S Roche

M.-C Ronzie

D Herbage

A.-M Freyria
email: am.freyria@ibcp.fr

Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering

Keywords: Articular cartilage repair, Collagen-based biomaterial, Collagen synthesis, Glycosaminoglycan deposition, Mineral deposition

Collagen-based biomaterials in the form of sponges (bovine type I collagen, both native and cross-linked by treatment with diphenylphosphorylazide, noted control and DPPA sponges respectively) were tested as three-dimensional sca!olds to support chondrocyte proliferation with maintenance of the phenotype in order to form neocartilage. Control and DPPA sponges were initially seeded with 10 or 10 foetal bovine epiphyseal chondrocytes and maintained for 4 weeks in culture under static conditions in RPMI/NCTC medium with 10% FCS and without addition of fresh ascorbic acid. Both supports were always present during the study and a partial decrease in size and weight was detected only with control sponges, both seeded and unseeded. Cell proliferation was only noted in the 10 cells-seeded sponges (4-fold increase after 4 weeks of culture). Speci"c cartilage collagens (types II and XI) were deposited in the matrix throughout the culture and traces of type I collagen were noticed only in the culture medium after 2}3 weeks and 4 weeks in the case of 10 and 10 cells-seeded sponges, respectively. Glycosaminoglycans accumulated in the matrix, up to 1.8 and 9.8% of total dry weight after one month with both seeding conditions, which was much lower than in the natural tissue. In the 10 cells-seeded sponges, mineral deposition, observed with unseeded sponges, was signi"cantly decreased (2-to 3-fold). These in vitro results indicate that both collagen matrices can support the development of tissue engineered cartilage.

Introduction

Mature articular cartilage does not repair adequately when injured as a consequence of trauma or diseases such as osteoarthritis. The integrity of the cartilage is maintained by the chondrocytes that both produce an extracellular matrix consisting mainly of collagens [START_REF] Petit | Cartilage collagens[END_REF] and proteoglycans [START_REF] Handley | Proteoglycan, hyaluronan, and noncollagenous matrix protein metabolism by chondrocytes[END_REF] and also control its composition and organization. These cells are ine!ective in responding to injury as wounds limited to cartilage stimulate only a slight reaction in the adjacent chondrocytes [START_REF] Hunziker | Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane[END_REF][START_REF] Caplan | Principles of cartilage repair and regeneration[END_REF]. Moreover, after a full-thickness injury the observed "brocartilaginous response due to the subchondral bone is short lived and has inferior mechanical properties to those of natural cartilage [START_REF] Shapiro | Cell origin and di!erentiation in the repair of full-thickness defects of articular cartilage[END_REF]. During the past decade many investigations have pursued techniques to stimulate articular cartilage repair or regeneration (for references see reviews [6}8]). Reports have described the replacement of damaged or lost cartilage with tissue grafts (periosteum, perichondrium or cartilage) [START_REF] Bentley | Grafts and implants for cartilage repair and replacement[END_REF][START_REF] Homminga | Perichondrial grafting for cartilage lesions of the knee[END_REF] or with delivery of chondrocytes or mesenchymal stem cell suspensions [11}17]. Stimulation of cartilage formation by the local injection of factors with chondrogenic potential such as transforming growth factor beta-1, insulin growth factor, bone morphogenic proteins was also investigated [START_REF] Hunziker | Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane[END_REF][START_REF] Otsuka | Requirement of "broblast growth factor signaling for regeneration of epiphyseal morphology in rabbit full-thickness defects of articular cartilage[END_REF][START_REF] Sellers | The e!ect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage[END_REF]. Successful results of autologous chondrocyte transplantation have led to the commercial development of this protocol in young adults with localized cartilage defects [START_REF] Brittberg | Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation[END_REF][START_REF] Peterson | Articular cartilage injuries treated with autologous chondrocyte transplantation in the human knee[END_REF]. Brie#y, chondrocytes were harvested from an area of the patient's normal articular cartilage, grown in culture and injected in the defect under a #ap of periosteum. This procedure has produced satisfactory results in more than 90% of patients but its application seems to be limited to patients younger than 40 and without osteoarthritic pathologies.

Another approach to introducing a new cell population into a cartilage defect was to deliver it in a sca!old to facilitate cell carrying and surgical handling for implantation and also to support cell growth and new matrix formation. These tissue engineering techniques [START_REF] Langer | Tissue engineering[END_REF] are based on the use of various biocompatible vehicles for grafting cartilage defects such as synthetic polymers: polyglycolic acid (PGA) [23}27], polylactic acid (PLA) [START_REF] Chu | Articular cartilage repair using allogeneic perichondrocyte-seeded biodegradable porous polylactic acid (PLA): a tissue-engineered study[END_REF], polyglycolic acid-poly-L-lactic acid copolymer (PGA-PLA) [START_REF] Sittinger | Resorbable polyesters in cartilage engineering: A$nity and biocompatibility of polymer "ber structures to chondrocytes[END_REF][START_REF] Britt | Autogenous tissue-engineered cartilage. Evaluation as an implant material[END_REF]], polyethylene [START_REF] Livecchi | In vitro chondrocyte collagen deposition within porous HDPE: Substrate microstructure and wettability e!ects[END_REF], polyethylene oxide [START_REF] Sims | Injectable cartilage using polyethylene oxide polymer substrates[END_REF]. Carrier materials of natural origin have also been used: alginate [START_REF] Van Susante | Culture of chondrocytes in alginate and collagen carrier gels[END_REF], "brin [34}36], hyaluronic acid [START_REF] Robinson | Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance[END_REF][START_REF] Aigner | Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester[END_REF], gelatin [START_REF] Stanton | The growth of chondrocytes using Gelfoam as a biodegradable sca!old[END_REF], collagen gel [START_REF] Wakitini | Mesenchymal cell-based repair of large fullthickness defects of articular cartilage[END_REF][START_REF] Van Susante | Culture of chondrocytes in alginate and collagen carrier gels[END_REF][START_REF] Schuman | Chondrocyte behaviour within di!erent types of collagen gel in vitro[END_REF], collagen spheres [START_REF] Frondoza | Human chondrocyte proliferate and produce matrix components in microcarrier suspension culture[END_REF], collagen}glycosaminoglycan matrices [START_REF] Nehrer | Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro[END_REF] and collagen sponges [25,43}46].

The use of collagen sponges as sca!olds for culture is facilitated by the fact that they are mechanically stable, have a pore structure that allows cell colonization, have low immunogenicity and are easily con"gured into di!erent shapes [START_REF] Silver | Cell growth on collagen: a review of tissue engineering using sca!olds containing extracellular matrix. Long-term E!ects[END_REF][START_REF] Chevallay | Collagen-based biomaterials and tissue engineering[END_REF]. Collagen sponges (native type I collagen) can be chemically cross-linked by di!erent techniques such as the acyl azide method using diphenylphosphorylazide (DPPA) developed in our laboratory [START_REF] Petite | Use of diphenylphosphorylazide for cross-linking collagen-based biomaterials[END_REF][START_REF] Rault | Evaluation of di!erent chemical methods for cross-linking collagen gel, "lm and sponges[END_REF]. This treatment results in collagen biomaterials with excellent biocompatibility, increased mechanical stability and decreased biodegradability, which are currently used as membranes for tissue guided regeneration [START_REF] Zahedi | A 2-year clinical evaluation of a diphenylphosphorylazide-cross-linked collagen membrane for the treatment of buccal gingival recession[END_REF]. We have recently described the behaviour of mouse and human "broblasts in these native and DPPA-crosslinked sponges [START_REF] Chevallay | Mouse "broblasts in long-term culture within collagen three-dimensional sca!olds: in#uence of cross-linking with diphenylphosphorylazide on matrix reorganization, growth, biosynthetic and proteolytic activities[END_REF][START_REF] Vaissiere | Comparative analysis of di!erent collagen-based biomaterials as sca!olds for long-term culture of human "broblasts[END_REF].

The objective of this study was to evaluate in vitro the ability of these three-dimensional matrices, seeded with fetal bovine epiphyseal chondrocytes, to support formation of a cartilage construct with a composition as close as possible to that of natural cartilage. In the current protocol of transplantation of autologous chondrocytes in humans the cells are propagated in monolayer culture on plastic where they undergo a rapid dedi!erentiation. We have studied here the possibility of multiplying chondrocytes directly into the collagen sponges without changes in their initial phenotype. Fetal bovine epiphyseal chondrocytes, which we previously studied in long term culture at high density [54}56], were seeded on these supports at di!erent initial densities and their metabolic activity and phenotype were followed during 4 weeks in culture. Total protein and collagen syntheses, proteoglycan content and mineral deposition were determined at di!erent times in culture as well as the biochemical composition of the cartilage construct at the end of the experiment.

Materials and methods

Collagen sponges

Collagen sponges, manufactured by Coletica (Lyon, France), were made up of collagen extracted from the skin of young calves containing 90}95% (dry weight) native type I collagen and 4}9% type III collagen. Sponges were prepared from a collagen solution (0.7% w/v) freeze-dried to produce a porous matrix with 58$24 m mean pore size (measured from 50 pores on SEM photographs as described by Matsuda et al. [START_REF] Matsuda | In#uence of glycosaminoglycans on the collagen sponge component of a bilayer arti"cial skin[END_REF]) and 6 mm thickness. Individual discs were cut out with a 10 mm diameter punch before being sterilized with 15 kGy radiation. Such sponges exhibited thermal transition temperatures measured by di!erential scanning calorimetry (Setaram, Lyon, France) of 36.3$0.13C (onset) and 44$0.53C (peak) and were referred to as control sponges. To increase their stability, some control sponges were cross-linked using the diphenylphosphorylazide (DPPA) technique described by [START_REF] Petite | Use of diphenylphosphorylazide for cross-linking collagen-based biomaterials[END_REF] [START_REF] Petite | Use of diphenylphosphorylazide for cross-linking collagen-based biomaterials[END_REF]. Brie#y, they were soaked in distilled water and then in dimethylformamide (DMF) and left for 24 h at room temperature in a solution of DMF containing 25 l of DPPA per 0.8 g of sponge. Such sponges, referred to as DPPA sponges, punched and sterilized like the control sponges, exhibited thermal transition temperatures of 42.9$3.43C (onset) and 49.7$2.23C (peak).

