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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 with high number of load cycles. As a result, there is a lack of knowledge of how isolated overload leads to degeneration. 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 and shape changes were evaluated by longitudinal MR imaging up to 8-weeks post-surgery. Tissue-scale structural changes were evaluated by semi-quantified 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, 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.

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.

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.

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, greatly increasing the risk for developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, this 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. However, many studies of meniscus function required opening the joint, which alters kinematics. The objective of this study was to use novel MRI methods to image the intact joint under axial load and measure the acute meniscus and femur displacements in an intact joint, after a meniscus root tear, and after suture repair in the porcine knee, a frequently used in vivo model. We found that anterior root tear led to large meniscus and femur 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 rotated in the axial plane. This technique can be applied to evaluate the effect of knee injuries and to develop improved repair strategies to restore joint kinematics.

Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Determining tissue biomechanical material properties from mechanical test data is frequently required in a variety of applications. However, the validity of the resulting constitutive model parameters is the subject of debate in the field. Parameter optimization in tissue mechanics often comes down to the “identifiability” or “uniqueness” of constitutive model parameters; however, despite advances in formulating complex constitutive relations and many classic and creative curve-fitting approaches, there is currently no accessible framework to study the identifiability of tissue material parameters. Our objective was to assess the identifiability of material parameters for established constitutive models of fiber-reinforced soft tissues, biomaterials, and tissue-engineered constructs and establish a generalizable procedure for other applications. To do so, we generated synthetic experimental data by simulating uniaxial tension and compression tests, commonly used in biomechanics. We then fit this data using a multi-start optimization technique based on the nonlinear least-squares method with multiple initial parameter guesses. We considered tendon and sclera as example tissues, using constitutive models that describe these fiber-reinforced tissues. We demonstrated that not all the model parameters of these constitutive models were identifiable from uniaxial mechanical tests, despite achieving virtually identical fits to the stress-stretch response. We further show that when the lateral strain was considered as an additional fitting criterion, more parameters are identifiable, but some remain unidentified. This work provides a practical approach for addressing parameter identifiability in tissue mechanics.

The knee menisci are comprised of two orthogonal collagenous networks – circumferential and radial – that combine to enable efficient load bearing by the tissue in adults. Here, we assessed how the structural and functional characteristics of these networks developed over the course of skeletal maturation and determined the role of these fiber networks in defect tolerance with tissue injury. Imaging of the radial tie fiber (RTF) collagen structure in medial bovine menisci from fetal, juvenile, and adult specimens showed increasing heterogeneity, anisotropy, thickness, and density with skeletal development. Mechanical analysis showed that the tensile modulus in the radial direction did not change with skeletal development, though the resilience (in the radial direction) increased and the tolerance to defects in the circumferential direction decreased, in adult compared to fetal tissues. This loss of defect tolerance correlated with increased order in the RTF network in adult tissue. These data provide new insights into the role of the radial fiber network in meniscus function, will lead to improved clinical decision-making in the presence of a tear, and may improve engineering efforts to reproduce this critical load-bearing structure in the knee.

The meniscus plays a central load bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis. In small animal models, progressive degenerative changes occur with unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of disease has not yet been defined in these models. Thus, the goal of this study was to develop and validate a large-animal model of surgically-induced destabilization of the medial meniscus and to use multi-modal (mechanical, histological, and MRI) and multi-scale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in thirteen Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By three months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at one month compared to Sham operated animals; these deleterious changes persisted through three months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to Sham menisci at both one and three months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provides a new large animal test bed in which to evaluate therapeutics and interventions to treat post-traumatic osteoarthritis (PTOA) that originates from meniscal injury.

