GENERATION OF OSTEOCLASTS IN VITRO - Journal of Cell Science

J. Cell Sci. 47, 127-137 (1981) 127Printed in Great Britain © Company of Biologists Limited 1981

GENERATION OF OSTEOCLASTS IN VITRO

N. G. TESTA, T. D. ALLEN, L. G. LAJTHAPaterson Laboratories, Christie Hospital and Holt Radium Institute, Withington,Manchester M20 9BX, U.K.

D. ONIONS AND O. JARRETUniversity of Glasgow, Veterinary School, Glasgow, U.K.

INTRODUCTION

Multinucleate osteoclasts, the classic cells implicated in bone resorption, have beenelusive cells to study. Although there is general agreement that osteoclasts are derivedfrom haemopoietic stem cells (Owen, 1978; Loutit & Nisbet, 1979) the formaldemonstration of such origin has only recently been achieved (Ash, Loutit & Town-send, 1980). Information on these cells has been limited by the experimental techni-ques available (Addison, 1978; Chambers, 1978). In vitro systems in which osteo-clasts harvested from fresh tissue could survive for a limited period have been used(Chambers, 1979)- However, the generation of osteoclasts in vitro has not been re-ported. In vitro systems developed in the last few years have resulted in remarkableadvances in our understanding of the processes which regulate the proliferation,maturation and function of other haemopoietic cells. Functionally normal granu-locytes, macrophages and erythrocytes can now be grown in culture (Bradley &Metcalf, 1966; Stephenson, Axelrad, McLeod & Shreeve, 1971). We describe here abone marrow culture technique which allows the formation and maintainance of cellswith the characteristics of osteoclasts in vitro for several weeks and which can beused to study their formation, physiology and fate.

MATERIALS AND METHODS

Bone marrow cultures

Cats from the colony maintained at the Veterinary School, University of Glasgow, werekilled by an injection of sodium barbiturate. The femurs were dissected and after cutting theephiphyses, the bone marrow cells were flushed out using alpha medium plus 2 % foetal calfserum (FCS). Dispersed cell suspensions were prepared by repeated pippetting and thecultures were established by inoculating into tissue culture flasks (Sterilin) io7 cells in 10 ml ofalpha medium (Gibco Biocult) supplemented with 30 % horse serum (Flow Laboratories),500 U per ml of penicillin and 50 fig per ml of streptomycin. The cultures were gassed withS % COa in air and incubated at 37 °C. At weekly intervals, they were fed by replacing half thegrowth medium with fresh medium. Cells in suspension, removed together with the growthmedium, were not replaced.

Microscopical examination

For optical microscopy, the cells attached to the surface of the culture flasks were rinsedtwice with saline, fixed with absolute methanol and stained with May Grunwald-Giemsa.Acid phosphatase staining was performed as reported by Li, Yam & Lam (1970).

128 N. G. Testa and others

For both scanning and transmission electron microscopy cells were fixed in situ in 3 %glutaraldehyde in M/15 Sorenson's buffer (pH 7-2) for 1 h, followed by 3 brief buffer washesand postfixation in 1 % OsO4 in the same buffer for 1 h.

After 3 further washes areas of one culture vessel were selected and bored out with a gentlyheated 20-mm diameter cork borer, dehydrated and critical-point dried with COa direct fromabsolute ethanol. The plastic disks were then mounted on stubs, sputter coated with 20 nm ofgold and viewed in a Cambridge S4-10 scanning electron microscope at an accelerating voltageof 20 kV.

For transmission electron microscopy, the other flask of the pair was dehydrated andembedded in situ, using HPMA as the resin solvent in place of propylene oxide to avoiddissolution of the plastic. The Epon was polymerized in situ and subsequently snapped freefrom the culture vessel. Sections were cut either perpendicular to, or parallel with the growingsurface. The sections were stained with uranyl acetate and lead citrate and viewed in anAEI 801A TEM at an accelerating voltage of 80 kV.

Virus assays

Bone marrow cells were assayed for the presence of Feline Syncitium Forming Virus(FSFV) by syncitium formation on feline embryo cells (FEA). One million nucleated bonemarrow cells were plated in 4 ml Eagle's MEM plus 10% FCS on to subconfiuent FEAcultures in 5-cm plates. After 48 h the non-adherent bone marrow cells were removed and thecultures were refed. The FEA cultures were passaged when confluent and were examinedtwice a week for 3 weeks for the presence of syncitia.

