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Cell Biology International (2006) 30, 288–294 (Printed in Great Britain)
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A novel terminal differentiation model of human articular chondrocytes in three-dimensional cultures mimicking chondrocytic changes in osteoarthritis
Mei‑Ling Hoac, Je‑Ken Changbcd, Shun‑Cheng Wuac, Ya‑Hui Chungc, Chung‑Hwan Chenbcd, Shao‑Hung Hungcef and Gwo‑Jaw Wangbcd*
aDepartment of Physiology, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
bDepartment of Orthopaedics, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
cOrthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan
dDepartment of Orthopedics, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
eDepartment of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
fDepartment of Orthopaedic Surgery, Fooyin University Hospital, Ping-Tung County, Taiwan

Abstract

This study establishes a cell culture model mimicking the terminal differentiation occurring in osteoarthritic chondrocytes. Normal articular chondrocytes obtained from human knees treated with 5-azacytidine (Aza-C) were harvested 3, 7 and 14 days after treatment. Phenotypic and genetic changes of articular chondrocytes were detected. The results show that mRNA expression of collagen type II, a marker for normal functional articular chondrocytes, was significantly decreased after Aza-C treatment in comparison to the control cultures, while those of collagen type X and ALP, markers for hypertrophic chondrocytes, were significantly increased. Cell size and apoptotic rate of articular chondrocytes showed significant increases compared to the control after 14 days of Aza-C treatment. Terminal differentiation is shown by this model of three-dimensional cultured human articular chondrocytes, which could apply to the studies of the cellular mechanisms of osteoarthritis.

Keywords: Human articular chondrocytes, Terminal differentiation, Three-dimensional cell culture, Aza-C.

*Corresponding author. Department of Orthopaedics, School of Medicine, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: +886 7 3121101x2553; fax: +886 7 3219452.

1 Introduction

Osteoarthritis (OA) is a joint disease with a gradual degradation of articular cartilage, especially in the aged population (Hamerman, 1995). Previous reports indicated that 10% of the elderly population (>60 years of age) suffered from OA in the United States (Hamerman, 1989; Peat et al., 2001). Furthermore, a previous report indicated that females with OA mostly suffered from the onset symptoms at perimenopausal stage (Nadkar et al., 1999). Therefore, it is quite necessary to study the strategies for preventing disease progress at the early stage of OA.

Several previous studies from OA patients indicated the biological changes of articular chondrocytes during the progress of OA including terminal differentiation, mineralisation and eventually apoptosis (Blanco et al., 1998; Heraud et al., 2000; Kirsch et al., 2000). Osteoarthritic chondrocytes were found to express annexins, alkaline phosphatase and collagen type X (Kirsch et al., 2000). Expressions of collagen type X and annexin V reflect the characteristics of hypertrophic chondrocytes as a mature differentiation. Chondrocytic apoptosis was also found in OA cartilage suggesting that this might be associated with the decrease of cellularity and abnormal mineralisation in OA cartilage (Blanco et al., 1998; Hashimoto et al., 1998b; Heraud et al., 2000). Accordingly, investigators indicated that the osteoarthritic chondrocytes resume the genetic and phenotypic changes similar to the terminal differentiation of chondrocytes in epiphyseal growth plates (Kirsch et al., 2000). One of the prospects of managing OA is to suppress the terminal differentiation of articular chondrocytes and eventually stop the disease progress in the very early stage of OA. However, developing a terminal differentiation model in cultured articular chondrocytes for studying the mechanism of OA has rarely been reported.

It has been reported that Aza-C (5-azacytidine), which suppresses cytidine methylation at gene transcription, changes differentiation in several cell lines (Christman et al., 1983; Jones and Taylor, 1980; Jones et al., 1983; Tarella et al., 1982; Walker et al., 1984). It was also reported that Aza-C induces terminal differentiation changes of cultured epiphyseal chondrocytes as they occur during endochondral ossification (Cheung et al., 2001). A recent report indicates that Aza-C also induces maturation of articular chondrocytes in chickens (Zuscik et al., 2004). However, this model has not been applied in cultured human articular chondrocytes.

This study was designed to develop a novel three-dimensional cell culture model of human articular chondrocytes with induced terminal differentiation changes. This model is for mimicking the osteoarthritic changes of articular chondrocytes and can be applied to the studies of the cellular mechanisms of OA.

