Redifferentiation of dedifferentiated human chondrocytes in high ...

Cell Tissue Res (2002) 308:371–379 DOI 10.1007/s00441-002-0562-7

REGULAR ARTICLE

G. Schulze-Tanzil · P. de Souza · H. Villegas Castrejon T. John · H.-J. Merker · A. Scheid · M. Shakibaei

Redifferentiation of dedifferentiated human chondrocytes in high-density cultures Received: 10 January 2002 / Accepted: 26 March 2002 / Published online: 18 May 2002 © Springer-Verlag 2002

Abstract High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting about 3 days) in monolayer culture. At each passage, monolayer cells were removed and allowed to grow in high-density cultures at the medium-air interface and subsequently investigated with morphological, immunolocalization and biochemical methods for the production of cartilage-specific markers, i.e. collagen type II and cartilage-specific proteoglycans. When such dedifferentiated chondrocytes from monolayer passages P1–P4 were introduced in high-density culture, they regained a chondrocyte phenotype and formed cartilage nodules surrounded by fibroblast-like cells. Cells were interconnected by typical gap junctions and after a few days in culturing produced cartilage-specific extracellular matrix, notably collagen type II and cartilage-specific proteoglycans. In contrast, cells taken from monolayer passages P5–P8 did not produce these chondrocyte-specific extracellular materials when grown in high-density culture. We conclude that the growth of dedifferentiated chondrocytes in high-density This work was supported by the Deutsche Forschungsgemeinschaft (DFG grants Sh 48/2–4, Sh 48/2–5) G. Schulze-Tanzil · P. de Souza · H. Villegas Castrejon H.-J. Merker · M. Shakibaei (✉) Institute of Anatomy, Freie Universität Berlin, Königin-Luise-Strasse 15, 14195 Berlin, Germany e-mail: [email protected] T. John Univ. Med. Ctr. Benjamin Franklin, Department for Trauma Surgery, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany A. Scheid Department of Surgery, University Childrens’ Hospital of Zürich, Zürich, Switzerland

culture promotes their redifferentiation and reveals their chondrogenic potential. Such high-density cultures might serve as a model system to initiate and promote the redifferentiation of chondrocytes and to provide sufficient quantities of differentiated chondrocytes for autologous chondrocyte transplantation. Keywords Chondrocyte · High-density cultures · Redifferentiation · Dedifferentiation · Collagen type II · Human

Introduction The acquisition of human differentiated chondrocytes for transplantation and cartilage coverage presents a major problem as these cells dedifferentiate rapidly during proliferation in monolayer culture (for mammalian chondrocytes in general, see Kuettner et al. 1982a, 1982b; Shakibaei 1995; Shakibaei et al. 1997). Dedifferentiated chondrocytes change their shape as well as their metabolic state and their program of matrix biosynthesis (Benya and Shaffer 1982; Watt 1988; Shakibaei 1995; Shakibaei et al. 1997; Martin et al. 1999). Generally, downregulated synthesis of cartilage-specific markers such as collagen type II and cartilage-specific proteoglycans (CSPG) and changes in their cell surface receptor complement are seen as signs of chondrocyte dedifferentiation (Benya and Shaffer 1982; Shakibaei 1995). After prolonged monolayer culture, dedifferentiated chondrocytes proliferate but appear to lose their chondrogenic potential irreversibly (Martin et al. 1999). High-density cultures promote chondrogenic differentiation since they support cell-cell interactions (Zimmermann et al. 1992; Denker et al. 1999; Haas and Tuan 1999). These interactions are mediated by cell adhesion molecules such as N-cadherin and by integrins (Shakibaei 1998; Haas and Tuan 1999) and also play an important role in cell condensation during chondrogenesis, which precedes conversion of cells to prechondroblasts (Hall and Miyake 1995; Loty et al. 2000). High-density cul-

