JOURNAL OF CELLULAR PHYSIOLOGY 203:573–582 (2005)

Human Trabecular Bone-Derived Osteoblasts

Support Human Osteoclast Formation In Vitro
in a Defined, Serum-Free Medium
GERALD J. ATKINS,1* PANAGIOTA KOSTAKIS,1,2 KATIE J. WELLDON,1 CRISTINA VINCENT,1
DAVID M. FINDLAY,1 AND ANDREW C.W. ZANNETTINO2
1
Department of Orthopaedics and Trauma, University of Adelaide,
and the Hanson Institute, Adelaide, South Australia, Australia
2
Division of Haematology, Institute of Medical and Veterinary Science,
and the Hanson Institute, Adelaide, South Australia, Australia

While it has been assumed that osteoblasts in the human support osteoclast formation, in vitro evidence of this is currently lacking.
We tested the ability of normal human trabecular bone-derived osteoblasts (NHBCs) to support osteoclast formation from human
peripheral blood mononuclear cells (PBMC) in response to treatment with either 1a,25-dihydroxyvitamin D3 (1,25D) or parathyroid
hormone (PTH), using a serum-replete medium previously used to support human osteoclast formation on a stroma of murine ST-2
cells. Under these conditions, NHBC did not support osteoclast formation, as assessed by morphological, histochemical, and
functional criteria, despite our previous results demonstrating a link between induction of RANKL mRNA expression and NHBC
phenotype in these media. We next tested a defined, serum-free medium (SDM) on NHBC phenotype, their expression of RANKL
and OPG, and their ability to support osteoclast formation. SDM, containing dexamethasone (DEX) and 1,25D, induced phenotypic
maturation of NHBC, based on the expression of STRO-1 and the bone/liver/kidney isoform of alkaline phosphatase (AP). PTH as a
single factor did not induce phenotypic change. 1,25D and DEX induced the greatest ratio of RANKL:OPG mRNA, predictive of
supporting osteoclast formation. Consistent with this, co-culture of NHBC with CD14þ PBMC, or bone marrow mononuclear cell
(BMMC), or CD34þ BMMC precursors in SDM þ 1,25D þ DEX, resulted in functional osteoclast formation. Osteoclast formation
also occurred in PTH þ DEX stimulated co-cultures. Interestingly, SDM supplemented with recombinant RANKL (25–100 ng/ml)
and M-CSF (25 ng/ml), did not induce osteoclast formation from any of the osteoclast precursor populations in stromal-free cultures,
unlike serum-replete medium. This study demonstrates that under the appropriate conditions, adult human primary osteoblasts
can support de novo osteoclast formation, and this model will enable the detailed study of the role of both cell types in this
process. J. Cell. Physiol. 203: 573–582, 2005. ß 2004 Wiley-Liss, Inc.

Cells of the osteoblast lineage perform a number of
functions, including the formation of new bone during
modelling and remodelling (Parfitt, 1984), organic
matrix degradation at the initiation of remodelling
(Everts et al., 2002), support of haemopoiesis (Taichman
and Emerson, 1998), and notably, osteoclastogenesis
(Hofbauer et al., 2000). However, the way in which
these seemingly diverse functional states of osteoblasts
interrelate, and the phenotypic identity of the osteo-
blasts involved, has not been elucidated, particularly for
human osteoblasts. Evidence linking the osteogenic and
osteoclast-inductive properties of osteoblasts is sparse,
despite the long held view that the processes of bone
resorption and bone formation are intimately coupled
(Parfitt, 2000). Normal human trabecular bone-derived
osteoblastic cells (NHBCs) provide a potentially valu-
able in vitro model system, with which to investigate the
relationship between these diverse functional proper-
ties. It has been shown that NHBC retain phenotype
plasticity in the absence of an osteoinductive stimulus,
and are able to differentiate into several mesenchymal
lineages given the appropriate growth stimuli (Sottile
et al., 2002). However, culture in the continuous pre-
sence of long-acting ascorbate (ascorbate-2-phosphate,
AS2P) confers an osteoblastic phenotype and NHBC
have been extensively characterised from the viewpoint
of osteogenesis (Gronthos et al., 1999; Stewart et al.,
1999; Sottile et al., 2002). In contrast, little is known
regarding the ability of these cells to support osteoclast
formation from naive precursors. In fact, the identifica-
tion of the human osteoblast phenotype that promotes
osteoclastogenesis has been elusive and an in vitro
ß 2004 WILEY-LISS, INC.
model for the differentiation of human osteoclasts using
human osteoblasts as a stromal layer has, until now, not
been described.
Models of human osteoclast formation described
previously include those in which human osteoclast
precursors are cultured together with rodent cell lines,
such as mouse ST-2 cells (Fujikawa et al., 1996, 2001)
or rat UMR-106 cells (Quinn et al., 1998), as stroma.
Whilst these assays are useful in examining interactions
between osteoclast precursors and osteoblast-lineage
cells (Haynes et al., 1999; Atkins et al., 2000; Fujikawa
et al., 2001), it is not certain that these assays can
accurately model human osteoclastogenesis. The limita-
tions of these models include the use of transformed cell
lines, as opposed to primary cells, as stromal layers and
the demonstrated differences that exist between rodent
and human osteoblast differentiation (Siggelkow et al.,
Contract grant sponsor: National Health and Medical Research
Council of Australia; Contract grant sponsor: Eli Lilly Australia
Ltd.; Contract grant sponsor: EISAI Co. Ltd., Japan; Contract
grant sponsor: Department of Orthopaedic Surgery and Trauma,
University of Adelaide.
*Correspondence to: Gerald J. Atkins, Department of Orthopae-
dics and Trauma, University of Adelaide, North Terrace,
Adelaide, South Australia, Australia, 5000.