Chondrocytes

Chondrocytes were obtained by enzymatic digestion from the reserve zone of the distal femoral epiphyses of four-month-old bovine fetuses (six di!erent animals were used in this study) after careful removal of the articular surface and the growth plate [START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF]. The isolated cells were kept in RPMI/NCTC (v/v) medium (Seromed , Strasbourg, France/Sigma, Saint Quentin Fallavier, France) containing 10% foetal calf serum (Seromed ), 50 U/ml penicillin, 50 g/ml streptomycin and 1 ng/ml amphotericin. No fresh ascorbic acid was added during this study.

Cell culture

Before seeding, the sponges were placed in 24-well culture plates for 2 h at room temperature in complete culture medium. After removing the medium, each sponge was seeded with 10 or 10 chondrocytes deposited onto the sponge in a volume of 50 l and further incubated at 373C and 5% CO for 2 h. After this, 2 ml of complete culture medium were added to each well. After 24 h in culture, to remove the cells present on the bottom of the well, especially in the 10 cells-seeded supports, the sponges were placed in new culture plates. The sponges were kept in culture for one month with changes of medium every 3 days. During the study, unseeded sponges were kept in culture as controls.

Cell proliferation

DNA content of the sponge was measured by the #uorimetric method of Lipman [START_REF] Lipman | Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258[END_REF] on the cyto#uor 4000 (Millipore, Saint-Quentin Yvelines, France) and with DNA calf thymus as standard. Cell number was calculated by using a converting factor of 8 pg DNA per chondrocyte [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF].

Sponge weight and surface area

Each week, the weight of seeded and unseeded sponges was measured on an electric microbalance (Mettler-Toledo, Giro#ay, France) after rinsing and freeze drying. The growth of the sponge diameter (average size of the construct measured on at least three di!erent sponges) was determined each week on photomicrographs.

Histologic examination

Samples were "xed at 43C overnight with 2% paraformaldehyde in 0.1 M cacodylate. Sponges were dehydrated in a graded series of ethanol baths and then impregnated and embedded in Histo-Resin GMA (Leica, Bron, France) at 43C according to the manufacturer's instruction. 4 m thick sections were cut with a tungsten knife on a Reichert/Jung 1040 Autocut rotary microtome (Leica). Sections were stained with Azure II/methylene blue.

Analysis of extracellular matrix components 2.7.1. Cell metabolic labeling and total protein and collagen syntheses

Total protein and collagen syntheses were measured by labeling the culture with 2 Ci per sponge of C Proline (290 mCi/mmol, NEN, Le Blanc Mesnil, France) for 24 h. After labeling, medium and washed sponges were separately dialyzed against water to eliminate unincorporated tracers. The samples were frozen, lyophilized and submitted separately to limited pepsin digestion. Culture media were treated as previously described [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF]. Neosynthesized proteins in the sponges were digested by pepsin A from porcine stomach mucosa (Sigma, Saint Quentin Falavier, France) for 24 h at 43C with magnetic stirring and at a 1/10, w/w pepsin/protein ratio. More than 92% of radiolabeled proteins were extracted by this procedure. The content of dpm in the pepsin-treated samples gave the level of synthesis of total protein. Pepsin-resistant proteins, assumed to be collagenous, were obtained after dialysis of the samples. After lyophilization the samples were dissolved in Laemmli's bu!er and counted for their radioactive content. Total neosynthesized collagen content was expressed as total dpm/DNA deposited in the cell layer and in culture medium, or as percentage of total protein synthesis after correction for the enrichment of proline in collagen with respect to the non-collagen proteins [START_REF] Peterkofsky | Bacterial collagenase[END_REF].

Collagen typing

Neosynthesized pepsin-soluble collagens deposited in the cell layer and secreted in the culture medium after 24 h labeling were analyzed by SDS-PAGE in a 7% separating gel under non-reducing conditions. Extracts containing the same amount of dpm (10 000) were precipitated by ethanol, redissolved in electrophoresis bu!er and loaded on each lane of the gel. The bands corresponding to type I collagen (heterotrimer composed of 2 1+I, chains and one 2+I, chain), type II collagen (homotrimer composed of 3 1+II, chains) and type XI collagen (heterotrimer composed of 1+XI,, 2+XI,, 3+XI, chains) were visualized by #uorography and identi"ed by comparison with collagen standards and western blotting with speci"c antibodies as previously described [START_REF] Ronzie | Comparative analysis of collagen solubilized from human foetal and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen[END_REF].

Glycosaminoglycan content

A modi"cation of the dimethylmethylene blue (DMMB) method of Farndale et al. [START_REF] Farndale | Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue[END_REF] was used to quantify sulphated GAG content in the cultured sponges. Brie#y, the samples were digested in 0.1% papain #0.1% proteinase K in 1 mM CaCl , 10 mM Tris HCl, pH 8 at 603C for 24 h with low stirring. A 100-l aliquot of the digest solution was assayed for total GAG content by addition of 2.5 ml of DMMB solution. The absorbance was measured at 525 nm and the proteoglycan content calculated by comparison with shark chondroitin sulfate (Sigma) standard curve.

Glycosaminoglycans identixcation

The separation of the di!erent GAG contained in the sponges was performed using an electrophoretic technique described by Bertolotto et al. [START_REF] Bertolloto | Biochemical, histochemical and immunohistochemical study of glycosaminoglycans in human meningiomas[END_REF]. The samples were prepared as for GAG content analysis and Titan III Zip Zone cellulose acetate plates (Helena Laboratories, Saint-Leu la Fore( t, France) and 0.1 M barium acetate, pH 5 were used. Bovine placenta dermatan sulfate (Coletica), shark cartilage chondroitin sulfate (Sigma) and hyaluronic acid (Bomann Laboratories, St Etienne de Crossey, France) were used as standards. Glycosaminoglycans were visualized after alcian blue (0.1%) staining followed by a destaining in acetic acid (10%) and methanol (50%). Electrophoretograms were scanned using a Personal Densitometer2+ (Molecular Dynamics).

Amino acid analysis

Sponges seeded with chondrocytes, collected after one month in culture, were freeze-dried and hydrolysed with 6 N HCl for 24 h at 1103C under vacuum in a Picotag2+ work station (Waters). The amino acid content was established with an automatic System 6300 analyser (Beckman). The percentage of collagen was estimated from the hydroxyproline level. Results were reported as the collagen percentage on a dry weight basis.

Sponge mineralization

Calcium and phosphorus contents of the sponges after one week and four weeks in culture were determined on lyophilized samples by induction coupling plasmaatomic emission spectroscopy at the Service Central d'Analyse du CNRS (Solaize, France).

Results

Macroscopic characteristics of the sponge

When compared to their initial dry dimensions, a decrease in sponge diameter was observed in the "rst days of culture for control sponges (30.8$3%) either unseeded or seeded (Fig. 1A). During the rest of the culture, no additional changes in diameter were observed. Unseeded and seeded DPPA sponges did not present signi"cant variations in comparison with their initial diameter. These variations were con"rmed by histological analysis of the remnant network: its size decreased by 1/3 in the native sponges and was not modi"ed in the DPPA-cross-linked sponges (Fig. 2).

After "ve days of culture the unseeded control sponges had lost 29% of their initial weight and this decrease reached 40% after 4 weeks in culture (Fig. 1B). In the presence of 10 cells, similar changes were observed over this time. A smaller decrease (20%) was measured after 4 weeks in the 10 cells-seeded sponges. DPPA sponges, either unseeded or seeded, exhibited at each measurement larger variations in weight than control sponges but did not decrease in weight after one month in culture.

Histological analysis

In Fig. 2, we report the results with DDPA sponges only, as the same di!erences were obtained with the two types of support seeded with 10 and 10 cells, respectively. Over the 4 week time period the pore sizes (58$24 m) were constant in the unseeded sponges (Fig. 2a andb), cultured at 373C. At the start of the culture both supports showed a progressive penetration of predominantly elongated cells throughout the collagen network (Fig. 2c ande). After 1 week in culture, chondrocytes remained #at and attached to the walls of the sponge in the center of the 10 cells seeded sponges (Fig. 2c). The cells became round and isolated and were surrounded by a new intensely stained extracellular matrix after 1 week in culture in the 10 cells-seeded sponges (Fig. 2e) and after 4 weeks in the 10 cells-seeded sponges (Fig. 2d). On the sponge surface many cells became larger and round (Fig. 2d andf ) and formed a 3}4 -cells thick layer after 4 weeks of culture. With time in culture a dense extracellular matrix was formed in both types of sponge with cell density decreasing from the sponge surface to the center.

