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There is an abrupt increase in the multiscale mechanical properties and load-bearing capabilities of tendon during development. While prior work has identified numerous changes that occur within the collagenous structure during this developmental period, the primary structural elements that give rise to this abrupt increase in mechanical functionality, and their mechanobiological sensitivity, remain unclear. To address this knowledge gap, we used a shear lag model along with ultrastructural imaging, biochemical/thermodynamic assays, and multiscale mechanical testing to investigate the dynamic structure-function relationships during late-stage embryonic chick development and to establish their sensitivity to mechanical stimulation. Mechanical testing and modeling suggested that the rapid increase in multiscale mechanics can be explained by increases in fibril length, intrafibrillar crosslinking, and fibril area fraction. To partially test this, we inhibited collagen crosslinking during development and observed a drastic reduction in multiscale mechanical behavior that was explained by a reduction in both fibril modulus and length. Using muscle paralysis to investigate mechanosensitivity, we observed a significantly impaired multiscale mechanical response despite minimal changes in fibril diameter and fibril area fraction. Additionally, the shear lag model found a trend toward lower fibril lengths with paralysis and experimental data found decreased crosslinking and fibril modulus values following flaccid paralysis. Together, these data suggest that both intrafibrillar crosslink formation and fibril elongation are critical to the formation of load-bearing capabilities in tenogenesis and are sensitive to mechanical loading. These findings provide critical insights into the biological and structural mechanisms that give rise to tensile load-bearing soft tissue. STATEMENT OF SIGNIFICANCE: Despite prior work investigating the structural and mechanical changes that occur during tendon development, there has not been a comprehensive analysis of how these simultaneous changes in structure and function are connected. In this study, we performed a comprehensive battery of mechanical and structural assessments of embryonic chick tendons and input these data into a shear lag model to estimate the individual importance of each structural change to the tendon mechanical properties. Additionally, we inhibited muscle activity in the embryos to evaluate the impact of mechanical stimulation on these evolving structure-function relationships during tendon development. These data provide insight into the primary structural elements that produce the tensile load-bearing capabilities of tendon, which will inform efforts to produce tissue engineered tendon replacements., 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 © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Introduction: Following early cell specification and tenocyte differentiation at the sites of future tendons, very little is known about how tendon maturation into robust load-bearing tissue is regulated. Between embryonic day (E)16 and E18 in the chick, there is a rapid change in mechanical properties which is dependent on normal embryo movement. However, the tissue, cellular and molecular changes that contribute to this transition are not well defined., Methods: Here we profiled aspects of late tendon development (collagen fibre alignment, cell organisation and Yap pathway activity), describing changes that coincide with tissue maturation. We compared effects of rigid (constant static loading) and flaccid (no loading) immobilisation to gain insight into developmental steps influenced by mechanical cues., Results: We show that YAP signalling is active and responsive to movement in late tendon. Collagen fibre alignment increased over time and under static loading. Cells organise into end-to-end stacked columns with increased distance between adjacent columns, where collagen fibres are deposited; this organisation was lost following both types of immobilisation., Discussion: We conclude that specific aspects of tendon maturation require controlled levels of dynamic muscle-generated stimulation. Such a developmental approach to understanding how tendons are constructed will inform future work to engineer improved tensile load-bearing tissues., 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 Rolfe, Bastürkmen, Sliney, Hayden, Dunne, Buckley, McCarthy, Szczesny and Murphy.)

Anterior cruciate ligament (ACL) injuries are historically thought to be a result of a single acute overload or traumatic event. However, recent studies suggest that ACL failure may be a consequence of fatigue damage. Additionally, the remodeling response of ACLs to fatigue loading is unknown. Therefore, the objective of this study was to investigate the remodeling response of ACLs to cyclic loading. Furthermore, given that women have an increased rate of ACL rupture, we investigated whether this remodeling response is sex specific. ACLs were harvested from male and female New Zealand white rabbits and cyclically loaded in a tensile bioreactor mimicking the full range of physiological loading (2, 4, and 8 MPa). Expression of markers for anabolic and catabolic tissue remodeling, as well as inflammatory cytokines, was quantified using quantitative reverse transcription polymerase chain reaction. We found that the expression of markers for tissue remodeling of the ACL is dependent on the magnitude of loading and is sex specific. Male ACLs activated an anabolic response to cyclic loading at 4 MPa but turned off remodeling at 8 MPa. These data support the hypothesis that noncontact ACL injury may be a consequence of failed tissue remodeling and inadequate repair of microtrauma resulting from elevated loading. Compared to males, female ACLs failed to increase anabolic gene expression with loading and exhibited higher expression of catabolic genes at all loading levels, which may explain the increased rate of ACL tears in women. Together, these data provide insight into load-induced ACL remodeling and potential causes of tissue rupture., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