E-mail: gerald.atkins@adelaide.edu.au
Received 13 August 2004; Accepted 6 October 2004
DOI: 10.1002/jcp.20255
574 ATKINS ET AL.
1999). The development of human stroma-driven models
of osteoclast differentiation is therefore highly desir-
able. Mbalaviele et al. (1999) have described the use of
undifferentiated mesenchymal stem cells and CD34þ
bone marrow (BM) cells to generate human osteoclasts
in vitro. A number of investigators have used trans-
formed human osteosarcoma cell lines to support human
osteoclast formation (Blair et al., 2000; Itoh et al.,
2000; Miyamoto et al., 2002). Neale et al. (2000) used
NHBC as stroma to support functional osteoclast-like
cells formation from the synovial tissue associated with
peri-prosthetic osteolysis. However, this tissue repre-
sents a rich source of late-stage, committed osteoclast
progenitors. To our knowledge, functional osteoclast
formation from a source of relatively immature osteo-
clast precursors, using NHBC, has not been described
previously.
We recently reported (Atkins et al., 2003) a link
between the induced expression by NHBC of RANKL, a
key factor required for osteoclast formation (Lacey et al.,
1998; Tsukii et al., 1998; Hofbauer et al., 2000; Udagawa
et al., 2000), and the cell-surface expression of STRO-1
and AP, phenotypic markers of osteoblast differen-
tiation (Gronthos et al., 1999; Stewart et al., 1999).
Induction of RANKL expression in response to 1,25D
and DEX correlated positively with an induced increase
in the percentage of cells expressing STRO-1 and nega-
tively with the percentage of cells expressing AP (Atkins
et al., 2003). On the basis of those results, we hypo-
thesised that support of osteoclast formation by osteo-
blasts occurs as a function of their differentiation state,
as defined by STRO-1/AP (Gronthos et al., 1999; Stewart
et al., 1999).
In this study, we sought to define the conditions under
which NHBC support osteoclast formation. Although we
were unable to generate osteoclasts in serum-replete
media in co-cultures, we were able to successfully gen-
erate functional human osteoclasts from precursors in
human peripheral blood or BM cells in a defined, serum-
free medium based on that developed for the efficient
growth of BM colony forming units-fibroblast (CFU-F)
(Gronthos and Simmons, 1995; Gronthos et al., 2003).
The ability to support osteoclast formation was in-
dependent of the addition of exogenous RANKL and
M-CSF, and was associated with the rapid phenotypic
differentiation of the NHBC. Thus, under hormonal sti-
mulation, NHBC appear capable of providing all of the
necessary factors for the support of complete osteoclas-
togenesis from CD14þ monocytic cells (Massey and
Flanagan, 1999) derived from peripheral blood or BM,
and from immature CD34þ BM cells. This novel model of
adult human primary osteoblasts driving human osteo-
clast differentiation will enable the in vitro study of the
role of both cell types in this process. In addition, the use
of a defined, serum-free medium will allow the study of
putative regulators of bone remodelling in the absence of
the potentially complex influence of serum.
MATERIALS AND METHODS
NHBC
All human donor material in this study was used after
informed donor consent, which was approved by the Ethics
committees of the Institute of Medical and Veterinary Science
and the Royal Adelaide Hospital. NHBC were derived from
trabecular bone fragments and cultured, as we have described
previously (Atkins et al., 2003). NHBC were cultured to con-
fluence in 75 cm2 tissue-culture flasks in alpha-minimal essen-
tial medium (a-MEM: Flow Laboratories, Irvine, Scotland)
supplemented with 10% foetal calf serum (FCS, Thermotrace,
Noble Park, VIC, Australia), 2 mM L-glutamine and 100 mM
L-ascorbate-2-phosphate (AS2P, Novachem, Melbourne, VIC,
Australia) (a-MEM-10). Single cell suspensions were obtained
from confluent primary NHBC cultures by enzymatic digestion
with collagenase (3 mg/ml) (Collagenase Type I; Worthington
Biochemical Co., Freehold, NJ) and dispase (4 mg/ml) (Neutral
Protease Grade II; Boehringer Mannheim, GMBH, Germany)
for 30 min at 378C, followed by trypsin digestion for 5 min at
378C.
Isolation of CD34þ bone marrow
mononuclear cell (BMMC)
BMMC were enriched from BM aspirates from normal
healthy volunteers by diluting whole marrow 1:3 in Hank’s
balanced salt solution (HBSS) and layering onto Ficoll–Paque
separation media (Pharmacia Biotech, Uppsala, Sweden).
After centrifugation at 400g for 25 min, mononuclear cells
were retrieved and washed twice in HBSS containing 10%
FCS. An aliquot of cells was removed for CD34 staining by
immunofluorescence. All subsequent steps were performed on
ice or at 48C, unless otherwise specified, and under sterile
conditions. CD34þ cells were isolated, essentially as described
previously (Zannettino et al., 1998). Briefly, CD34-detacha-
beads (4
107, 100 ml/ml cells, Dynal Biotech, Oslo, Norway)
were washed twice in 1 ml HHF medium (HBSS, 20 mM Hepes,
10% FCS) before addition to the BMMC suspension. The cell-
detachabead suspension was mixed for 30 min with rotation.
Isolation buffer was added to the cells, which were then placed
in a magnet apparatus for 2 min. The supernatant containing
CD34 BMMC was removed, washed three times in HHF and
then processed for CD14, as described below. The remaining
CD34þ BMMC complexed to beads were washed three times in
isolation buffer and then resuspended at 4
108 cells/ml in
HHF. To this suspension was added two volumes of detacha-
bead buffer, and the cells were then mixed using a vortex
mixer. The mixture was incubated at 378C with rotation for the
detachment of the beads from the cells, after which time the
volume of the tube was increased by 2 ml with HHF. The cell
suspension was vigorously mixed and then placed in a magnet
apparatus for 2 min. Supernatant containing detached cells
was collected and the wash process was repeated three times.