Cell proliferation

Chondrocyte proliferation, depending on the initial number of cells seeded in the sponges, was not a!ected by the type of sponge throughout the present study as shown in Fig. 3. A decrease in cell number (30$4%) was observed during the "rst week in culture for the sponges initially seeded with 10 chondrocytes. This corresponded to the detachment of cells from the sponge probably due to the high cell number/sponge volume ratio. Thereafter, a constant cell number (7$0.4;10) was observed throughout the culture. When the initial seeding was 10 chondrocytes, the cell number consistently increased reaching 4$0.4;10 cells after 4 weeks in culture.

Analysis of matrix components 3.4.1. Collagen synthesis

Collagen synthesis after 24 h labeling, expressed per cell or as a percentage of total protein synthesis, presented slight variations throughout the study in the two types of support and for the two seeding conditions (Table 1). The deposition in the cell layer of neosynthesized pepsin-resistant material increased with time in the 10 cells-seeded sponges in parallel with the increase in cell number. In the 10 cells-seeded sponges this material was deposited constantly as there was no change in cell density.

Collagen typing

When the neosynthesized pepsin-resistant and -soluble collagens of the sponge compartment were examined by SDS-PAGE analysis, type II collagen was the most abundant protein extracted from the two types of sponge seeded with 10 or 10 cells (Fig. 4a}d). Type XI was detected in small amounts both in the sponge compartment and in the culture medium. Traces of type I collagen were present in the culture medium (Fig. 4e}h) only after 2}3 weeks in culture in sponges seeded with 10 cells and only after 4 weeks with the 10 cells-seeded sponges.

GAG content

Sulfated glycosaminoglycan content was similar in both control and DPPA sponges and was dependent on the initial cell density, as shown in Fig. 5. Amounts in both types of sponge seeded with 10 cells reached a maximum of 900 g per sponge in 2 weeks and remained stable from day 14 to day 25. In the sponges seeded with 10 cells, the amount of glycosaminoglycan had reached only 150 g per sponge at day 30.

GAG identixcation

Glycosaminoglycans, extracted from the di!erent constructs and from fetal bovine cartilage, were separated by electrophoresis on cellulose acetate (Fig. 6). Chondroitin sulfate and hyaluronic acid, the major GAGs of cartilage, were present in the two types of sponge seeded with 10 and 10 cells after 4 weeks of culture. Traces of dermatan sulfate were detected in fetal bovine cartilage and after 4 weeks of culture in DPPA sponges seeded with 10 and 10 cells (Fig. 6, lanes 5, 9 and 10).

Sponge mineralization

The initial amounts of Ca and P were 10 times less in DPPA sponges than in control sponges due to the long washing step performed at the end of the DPPA treatment (Table 2). In the unseeded sponges, the mineral content increased with time in culture to reach similar values in both types of sponge. In the presence of 10 cells, the Ca and P values were no di!erent from the values found in the unseeded sponges. When seeded with The values in % (w/w) represent the mean $S.D. for n"3 10 cells a 2}2.5 fold decrease in the mineral content was observed in the two types of sponge. The Ca/P ratios measured in the di!erent conditions varied between 1.5 and 2.

Biochemical characteristics of the sponges

The composition of the cartilage constructs after one month in culture is presented in Table 3 in comparison with natural bovine fetal epiphyseal and neo-natal articular cartilage. Amino acid analysis indicated that, with time in culture, the protein content of the sponges decreased towards that of natural cartilage. This was also true for the collagen level (including both sponge collagen and neosynthesized collagens), especially for the 10 cells-seeded sponges. GAG content in the 4-week constructs was dependent on cell number and represented 25% (for the 10 cells-seeded sponge) of natural bovine cartilage. Cellularities were comparable to the value found in natural articular new-born cartilage.

Discussion

In this study, sponges, made of native and diphenylphosphorylazide cross-linked collagen, were tested as sca!olds to maintain bovine chondrocytes in in vitro culture and to determine the best culture conditions for the production of a functional cartilage construct. We demonstrated that fetal bovine epiphyseal chondrocytes were able to proliferate, to populate and to deposit an extracellular matrix without disintegration of the support during one month in culture in these sca!olds. The variability in response was dependent mainly on the number of cells initially seeded in the sponges and not on the composition of the support, i.e. native or cross-linked sponges. Indeed the main di!erence observed between these two matrices was a rapid partial drop in weight and size of the control sponges (unseeded or seeded) after 5 days incubation at 373C. This probably corresponded to partial denaturation of the collagen triple helix as the onset denaturation temperature of these control sponges was below 373C as compared to 433C for the DPPA sponges. We have previously demonstrated that "broblasts were not able to contract these same sponges [START_REF] Chevallay | Mouse "broblasts in long-term culture within collagen three-dimensional sca!olds: in#uence of cross-linking with diphenylphosphorylazide on matrix reorganization, growth, biosynthetic and proteolytic activities[END_REF] in contrast to what was usually observed with free-#oating collagen gels.

Under our culture conditions, characterized by the absence of the addition of fresh ascorbic acid to the culture medium and static #asks, and when seeded, with a low initial cell number [START_REF] Homminga | Perichondrial grafting for cartilage lesions of the knee[END_REF], chondrocytes proliferated in the sponges over time, showing a 4-fold increase after 4 weeks, with maintenance of a di!erentiated phenotype. Indeed the neosynthesized collagen deposited in the sponge was composed essentially of type II collagen and traces of type I collagen were only detected in the culture medium. With an initial cell number 10 times higher [START_REF] Homminga | Perichondrial grafting for cartilage lesions of the knee[END_REF], chondrocytes populated the sponges without proliferation over time but with the rapid formation of an The dry weight of one chondrocyte was estimated to 10\ g [START_REF] Riesle | Collagen in tissue-engineered cartilage: types, structure and crosslinks[END_REF]. ND: not determined.

abundant extracellular matrix composed of type II collagen and glycosaminoglycans. Thus, di!erentiated chondrocytes seeded into a type I collagen matrix maintain their phenotype if the culture conditions are appropriate, indeed the addition of fresh ascorbic acid to the culture medium led to a rapid dedi!erentiation of the cells (unpublished data). Similar observations were obtained with the same cells and the same culture medium composition (with or without ascorbic acid) in long-term culture at high density on plastic [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF]. Therefore, it does not seem necessary to use type-II collagen matrices as proposed by Nehrer et al. [START_REF] Nehrer | Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro[END_REF], who showed that chondrocytes, dedi!erentiated after proliferation in a monolayer culture, presented a higher level of redi!erentiation after seeding in type-II versus type-I collagen sponges. Furthermore, the arthritogenic potential of type-II collagen does not favor its clinical use in humans. After 4 weeks, cells in both sca!olds presented a round shape but with a higher density at the periphery than at the center of the sponges, certainly re#ecting the access to nutrient and gas transfer as also reported using collagen disks seeded with horse chondrocytes [START_REF] Nixon | Temporal matrix synthesis and histologic features of a chondrocyte-laden porous cartilage analogue[END_REF] and PGA disks seeded with new-born bovine chondrocytes [START_REF] Grande | Evaluation of matrix sca!olds for tissue engineering of articular cartilage grafts[END_REF].

As the presence of mineral may be a problem for an optimum biofunctionality of cartilage constructs and as acellular apatite deposition in our collagen sponges incubated in di!erent cell culture media was recently reported [START_REF] Chevallay | Mouse "broblasts in long-term culture within collagen three-dimensional sca!olds: in#uence of cross-linking with diphenylphosphorylazide on matrix reorganization, growth, biosynthetic and proteolytic activities[END_REF][START_REF] Andre-Frei | Acellular mineral deposition in collagen-based biomaterials incubated in cell culture media[END_REF], we measured the variation in the Ca and P content in the di!erent sponges, seeded and unseeded with chondrocytes, during the culture period. Mineral deposition in the unseeded collagen supports was previously reported to be related "rstly to the sponge residual P content and secondly to Ca and P concentration in the incubation medium [START_REF] Andre-Frei | Acellular mineral deposition in collagen-based biomaterials incubated in cell culture media[END_REF]. The culture medium used in this study (NCTC/RPMI) contains a low calcium concentration (1.18 mM) and in its presence we observed after 4 weeks in culture, a calcium content in the unseeded control and DPPA sponges (4.9}4.6%) much lower than the content (18}14%) previously [START_REF] Chevallay | Mouse "broblasts in long-term culture within collagen three-dimensional sca!olds: in#uence of cross-linking with diphenylphosphorylazide on matrix reorganization, growth, biosynthetic and proteolytic activities[END_REF] reported after incubation in DMEM (Ca concentration : 1.96 mM). This mineralisation was signi"cantly decreased (2-to 3-fold) for the sponges seeded with a high cell density but in the 4-week constructs the calcium content (1.2}2.4%) was still much higher than in natural cartilage. Our "ndings demonstrated that this parameter, not usually considered by other authors, should not be neglected.

Comparison of the global biochemical composition of our cartilage constructs, obtained after 4 weeks of culture, with that of natural articular cartilage, demonstrated the presence, in the extracellular matrix, of the same individual components (collagens, glycosaminoglycans) but with di!erences in their relative proportions, especially too low a glycosaminoglycan content.

It is interesting to compare these results with those described by the MIT group using polyglycolic acid as a sca!old [17,24,26,27,64}66]. These constructs prepared under optimized conditions [START_REF] Vunjak-Novakovic | Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage[END_REF][START_REF] Obradovic | Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage[END_REF] more closely approximated cartilage with respect to composition and function but still without quite reaching the properties of the natural tissue (for example, a lower collagen concentration and cross-link content) [START_REF] Riesle | Collagen in tissue-engineered cartilage: types, structure and crosslinks[END_REF].

