Chondrogenesis of expanded adult human articular chondrocytes is enhanced by specific prostaglandins
M. Jakob1,
O. Démarteau1,
R. Suetterlin2,
M. Heberer1 and
I. Martin1
1 Departments of Surgery and of Research, University Hospital and 2 M. E. Mueller Institute, Biozentrum, University of Basel, Basel, Switzerland.
Correspondence to: M. Jakob, Institute for Surgical Research and Hospital Management, University Hospital Basel, Hebelstrasse 20, ZLF, Room 405, 4031 Basel, Switzerland. E-mail: mjakob{at}uhbs.ch
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Abstract
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Objective. To investigate the effects of the cyclooxygenase-2 (cox-2)-dependent prostaglandins D2 (PGD2), E2 (PGE2) and F2
(PGF2
) on the redifferentiation and cartilage matrix production of dedifferentiated articular chondrocytes.
Methods. Human articular chondrocytes from three adult donors were dedifferentiated by monolayer expansion and induced to redifferentiate by culture as 3D pellets in a defined serum-free medium containing TGF-ß1 and dexamethasone, without or with further supplementation with PGD2, PGE2 or PGF2
. After 2 weeks, pellets were assessed histologically, immunohistochemically, biochemically and by real-time quantitative reverse transcriptasepolymerase chain reaction.
Results. All three PGs, but predominantly PGE2, reduced the staining intensity of pellets for collagen type I, whereas PGD2 and PGF2
increased the staining intensity of pellets for collagen type II and glycosaminoglycans (GAG). The GAG/DNA content of pellets was not affected by PGE2 but was increased 1.5- and 2.1-fold by PGD2 and PGF2
respectively. PGE2 reduced the expression of collagen type I mRNA (9.0-fold), whereas PGD2 and PGF2
increased the mRNA expression of collagen type II (6.2- and 4.1-fold respectively) and aggrecan (29.8- and 10.7-fold respectively).
Conclusion. In contrast to PGE2, PGD2 and PGF2
enhanced chondrogenic differentiation and hyaline cartilage matrix deposition by expanded human articular chondrocytes, and could thus be used to improve in vitro or in vivo cartilage regeneration approaches based on these cells.
KEY WORDS: Tissue engineering, Cartilage repair, Cell differentiation, Cartilage degeneration, Joint inflammation
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Introduction
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Prostaglandins (PGs) are important regulatory factors in normal cartilage metabolism and in the pathogenesis of OA and inflammatory joint disorders [1, 2]. The cyclooxygenase-2 (cox-2) dependent PGE2 was found to be the most abundant PG in the synovial fluid of RA and OA patients and has been demonstrated to mediate joint inflammation and degradation of cartilage matrix components [1, 3]. Interestingly, an alternative set of PGs, namely PGD2 and its metabolites, was recently proposed to have anti-inflammatory properties [4], and to act as a negative feedback loop for joint inflammation and cartilage catabolism [5]. These observations increase scientific interest in the possible differential effects of PGs on chondrogenesis, with potential implications in the treatment of cartilage pathologies.
So far, studies investigating the effects of PGs during in vitro chondrogenesis have been mostly limited to the use of PGE2, and have reported varied and sometimes contradictory results [69], probably due to the variety of model systems employed. In the few available investigations, other PGs have been reported to have beneficial short-term effects on chondrocytes. In particular, PGF2
stimulated aggrecan synthesis in a rat chondrocyte cell line [10], promoted type II collagen mRNA expression and counteracted its IL-1ß-induced suppression in human chondrocytes [11]. In addition, PGD2 metabolites, such as 15-deoxy-
12,14-prostaglandin J2 (PGJ2), decreased IL-1ß-induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes [12]. In the present study, we investigated the effects of prolonged exposure to PGs on human chondrocyte differentiation, using an in vitro model system which mimics some of the processes occurring during in vivo cartilage degeneration and repair.