The recovered cells were pooled, washed twice by centrifuga-
tion at 400g in HHF, resuspended in thaw media (HBSS con-
taining Dnase 1) and rotated at 48C until used.
Isolation of CD14þ BMMC and peripheral blood
mononuclear cells (PBMCs)
PBMC from the peripheral blood of healthy donors were
isolated, as described for BMMC, and both populations were
sorted by magnetic activation cell sorting (MACS) for CD14þ
cells. First, an aliquot of cells was taken to assess input CD14
levels by immunostaining with the CD14 mouse monoclonal
antibody (MAb) FMC-17 (kindly provided by Prof. Peter
McCardle, Flinders Medical Centre, Bedford Park, South
Australia, Australia). All remaining steps were performed at
48C or on ice. The remaining cells were incubated in blocking
buffer, consisting of 10% v/v normal rabbit serum in HHF,
for 20 min. After this time, cells were incubated in FMC-17
supernatant for 1 h, and then washed twice in HHF by centri-
fugation at 200g. After this, a 1/50 dilution of goat anti-mouse
g-biotin (Southern Biotechnology Associates, Birmingham,
UK) in HHF buffer was added and the cells incubated for 1 h.
Cells were then washed twice by centrifugation in MACS
buffer (Ca2þ/Mn2þ-free PBS supplemented with 1% BSA, 5 mM
EDTA, and 0.01% sodium azide). Cells were resuspended in
0.9 ml of MACS buffer and to these was added 0.1 ml
streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach,
Germany), which were left for 15 min to attach. Cells were then
washed twice and then resuspended in 0.5 ml of MACS buffer.
The cell suspension was then loaded onto a mini MACS column
(Miltenyi Biotec), which was then washed through twice with
0.5 ml MACS buffer to retrieve the CD14 cells. After addition
of a further 0.5 ml MACS buffer, the column was removed from
the magnet and the CD14þ cells aspirated using a syringe
plunger. An aliquot of cells from each fraction was stained with
streptavidin-FITC to assess purity by flow cytometry. By this
 HUMAN OSTEOBLAST SUPPORT OF OSTEOCLASTOGENESIS
method, greater than 95% purity of CD14þ cells was routinely
achieved (data not shown).
Human–human osteoclast forming co-culture
Semi-confluent cultures of NHBC were digested with
dispase/collagenase/trypsin, and single cell suspensions were
then washed twice and resuspended in media for co-culture.
Co-cultures were performed using media used previously to
support human osteoclast formation on stroma of murine ST-2
cells (Fujikawa et al., 1996; Haynes et al., 1999; Atkins et al.,
2000). This consisted of a-MEM supplemented with 10% FCS,
L-ascorbate-2-phosphate (100 mM, Novachem), DEX (10 nM),
L-glutamine (2 mM), 1,25D (20 nM, Wako Pure Chemical
Industries, Osaka, Japan), and recombinant human M-CSF
(25 ng/ml, Genetics Institute, Cambridge, MA). Co-cultures
were also performed in serum-deprived medium (SDM), based
on a medium shown previously to support the growth of human
CFU-F (Gronthos and Simmons, 1995). SDM consisted of
a-MEM (Flow Laboratories), supplemented with 1% BSA (Cat.
No. A-2153, Sigma Chemical Co., St. Louis, MO), transferrin
(100 mg/ml), insulin (10 mg/ml), b-mercaptoethanol (5
105M),
human low density lipoprotein (LDL, 20 mg/ml, a kind gift from
A/Prof. Kerry-Anne Rye, Heart Research Institute, Sydney,
Australia), L-ascorbate-2-phosphate (100 mM, Novachem),
epidermal growth factor (EGF, 10 nM, Peprotech, Inc.,
Rocky Hill, NJ), PDGF-BB (10 nM, Peprotech), DEX (10 nM,
Fauldings, Adelaide, Australia), L-glutamine (2 mM), and
Hepes (20 mM). NHBC were then seeded onto sterilised 4 mm2
slices (0.1 mm thick) of human cortical bone, or elephant tusk
dentine in a 96-well plate, at a density of 2–4
105 cells/ml
in 0.1 ml volumes, and cultured overnight in 5% CO2 in air
at 378C. Following this, CD14þ BMMC, CD14þ PBMC, or
CD34þ BMMC, isolated as described above, were washed twice
in SDM, counted and resuspended at 6
105 cells/ml (for
CD14þ cells) or 1
105 cells/ml (for CD34þ cells), and added
to wells containing NHBC or onto virgin bone/dentine. The
cells were allowed to attach for 2 h at 378C, and then four bone/
dentine slices were transferred into wells of a 24-well plate
containing 1 ml SDM, additionally supplemented with either
recombinant peptide PTH1 –34 (20 nM, Auspep Pty. Ltd.,
Parkville, VIC, Australia) or 1,25D (20 nM, Roche, Uppsala,
Sweden), and recombinant human M-CSF (25 ng/ml, Genetics
Institute), as indicated. In some experiments, media were
supplemented with recombinant human RANKL or OPG
(Peprotech, Inc.) at the concentrations indicated. Cultures
were maintained for 21 days and media were replaced every
3 days.
ST-2 osteoclast-forming co-culture
Human osteoclasts were derived from PBMC on a stromal
support of ST-2 cells, essentially as described previously
(Fujikawa et al., 1996; Haynes et al., 1999; Atkins et al.,
2000). PBMC (2
105 cells) or CD14þ PBMC (6
104 cells)
were plated onto seeded layers of 8
103 ST-2 cells on dentine
slices. Non-adherent cells were removed by washing three
times with HBSS after 1 h at 378C, and pairs of dentine slices
were placed in 16 mm diameter wells containing 1 ml of a-
MEM medium with 10% v/v FCS, 20 nM 1,25D, 10 nM DEX
(Fauldings), and 25 ng/ml M-CSF. Media were replenished
every 3 days throughout the experiment.
