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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.)

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.)

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.)

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.)

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.