The superficial zone in cartilage adapts with stiffening when challenged tribologically

Purpose: In osteoarthritis, the superficial zone of articular cartilage is compromised by fissures and cracks and, thus, may lose its intended function. While there is agreement that the superficial zone is important for the load bearing capacity of the tissue, its mechanical response upon articulation is less investigated. In previous studies, using fully intact cartilage samples, we have shown that the cartilage surface undergoes a transient stiffening response upon articulation. Here, we hypothesize that this response is lost when the surface is removed. In order to address this question we used a tribological bioreactor and articulated on cartilage samples with and without superficial zone. Further, we started to study structural changes at the surface with an atomic force microscope (AFM) before and after the application of tribological stress. Methods: Twenty-four 14x20x3 mm oval bovine cartilage explants that had undergone one freeze-thaw cycle were distributed into four groups per animal: (1) intact surface and articular loading; (2) removed surface and articular loading; (3) intact free-swelling-control (FSC); 4) removed surface FSC. Thus, each group had n=6 samples. A vibratome was used to remove the superficial zone of the explants (at least 20% of full sample thickness). Microindentation stress-relaxation tests were performed in the center of explants using a Hysitron TI 950 with a 20 μm spherical indenter and an 8 μm indent depth. All indentation on explants was performed submerged in 1× PBS to maintain tissue hydration. Following initial characterization, cartilage explants were removed from the indentation sample holder and placed into the bioreactor. Cartilage explants were confined in porous polyethylene scaffold rings and fixed in a well. Using a 32-mm ceramic ball (Ra ∼10 nm), a 40 N static confined compression was applied that lead to approx. 2 MPa contact stress. The ball oscillated at 0.5 Hz, and was migrated at a speed of 1mm/s across the cartilage surface generating a wear scar length of 10 mm. Following 1-hr of bioreactor testing, microindentation was repeated in the center/wear scar. The reduced modulus was obtained using the Oliver-Pharr method on 80-95% of the unloading curve. Cartilage surfaces removed by vibratome were prepared for AFM analysis (Nanowizard Ultra, JPK Instruments, Germany). All measurements were conducted in 1× PBS. Using QI imaging, a force sampling mode, an initial image of 256 x 256 pixels (approx. 10 x 10 μm2) was obtained. The AFM was then put into contact mode, and an 80 nm silicon tip was repeatedly (>850 times) slid linearly across the cartilage surface. Following these sliding experiments, the tissue was reimaged in QI mode. For data analysis, the ratio of post- to pre-bioreactor reduced modulus was calculated and analyzed using Shapiro-Wilks tests for normality and Paired Student’s T-tests. Results: Our results show that intact surface groups exhibited significantly increased stiffening following articular loading compared to the FSC (p=0.006). For the removed surface groups, no significant difference between articulated and FSC explants was detected (p=0.927) (Fig 1). Also in the AFM the cartilage surface demonstrated topographical and stiffness related changes upon sliding that are shown in Figure 2. Conclusions: Surface stiffening following tribological loading may be an important biomechanical function of load transfer during articulation. Surface removal leads to a loss of this adaptive response. The stiffening response may be related to a reorganization of matrix molecules that are unique to the superficial zone. In the future we plan to investigate the influencing parameters of this stiffening response and identify the matrix molecules that are involved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)