Quantification of Cartilage Poroelastic Material Properties Via Analysis of Loading-Induced Cell Death.

Articular cartilage (AC) is a load-bearing tissue that covers long bones in synovial joints. The biphasic/poroelastic mechanical properties of AC help it to protect joints by distributing loads, absorbing impact forces, and reducing friction. Unfortunately, alterations in these mechanical properties adversely impact cartilage function and precede joint degeneration in the form of osteoarthritis (OA). Thus, understanding what factors regulate the poroelastic mechanical properties of cartilage is of great scientific and clinical interest. Transgenic mouse models provide a valuable platform to delineate how specific genes contribute to cartilage mechanical properties. However, the poroelastic mechanical properties of murine articular cartilage are challenging to measure due to its small size (thickness ∼ 50 microns). In the current study, our objective was to test whether the poroelastic mechanical properties of murine articular cartilage can be determined based solely on time-dependent cell death measurements under constant loading conditions. We hypothesized that in murine articular cartilage subjected to constant, sub-impact loading from an incongruent surface, cell death area and tissue strain are closely correlated. We further hypothesized that the relationship between cell death area and tissue strain can be used-in combination with inverse finite element modeling-to compute poroelastic mechanical properties. To test these hypotheses, murine cartilage-on-bone explants from different anatomical locations were subjected to constant loading conditions by an incongruent surface in a custom device. Cell death area increased over time and scaled linearly with strain, which rose in magnitude over time due to poroelastic creep. Thus, we were able to infer tissue strain from cell death area measurements. Moreover, using tissue strain values inferred from cell death area measurements, we applied an inverse finite element modeling procedure to compute poroelastic material properties and acquired data consistent with previous studies. Collectively, our findings demonstrate in the key role poroelastic creep plays in mediating cell survival in mechanically loaded cartilage and verify that cell death area can be used as a surrogate measure of tissue strain that enables determination of murine cartilage mechanical properties.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing.

Increased Ca2+ signaling through CaV 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone, and cartilage development and homeostasis, knowledge about Ca2+ signaling and the source of Ca2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca2+ signaling through CaV 1.2 voltage-gated Ca2+ channel in tendon formation. Using a reporter mouse, we found that CaV 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;CaV 1.2TS mice that express a gain-of-function mutant CaV 1.2 in tendon. We found that mutant tendons were hypertrophic, with more tendon fibroblasts but decreased cell density. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendons. Biomechanical testing revealed that the hypertrophic tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomic analysis showed no significant difference in the abundance of type I and III collagens, but mutant tendons had about two-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2, and cathepsin K. Taken together, these data highlight roles for increased Ca2+ signaling through CaV 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

Increased Ca 2+ signaling through Ca V 1.2 induces tendon hypertrophy with increased collagen fibrillogenesis and biomechanical properties.

Tendons are tension-bearing tissues transmitting force from muscle to bone for body movement. This mechanical loading is essential for tendon development, homeostasis, and healing after injury. While Ca 2+ signaling has been studied extensively for its roles in mechanotransduction, regulating muscle, bone and cartilage development and homeostasis, knowledge about Ca 2+ signaling and the source of Ca 2+ signals in tendon fibroblast biology are largely unknown. Here, we investigated the function of Ca 2+ signaling through Ca V 1.2 voltage-gated Ca 2+ channel in tendon formation. Using a reporter mouse, we found that Ca V 1.2 is highly expressed in tendon during development and downregulated in adult homeostasis. To assess its function, we generated ScxCre;Ca V 1.2 TS mice that express a gain-of-function mutant Ca V 1.2 channel (Ca V 1.2 TS ) in tendon. We found that tendons in the mutant mice were approximately 2/3 larger and had more tendon fibroblasts, but the cell density of the mutant mice decreased by around 22%. TEM analyses demonstrated increased collagen fibrillogenesis in the hypertrophic tendon. Biomechanical testing revealed that the hypertrophic Achilles tendons display higher peak load and stiffness, with no changes in peak stress and elastic modulus. Proteomics analysis reveals no significant difference in the abundance of major extracellular matrix (ECM) type I and III collagens, but mutant mice had about 2-fold increase in other ECM proteins such as tenascin C, tenomodulin, periostin, type XIV and type VIII collagens, around 11-fold increase in the growth factor of TGF-ß family myostatin, and significant elevation of matrix remodeling proteins including Mmp14, Mmp2 and cathepsin K. Taken together, these data highlight roles for increased Ca 2+ signaling through Ca V 1.2 on regulating expression of myostatin growth factor and ECM proteins for tendon collagen fibrillogenesis during tendon formation.

