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Laboratorio di Differenziamento Cellulare (R.Q., G.C., R.C., B.D.), Istituto Nazionale per la Ricerca sul Cancro, Centro di Biotecnologie Avanzate; and Dipartimento di Oncologia Clinica e Sperimentale (R.C.), Universita’ di Genova, 16132 Genova, Italy
Address all correspondence and requests for reprints to: Beatrice Dozin, Ph.D., Laboratorio di Differenziamento Cellulare, Istituto Nazionale per la Ricerca sul Cancro, Centro di Biotecnologie Avanzate, Largo Rosanna Benzi n.10, 16132 Genova, Italy.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precise nature of the factors directly responsible for cartilage maturation is still poorly understood. An excess or deficiency of most hormones and/or some growth factors deeply affects statural growth, as shown by in vivo studies and clinical surveys (2, 3). With the development of various in vitro models (including organ culture, primary culture of prechondrogenic cells or differentiated chondrocytes, established cell lines and the more recent animal models carrying gene knock-outs) most of the emphasis has been placed on molecules such as GH, thyroid hormones, glucocorticoids, sex steroids, insulin (Ins), and basic fibroblast growth factor (FGF-2) (3, 4, 5, 6, 7). However, the effects elicited in vivo or in tissue cultures are often difficult to interpret because of the interference of circulating components or the addition of animal serum to the medium. Over the last few years, we have developed a culture system of chicken embryo tibia chondrocytes that are able to reproduce in vitro the full differentiation program of chondrogenesis as it occurs in vivo (8). When plated in anchorage-dependent conditions, primary chondrocytes highly proliferate, revert their phenotype, and synthesize mainly type I collagen. In view of their morphological and biochemical properties, these cells have been widely considered and recognized as dedifferentiated cells with chondrogenic potential comparable with the mesenchymal prechondrogenic precursors. Indeed, the differentiation pathway can be reinduced by transferring the dedifferentiated cells in suspension culture. In vitro chondrogenesis is characterized by an initial cell aggregation, the switch to type II and type IX collagen synthesis, and the ultimate state of cellular hypertrophy evidenced by the expression of type X collagen. In the attempt to transpose the system to serum-free conditions, we also have demonstrated that the differentiation can be fully induced and supported by substituting the serum with three hormones: Ins, triiodothyronine (T3), and dexamethasone (Dex). However, this basic hormonal medium failed to support the proliferation of the dedifferentiated cells (9). The aim of this study was to identify the growth promoting factor(s) able to replace serum components to support the proliferation of chondrogenic cells, while maintaining their metabolic properties and their differentiation potential. Our study evidences the major role of FGF-2 on the proliferation of prechondrogenic dedifferentiated cells, as compared with other growth factors, and the dissociation between Ins-like growth factor I (IGF-I) and Ins effects on growth and differentiation, respectively. Our serum-free culture experiments also support the concept of the instability of the chondrogenic phenotype and suggest that a population of dedifferentiated chondrocytes can reacquire a multipotent state, being able to enter the adipocytic, chondrogenic, or osteogenic lineage (depending on the culture conditions established).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Metabolic labeling and SDS-PAGE analysis
Cells were washed in PBS (pH 7.2), transferred to
methionine-free Coon’s modified Ham’s F12 supplemented with 50
µg/ml ascorbic acid, and incubated for 2 h at 37 C.
35S-methionine was added at a concentration of 100
µCi/ml, and the incubation was resumed for two h. Supernatants were
collected and clarified by low-speed centrifugation, and the labeled
proteins were analyzed by electrophoresis in reducing conditions on
12.5% SDS-polyacrylamide gels. In some cases, samples were subjected
to immunoprecipitation for further identification of the secreted
proteins. The antibodies used were rabbit antisera raised against type
I, II, and X collagens (Pasteur Institute, Lyon, France) to Ex-FABP
protein (the extracellular fatty acid-binding protein previously named
Ch21) (14) and to ovotransferrin (15). The immunoprecipitation products
were analyzed by SDS-PAGE, as described above.
