Research Article
2949
Formation of a chondro-osseous rudiment in
micromass cultures of human bone-marrow stromal
cells
Anita Muraglia1, Alessandro Corsi2,3, Mara Riminucci2,3, Maddalena Mastrogiacomo1, Ranieri Cancedda1,4,
Paolo Bianco3,5 and Rodolfo Quarto1,4,*
1Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy
2Dipartimento di Medicina Sperimentale, Universita’ dell’Aquila, Italy
3Dipartimento di Medicina Sperimentale e Patologia, Universita’ La Sapienza,
4Dipartimento di Oncologia, Biologia e Genetica, Universita’ di Genova, Italy
5Parco Scientifico Biomedico San Raffaele Roma, Italy
Roma, Italy
*Author for correspondence (e-mail: [email protected])
Accepted 1 April 2003
Journal of Cell Science 116, 2949-2955 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00527
Summary
Bone-marrow stromal cells can differentiate into multiple
mesenchymal lineages including cartilage and bone. When
these cells are seeded in high-density ‘pellet culture’,
they undergo chondrogenesis and form a tissue that is
morphologically and biochemically defined as cartilage.
Here, we show that dual chondro-osteogenic differentiation
can be obtained in the same micromass culture of human
bone-marrow stromal cells. Human bone-marrow stromal
cells were pellet cultured for 4 weeks in chondro-inductive
medium. Cartilage ‘beads’ resulting from the micromass
culture were then subcultured for further 1-3 weeks in
osteo-inductive medium. This resulted in the formation of
a distinct mineralized bony collar around hyaline cartilage.
During the chondrogenesis phase, type I collagen and bone
sialoprotein were produced in the outer portion of the
Introduction
Probing the differentiation potential of human bone-marrow
stromal stem cells (BMSCs) employs a variety of experimental
approaches. Most commonly, single or multiple differentiation
capabilities (e.g. osteo-, chondro- or adipogenic) are ascribed
to either clonal or non-clonal stromal cell populations based on
the assessment of tissue-specific differentiation markers in
culture. Nonetheless, several considerations limit the value and
significance of such assays, without detracting from their
empirical value or from their desirable amenability to routine
use. For example, the expression of an in vitro phenotype
reminiscent of osteogenic cells does not necessarily predict the
ability of a given cell strain to generate bone upon in vivo
transplantation (Kuznetsov et al., 1997; Satomura et al., 2000);
likewise, marked phenotypic variability is observed over time
in clonal cell strains in culture (Deryugina et al., 1994;
Muraglia et al., 2000). The use of in vivo transplantation assays
(Friedenstein et al., 1987; Goshima et al., 1991; Gundle et al.,
1995; Haynesworth et al., 1992; Krebsbach et al., 1997; Martin
et al., 1997; Quarto et al., 1995) has become a valuable
standard for assessing the osteogenic potential of marrow
stromal cells. Not only do these assays probe the physiological
cartilage bead, which, upon subsequent exposure to
β-glycerophosphate, mineralized and accumulated
extracellular bone sialoprotein and osteocalcin. Our
modification of the pellet culture system results in the
formation of a chondro-osseous ‘organoid’ structurally
reminiscent of pre-invasion endochondral rudiments, in
which a bony collar forms around hyaline cartilage. The
transition from a cell culture to an organ culture dimension
featured by our system provides a suitable model for the
dissection of molecular determinants of endochondral bone
formation, which unfolds in a precisely defined spatial and
temporal frame
Key words: Osteoprogenitors, Osteogenesis, Chondrogenesis, Bonemarrow stromal cells, Condensation
osteogenic function in vivo, they also provide the simplest and
most convincing readout (the formation of histologically
proved true bone tissue). The micromass culture [‘pellet
culture’ (Johnstone et al., 1998)] of human BMSCs provides
an assay of similar significance for chondrogenesis. Culturing
BMSCs after inducing an artificial condensation event and
exposing them to a chondrogenic cytokine milieu results in the
generation of a three-dimensional structure that is directly
reminiscent of true hyaline cartilage (Johnstone et al., 1998).
Recent work has shown that the system can be advantageously
used with human BMSCs (Johnstone et al., 1998; Mackay et
al., 1998; Mastrogiacomo et al., 2001; Sekiya et al., 2001;
Sekiya et al., 2002; Yoo et al., 1998) as well as with BMSCs
from other species (Johnstone et al., 1998), and has
emphasized the value of the system for dissecting molecular
determinants of chondrogenesis in a relatively simple and
defined set of experimental conditions (Sekiya et al., 2002).
