stained with uranyl and

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lead citrate, and then examined with a Transmission Electron Microscope Jeol model 100S (JEOL, Peabody, MA, USA) operated at 80 kV. Scanning Electron Microscopy For scanning electron microscopy (SEM) analysis, cells were grown in the same conditions as the optical microscopy technique. Cell cultures were fixed with 2.5% glutaraldehyde in 0.1 buffered phosphate (pH 7.3) for 30 min at 4°C, washed in phosphate buffer and post-fixed with 1% osmium tetroxide in the same buffer for 1 h at room temperature. The cells were then washed in distilled water, dehydrated in ethanol and dried by the critical-point method with a CPD Balzers Union. Finally, the cultures were sputter-coated with gold and observed under a scanning electron microscope Leica Stereoscan 430i.

Results Osteoclasts were identified in the mixed population derived from the co-culture model described above by scoring the appearance of TRAP+ multinucleated cells within 4 days in the absence of cytokines (Fig. 1). Since OC and their precursors are surrounded by many osteoblasts on the bone surface in vivo, it has been speculated from a morphological standpoint that osteoblasts contribute to osteoclast cytodifferentiation. Figure 2a shows the networking between OC and the other cell types. We have frequently observed numerous interconnections between monocytes and osteoblasts, and thus can assume that cell-to-cell contact of the osteoblast lineage and hematopoietic cells is necessary for inducing differentiation of OC. In particular,

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Fig. 2b shows an osteoclast with three nuclei visualized by phase-contrast light microscope (high-magnification), while Fig. 2d shows an osteoclast with characteristic dorsal microvilli examined by SEM. The morphological differences between the osteoblasts can be noted, characterized by the flat, smooth surface with some cytoplasmic processes stemming in various directions, and the rounded mononuclear monocyte cells surrounding the OC (Fig. 2c). Figure 3a, b shows the expression of TRAP on co-culture 4 days after differentiation. Clearly, TRAP staining increases after 4 days, confirming the maturation toward the osteclastogenic lineage. The subsequent group of experiments focused on the organization of actin during the osteoclast differentiation in the presence or in the absence of a dentin slice. The actin network organization in multinucleated OC is rather different than in other cell types because we can describe at least two different structures, i.e., podosomes and actin rings (Saltel et al. 2004). Figure 3c, d shows individual self-organized podosomes during osteoclast formation in OC seeded on glass. In agreement with Destaing et al. (2003), podosome clusters develop at 2 days of differentiation (Fig. 3c), evolve at the intermediate stage (3 days of differentiation) into a dynamic actin ring (Fig. 3d), and end up forming a peripheral podosome belt at 4 days after differentiation. Furthermore, in agreement with Linder and Aepfelbacher (2003), after 4 days of differentiation we observe actin stress fibers necessary to adhere to a substrate or to migrate (Fig. 3e). To better investigate the morphology of mature OC, a semi-thin section was performed analyzing the total organization of the cytoplasmic organelles (Fig. 3f). Several nuclei can be seen in the marginal region of the cell, with a large

Fig. 1 Differentiation quantification of CRL12557 and CRL 2278 cocultured at 2, 4, 7, and 10 days. Figure shows percentage polynucleated cells at different time of differentiation

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Fig. 2 Comparative morphological analysis of coculture model of CRL-12257 and CRL-2278 at 4 days of differentiation by phase contrast microscopy (a–b) and SEM (c–d). Scale bars = a–c 10 lm B: 20 lm; D: 2 lm

Fig. 3 Coculture of osteoblasts CRL 12257 with mouse monocytes CRL 2278 after 4 days of differentiation. (a, b): Rapresentative fields of the tartrate-resistant acid phosphatase (TRAP+) cells. Scale bars = 2A 100 lm; 2B 50 lm. (c): Immunostaining for actin shows a podosome clusters developed at 2 days of differentiation Bar 50 lm. (d): Dynamic actin ring forming peripheral podosome belt at 4 days of differentiation Bar 50 lm. (e): Actin stress fibers developed at 4 days of differentiation during osteoclats migration Bar 50 lm. (f): Semi-thin section of mature osteoclast at 4 days of differentiation. Bar: 10 lm

number of irregular nucleoli, numerous electrondense vesicles, and probable primary lysosomes. In a parallel group of experiments we compared the

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morphology of OC grown on glass and OC seeded on a dentin surface. Using Transmission electron microscopy (TEM) analysis, we compared the mor-

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phology of OC co-cultured on a dentin slice (Fig. 4a) with control cultures with no additive (Fig. 4d). In the osteoclast cultures we observed round TRAP positive mononuclear cells pre-osteoclasts (p-OCs) and multinuclear cells distributed on the cover slips. These mononuclear p-OCs showed smooth cell surfaces and extended pseudopodia at the basolateral cell surfaces (Fig. 2d). In the Golgi apparatus of the cell cytoplasm were observed a considerable number of mitochondria and cisterns of rough-surfaced endoplasmic reticulum (RER), and many electron-dense lysosomal bodies (Fig. 4d, e). Among the osteoclast population grown on a dentin slice, many TRAP-positive mononuclear and multinucleated cells of various size and configuration was observed (data not shown). These multinucleated OC were structurally characterized by: (a) the distribution of a large number of mitochondria (greater than in OC grown in the absence of dentin) and RER cisterns throughout the cytoplasm; (b) localization of stalks of Golgi lamellae and vesicles in the perinuclear cytoplasm; and (c) development of a ruffled border in apposition to the dentin slice, and presence of pale vacuoles and lysosomes in the cytoplasm proximal to the ruffled border

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(Fig. 4a–c). Our remarks should be considered in the context of several previous studies (Miyazaki et al. 2006), which reported that the large number of mitochondria is related to the osteoclast resorbtion activity.

