Functional Assessment of Human Articular Cartilage Using Second Harmonic Generation (SHG) Imaging: A Feasibility Study.

Many arthroscopic tools developed for knee joint assessment are contact-based, which is challenging for in vivo application in narrow joint spaces. Second harmonic generation (SHG) laser imaging is a non-invasive and non-contact method, thus presenting an attractive alternative. However, the association between SHG-based measures and cartilage quality has not been established systematically. Here, we investigated the feasibility of using image-based measures derived from SHG microscopy for objective evaluation of cartilage quality as assessed by mechanical testing. Human tibial plateaus harvested from nine patients were used. Cartilage mechanical properties were determined using indentation stiffness (Einst) and streaming potential-based quantitative parameters (QP). The correspondence of the cartilage electromechanical properties (Einst and QP) and the image-based measures derived from SHG imaging, tissue thickness and cell viability were evaluated using correlation and logistic regression analyses. The SHG-related parameters included the newly developed volumetric fraction of organised collagenous network (Φcol) and the coefficient of variation of the SHG intensity (CVSHG). We found that Φcol correlated strongly with Einst and QP (ρ = 0.97 and - 0.89, respectively). CVSHG also correlated, albeit weakly, with QP and Einst, (|ρ| = 0.52-0.58). Einst and Φcol were the most sensitive predictors of cartilage quality whereas CVSHG only showed moderate sensitivity. Cell viability and tissue thickness, often used as measures of cartilage health, predicted the cartilage quality poorly. We present a simple, objective, yet effective image-based approach for assessment of cartilage quality. Φcol correlated strongly with electromechanical properties of cartilage and could fuel the continuous development of SHG-based arthroscopy.

Chondrocyte morphology as an indicator of collagen network integrity.

Osteochondral allograft (OCA) transplantation offers an attractive treatment option as it can be used to repair large cartilage defects that otherwise would not heal. The currently accepted criterion for OCA selection for joint reconstruction is the percentage of viable chondrocytes, but this criterion alone may not be sufficient to ensure structural integrity and functional performance of allografts following transplantation. We sought to determine an additional parameter that indicates matrix integrity. We used multi-photon microscopy to quantitatively assess chondrocyte viability, chondrocyte shape, and collagen structure of articular cartilage of OCAs. Chondrocyte shape varied considerably in otherwise macroscopically healthy-looking OCAs with good (>90%) cell viability. Shape varied from the expected ellipsoidal form found in healthy cartilage, to excessively elongated and flattened cells that often contained multiple cytoplasmic processes reminiscent of those observed in fibroblasts. Chondrocytes with abnormal morphology were associated with degradation of their pericellular matrix and disruption of the collagen fiber orientation, reflected by an increase in heterogeneity of second harmonic signal intensity. Cell shape may be an important marker for collagen network integrity in articular cartilage in general and OCAs specifically. We propose that, aside from cell viability, cell shape may be used as an additional criterion measure for the selection of OCAs. OCAs selected for transplantation based on these criteria showed good graft-host integration post-operation. In view of the rapid and nondestructive nature of the current approach, it may be suitable for clinical application in the future.

Evolution of a Novel Tissue Preservation Protocol to Optimize Osteochondral Transplantation Outcomes.

OBJECTIVE: Osteochondral allograft transplantation is a procedure to treat focal osteochondral lesions (OCLs), but is limited by tissue availability, the quality of transplanted tissue, and inconsistent storage protocols. The objective of this study was to assess the clinical outcomes of a novel tissue procurement, storage, and quality control protocol in treating OCLs. DESIGN: Prospective case series. Donor cadaveric tissue was processed, stored, and the tissue quality analyzed using the unique tissue preservation protocol developed at our institution. Advanced cross-sectional imaging was used to size match donor tissue with recipient patients. Osteochondral allografts were transplanted using the Arthrex Allograft OATS. Patients were evaluated with the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Knee injury and Osteoarthritis Outcome Score (KOOS), visual analog scale (VAS), and 36-Item Short Form Survey (SF-36) preoperatively and at 1 year and 2 years postoperatively. RESULTS: Twenty patients (17 knees, 3 shoulders) were included in the study. There was a significant improvement in the following scores: overall WOMAC score, WOMAC function and pain subcategories; KOOS pain, knee-related symptoms, activities of daily living, sports and recreation, and quality of life; SF-36 physical functioning, physical role, pain, and social functioning subcategories; and VAS at all time points postoperatively. There was a significant improvement in WOMAC stiffness at 2 years postoperatively. There were 2 failures, defined by graft subsidence and persistent pain requiring reoperation. CONCLUSION: The protocol developed at our institution for OAT resulted in significant clinical improvement in patients with OCLs and is an improvement on existing tissue storage techniques.

Chondrocyte Deformations Under Mild Dynamic Loading Conditions.

