Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression
© Bougault et al; licensee BioMed Central Ltd. 2008
Received: 02 May 2008
Accepted: 15 September 2008
Published: 15 September 2008
Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice.
Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell). After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK) in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the biological response of mouse chondrocytes to the dynamic compression at the transcriptional level, we also developed an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs. A decrease in promoter activity of the gene coding for type II procollagen, the most abundant protein in cartilage, was observed in response to dynamic loading.
The protocols developed here offer the possibility to perform an integrated analysis of the molecular mechanisms of mechanotransduction in chondrocytes, at the gene and protein level.
Articular cartilage serves as a bearing surface for joints and is routinely exposed to mechanical loading. The mechanical properties of the tissue are due to its extracellular matrix particular composition. On the one hand, the load-bearing function is based on the high osmotic pressure created by the negatively charged glycosaminoglycans. These polyanions attract an excess of sodium ions into the tissue, resulting in an influx of water. On the other hand, the fibrillar collagen network, with type II collagen representing the major protein of cartilage, provides the tissue with its tensile strength. Thus, cartilage is a highly specialized tissue with relatively few cells, the chondrocytes, embedded in it. In this context, mechanical forces are thought to play an important role in regulating chondrocyte physiology. Several studies using cartilage explants or chondrocytes seeded in three-dimensional (3D) scaffolds have shown that mechanical compressive loading affects the chondrocyte metabolic activity [1–10]. However little is currently known regarding the biochemical pathways involved in mechanotransduction signalling in cartilage.
Numerous previous studies have examined the phenotypic responsiveness of chondrocytes to mechanical stress using the well-characterized experimental system consisting of isolated chondrocytes embedded within agarose hydrogels [11–15]. The ability of this 3D model system to maintain or re-establish the differentiation state of chondrocytes over extended culture periods has been extensively studied [16, 17]. Also, of the 3D matrices, hydrogels may prove most suitable for analysis of mechanical response as they are homogeneous from the outset and fully surround cells embedded within. Additionally, the cell-agarose model could be used to examine the interactions of growth factors, such as Bone Morphogenetic Proteins, and mechanical stimuli on chondrogenic activity. Furthermore, it has been suggested that mechanical conditioning may be useful in cartilage-engineering context to stimulate in vitro biosynthesis by chondrocytes within 3D scaffolds prior to implantation. Considering that agarose or agarose/alginate hydrogels are clinically potential scaffolds for autologous chondrocyte implantation , the chondrocyte/agarose construct under compression is a model that could also provide insight into mechanotransduction within cartilage-engineered constructs.
Since the most recent studies indicate that the molecular mechanisms by which chondrocytes sense and respond to mechanical stimuli lead to changes at the levels of transcription, translation, post-translational modification, and matrix synthesis or degradation, there is a need to develop protocols to extract from agarose gels high quality RNA and proteins. We provide here general protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the influence of cell compression on the steady-state level of mRNA, gene transcription using reporter promoter construct, and signalling molecules phosphorylation state. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice.
Results and Discussion
Preparation of chondrocyte/agarose constructs
Timetable of progressive deprivation of serum over the 6-day culture period of chondrocyte/agarose constructs.
Choice of compression system
Protein extraction and analysis: Mechanical loading triggers MAPK activation
RNA extraction and gene expression analysis: Mechanical loading stimulates cFos and cJun expression
Total RNAs from cell/agarose constructs were extracted using the RNeasy-mini kit (Qiagen). To facilitate agarose dissolution, QG buffer (Qiagen) was added to the classical RLT lysis buffer, and agarose was diluted in large volumes, as described in "Methods". According to the manufacturer's protocol, a DNase treatment was included to remove any contaminating genomic DNA. After assessment of the quality of RNA by capillary electrophoresis (Figure 4B), steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as an immediate response to compression (Figure 4B). Interestingly, it has already been shown that gene expression levels of cFos and cJun increase within 1 h of a variety of intact cartilage loading regimen [5, 24, 25]. It has also been shown that the binding of activating protein-1, the heterodimer of cFos and cJun, increases in response to dynamic compression of a tissue engineered construct . Thus, the up-regulation of cFos and cJun mRNAs observed here in the chondrocyte/agarose constructs that have been submitted to compression is in agreement with the mechanosensitive character of these two genes.
