Abstract
Introduction
Mechanical loading has been hypothesized to play an important role in the development, remodeling and in diseases of many skeletal tissues, including cartilage. In order to study the metabolic response of cartilage to physical forces, in vitro systems have often been used because of the precise control with which mechanical loads can be applied. We developed a new mechanical loading system, in which we were able to load the intact femoral condyle in order to preserve the native cartilage/subchondral bone structure. This system represents a more ‚in vivo‘ situation than cartilage explants or chondrocyte cell culture systems.
Our approach focused on changes in mRNA expression of type II collagen, type VI collagen, and aggrecan in loaded versus adjacent unloaded cartilage in order to analyse the early response of chondrocytes to well-defined mechanical stresses.
Methods
Femoral condyles were obtained from two-year-old cows. The integrity of the cartilage surface was controlled by staining with safranin O. The femoral condyles were compressed in an Instron 8501 material testing machine. Cyclic compression pressure was applied for 2000 cycles in a sinusoidal waveform of 0. 5 Hz-frequency with a peak stress of 0. 2 to12. 5 MPa. Following loading, full depth cartilage sections were cut out and one half immediately frozen in liquid nitrogen for RNA isolation and the other half soaked in 4% paraformaldehyde for paraffin embedding. As control, the adjacent unloaded cartilage was collected and treated in the same way. Total RNA was isolated and changes in mRNA expression were quantitated by competitive quantitative PCR, using an internal standard of a C-terminal truncated version of the corresponding genes. The PCR-reactions were separated by agarose gel electrophoresis and amplified fragments quantified using video-densitometry analysis. The results were expressed as the ratio of mRNA from loaded to unloaded cartilage
Results
Cyclic compression with peak stresses of 12. 5, 6. 3, 2. 5 and 0. 6 MPa lead to a two-fold decrease in the mRNA expression of type II collagen and aggrecan and a threefold decrease of type VI collagen, in consideration of the intra-assay variability of about 30%. Compression with peak stresses of 0. 3 and 0. 2 MPa lead to a three-fold increase of the mRNA expression of type II collagen, a four-fold increase of aggrecan and a slight decrease of type VI collagen.
Low compression strength leads to an increase of the mRNA expression of the major components of cartilage, type II collagen and aggrecan, whereas high loading leads to a decrease of the mRNA expression.
Conclusion
The results show that our system can be used to analyze early responses of chondrocytes to well-defined mechanical stresses in an intact cartilage/bone-system and therefore will enable us to investigate the role of physiological and non-physiological high loading on the induction of cartilage degradation and regeneration in joint trauma and osteoarthritis. Since the cartilage/bone samples are incubated in medium during the experiment, this system will also offer us the opportunity to investigate additives to the medium as potential pharmacological therapeutics in osteoarthritis.