Cats were examined for FeLV viraemia by a modification of the C81 test for the rescue ofsarcoma virus from feline non-producer cells (Fischinger, Blevins & Nomura, 1974; Jarret &Russell, 1978). A mixture of 3 x io5 FEA and 3 x 10* C81 (non-producer) cells were seededin s-cm plates in 4 ml of Eagle's MEM plus 10% FCS. After 24 h, the medium wasremoved and the plasma adsorbed for 00 mins in fifty per cent medium containing a finalpolybrene concentration of 4/Jg/ml. The inoculum was then replaced with 4 ml of freshmedium. The medium was changed at 3 days and the plates examined for foci on the 6th day.

RESULTS

Morphology and ultrastructure

Initially, granulocyte, erythroid and megakaryocytic cells in all stages of maturationwere recognizable in the bone marrow cultures. However, after 1-2 weeks only mono-or binucleated macrophages remained. At that time multinucleated cells containingup to 45 nuclei per cell, not observed in the original cell suspension, began to appearand by 2-3 weeks were observed in all cultures. Microscopical examination (Fig. 1)showed that they measured up to 300 /«m diameter. Their nuclei were congregatedaround a central area containing vacuoles, or in 2 or more distinct areas, but withindividual nuclei remaining clearly discrete. Neither mitotic figures, nor nucleoplasmicbridges between nuclei have been observed. All nuclei were of similar size, round,with evenly dispersed condensed peripheral chromatin, and with one, or occasionallytwo, prominent nucleoli. The cytoplasm presented a perinuclear zone which staineddeeply with May Grunwald-Giemsa and contained azurophilic granulations and amuch clearer peripheral area. The reaction for acid phosphatase (Li et al. 1970)performed after 2 and 3 weeks of culture was positive. There was variation in theintensity of staining reaction in individual cells, which showed irregular concentra-tions of positive granules in different areas of the cytoplasm.

Generation of osteoclasts in vitro 129

Most cells presented a regular oval or round shape with smooth borders, but someshowed slender cytoplasmic extensions. They were often found in contact with eachother, in which case the peripheral membranes were in close juxtaposition. Theirmorphological and cytochemical features allowed the characterization of thesemultinucleated cells as osteoclasts.

This characterization was strengthened by ultrastructural studies. Parallel sectionsdisplayed the overall similarity of nuclear profiles with prominent nucleoli and a thin

1m +

Fig. 1. In titu preparation of osteoclasts and macrophages stained with May Grun-wald-Giemsa 3 weeks post plating. Mono- and binucleate precursors are arrowed.Osteoclast nuclei may be in individual groups possibly suggesting recent fusion,x 140.

rim of peripheral condensed chromatin (Fig. 2). The cytoplasm was characterized bya high mitochondrial population and a well developed Golgi apparatus with numerousdictyosomes. The endoplasmic reticulum was also well developed and parallel sectionsrevealed large areas of typical polyribosomal whorls. The most convincing ultra-structural feature of these cells, however, were the areas of ruffled border also revealedby the parallel sections cut at or near the cell underside and the characteristic clearzone (Fig. 3). These regions showed close similarity to both the ultrastructure ofosteoclasts in vivo (Miller, 1977, 1978) and those also briefly maintained (48 h) inorgan culture (Holtrop, Raisz& Simmons, 1974).

Scanning-microscope examination of regions of the growing surface confirmed the


large size of the osteoclasts, measuring up to 300 /im in diameter. The periphery ofthese cells was characterized by an annular band, some 10—20/tm wide, which waslacking in surface features, possibly representing the surface of the clear zone. Theremainder of the cell surface was covered in deeply sculptured and tightly packedridges (Fig. 4), not dissimilar to those described from sections of the non-bone-apposed cell side in vitro (Holtrop et al. 1974). The extreme periphery of the cellswas characterized by numerous fine filopodia resulting in a 'fringed appearance'.

•V-7J.-.S

i.

Fig. 2. TEM preparation fixed and embedded in situ and cut parallel to the substratum.This portion of the cell displays circular nuclear profiles, prominent nucleoli andnumerous mitochondria, x 2200.

Cells in close contact, possibly representing an early stage of fusion (see Discussion),showed numerous cell-cell processes of similar appearance, suggesting that thefilopodia may play a role in contact and recognition in the fusion process (Fig. 5).

Kinetics of osteoclasts in vitro

After 3 weeks, 2-5 x io4 osteoclasts per culture were found attached to the surfaceof the culture flasks (Table 1). They were also recovered in suspension in the growthmedium: from the numbers harvested in the spent growth medium from Culture 2 at

Generation of osteoclasts in vitro

Table 1. Osteoclasts in cat bone marrow cultures

Culture

. j

2

3

4

Age of donor,months

42

64

Virus determinations(FSFV and FeLV)

ND

Negative

NegativeNegative

Weeks ofculture

18

2

38

2

678

i fND: not determined.

