Ziad Abusara

Tie-fibre structure and organization in the knee menisci

The collagenous structure of the knee menisci is integral to the mechanical integrity of the tissue and the knee joint. The tie‐fibre structure of the tissue has largely been neglected, despite previous studies demonstrating its correlation with radial stiffness. This study has evaluated the structure of the tie‐fibres of bovine menisci using 2D and 3D microscopy techniques. Standard collagen and proteoglycan (PG) staining and 2D light microscopy techniques were conducted. For the first time, the collagenous structure of the menisci was evaluated using 3D, second harmonic generation (SHG) microscopy. This technique facilitated the imaging of collagen structure in thick sections (50–100 μm). Imaging identified that tie‐fibres of the menisci arborize from the outer margin of the meniscus toward the inner tip. This arborization is associated with the structural arrangement of the circumferential fibres. SHG microscopy has definitively demonstrated the 3D organization of tie‐fibres in both sheets and bundles. The hierarchy of the structure is related to the organization of circumferential fascicles. Large tie‐fibre sheets bifurcate into smaller sheets to surround circumferential fascicles of decreasing size. The tie‐fibres emanate from the lamellar layer that appears to surround the entire meniscus. At the tibial and femoral surfaces these tie‐fibre sheets branch perpendicularly into the meniscal body. The relationship between tie‐fibres and blood vessels in the menisci was also observed in this study. Tie‐fibre sheets surround the blood vessels and an associated PG‐rich region. This subunit of the menisci has not previously been described. The size of tie‐fibre sheets surrounding the vessels appeared to be associated with the size of blood vessel. These structural findings have implications in understanding the mechanics of the menisci. Further, refinement of the complex structure of the tie‐fibres is important in understanding the consequences of injury and disease in the menisci. The framework of meniscus architecture also defines benchmarks for the development of tissue‐engineered replacements in the future.

In vivo Sarcomere Lengths and Sarcomere Elongations Are Not Uniform across an Intact Muscle

Sarcomere lengths have been a crucial outcome measure for understanding and explaining basic muscle properties and muscle function. Sarcomere lengths for a given muscle are typically measured at a single spot, often in the mid-belly of the muscle, and at a given muscle length. It is then assumed implicitly that the sarcomere length measured at this single spot represents the sarcomere lengths at other locations within the muscle, and force-length, force-velocity, and power-velocity properties of muscles are often implied based on these single sarcomere length measurements. Although, intuitively appealing, this assumption is yet to be supported by systematic evidence. The objective of this study was to measure sarcomere lengths at defined locations along and across an intact muscle, at different muscle lengths. Using second harmonic generation (SHG) imaging technique, sarcomere patterns in passive mouse tibialis anterior (TA) were imaged in a non-contact manner at five selected locations (“proximal,” “distal,” “middle,” “medial,” and “lateral” TA sites) and at three different lengths encompassing the anatomical range of motion of the TA. We showed that sarcomere lengths varied substantially within small regions of the muscle and also for different sites across the entire TA. Also, sarcomere elongations with muscle lengthening were non-uniform across the muscle, with the highest sarcomere stretches occurring near the myotendinous junction. We conclude that muscle mechanics derived from sarcomere length measured from a small region of a muscle may not well-represent the sarcomere length and associated functional properties of the entire muscle.

In situ chondrocyte viscoelasticity

It has been proposed, based on theoretical considerations, that the strain rate-dependent viscoelastic response of cartilage reduces local tissue and cell deformations during cyclic compressions. However, experimental studies have not addressed the in situ viscoelastic response of chondrocytes under static and dynamic loading conditions. In particular, results obtained from experimental studies using isolated chondrocytes embedded in gel constructs cannot be used to predict the intrinsic viscoelastic responses of chondrocytes in situ or in vivo. Therefore, the purpose of this study was to investigate the viscoelastic response of chondrocytes in their native environment under static and cyclic mechanical compression using a novel in situ experimental approach. Cartilage matrix and chondrocyte recovery in situ following mechanical compressions was highly viscoelastic. The observed in situ behavior was consistent with a previous study on in vivo chondrocyte mechanics which showed that it took 5-7 min for chondrocytes to recover shape and volume following virtually instantaneous cell deformations during muscular loading of the knee in live mice. We conclude from these results that the viscoelastic properties of cartilage minimize chondrocyte deformations during cyclic dynamic loading as occurs, for example, in the lower limb joints during locomotion, thereby allowing the cells to reach mechanical and metabolic homeostasis even under highly dynamic loading conditions.