The wide variety of cell and tissue culture systems used to study and engineer tendons can make it difficult to choose the best approach and "optimal" culture conditions to test a given hypothesis. Therefore, a breakout session was organized at the 2022 ORS Tendon Section Meeting that focused on establishing a set of guidelines for conducting cell and tissue culture studies of tendon. This paper summarizes the outcomes of that discussion and presents recommendations for future studies. In the case of studying tendon cell behavior, cell and tissue culture systems are reductionist models in which the culture conditions should be strictly defined to approximate the in vivo condition as closely as possible. In contrast, for tissue engineering tendon replacements, the culture conditions do not need to replicate native tendon, but the outcome measures for success should be narrowly defined for the specific clinical application. Common recommendations for both applications are that researchers should perform a baseline phenotypic characterization of the cells that are ultimately used for experimentation. For models of tendon cell behavior, culture conditions should be well justified by existing literature and meticulously reported, tissue explant viability should be assessed, and comparisons to in vivo conditions should be made to determine baseline physiological relevance. For tissue engineering applications, the functional/structural/compositional outcome targets should be defined by the specific tendons they seek to replace, with key biologic and material properties prioritized for construct assessment. Lastly, when engineering tendon replacements, researchers should utilize clinically approved cGMP materials to facilitate clinical translation., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Cell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell-based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN 2 ). This method allows high-capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN 2 is a promising platform for advancing cell-based applications., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

While overuse is a prominent risk factor for tendinopathy, the fatigue-induced structural damage responsible for initiating tendon degeneration remains unclear. Denaturation of collagen molecules and collagen fiber disorganization have been observed within certain tendons in response to fatigue loading. However, no studies have investigated whether these forms of tissue damage occur in Achilles tendons, which commonly exhibit tendinopathy. Therefore, the objective of this study was to determine whether mouse Achilles tendons undergo collagen denaturation and collagen fiber disorganization when cyclically loaded to failure. Consistent with previous testing of other energy-storing tendons, we found that cyclic loading of mouse Achilles tendons produced collagen disorganization but minimal collagen denaturation. To determine whether the lack of collagen denaturation is unique to mouse Achilles tendons, we monotonically loaded the Achilles and other mouse tendons to failure. We found that the patellar tendon was also resistant to collagen denaturation, but the flexor digitorum longus (FDL) tendon and tail tendon fascicles were not. Furthermore, the Achilles and patellar tendons had a lower tensile strength and modulus. While this may be due to differences in tissue structure, it is likely that the lack of collagen denaturation during monotonic loading in both the Achilles and patellar tendons was due to failure near their bony insertions, which were absent in the FDL and tail tendons. These findings suggest that mouse Achilles tendons are resistant to collagen denaturation in situ and that Achilles tendon degeneration may not be initiated by mechanically-induced damage to collagen molecules., 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.)

There is considerable scientific interest in understanding the strains that tendon cells experience in situ and how these strains influence tissue remodeling. Based on this interest, several analytical techniques have been developed to measure local tissue strains within tendon explants during loading. However, in several cases, the accuracy and sensitivity of these techniques have not been reported, and none of the algorithms are publicly available. This has made it difficult for the more widespread measurement of local tissue strains in tendon explants. Therefore, the objective of this paper was to create a validated analysis tool for measuring local tissue strains in tendon explants that is readily available and easy to use. Specifically, a publicly available augmented-Lagrangian digital image correlation (ALDIC) algorithm was adapted for measuring 2D strains by tracking the displacements of cell nuclei within mouse Achilles tendons under uniaxial tension. Additionally, the accuracy of the calculated strains was validated by analyzing digitally transformed images, as well as by comparing the strains with values determined from an independent technique (i.e., photobleached lines). Finally, a technique was incorporated into the algorithm to reconstruct the reference image using the calculated displacement field, which can be used to assess the accuracy of the algorithm in the absence of known strain values or a secondary measurement technique. The algorithm is capable of measuring strains up to 0.1 with an accuracy of 0.00015. The technique for comparing a reconstructed reference image with the actual reference image successfully identified samples that had erroneous data and indicated that, in samples with good data, approximately 85% of the displacement field was accurate. Finally, the strains measured in mouse Achilles tendons were consistent with the prior literature. Therefore, this algorithm is a highly useful and adaptable tool for accurately measuring local tissue strains in tendons.

The 2021 Summer Biomechanics, Bioengineering, and Biotransport Conference (SB3C) featured a workshop titled "The Elephant in the Room: Nuclear Mechanics and Mechanobiology." The goal of this workshop was to provide a perspective from experts in the field on the current understanding of nuclear mechanics and its role in mechanobiology. This paper reviews the major themes and questions discussed during the workshop, including historical context on the initial methods of measuring the mechanical properties of the nucleus and classifying the primary structures dictating nuclear mechanics, physical plasticity of the nucleus, the emerging role of the linker of nucleoskeleton and cytoskeleton (LINC) complex in coupling the nucleus to the cytoplasm and driving the behavior of individual cells and multicellular assemblies, and the computational models currently in use to investigate the mechanisms of gene expression and cell signaling. Ongoing questions and controversies, along with promising future directions, are also discussed., (Copyright © 2022 by ASME.)