These results suggest several possibilities to improve the quality of our cartilage construct, for example, by the use of a rotating bioreactor instead of static #asks, by increasing the culture time or by addition of speci"c factors such as TGF 1 [START_REF] Hunziker | Repair of partial-thickness defects in articular cartilage: cell recruitment from the synovial membrane[END_REF][START_REF] Athanasiou | Biodegradable implants for the treatment of osteochondral defects in a goat model[END_REF][START_REF] Bradham | In vivo cartilage formation from growth factor modulated articular chondrocytes[END_REF], FGF-2 [START_REF] Martin | In vitro di!erentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues[END_REF][START_REF] Toolan | E!ects of growth-factor-enhanced culture on a chondrocyte-collagen implant for cartilage repair[END_REF] or BMP-2 [START_REF] Sellers | The e!ect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage[END_REF] whose mitogenic and/or chondrogenic effects were previously tested for regeneration of articular cartilage. Characterization of the exact nature of the proteoglycans synthesized and of the mechanical properties of the construct also appears necessary for a better evaluation of the biofunctionality of the "nal product.

Parallel studies are also performed with mature human chondrocytes to obtain neocartilage of potential use in clinical human application.

In conclusion, these results and encouraging preliminary "ndings (unpublished results) in the repair of a partial thickness cartilage defect in rabbit knee with DPPA cross-linked collagen sponges seeded with allogenic rabbit chondrocytes, demonstrate the suitability of collagen-based sca!olds for cartilage tissue engineering.

Fig. 1 .

 1 Fig. 1. Changes of dry weight (A) and diameter (B) (expressed as percentage of initial dry value) of sponges during 1 month of culture. Unseeded control (#) and DPPA sponges (). Cells were seeded in either control sponges (ᮀ for 10 cells and ᭜ for 10 cells) or in DPPA sponges (* 10 cells and ᭡ for 10 cells). Data are presented as the mean $s.d. of triplicate samples.

Fig. 2 .

 2 Fig. 2. Light microscopic analysis of collagen DPPA sponges grown in culture. (a) and (b) sponges without cells; (c) and (d) sponges seeded with 10 chondrocytes; (e) and (f) sponges seeded with 10 cells. (a), (c) and (e) after 1 week of culture and (b), (d) and (f) after 4 weeks of culture. Histo-Resin GMA embedded section stained with Azure II/methylene blue. Bar"100 m.

Fig. 3 .

 3 Fig. 3. Proliferation of bovine chondrocytes seeded in collagen sponges. Cells were seeded in either control sponges (ᮀ for 10 cells and ᭜ for 10 cells) or in DPPA sponges (* for 10 cells and ᭡ for 10 cells). Cell numbers were measured by the DNA content of the sponges every week during the culture. Data are presented as the mean $S.D. of triplicate samples.

Fig. 4 .

 4 Fig. 4. Fluorographs of C-labeled pepsin-soluble collagens neosynthesized by bovine chondrocytes for the last 24 h, deposited in the cell layer (a}d) and secreted in the culture medium (e}h), after 1, 2, 3 and 4 weeks of culture in sponges and separated by SDS-PAGE. Control sponges (a and e: 10 cells; b and f : 10 cells) and DPPA sponges (c and g : 10 cells; d and h : 10 cells). Data are representative of three separate experiments.

Fig. 5 .

 5 Fig. 5. Total sulphated glycosaminoglycan (GAG) content in collagen sponges seeded with bovine chondrocytes grown during 4 weeks of culture. Cells were seeded in either control sponges (ᮀ for 10 cells and ᭜ for 10 cells) or in DPPA sponges (* for 10 cells and ᭡ or 10 cells). GAG content was measured by a colorimetric method with dimethylmethylene blue. Data are presented as the mean $S.D. of triplicate samples. All values are on a dry weight basis.

Fig. 6 .

 6 Fig. 6. Electrophoretogram of GAG contained in collagen sponges seeded with bovine chondrocytes. GAG were extracted by papain digestion of the sponges and the cartilage pieces and visualized after Alcian blue staining as described in Methods. Control sponges (lanes 2 and 3: seeded with 10 cells; lanes 6 and 7: seeded with 10 cells) and DPPA sponges (lanes 4 and 5: seeded with 10 cells; lanes 8 and 9: seeded with 10 cells) after 2 days (lanes 2-4-6-8) and 27 days (lanes 3-5-7-9) in culture. For comparison lane 1 received a mixture of dermatan sulfate (DS), chondroitin sulfate (CS) and hyaluronic acid (HA) and lane 10 received the GAG extracted from fetal bovine articular cartilage. Data are representative of three separate experiments.

Table 1 Changes

 1

	Control sponge
	10 cells	4.3 (8%)	3 (11%)	1.8 (8%)	1.3 (7%)
	10 cells	2.4 (5%)	1.8 (7%)	2.6 (5%)	2.4 (4%)
	DPPA sponge
	10 cells	6.3 (6%)	1.2 (4%)	2.3 (12%)	2.1 (13%)
	10 cells	4.2 (6%)	1.4 (5%)	2.4 (5%)	1.6 (3%)
	Pepsin-resistant protein deposited in the sponges (dpm;10\ 3 )
	Control sponge
	10 cells	9.8	21.3	46.6	32.8
	10 cells	82.5	58.9	117.3	115.7
	DPPA sponge
	10 cells	11.6	17.3	72.7	48.5
	10 cells	60.3	35.3	117.4	82.7
	Values represent the data from one representative single experiment.
	Comparable results were obtained with chondrocytes isolated from
	another fetus.

in the level of neosynthesized pepsin-resistant proteins (24 hlabeling) by bovine chondrocytes during one month in culture Days 7 13 20 27

Total pepsin-resistant proteins (deposited in the sponge and secreted in the culture medium) per cell (dpm/ g DNA;10\

3

) and as a percentage (%) of total protein synthesis

Table 2

 2 Calcium and phosporous content in of control and DPPA sponges seeded with bovine chondrocytes

		Calcium content (%)	Phosphorous content (%)
		Control	DPPA	Control	DPPA
	Initial content	0.5$0.1	0.05$0.01 0.26	0.02
	of sponges
	Unseeded sponges
	Day 7	3.1	2	1.6	1.8
	Day 27	4.9$0.7	3.6$1.7	2.5$0.3	1.8$0.8
	Cell-seeded sponges (Day 27)
	1;10	6.5$0.5	2.8$0.4	3.5$0.3	1.6$0.2
	1;10	2.4$0.5	1.2$1.3	1.6$0.3	0.9$0.5

Table 3

 3 Biochemical characterization of tissue engineered constructs after one month in culture

		Amino acid	Collagen	S-GAG	Cell	Calcium
		(% dry weight)	(% dry weight)	(% dry weight)	(% dry weight)	(% dry weight)
	Control sponge
	Unseeded	90$19	88$5	0	0	4.9$0.7
	10 cells	60$10	48.6$6	1.3	3.9$0.5	6.5$0.5
	10 cells	64$4	4 9 $5.8	9.8	6.7$0.7	2.4$0.5
	DPPA sponge
	Unseeded	92$17	87$3	0	0	3.6$1.7
	10 cells	89$10	74.6$3	1.8	3.4$0.4	2.8$0.4
	10 cells	74$9	54.4$7.4	6.4	6.7$0.7	1.2$1.3
	Bovine cartilage
	fetal epiphyseal	53$2	36.4$4.8	40$9	2.1$0.6	0.061$0.001
	articular new born [26]	ND	51$5	3 4 $3	5 $0.5	ND
	Data represent mean $S.D. and n"3.

Acknowledgements

This work was supported by grant BIOMED 2 No. BMH4-CT95-O396 and by grant Emergence L094120401 from Rho( ne-Alpes ReH gion. The authors thank R Willems for expert technical assistance and C van Herrewhegge and A Bosch for assistance in preparing the manuscript.


Marie-Claire Ronzi

" Ere

St ! Ephane Roche

J ! Er # Ome Gouttenoire

Olivier D ! Emarteau

Daniel Herbage

Anne-Marie Freyria
email: am.freyria@ibcp.fr

Ascorbate modulation of bovine chondrocyte growth, matrix protein gene expression and synthesis in three-dimensional collagen sponges

Keywords: Ascorbate, Bovine chondrocytes, Collagen biomaterials, Cartilage engineering

This report completes a previous study on the growth and metabolism of fetal bovine epiphyseal chondrocytes cultured, within native or cross-linked collagen sponges carried out without the addition of fresh ascorbate. At low initial cell density (2.3 Â 10 6 cells/ cm 3 ) cell proliferation and a low matrix deposition were observed, whereas at high initial cell density (2.3 Â 10 7 cells/cm 3 ) there was an absence of cell proliferation, but the deposition of a cartilage-like matrix was measured. In both cases, only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. In this report, we observed, after 1 month in culture with ascorbate, in both type of scaffolds and initial cell densities, an increase in cell proliferation (2-fold) and in expression of genes encoding for collagen types I, II, X and MMP-2 and -13, but no change in the level of matrix deposition (collagen and GAG). With regard to the proteins present, the main differences with or without ascorbate concerned the increase of neosynthesised type I collagen (up to 35% of the total collagen deposited in the sponge) and of the MMP-2 active form. In conclusion, these results show that ascorbate is an important factor to consider when preparing cartilage constructs for its action on chondrocyte phenotype modulation and proliferation.

