Early Signs of Bone and Cartilage Changes Induced by Treadmill Exercise in Rats.

This study aims to investigate the earliest alterations of bone and cartilage tissues as a result of different exercise protocols in the knee joint of Wistar rats. We hypothesize that pretraining to a continuous intense running protocol would protect the animals from cartilage degeneration. Three groups of animals were used: (i) an adaptive (pretraining) running group that ran for 8 weeks with gradually increasing velocity and time of running followed by a constant running program (6 weeks of 1.12 km/hour running per day); (ii) a non-adaptive running (constant running) group that initially rested for 8 weeks followed by 6 weeks of constant running; and (iii) a non-running (control) group. At weeks 8, 14, and 20 bone and cartilage were analyzed. Both running groups developed mild symptoms of cartilage irregularities, such as chondrocyte hypertrophy and cell clustering in different cartilage zones, in particular after the adaptive running protocol. As a result of physical training in the adaptive running exercise a dynamic response of bone was detected at week 8, where bone growth was enhanced. Conversely, the thickness of epiphyseal trabecular and subchondral bone (at week 14) was reduced due to the constant running in the period between 8 and 14 weeks. Finally, the intermediate differences between the two running groups disappeared after both groups had a resting period (from 14 to 20 weeks). The adaptive running group showed an increase in aggrecan gene expression and reduction of MMP2 expression after the initial 8 weeks running. Thus, the running exercise models in this study showed mild bone and cartilage/chondrocyte alterations that can be considered as early-stage osteoarthritis. The pretraining adaptive protocol before constant intense running did not protect from mild cartilage degeneration. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Effects of non-enzymatic glycation on the micro- and nano-mechanics of articular cartilage.

The mechanical properties of articular cartilage depend on the quality of its matrix, which consists of collagens and glycosaminoglycans (GAGs). The accumulation of advanced glycation end products (AGEs) can greatly affect the mechanics of cartilage. In the current study, we simulated the accumulation of AGEs by using L-threose to cross-link collagen molecules in the cartilage matrix (in vitro). The resulting changes in the mechanical properties (stiffness) of cartilage are then measured both at the micrometer-scale (using micro-indenter) and nanometer-scale (using indentation-type atomic force microscopy). Non-enzymatic cross-linking within the cartilage matrix was confirmed by the browning of L-threose-treated samples. We observed > 3 times increase in the micro-scale stiffness and up to 12-fold increase in the nano-scale stiffness of the glycated cartilage in the peak pertaining to the collagen fibers, which is caused by cartilage network embrittlement. At the molecular level, we found that besides the collagen component, the glycation process also influenced the GAG macromolecules. Comparing cartilage samples before and after L-threose treatment revealed that artificially induced-AGEs also decelerate in vitro degradation (likely via matrix metalloproteinases), observed at both micro- and nano-scales. The combined observations suggest that non-enzymatic glycation may play multiple roles in mechanochemical functioning of articular cartilage.

Non-enzymatic cross-linking of collagen type II fibrils is tuned via osmolality switch.

An important aspect in cartilage ageing is accumulation of advanced glycation end products (AGEs) after exposure to sugars. Advanced glycation results in cross-links formation between the collagen fibrils in articular cartilage, hampering their flexibility and making cartilage more brittle. In the current study, we investigate whether collagen cross-linking after exposure to sugars depends on the stretching condition of the collagen fibrils. Healthy equine cartilage specimens were exposed to l-threose sugar and placed in hypo-, iso-, or hyper-osmolal conditions that expanded or shrank the tissue and changed the 3D conformation of collagen fibrils. We applied micro-indentation tests, contrast enhanced micro-computed tomography, biochemical measurement of pentosidine cross-links, and cartilage surface color analysis to assess the effects of advanced glycation cross-linking under these different conditions. Swelling of extracellular matrix due to hypo-osmolality made cartilage less susceptible to advanced glycation, namely, the increase in effective Young's modulus was approximately 80% lower in hypo-osmolality compared to hyper-osmolality and pentosidine content per collagen was 47% lower. These results indicate that healthy levels of glycosaminoglycans not only keep cartilage stiffness at appropriate levels by swelling and pre-stressed collagen fibrils, but also protect collagen fibrils from adverse effects of advanced glycation. These findings highlight the fact that collagen fibrils and therefore cartilage can be protected from further advanced glycation ("ageing") by maintaining the joint environment at sufficiently low osmolality. Understanding of mechanochemistry of collagen fibrils provided here might evoke potential ageing prohibiting strategies against cartilage deterioration. © 2018 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:1929-1936, 2018.

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 1mm2. 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/mm2 at the medial side versus a higher modulus of 4.2±2.6N/mm2 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.