Analysis of human COL2A1gene promoter activity in response to compression
To our knowledge, only two studies have attempted to monitor cartilaginous extracellular matrix gene transcription by chondrocytes in 3D culture in response to compression [3, 27]. To explore further the biological response of embryonic mouse chondrocytes to the dynamic compression at the transcriptional level, we developed here an approach for monitoring changes in gene transcription in agarose culture by using reporter promoter constructs.
Specifically, we used chimeric plasmid "p3" (pGL2-0.387 kb) to monitor promoter activity of the human COL2A1 gene, which codes for type II procollagen . "PGL2C" is a plasmid control carrying SV40 promoter instead of a fragment of COL2A1 promoter. These two plasmids are luciferase reporter vectors. We also used a plasmid encoding β-galactosidase under the control of Rous sarcoma virus (RSV) promoter to monitor transfection efficiency.
This study integrates protocols that allow analysis of the effects of compression on mouse chondrocytes embedded in agarose gels at the cellular, protein, and gene levels. Figure 3 summarizes the loading protocols we used for PCR, Western-blotting and gene promoter activity analyses but other types of loading regimen could be used for such analyses. The techniques presented here have been optimized so that the studies can be performed with chondrocytes isolated from a limited number of mouse embryos or new-borns and this point is important in regard of genetically modified mice. Besides, it is possible to apply these protocols to other types of chondrocytes, including human chondrocytes.
Preparation and Culture of chondrocyte/agarose constructs
Embryonic mouse chondrocytes were isolated from the ventral parts of the rib cages of 17.5 dpc mice. For each experiment, cells were pooled from all embryos from 2 or 3 OF-1 mouse litters. Mouse care and treatment were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (Authorization n°69387416 given by the French Prefecture du Department du Rhone). Agarose hydrogels were prepared by mixing 2.5% low melting agarose (Seaplaque, Cambrex BioScience) with 5 × Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) containing 10% FBS, 50 mM Hepes and 500 U/mL Penicillin and 500 μg/mL Streptomycin (all products from Gibco). Cells were suspended in this agarose solution with a density of 2 × 106 cells/mL. 700 μL of the mixture was poured into wells of 24-well plates, and chondrocyte/agarose constructs were allowed to gel at room temperature. Constructs were then punched to obtain cylindrical gels (13 mm diameter and 3 mm height) that were placed into wells of Biopress™ compression plates (Flexcell international), within the 13 mm diameter foam ring (Figure 2). A volume of 4 mL DMEM/F-12 culture medium containing 10% FBS and 100 U/mL Penicillin and 100 μg/mL Streptomycin was added to each well. The cell/agarose constructs were maintained in culture for 6 days in 5% CO2 at 37°C. The culture medium was changed every day with a progressive deprivation of serum as detailed in Table 1.
Samples were submitted to load with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell International). The strain regimen consisted either in (i) cyclical compression with pulses of 20 kPa at a frequency of 0.33 Hz (2 sec on, 1 sec off) superimposed on 20 kPa static offset pressure for 30 min, or in (ii) intermittent compression of 30 kPa using a sinusoid waveform at 0.2 Hz for 30 min, as indicated (Figure 3). As control, cell/agarose constructs were maintained under uncompressed conditions.
The chondrocyte/agarose constructs were fixed in 4% formaldehyde for at least 24 h, after which they were embedded in Paraffin, sectioned at 7 μm, and then deparaffinised. Prior to antibody treatment, the sections were sequentially incubated with hyaluronidase (Sigma, type I-S, 800 U/mL) for 1 h at 37°C and 0.1% Triton X-100 for 20 min at room temperature. The sections were then incubated overnight with 2B1 anti-type II collagen primary mouse antibody (kind gift from Richard Mayne)  at 4°C. After washing in Phosphate Buffer Saline, the sections were incubated with Cy™2-conjugated AffiniPure donkey anti-mouse IgG (Jackson Immunoresearch) for 1 h at room temperature.