Nos. per culture(attached cells only)

ND

1712S ±i°7S24125 ±825

ND

56915 ±2993 395 ±9572448 ±6343852145°47O5±472

Fig. 3. Section parallel to surface through in situ prepared material at the undersideperiphery of an osteoclast. The clear zone (cz) and adjacent ruffled border (rb) arereadily apparent, x 14000.


week 3, plus the numbers still attached, a total of 6-5 x 10* per culture was calculated.Osteoclasts were present in all the cultures to date until the time at which they wereterminated (Table 1). Only Culture 1 were allowed to continue until extensive celldeath and detachment were observed, commencing at weeks 15-16. All other cultureswere in good condition when terminated at the times stated. In one experiment, inwhich serial enumeration of attached osteoclasts was performed, the numbers foundbetween 6 and 13 weeks remained constant, but were lower than those present at 2

Fig. 4. SEM preparation showing two relatively small osteoclasts (o), surroundingmacrophages (w) and possible osteoclast precursors (/>). x 300.

weeks. The other cells found were mono or binucleated macrophages. The number ofthese macrophages found in suspension remained more or less stable during that timeinterval within a range of 2 to 4 x io5 cells per culture. As binucleated cells may be seenoccasionally in macrophage colonies derived from bone marrow (Testa, unpublishedobservation) only cells with a minimum of 3 nuclei were enumerated here (Table 1).Occasionally fibroblastoid cells were found, but only in small foci which did notovergrow the cultures.

Although the numbers of osteoclasts decreased between weeks 2 and 6, the numberof nuclei per cell increased with time. Fig. 6 shows that at 2 weeks, 73 % of the multi-


Fig. 5. Detail of adjacent cell membranes in close contact via numerous filopodialextensions, x 17500.

40r r^

30

20

10

25

m50 3 25

No. of nuclei

50 75 100101-300

Fig. 6. Distribution of the number of nuclei per cell in cultures of different ages:left-hand histogram, day 12; right-hand, day 43. 300 multinucleated cells scored insitu for each time point in stained preparations.


nucleated cells contained between 3 and 10 nuclei, but that proportion fell to 31%after 6 weeks. At that time, cells with an excess of 50 nuclei, which were not observedat week 2, formed a significant proportion of the population and cells with more than100 nuclei reached 13 % of the total.

Virus determination

Two retroviruses, feline syncitium-forming virus (FSFV) and feline leukaemiavirus (FeLV) may be isolated from a proportion of clinically normal cats. Bothviruses may replicate in and have effects upon haemopoietic cells. FSFV producedsyncitia in vitro (Riggs, Oshiro, Taylor & Lennette, 1969) whilst FeLV is oncogenicin vivo and has been associated with non-oncogenic conditions including osteosclerosis(Hoover & Kociba, 1974). Since FSFV may form syncitia in bone marrow culturesand RNA leukaemia viruses may directly or indirectly affect the function or formationof osteoclasts, cats known to be free from these viruses were used in all but the firstexperiment described (Table 1).

In addition to examining the bone marrow cells for FSFV infection at initiation ofthe cultures, supernatant osteoclasts and macrophages from 2 established cultureswere also assayed for FSFV at 3 and 4 weeks of culture. No FSFV was detected inthese cultures.

Furthermore, no virus particles were observed in TEM studies.

DISCUSSION

Several criteria for the identification of osteoclasts have been fulfilled. Opticalmicroscopy rendered a classic description of these cells (Chambers, 1979). In particu-lar, the pattern seen after acid phosphatase staining was identical to that found infreshly isolated kitten osteoclasts (Addison, 1978).

Ultrastructurally, the cells possessed the recorded features of osteoclasts eitherin vivo, or freshly isolated, and of those maintained for short term in organ culture(Holtrop et al. 1974; Holtrop, King, Cox & Reit, 1979; Miller, 1977). Nuclear andcytoplasmic morphology are identical and the retention of the peripheral clear zoneand ruffled border strongly suggests that these multinucleate cells are osteoclasts.Retention of albeit a reduced ruffled border against a non-bony surface is unusual, asin non-resorbing osteoclasts the ruffled border is largely absent (Miller, 1977). Thesurface morphology as observed in the scanning microscope is also similar to thesurface of the cells which is away from bone in vivo (Miller, 1977).

