Catharina Müller:
Studies on cartilage extracellular matrix – composition and functions in chondrocyte differentiation

Date: 08-06-2012    Supervisor: Anders Aspberg

The extracellular matrix (ECM) of cartilage is highly specialized to meet the specific needs of this tissue during development of the vertebrate skeleton and in the adult. During embryonic development cartilage serves as a scaffold for the majority of the developing bones and gets replaced by bone tissue in the process of endochondral ossification. In the adult, cartilage persists as a thin layer on the opposing bones of a synovial joint ensuring friction-free movement and balancing mechanical impact. Chondrocytes produce an extracellular matrix consisting mainly of collagens and proteoglycans but also a multitude of other proteins. Basement membrane (BM) components have been identified in cartilage forming a BM-like structure around chondrocytes. To date there is no complete overview about the arrangement of the matrix molecules in the cartilage ECM and the functional implications thereof.

The here presented studies focused on the investigation of BM molecules during chondrogenic differentiation and on the distribution of ECM molecules in articular cartilage. Additionally, functional aspects of ECM molecules in general, and BM molecules in particular, on chondrocyte differentiation were studied in vitro.

Gene expression studies on ATDC5 cells during chondrogenic differentiation revealed the expression of BM genes in differentiating chondrocytes with several genes being upregulated with hypertrophy. Immunofluorescence studies confirmed the deposition of BM proteins in the growth plate, some with specificity to certain differentiation stages.

Growing ATDC5 cells on different isolated laminins demonstrated an inhibitory effect of certain laminins on chondrogenic differentiation. In addition, culturing of chondrogenic ATDC5 cells or MC615 subclones on pre-laid, decellularised ECMs from cells at various differentiation stages influenced cell morphology and attachment. Furthermore, chondrogenic cells were cultured under limited diffusion conditions aiming to increase the local concentration of newly produced ECM molecules and facilitating ECM network assembly. This resulted in the formation of capsule-like ECM structures around chondrocytes, inhibition of nodularisation in the cell layer and the formation of an ECM resistant to guanidine hydrochloride extraction.

Quantitative mass spectrometry using the iTRAQ technology revealed unique patterns of protein distribution in a human lateral tibia plateau sample with groups of proteins showing preferential distribution in the superficial, middle or deep layers.

Taken together these studies increased the knowledge about the distribution of ECM proteins in growth plate and articular cartilage and indicated a functional role of BM proteins during chondrogenic differentiation. These results open up for further studies on the interaction of BM proteins with chondrocytes and the role of ECM molecules in tissue turnover in health and disease.