Ectopic tissue formation in nude mice
Primary chondrocytes were expanded for 2–3 weeks, in serum-free
conditions, in the presence of either Ins/FGF-2 or
Ins/T3/Dex/FGF-2. Cells were detached, resuspended in
aliquots of 1 x 106/20 µl Coon’s modified Ham’s
F12, and adsorbed on two opposite faces, either of 100% porous
hydroxyapatite (HA) ceramic cubes (50 mm3; Fin-Ceramica,
Faenza, Italy) or hemostatic sponges of cross-linked native bovine
collagen (60 mm3; Coletica, Lyon, France). For each culture
condition, an additional sample of 7 x 106 cells/100
µl also was prepared for direct implantation without scaffold.
One-month-old recipient nude mice (CD-1 nu/nu) were purchased from
Charles River Italia (Calco, Italy), kept in a controlled environment,
and given free access to food and water. The mice were cared for and
treated, according to institutional guidelines. The animals were
anesthetized by im injection of Xylazine (1 mg/50 µl) and Ketamine (3
mg/50 µl). HA and collagen sponge grafts were implanted on the back
of the mice while cell suspensions were directly injected sc. The
experiment was performed on duplicate mice, each carrying all six
sample types. The animals were killed 8 weeks after implantation, and
the grafts were removed and processed for histological and
immunohistochemical analysis.
Histological and immunohistochemical analysis
The grafts were fixed overnight in 4% formaldehyde in PBS,
decalcified in 0.5 M EDTA (only when the HA support was
used), paraffin-embedded, sectioned (5-µm thickness), and stained
with toluidine blue or hematoxylin-eosin. Samples of hypertrophic
cartilage, reconstituted in vitro, were processed in the
same way for toluidine or alcian blue staining. For each sample, at
least two different section levels and two histological sections for
each level were analyzed.
Selected sections of the grafts were further subjected to immunoperoxidase staining to determine the cellular origin of the tissue formed. To distinguish between host (mouse) and donor (chicken), the monoclonal antibody 3.2.B5 specific for avian nuclei was used (16). The immunostaining was performed, as described previously (17), with the only modification that the sections were first treated with 0.1 M Tris-HCl (pH 3.2) to elute residual mouse Igs. The sections were analyzed and photographed with a Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that FGF-2 was the most potent proliferative agent (column 2) and
promoted a cell growth rate statistically similar to the control
10%-FCS culture (column 1). EGF (column 3) and GH (column 4) were also
mitogenic, although to a lesser extent than FGF-2. PDGFbb (column 5)
produced very limited effects. The experiments were performed with an
initial concentration of 10 ng/ml of the various growth factors. The
use of 100 ng/ml for EGF, GH, and PDGFbb did not increase their
growth-promoting activity, whereas FGF-2 used at 1 ng/ml was as potent
as at higher concentrations up to 100 ng/ml (not shown). As reported,
the three hormones alone failed to stimulate any cell growth (column
7); likewise FGF-2 alone was not mitogenic (column 8). Both conditions
allowed only the survival of the culture, just as when the cells were
maintained in plain medium lacking any supplement (column 6).
Therefore, the proliferative effect of FGF-2 must depend on its
association with one or more of the three hormones present in the
medium. Columns 9 and 10 indicate that the synergism does not occur
through either T3 or Dex. Instead, full proliferation was
obtained when Ins was associated with FGF-2 (column 11), reaching
values statistically similar to the control 10%-FCS culture.
Furthermore, Ins could be fully replaced by IGF-I (column 12), with
similar results, regardless of the concentration (1–100 ng/ml). The
addition of T3 and Dex did not influence the proliferation
induced by IGF-I (column 13).
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illustrates the morphology of cells
grown in Ins/FGF-2 (panel A) or Ins/T3/Dex/FGF-2 (panel E).
In this second condition, the cells presented a more pronounced spindle
shape. At confluence, few centers of chondrogenesis could be observed,
in particular in the Ins/FGF-2 condition (panel B), as compared with
the Ins/T3/Dex/FGF-2 condition (panel F).
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) and biochemically by electrophoretic separation of the
35S-labeled and secreted proteins (Fig. 3). The identity of type I, type II, and
type X collagens and of transferrin (TF) was confirmed by
immunoprecipitation with the respective specific antibodies (Fig. 4).