A major risk factor for tendinopathy is tendon overuse (i.e., fatigue loading). Fatigue loading of tendon damages the extracellular matrix and induces tissue degeneration. However, the specific mechanisms linking tendon fatigue damage with tissue degeneration are unclear. While explant models of tendon fatigue loading have been used to address this knowledge gap, they predominantly employ bioreactors that apply cyclic displacements/strains rather than loads/stresses, which are more physiologically relevant. This is because of the technical complexity and cost of building a load-controlled bioreactor, which requires multiple motors, load cells, and computationally intensive feedback loops. Here, we present a novel, low-cost, load-controlled bioreactor that applies cyclic loading to multiple tendon explants by offloading weights from a single motorized stage. Using an optional load cell, we validated that the bioreactor can effectively provide load-controlled fatigue testing of mouse and rat tendon explants while maintaining tissue viability. Furthermore, all the design files, bill of materials, and operating software are available "open source"1 so that anyone can easily manufacture and use the bioreactor for their own research. Therefore, this novel load-controlled bioreactor will enable researchers to study the mechanisms driving fatigue-induced tendon degeneration in a more physiologically relevant and cost-effective manner., (Copyright © 2022 by ASME.)

During embryonic development, tendons transform into a hypocellular tissue with robust tensile load-bearing capabilities. Previous work suggests that this mechanical transformation is due to increases in collagen fibril length and is dependent on mechanical stimulation via muscle activity. However, the relationship between changes in the microscale tissue structure and changes in macroscale tendon mechanics is still unclear. Additionally, the specific effect of mechanical stimulation on the multiscale structure-function relationships of developing tendons is also unknown. Therefore, the objective of this study was to measure the changes in tendon mechanics and structure at multiple length scales during embryonic development with and without skeletal muscle paralysis. Tensile testing of tendons from chick embryos was performed to determine the macroscale tensile modulus as well as the magnitude of the fibril strains and interfibrillar sliding with applied tissue strain. Embryos were also treated with either decamethonium bromide or pancuronium bromide to produce rigid or flaccid paralysis. Histology was performed to assess changes in tendon size, spacing between tendon subunits, and collagen fiber diameter. We found that the increase in the macroscale modulus observed with development is accompanied by an increase in the fibril:tissue strain ratio, which is consistent with an increase in collagen fibril length. Additionally, we found that flaccid paralysis reduced the macroscale tendon modulus and the fibril:tissue strain ratio, whereas less pronounced effects that were not statistically significant were observed with rigid paralysis. Finally, skeletal paralysis also reduced the size of collagen fibril bundles (i.e., fibers). Together, these data suggest that more of the applied tissue strain is transmitted to the collagen fibrils at later embryonic ages, which leads to an increase in the tendon macroscale tensile mechanics. Furthermore, our data suggest that mechanical stimulation during development is necessary to induce structural and mechanical changes at multiple physical length scales. This information provides valuable insight into the multiscale structure-function relationships of developing tendons and the importance of mechanical stimulation in producing a robust tensile load-bearing soft tissue., 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 © 2021 Peterson, Rolfe, Kunselman, Murphy and Szczesny.)

While collagen fibrils are understood to be the primary load-bearing elements in tendon, controversy still exists on how fibrils functionally transmit load from muscle to bone. Specifically, it's unclear whether fibrils are structurally continuous along the tendon length and bear load independently, or if they are discontinuous and transfer load through interfibrillar shear forces. To address this question, we investigated whether the multiscale mechanics of rat tail tendon fascicles is dependent on sample gauge length. We hypothesized that as the grip-to-grip length is reduced and approaches the length of the collagen fibrils, tendon fascicles will adopt a multiscale mechanical response consistent with structurally continuous fibrils. Our findings show that, for gauge lengths of 20 mm or greater, the local fibril strains are less than the bulk tissue strains, which can be explained by relative sliding between discontinuous collagen fibrils. In contrast, at a 5 mm gauge length, the fibril strains are equivalent to the applied tissue strains, suggesting that the collagen fibrils are structurally continuous between the grips. Additionally, the macroscale tissue modulus is increased at gauge lengths of 5 and 10 mm. Together, these data support the hypothesis that collagen fibrils in rat tail tendon fascicles are discontinuous and also suggest that their length is between 5 and 10 mm. This fundamental information regarding tendon structure-function relationships underscores the importance of the tissue components that transmit load between fibrils and is critical for understanding tendon pathology as well as establishing structural benchmarks for suitable tissue engineered replacements., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure - but not local tissue mechanics - initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing., Competing Interests: The authors declare no conflict of interest.

Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies and tissue engineering applications given their multipotential differentiation capacity. However, MSC isolation and expansion are typically performed on super-physiologically stiff tissue culture plastic (TCP), which may alter their behavior and lead to unintended consequences upon implantation. In contrast, electrospun nanofibrous scaffolds possess physical and mechanical properties that are similar to that of native tissue. In this study, we investigated whether isolation and expansion of juvenile bovine MSCs directly onto electrospun nanofibrous scaffolds better preserves MSC phenotype and stemness compared to TCP. Our data show that culture of MSCs on electrospun scaffolds reduces proliferation, decreases cellular senescence, and better maintains stemness compared to cells isolated and expanded on TCP, likely due to a reduction in cell contractility. Furthermore, in contrast to electrospun scaffolds, TCP biased MSCs towards a fibrotic phenotype that persisted even after the cells were reseeded onto a different substrate. Cells pre-cultured on electrospun scaffolds exhibited a heightened response to mechanical stimuli and greater chondrogenesis in methacrylated hyaluronic acid hydrogels. These data suggest that alternative substrates that better approximate the native cell environment should be used to preserve endogenous MSC behavior and may improve their success in tissue engineering applications. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:808-815, 2018., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

To fully recapitulate tissue microstructure and mechanics, fiber crimping must exist within biomaterials used for tendon/ligament engineering. Existing crimped nanofibrous scaffolds produced via electrospinning are dense materials that prevent cellular infiltration into the scaffold interior. In this study, we used a sacrificial fiber population to increase the scaffold porosity and evaluated the effect on fiber crimping. We found that increasing scaffold porosity increased fiber crimping and ensured that the fibers properly uncrimped as the scaffolds were stretched by minimizing fiber-fiber interactions. Constitutive modeling demonstrated that the fiber uncrimping produced a nonlinear mechanical behavior similar to that of native tendon and ligament. Interestingly, fiber crimping altered strain transmission to the nuclei of cells seeded on the scaffolds, which may account for previously observed changes in gene expression. These crimped biomaterials are useful for developing functional fiber-reinforced tissues and for studying the effects of altered fiber crimping due to damage or degeneration., Competing Interests: Notes The authors declare no competing financial interest.

Collagen fibrils in tendon are believed to be discontinuous and transfer tensile loads through shear forces generated during interfibrillar sliding. However, the structures that transmit these interfibrillar forces are unknown. Various extrafibrillar tissue components (e.g., glycosaminoglycans, collagens XII and XIV) have been suggested to transmit interfibrillar loads by bridging collagen fibrils. Alternatively, collagen fibrils may interact directly through physical fusions and interfibrillar branching. The objective of this study was to test whether extrafibrillar proteins are necessary to transmit load between collagen fibrils or if interfibrillar load transfer is accomplished directly by the fibrils themselves. Trypsin digestions were used to remove a broad spectrum of extrafibrillar proteins and measure their contribution to the multiscale mechanics of rat tail tendon fascicles. Additionally, images obtained from serial block-face scanning electron microscopy were used to determine the three-dimensional fibrillar organization in tendon fascicles and identify any potential interfibrillar interactions. While trypsin successfully removed several extrafibrillar tissue components, there was no change in the macroscale fascicle mechanics or fibril:tissue strain ratio. Furthermore, the imaging data suggested that a network of smaller diameter fibrils (<150 nm) wind around and fuse with their neighboring larger diameter fibrils. These findings demonstrate that interfibrillar load transfer is not supported by extrafibrillar tissue components and support the hypothesis that collagen fibrils are capable of transmitting loads themselves. Conclusively determining how fibrils bear load within tendon is critical for identifying the mechanisms that impair tissue function with degeneration and for restoring tissue properties via cell-mediated regeneration or engineered tissue replacements. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2127-2134, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