Introduction

The inability of cartilage to self-repair to any significant degree has been extensively studied and reported by numerous investigators [START_REF] Bentley | Grafts and implants for cartilage repair and replacement[END_REF][START_REF] Minas | Current concepts in the treatment of articular cartilage defects[END_REF][START_REF] Caplan | Principles of cartilage repair and regeneration[END_REF][START_REF] Buckwalter | Articular cartilage repair and transplantation[END_REF][START_REF] Driscoll | The healing and regeneration of articular cartilage[END_REF][START_REF] Newman | Articular cartilage repair[END_REF][START_REF] Boyan | Bone and cartilage tissue engineering[END_REF]. Clinical procedures used to treat the articular cartilage defects include lavage and debridement, drilling, microfracture, and abrasion arthroplasty, all of which cause formation of a fibrocartilage lacking the mechanical properties of hyaline cartilage. Since the pioneering work of Brittberg et al. [START_REF] Brittberg | Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation[END_REF] autologous chondrocyte transplantation (ACT) is now a widely used technique for the treatment of deep cartilage defects of articular cartilage [START_REF] Peterson | Articular cartilage injuries treated with autologous chondrocyte transplantation in the human knee[END_REF][START_REF] Mayhew | Validation of a quality assurance program for autologous cultured chondrocyte implantation[END_REF][START_REF] Gillogly | Treatment of articular cartilage defects of the knee with autologous chondrocyte implantation[END_REF][START_REF] Richardson | Repair of human articular cartilage after implantation of autologous chondrocytes[END_REF]. In order to improve retention of cells in the defect and/or to transplant differentiated or in vitro redifferentiated chondrocytes, different scaffolds and matrices have been proposed for in vitro chondrocyte culture before transplantation. Several cell types (chondrocytes derived from auricular, nasal or articular cartilage or progenitor cells) have been cultured under different conditions after seeding in natural (fibrin [START_REF] Hendrickson | Chondrocyte-fibrin matrix transplants for resurfacing extensive articular cartilage defects[END_REF][START_REF] Brittberg | Influence of fibrin sealant (Tisseel R) on osteochondral defect repair in the rabbit knee[END_REF][START_REF] Fortier | Disparate chondrocyte metabolism in three-dimensional fibrin cultures derived from autogenous or commercially manufactured fibrinogen[END_REF][START_REF] Ting | In vitro prefabrication off human cartilage shapes using fibrin glue and human chondrocytes[END_REF][START_REF] Silverman | Injectable tissue-engineered cartilage using a fibrin glue polymer[END_REF][START_REF] Meinhart | Stabilization of fibrinchondrocyte constructs for cartilage reconstruction[END_REF][START_REF] Perka | Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants[END_REF], alginate [START_REF] Perka | Matrix-mixed culture: new methodology for chondrocyte culture and preparation of cartilage transplants[END_REF][START_REF] Van Susante | Culture of chondrocytes in alginate and collagen carrier gels[END_REF][START_REF] Loty | Cartilage formation by fetal rat chondrocytes cultured in alginate beads: a proposed model for investigating tissuebiomaterial interactions[END_REF][START_REF] Ragan | Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system[END_REF][START_REF] Fragonas | Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate[END_REF][START_REF] Van Osch | In vitro redifferentiation of culture-expanded rabbit and human articular auricular chondrocytes for cartilage reconstruction[END_REF][START_REF] Chang | Injection molding of chondrocyte/alginate constructs in the shape of facial implants[END_REF][START_REF] Miralles | Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage[END_REF], agarose [START_REF] Rahfoth | Transplantation of allograft chondrocytes embedded in agarose gel into cartilage defects of rabbits[END_REF][START_REF] Knight | The influence of elaborated pericellular matrix on the deformation of isolated articular chondrocytes cultured in agarose[END_REF], hyaluronan [START_REF] Robinson | Regenerating hyaline cartilage in articular defects of old chickens using implants of embryonal chick chondrocytes embedded in a new natural delivery substance[END_REF][START_REF] Aigner | Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester[END_REF][START_REF] Solchaga | Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage[END_REF][START_REF] Brun | Chondrocyte aggregation and reorganization into three-dimensional scaffolds[END_REF][START_REF] Grigolo | Transplantation of chondrocytes seeded on a hyaluronan derivative (Hyaff-11) into cartilage defects in rabbits[END_REF] or collagen [START_REF] Nixon | Temporal matrix synthesis and histologic features of a chondrocyte-laden porous cartilage analogue[END_REF][START_REF] Wakitani | Mesenchymal cell-based repair of large, fullthickness defects of articular cartilage[END_REF][START_REF] Schuman | Chondrocyte behaviour within different collagen gel in vitro[END_REF][START_REF] Matsuda | In vivo chondrogenesis in collagen sponge sandwiched by perichondrium[END_REF][START_REF] Ben-Yishay | Repair of articular cartilage defects with collagen-chondrocyte allografts[END_REF][START_REF] Stanton | The growth of chondrocytes using Gelfoam s as a biodegradable scaffold[END_REF][START_REF] Toolan | Effects of growth-factor-enhanced culture on a chondrocytecollagen implant for cartilage repair[END_REF][START_REF] Frondoza | Human chondrocyte proliferate and produce matrix components in microcarrier suspension culture[END_REF][START_REF] Nehrer | Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro[END_REF][START_REF] Kawasaki | Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels[END_REF][START_REF] Ehlers | Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I:III sponge[END_REF][START_REF] Uchio | Human chondrocyte proliferation and matrix synthesis cultured in Atellocollagen R gel[END_REF][START_REF] Ochi | Current concepts in tissue engineering technique for repair of cartilage defect[END_REF][START_REF] Mizuno | Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges[END_REF][START_REF] Kinner | Smooth muscle actin expression by human articular chondrocytes and their contraction of a collagenglycosaminoglycan matrix in vitro[END_REF][START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF][START_REF] Hunter | Mechanical compression alters gene expression and extracellular matrix synthesis by chondrocytes cultured in collagen I gels[END_REF]) or synthetic (PGA/PLA, polyethylene or polyethylene oxide or glycol) biomaterials [START_REF] Langer | Tissue engineering[END_REF][START_REF] Freed | Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers[END_REF][START_REF] Kim | Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers[END_REF][START_REF] Chu | Articular cartilage repair using allogeneic perichondrocyte-seeded biodegradable porous polylactic acid (PLA): a tissue-engineered study[END_REF][START_REF] Sittinger | Resorbable polyesters in cartilage engineering: affinity and biocompatibility of polymer fiber structures to chondrocytes[END_REF][START_REF] Riesle | Collagen in tissue-engineered cartilage: types, structure and crosslinks[END_REF][START_REF] Vunjak-Novakovic | Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage[END_REF][START_REF] Rotter | Behavior of tissue-engineered human cartilage after transplantation into nude mice[END_REF][START_REF] Schreiber | A method for tissue engineering of cartilage by cell seeding on bioresorbable scaffolds[END_REF][START_REF] Gugala | In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold[END_REF][START_REF] Lohman | Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vitro[END_REF][START_REF] Martin | Selective differentiation of mammalian bone marrow stromal cells cultured on threedimensional polymer foams[END_REF][START_REF] Britt | Autogenous tissue-engineered cartilage. Evaluation as an implant material[END_REF][START_REF] Livecchi | In vitro chondrocyte collagen deposition within porous HDPE: substrate microstructure and wettability effects[END_REF][START_REF] Sims | Injectable cartilage using polyethylene oxide polymer substrates[END_REF][START_REF] Bryant | The effects of scaffold thickness on tissue engineered cartilage in photocrosslinked poly (ethylene oxide) hydrogels[END_REF][START_REF] Temenoff | Effect of poly (ethylene glycol) molecular weight on tensile and swelling properties of oligo (poly (ethylene glycol) fumarate) hydrogels for cartilage tissue engineering[END_REF]. In a previous study [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF] we evaluated the ability of collagen-based matrices, in the form of native or cross-linked (DPPA) sponges, to support the growth of fetal bovine epiphyseal chondrocytes, without changes in their initial phenotype, leading to the formation of a cartilage construct with a composition as close as possible to that of natural cartilage. Under these culture conditions, characterised by the lack of addition of fresh ascorbic acid to the culture medium (RPMI/NCTC (v/v) containing 10% fetal calf serum) and the use of static flasks, we observed after 4 weeks (i) a 4-fold increase in cell number and a low level of matrix deposition in sponges seeded with 10 6 cells, (ii) an absence of cell proliferation and the neoformation of a cartilage-like matrix in sponges seeded with 10 7 cells. In both cases only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. These findings demonstrated the suitability of collagenbased scaffolds for cartilage tissue engineering as well as the necessity to improve the culture conditions in order to obtain a better quality cartilage construct (for example higher collagen and glycosaminoglycan contents for sponges seeded with small numbers of cells). In another set of experiments [START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF][START_REF] Freyria | Two dimensional electrophoresis of intracellular and secreted protein synthesised by fetal bovine chondrocytes in high-density culture[END_REF][START_REF] Ronzi | Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture[END_REF][START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF] using high density culture (10 6 cells/cm 2 ) of the same cells (fetal bovine chondrocytes) on plastic, we noted [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF] that the addition of fresh ascorbic acid (25 mg/ml) to the culture induced increases in both cell proliferation and total protein and collagen synthesis as well as a progressive cell maturation (type I and X collagen synthesis after 30 days). After comparing these results [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF][START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF], we decided to examine the influence of fresh ascorbic acid addition on fetal bovine chondrocyte proliferation and metabolic response after culture for 1 month within native and DPPA-treated type I collagen sponges. Here we describe the chondrocyte response (with initial seedings of 10 6 and 10 7 cells per sponge) by quantitation of DNA content as well as gene expression of collagens I, II and X and matrix metalloproteinases-2 and -13 (MMP-2 and -13), by RT-PCR on RNA isolated after 1 month. We also measured the levels of total collagen and collagens I and II neosynthesised after 7 and 30 days, the level of active and inactive MMP-2 after 7, 15 and 30 days and the content of sulphated glycosaminoglycans deposited in the matrix after 30 days.