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). After 8 days of culture, all cells
had homogeneously reached the state of hypertrophy (Fig. 2D). At a
biochemical level, the adherent cells synthesized mainly fibronectin
(FN) and type I collagen (Fig. 3A, lane 1). They also synthesized a
large amount of TF and of the Ex-FABP (Ch21) protein that we originally
identified in serum-containing cultures as a main product of
differentiating chondrocytes (14). Differentiation in suspension
culture was evidenced by the passage from type I to type II collagen
synthesis and the accumulation of type X collagen, a specific marker of
the hypertrophic stage (Fig. 3A, lane 2). The addition of BMP-2 to the
suspension culture allowed the cells to reach hypertrophy in a shorter
time but did not modify the differentiated protein pattern (Fig. 3A, lane 3). Cells expanded in Ins/Dex/FGF-2 behaved in monolayer and in
suspension culture exactly as the cells deprived of Dex (not
shown).
Cells expanded in Ins/T3/Dex/FGF-2 did not proceed further
to the condensation phase and remained clustered for up to 3 weeks in
suspension culture (Fig. 2G). However, when BMP-2 was added to the
medium at the time of transfer to suspension, the cells overcame this
blockage and reached full hypertrophy within 8 days (Fig. 2H). The
protein analysis confirmed these data. The aggregated cells maintained
the predominant synthesis of FN, type I collagen, TF, and Ex-FABP
(Ch21) protein (Fig. 3B, lane 2) as they were expressed by adherent
cells (Fig. 3B, lane 1). The addition of BMP-2 induced the synthesis of
type II and X collagens (Fig. 3B, lane 3). Cells expanded in the
presence of T3 in other combinations (i.e.
T3/Dex/FGF-2 or T3/IGF-I/Dex/FGF-2) always
presented a similar arrest of differentiation in suspension culture,
thereby suggesting that the inhibition is probably caused by the
presence of thyroid hormone in the phase of proliferation in adherent
conditions.
In Fig. 1, we have shown that during proliferation, Ins could be fully
replaced by IGF-I. In this condition, both the fibroblast-like
morphology (not shown) and the metabolic properties of the cells were
preserved as shown in Fig. 3C (lane 1); where again, FN, type I
collagen, TF, and Ex-FABP (Ch21) protein were the major metabolic
products. Also, the differentiation pattern in suspension culture was
not modified, as compared with the one described for cells expanded in
Ins/FGF-2 showing the switch to the synthesis of type II and X
collagens (Fig. 3C, lane 2), regardless of the addition of BMP-2 (Fig. 3C, lane 3). However, if IGF-I replaced Ins in the differentiation
phase, the cells remained aggregated and continued to synthesize type I
collagen, TF, and Ex-FABP (Ch21) protein (Fig. 3C, lane 4). The
apparent lower levels of TF and Ex-FABP (Ch21) protein, as compared
with the dedifferentiated cells, may indicate an initiation of
reversion toward the chondrogenic phenotype. The introduction of BMP-2
in the suspension culture fully stimulated the reversion, leading to a
rapid conversion to type II and type X collagen expression (Fig. 3C, lane 5). These data suggest the existence of a sequential involvement
of IGF-I and Ins receptors in chondrocyte metabolism, with IGF-I
supporting cell proliferation and Ins being essential for
differentiation to hypertrophy.
In vitro reconstitution of cartilage
The data presented above indicated that the conditions of in
vitro expansion of prechondrogenic cells could significantly
influence their capacity to undergo the chondrogenic pathway, Ins/FGF-2
being permissive and Ins/T3/Dex/FGF-2 preventing further
differentiation. The various types of collagen proteins synthesized by
the cells, although properly secreted as shown in Fig. 3, could not be
organized in either of these conditions. Therefore, to assess the
importance of an organized extracellular matrix for proper
differentiation, the suspension cultures were repeated in the constant
presence of ascorbic acid, an obligatory cofactor of the hydroxylases
required for the correct tridimensional assembly of collagen fibrils.