Starting from previous in vivo and in vitro observations
indicating that chondrogenic differentiation does not preclude
the further development of an osteoblast-like phenotype in
vitro (Bianco et al., 1998; Galotto et al., 1994; Gentili et al.,
1993; Jimenez et al., 2001), we asked whether this could be
2950
Journal of Cell Science 116 (14)
effectively investigated taking advantage of the histological
dimension and the three-dimensional nature of the pellet
culture system of marrow-derived skeletal progenitor cells. We
report here that, by appropriate manipulation of the system, not
only can true bone formation be obtained but also specific
spatial and temporal patterns of chondro- and osteogenesis are
obtained in vitro that culminate in the generation of an in vitro
formed ‘organoid’ that directly mimics the formation of the
bony collar around cartilage anlagen that occurs during
embryonic bone development.
Materials and Methods
Chemicals
Recombinant human fibroblast growth factor 2 (rh FGF-2) and
recombinant human transforming growth factor β1 (rhTGFβ1) were
from Austral Biologicals (San Ramon, CA). Foetal calf serum (FCS)
was purchased from Kallergen (Settimo Milanese, Italy). All other
chemicals were from Sigma (St Louis, MO).
Cell culture
BMSCs were obtained from iliac crest marrow aspirates of healthy
donors (age range 1-60 years). All the procedures were approved by
an institutional ethical review committee. After washing in PBS,
mononucleated cells were stained with 0.1% methyl violet in 0.1 M
citric acid and counted. The cells were then suspended in Coon’s
modified Ham’s F12 medium supplemented with 10% FCS and 1 ng
ml–1 rhFGF-2 and plated at between 2×106 and 5×106 cells per 100
mm dish. Medium was changed 3 days after plating and then twice a
week thereafter.
Pellet culture
Cells from confluent cultures (20-25 days in culture, passage 0,
corresponding to 12-15 doublings) were released by 0.05% trypsin in
0.01% EDTA, counted and used to generate pellet cultures conducive
for chondrogenesis in vitro, essentially as previously described
(Johnstone et al., 1998). Briefly, 2.5×105 cells were centrifuged at
500 g in 15 ml polypropylene conical tubes and the resulting pellets
were cultured for 4-7 weeks. Control cultures were grown in a serumfree chemically defined medium consisting of Coon’s modified Ham’s
F12 medium supplemented with 10–6 M bovine insulin, 8×10–8 M
human apo-transferrin, 8×10–8 M bovine serum albumin, 4×10–6 M
linoleic acid, 10–3 M sodium pyruvate (control medium). To induce
chondrogenic differentiation, the control medium was supplemented
with 10 ng ml–1 rhTGFβ1, 10–7 M dexamethasone and 2.5×10–4 M
ascorbic acid.
Cultures were incubated for 4 weeks at 37°C in an atmosphere
containing 5% CO2; the medium was changed every 4-5 days and
ascorbic acid added three times a week. To monitor chondrogenesis,
cultures were harvested at 1-4 weeks and processed for histology
(see below). At the end of the 4-week culture, some cultures
were incubated for a further 2-3 weeks in a medium conducive for
in vitro mineralization (control medium containing 7.0×10–3 M βglycerophosphate, 10–8 M dexamethasone and 2.5×10–4 M ascorbic
acid), then fixed and processed for histology.
Histology
The cell aggregates were fixed with 4% formaldehyde in PBS for 1015 minutes and routinely embedded in paraffin. Paraffin sections were
stained with haematoxylin-eosin, toluidine blue, alcian blue and
alizarin red S, and viewed in transmitted and polarized light
microscopy.
Antibodies
Monoclonal antibodies against type I and type II collagen (SP1D8 and
CIICI, respectively) were obtained from the Developmental Studies
Hybridoma Bank (Department of Biological Sciences, University of
Iowa). Supernatants from hybridoma cultures were used undiluted
(SP1D8) or concentrated ten times (CIICI). A monoclonal antibody
to human recombinant type X collagen (X53) was kindly provided by
K. von der Mark (Institute of Experimental Medicine, Friedrich
Alexander University of Erlangen, Germany); supernatant from
hybridoma culture was used undiluted. A rabbit antiserum raised
against human bone sialoprotein [BSP, LF6 – (Fisher et al., 1995)]
was kindly provided by L. W. Fisher (NIDCR, NIH, Bethesda, MD)
and was used at a dilution of 1:100 in PBS, 0.1% bovine serum
albumin (BSA). A rabbit antiserum raised against bovine osteocalcin
(cross-reactive with the human protein) was kindly provided by S.