Protein Extraction and Analysis
Antibodies used for Western blot analysis
p38 MAP kinase
Cell Signaling #9212
p44/42 MAP kinase
Cell Signaling #9102
Phospho-p38 MAP kinase
Cell Signaling #9211
Phospho-p44/42 MAP kinase
Cell Signaling #9101
The protocol of RNA extraction was modified from the RNeasy-mini kit (Qiagen) procedure. Briefly, each chondrocyte/agarose construct was diced in a mixture of 1.5 mL QG buffer (Qiagen) and 2 mL RLT buffer (Qiagen kit) and the samples were kept at room temperature until complete dissolution, and then stored at -20°C. After defrosting, the samples were homogenized and supplemented with 2 mL 70% ethanol. The subsequent RNA purification steps were performed as described by the manufacturer. RNA quantity and quality were assessed by using a capillary electrophoresis system (RNA Nano kit and 2100 BioAnalyzer, Agilent).
Reverse transcription and Real time PCR
Primers used for real time PCR analysis
RPL13a (reference gene)
Forward : atccctccaccctatgacaa
Reverse : gccccaggtaagcaaactt
Forward : gggacagcctttcctactacc
Reverse : gatctgcgcaaaagtcctgt
Forward : agggacccatggaagttttt
Reverse : tttttctaggagttgtcagattcaaa
Prior to transfection, chondrocytes were amplified in monolayer culture for one week. After trypsinization, 3 × 106 cells were suspended in 100 μL of electroporation buffer and mixed with 4 μg plasmid of interest and 1 μg plasmid encoding β-galactosidase. The plasmids were transfected by using the Human Chondrocyte Nucleofector kit (Amaxa) according to the manufacturer's protocol (Program U-24). Although nucleofection leads to about 60% cell mortality, this method provides high transfection efficiency (around 80%) for the viable cells. This transfection efficiency was assessed by monitoring synthesis of green fluorescent protein in chondrocytes observed under a fluorescence microscope, after transfection of the corresponding expression vector (data not shown).
Measure of COL2A1promoter activity
Immediately after nucleofection, chondrocytes were embedded in agarose and the cell/agarose constructs were submitted to compression the following day. At the end of the compression regimen (20 h post-transfection), the constructs were frozen in liquid nitrogen and stored at -80°C. The frozen samples were then freeze-dried for 12 h before being grinded in TissueLyser (Qiagen) for 1 min at 20 Hz. The resulting powder was rehydrated in 200 μL of the kit lysis solution for 1 h at 4°C and reporter assays were performed using Dual-Light® combined reporter gene assay system for detection of luciferase and β-galactosidase (Applied Biosystems), as described by the manufacturer, and MLX™ Microtiter® plate luminometer (Dynex). COL2A1 promoter activity was obtained as a ratio of luciferase to β-galactosidase luminescence.
Quantification and statistical analysis
For Western-blotting analysis, band intensity was quantified by densitometry using ImageQuant software (v5.2, Molecular Dynamics). The ratio of phospho-MAPK to total-MAPK band intensity was calculated and mechanically-induced phosphorylation was normalized to uncompressed controls. For the analysis of the compression effects on mRNA levels and promoter activity, data represent the mean and standard deviation values of 3 independent experiments. Statistical analysis was performed using a Student's paired, two-tailed t-test. Significance was reported at the 90% (*) or 95% (**) confidence level.
We are grateful to Karine Duroure for technical assistance. We also thank Novotec (Lyon) for casting the chondrocyte/agarose constructs into Paraffin blocks, and the technical facilities of IFR 128 (BioSciences Gerland – Lyon Sud) for the quantitative PCR analyses. We are grateful to Professor Philippe Galéra (Caen University) for the gift of the p3 plasmid carrying a fragment of the human type II collagen gene promoter. Our studies were funded by Rhône-Alpes Region (Emergence 2005), ANR (PROMOCART 2006), CNRS and Lyon 1 University. CB is supported by the French Ministère de la Recherche.
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