The model system identified for this work consists in the redifferentiation in 3D pellet cultures of monolayer expanded, dedifferentiated human articular chondrocytes. The choice of the model system has been motivated by the facts that (i) dedifferentiated articular chondrocytes assume a prechondrogenic phenotype, with a gene expression profile resembling that of OA chondrocytes [13, 14]; (ii) during the redifferentiation of expanded chondrocytes, an initially fibrocartilaginous tissue matures into a hyaline-like cartilaginous structure [15, 16], thus resembling the remodelling process reported during cartilage repair following autologous chondrocyte implantation [17]; (iii) chondrocyte redifferentiation in pellet cultures can be induced in a defined serum-free medium containing dexamethasone [16, 18], which is a selective cox-2 inhibitor [19]; therefore, in this model only basal concentrations of PGs, necessary for normal physiological processes [1], are expected to be produced through the constitutive expression of cox-1. Using the described system as a model of chondrogenesis, we tested the hypothesis that long-term exposure to PGD2 and PGF2
, but not to PGE2, will stimulate in vitro redifferentiation and cartilage tissue development by dedifferentiated human articular chondrocytes.
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Materials and methods
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Culture of adult human articular chondrocytes
Cartilage specimens
Cartilage samples were collected from post-mortem knee articular cartilage of three adult human males (mean age 54.5, range 4862 yr old) with no history of joint disease, after informed consent by relatives and in accordance with the local ethics committee (University Hospital Bern, Switzerland). Mankin scores of the collected tissues were below levels generally associated with an overt degenerative disease (average, 2.9±1.3). Full-thickness cartilage samples were removed from the femoral condyle or tibial plateau within 24 h after donor death and processed as described below.
Cell isolation and monolayer expansion
Chondrocytes were isolated by digestion with 0.15% type II collagenase for 22 h and expanded in monolayer cultures for two passages, as described previously [18]. Throughout the expansion phase, cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 4.5 mg/ml D-glucose, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mM HEPES buffer, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.29 mg/ml L-glutamine, further supplemented with 1 ng/ml transforming growth factor ß1 (TGF-ß1), 5 ng/ml fibroblast growth factor 2 (FGF-2) and 10 ng/ml platelet-derived growth factor-bb (PDGFbb) (all from R&D Systems, Abingdon, UK). This combination of growth factors used during human articular chondrocyte expansion was previously reported to markedly increase not only cell proliferation rate and dedifferentiation, but also the capacity of the expanded cells to redifferentiate when transferred to a 3D culture system [18]. During expansion, chondrocytes underwent a total of seven to nine doublings; doubling time averaged 18.3±1.8 h.
Cell cultivation in pellets
Expanded chondrocytes were suspended in a chemically defined chondrogenic serum-free medium consisting of DMEM supplemented with ITS+1 (Sigma Chemical, Buchs, Switzerland; 10 µg/ml insulin, 5.5 µg/ml transferrin, 5 ng/ml selenium, 0.5 mg/ml bovine serum albumin, 4.7 µg/ml linoleic acid), 0.1 mM ascorbic acid 2-phosphate, 107 M dexamethasone, 10 ng/ml TGF-ß1 and 1.25 µg/ml human serum albumin [16]. Aliquots of 5 x 105 cells in 0.5 ml of medium were centrifuged in polypropylene conical tubes (Sarstedt, Numbrecht, Germany) to form spherical pellets, which were placed onto a 3D orbital shaker (Bioblock Scientific, Frenkendorf, Switzerland) at 30 r.p.m. in a humidified incubator at 37°C, 5% CO2. Using this system, we previously reported that medium supplementation with TGF-ß1 is crucial for chondrocyte redifferentiation, although cells still express high levels of collagen type I and the resulting tissues do not fully reach the typical properties of hyaline cartilage [16]. Pellets were cultured in the serum-free medium, without (control) or with daily supplementation with 3.0 µM PGD2, PGE2 or PGF2
. The specific concentration was selected in the range of the highest levels of activity reported in previous doseresponse studies [10, 12]. After 2 weeks, pellets were processed for histological, immunohistochemical, biochemical or mRNA analysis as described below. Each analysis was performed independently in at least two entire pellets for each primary culture and cultivation condition.