To differentiate between osteoclasts and giant multinucleated cells is difficult: bothare produced by fusion of macrophages and conserve the capacity to phagocytose.Acid phosphatase activity and some of the ultrastructural characteristics describedhere are also found in inflammatory giant multinucleated cells (Ham, 1974). However,the ruffled border and the adjacent clear zone are thought to be definitive for osteo-clasts. Whether they are features which depend on the nature of the substratum, or aspecialized adaptation of the osteoclast, is not known.

Two reports (Chambers, 1979; Sanger, Frederickson & Morrill, 1966) indicate


that osteoclasts isolated from fresh tissue do not express Fc or C3 receptors whileinflammatory giant cells are positive for both. However, whether this represents a truespecialization of osteoclasts is not known, as it is not unreasonable to expect thatnearly formed osteoclasts may conserve these receptors, which are present in theirmononuclear precursors, but may lose them as they age.

The presence or absence of these receptors may well represent the fast or slowturnover of the multinucleate cells, rather than an intrinsic characteristic of their celltype.

Osteoclasts in vivo are generally considered to be formed by cell fusion (Loutit &Nisbet, 1979; Chambers, 1979). Preliminary time-lapse observations suggest that thisis also the case in vitro (Testa & Allen, unpublished). Furthermore, the failure toobserve mitotic figures in the multinucleated cells agrees with the concept. In thesecultures, 2 processes may be occurring. Firstly, continuous formation from theirimmediate precursors, the mono- and binucleated cells present in the cultures: theobservations that the number of mono- plus binucleated cells in suspension remainedstable in spite of the weekly half depopulation by feeding, indicates that they aremultiplying, albeit at a low rate. They may fuse to produce new polykaryons, or maycontribute to the increase in multinuclearity of the larger cells. Secondly, the multi-nucleated cells themselves may fuse. The grouping of the nuclei in 3 or 4 distinct areasin some cells (shown in Fig. 1) is compatible with this concept, which may also explainthe decrease in cell numbers which accompanies the increase in nuclei per cell.

The observation that cells with 3-10 nuclei are still present, together with the widespectrum observed in the number of nuclei per cell, suggest that new multinucleatedcells are still being produced at week 6.

The culture system described here may be of value in investigating the nature ofthe bone lesions induced by RNA leukaemia viruses. Both osteopetrosis (Sangeret al. 1966) and osteosclerosis (Stubbs & Furth, 1932) have been reported in chickensinfected with avian leukosis virus. In the cat, medullary osteosclerosis can be producedby infection with strains of FeLV which induce non-regenerative anaemia (Hoover &Kociba, 1974). This association between anaemia-inducing viruses and bone lesions isalso found in the chicken (Paterson & Smith, 1978).

The production of anaemia in the cat is not secondary to myelophthisis and whilstthe medullary sclerosis could be enhanced by the associated anaemia, the changes inosteoclast numbers in infected cats suggest that FeLV may directly affect the forma-tion of these cells.

The culture system reported should prove useful in the investigation of severalquestions concerned with the pathophysiology of bone resorption: (a) what are thefunctional characteristics of the multinucleate osteoclasts, compared to those ofmononuclear cells which have also been implicated in bone resorption (Mundy,Altman, Gondek & Bandelin, 1977) and what is their relative importance in suchprocess both in physiological and pathological situations ? (b) what are the effects onisolated osteoclasts of humoral and local factors which regulate bone resorption ?(c) are cell interactions important in resorption ? In particular, do thymus cellswhich may be regulatory cells in haemopoiesis (Goodman & Grubbs, 1970; Testa,


Schofield & Eliason, 1980) and which may be defective in osteopetrotic rats (Milhaud& Labat, 1978) play a role in this system ? (d) are there differences in osteoclastformation and function during growth, at maturity and at old age, stages at which theratio of apposition to resorption of bone changes ?

Preliminary experiments suggest that the culture system described here may beadapted for other species: we have observed polykarons in cultures of mouse,human and chicken bone marrow. The recent demonstration that osteoclasts derivefrom the haemopoietic stem cells (Ash et al. 1980) opens the possibility of investigatingthe process of development from multipotential cells, which can be maintained in vitro(Dexter & Testa, 1976; Moore et al. 1980), to the mature osteoclasts. If that processcan be unravelled, the nature of osteopetrosis as well as other problems related toquantitative or qualitative defects in osteoclast function may be amenable to study.

This work was supported by the Cancer Research Campaign, the Medical Research Counciland the Leukaemia Research Fund.

We thank G. Molineux, G. R. Bennion and P. E. Young for expert technical assistance.

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(Received 23 June 1980)

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GENERATION OF OSTEOCLASTS IN VITRO - Journal of Cell Science