2 Methods

2.1 Three-dimensional human articular chondrocyte cultures and Aza-C treatment

Normal human articular chondrocytes (NHAC-kn, Clonic®; BioWhittaker, Walkersville, MD) were obtained from the knee of a 50-year-old Caucasian male. According to the manufacturer's guidelines, NHAC-kn cultures are established for application to the studies of formation, breakdown and regeneration of hyaline cartilage, osteoarthritis research and the proliferation and differentiation of chondrocytes. Another normal human articular cartilage (KMU-AC) was obtained from fresh cadaver-knees of a 23-year-old Asian male, which are supplied by the Hospital of Kaohsiung Medical University. The cartilage was minced and sequentially digested by hyaluronidase (0.5mg/ml), pronase (1mg/ml) and collagenase (1 mg/ml). Chondrocytes were encapsulated in alginate beads as previously described (Sanchez et al., 2002). Briefly, cells were suspended in 1.2% alginate solution in 0.9% NaCl, at a density of 1×106cells/ml, which was slowly dropped into a 102mM CaCl2 solution through a yellow pipette tip. Alginate beads that formed were allowed to polymerise further for 10min in the CaCl2 solution at room temperature. After washing with saline solution, 15 beads were cultured in 5ml of culture medium per well in a 6-well plate. Culture medium was the chondrocyte basal medium (Bulletkit, Clonic®; BioWhittaker, Walkersville, MD) with a supplement of the chondrocyte growth medium, containing R3-IGF1, bFGF, transferrin, insulin, fetal bovine serum and gentamicin/amphotericin-B (SingleQuots, Clonic®; BioWhittaker, Walkersville, MD). The beads were cultured for 7 days at 37°C in a humidified 5% CO2 incubator and the culture medium was changed every 3 days. Cultures were then treated with 15μg/ml of 5-azacytidine (Aza-C) (Sigma, St. Louis, MO) for 48h. The cultures were maintained in media without Aza-C for another 2 weeks. Cells were harvested at the 3rd, 7th and 14th day after Aza-C treatment. The control cultures were cultivated and harvested in the same condition as the experiment cultures except no Aza-C was treated. Chondrocytes were released from alginate beads by dissolving the beads in a 0.9% NaCl solution containing 0.05M Na2 citrate and 0.03M Na2 EDTA at pH 7.4. Cells were collected for each experiment by low-speed centrifugation at 1000rpm for 5min. NHAC-kn cultures were used in the following experiments except real-time polymerase chain reaction (real-time PCR). The chondrocyte cultures from KMU-AC were used to quantify mRNA expressions by real-time PCR, which was to further confirm the effect of Aza-C on gene expressions of human articular chondrocytes.

2.2 Reverse transcription-polymerase chain reaction (RT-PCR)

At the 3rd, 7th and 14th day after Aza-C treatment, expressions of the mRNA of type II and type X collagens and alkaline phosphatase (ALP) of Aza-C treated and non-treated cells were measured by RT-PCR. Total RNA was isolated from chondrocytes by using the Trizol reagent (Gibco BRL, Rockville, MD). The first strand cDNA was converted from 1μg RNA by adding the Moloney murine leukemia virus reverse transcriptase and the oligo(dT) primer. PCR was performed with an Applied Biosystems GeneAmp 9600 PCR system (Applied Biosystems, Foster City, CA). The PCR reaction was carried out with the specific primers of each gene and with the following cycling conditions: denaturing at 94°C for 4min, followed by 40 cycles at 94°C for 30s, annealing at 60°C for 45s, and extension at 72°C for 45s. The products of PCR were resolved by electrophoresis on a 1.4% agarose gel and visualised with ethidium bromide. The optical densities of the resolved bands were semi-quantified by a Bioimaging System (UVP Inc., Upland, CA). The 18S rRNA was used as a control gene for normalisation. The intensity ratios of PCR products to 18S were calculated for comparison. The sequences of the PCR primers for following genes from human are as follows:

Collagen type IIα1 (621 bp product):

Forward primer: 5′-AAC TGG CAA GCA AGG AGA CA-3′

Reverse primer: 5′-AGT TTC AGG TCT CTG CAG GT-3′

Collagen type Xα1 (387 bp product):

Forward primer: 5′-AGC CAG GGT TGC CAG GAC CA-3′

Reverse primer: 5′-TTT TCC CAC TCC AGG AGG GC-3′

Alkaline phosphatase (367 bp product):