Analytical methods
Histological and immunohistochemical analyses
Cell pellets were embedded in paraffin and cross-sectioned (5 mm thick). For histological evaluation, sections were stained with safranin-O for sulphated glycosaminoglycans (GAG). For immunohistochemical analysis, sections were double-labelled with antibodies against collagen type I (T40111R; Biodesign International, Saco, ME, USA) and type II (II-II6B3; Developmental Studies Hybridoma Bank, Baltimore, MD, USA), followed by secondary antibodies conjugated respectively with Alexa 488 (Molecular Probes, Eugene, OR, USA) and Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA, USA). Specimens were imaged by acquiring optical sections in parallel in the 488 and 568 nm channels, using a confocal laser scanning microscope (TCS 4-D CLSM; Leica AG, Heidelberg, Germany).
Biochemical analyses
Chondrocyte pellets were digested with protease K (0.5 ml of 1 mg/ml protease K in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 µg/ml pepstatin-A for 15 h at 56°C). GAG content was measured spectrophotometrically using dimethylmethylene blue, with chondroitin sulphate as a standard, and normalized to the DNA amount, measured spectrofluorometrically using the CyQuant® Kit (Molecular Probes), using calf thymus DNA as a standard.
Real-time quantitative reverse transcriptasepolymerase chain reaction (RT-PCR) assays
RNA was extracted from pellets using TRIzol (Life Technologies, Basel, Switzerland), treated with DNase I using the DNA-free kit (Ambion, Austin, TX, USA), and used to generate cDNA as described previously [18]. PCRs were performed and monitored using a ABI Prism 7700 Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland), using a multiplex approach (Perkin-Elmer User Bulletin N. 2) with 18S ribosomal RNA as reference gene. The assays were used to quantify the mRNA expression of collagen type II and aggrecan (typical markers of differentiated chondrocytes in hyaline cartilage), and of collagen type I and versican (typical markers of dedifferentiated chondrocytes). The sequences of primers and probes and the PCR operating conditions have been described previously [18, 20]. For each cDNA sample, the threshold cycle (Ct) value of each target sequence was subtracted from the Ct value of the reference gene, to derive
Ct. The level of expression of each target gene was then calculated as 2
Ct. Each sample was assessed at least twice for each gene of interest.
Statistical analysis
Unless otherwise stated, all experiments were performed at least three times with cells from different donors. In order to better identify common trends in the different experiments, quantitative data obtained using cells from different donors were normalized to the average values of the corresponding control condition (i.e. without supplementation with a PG). Values are presented as mean ± S.D. Differences among experimental groups were assessed with non-parametric MannWhitney tests. A P value of <0.05 was considered to indicate a statistically significant difference.
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Results
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Histological and immunohistochemical analyses
Medium supplementation with PGD2 or PGF2
induced the formation of a cartilaginous matrix more intensely stained by safranin-O compared with the control condition (Fig. 1). Pellets cultured in the presence of PGF2
(Fig. 1D) adopted a more rounded shape, typically associated with the differentiated chondrocyte phenotype. The extracellular matrix of pellets cultured in medium supplemented with PGE2 was poorly stained by safranin-O (Fig. 1C).

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FIG. 1. Histological appearance of pellets based on expanded human articular chondrocytes and redifferentiated in control medium without supplementation (A) or supplemented with PGD2 (B), PGE2 (C) or PGF2 (D). Sections were stained with safranin-O for glycosaminoglycans (GAG). Scale bars = 100 µm.
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Immunohistochemical analysis (Fig. 2) revealed that chondrocytes cultured in the presence of PGD2 and PGF2
deposited high amounts of both collagen types I and II. In these pellets, staining for collagen I was slightly weaker than in control pellets (Fig. 2B, I and D, II), whereas staining for collagen II was much stronger and uniform (Fig. 2B, II and D, II). Low staining intensities for both collagens were detected in pellets cultured with PGE2 and were mostly localized at the periphery of the tissues (Fig. 2C).

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FIG. 2. Immunostaining for collagen type I (I) and type II (II), and overlay of the two channels (III) for pellets based on expanded human articular chondrocytes and redifferentiated in control medium without supplementation (A) or supplemented with PGD2 (B), PGE2 (C) or PGF2 (D). Scale bar = 100 µm.