A buffer solution is often used to maintain tissue hydration during mechanical testing. The most commonly used buffer solution is a physiological concentration of phosphate buffered saline (PBS); however, PBS increases the tissue's water content and decreases its tensile stiffness. In addition, solutes from the buffer can diffuse into the tissue and interact with its structure and mechanics. These bathing solution effects can confound the outcome and interpretation of mechanical tests. Potential bathing solution artifacts, including solute diffusion, and their effect on mechanical properties, are not well understood. The objective of this study was to measure the effects of long-term exposure of rat tail tendon fascicles to several concentrations (0.9-25%) of NaCl, sucrose, polyethylene glycol (PEG), and SPEG (NaCl+PEG) solutions on water content, solute diffusion, and mechanical properties. We found that with an increase in solute concentration the apparent water content decreased for all solution types. Solutes diffused into the tissue for NaCl and sucrose, however, no solute diffusion was observed for PEG or SPEG. The mechanical properties changed for both NaCl solutions, in particular after long-term (8h) incubation the modulus and equilibrium stress decreased compared to short-term (15min) for 25% NaCl, and the cross sectional area increased for 0.9% NaCl. However, the mechanical properties were unchanged for both PEG and SPEG except for minor alterations in stress relaxation parameters. This study shows that NaCl and sucrose buffer solutions are not suitable for long-term mechanical tests. We therefore propose using PEG or SPEG as alternative buffer solutions that after long-term incubation can maintain tissue hydration without solute diffusion and produce a consistent mechanical response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tendon pathology is associated with damage. While tendon damage is likely initiated by mechanical loading, little is known about the specific etiology. Damage is defined as an irreversible change in the microstructure that alters the macroscopic mechanical parameters. In tendon, the link between mechanical loading and microstructural damage, resulting in macroscopic changes, is not fully elucidated. In addition, tendon damage at the macroscale has been proposed to initiate when tendon is loaded beyond a strain threshold, yet the metrics to define the damage threshold are not determined. We conducted multi-scale mechanical testing to investigate the mechanism of tendon damage by simultaneously quantifying macroscale mechanical and microstructural changes. At the microscale, we observe full recovery of the fibril strain and only partial recovery of the interfibrillar sliding, indicating that the damage initiates at the interfibrillar structures. We show that non-recoverable sliding is a mechanism for tendon damage and is responsible for the macroscale decreased linear modulus and elongated toe-region observed at the fascicle-level, and these macroscale properties are appropriate metrics that reflect tendon damage. We concluded that the inflection point of the stress-strain curve represents the damage threshold and, therefore, may be a useful parameter for future studies. Establishing the mechanism of damage at multiple length scales can improve prevention and rehabilitation strategies for tendon pathology., Statement of Significance: Tendon pathology is associated with mechanically induced damage. Damage, as defined in engineering, is an irreversible change in microstructure that alters the macroscopic mechanical properties. Although microstructural damage and changes to macroscale mechanics are likely, this link to microstructural change was not yet established. We conducted multiscale mechanical testing to investigate the mechanism of tendon damage by simultaneously quantifying macroscale mechanical and microstructural changes. We showed that non-recoverable sliding between collagen fibrils is a mechanism for tendon damage. Establishing the mechanism of damage at multiple length scales can improve prevention and rehabilitation strategies for tendon pathology., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The meniscus is comprised of circumferentially aligned fibers that resist the tensile forces within the meniscus (i.e., hoop stress) that develop during loading of the knee. Although these circumferential fibers are severed by radial meniscal tears, tibial contact stresses do not increase until the tear reaches ∼90% of the meniscus width, suggesting that the severed circumferential fibers still bear load and maintain the mechanical functionality of the meniscus. Recent data demonstrates that the interfibrillar matrix can transfer strain energy to disconnected fibrils in tendon fascicles. In the meniscus, interdigitating radial tie fibers, which function to stabilize and bind the circumferential fibers together, are hypothesized to function in a similar manner by transmitting load to severed circumferential fibers near a radial tear. To test this hypothesis, we developed an engineered fibrous analog of the knee meniscus using poly(ε-caprolactone) to create aligned scaffolds with variable amounts of non-aligned elements embedded within the scaffold. We show that the tensile properties of these scaffolds are a function of the ratio of aligned to non-aligned elements, and change in a predictable fashion following a simple mixture model. When measuring the loss of mechanical function in scaffolds with a radial tear, compared to intact scaffolds, the decrease in apparent linear modulus was reduced in scaffolds containing non-aligned layers compared to purely aligned scaffolds. Increased strains in areas adjacent to the defect were also noted in composite scaffolds. These findings indicate that non-aligned (disorganized) elements interspersed within an aligned network can improve overall mechanical function by promoting strain transfer to nearby disconnected fibers. This finding supports the notion that radial tie fibers may similarly promote tear tolerance in the knee meniscus, and will direct changes in clinical practice and provide guidance for tissue engineering strategies., Statement of Significance: The meniscus is a complex fibrous tissue, whose architecture includes radial tie fibers that run perpendicular to and interdigitate with the predominant circumferential fibers. We hypothesized that these radial elements function to preserve mechanical function in the context of interruption of circumferential bundles, as would be the case in a meniscal tear. To test this hypothesis, we developed a biomaterial analog containing disorganized layers enmeshed regularly throughout an otherwise aligned network. Using this material formulation, we showed that strain transmission is improved in the vicinity of defects when disorganized fiber layers were present. This supports the idea that radial elements within the meniscus improve function near a tear, and will guide future clinical interventions and the development of engineered replacements., (Copyright © 2017 Acta Materialia Inc. All rights reserved.)

