To identify genes showing altered expression in osteoarthritic (OA) cartilage and synovium. Dkk3, a member of the Dickoppf family of Wnt signalling inhibitors was overexpressed and this work highlights the potential function of Dkk3 in OA. Real-time PCR was used to compare the expression of 270 cytokines, chemokines and their receptors in cartilage and synovium from OA and non-OA patients. Expression of Dkk3 was also measured in ATDC5 cells and in bovine nasal cartilage (BNC) explants treated with inflammatory cytokines. The effect of Dkk3 on hydroxyproline and GAG release was measured in BNC explant cultures. To assess the distribution of Dkk3 in OA cartilage immunohistochemistry was carried out on anteromedial gonarthrosis specimens. The level of Dkk3 in synovial fluid tricompartmental and unicompartmental cartilage lesions was measured using ELISA.Purpose
Methods
Anteromedial Osteoarthritis of the Knee (AMOA) is a distinct phenotype of OA. Within this pattern of disease, the anterior third of the medial tibial plateau exhibits full thickness cartilage loss. The middle third has damaged partial thickness cartilage, and the posterior third has retained cartilage, which is seen on macroscopic visual assessment to be normal. This study investigates the molecular features of progressive severities of cartilage damage within this phenotype. Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content. Real time PCR compared gene expression between areas T and N. There was a decrease in OARSI grade across the four areas, with progressively less fibrillation between areas T1, T2 and T3. Area N had a grade of 0 (normal). The GAG immunoassay showed decreased levels with increasing severity of cartilage damage (p<
0.0001). Proliferation and apoptosis, as expected, were increased in the more damaged areas. There was no significant difference in the Collagen II content or gene expression between areas. The Collagen I immunohistochemistry showed increased staining within chondrocyte pericellular areas in the undamaged region (N) and immunoassays showed that the Collagen I content of this macroscopically and histologically normal cartilage, was significantly higher than the damaged areas (p<
0.0001). Furthermore, real time PCR showed a significant increase in Collagen I expression in the macroscopically normal areas compared to the damaged areas (p=0.04). We conclude that in this phenotype the Collagen I increase, in areas of macroscopically and histologically normal cartilage, may represent very early changes of the cartilage matrix within the osteoarthritic disease process. This may be able to be used as an assay of early disease and as a therapeutic target for disease modification or treatment.
The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H&
E staining) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content. Real time PCR compared gene expression between areas T and N.
The GAG immunoassay showed decreased levels with increasing severity of cartilage damage (ANOVA P<
0.0001). There was no significant difference in the Collagen II content or gene expression between areas. The Collagen I immunohistochemistry showed increased staining within chondrocyte pericellular areas in the undamaged region (N) and immunoassays showed that the Collagen I content of this macroscopically and histologically normal cartilage, was significantly higher than the damaged areas (ANOVA P<
0.0001). Furthermore, real time PCR showed that there was a significant difference in Collagen I expression between the damaged and macroscopically normal areas (p=0.04).
