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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 (E inst ) and streaming potential-based quantitative parameters (QP). The correspondence of the cartilage electromechanical properties (E inst 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 (Φ col ) and the coefficient of variation of the SHG intensity (CV SHG ). We found that Φ col correlated strongly with E inst and QP (ρ = 0.97 and - 0.89, respectively). CV SHG also correlated, albeit weakly, with QP and E inst , (|ρ| = 0.52-0.58). E inst and Φ col were the most sensitive predictors of cartilage quality whereas CV SHG 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. Φ col correlated strongly with electromechanical properties of cartilage and could fuel the continuous development of SHG-based arthroscopy., (© 2024. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Osteoarthritis (OA) is a multifactorial joint disease characterized by articular cartilage degradation. Risk factors for OA include joint trauma, obesity, and inflammation, each of which can affect joint health independently, but their interaction and the associated consequences of such interaction were largely unexplored. Here, we studied compositional and structural alterations in knee joint cartilages of Sprague-Dawley rats exposed to two OA risk factors: joint injury and diet-induced obesity. Joint injury was imposed by surgical transection of anterior cruciate ligaments (ACLx), and obesity was induced by a high fat/high sucrose diet. Depth-dependent proteoglycan (PG) content and collagen structural network of cartilage were measured from histological sections collected previously in Collins et al.. (2015). We found that ACLx primarily affected the superficial cartilages. Compositionally, ACLx led to reduced PG content in lean animals, but increased PG content in obese rats. Structurally, ACLx caused disorganization of collagenous network in both lean and obese animals through increased collagen orientation in the superficial tissues and a change in the degree of fibrous alignment. However, the cartilage degradation attributed to joint injury and obesity was not necessarily additive when the two risk factors were present simultaneously, particularly for PG content and collagen orientation in the superficial tissues. Interestingly, sham surgeries caused a through-thickness disorganization of collagen network in lean and obese animals. We conclude that the interactions of multiple OA risk factors are complex and their combined effects cannot be understood by superposition principle. Further research is required to elucidate the interactive mechanism between OA subtypes.

Sarcomere lengths are non-uniform on all structural levels of mammalian skeletal muscle. These non-uniformities have been associated with a variety of mechanical properties, including residual force enhancement and depression, creep, increased force capacity, and extension of the plateau of the force-length relationship. However, the nature of sarcomere length non-uniformities has not been explored systematically. The purpose of this study was to determine the properties of sarcomere length non-uniformities in active and passive muscle. Single myofibrils of rabbit psoas ( n = 20; with 412 individual sarcomeres) were subjected to three activation/deactivation cycles and individual sarcomere lengths were measured at 4 passive and 3 active points during the activation/deactivation cycles. The myofibrils were divided into three groups based on their initial average sarcomere lengths: short, intermediate, and long average sarcomere lengths of 2.7, 3.2, and 3.6 µm. The primary results were that sarcomere length non-uniformities did not occur randomly but were governed by some structural and/or contractile properties of the sarcomeres and that sarcomere length non-uniformities increased when myofibrils went from the passive to the active state. We propose that the mechanisms that govern the systematic sarcomere lengths non-uniformities observed in active and passive myofibrils may be associated with the variable number of contractile proteins and the variable number and the adjustable stiffness of titin filaments in individual sarcomeres., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Leonard, Han, Moo and Herzog.)

