Mechanobiology of articular cartilage : Consequences of physical activity and advanced glycation

Osteoarthritis (OA) is a type of degenerative arthritis that limits daily activity and quality of life for more than 10% of the people worldwide. Having a better insight into the mechanisms of OA pathology is a key to better diagnostic strategies and improved therapies. OA patients experience different stages of disease progressing from a pre-OA to early and eventually end-stage OA. Irreversible structural damage is mainly detectable in the progressed stages of OA at tissue level, while early degradations are initiated at a small molecular-scale from the pre-OA stage. This underscores the importance of detecting the early signs of OA especially on a molecular-scale. Articular cartilage is a highly specialized load-bearing tissue that reduces friction and distributes forces over the underlying bone. Articular cartilage has a hierarchical structural architecture which mainly consists of proteins and polysaccharides such as type II collagen and proteoglycans that make up the extracellular matrix (ECM), and chondrocytes. Chondrocytes are responsible for tissue homeostasis, which is an outcome of a series of phenomena that exists in the cartilage matrix. Mechanical properties (especially elasticity) can be considered as a key determinant of cartilage health. This PhD dissertation aims to provide detailed knowledge of mechanobiological mechanisms contributing to the age- and overload-related deterioration of articular cartilage. To address the general hypothesis, we applied a set of multi-scale biomechanics experimental tools on articular cartilage of various species. We sought to determine the mechanical contributions of its main components, such as collagen fibers and proteoglycans, especially at the superficial zone where the osteoarthritis-related biomechanical abnormalities likely initiate. The obtained indentation-based mechanical properties from the molecular (Atomic Force Microscopy) to more tissue level (nano-indenter), together revealed the contribution of cartilage specific components to its overall mechanics. We also found that the cartilage stiffness as determined from indentation tests is highly dependent on indentation and indenter characteristics has a very high spatial variability, necessitating a large number of indentation tests to provide a representative characteristic of the mechanical response of cartilage under investigation. Subsequently, we investigated the effect of mechanical loading on the knee joint and the resulting cartilage and bone adaptation at both cell and tissue levels, in an experiment with rats. Our observations proved the dynamic nature of bone turnover which continuously reacts to forces as well as cartilage/chondrocyte mechano-sensitive responses to mechanical stimuli. Then, we analyzed artificially induced ‘age’-related changes regarding advanced glycation in both equine and rodent articular cartilage from the mechanobiological viewpoint. Micro- and nano-scale indentation experiments indicate that AGE crosslinks enhance cartilage overall stiffness, which is associated with increased cross-linking density of the collagen fibers. In addition, we showed that it is possible to tailor the efficiency of AGEs-related cross-linking by manipulation of the collagen fiber pre-stress induced via osmotic pressure

Guidelines for an optimized indentation protocol for measurement of cartilage stiffness : The effects of spatial variation and indentation parameters

Mechanical properties of articular cartilage that are vital to its function are often determined by indentation tests, which can be performed at different scales. Cartilage tissue exhibits various types of structural, geometrical, and spatial variations that pose strict demands on indentation protocols. This study aims to define a reproducible micro-indentation protocol for measuring the effective (average) stiffness of the cartilage surface in a region around 1mm(2). We elucidated how different parameters such as indenter size, indenter depth, and the location of the indentation influence the effective elastic modulus measured in micrometer scale on rat knee cartilage. When an indentation was performed (50μm radial probe, ≈10μm indentation depth) at exactly the same location, the variability was less than 10%, even with a recovery period of 30s. However, there was a high spatial variation and a small change of around 60μm in location could change the modulus values up to as much as 10-20 fold. The effective elastic modulus of cartilage surface layer cannot therefore be reproducibly determined from a few indentations on a cartilage sample, and requires at least 144 (12×12) indentations for a soft spherical probe with a 50μm radius. With higher depths, the spatial variation is slightly lower, allowing slightly lower number of indentations (≈80 measurements or a 9×9 frame) to provide a representative elastic modulus. Using this protocol, we determined an elastic modulus of 2.6±1.9N/mm(2) at the medial side versus a higher modulus of 4.2±2.6N/mm(2) at the lateral side of the tibia of 12 weeks old Wistar rats. Optimized indentation protocols similar to the one presented here are required for revealing such variations in the mechanical properties of cartilage with anatomical location