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tures have also been recommended as convenient models for studying chondrogenesis and enchondral ossification since under these conditions mesenchymal cells of mouse limb buds differentiate into chondrocytes in vitro (Zimmermann et al. 1992; Mello and Tuan 1999; Kulyk et al. 2000). Furthermore, as a possible way for intercellular communication by exchange of small molecules, gap junctions can be observed in such chondrocytes during differentiation (Loty et al. 2000) and also in high-density cultures of mouse limb bud mesenchymal cells as specific contacts (Zimmermann et al. 1992). Since cartilage is avascular and mature chondrocytes do not proliferate, cartilage lesions do not possess the capacity to heal. Autologous chondrocyte transplantation (ACT) is a relatively new therapeutic approach to covering full-thickness articular cartilage defects by in vitro grown chondrocytes from the same joint of the same patient (autologous). These chondrocytes are taken by a biopsy specimen, cultured and multiplied for about 3 weeks in vitro in monolayer culture. The cultured chondrocytes are then injected under a periosteal flap sutured over the defect of the articular cartilage (Brittberg et al. 1994). The success of ACT correlates with the capacity of transplanted chondrocytes to produce a hyaline matrix containing collagen type II (Peterson et al. 2000). The present study demonstrates that proliferation of alginate-derived chondrocytes in monolayer culture and redifferentiation in high-density culture would be useful as a means to obtain a sufficiently high density of chondrocytes for ACT. Furthermore chondrocytes that emigrate from alginate beads may be cultured for as long as four passages in monolayer culture without losing their ability to redifferentiate in high-density culture and remain available for ACT for up to 2 weeks or longer. We have found that these redifferentiated chondrocytes produce collagen type II and cartilage-specific proteoglycans that are indistinguishable from that of primary chondrocytes.

Materials and methods Antibodies Monoclonal and polyclonal antibodies against collagen type II (MAB1330; AB746), monoclonal antibody against cartilage-specific proteoglycan (MAB2015), alkaline phosphatase-conjugated sheep anti-mouse (AP303A) and sheep against rabbit (AP304A) secondary antibodies for immunoblotting were purchased from Chemicon International Inc. (Temecula, CA). Dual-system-APAAP complex was obtained from Dianova (Hamburg, Germany). Growth medium [Hams F-12/DMEM (50/50) containing 10% fetal calf serum, 25 µg/ml ascorbic acid, 50 IU/ml streptomycin, 50 IU/ml penicillin, 1% gentamicin, 1% essential amino acids and 2.5 µg/ml amphotericin B] was obtained from Seromed (Munich, Germany). Epon and LR-white (London resin) were obtained from Plano (Marburg, Germany). The secondary gold-labeled anti-bodies were purchased from Amersham (Brunswick, Germany). OCT compound embedding medium for frozen tissue specimen, trypsin/EDTA (EG 3.4.21.4) and collagenase were purchased from Sigma (Munich, Germany), pronase from Roche (Mannheim, Germany) and Kaiser’s glycerol gelatin from Merck (Darmstadt, Germany).

Chondrocyte culture Primary cultures of human chondrocytes were prepared from articular cartilage of femoral heads obtained during joint replacement surgery for femoral neck fractures as described (Shakibaei et al. 1999, 2001). Briefly, cartilage was cut into small slices, incubated and washed in medium before slices were digested first with 1% pronase (2 h at 37°C) and then with 0.2% (v/v) collagenase (4 h at 37°C). The cells were suspended in growth medium and homogeneously distributed by repeated pipetting. The cells (2×106 cells/ml) were cultivated in alginate beads (Shakibaei and de Souza 1997). After a few days of culture, cells continuously emigrated from alginate beads and adhered to Petri dishes. Three days later, when cell density had approached confluence, these cells were removed using 0.05% trypsin/1.0 mM ethylenediaminetetraacetic acid (EDTA) and replated in culture flasks. Every 3 days the confluent monolayer culture cells were passaged until passage P8. Cells from each monolayer passage were allowed to grow in high-density cultures as described in detail by Zimmermann et al. (1992) and Shakibaei et al. (1999). Briefly, cells were washed twice in growth medium and pelleted by centrifugation (600 rpm for 10 min). Ten microliters of the cell sediment (~2×106 cells/10 µl) was pipetted onto a membrane filter with a pore diameter of 0.2 µm (Sartorius, Göttingen, Germany) on top of a stainless steel grid at the medium-air interface in a Petri dish. Cultures were grown at 37°C in a humidified atmosphere with 5% CO2 and medium was changed every 3 days. Alcian blue staining The high-density cultures were fixed in toto with 3.7% formaldehyde and stained with 0.05% Alcian blue in 3% acetic acid (pH 1.5) containing 0.3 M MgCl2 for 24 h (Scott and Dorling 1965). The fixed cultures were then washed in 5% acetic acid, followed by gradual dehydration in ethanol and xylene. Then preparations were examined using a light microscope (Axiophot 100, Zeiss, Germany). Immunoblotting Immunoblotting was performed as described (Shakibaei et al. 1999, 2001). Cell proteins were extracted with lysis buffer (50 mM TRIS/HCl, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 µg/ml pepstatin A, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, PMSF) on ice for 30 min. Total protein concentration of samples was adjusted before separating them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (7.5% gels) under reducing conditions. Separated proteins were transferred onto nitrocellulose membranes. Membranes were incubated in blocking buffer [5% (w/v) skimmed milk powder in phosphatebuffered saline (PBS)/0.1% Tween 20] for 1 h and incubated with primary antibodies for 1 h at room temperature (RT). After thorough washing the samples were incubated with alkaline phosphatase conjugated secondary antibodies for 30 min. Membranes were first washed in blocking buffer and then 3 times in 0.1 M TRIS, pH 9.5, containing 0.05 M MgCl2 and 0.1 M NaCl. Specific antigen-antibody complexes were revealed using nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate (p-toluidine salt; Pierce, Rockford, IL) as substrates for alkaline phosphatase. Protein determination was done with the bicinchoninic acid system (Pierce) using bovine serum albumin (BSA) as a standard. Alkaline phosphatase/anti-alkaline phosphatase (APAAP) method The cultures were immersed in OCT embedding medium and immediately frozen in liquid nitrogen. Eight-micrometer-thick sections were cut. Sections of the high-density cultures were