The actin cytoskeleton is a central mediator between mechanical force and cellular phenotype. In tendon, it is speculated that mechanical stress deprivation regulates gene expression by filamentous (F−) actin destabilization. However, the molecular mechanisms that stabilize tenocyte F-actin networks remain unclear. Tropomyosins (Tpms) are master regulators of F-actin networks. There are over 40 mammalian Tpm isoforms, with each isoform having the unique capability to stabilize F-actin sub-populations. Thus, the specific Tpm(s) expressed by a cell defines overall F-actin organization. Here, we investigated F-actin destabilization by stress deprivation of tendon and tested the hypothesis that stress fiber-associated Tpm(s) stabilize tenocyte F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon fascicles downregulated tenocyte genes (collagen-I, tenascin-C, scleraxis, α-smooth muscle actin) and upregulated matrix metalloproteinase-3. Concomitant with mRNA modulation were increases in DNAse-I/Phallodin (G/F-actin) staining, confirming F-actin destabilization by tendon stress deprivation. To investigate the molecular regulation of F-actin stabilization, we first identified the Tpms expressed by mouse tendons. Tendon cells from different origins (tail, Achilles, plantaris) express three isoforms in common: Tpm1.6, 3.1, and 4.2. We examined the function of Tpm3.1 since we previously determined that it stabilizes F-actin stress fibers in lens epithelial cells. Tpm3.1 associated with F-actin stress fibers in native and primary tendon cells. Inhibition of Tpm3.1 depolymerized F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin stability in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Summary Objectives The objective of this study was to perform a quantitative analysis of the structural and functional alterations in the intervertebral disc during in vivo degeneration, using emerging tools that enable rigorous assessment from the microscale to the macroscale, as well as to correlate these outcomes with noninvasive, clinically relevant imaging parameters. Design Degeneration was induced in a rabbit model by puncturing the annulus fibrosus (AF) with a 16-gauge needle. 2, 4, 8, and 12 weeks following puncture, degenerative changes in the discs were evaluated via magnetic resonance imaging (MRI), whole motion segment biomechanics, atomic force microscopy, histology and polarized light microscopy, immunohistochemistry, biochemical content, and second harmonic generation imaging. Results Following puncture, degeneration was evident through marked changes in whole disc structure and mechanics. Puncture acutely compromised disc macro and microscale mechanics, followed by progressive stiffening and remodeling. Histological analysis showed substantial anterior fibrotic remodeling and osteophyte formation, as well as an overall reduction in disc height, and disorganization and infolding of the AF lamellae into the NP space. Increases in NP collagen content and aggrecan breakdown products were also noted within 4 weeks. On MRI, NP T2 was reduced at all post-puncture time points and correlated significantly with microscale indentation modulus. Conclusion This study defined the time dependent changes in disc structure–function relationships during IVD degeneration in a rabbit annular injury model and correlated degeneration severity with clinical imaging parameters. Our findings identified AF infolding and occupancy of the space as a principle mechanism of disc degeneration in response to needle puncture, and provide new insights to direct the development of novel therapeutics.

In fibrous tissues, prestressed boundary constraints at bone interfaces instil residual strain throughout the tissue, even when unloaded. For example, internal swelling pressures in the central nucleus pulposus of the intervertebral disc generate prestrain in the outer annulus fibrosus. With injury and depressurization, these residual strains are lost. Here we show that the loss of residual strains in the intervertebral disc alters the microenvironment and instigates aberrant tissue remodelling and the adoption of atypical cellular phenotypes. By using puncture surgery of the annulus fibrosus in rabbits, ex vivo puncture experiments and electrospun nanofibrous scaffolds recapitulating these evolving boundary constraints, we show that the loss of residual strain promotes short-term apoptosis and the emergence of a fibrotic phenotype. We also show that local fibre organization and cellular contractility mediate this process and that the aberrant cellular changes could be abrogated by targeting the cell-mechanosensing machinery with small molecules. Our findings indicate that injury to dense connective tissues under prestrain alters boundary constraints and residual strain; this leads to aberrant mechanosensing, which in turn promotes disease progression.

Tendinopathy, degeneration of the tendon that leads to pain and dysfunction, is common in both sports and occupational settings, but multi-scale mechanisms for tendinopathy are still unknown. We recently showed that micro-scale sliding (shear) is responsible for both load transfer and damage mechanisms in the rat tail tendon; however, the rat tail tendon is a specialized non-load-bearing tendon, and thus the load transfer and damage mechanisms are still unknown for load-bearing tendons. The objective of this study was to investigate the load transfer and damage mechanisms of load-bearing tendons using the rat plantaris tendon. We demonstrated that micro-scale sliding is a key component for both mechanisms in the plantaris tendon, similar to the tail tendon. Namely, the micro-scale sliding was correlated with applied strain, demonstrating that load was transferred via micro-scale sliding in the plantaris and tail tendons. In addition, while the micro-scale strain fully recovered, the micro-scale sliding was non-recoverable and strain-dependent, and correlated with tissue-scale mechanical parameters. When the applied strain was normalized, the % magnitudes of non-recoverable sliding was similar between the plantaris and tail tendons. Statement of clinical significance: Understanding the mechanisms responsible for the pathogenesis and progression of tendinopathy can improve prevention and rehabilitation strategies and guide therapies and the design of engineered constructs. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1827-1837, 2019.

