The pericellular matrix (PCM) is a distinct zone of matrix that surrounds individual chondrocytes throughout articular cartilage. Although not much is known about its function, our group has shown that it has a role in mechanotransduction by controlling the bioavailability of perlecan-bound FGF-2. The aim of this project was to determine the other structural components of the PCM, and to search for other heparin binding growth factors which might be sequestered in the matrix. By confocal microscopy I confirmed that the PCM was rich in type VI collagen and perlecan, although devoid of type II collagen and aggrecan. Isolation of individual chondrons (the chondrocyte together with its PCM) was performed by partial digestion of the matrix using dispase and collagenase, and proteomic analysis using mass spectrometry was performed to identify new proteins. As this method was only partially successful I looked for the presence of a known heparin binding growth factor, connective tissue growth factor (CCN2), in chondron preparations. CCN2, which is an abundant secreted protein of articular cartilage, was present in both the chondron preparation by western blot, and was visualised in the PCM of porcine and human articular cartilage by confocal microscopy.
CCN2 is not commercially available so His-tagged recombinant protein was stably expressed and purified using nickel affinity chromatography. Biological activity of the purified protein was investigated in a number of established assays. No biological activity was demonstrated when purified CCN2 was used alone on murine mesenchymal stem cells, but was evident when assayed in combination with low dose TGF-β. The effect of exogenous CCN2 on fibroblasts was limited by the significant release of endogenous CCN2. High constitutive expression of CCN2 in articular cartilage may limit the effects seen by exogenous CCN2. However, the results presented in this thesis support the role of CCN2 as a modulator of TGF beta signalling, and suggest that, through potentiating TGF beta it may regulate matrix turnover in articular cartilage.