Forward primer: 5′-GCG AAC GTA TTT CTC CAG ACC CAG-3′

Reverse primer: 5′-TTC CAA ACA GGA GAG TCG CTT CAA-3′

2.3 Quantitative real-time PCR

Real-time PCR was also performed to confirm the finding from RT-PCR. At the 3rd, 7th and 14th day after Aza-C treatment, the mRNA expressions of type II and type X collagens and ALP of Aza-C treated and non-treated cells were measured. The processes for total RNA isolation and the first strand cDNA conversion were the same as those for RT-PCR. The quantitative RT-PCR was performed in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA) using the SYBR Green PCR Master Mix Reagent containing the Amplitag Gold DNA polymerase, dNTP mixed with dUTP, SYBR Green I, passive reference and reaction buffer (Applied Biosystems, Foster City, CA). Reactions were performed in a 25μl of mixture containing cDNA, specific primers of each gene and the SYBR Green PCR Master Mix reagent. The cycling conditions were as follows: for collagen type IIα1 were 1 cycle at 50°C for 2min and 95°C for 10min, followed by 40 cycles of 95°C for 15s and 62°C for 1min; for collagen type Xα1 were 1 cycle at 50°C for 2min and 95°C for 10min, followed by 40 cycles of 95°C for 15s, 52°C for 20s and 72°C for 20s; for ALP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 1 cycle at 50°C for 2min and 95°C for 10min, followed by 40 cycles of 95°C for 15s and 60°C for 1min.

Primer sequences were as follows:

Collagen type IIα1 (71 bp product):

Forward primer: 5′-CAA CAC TGC CAA CGT CCA GAT-3′

Reverse primer: 5′-TCT TGC AGT GGT AGG TGA TGT TCT-3′

Collagen type Xα1 (85 bp product):

Forward primer: 5′-CAG ATT TGA GCT ATC AGA CCA ACA A-3′

Reverse primer: 5′-AAA TTC AAG AGA GGC TTC ACA TAC G-3′

GAPDH (126 bp product):

Forward primer: 5′-TCT CCT CTG ACT TCA ACA GCG AC-3′

Reverse primer: 5′-CCC TGT TGC TGT AGC CAA ATT C-3′

Alkaline phosphatase (72 bp product):

Forward primer: 5′-GGA GGC CGA AAG TAC ATG TTT C-3′

Reverse primer: 5′-GAA ACA TGT ACT TTC GGC CTC C-3′

The specific PCR products were detected by the fluorescence of SYBR Green, the double stranded DNA binding dye (Morrison et al., 1998). The relative mRNA expression level was calculated from the threshold cycle (Ct) value of each PCR product and normalised with that of GAPDH by using comparative Ct method (Livak and Schmittgen, 2001). The relative quantity of the expression of each gene from the control cells at the 3rd day after Aza-C treatment was set to 100%, and all the others were transformed to a percentage change to it. After PCR reaction, a dissociation (melting) curve was generated to check the specificity of PCR reaction. All the PCR amplifications were performed in triplicate, and experiments were repeated at least three times.

2.4 Flow cytometry

The cell size of articular chondrocytes from the control and Aza-C treated cultures was analysed by flow cytometry at the 14th day after Aza-C treatment. The method for releasing cells from alginate beads was as the description above. Cells (1×106) were then suspended in phosphate buffered saline and analysed immediately on a laser flow cytometer (EPICS Elite; Coulter, Hialeah, FL). The intensity in forward scatter was measured by using an argon laser (488nm) as a probing beam. The relative intensity represents the cell size of a chondrocyte. Five to six thousand chondrocytes from the control and Aza-C treated cultures were measured. The data were analysed by the Winmidi software (EPICS Elite; Coulter, Hialeah, FL).