Robins (Rowett Research Institute, Aberdeen, UK) and was used at a
dilution of 1:500 in PBS, 10% goat serum.
Immunohistochemistry
Deparaffinized and rehydrated 5 µm sections were incubated with 3%
hydrogen peroxide in methanol for 30 minutes to inhibit endogenous
peroxidase activity. Some sections were subjected to digestion with 1
mg ml–1 hyaluronidase in PBS, pH 6.0 for 15 minutes at 37°C prior
to use. Sections were exposed to normal goat or pig serum (Dako,
Glostrup, Denmark) diluted 1:10 in PBS, 0.1% BSA for 30 minutes
before incubation with the primary antibodies. Slides were then
washed with PBS, 0.01% Triton X-100 (Sigma, St Louis, MO) (four
times for 5 minutes each), incubated with the secondary biotinylated
antibodies (1:200 or 1:500 in PBS, 0.1% BSA) for 30 minutes, rinsed
in PBS, 0.01% Triton X-100 (four times for 5 minutes each) and
incubated with peroxidase-conjugated ExtrAvidin (1:50 in PBS, 0.1%
BSA) for 30 minutes. The peroxidase reaction was developed using
either 3-amino-9-ethylcarbazole (AEC) or 3,3′-diaminobenzidine
tetrahydrochloride (DAB) as chromogens. All incubations were
performed at room temperature. After rinsing in distilled water,
sections were dehydrated in ascending ethanol solutions, cleared in
xylene and mounted.
Transmission electron microscopy
In vitro generated tissues were decalcified in neutral buffered 10%
EDTA or left undecalcified. After washing in PBS, samples were
postfixed for 1 hour at 4°C in 1% osmium tetroxide in cacodylate
buffer, rinsed in water, dehydrated through graded ethanol solutions,
transferred in propylene oxide, and embedded in epoxy resin
(Araldite™). Semithin sections were stained with Azur II-Methylene
Blue to select appropriate fields; ultrathin sections were cut with
diamond knives, placed on uncoated grids, contrasted with uranyl
acetate and lead cytrate and examined with a CM 10 Philips
transmission electron microscope.
Results
Chondrogenesis in BMSC pellet cultures
BMSC pellets cultured in the presence of TGFβ1 generated a
solid three-dimensional tissue structure that could be harvested
and processed intact for histology. By day 7, a clear-cut
chondroid morphology (substantial amounts of basophilic,
alcianophilic, metachromatic matrix containing cells encased
in chondrocytic lacunae) was formed (Fig. 1). Strong diffuse
immunoreactivity for type II collagen was seen as early as
day 7 (Fig. 2). Interestingly, a thin peripheral rim of matrix
remained essentially unlabelled.
The in vitro generated cartilage ‘beads’ grew in size
Perichondral bone formation by stromal cells
2951
Fig. 1. Chondrogenesis in pellet cultures. Cartilage beads
formed by BMSCs cultured under chondrogenic conditions for
1 (a,b), 2 (c,d) and 3 (e,f) weeks. (a,c,e) Toluidine-blue
staining. (b,d,f) Alcian-blue staining. Overall morphology,
alcianophilia and metachromatic staining with toluidine blue
are typical of cartilage. Bar, 100 µm.
Immunoreactivity for type X collagen was not seen
until day 21, at which point it became distinctive in
individual cells and throughout the extracellular matrix
in the central cartilaginous region of the tissue bead (data
not shown), indicating progression to hypertrophy of
chondrocytes differentiated in pellet culture.
BMSC pellets cultured in the absence of TGF-β1
(control cultures) did not grow in size, nor did they
generate any tissue structure reminiscent of cartilage
(data not shown).
progressively, reaching a plateau at day 14 (Fig. 2).
Alcianophilia and metachromasia, two histochemical features
of proteoglycan content in cartilage, were uniform throughout
the cross-sectional areas of the cartilage ‘beads’ up until day
14, when the beads reached their maximum size. After day 14,
both alcianophilia and metachromasia continued to increase in
intensity in the central region of the beads. After day 21, they
began to disappear from a progressively wider peripheral zone.
By day 28, a clear-cut zonal pattern had formed, with a nonbasophilic collar of cellular tissue, up to 300 µm thick,
encasing a central core of histologically and histochemically
well-defined hyaline cartilage.