Tissue swelling represents an early sign of osteoarthritis, reflecting osmolarity changes from iso- to hypo-osmotic in the diseased joints. Increased tissue hydration may drive cell swelling. The opposing cartilages in a joint may swell differently, thereby predisposing the more swollen cartilage and cells to mechanical injuries. However, our understanding of the tissue-cell interdependence in osmotically loaded joints is limited as tissue and cell swellings have been studied separately. Here, we measured tissue and cell responses of opposing patellar (PAT) and femoral groove (FG) cartilages in lapine knees exposed to an extreme hypo-osmotic challenge. We found that the tissue matrix and most cells swelled during the hypo-osmotic challenge, but to a different extent (tissue: <3%, cells: 11%-15%). Swelling-induced tissue strains were anisotropic, showing 2%-4% stretch and 1%-2% compression along the first and third principal directions, respectively. These strains were amplified by 5-8 times in the cells. Interestingly, the first principal strains of tissue and cells occurred in different directions (60-61° for tissue vs. 8-13° for cells), suggesting different mechanisms causing volume expansion in the tissue and the cells. Instead of the continuous swelling observed in the tissue matrix, >88% of cells underwent regulatory volume decrease to return to their pre-osmotic challenge volumes. Cell shapes changed in the early phase of swelling but stayed constant thereafter. Kinematic changes to tissue and cells were larger for PAT cartilage than for FG cartilage. We conclude that the swelling-induced deformation of tissue and cells is anisotropic. Cells actively restored volume independent of the surrounding tissues and seemed to prioritize volume restoration over shape restoration. Our findings shed light on tissue-cell interdependence in changing osmotic environments that is crucial for cell mechano-transduction in swollen/diseased tissues., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Fluid pressure develops transiently within mechanically-loaded, cell-embedding hydrogels, but its magnitude depends on the intrinsic material properties of the hydrogel and cannot be easily altered. The recently developed melt-electrowriting (MEW) technique enables three-dimensional printing of structured fibrous mesh with small fibre diameter (20 μm). The MEW mesh with 20 μm fibre diameter can synergistically increase the instantaneous mechanical stiffness of soft hydrogels. However, the reinforcing mechanism of the MEW meshes is not well understood, and may involve load-induced fluid pressurisation. Here, we examined the reinforcing effect of MEW meshes in three hydrogels: gelatin methacryloyl (GelMA), agarose and alginate, and the role of load-induced fluid pressurisation in the MEW reinforcement. We tested the hydrogels with and without MEW mesh (i.e., hydrogel alone, and MEW-hydrogel composite) using micro-indentation and unconfined compression, and analysed the mechanical data using biphasic Hertz and mixture models. We found that the MEW mesh altered the tension-to-compression modulus ratio differently for hydrogels that are cross-linked differently, which led to a variable change to their load-induced fluid pressurisation. MEW meshes only enhanced the fluid pressurisation for GelMA, but not for agarose or alginate. We speculate that only covalently cross-linked hydrogels (GelMA) can effectively tense the MEW meshes, thereby enhancing the fluid pressure developed during compressive loading. In conclusion, load-induced fluid pressurisation in selected hydrogels was enhanced by MEW fibrous mesh, and may be controlled by MEW mesh of different designs in the future, thereby making fluid pressure a tunable cell growth stimulus for tissue engineering involving mechanical stimulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cartilage cracks disrupt tissue mechanics, alter cell mechanobiology, and often trigger tissue degeneration. Yet, some tissue cracks heal spontaneously. A primary factor determining the fate of tissue cracks is the compression-induced mechanics, specifically whether a crack opens or closes when loaded. Crack deformation is thought to be affected by tissue structure, which can be probed by quantitative polarized light microscopy (PLM). It is unclear how the PLM measures are related to deformed crack morphology. Here, we investigated the relationship between PLM-derived cartilage structure and mechanical behavior of tissue cracks by testing if PLM-derived structural measures correlated with crack morphology in mechanically indented cartilages., Methods: Knee joint cartilages harvested from mature and immature animals were used for their distinct collagenous fibrous structure and composition. The cartilages were cut through thickness, indented over the cracked region, and processed histologically. Sample-specific birefringence was quantified as two-dimensional (2D) maps of azimuth and retardance, two measures related to local orientation and degree of alignment of the collagen fibers, respectively. The shape of mechanically indented tissue cracks, measured as depth-dependent crack opening, were compared with azimuth, retardance, or "PLM index," a new parameter derived by combining azimuth and retardance., Results: Of the three parameters, only the PLM index consistently correlated with the crack shape in immature and mature tissues., Conclusion: In conclusion, we identified the relative roles of azimuth and retardance on the deformation of tissue cracks, with azimuth playing the dominant role. The applicability of the PLM index should be tested in future studies using naturally-occurring tissue cracks.

The deformation of articular cartilage and its cells at the micro-scale during dynamic activities such as gait has high mechanoregulatory importance. Measuring the cellular geometries during such dynamics has been limited by the rate of microscopic image acquisition. The introduction of resonating mirrors for image rasterization (resonant scanning), rather than the conventional servo control (galvano scanning), has significantly improved the scanning rate by more than 100×. However, the high scanning rate comes at the cost of image quality, thereby posing challenges in image processing. Here, resonance-driven 3-D laser microscopy is used to observe the transient, micro-scale deformation of articular cartilage and its cells under osmotic challenge conditions. Custom image segmentation and deformable registration software were implemented for analysis of the resonance-scanned microscopy data. The software exhibited robust and accurate performance on the osmotic swelling measurements, as well as quantitative validation testing. The resonance-scanning protocol and developed analysis software allow for simultaneous strain calculation of both the local tissue and cells, and are thus a valuable tool for real-time probing of the cell-matrix interactions that are highly relevant in the fields of orthopedic biomechanics, cell mechanobiology, and functional tissue engineering., (Copyright © 2023 by ASME.)

