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Isolation of osteoclasts from pagetic bone tissue morphometry and cytochemistry on isolated cells
Bone, 9, l-6 (1988) Printed in the USA. A11 rights reserved.
Copyright
8756-3282188 $3.00 + .OO 6 1988 Pergamon Press plc
Isolation of Osteoclasts from Pagetic Bone Tissue Morphometry and Cytochemistry on Isolated Cells M.F. BASLE,
P. MAZAUD,
K. MALKANI,
M.F. CHRETIEN,
Lcrhorutoire d’Histologie-Emhr??c,logir, FucultC de MPdecine, Address
for Corresnondence
Recult!e,d49045 A&ERS
and Reorints:
M.F. MOREAU,
and A. REBEL
UniversitL; d’Angers-France
M.E Basle. Laboratoire d’Histologie-Embryologie
Fact&e de Mkdecine, Rue Haute de
CCdex, FLnce
Abstract Giant osteoclasts and other ceils were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget’s disease. The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes. The number of nuclei in isolated osteoclasts (33.85 f 20.92 nuclei/osteoclast) correlates fairly well (p < 0.02) with the number of nuclei counted on histologic sections (15.88 +11.80 nuclei/osteoclast) for samples from each patient. Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes. Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteocfasts, osteoblasts and monocytes. Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages. Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin. Key Words: Paget’s disease, osteoclast-Cell Morphometry-Enzyme cytochemistry.
only osteoclasts show morphologic and immunocytologic modifications appears to be of significance in Paget’s bone disease (Basle et al., 1987). Many questions however, remain unanswered, particularly concerning the role of cellular viral infection in the onset and spread of the disease. It is not known whether the presence of the virus is a cause or a consequence of the disorder or even whether osteoclasts can communicate viral genetic information to other cells. Attempts to solve such problems have been frustrated by the nature of bone tissue which makes it difficult to study different bone cell populations separately. In our study, we have extracted and isolated osteociasts from fragments of Pagetic bone. This report presents preliminary results from observations on 8 cases of Pagefs bone disease. The aim of this work was to isolate Pagetic osteoclasts, to compare their morphology to that of in situ osteoclasts and to test their enzymatic activity. Material and Methods
isolation-
Bone in Paget’s disease is characterized by abnormal osteoclastic resorption reflected by the number and the spread of the active foci, the increase in the size of osteoclasts and the number of nuclei they contain. The giant multinucleated osteoclast in Paget’s bone disease has attracted much attention as a possible key cell in the etiology and the pathogenesis of the disorder. This approach has been substantiated by: a) the observation that the initial event is an intense focal bone resorption by large abnormal osteoclasts (Krane, 1986), b) the discovery of microcylindric inclusions in osteoclast nuclei and cytoplasm strongly resembling paramyxovirus nucleocapsids (Rebel et al., 1974; Mills and Singer, 1976): c) the demonstration of paramyxovirus antigens in Pagetic osteoclasts (Rebel et al., 1980; Mills et al., 1984; Basle et al., 1985); d) the detection of measles RNA viral sequences both in osteoclasts as well as in other bone cells (Basle et al., 1986). The finding that among bone cells containing viral information
Bone samples were obtained with a trephine or during surgery from radiologically identified Pagetic zones in 8 patients, 7 untreated patients and I patient who had received 20 mglkgiday of ethane-1. hydroxy-I, I diphosphonate (EHDP) for two months before biopsy (Obs. I, Table 1). In each cases 5 or 6 fragments of bone, 5 to 10 mm3, were fixed 48 h in a solution of 75% picric acid, 20% formaldehyde and 5% acetic acid and then decalcified for 15 days in 0. I M ethylene diamine tetra-acetic acid (EDTA, Sigma) in a 7.4 pH phosphate buffer. The bone samples were embedded in paraffin and the sections, 7 km thick, were stained with hematoxylin eosin. The number of osteoclasts per unit surface of section was determined. The mean number of nuclei in the osteoclasts was obtained by counting nuclei in 50 osteoclasts in each case. All measurements were made with a Zeiss photomicroscope using a 20 x objective. Isolation
prowdrrres
md
cytologic~ stains
The osteoclasts were isolated according to a method previously described (Hefley and Stern. 1982) with some mod-
2
M.F.
ifications. Immediately after biopsy, the bone tissue was broken down into 1 to 2 mm3 fragments, rinsed in phosphate buffer, resuspended in a solution made up of dimethylsulfoxide 10% (Sigma) in RPM1 1640 medium (Eurobio) and then gradually frozen and stored in liquid nitrogen using a liquid nitrogen cooled mill (Nicool LM IO, Compagnie Francaise des Produits Oxygen&). Before use the bone tissue fragments were slowly thawed and kept at 4°C awaiting further treatment. The isolation solution was a 7.2 pH phosphate buffer, containing 136.8 mM NaCI, 2.68 mM KCI, 4.00 mM Na2 HP04, I .47 mM KH2 PO4 and EDTA at a final concentration of 0.5 mM. Preliminary comparative trials with hyaluronidase (Flucka) at 2 mgiml, trypsin (Eurobio) at 0.25%, collagenase IA (Sigma) or II (Sigma) at I mglml and EDTA at 0.5 mM led us to prefer EDTA because of its higher efficiency and better preservation of the morphology of cells. The EDTA solution was filtered through on a 0.2 pm cellulose acetate filter (Sartorius), stored at 4°C and preheated to 37°C just before use. The isolation procedure was carried out in closed 50 ml polypropylene tubes (Falcon) placed on a rotary agitator (60 rpm) at 37°C. Each tube containing about 15 bone fragments, I to 3 mm3 in IO ml of 0.5 mM EDTA, was incubated for 45 min at 37°C. After incubation, 30 ml of 7.2 pH phosphate buffer at 37°C were added and the tubes were centrifuged for 10 min at 1000 rpm. The supernatants were discarded; the pellets were resuspended in 40 ml of 7.2 pH phosphate buffer at 37°C and centrifuged for IO min at 1000 rpm. The pellets were resuspended in 30 ml of the phosphate buffer and the tubes were then held in a vertical position for 20 to 30 set to allow sedimentation of the bone fragments. The supernatant was pelleted and resuspended in 3 ml of phosphate buffer. Trypan blue exclusion was used to test the integrity of the cells. After rinsing, 3 drops of the cell suspension were deposited on a slide and I drop of a 0.4% trypan blue solution in phosphate buffer was added. Ability of the cells to exclude the dye was observed after 5 min. Slides were prepared by cytocentrifugation (Cytospin 2 Shandon) at 600 rpm for 6 min. Two to 5 slides per case were air dried and stained with a l/l mixture of methylene blue and azure blue. The mean number of osteoclasts per slide, the number of nuclei per osteoclast and the mean diameter of each osteoclast were determined, counting at least 50 osteoclasts in each case. The observations were made on a Zeiss photomicroscope equipped with an integrating eyepiece bearing a numerical scale and a 20 x objective. Hisrochemid