Finite element models of the intervertebral disc are used to address research questions that cannot be tested through typical experimentation. A disc model requires complex geometry and tissue properties to be accurately defined to mimic the physiological disc. The physiological disc possesses residual strain in the annulus fibrosus (AF) due to osmotic swelling and due to inherently pre‐strained fibers. We developed a disc model with residual contributions due to swelling‐only, and a multigeneration model with residual contributions due to both swelling and AF fiber pre‐strain and validated it against organ‐scale uniaxial, quasi‐static and multiaxial, dynamic mechanical tests. In addition, we demonstrated the models' ability to mimic the opening angle observed following radial incision of bovine discs. Both models were validated against organ‐scale experimental data. While the swelling only model responses were within the experimental 95% confidence interval, the multigeneration model offered outcomes closer to the experimental mean and had a bovine model opening angle within one SD of the experimental mean. The better outcomes for the multigeneration model, which allowed for the inclusion of inherently pre‐strained fibers in AF, is likely due to its uniform fiber contribution throughout the AF. We conclude that the residual contribution of pre‐strained fibers in the AF should be included to best simulate the physiological disc and its behaviors., We validated a disc model with residual contributions due to swelling‐only and a multigeneration model with residual contributions due to both swelling and AF fiber pre‐strain against organ‐scale uniaxial, quasi‐static and multiaxial, dynamic mechanical tests as well as tested the models’ ability to mimic the opening angle observed following radial incision of bovine discs. The swelling only model was within the confidence interval, though the multigeneration model was closer to the experimental mean; the better outcomes for the multigeneration model is likely due to its uniform fiber contribution throughout the AF. We conclude that the residual contribution of pre‐strained fibers in the AF should be included to best simulate the physiological disc and its behaviors.

Macro-scale changes in longitudinal bone growth resulting from mechanical loading were shown in Part 1 of this review to depend on load magnitude, anatomical location, and species. While no significant effect on longitudinal growth was observed by varying frequency and amplitude of cyclic loading, such variations, in addition to loading duration and species, were shown to affect the morphology, viability, and gene and protein expression within the growth plate. Intermittent compression regimens were shown to preserve or increase growth plate height while stimulating increased chondrocyte presence in the hypertrophic zone relative to persistent and static loading regimens. Gene and protein expressions related to matrix synthesis and degradation, as well as regulation of chondrocyte apoptosis were shown to exhibit magnitude-, frequency-, and duration-dependent responses to loading regimen. Chondrocyte viability was shown to be largely preserved within physiological bounds of magnitude, frequency, amplitude, and duration. Persistent static loading was shown to be associated with overall growth plate height in tension only, reducing it in compression, while affecting growth plate zone heights differently across species and encouraging mineralization relative to intermittent cyclic loading. Lateral loading of the growth plate, as well as microfluidic approaches are relatively understudied, and age, anatomical location, and species effects within these approaches are undefined. Understanding the micro-scale effects of varied loading regimes can assist in the development of growth modulation methods and device designs optimized for growth plate viability preservation or mineralization stimulation based on patient age and anatomical location.

Determining tissue biomechanical material properties from mechanical test data is frequently required in a variety of applications, e.g. tissue engineering. However, the validity of the resulting constitutive model parameters is the subject of debate in the field. Common methods to perform fitting, such as nonlinear least-squares, are known to be subject to several limitations, most notably the uniqueness of the fitting results. Parameter optimization in tissue mechanics often comes down to the “identifiability” or “uniqueness” of constitutive model parameters; however, despite advances in formulating complex constitutive relations and many classic and creative curve-fitting approaches, there is no accessible framework to study the identifiability of tissue material parameters. Our objective was to assess the identifiability of material parameters for established constitutive models of fiber-reinforced soft tissues, biomaterials, and tissue-engineered constructs. To do so, we generated synthetic experimental data by simulating uniaxial tension and compression tests, commonly used in biomechanics. We considered tendon and sclera as example tissues, using constitutive models that describe these fiber-reinforced tissues. We demonstrated that not all of the model parameters of these constitutive models were identifiable from uniaxial mechanical tests, despite achieving virtually identical fits to the stress-stretch response. We further show that when the lateral strain was considered as an additional fitting criterion, more parameters are identifiable, but some remain unidentified. This work provides a practical approach for addressing parameter identifiability in tissue mechanics.Statement of SignificanceData fitting is a powerful technique commonly used to extract tissue material parameters from experimental data, and which thus has applications in tissue biomechanics and engineering. However, the problem of “uniqueness” or “identifiability” of the fit parameters is a significant issue, limiting the fit results’ validity. Here we provide a novel method to evaluate data fitting and assess the uniqueness of results in the tissue mechanics constitutive models. Our results indicate that the uniaxial stress-stretch experimental data are not adequate to identify all the tissue material parameters. This study is of potential interest to a wide range of readers because of its application for the characterization of other engineering materials, while addressing the problem of uniqueness of the fitted results.

