This study focused on the development of a novel technique to investigate the depth-dependent biaxial mechanical properties of native and tissue engineering articular cartilage. Samples were stained fluorescently and tested in a custom designed instrument, which was used to apply displacement in compression and shear. It was also enhanced with a 6 DOF loadcell, which measured applied compressive and shear forces. The depthdependent compressive and shear strains were determined by analyzing fluorescently stained images of the cartilage obtained and analyzed using a confocal microscope and a custom Matlab code. In this method, displacement was determined from images of deformed lines photobleached as markers through the entire thickness of the samples, and strain was obtained from the derivative of the displacement. We investigated the feasibility of an alternative systematic approach to numerical differentiation for computing the shear strain that was based on fitting a continuous function to the shear displacement. Three models for a continuous shear displacement function were evaluated:​ polynomials, cubic splines, and non-parametric locally weighted scatter plot curves. Four independent approaches were then applied to identify the best-fit model and the accuracy of the first derivative. One approach was based on the Akaiki Information Criteria, and the Bayesian Information Criteria. The second was based on a method developed to smooth and differentiate digitized data from human motion. The third method was based on photobleaching a predefined circular area with a specific radius. Finally, we integrated the shear strain and compared it with the total shear deflection of the sample measured experimentally. Results showed that 6th and 7th order polynomials are the best models for the shear displacement and its first derivative. In addition, failure of tissue-engineered cartilage, consistent with previous results, demonstrated the qualitative value of this imaging approach. By measuring the depth-dependent displacements of the cartilage under defined biaxial deformations and forces, the local mechanical behavior of the tissue was determined and related to the collagen fibers structure using compensated polarized microscopy techniques. We found that depthdependent shear behavior of mature AC was consistent with the micro-architectural structure of AC, indicating the role of collagen structure in mechanical properties of AC. However, based on the structural variation between mature and immature AC, their mechanical behavior differences could be tenable. This suggested that age, species and anatomic location needed to be considered when reporting mechanical behavior results.

Further investigations regarding the samples’ configurations (boneless versus with bone) and anatomical locations effects on depth-dependent shear behavior have been performed. We also investigated compressive deflection on shear behavior of the samples and shear effects on axial behavior.