staining
Bone remodeling is mainly due to osteoclasts (bone resorption) and to osteoblasts (bone formation). A considerable number of enzymes, including phosphatases, dehydrogenases and esterases, have been demonstrated in these bone cells (Vaughan 1970). The detection of enzyme activity in isolated cells was carried out on slides obtained by cytocentrifugation as described above. 1. Phosphatases were demonstrated by techniques derived from Burstone (1958, 1962). For the demonstration of alkaline phosphatase, a marker for osteoblasts, the cells were fixed 2 min at 4°C in an aqueous solution containing 4% formaldehyde and 90% methanol and incubated for 60 min at 20°C in a medium made up of Fast Blue BB salt (Sigma) and Naphthol AS-MX phosphate (Sigma) in
Bade
et al.: Isolation
of osteoclasts
from Pagetic bone
aqueous solution in pH 8.6. prepared just before use. For the demonstration of acid phosphatase, which is associated with osteoclastic resorption processes, the cells were fixed for 2 min at 4°C in an aqueous solution containing 8% formaldehyde and 50% acetone and incubated for 60 min at 20°C in a medium made up of Fast Garnet GBC salt (Fluka) and Naphthol AS-MX phosphate (Sigma) in aqueous solution at pH 5.2, prepared just before use. 2. Succino-dehydrogenase was detected according to Nachlas et al. (1957). Unfixed cells were incubated fat 15 min at 37°C in a medium made up of succinate of sodium nitrotetrazolium blue chloride (Fluka) in a 7.6 pH phosphate buffer. After rinsing in distilled water the cells were fixed in 10% formaldehyde in a 7.2 pH phosphate buffer. 3. A nonspecific esterase, aliesterase, was detected according to Loeffler and Schubert (1961). The slides were fixed for 5 min in 40%’ formol vapor, rinsed in distilled water and air-dried. They were then incubated for 60 min at 20°C in a medium prepared just before use made up of naphthyl acetate (Sigma) and pararosaniline hexazonium salt (Sigma) in a 0.2 M, 7. I pH Sorensen phosphate buffer. and then stained with Mayer’s hematoxylin (Merck). 4. Specific naphthol AS-D acetate esterase (NASDA) was identified by a technique derived from Burstone (1962). After fixation for 5 min in 40% formol vapor the slides were rinsed in distilled water, air-dried and incubated for 70 min at 20°C in a solution of naphthol AS-D acetate (Sigma) and Fast Blue BB Salt (Sigma) in a 0.2 M. 7. I pH Sorensen phosphate buffer. The addition of 35 mM sodium fluoride to the incubation medium was used to test inhibition of NASDA esterase activity in monocytes.
Results
Histology of bone sections confirmed that tissue samples had indeed been taken from zones affected by Paget’s bone disease. Observations showed characteristic lesions with irregular woven-type bone, large amounts of fibrotic tissue in the medullary spaces. absence of hematogenous clusters and considerably increased cellular bone remodeling. Resorption activity. evaluated by counting osteoclasts per unit surface, appeared increased (3.67 -C 2.60 (SD) osteoclastsimm’) but varied from one case to another (from I .2X to 8.25 osteoclastsimm’). The osteoclasts were voluminous and contained a large number of nuclei: 15.88 2 I I .80 (SD) (range 3 - 123). The morphology of the osteoclasts was fairly variable. Some were spread on the endosteal surface, closely following the irregular contours. while others had only small zones of contact with bone. Some osteoclasts, even very large ones, were found situated away from the bone surface, lying deep within fibrotic tissue, without any contact with bone, as demonstrated using serial sections. The number of isolated osteoclasts on each slide after cytocentrifugation varied greatly from sample to sample. Using identical conditions with respect to final suspension volume and number of cytocentrifuged drops per slide. we found less than IO osteoclasts per slide (cases 6 and 7). IO to 20 osteoclasts per slide (cases I, 4 and 5), more than 20 osteoclasts per slide (cases 2 and 3) and even as many as 60 to 70 osteoclasts per slide in one instance (case 8). After
M.F. Basle et al.: Isolation of osteoclasts from Pagetic bone
2 Fig. 2. After 5 min exposure to trypan blue, the dye is concentrated more in the nuclei than in the cytoplasm of the isolated osteoclast. 460 x
1 Fig. 1. Isolated Pagetic osteoclast obtained by treatment of bone sample with 05 mM EDTA. Methylene blue/Azure blue staining. 580x.
good correlation between the diameter of isolated osteoclasts and the number of nuclei they contain (p < 0.001). Enzymology
staining with a mixture of methylene blue and azure blue, most of the osteoclasts, isolated and cytocentrifuged, appeared disc shaped or ovoid. Considerable differences in osteoclast size were noted (Fig. 1). The mean diameter of isolated osteoclasts was 90.8 p,rn 2 28.39 (SD) (range 45 172 pm) and the number of nuclei was high: 33.85 rf~20.92 (SD) (range 6 - 192) (Table I). A few isolated nuclei or bits of cytoplasm without nuclei, very likely belonging to osteoclasts broken up during cytocentrifugation, were observed. The number of naked single nuclei was estimated to be about 2% of the number of nuclei in isolated osteoclasts. After 5 min exposure to trypan blue we observed that isolated osteoclasts failed to exclude the dye. The dye was found uniformly concentrated in nuclei with dark nucleoli. In cytoplasm the staining was slight and regular but after 5 min more it was concentrated in small granulations scattered throughout the cytoplasm (Fig. 2). The osteoclast nuclei were round shaped, generally smooth contoured, with a large eccentric nucleolus. There was good correlation (p < 0.02) between the number of nuclei in osteoclasts studied on histological sections and in osteoclasts isolated and cytocentrifuged on slides (Table I). There was also
Acid phosphatases were identified in osteoclasts in the form of numerous red granulations spread through the cytoplasm. In the absence of nuclear staining, the location of the multiple nuclei was easily identified because of the decreased density of the coloured spots (Fig. 3a). Fragments
Table I. The mean number ‘_ SD of nuclei per osteoclast was determined by counting nuclei in 50 osteoclasts in each case. There is a significant correlation (p < 0.02) between the number of nuclei per osteoclast in histologic sections and in isolated fractions. Mean number 2 SD of nuclei per osteoclast Obs. I
2 3 4 5
6 7 8
Histologic sections 17.32 24.70 15.30 15.18 12.96 7.68 17.08 16.88
2 10.70 2 21.70 k 6.86 k 8.59 ‘- 6.30 k 4.51 k 8.69 t 10.69
Isolated osteoclasts 30.11 46.40 41.48 28.91 35.44 19.92 32.47 36.10
k 21.17 of- 30.78 k 26.33 -+ 19.31 r 22.34 2 12.42 ” 21.63 i 18.50
Fig. 3. Acid phosphatase activity in cells isolated from Pagetic bone tissue. (a) 2 osteoclasts show numerous granulations throughout the cytoplasm. The location of nuclei is easily identified (c) 450 x (b) A mononuclear cell, probably of the monocyte line, shows acid phosphatase activity. 1300 X
M.F. Basle et al.: Isolation of osteoclasts
4
I
from Pagetic bone
’ ..
1% ‘!
