Site- and Zone-Dependent Changes in Proteoglycan Content and Biomechanical Properties of Bluntly and Sharply Grooved Equine Articular Cartilage

In this study, we mapped and quantified changes of proteoglycan (PG) content and biomechanical properties in articular cartilage in which either blunt or sharp grooves had been made, both close to the groove and more remote of it, and at the opposing joint surface (kissing site) in equine carpal joints. In nine adult Shetland ponies, standardized blunt and sharp grooves were surgically made in the radiocarpal and middle carpal joints of a randomly chosen front limb. The contralateral control limb was sham-operated. At 39 weeks after surgery, ponies were euthanized. In 10 regions of interest (ROIs) (six remote from the grooves and four directly around the grooves), PG content as a function of tissue-depth and distance-to-groove was estimated using digital densitometry. Biomechanical properties of the cartilage were evaluated in the six ROIs remote from the grooves. Compared to control joints, whole tissue depth PG loss was found in sites adjacent to sharp and, to a larger extent, blunt grooves. Also, superficial PG loss of the surgically untouched kissing cartilage layers was observed. Significant PG loss was observed up to 300 µm (sharp) and at 500 µm (blunt) from the groove into the surrounding tissue. Equilibrium modulus was lower in grooved cartilage than in controls. Grooves, in particular blunt grooves, gave rise to severe PG loss close to the grooved sites and to mild degeneration more remote from the grooves in both sharply and bluntly grooved cartilage and at the kissing sites, resulting in loss of mechanical strength over the 9-month period

Dual-contrast micro-CT enables cartilage lesion detection and tissue condition evaluation ex vivo

Background: Post-traumatic osteoarthritis is a frequent joint disease in the horse. Currently, equine medicine lacks effective methods to diagnose the severity of chondral defects after an injury. Objectives: To investigate the capability of dual-contrast-enhanced computed tomography (dual-CECT) for detection of chondral lesions and evaluation of the severity of articular cartilage degeneration in the equine carpus ex vivo. Study design: Pre-clinical experimental study. Methods: In nine Shetland ponies, blunt and sharp grooves were randomly created (in vivo) in the cartilage of radiocarpal and middle carpal joints. The contralateral joint served as control. The ponies were subjected to an 8-week exercise protocol and euthanised 39 weeks after surgery. CECT scanning (ex vivo) of the joints was performed using a micro-CT scanner 1 hour after an intra-articular injection of a dual-contrast agent. The dual-contrast agent consisted of ioxaglate (negatively charged, q = −1) and bismuth nanoparticles (BiNPs, q = 0, diameter ≈ 0.2 µm). CECT results were compared to histological cartilage proteoglycan content maps acquired using digital densitometry. Results: BiNPs enabled prolonged visual detection of both groove types as they are too large to diffuse into the cartilage. Furthermore, proportional ioxaglate diffusion inside the tissue allowed differentiation between the lesion and ungrooved articular cartilage (3 mm from the lesion and contralateral joint). The mean ioxaglate partition in the lesion was 19 percentage points higher (P < 0.001) when compared with the contralateral joint. The digital densitometry and the dual-contrast CECT findings showed good subjective visual agreement. Main limitations: Ex vivo study protocol and a low number of investigated joints. Conclusions: The dual-CECT methodology, used in this study for the first time to image whole equine joints, is capable of effective lesion detection and simultaneous evaluation of the condition of the articular cartilage

Composition of the pericellular matrix modulates the deformation behaviour of chondrocytes in articular cartilage under static loading

The aim was to assess the role of the composition changes in the pericellular matrix (PCM) for the chondrocyte deformation. For that, a three-dimensional finite element model with depth-dependent collagen density, fluid fraction, fixed charge density and collagen architecture, including parallel planes representing the split-lines, was created to model the extracellular matrix (ECM). The PCM was constructed similarly as the ECM, but the collagen fibrils were oriented parallel to the chondrocyte surfaces. The chondrocytes were modelled as poroelastic with swelling properties. Deformation behaviour of the cells was studied under 15% static compression. Due to the depth-dependent structure and composition of cartilage, axial cell strains were highly depth-dependent. An increase in the collagen content and fluid fraction in the PCMs increased the lateral cell strains, while an increase in the fixed charge density induced an inverse behaviour. Axial cell strains were only slightly affected by the changes in PCM composition. We conclude that the PCM composition plays a significant role in the deformation behaviour of chondrocytes, possibly modulating cartilage development, adaptation and degeneration. The development of cartilage repair materials could benefit from this information

Structural, compositional, and functional effects of blunt and sharp cartilage damage on the joint: a 9-month equine groove model study

