Matrilin-3 is a member of the recently described matrilin family of extracellular matrix proteins containing von Willebrand factor A-like domains. The matrilin-3 subunit can form homotetramers as well as hetero-oligomers together with subunits of matrilin-1 (cartilage matrix protein). It has a restricted tissue distribution and is strongly expressed in growing skeletal tissues. Detailed information on expression and distribution of extracellular matrix proteins is important to understand cartilage function in health and in disease like osteoarthritis.
Matrilin-3 expression was analysed on decalcified normal cartilage/bone sections (N = 5) and decalcified cartilage/ bone sections with minor (N= 10), moderate (N = 10), and severe osteoarthritic lesions (N = 10). Osteoarthritic changes were classified histomorphologically, using the grading system of Mankin. Matrilin-3 expression was investigated by immunohistochemistry, in situ hybridization, Western blot analysis, and quantitative PCR. For immunohistochemistry, a polyclonal antibody against matrilin-3 was used. For Western blot analysis, cartilage extracts were obtained from normal and osteoarthritic samples, partially purified, and separated in SDS poly-acrylamide gelelectrophoreses. After blotting onto nitro-cellulose, matrilin-3 was visualized by incubation with the polyclonal anti-matrilin-3 antibody and chemiluminescence detection. Matrilin-3 -mRNA expression was determined by in situ hybridization using a digoxigenin-labeled anti-sense probe.
Our results indicate that matrilin-3 is a mandatory component of mature articular cartilage with its expression being restricted to chondrocytes from the tangential zone and the upper middle cartilage zone. Osteoarthritic cartilage samples with only moderate morphological osteoarthritic destructions have elevated levels of matrilin-3 mRNA. In parallel, we found an increased deposition of matrilin-3 protein in the cartilage matrix. Matrilin-3 staining was diffusely distributed in the cartilage matrix, with no cellular staining being detectable. In cartilage samples with minor osteoarthritic lesions, matrilin-3 deposition was restricted to the middle zone and to the upper deep zone. A strong correlation was found between enhanced matrilin-3 gene and protein expression and the extent of tissue damage. Sections with severe osteoarthritic destruction showed the highest amount of matrilin-3 mRNA, strong signals in in situ hybridization, and prominent protein deposition in the middle and deep cartilage zone.
We conclude that matrilin-3 is an integral component of human articular cartilage matrix and that the enhanced expression of matrilin-3 in osteoarthritis may be a cellular response to the modified microenvironment in the disease.
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.
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
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.
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.