4 Fig. 4. Alkaline phosphatase activity in the osteoblast, around the nucleolus and in the Golgi zone. 1200 x
of osteoclast cytoplasm, with no detectable nuclei even after nuclear staining, probably broken up during cytocentrifugation, were also acid phosphatase positive. Some mononucleated cells, perhaps monocytes, also revealed high acid phosphatase activity in the form of several, large red granulations spread uniformly over the cytoplasm (Fig. 3b). Alkaline phosphatase was not found in Pagetic osteoclasts. However, osteoblasts, recognizable by the juxtanuclear Golgi zone, showed high alkaline phosphatase activity especially concentrated around the nucleus (Fig. 4). High succinate dehydrogenase activity was demonstrated in the osteoclasts. The stain, in the form of violet granulations of various sizes, was uniformly distributed in the whole of the cytoplasm including the peripheral regions where there was no nuclei (Fig. 5). Similar staining was also observed in mononucleated cells, osteoblasts and moncytes. However, the intensity of the reaction, as judged by the density of the coloured grains, was clearly of a lesser degree in mononucleated cells than in osteoclasts. Nonspecific esterase, aliesterase, and specific naphthol AS-D acetate esterase were also identified in the osteoclasts. Nonspecific esterase activity was uniformly observed through the cytoplasm of osteoclasts and macro-
5 Fig. 5. High succinate dehydrogenase activity clearly visible in this isolated osteoclast. The nuclei are not stained but are apparent. 850 X
6 Fig. 6. Nonspecific
esterase, aliesterase, activity was detected in isolated osteoclasts an‘d macrophage-like cells. In this osteocla5t enzyme activity is identified throughout the cytoplasm. Nuclei are stained with Mayer hematoxylin 900 x
phage-like cells (Fig. 6). The naphthol AS-D acetate esterase stain appeared in the form of abundant blue granulations in some mononucleated cells (Fig. 7a) but considerably fewer granulations were observed in the osteoclasts (Fig. 7b). The addition of sodium fluoride eliminated naphthol AS-D acetate esterase activity in the osteoclasts and in the mononucleated cells (Fig. 7~).
Discussion
Several experimental techniques, such as microdissection, mechanical dispersion and enzyme digestion, have been developed for the isolation of the different cell populations found in the medullary space and at the endosteal surface of bone (Walker, 1971; Hefley and Stern, 1982; Osdoby et al., 1982; Ries and Gong, 1982; Zambonin-Zallone et al.. 1982; Wong and Kocour, 1983; Boyde et al., 1984: Chambers et al., 1984; Finkelson et al., 1984; Ries, 1984). The same methods have been applied to osteoclasts in animal bone but cannot. unfortunately, be appleid directly to human bone tissue. Indeed, most of the experiments follow stimulation of bone cell activity, by hormones or by fasting, to obtain samples rich in osteoblasts or osteoclasts. Moreover, in animal bone samples, it is possible to choose privileged zones with a high remodeling rate. such as ossification points or embryonic bone. Furthermore, the abundance of experimental bone tissue allows selective enrichment of cell suspensions by filtration or sedimentation in density gradients (Nelson and Bauer, 1977; Wong, 1982). The large amount of fibrotic tissue in the medullary space of Pagetic bone is a limiting factor in the isolation of osteoclasts. Enzyme digestion gives poor results: for example, collagenase, an enzyme particularly adapted to the digestion of fibrotic tissue, seems to have a destructive action on certain cells as well, especially on large osteoclasts , (Hefley and Stern, 1982). In normal human bone, the number of osteoclasts in histological sections is usually less than 0.20/mm2 and the mean number of nuclei per cell section is about 4.0 ? 3.0 (SD) (Rasmussen and Bordier, 1974). In Paget’s bone disease, we have observed 3.67 2 2.6 (SD) osteoclastsimm* and a mean of 15.88 f I I .80 (SD) nuclei per osteoclast section. These results are comparable with previous reports (Rasmussen and Bordier, 1974; Meunier et al.. 1980).
M.F. Basle et al.: Isolation of osteoclasts
from Pagetic bone
Fig. 7. Specific naphthol AS-D acetate esterase activity was present in isolated osteoclasts and mononuclear cells. (a) This macrophage-like cell shows numerous darkly stained granulations scattered within the cytoplasm. Note the oval shaped eccentric nucleus (t) 1175 x (b) In the osteoclast considerably fewer granulations are observed 920x. (c) The addition of sodium fluoride to the incubation medium inhibits the enzyme activity in the osteoclast 920 x
5
The increase in the number of nuclei per cell is all the more significant in osteoclasts in Paget’s disease that in normal subjects aged over 70, the mean number of nuclei per osteoclast section is only 2.25 f 1.3 (SD) (Rasmussen and Bordier, 1974). Small fragments of Pagetic bone tissue treated with EDTA produced cell suspensions made up of different cell populations including osteoblasts, monocytes, fibroblasts and osteoclasts. Since no selective enrichment could be carried out, the number of osteoclasts in the isolated cell fraction reflected the number of osteoclasts present in the bone fragments used. However we do not know whether isolation procedures provide a representative sampling of the osteoclast population of Pagetic bone. With the technique we used we observed that isolated cells, including osteoclasts, were stained by trypan blue. Such findings were also reported in experimental conditions even when the cells were obtained from freshly dissected bone fragments (Hefley and Stern, 1982). These results suggest that enzymatic and chemical isolation methods lead to cell membrane damage, probably aggravated by freezing. Mechanical isolation procedures may be expected to yield viable bone cells but it is difficult to apply these techniques to fresh bone tissue samples. The study of isolated osteoclasts provides some additional information. The size of the cells is considerable, frequently of the order of 100 km. The number of nuclei contained in each cell is very high, 33.85 on the average, a figure of course much greater than that found on histological sections. In fact, Pagetic osteoclasts with more than a hundred nuclei were often observed. Enzyme histology shows that isolated Pagetic osteoclasts contain a fairly complete set of enzymes. Most of the enzymes involved in the degradation of mineralized bone, particularly acid phosphatases, are found in the isolated osteoclasts from Paget’s bone tissue. However, we detected no alkaline phosphatase activity in these osteoclasts. Alkaline phosphatases are present in osteoblasts which are the cells associated with the synthesis of bone matrix and perhaps mineralization (Krane, 1986). Succinate dehydrogenase, which intervenes in the citric acid cycle, is normally found in bone cells (Vaughan, 1970). In Pagetic bone, this enzyme activity appears higher in osteoclasts than in mononucleated cells. However, differences in staining have been noted among the various types of bone cells. Osteoclasts stain more quickly and strongly than other cells for most dehydrogenases, and especially so for succinate dehydrogenase. Thus the time required for detection of staining with formazan is 5-10 min for osteoclasts, lo-20 min for osteoblasts and 15-30 min, for osteocytes (Vaughan, 1970). In our study, the cells were incubated for 15 min and this may explain the weak staining of mononucleated cells. Enzyme activity for nonspecific esterase, aliesterase, and for specific naphthol AS-D acetate esterase, was found in osteoclasts and in some mononucleated cells, probably of monocytic origin, isolated from Pagetic bone. In the osteoclasts, aliesterase activity, more specific to monocyte esterases, appears to be much stronger than the aotivity of naphthol AS-D acetate esterase, more specific to granulocyte esterases. Besides, the inhibition of naphthol AS-D acetate esterase by the addition of sodium fluoride shows that this low enzyme activity in Pagetic osteoclasts is due more particularly to monocyte esterases. The presence of
