Chondrocyte density, proteoglycan content and gene expressions from native cartilage are species specific and not dependent on cartilage thickness: a comparative analysis between rat, rabbit and goat

Abstract

Background: In many pre-clinical studies of cartilage tissue, it has been generally assumed that the major difference of the tissue between the species is the tissue thickness, which is related to the size of the animal itself. At present, there appear to be lack of studies demonstrating the relationship between chondrocyte densities, protein content, gene expressions and cartilage thickness in the various animal models that are commonly used. The present study was conducted to determine whether or not chondrocyte density, proteoglycan/protein content and selective chondrocyte gene expression are merely related to the cartilage thickness (thus animal size), and not the intrinsic nature of the species being investigated. Mature animals (rabbit, rats and goats) were sacrificed for their hind knee cartilages. Image analyses were performed on five consecutive histological sections, sampled from three pre-defined locations at the lateral and medial femoral condyles. Cartilage thickness, chondrocyte density, Glycosaminoglycan (GAGs)/protein content and gene expression levels for collagen II and SOX-9 were compared across the groups. Correlation analysis was done between cartilage thickness and the other variables.

Results: The mean cartilage thickness of rats, rabbits and goats were 166.5 ± 10.9, 356.2 ± 25.0 907.5 ± 114.6 μm, respectively. The mean cartilage cell densities were 3.3 ± 0.4×10(-3) for rats, 2.6 ± 0.3×10(-3) for rabbits and 1.3 ± 0.2×10(-3) cells/μm2 for goats. The mean μg GAG/mg protein content were 23.8 ± 8.6 in rats, 20.5 ± 5.3 in rabbits and 328.7 ± 64.5 in goats; collagen II gene expressions were increased by 0.5 ± 0.1 folds in rats; 0.6 ± 0.1 folds in rabbits, and 0.1 ± 0.1 folds in goats, whilst the fold increase of SOX-9 gene expression was 0.5 ± 0.1 in rats, 0.7 ± 0.1 in rabbits and 0.1 ± 0.0 in goats. Cartilage thickness correlated positively with animals' weight (R2 =0.9856, p = 0.001) and GAG/protein content (R2 =0.6163, p = <0.001). Whereas, it correlates negatively with cell density (R2 = 0.7981, p < 0.001) and cartilage gene expression levels (R2 = 0.6395, p < 0.001).

Conclusion: There are differences in the composition of the articular cartilage in diverse species, which are not directly dependent on the cartilage thickness of these animals but rather the unique characteristics of that species. Therefore, the species-specific nature of the cartilage tissue should be considered during any data interpretation.

Figure 1

Cartilage Thickness. Measurement of cartilage thickness of (A) rat, (B) rabbit and (C) goat. Mean cartilage thickness was derived from a minimum five readings in each slide. Representative slides were obtained from the posterolateral condyle of left distal femur. Surface area is calculated in 500 μm (five boxes) in length and full thickness in depth. (Safranin O staining; magnification rat and rabbit 10×, goat 4× objective lens).

Figure 2

Correlation for cartilage thickness, cell density and animal weight. The figure demonstrates: (A) An inverse relationship between the cartilage thickness and the cell density. (B) Positive correlation of the cartilage thickness and the animal weight. (C) An inverse relationship between chondrocytes density and animal weight.

Figure 3

Comparison in the Glycosaminoglycan/protein content. Goat cartilage contained the highest amount of GAGs/protein (μg/mg) followed by rat and rabbit.

Figure 4

Correlation for GAGs/protein content and cartilage thickness/cell density/animal weight. Figures demonstrate: (A) GAGs/protein and the weight of the animals. (B) GAGs/protein and cartilage thickness. (C) GAGs/protein and cell density.

Figure 5

Gene expression analyses. Gene expression analysis of both collagen II and SOX9 in rat, rabbit and goat demonstrate significant variation between species.

Figure 6

Correlation for gene expression pattern. Correlation of gene expression patterns for Collagen II and SOX-9: (A) An inverse relationship was observed between the genes expressed and animal weight. (B) Thicker cartilage had low levels of gene expression as compared to thin articular cartilage as depicted by a downward non-linear line in this graph. (C) The upward line in the graph demonstrates that cartilage with less cell count per surface area has low levels of gene expression, possibly due to the low concentration of RNA in the chondrocytes of the articular cartilage. (D) The inverse line in the graph suggests that cartilage with low level of gene expression had greater amounts of GAGs/protein.