Noninvasive assessments of intervertebral disc health and degeneration are critical for addressing disc degeneration and low back pain. Magnetic resonance imaging (MRI) is exceptionally sensitive to tissue with high water content, and measurement of the MR transverse relaxation time, T 2, has been applied as a quantitative, continuous, and objective measure of disc degeneration that is linked to the water and matrix composition of the disc. However, T 2 measurement is susceptible to inaccuracies due to Rician noise, T 1 contamination, and stimulated echo effects. These error generators can all be controlled for with proper data collection and fitting methods. The objective of this study was to identify sequence parameters to appropriately acquire MR data and to establish curve fitting methods to accurately calculate disc T 2 in the presence of noise by correcting for Rician noise. To do so, we compared T 2 calculated from the typical monoexponential (MONO) fits and noise corrected exponential (NCEXP) fits. We examined how the selected sequence parameters altered the calculated T 2 in silico and in vivo. Typical MONO fits were frequently poor due to Rician noise, and NCEXP fits were more likely to provide accurate T 2 calculations. NCEXP is particularly less biased and less uncertain at low SNR. This study showed that the NCEXP using sequences with data from 20 echoes out to echo times of ~300 ms is the best method for calculating T 2 of discs. By acquiring signal data out to longer echo times and accounting for Rician noise, the curve fitting is more robust in calculating T 2 despite the noise in the data. This is particularly important when considering degenerate discs or AF tissue because the SNR of these regions is lower.

Intervertebral disc degeneration is a cascade of cellular, structural, and biomechanical changes that is strongly implicated as a cause of low-back pain. Current treatment strategies have poor long-term efficacy as they seek only to alleviate symptoms without preserving or restoring native tissue structure and function. The objective of this study was to evaluate the efficacy of a combined triple interpenetrating network hydrogel (comprising dextran, chitosan, and teleostean) and mesenchymal stem cell (MSC) therapy targeting moderate-severity disc degeneration in a clinically relevant goat model. Degeneration was induced in lumbar discs of 10 large frame goats by injection of chondroitinase ABC. After 12 weeks, degenerate discs were treated by injection of either hydrogel alone or hydrogel seeded with allogeneic, bone marrow-derived MSCs. Untreated healthy and degenerate discs served as controls, and animals were euthanized 2 weeks after treatment. Discs exhibited a significant loss of disc height 12 weeks after degeneration was induced. Two weeks after treatment, discs that received the combined hydrogel and MSC injection exhibited a significant, 10% improvement in disc height index, as well as improvements in histological condition. Discs that were treated with hydrogel alone exhibited reduced tumor necrosis factor-α expression in the nucleus pulposus (NP). Microcomputed tomography imaging revealed that the hydrogel remained localized to the central NP region of all treated discs after 2 weeks of unrestricted activity. These encouraging findings motivate further, longer term studies of therapeutic efficacy of hydrogel and MSC injections in this large animal model. IMPACT STATEMENT: Low-back pain is the leading cause of disability worldwide, and degeneration of the intervertebral discs is considered to be one of the most common reasons for low-back pain. Current treatment strategies focus solely on alleviation of symptoms, and there is a critical need for new treatments that also restore disc structure and function. In this study, using a clinically relevant goat model of moderate-severity disc degeneration, we demonstrate that a combined interpenetrating network hydrogel and mesenchymal stem cell therapy provides acute improvements in disc height, histological condition, and local inflammation.

