Off-Axis Loading Fixture for Spine Biomechanics: Combined Compression and Bending.

The spine is a multi-tissue musculoskeletal system that supports large multi-axial loads and motions during physiological activities. The healthy and pathological biomechanical function of the spine and its subtissues are generally studied using cadaveric specimens that often require multi-axis biomechanical test systems to mimic the complex loading environment of the spine. Unfortunately, an off-the-shelf device can easily exceed 200,000 USD, while a custom device requires extensive time and experience in mechatronics. Our goal was to develop a cost-appropriate compression and bending (flexion-extension and lateral bending) spine testing system that requires little time and minimal technical knowledge. Our solution was an off-axis loading fixture (OLaF) that mounts to an existing uni-axial test frame and requires no additional actuators. OLaF requires little machining, with most components purchased off-the-shelf, and costs less than 10,000 USD. The only external transducer required is a six-axis load cell. Furthermore, OLaF is controlled using the existing uni-axial test frame's software, while the load data is collected using the software included with the six-axis load cell. Here we provide the design rationale for how OLaF develops primary motions and loads and minimizes off-axis secondary constraints, verify the primary kinematics using motion capture, and demonstrate that the system is capable of applying physiologically relevant, noninjurious, axial compression and bending. While OLaF is limited to compression and bending studies it produces repeatable physiologically relevant biomechanics, with high quality data, and minimal startup costs.

Volume Loss and Recovery in Bovine Knee Meniscus Loaded in Circumferential Tension.

Load-induced volume change is an important aspect of knee meniscus function because volume loss creates fluid pressure, which minimizes friction and helps support compressive loads. The knee meniscus is unusual amongst cartilaginous tissues in that it is loaded not only in axial compression, but also in circumferential tension between its tibial attachments. Despite the physiologic importance of the knee meniscus' tensile properties, its volumetric strain in tension has never been directly measured, and predictions of volume strain in the scientific literature are inconsistent. In this study, we apply uniaxial tension to bovine knee meniscus and use biplanar imaging to directly observe the resulting three-dimensional volume change and unloaded recovery, revealing that tension causes volumetric contraction. Compression is already known to also cause contraction; therefore, all major physiologic loads compress and pressurize the meniscus, inducing fluid outflow. Although passive unloaded recovery is often described as slow relative to loaded loss, here we show that at physiologic strains the volume recovery rate in the meniscus upon unloading is faster than the rate of volume loss. These measurements of volumetric strain are an important step toward a complete theory of knee meniscus fluid flow and load support.

Acute Repair of Meniscus Root Tear Partially Restores Joint Displacements as Measured With Magnetic Resonance Images and Loading in a Cadaveric Porcine Knee.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, increasing the risk of developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post-injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. The objective of this study was to quantify joint kinematics in an intact joint, after a meniscus root tear, and after suture repair in cadaveric porcine knees, a frequently used in vivo model. We utilized an magnetic resonance images-compatible loading device and novel use of a T1 vibe sequence to measure meniscus and femur displacements under physiological axial loads. We found that anterior root tear led to large meniscus displacements under physiological axial loading and that suture anchor repair reduced these displacements but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus pivoted in the axial plane. Methods from this study can be applied to assess altered joint kinematics following human knee injuries and evaluate repair strategies aimed to restore joint kinematics.

Overload in a Rat In Vivo Model of Synergist Ablation Induces Tendon Multiscale Structural and Functional Degeneration.