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Biochemical analyses
Within each experiment, the DNA content was similar in all culture conditions (values obtained from all experimental groups averaged 4.6 ± 1.2 µg). All values of GAG deposited in the pellets were thus normalized to the DNA content (GAG/DNA). The amount of GAG/DNA accumulated in pellets cultured in the presence of PGD2 or PGF2
was higher (respectively 1.5- and 2.1-fold) than in pellets cultured in control medium, although the difference was statistically significant only for PGF2
(P = 0.01).
Real-time RT-PCR assays
The expression levels of the genes analysed were differentially affected by the PGs (Fig. 3). PGE2 induced a 9.0-fold decrease (P<0.001) in the mRNA expression of collagen type I. PGD2 and PGF2
significantly increased the mRNA expression of collagen type II (PGD2, 6.2-fold, P<0.03; PGF2
, 29.8-fold, P<0.01) and of aggrecan (PGD2, 4.12-fold, P<0.02; PGF2
, 10.7-fold, P<0.03). Versican gene expression was slightly increased by the three PGs but only the increase induced by PGE2 was statistically significant (3.22-fold, P<0.04).

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FIG. 3. Levels of mRNA expression of collagen type I, collagen type II, aggrecan and versican in pellets based on expanded human articular chondrocytes and redifferentiated in medium without supplementation (control) or supplemented with PGD2, PGE2 or PGF2 . In order to better identify common trends using cells from different donors, for each experiment all values were normalized to those of the corresponding control condition. *Statistically significant difference from the control condition.
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Discussion
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In the present study, we demonstrated that PGs differentially modulated redifferentiation and cartilage tissue development by culture-expanded human articular chondrocytes. Specifically, PGE2 induced a decrease in the mRNA expression and deposition of collagen type I (Figs 3A and 2C, I), which paralleled a more rounded (less fibroblastic) cell morphology but not increased deposition of cartilage specific extracellular matrix (Figs 1C and 2C, II). PGD2 and PGF2
enhanced the process of chondrogenic differentiation, as assessed by the increase in the mRNA expression of collagen type II and aggrecan (Fig. 3B, C). Moreover, both PGs yielded tissues more intensely stained for collagen type II (Fig. 2B, II and D, II) and GAG (Fig. 1B, D), although only PGF2
induced a statistically significant increase in the biochemically measured amount of accumulated GAG.
The enhancement of chondrogenesis and cartilage tissue formation by the cox-2-dependent PGD2 and PGF2
appeared to be mediated by the up-regulation of extracellular matrix genes, although the process could have been supported by a concomitant reduction in catabolic events (e.g. decreased expression of matrix metalloproteinases) [12]. Given the fact that changes in the chondrocyte phenotype in our model system resemble some aspects of in vivo cartilage repair following degeneration, our results indicate possible beneficial effects of specific PGs during the progression of cartilage disorders, not only by reducing inflammation [5] but also by stimulating cell differentiation and extracellular matrix deposition, thus promoting appropriate cartilage repair [11]. This highlights the potential limits of therapeutic interventions for joint diseases that reduce the production of all PGs for prolonged periods by continuous application of cox-2 inhibitors [21], and may help to explain the lack of long-term disease-modifying activity in patients receiving NSAIDs (selective or non-selective cox-2 inhibitors) for painful arthropathies [4].
In the clinical setting, grafting of dedifferentiated articular chondrocytes, either as cell suspensions (i.e. autologous chondrocyte implantation) or loaded into porous polymeric scaffolds [22], is currently being used to promote the healing of joint surface defects. The finding that certain PGs support cell redifferentiation and cartilage tissue formation by dedifferentiated human articular chondrocytes prompts further investigation of the possible combination of autologous chondrocyte implantation with a molecular therapy (i.e. the delivery in the joint of specific PGs) and on the potential use of PGs as medium supplements for the in vitro generation of engineered cartilage grafts.
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Acknowledgments
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We are grateful to Dr P. Mainil-Varlet for provision of human cartilage specimens. This work was supported by the Department of Surgery, University Hospital Basel.
The authors have declared no conflicts of interest.
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Submitted 5 February 2004;
revised version accepted 16 March 2004.