Materials and methods

Collagen sponges

Collagen scaffolds were made as previously described [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF]. In brief, collagen sponges, manufactured by Coletica (Lyon, France), were made up of collagen extracted from the skin of young calves containing 90-95% (dry weight) native type I collagen and 4-9% type III collagen. Control sponges (denoted by H) and DPPA sponges (control sponges cross-linked using the diphenylphosphorylazide method [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF][START_REF] Petite | Use of diphenylphosphorylazide for cross-linking collagen-based biomaterials[END_REF]; denoted by D) were used in this study. Individual discs were cut out with a 10 mm diameter punch before being sterilised with 15 kGy b radiation.

Chondrocyte seeding onto collagen scaffolds

After enzymatic isolation, fetal bovine chondrocytes were cultured as previously described [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF]. Briefly, each sponge (10 mm diameter  6 mm thick disc) was seeded with 10 6 or 10 7 chondrocytes deposited onto the sponge in a volume of 50 ml, then further incubated in 24-well culture plates at 371C with 5% CO 2 for 2 h. Subsequently, 2 ml of complete culture medium [RPMI/ NCTC (v/v) medium (Seromed s , Strasbourg, France/ Sigma, Saint Quentin Fallavier, France) containing 10% fetal calf serum (Seromed s ), 50 U/ml penicillin, 50 mg/ ml streptomycin and 1 ng/ml amphotericin] were added to each well. The sponges were maintained in culture for 48 h to allow the cells to attach to the scaffold.

Cell-seeded sponge cultivation

Sponges were cultured in 24-well plates for 1 month, changing the medium every 3 days. In some experiments, fresh l-ascorbic acid was added to the medium throughout the culture at various concentrations: 8 mg/l (0.05 mm) for the first 24 h, 10 mg/l (0.06 mm) between 24 and 48 h and 25 mg/l (0.14 mm) thereafter. Throughout the study, unseeded sponges were maintained in culture as controls.

Cell proliferation

Numbers of chondrocytes per sponge were calculated from the amounts of DNA measured using the Hoechst 33258 dye [START_REF] Lipman | Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258[END_REF], based on the known DNA content per chondrocyte of 8 pg [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF].

Collagen synthesis and typing

Collagen synthesis was measured on days 7 and 30 by labelling the culture with 20 mCi per sponge of 35 S methionine (1000 Ci/mmol, SJ 204 Amersham Biosciences, Orsay, France) for 14 h in fresh RPMI lacking methionine and in the absence of fetal calf serum, as previously described [START_REF] Freyria | Two dimensional electrophoresis of intracellular and secreted protein synthesised by fetal bovine chondrocytes in high-density culture[END_REF]. After labelling, the medium and washed sponges were dialysed separately against water to eliminate unincorporated radiolabel. Samples were frozen, freeze-dried and submitted separately to limited pepsin digestion as previously described [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF]. Pepsin treatment with magnetic stirring almost totally solubilised the sponges. The content of dpm in the pepsin-treated samples gave the level of total protein synthesis while pepsin-resistant proteins, assumed to be collagenous, were retained following dialysis. After lyophilisation or acetone precipitation (volume ratio 1:5) samples were dissolved in Laemmli's buffer and counted for their radioactive content.

Pepsin-digested samples were analysed by SDS-PAGE using 6% separating gels under non-reducing conditions, as previously described [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF]. Volumes corresponding to the same amount of material were loaded on each lane of the gel. Bands corresponding to types I, II and XI collagens were visualised by fluorography and their relative proportions were quantified densitometrically on the fluorographs with a Personal Densitometer (Amersham Biosciences) using Image Quant software as previously described [START_REF] Freyria | Two dimensional electrophoresis of intracellular and secreted protein synthesised by fetal bovine chondrocytes in high-density culture[END_REF].

Glycosaminoglycan content

Amounts of sulphated GAG deposited in the cultured sponges were determined spectrophotometrically at 525 nm using dimethylmethylene blue dye and shark chondroitin sulphate (Sigma) as standard [START_REF] Farndale | Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue[END_REF].

Metalloproteinase analysis

Gelatinase activity was measured by gelatin-substrate zymography as described [START_REF] Chevallay | Mouse fibroblasts in long-term culture within collagen three-dimensional scaffolds: influence of cross-linking with diphenylphosphorylazide on matrix reorganization, growth, biosynthetic and proteolytic activities[END_REF] using the culture medium from bovine chondrocytes grown in collagen sponges for 24 or 48 h in RPMI/NCTC medium without fetal calf serum. From each culture medium 5-10 ml samples were mixed with 4X Laemmli's loading buffer and subjected to electrophoresis in 8% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, gels were washed with 50 mm Tris-HCl buffer (pH 7.5) containing 50 mm NaCl and 2.5% Triton X100 then incubated at 371C for 16 h in 50 mm Tris-HCl buffer (pH 7.5) containing 50 mm NaCl, 10 mm CaCl 2 . Gels were stained with 0.5% Coomassie brillant blue R-250, then destained and analysed using a Personal densitometer and Image Quant software (Amersham Biosciences).

Histology and immunochemistry

Samples were extensively rinsed in phosphate-buffered saline (PBS), fixed for 24 h with 2.5% paraformaldehyde in PBS prior to inclusion in Tissue Tek OCT. For general evaluation, sections (5-7 mm thick) were stained with hematoxylin-eosin. Proteoglycan staining was performed using safranin-O. For immunofluorescence staining, cryostat sections were post-fixed in 2.5% paraformaldehyde in PBS for 1 h, rinsed in PBS, treated with 0.2% hyaluronidase (Sigma Type III, 800 U/mg) in PBS for 1 h at 371C then incubated for 1 h in 1% bovine serum albumin before treatment with monoclonal antibodies. Antibodies against chicken type-II collagen (Neomarkers, Interchim, Montlu-con, France) which also recognise bovine protein and monoclonal antibodies against bovine type-I collagen (Sigma) were used in this study. Samples were then incubated for 1 h at room temperature with the primary antibodies diluted in PBS/ 1% BSA (1:100 for anti-type-II and anti-type-I collagen), then washed with PBS and incubated with fluorescein-conjugated goat anti-mouse serum. Samples mounted in glycerol/PBS (1/1) were examined using a Zeiss Universal microscope equipped for epifluorescence.

Total RNA extraction

RNA was extracted from the construct after 1 month in culture using a RNeasy kit (Qiagen, Courtaboeuf, France). Additional steps included protein digestion using proteinase K and on-column DNase digestion using RNase-free DNase set (Qiagen). RNA was eluted from the membrane with diethylpyrocarbonate-treated water. RNA purity was confirmed spectrophotometrically (260/280 ratio) and ranged between 1.6 and 2.1.

Reverse transcription and polymerase chain reaction (RT-PCR)

Reverse transcription of 1 mg of total RNA was carried out in a 40 ml reaction volume with 200 U of SuperScript II RNase H À (GibcoBRL/Life Technologies, Cergy Pontoise, France) under the following conditions, previously described [START_REF] Bluteau | Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis[END_REF]: 421C for 50 min and 701C for 15 min, cDNA was then submitted to RNase H digestion using 4 units of enzyme (GibcoBRL/ Life Technologies) for 30 min at 371C. For PCR, specific primers were designed for the Col1a1, Col1a2, Col2a1, Col10a1, MMP-2, MMP-13 and Glyceraldehyde-3phosphate dehydrogenase (GAPDH) genes according to their respective sequences (see Table 1). Each 50 ml volume reaction contained: 1 ml RT product, 50 mm of each dNTP, 0.2 mm each primer, 0.5-1.5 mm MgCl 2 and 2.5 U Amplitaq DNA polymerase (Applied Biosystems, Les Ulis, France). A denaturation step of 2 min at 941C was carried out before the amplification step which consisted of 23-40 cycles (23 cycles for GAPDH, Col1a2 and Col2a1, 31 cycles for MMP-2 and 40 cycles for MMP-13, Col10a1 and Col1a1) of 30 s at 941C, 30-45 s at the annealing temperature, 45 s at 721C followed by a final extension step of 5 min at 721C. All PCR products were subcloned into the pCR 2.1-TOPO vector (Invitrogen, Groningen, The Netherlands) and subjected to sequencing for their identification (Genome Express SA, Grenoble, France). Amplified products were analysed by electrophoresis on 2% agarose gels followed by staining with ethidium bromide. The amplification signal obtained for the GAPDH gene was used to normalise the amount of cDNA in each sample.

Results

Cell proliferation

Total amounts of DNA were determined in sponges seeded with 10 6 and 10 7 chondrocytes cultured in control (H) and cross-linked (D) scaffolds and in medium either supplemented or not with ascorbic acid. No significant differences were observed between control and DPPA sponges. With 10 6 cells per sponge (Fig. 1), chondrocyte numbers consistently increased with time reaching, in the absence of ascorbic acid and after 4 weeks in culture in the control and cross-linked sponges, 3.5 Â 10 6 and 4.9 Â 10 6 cells respectively. In contrast, in the presence of ascorbic acid, almost 2-fold increases in cell number were observed, reaching 6.5 Â 10 6 and 8.5 Â 10 6 cells, respectively. With 10 7 cells per sponge, after an initial decrease, cell numbers remained constant throughout culture, again with higher values in the presence of ascorbate (12-15 Â 10 6 cells in cross-linked sponges).