Given the significance of hydrogels as cell-instructive materials, it is important to understand how differences in their chemical and physical properties are able to direct cell fate. For example, it remains unclear how different hydrogel cross-linking chemistries and gelation mechanisms influence cell behavior. Here, we report on hyaluronan-tyramine (HA-Tyr) hydrogels prepared either with enzymatic cross-linking using horseradish peroxidase and H 2 O 2 or with visible light (500 nm) triggered gelation. We demonstrate that when hydrogels are polymerized to equivalent Young's moduli, the specific cross-linking chemistry of HA-Tyr hydrogels can have a substantial impact on mesenchymal stem cell (MSC) behavior. MSCs cultured on HA-Tyr hydrogels exhibit increased cell spread areas on enzymatically formed substrates relative to photo-cross-linked matrices. While enzymatically formed hydrogels led to MSCs exhibiting greater cell focal adhesion length, MSCs cultured on the photo-cross-linked matrices exhibited smaller cell spread area and shorter focal adhesion length but generated increased traction stress. These findings highlight the importance of understanding hydrogel cross-linking chemistries when the role of biophysical cues in regulating stem cell fate is investigated.

Biophysical stimuli presented to cells via microenvironmental properties (e.g., alignment and stiffness) or external forces have a significant impact on cell function and behavior. Recently, the cell nucleus has been identified as a mechanosensitive organelle that contributes to the perception and response to mechanical stimuli. However, the specific mechanotransduction mechanisms that mediate these effects have not been clearly established. Here, we offer a comprehensive review of the evidence supporting (and refuting) three hypothetical nuclear mechanotransduction mechanisms: physical reorganization of chromatin, signaling at the nuclear envelope, and altered cytoskeletal structure/tension due to nuclear remodeling. Our goal is to provide a reference detailing the progress that has been made and the areas that still require investigation regarding the role of nuclear mechanotransduction in cell biology. Additionally, we will briefly discuss the role that mathematical models of cell mechanics can play in testing these hypotheses and in elucidating how biophysical stimulation of the nucleus drives changes in cell behavior. While force-induced alterations in signaling pathways involving lamina-associated polypeptides (LAPs) (e.g., emerin and histone deacetylase 3 (HDAC3)) and transcription factors (TFs) located at the nuclear envelope currently appear to be the most clearly supported mechanism of nuclear mechanotransduction, additional work is required to examine this process in detail and to more fully test alternative mechanisms. The combination of sophisticated experimental techniques and advanced mathematical models is necessary to enhance our understanding of the role of the nucleus in the mechanotransduction processes driving numerous critical cell functions.

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement.

Despite current knowledge of tendon structure, the fundamental deformation mechanisms underlying tendon mechanics and failure are unknown. We recently showed that a shear lag model, which explicitly assumed plastic interfibrillar load transfer between discontinuous fibrils, could explain the multiscale fascicle mechanics, suggesting that fascicle yielding is due to plastic deformation of the interfibrillar matrix. However, it is unclear whether alternative physical mechanisms, such as elastic interfibrillar deformation or fibril yielding, also contribute to fascicle mechanical behavior. The objective of the current work was to determine if plasticity of the interfibrillar matrix is uniquely capable of explaining the multiscale mechanics of tendon fascicles including the tissue post-yield behavior. This was examined by comparing the predictions of a continuous fibril model and three separate shear lag models incorporating an elastic, plastic, or elastoplastic interfibrillar matrix with multiscale experimental data. The predicted effects of fibril yielding on each of these models were also considered. The results demonstrated that neither the continuous fibril model nor the elastic shear lag model can successfully predict the experimental data, even if fibril yielding is included. Only the plastic or elastoplastic shear lag models were capable of reproducing the multiscale tendon fascicle mechanics. Differences between these two models were small, although the elastoplastic model did improve the fit of the experimental data at low applied tissue strains. These findings suggest that while interfibrillar elasticity contributes to the initial stress response, plastic deformation of the interfibrillar matrix is responsible for tendon fascicle post-yield behavior. This information sheds light on the physical processes underlying tendon failure, which is essential to improve our understanding of tissue pathology and guide the development of successful repair., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The material properties of myocardium are an important determinant of global left ventricular function. Myocardial infarction results in a series of maladaptive geometric alterations which lead to increased stress and risk of heart failure. In vivo studies have demonstrated that material injection can mitigate these changes. More importantly, the material properties of these injectates can be tuned to minimize wall thinning and ventricular dilation. The current investigation combines experimental data and finite element modeling to correlate how injectate mechanics and volume influence myocardial wall stress. Experimentally, mechanics were characterized with biaxial testing and injected hydrogel volumes were measured with magnetic resonance imaging. Injection of hyaluronic acid hydrogel increased the stiffness of the myocardium/hydrogel composite region in an anisotropic manner, significantly increasing the modulus in the longitudinal direction compared to control myocardium. Increased stiffness, in combination with increased volume from hydrogel injection, reduced the global average fiber stress by ~14% and the transmural average by ~26% in the simulations. Additionally, stiffening in an anisotropic manner enhanced the influence of hydrogel treatment in decreasing stress. Overall, this work provides insight on how injectable biomaterials can be used to attenuate wall stress and provides tools to further optimize material properties for therapeutic applications.