M.F. Basle et al.: Isolation of osteoclasts
6
been reare more abundant in osteoclasts and in some mononucleated cells, probably of the pre-osteoclast type, than in macrophages (Ries, 1984). This observation confirms an association between some monocyte lines and the multinucleated osteoclast. The fusion of mononucleated cells, possibly of monocytic origin is a currently accepted hypothesis to explain the formation of multinucleated osteoclasts (Burger et al., 1982; Marks, 1983; Severson, 1983; Zambonin-Zallone and Teti, 1985). Techniques for the isolation of cell populations from bone tissue have led to a better understanding of the contribution of the different cells towards bone remodeling and regulation. Of course, it cannot yet be claimed that the behaviour of isolated cells, maintained in culture conditions, is identical to that of bone cells in a normal physiological context. However, in certain pathological situations, such as in Paget’s bone disease, the in vitro study of isolated bone cells appears to be a promising approach to the mechanisms of abnormal osteoclast and osteoblast activity. Our work demonstrates the feasibility of isolating osteoclasts from Pagetic bone tissue and shows that the cells retain good levels of enzyme activity. Ultrastructural observations and attempts to establish osteoclast cell cultures are now under way to further this preliminary study. Cultured isolated osteoclasts would surely make an interesting in vitro model for important aspects of Paget’s bone disease. monocyte esterases ported and it would
in osteoclasts has already appear that these enzymes
from Pagetic bone
Finkelson M.D., Feldman R.S.. Sokamoto M. and Sokamoto S.: Isolation and maintenance of osteoclasts in culture. IRCS Med. Sri. 12:238-2X 1984. Hefley T.J. and Stern P.H.: Isolation of osteoclasta from fetal rat long bones. Ca/c$ Tissrrr Int. 34:480-487. 1982. Krane S.: Paget’s disease of bone. C&if. Tissue Int. 38:309-317, 1986. Loeftler H. and Schubert J.C.: Cytochemical proof of non specific esterabe\ in smears: contribution on the technic and findings in human blood smears. Klin. Wschr. 39: 1220, 1961. Marks S.C.: The origin of osteoclasts. J. Patho/. 12:226-256. 1983. Meunier P.J.. Coindre J.M.. Edouard M.C. and Arlot M.E.: Bone hiatomorphometry in Paget‘s disease. Art/t. Rhurrm. 23: 1095- 1103. 1980. Mills B.G. and Singer F.R.: Nuclear inclusion in Paget’s disease of bone Science. 194:201-202. 1976. Mills, B.G., Singer ER.. Weiner L.P.. Suffen S.C.. Stabile E. and Hol\t P.: Evidence for both respiratory ayncytial virus and measles virus antigens in osteoclasts of patients with Paget’s disease of bone. C/in. Orthop. 183:303-311. 1984. Nachlas M.M.. Tsou K.C.. De Souza E.. Cheng C.S. and Seligman A.M.. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Hisrochc~m. Cytoc~hrv~. 5:420-436. 1957. Nelson R.L.. and Bauer G.E.: Isolation of osteoclasts by velocity sedimentation at unit gravity. C&if, 7kue RPS. 22303-313. 1977. Osdoby P., Martini M.C. and Caplan A.I.: Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture. J. Erp. Zoo/. 224:331-344, 1982. Rasmussen H. and Bordier P.: Paget’s disease of bone. In: The physiological and cellular basis of metabolic bone disease. William and Wilkin\. Baltimore, 1974, pp. 292-303. Rebel A., Basle M.F., Pouplard A.. Malkani K., Filmon R. and Le Patezour A.: Bone tissue in Paget’s disease of bone. Arfh. Rheum. 23: I ItI& I 114, 1980. Rebel A., Malkani K. and Basle M.F.: Anomalies nucltaires des oat&clastes de la maladie osseuse de Paget. Noun. Prene. Med. 3:1?991301. 1974.
This work was supported by grants from the Inserm (CRL 86-4013) and the Langlois Foundation. The authors thank M. Fuentes and N. Thierry for secretarial work and P. Pilet and R. Filmon for expert technical assistance.
Acknow/edgment:
References Basle M.F., Fournier J.G., Rozenblatt S., Rebel A. and Bouteille M.: Measles virus RNA detected in Paget’s disease bone tissue by in situ hybridization. J. Gen. Viral. 67:907-913, 1986. Basle M.F., Rebel A., Fournier J.G.. Russell W.C. and Malkani K.: On the track of paramyxoviruses in Paget’s disease of bone C/in. Orthop. 217:9-15, 1987. Basle M.F., Russell W.C., Goswami K.K., Rebel A., Giraudon P.. Wild E and Filmon R.: Paramyxovirus antigens in osteoclasts from Paget’s bone tissue detected by monoclonal antibodies. J. Gcn. Vim/. 66:21032110, 1985. Boyde A., Ali N.N. and Jones S.J.: Resorption of dentine by isolated osteoclasts in vitro. Brir. Dent. J. 156:216-220. 1984. Burger E.H., Van Der Meer J.W.. Van De Gevel J.S., Gribnau J.C.. Thesingh C.W. and Van Furth R.: In vitro formation of osteoclasts from long term cultures of bone marrow mononuclear phagocytes. J. Exp. Med. 156: 16@4- 1614, 1982 Burstone M.S.: Histochemical demonstration of acid phosphatases with naphthol AS-phosphates. J. Nut/. Cancer. Inst. 21:523-539, 1958. Burstone M.S.: Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York, 1962, p. 513. Chambers T.J., Revel1 P.A., Fuller K. and Athanasou N.A.: Resorption of bone by isolated rabbit osteoclasts. J. Cell. Sci. 66:383-399. 1984.
Ries W.L.: Osteogenic periosteum esterase activity: A comparative morphological and cytochemical study of bone cells in situ. on rat proximal tibia and in smears. J. Histochem. Cytochm. 32:55-62. 1984. Ries W.L.. and Gong J.K.: A comparative study of osteoclasts: In situ versus smear specimens. Amt. Rec. 203:221-232, 1982. Severson A.R.: Differentiation of mononuclear cells into multinucleated osteoclast-like cells. E.rp. Cc//. Rio/. 51:267-274. 1983. Vaughan J.M.: Enzymes of bone and cartilage. The Physiology of Bone, Clarendon Press, Oxford, 1970. pp. 246-253. Walker D.G.: Enzymatic and electron microscopic analysis of isolated o\teoclasts. C&if. Es. RES. 9:296-309. 1971. Wong G.L.: Characteristic5 of subpopulation of OC and OB bone cells obtained by sedimentation at unit gravity. Co/c(f. Tissrrr Int. 34:67-75. 1982. Wong G.L. and Kocour B.A.: Differential serum dependence of cultured osteoclastic and osteoblastic bone cells. C&if. Tissue Irlt. 35:778-782. 1983. Zambonin-Zallone A. and Teti A.: Autoradiographic demonstration of m vitro fusion of blood monocytes with osteoclasts. Bus. Appl. Hisfc~them. 29:45-48,
1985.
Zambonin-Zallone A., Teti A.. and Primavera M.V.: Isolated osteoclaata m primary culture: first observations on structure and survival in culture media. Amu. EmhryL 165:405-413, 1982.
