Chondrocyte culture in agarose: age dependence and characterization of interfacial growth [English translation]

Research on articular cartilage allows a better understanding of articular diseases, like osteoarthritis, and the elaboration of new regenerating therapies. Even if most of the cartilage-related troubles are linked to aging, almost all of the in vitro studies use chondrocytes isolated from immature animals. It is already known that age modulates chondrocyte metabolism. However, few data are available concerning the role of donor age when chondrocytes are cultivated in tridimensional scaffolds, which are broadly used in tissue engineering.

The initial hypothesis of the present work was that cell proliferation, extracellular matrix synthesis and accumulation are reduced with increased age of the donor. To test this hypothesis, bovine chondrocytes from fetus, young adults and aged animals were cultivated in an agarose gel. After more than three weeks in culture, cell density as well as matrix content and synthesis rates were measured. We observed reduced cell proliferation and matrix accumulation for chondrocytes from aged animals, compared to chondrocytes from younger ones. Collagen synthesis was reduced by a more pronounced manner than proteoglycan synthesis. Stress-relaxation tests, performed by unconfined compressions, showed that the mechanical parameters extracted with the fibril-reinforced biphasic model are age-dependant. The latter parameters correlated significantly with matrix content.

At the end of the culture, we noted the presence of a tissue on the surface of agarose constructs. Characterization of these outgrowths at the agarose-media interface showed that the tissue biochemical composition was modulated with age. Cell content was the greatest in young adult samples, which produced a matrix rich in collagen II and in glycosaminoglycan. Interfacial tissue produced by fetal cells contained both collagen types I and II, but no glycosaminoglycan. Cells from aged donors produced matrix-poor cell clusters. In the central regions of young adult outgrowths, cell morphology was round and representative of normal articular chondrocytes. However, cells were elongated at the periphery of the outgrowths, where cell proliferation occured. In order to observe cellular dynamics at the interface, we developed a new live cell imaging technique, using time-lapsed images acquired by confocal microscopy in the presence of low doses of acridine orange. With this method, we could observe that cell migration took place on the agarose surface, and we could directly monitor cell division.

With this work, we verified the initial hypothesis, and strongly recommend to carefully evaluate the effect of donor age when functional properties or behavior of chondrocytes is to be assessed. Furthermore, it led us to the characterization of outgrowing interfacial tissue. In the light of our results, we suggest agarose interface culture as a novel culture system, which supports both cell proliferation and de novo hyaline tissue formation. Finally, the imaging technique developed in the last part of the project allows the observation of dynamic phenomenon happening in the outgrowths, and offers a new tool to analyze living cells cultured in 3-dimensional systems.