2.5 TUNEL stain (terminal deoxy-nucleotidyl transferase mediated dUTP nick end labeling stain)

Fragmented DNA of an apoptotic cell was TUNEL (terminal deoxy-nucleotidyl transferase mediated dUTP nick end labeling) stained by using an In Situ Cell Death Detection Kit, TMR red (Roche, Germany). According to the manufacturer's guidelines, cells were fixed with 4% of paraformaldehyde in phosphate buffered solution (PBS) at a cell density of 1×106/ml and incubated at room temperature for 10min. After centrifugation cells were fixed in 80% ethanol. Cells were then settled on a slide by centrifugation at a speed of 2000rpm for 5min by using a cytospin (Cytospin 3; Shandon, UK). Slides were rinsed twice with PBS, and cells were permeabilized by incubating in permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2min on ice. TUNEL reaction mixture, containing terminal deoxy-nucleotidyl transferase and rhodamine (the labeling dye), was added to slides and incubated for 60min at 37°C in a humidified chamber in the dark. The reaction was stopped by blocking buffer (0.1% Triton X-100/0.5% BSA in PBS). Cells were counter-stained by 4′,6-diamidino-2-phenylindole (DAPI). Slides were observed on a fluorescence microscope with an excitation wavelength of 580nm for rhodamine and 365nm for DAPI. Cell nuclei were stained blue by DAPI, while only apoptotic cells were stained red by rhodamine. Stained cells were counted in 5 microscopic fields for each slide. Data were analysed by using Image-Pro® Plus analysis software (Media Cybernetics, Sliver Spring, MD). The ratio of red stained cells (apoptotic cells) to blue stained cells (total cells) was defined as the apoptotic rate of chondrocytes.

2.6 Statistic analysis

Data from RT-PCR, real-time PCR and TUNEL stain are expressed as the means±SEM of 4 wells from representative experiments. Statistical significance was evaluated by Mann–Whitney U-test. Data from flow cytometry for cell size measurement are shown as the means±SEM of 5000–6000 cells in each group. Statistical significance was evaluated by Student's t-test. All the experiments were repeated at least three times. p<0.05 was considered significant.

3 Results

3.1 Reverse transcription-polymerase chain reaction (RT-PCR)

Our results showed that the mRNA expression of collagen type II was decreased while type X was increased gradually with time duration of cultivation after Aza-C treatment (Figs. 1 and 2). In comparison with the control cultures, the mRNA expression of collagen type II in Aza-C treated cultures was significantly decreased at the 7th and 14th day after Aza-C treatment (p<0.01) (Fig. 1), while the collagen type X expression was significantly increased at the 3rd, 7th and 14th day (p<0.01) (Fig. 2). The mRNA expression of ALP was significantly induced at the 7th and 14th day after Aza-C treatment (p<0.01), while that of the control cultures was hardly detectable (Fig. 3).

Fig. 1

Changes of the mRNA expression of collagen type II at 3, 7 and 14 days after a 48h treatment of Aza-C. mRNA expression of collagen type II was semi-quantified by RT-PCR, and normalised relative to the expression of 18S rRNA. The representative images assessed from the control and Aza-C treated cultures are shown in (A). The ratio of the optical densities of collagen type II to 18S (% optical density collagen II/18S) was calculated for comparison (B). Each bar represents the mean±SEM of 4 replica cultures. Data were evaluated by Mann–Whitney U-test. Key: **p<0.01, in comparison with the control culture of the same day after Aza-C treatment.

Fig. 2

Changes of the mRNA expression of collagen type X at 3, 7 and 14 days after a 48h treatment of Aza-C. The mRNA expression of collagen type X was semi-quantified by RT-PCR, and normalised relative to the expression of 18S rRNA. The representative images of the bands assessed from the control and Aza-C treated cultures are shown in (A). The ratio of the optical densities of collagen type X to 18S (% optical density collagen II/18S) was calculated for comparison (B). Each bar represents the mean±SEM of 4 replicated cultures. Data were evaluated by Mann–Whitney U-test. Key: **p<0.01, in comparison with the control culture of the same day after Aza-C treatment.

Fig. 3

Changes of the mRNA expression of alkaline phosphatase at 3, 7 and 14 days after a 48h treatment of Aza-C. The mRNA expression of alkaline phosphatase was semi-quantified by RT-PCR, and normalised relative to the expression of 18S rRNA. The representative images of the bands assessed from the control and Aza-C treated cultures are shown in (A). The ratio of the optical densities of alkaline phosphatase to 18S (% optical density ALP/18S) was calculated for comparison (B). Each bar represents the mean±SEM of four replicated cultures. Data were evaluated by Mann–Whitney U-test. Key: **p<0.01, in comparison with the control culture of the same day after Aza-C treatment.

3.2 Quantitative real-time PCR

The representative real-time PCR profiles from the quantization of ALP and GAPDH mRNA expressions were shown (Fig. 4, left panel), and the dissociation curves of these two PCR products were also shown (Fig. 4, right panel). The results from real-time PCR showed that the mRNA expressions of collagen type II significantly decreased in Aza-C treated cultures compared to the control cultures at the 3rd (p<0.01), 7th (p<0.01) and 14th (p<0.05) day after Aza-C treatment (Table 1). Collagen type X expression of chondrocytes was significantly induced in Aza-C treated cultures at the 3rd (p<0.05), 7th (p<0.01) and 14th (p<0.05) day after Aza-C treatment (Table 1). ALP mRNA expression of chondrocytes was significantly induced in Aza-C treated cultures at the 7th and 14th day after Aza-C treatment (p<0.01) (Table 1).