Tendon degeneration is typically described as an overuse injury with little distinction made between magnitude of load (overload) and number of cycles (overuse). Further, in vivo, animal models of tendon degeneration are mostly overuse models, where tendon damage is caused by a high number of load cycles. As a result, there is a lack of knowledge of how isolated overload leads to degeneration in tendons. A surgical model of synergist ablation (SynAb) overloads the target tendon, plantaris, by ablating its synergist tendon, Achilles. The objective of this study was to evaluate the structural and functional changes that occur following overload of plantaris tendon in a rat SynAb model. Tendon cross-sectional area (CSA) and shape changes were evaluated by longitudinal MR imaging up to 8 weeks postsurgery. Tissue-scale structural changes were evaluated by semiquantified histology and second harmonic generation microscopy. Fibril level changes were evaluated with serial block face scanning electron microscopy (SBF-SEM). Functional changes were evaluated using tension tests at the tissue and microscale using a custom testing system allowing both video and microscopy imaging. At 8 weeks, overloaded plantaris tendons exhibited degenerative changes including increases in CSA, cell density, collagen damage area fraction (DAF), and fibril diameter, and decreases in collagen alignment, modulus, and yield stress. To interpret the differences between overload and overuse in tendon, we introduce a new framework for tendon remodeling and degeneration that differentiates between the inputs of overload and overuse. In summary, isolated overload induces multiscale degenerative structural and functional changes in plantaris tendon.

Comparative multi-scale hierarchical structure of the tail, plantaris, and Achilles tendons in the rat.

Rodent tendons are widely used to study human pathologies such as tendinopathy and repair, and to address fundamental physiological questions about development, growth, and remodeling. However, how the gross morphology and multi-scale hierarchical structure of rat tendons, such as the tail, plantaris, and Achilles tendons, compare with that of human tendons are unknown. In addition, there remains disagreement about terminology and definitions. Specifically, the definitions of fascicle and fiber are often dependent on diameter sizes, not their characteristic features, and these definitions impair the ability to compare hierarchical structure across species, where the sizes of the fiber and fascicle may change with animal size and tendon function. Thus, the objective of the study was to select a single species that is commonly used for tendon research (rat) and tendons with varying mechanical functions (tail, plantaris, Achilles) to evaluate the hierarchical structure at multiple length scales using histology, SEM, and confocal imaging. With the exception of the specialized rat tail tendon, we confirmed that in rat tendons there are no fascicles and the fiber is the largest subunit. In addition, we provided a structurally based definition of a fiber as a bundle of collagen fibrils that is surrounded by elongated cells, and this definition was supported by both histologically processed and unprocessed samples. In all rat tendons studied, the fiber diameters were consistently between 10 and 50 µm, and this diameter range appears to be conserved across larger species. Specific recommendations were made highlighting the strengths and limitations of each rat tendon as a research model. Understanding the hierarchical structure of tendon can advance the design and interpretation of experiments and development of tissue-engineered constructs.

A Reactive Inelasticity Theoretical Framework for Modeling Viscoelasticity, Plastic Deformation, and Damage in Fibrous Soft Tissue.

Fibrous soft tissues are biopolymeric materials that are made of extracellular proteins, such as different types of collagen and proteoglycans, and have a high water content. These tissues have nonlinear, anisotropic, and inelastic mechanical behaviors that are often categorized into viscoelastic behavior, plastic deformation, and damage. While tissue's elastic and viscoelastic mechanical properties have been measured for decades, there is no comprehensive theoretical framework for modeling inelastic behaviors of these tissues that is based on their structure. To model the three major inelastic mechanical behaviors of tissue's fibrous matrix, we formulated a structurally inspired continuum mechanics framework based on the energy of molecular bonds that break and reform in response to external loading (reactive bonds). In this framework, we employed the theory of internal state variables (ISV) and kinetics of molecular bonds. The number fraction of bonds, their reference deformation gradient, and damage parameter were used as state variables that allowed for consistent modeling of all three of the inelastic behaviors of tissue by using the same sets of constitutive relations. Several numerical examples are provided that address practical problems in tissue mechanics, including the difference between plastic deformation and damage. This model can be used to identify relationships between tissue's mechanical response to external loading and its biopolymeric structure.

Evaluating Plastic Deformation and Damage as Potential Mechanisms for Tendon Inelasticity Using a Reactive Modeling Framework.