The histological sections presented in Fig. 5 are representative of three independent
experiments. In the presence of ascorbic acid, differentiating cells
remained aggregated without releasing single hypertrophic cells into
the medium. They reconstituted in vitro a well-organized
structure closely resembling hypertrophic cartilage, where mature
chondrocytes were contained in lacunae embedded in a metachromatic
matrix. Panel A shows that the organization of the matrix allowed full
differentiation of cells expanded in Ins/T3/Dex/FGF-2,
which otherwise would not have undergone maturation. The hypertrophic
cartilage thus formed was identical to the structure obtained with
cells expanded in Ins/FGF-2, retaining full chondrogenic potential
(panel B). In neither case was the addition of BMP-2 necessary to
promote cartilage formation, nor did it modify the histological
appearance of the structure, which remained highly positive for
toluidine and alcian blue staining.
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summarizes the types of tissues
neoformed according to cell culture and implantation conditions.
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. A higher
magnification of the tissue (Fig. 6B) clearly shows that chondrocytes
matured to hypertrophy, secreting a metachromatic extracellular matrix
that embedded the cells in individual lacunae. Moreover, the
cartilaginous structures were infiltrated with a tissue histologically
similar to hemopoietic marrow (Fig. 6A, arrowheads),
comprising adipocytes, fibroblast-like cells, and elements of the
hematopoietic compartment (Fig. 6C).
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, D and E).
Cells grown in Ins/FGF-2 formed mainly cartilage (Fig. 6D), whereas the
expansion in Ins/T3/Dex/FGF-2 resulted in a major
accumulation of adipocytes (Fig. 6E). In both cases, the cartilage
structures seemed to have reached an overall lower grade of maturation,
as compared with cells directly implanted without scaffold.
When cells expanded in Ins/FGF-2 were implanted on HA, they formed
cartilage and adipose tissue (Table 1) with a histological pattern
similar to the one presented in Fig. 6D. However, when HA was loaded
with cells expanded in Ins/T3/Dex/FGF-2, we observed the
generation of adipocytic centers, cartilage nodules, and bone tissue
(Table 1; Fig. 7, A and C). The cartilage
nodules seemed rather mature, with some hypertrophic chondrocytes in
lacunae surrounded by extracellular matrix (Fig. 7A). The bone tissue
was composed of osteoblasts proliferating at the edge of the structure
(Fig. 7C, arrowheads) and osteocytes embedded in lacunae
within a well-formed bone matrix (Fig. 7C, arrows). Parallel
controls, performed with HA cubes or collagen sponges implanted without
adsorbed cells, showed only the formation of a very loose connective
tissue, presumably of host origin, but no evidence of newly formed
adipose tissue, cartilage, or bone (not shown).
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, B and D, correspond to serial sections of the cartilage and
bone tissues illustrated, respectively, in Fig. 7, A and C. They show a
clear nuclear coloration of the chondrocytes and osteocytes, thereby
demonstrating the chicken origin of both cell types. A similar positive
staining also was obtained on cartilage tissue, formed either by direct
injection of the cells or after adsorption on collagen sponge (not
shown). To further confirm the specie specificity of the antibody, a
longitudinal section across the leg of a 7-day-old mouse embryo was
processed in parallel. The histological appearance of the mouse
cartilage is illustrated in Fig. 7E. On a serial section treated with
the avian antibody, no nuclear staining was observed (Fig. 7F),
therefore excluding any cross-reactivity with mouse antigen. The
adipocytes formed on HA and on collagen sponges also were of chicken
origin, whereas those accumulated in the bone marrow were of mouse
origin (not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we have attempted to identify the hormones and growth factors essential to sustain, in complete serum-free conditions, the full process of chondrogenesis from prechondrogenic to hypertrophic cells. We have further assigned to some of these factors a primary role, either in the proliferation or the differentiation of the chondrocytes, and in a possible reversion of the chondrogenic phenotype toward alternative lineages of the mesenchymal compartment.