Scleraxis-lineage cells are required for tendon homeostasis and their depletion induces an accelerated extracellular matrix aging phenotype.

Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage cells in young mice (Scx-DTR). Scx-DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single-cell RNA sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and Scx-DTR tendons, identifying the requirement for Scleraxis-lineage cells during homeostasis. However, the remaining cells in aged and Scx-DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, tenocytes from Scx-DTR tendons demonstrate enhanced remodeling capacity. Collectively, this study defines Scx-DTR as a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan.

Depletion of Scleraxis-lineage cells during tendon healing transiently impairs multi-scale restoration of tendon structure during early healing.

Tendons are composed of a heterogeneous cell environment, with Scleraxis-lineage (ScxLin) cells being the predominant population. Although ScxLin cells are required for maintenance of tendon homeostasis, their functions during tendon healing are unknown. To this end, we first characterized the spatiotemporal dynamics of ScxLin cells during tendon healing, and identified that the overall ScxLin pool continuously expands up to early remodeling healing phase. To better define the function of ScxLin cells during the late proliferative phase of healing, we inducibly depleted ScxLin cells from day 14-18 post-surgery using the Scx-Cre; Rosa-DTR mouse model, with local administration of diphtheria toxin inducing apoptosis of ScxLin cells in the healing tendon. At D28 post-surgery, ScxLin cell depleted tendons (DTRScxLin) had substantial impairments in structure and function, relative to WT, demonstrating the importance of ScxLin cells during tendon healing. Next, bulk RNAseq was utilized to identify the underlying mechanisms that were impaired with depletion and revealed that ScxLin depletion induced molecular and morphological stagnation of the healing process at D28. However, this stagnation was transient, such that by D56 tendon mechanics in DTRScxLin were not significantly different than wildtype repairs. Collectively, these data offer fundamental knowledge on the dynamics and roles of ScxLin cells during tendon healing.

Characterization of scar tissue biomechanics during adult murine flexor tendon healing.

Tendon injuries are very common and result in significant impairments in mobility and quality of life. During healing, tendons produce a scar at the injury site, characterized by abundant and disorganized extracellular matrix and by permanent deficits in mechanical integrity compared to healthy tendon. Although a significant amount of work has been done to understand the healing process of tendons and to develop potential therapeutics for tendon regeneration, there is still a significant gap in terms of assessing the direct effects of therapeutics on the functional and material quality specifically of the scar tissue, and thus, on the overall tendon healing process. In this study, we focused on characterizing the mechanical properties of only the scar tissue in flexor digitorum longus (FDL) tendons during the proliferative and early remodeling healing phases and comparing these properties with the mechanical properties of the composite healing tissue. Our method was sensitive enough to identify significant differences in structural and material properties between the scar and tendon-scar composite tissues. To account for possible inaccuracies due to the small aspect ratio of scar tissue, we also applied inverse finite element analysis (iFEA) to compute mechanical properties based on simulated tests with accurate specimen geometries and boundary conditions. We found that the scar tissue linear tangent moduli calculated from iFEA were not significantly different from those calculated experimentally at all healing timepoints, validating our experimental findings, and suggesting the assumptions in our experimental calculations were accurate. Taken together, this study first demonstrates that due to the presence of uninjured stubs, testing composite healing tendons without isolating the scar tissue overestimates the material properties of the scar itself. Second, our scar isolation method promises to enable more direct assessment of how different treatment regimens (e.g., cellular ablation, biomechanical and/or biochemical stimuli, tissue engineered scaffolds) affect scar tissue function and material quality in multiple different types of tendons.

Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse.

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth's Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.

Ultrasound strain mapping of the mouse Achilles tendon during passive dorsiflexion.