Current concepts on structure–function relationships in the menisci

ABSTRACT The menisci are intricately organized structures that perform many tasks in the knee. We review their structure and function and introduce new data about their tibial and femoral surfaces. As the femur and tibia approach each other when the knee is bearing load, circumferential tension develops in the menisci, enabling the transmission of compressive load between the femoral and tibial cartilage layers. A low shear modulus is necessary for the tissue to adapt its shape to the changing radius of the femur as that bone moves relative to the tibia during joint articulation. The organization of the meniscus facilitates its functions. In the outer region of the menisci, intertwined collagen fibrils, fibers, and fascicles with predominantly circumferential orientation are prevalent; these structures are held together by radial tie fibers and sheets. Toward the inner portion of the menisci, there is more proteoglycan and the structure becomes more cartilage-like. The transition between these structural forms is gradual and seamless. The flexible roots, required for rigid body motion of the menisci, meld with both the tibia and the outer portion of the menisci to maintain continuity for resistance to the circumferential tension. Our new data demonstrate that the femoral and tibial surfaces of the menisci are structurally analogous to the surfaces of articular cartilage, enabling consistent modes of lubrication and load transfer to occur at the interfacing surfaces throughout motion. The structure and function of the menisci are thus shown to be strongly related to one another: form clearly complements function.

In Vivo Dynamic Deformation of Articular Cartilage in Intact Joints Loaded by Controlled Muscular Contractions

When synovial joints are loaded, the articular cartilage and the cells residing in it deform. Cartilage deformation has been related to structural tissue damage, and cell deformation has been associated with cell signalling and corresponding anabolic and catabolic responses. Despite the acknowledged importance of cartilage and cell deformation, there are no dynamic data on these measures from joints of live animals using muscular load application. Research in this area has typically been done using confined and unconfined loading configurations and indentation testing. These loading conditions can be well controlled and allow for accurate measurements of cartilage and cell deformations, but they have little to do with the contact mechanics occurring in a joint where non-congruent cartilage surfaces with different material and functional properties are pressed against each other by muscular forces. The aim of this study was to measure in vivo, real time articular cartilage deformations for precisely controlled static and dynamic muscular loading conditions in the knees of mice. Fifty and 80% of the maximal knee extensor muscular force (equivalent to approximately 0.4N and 0.6N) produced average peak articular cartilage strains of 10.5±1.0% and 18.3±1.3% (Mean ± SD), respectively, during 8s contractions. A sequence of 15 repeat, isometric muscular contractions (0.5s on, 3.5s off) of 50% and 80% of maximal muscular force produced cartilage strains of 3.0±1.1% and 9.6±1.5% (Mean ± SD) on the femoral condyles of the mouse knee. Cartilage thickness recovery following mechanical compression was highly viscoelastic and took almost 50s following force removal in the static tests.

Menisci protect chondrocytes from load-induced injury

Menisci in the knee joint are thought to provide stability, increased contact area, decreased contact pressures, and offer protection to the underlying articular cartilage and bone during joint loading. Meniscal loss or injury is typically accompanied by degenerative changes in the knee, leading to an increased risk for osteoarthritis in animals including humans. However, the detailed mechanisms underlying joint degeneration and the development of osteoarthritis remain largely unknown, and the acute effects of meniscal loss have not been studied systematically. We developed a microscopy-based system to study microscale joint mechanics in living mice loaded by controlled muscular contractions. Here, we show how meniscal loss is associated with rapid chondrocyte death (necrosis) in articular cartilage within hours of injury, and how intact menisci protect chondrocytes in vivo in the presence of intense muscle-based joint loading and/or injury to the articular cartilage. Our findings suggest that loading the knee after meniscal loss is associated with extensive cell death in intact and injured knees, and that early treatment interventions should be aimed at preventing chondrocyte death.