Identification of the deformation mechanisms and specific components underlying the mechanical function of biological tissues requires mechanical testing at multiple levels within the tissue hierarchical structure. Dichlorotriazinylaminofluorescein (DTAF) is a fluorescent dye that is used to visualize microscale deformations of the extracellular matrix in soft collagenous tissues. However, the DTAF concentrations commonly employed in previous multiscale experiments (≥2000 µg/ml) may alter tissue mechanics. The objective of this study was to determine whether DTAF affects tendon fascicle mechanics and if a concentration threshold exists below which any observed effects are negligible. This information is valuable for guiding the continued use of this fluorescent dye in future experiments and for interpreting the results of previous work. Incremental strain testing demonstrated that high DTAF concentrations (≥100 µg/ml) increase the quasi-static modulus and yield strength of rat tail tendon fascicles while reducing their viscoelastic behavior. Subsequent multiscale testing and modeling suggests that these effects are due to a stiffening of the collagen fibrils and strengthening of the interfibrillar matrix. Despite these changes in tissue behavior, the fundamental deformation mechanisms underlying fascicle mechanics appear to remain intact, which suggests that conclusions from previous multiscale investigations of strain transfer are still valid. The effects of lower DTAF concentrations (≤10 µg/ml) on tendon mechanics were substantially smaller and potentially negligible; nevertheless, no concentration was found that did not at least slightly alter the tissue behavior. Therefore, future studies should either reduce DTAF concentrations as much as possible or use other dyes/techniques for measuring microscale deformations.

Despite the critical role tendons play in transmitting loads throughout the musculoskeletal system, little is known about the microstructural mechanisms underlying their mechanical function. Of particular interest is whether collagen fibrils in tendon fascicles bear load independently or if load is transferred between fibrils through interfibrillar shear forces. We conducted multiscale experimental testing and developed a microstructural shear lag model to explicitly test whether interfibrillar shear load transfer is indeed the fibrillar loading mechanism in tendon. Experimental correlations between fascicle macroscale mechanics and microscale interfibrillar sliding suggest that fibrils are discontinuous and share load. Moreover, for the first time, we demonstrate that a shear lag model can replicate the fascicle macroscale mechanics as well as predict the microscale fibrillar deformations. Since interfibrillar shear stress is the fundamental loading mechanism assumed in the model, this result provides strong evidence that load is transferred between fibrils in tendon and possibly other aligned collagenous tissues. Conclusively establishing this fibrillar loading mechanism and identifying the involved structural components should help develop repair strategies for tissue degeneration and guide the design of tissue engineered replacements., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The annulus fibrosus (AF) of the intervertebral disk plays a critical role in vertebral load transmission that is heavily dependent on the microscale structure and composition of the tissue. With degeneration, both structure and composition are compromised, resulting in a loss of AF mechanical function. Numerous tissue engineering strategies have addressed the issue of AF degeneration, but few have focused on recapitulation of AF microstructure and function. One approach that allows for generation of engineered AF with appropriate (+/-)30° lamellar microstructure is the use of aligned electrospun scaffolds seeded with mesenchymal stem cells (MSCs) and assembled into angle-ply laminates (APL). Previous work indicates that opposing lamellar orientation is necessary for development of near native uniaxial tensile properties. However, most native AF tensile loads are applied biaxially, as the disk is subjected to multi-axial loads and is constrained by its attachments to the vertebral bodies. Thus, the objective of this study was to evaluate the biaxial mechanical response of engineered AF bilayers, and to determine the importance of opposing lamellar structure under this loading regime. Opposing bilayers, which replicate native AF structure, showed a significantly higher modulus in both testing directions compared to parallel bilayers, and reached ∼60% of native AF biaxial properties. Associated with this increase in biaxial properties, significantly less shear, and significantly higher stretch in the fiber direction, was observed. These results provide additional insight into native tissue structure-function relationships, as well as new benchmarks for engineering functional AF tissue constructs., (Copyright © 2013 Orthopaedic Research Society.)

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.