373 Fig. 1 Light-microscopic demonstration of chondrocytes grown in monolayer from passage P1 and stained with methylene blue. Two cell populations can be distinguished: a group of large round, chondrocytelike cells and a group of polymorphic bipolar, reticular or star-shaped fibroblast-like cells. ×240

fixed with acetone (10 min) and washed twice (5 min) in TBS (0.05 M TRIS, 0.15 M NaCl, pH 7.6) at RT. Sections of cultures were overlaid with serum (1:20) in TBS (RT, 10 min) and incubated with primary antibodies (1:30) in a moist chamber overnight at 4°C. They were rinsed several times in TBS, incubated with mouse antibodies to rabbit IgG (1:50) for 30 min at RT and incubated with dual system bridge antibodies (1:50). Before and after incubation with the dual-system APAAP complex (1:50) for 30 min at RT, cells were thoroughly washed and stained with new fuchsin for 30 min at RT. Cultures were washed, air dried, covered with Kaiser’s glycerol gelatin and examined with a light microscope (Axiophot 100, Zeiss, Germany). Transmission electron microscopy After fixation and postfixation in 1% tannic acid (0.1 M phosphate buffer) and 1% OsO4 solution (0.1 M phosphate buffer), cartilage high-density cultures were rinsed and dehydrated in ascending alcohol series. They were embedded in Epon, cut on a Reichert Ultracut, followed by contrasting with 2% uranyl acetate/lead citrate. A transmission electron microscope (TEM 10, Zeiss, Germany) was used to examine the cultures. Immunoelectron microscopy A detailed description of the culture technique used for the following experiments has already been published (Shakibaei 1995, 1998). After fixation in 3% formaldehyde freshly prepared from paraformaldehyde plus 0.25% glutaraldehyde in PBS for 1 h, the cultures were washed with PBS/1% BSA, dehydrated in ethanol and embedded in LR-white. Ultrathin sections were cut and treated with the following solutions: (1) 1% BSA at RT for 30 min; (2) testicular hyaluronidase (5,000 U/ml) for 5 min at RT to unmask epitopes; (3) PBS/1% BSA/0.5% Tween 20 2×5 min at RT; (4) primary antibodies (1:50 in PBS/1% BSA/0.5% Tween 20) overnight at 4°C; (5) PBS/BSA/Tween for 2×5 min at RT; (6) secondary antibody conjugated with goat anti-rabbit immunoglobulin with 10-nm gold particles (1:50 for 30 min) at RT. (7) After rinsing for 2×5 min at RT, (8) contrasting was carried out with 1% tannic acid for 20 min at RT, with OsO4 for 10 min and with 2% uranyl acetate for 30 min. Finally, the sections were rinsed and examined under a transmission electron microscope (TEM 10, Zeiss, Germany).

