|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2006) 30, 288294 (Printed in Great Britain)
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
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.
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.
2.1 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 (0.5
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 1 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′
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′
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′
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′
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 25
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′
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′
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′
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′
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′
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 (C
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
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
2.6 Statistic analysis
Data from RT-PCR, real-time PCR and TUNEL stain are expressed as the means
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
Changes of the mRNA expression of collagen type II at 3, 7 and 14 days after a 48
Changes of the mRNA expression of collagen type X at 3, 7 and 14 days after a 48
Changes of the mRNA expression of alkaline phosphatase at 3, 7 and 14 days after a 48
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
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.
Percentage change of the mRNA expressions of type II collagen, type X collagen and ALP by quantitative real-time PCR
3.3 Flow cytometry
The cell size of articular chondrocytes in Aza-C treated cultures (509.20
Changes of the cell size at 14 days after a 48
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
Induction of apoptosis of human articular chondrocytes at 14 days after a 48
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 48
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.
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 2005doi:10.1016/j.cellbi.2005.11.009