Engineered cartilage constructs subject to very low regimens of interstitial perfusion

We have studied an in vitro engineered cartilage model, consisting of bovine articular chondrocytes seeded on micro-porous scaffolds and perfused with very low regimens of interstitial flow. Our previous findings suggested that synthesis of sulphated glycosaminoglycans (sGAG) was promoted in this model, if the level of shear generated on cells was maintained below 10 mPa (0.1 dyn/cm2). Constructs were stimulated with a median shear stress of 1.2 and 6.7 mPa using two independent culture chambers. Quantification of the applied stresses and of oxygen consumption rates was obtained from computational modelling. Experimentally, we set a time zero reference at 24 hours after cell seeding and total culture time at two weeks. The cell metabolic activity, measured by MTT, was significantly lower in all constructs at two weeks (-73% in static controls, -66% in the 1.2 mPa group and -60% in the 6.7 mPa group) vs. the time zero group, and significantly higher (+33%) in the 7 mPa group vs. static controls. The ratio between synthesis of collagen type II/type I, measured by Western Blot, was significantly higher in the 1.2 mPa constructs (+109% vs. the 6.7 mPa group, +120% vs. the time zero group and +286% vs. static controls). A trend of decreased alpha-actin expression was observed with increased ratio of type II to type I collagen, in all groups. These results reinforce the notion that, at early time points in culture, hydrodynamic shear below 10 mPa may promote formation of extra-cellular matrix specific to hyaline cartilage in chondrocyte-seeded constructs.