Receiwd: April 7, 1987 Revised: August 12, 1987 Accepred: August 18. 1987
Copyright
8756-3282188 $3.00 + .OO 6 1988 Pergamon Press plc
Isolation of Osteoclasts from Pagetic Bone Tissue Morphometry and Cytochemistry on Isolated Cells M.F. BASLE,
P. MAZAUD,
K. MALKANI,
M.F. CHRETIEN,
Lcrhorutoire d’Histologie-Emhr??c,logir, FucultC de MPdecine, Address
for Corresnondence
Recult!e,d49045 A&ERS
and Reorints:
M.F. MOREAU,
and A. REBEL
UniversitL; d’Angers-France
M.E Basle. Laboratoire d’Histologie-Embryologie
Fact&e de Mkdecine, Rue Haute de
CCdex, FLnce
Abstract Giant osteoclasts and other ceils were isolated from Pagetic bone tissue using 0.5 mM ethylene diamine tetraacetic acid on bone samples from 8 patients with Paget’s disease. The cell suspension contained osteoclasts and osteoblasts as well as some mononuclear cells such as monocytes. The number of nuclei in isolated osteoclasts (33.85 f 20.92 nuclei/osteoclast) correlates fairly well (p < 0.02) with the number of nuclei counted on histologic sections (15.88 +11.80 nuclei/osteoclast) for samples from each patient. Enzyme histochemistry demonstrated acid phosphatase activity in isolated osteoclasts and in mononucleated cells, such as monocytes. Alkaline phosphatase was detected only in osteoblasts while succinate dehydrogenase was observed in osteocfasts, osteoblasts and monocytes. Esterases, such as nonspecific aliesterase and specific naphthol AS-D acetate esterase, were identified in osteoclasts and in macrophages. Inhibition of specific naphthol AS-D acetate esterase in osteoclasts by addition of sodium fluoride suggests that the enzyme could be of monocytic origin. Key Words: Paget’s disease, osteoclast-Cell Morphometry-Enzyme cytochemistry.
only osteoclasts show morphologic and immunocytologic modifications appears to be of significance in Paget’s bone disease (Basle et al., 1987). Many questions however, remain unanswered, particularly concerning the role of cellular viral infection in the onset and spread of the disease. It is not known whether the presence of the virus is a cause or a consequence of the disorder or even whether osteoclasts can communicate viral genetic information to other cells. Attempts to solve such problems have been frustrated by the nature of bone tissue which makes it difficult to study different bone cell populations separately. In our study, we have extracted and isolated osteociasts from fragments of Pagetic bone. This report presents preliminary results from observations on 8 cases of Pagefs bone disease. The aim of this work was to isolate Pagetic osteoclasts, to compare their morphology to that of in situ osteoclasts and to test their enzymatic activity. Material and Methods
isolation-
Bone in Paget’s disease is characterized by abnormal osteoclastic resorption reflected by the number and the spread of the active foci, the increase in the size of osteoclasts and the number of nuclei they contain. The giant multinucleated osteoclast in Paget’s bone disease has attracted much attention as a possible key cell in the etiology and the pathogenesis of the disorder. This approach has been substantiated by: a) the observation that the initial event is an intense focal bone resorption by large abnormal osteoclasts (Krane, 1986), b) the discovery of microcylindric inclusions in osteoclast nuclei and cytoplasm strongly resembling paramyxovirus nucleocapsids (Rebel et al., 1974; Mills and Singer, 1976): c) the demonstration of paramyxovirus antigens in Pagetic osteoclasts (Rebel et al., 1980; Mills et al., 1984; Basle et al., 1985); d) the detection of measles RNA viral sequences both in osteoclasts as well as in other bone cells (Basle et al., 1986). The finding that among bone cells containing viral information
Bone samples were obtained with a trephine or during surgery from radiologically identified Pagetic zones in 8 patients, 7 untreated patients and I patient who had received 20 mglkgiday of ethane-1. hydroxy-I, I diphosphonate (EHDP) for two months before biopsy (Obs. I, Table 1). In each cases 5 or 6 fragments of bone, 5 to 10 mm3, were fixed 48 h in a solution of 75% picric acid, 20% formaldehyde and 5% acetic acid and then decalcified for 15 days in 0. I M ethylene diamine tetra-acetic acid (EDTA, Sigma) in a 7.4 pH phosphate buffer. The bone samples were embedded in paraffin and the sections, 7 km thick, were stained with hematoxylin eosin. The number of osteoclasts per unit surface of section was determined. The mean number of nuclei in the osteoclasts was obtained by counting nuclei in 50 osteoclasts in each case. All measurements were made with a Zeiss photomicroscope using a 20 x objective. Isolation
prowdrrres
md
cytologic~ stains
The osteoclasts were isolated according to a method previously described (Hefley and Stern. 1982) with some mod-
2
M.F.
ifications. Immediately after biopsy, the bone tissue was broken down into 1 to 2 mm3 fragments, rinsed in phosphate buffer, resuspended in a solution made up of dimethylsulfoxide 10% (Sigma) in RPM1 1640 medium (Eurobio) and then gradually frozen and stored in liquid nitrogen using a liquid nitrogen cooled mill (Nicool LM IO, Compagnie Francaise des Produits Oxygen&). Before use the bone tissue fragments were slowly thawed and kept at 4°C awaiting further treatment. The isolation solution was a 7.2 pH phosphate buffer, containing 136.8 mM NaCI, 2.68 mM KCI, 4.00 mM Na2 HP04, I .47 mM KH2 PO4 and EDTA at a final concentration of 0.5 mM. Preliminary comparative trials with hyaluronidase (Flucka) at 2 mgiml, trypsin (Eurobio) at 0.25%, collagenase IA (Sigma) or II (Sigma) at I mglml and EDTA at 0.5 mM led us to prefer EDTA because of its higher efficiency and better preservation of the morphology of cells. The EDTA solution was filtered through on a 0.2 pm cellulose acetate filter (Sartorius), stored at 4°C and preheated to 37°C just before use. The isolation procedure was carried out in closed 50 ml polypropylene tubes (Falcon) placed on a rotary agitator (60 rpm) at 37°C. Each tube containing about 15 bone fragments, I to 3 mm3 in IO ml of 0.5 mM EDTA, was incubated for 45 min at 37°C. After incubation, 30 ml of 7.2 pH phosphate buffer at 37°C were added and the tubes were centrifuged for 10 min at 1000 rpm. The supernatants were discarded; the pellets were resuspended in 40 ml of 7.2 pH phosphate buffer at 37°C and centrifuged for IO min at 1000 rpm. The pellets were resuspended in 30 ml of the phosphate buffer and the tubes were then held in a vertical position for 20 to 30 set to allow sedimentation of the bone fragments. The supernatant was pelleted and resuspended in 3 ml of phosphate buffer. Trypan blue exclusion was used to test the integrity of the cells. After rinsing, 3 drops of the cell suspension were deposited on a slide and I drop of a 0.4% trypan blue solution in phosphate buffer was added. Ability of the cells to exclude the dye was observed after 5 min. Slides were prepared by cytocentrifugation (Cytospin 2 Shandon) at 600 rpm for 6 min. Two to 5 slides per case were air dried and stained with a l/l mixture of methylene blue and azure blue. The mean number of osteoclasts per slide, the number of nuclei per osteoclast and the mean diameter of each osteoclast were determined, counting at least 50 osteoclasts in each case. The observations were made on a Zeiss photomicroscope equipped with an integrating eyepiece bearing a numerical scale and a 20 x objective. Hisrochemid