Tartrate resistant acid phosphatase (TRAP) staining
and measurement of bone resorption
Cultures on slices of dentine were fixed and stained for
TRAP, as recommended by the manufacturer (Sigma Chemical
Company, Castle Hill, Australia). TRAP positive cells were
visualised by light microscopy and images taken with a Nikon
DIH digital camera. Dentine slices were then treated with 6M
ammonium hydroxide for at least 1 h, and then the fixed
adherent cells were removed with an artist’s brush. Following
this, the bone slices were washed four times in deionised water,
dehydrated in 100% ethanol, air-dried and then processed for
scanning electron microscopy (Haynes et al., 1999).
575
Preparation of total RNA and real-time reverse
transcription PCR
Total RNA was prepared from NHBC, and RT-PCR per-
formed, as we have recently reported (Atkins et al., 2000).
Briefly, cells were dissolved in Trizol reagent (Life Technolo-
gies, Gaithersburg, MD). Total RNA was then prepared as per
manufacturer’s instructions. RNA was reverse transcribed
from 1 mg of total RNA from each sample, using a cDNA syn-
thesis kit, as per manufacturer’s instructions (Promega Corp.,
Madison, WI). RANKL, OPG, and GAPDH mRNA expression
was analysed by real-time PCR using the SYBR GREEN-1
(Molecular Probes, Eugene, OR) detection system. For RANKL
expression, the following primer sequences were used: 50 -
TCAGCCTTTTGCTCATCTCACTAT-30 (forward); 50 -CCAC-
CCCCGATCATGGT-30 (reverse). OPG and GAPDH cDNAs
were amplified using our previously published primer sequen-
ces (Atkins et al., 2000). cDNA was amplified using AmpliTaq
Gold DNA polymerase (Perkin Elmer, Norwalk, CT) ampli-
fication, as described (Atkins et al., 2000), on a Rotor-Gene
thermocycler (Corbett Research, Mortlake, NSW, Australia),
with the addition to each reaction of SYBR GREEN-1
(1:100,000 dilution of the commercial stock, Molecular Probes).
All PCRs were validated by the presence of a single peak in the
melt curve analysis and amplification of a single specific
product was further confirmed by electrophoresis on a 2.5% w/v
agarose gel. Relative quantification of RANKL and OPG
mRNA expression between samples was calculated using the
comparative cycle threshold (CT) method (DCT) (Anonymous,
1997). Briefly, the formula XN ¼ 2DCT was used, where XN is
the relative amount of target gene in question and DCT is the
difference between the CT of the gene in question and the CT of
the housekeeping gene, GAPDH, for a given sample.
Flow cytometric analysis
NHBC were analysed by flow cytometry for the expression of
STRO-1 (Simmons and Torok-Storb, 1991) and AP, as des-
cribed previously (Atkins et al., 2003). Analysis was performed
on an Epics (Beckman Coulter, Fullerton, CA) flow cytometer.
Statistical analysis
Parametric data sets were analysed using Student’s t-test.
Multiple and non-parametric datasets were analysed for dif-
ferences by one way analysis of variance (ANOVA) and Tukey’s
Multiple Comparison using GraphPad Prism V4 software
(GraphPad Software Ltd., San Diego, CA). P values <0.05 were
considered to be significant.
RESULTS
Comparison of ST-2 and NHBC in supporting
human osteoclast formation
Initial experiments examining the osteoclastogenic
potential of NHBC were closely modelled on a serum-
replete culture system described by Fujikawa et al.
(1996), except that we substituted NHBC for mouse ST-2
cells. Because NHBC proliferate slowly compared with
ST-2 cells, we titrated the number of NHBC added to
these co-cultures, starting from 8
103 per dentine slice,
as used for ST-2 cells, up to 4
104 per slice. Under these
conditions, ST-2 cells supported robust osteoclast forma-
tion from unfractionated human PBMC, as determined
by TRAPþ multinucleated cell (MNC) and resorption pit
formation on dentine. In contrast, neither TRAPþ cells
nor functional osteoclasts were generated using NHBC
isolated from four individual donors as a stromal sup-
port, under otherwise identical culture conditions (data
not shown).
Comparison of RANKL and OPG expression in
SDM versus serum-replete medium
As an alternative to the serum-replete medium used
in ST-2 co-cultures, we next proposed to test osteoclast
formation supported by NHBC in a defined SDM. Since
576 ATKINS ET AL.

the ratio of RANKL to OPG mRNA is a major determi-

nant of the propensity to support osteoclast formation,
we initially tested the expression of RANKL and OPG
mRNA by NHBC cultured in the absence of PBMC,
in media containing combinations of the components
in SDM. NHBC were cultured for 72 h in basal SDM
(B-SDM), comprising a-MEM supplemented with in-
sulin, transferrin, BSA, and human LDL, being the
basic requirements for growth in the absence of serum
(Gronthos and Simmons, 1995). Other components of
the previously published formula for SDM, EGF, and
PDGF-BB (Gronthos and Simmons, 1995), and factors
that were pro-osteoclastogenic in the serum replete co-
culture medium used with ST-2 cells, DEX and 1,25D
(Fujikawa et al., 1996), were then added alone or in com-
bination. These media were compared with the serum-
replete medium, used previously to examine changes in
NHBC gene expression and phenotype (Atkins et al.,
2003). Of the conditions tested, only culture in media
containing both EGF and PDGF-BB (i.e., complete SDM),
complete SDM containing DEX, and those additionally
supplemented with 1,25D, gave appreciable increases in
RANKL mRNA expression (Fig. 1A). Conditions that
favoured RANKL mRNA expression were also associat-
ed with lower OPG mRNA expression (Fig. 1B), and
thereby a higher RANKL:OPG mRNA ratio (Fig. 1C).