The function of articular cartilage as a load-bearing connective tissue is derived primarily from a balanced interaction between the swelling proteoglycan (PG) matrix and tension-resistant collagen fibrous network. Such balance is compromised during joint disease such as osteoarthritis (OA) due to degradation to PGs and/or collagens. While the PG degradation is generally thought to be related to a loss of protein abundance, the collagenous degradation is more complex as it can be caused independently by a decrease of collagen content, disorganisation of fibrous structure and softening of individual collagen fibrils. A comprehensive understanding of the initial trajectories of degradation of PGs and collagen network can improve our chance of finding potential therapeutic solutions for OA. Here, we developed geometrically, structurally, and compositionally realistic and sample-specific Finite Element (FE) models under the framework of multiphasic mixture theory, from which the elastic moduli of collagen fibres and the PG load-bearing quality in healthy and diseased cartilages were estimated by numerical optimisation of the multi-step indentation stress relaxation force-time curves. We found the intrinsic quality of collagen fibres, measured by their elastic moduli, to stay constant for healthy and diseased cartilages. Combining with previous findings which show unaltered collagen content during early stages of OA, our results suggest the disorganisation of collagen fibrous network as the first form of collagenous degradation in osteoarthritic cartilage. We also found that PG degradation involves not only a loss of protein abundance, but also the quality of the remaining PGs in generating sufficient osmotic pressure for load bearing. This study sheds light on the mechanism of OA pathogenesis and highlights the restoration of collageneous organisation in cartilage as key medical intervention for OA. STATEMENT OF SIGNIFICANCE: Collagen network in articular cartilage consists of individual fibres that are organised into depth-dependent structure specialised for joint load-bearing and lubrication. During osteoarthritis, the collagen network undergoes mechanical degradation, but it is unclear if a loss of content, disorganisation of fibrous structure, or softening of individual fibres causes this degeneration. Using mechanical indentation, Finite Element modelling, and numerical optimisation methods, we determined that individual fibres did not soften in early disease stage. Together with previous findings showing unaltered collagen content, our results pinpoint the disorganisation of collagen structure as the main culprit for early collagenous degradation in osteoarthritic cartilage. Thus, early restoration in cartilage of collagen organisation, instead of individual fibre quality, may be key to slow osteoarthritis development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Histochemical characterization of proteoglycan content in articular cartilage is important for the understanding of osteoarthritis pathogenesis. However, cartilage cells may interfere with the measurement of matrix proteoglycan content in small animal models (e.g. mice and rats) due to the high cell volume fraction (38%) in mice compared to human tissue (~1%). We investigated whether excluding the cells from image analysis affects the histochemically measured proteoglycan content of rat knee joint cartilage and assessed the effectiveness of a deep learning algorithm-based tool named U-Net in cell segmentation., Design: Histological sections were stained with Safranin-O, after which optical densities were measured using digital densitometry to estimate proteoglycan content. U-Net was trained with 600 annotated Safranin-O cartilage images for exclusion of cells from the cartilage extracellular matrix. Optical densities of the ECM with and without cells were compared as a function of normalized tissue depth., Results: U-Net cell segmentation was accurate, with the measured cell area fraction following largely that of ground-truth images (average difference: 4.3%). Cell area fraction varied as a function of tissue depth and took up 8-21% of the tissue area. The exclusion of cells from the analysis led to an increase in the analyzed depth-dependent optical density of cartilage by approximately 0.6-1.8% ( p < 0.01)., Conclusions: Although the effect of cells on the analyzed proteoglycan content is small, it should be considered for improved sensitivity, especially at the onset of the disease during which cells may proliferate in small animals.

Degeneration of articular cartilage is often triggered by a small tissue crack. As cartilage structure and composition change with age, the mechanics of cracked cartilage may depend on the tissue age, but this relationship is poorly understood. Here, we investigated cartilage mechanics and crack deformation in immature and mature cartilage exposed to a full-thickness tissue crack using indentation testing and histology, respectively. When a cut was introduced, tissue cracks opened wider in the mature cartilage compared to the immature cartilage. However, the opposite occurred upon mechanical indentation over the cracked region. Functionally, the immature-cracked cartilages stress-relaxed faster, experienced increased tissue strain, and had reduced instantaneous stiffness, compared to the mature-cracked cartilages. Taken together, mature cartilage appears to withstand surface cracks and maintains its mechanical properties better than immature cartilage and these superior properties can be explained by the structure of their collagen fibrous network., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

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.