Low back pain arising from disc degeneration is one of the most common causes of limited function in adults. A number of tissue engineering strategies have been used to develop composite tissue engineered total disc replacements to restore native tissue structure and function. In this study we fabricated a composite engineered disc based on the combination of a porous polycaprolactone (PCL) foam annulus fibrosus (AF) and a hyaluronic acid (HA) hydrogel nucleus pulposus (NP). To evaluate whether native tissue cells or mesenchymal stem cells (MSCs) would perform better, constructs were seeded with native AF/NP cells or with MSCs in the foam and/or gel region. Maturation of these composite engineered discs was evaluated for 9 weeks in vitro culture by biochemical content, histological analysis and mechanical properties. To evaluate the performance of these constructs in the in vivo space, engineered discs were implanted into the caudal spines of athymic rats for 5 weeks. Our findings show that engineered discs comprised of AF/NP cells and MSCs performed similarly and maintained their structure after 5 weeks in vivo. However, for both cell types, loss of proteoglycan was evident in the NP region. These data support the continued development of the more clinically relevant MSCs population for disc replacement applications. STATEMENT OF SIGNIFICANCE: A number of tissue engineering strategies have emerged that are focused on the creation of a composite disc replacement. We fabricated a composite engineered disc based on the combination of a porous foam AF and a HA gel NP. We used these constructs to determine whether the combination of AF/NP cells or MSCs would mature to a greater extent in vitro and which cell type would best retain their phenotype after implantation. Engineered discs comprised of AF/NP cells and MSCs performed similarly, maintaining their structure after 5 weeks in vivo. These data support the successful fabrication and in vivo function of an engineered disc composed of a PCL foam AF and a hydrogel NP using either disc cells or MSCs.

Background: The corrective procedures for meniscal injury are dependent on tear type, severity, and location. Vertical longitudinal tears are common in young and active individuals, but their natural progression and impact on osteoarthritis (OA) development are not known. Root tears are challenging and they often indicate poor outcomes, although the timing and mechanisms of initiation of joint dysfunction are poorly understood, particularly in large-animal and human models. Purpose/Hypothesis: In this study, vertical longitudinal and root tears were made in a large-animal model to determine the progression of joint-wide dysfunction. We hypothesized that OA onset and progression would depend on the extent of injury-based load disruption in the tissue, such that root tears would cause earlier and more severe changes to the joint. Study Design: Controlled laboratory study. Methods: Sham surgeries and procedures to create either vertical longitudinal or root tears were performed in juvenile Yucatan mini pigs through randomized and bilateral arthroscopic procedures. Animals were sacrificed at 1, 3, or 6 months after injury and assessed at the joint and tissue level for evidence of OA. Functional measures of joint load transfer, cartilage indentation mechanics, and meniscal tensile properties were performed, as well as histological evaluation of the cartilage, meniscus, and synovium. Results: Outcomes suggested a progressive and sustained degeneration of the knee joint and meniscus after root tear, as evidenced by histological analysis of the cartilage and meniscus. This occurred in spite of spontaneous reattachment of the root, suggesting that this reattachment did not fully restore the function of the native attachment. In contrast, the vertical longitudinal tear did not cause significant changes to the joint, with only mild differences compared with sham surgery at the 6-month time point. Conclusion: Given that the root tear, which severs circumferential connectivity and load transfer, caused more intense OA compared with the circumferentially stable vertical longitudinal tear, our findings suggest that without timely and mechanically competent fixation, root tears may cause irreversible joint damage. Clinical Relevance: More generally, this new model can serve as a test bed for experimental surgical, scaffold-based, and small molecule–driven interventions after injury to prevent OA progression.

Total disc replacement with an engineered substitute is a promising avenue for treating advanced intervertebral disc disease. Toward this goal, we developed cell-seeded disc-like angle ply structures (DAPS) and showed through in vitro studies that these constructs mature to match native disc composition, structure, and function with long-term culture. We then evaluated DAPS performance in an in vivo rat model of total disc replacement; over 5 weeks in vivo, DAPS maintained their structure, prevented intervertebral bony fusion, and matched native disc mechanical function at physiologic loads in situ. However, DAPS rapidly lost proteoglycan post-implantation and did not integrate into adjacent vertebrae. To address this, we modified the design to include polymer endplates to interface the DAPS with adjacent vertebrae, and showed that this modification mitigated in vivo proteoglycan loss while maintaining mechanical function and promoting integration. Together, these data demonstrate that cell-seeded engineered discs can replicate many characteristics of the native disc and are a viable option for total disc arthroplasty.