Significantly, the RANKL:OPG mRNA ratio in SDM
media was markedly higher than that seen in serum-
replete medium containing DEX and 1,25D (Fig. 1C).
Based on these data, complete SDM with the addition of
1,25D and DEX was predicted to be the medium most
favourable for osteoclast formation.
Comparison of NHBC phenotype in SDM
versus serum-replete medium
We previously showed a link between osteoblast phe-
notype, as defined by STRO-1 and AP expression, and
RANKL expression when experiments were performed
in serum-replete medium (Atkins et al., 2003). The
phenotype of NHBC cultured in SDM has not been
previously reported. Thus, we next tested the effect
on osteoblast phenotype of culture in media consisting
of various combinations of the components of SDM.
NHBC were cultured for 72 h, as for the gene expression
studies described above, and analysed for STRO-1 and
AP expression by flow cytometry. The expression of
STRO-1/AP in response to culture in B-SDM is shown in
Figure 2A. All additions to this basal medium signifi-
cantly affected the phenotypic profile. EGF decreased Fig. 1. Real-time RT-PCR analysis of (A) RANKL and (B) OPG
mRNA expression, and (C) the resulting RANKL:OPG mRNA ratio, in
the STRO-1þ populations and increased the AP popula- normal human trabecular bone-derived osteoblasts (NHBC) cultured
tion, consistent with a differentiative effect (Fig. 2B). in the absence of peripheral blood mononuclear cells (PBMC), under
Culture in PDGF-BB as a single factor increased the serum-free conditions for 72 h in various components of serum-
proportion of osteoprogenitor cells (%STRO-1þ/AP) deprived medium (SDM). Data are expressed as the DCT values
relative to the expression of GAPDH (A and B) or the ratio of these
(Fig. 2C). Culture in B-SDM with EGF and PDGF-BB values (C), and are means of triplicate reactions  standard errors of
together (complete SDM) increased the %STRO-1APþ the mean (SEM). Similar results were obtained in two independent
and %STRO-1/AP, consistent with a net osteogenic experiments.
effect of these factors (Fig. 2D). DEX in complete SDM
dramatically enhanced the %STRO-1APþ (Fig. 2E).
The addition of 1,25D in complete SDM increased the phenotype over the 72 h period at 20 nM (compare
proportion of STRO-1þ/APþ and the STRO-1/APþ Fig. 2D with 2I), nor was the phenotype affected further
populations, compared with complete SDM alone (com- by a combination of PTH and DEX in (Fig. 2J). Together,
pare parts D and F). Culture of NHBC in complete SDM these data are consistent with a stronger osteogenic
with the addition of DEX and 1,25D resulted in a further effect and more rapid phenotypic maturation in SDM
increase in the percentage of STRO-1/APþ cells than in serum-replete media.
(%STRO-1APþ) (Fig. 2G), and greatly enhanced the
differentiation-associated progression into the STRO- Osteoclast formation in SDM
1/APþ population when compared with culture in DEX Having established the composition of the medium
and 1,25D-supplemented standard serum-replete med- that resulted in the greatest ratio of RANKL:OPG
ium (Fig. 2H). PTH had no additional effect on NHBC mRNA, and which would therefore most likely favour
 HUMAN OSTEOBLAST SUPPORT OF OSTEOCLASTOGENESIS
Fig. 2. Flow cytometry dot plots showing the expression of STRO-1
(Y-axis) and AP (X-axis) by NHBC cultured for 72 h in (A) basal SDM
(B-SDM) (SDM without added EGF or PDGF-BB); (B) B-SDM con-
taining EGF (10 mg/ml); (C) B-SDM containing PDGF-BB (10 mg/ml);
(D) B-SDM containing EGF and PDGF-BB together (complete SDM);
(E) complete SDM containing DEX (10 nM); (F) complete SDM con-
taining 1,25D (20 nM); (G) complete SDM containing DEX (10 nM) in
osteoclast formation, we next tested the ability of NHBC
to support complete osteoclast formation in SDM. To
more stringently define the system, we used MACS-
purified CD14þ PBMC, shown previously to contain the
osteoclast precursor population (Massey and Flanagan,
1999). NHBC were co-cultured with purified CD14þ
PBMC on dentine slices in SDM containing DEX, with or
without 1,25D. After culture for 21 days, large TRAPþ
cells were detected in co-cultures stimulated with 1,25D
(Fig. 3A,B). It has been our experience with human
cells that TRAP positivity and/or multinucleation does
not always equate to functional osteoclast formation
(unpublished data). It was therefore important in these
cultures to demonstrate the resorbing capability of these
cells. SEM analysis revealed the presence of typical
resorption lacunae underlying regions of TRAPþ cells
(Fig. 3C). It was noted that resorption was frequently
associated with pre-existing topological features of the
substrate, such as the edges of haversian canals when
human cortical bone was used, small (approximately
1 mm) pores, likely to be microvessels, or alternatively,
577
combination with 1,25D (20 nM); (H) serum-replete a-MEM contain-
ing 1,25D (20 nM) and DEX (10 nM); (I) complete SDM containing
PTH (20 nM); (J) complete SDM containing PTH (20 nM) and DEX
(10 nM) in combination. Percentages of cells in each quadrant are
indicated. Staining patterns shown are representatives of those ob-
tained using two independent donors’ cells.