Immediately prior to inserting into bone, many healthy tendons experience impingement from nearby bony structures. However, super-physiological levels of impingement are implicated in insertional tendinopathies. Unfortunately, the mechanisms underlying the connection between impingement and tendon pathology remain poorly understood, in part due to the shortage of well-characterized animal models of impingement at clinically relevant sites. As a first step towards developing a model of excessive tendon impingement, the objective of this study was to characterize the mechanical strain environment in the mouse Achilles tendon insertion under passive dorsiflexion and confirm that - like humans - mice experience impingement of the tendon insertion from the calcaneus (heel bone) in dorsiflexed ankle positions. Based on previous work in humans, we hypothesized that during dorsiflexion, the mouse Achilles tendon insertion would experience high levels of transverse compressive strain due to calcaneal impingement. A custom-built loading platform was used to apply passive dorsiflexion, while an ultrasound transducer positioned over the Achilles tendon captured radiofrequency images. A non-rigid image registration algorithm was then used to map the transverse compressive strain based on the acquired ultrasound image sequences. Our results demonstrate that during passive dorsiflexion, transverse compressive strains were produced throughout the Achilles tendon, with significantly larger strain magnitudes at the tendon insertion than at the midsubstance. Furthermore, there was increasing transverse compressive strain observed within the Achilles tendon as a function of increasing dorsiflexion angle. This study enhances our understanding of the unique mechanical loading environment of the Achilles tendon under physiologically relevant conditions.

Impingement in Insertional Achilles Tendinopathy Occurs Across a Larger Range of Ankle Angles and Is Associated With Increased Tendon Thickness.

BACKGROUND: Insertional Achilles tendinopathy (IAT) is characterized by tendon degeneration and thickening near the tendon-bone insertion.11 Calcaneal impingement is believed to contribute to the pathogenesis of IAT.5 However, it is unclear how increased tendon thickness in individuals with IAT influences impingement. This study aimed to compare Achilles tendon impingement in individuals with and without IAT. METHODS: Eight healthy adults and 12 adults with clinically diagnosed symptomatic IAT performed a passive flexion exercise during which ankle flexion angle, anterior-posterior (A-P) thickness, and an ultrasonographic image sequence of the Achilles tendon insertion were acquired. The angle of ankle plantarflexion at which the calcaneus first impinges the Achilles tendon, defined as the impingement onset angle, was identified by (1) a anonymized observer (visual inspection method) and (2) a computational image deformation-based approach (curvature method). RESULTS: Although the 2 methods provided different impingement onset angles, the measurements were strongly correlated (R2 = 0.751, P < .05). The impingement onset angle and the thickness of the Achilles tendon insertion were greater in subjects with clinically diagnosed IAT (P = .0048, P = .0047). Furthermore, impingement onset angle proved to have a moderate correlation with anterior-posterior thickness (R2 = 0.454, P < .05). CONCLUSION: Our findings demonstrated that increased tendon thickness in IAT patients is associated with larger impingement onset angles, raising the range of ankle angles over which the tendon is exposed to impingement. CLINICAL RELEVANCE: Increased susceptibility to impingement may exacerbate or perpetuate the pathology, highlighting the need for clinical strategies to reduce impingement in IAT patients.

Scleraxis-lineage cell depletion improves tendon healing and disrupts adult tendon homeostasis.

Despite the requirement for Scleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLin cells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTR mice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLin cells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTR mice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLin cell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

Naked mole-rats are extremely resistant to post-traumatic osteoarthritis.

Osteoarthritis (OA) is the most prevalent disabling disease, affecting quality of life and contributing to morbidity, particularly during aging. Current treatments for OA are limited to palliation: pain management and surgery for end-stage disease. Innovative approaches and animal models are needed to develop curative treatments for OA. Here, we investigated the naked mole-rat (NMR) as a potential model of OA resistance. NMR is a small rodent with the maximum lifespan of over 30 years, resistant to a wide range of age-related diseases. NMR tissues accumulate large quantities of unique, very high molecular weight, hyaluronan (HA). HA is a major component of cartilage and synovial fluid. Importantly, both HA molecular weight and cartilage stiffness decline with age and progression of OA. As increased polymer length is known to result in stiffer material, we hypothesized that NMR high molecular weight HA contributes to stiffer cartilage. Our analysis of biomechanical properties of NMR cartilage revealed that it is significantly stiffer than mouse cartilage. Furthermore, NMR chondrocytes were highly resistant to traumatic damage. In vivo experiments using an injury-induced model of OA revealed that NMRs were highly resistant to OA. While similarly treated mice developed severe cartilage degeneration, NMRs did not show any signs of OA. Our study shows that NMRs are remarkably resistant to OA, and this resistance is likely conferred by high molecular weight HA. This work suggests that NMR is a useful model to study OA resistance and NMR high molecular weight HA may hold therapeutic potential for OA treatment.