While tendons typically undergo primary tensile loading, the human supraspinatus tendon (SST) experiences substantial amounts of tension, compression, and shear in vivo. As a result, the functional roles of the extracellular matrix components, in particular the proteoglycans (PGs), are likely complex and important. The goal of this study was to determine the PG content in specific regions of the SST that exhibit differing mechanical function. The concentration of aggrecan, biglycan, and decorin was determined in six regions of the human SST using immunochemical techniques. We hypothesized that aggrecan concentrations would be highest in areas where the tendon likely experiences compression; biglycan levels would be highest in regions likely subjected to injury and/or active remodeling such as the anterior regions; decorin concentrations would be highest in regions of greatest tensile stiffness. Our results generally supported these hypotheses and demonstrated that aggrecan and biglycan share regional variability, with increased concentration in the anterior and posterior regions and smaller concentration in the medial regions. Decorin, however, was in high concentration throughout all regions. The data presented in this study represent the first regional measurements of PG in the SST. Together with our previous regional measurements of mechanical properties, these data can be used to evaluate SST structure-function relationships. With knowledge of the differences in specific PG content, their spatial variations in the SST, and their relationships to tendon mechanics, we can begin to associate defects in PG content with specific pathology, which may provide guidance for new therapeutic interventions.

Orthopedic tissues respond to mechanical loads to maintain normal homeostasis and in response to injury. As the body of work on this continues to grow, it is important to synthesize the recent studies across tissues and specialties with one another and with past studies. Hence, this review highlights the knowledge gained since 2000, with only few exceptions, concerning the effects of mechanical load and biologics on remodeling and repair of orthopedic tissue.

Orthopedic tissues respond to mechanical loads to maintain normal homeostasis and in response to injury. As the body of work on this continues to grow, it is important to synthesize the recent studies across tissues and specialties with one another and with past studies. Hence, this review highlights the knowledge gained since 2000, with only few exceptions, concerning the effects of mechanical load and biologics on remodeling and repair of orthopedic tissue.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disorder characterized by a deficiency in beta-glucuronidase activity, leading to systemic accumulation of poorly degraded glycosaminoglycans (GAG). Along with other morbidities, MPS VII is associated with pediatric spinal deformity. The objective of this study was to examine potential associations between abnormal lumbar spine matrix structure and composition in MPS VII, and spine segment and tissue-level mechanical properties, using a naturally occurring canine model with a similar clinical phenotype to the human form of the disorder. Segments from juvenile MPS VII and unaffected dogs were allocated to: radiography, gross morphology, histology, biochemistry, and mechanical testing. MPS VII spines had radiolucent lesions in the vertebral body epiphyses. Histologically, this corresponded to a GAG-rich cartilaginous region in place of bone and elevated GAG staining was seen in the annulus fibrosus. Biochemically, MPS VII samples had elevated GAG in the outer annulus fibrosus and epiphyses, low calcium in the epiphyses, and high water content in all regions except the nucleus pulposus. MPS VII spine segments had higher range of motion and lower stiffness than controls. Endplate indentation stiffness and failure loads were significantly lower in MPS VII samples, while annulus fibrosus tensile mechanical properties were normal. Vertebral body lesions in MPS VII spines suggest a failure to convert cartilage to bone during development. Low stiffness in these regions likely contributes to mechanical weakness in motion segments and is a potential factor in the progression of spinal deformity., (Copyright (c) 2009 Orthopaedic Research Society.)

Head trauma is the leading cause of death and debilitating injury in children. Computational models are important tools used to understand head injury mechanisms but they must be validated with experimental data. In this communication we present in situ measurements of brain deformation during rapid, nonimpact head rotation in juvenile pigs of different ages. These data will be used to validate computational models identifying age-dependent thresholds of axonal injury. Fresh 5 days (n=3) and 4 weeks (n=2) old piglet heads were transected horizontally and secured in a container. The cut surface of each brain was marked and covered with a transparent, lubricated plate that allowed the brain to move freely in the plane of rotation. For each brain, a rapid (20-28 ms) 65 deg rotation was applied sequentially at 50 rad/s, 75 rad/s, and 75 rad/s. Each rotation was digitally captured at 2500 frames/s (480x320 pixels) and mark locations were tracked and used to compute strain using an in-house program in MATLAB. Peak values of principal strain (E(peak)) were significantly larger during deceleration than during acceleration of the head rotation (p<0.05), and doubled with a 50% increase in velocity. E(peak) was also significantly higher during the second 75 rad/s rotation than during the first 75 rad/s rotation (p<0.0001), suggesting structural alteration at 75 rad/s and the possibility that similar changes may have occurred at 50 rad/s. Analyzing only lower velocity (50 rad/s) rotations, E(peak) significantly increased with age (16.5% versus 12.4%, p<0.003), which was likely due to the larger brain mass and smaller viscoelastic modulus of the 4 weeks old pig brain compared with those of the 5 days old. Strain measurement error for the overall methodology was estimated to be 1%. Brain tissue strain during rapid, nonimpact head rotation in the juvenile pig varies significantly with age. The empirical data presented will be used to validate computational model predictions of brain motion under similar loading conditions and to assist in the development of age-specific thresholds for axonal injury. Future studies will examine the brain-skull displacement and will be used to validate brain-skull interactions in computational models.