Inelastic behaviors, such as softening, a progressive decrease in modulus before failure, occur in tendon and are important aspects in degeneration and tendinopathy. These inelastic behaviors are generally attributed to two potential mechanisms: plastic deformation and damage. However, it is not clear which is primarily responsible. In this study, we evaluated these potential mechanisms of tendon inelasticity by using a recently developed reactive inelasticity model (RIE), which is a structurally inspired continuum mechanics framework that models tissue inelasticity based on the molecular bond kinetics. Using RIE, we formulated two material models, one specific to plastic deformation and the other to damage. The models were independently fit to published macroscale experimental tensile tests of rat tail tendons. We quantified the inelastic effects and compared the performance of the two models in fitting the mechanical response during loading, relaxation, unloading, and reloading phases. Additionally, we validated the models by using the resulting fit parameters to predict an independent set of experimental stress-strain curves from ramp-to-failure tests. Overall, the models were both successful in fitting the experiments and predicting the validation data. However, the results did not strongly favor one mechanism over the other. As a result, to distinguish between plastic deformation and damage, different experimental protocols will be needed. Nevertheless, these findings suggest the potential of RIE as a comprehensive framework for studying tendon inelastic behaviors.

Human Disc Nucleotomy Alters Annulus Fibrosus Mechanics at Both Reference and Compressed Loads.

Nucleotomy is a common surgical procedure and is also performed in ex vivo mechanical testing to model decreased nucleus pulposus (NP) pressurization that occurs with degeneration. Here, we implement novel and noninvasive methods using magnetic resonance imaging (MRI) to study internal 3D annulus fibrosus (AF) deformations after partial nucleotomy and during axial compression by evaluating changes in internal AF deformation at reference loads (50 N) and physiological compressive loads (∼10% strain). One particular advantage of this methodology is that the full 3D disc deformation state, inclusive of both in-plane and out-of-plane deformations, can be quantified through the use of a high-resolution volumetric MR scan sequence and advanced image registration. Intact grade II L3-L4 cadaveric human discs before and after nucleotomy were subjected to identical mechanical testing and imaging protocols. Internal disc deformation fields were calculated by registering MR images captured in each loading state (reference and compressed) and each condition (intact and nucleotomy). Comparisons were drawn between the resulting three deformation states (intact at compressed load, nucleotomy at reference load, nucleotomy at compressed load) with regard to the magnitude of internal strain and direction of internal displacements. Under compressed load, internal AF axial strains averaged -18.5% when intact and -22.5% after nucleotomy. Deformation orientations were significantly altered by nucleotomy and load magnitude. For example, deformations of intact discs oriented in-plane, whereas deformations after nucleotomy oriented axially. For intact discs, in-plane components of displacements under compressive loads oriented radially outward and circumferentially. After nucleotomy, in-plane displacements were oriented radially inward under reference load and were not significantly different from the intact state at compressed loads. Re-establishment of outward displacements after nucleotomy indicates increased axial loading restores the characteristics of internal pressurization. Results may have implications for the recurrence of pain, design of novel therapeutics, or progression of disc degeneration.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Strain Distribution of Intact Rat Rotator Cuff Tendon-to-Bone Attachments and Attachments With Defects.

This study aimed to experimentally track the tissue-scale strains of the tendon-bone attachment with and without a localized defect. We hypothesized that attachments with a localized defect would develop strain concentrations and would be weaker than intact attachments. Uniaxial tensile tests and digital image correlation were performed on rat infraspinatus tendon-to-bone attachments with defects (defect group) and without defects (intact group). Biomechanical properties were calculated, and tissue-scale strain distributions were quantified for superior and inferior fibrous and calcified regions. At the macroscale, the defect group exhibited reduced stiffness (31.3±3.7 N/mm), reduced ultimate load (24.7±3.8 N), and reduced area under the curve at ultimate stress (3.7±1.5 J/m2) compared to intact attachments (42.4±4.3 N/mm, 39.3±3.7 N, and 5.6±1.4 J/m2, respectively). Transverse strain increased with increasing axial load in the fibrous region of the defect group but did not change for the intact group. Shear strain of the superior fibrous region was significantly higher in the defect group compared to intact group near yield load. This work experimentally identified that attachments may resist failure by distributing strain across the interface and that strain concentrations develop near attachment defects. By establishing the tissue-scale deformation patterns of the attachment, we gained insight into the micromechanical behavior of this interfacial tissue and bolstered our understanding of the deformation mechanisms associated with its ability to resist failure.