Results Light microscopy Cell culture After a few days of culture some cells emigrated from the alginate beads and adhered to the culture dish, hence forming a monolayer (P1). This monolayer (Fig. 1) was passaged every 3 days until passage P8. The emigrated cells consisted of two morphologically distinct cell populations: a group of large and round cells with a large euchromatic nucleus, several nucleoli and small vacuoles and a group of morphologically heterogeneous small fibroblast-like cells that had a polymorphic cell shape: varying from round or bipolar, to reticular or star shaped (Fig. 1). Alcian blue staining Alcian blue binds to anionic groups of macromolecules and selectively stains cartilage-specific matrix. Cells, which had been grown for less than five passages in monolayer, were re-cultured in high-density culture and these survived and synthesized CSPG-rich matrix as revealed by Alcian-blue-stained cartilage nodules (Fig. 2A–D). Cells from monolayer passages P5–P8 did not redifferentiate after transition into high-density culture. Alcian blue staining of these cells remained negative (data not shown). Alkaline phosphatase/anti-alkaline phosphatase (APAAP) method Cells from monolayer passages P1–P4 were recultivated for 1, 3, 4, 5, 7 and 9 days in high-density culture and

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Fig. 2 Chondrocytes from monolayer passage (P3) recultivated for 3 (A), 5 (B), 7 (C) and 9 (D) days in high-density culture were stained with Alcian blue. After 3 days, faintly blue stained areas appeared. After about 7 days, the round cells were surrounded by Alcian-blue-positive matrix (A–D). ×160

immunolabeled with anti-collagen type II (Fig. 3A–F) and -CSPG antibodies (data not shown). On day 3, collagen type II and CSPG labeling was clearly positive within the nodules of cultures and increased until day 9 (Fig. 3A–F). Cells from the internodular space remained collagen type II- and CSPG-negative. Cells from monolayer passages P5–P8 remained collagen type II- and CSPG-negative for the entire culture period in high-density culture (data not shown).

Collagen fibrils became thicker and the width of intercellular spaces and the quantity of extracellular matrix increased (Fig. 4D). The matrix was closely attached to the cell membrane. On day 7 (Fig. 4E), cells appeared like typical chondrocytes, mainly round or ellipsoid with small cuspidal processes and large cell organelles. Glycogen granula were visible. High-density cultures resulting from monolayer passages P5–P8 differed from the above. These cultures exhibited mainly cell debris, dying cells and a few fibroblast-like cells. On days 1–3 of culture, cell organelles were swollen, and fragmentation of cell membranes and lipid vacuoles were visible. In addition to these findings, we observed reduced matrix with matrix-free pericellular areas around the cells (Fig. 4F). Immunoelectron microscopy

Transmission electron microscopy Chondrocytes from monolayer passages P1–P4 recultivated in high-density cultures exhibited the following characteristics: During the first 3 days of culture, cells were densely packed and formed tight contacts (Fig. 4A). On day 2 of culture, intercellular spaces between cells widened (Fig. 4B). After 2–3 days, the first signs of an extracellular matrix appeared: small, singularly and irregularly running fibrils in the intercellular spaces appeared near the cell membrane. Organelles of some cells became increasingly visible and were identifiable as rough endoplasmic reticulum (rER), vacuoles and mitochondria (Fig. 4B, C). On day 5 of culture, cells had lost most of their contacts.

When cells from monolayer passages P1–P4 were recultivated in high-density culture and immunolabeled with anti-collagen type II (Fig. 5A) and -CSPG (Fig. 5B) gold-linked antibodies, immunomorphological studies at the electron-microscopic level revealed gold particles distributed throughout the typical cartilage matrix in the nodules. Collagen type II immunogold-labeling (A) was evenly distributed on the irregularly running fibrils of extracellular cartilage matrix. Anti-CSPG-labeled gold particles (B) formed clusters distributed irregularly in the matrix. In contrast, in dedifferentiated cells from P5–P8 of monolayer, no specific immunostaining was detectable (data not shown).