and most frequently, the edge of the bone or dentine slice
(e.g., Fig. 3B,C, and E). Significantly, cultures grown
simultaneously in media containing 10% FCS failed to
generate osteoclast-like cells in the presence of either
1,25D or PTH (not shown). In experiments where both
peripheral blood and BM precursors were isolated from
the same donor, functional osteoclasts were derived
successfully from CD14þ PBMC (Fig. 3D), CD14þ
BMMC, with similar growth kinetics and appearance
(Fig. 3E), and from immature CD34þ BMMC. In the case
of CD34þ BMMC, the resulting resorption sites
appeared as rosette-like clusters (Fig. 3F), perhaps
resulting from discrete colonies of mononuclear pro-
geny. While the addition of recombinant human M-CSF
is a necessary addition to the ST-2 co-cultures because of
the lack of species cross-reactivity, exogenous M-CSF in
this human-human system was not a requirement
(Fig. 3G) most likely due to its endogenous expression
by NHBC (e.g., Yao et al., 1998 and confirmed in our
laboratory; data not shown). Similar resorption data
were obtained from experiments, in which PTH1–34 was
578 ATKINS ET AL.
Fig. 3. Human–human osteoclast forming co-cultures in serum-free
conditions after 21 days of culture. Unless otherwise stated, co-
cultures included 1,25D (20 nM) and DEX (10 nM). A: A transmission
image collected on a confocal microscope of tartrate resistant acid
phosphatase (TRAPþ) multinucleate cells (arrows) derived from
CD14þ PBMC. B: Light microscopic image of typical ‘‘edge’’ staining
of osteoclasts, in this case from CD14þ BMMC and (C) the same region
analysed by scanning electron microscopy for resorption pits. D–F:
Showing resulting electron microscopic images of co-cultures of (D)
CD14þ PBMC, (E) CD14þ BMMC and (F) CD34þ BMMC, all isolated
from the same donor. Resorption lacunae were also derived from a co-
culture of the same CD14þ BMMC, as in (E), except in (G) the absence
of added M-CSF and (H) the absence of added M-CSF and 1,25D. In all
cases, the white bar represents 200 mm. I: Quantitation of resorption
area of experiment depicted in D–H. Average resorption  SEM was
calculated using Imagequant software (Molecular Dynamics) of mul-
tiple.TIF images encompassing the entire surface area of four 4 mm2
cortical bone slices per co-culture. One-way analysis of variance anal-
ysis (ANOVA) revealed no significant difference existed between co-
cultures, except in the case of no added M-CSF and 1,25D (*P < 0.05).
Similar resorption was obtained using NHBC isolated from three
donors.
 HUMAN OSTEOBLAST SUPPORT OF OSTEOCLASTOGENESIS
utilised as a stimulus in place of 1,25D (data not shown).
Occasionally, osteoclasts formed in co-cultures where no
M-CSF, 1,25D, or PTH was added, although the extent
and number of resorption pits was always small
compared with hormone treated cells (Fig. 3H,I). Under
stimulation with 1,25D, the extent of resorption result-
ing from all of CD14þ PBMC and BMMC, and CD34þ
BMMC populations, under the conditions described,
was found to be similar (Fig. 3I). Together, these are the
first data to demonstrate that, given the appropriate
stimuli, NHBC can support de novo osteoclast formation
from relatively mature (CD14þ PBMC and BMMC) and
immature (CD34þ BMMC) precursors.
Effect of exogenous recombinant RANKL
on co-cultures
Our previous experiments indicated that OPG was
secreted by NHBC in the range of 2–5 ng/ml over a 24 h
period, and 10–15 ng/ml after 72 h, in sub-confluent
cultures (Atkins et al., 2002). We hypothesised that the
production of OPG, as well as other potential inhibitors,
could account for the observed variability in the ability
of NHBC from different donors to support osteoclast
formation in this system. To test the influence of endo-
genous OPG production, co-cultures were established in
SDM, as above, in which sub-optimal concentrations
of recombinant human (rh) RANKL (1–10 ng/ml) were
added, and the resulting osteoclast formation examin-
ed. This was compared with co-cultures to which no
rhRANKL was added, or to RANKL-treated mono-
cultures of PBMC. In all cases, 25 ng/ml rhM-CSF,
10 nM 1,25D, and 10 nM dexamethasone were added
to the cultures. Recombinant human RANKL induc-
ed a dose-dependent increase in osteoclast formation in
co-cultures to a significantly greater extent than in
cultures of PBMC alone, at all concentrations tested
(Fig. 4). These results suggest that endogenous OPG is
an inhibitory influence on osteoclast formation in this
model.
Fig. 4. Augmentation of osteoclast formation in co-cultures by exo-
genous rhRANKL. NHBC-CD14þ PBMC co-cultures or CD14þ PBMC
cultured alone were performed in the presence or absence of rhRANKL
ranging from 0 to 10 ng/ml, all in the presence of rhM-CSF (25 ng/ml),
as described. Average resorption  SEM was calculated using Image-
quant software (Molecular Dynamics) of multiple.TIF images encom-
passing the entire surface area of four 4 mm2 cortical bone slices per
co-culture. Significance was determined using one-way ANOVA and
Tukey’s multiple comparison of non-parametric data. *P < 0.05. Simi-
lar data were reproduced in two independent experiments.
579
Recombinant RANKL/M-CSF do not support
stroma-free osteoclast formation in SDM
All osteoclastogenesis assays included, as a control,
the respective osteoclast precursor populations cultured
in the absence of stroma with the addition of recombi-
nant RANKL (20–30 ng/ml) and M-CSF (25 ng/ml).
PBMC cultured in SDM under these conditions gave rise
only to few, weakly TRAPþ, cells. In addition, these cells
did not form resorption lacunae. However, the same cells
cultured in serum-replete a-MEM containing RANKL
and M-CSF produced large numbers of functional
osteoclasts (data not shown). To investigate this pheno-
menon further, we added FCS to SDM containing 1,25D
and DEX, and tested the ability of this medium to sup-
port osteoclastogenesis. As seen in Figure 5, culture
in SDM containing 0, 25, or 100 ng/ml recombinant
human RANKL, in the presence of 25 ng/ml recombi-
nant human M-CSF, did not result in the formation of
functional osteoclasts. Consistent with our co-culture
results, some (atypical) TRAPþ MNC were observed in
SDM cultures, however, these cells did not arise in a
RANKL-dependent fashion, nor did they resorb dentine.