A compression system for studying depth-dependent mechanical properties of articular cartilage under dynamic loading conditions

The biological activities of chondrocytes are influenced by the mechanical characteristics of their environment. The overall real-time mechanical response of cartilage has been investigated earlier. However, the instantaneous local mechano-biology of cartilage has not been investigated in detail under dynamic loading conditions. In order to address this gap in the literature, we designed a compression testing device and implemented a dual photon microscopy technique with the goal of measuring local mechanical and biological responses of articular cartilage under dynamic loading conditions. The details of the compression system and results of a pilot study are presented here. A 15% ramp compression at a rate of 0.003/s with a subsequent stress relaxation phase was applied to the cartilage explant samples. The extra cellular matrix was imaged throughout the entire thickness of the cartilage sample, and local tissue strains were measured during the compression and relaxation phase. The axial compressive strains in the middle and superficial zones of cartilage were observed to increase during the relaxation phase: this was a new finding, suggesting the importance of further investigations on the real-time local behavior of cartilage. The compression system showed promising results for investigating the dynamic, real-time mechanical response of articular cartilage, and can now be used to reveal the instantaneous mechanical and biological responses of chondrocytes in response to dynamic loading conditions.

Fibril deformation under load of the rabbit Achilles tendon and medial collateral ligament femoral entheses: ENTHESIS MICROSCOPIC LOAD RESPONSE

Microscopic visualization under load of the region connecting ligaments/tendons to bone, the enthesis, has been performed previously; however, specific investigation of individual fibril deformation may add insight to such studies. Detailed visualization of fibril deformation would inform on the mechanical strategies employed by this tissue in connecting two mechanically disparate materials. Clinically, an improved understanding of enthesis mechanics may help guide future restorative efforts for torn or injured ligaments/tendons, where the enthesis is often a point of weakness. In this study, a custom ligament/tendon enthesis loading device was designed and built, a unique method of sample preparation was devised, and second harmonic and two‐photon fluorescence microscopy were used to capture the fibril‐level load response of the rabbit Achilles tendon and medial collateral ligament femoral entheses. A focus was given to investigation of the mechanical problem of fibril embedment. Resultant images indicate a rapid (occurring over approximately 60 μm) change in fibril orientation at the interface of ligament/tendon and calcified fibrocartilage early in the loading regime, before becoming relatively constant. Such a change in fibril angle helps confirm the materially graded region demonstrated by others, while, in this case, providing additional insight into fibril bending. We speculate that the scale of the mechanical problem (i.e., fibril diameters being on the order of 250 nm) allows fibrils to bend over the small (relative to the imaging field of view, but large relative to fibril diameter) distances observed; thus, potentially lessening required embedment lengths. Nevertheless, this behavior merits further investigation to be confirmed.

In-vivo Cartilage Mechano-Biology: How to Make Progress in Osteoarthritis Research

Cartilage mechano-biology has typically been performed in isolated tissue explants exposed to hydrostatic pressure, or subjected to confined or unconfined loading conditions. Although these approaches offer great control over the experiments, they do not reflect the physiological loading and boundary conditions of cartilage in the intact joint. Here, we will describe recent approaches that allow for evaluation of cartilage and chondrocyte biomechanics and signaling in the intact cartilage and intact joint of live animals. Although not as well controlled as experiments performed on tissue explants, the in vivo work offers the opportunity to study chondrocyte mechanics and signaling as well as tissue biomechanics for physiologically relevant loading situations and with natural boundary conditions.