Analysis of matrix components

Chondrocyte gene expression in the presence or absence of ascorbic acid

The effects of ascorbic acid in the culture medium using sponges seeded with 10 6 and 10 7 cells were examined on day 30 by comparing relative gene transcript levels in chondrocytes grown in treated or untreated cultures. As shown in Fig. 2, on the whole, increases were noted in the expression of all genes studied (types II, I (a1 and a2 chains), X collagens and MMPs-2 and -13) as a result of ascorbic acid treatment for both types of scaffold and for sponges seeded with 10 6 and 10 7 cells. In particular, the greatest increase in gene expression as a result of ascorbic acid treatment was observed with the sponges seeded with 10 6 cells for type I (a2 chain) collagen and MMPs-2 and -13. Type X collagen mRNA expression was detected in the DPPAtreated sponges (after 40 PCR cycles) in the presence of ascorbic acid.

Collagen synthesis and immunolocalisation

In order to confirm the data obtained at the gene level, we measured neosynthesis of total collagen as well as individual types I and II collagens, expressed per mg of DNA and either deposited in the sponge or secreted into the culture medium, on days 7 and 30 of culture after labelling for 14 h with 35 S methionine (Fig. 3). Whatever the culture conditions used (both types of support, under both seeding conditions and with or without ascorbate) collagen synthesis levels (per cell) and the percentage of neosynthesised collagen deposited in the sponge (75-85% of the total) showed only slight and insignificant variations with the exception of a net decrease in the collagen synthesised per cell after 30 days in sponges seeded with 10 6 cells in the presence of ascorbate (Fig. 3 B1).

The main differences observed between cultures with or without ascorbate concerned the neosynthesised type I/type II collagen ratio. In the absence of ascorbate (Fig. 3 C1) small amounts of type I collagen (5-15% of total) were observed mostly after 30 days and especially in the culture medium. In the presence of ascorbate, these values reached 10-35% of the collagen deposited in the sponge (Fig. 3 D1) and 30-45% of the collagen secreted into the medium (Fig. 3 D2). Immunohistology with antibodies against types II and I collagens (Fig. 4) demonstrated that type II collagen was located mostly where cells were forming aggregates in both types of scaffold and was more abundant (even in the presence of ascorbate (Fig. 4a)) than type I collagen, present either in small patches throughout the sponges (Fig. 4b) or at the periphery of the scaffold. It is interesting to note that the initial scaffold, still clearly detectable after 1 month culture, was not recognised by the antibodies against native type I collagen.

GAG content

Sulphated glycosaminoglycan (sGAG) content was similar in both control and cross-linked sponges and was dependent on initial cell density (Fig. 5). After 30 days in culture in absence of ascorbic acid, the amounts of sGAG in both types of sponge reached a maximum of 0.9 mg in sponges initially seeded with 10 7 cells and only 0.15 mg in sponges initially seeded with 10 6 cells. This low deposition of sGAG in the latter case, was not modified by the addition of ascorbic acid, whereas after 1 month in culture a slight increase in sGAG was shown in native and cross-linked scaffolds for sponges seeded with 10 7 cells (+31 and +17% respectively) as compared with untreated cultures. The net difference in sGAG content in the sponges seeded with 10 6 and 10 7 cells was clearly demonstrated by histology using Safranin O staining (Fig. 4c andd).

Metalloproteinase analysis

To investigate the involvement of MMPs in neomatrix production, the culture media were analysed for gelatinase activity (MMP-2 and-9) by gelatin substrate zymography (Fig. 6A). Traces of proMMP-9 were only detected for cultures in native sponges. Most of the MMP-2 was present as inactive forms in spent media and the total content of the pro-and active forms increased with time to reach values that were not significantly different either without ascorbic acid (Fig. 6 B1) or with (Fig. 6 B2). However, a higher percentage of an active form of MMP-2) was noted after 30 days in the presence of ascorbic acid (Fig. 6 C2) than in its absence (Fig. 6 C1) (12-18% instead of 8-10% respectively). MMP-13 activity was not detected in the medium tested for MMP-2 even after a 10-fold concentration of the medium.

Discussion

Many physiological and biochemical processes appear to be influenced by ascorbate [START_REF] Englard | The biochemical functions of ascorbic acid[END_REF], this being an essential requirement in normal connective tissue metabolism and collagen formation. As a reducing agent, ascorbate is a cofactor for prolyl and lysyl hydroxylase and its absence prevents the efficient secretion of procollagen. Furthermore, ascorbate is also thought to enhance the transcription of procollagen mRNA in fibroblasts [START_REF] Geesin | Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human skin fibroblasts[END_REF] without changing the rate of intracellular degradation. Ascorbate, in the form of stable derivatives such as vitamin C-phosphate, was also shown to improve cell proliferation when added in culture of rabbit renal proximal tubular cells [START_REF] Nowak | l-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture[END_REF], in combination with dexamethasone in culture of human anterior cruciate ligament cells [START_REF] Fermor | Proliferation and collagen synthesis of human anterior cruciate ligament cells in vitro: effects of ascorbate-2-phosphate, dexamethasone and oxygen tension[END_REF] or in combination with bFGF in culture of human vascular myofibroblasts in PGA-P4HB coated scaffold [START_REF] Hoerstrup | Optimized growth conditions for tissue engineering of human cardiovascular structures[END_REF]. Together with several others, our group has examined the effects of ascorbate on growth and synthesis of matrix components by cultured chondrocytes of different origins when plated both at low [START_REF] Sandell | Effects of ascorbic acid on collagen mRNA levels in short term chondrocyte cultures[END_REF][START_REF] Pacifici | Independent secretion of proteoglycans and collagens in chick chondrocytes cultures during acute ascorbic acid treatment[END_REF][START_REF] Dozin | In vitro differentiation of mouse chondrocytes: requirement for ascorbic acid[END_REF][START_REF] Sullivan | Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion[END_REF][START_REF] Farquharson | Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D[END_REF][START_REF] Venezian | Modulation of chondrocyte proliferation by ascorbic acid and BMP-2[END_REF] and high [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF][START_REF] Daniel | Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III Effects of ascorbate[END_REF][START_REF] Hering | Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension[END_REF] cell densities and as explants [START_REF] Benya | The capacity of chondrocytes to respond to serum is enhanced by organ culture in the absence of serum, stimulated by serum, and modified by ascorbate[END_REF]. In addition, chondrocyte culture in a 3D environment (within natural or synthetic matrices) is now an active domain of research in the preparation of cell-seeded matrices for use as implants to facilitate cartilage repair. Among the bioactive factors thought to be involved in the generation of cartilaginous constructs in vitro several growth factors (PDGF, EGF, TGF-b, FGF, IGF) have been tested [START_REF] Fortier | Disparate chondrocyte metabolism in three-dimensional fibrin cultures derived from autogenous or commercially manufactured fibrinogen[END_REF][START_REF] Van Osch | In vitro redifferentiation of culture-expanded rabbit and human articular auricular chondrocytes for cartilage reconstruction[END_REF][START_REF] Toolan | Effects of growth-factor-enhanced culture on a chondrocytecollagen implant for cartilage repair[END_REF][START_REF] Lohman | Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vitro[END_REF][START_REF] Yaeger | Synergistic action of TGF-b and IGF-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes[END_REF][START_REF] Jakob | Specific growth factors during the expansion and redifferentiation of adult human articular chondrocytes enhance chondrogenesis and cartilaginous tissue formation in vitro[END_REF], while the specific influence of ascorbate has not yet been investigated in detail, even though most of the culture medium usually contains ascorbic acid.

We have thus completed our previous study [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF] on the growth and metabolism of fetal epiphyseal chondrocytes cultured within collagen sponges but carried out without the addition of fresh ascorbic acid. Several Fig. 3. Effect of ascorbic acid on the levels of collagens neosynthesised by bovine chondrocytes grown in collagen sponges and incubated with 35 S methionine for the last 14 h of culture on days 7 and day 30. Neosynthesised collagen (expressed in dpm/mg DNA Â 10 À4 ) deposited in the sponges in the absence of ascorbic acid (A1) and in its presence (B1). Neosynthesised collagen secreted into the culture medium in the absence of ascorbic acid (A2) and in its presence (B2). Levels of types II and I collagen (expressed as % of collagen types XI, II and I separated on SDS-PAGE and quantified as described in Materials and Methods) deposited in the sponges in the absence of ascorbic acid (C1) and in its presence (D1). Levels of types II and I collagen secreted into the culture medium in the absence of ascorbic acid (C2) and in its presence (D2).

interesting results have been obtained regarding these parameters in this study. First, the addition of ascorbate induced a net increase in cell proliferation (almost a 2fold increase after 1 month culture when compared to culture without ascorbate), independent of the initial cell-seeding density and of the nature of the collagen sponge (cross-linked or not). As previously observed [START_REF] Roche | Native and DPPA cross-linked collagen sponges seeded with fetal bovine epiphyseal chondrocytes used for cartilage tissue engineering[END_REF] chondrocyte growth rate was dependent on initial cellseeding density. With a low initial cell-seeding density (2.3 Â 10 6 cells/cm 3 ) and after 1 month culture, we observed an up to 8.5-fold increase in cell numbers within the sponge. This result can be compared to growth rates obtained by different authors for chondrocytes cultured at similar low initial cell-seeding densities (0.5-2 Â 10 6 cells/cm 3 ) and in the presence of ascorbate but in different 3D environments: 5-fold and 2.25-fold increases with rabbit [START_REF] Kawasaki | Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels[END_REF] and human [START_REF] Uchio | Human chondrocyte proliferation and matrix synthesis cultured in Atellocollagen R gel[END_REF] chondrocytes, respectively, after 1 month culture in collagen gels, a 2.2-fold increase with bovine chondrocytes after 57 days in an alginate disk [START_REF] Ragan | Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system[END_REF], and a 2.2-fold with sheep articular chondrocytes after 9 weeks in a poly (l/dl-lactide) porous scaffold [START_REF] Gugala | In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold[END_REF]. With a high initial cell-seeding density (2.3 Â 10 7 cells/cm 3 ) cell losses were observed at the beginning of the culture followed by limited cell growth (maximum 1.5-fold increase after 1 month with ascorbate) as previously noted by several authors using collagen-GAG [START_REF] Nehrer | Canine chondrocytes seeded in type I and type II collagen implants investigated in vitro[END_REF], polyglycolic acid [START_REF] Freed | Neocartilage formation in vitro and in vivo using cells cultured on synthetic biodegradable polymers[END_REF] or fibrin [START_REF] Fortier | Disparate chondrocyte metabolism in three-dimensional fibrin cultures derived from autogenous or commercially manufactured fibrinogen[END_REF] scaffolds and similar or higher initial cellseeding densities. These culture conditions gave proliferation levels similar to those we obtained previously with the same chondrocytes plated at a high cell density (0.8 Â 10 6 cells/cm 2 ) on plastic [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF].