Degeneration of the intervertebral discs is a progressive cascade of cellular, compositional and structural changes that is frequently associated with low back pain. As the first signs of disc degeneration typically arise in the disc’s central nucleus pulposus (NP), augmentation of the NP via hydrogel injection represents a promising strategy to treat early to mid-stage degeneration. The purpose of this study was to establish the translational feasibility of a triple interpenetrating network hydrogel composed of dextran, chitosan, and teleostean (DCT) for augmentation of the degenerative NP in a preclinical goat model. Ex vivo injection of the DCT hydrogel into degenerated goat lumbar motion segments restored range of motion and neutral zone modulus towards physiologic values. To facilitate non-invasive assessment of hydrogel delivery and distribution, zirconia nanoparticles were added to make the hydrogel radiopaque. Importantly, the addition of zirconia did not negatively impact viability or matrix producing capacity of goat mesenchymal stem cells or NP cells seeded within the hydrogel in vitro. In vivo studies demonstrated that the radiopaque DCT hydrogel was successfully delivered to degenerated goat lumbar intervertebral discs, where it was distributed throughout both the NP and annulus fibrosus, and that the hydrogel remained contained within the disc space for two weeks without evidence of extrusion. These results demonstrate the translational potential of this hydrogel for functional regeneration of degenerate intervertebral discs. Statement of Significance The results of this work demonstrate that a radiopaque hydrogel is capable of normalizing the mechanical function of the degenerative disc, is supportive of disc cell and mesenchymal stem cell viability and matrix production, and can be maintained in the disc space without extrusion following intradiscal delivery in a preclinical large animal model. These results support evaluation of this hydrogel as a minimally invasive disc therapeutic in long-term preclinical studies as a precursor to future clinical application in patients with disc degeneration and low back pain.

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 the 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% to 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 of NaCl solutions, in particular after long-term (8 hr) incubation the modulus and equilibrium stress decreased compared to short-term (15 min) 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.

One of the most common bath solutions used in musculoskeletal mechanical testing is phosphate buffered saline (PBS). In tendon, swelling induced by physiological PBS results in decreased tendon modulus and induces microstructural changes. It is critical to evaluate the multiscale mechanical behavior of tendon under swelling to interpret prior work and provide information to design future studies. We compared the effects of physiological PBS and 8% polyethylene glycol and saline bathing solutions on tendon multiscale tendon mechanics and damage as well as microstructure with TEM in order to understand the effect of swelling on tendon. At the tissue level, tendons in PBS had a lower modulus than SPEG samples. PBS samples also showed an increased amount of non-recoverable sliding, which is an analog for microscale damage. SPEG had a higher microscale to tissue-scale strain ratio, showing the fibrils experienced less strain attenuation. From the TEM data, we showed the fibril spacing of SPEG samples was more similar to fresh control than PBS. We concluded that swelling alters multiscale mechanics and damage in addition to tendon microstructure. Future mechanical testing should consider using SPEG as a bath solution with an osmotic pressure which preserves fresh tissue water content.

Intervertebral disc degeneration is implicated as a leading cause of low back pain. Persistent, local inflammation within the disc nucleus pulposus (NP) and annulus fibrosus (AF) is an important mediator of disc degeneration and negatively impacts the performance of therapeutic stem cells. There is a lack of validated large animal models of disc degeneration that recapitulate clinically relevant local inflammation. We recently described a goat model of disc degeneration in which increasing doses of chondroitinase ABC (ChABC) were used to reproducibly induce a spectrum of degenerative changes. The objective of this study was to extend the clinical relevance of this model by establishing whether these degenerative changes are associated with the local expression of inflammatory cytokines and catabolic enzymes. Degeneration was induced in goat lumbar discs using ChABC at different doses. After 12 weeks, degeneration severity was determined histologically and using quantitative magnetic resonance imaging (MRI). Expression levels of inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], and IL-6) and catabolic enzymes (matrix metalloproteinases-1 [MMPs-1] and 13, and a disintegrin and metalloproteinase with thrombospondin type-1 motifs-4 [ADAMTS-4]) were assessed as the percentage of immunopositive cells in the NP and AF. With the exception of MMP-1, cytokine, and enzyme expression levels were significantly elevated in ChABC-treated discs in the NP and AF. Expression levels of TNF-α, IL1-β, and ADAMTS-4 were positively correlated with histological grade, while all cytokines and ADAMTS-4 were negatively correlated with MRI T2 and T1ρ scores. These results demonstrate that degenerate goat discs exhibit elevated expression of clinically relevant inflammatory mediators, and further validate this animal model as a platform for evaluating new therapeutic approaches for disc degeneration.

