OA is a complex disease of which the ethiopathology is not completely known and therapies to repair cartilage are still under investigation. The increase of collagen type II expression in osteoarthritic cartilage suggests an activated repair mechanism that is however ineffective in repairing or maintaining the ECM homeostasis. We therefore investigated the ability to modulate the formation of a functional collagen type II network that can ultimately contribute to innovation of cartilage repair in OA. To do so we used different approaches: addition of growth factors, inhibition of collagen cross-links, inhibition of proteoglycan formation, overexpression of cartilage oligomeric matrix protein (COMP) and knock-down of COMP and collagen IX, Of the growth factors used in this thesis, IGF1 had positive effects on the parameters in our chondrocyte alginate cultures. It stimulated chondrocytes to deposit more collagen and proteoglycans without affecting collagen cross-linking, it increased mechanical functioning and decreased MMP-1 gene expression. FGF2 on the other hand had no positive effects in our culture system: It lowered the collagen deposition and did not affect the proteoglycan deposition. The effect TGFb had on chondrocytes in culture was more complex. Addition of TGFb to chondrocytes in alginate had no large effect on proteoglycan and collagen deposition but did lower the number of cross-links per collagen molecule and reduced the fibril thickness. The effect of TGF-beta appeared to depend on the physical environment of the cell as concluded from our comparison between chondrocytes cultured in monolayer on plastic or in alginate beads. We inhibited crosslink formation by addition of beta-aminopropionitrile (BAPN), which inhibits one of the key enzymes for crosslink formation, lysyloxidase, via covalent binding. As a result, the chondrocyte produced more collagen (as shown by increased collagen type II gene expression) and also more collagen was deposited. Furthermore we were also interested in the effect of other extracellular matrix components on collagen network formation. Addition of FGF2 decreased and addition of TGF-beta increased COMP deposition. We overexpressed COMP in chondrocytes and found no effect on matrix deposition. However, COMP overexpression also resulted in collagen fibrils with a smaller diameter, like we saw after addition of TGFb. By inhibiting the formation of a proteoglycan network using para-nitrophenyl-beta-D-xyloside (PNPX) and therefore proteoglycan deposition, collagen deposition was decreased. The absence of an intact proteoglycan network lowered the collagen retention in our culture system. Cultures of collagen type IX deficient chondrocytes also had altered collagen retention. Since collagen type IX forms a network together with collagen type II and other matrix molecules, the absence of this network probably also reduced collagen retention in the alginate bead. However, GAG distribution was also altered when collagen type IX was absent in the newly formed matrix. Which in turn could also have contributed to altered collagen retention. Interestingly, the absence of COMP in addition to collagen type IX deficiency did not alter matrix production and distribution. This leads to the conclusion that collagen type IX is more important in the matrix interaction than COMP. Taken together, it seems that for the production of collagen in cartilage, focussing on the formation of an intact cartilage network is as important as stimulating collagen type II production. This network formation is important to retain the collagen type II in the newly formed matrix. Growth factors can influence collagen network formation, either directly or via their effect on other extracellular matrix components.

, , , , , ,
J.A.N. Verhaar (Jan)
Erasmus University Rotterdam
TNO, Rheumafonds, NVMB, Anna Fonds, CellCoTec
Erasmus MC: University Medical Center Rotterdam

Bastiaansen-Jenniskens, Y.M. (2009, June 4). A Novel Approach to Stimulate Cartilage Repair: Targeting Collagen Turnover. Erasmus University Rotterdam. Retrieved from http://hdl.handle.net/1765/17730