FGF-2 is the major mitogen for primary cells, as compared with the other growth factors analyzed, which reinforces indications previously reported in the literature on the role of FGF-2 on chondrocyte proliferation. It is known that cartilage in vivo contains a large amount of FGF-2 and that it therefore is highly responsive to this factor. In vitro studies have suggested that FGF-2 acts primarily on the growth of cartilage, rather than on its maturation. High levels of colony formation have been obtained by stimulating rabbit or chicken chondrocytes with FGF-2 in soft agar culture (25). On the other hand, FGF-2 has been shown to inhibit chondrocyte maturation (26) and to prevent type X collagen synthesis and terminal differentiation (27). It should be noted that the number of FGF-2 receptors decreases as the cells become hypertrophic (28), which supports the concept of a higher sensitivity of immature proliferating cells to the factor. Our data also show that FGF-2 does not induce cell proliferation by itself but must be associated with Ins, thereby demonstrating a synergistic effect of the two factors. This synergism enters into the general concept of the required combination of competence and progression factors to trigger cell division, the former (i.e. EGF, PDGF, or FGF) allowing the passage from Go to G1 at the entry in the cell cycle, the latter (i.e. IGF-I, Ins) committing the cells to DNA synthesis (29). Ins could be fully replaced by IGF-I. Consequently, chondrocyte proliferation must depend primarily on the IGF-I receptor pathway, rather than on that of Ins. A prominent role of IGF-I on cartilage metabolism is long recognized. It stimulates extracellular matrix deposition in cartilage explants (30), can substitute GH during development or growth of hypophysectomized animals (31), and can support the in vitro proliferation of prechondrogenic limb bud cells (32) and growth plate or articular chondrocytes (22, 33, 34). The precise mode of action of IGF-I on cartilage metabolism is still unclear. Chondrocytes, themselves, secrete significant amounts of IGF-I (35). Furthermore, changes in the number (36) and in the distribution (37) of IGF-I receptors have been correlated to the level and distribution of the growth factor. These observations suggest that cartilage metabolism relies more on an autocrine/paracrine activity of locally produced GH-independent IGF-I, rather than on the circulating counterpart. Our observation that IGF-I induces full proliferation of prechondrogenic cells, whereas GH has only a limited effect, supports this hypothesis.
The expansion of primary chondrocytes, in the presence of FGF-2, led (after several passages) to a homogeneous population of dedifferentiated cells characterized by a fibroblast-like phenotype. This phenotype was not significantly modulated by the association of FGF-2, either with Ins, IGF-I, T3, or Dex. Cells grown in any condition secreted mainly type I collagen, FN, TF, and Ex-FABP (Ch21) protein. Type I collagen is the major marker of the prechondrogenic stage, and its appearance, therefore, confirmed the dedifferentiation process. The high level of FN is compatible with the presence of FGF-2, which has been shown to increase FN synthesis (38). TF and Ex-FABP (Ch21) protein are normally synthesized by maturating or hypertrophic chondrocytes (14, 15). Their significant level of expression by dedifferentiated cells in serum-free conditions suggests that additional serum components modulate their synthesis during chondrogenesis. This hypothesis is currently under investigation.
The differentiation potential of the primary cells, expanded in defined conditions, was next analyzed by transferring them into suspension culture in the presence of only Ins, T3, and Dex (9). The morphological, biochemical, and histological data clearly indicate that the conditions of expansion of the dedifferentiated prechondrogenic cells modulate their capacity to reenter the differentiation program. Cells expanded in the presence of FGF-2, associated either with Ins or IGF-I, underwent normal differentiation. When T3 was included in the proliferation medium, the cells failed to differentiate, but this apparent blockage of chondrogenesis was overcome by the addition of either BMP-2 or ascorbic acid in the differentiation medium. The first factor led to cell hypertrophy and type X collagen synthesis, whereas the second allowed reconstitution of hypertrophic cartilage in the culture dish. The effect obtained with BMP-2 was not extremely surprising, because all BMPs are known to be potent inducers of chondrogenesis and osteogenesis. In vivo, these growth factors are thought to regulate the early commitment of mesenchymal cells to the chondrogenic and osteogenic lineages, and to further support the proliferation and/or differentiation of the committed cells (39, 40). In vitro, BMP-2 induces undifferentiated mesenchymal progenitors to differentiate into osteoblasts, chondrocytes, and adipocytes and also stimulates the committed-osteo-chondroprogenitors to differentiate into more mature cell types (41). Likewise, osteogenic protein-1 (OP-1 or BMP-7) has been shown to trigger chondroblastic, osteoblastic, and/or adipocytic differentiation along elements of the endochondral ossification pathway, depending on the stage and potential of the target cells (42). Our data on the inductive effect of BMP-2 are along the same lines; in fact, upon stimulation, dedifferentiated cells that apparently have lost their capability to differentiate in vitro can reacquire the hypertrophic phenotype. Regarding the differentiation effect of ascorbic acid, two explanations must be considered. First, the organization of the extracellular matrix by ascorbic acid may create a particular microenvironment, where growth and differentiation factors, diffusable from exogenous source or locally secreted, are bound to and sequestered by matrix components that can play a role in the clearance or storage of important biological ligands. Alternatively, chondrocyte differentiation and gene expression can be modulated by cell-cell contacts and cell-matrix interactions that are favored by the organization of the collagen fibrils (43, 44).