Addition of FCS at either 2% v/v or 10% v/v to SDM did
not induce TRAPþ MNC formation and did not give rise
to functional osteoclast formation at concentrations of
RANKL up to 100 ng/ml. In contrast, CD14þ PBMC
Fig. 5. Stromal-free osteoclastogenesis assays of CD14þ PBMC
cultured in the presence of M-CSF (25 ng/ml) and the presence or
absence of rhRANKL (25 or 100 ng/ml). Cells were either cultured in
SDM, SDM supplemented with 2% v/v FCS (SDM-2), SDM supple-
mented with 10% v/v FCS (SDM-10), or a-MEM supplemented with
10% FCS (a-MEM-10). Upper part: TRAPþ multinucleate cell (MNC)
formation in wells containing cells cultured on plastic. Data shown are
mean  SEM of quadruplicate wells of a 96-well plate. Lower part:
Resorption pit formation on dentine slices (mm2 per 2
106 mm2). Data
shown are mean  SEM of four representative fields taken at 50

magnification. Similar results were obtained from two independent
experiments. *P < 0.03.
580 ATKINS ET AL.
cultured in serum-replete a-MEM under otherwise iden-
tical culture conditions gave rise to TRAPþ MNC
capable of extensive resorption of dentine, in a strictly
RANKL-dependent manner (Fig. 5).
DISCUSSION
To date, little is known about which cells of the
osteoblast lineage are capable of supporting osteoclast
formation. Although various reports have demonstrated
the ability of human osteosarcoma cell lines to support
human osteoclast formation (Blair et al., 2000; Itoh et al.,
2000; Miyamoto et al., 2002), understanding the
requirements for normal human osteoclast formation
has proved elusive. Fujikawa et al. (1996) adapted the
use of the pre-adipocytic cell line ST-2 model to sup-
port human osteoclast formation from PBMC. While
our laboratory has successfully reproduced this model
system (Haynes et al., 1999; Atkins et al., 2000), our
numerous attempts to transfer those culture condi-
tions to an all-human osteoclastogenesis model, where
NHBC are substituted for ST-2 cells as the stromal
element, were unsuccessful. However, the use of a de-
fined SDM, shown previously to support the growth of
CFU-F (Gronthos and Simmons, 1995) and which has
been used in our laboratory to grow NHBC routinely,
was tested and found to give enhanced expression of
RANKL relative to OPG mRNA, particularly with the
addition of 1,25D and DEX. SDM supplemented with
1,25D and DEX was found to promote fully functional
human osteoclast formation from CD14þ PBMC and
BMMC, and from CD34þ BMMC, using NHBC as a
stromal layer.
The finding that SDM rather than serum-replete
a-MEM is permissive for osteoclast formation is in-
triguing. One explanation may simply be the greater
induction of RANKL relative to OPG expression in
NHBC when exposed to 1,25D and DEX in SDM. Based
on our previous study (Atkins et al., 2003), we hypo-
thesise that this property is intrinsically related to
osteoblast differentiation. NHBC are heterogeneous
and display a range of phenotypes representing various
stages of osteogenic maturation (Gronthos et al., 1999;
Stewart et al., 1999). In our previous study, we showed
that when grown in medium containing FCS, NHBC
differentiation followed a distinct pattern of phenotypic
change upon exposure to 1,25D and DEX, with respect to
the expression of the phenotypic markers STRO-1/AP
and RANKL expression (Atkins et al., 2003). Whilst
positive changes in the RANKL:OPG ratio were ob-
served in that study, driven mainly by 1,25D induction
of RANKL, it now seems that these changes are not
adequate to allow osteoclastogenesis to proceed. Simi-
larly, the phenotypic changes observed in that study
were mild when compared to the dramatic and rapid
progression to the mature (STRO-1APþ) differentia-
tion state observed in a culture background of SDM, as
shown in this study. From the studies to date, it is not
possible to conclude which is the phenotype of osteoblast
that best supports osteoclast formation, an issue made
difficult to resolve because of the transient nature of
NHBC phenotype. However, progression through the
osteoblast differentiation pathway does occur and is
likely necessary to provide a window in which osteoclast
formation can proceed. Interestingly, differentiation of
ST-2 cells also occurs during the osteoclast-forming co-
culture. ST-2 cells represent an immature osteoblastic
cell line, and are capable of osteoblastic differentia-
tion under stimulation by bone morphogenetic proteins
(BMPs) or ascorbate (Ogawa et al., 1988; Otsuka et al.,
1999). The culture medium for that assay contains both
ascorbate and DEX, potent osteoblast differentiation
factors. We have shown that under conditions sup-
portive of human osteoclast formation, in the additional
presence of 1,25D, the expression of RANKL mRNA
relative to OPG increases during the co-culture period
(Atkins et al., 2000).