Individualized Characterization of the Distribution of Collagen Fibril Dispersion Using Optical Aberrations of the Cornea for Biomechanical Models.

Purpose: The spatial distribution of collagen fibril dispersion has a significant impact on both corneal biomechanical and optical behaviors. The goal of this study was to demonstrate a novel method to characterize collagen fibril dispersion using intraocular pressure (IOP)-induced changes in corneal optical aberrations for individualized finite-element (FE) modeling. Methods: The method was tested through both numerical simulations and ex vivo experiments. Inflation tests were simulated in FE models with three assumed patterns of collagen fibril dispersion and experimentally on three rhesus monkey corneas. Geometry, matrix stiffness, and the IOP-induced changes in wavefront aberrations were measured, and the collagen fibril dispersion was characterized. An individualized corneal model with customized collagen fibril dispersion was developed, and the estimated optical aberrations were compared with the measured data. Results: For the theoretical investigations, three assumed distributions of fibril dispersion were all successfully characterized. The estimated optical aberrations closely matched the measured data, with average root-mean-square (RMS) differences of 0.29, 0.24, and 0.10 µm for the three patterns, respectively. The overall features of the IOP-induced changes in optical aberrations were estimated for two ex vivo monkey corneas, with average RMS differences of 0.57 and 0.43 µm. Characterization of the fibril dispersion in the third cornea might have been affected by corneal hydration, resulting in an increased RMS difference, 0.8 µm. Conclusions: A more advanced corneal model with individualized distribution of collagen fibril dispersion can be developed and used to improve our ability to understand both biomechanical and optical behaviors of the cornea.

Tensile mechanical changes in the Achilles tendon due to Insertional Achilles tendinopathy.

Insertional Achilles tendinopathy (IAT) is a painful condition that is challenging to treat non-operatively. Although previous studies have characterized the gross histological features, in vivo strain patterns and transverse compressive mechanical properties of tissue affected by IAT, it is not known how IAT impacts the tensile mechanical properties of the Achilles tendon insertion along the axial/longitudinal direction (i.e., along the predominant direction of loading). To address this knowledge gap, the objectives of this study were to 1) apply ex vivo mechanical testing, nonlinear elastic analysis and quasilinear viscoelastic (QLV) analysis to compare the axial tensile mechanical properties of the Achilles tendon insertion in individuals with and without IAT; and 2) use biochemical analysis and second harmonic generation (SHG) imaging to assess structural and compositional changes induced by IAT in order to help explain IAT-associated tensile mechanical changes. Tissue from the Achilles tendon insertion was acquired from healthy donors and from patients undergoing debridement surgery for IAT. Tissue specimens were mechanically tested using a uniaxial tensile (stress relaxation) test applied in the axial direction. A subset of the donor specimens was used for SHG imaging and biochemical analysis. Linear and non-linear elastic analyses of the stress relaxation tests showed no significant tensile mechanical changes in IAT specimens compared to healthy controls. However, SHG analysis showed that fibrillar collagen was significantly more disorganized in IAT tissue as compared with healthy controls, and biochemical analysis showed that sulfated glycosaminoglycan (sGAG) content and water content were higher in IAT specimens. Collectively, these findings suggest that conservative interventions for IAT should target restoration of ultrastructural organization, reduced GAG content, and reduced resistance to transverse compressive strain.

Evidence that reduction in volume protects in situ articular chondrocytes from mechanical impact.