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

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.

Theory of MRI contrast in the annulus fibrosus of the intervertebral disc.

OBJECTIVE: Here we develop a three-dimensional analytic model for MR image contrast of collagen lamellae in the annulus fibrosus of the intervertebral disc of the spine, based on the dependence of the MRI signal on collagen fiber orientation. MATERIALS AND METHODS: High-resolution MRI scans were performed at 1.5 and 7 T on intact whole disc specimens from ovine, bovine, and human spines. An analytic model that approximates the three-dimensional curvature of the disc lamellae was developed to explain inter-lamellar contrast and intensity variations in the annulus. The model is based on the known anisotropic dipolar relaxation of water in tissues with ordered collagen. RESULTS: Simulated MRI data were generated that reproduced many features of the actual MRI data. The calculated inter-lamellar image contrast demonstrated a strong dependence on the collagen fiber angle and on the circumferential location within the annulus. CONCLUSION: This analytic model may be useful for interpreting MR images of the disc and for predicting experimental conditions that will optimize MR image contrast in the annulus fibrosus.

Advances in Quantification of Meniscus Tensile Mechanics Including Nonlinearity, Yield, and Failure.

The meniscus provides crucial knee function and damage to it leads to osteoarthritis of the articular cartilage. Accurate measurement of its mechanical properties is therefore important, but there is uncertainty about how the test procedure affects the results, and some key mechanical properties are reported using ad hoc criteria (modulus) or not reported at all (yield). This study quantifies the meniscus' stress-strain curve in circumferential and radial uniaxial tension. A fiber recruitment model was used to represent the toe region of the stress-strain curve, and new reproducible and objective procedures were implemented for identifying the yield point and measuring the elastic modulus. Patterns of strain heterogeneity were identified using strain field measurements. To resolve uncertainty regarding whether rupture location (i.e., midsubstance rupture versus at-grip rupture) influences the measured mechanical properties, types of rupture were classified in detail and compared. Dogbone (DB)-shaped specimens are often used to promote midsubstance rupture; to determine if this is effective, we compared DB and rectangle (R) specimens in both the radial and circumferential directions. In circumferential testing, we also compared expanded tab (ET) specimens under the hypothesis that this shape would more effectively secure the meniscus' curved fibers and thus produce a stiffer response. The fiber recruitment model produced excellent fits to the data. Full fiber recruitment occurred approximately at the yield point, strongly supporting the model's physical interpretation. The strain fields, especially shear and transverse strain, were extremely heterogeneous. The shear strain field was arranged in pronounced bands of alternating positive and negative strain in a pattern similar to the fascicle structure. The site and extent of failure showed great variation, but did not affect the measured mechanical properties. In circumferential tension, ET specimens underwent earlier and more rapid fiber recruitment, had less stretch at yield, and had greater elastic modulus and peak stress. No significant differences were observed between R and DB specimens in either circumferential or radial tension. Based on these results, ET specimens are recommended for circumferential tests and R specimens for radial tests. In addition to the data obtained, the procedural and modeling advances made in this study are a significant step forward for meniscus research and are applicable to other fibrous soft tissues.

Evaluation of an In Situ Gelable and Injectable Hydrogel Treatment to Preserve Human Disc Mechanical Function Undergoing Physiologic Cyclic Loading Followed by Hydrated Recovery.