staining
Bone remodeling is mainly due to osteoclasts (bone resorption) and to osteoblasts (bone formation). A considerable number of enzymes, including phosphatases, dehydrogenases and esterases, have been demonstrated in these bone cells (Vaughan 1970). The detection of enzyme activity in isolated cells was carried out on slides obtained by cytocentrifugation as described above. 1. Phosphatases were demonstrated by techniques derived from Burstone (1958, 1962). For the demonstration of alkaline phosphatase, a marker for osteoblasts, the cells were fixed 2 min at 4°C in an aqueous solution containing 4% formaldehyde and 90% methanol and incubated for 60 min at 20°C in a medium made up of Fast Blue BB salt (Sigma) and Naphthol AS-MX phosphate (Sigma) in
Bade
et al.: Isolation
of osteoclasts
from Pagetic bone
aqueous solution in pH 8.6. prepared just before use. For the demonstration of acid phosphatase, which is associated with osteoclastic resorption processes, the cells were fixed for 2 min at 4°C in an aqueous solution containing 8% formaldehyde and 50% acetone and incubated for 60 min at 20°C in a medium made up of Fast Garnet GBC salt (Fluka) and Naphthol AS-MX phosphate (Sigma) in aqueous solution at pH 5.2, prepared just before use. 2. Succino-dehydrogenase was detected according to Nachlas et al. (1957). Unfixed cells were incubated fat 15 min at 37°C in a medium made up of succinate of sodium nitrotetrazolium blue chloride (Fluka) in a 7.6 pH phosphate buffer. After rinsing in distilled water the cells were fixed in 10% formaldehyde in a 7.2 pH phosphate buffer. 3. A nonspecific esterase, aliesterase, was detected according to Loeffler and Schubert (1961). The slides were fixed for 5 min in 40%’ formol vapor, rinsed in distilled water and air-dried. They were then incubated for 60 min at 20°C in a medium prepared just before use made up of naphthyl acetate (Sigma) and pararosaniline hexazonium salt (Sigma) in a 0.2 M, 7. I pH Sorensen phosphate buffer. and then stained with Mayer’s hematoxylin (Merck). 4. Specific naphthol AS-D acetate esterase (NASDA) was identified by a technique derived from Burstone (1962). After fixation for 5 min in 40% formol vapor the slides were rinsed in distilled water, air-dried and incubated for 70 min at 20°C in a solution of naphthol AS-D acetate (Sigma) and Fast Blue BB Salt (Sigma) in a 0.2 M. 7. I pH Sorensen phosphate buffer. The addition of 35 mM sodium fluoride to the incubation medium was used to test inhibition of NASDA esterase activity in monocytes.
Results
Histology of bone sections confirmed that tissue samples had indeed been taken from zones affected by Paget’s bone disease. Observations showed characteristic lesions with irregular woven-type bone, large amounts of fibrotic tissue in the medullary spaces. absence of hematogenous clusters and considerably increased cellular bone remodeling. Resorption activity. evaluated by counting osteoclasts per unit surface, appeared increased (3.67 -C 2.60 (SD) osteoclastsimm’) but varied from one case to another (from I .2X to 8.25 osteoclastsimm’). The osteoclasts were voluminous and contained a large number of nuclei: 15.88 2 I I .80 (SD) (range 3 - 123). The morphology of the osteoclasts was fairly variable. Some were spread on the endosteal surface, closely following the irregular contours. while others had only small zones of contact with bone. Some osteoclasts, even very large ones, were found situated away from the bone surface, lying deep within fibrotic tissue, without any contact with bone, as demonstrated using serial sections. The number of isolated osteoclasts on each slide after cytocentrifugation varied greatly from sample to sample. Using identical conditions with respect to final suspension volume and number of cytocentrifuged drops per slide. we found less than IO osteoclasts per slide (cases 6 and 7). IO to 20 osteoclasts per slide (cases I, 4 and 5), more than 20 osteoclasts per slide (cases 2 and 3) and even as many as 60 to 70 osteoclasts per slide in one instance (case 8). After
M.F. Basle et al.: Isolation of osteoclasts from Pagetic bone
2 Fig. 2. After 5 min exposure to trypan blue, the dye is concentrated more in the nuclei than in the cytoplasm of the isolated osteoclast. 460 x
1 Fig. 1. Isolated Pagetic osteoclast obtained by treatment of bone sample with 05 mM EDTA. Methylene blue/Azure blue staining. 580x.
good correlation between the diameter of isolated osteoclasts and the number of nuclei they contain (p < 0.001). Enzymology
staining with a mixture of methylene blue and azure blue, most of the osteoclasts, isolated and cytocentrifuged, appeared disc shaped or ovoid. Considerable differences in osteoclast size were noted (Fig. 1). The mean diameter of isolated osteoclasts was 90.8 p,rn 2 28.39 (SD) (range 45 172 pm) and the number of nuclei was high: 33.85 rf~20.92 (SD) (range 6 - 192) (Table I). A few isolated nuclei or bits of cytoplasm without nuclei, very likely belonging to osteoclasts broken up during cytocentrifugation, were observed. The number of naked single nuclei was estimated to be about 2% of the number of nuclei in isolated osteoclasts. After 5 min exposure to trypan blue we observed that isolated osteoclasts failed to exclude the dye. The dye was found uniformly concentrated in nuclei with dark nucleoli. In cytoplasm the staining was slight and regular but after 5 min more it was concentrated in small granulations scattered throughout the cytoplasm (Fig. 2). The osteoclast nuclei were round shaped, generally smooth contoured, with a large eccentric nucleolus. There was good correlation (p < 0.02) between the number of nuclei in osteoclasts studied on histological sections and in osteoclasts isolated and cytocentrifuged on slides (Table I). There was also
Acid phosphatases were identified in osteoclasts in the form of numerous red granulations spread through the cytoplasm. In the absence of nuclear staining, the location of the multiple nuclei was easily identified because of the decreased density of the coloured spots (Fig. 3a). Fragments
Table I. The mean number ‘_ SD of nuclei per osteoclast was determined by counting nuclei in 50 osteoclasts in each case. There is a significant correlation (p < 0.02) between the number of nuclei per osteoclast in histologic sections and in isolated fractions. Mean number 2 SD of nuclei per osteoclast Obs. I
2 3 4 5
6 7 8
Histologic sections 17.32 24.70 15.30 15.18 12.96 7.68 17.08 16.88
2 10.70 2 21.70 k 6.86 k 8.59 ‘- 6.30 k 4.51 k 8.69 t 10.69
Isolated osteoclasts 30.11 46.40 41.48 28.91 35.44 19.92 32.47 36.10
k 21.17 of- 30.78 k 26.33 -+ 19.31 r 22.34 2 12.42 ” 21.63 i 18.50
Fig. 3. Acid phosphatase activity in cells isolated from Pagetic bone tissue. (a) 2 osteoclasts show numerous granulations throughout the cytoplasm. The location of nuclei is easily identified (c) 450 x (b) A mononuclear cell, probably of the monocyte line, shows acid phosphatase activity. 1300 X
M.F. Basle et al.: Isolation of osteoclasts
4
I
from Pagetic bone
’ ..
1% ‘!