Osteogenesis in BMSC pellet cultures
BMSC pellets which had been cultured in the presence
of TGF-β1 for 28 days were transferred to mineralization
medium and cultured for an additional 1-3 week period.
Within one week of culture under conditions conducive
to in vitro mineralization, the peripheral collar of tissue
had turned into a tissue histologically reminiscent of
bone (Fig. 3). The tissue was fully mineralized, whereas the
central core of hyaline cartilage had remained uncalcified,
as demonstrated by alizarin red S or von Kossa staining.
Apparently, viable cells were encased in lacunar spaces within
the fully calcified bone-like matrix, reminiscent of osteocytes.
The bone-like matrix appeared as woven bone in polarized
light microscopy. In essence, a bony collar had formed around
a cartilage core, closely mimicking the events occurring during
the early phases of endochondral ossification.
Electron microscopic analysis (Fig. 4) confirmed the
occurrence of mineralization and demonstrated features typical
of genuine cartilage and bone matrix. Central areas of the
cartilage beads, exhibiting histological features of hyaline
cartilage, contained widely spaced, thin, non-banded collagen
fibrils and abundant proteoglycan granules. At the transition
between the central areas and the outer collar of non-chondroid
tissue, mineralization nodules appeared in the context of a
typical cartilaginous matrix structure. More peripherally, thick
and periodically banded collagen fibrils became predominant.
In the outer portion of the peripheral collar, dense bundles of
banded fibrils and extensive calcification were observed.
Spatial and temporal patterns of expression of bone
matrix proteins in BMSC pellet cultures
Because both bone- and cartilage-like matrix formed in our
Fig. 2. Chondrogenesis in pellet cultures. (a,b) Immunoreactivity for
type II collagen at 7 days (a) and 14 days (b) of culture. Notice the
diffuse staining, which spares a thin rim of matrix at the periphery of
the tissue ‘bead’. (c) Growth in size of the tissue bead over time. A
plateau is reached at day 14. The arrow indicates the switch to
medium conducive to in vitro mineralization. Each point represents
the mean of the cross-sectional diameter measured in three samples
per time point. Bar, 125 µm.
2952
Journal of Cell Science 116 (14)
Fig. 3. Osteogenesis in pellet cultures. BMSCs were cultured under
chondrogenic conditions for 4 weeks and subsequently in
mineralization-inducing medium for 1 week. Histological sections of
the cultured tissue harvested at 35 days of culture. (a) Toluidine-blue
staining. Notice the absence of metachromatic staining (purple) in a
peripheral rind of tissue. (b) Alizarin-red-S staining of a comparable
section shows that the peripheral rind of non-chondroid tissue has
accumulated calcium, which indicates mineralization.
(c) Haematoxylin and eosin staining of another section,
demonstrating a central core of histologically typical hyaline
cartilage (hc) surrounded by a transition zone (tz) where basophilia
(blue staining) fades out and an outermost layer of mineralized bonelike tissue (b) containing individual cells encased in lacunar spaces
(d). Bar: a,b, 100 µm; c, 50 µm; d, 12.5 µm.
system, we investigated the expression of characteristic bone
matrix proteins during the process leading to the in vitro
generation of an osteochondral ‘organoid’. To investigate the
expression of type I collagen, we used a monoclonal antibody
recognizing the N-propeptide of type I procollagen, which is
cleaved extracellularly following secretion of procollagen
molecules. Thus, intracellular immunoreactivity could be
taken as evidence of procollagen synthesis and extracellular
immunoreactivity as indicative of sites of initial deposition
associated with freshly secreted procollagen molecules.
Expression and initial deposition of type I collagen were
spatially restricted to an outer layer of tissue that progressively
Fig. 4. Electron microscopic analysis of cartilage and bone tissues
formed in pellet culture. The culture was harvested at 35 days (28
days of culture under chondrogenic conditions and 7 days of culture
in mineralization-inducing medium). (a) Azur II / methylene blue
stained semithin section demonstrating the relative position of the
electron-microscopy fields imaged in b-e. Mineralized tissue is on
the left and hyaline cartilage on the right. (b) Bone-like matrix
containing thick, periodically banded collagen fibrils and areas of
mineral deposition (arrows) (undecalcified section, original
magnification 28500×). (c) Transition zone containing alternate
thick, periodically banded, and thin, non-banded collagen fibrils.
Arrow indicates mineral clusters. No proteoglycan granules are seen
(undecalcified section, original magnification 28500×).