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.

The kinematics of the intervertebral disc are defined by six degrees of freedom (DOF): three translations (Tz: axial compression, Tx: lateral shear, and Ty: anterior‐posterior shear) and three rotations (Rz: torsion, Rx: flexion‐extension, and Ry: lateral bending). There is some evidence that the six DOFs are mechanically coupled, such that loading in one DOF affects the mechanics of the other five “off‐axis” DOFs, however, most studies have not controlled and/or measured all six DOFs simultaneously. Additionally, the relationships between disc geometry and disc mechanics are important for evaluation of data from different sized donor and patient discs. The objectives of this study were to quantify the mechanical behavior of the intervertebral disc in all six degrees of freedom (DOFs), measure the coupling between the applied motion in each DOF with the resulting off‐axis motions, and test the hypothesis that disc geometry influences these mechanical behaviors. All off‐axis displacements and rotations were significantly correlated with the applied DOF and were of similar magnitude as physiologically relevant motion, confirming that off‐axis coupling is an important mechanical response. Interestingly, there were pairs of DOFs that were especially strongly coupled: lateral shear (Tx) and lateral bending (Ry), anterior‐posterior shear (Ty) and flexion‐extension (Rx), and compression (Tz) and torsion (Rz). Large off‐axis shears may contribute to injury risk in bending and flexion. In addition, the disc responded to shear (Tx, Ty) and rotational loading (Rx, Ry, and Rz) by increasing in disc height in order to maintain the applied compressive load. Quantifying these mechanical behaviors across all six DOF are critical for designing and testing disc therapies, such as implants and tissue engineered constructs, and also for validating finite element models.

Tendon’s hierarchical structure allows for load transfer between its fibrillar elements at multiple length scales. Tendon microstructure is particularly important, because it includes the cells and their surrounding collagen fibrils, where mechanical interactions can have potentially important physiological and pathological contributions. However, the three-dimensional microstructure and the mechanisms of load transfer in that length scale are not known. It has been postulated that interfibrillar matrix shear or direct load transfer via the fusion/branching of small fibrils are responsible for load transfer, but the significance of these mechanisms is still unclear. Alternatively, the helical fibrils that occur at the microstructural scale in tendon may also mediate load transfer, however, these structures are not well studied due to the lack of a three-dimensional visualization of tendon microstructure. In this study, we used serial block-face scanning electron microscopy (SBF-SEM) to investigate the threedimensional microstructure of fibrils in rat tail tendon. We found that tendon fibrils have a complex architecture with many helically wrapped fibrils. We studied the mechanical implications of these helical structures using finite element modeling and found that frictional contact between helical fibrils can induce load transfer even in the absence of matrix bonding or fibril fusion/branching. This study is significant in that it provides a three-dimensional view of the tendon microstructure and suggests friction between helically wrapped fibrils as a mechanism for load transfer, which is an important aspect of tendon biomechanics.

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 (

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.

Geometry is an important indicator of disc mechanical function and degeneration. While the geometry and associated degenerative changes in the nucleus pulposus and the annulus fibrosus are well-defined, the geometry of the cartilage endplate (CEP) and its relationship to disc degeneration are unknown. The objectives of this study were to quantify CEP geometry in three dimensions using an MRI FLASH imaging sequence and evaluate relationships between CEP geometry and age, degeneration, spinal level, and overall disc geometry. To do so, we assessed the MRI-based measurements for accuracy and repeatability. Next, we measured CEP geometry across a larger sample set and correlated CEP geometric parameters to age, disc degeneration, level, and disc geometry. The MRI-based measures resulted in thicknesses (0.3-1 mm) that are comparable to prior measurements of CEP thickness. CEP thickness was greatest at the anterior/posterior (A/P) margins and smallest in the center. The CEP A/P thickness, axial area, and lateral width decreased with age but were not related to disc degeneration. Age-related, but not degeneration-related, changes in geometry suggest that the CEP may not follow the progression of disc degeneration. Ultimately, if the CEP undergoes significant geometric changes with aging and if these can be related to low back pain, a clinically feasible translation of the FLASH MRI-based measurement of CEP geometry presented in this study may prove a useful diagnostic tool. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1410-1417, 2016.