Abnormal loading of the knee due to injuries or obesity is thought to contribute to the development of osteoarthritis (OA). Small animal models have been used for studying OA progression mechanisms. However, numerical models to study cartilage responses under dynamic loading in preclinical animal models have not been developed. Here we present a musculoskeletal finite element model of a rat knee joint to evaluate cartilage biomechanical responses during a gait cycle. The rat knee joint geometries were obtained from a 3-D MRI dataset and the boundary conditions regarding loading in the joint were extracted from a musculoskeletal model of the rat hindlimb. The fibril-reinforced poroelastic (FRPE) properties of the rat cartilage were derived from data of mechanical indentation tests. Our numerical results showed the relevance of simulating anatomical and locomotion characteristics in the rat knee joint for estimating tissue responses such as contact pressures, stresses, strains, and fluid pressures. We found that the contact pressure and maximum principal strain were virtually constant in the medial compartment whereas they showed the highest values at the beginning of the gait cycle in the lateral compartment. Furthermore, we found that the maximum principal stress increased during the stance phase of gait, with the greatest values at midstance. We anticipate that our approach serves as a first step towards investigating the effects of gait abnormalities on the adaptation and degeneration of rat knee joint tissues and could be used to evaluate biomechanically-driven mechanisms of the progression of OA as a consequence of joint injury or obesity., Competing Interests: The authors have declared that no competing interests exist.

The specialized pericellular matrix (PCM) surrounding chondrocytes within articular cartilage is critical to the tissue's health and longevity. Growing evidence suggests that PCM alterations are ubiquitous across all trajectories of osteoarthritis, a crippling and prevalent joint disease. The PCM geometry is of particular interest as it influences the cellular mechanical environment. Observations of asymmetrical PCM thickness have been reported, but a quantified characterization is lacking. To this end, a novel microscopy protocol was developed and applied to acquire images of the PCM surrounding live cells. Morphometric analysis indicated a statistical bias towards thicker PCM on the inferior cellular surface. The mechanical effects of this bias were investigated with multiscale modelling, which revealed potentially damaging, high tensile strains in the direction perpendicular to the membrane and localized on the inferior surface. These strains varied substantially between PCM asymmetry cases. Simulations with a thicker inferior PCM, representative of the observed geometry, resulted in strain magnitudes approximately half of those calculated for a symmetric geometry, and a third of those with a thin inferior PCM. This strain attenuation suggests that synthesis of a thicker inferior PCM may be a protective adaptation. PCM asymmetry may thus be important in cartilage development, pathology, and engineering., (© 2021. The Author(s) under exclusive licence to Biomedical Engineering Society.)

Cracks in articular cartilage compromise tissue integrity and mechanical properties and lead to chondral lesions if untreated. An understanding of the mechanics of cracked cartilage may help in the prevention of cartilage deterioration and the development of tissue-engineered substitutes. The degeneration of cartilage in the presence of cracks may depend on the ultrastructure and composition of the tissue, which changes with aging, disease and habitual loading. It is unknown if the structural and compositional differences between immature and mature cartilage affect the mechanics of cartilage cracks, possibly predisposing one to a greater risk of degeneration than the other. We used a fibre-reinforced poro-viscoelastic swelling material model that accounts for large deformations and tension-compression non-linearity, and the finite element method to investigate the role of cartilage structure and composition on crack morphology and tissue mechanics. We demonstrate that the crack morphology predicted by our theoretical model agrees well with the histo-morphometric images of young and mature cracked cartilages under indentation loading. We also determined that the crack morphology was primarily dependent on collagen fibre orientation which differs as a function of cartilage depth and tissue maturity. The arcade-like collagen fibre orientation, first discussed by Benninghoff in his classical 1925 paper, appears to be beneficial for slowing the progression of tissue cracks by 'sealing' the crack and partially preserving fluid pressure during loading. Preservation of the natural load distribution between solid and fluid constituents of cartilage may be a key factor in slowing or preventing the propagation of tissue cracks and associated tissue matrix damage. STATEMENT OF SIGNIFICANCE: Cracks in articular cartilage can be detrimental to joint health if not treated, but it is not clear how they propagate and lead to tissue degradation. We used an advanced numerical model to determine the role of cartilage structure and composition on crack morphology under loading. Based on the structure and composition found in immature and mature cartilages, our model successfully predicts the crack morphology in these cartilages and determines that collagen fibre as the major determinant of crack morphology. The arcade-like Benninghoff collagen fibre orientation appears to be crucial in 'sealing' the tissue crack and preserves normal fluid-solid load distribution in cartilage. Inclusion of the arcade-like fibre orientation in tissue-engineered construct may help improve its integration within the host tissue., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The seemingly uniform striation pattern of skeletal muscles, quantified in terms of sarcomere lengths (SLs), is inherently non-uniform across all hierarchical levels. The SL non-uniformity theory has been used to explain the force creep in isometric contractions, force depression following shortening of activated muscle, and residual force enhancement following lengthening of activated muscle. Our understanding of sarcomere contraction dynamics has been derived primarily from in vitro experiments using regular bright-field light microscopy or laser diffraction techniques to measure striation/diffraction patterns in isolated muscle fibers or myofibrils. However, the collagenous extracellular matrices present around the muscle fibers, as well as the complex architecture in the whole muscles may lead to different contraction dynamics of sarcomeres than seen in the in vitro studies. Here, we used multi-photon excitation microscopy to visualize in situ individual sarcomeres in intact muscle tendon units (MTUs) of mouse tibialis anterior (TA), and quantified the temporal changes of SL distribution as a function of SLs in relaxed and maximally activated muscles for quasi-steady state, fixed-end isometric conditions. The corresponding muscle forces were simultaneously measured using a force transducer. We found that SL non-uniformity, quantified by the coefficient of variation (CV) of SLs, decreased at a rate of 1.9-3.1%/s in the activated muscles, but remained constant in the relaxed muscles. The force loss during the quasi-steady state likely did not play a role in the decrease of SL non-uniformity, as similar force losses were found in the activated and relaxed muscles, but the CV of SLs in the relaxed muscles underwent negligible change over time. We conclude that sarcomeres in the mid-belly of maximally contracting whole muscles constantly re-organize their lengths into a more uniform pattern over time. The molecular mechanisms accounting for SL non-uniformity appear to differ in active and passive muscles, and need further elucidation, as do the functional implications of the SL non-uniformity., (Copyright © 2020 Moo and Herzog.)

