Atomic force microscopy to investigate spatial patterns of response to interleukin-1beta in engineered cartilage...

Atomic force microscopy (AFM) has been proposed as a tool to evaluate the structural and mechanical properties of cartilage tissue. Here, we aimed at assessing whether AFM can be employed to quantify spatially resolved elastic response of tissue engineered cartilage (TEC) to short exposure to IL-1β, thus mimicking the initially inflammatory implantation site. TEC generated by 14 days of pellet-culture of expanded human chondrocytes was left untreated (ctr) or exposed to IL-1β for 3 days. TEC pellets were then cut in halves that were glued on a Petri dish. Profiles of elasticity were obtained by sampling with a nanometer sized, pyramidal indenting tip, with 200µm step resolution, the freshly exposed surfaces along selected directions. Replicate TECs were analyzed biochemically and histologically. GAG contents and elasticity of pellets decreased (1.4- and 2.6-fold, respectively, p<0.05) following IL-1β stimulation. Tissue quality was evaluated by scoring histological pictures taken at 200μm intervals, using the Bern-score grading system. At each distance, scores of ctr TEC were higher than those IL-1β treated, with the largest differences between the two groups observed in the central regions. Consistent with the histological results, elasticity of IL-1β-treated TEC was lower than in ctr pellets (up to 3.4-fold at 200μm from the center). IL-1β treated but not ctr TEC was intensely stained for MMP-13 and DIPEN (cryptic fragment of aggrecan) especially in the central regions. The findings indicate the potential of AFM to investigate structure/function relationships in TEC and to perform tests aimed at predicting the functionality of TEC upon implantation.