(d) Mineralizing cartilage. Arrows indicate mineral clusters. The
remaining matrix has typical features of hyaline cartilage (thin,
randomly oriented collagen fibrils and proteoglycan granules)
(decalcified section, original magnification 11500×). (e) Hyaline
cartilage (decalcified section, original magnification 11500×). Bar:
a, 25 µm; b,c, 6 µm; d,e, 2.3 µm.
increased in thickness over time (Fig. 5). Extracellular
immunoreactivity was restricted at all times to a thin rim of
matrix marking the outer boundary of the hyaline cartilage
region. Intracellular immunoreactivity for type I collagen N
propeptide was detected in a progressively thicker region
of peripheral tissue. From day 14 onward, extracellular
immunoreactivity for type I collagen N-propeptide was lost in
the same region, probably as a result of matrix maturation,
whereas immunolabelling of procollagen producing cells
remained distinctly detectable.
Bone sialoprotein is specifically expressed during early
stages of embryonic bone formation, and so we also analysed
the production of BSP during in vitro perichondral
Perichondral bone formation by stromal cells
Fig. 5. Immunoreactivity for type I
procollagen N-terminal propeptide
at 7 (a), 14 (b) and 28 (c) days of
culture under chondrogenic
conditions. Notice the growing
peripheral region of type I collagen
production (double arrows).
Extracellular immunoreactivity,
denoting the initial deposition (id)
of collagen, marks the boundary of
cartilage. Intracellular labelling
(arrows) is restricted to a
peripheral rind of tissue where
immunoreactivity is lost in the
extracellular matrix, consistent
with zonally defined matrix
maturation events (m). Bar, 50 µm.
2953
osteogenesis in the pellet culture system (Fig. 6). BSPproducing cells were detected as early as day 7 and increased
in number thereafter. At all time points, BSP-producing cells
were spatially confined to the outer portion of the cartilage
‘beads’. At day 14, they formed a distinct peripheral region of
BSP production. Between days 21 and 28, when physical
growth of the beads had ceased, production of BSP shifted
from the outermost portion of the beads to an adjacent, more
central region. Overall, the spatial and temporal pattern of
cellular immunolabelling for BSP and type I collagen N
propeptide were closely similar to one another. Extracellular
immunoreactivity for BSP only appeared in cultures exposed
to mineralization-conducive conditions (day 28-42).
No immunoreactivity for osteocalcin was observed at any
time during the chondrogenic period (days 1-28). Abundant
extracellular immunoreactivity for osteocalcin was, by
contrast, seen in cultures harvested during the mineralization
period (days 35-49) within the peripheral rind of bone-like
tissue (Fig. 6).
Discussion
Direct evidence of osteogenesis in pellet cultures of BMSCs
(Johnstone et al., 1998; Mackay et al., 1998; Mastrogiacomo
et al., 2001; Sekiya et al., 2001; Yoo et al., 1998) has never
been reported before. Likewise, so-called ‘mineralization
nodules’ (Beresford et al., 1993; Jaiswal et al., 1997;
Maniatopoulos et al., 1988; Martin et al., 1997) but not
histologically proved, three-dimensional bony structures form
in cultures of adherent osteogenic cells (reviewed by Bianco
and Robey, 1999; Bianco and Gehron
Robey, 2000) and in vivo
transplantation assays (Beresford,
1989; Goshima et al., 1991;
Krebsbach et al., 1997; Kuznetsov et
al., 1997; Martin et al., 1997) are
Fig. 6. Spatial and temporal expression
of BSP and osteocalcin in BMSC pellet
cultures. Haematoxylin and eosin stained
section (a-d), BSP immunolabelling (e,f)
and osteocalcin immunolabelling (g-l) at
7, 14 and 28 days of culture
(chondrogenic conditions), and at 35
days of culture (28 days under
chondrogenic conditions and 7 days
under mineralization-permissive
conditions). Concurrent with a
morphological change in the outer layer
of tissue (a-d), BSP production is
restricted to a progressively thicker rind
of tissue during the chondrogenic phase
(e-g). Deposition of extracellular BSP (h,
arrows) and osteocalcin (l) is only
observed when mineralization has
occurred (h, arrows). Notice the similar
patterns of production of BSP and type I
procollagen (Fig. 5), including the
apparent centripetal shift of the cellular
production of either protein between day
14 and 28 (f,g, double arrows for BSP).
Bar, 50 µm.