The periodic striation pattern in skeletal muscle reflects the length of the basic contractile unit: the sarcomere. More than half a century ago, Gordon, Huxley and Julian provided strong support for the 'sliding filament' theory through experiments with single muscle fibres. The sarcomere force-length (FL) relationship has since been extrapolated to whole muscles in an attempt to unravel in vivo muscle function. However, these extrapolations were frequently associated with non-trivial assumptions, such as muscle length changes corresponding linearly to SL changes. Here, we determined the in situ sarcomere FL relationship in a whole muscle preparation by simultaneously measuring muscle force and individual SLs in an intact muscle-tendon unit (MTU) using state-of-the-art multi-photon excitation microscopy. We found that despite great SL non-uniformity, the mean value of SLs measured from a minute volume of the mid-belly, equivalent to about 5×10 -6 % of the total muscle volume, agrees well with the theoretically predicted FL relationship, but only if the precise contractile filament lengths are known, and if passive forces from parallel elastic components and activation-associated sarcomere shortening are considered properly. As SLs are not uniformly distributed across the whole muscle and changes in SL with muscle length are location dependent, our results may not be valid for the proximal or distal parts of the muscle. The approach described here, and our findings, may encourage future studies to determine the role of SL non-uniformity in influencing sarcomere FL properties in different muscles and for different locations within single muscles., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Sarcomere length (SL) instability and SL non-uniformity have been used to explain fundamental properties of skeletal muscles, such as creep, force depression following active muscle shortening and residual force enhancement following active stretching of muscles. Regarding residual force enhancement, it has been argued that active muscle stretching causes SL instability, thereby increasing SL non-uniformity. However, we recently showed that SL non-uniformity is not increased by active muscle stretching, but it remains unclear if SL stability is affected by active stretching. Here, we used single myofibrils of rabbit psoas muscle and measured SL non-uniformity and SL instability during isometric contractions and for isometric contractions following active stretching at average SLs corresponding to the descending limb of the force-length relationship. We defined isometric contractions as contractions during which mean SL remained constant. SL instability was quantified by the rate of change of individual SLs over the course of steady-state isometric force and SL non-uniformity was defined as deviations of SLs from the mean SL at an instant of time. We found that whereas the mean SL remained constant during isometric contraction, by definition, individual SLs did not. SLs were more stable in the force-enhanced, isometric state following active stretching compared with the isometric reference state. We also found that SL instability was not correlated with the rate of change of SL non-uniformity. Also, SL non-uniformity was not different in the isometric and the post-stretch isometric contractions. We conclude that since SL is more stable but similarly non-uniform in the force-enhanced compared with the corresponding isometric reference contraction, it appears unlikely that either SL instability or SL non-uniformity contribute to the residual force enhancement property of skeletal muscle., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The instantaneous sarcomere length (SL) is regarded as an important indicator of the functional properties of striated muscle. Previously, we found greater sarcomere elongations at the distal end compared to the mid-portion in the mouse tibialis anterior (TA) when the muscle was stretched passively. Here, we wanted to see if SL dispersions increase with activation, as has been observed in single myofibrils, and if SL dispersions differ for different locations in a muscle. Sarcomere lengths were measured at a mid- and a distal location of the TA in live mice using second harmonic generation imaging. Muscle force was measured using a tendon force transducer. We found that SL dispersions increased substantially from the passive to the active state, and were the same for the mid- and distal portions of TA. Sarcomere length non-uniformities within a segment of ~30 serial sarcomeres were up to 1.0 µm. We conclude from these findings that passive, mean SLs obtained from a single location are not necessarily representative of the distribution of SL in active muscle, and thus may be misinterpreted when deriving muscle mechanical properties, such as the force-length relationship. In view of these findings, it seems crucial to determine how SL distributions within a muscle relate to the most fundamental properties of muscle, such as the maximal isometric force.

Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues., Statement of Significance: We presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro-strain analysis that allowed for detailed qualitative and quantitative assessment of the 3D tissue kinematics. The approach presented here can also be applied to other biological tissues such as meniscus and annulus fibrosus, as well as tissue-engineered tissues for the characterization of their mechanical properties. This imaging technique opens doors for experimental and theoretical investigation on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The sarcomere force-length relationship has been extensively used to predict muscle force potential. The common practice is to measure the mean sarcomere length (SL) in a relaxed muscle at a single location and at a given length, and this mean SL is assumed to represent the SLs at other locations across the muscle. However, in a previous study, we found that SLs are highly non-uniform across an intact passive muscle. Moreover, SL non-uniformity increases during activation in single myofibril experiments. Myofibrils lack some structural proteins that comprise an intact muscle, and therefore, the increased SL dispersion upon activation seen in myofibrils may not occur in intact whole muscle. The objectives of the current study were (i) to measure the distribution of SLs in an activated intact muscle; and (ii) to assess the feasibility of using the mean SL measured at a specific location of the muscle to predict muscle force. Using state-of-the-art multi-photon microscopy and a miniature tendon force transducer, in vivo sarcomeres in the mouse tibialis anterior were imaged simultaneously with muscle force during isometric tetanic contractions. We found that in vivo SL dispersion increased substantially during activation and reached average differences of ~1.0 μm. These differences in SL are associated with theoretical force differences of 70-100% of the maximal isometric force. Furthermore, SLs measured at a single location in the passive muscle were poor predictors of active force potential. Although mean SLs in the activated muscle were better predictors of force potential, predicted forces still differed by as much as 35% from the experimentally measured maximal isometric forces.

In actively stretched skeletal muscle sarcomeres, titin-based force is enhanced, increasing the stiffness of active sarcomeres. Titin force enhancement in sarcomeres is vastly reduced in mdm , a genetic mutation with a deletion in titin. Whether loss of titin force enhancement is associated with compensatory mechanisms at higher structural levels of organization, such as single fibres or entire muscles, is unclear. The aim of this study was to determine whether mechanical deficiencies in titin force enhancement are also observed at the fibre level, and whether mechanisms compensate for the loss of titin force enhancement. Single skinned fibres from control and mutant mice were stretched actively and passively beyond filament overlap to observe titin-based force. Mutant fibres generated lower contractile stress (force divided by cross-sectional area) than control fibres. Titin force enhancement was observed in control fibres stretched beyond filament overlap, but was overshadowed in mutant fibres by an abundance of collagen and high variability in mechanics. However, titin force enhancement could be measured in all control fibres and most mutant fibres following short stretches, accounting for ∼25% of the total stress following active stretch. Our results show that the partial loss of titin force enhancement in myofibrils is not preserved in all mutant fibres and this mutation likely affects fibres differentially within a muscle. An increase in collagen helps to reestablish total force at long sarcomere lengths with the loss in titin force enhancement in some mutant fibres, increasing the overall strength of mutant fibres., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Frogs' outstanding jumping ability has been associated with a high power output from the leg extensor muscles. Two main theories have emerged to explain the high power output of the frog leg extensor muscles, either (i) the contractile conditions of all leg extensor muscles are optimized in terms of muscle length and speed of shortening, or (ii) maximal power is achieved through a dynamic catch mechanism that uncouples fibre shortening from the corresponding muscle-tendon unit shortening. As in vivo instantaneous power generation in frog hind limb muscles during jumping has never been measured directly, it is hard to distinguish between the two theories. In this study, we determined the instantaneous variable power output of the plantaris longus (PL) of Lithobates pipiens (also known as Rana pipiens), by directly measuring the in vivo force, length change, and speed of muscle and fibre shortening in near maximal jumps. Fifteen near maximal jumps (> 50cm in horizontal distance) were analyzed. High instantaneous peak power in PL (536 ± 47 W/kg) was achieved by optimizing the contractile conditions in terms of the force-length but not the force-velocity relationship, and by a dynamic catch mechanism that decouples fascicle shortening from muscle-tendon unit shortening. We also found that the extra-muscular free tendon likely amplifies the peak power output of the PL by modulating fascicle shortening length and shortening velocity for optimum power output, but not by releasing stored energy through recoiling as the tendon only started recoiling after peak PL power had been achieved.