Second, metabolic activities of the chondrocytes were compared, with or without ascorbate, by analysing the expression and synthesis of different collagens and MMPs. With ascorbate, we observed an increase in the expression of genes encoding collagen types I, II and X and MMPs-2 and 13 after normalisation to a reference mRNA (GAPDH) but non-significant increases in global collagen synthesis (dpm/mg DNA) and sulphated glycosaminoglycan content of the sponges. After metabolic labelling of the cultures with 35 S methionine, pepsin treatment and SDS-PAGE, we confirmed the relative increase with ascorbate of type I collagen synthesis with a 2-3-fold increase in the ratio of neosynthesised collagen types I to II. Even though type II collagen remained the main collagen synthesised and present in the sponge (as shown by immunohistology) after 1 month culture with ascorbate, the high amounts of type I collagen produced (up to 35% of total collagen deposited in the sponge and up to 45% of total collagen secreted in the medium) demonstrate the induction of a partial loss of the cartilaginous phenotype by ascorbate. Furthermore, as traces of type X collagen mRNA were detected in cross-linked sponges after 1 month with ascorbate, we confirm here that the gene expression of chondrocytes cultured in a 3D environment may be significantly influenced by ascorbate as previously shown with the same cells [START_REF] Freyria | Regulation of growth, protein synthesis and maturation of foetal bovine chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid and dihydrocytochalasin B[END_REF] or with avian, mouse or bovine chondrocytes [START_REF] Dozin | In vitro differentiation of mouse chondrocytes: requirement for ascorbic acid[END_REF][START_REF] Sullivan | Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion[END_REF][START_REF] Farquharson | Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D[END_REF][START_REF] Venezian | Modulation of chondrocyte proliferation by ascorbic acid and BMP-2[END_REF][START_REF] Daniel | Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III Effects of ascorbate[END_REF][START_REF] Hering | Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension[END_REF] plated at low or high cell densities on plastic. This ascorbate induced maturation was not accompanied by increased synthesis of total collagen and proteoglycan and deposition of a more abundant extracellular matrix within the collagen sponges. It was previously suggested that ascorbate modulation of chondrocyte gene expression was independent of its role in collagen secretion [START_REF] Sullivan | Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion[END_REF].

Matrix metalloproteinases (MMPs) play important roles in the turnover of matrix components in normal cartilage. As several data support the hypothesis whereby matrix degradation in degenerative articular cartilage could be promoted by an imbalance between the levels of MMPs and their inhibitors, we analysed the influence of ascorbate on gene expression and synthesis of MMPs-2, -9 and -13 by chondrocytes in our 3D culture conditions. Ascorbate induced a net increase in both MMP-2 and MMP-13 mRNA, but at the protein level, only MMP-2 was detected by gelatin zymography with an increase in its active form (12-18% of total) whereas MMP-13 activity was undetectable. Parallel induction by ascorbate of collagen X and MMP-2 and -13 mRNA is consistent with a previous report [START_REF] Angelo | MMP-13 is induced during chondrocyte hypertrophy[END_REF] that mature hypertrophic chondrocytes are characterised by high MMP-2 and MMP-13 mRNA. However, although MMP-2 can activate pro-MMP-13 [START_REF] Kn | Cellular mechanisms for human MMP-13 activation-evidence that MT1-MMP (MMP-14) and MMP-2 are able to generate active enzyme[END_REF], its low levels as an active form under our culture conditions, even in the presence of ascorbate, could explain the absence of MMP-13 as an active enzyme.

In conclusion, evidence is provided of an important role for ascorbic acid in the regulation of chondrocyte development and matrix formation during culture in porous 3D collagen sponges. Under these conditions ascorbate stimulates chondrocyte proliferation but does not increase cartilage matrix synthesis (at least after 1 month culture) and progressively alters the phenotypic expression of these cells. Thus further studies to evaluate the potential of various scaffolds and culture conditions (for example the effects of growth factors and/or mechanical stimuli) in preparing functional cartilage implants should take into account, contrary to most recent studies, the presence or absence of ascorbic acid in the culture medium as an important biological parameter.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Effect of ascorbic acid on the proliferation of bovine chondrocytes seeded in collagen sponges. Cells were seeded in either control sponges (H) or in DPPA sponges (D) at one million (10 6 ) and 10 million (10 7 ) cells per sponge. Cell numbers were quantified by measuring the DNA content of the sponges after 7, 13 and 30 days of culture in absence and in the presence of ascorbic acid. Values represent the average levels 7 SEM of three separate experiments. 1po0.07; 11po0.03 and 111po0.01 when untreated cultures were compared with treated cultures.

Fig. 6 .

 6 Fig. 6. Effect of the presence of ascorbic acid throughout the culture on the secretion of MMPs-2 and -9 into the medium by bovine chondrocytes grown in collagen sponges. Cells were seeded at 10 6 and 10 7 per control (H) and DPPA (D) sponges. The sponges were transferred to serum-free medium 48 h before harvesting the conditioned media samples at days 7, 15 and 30. (A) Culture media, harvested on day 30, subjected to gelatin zymography. (B) Histograms showing expression levels in arbitrary units (intensity per cell) of total MMP-2 (pro-MMP-2 and active MMP-2) in culture in the absence (B1) or presence (B2) of ascorbic acid. (C) Histograms showing the percent active MMP-2 compared with total MMP-2 in the culture medium in the absence (C1) or presence (C2) of ascorbic acid.

Fig. 4 .

 4 Fig. 4. Immunohistology of collagen types II and I and glycosaminoglycan staining of native collagen sponges initially seeded with fetal bovine chondrocytes and grown for 1 month with ascorbic acid. (a) immunolabelling with antibodies against type II collagen in sponges initially seeded with 10 7 cells ( Â 150); (b) immunolabelling with antibodies against type I collagen in sponges initially seeded with 10 7 cells ( Â 150). Glycosaminoglycan staining with safranin O, in sponges initially seeded with (c) 10 6 cells and (d) 10 7 cells ( Â 150). Bar=100 mm. Arrow heads indicate the sponge and stars the deposition of GAG.

Fig. 5 .

 5 Fig. 5. Effect of ascorbic acid added throughout the culture on the total sulphated glycosaminoglycan (sGAG) content in collagen sponges. Cells were seeded in either control (H) or in DPPA (D) sponges at 10 6 and 10 7 cells per sponge. sGAG content was measured by a colorimetric method with dimethylmethylene blue. Data are presented as the mean7SD of triplicate samples.

Table 1

 1 Nucleotide sequences of primers used for RT-PCR

	Genes	Primers	Strand	Product size (bp)	Annealing	Reference
					temperature (1C)
	GAPDH	ATCACTGCCACCCAGAAGAC	+	443	57	Bluteau et al. (2001)
		ATGAGGTCCACCACCCTGTT	-
	Col1a1	CACCTACCACTGCAAGAACAG	+	513	45	Personal data
		GAATGCACTTTTGGTTTTTGGTG	-
	Col1a2	GGTTACTACTGGATTGACCC	+	425	55	AB008683
		GCAGCCATCTACAAGAACAG	-
	Col2a1	GATCCGCAACATGGAGACTGGCGA	+	527	75	Personal data
		CAAGAAGCAGACAGGCCCTATGTCCAC	-
	Col10a1	GCAACAGCATTATGACCCA	+	340	51	BTCOL10A1
		CACCAAAGGAAGCCATCG	-
	MMP-2	GGAACAGATCACATACAGG	+	414	51	AF135231
		CACCAAAGGAAGCCATCG	-
	MMP-13	GATGCCATAACCAGTCTCC	+	488	51	AF072685
		GCTGTATTCAAACTGTATGG	-

Primers are presented in the 5 0 -3 0 orientation. No sequences for Col1a1 and Col2a1 are available in the databank; oligonucleotides were designed from sequences determined in our laboratory. GAPDH primers were obtained from the literature.

Acknowledgements

This work was funded by a ''Programme Th! ematique Prioritaire'' (085113) from the Rh # one-Alpes region, a CNRS-INSERM grant ''Ing! enierie Tissulaire'' (2000-03) and two grants from ANVAR (J0011507 V and J0110415 V). The authors thank R Willems and Qin Lu-Wang for expert technical assistance and C van Herrewegge and A Bosch for assistance in preparing the manuscript.