2954
Journal of Cell Science 116 (14)
ultimately required to prove the osteogenic capacity of a
test cell strain. We have provided evidence that a fully
mineralized tissue with the histological, ultrastructural and
immunohistochemical characteristics of bone (as observed
in vivo) does form in pellet cultures of BMSCs exposed
sequentially to chondrogenic and mineralization-conducive
conditions. Thus, our modification of the pellet-culture system
provides a relatively simple in vitro assay for testing the
osteogenic capacity of putative osteogenic cells strains.
Considering that the pellet-derived tissue beads stop
growing in size around day 14, the subsequent deposition of
peripheral rind of bone-like matrix must occur at the expenses
of pre-existing cartilage. Thus, an internal remodelling of
cartilage to bone matrix occurs once the pellet-derived tissue
has reached its maximum size. Assuming a roughly spherical
shape for the tissue bead formed in vitro, approximately onethird of the volume of unmineralized cartilage existing at day
14 is physically replaced by an equal volume of bone-like
tissue. Given the very different water contents of unmineralized
hyaline cartilage and mineralized bone, the actual matrix
remodelling taking place is even greater. The internal
remodelling of cartilage into bone in our system might reflect
either a direct phenotypic conversion of chondrogenic cells to
an osteogenic phenotype or the local selection over time of an
osteogenic population replacing a chondrogenic one. The
former possibility would be consistent with prior in vitro and
in vivo evidence (Berry et al., 1992; Gentili et al., 1993;
Galotto et al., 1994), suggesting an eventual osteogenic fate of
hypertrophic chondrocytes. The non-clonal nature of our cell
population prevents inferences about a direct conversion of
chondrocytes to osteoblasts. For the same reason, whether
chondrogenesis and osteogenesis in our cultures reflect the dual
differentiation potential, the existence of a single stromal
progenitor cannot be conclusively stated. The specific point of
interest in our present results rather rests upon the precise
spatial and temporal pattern that chondro- and osteogenesis
obey in the pellet-culture system.
In vivo, embryonic bone formation occurs around
cartilaginous anlagen (rudiments) prior to the onset of
endochondral ossification proper. This peculiar spatial
arrangement is closely mimicked in our culture system, in
which a rind of bone-like tissue forms around a core of hyaline
and mineralizing cartilage. Our system thus directly models the
precise spatial determinants of cell differentiation operating in
development. Interestingly, whereas cartilage only forms
occasionally in open transplants of BMSCs (Krebsbach et al.,
1997; Kuznetsov et al., 1997; Martin et al., 1997), cartilage and
bone tissues that do form in closed transplantation systems
(diffusion chambers) (Ashton et al., 1980; Bab et al., 1984;
Gundle et al., 1995) are organized in a spatial pattern similar to
the one we observe in vitro (cartilage in the interior, bone in the
periphery). It is plausible that a gradient of oxygen tension
(Ashton et al., 1980; Scott, 1992) might be directly involved in
the generation of the spatial pattern observed in both systems.
Although chondrogenesis and osteogenesis are spatially
segregated in our system, they partially overlap temporally.
Initiation of osteogenesis occurs simultaneously with
chondrogenic differentiation but can only be completed upon
switching to mineralization-conducive conditions. During the
chondrogenesis phase, type I collagen and BSP are actively
synthesized and the proteoglycan content is reduced in the
region that is to become mineralized, bone-like tissue. By
contrast, it is the exposure to a mineralization-conducive
environment that induces the production and deposition of
osteocalcin, and the deposition (but not the production) of BSP.
In vivo, production of BSP by differentiating osteoblasts in
the presumptive bony collar and in the adjacent outermost
chondrocytes occurs simultaneously and ‘primes’ the
subsequent deposition of the bony collar (Bianco et al., 1991;
Bianco et al., 1993; Bianco et al., 1998; Riminucci et al., 1998).
Just as our system models the formation of the bony collar in
vitro, so our data on the localization of BSP recapitulate the
specific spatial and temporal pattern of expression of BSP
associated with the in vivo events.
The pellet culture of human BMSCs is generally taken as a
good in vitro model of chondrogenesis. By showing the
occurrence of bone deposition in the same system, our data
highlight a hitherto unrecognized experimental benefit of the
system. That is, the direct transition, in vitro, from a cell culture
to an organ culture dimension. A number of specific,
temporally defined events of bone morphogenesis, such as
endochondral bone formation proper or ontogeny of the bone
marrow, could in principle be modelled and dissected in terms
of their molecular determinants by further exploring the
experimental flexibility of the system.
This work was supported by grants from MIUR, Ministero della
Sanita’ and funds from the Italian (ASI) and European (ESAERISTO) Space Agencies.
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