Microindentation of cartilage before and after articular loading in a bioreactor: assessment of length-scale dependency using two analysis methods.

BACKGROUND: Microindentation is a technique with high sensitivity and spatial resolution, allowing for measurements at small-scale indentation depths. Various methods of indentation analysis to determine output properties exist. OBJECTIVE: Here, the Oliver-Pharr Method and Hertzian Method were compared for stiffness analyses of articular cartilage at varying length-scales before and after bioreactor loading. METHODS: Using three different conospherical tips with varying radii (20, 100, 793.75 μm), a bioreactor-indenter workflow was performed on cartilage explants to assess changes in stiffness due to articular loading. For all data, both the Oliver-Pharr Method and Hertzian Method were applied for indentation analysis. RESULTS: The reduced moduli calculated by the Hertzian Method were found to be similar to those of the Oliver-Pharr Method when the 20 μm tip size was used. The reduced moduli calculated using the Hertzian Method were found to be consistent across the varying length-scales, whereas for the Oliver-Pharr Method, adhesion/suction led to the largest tip exhibiting an increased average reduced modulus compared to the two smaller tips. Loading induced stiffening of articular cartilage was observed consistently, regardless of tip size or indentation analysis applied. CONCLUSIONS: Overall, geometric linearity is preserved across all tip sizes for the Hertzian Method and may be assumed for the two smaller tip sizes using the Oliver-Pharr Method. These findings further validate the previously described stiffening response of the superficial zone of cartilage after articular loading and demonstrate that the finding is length-scale independent.