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Endocrinology Vol. 138, No. 11 4966-4976
Copyright © 1997 by The Endocrine Society

ARTICLES

Modulation of Commitment, Proliferation, and Differentiation of Chondrogenic Cells in Defined Culture Medium1

Rodolfo Quarto, Giuliano Campanile, Ranieri Cancedda and Beatrice Dozin

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LONG bone formation initiates from the proliferation and condensation of a mesenchymal cellular compartment committed to prechondrogenic cells that will eventually produce a template of hypertrophic cartilage for endochondral ossification. This process is characterized by spatial and temporal stage specific modifications in the morphology and the biochemical properties of the maturating cells and in the structure and composition of the extracellular matrix they secrete. The molecular basis of the progression from undifferentiated stem cell to hypertrophic chondrocyte involves both the genomic potential of the cells (homeo- and controlling genes) and their local microenvironment created by the extracellular matrix, systemic factors and other signaling molecules (hormones, growth factors, and cytokines) that can modulate the cellular metabolism in an autocrine/paracrine manner (1).

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).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
To establish a primary culture of chondrocytes, stage H.H. 28–30 (10) chick embryo tibiae were removed, cleaned of muscular and connective tissues, and digested for 15 min at 37 C with 400 U/ml collagenase I (Worthington Biochemical Corp.) and 0.25% trypsin (Gibco, Ltd.). After sedimentation, the supernatant, containing tissue debris and perichondrium, was discarded, and the tibiae were digested until complete dissociation with the same enzymes supplemented with 1000 U/ml collagenase II (Worthington Biochemical Corp.). Cells were counted and plated at a minimal density of 5000 cells/cm2 in anchorage-dependent conditions on regular tissue culture dishes. Culture medium was Coon’s modified Ham’s F12 (11). For serum-free culture, this medium was supplemented with various combinations of the following hormones and growth factors: T3 (10-9 M), Dex (10-10 M), Ins (60 ng/ml), GH, FGF-2, IGF-I, platelet-derived growth factor bb (PDGFbb), and epithelial growth factor (EGF). Details are given in the legends of the figures. The growth factors were human recombinants (Austral Biological, San Ramon, CA) and used at concentrations ranging from 1–100 ng/ml. FGF-2, PDGFbb, Ins, and EGF were tested as known mitogens for cells of mesenchymal origin. GH was used as long-bone specific growth factor. IGF-I was used to distinguish between Ins receptor and IGF-I receptor pathways along the cellular metabolic response. Dex was included because it maintains the capacity of proliferating precursors to differentiate into different phenotypes, including cartilage (12), and increases the viability of differentiating chondrocytes in serum-free culture conditions (9). T3 was used as chondrogenic inducer. Control adherent cultures contained Coon’s modified Ham’s F12 medium, supplemented with 10% FCS (Mascia Brunelli, Milano, Italy). To induce differentiation, adherent cells were transferred into suspension culture on a 1% (wt/vol) agarose layer. The standard differentiation medium was Coon’s modified Ham’s F12 containing T3, Dex, and Ins at the concentrations mentioned above, as previously described for chondrogenesis in serum-free conditions (9). When indicated, the medium also was supplemented with bone morphogenetic protein 2 (BMP-2, 10 ng/ml; Genetics Institute, Cambridge, MA). For the reconstruction of hypertrophic cartilage in vitro, ascorbic acid was added daily at 100 µg/ml (13).