Despite the prevalence of disc degeneration and its contributions to low back problems, many current treatments are palliative only and ultimately fail. To address this, nucleus pulposus replacements are under development. Previous work on an injectable hydrogel nucleus pulposus replacement composed of n-carboxyethyl chitosan, oxidized dextran, and teleostean has shown that it has properties similar to native nucleus pulposus, can restore compressive range of motion in ovine discs, is biocompatible, and promotes cell proliferation. The objective of this study was to determine if the hydrogel implant will be contained and if it will restore mechanics in human discs undergoing physiologic cyclic compressive loading. Fourteen human lumbar spine segments were tested using physiologic cyclic compressive loading while intact, following nucleotomy, and again following treatment of injecting either phosphate buffered saline (PBS) (sham, n = 7) or hydrogel (implant, n = 7). In each compressive test, mechanical parameters were measured immediately before and after 10,000 cycles of compressive loading and following a period of hydrated recovery. The hydrogel implant was not ejected from the disc during 10,000 cycles of physiological compression testing and appeared undamaged when discs were bisected following all mechanical tests. For sham samples, creep during cyclic loading increased (+15%) from creep during nucleotomy testing, while for implant samples creep strain decreased (-3%) toward normal. There was no difference in compressive modulus or compressive strains between implant and sham samples. These findings demonstrate that the implant interdigitates with the nucleus pulposus, preventing its expulsion during 10,000 cycles of compressive loading and preserves disc creep within human L5-S1 discs. This and previous studies provide a solid foundation for continuing to evaluate the efficacy of the hydrogel implant.

The shear modulus of the nucleus pulposus measured using magnetic resonance elastography: a potential biomarker for intervertebral disc degeneration.

PURPOSE: This study aims to: (1) measure the shear modulus of nucleus pulposus (NP) in intact human vertebra-disc-vertebra segments using a magnetic resonance elastography setup for a 7T whole-body scanner, (2) quantify the effect of disc degeneration on the NP shear modulus measured using magnetic resonance elastography, and (3) compare the NP shear modulus to other magnetic resonance-based biomarkers of dis degeneration. METHODS: Thirty intact human disc segments were classified as normal, mild, or severely degenerated. The NP shear modulus was measured using a custom-made setup that included a novel inverse method less sensitive to noisy displacements. T2 relaxation time was measured at 7T. The accuracy of these parameters to classify different degrees of degeneration was evaluated using receiver operating characteristic curves. RESULTS: The magnetic resonance elastography measure of shear modulus in the NP was able to differentiate between normal, mild degeneration, and severe degeneration. The T2 relaxation time was able to differentiate between normal and mild degeneration, but it could not distinguish between mild and severe degeneration. CONCLUSIONS: This study shows that the NP shear modulus measured using magnetic resonance elastography is sensitive to disc degeneration and has the potential of being used as a clinical tool to quantify the mechanical integrity of the intervertebral disc.

Internal three-dimensional strains in human intervertebral discs under axial compression quantified noninvasively by magnetic resonance imaging and image registration.

Study objectives were to develop, validate, and apply a method to measure three-dimensional (3D) internal strains in intact human discs under axial compression. A custom-built loading device applied compression and permitted load-relaxation outside of the magnet while also maintaining compression and hydration during imaging. Strain was measured through registration of 300 µm isotropic resolution images. Excellent registration accuracy was achieved, with 94% and 65% overlap of disc volume and lamellae compared to manual segmentation, and an average Hausdorff, a measure of distance error, of 0.03 and 0.12 mm for disc volume and lamellae boundaries, respectively. Strain maps enabled qualitative visualization and quantitative regional annulus fibrosus (AF) strain analysis. Axial and circumferential strains were highest in the lateral AF and lowest in the anterior and posterior AF. Radial strains were lowest in the lateral AF, but highly variable. Overall, this study provided new methods that will be valuable in the design and evaluation surgical procedures and therapeutic interventions.

Accurate Prediction of Stress in Fibers with Distributed Orientations Using Generalized High-Order Structure Tensors.

