Chondrosarcomas are hyaline cartilage-forming tumours which can be classified according to malignancy through histological grading. Grade I chondrosarcomas rarely metastasize whereas in grade III chondrosarcoma metastasis is observed in 71% of cases. There is, so far, no clear molecular marker allowing an objective classification of chondrosarcoma. The aim of this project was to identify such marker genes through the comparison of gene expression of chondrosarcoma and normal hyaline cartilage and through the correlation of expression profiles to histological grading.

The mRNA of 19 chondrosarcomas with different histological grades and of eight normal cartilage samples was analysed. Gene expression profiles were assessed on a customised cDNA array including 230 cartilage- and stem cell-relevant genes. Data were analysed by hierarchical clustering and significance analysis of microarrays. Results were confirmed by real-time RT-PCR.

Gene expression profiles clearly discriminated between normal and neoplastic cartilage. Between them, 73 differentially expressed genes were identified. The genes higher expressed in cartilage included several genes encoding matrix proteins. Among the genes higher expressed in chondrosarcoma, molecules involved in PTH and BMP signalling were found. Genes differentially expressed between tumours of different grade were identified. Among others, galectin 1 was significantly higher expressed in highly malignant tumours compared to grade I tumours. This correlation could be confirmed at protein level by immunohistological analysis.

The comparative analysis of normal cartilage and chondrosarcoma gene expression showed that there are important molecular differences between the matrix of normal and neoplastic cartilage. Our results furthermore confirm that genes implicated in the regulation of the growth plate were expressed in chondrosarcoma. Remarkably, we identified galectin 1 as a marker correlating to malignancy on the level of gene and protein expression. More extended studies on this functionally polyvalent molecule would be necessary to establish it as a marker for malignancy in chondrosarcoma.

Monolayer expansion of human articular chondrocytes (HAC) is known to result in progressive dedifferentiation and loss of stable cartilage formation capacity in vivo. For optimal outcome of chondrocyte based repair strategies, HAC capable of ectopic cartilage formation may be required. Thus, the aim of this study was to establish appropriate quality control measures capable to predict the ectopic cartilage formation capacity of HAC from culture supernatants. This strategy would avoid the waste of cells for quality control purposes, in order to improve cell therapy and tissue-engineering approaches for the repair of joint surface lesions.

Standardized medium supernatants (n=5) of freshly isolated HAC and chondrocytes expanded for 2 (PD2) or 6 population doublings (PD6) were screened for 15 distinct interleukins, 8 MMPs and 11 miscellaneous soluble factors by a multiplexed immunoassay. Cartilage differentiation markers like COMP and YKL-40 were determined by ELISA. Corresponding HAC were subcutaneously transplanted into SCID-mice and their capacity to form stable ectopic cartilage was examined histologically 4 weeks later.

While freshly isolated chondrocytes generated stable ectopic cartilage positive for collagen type II, none of the PD6 transplants formed cartilaginous matrix. Loss of ectopic stable cartilage formation capacity between PD0 and PD6 correlated with a drop of MMP3 secretion to < 10% of initial levels, while changes for other investigated molecules were not predictive. Chondrocytes from donors with low MMP3 levels (< 10%) at PD2 failed to regenerate ectopic cartilage at PD2, indicating that MMP3 levels of cultured chondrocytes, independent of the number of cell doublings and the time in culture, predicted ectopic cartilage formation.

In conclusion, loss of stable ectopic cartilage formation capacity in the course of HAC dedifferentiation can be predicted by determination of relative MMP3 levels demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.