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Fig. 3 Cells from monolayer passage (P3) recultivated for 1 (A), 3 (B), 4 (C), 5 (D), 7 (E) and 9 (F) days in high-density culture were immunolabeled with anti-collagen type II antibody. On day 3 (B), collagen type II labeling was positive in the nodules of highdensity cultures and increased until day 9 (F). Cells from internodular space remained collagen type II-negative. ×160

Immunoblotting Western blotting was used to examine the synthesis of cartilage-specific extracellular matrix proteins in chondrocyte monolayer P1–P8. Cells from monolayer passages P1–P4 produced collagen type II (Fig. 6) and CSPG (data not shown). In cells from monolayer passages P5–P6, immunolabeling of collagen type II and CSPG was barely detectable and not detectable in later passages.

Discussion The major findings of the present study are as follows: the emigrated cells from alginate beads were passaged up to 8 times and monolayer cells from each passage were removed and allowed to grow in high-density cultures, (1) until the fourth monolayer passage such dedifferentiated chondrocytes were able to redifferentiate in high-density culture producing cartilage-specific markers. (2) Cells from monolayer passages P5–P8 did not redifferentiate in high-density culture. These cells remained fibroblast-like or underwent cell death and did not produce any cartilage-specific markers. (3) Morphological features of redifferentiation (cells from P1–P4) were similar to those seen during chondrogenesis of limb bud mesenchymal cells.

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Fig. 4 Electron-microscopic demonstration of recultivated chondrocytes (C) in high-density culture from passage (P3) after 1 (A), 2 (B), 3 (C), 5 (D), 7 (E) days and from passage (P5) after 3 (F) days. On day 1 fibroblast-like cells lay in close contact together (triangle), on day 2 intercellular spaces began to widen (*) and extracellular matrix consisting of thin irregularly running fibrils was visible (arrows). During the next few days, an increasing number of typically round/oval chondrocytes (C) with small pro-

cesses embedded in a network of extracellular matrix fibrils (M) developed after recultivation in high-density culture. Cells contained a well-developed rER, mitochondria, other cell organelles, vacuoles and granules. High-density cultures resulting from monolayer passage (P5) (F) showed swollen cell organelles, fragmentation of cell membrane and necrotic cells with reduced extracellular matrix (M) and matrix-free pericellular areas (*). ×5,000 (A–F)

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Fig. 5 Immunoelectron-microscopic studies of recultivated chondrocytes (C) in high-density culture from passage (P3) after 7 days with immunogold-labeled anti-collagen type II (A) or anti-cartilage-specific proteoglycan antibodies (B). Anti-collagen type II immunogold-labeling (A) was evenly distributed on irregularly running fibrils of extracellular matrix (arrows). Antibodies to cartilage-specific proteoglycan-labeled gold particles (B) formed clusters distributed irregularly in the extracellular matrix and on the cell surface (arrows). ×10,000 (A, B)

One problem with autologous chondrocyte transplantation (ACT) is attaining sufficient quantities of differentiated chondrocytes (usually 4.5×106; Peterson et al. 2000) to cover cartilage lesions and at the same time avoid irreversible dedifferentiation of these cells during prolonged passages in monolayer cultures. In alginate culture, only prechondrogenic mesenchymal cells (cells with a chondrogenic potential) survive and differentiate to chondrocytes (Shakibaei and de Souza 1997). Chondrocytes that emigrated from alginate

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Fig. 6 Collagen type II synthesis in chondrocytes from monolayer passages P1–P8 demonstrated by Western blotting. Cells from monolayer passages P1–P4 revealed clear synthesis of collagen type II. In the fifth monolayer passage (P5) there was weak collagen type II production. Cells from P6–P8 were collagen type II negative. The arrow in the right margin indicates the relative position of the collagen type II α-chain [α1(II)]