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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mitogenic effect of growth factors on dedifferentiated chondrogenic cells: monolayer cultures
Dedifferentiated chondrocytes from chicken embryo tibiae, expanded in monolayer in the presence of 10% FCS, can fully differentiate in suspension culture in serum-free medium containing only Ins, T3, and Dex but cannot grow in this supplement (9). Therefore, we first attempted to associate the hormones with additional growth-promoting factors known to act in vivo on cells of mesenchymal origin. The effects of FGF-2, EGF, PDGFbb, and GH were analyzed and compared with a control culture grown in the presence of 10% FCS. Cells were plated at a starting density of 5000 cells/cm2. When the control 10%-FCS culture reached confluence, all dishes were trypsinized, and the final cell number was determined in each of them. Figure 1Go 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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  		Figure 1. Expansion of primary chondrocytes in defined medium, in response to growth factors and hormones. Primary cells were seeded at an initial density of 5000/cm2 in the following defined conditions: column 1, 10% FCS (control serum-supplemented culture); columns 2–5, Ins/T3/Dex associated either with FGF-2 (column 2) or EGF (column 3) or GH (column 4) or PDGFbb (column 5); column 6, plain medium without supplement; column 7, Ins/T3/Dex alone; column 8, FGF-2 alone; column 9, T3/FGF-2; column 10, Dex/FGF-2; column 11, Ins/FGF-2; column 12, T3/Dex/IGF-I/FGF-2; column 13, IGF-I/FGF-2. Plating efficiency was approximately 95% in any condition. When the control 10%-FCS culture reached confluence, all cells were trypsinized and counted, and the final cell number (obtained in each condition) was reported to the initial plating number. Concentrations of the various factors were: Ins, 60 ng/ml; T3, 10-9 M; Dex, 10-10 M; and all other growth factors were used at 10 ng/ml. Data represent the mean values ± SE of four to six independent experiments. *, Student’s t test: P = 0.084, 0.171, 0.116, and 0.161 for columns 2, 11, 12, and 13, respectively.

 
In the various serum-free conditions, the cells presented the typical fibroblast-like phenotype of dedifferentiated cells, as described for cultures maintained in 10% FCS (8). As an example, Fig. 2Go 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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  		Figure 2. Comparative morphology of primary cells expanded and further differentiated in defined conditions as follows: primary cells were first expanded for 2–3 weeks in anchorage-dependent conditions in Ins/FGF-2 (A and B) or in Ins/T3/Dex/FGF-2 (E and F), displaying the fibroblast-like phenotype typical of dedifferentiated cells. At confluence, few centers of chondrogenesis could be observed (B and F). To induce differentiation, the adherent cells were transferred into suspension in medium containing only Ins/T3/Dex (C, D, G, and H). All factors were used at the concentrations indicated under Fig. 1Go. C and D, Suspension cultures of cells expanded in Ins/FGF-2, whereas G and H correspond to suspension cultures of cells expanded in Ins/T3/Dex/FGF-2. The culture in H also contained 10 ng/ml BMP-2. Times of culture were 4 days (A and E), 5 days (C), 8 days (D and H), 10 days (B and F), and 18 days (G). Bar, 100 µm.

 
In vitro differentiation potential: suspension cultures
The differentiation of cells, expanded in selected serum-free conditions, was analyzed by transferring them into a permissive anchorage-independent condition. The culture medium was thus depleted of FGF-2 and contained only Ins, T3, and Dex. Differentiation was evaluated morphologically by microscopic evaluation (Fig. 2Go) and biochemically by electrophoretic separation of the 35S-labeled and secreted proteins (Fig. 3Go). 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. 4Go).



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  		Figure 3. Comparative electrophoretic profile of 35S-labeled proteins secreted by dedifferentiated and maturating chondrocytes in the following culture conditions: primary cells were expanded in anchorage-dependent conditions in Ins/FGF-2 (A), Ins/T3/Dex/FGF-2 (B), or IGF-I/FGF-2 (C). The electrophoretic pattern of the adherent cells is presented in lane 1 of the respective panels. To induce differentiation, cells were transferred into suspension in medium containing Ins/T3/Dex, and the secreted proteins were analyzed after 15 days of culture (lanes 2). In lanes 4 and 5, Ins was replaced by IGF-I. In lanes 3 of all panels and in lane 5 of panel C, the medium also contained 10 ng/ml BMP-2. Equal amounts of counts were loaded on each lane, and the electrophoretic resolution was performed in reducing conditions on 12.5% SDS-polyacrylamide gels. Bars on the right refer to the migration of molecular mass standards, whose size expressed in kDa was, from top to bottom: 200, 92, 69, 46, and 30.