The inter-lamellar matrix (ILM) has an average thickness of less than 30 µm and lies between adjacent lamellae in the annulus fibrosus (AF). The microstructure and composition of the ILM have been studied in various anatomic regions of the disc; however, their contribution to AF mechanical properties and structural integrity is unknown. It was suggested that the ILM components, mainly elastic fibers and cross-bridges, play a role in providing mechanical integrity of the AF. Therefore, the manner in which they respond to different loadings and stabilize adjacent lamellae structure will influence AF tear formation and subsequent herniation. This review paper summarizes the composition, microstructure, and potential role of the ILM in the progression of disc herniation, clarifies the micromechanical properties of the ILM, and proposes critical areas for future studies. There are a number of unknown characteristics of the ILM, such as its mechanical role, impact on AF integrity, and ultrastructure of elastic fibers at the ILM-lamella boundary. Determining these characteristics will provide important information for tissue engineering, repair strategies, and the development of more-physiological computational models to study the initiation and propagation of AF tears that lead to herniation and degeneration. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1307-1315, 2016.

Tendon’s viscoelastic behaviors are important to the mechanical function and mechanobiology. When loaded in longitudinal tension, tendons often have a very large Poisson’s ratio (ν > 2) that exceeds the limit of incompressibility for isotropic material (ν = 0.5), indicating that tendon experiences volume loss, inducing poroelastic fluid exudation in the transverse direction. Therefore, transverse poroelasticity is an important contributor to tendon material behavior. Tendon hydraulic permeability which is required to evaluate the fluid flow contribution to viscoelasticity, is mostly unavailable, and where available, varies by several orders of magnitude. In this manuscript, we quantified the transverse poroelastic material parameters of rat tail tendon fascicles by conducting transverse osmotic loading experiments, in both tension and compression. We used a multi-start optimization method to evaluate the parameters based on biphasic finite element modeling. Our tendon samples had a transverse hydraulic permeability of 10(−4) to 10(−5) mm(4) (Ns)(−1) and showed a significant tension-compression nonlinearity in the transverse direction. Further, using these results, we predict hydraulic permeability during longitudinal (fiber-aligned) tensile loading, and the spatial distribution of fluid flow during osmotic loading. These results reveal novel aspects of tendon mechanics and can be used to study the physiomechanical response of tendon in response to mechanical loading.

Tissue engineering holds great promise for the treatment of advanced intervertebral disc degeneration. However, assessment of in vivo integration and mechanical function of tissue engineered disc replacements over the long term, in large animal models, will be necessary to advance clinical translation. To that end, we developed tissue engineered, endplate-modified, disc-like angle ply structures (eDAPS) sized for the rat caudal and goat cervical spines that recapitulate the hierarchical structure of the native disc. Here, we demonstrate functional maturation and integration of these eDAPS in a rat caudal disc replacement model, with compressive mechanical properties reaching native values after 20 weeks in vivo and evidence of functional integration under physiologic loads. To further this therapy towards clinical translation, we implanted eDAPS sized for the human cervical disc space in a goat cervical disc replacement model. Our results demonstrate maintenance of eDAPS composition and structure up to 8 weeks in vivo in the goat cervical disc space, and maturation of compressive mechanical properties to match native levels. These results demonstrate the translational feasibility of disc replacement with a tissue engineered construct for the treatment of advanced disc degeneration.

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.

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.

Inelastic behaviors, such as softening, a progressive decrease in modulus before failure, occur in tendon andare important aspect in degeneration and tendinopathy. These in elastic 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 in elasticity by using a recently developed reactive in elasticity model (RIE), which is a structurally-inspired continuum mechanics frame work that models tissue in elasticity based on the molecular bond kinetics. Using RIE, we formulated two material models, one specific toplastic deformation and the other to damage. The models were independently fit to published experimental tensiletests of rat tail tendons. We quantified the inelastic effects and compared the performance of the two models infitting 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-straincurves 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.

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. 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. 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. 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.