Impact loading results in chondrocyte death. Previous studies implicated high tensile strain rates in chondrocyte membranes as the cause of impact-induced cell deaths. However, this hypothesis relies on the untested assumption that chondrocyte membranes unfold in vivo during physiological tissue compression, but do not unfold during impact loading. Although membrane unfolding has been observed in isolated chondrocytes during osmotically induced swelling and mechanical compression, it is not known if membrane unfolding also occurs in chondrocytes embedded in their natural extracellular matrix. This study was aimed at quantifying changes in membrane morphology of in situ superficial zone chondrocytes during slow physiological cartilage compression. Bovine cartilage-bone explants were loaded at 5 μm/s to nominal compressive strains ranging from 0% to 50%. After holding the final strains for 45 min, the loaded cartilage was chemically pre-fixed for 12 h. The cartilage layer was post-processed for visualization of cell ultrastructure using electron microscopy. The changes in membrane morphology in superficial zone cells were quantified from planar electron micrographs by measuring the roughness and the complexity of the cell surfaces. Qualitatively, the cell surface ruffles that existed before loading disappeared when cartilage was loaded. Quantitatively, the roughness and complexity of cell surfaces decreased with increasing load magnitudes, suggesting a load-dependent use of membrane reservoirs. Chondrocyte membranes unfold in a load-dependent manner when cartilage is compressed. Under physiologically meaningful loading conditions, the cells likely expand their surface through unfolding of the membrane ruffles, and therefore avoid direct stretch of the cell membrane. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:304-310, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

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.

Background: Impact loading causes cartilage damage and cell death. Pre-loading prior to impact loading may protect cartilage and chondrocytes. However, there is no systematic evidence and understanding of the effects of pre-load strategies on cartilage damage and chondrocyte death. This study aimed at determining the effects of the pre-load history on impact-induced chondrocyte death in an intact joint., Methods: Patellofemoral joints from 42 rabbits were loaded by controlled quadriceps muscle contractions and an external impacter. Two extreme muscular loading conditions were used: (i) a short-duration, high intensity, static muscle contraction, and (ii) a long-duration, low-intensity, cyclic muscle loading protocol. A 5-Joule centrally-oriented, gravity-accelerated impact load was applied to the joints. Chondrocyte viability was quantified following the muscular loading protocols, following application of the isolated impact loads, and following application of the impact loads that were preceded by the muscular pre-loads. Joint contact pressures were measured for all loading conditions by a pressure-sensitive film., Findings: Comparing to cartilage injured by impact loading alone, cartilage pre-loaded by static, maximal intensity, short-term muscle loads had lower cell death, while cartilage pre-loaded by repetitive, low-intensity, long-term muscular loads has higher cell death. The locations of peak joint contact pressures were not strongly correlated with the locations of greatest cell death occurrence., Interpretation: Static, high intensity, short muscular pre-load protected cells from impact injury, whereas repetitive, low intensity, prolonged muscular pre-loading to the point of muscular fatigue left the chondrocytes vulnerable to injury. However, cell death seems to be unrelated to the peak joint pressures., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cartilage lesions change the microenvironment of cells and may accelerate cartilage degradation through catabolic responses from chondrocytes. In this study, we investigated the effects of structural integrity of the extracellular matrix (ECM) on chondrocytes by comparing the mechanics of cells surrounded by an intact ECM with cells close to a cartilage lesion using experimental and numerical methods. Experimentally, 15% nominal compression was applied to bovine cartilage tissues using a light-transmissible compression system. Target cells in the intact ECM and near lesions were imaged by dual-photon microscopy. Changes in cell morphology (N(cell)=32 for both ECM conditions) were quantified. A two-scale (tissue level and cell level) Finite Element (FE) model was also developed. A 15% nominal compression was applied to a non-linear, biphasic tissue model with the corresponding cell level models studied at different radial locations from the centre of the sample in the transient phase and at steady state. We studied the Green-Lagrange strains in the tissue and cells. Experimental and theoretical results indicated that cells near lesions deform less axially than chondrocytes in the intact ECM at steady state. However, cells near lesions experienced large tensile strains in the principal height direction, which are likely associated with non-uniform tissue radial bulging. Previous experiments showed that tensile strains of high magnitude cause an up-regulation of digestive enzyme gene expressions. Therefore, we propose that cartilage degradation near tissue lesions may be due to the large tensile strains in the principal height direction applied to cells, thus leading to an up-regulation of catabolic factors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Impact loading of articular cartilage causes extensive chondrocyte death. Cell membranes have a limited elastic range of 3-4% strain but are protected from direct stretch during physiological loading by their membrane reservoir, an intricate pattern of membrane folds. Using a finite-element model, we suggested previously that access to the membrane reservoir is strain-rate-dependent and that during impact loading, the accessible membrane reservoir is drastically decreased, so that strains applied to chondrocytes are directly transferred to cell membranes, which fail when strains exceed 3-4%. However, experimental support for this proposal is lacking. The purpose of this study was to measure the accessible membrane reservoir size for different membrane strain rates using membrane tethering techniques with atomic force microscopy. We conducted atomic force spectroscopy on isolated chondrocytes (n = 87). A micron-sized cantilever was used to extract membrane tethers from cell surfaces at constant pulling rates. Membrane tethers could be identified as force plateaus in the resulting force-displacement curves. Six pulling rates were tested (1, 5, 10, 20, 40, and 80 μm/s). The size of the membrane reservoir, represented by the membrane tether surface areas, decreased exponentially with increasing pulling rates. The current results support our theoretical findings that chondrocytes exposed to impact loading die because of membrane ruptures caused by high tensile membrane strain rates., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Experimental findings indicate that in-situ chondrocytes die readily following impact loading, but remain essentially unaffected at low (non-impact) strain rates. This study was aimed at identifying possible causes for cell death in impact loading by quantifying chondrocyte mechanics when cartilage was subjected to a 5% nominal tissue strain at different strain rates. Multi-scale modelling techniques were used to simulate cartilage tissue and the corresponding chondrocytes residing in the tissue. Chondrocytes were modelled by accounting for the cell membrane, pericellular matrix and pericellular capsule. The results suggest that cell deformations, cell fluid pressures and fluid flow velocity through cells are highest at the highest (impact) strain rate, but they do not reach damaging levels. Tangential strain rates of the cell membrane were highest at the highest strain rate and were observed primarily in superficial tissue cells. Since cell death following impact loading occurs primarily in superficial zone cells, we speculate that cell death in impact loading is caused by the high tangential strain rates in the membrane of superficial zone cells causing membrane rupture and loss of cell content and integrity.