4 Fig. 4. Alkaline phosphatase activity in the osteoblast, around the nucleolus and in the Golgi zone. 1200 x
of osteoclast cytoplasm, with no detectable nuclei even after nuclear staining, probably broken up during cytocentrifugation, were also acid phosphatase positive. Some mononucleated cells, perhaps monocytes, also revealed high acid phosphatase activity in the form of several, large red granulations spread uniformly over the cytoplasm (Fig. 3b). Alkaline phosphatase was not found in Pagetic osteoclasts. However, osteoblasts, recognizable by the juxtanuclear Golgi zone, showed high alkaline phosphatase activity especially concentrated around the nucleus (Fig. 4). High succinate dehydrogenase activity was demonstrated in the osteoclasts. The stain, in the form of violet granulations of various sizes, was uniformly distributed in the whole of the cytoplasm including the peripheral regions where there was no nuclei (Fig. 5). Similar staining was also observed in mononucleated cells, osteoblasts and moncytes. However, the intensity of the reaction, as judged by the density of the coloured grains, was clearly of a lesser degree in mononucleated cells than in osteoclasts. Nonspecific esterase, aliesterase, and specific naphthol AS-D acetate esterase were also identified in the osteoclasts. Nonspecific esterase activity was uniformly observed through the cytoplasm of osteoclasts and macro-
5 Fig. 5. High succinate dehydrogenase activity clearly visible in this isolated osteoclast. The nuclei are not stained but are apparent. 850 X
6 Fig. 6. Nonspecific
esterase, aliesterase, activity was detected in isolated osteoclasts an‘d macrophage-like cells. In this osteocla5t enzyme activity is identified throughout the cytoplasm. Nuclei are stained with Mayer hematoxylin 900 x
phage-like cells (Fig. 6). The naphthol AS-D acetate esterase stain appeared in the form of abundant blue granulations in some mononucleated cells (Fig. 7a) but considerably fewer granulations were observed in the osteoclasts (Fig. 7b). The addition of sodium fluoride eliminated naphthol AS-D acetate esterase activity in the osteoclasts and in the mononucleated cells (Fig. 7~).
Discussion
Several experimental techniques, such as microdissection, mechanical dispersion and enzyme digestion, have been developed for the isolation of the different cell populations found in the medullary space and at the endosteal surface of bone (Walker, 1971; Hefley and Stern, 1982; Osdoby et al., 1982; Ries and Gong, 1982; Zambonin-Zallone et al.. 1982; Wong and Kocour, 1983; Boyde et al., 1984: Chambers et al., 1984; Finkelson et al., 1984; Ries, 1984). The same methods have been applied to osteoclasts in animal bone but cannot. unfortunately, be appleid directly to human bone tissue. Indeed, most of the experiments follow stimulation of bone cell activity, by hormones or by fasting, to obtain samples rich in osteoblasts or osteoclasts. Moreover, in animal bone samples, it is possible to choose privileged zones with a high remodeling rate. such as ossification points or embryonic bone. Furthermore, the abundance of experimental bone tissue allows selective enrichment of cell suspensions by filtration or sedimentation in density gradients (Nelson and Bauer, 1977; Wong, 1982). The large amount of fibrotic tissue in the medullary space of Pagetic bone is a limiting factor in the isolation of osteoclasts. Enzyme digestion gives poor results: for example, collagenase, an enzyme particularly adapted to the digestion of fibrotic tissue, seems to have a destructive action on certain cells as well, especially on large osteoclasts , (Hefley and Stern, 1982). In normal human bone, the number of osteoclasts in histological sections is usually less than 0.20/mm2 and the mean number of nuclei per cell section is about 4.0 ? 3.0 (SD) (Rasmussen and Bordier, 1974). In Paget’s bone disease, we have observed 3.67 2 2.6 (SD) osteoclastsimm* and a mean of 15.88 f I I .80 (SD) nuclei per osteoclast section. These results are comparable with previous reports (Rasmussen and Bordier, 1974; Meunier et al.. 1980).
M.F. Basle et al.: Isolation of osteoclasts
from Pagetic bone
Fig. 7. Specific naphthol AS-D acetate esterase activity was present in isolated osteoclasts and mononuclear cells. (a) This macrophage-like cell shows numerous darkly stained granulations scattered within the cytoplasm. Note the oval shaped eccentric nucleus (t) 1175 x (b) In the osteoclast considerably fewer granulations are observed 920x. (c) The addition of sodium fluoride to the incubation medium inhibits the enzyme activity in the osteoclast 920 x
5
The increase in the number of nuclei per cell is all the more significant in osteoclasts in Paget’s disease that in normal subjects aged over 70, the mean number of nuclei per osteoclast section is only 2.25 f 1.3 (SD) (Rasmussen and Bordier, 1974). Small fragments of Pagetic bone tissue treated with EDTA produced cell suspensions made up of different cell populations including osteoblasts, monocytes, fibroblasts and osteoclasts. Since no selective enrichment could be carried out, the number of osteoclasts in the isolated cell fraction reflected the number of osteoclasts present in the bone fragments used. However we do not know whether isolation procedures provide a representative sampling of the osteoclast population of Pagetic bone. With the technique we used we observed that isolated cells, including osteoclasts, were stained by trypan blue. Such findings were also reported in experimental conditions even when the cells were obtained from freshly dissected bone fragments (Hefley and Stern, 1982). These results suggest that enzymatic and chemical isolation methods lead to cell membrane damage, probably aggravated by freezing. Mechanical isolation procedures may be expected to yield viable bone cells but it is difficult to apply these techniques to fresh bone tissue samples. The study of isolated osteoclasts provides some additional information. The size of the cells is considerable, frequently of the order of 100 km. The number of nuclei contained in each cell is very high, 33.85 on the average, a figure of course much greater than that found on histological sections. In fact, Pagetic osteoclasts with more than a hundred nuclei were often observed. Enzyme histology shows that isolated Pagetic osteoclasts contain a fairly complete set of enzymes. Most of the enzymes involved in the degradation of mineralized bone, particularly acid phosphatases, are found in the isolated osteoclasts from Paget’s bone tissue. However, we detected no alkaline phosphatase activity in these osteoclasts. Alkaline phosphatases are present in osteoblasts which are the cells associated with the synthesis of bone matrix and perhaps mineralization (Krane, 1986). Succinate dehydrogenase, which intervenes in the citric acid cycle, is normally found in bone cells (Vaughan, 1970). In Pagetic bone, this enzyme activity appears higher in osteoclasts than in mononucleated cells. However, differences in staining have been noted among the various types of bone cells. Osteoclasts stain more quickly and strongly than other cells for most dehydrogenases, and especially so for succinate dehydrogenase. Thus the time required for detection of staining with formazan is 5-10 min for osteoclasts, lo-20 min for osteoblasts and 15-30 min, for osteocytes (Vaughan, 1970). In our study, the cells were incubated for 15 min and this may explain the weak staining of mononucleated cells. Enzyme activity for nonspecific esterase, aliesterase, and for specific naphthol AS-D acetate esterase, was found in osteoclasts and in some mononucleated cells, probably of monocytic origin, isolated from Pagetic bone. In the osteoclasts, aliesterase activity, more specific to monocyte esterases, appears to be much stronger than the aotivity of naphthol AS-D acetate esterase, more specific to granulocyte esterases. Besides, the inhibition of naphthol AS-D acetate esterase by the addition of sodium fluoride shows that this low enzyme activity in Pagetic osteoclasts is due more particularly to monocyte esterases. The presence of