Fig. 4

Representative amplification profiles (left panel) and dissociation curves (right panel) from quantitative real-time PCR for examining mRNA expression of alkaline phosphatase (ALP) are shown. GAPDH is an house keeping gene for normalisation.

Table 1.

Percentage change of the mRNA expressions of type II collagen, type X collagen and ALP by quantitative real-time PCR

mRNATreatmentDay 3 (%)Day 7 (%)Day 14 (%)
CollagenControl100.0 ± 1.6125.0 ± 1.210.0 ± 6.3
Type IIAza-C7.3 ± 1.2**1.0 ± 0.4**3.0 ± 0.9*

CollagenControl100.0 ± 9.171.0 ± 17.5159.0 ± 21.8
Type XAza-C398.0 ± 26.3*64013.0 ± 5.0**335.0 ± 1.4*

ALPControl100.0 ± 9.34.0 ± 1.118.0 ± 4.7
Aza-C175.8 ± 13.531900.0 ± 4.7**735.6 ± 3.2**

3.3 Flow cytometry

The cell size of articular chondrocytes in Aza-C treated cultures (509.20±1.93) was significantly increased (a 26% increase) in comparison to the control cultures (404.41±1.45) at 14th day after Aza-C treatment (p<0.01) (Fig. 5).

Fig. 5

Changes of the cell size at 14 days after a 48h treatment of Aza-C. The representative cell size distribution profile measured from flow cytometry is shown. The relative intensity represents the cell size of a chondrocyte. Mean±SEM of the relative intensity from the control cultures (404.41±1.45) and Aza-C treated cultures (509.20±1.93) were shown. Data were evaluated by Student's t-test. **p<0.01, in comparison with the control culture.

3.4 TUNEL stain

In comparison to the control cultures, Aza-C treated cultures significantly increased the apoptotic rate of articular chondrocytes at 14th day after Aza-C treatment (p<0.01) (Fig. 6).

Fig. 6

Induction of apoptosis of human articular chondrocytes at 14 days after a 48h treatment of Aza-C. The representative images of the TUNEL stained cells (stained red, defined apoptoic cells) and DAPI counter-stained cells (stained blue, defined total cells) are shown (×100 magnification). The apoptotic rate of the control and Aza-C treated cells were compared. Each bar represents the mean±SEM of 4 replica cultures. Data were evaluated by Mann–Whitney U-test. Key: **p<0.01, in comparison with the control culture of the same day after Aza-C treatment.

4 Discussion

Endochondral bone formation physiologically occurs during the development of embryonic long bones and the post-natal longitudinal bone growth in epiphyseal growth plate. During this process, chondrocytes play important roles according to the temporal and spatial signals in the internal environment of the body. Chondrocytes pass through several stages including proliferation, maturation, hypertrophy, calcification and eventually apoptosis (Kronenberg, 2003; Shum and Nuckolls, 2002). Proliferation and matrix production of chondrocytes result in cartilage enlargement, and the specific marker molecules are collagen type II and glucosaminoglycan in this stage. Afterward chondrocytes undergo terminal differentiation: cells stop proliferating, enlarge (hypertrophy), and express collagen type X; subsequently cells express annexins, Indian hedgehog (Ihh), alkaline phosphatase and deposit mineral; and finally hypertrophic chondrocytes undergo apoptosis (Kronenberg, 2003; Long et al., 2001; Noonan et al., 1998). It has been reported that hypertrophic chondrocytes play roles in directing the mineralisation, vascularisation and osteoblastogenesis in the surrounding area through factors secreted from hypertrophic chondrocytes (Kronenberg, 2003). However, the process of endochondral ossification does not occur in articular chondrocytes, which maintain chondrocytic functions for one's whole life.