Introduction: Although previous studies have been performed on cartilage explant cultures, the generalized dynamics of cartilage metabolism after extraction from the host are still poorly understood due to differences in the experimental setups across studies, which in turn prevent building a complete picture., Methods: In this study, we investigated the response of cartilage to the trauma sustained during extraction and determined the time needed for the cartilage to stabilize. Explants were extracted aseptically from bovine metacarpal-phalangeal joints and cultured for up to 17 days., Results: The cell viability, cell number, proteoglycan content, and collagen content of the harvested explants were analyzed at 0, 2, 10, and 17 days after explantation. A high percentage of the cartilage explants were found to be viable. The cell density initially increased significantly but stabilized after two days. The proteoglycan content decreased gradually over time, but it did not decrease to a significant level due to leakage through the distorted peripheral collagen network and into the bathing medium. The collagen content remained stable for most of the culture period until it dropped abruptly on day 17., Conclusion: Overall, the tested cartilage explants were sustainable over long-term culture. They were most stable from day 2 to day 10. The degradation of the collagen on day 17 did not reach diseased levels, but it indicated the potential of the cultures to develop into degenerated cartilage. These findings have implications for the application of cartilage explants in pathophysiological fields.

Conventionally, patellar tendon-bearing (PTB) sockets, which need high dexterity of prosthetist, are widely used. Lack of chartered and experienced prosthetist has often led to painful experience of wearing prosthesis and this will in turn deter the patients to wear the prosthesis, which will further aggravate stump shrinkage. Thus, the hydrostatic socket which demands relatively lower level of fabricating skill is proposed to replace the PTB socket in order to produce the equivalent, if not better, quality of support to the amputee patients. Both sockets' pressure profiles are studied and compared using finite element analysis (FEA) software. Three-dimensional models of both sockets were developed using MIMICS software. The analysis results showed that hydrostatic socket did exhibit more uniform pressure profiles than that of PTB socket. PTB socket showed pressure concentration near the proximal brim of the socket and also at the distal fibula. It was also found that the pressure magnitude in hydrostatic socket is relatively lower than that of PTB socket.