Discussion Many studies of the past decade have focused on the mechanisms by which osteoblasts regulate osteoclast formation (Vesely et al. 1992; Boyle et al. 2003). Bone is continuously remodeled by both bone formation and resorption processes; this cooperative bone metabolism is tightly regulated to maintain tissue homeostasis. Following the recent commercial availability of a recombinant RANKL protein, a new technique of osteoclast generation in vitro has been described and is now routinely used that avoids the need for osteoblasts in culture. These new techniques for osteoclast generation allow the study of both osteoclast formation and resorption. Although these procedures are highly valuable in that they provide a constant supply of human OC, they are also time consuming, since

Fig. 4 Transmission electron microphotographies of coculture at 4 days of differentiation cultured in presence (a–c) or in absence (d, e) of dentin slice. (a): An active osteoclast with numerous mitochondria, vescicles, and organized ruffled border. Bar: 1 lm; (b): Detail of resorption zone showing convolute plasma (ruffled border) membrane of active osteoclast. Bar 2 lm. (c) Detail of mature resorption zone showing large as well as smaller vescicles and secondary lysosomes. Bar 1 lm. (d): The cytoplasm presents mitochondria and electrondense lysosomal bodies. There are no differences in the number and in the morphology of the nuclei. Bar 1 lm. (e): Detail of microvilli on the dorsal surface on cells. Bar 2 lm

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osteoclast formation requires from 7 to 10 days in culture. As a model of osteoclastogenesis we used monocyte/macrophage cell line RAW 264.7 type CRL 2278, based on our recent study that showed this clone to undergo faster osteoclast differentiation than the other clone RAW 264.7 type TIB-71 (Nicolin et al. 2005). Furthermore, the cell co-culture described was chosen (using the CRL-2278 clone and the murine osteoblasts CRL-12257) because we considered this latter system to be more physiological compared to that induced by the addition of cytokine in culture. Our study shows that RAW 264.7 cell line co-cultured with murine osteoblasts, in the absence of exogenous factors, is able to differentiate into functional OC capable of resorbing the dentin surface. In the bone microenvironment, osteoclast formation requires the cell–cell interaction of osteoclast precursor cells with osteoblasts and can be achieved in vitro by co-culturing monocyte precursor cells with osteoblast/stromal cells. In this study we underline the importance of contact interactions between monocytes and osteoblasts confirmed by rapid differentiation of OC. In fact, mouse OC are formed within 4 days using a co-culture of hematopoietic precursors and mouse osteoblasts, whereas, a longer culture period (10 days) is required for osteoclast formation in the absence of osteoblast cells. Our results should be considered in the context of several previous studies, which have reported contrasting and sometimes opposing roles of the actin ring during the osteoclastogenesis process. In fact, some literature reports that actin rings are not seen on cells grown on a plastic surface because the resulting OC are functionally immature (Lader et al. 2001; Kirstein et al. 2006). In this study we show that a substantial presence of osteoblasts and pre-OC is necessary for osteoclast maturation in vitro. This suggests that cell–cell contact in the co-culture model might be essential to differentiate and activate functionally mature OC and to develop the actin ring in multinucleated cells seeded on glass. Moreover, we observed that the differentiation of OC can be increased by the presence of a dentin slice. In accordance with Sabokbar et al. (2001), we suggest that hydroxyapatite particles induce microenvironment factors capable of p-OC differentiation. In keeping with this hypothesis we postulate, in agreement with Saltel et al. (2004), the presence of a specific receptor for mineral crystals at the membrane of OC grown in our co-culture model. In fact, a dentin slice induces morphological changes of OC by the formation of organized ruffled-border and a large number of vesicles. Mature

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OC grown on dentin slices present a very flat outline, many vacuoles, mitochondria, and rough surface reticulum. On the contrary, OC grown on glass coverslips have a round shape, microvilli on the dorsal surface of the cell, and vesicles. In conclusion, the data obtained in this study underline the importance of inducing ostoclastogenesis in cell–cell interactions between osteoblasts and monocytes in vitro; most likely osteoblasts play a similar, key role in supporting osteoclastogenesis on bone reformation even in vivo. The differentiation and function of OC are regulated by osteoblast-derived factors such as RANKL that stimulates osteoclast formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. Our results suggest that the critical role of p-OC differentiation is not only induced by RANKL secretion, but also induced by the presence of mineralized matrix. Thus, the effects of mineralized matrix on p-OC are the stimulation of cell differentiation and modulation of cell phenotype. Moreover, we suggest that the dual role of osteoblasts, in supporting osteoclastogenesis or forming bone, might be performed by the same lineage of cells at different stages of their maturation. Finally, we propose the hypothesis that osteoblasts could express RANKL, which is a membrane-bound factor, to promote differentiation of osteoclast progenitors into OC through a mechanism involving cell-to-cell contact. The RANKL/RANK/OPG system is implicated in various skeletal and immune-mediated diseases characterized by increased bone resorption and bone loss, including several forms of osteoporosis, bone metastasis, periodontal disease, and rheumatoid arthritis. The identification of the relationship among these cells (monocyte-osteoblasts-OC) establishes new approaches for future research aimed to set up a potential pharmacological control of these cells and related tissues in health and disease. Acknowledgements The financial support comes from grants from Fondo per gli Investimenti della Ricerca di Base (FIRB) RBNEø1SP72.

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