The differentiation potential of the cells was also investigated in vivo by ectopic tissue formation in nude mice. Cells expanded in Ins/FGF-2 (maintaining chondrogenicity in vitro) reconstituted cartilage in any of the three conditions of implantation. Cells expanded in Ins/T3/Dex/FGF-2 (losing chondrogenic potential in vitro) gave similar results, except after implantation on HA, where several centers of bone formation were observed, in addition to the cartilaginous structures. In any condition of cell expansion and implantation, cartilage and bone were surrounded by numerous adipocytes.
Taken together, the in vitro and in vivo data presented here may shed light on some molecular mechanisms regulating the chondrogenic phenotype and its commitment. In agreement with previous reports (45, 46), our data suggest that primary chondrocytes serially grown in vitro in monolayer gradually tend to lose the cartilage-specific characteristics and that the phenotype can be recovered by culturing cells in appropriate differentiation permissive conditions. On the other hand, it is also known that in vivo chondrogenic cells emerge from an undifferentiated mesenchymal compartment that can potentially give rise to chondroblasts, osteoblasts, and adipocytes. This concept of the pluripotential capacity of the progenitor cells has been experimentally verified in vitro by inducing the mouse fibroblast cell line C3H-10T1/2, upon treatment with 5-azacytidine, to differentiate in four different mesenchymal lineages (47). Our data suggest that according to the nature of the systemic factors introduced into the medium of expansion of prechondrogenic cells, a loss of chondrogenic phenotype could be accompanied by the acquisition of new differentiation pathways. These alternative lines of development, adipogenesis and osteogenesis, did not occur spontaneously in vitro but were probably induced by the in vivo microenvironment to which the implanted cells were exposed in the host. Concerning bone formation, the mineralized surface of the ceramic cube probably contributed to the process by serving as a primer for bone matrix deposition. With respect to the commitment of undifferentiated cells toward adipogenesis, chondrogenesis, or osteogenesis, a few attempts have been made to determine the sequence of branching of these lineages, by analogy to what has been already successfully established for the cellular components of the hematopoietic system. For instance, Zull et al. (48) have suggested that the commitment to the chondrogenic lineage could be coincident or, at the most, follow the osteogenic lineage branching. To date, the branching toward adipogenesis has not been explored. The data presented in this study do not suggest a defined lineage branching, nor do they allow us to determine whether such branching actually exists during mesenchymal cell differentiation. Our data does, however, emphasize the concept of the instability of the chondrogenic phenotype and point out that the fate of the chondrocyte (as versatile as it seems to be) can be determined by both the in vivo and the in vitro history of the cell. Indeed, our study has been performed with primary cells originally harvested from a newly bone-forming structure, where chondrocytes had been already induced to develop into hypertrophic cartilage, although maintaining some phenotype plasticity. This in vivo stamped memory of the cells could have been modulated thereafter (in vitro) by the cell culture conditions, which triggered the unstable phenotype to reexpress (after implantation in vivo) alternative differentiated properties, depending on the microenvironment encountered in the host mouse. Studies on cloned primary chondrocytes are currently in progress to verify the possible reversion and modulation of chondrogenic development at the single-cell level.
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Acknowledgments |
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Footnotes |
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Received March 21, 1997.
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