For all donors’ NHBC tested, the extent of osteo-
clast formation in our co-culture system appeared to be
tightly regulated, evidenced by the highly focal nature
of osteoclast generation, often associated with a pre-
existing physical feature of the bone or dentine, such as
microvascular pores or the cut edge of the material. This
is in contrast to the widespread osteoclast formation
observed in the Fujikawa assay (Fujikawa et al., 1996),
where, in our hands, resorption events routinely exceed
500 per 105 input PBMC (data not shown), compared
with 1–300 resorption events per 105 CD14þ PBMC in
NHBC co-cultures. The reasons for NHBC donor vari-
ability and lower efficiency of osteoclast formation in the
all-human co-culture are not yet known. Unlike ST-2
cells, which are clonal in origin, the NHBC cultures
represent a mixed population of cells with likely dif-
ferential potential for either osteoclast support or bone
formation. Our previous study identified that 1,25D-
induced RANKL mRNA expression was positively asso-
ciated with an induced change in the percentage of
cells expressing STRO-1, implying that in the hetero-
geneous population of NHBC only a proportion of the
cells would likely positively influence osteoclast forma-
tion (Atkins et al., 2003). In that study also, the ex-
pression of OPG mRNA did not appear to be regulated
in a sub-population dependent fashion, implying that
the majority of osteoblasts potentially contribute to the
active suppression of osteoclast formation under normal
conditions (Atkins et al., 2003). In the co-culture system
described here, the endogenous production of OPG, or
other factors, appeared to be readily overcome by the
addition of sub-optimal concentrations of exogenous re-
combinant RANKL, which were significantly less effec-
tive in inducing osteoclast formation from cultures of
PBMC in the absence of stromal cells. We cannot rule out
that as well as overcoming the effect of endogenous OPG,
the effect of rhRANKL in these experiments was to
synergise with other co-stimulators of osteoclastogen-
esis, such has been shown for TNF-a (Fuller et al., 2002).
In addition, we have shown that NHBC express abund-
ant mRNA for the newly described osteoclast inhibitory
lectin (OCIL) (Hu et al., 2004), and this, too, was expres-
sed by all sub-populations of NHBC (data not shown). It
should be noted that variability also exists in the ability
of various donors’ cells to mineralise in vitro (Findlay
et al., 2004); the possibility exists that the apparent dif-
ferences in both the bone forming and osteoclast sup-
porting abilities of NHBC reflect inherent differences in
the bone metabolism of these individuals.
To date, we have derived human osteoclast precursors
from peripheral blood or BM cells in response to 1,25D
and to PTH. While the expression of RANKL and OPG
in response to 1,25D was reflected in the effects of this
agent on the phenotype of NHBC, the same relation-
ships were not seen with PTH. It should be noted that
while in all donors’ cells tested, RANKL expression
increased to some extent in response to 1,25D, PTH did
not consistently up-regulate RANKL expression. Con-
sistent with this, we have found that the PTH/PTHrP
receptor is not always detectable in NHBC cultures by
RT-PCR (unpublished data). Nevertheless, in some ins-
tances donors’ cells did respond positively to PTH in the
 HUMAN OSTEOBLAST SUPPORT OF OSTEOCLASTOGENESIS
osteoclast-forming assay, indicating heterogeneity be-
tween different donors’ cells in this respect also. Having
developed this model, it will now be of interest to further
explore the effects of other agents, including PTH, in
promoting human osteoclast formation in a defined
medium.
An interesting aspect of this study was the attempt
to generate osteoclasts from PBMC precursors in a
stromal- and serum-free system. Recombinant RANKL
in combination with M-CSF has been widely used to
generate osteoclasts from PBMC in stromal-free sys-
tems but always in serum-replete media. We were un-
able to generate osteoclasts from PBMC or BMMC in
stromal-free cultures in SDM, or in two commercially
available serum-free media (AIM V (Invitrogen, Carls-
bad, CA) and Nutridoma (Roche), data not shown), even
when concentrations of up to 100 ng/ml rhRANKL were
used in combination with 25 ng/ml rhM-CSF. Further-
more, the addition of FCS to SDM did not recover the
ability of RANKL and M-CSF to stimulate functional
osteoclast formation. TRAPþ MNC that did form in SDM
were atypical in appearance, did not resorb dentine, did
not form in a RANKL-dependent fashion, and were
reminiscent of TRAPþ macrophage polykaryons
described by Guicheux et al. (1998). The reasons why
RANKL-mediated osteoclastogenesis is not observed in
SDM are currently unclear. We can conclude, however,
that complete SDM used for the generation of osteo-
clasts in our human co-culture contains, in addition to
osteogenic factors, anti-osteoclastogenic factors that
cannot be overcome by the addition of exogenous
RANKL/M-CSF. Also, NHBC cultured in SDM must
produce factors that overcome the inhibitory influence of
the medium on the osteoclast precursor, allowing
osteoclast formation to proceed. It is likely that the
other serum-free media used in this study (AIM V and
Nutridoma) both contain combinations of growth factors
that divert differentiation of precursors from the
osteoclastogenic pathway. FCS, on the other hand,
contains factors supportive of osteoclast formation in
stromal-free systems but with the required presence of
recombinant RANKL/M-CSF. Our results indicate that
FCS must also contain factors that negatively influence
the ability of human osteoblasts to support osteoclast
formation in co-culture, highlighting both the complex-
ity of this common additive to cell culture systems
(Leffert, 1974) and the desirability for the establishment
of defined serum-free assays. Elucidation of these
phenomena will require a more substantive breakdown
of SDM and testing the effects of individual components
alone and in combination on osteoclast formation. This is
the first report, to our knowledge, that shows RANKL
and M-CSF do not always stimulate osteoclast forma-
tion and that other growth factors may divert precursors
from this pathway.
In summary, we report the development of an ‘‘all
human’’ culture system in which fully functional human
osteoclasts can be formed by co-culture of normal,
phenotypically-defined osteoclast peripheral blood- and
BM-derived precursors with normal human trabecular
bone-derived osteoblasts as a stromal layer. Further-
more, it is the first report of human osteoclast formation
in a serum-free system. This model system provides
a basis for the study of both the osteoclast and its
precursor, and the normal osteoblast that likely carries
out this function in vivo. We contend that the con-
tributions of both cell types need to be taken into
account to fully elucidate the intricacies of human bone
remodelling.
581
ACKNOWLEDGMENTS
The authors wish to thank the surgeons and nursing
staff of the Department of Orthopaedic Surgery and
Trauma, Royal Adelaide Hospital, for the provision of
surgical bone samples.
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