Effect of moderate increasing exercise on the mechanical balance of the knee joint in young rats

Purpose: It is hypothesized that the amount, duration and magnitude of mechanical loading are important factors that maintain the cartilage tissue in physiological condition. The underlying subchondral bone attached to the cartilage tissue and the cartilage-bone interface are influenced by the mechanical loading as well. In this study, we aimed to investigate adaptation of the rat knee joint to the mechanical demands. For this purpose, we applied load in the form of exercise using a moderate-intensity increasing running protocol. A series of analyses was performed to elucidate the response of cartilage and bone to this physical activity. Methods: Male Wistar rats (Charles River, Germany) with an age of 8 weeks were placed in 2 groups: a moderate running group that runs for 8 weeks with a slowly increasing running velocity - from 10 m/min for 10 min, up to 20 m/min for 1 hour (n = 10), and a control group without running (n = 10). Running takes place on a 5 lane motorized rodent treadmill (LE-8700; Panlab Harvard Apparatus). At starting point and after 8 weeks cartilage qPCR, micro-CT, histology and plasma FIB 3-2 (Artialis) was performed. Results: A total 24 km running within 8 weeks of this running protocol illustrates chondrocyte sensitivity and cartilage response to the mechanical loading (Fig. 1). At the end of the experiment, aggrecan was 1.55-fold up-regulated while MMP-2 was 2.38-fold down-regulated (P < 0.05) (Fig. 1A). The histological appearance of the chondrocytes also showed load-dependency, with more hypercellularity and hypertrophy in the running group (Fig. 1B). FIB3-2 as a plasma biomarker, interacts with the tissue inhibitor of metalloproteinase 3 (TIMP-3) and the elevated amount of FIB3-2 is expected in osteoarthritis samples. FIB3-2 is also known to be cleaved by several MMPs family including MMP-2. FIB3-2 levels dropped in the running group (from 52.7 ± 13.2 nM to 30.2 ± 8.4 nM) as compared to the control group (45.3 ± 15.0 nM to 33.3 ± 9.7 nM) (Fig. 1C). MicroCT analysis revealed an enhancement in bone response as a result of early moderate physical training where epiphysis bone parameters in the running group including thickness and bone volume fraction of subchondral bone tibia plateau as well as trabecular bone mass significantly increased compared with control animals (Fig. 2). Conclusions: Gradual increase of running up to a moderate level to 1120 m/h for one hour enhances aggrecan expression, reduces catabolic enzymatic activity of MMP-2 as well as increases subchondral bone thickness in the epiphysis area and leads to hypercellularity and hypertrophy of the chondrocytes. Conclusively, a moderate exercise program can significantly influence both bone remodeling and cartilage tissue adaptation

Quantifying the Effects of Different Treadmill Training Speeds and Durations on the Health of Rat Knee Joints

Abstract Background Walking and running provide cyclical loading to the knee which is thought essential for joint health within a physiological window. However, exercising outside the physiological window, e.g. excessive cyclical loading, may produce loading conditions that could be detrimental to joint health and lead to injury and, ultimately, osteoarthritis. The purpose of this study was to assess the effects of a stepwise increase in speed and duration of treadmill training on knee joint integrity and to identify the potential threshold for joint damage. Methods Twenty-four Sprague-Dawley rats were randomized into four groups: no exercise, moderate duration, high duration, and extra high duration treadmill exercise. The treadmill training consisted of a 12-week progressive program. Following the intervention period, histologic serial sections of the left knee were graded using a modified Mankin Histology Scoring System. Mechanical testing of the tibial plateau cartilage and RT-qPCR analysis of mRNA from the fat pad, patellar tendon, and synovium were performed for the right knee. Kruskal-Wallis testing was used to assess differences between groups for all variables. Results There were no differences in cartilage integrity or mechanical properties between groups and no differences in mRNA from the fat pad and patellar tendon. However, COX-2 mRNA levels in the synovium were lower for all animals in the exercise intervention groups compared to those in the no exercise group. Conclusions Therefore, these exercise protocols did not exceed the joint physiological window and can likely be used safely in aerobic exercise intervention studies without affecting knee joint health