 


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  		Figure 4. Immunoprecipitation of 35S-labeled proteins secreted by dedifferentiated and maturating chondrocytes. The labeled proteins secreted by dedifferentiated and maturating chondrocytes and corresponding, respectively, to the lanes 1 and 3 of panel B in Fig. 3Go were immunoprecipitated with polyclonal antibodies raised against type I collagen, type II collagen, type X collagen, and TF and analyzed in reducing conditions on a 12.5% SDS-polyacrylamide gel. Lanes A1, B2, C1, and C4 in Fig. 3Go gave the same immunoprecipitation products as lane B1. Lanes A2, A3, C2, C3, and C5 in Fig. 3Go gave the same immunoprecipitation profile as lane B3.

 
Cells expanded in Ins/FGF-2 underwent a rapid differentiation evidenced, after 5 days of suspension culture, by the flourishing of initial aggregates and their release into the medium of single hypertrophic chondrocytes (Fig. 2CGo). After 8 days of culture, all cells had homogeneously reached the state of hypertrophy (Fig. 2DGo). At a biochemical level, the adherent cells synthesized mainly fibronectin (FN) and type I collagen (Fig. 3AGo, 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. 3AGo, 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. 3AGo, 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. 2GGo). 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. 2HGo). 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. 3BGo, lane 2) as they were expressed by adherent cells (Fig. 3BGo, lane 1). The addition of BMP-2 induced the synthesis of type II and X collagens (Fig. 3BGo, 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. 1Go, 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. 3CGo (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. 3CGo, lane 2), regardless of the addition of BMP-2 (Fig. 3CGo, 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. 3CGo, 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. 3CGo, 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. 3Go, 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. 5Go 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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  		Figure 5. Histological sections of in vitro reconstituted cartilage. Primary cells were expanded, either in Ins/T3/Dex/FGF-2 (A) or Ins/FGF-2 (B), for 2 weeks. The expanded cells were then transferred into suspension culture and maintained for 13 days in medium containing Ins/T3/Dex and daily supplemented with 100 µg/ml ascorbic acid. The cell aggregates obtained were fixed, paraffin embedded, sectioned, and stained with toluidine blue. Bar, 16 µm.

 
In vivo differentiation potential: ectopic tissue formation in nude mice
As in vivo embryonal cartilage is an avascularized tissue, chondrocyte maturation is largely conditioned by the appropriate endogenous microenvironment surrounding the cells. Because the in vitro culture conditions, as defined above, did not necessarily adequately recreate the in vivo situation, we implanted sc, in nude mice, dedifferentiated prechondrogenic cells, expanded either in Ins/FGF-2 or Ins/T3/Dex/FGF-2, to assess their differentiation potential in vivo. The expanded cells were loaded in the animals either by direct injection, as a dense cell suspension, or after adsorption on porous HA cubes or collagen sponges. After 8 weeks, the grafts were harvested, paraffin-embedded, and processed for histological staining and immunohistochemical analysis. Table 1Go summarizes the types of tissues neoformed according to cell culture and implantation conditions.


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  		Table 1. Types of tissues neoformed according to cell culture and implantation conditions

 
When cells, expanded in either of the conditions, were implanted as a cell suspension, they led to the formation of cartilage tissue, as illustrated in Fig. 6AGo. A higher magnification of the tissue (Fig. 6BGo) 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. 6AGo, arrowheads), comprising adipocytes, fibroblast-like cells, and elements of the hematopoietic compartment (Fig. 6CGo).