beads therefore present a pure chondrocyte population when introduced into monolayer culture. Cells from monolayer passages P1–P4, as opposed to cells from P5–P8, redifferentiated into chondrocytes after transfer to high-density culture, and produced cartilage-specific matrix, i.e., collagen type II and cartilage-specific proteoglycans. On the other hand, we cannot exclude the presence of other cartilage-specific markers in this chondrocyte culture. Dedifferentiated cells from monolayer passages P5–P8 were not able to redifferentiate in high-density cultures and, as observed in another study, were not able to redifferentiate in alginate cultures (unpublished observations: Schulze-Tanzil and coworkers). These cells remained fibroblast-like or underwent cell death and did not produce any cartilage-specific markers, either in monolayer culture or in high-density culture. Firm cell-cell contacts play an important role during the first phase of chondrocyte differentiation in chondrogenesis, a phase known as the cell condensation phase (Denker et al. 1999; Loty et al. 2000). This phase also occurs at the early stages of high-density culture. Redifferentiation of dedifferentiated chondrocytes in high-density cultures closely resembles primary chondrogenic differentiation of mesenchymal cells, which can be simulated in vitro by culturing mouse limb buds in high-density culture (Zimmermann et al. 1992; Loty et al. 2000). Mesenchymal cells from mouse limb buds lost their chondrogenic potential after only 3 or 4 days in monolayer culture (Zimmermann et al. 1992). Loss of chondrogenic potential in chondrocytes from frequently passaged monolayer cultures has also been described by other authors (Martin et al. 1999). In the present study chondrocytes that emigrated from alginate beads maintained their chondrogenic potential throughout four passages (about 2 weeks) in monolayer culture. However, the reason for the loss of chondro-

genic potential in chondrocytes is still unknown at present. It has been observed that growth factors such as FGF-2 and BMP-2 which can promote (re)differentiation (Denker et al. 1999; Martin et al. 1999) increase the expression of transcription factor sox9 (EnomotoIwamoto et al. 1998; Healy et al. 1999; Zehentner et al. 1999). Activation of sox9 gene seems to be mediated by mitogen-activated protein kinase (MAPK) pathway (Murakami et al. 2000; de Crombrugghe et al. 2000). Furthermore, recent studies have reported that specific inhibition of the MAPK pathway leads to apoptosis of human chondrocytes in vitro (Shakibaei et al. 2001). Sox9 promotes the expression of chondrocyte marker genes (Zehentner et al. 1999) and is required for the initiation of cartilage differentiation during embryogenesis. The gene sox9 was found to be activated in chondrogenic condensations during chondrogenesis in mice (Wright et al. 1995; Ng et al. 1997; Healy et al. 1999) and might also control expression of cell surface proteins essential for mesenchymal condensations (de Crombrugghe et al. 2000). Sox9 is also expressed in high-density cultures (Kulyk et al. 2000) and may be important for redifferentiation of dedifferentiated chondrocytes. We can assume that loss or modification of cellmatrix interactions may be a reason for loss of chondrogenic potential (Chen et al. 1995). Cell-matrix interaction is mediated by specific receptors, e.g., integrins (Dürr et al. 1993; Enomoto et al. 1993; Shakibaei et al. 1997, 1999; Mobasheri et al. 2002). Integrins play a crucial role in early cartilage differentiation, since the presence of anti-integrin antibodies is able to inhibit differentiation of blastemal cells to chondroblasts (Shakibaei 1998). During dedifferentiation in monolayer culture, and also in ageing cartilage, chondrocytes switch from producing collagen type II to type I and synthesis of cell surface receptors (integrins) changes from α3- to α1integrin (Shakibaei et al. 1993, 1995). During early chondrogenesis there is a switch from collagen type I to type II and from cell surface α1- to α3-integrin (Shakibaei et al. 1995). High-density cultures are a suitable in vitro system to promote and further investigate chondrocyte redifferentiation. In high-density cultures redifferentiated chondrocytes remain available in a differentiated state for up to 2 weeks and may be used for ACT. We propose here a model system for ACT: (1) the alginate culture allows the “storage” of chondrocytes for a few days or weeks before (2) their multiplication in monolayer culture, (3) followed by redifferentiation in high-density culture revealed by their ability to synthesize collagen type II and CSPG. With regard to ACT the ongoing production of collagen type II by transplanted chondrocytes is of major importance because the collagen type II fibers (unlike collagen type I fibers) stabilize the macromolecular framework of the new ECM in the cartilage lesions (Brittberg et al. 1994).

379 Acknowledgement The authors are indebted to Dr. A. Mobasheri and Dr. S. Carter (Connective Tissue Research Group, Liverpool, UK) for reviewing the manuscript. Mrs. Angelika Hartjie’s – Mrs. Angelika Steuer’s and Mr. Jörg Romahn’s technical assistance is gratefully acknowledged.

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