Dynamic deformation of chondrocytes are associated with cell mechanotransduction and thus may offer a new understanding of the mechanobiology of articular cartilage. Despite extensive research on chondrocyte deformations for static conditions, work for dynamic conditions remains rare. However, it is these dynamic conditions that articular cartilage in joints are exposed to everyday, and that seem to promote biological signaling in chondrocytes. Therefore, the objective of this study was to develop an experimental technique to determine the in situ deformations of chondrocytes when the cartilage is dynamically compressed. We hypothesized that dynamic deformations of chondrocytes vastly differ from those observed under steady-state static strain conditions. Real-time chondrocyte geometry was reconstructed at 10, 15, and 20% compression during ramp compressions with 20% ultimate strain, applied at a strain rate of 0.2% s-1, followed by stress relaxation. Dynamic compressive chondrocyte deformations were non-linear as a function of nominal strain, with large deformations in the early and small deformations in the late part of compression. Early compression (up to about 10%) was associated with chondrocyte volume loss, while late compression (> ~ 10%) was associated with cell deformation but minimal volume loss. Force continued to decrease for 5 min in the stress-relaxation phase, while chondrocyte shape/volume remained unaltered after the first minute of stress-relaxation.

The influence of maximal and submaximal cyclic concentric and eccentric exercise on chondrocyte death and synovial fluid proteins in the rabbit knee.

BACKGROUND: Mechanical stimulation of joints regulates the biosynthetic activity of chondrocytes. It has been argued that excessive loading might cause chondrocyte death, leading to degeneration of cartilage and cause osteoarthritis. The aims of this study were to apply a high, short-term loading, and a low intensity, long-term loading protocol to intact joints in life animals and determine changes in synovial fluid and the percentage of dead cells in rabbit knee cartilage. METHOD: Nine rabbits were subjected to unilateral exercise loading consisting of five sets of 10 maximal eccentric knee contractions. Another 6 rabbits were subjected to submaximal concentric contractions for 30 min at 20% of the maximum isometric knee extensor force. Contralateral joints served as unloaded controls. Cell viability was assessed using confocal microscopy. Synovial fluid was analyzed for total protein concentration and total number of identifiable proteins and was compared to protein content of control rabbits (n = 4). FINDINGS: Neither the high-intensity, short-term nor the low-intensity, long-term loading protocol caused increased chondrocyte death compared to the unloaded control joints. Total synovial fluid protein concentration was the same before and after exercise. Following the high-intensity exercise protocol, the number of identifiable proteins was decreased, while following the low-intensity exercise protocol, the number of identifiable proteins was increased compared to control. INTERPRETATION: Chondrocytes are well protected in the intact joint and withstood maximal eccentric muscular loading, and maximal endurance loading. Synovial fluid protein content was changed after exercise, and these changes depended crucially on the type of loading.

Fluorescence recovery after photobleaching: direct measurement of diffusion anisotropy.

Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying diffusion in biological tissues. Most of the existing approaches for the analysis of FRAP experiments assume isotropic diffusion, while only a few account for anisotropic diffusion. In fibrous tissues, such as articular cartilage, tendons and ligaments, diffusion, the main mechanism for molecular transport, is anisotropic and depends on the fibre alignment. In this work, we solve the general diffusion equation governing a FRAP test, assuming an anisotropic diffusivity tensor and using a general initial condition for the case of an elliptical (thereby including the case of a circular) bleaching profile. We introduce a closed-form solution in the spatial coordinates, which can be applied directly to FRAP tests to extract the diffusivity tensor. We validate the approach by measuring the diffusivity tensor of [Formula: see text] FITC-Dextran in porcine medial collateral ligaments. The measured diffusion anisotropy was [Formula: see text] (SE), which is in agreement with that reported in the literature. The limitations of the approach, such as the size of the bleached region and the intensity of the bleaching, are studied using COMSOL simulations.

Effect of strain rate on transient local strain variations in articular cartilage.

The non-homogeneous, anisotropic material properties, and triphasic nature of articular cartilage enables diarthrodial joints to withstand large and complex physiological loading conditions. To develop biomaterials that provide similar functional properties as those found in articular cartilage, it is vital to have knowledge of the strain distributions in cartilage for a large range of loading conditions. Applied stress vs. strain properties of articular cartilage have been measured primarily for static conditions, but the dynamic properties are thought to be more relevant for joint function and cartilage biosynthesis. Furthermore, the dynamic stress-strain properties are expected to vary significantly from those obtained for static, steady-state conditions. Here, we present a method for the determination of axial strain fields throughout the depth of articular cartilage for static loading conditions and dynamic conditions performed at different loading rates. For the conditions tested here, the strain distributions throughout the cartilage depth were more uniform for the dynamic than the static loading conditions, and more uniform for high compared to low strain rates.

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.

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. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2506-2515, 2018.

Current concepts on structure-function relationships in the menisci.

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 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 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.

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.

Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in cell responses to mechanical stimuli that depend on the microscopic approach and the thresholding methods used and may result in contradictory interpretations.

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.