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  		Figure 6. Representative fields of histological sections of cell specimens ectopically implanted in nude mice. A, Cells expanded in Ins/FGF-2 and implanted by direct sc injection (arrowheads denote the presence of hemopoietic marrow); B and C, particulars of A (at a higher magnification) showing cartilage (B) and marrow (C); D and E, collagen sponges loaded with cells expanded either in Ins/FGF-2 (D) or Ins/T3/Dex/FGF-2 (E); c, chondrocytes; a, adipocyte clusters. A, B, D, and E, toluidine blue staining; C, hematoxylin-eosin staining. Bar, 100 µm (A) or 25 µm (B, C, D, and E).

 
When adsorbed on collagen sponges, cells expanded in either of the conditions gave rise to cartilage and adipose tissue (Fig. 6Go, D and E). Cells grown in Ins/FGF-2 formed mainly cartilage (Fig. 6DGo), whereas the expansion in Ins/T3/Dex/FGF-2 resulted in a major accumulation of adipocytes (Fig. 6EGo). 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 1Go) with a histological pattern similar to the one presented in Fig. 6DGo. 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 1Go; Fig. 7Go, A and C). The cartilage nodules seemed rather mature, with some hypertrophic chondrocytes in lacunae surrounded by extracellular matrix (Fig. 7AGo). The bone tissue was composed of osteoblasts proliferating at the edge of the structure (Fig. 7CGo, arrowheads) and osteocytes embedded in lacunae within a well-formed bone matrix (Fig. 7CGo, 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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  		Figure 7. Histological and immunohistochemical staining of sections of cell specimens ectopically implanted in nude mice after adsorption on HA ceramic cubes (A–D) and of 7-day-old mouse embryo leg cartilage as control (E and F). HA cubes were loaded with cells expanded in Ins/T3/Dex/FGF-2. A and E, Toluidine blue staining; C, hematoxylin-eosin staining (arrowheads denote alignment of osteoblasts and arrows indicate osteocytes in the bone matrix); B, D, and F, peroxidase staining driven by the avian-specific monoclonal antibody described under Materials and Methods; A, C, and E, regular light microscopy; B, D, and F, differential interference contrast (DIC); Bar, 16 µm.

 
To determine the cellular origin of the different structures obtained, an additional immunohistochemical analysis was performed with a monoclonal antibody known to specifically recognize a 170-kDa nuclear antigen present in cells of avian origin but not in mammals or other classes of vertebrates (16). The immunoperoxidase stainings, presented in Fig. 7Go, B and D, correspond to serial sections of the cartilage and bone tissues illustrated, respectively, in Fig. 7Go, 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. 7EGo. On a serial section treated with the avian antibody, no nuclear staining was observed (Fig. 7FGo), 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although considerable advances recently have been made in understanding some of the fundamental processes involved in endochondral bone formation, a more comprehensive knowledge about the hormonal and growth factor control of cartilage development and function is still needed. In vivo, chondrogenesis occurs through a progression from mesenchymal precursors to hypertrophic chondrocytes, each step along the way being tagged by the biochemical properties of the cells. To investigate the basic requirement for these properties to be sequentially expressed first by proliferating, then by maturating, and finally by hypertrophic calcifying chondrocytes, in vitro culture systems, based on chemically defined media, have been developed (9, 18, 19, 20, 21, 22, 23, 24). However, all these studies reported culture conditions limited to the control of the proliferation or of particular stages of differentiation of chondrogenic cells.

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.


   Acknowledgments
 
We thank Prof. G. Cossu for giving us the avian specific monoclonal antibody 3.2.B5. We also thank the Genetics Institute for the generous supply of BMP-2, Fin-Ceramica Faenza for the bioceramic specimens, and Coletica for the collagen sponges.


   Footnotes
 
1 This work was partially supported by funds from Agenzia Spaziale Italiana, from Associazione Nazionale per la Ricerca sul Cancro, and from Istituto Superiore di Sanita’. Back

Received March 21, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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