The orientation of collagen fibers plays an important role on the mechanics of connective tissues. Connective tissues have fibers with different orientation distributions. The angular integration formulation used to model the mechanics of fibers with distributed orientation is accurate, but computationally expensive for numerical methods such as finite elements. This study presents a formulation based on pre-integrated Generalized High-Order Structure Tensors (GHOST) which greatly improves the accuracy of the predicted stress. Simplifications of the GHOST formulation for transversely-isotropic and planar fiber distributions are also presented. Additionally, the GHOST and the angular integration formulations are compared for different loading conditions, fiber orientation functions, strain energy functions and degrees of fiber non-linearity. It was found that the GHOST formulation predicted the stress of the fibers with an error lower than 10% for uniaxial and biaxial tension. Fiber non-linearity increased the error of the GHOST formulation; however, the error was reduced to negligible values by considering higher order structure tensors. The GHOST formulation produced lower errors when used with an elliptical fiber density function and a binomial strain energy function. In conclusion, the GHOST formulation is able to accurately predict the stress of fibers with distributed orientation without requiring numerous integral calculations. Consequently, the GHOST formulation may reduce the computational effort needed to analyze the mechanics of fibrous tissues with distributed orientations.

Geometric determinants of the mechanical behavior of image-based finite element models of the intervertebral disc.

The intervertebral disc is an important structure for load transfer through the spine. Its injury and degeneration have been linked to pain and spinal fractures. Disc injury and spine fractures are associated with high stresses; however, these stresses cannot be measured, necessitating the use of finite element (FE) models. These models should include the disc's complex structure, as changes in disc geometry have been linked to altered mechanical behavior. However, image-based models using disc-specific structures have yet to be established. This study describes a multiphasic FE modeling approach for noninvasive estimates of subject-specific intervertebral disc mechanical behavior based on medical imaging. The models (n = 22) were used to study the influence of disc geometry on the predicted global mechanical response (moments and forces), internal local disc stresses, and tractions at the interface between the disc and the bone. Disc geometry was found to have a strong influence on the predicted moments and forces on the disc (R2 = 0.69-0.93), while assumptions regarding the side curvature (bulge) of the disc had only a minor effect. Strong variability in the predicted internal disc stresses and tractions was observed between the models (mean absolute differences of 5.1%-27.7%). Disc height had a systematic influence on the internal disc stresses and tractions at the disc-to-bone interface. The influence of disc geometry on mechanics highlights the importance of disc-specific modeling to estimate disc injury risk, loading on the adjacent vertebral bodies, and the mechanical environment present in disc tissues.

Can axial loading restore in vivo disc geometry, opening pressure, and T2 relaxation time?

Background: Cadaveric intervertebral discs are often studied for a variety of research questions, and outcomes are interpreted in the in vivo context. Unfortunately, the cadaveric disc does not inherently represent the LIVE condition, such that the disc structure (geometry), composition (T2 relaxation time), and mechanical function (opening pressure, OP) measured in the cadaver do not necessarily represent the in vivo disc. Methods: We conducted serial evaluations in the Yucatan minipig of disc geometry, T2 relaxation time, and OP to quantify the changes that occur with progressive dissection and used axial loading to restore the in vivo condition. Results: We found no difference in any parameter from LIVE to TORSO; thus, within 2 h of sacrifice, the TORSO disc can represent the LIVE condition. With serial dissection and sample preparation the disc height increased (SEGMENT height 18% higher than TORSO), OP decreased (POTTED was 67% lower than TORSO), and T2 time was unchanged. With axial loading, an imposed stress of 0.20-0.33 MPa returned the disc to in vivo, LIVE disc geometry and OP, although T2 time was decreased. There was a linear correlation between applied stress and OP, and this was conserved across multiple studies and species. Conclusion: To restore the LIVE disc state in human studies or other animal models, we recommend measuring the OP/stress relationship and using this relationship to select the applied stress necessary to recover the in vivo condition.

Macro- to microscale strain transfer in fibrous tissues is heterogeneous and tissue-specific.

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35-70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15-25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.