In OA cartilage, collagen type II that normally exists in articular cartilage was decreased, but the reparative collagen, collagen type X, was expressed (Adam and Deyl, 1983; Aigner et al., 1993; Kirsch et al., 2000). It was also reported that OA articular chondrocytes resumed the terminal differentiation that was similar to the epiphyseal growth plate, expressing annexins and ALP, and also promote mineralisation and finally apoptosis (Kirsch et al., 2000). It was also reported that the decrease of cellularity of articular cartilage is consistent with aging and the occurrence of OA in human subjects (Abyad and Boyer, 1992; Adams and Horton, 1998; Blanco et al., 1998). Apoptotic chondrocytes were found in the superficial and middle zones of articular cartilage in OA patients (Blanco et al., 1998; Hashimoto et al., 1998a,c; Heraud et al., 2000; Horton et al., 1998; Kirsch et al., 2000). Researchers suggested that apoptosis of articular chondrocytes might play a very important role in the loss of cellularity of chondrocytes (Lotz et al., 1999). Subsequently, the decrease of cellularity of articular chondrocytes may result in an inability to maintain the appropriate formation of extracellular matrix, and eventually OA develops (Lotz et al., 1999). Accordingly, several strategies to prevent the progress of OA from the aspect of chondrocyte biology have been suggested, such as blocking apoptosis of chondrocytes (Blanco et al., 1995; Hashimoto et al., 1997; Heraud et al., 2000). Suppressing the terminal differentiation of articular chondrocytes may also be a helpful strategy for preventing the OA progress. It is useful to test the strategies in cultured chondrocytes before in vivo study. Therefore, this study was to establish a terminal differentiation model in cultured human articular chondrocytes that could mimic the biologic changes of OA chondrocytes.

The results from this study indicated that the terminal differentiation of human articular chondrocytes in a three-dimensional culture system could be induced. We found that a 48h treatment of Aza-C could induce the expressions of collagen type X and suppress that of collagen type II starting from the 3rd day after induction. Subsequently, ALP expression was induced at the 7th day after Aza-C treatment. Furthermore, hypertrophy and even apoptosis of articular chondrocytes were also observed at 14 days after this Aza-C induction. The down-regulation of the normal functional gene, collagen type II, of articular chondrocytes and the up-regulation of marker genes of hypertrophy (type X collagen) and mineralisation (ALP) of chondrocytes demonstrated that the articular chondrocytes underwent terminal differentiation. The cell enlargement of Aza-C treated cells analysed by flow cytometry further demonstrated the hypertrophy of articular chondrocytes. Moreover, the increase of apoptotic rate of Aza-C treated cells indicated that the articular chondrocytes underwent the final process of terminal differentiation. Our results demonstrated that the genetic and phenotypic properties of human articular chondrocytes could be induced into terminal differentiation in this in vitro model.

The results of quantitative real-time PCR from chondrocytes of KMU-AC further provided a more promised quantification of mRNA expressions of collagen type II, collagen type X and ALP. Although we were unable to determine the protein levels encoded from these genes at this step, we further demonstrated the influence of Aza-C on mRNA expressions of these genes. Moreover, this result showed that the Aza-C effects on decreasing collagen type II expression but increasing the expressions of marker genes in terminal differentiation of articular chondrocytes does not particularly occur in the NHAC-kn chondrocytes. However, gene expression changes of chondrocytes from NHAC-kn and KMU-AC, which affected by Aza-C, were not consistent with the temporal changes. The maximal effect of Aza-C treatment on gene expressions of KMU-AC was at the 7th day, while that of NHAC-kn was at the 14th day in this study. It might be due to the individual variation or age related reasons; however, a definitive conclusion was not possible with only 2 subjects to work from. Nevertheless, we suggest that characterizing a temporal profile of the gene expression changes that affected by Aza-C is needed before using this in vitro terminal differentiation model for articular chondrocytes.

Previous reports indicated that monolayer cultured chondrocytes would undergo dedifferentiation where type II collagen synthesis might be eliminated, while the three-dimensional cultured chondrocytes expressed their differentiation functions like those in vivo (Domm et al., 2002; Malda et al., 2003a,b). Accordingly, in the present study, the chondrocytes were cultivated in alginate beads for maintaining their characteristics, as they exist in vivo. In this way, we established a novel cell culture model to mimic the biologic changes of articular chondrocytes that undergo terminal differentiation. This model could apply to the studies of the molecular and cellular mechanisms of osteoarthritis.

Acknowledgements

This study was supported by grants from the National Science Council (NSC91-2314-B-037-291-) and the Department of Industrial Technology, Economic Affairs (91-EC-17-A-17-SI-0009) in Taiwan, ROC.

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Received 13 December 2004/16 November 2005; accepted 20 November 2005

doi:10.1016/j.cellbi.2005.11.009
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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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