Chondrocytes, the resident cells in articular cartilage, carry the burden of producing and maintaining the extracellular matrix (ECM). However, as these cells have a low proliferative capacity and are not readily replaced, chondrocyte death due to extreme forces may contribute to the pathogenesis of osteoarthritis (OA) after injury or may inhibit healing after osteochondral transplantation, a restorative procedure for damaged cartilage that requires a series of mechanical impacts to insert the graft. Consequently, there is a need to understand what factors influence the vulnerability of in situ chondrocytes to mechanical trauma. To this end, the objective of this study was to investigate how altering cell volume by different means (hydrostatic pressure, uniaxial load, and osmotic challenge with and without inhibition of regulatory volume decrease) affects the vulnerability of in situ chondrocytes to extreme mechanical forces. Using a custom experimental platform enabling testing of viable and intact murine cartilage-on-bone explants, we established a strong correlation between chondrocyte volume and vulnerability to impact injury wherein reduced volume was protective. Moreover, we found that the volume-perturbing interventions did not affect cartilage ECM mechanical properties, suggesting that their effects on chondrocyte vulnerability occurred at the cellular level. The findings of this study offer new avenues for novel strategies aimed at preventing chondrocyte loss during osteochondral grafting or to halting the progression of cell death after a joint destabilizing injury.

Tendon Biomechanics and Crimp Properties Following Fatigue Loading Are Influenced by Tendon Type and Age in Mice.

In tendon, type-I collagen assembles together into fibrils, fibers, and fascicles that exhibit a wavy or crimped pattern that uncrimps with applied tensile loading. This structural property has been observed across multiple tendons throughout aging and may play an important role in tendon viscoelasticity, response to fatigue loading, healing, and development. Previous work has shown that crimp is permanently altered with the application of fatigue loading. This opens the possibility of evaluating tendon crimp as a clinical surrogate of tissue damage. The purpose of this study was to determine how fatigue loading in tendon affects crimp and mechanical properties throughout aging and between tendon types. Mouse patellar tendons (PT) and flexor digitorum longus (FDL) tendons were fatigue loaded while an integrated plane polariscope simultaneously assessed crimp properties at P150 and P570 days of age to model mature and aged tendon phenotypes (N = 10-11/group). Tendon type, fatigue loading, and aging were found to differentially affect tendon mechanical and crimp properties. FDL tendons had higher modulus and hysteresis, whereas the PT showed more laxity and toe region strain throughout aging. Crimp frequency was consistently higher in FDL compared with PT throughout fatigue loading, whereas the crimp amplitude was cycle dependent. This differential response based on tendon type and age further suggests that the FDL and the PT respond differently to fatigue loading and that this response is age-dependent. Together, our findings suggest that the mechanical and structural effects of fatigue loading are specific to tendon type and age in mice. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:36-42, 2020.

Design of a Bioreactor to Assess the Effect of Passive Joint Loading in a Live Chick Embryo In Ovo.

There is increasing interest in understanding how mechanical cues (e.g., physical forces due to kicking and other movements) influence the embryological development of tissues and organs. For example, recent studies from our laboratory and others have used the chick embryo model to demonstrate that the compositional and mechanical properties of developing tendons are strongly regulated by embryo movement frequency. However, current research tools for manipulating embryological movements and in ovo (or in utero) mechanical forces are generally limited to chemical treatments that either paralyze or overstimulate muscles without allowing for precise control of physical cues. Thus, in this study, we introduce an instrument that enables application of passive, dynamic ankle flexion at prescribed amplitudes and frequencies in live, developing chick embryos. This device meets the design goals of allowing for precise (<1.5°) control of different waveforms of ankle motion at a physiologically relevant frequency (0.17 Hz) across a range of ankle angles (0-90° plantarflexion) with maintenance of embryo viability comparable to other methods. Impact Statement We describe the design and implementation of a novel bioreactor to precisely control ankle motion in a chick embryo within its physiological environment. The chick embryo has been used for decades to study mechanobiology of musculoskeletal tissue development and regeneration, but approaches have been limited to chemical treatments that either paralyze or overstimulate muscles without allowing for precise control of physical cues. Thus, this novel instrument is a major advancement over current research tools for manipulating chick embryological movements in ovo (or in utero).