This study aimed to quantify the long-term progression of blunt and sharp cartilage defects and their effect on joint homeostasis and function of the equine carpus. In nine adult Shetland ponies, the cartilage in the radiocarpal and middle carpal joint of one front limb was grooved (blunt or sharp randomized). The ponies were subjected to an 8-week exercise protocol and sacrificed at 39 weeks. Structural and compositional alterations in joint tissues were evaluated in vivo using serial radiographs, synovial biopsies, and synovial fluid samples. Joint function was monitored by quantitative gait analysis. Macroscopic, microscopic, and biomechanical evaluation of the cartilage, and assessment of subchondral bone parameters were performed ex vivo. Grooved cartilage showed higher OARSI microscopy scores than the contra-lateral sham-operated controls (p <0.0001). Blunt-grooved cartilage scored higher than sharp-grooved cartilage (p = 0.007) and fixed charge density around these grooves was lower (p = 0.006). Equilibrium and instantaneous moduli trended lower in grooved cartilage than their controls (significant for radiocarpal joints). Changes in other tissues included a 3 to 7-fold change in IL-6 expression in synovium from grooved joints at week 23 (p = 0.042) and an increased CPII/C2C ratio in synovial fluid from blunt-grooved joints at week 35 (p = 0.010). Gait analysis outcome revealed mild, gradually increasing lameness. In conclusion, blunt and, to a lesser extent, sharp grooves in combination with a period of moderate exercise, lead to mild degeneration in equine carpal cartilage over a 9-month period, but the effect on overall joint health remains limited. This article is protected by copyright. All rights reserved

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The effect of body configuration on the strain magnitude and distribution within the acetabulum during sideways falls:a finite element approach

Abstract While the incidence of hip fractures has declined during the last decades, the incidence of acetabular fractures resulting from low-energy sideways falls has increased, and the mechanisms responsible for this trend remain unknown. Previous studies have suggested that body configuration during the impact plays an important role in a hip fracture. Thus, the aim of this study was to investigate the effect of body configuration angles (trunk tilt angle, trunk flexion angle, femur horizontal rotation angle, and femur diaphysis angle) on low-energy acetabular fractures via a parametric analysis. A computed tomography–based (CT) finite element model of the ground–proximal femur–pelvis complex was created, and strain magnitude, time-history response, and distribution within the acetabulum were evaluated. Results showed that while the trunk tilt angle and femur diaphysis angle have the greatest effect on strain magnitude, the direction of the fall (lateral vs. posterolateral) contributes to strain distribution within the acetabulum. The results also suggest that strain level and distribution within the proximal femur and acetabulum resulting from a sideways fall are not similar and, in some cases, even opposite. Taken together, our simulations suggest that a more horizontal trunk and femoral shaft at the impact phase can increase the risk of low-energy acetabular fractures

Experimental mechanical strain measurement of tissues

Abstract Strain, an important biomechanical factor, occurs at different scales from molecules and cells to tissues and organs in physiological conditions. Under mechanical strain, the strength of tissues and their micro- and nanocomponents, the structure, proliferation, differentiation and apoptosis of cells and even the cytokines expressed by cells probably shift. Thus, the measurement of mechanical strain (i.e., relative displacement or deformation) is critical to understand functional changes in tissues, and to elucidate basic relationships between mechanical loading and tissue response. In the last decades, a great number of methods have been developed and applied to measure the deformations and mechanical strains in tissues comprising bone, tendon, ligament, muscle and brain as well as blood vessels. In this article, we have reviewed the mechanical strain measurement from six aspects: electro-based, light-based, ultrasound-based, magnetic resonance-based and computed tomography-based techniques, and the texture correlation-based image processing method. The review may help solving the problems of experimental and mechanical strain measurement of tissues under different measurement environments

Ultrasound assessment of human meniscus

Abstract The aim of the present study was to evaluate the applicability of ultrasound imaging to quantitative assessment of human meniscus in vitro. Meniscus samples (n = 26) were harvested from 13 knee joints of non-arthritic human cadavers. Subsequently, three locations (anterior, center and posterior) from each meniscus were imaged with two ultrasound transducers (frequencies 9 and 40 MHz), and quantitative ultrasound parameters were determined. Furthermore, partial-least-squares regression analysis was applied for ultrasound signal to determine the relations between ultrasound scattering and meniscus integrity. Significant correlations between measured and predicted meniscus compositions and mechanical properties were obtained (R² = 0.38–0.69, p &lt; 0.05). The relationship between conventional ultrasound parameters and integrity of the meniscus was weaker. To conclude, ultrasound imaging exhibited a potential for evaluation of meniscus integrity. Higher ultrasound frequency combined with multivariate analysis of ultrasound backscattering was found to be the most sensitive for evaluation of meniscus integrity