M.F. Basle et al.: Isolation of osteoclasts
6
been reare more abundant in osteoclasts and in some mononucleated cells, probably of the pre-osteoclast type, than in macrophages (Ries, 1984). This observation confirms an association between some monocyte lines and the multinucleated osteoclast. The fusion of mononucleated cells, possibly of monocytic origin is a currently accepted hypothesis to explain the formation of multinucleated osteoclasts (Burger et al., 1982; Marks, 1983; Severson, 1983; Zambonin-Zallone and Teti, 1985). Techniques for the isolation of cell populations from bone tissue have led to a better understanding of the contribution of the different cells towards bone remodeling and regulation. Of course, it cannot yet be claimed that the behaviour of isolated cells, maintained in culture conditions, is identical to that of bone cells in a normal physiological context. However, in certain pathological situations, such as in Paget’s bone disease, the in vitro study of isolated bone cells appears to be a promising approach to the mechanisms of abnormal osteoclast and osteoblast activity. Our work demonstrates the feasibility of isolating osteoclasts from Pagetic bone tissue and shows that the cells retain good levels of enzyme activity. Ultrastructural observations and attempts to establish osteoclast cell cultures are now under way to further this preliminary study. Cultured isolated osteoclasts would surely make an interesting in vitro model for important aspects of Paget’s bone disease. monocyte esterases ported and it would
in osteoclasts has already appear that these enzymes
from Pagetic bone
Finkelson M.D., Feldman R.S.. Sokamoto M. and Sokamoto S.: Isolation and maintenance of osteoclasts in culture. IRCS Med. Sri. 12:238-2X 1984. Hefley T.J. and Stern P.H.: Isolation of osteoclasta from fetal rat long bones. Ca/c$ Tissrrr Int. 34:480-487. 1982. Krane S.: Paget’s disease of bone. C&if. Tissue Int. 38:309-317, 1986. Loeftler H. and Schubert J.C.: Cytochemical proof of non specific esterabe\ in smears: contribution on the technic and findings in human blood smears. Klin. Wschr. 39: 1220, 1961. Marks S.C.: The origin of osteoclasts. J. Patho/. 12:226-256. 1983. Meunier P.J.. Coindre J.M.. Edouard M.C. and Arlot M.E.: Bone hiatomorphometry in Paget‘s disease. Art/t. Rhurrm. 23: 1095- 1103. 1980. Mills B.G. and Singer F.R.: Nuclear inclusion in Paget’s disease of bone Science. 194:201-202. 1976. Mills, B.G., Singer ER.. Weiner L.P.. Suffen S.C.. Stabile E. and Hol\t P.: Evidence for both respiratory ayncytial virus and measles virus antigens in osteoclasts of patients with Paget’s disease of bone. C/in. Orthop. 183:303-311. 1984. Nachlas M.M.. Tsou K.C.. De Souza E.. Cheng C.S. and Seligman A.M.. Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Hisrochc~m. Cytoc~hrv~. 5:420-436. 1957. Nelson R.L.. and Bauer G.E.: Isolation of osteoclasts by velocity sedimentation at unit gravity. C&if, 7kue RPS. 22303-313. 1977. Osdoby P., Martini M.C. and Caplan A.I.: Isolated osteoclasts and their presumed progenitor cells, the monocyte, in culture. J. Erp. Zoo/. 224:331-344, 1982. Rasmussen H. and Bordier P.: Paget’s disease of bone. In: The physiological and cellular basis of metabolic bone disease. William and Wilkin\. Baltimore, 1974, pp. 292-303. Rebel A., Basle M.F., Pouplard A.. Malkani K., Filmon R. and Le Patezour A.: Bone tissue in Paget’s disease of bone. Arfh. Rheum. 23: I ItI& I 114, 1980. Rebel A., Malkani K. and Basle M.F.: Anomalies nucltaires des oat&clastes de la maladie osseuse de Paget. Noun. Prene. Med. 3:1?991301. 1974.
This work was supported by grants from the Inserm (CRL 86-4013) and the Langlois Foundation. The authors thank M. Fuentes and N. Thierry for secretarial work and P. Pilet and R. Filmon for expert technical assistance.
Acknow/edgment:
References Basle M.F., Fournier J.G., Rozenblatt S., Rebel A. and Bouteille M.: Measles virus RNA detected in Paget’s disease bone tissue by in situ hybridization. J. Gen. Viral. 67:907-913, 1986. Basle M.F., Rebel A., Fournier J.G.. Russell W.C. and Malkani K.: On the track of paramyxoviruses in Paget’s disease of bone C/in. Orthop. 217:9-15, 1987. Basle M.F., Russell W.C., Goswami K.K., Rebel A., Giraudon P.. Wild E and Filmon R.: Paramyxovirus antigens in osteoclasts from Paget’s bone tissue detected by monoclonal antibodies. J. Gcn. Vim/. 66:21032110, 1985. Boyde A., Ali N.N. and Jones S.J.: Resorption of dentine by isolated osteoclasts in vitro. Brir. Dent. J. 156:216-220. 1984. Burger E.H., Van Der Meer J.W.. Van De Gevel J.S., Gribnau J.C.. Thesingh C.W. and Van Furth R.: In vitro formation of osteoclasts from long term cultures of bone marrow mononuclear phagocytes. J. Exp. Med. 156: 16@4- 1614, 1982 Burstone M.S.: Histochemical demonstration of acid phosphatases with naphthol AS-phosphates. J. Nut/. Cancer. Inst. 21:523-539, 1958. Burstone M.S.: Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York, 1962, p. 513. Chambers T.J., Revel1 P.A., Fuller K. and Athanasou N.A.: Resorption of bone by isolated rabbit osteoclasts. J. Cell. Sci. 66:383-399. 1984.
Ries W.L.: Osteogenic periosteum esterase activity: A comparative morphological and cytochemical study of bone cells in situ. on rat proximal tibia and in smears. J. Histochem. Cytochm. 32:55-62. 1984. Ries W.L.. and Gong J.K.: A comparative study of osteoclasts: In situ versus smear specimens. Amt. Rec. 203:221-232, 1982. Severson A.R.: Differentiation of mononuclear cells into multinucleated osteoclast-like cells. E.rp. Cc//. Rio/. 51:267-274. 1983. Vaughan J.M.: Enzymes of bone and cartilage. The Physiology of Bone, Clarendon Press, Oxford, 1970. pp. 246-253. Walker D.G.: Enzymatic and electron microscopic analysis of isolated o\teoclasts. C&if. Es. RES. 9:296-309. 1971. Wong G.L.: Characteristic5 of subpopulation of OC and OB bone cells obtained by sedimentation at unit gravity. Co/c(f. Tissrrr Int. 34:67-75. 1982. Wong G.L. and Kocour B.A.: Differential serum dependence of cultured osteoclastic and osteoblastic bone cells. C&if. Tissue Irlt. 35:778-782. 1983. Zambonin-Zallone A. and Teti A.: Autoradiographic demonstration of m vitro fusion of blood monocytes with osteoclasts. Bus. Appl. Hisfc~them. 29:45-48,
1985.
Zambonin-Zallone A., Teti A.. and Primavera M.V.: Isolated osteoclaata m primary culture: first observations on structure and survival in culture media. Amu. EmhryL 165:405-413, 1982.
Receiwd: April 7, 1987 Revised: August 12, 1987 Accepred: August 18. 1987